HMGB1 binds to activated platelets via the receptor for advanced glycation end products and is present in platelet rich human coronary artery thrombi.

PubMed

Ahrens, Ingo; Chen, Yung-Chih; Topcic, Danijal; Bode, Michael; Haenel, David; Hagemeyer, Christoph E; Seeba, Hannah; Duerschmied, Daniel; Bassler, Nicole; Jandeleit-Dahm, Karin A; Sweet, Matthew J; Agrotis, Alex; Bobik, Alex; Peter, Karlheinz

2015-11-01

High mobility group box 1 (HMGB1) acts as both a nuclear protein that regulates gene expression, as well as a pro-inflammatory alarmin that is released from necrotic or activated cells. Recently, HMGB1-expression in human atherosclerotic plaques was identified. Therapeutic blockade of HMGB1 reduced the development of diet-induced atherosclerosis in ApoE knockout mice. Thus, we hypothesised an interaction between HMGB1 and activated platelets. Binding of recombinant HMGB1 to platelets was assessed by flow cytometry. HMGB1 bound to thrombin-activated human platelets (MFI 2.49 vs 25.01, p=0.0079). Blood from wild-type, TLR4 and RAGE knockout mice was used to determine potential HMGB1 receptors on platelets. HMGB1 bound to platelets from wild type C57Bl6 (MFI 2.64 vs 20.3, p< 0.05), and TLR4-/- mice (MFI 2.11 vs 25.65, p< 0.05) but failed to show binding to platelets from RAGE-/- mice (p > 0.05). RAGE expression on human platelets was detected by RT-PCR with mRNA extracted from highly purified platelets and confirmed by Western blot and immunofluorescence microscopy. Platelet activation increased RAGE surface expression (MFI 4.85 vs 6.74, p< 0.05). Expression of HMGB1 in human coronary artery thrombi was demonstrated by immunohistochemistry and revealed high expression levels. Platelets bind HMGB1 upon thrombin-induced activation. Platelet specific expression of RAGE could be detected at the mRNA and protein level and is involved in the binding of HMGB1. Furthermore, platelet activation up-regulates platelet surface expression of RAGE. HMGB1 is highly expressed in platelet-rich human coronary artery thrombi pointing towards a central role for HMGB1 in atherothrombosis, thereby suggesting the possibility of platelet targeted anti-inflammatory therapies for atherothrombosis.

Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2.

PubMed

Konopatskaya, Olga; Matthews, Sharon A; Harper, Matthew T; Gilio, Karen; Cosemans, Judith M E M; Williams, Christopher M; Navarro, Maria N; Carter, Deborah A; Heemskerk, Johan W M; Leitges, Michael; Cantrell, Doreen; Poole, Alastair W

2011-07-14

Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

Compression force sensing regulates integrin αIIbβ3 adhesive function on diabetic platelets.

PubMed

Ju, Lining; McFadyen, James D; Al-Daher, Saheb; Alwis, Imala; Chen, Yunfeng; Tønnesen, Lotte L; Maiocchi, Sophie; Coulter, Brianna; Calkin, Anna C; Felner, Eric I; Cohen, Neale; Yuan, Yuping; Schoenwaelder, Simone M; Cooper, Mark E; Zhu, Cheng; Jackson, Shaun P

2018-03-14

Diabetes is associated with an exaggerated platelet thrombotic response at sites of vascular injury. Biomechanical forces regulate platelet activation, although the impact of diabetes on this process remains ill-defined. Using a biomembrane force probe (BFP), we demonstrate that compressive force activates integrin α IIb β 3 on discoid diabetic platelets, increasing its association rate with immobilized fibrinogen. This compressive force-induced integrin activation is calcium and PI 3-kinase dependent, resulting in enhanced integrin affinity maturation and exaggerated shear-dependent platelet adhesion. Analysis of discoid platelet aggregation in the mesenteric circulation of mice confirmed that diabetes leads to a marked enhancement in the formation and stability of discoid platelet aggregates, via a mechanism that is not inhibited by therapeutic doses of aspirin and clopidogrel, but is eliminated by PI 3-kinase inhibition. These studies demonstrate the existence of a compression force sensing mechanism linked to α IIb β 3 adhesive function that leads to a distinct prothrombotic phenotype in diabetes.

Influence of Pre-Analytical Factors on Thymus- and Activation-Regulated Chemokine Quantitation in Plasma

PubMed Central

Zhao, Xuemei; Delgado, Liliana; Weiner, Russell; Laterza, Omar F.

2015-01-01

Thymus- and activation-regulated chemokine (TARC) in serum/plasma associates with the disease activity of atopic dermatitis (AD), and is a promising tool for assessing the response to the treatment of the disease. TARC also exists within platelets, with elevated levels detectable in AD patients. We examined the effects of pre-analytical factors on the quantitation of TARC in human EDTA plasma. TARC levels in platelet-free plasma were significantly lower than those in platelet-containing plasma. After freeze-thaw, TARC levels increased in platelet-containing plasma, but remained unchanged in platelet-free plasma, suggesting TARC was released from the platelets during the freeze-thaw process. In contrast, TARC levels were stable in serum independent of freeze-thaw. These findings underscore the importance of pre-analytical factors to TARC quantitation. Plasma TARC levels should be measured in platelet-free plasma for accurate quantitation. Pre-analytical factors influence the quantitation, interpretation, and implementation of circulating TARC as a biomarker for the development of AD therapeutics. PMID:28936246

A systems approach to hemostasis: 3. Thrombus consolidation regulates intrathrombus solute transport and local thrombin activity

PubMed Central

Welsh, John D.; Tomaiuolo, Maurizio; Wu, Jie; Colace, Thomas V.; Diamond, Scott L.

2014-01-01

Hemostatic thrombi formed after a penetrating injury have a distinctive structure in which a core of highly activated, closely packed platelets is covered by a shell of less-activated, loosely packed platelets. We have shown that differences in intrathrombus molecular transport emerge in parallel with regional differences in platelet packing density and predicted that these differences affect thrombus growth and stability. Here we test that prediction in a mouse vascular injury model. The studies use a novel method for measuring thrombus contraction in vivo and a previously characterized mouse line with a defect in integrin αIIbβ3 outside-in signaling that affects clot retraction ex vivo. The results show that the mutant mice have a defect in thrombus consolidation following vascular injury, resulting in an increase in intrathrombus transport rates and, as predicted by computational modeling, a decrease in thrombin activity and platelet activation in the thrombus core. Collectively, these data (1) demonstrate that in addition to the activation state of individual platelets, the physical properties of the accumulated mass of adherent platelets is critical in determining intrathrombus agonist distribution and platelet activation and (2) define a novel role for integrin signaling in the regulation of intrathrombus transport rates and localization of thrombin activity. PMID:24951426

Distinct Pathways Regulate Syk Protein Activation Downstream of Immune Tyrosine Activation Motif (ITAM) and hemITAM Receptors in Platelets*

PubMed Central

Manne, Bhanu Kanth; Badolia, Rachit; Dangelmaier, Carol; Eble, Johannes A.; Ellmeier, Wilfried; Kahn, Mark; Kunapuli, Satya P.

2015-01-01

Tyrosine kinase pathways are known to play an important role in the activation of platelets. In particular, the GPVI and CLEC-2 receptors are known to activate Syk upon tyrosine phosphorylation of an immune tyrosine activation motif (ITAM) and hemITAM, respectively. However, unlike GPVI, the CLEC-2 receptor contains only one tyrosine motif in the intracellular domain. The mechanisms by which this receptor activates Syk are not completely understood. In this study, we identified a novel signaling mechanism in CLEC-2-mediated Syk activation. CLEC-2-mediated, but not GPVI-mediated, platelet activation and Syk phosphorylation were abolished by inhibition of PI3K, which demonstrates that PI3K regulates Syk downstream of CLEC-2. Ibrutinib, a Tec family kinase inhibitor, also completely abolished CLEC-2-mediated aggregation and Syk phosphorylation in human and murine platelets. Furthermore, embryos lacking both Btk and Tec exhibited cutaneous edema associated with blood-filled vessels in a typical lymphatic pattern similar to CLEC-2 or Syk-deficient embryos. Thus, our data show, for the first time, that PI3K and Tec family kinases play a crucial role in the regulation of platelet activation and Syk phosphorylation downstream of the CLEC-2 receptor. PMID:25767114

Regulating thrombus growth and stability to achieve an optimal response to injury

PubMed Central

Brass, Lawrence F.; Wannemacher, Kenneth M.; Ma, Peisong; Stalker, Timothy J.

2012-01-01

An optimal platelet response to injury can be defined as one in which blood loss is restrained and haemostasis is achieved without the penalty of further tissue damage caused by unwarranted vascular occlusion. This brief review considers some of the ways in which thrombus growth and stability can be regulated so that an optimal platelet response can be achieved in vivo. Three related topics are considered. The first focuses on intracellular mechanisms that regulate the early events of platelet activation downstream of G protein coupled receptors for agonists such as thrombin, thromboxane A2 and ADP. The second considers the ways in which signalling events that are dependent on stable contacts between platelets can influence the state of platelet activation and thus affect thrombus growth and stability. The third focuses on the changes that are experienced by platelets as they move from their normal environment in freely-flowing plasma to a very different environment within the growing haemostatic plug, an environment in which the narrowing gaps and junctions between platelets not only facilitate communication, but also increasingly limit both the penetration of plasma and the exodus of platelet-derived bioactive molecules. PMID:21781243

Platelet-derived-growth-factor-induced signalling in human platelets: phosphoinositide-3-kinase-dependent inhibition of platelet activation.

PubMed Central

Selheim, F; Fukami, M H; Holmsen, H; Vassbotn, F S

2000-01-01

Human platelets release platelet-derived growth factor (PDGF) from alpha-granules during platelet activation. We have previously shown that platelets have PDGF alpha-receptors, a transmembrane tyrosine kinase that takes part in negative feedback regulation during platelet activation. Here we have described a study of PDGF-induced tyrosine phosphorylation of platelet substrates and phosphoinositide 3-kinase (PI-3K) activity in collagen-stimulated platelets. By immunoblotting with phosphotyrosine antibodies of collagen-activated platelets we found that PDGF increased the phosphorylation of several platelet substrates, e.g. pp140, pp120 and pp85. PDGF inhibited collagen-induced platelet activation in the presence of inhibitors of autocrine stimulation, thus blocking the pure collagen-induced signal transduction. PDGF enhanced the collagen-induced formation of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) as measured by HPLC. Wortmannin and LY294002, two unrelated inhibitors of PI-3K, were used to investigate the role of PI-3K in PDGF-induced platelet signalling. Incubation of platelets with wortmannin and LY294002 blocked the formation of three phosphorylated inositides as well as the inhibitory effect of PDGF on collagen-induced platelet activation. We conclude that the inhibitory effect of PDGF on platelet activation is PI-3K dependent. This is the first demonstration of a negative regulatory function of 3-phosphorylated inositides in platelets. PMID:10947961

Platelet-derived-growth-factor-induced signalling in human platelets: phosphoinositide-3-kinase-dependent inhibition of platelet activation.

PubMed

Selheim, F; Fukami, M H; Holmsen, H; Vassbotn, F S

2000-09-01

Human platelets release platelet-derived growth factor (PDGF) from alpha-granules during platelet activation. We have previously shown that platelets have PDGF alpha-receptors, a transmembrane tyrosine kinase that takes part in negative feedback regulation during platelet activation. Here we have described a study of PDGF-induced tyrosine phosphorylation of platelet substrates and phosphoinositide 3-kinase (PI-3K) activity in collagen-stimulated platelets. By immunoblotting with phosphotyrosine antibodies of collagen-activated platelets we found that PDGF increased the phosphorylation of several platelet substrates, e.g. pp140, pp120 and pp85. PDGF inhibited collagen-induced platelet activation in the presence of inhibitors of autocrine stimulation, thus blocking the pure collagen-induced signal transduction. PDGF enhanced the collagen-induced formation of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) as measured by HPLC. Wortmannin and LY294002, two unrelated inhibitors of PI-3K, were used to investigate the role of PI-3K in PDGF-induced platelet signalling. Incubation of platelets with wortmannin and LY294002 blocked the formation of three phosphorylated inositides as well as the inhibitory effect of PDGF on collagen-induced platelet activation. We conclude that the inhibitory effect of PDGF on platelet activation is PI-3K dependent. This is the first demonstration of a negative regulatory function of 3-phosphorylated inositides in platelets.

Dual Regulation of Glycogen Synthase Kinase 3 (GSK3)α/β by Protein Kinase C (PKC)α and Akt Promotes Thrombin-mediated Integrin αIIbβ3 Activation and Granule Secretion in Platelets*

PubMed Central

Moore, Samantha F.; van den Bosch, Marion T. J.; Hunter, Roger W.; Sakamoto, Kei; Poole, Alastair W.; Hers, Ingeborg

2013-01-01

Glycogen synthase kinase-3 is a Ser/Thr kinase, tonically active in resting cells but inhibited by phosphorylation of an N-terminal Ser residue (Ser21 in GSK3α and Ser9 in GSK3β) in response to varied external stimuli. Recent work suggests that GSK3 functions as a negative regulator of platelet function, but how GSK3 is regulated in platelets has not been examined in detail. Here, we show that early thrombin-mediated GSK3 phosphorylation (0–30 s) was blocked by PKC inhibitors and largely absent in platelets from PKCα knock-out mice. In contrast, late (2–5 min) GSK3 phosphorylation was dependent on the PI3K/Akt pathway. Similarly, early thrombin-mediated inhibition of GSK3 activity was blocked in PKCα knock-out platelets, whereas the Akt inhibitor MK2206 reduced late thrombin-mediated GSK3 inhibition and largely prevented GSK3 inhibition in PKCα knock-out platelets. More importantly, GSK3 phosphorylation contributes to platelet function as knock-in mice where GSK3α Ser21 and GSK3β Ser9 were mutated to Ala showed a significant reduction in PAR4-mediated platelet aggregation, fibrinogen binding, and P-selectin expression, whereas the GSK3 inhibitor CHIR99021 enhanced these responses. Together, these results demonstrate that PKCα and Akt modulate platelet function by phosphorylating and inhibiting GSK3α/β, thereby relieving the negative effect of GSK3α/β on thrombin-mediated platelet activation. PMID:23239877

Deciphering of ADP-induced, phosphotyrosine-dependent signaling networks in human platelets by Src-homology 2 region (SH2)-profiling.

PubMed

Schweigel, Hardy; Geiger, Jörg; Beck, Florian; Buhs, Sophia; Gerull, Helwe; Walter, Ulrich; Sickmann, Albert; Nollau, Peter

2013-03-01

Tyrosine phosphorylation plays a central role in signal transduction controlling many important biological processes. In platelets, the activity of several signaling proteins is controlled by tyrosine phosphorylation ensuring proper platelet activation and aggregation essential for regulation of the delicate balance between bleeding and hemostasis. Here, we applied Src-homology 2 region (SH2)-profiling for deciphering of the phosphotyrosine state of human platelets activated by adenosine diphosphate (ADP). Applying a panel of 31 SH2-domains, rapid and complex regulation of the phosphotyrosine state of platelets was observed after ADP stimulation. Specific inhibition of platelet P2Y receptors by synthetic drugs revealed a major role for the P2Y1 receptor in tyrosine phosphorylation. Concomitant activation of protein kinase A (PKA) abolished ADP-induced tyrosine phosphorylation in a time and concentration-dependent manner. Given the fact that PKA activity is negatively regulated by the P2Y12 receptor, our data provide evidence for a novel link of synergistic control of the state of tyrosine phosphorylation by both P2Y receptors. By SH2 domain pull down and MS/MS analysis, we identified distinct tyrosine phosphorylation sites in cell adhesion molecules, intracellular adapter proteins and phosphatases suggesting a major, functional role of tyrosine phosphorylation of theses candidate proteins in ADP-dependent signaling in human platelets. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular mechanisms of platelet P2Y(12) receptor regulation.

PubMed

Cunningham, Margaret R; Nisar, Shaista P; Mundell, Stuart J

2013-02-01

Platelets are critical for haemostasis, however inappropriate activation can lead to the development of arterial thrombosis, which can result in heart attack and stroke. ADP is a key platelet agonist that exerts its actions via stimulation of two surface GPCRs (G-protein-coupled receptors), P2Y(1) and P2Y(12). Similar to most GPCRs, P2Y receptor activity is tightly regulated by a number of complex mechanisms including receptor desensitization, internalization and recycling. In the present article, we review the molecular mechanisms that underlie P2Y(1) and P2Y(12) receptor regulation, with particular emphasis on the structural motifs within the P2Y(12) receptor, which are required to maintain regulatory protein interaction. The implications of these findings for platelet responsiveness are also discussed.

Effects of the NO/soluble guanylate cyclase/cGMP system on the functions of human platelets.

PubMed

Makhoul, Stephanie; Walter, Elena; Pagel, Oliver; Walter, Ulrich; Sickmann, Albert; Gambaryan, Stepan; Smolenski, Albert; Zahedi, René P; Jurk, Kerstin

2018-06-01

Platelets are circulating sentinels of vascular integrity and are activated, inhibited, or modulated by multiple hormones, vasoactive substances or drugs. Endothelium- or drug-derived NO strongly inhibits platelet activation via activation of the soluble guanylate cyclase (sGC) and cGMP elevation, often in synergy with cAMP-elevation by prostacyclin. However, the molecular mechanisms and diversity of cGMP effects in platelets are poorly understood and sometimes controversial. Recently, we established the quantitative human platelet proteome, the iloprost/prostacyclin/cAMP/protein kinase A (PKA)-regulated phosphoproteome, and the interactions of the ADP- and iloprost/prostacyclin-affected phosphoproteome. We also showed that the sGC stimulator riociguat is in vitro a highly specific inhibitor, via cGMP, of various functions of human platelets. Here, we review the regulatory role of the cGMP/protein kinase G (PKG) system in human platelet function, and our current approaches to establish and analyze the phosphoproteome after selective stimulation of the sGC/cGMP pathway by NO donors and riociguat. Present data indicate an extensive and diverse NO/riociguat/cGMP phosphoproteome, which has to be compared with the cAMP phosphoproteome. In particular, sGC/cGMP-regulated phosphorylation of many membrane proteins, G-proteins and their regulators, signaling molecules, protein kinases, and proteins involved in Ca 2+ regulation, suggests that the sGC/cGMP system targets multiple signaling networks rather than a limited number of PKG substrate proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

PKCalpha regulates platelet granule secretion and thrombus formation in mice.

PubMed

Konopatskaya, Olga; Gilio, Karen; Harper, Matthew T; Zhao, Yan; Cosemans, Judith M E M; Karim, Zubair A; Whiteheart, Sidney W; Molkentin, Jeffery D; Verkade, Paul; Watson, Steve P; Heemskerk, Johan W M; Poole, Alastair W

2009-02-01

Platelets are central players in atherothrombosis development in coronary artery disease. The PKC family provides important intracellular mechanisms for regulating platelet activity, and platelets express several members of this family, including the classical isoforms PKCalpha and PKCbeta and novel isoforms PKCdelta and PKCtheta. Here, we used a genetic approach to definitively demonstrate the role played by PKCalpha in regulating thrombus formation and platelet function. Thrombus formation in vivo was attenuated in Prkca-/- mice, and PKCalpha was required for thrombus formation in vitro, although this PKC isoform did not regulate platelet adhesion to collagen. The ablation of in vitro thrombus formation in Prkca-/- platelets was rescued by the addition of ADP, consistent with the key mechanistic finding that dense-granule biogenesis and secretion depend upon PKCalpha expression. Furthermore, defective platelet aggregation in response to either collagen-related peptide or thrombin could be overcome by an increase in agonist concentration. Evidence of overt bleeding, including gastrointestinal and tail bleeding, was not seen in Prkca-/- mice. In summary, the effects of PKCalpha ablation on thrombus formation and granule secretion may implicate PKCalpha as a drug target for antithrombotic therapy.

The role of Nox1 and Nox2 in GPVI-dependent platelet activation and thrombus formation☆

PubMed Central

Walsh, T.G.; Berndt, M.C.; Carrim, N.; Cowman, J.; Kenny, D.; Metharom, P.

2014-01-01

Background Activation of the platelet-specific collagen receptor, glycoprotein (GP) VI, induces intracellular reactive oxygen species (ROS) production; however the relevance of ROS to GPVI-mediated platelet responses remains unclear. Objective The objective of this study was to explore the role of the ROS-producing NADPH oxidase (Nox)1 and 2 complexes in GPVI-dependent platelet activation and collagen-induced thrombus formation. Methods and results ROS production was measured by quantitating changes in the oxidation-sensitive dye, H2DCF-DA, following platelet activation with the GPVI-specific agonist, collagen related peptide (CRP). Using a pharmacological inhibitor specific for Nox1, 2-acetylphenothiazine (ML171), and Nox2 deficient mice, we show that Nox1 is the key Nox homolog regulating GPVI-dependent ROS production. Nox1, but not Nox2, was essential for CRP-dependent thromboxane (Tx)A2 production, which was mediated in part through p38 MAPK signaling; while neither Nox1 nor Nox2 was significantly involved in regulating CRP-induced platelet aggregation/integrin αIIbβ3 activation, platelet spreading, or dense granule and α-granule release (ATP release and P-selectin surface expression, respectively). Ex-vivo perfusion analysis of mouse whole blood revealed that both Nox1 and Nox2 were involved in collagen-mediated thrombus formation at arterial shear. Conclusion Together these results demonstrate a novel role for Nox1 in regulating GPVI-induced ROS production, which is essential for optimal p38 activation and subsequent TxA2 production, providing an explanation for reduced thrombus formation following Nox1 inhibition. PMID:24494191

Distinct pathways regulate Syk protein activation downstream of immune tyrosine activation motif (ITAM) and hemITAM receptors in platelets.

PubMed

Manne, Bhanu Kanth; Badolia, Rachit; Dangelmaier, Carol; Eble, Johannes A; Ellmeier, Wilfried; Kahn, Mark; Kunapuli, Satya P

2015-05-01

Tyrosine kinase pathways are known to play an important role in the activation of platelets. In particular, the GPVI and CLEC-2 receptors are known to activate Syk upon tyrosine phosphorylation of an immune tyrosine activation motif (ITAM) and hemITAM, respectively. However, unlike GPVI, the CLEC-2 receptor contains only one tyrosine motif in the intracellular domain. The mechanisms by which this receptor activates Syk are not completely understood. In this study, we identified a novel signaling mechanism in CLEC-2-mediated Syk activation. CLEC-2-mediated, but not GPVI-mediated, platelet activation and Syk phosphorylation were abolished by inhibition of PI3K, which demonstrates that PI3K regulates Syk downstream of CLEC-2. Ibrutinib, a Tec family kinase inhibitor, also completely abolished CLEC-2-mediated aggregation and Syk phosphorylation in human and murine platelets. Furthermore, embryos lacking both Btk and Tec exhibited cutaneous edema associated with blood-filled vessels in a typical lymphatic pattern similar to CLEC-2 or Syk-deficient embryos. Thus, our data show, for the first time, that PI3K and Tec family kinases play a crucial role in the regulation of platelet activation and Syk phosphorylation downstream of the CLEC-2 receptor. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Association of membrane/lipid rafts with the platelet cytoskeleton and the caveolin PY14: participation in the adhesion process.

PubMed

Cerecedo, Doris; Martínez-Vieyra, Ivette; Maldonado-García, Deneb; Hernández-González, Enrique; Winder, Steve J

2015-11-01

Platelets are the most prominent elements of blood tissue involved in hemostasis at sites of blood vessel injury. Platelet cytoskeleton is responsible for their shape modifications observed during activation and adhesion to the substratum; therefore the interactions between cytoskeleton and plasma membrane are critical to modulate blood platelet functions. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to membrane/lipid rafts (MLR) and regulate lateral diffusion of membrane proteins and lipids. Resting, thrombin-activated, and adherent human platelets were processed for biochemical studies including western-blot and immunprecipitation assays and confocal analysis were performed to characterize the interaction of MLR with the main cytoskeleton elements and β-dystroglycan as well as with the association of caveolin-1 PY14 with focal adhesion proteins. We transfected a megakaryoblast cell line (Meg-01) to deplete β-dystroglycan, subsequent to their differentiation to the platelet progenitors. Our data showed a direct interaction of the MLR with cytoskeleton to regulate platelet shape, while an association of caveolin-1 PY14 with vinculin is needed to establish focal adhesions, which are modulated for β-dystroglycan. In conclusion, caveolin-1 PY14 in association with platelet cytoskeleton participate in focal adhesions dynamics. © 2015 Wiley Periodicals, Inc.

The PPAR-Platelet Connection: Modulators of Inflammation and Potential Cardiovascular Effects

PubMed Central

Spinelli, S. L.; O'Brien, J. J.; Bancos, S.; Lehmann, G. M.; Springer, D. L.; Blumberg, N.; Francis, C. W.; Taubman, M. B.; Phipps, R. P.

2008-01-01

Historically, platelets were viewed as simple anucleate cells responsible for initiating thrombosis and maintaining hemostasis, but clearly they are also key mediators of inflammation and immune cell activation. An emerging body of evidence links platelet function and thrombosis to vascular inflammation. peroxisome proliferator-activated receptors (PPARs) play a major role in modulating inflammation and, interestingly, PPARs (PPARβ/δ and PPARγ) were recently identified in platelets. Additionally, PPAR agonists attenuate platelet activation; an important discovery for two reasons. First, activated platelets are formidable antagonists that initiate and prolong a cascade of events that contribute to cardiovascular disease (CVD) progression. Dampening platelet release of proinflammatory mediators, including CD40 ligand (CD40L, CD154), is essential to hinder this cascade. Second, understanding the biologic importance of platelet PPARs and the mechanism(s) by which PPARs regulate platelet activation will be imperative in designing therapeutic strategies lacking the deleterious or unwanted side effects of current treatment options. PMID:18288284

Quantitative Glycoproteomic Analysis Identifies Platelet-Induced Increase of Monocyte Adhesion via the Up-Regulation of Very Late Antigen 5.

PubMed

Huang, Jiqing; Kast, Juergen

2015-08-07

Physiological stimuli, such as thrombin, or pathological stimuli, such as lysophosphatidic acid (LPA), activate platelets circulating in blood. Once activated, platelets bind to monocytes via P-selectin-PSGL-1 interactions but also release the stored contents of their granules. These platelet releasates, in addition to direct platelet binding, activate monocytes and facilitate their recruitment to atherosclerotic sites. Consequently, understanding the changes platelet releasates induce in monocyte membrane proteins is critical. We studied the glyco-proteome changes of THP-1 monocytic cells affected by LPA- or thrombin-induced platelet releasates. We employed lectin affinity chromatography combined with filter aided sample preparation to achieve high glyco- and membrane protein and protein sequence coverage. Using stable isotope labeling by amino acids in cell culture, we quantified 1715 proteins, including 852 membrane and 500 glycoproteins, identifying the up-regulation of multiple proteins involved in monocyte extracellular matrix binding and transendothelial migration. Flow cytometry indicated expression changes of integrin α5, integrin β1, PECAM-1, and PSGL-1. The observed increase in monocyte adhesion to fibronectin was determined to be mediated by the up-regulation of very late antigen 5 via a P-selectin-PSGL-1 independent mechanism. This novel aspect could be validated on CD14+ human primary monocytes, highlighting the benefits of the improved enrichment method regarding high membrane protein coverage and reliable quantification.

Relationship between Platelet PPARs, cAMP Levels, and P-Selectin Expression: Antiplatelet Activity of Natural Products.

PubMed

Fuentes, Eduardo; Palomo, Iván

2013-01-01

Platelets are no longer considered simply as cells participating in thrombosis. In atherosclerosis, platelets are regulators of multiple processes, with the recruitment of inflammatory cells towards the lesion sites, inflammatory mediators release, and regulation of endothelial function. The antiplatelet therapy has been used for a long time in an effort to prevent and treat cardiovascular diseases. However, limited efficacy in some patients, drug resistance, and side effects are limitations of current antiplatelet therapy. In this context, a large number of natural products (polyphenols, terpenoids, alkaloids, and fatty acids) have been reported with antiplatelet activity. In this sense, the present paper describes mechanisms of antiplatelet action of natural products on platelet P-selectin expression through cAMP levels and its role as peroxisome proliferator-activated receptors agonists.

DREAM plays an important role in platelet activation and thrombogenesis

PubMed Central

Kim, Kyungho; Tseng, Alan; Barazia, Andrew; Italiano, Joseph E.

2017-01-01

Downstream regulatory element antagonist modulator (DREAM), a transcriptional repressor, is known to modulate pain responses. However, it is unknown whether DREAM is expressed in anucleate platelets and plays a role in thrombogenesis. By using intravital microscopy with DREAM-null mice and their bone marrow chimeras, we demonstrated that both hematopoietic and nonhematopoietic cell DREAMs are required for platelet thrombus formation following laser-induced arteriolar injury. In a FeCl3-induced thrombosis model, we found that compared with wild-type (WT) control and nonhematopoietic DREAM knockout (KO) mice, DREAM KO control and hematopoietic DREAM KO mice showed a significant delay in time to occlusion. Tail bleeding time was prolonged in DREAM KO control mice, but not in WT or DREAM bone marrow chimeric mice. In vivo adoptive transfer experiments further indicated the importance of platelet DREAM in thrombogenesis. We found that DREAM deletion does not alter the ultrastructural features of platelets but significantly impairs platelet aggregation and adenosine triphosphate secretion induced by numerous agonists (collagen-related peptide, adenosine 5′-diphosphate, A23187, thrombin, or U46619). Biochemical studies revealed that platelet DREAM positively regulates phosphoinositide 3-kinase (PI3K) activity during platelet activation. Using DREAM-null platelets and PI3K isoform-specific inhibitors, we observed that platelet DREAM is important for α-granule secretion, Ca2+ mobilization, and aggregation through PI3K class Iβ (PI3K-Iβ). Genetic and pharmacological studies in human megakaryoblastic MEG-01 cells showed that DREAM is important for A23187-induced Ca2+ mobilization and its regulatory function requires Ca2+ binding and PI3K-Iβ activation. These results suggest that platelet DREAM regulates PI3K-Iβ activity and plays an important role during thrombus formation. PMID:27903531

Human Cancer and Platelet Interaction, a Potential Therapeutic Target.

PubMed

Wang, Shike; Li, Zhenyu; Xu, Ren

2018-04-20

Cancer patients experience a four-fold increase in thrombosis risk, indicating that cancer development and progression are associated with platelet activation. Xenograft experiments and transgenic mouse models further demonstrate that platelet activation and platelet-cancer cell interaction are crucial for cancer metastasis. Direct or indirect interaction of platelets induces cancer cell plasticity and enhances survival and extravasation of circulating cancer cells during dissemination. In vivo and in vitro experiments also demonstrate that cancer cells induce platelet aggregation, suggesting that platelet-cancer interaction is bidirectional. Therefore, understanding how platelets crosstalk with cancer cells may identify potential strategies to inhibit cancer metastasis and to reduce cancer-related thrombosis. Here, we discuss the potential function of platelets in regulating cancer progression and summarize the factors and signaling pathways that mediate the cancer cell-platelet interaction.

Platelets regulate lymphatic vascular development through CLEC-2-SLP-76 signaling.

PubMed

Bertozzi, Cara C; Schmaier, Alec A; Mericko, Patricia; Hess, Paul R; Zou, Zhiying; Chen, Mei; Chen, Chiu-Yu; Xu, Bin; Lu, Min-min; Zhou, Diane; Sebzda, Eric; Santore, Matthew T; Merianos, Demetri J; Stadtfeld, Matthias; Flake, Alan W; Graf, Thomas; Skoda, Radek; Maltzman, Jonathan S; Koretzky, Gary A; Kahn, Mark L

2010-07-29

Although platelets appear by embryonic day 10.5 in the developing mouse, an embryonic role for these cells has not been identified. The SYK-SLP-76 signaling pathway is required in blood cells to regulate embryonic blood-lymphatic vascular separation, but the cell type and molecular mechanism underlying this regulatory pathway are not known. In the present study we demonstrate that platelets regulate lymphatic vascular development by directly interacting with lymphatic endothelial cells through C-type lectin-like receptor 2 (CLEC-2) receptors. PODOPLANIN (PDPN), a transmembrane protein expressed on the surface of lymphatic endothelial cells, is required in nonhematopoietic cells for blood-lymphatic separation. Genetic loss of the PDPN receptor CLEC-2 ablates PDPN binding by platelets and confers embryonic lymphatic vascular defects like those seen in animals lacking PDPN or SLP-76. Platelet factor 4-Cre-mediated deletion of Slp-76 is sufficient to confer lymphatic vascular defects, identifying platelets as the cell type in which SLP-76 signaling is required to regulate lymphatic vascular development. Consistent with these genetic findings, we observe SLP-76-dependent platelet aggregate formation on the surface of lymphatic endothelial cells in vivo and ex vivo. These studies identify a nonhemostatic pathway in which platelet CLEC-2 receptors bind lymphatic endothelial PDPN and activate SLP-76 signaling to regulate embryonic vascular development.

RNA sequencing enables systematic identification of platelet transcriptomic alterations in NSCLC patients.

PubMed

Zhang, Qun; Hu, Huan; Liu, Hongda; Jin, Jiajia; Zhu, Peiyuan; Wang, Shujun; Shen, Kaikai; Hu, Yangbo; Li, Zhou; Zhan, Ping; Zhu, Suhua; Fan, Hang; Zhang, Jianya; Lv, Tangfeng; Song, Yong

2018-05-29

Platelets are implicated as key players in the metastatic dissemination of tumor cells. Previous evidence demonstrated platelets retained cytoplasmic RNAs with physiologically activity, splicing pre-mRNA to mRNA and translating into functional proteins in response to external stimulation. Recently, platelets gene profile of healthy or diseased individuals were characterized with the help of RNA sequencing (RNA-Seq) in some studies, leading to new insights into the mechanisms underlying disease pathogenesis. In this study, we performed RNA-seq in platelets from 7 healthy individuals and 15 non-small cell lung cancer (NSCLC) patients. Our data revealed a subset of near universal differently expressed gene (DEG) profiles in platelets of metastatic NSCLC compared to healthy individuals, including 626 up-regulated RNAs (mRNAs and ncRNAs) and 1497 down-regulated genes. The significant over-expressed genes showed enrichment in focal adhesion, platelets activation, gap junction and adherens junction pathways. The DEGs also included previously reported tumor-related genes such as PDGFR, VEGF, EGF, etc., verifying the consistence and significance of platelet RNA-Seq in oncology study. We also validated several up-regulated DEGs involved in tumor cell-induced platelet aggregation (TCIPA) and tumorigenesis. Additionally, transcriptomic comparison analyses of NSCLC subgroups were conducted. Between non-metastatic and metastatic NSCLC patients, 526 platelet DEGs were identified with the most altered expression. The outcomes from subgroup analysis between lung adenocarcinoma and lung squamous cell carcinoma demonstrated the diagnostic potential of platelet RNA-Seq on distinguishing tumor histological types. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

A dual role for integrin-linked kinase in platelets: regulating integrin function and α-granule secretion

PubMed Central

Sage, Tanya; Stevens, Joanne M.; Jordan, Peter A.; Jones, Sarah; Barrett, Natasha E.; St-Arnaud, Rene; Frampton, Jonathan; Dedhar, Shoukat; Gibbins, Jonathan M.

2008-01-01

Integrin-linked kinase (ILK) has been implicated in the regulation of a range of fundamental biological processes such as cell survival, growth, differentiation, and adhesion. In platelets ILK associates with β1- and β3-containing integrins, which are of paramount importance for the function of platelets. Upon stimulation of platelets this association with the integrins is increased and ILK kinase activity is up-regulated, suggesting that ILK may be important for the coordination of platelet responses. In this study a conditional knockout mouse model was developed to examine the role of ILK in platelets. The ILK-deficient mice showed an increased bleeding time and volume, and despite normal ultrastructure the function of ILK-deficient platelets was decreased significantly. This included reduced aggregation, fibrinogen binding, and thrombus formation under arterial flow conditions. Furthermore, although early collagen stimulated signaling such as PLCγ2 phosphorylation and calcium mobilization were unaffected in ILK-deficient platelets, a selective defect in α-granule, but not dense-granule, secretion was observed. These results indicate that as well as involvement in the control of integrin affinity, ILK is required for α-granule secretion and therefore may play a central role in the regulation of platelet function. PMID:18772455

PPARγ ligands decrease hydrostatic pressure-induced platelet aggregation and proinflammatory activity.

PubMed

Rao, Fang; Yang, Ren-Qiang; Chen, Xiao-Shu; Xu, Jin-Song; Fu, Hui-Min; Su, Hai; Wang, Ling

2014-01-01

Hypertension is known to be associated with platelet overactivity, but the direct effects of hydrostatic pressure on platelet function remain unclear. The present study sought to investigate whether elevated hydrostatic pressure is responsible for platelet activation and to address the potential role of peroxisome proliferator-activated receptor-γ (PPARγ). We observed that hypertensive patients had significantly higher platelet volume and rate of ADP-induced platelets aggregation compared to the controls. In vitro, Primary human platelets were cultured under standard (0 mmHg) or increased (120, 180, 240 mmHg) hydrostatic pressure for 18 h. Exposure to elevated pressure was associated with morphological changes in platelets. Platelet aggregation and PAC-1 (the active confirmation of GPIIb/IIIa) binding were increased, CD40L was translocated from cytoplasm to the surface of platelet and soluble CD40L (sCD40L) was released into the medium in response to elevated hydrostatic pressure (180 and 240 mmHg). The PPARγ activity was up-regulated as the pressure was increased from 120 mmHg to 180 mmHg. Pressure-induced platelet aggregation, PAC-1 binding, and translocation and release of CD40L were all attenuated by the PPARγ agonist Thiazolidinediones (TZDs). These results demonstrate that platelet activation and aggregation are increased by exposure to elevated pressure and that PPARγ may modulate platelet activation induced by high hydrostatic pressure.

PPARγ Ligands Decrease Hydrostatic Pressure-Induced Platelet Aggregation and Proinflammatory Activity

PubMed Central

Chen, Xiao-Shu; Xu, Jin-Song; Fu, Hui-Min; Su, Hai; Wang, Ling

2014-01-01

Hypertension is known to be associated with platelet overactivity, but the direct effects of hydrostatic pressure on platelet function remain unclear. The present study sought to investigate whether elevated hydrostatic pressure is responsible for platelet activation and to address the potential role of peroxisome proliferator-activated receptor-γ (PPARγ). We observed that hypertensive patients had significantly higher platelet volume and rate of ADP-induced platelets aggregation compared to the controls. In vitro, Primary human platelets were cultured under standard (0 mmHg) or increased (120, 180, 240 mmHg) hydrostatic pressure for 18 h. Exposure to elevated pressure was associated with morphological changes in platelets. Platelet aggregation and PAC-1 (the active confirmation of GPIIb/IIIa) binding were increased, CD40L was translocated from cytoplasm to the surface of platelet and soluble CD40L (sCD40L) was released into the medium in response to elevated hydrostatic pressure (180 and 240 mmHg). The PPARγ activity was up-regulated as the pressure was increased from 120 mmHg to 180 mmHg. Pressure-induced platelet aggregation, PAC-1 binding, and translocation and release of CD40L were all attenuated by the PPARγ agonist Thiazolidinediones (TZDs). These results demonstrate that platelet activation and aggregation are increased by exposure to elevated pressure and that PPARγ may modulate platelet activation induced by high hydrostatic pressure. PMID:24586940

Role of IκB kinase β in regulating the remodeling of the CARMA1-Bcl10-MALT1 complex.

PubMed

Karim, Zubair A; Hensch, Nicole R; Qasim, Hanan; Alshbool, Fatima Z; Khasawneh, Fadi T

2018-06-02

The current work investigates the notion that inducible clustering of signaling mediators of the IKK pathway is important for platelet activation. Thus, while the CARMA1, Bcl10, and MALT1 (CBM) complex is essential for triggering IKK/NF-κB activation upon platelet stimulation, the signals that elicit its formation and downstream effector activation remain elusive. We demonstrate herein that IKKβ is involved in membrane fusion, and serves as a critical protein kinase required for initial formation and the regulation of the CARMA1/MALT1/Bcl10/CBM complex in platelets. We also show that IKKβ regulates these processes via modulation of phosphorylation of Bcl10 and IKKγ polyubiquitination. Collectively, our data demonstrate that IKKβ regulates membrane fusion and the remodeling of the CBM complex formation. Copyright © 2018 Elsevier Inc. All rights reserved.

Relationship between Platelet PPARs, cAMP Levels, and P-Selectin Expression: Antiplatelet Activity of Natural Products

PubMed Central

Fuentes, Eduardo; Palomo, Iván

2013-01-01

Platelets are no longer considered simply as cells participating in thrombosis. In atherosclerosis, platelets are regulators of multiple processes, with the recruitment of inflammatory cells towards the lesion sites, inflammatory mediators release, and regulation of endothelial function. The antiplatelet therapy has been used for a long time in an effort to prevent and treat cardiovascular diseases. However, limited efficacy in some patients, drug resistance, and side effects are limitations of current antiplatelet therapy. In this context, a large number of natural products (polyphenols, terpenoids, alkaloids, and fatty acids) have been reported with antiplatelet activity. In this sense, the present paper describes mechanisms of antiplatelet action of natural products on platelet P-selectin expression through cAMP levels and its role as peroxisome proliferator-activated receptors agonists. PMID:24324520

Novel insights into the regulation of cyclooxygenase-2 expression by platelet-cancer cell cross-talk

PubMed Central

Dovizio, Melania; Alberti, Sara; Sacco, Angela; Guillem-Llobat, Paloma; Schiavone, Simone; Maier, Thorsten J.; Steinhilber, Dieter; Patrignani, Paola

2015-01-01

Platelets are activated by the interaction with cancer cells and release enhanced levels of lipid mediators [such as thromboxane (TX)A2 and prostaglandin (PG)E2, generated from arachidonic acid (AA) by the activity of cyclooxygenase (COX)-1], granule content, including ADP and growth factors, chemokines, proteases and Wnt proteins. Moreover, activated platelets shed different vesicles, such as microparticles (MPs) and exosomes (rich in genetic material such as mRNAs and miRNAs). These platelet-derived products induce several phenotypic changes in cancer cells which confer high metastatic capacity. A central event involves an aberrant expression of COX-2 which influences cell-cycle progression and contribute to the acquisition of a cell migratory phenotype through the induction of epithelial mesenchymal transition genes and down-regulation of E-cadherin expression. The identification of novel molecular determinants involved in the cross-talk between platelets and cancer cells has led to identify novel targets for anti-cancer drug development. PMID:26551717

Platelet Surface-Associated Activation and Secretion-Mediated Inhibition of Coagulation Factor XII

PubMed Central

Zakharova, Natalia V.; Artemenko, Elena O.; Podoplelova, Nadezhda A.; Sveshnikova, Anastasia N.; Demina, Irina A.; Ataullakhanov, Fazly I.; Panteleev, Mikhail A.

2015-01-01

Coagulation factor XII (fXII) is important for arterial thrombosis, but its physiological activation mechanisms are unclear. In this study, we elucidated the role of platelets and platelet-derived material in fXII activation. FXII activation was only observed upon potent platelet stimulation (with thrombin, collagen-related peptide, or calcium ionophore, but not ADP) accompanied by phosphatidylserine exposure and was localised to the platelet surface. Platelets from three patients with grey platelet syndrome did not activate fXII, which suggests that platelet-associated fXII-activating material might be released from α-granules. FXII was preferentially bound by phosphotidylserine-positive platelets and annexin V abrogated platelet-dependent fXII activation; however, artificial phosphotidylserine/phosphatidylcholine microvesicles did not support fXII activation under the conditions herein. Confocal microscopy using DAPI as a poly-phosphate marker did not reveal poly-phosphates associated with an activated platelet surface. Experimental data for fXII activation indicates an auto-inhibition mechanism (k i/k a = 180 molecules/platelet). Unlike surface-associated fXII activation, platelet secretion inhibited activated fXII (fXIIa), particularly due to a released C1-inhibitor. Platelet surface-associated fXIIa formation triggered contact pathway-dependent clotting in recalcified plasma. Computer modelling suggests that fXIIa inactivation was greatly decreased in thrombi under high blood flow due to inhibitor washout. Combined, the surface-associated fXII activation and its inhibition in solution herein may be regarded as a flow-sensitive regulator that can shift the balance between surface-associated clotting and plasma-dependent inhibition, which may explain the role of fXII at high shear and why fXII is important for thrombosis but negligible in haemostasis. PMID:25688860

Glutamate mediates platelet activation through the AMPA receptor

PubMed Central

Morrell, Craig N.; Sun, Henry; Ikeda, Masahiro; Beique, Jean-Claude; Swaim, Anne Marie; Mason, Emily; Martin, Tanika V.; Thompson, Laura E.; Gozen, Oguz; Ampagoomian, David; Sprengel, Rolf; Rothstein, Jeffrey; Faraday, Nauder; Huganir, Richard; Lowenstein, Charles J.

2008-01-01

Glutamate is an excitatory neurotransmitter that binds to the kainate receptor, the N-methyl-D-aspartate (NMDA) receptor, and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR). Each receptor was first characterized and cloned in the central nervous system (CNS). Glutamate is also present in the periphery, and glutamate receptors have been identified in nonneuronal tissues, including bone, heart, kidney, pancreas, and platelets. Platelets play a central role in normal thrombosis and hemostasis, as well as contributing greatly to diseases such as stroke and myocardial infarction. Despite the presence of glutamate in platelet granules, the role of glutamate during hemostasis is unknown. We now show that activated platelets release glutamate, that platelets express AMPAR subunits, and that glutamate increases agonist-induced platelet activation. Furthermore, we demonstrate that glutamate binding to the AMPAR increases intracellular sodium concentration and depolarizes platelets, which are important steps in platelet activation. In contrast, platelets treated with the AMPAR antagonist CNQX or platelets derived from GluR1 knockout mice are resistant to AMPA effects. Importantly, mice lacking GluR1 have a prolonged time to thrombosis in vivo. Our data identify glutamate as a regulator of platelet activation, and suggest that the AMPA receptor is a novel antithrombotic target. PMID:18283118

The antiplatelet activity of magnolol is mediated by PPAR-β/γ.

PubMed

Shih, Ching-Yu; Chou, Tz-Chong

2012-09-15

Activation of peroxisome proliferator-activated receptor (PPAR) isoforms (α, β/δ, and γ) is known to inhibit platelet aggregation. In the present study, we examined whether PPARs-mediated pathways contribute to the antiplatelet activity of magnolol, a compound purified from Magnolia officinalis. Magnolol (20-60 μM) dose-dependently enhanced the activity and intracellular level of PPAR-β/γ in platelets. In the presence of selective PPAR-β antagonist (GSK0660) or PPAR-γ antagonist (GW9662), the inhibition of magnolol on collagen-induced platelet aggregation and intracellular Ca(2+) mobilization was significantly reversed. Moreover, magnolol-mediated up-regulation of NO/cyclic GMP/PKG pathway and Akt phosphorylation leading to increase of eNOS activity were markedly abolished by blocking PPAR-β/γ activity. Additionally, magnolol significantly inhibited collagen-induced PKCα activation through a PPAR-β/γ and PKCα interaction manner. The arachidonic acid (AA) or collagen-induced thromboxane B(2) formation and elevation of COX-1 activity caused by AA were also markedly attenuated by magnolol. However, these above effects of magnolol on platelet responses were strongly reduced by simultaneous addition of GSK0660 or GW9662, suggesting that PPAR-β/γ-mediated processes may account for magnolol-regulated antiplatelet mechanisms. Similarly, administration of PPAR-β/γ antagonists remarkably abolished the actions of magnolol in preventing platelet plug formation and prolonging bleeding time in mice. Taken together, we demonstrate for the first time that the antiplatelet and anti-thrombotic activities of magnolol are modulated by up-regulation of PPAR-β/γ-dependent pathways. Copyright © 2012 Elsevier Inc. All rights reserved.

Megakaryocytic Smad4 Regulates Platelet Function through Syk and ROCK2 Expression.

PubMed

Wang, Yanhua; Jiang, Lirong; Mo, Xi; Lan, Yu; Yang, Xiao; Liu, Xinyi; Zhang, Jian; Zhu, Li; Liu, Junling; Wu, Xiaolin

2017-09-01

Smad4, a key transcription factor in the transforming growth factor- β signaling pathway, is involved in a variety of cell physiologic and pathologic processes. Here, we characterized megakaryocyte/platelet-specific Smad4 deficiency in mice to elucidate its effect on platelet function. We found that megakaryocyte/platelet-specific loss of Smad4 caused mild thrombocytopenia and significantly extended first occlusion time and tail bleeding time in mice. Smad4-deficient platelets showed reduced agonist-induced platelet aggregation. Further studies showed that a severe defect was seen in integrin α IIb β 3 -mediated bidirectional (inside-out and outside-in) signaling in Smad4-deficient platelets, as evidenced by reduced fibrinogen binding and α -granule secretion, suppressed platelet spreading and clot retraction. Microarray analysis showed that the expression levels of multiple genes were altered in Smad4-deficient platelets. Among these genes, spleen tyrosine kinase (Syk) and Rho-associated coiled-coil containing protein kinase 2 (ROCK2) were downregulated several times as confirmed by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Further research showed that Smad4 directly regulates ROCK2 transcription but indirectly regulates Syk. Megakaryocyte/platelet-specific Smad4 deficiency caused decreased expression levels of Syk and ROCK2 in platelets. These results suggest potential links among Smad4 deficiency, attenuated Syk, and ROCK2 expression and defective platelet activation. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Elucidation of flow-mediated tumour cell-induced platelet aggregation using an ultrasound standing wave trap.

PubMed

Bazou, D; Santos-Martinez, M J; Medina, C; Radomski, M W

2011-04-01

Tumour cells activate and aggregate platelets [tumour cell-induced platelet aggregation (TCIPA)] and this process plays an important role in the successful metastasis of cancer cells. To date, most studies on TCIPA have been conducted under no-flow conditions. In this study, we have investigated TCIPA in real time under flow conditions, using an ultrasound standing wave trap that allows formation and levitation of cancer cell clusters in suspension, thus mimicking the conditions generated by flowing blood. Using 59M adenocarcinoma and HT1080 fibrosarcoma cells and human platelets, cancer cell cluster-platelet aggregates were imaged in real time using epi-fluorescence microscopy (F-actin) and investigated in detail using confocal microscopy (matrix metalloproteinase-2-GPIIb/IIIa co-localization) and scanning electron and helium-ion microscopy (<1 nm resolution). The release of gelatinases from aggregates was studied using zymography. We found that platelet activation and aggregation takes place on the surface of cancer cells (TCIPA), leading to time-dependent disruption of cancer cell clusters. Pharmacological modulation of TCIPA revealed that EDTA, prostacyclin, o-phenanthroline and apyrase significantly down-regulated TCIPA and, in turn, delayed cell cluster disruption, However, EGTA and aspirin were ineffective. Pharmacological inhibition of TCIPA correlated with the down-regulation of platelet activation as shown by flow-cytometry assay of platelet P-selectin. Our results show for the first time, that during TCIPA, platelet activation disrupts cancer cell clusters and this can contribute to metastasis. Thus, selective targeting of platelet aggregate-cancer cell clusters may be an important strategy to control metastasis. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Elucidation of flow-mediated tumour cell-induced platelet aggregation using an ultrasound standing wave trap

PubMed Central

Bazou, D; Santos-Martinez, MJ; Medina, C; Radomski, MW

2011-01-01

BACKGROUND AND PURPOSE Tumour cells activate and aggregate platelets [tumour cell-induced platelet aggregation (TCIPA)] and this process plays an important role in the successful metastasis of cancer cells. To date, most studies on TCIPA have been conducted under no-flow conditions. In this study, we have investigated TCIPA in real time under flow conditions, using an ultrasound standing wave trap that allows formation and levitation of cancer cell clusters in suspension, thus mimicking the conditions generated by flowing blood. EXPERIMENTAL APPROACH Using 59M adenocarcinoma and HT1080 fibrosarcoma cells and human platelets, cancer cell cluster–platelet aggregates were imaged in real time using epi-fluorescence microscopy (F-actin) and investigated in detail using confocal microscopy (matrix metalloproteinase-2-GPIIb/IIIa co-localization) and scanning electron and helium-ion microscopy (<1 nm resolution). The release of gelatinases from aggregates was studied using zymography. KEY RESULTS We found that platelet activation and aggregation takes place on the surface of cancer cells (TCIPA), leading to time-dependent disruption of cancer cell clusters. Pharmacological modulation of TCIPA revealed that EDTA, prostacyclin, o-phenanthroline and apyrase significantly down-regulated TCIPA and, in turn, delayed cell cluster disruption, However, EGTA and aspirin were ineffective. Pharmacological inhibition of TCIPA correlated with the down-regulation of platelet activation as shown by flow-cytometry assay of platelet P-selectin. CONCLUSION AND IMPLICATIONS Our results show for the first time, that during TCIPA, platelet activation disrupts cancer cell clusters and this can contribute to metastasis. Thus, selective targeting of platelet aggregate–cancer cell clusters may be an important strategy to control metastasis. PMID:21182493

Rho Associated Coiled-Coil Kinase-1 Regulates Collagen-Induced Phosphatidylserine Exposure in Platelets

PubMed Central

Dasgupta, Swapan K.; Le, Anhquyen; Haudek, Sandra B.; Entman, Mark L.; Rumbaut, Rolando E.; Thiagarajan, Perumal

2013-01-01

Background The transbilayer movement of phosphatidylserine mediates the platelet procoagulant activity during collagen stimulation. The Rho-associated coiled-coil kinase (ROCK) inhibitor Y-27632 inhibits senescence induced but not activation induced phosphatidylserine exposure. To investigate further the specific mechanisms, we now utilized mice with genetic deletion of the ROCK1 isoform. Methods and Results ROCK1-deficient mouse platelets expose significantly more phosphatidylserine and generate more thrombin upon activation with collagen compared to wild-type platelets. There were no significant defects in platelet shape change, aggregation, or calcium response compared to wild-type platelets. Collagen-stimulated ROCK1-deficient platelets also displayed decreased phosphorylation levels of Lim Kinase-1 and cofilin-1. However, there was no reduction in phosphorylation levels of myosin phosphatase subunit-1 (MYPT1) or myosin light chain (MLC). In an in vivo light/dye-induced endothelial injury/thrombosis model, ROCK1-deficient mice presented a shorter occlusion time in cremasteric venules when compared to wild-type littermates (3.16 ± 1.33 min versus 6.6 ± 2.6 min; p = 0.01). Conclusions These studies define ROCK1 as a new regulator for collagen-induced phosphatidylserine exposure in platelets with functional consequences on thrombosis. This effect was downstream of calcium signaling and was mediated by Lim Kinase-1 / cofilin-1-induced cytoskeletal changes. PMID:24358370

Impaired Production and Diurnal Regulation of Vascular RvDn-3 DPA Increase Systemic Inflammation and Cardiovascular Disease.

PubMed

Colas, Romain A; Souza, Patricia R; Walker, Mary E; Burton, Maudrian; Zasłona, Zbigniew; Curtis, Annie M; Marques, Raquel M; Dalli, Jesmond

2018-03-16

Diurnal mechanisms are central to regulating host responses. Recent studies uncovered a novel family of mediators termed as specialized proresolving mediators that terminate inflammation without interfering with the immune response. Herein, we investigated the diurnal regulation of specialized proresolving mediators in humans and their role in controlling peripheral blood leukocyte and platelet activation. Using lipid mediator profiling and healthy volunteers, we found that plasma concentrations of n-3 docosapentaenoic acid-derived D-series resolvins (RvD n-3 DPA ) were regulated in a diurnal manner. The production and regulation of these mediators was markedly altered in patients at risk of myocardial infarct. These changes were associated with decreased 5-lipoxygenase expression and activity, as well as increased systemic adenosine concentrations. We also found a significant negative correlation between plasma RvD n-3 DPA and markers of platelet, monocyte, and neutrophil activation, including CD63 and CD11b. Incubation of RvD n-3 DPA with peripheral blood from healthy volunteers and patients with cardiovascular disease significantly and dose-dependently decreased platelet and leukocyte activation. Furthermore, administration of RvD5 n-3 DPA to ApoE -/- (apolipoprotein E deficient) mice significantly reduced platelet-leukocyte aggregates, vascular thromboxane B 2 concentrations, and aortic lesions. These results demonstrate that peripheral blood RvD n-3 DPA are diurnally regulated in humans, and dysregulation in the production of these mediators may lead to cardiovascular disease. © 2018 The Authors.

Deletion of Crry and DAF on murine platelets stimulates thrombopoiesis and increases factor H-dependent resistance of peripheral platelets to complement attack.

PubMed

Barata, Lidia; Miwa, Takashi; Sato, Sayaka; Kim, David; Mohammed, Imran; Song, Wen-Chao

2013-03-15

Complement receptor 1-related gene/protein y (Crry) and decay-accelerating factor (DAF) are two murine membrane C3 complement regulators with overlapping functions. Crry deletion is embryonically lethal whereas DAF-deficient mice are generally healthy. Crry(-/-)DAF(-/-) mice were viable on a C3(-/-) background, but platelets from such mice were rapidly destroyed when transfused into C3-sufficient mice. In this study, we used the cre-lox system to delete platelet Crry in DAF(-/-) mice and studied Crry/DAF-deficient platelet development in vivo. Rather than displaying thrombocytopenia, Pf4-Cre(+)-Crry(flox/flox) mice had normal platelet counts and their peripheral platelets were resistant to complement attack. However, chimera mice generated with Pf4-Cre(+)-Crry(flox/flox) bone marrows showed platelets from C3(-/-) but not C3(+/+) recipients to be sensitive to complement activation, suggesting that circulating platelets in Pf4-Cre(+)-Crry(flox/flox) mice were naturally selected in a complement-sufficient environment. Notably, Pf4-Cre(+)-Crry(flox/flox) mouse platelets became complement susceptible when factor H function was blocked. Examination of Pf4-Cre(+)-Crry(flox/flox) mouse bone marrows revealed exceedingly active thrombopoiesis. Thus, under in vivo conditions, Crry/DAF deficiency on platelets led to abnormal platelet turnover, but peripheral platelet count was compensated for by increased thrombopoiesis. Selective survival of Crry/DAF-deficient platelets aided by factor H protection and compensatory thrombopoiesis demonstrates the cooperation between membrane and fluid phase complement inhibitors and the body's ability to adaptively respond to complement regulator deficiencies.

Platelet-Derived MRP-14 Induces Monocyte Activation in Patients With Symptomatic Peripheral Artery Disease.

PubMed

Dann, Rebecca; Hadi, Tarik; Montenont, Emilie; Boytard, Ludovic; Alebrahim, Dornaszadat; Feinstein, Jordyn; Allen, Nicole; Simon, Russell; Barone, Krista; Uryu, Kunihiro; Guo, Yu; Rockman, Caron; Ramkhelawon, Bhama; Berger, Jeffrey S

2018-01-02

Peripheral artery disease (PAD), a diffuse manifestation of atherothrombosis, is a major cardiovascular threat. Although platelets are primary mediators of atherothrombosis, their role in the pathogenesis of PAD remains unclear. The authors sought to investigate the role of platelets in a cohort of symptomatic PAD. The authors profiled platelet activity, mRNA, and effector roles in patients with symptomatic PAD and in healthy controls. Patients with PAD and carotid artery stenosis were recruited into ongoing studies (NCT02106429 and NCT01897103) investigating platelet activity, platelet RNA, and cardiovascular disease. Platelet RNA sequence profiling mapped a robust up-regulation of myeloid-related protein (MRP)-14 mRNA, a potent calcium binding protein heterodimer, in PAD. Circulating activated platelets were enriched with MRP-14 protein, which augmented the expression of the adhesion mediator, P-selectin, thereby promoting monocyte-platelet aggregates. Electron microscopy confirmed the firm interaction of platelets with monocytes in vitro and colocalization of macrophages with MRP-14 confirmed their cross talk in atherosclerotic manifestations of PAD in vivo. Platelet-derived MRP-14 was channeled to monocytes, thereby fueling their expression of key PAD lesional hallmarks and increasing their directed locomotion, which were both suppressed in the presence of antibody-mediated blockade. Circulating MRP-14 was heightened in the setting of PAD, significantly correlated with PAD severity, and was associated with incident limb events. The authors identified a heightened platelet activity profile and unraveled a novel immunomodulatory effector role of platelet-derived MRP-14 in reprograming monocyte activation in symptomatic PAD. (Platelet Activity in Vascular Surgery and Cardiovascular Events [PACE]; NCT02106429; and Platelet Activity in Vascular Surgery for Thrombosis and Bleeding [PIVOTAL]; NCT01897103). Copyright © 2018 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Computational biology analysis of platelet signaling reveals roles of feedbacks through phospholipase C and inositol 1,4,5-trisphosphate 3-kinase in controlling amplitude and duration of calcium oscillations.

PubMed

Balabin, Fedor A; Sveshnikova, Anastasia N

2016-06-01

Blood platelet activation is required to allow their participation in hemostasis and thrombosis. It is regulated by a complicated signaling network, whose functioning has been recently attracting attention for basic research and pharmacological purposes. Phospholipase С (PLC) is an enzyme playing an important role in platelet calcium signaling and responsible for release of inositol triphosphate (IP3) into platelet cytoplasm thus controlling intracellular calcium concentration. Using a comprehensive computational model of platelet calcium signaling, we studied the influence of the positive feedback executed by cytosolic calcium on the PLC isoform β2 during platelet activation. With the positive feedback, the model predicted hyperintensive response to platelet activation by thrombin, where non-physiologically high calcium concentrations arose. However, if one took into account a negative feedback determined by IP3 3-kinase (IP3K), combination of the feedback resulted in the formation of a stepped response (with a stable oscillation amplitude and activation-dependent duration). Stochastic simulations confirmed that PLC and IP3K should act in pair to ensure platelet's "all-or-none" response to activation, when the activation level sets the probability of platelet activation, but not its intensity. Copyright © 2016 Elsevier Inc. All rights reserved.

Antiplatelet effects of Rhus verniciflua stokes heartwood and its active constituents--fisetin, butein, and sulfuretin--in rats.

PubMed

Lee, Jun-Hyeong; Kim, Mikyung; Chang, Kyung-Hwa; Hong, Cheol Yi; Na, Chun-Soo; Dong, Mi-Sook; Lee, Dongho; Lee, Moo-Yeol

2015-01-01

Rhus verniciflua stokes (RVS) is known to promote blood circulation by preventing blood stasis, although the active ingredients and the underlying mechanism are unclear. Platelets are the primary cells that regulate circulation and contribute to the development of diverse cardiovascular diseases by aggregation and thrombosis. The study assessed the antiplatelet activity of RVS and sought to identify the active constituents. Pretreatment of washed platelets with RVS heartwood extract blunted the aggregatory response of platelets to collagen. In the subfractions, fisetin, butein, and sulfuretin were identified as effective inhibitors of platelet aggregation by collagen, thrombin, and adenosine-5'-diphosphate. Antiplatelet activities of all three compounds were concentration dependent, and fisetin had longer in vitro duration of action compared with butein or sulfuretin. Extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase activation by collagen was prevented by fisetin, whereas butein and sulfuretin failed to inhibit ERK and p38 activation was not affected by any of the compounds. Rats orally administered 100 mg/(kg·day(-1)) fisetin for 7 days were resistant to arterial thrombosis, although total extract of RVS heartwood exhibited little effect at a dose of 1000 mg/(kg·day(-1)). RVS heartwood may have cardiovascular protective activity by inhibiting platelet aggregation. The active constituents are fisetin, butein, and sulfuretin, and fisetin is orally effective against thrombosis.

Amarogentin, a secoiridoid glycoside, abrogates platelet activation through PLC γ 2-PKC and MAPK pathways.

PubMed

Yen, Ting-Lin; Lu, Wan-Jung; Lien, Li-Ming; Thomas, Philip Aloysius; Lee, Tzu-Yin; Chiu, Hou-Chang; Sheu, Joen-Rong; Lin, Kuan-Hung

2014-01-01

Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60  μM) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC) γ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLC γ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders.

Amarogentin, a Secoiridoid Glycoside, Abrogates Platelet Activation through PLCγ2-PKC and MAPK Pathways

PubMed Central

Yen, Ting-Lin; Lu, Wan-Jung; Lien, Li-Ming; Thomas, Philip Aloysius; Lee, Tzu-Yin; Chiu, Hou-Chang; Sheu, Joen-Rong

2014-01-01

Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60 μM) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLCγ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders. PMID:24868545

ARF6-dependent regulation of P2Y receptor traffic and function in human platelets.

PubMed

Kanamarlapudi, Venkateswarlu; Owens, Sian E; Saha, Keya; Pope, Robert J; Mundell, Stuart J

2012-01-01

Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1) and P2Y(12) purinoceptors. Recently, we demonstrated that P2Y(1) and P2Y(12) purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6) in the internalization and function of P2Y(1) and P2Y(12) purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1) or P2Y(12) purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP) kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.

Talin-dependent integrin activation is required for fibrin clot retraction by platelets

PubMed Central

Haling, Jacob R.; Monkley, Susan J.; Critchley, David R.

2011-01-01

Talin functions both as a regulator of integrin affinity and as an important mechanical link between integrins and the cytoskeleton. Using genetic deletion of talin, we show for the first time that the capacity of talin to activate integrins is required for fibrin clot retraction by platelets. To further dissect which talin functions are required for this process, we tested clot retraction in platelets expressing a talin1(L325R) mutant that binds to integrins, but exhibits impaired integrin activation ascribable to disruption of the interaction between talin and the membrane-proximal region (MPR) in the β-integrin cytoplasmic domain. Talin-deficient and talin1(L325R) platelets were defective in retracting fibrin clots. However, the defect in clot retraction in talin1(L325R) platelets, but not talin-deficient platelets, was rescued by extrinsically activating integrins with manganese, thereby proving that integrin activation is required and showing that talin1(L325R) can form functional links to the actin cytoskeleton. PMID:20971947

Staurosporine potentiates platelet activating factor stimulated phospholipase C activity in rabbit platelets but does not block desensitization by platelet activating factor.

PubMed

Morrison, W J; Dhar, A; Shukla, S D

1989-01-01

The possible involvement of protein kinase C activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets. PAF (100 nM for 5 seconds) stimulated incorporation of 32P into proteins and caused [3H]InsP3 levels to increase about 260% of control. These responses were compared after platelets were pretreated with either PAF, phorbol 12-myristate 13-acetate (PMA) or staurosporine and also after pretreatments with staurosporine followed by PAF or PMA. Pretreating platelets with staurosporine potentiated PAF-stimulated [3H]InsP3 levels by 54% and blocked protein phosphorylation. Pretreatments with PAF and PMA caused PAF-stimulated [3H]InsP3 levels to decrease to 115 and 136%, respectively. Staurosporine pretreatment blocked the decrease caused by the PMA pretreatment but not that by PAF. This study demonstrates that PAF-stimulated PLC activity is negatively affected by protein kinase C (PKC) activation and that inhibition of PKC activity did not prevent desensitization of PLC by PAF.

A critical role for the regulation of Syk from agglutination to aggregation in human platelets.

PubMed

Shih, Chun-Ho; Chiang, Tin-Bin; Wang, Wen-Jeng

2014-01-10

Agglucetin, a tetrameric glycoprotein (GP) Ibα agonist from Formosan Agkistrodon acutus venom, has been characterized as an agglutination inducer in human washed platelets (WPs). In platelet-rich plasma (PRP), agglucetin dramatically elicits a biphasic response of agglutination and subsequent aggregation. For clarifying the intracellular signaling events from agglutination to aggregation in human platelets, we examined the essential signaling molecules involved through the detection of protein tyrosine phosphorylation (PTP). In WPs, an anti-GPIbα monoclonal antibody (mAb) AP1, but not a Src kinase inhibitor PP1, completely inhibited agglucetin-induced agglutination. However, PP1 but not AP1 had a potent suppression on platelet aggregation by a GPVI activator convulxin. The PTP analyses showed agglucetin alone can cause a weak pattern involving sequential phosphorylation of Lyn/Fyn, Syk, SLP-76 and phospholipase Cγ2 (PLCγ2). Furthermore, a Syk-selective kinase inhibitor, piceatannol, significantly suppressed the aggregating response in agglucetin-activated PRP. Analyzed by flow cytometry, the binding capacity of fluorophore-conjugated PAC-1, a mAb recognizing activated integrin αIIbβ3, was shown to increase in agglucetin-stimulated platelets. Again, piceatannol but not PP1 had a concentration-dependent suppression on agglucetin-induced αIIbβ3 exposure. Moreover, the formation of signalosome, including Syk, SLP-76, VAV, adhesion and degranulation promoting adapter protein (ADAP) and PLCγ2, are required for platelet aggregation in agglucetin/fibrinogen-activated platelets. In addition, GPIbα-ligation via agglucetin can substantially promote the interactions between αIIbβ3 and fibrinogen. Therefore, the signal pathway of Lyn/Fyn/Syk/SLP-76/ADAP/VAV/PLCγ2/PKC is sufficient to trigger platelet aggregation in agglucetin/fibrinogen-pretreated platelets. Importantly, Syk may function as a major regulator for the response from GPIbα-initiated agglutination to integrin αIIbβ3-dependent aggregation in human platelets. Copyright © 2013 Elsevier Inc. All rights reserved.

Platelets lacking PIP5KIγ have normal integrin activation but impaired cytoskeletal-membrane integrity and adhesion

PubMed Central

Wang, Yanfeng; Zhao, Liang; Suzuki, Aae; Lian, Lurong; Min, Sang H.; Wang, Ziqian; Litvinov, Rustem I.; Stalker, Timothy J.; Yago, Tadayuki; Klopocki, Arkadiusz G.; Schmidtke, David W.; Yin, Helen; Choi, John K.; McEver, Rodger P.; Weisel, John W.; Hartwig, John H.; Abrams, Charles S.

2013-01-01

Three isoforms of phosphatidylinositol-4-phosphate 5-kinase (PIP5KIα, PIP5KIβ, and PIP5KIγ) can each catalyze the final step in the synthesis of phosphatidylinositol-4,5-bisphosphate (PIP2), which in turn can be either converted to second messengers or bind directly to and thereby regulate proteins such as talin. A widely quoted model speculates that only p90, a longer splice form of platelet-specific PIP5KIγ, but not the shorter p87 PIP5KIγ, regulates the ligand-binding activity of integrins via talin. However, when we used mice genetically engineered to lack only p90 PIP5KIγ, we found that p90 PIP5KIγ is not critical for integrin activation or platelet adhesion on collagen. However, p90 PIP5KIγ-null platelets do have impaired anchoring of their integrins to the underlying cytoskeleton. Platelets lacking both the p90 and p87 PIP5KIγ isoforms had normal integrin activation and actin dynamics, but impaired anchoring of their integrins to the cytoskeleton. Most importantly, they formed weak shear-resistant adhesions ex vivo and unstable vascular occlusions in vivo. Together, our studies demonstrate that, although PIP5KIγ is essential for normal platelet function, individual isoforms of PIP5KIγ fulfill unique roles for the integrin-dependent integrity of the membrane cytoskeleton and for the stabilization of platelet adhesion. PMID:23372168

Quantitative Protein Sulfenic Acid Analysis Identifies Platelet Releasate-Induced Activation of Integrin β2 on Monocytes via NADPH Oxidase.

PubMed

Li, Ru; Klockenbusch, Cordula; Lin, Liwen; Jiang, Honghui; Lin, Shujun; Kast, Juergen

2016-12-02

Physiological stimuli such as thrombin, or pathological stimuli such as lysophosphatidic acid (LPA), activate platelets. The activated platelets bind to monocytes through P-selectin-PSGL-1 interactions but also release the contents of their granules, commonly called "platelet releasate". It is known that monocytes in contact with platelet releasate produce reactive oxygen species (ROS). Reversible cysteine oxidation by ROS is considered to be a potential regulator of protein function. In a previous study, we used THP-1 monocytic cells exposed to LPA- or thrombin-induced platelet releasate and a modified biotin switch assay to unravel the biological processes that are influenced by reversible cysteine oxidation. To gain a better understanding of the redox regulation of monocytes in atherosclerosis, we have now altered the modified biotin switch to selectively quantify protein sulfenic acid, a subpopulation of reversible cysteine oxidation. Using arsenite as reducing agent in the modified biotin switch assay, we were able to quantify 1161 proteins, in which more than 100 sulfenic acid sites were identified. Bioinformatics analysis of the quantified sulfenic acid sites highlighted the relevant, previously missed biological process of monocyte transendothelial migration, which included integrin β 2 . Flow cytometry validated the activation of LFA-1 (α L β 2 ) and Mac-1 (α M β 2 ), two subfamilies of integrin β 2 complexes, on human primary monocytes following platelet releasate treatment. The activation of LFA-1 was mediated by ROS from NADPH oxidase (NOX) activation. Production of ROS and activation of LFA-1 in human primary monocytes were independent of P-selectin-PSGL-1 interaction. Our results proved the modified biotin switch assay to be a powerful tool with the ability to reveal new regulatory mechanisms and identify new therapeutic targets.

Platelet activation, adhesion, inflammation, and aggregation potential are altered in the presence of electronic cigarette extracts of variable nicotine concentrations.

PubMed

Hom, Sarah; Chen, Li; Wang, Tony; Ghebrehiwet, Berhane; Yin, Wei; Rubenstein, David A

2016-11-01

Tobacco smoke extracts prepared from both mainstream and sidestream smoking have been associated with heightened platelet activation, aggregation, adhesion, and inflammation. Conversely, it has been shown that pure nicotine inhibits similar platelet functions. In this work, we 1) evaluated the effects of e-cigarette extracts on platelet activities and 2) elucidated the differences between the nicotine-dependent and non-nicotine dependent (e.g. fine particulate matter or toxic compounds) effects of tobacco and e-cigarette products on platelet activities. To accomplish these goals, platelets from healthy volunteers (n = 50) were exposed to tobacco smoke extracts, e-cigarette vapor extracts, and pure nicotine and changes in platelet activation, adhesion, aggregation, and inflammation were evaluated, using optical aggregation, flow cytometry, and ELISA methods. Interestingly, the exposure of platelets to e-vapor extracts induced a significant up-regulation in the expression of the pro-inflammatory gC1qR and cC1qR and induced a marked increase in the deposition of C3b as compared with traditional tobacco smoke extracts. Similarly, platelet activation, as measured by a prothrombinase based assay, and platelet aggregation were also significantly enhanced after exposure to e-vapor extracts. Finally, platelet adhesion potential toward fibrinogen, von Willebrand factor, and other platelets was also enhanced after exposure to e-cigarette vapor extracts. In the presence of pure nicotine, platelet functions were observed to be inhibited, which further suggests that other constituents of tobacco smoke and electronic vapor can antagonize platelet functions, however, the presence of nicotine in extracts somewhat perpetuated the platelet functional changes in a dose-dependent manner.

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) plays an agonist specific role in platelet function through RhoA activation

PubMed Central

Zou, Siying; Teixeira, Alexandra M.; Yin, Mingzhu; Xiang, Yaozu; Xavier-Ferruccio, Juliana; Zhang, Ping-xia; Hwa, John; Min, Wang; Krause, Diane S.

2018-01-01

Summary Leukemia-Associated RhoGEF (LARG) is highly expressed in platelets, which are essential for maintaining normal hemostasis. We studied the function of LARG in murine and human megakaryocytes and platelets with Larg knockout, shRNA-mediated knockdown and small molecule-mediated inhibition. We found that LARG is important for human, but not murine, megakaryocyte maturation. Larg KO mice exhibit macrothrombocytopenia, internal bleeding in the ovaries and prolonged bleeding times. KO platelets have impaired aggregation, α-granule release and integrin α2bβ3 activation in response to thrombin and thromboxane, but not to ADP. The same agonist-specific reductions in platelet aggregation occur in human platelets treated with a LARG inhibitor. Larg KO platelets have reduced RhoA activation and myosin light chain phosphorylation, suggesting that Larg plays an agonist-specific role in platelet signal transduction. Using 2 different in vivo assays, Larg KO mice are protected from in vivo thrombus formation. Together, these results establish that LARG regulates human megakaryocyte maturation, and is critical for platelet function in both humans and mice. PMID:27345948

Leukaemia-associated Rho guanine nucleotide exchange factor (LARG) plays an agonist specific role in platelet function through RhoA activation.

PubMed

Zou, Siying; Teixeira, Alexandra M; Yin, Mingzhu; Xiang, Yaozu; Xavier-Ferrucio, Juliana; Zhang, Ping-Xia; Hwa, John; Min, Wang; Krause, Diane S

2016-08-30

Leukemia-Associated RhoGEF (LARG) is highly expressed in platelets, which are essential for maintaining normal haemostasis. We studied the function of LARG in murine and human megakaryocytes and platelets with Larg knockout (KO), shRNA-mediated knockdown and small molecule-mediated inhibition. We found that LARG is important for human, but not murine, megakaryocyte maturation. Larg KO mice exhibit macrothrombocytopenia, internal bleeding in the ovaries and prolonged bleeding times. KO platelets have impaired aggregation, α-granule release and integrin α2bβ3 activation in response to thrombin and thromboxane, but not to ADP. The same agonist-specific reductions in platelet aggregation occur in human platelets treated with a LARG inhibitor. Larg KO platelets have reduced RhoA activation and myosin light chain phosphorylation, suggesting that Larg plays an agonist-specific role in platelet signal transduction. Using two different in vivo assays, Larg KO mice are protected from in vivo thrombus formation. Together, these results establish that LARG regulates human megakaryocyte maturation, and is critical for platelet function in both humans and mice.

Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation

PubMed Central

2010-01-01

Background 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin) may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved. Methods The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin. Results Simvastatin (20-50 μM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin). Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TxA2) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC), and mitogen-activated protein kinases (i.e., p38 MAPK, JNKs) phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP) phosphorylation, and endothelial nitric oxide synthase (eNOS) expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCγ2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation. Conclusion The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP-eNOS/NO-cyclic GMP pathway, resulting in inhibition of the PLCγ2-PKC-p38 MAPK-TxA2 cascade, and finally inhibition of platelet aggregation. PMID:20525309

Amifostine, a reactive oxigen species scavenger with radiation- and chemo-protective properties, inhibits in vitro platelet activation induced by ADP, collagen or PAF.

PubMed

Porta, C; Maiolo, A; Tua, A; Grignani, G

2000-08-01

Reactive oxygen species (ROS) generation has been suggested to represent an important regulatory mechanism of platelet reactivity in both physiologic and pathologic conditions; consistent with this hypothesis is the observation that free-radical scavengers may inhibit platelet activation, thus contributing to the regulation of their reactivity. The purpose of the present study is to study the in vitro effects of amifostine (WR-2721, ethyol ), a selective cytoprotective agent for normal tissues against the toxicities of chemotherapy and radiation, on platelet activation induced by the physiologic agonists ADP, collagen and PAF. The effect of amifostine, added to the experimental system at final concentrations ranging from 10(-7) M to 10(-5) M, was studied on platelet aggregation induced by the following physiologic agonists at the given concentrations: ADP (1 microM), collagen (2 microg/mL), and PAF (0.1 microg/mL). Platelet aggregation was investigated using a platelet ionized calcium aggregometer and was expressed as the percentage change in light transmission. Furthermore, thromboxane B((2)) (TxB((2))) levels and nitric oxide (NO) production were determined by radioimmunoassay and by evaluating the total nitrite/nitrate concentration using a commercially available colorimetric kit, respectively, both in the control system and after the addition of amifostine. Amifostine inhibited both platelet aggregation and TxB((2)) production induced by ADP, collagen and PAF, in a dose-dependent manner. Amifostine proved to be an effective inhibitor of platelet function and the effect was more pronounced if platelets were stimulated with ADP, intermediate when collagen was the chosen agonist, and less evident, though present, when PAF was used. Platelets stimulated with ADP, collagen or PAF produced significant amounts of NO over the baseline. When amifostine was added at a final concentration of 5 microM, it significantly increased ADP, collagen and PAF-induced NO production, which suggests that NO release by activated platelets was involved in the inhibitory effect of amifostine. Amifostine proved to be an effective inhibitor of platelet activation induced in vitro by physiologic inducers. This previously unrecognized effect was more evident with the weak agonist ADP and was related to reduced NO consumption by free radicals generated during platelet activation. Amifostine proved to be not only a powerful cytoprotectant, but, more generally, a therapeutic agent endowed with several relevant, though largely unknown, biological effects. Finally, our data once again support the concept that oxidative balance is of crucial importance in regulating platelet reactivity in both health and disease.

RhoG protein regulates platelet granule secretion and thrombus formation in mice.

PubMed

Goggs, Robert; Harper, Matthew T; Pope, Robert J; Savage, Joshua S; Williams, Christopher M; Mundell, Stuart J; Heesom, Kate J; Bass, Mark; Mellor, Harry; Poole, Alastair W

2013-11-22

Rho GTPases such as Rac, RhoA, and Cdc42 are vital for normal platelet function, but the role of RhoG in platelets has not been studied. In other cells, RhoG orchestrates processes integral to platelet function, including actin cytoskeletal rearrangement and membrane trafficking. We therefore hypothesized that RhoG would play a critical role in platelets. Here, we show that RhoG is expressed in human and mouse platelets and is activated by both collagen-related peptide (CRP) and thrombin stimulation. We used RhoG(-/-) mice to study the function of RhoG in platelets. Integrin activation and aggregation were reduced in RhoG(-/-) platelets stimulated by CRP, but responses to thrombin were normal. The central defect in RhoG(-/-) platelets was reduced secretion from α-granules, dense granules, and lysosomes following CRP stimulation. The integrin activation and aggregation defects could be rescued by ADP co-stimulation, indicating that they are a consequence of diminished dense granule secretion. Defective dense granule secretion in RhoG(-/-) platelets limited recruitment of additional platelets to growing thrombi in flowing blood in vitro and translated into reduced thrombus formation in vivo. Interestingly, tail bleeding times were normal in RhoG(-/-) mice, suggesting that the functions of RhoG in platelets are particularly relevant to thrombotic disorders.

Platelet activation by Histophilus somni and its lipooligosaccharide induces endothelial cell proinflammatory responses and platelet internalization.

PubMed

Kuckleburg, Christopher J; McClenahan, Dave J; Czuprynski, Charles J

2008-02-01

Histophilus somni is a gram-negative coccobacillus that causes respiratory and reproductive disease in cattle. The hallmark of systemic H. somni infection is diffuse vascular inflammation that can lead to an acute central nervous system disease known as thrombotic meningoencephalitis. Previously, we demonstrated that H. somni and its lipooligosaccharide (LOS) activate bovine platelets, leading to expression of P selectin, CD40L, and FasL. Because activated platelets have been reported to induce endothelial cell cytokine production and adhesion molecule expression, we sought to determine if bovine platelets induce proinflammatory and procoagulative changes in bovine pulmonary artery endothelial cells. Endothelial cells were incubated with platelets activated with adenosine diphosphate, H. somni, or H. somni LOS. Incubation with activated bovine platelets significantly increased expression of in adhesion molecules (intercellular adhesion molecule 1, E selectin) and tissue factor, as measured by flow cytometry, real-time polymerase chain reaction, and Western blot analysis. Activated platelets also up-regulated expression of endothelial cell IL-1beta, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1alpha as determined by real-time polymerase chain reaction and an IL-1beta enzyme-linked immunosorbent assay. An interesting and surprising finding was that bovine platelets activated by H. somni or its LOS were internalized by bovine endothelial cells as visualized by transmission electron microscopy. This internalization seemed to correlate with endothelial cell activation and morphological changes indicative of cell stress. These findings suggest that activated platelets might play a role in promoting vascular inflammation during H. somni infection.

Maintenance of murine platelet homeostasis by the kinase Csk and phosphatase CD148

PubMed Central

Di Nunzio, Giada; Smith, Christopher W.; Al Ghaithi, Rashid; van Geffen, Johanna P.; Heising, Silke; Tullemans, Bibian M. E.; Tee, Louise; Heemskerk, Johan W. M.; Tarakhovsky, Alexander

2018-01-01

Src family kinases (SFKs) coordinate the initiating and propagating activation signals in platelets, but it remains unclear how they are regulated. Here, we show that ablation of C-terminal Src kinase (Csk) and receptor-like protein tyrosine-phosphatase CD148 in mice results in a dramatic increase in platelet SFK activity, demonstrating that these proteins are essential regulators of platelet reactivity. Paradoxically, Csk/CD148-deficient mice exhibit reduced in vivo and ex vivo thrombus formation and increased bleeding following injury rather than a prothrombotic phenotype. This is a consequence of multiple negative feedback mechanisms, including downregulation of the immunoreceptor tyrosine-based activation motif (ITAM)– and hemi-ITAM–containing receptors glycoprotein VI (GPVI)-Fc receptor (FcR) γ-chain and CLEC-2, respectively and upregulation of the immunoreceptor tyrosine-based inhibition motif (ITIM)–containing receptor G6b-B and its interaction with the tyrosine phosphatases Shp1 and Shp2. Results from an analog-sensitive Csk mouse model demonstrate the unconventional role of SFKs in activating ITIM signaling. This study establishes Csk and CD148 as critical molecular switches controlling the thrombotic and hemostatic capacity of platelets and reveals cell-intrinsic mechanisms that prevent pathological thrombosis from occurring. PMID:29301754

Platelets Play Differential Role During the Initiation and Progression of Autoimmune Neuroinflammation

PubMed Central

Starossom, Sarah C.; Veremeyko, Tatyana; Yung, Amanda W. Y.; Dukhinova, Marina; Au, Cheryl; Lau, Alexander Y.; Weiner, Howard L.; Ponomarev, Eugene D.

2015-01-01

Rationale Platelets are known to participate in vascular pathologies; however, their role in neuroinflammatory diseases such as multiples sclerosis (MS) is unknown. Autoimmune CD4 T cells have been the main focus of studies of MS, although the factors that regulate T cell differentiation towards pathogenic Th1/Th17 phenotypes are not completely understood. Objectives We investigated the role of platelets in the modulation of CD4 T cell functions in MS patients and in mice with experimental autoimmune encephalitis (EAE), an animal model for MS. Methods and Results We found that early in MS and EAE platelets degranulated and produced a number of soluble factors serotonin (5HT), PF4 and PAF, which specifically stimulated differentiation of T cells towards pathogenic Th1, Th17 and IFN-γ/IL-17-producing CD4 T cells. At the later stages of MS and EAE platelets became exhausted in their ability to produce proinflammatory factors and stimulate CD4 T cells, but substantially increased their ability to form aggregates with CD4 T cells. Formation of platelet-CD4 T cell aggregates involved interaction of CD62P on activated platelets with adhesion molecule CD166 on activated CD4 T cells, contributing to downmodulation of CD4 T cell activation, proliferation and production of IFN-γ. Blocking of formation of platelet-CD4 T cell aggregates during progression of EAE substantially enhanced proliferation of CD4 T cell in the CNS and the periphery leading to exacerbation of the disease. Conclusion Our study indicates differential roles for platelets in the regulation of functions of pathogenic CD4 T cells during initiation and progression of CNS autoimmune inflammation. PMID:26294656

Lymphocyte-mediated regulation of platelet activation during desensitization in patients with hymenoptera venom hypersensitivity.

PubMed Central

Ledru, E; Pestel, J; Tsicopoulos, A; Joseph, M; Wallaert, B; Tonnel, A B; Capron, A

1988-01-01

T cells from peripheral blood of hymenoptera sensitive patients were studied before and after venom desensitization. Before treatment, T cells showed a variable but higher proliferative response to allergen than T cells of treated patients or controls. While before desensitization, T cell products, specifically released after in vitro allergen stimulation, were able to amplify the IgE-dependent platelet activity, we showed that after treatment of the same patients, T cell products strongly reduced platelet activation. Considering the modifications in platelet activation previously observed in patients treated by specific immunotherapy, the present results suggest that, through a modification of T cell reactivity to allergen, T cell functions are modulated by desensitization, and emphasize the involvement of T cell products in the desensitization mechanisms. PMID:3263227

JAM-A protects from thrombosis by suppressing integrin αIIbβ3-dependent outside-in signaling in platelets

PubMed Central

Naik, Meghna U.; Stalker, Timothy J.; Brass, Lawrence F.

2012-01-01

Mounting evidence suggests that agonist-initiated signaling in platelets is closely regulated to avoid excessive responses to injury. A variety of physiologic agonists induce a cascade of signaling events termed as inside-out signaling that culminate in exposure of high-affinity binding sites on integrin αIIbβ3. Once platelet activation has occurred, integrin αIIbβ3 stabilizes thrombus formation by providing agonist-independent “outside-in” signals mediated in part by contractile signaling. Junctional adhesion molecule A (JAM-A), a member of the cortical thymocyte marker of the Xenopus (CTX) family, was initially identified as a receptor for a platelet stimulatory mAb. Here we show that JAM-A in resting platelets functions as an endogenous inhibitor of platelet function. Genetic ablation of Jam-A in mice enhances thrombotic function of platelets in vivo. The absence of Jam-A results in increase in platelet aggregation ex vivo. This gain of function is not because of enhanced inside-out signaling because granular secretion, Thromboxane A2 (TxA2) generation, as well as fibrinogen receptor activation, are normal in the absence of Jam-A. Interestingly, integrin outside-in signaling such as platelet spreading and clot retraction is augmented in Jam-A–deficient platelets. We conclude that JAM-A normally limits platelet accumulation by inhibiting integrin outside-in signaling thus preventing premature platelet activation. PMID:22271446

Dark chocolate inhibits platelet isoprostanes via NOX2 down-regulation in smokers.

PubMed

Carnevale, R; Loffredo, L; Pignatelli, P; Nocella, C; Bartimoccia, S; Di Santo, S; Martino, F; Catasca, E; Perri, L; Violi, Francesco

2012-01-01

Dark chocolate is reported to decrease platelet activation but the underlying mechanism is still undefined. Dark chocolate is rich in polyphenols that could exert an antiplatelet action via inhibition of oxidative stress. The aim of the present study was to assess if dark chocolate inhibits platelet reactive oxidant species (ROS) formation and platelet activation. Twenty healthy subjects (HS) and 20 smokers were randomly allocated to receive 40 g of dark (cocoa > 85%) or milk chocolate (cocoa < 35%) in a cross-over, single-blind study. There was an interval of 7 days between the two phases of the study. At baseline and 2 h after chocolate ingestion, platelet recruitment (PR), platelet ROS, platelet isoprostane 8-ISO-prostaglandin F2α (8-iso-PGF2α), Thromboxane (TxA2) and platelet activation of NOX2, the catalytic sub-unit of NADPH oxidase, and serum epicatechin were measured. Compared with HS, smokers showed enhanced PR, platelet formation of ROS and eicosanoids and NOX2 activation. After dark chocolate, platelet ROS (-48%, P < 0.001), 8-iso-PGF2α (-10%, P < 0.001) and NOX2 activation (-22%, P < 0.001) significantly decreased; dark chocolate did not affect platelet variables in HS. No effect of milk chocolate was detected in both groups. Serum epicatechin increased after dark chocolate in HS (from 0.454 ± 0.3 nm to 118.3 ± 53.7 nm) and smokers (from 0.5 ± 0.28 nm to 120.9 ± 54.2 nm). Platelet incubation with 0.1-10 μm catechin significantly reduced PR, platelet 8-iso-PGF2α and ROS formation and NOX2 activation only in platelets from smokers. Dark chocolate inhibits platelet function by lowering oxidative stress only in smokers; this effect seems to be dependent on its polyphenolic content. © 2011 International Society on Thrombosis and Haemostasis.

Platelet-derived HMGB1 is a critical mediator of thrombosis.

PubMed

Vogel, Sebastian; Bodenstein, Rebecca; Chen, Qiwei; Feil, Susanne; Feil, Robert; Rheinlaender, Johannes; Schäffer, Tilman E; Bohn, Erwin; Frick, Julia-Stefanie; Borst, Oliver; Münzer, Patrick; Walker, Britta; Markel, Justin; Csanyi, Gabor; Pagano, Patrick J; Loughran, Patricia; Jessup, Morgan E; Watkins, Simon C; Bullock, Grant C; Sperry, Jason L; Zuckerbraun, Brian S; Billiar, Timothy R; Lotze, Michael T; Gawaz, Meinrad; Neal, Matthew D

2015-12-01

Thrombosis and inflammation are intricately linked in several major clinical disorders, including disseminated intravascular coagulation and acute ischemic events. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) is upregulated by activated platelets in multiple inflammatory diseases; however, the contribution of platelet-derived HMGB1 in thrombosis remains unexplored. Here, we generated transgenic mice with platelet-specific ablation of HMGB1 and determined that platelet-derived HMGB1 is a critical mediator of thrombosis. Mice lacking HMGB1 in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/hemorrhagic shock. Platelets were the major source of HMGB1 within thrombi. In trauma patients, HMGB1 expression on the surface of circulating platelets was markedly upregulated. Moreover, evaluation of isolated platelets revealed that HMGB1 is critical for regulating platelet activation, granule secretion, adhesion, and spreading. These effects were mediated via TLR4- and MyD88-dependent recruitment of platelet guanylyl cyclase (GC) toward the plasma membrane, followed by MyD88/GC complex formation and activation of the cGMP-dependent protein kinase I (cGKI). Thus, we establish platelet-derived HMGB1 as an important mediator of thrombosis and identify a HMGB1-driven link between MyD88 and GC/cGKI in platelets. Additionally, these findings suggest a potential therapeutic target for patients sustaining trauma and other inflammatory disorders associated with abnormal coagulation.

Inner Mitochondrial Membrane Disruption Links Apoptotic and Agonist-Initiated Phosphatidylserine Externalization in Platelets.

PubMed

Choo, Hyo-Jung; Kholmukhamedov, Andaleb; Zhou, ChengZing; Jobe, Shawn

2017-08-01

Phosphatidylserine exposure mediates platelet procoagulant function and regulates platelet life span. Apoptotic, necrotic, and integrin-mediated mechanisms have been implicated as intracellular determinants of platelet phosphatidylserine exposure. Here, we investigate (1) the role of mitochondrial events in platelet phosphatidylserine exposure initiated by these distinct stimuli and (2) the cellular interactions of the procoagulant platelet in vitro and in vivo. Key mitochondrial events were examined, including cytochrome c release and inner mitochondrial membrane (IMM) disruption. In both ABT-737 (apoptotic) and agonist (necrotic)-treated platelets, phosphatidylserine externalization was temporally correlated with IMM disruption. Agonist stimulation resulted in rapid cyclophilin D-dependent IMM disruption that coincided with phosphatidylserine exposure. ABT-737 treatment caused rapid cytochrome c release, eventually followed by caspase-dependent IMM disruption that again closely coincided with phosphatidylserine exposure. A nonmitochondrial and integrin-mediated mechanism has been implicated in the formation of a novel phosphatidylserine-externalizing platelet subpopulation. Using image cytometry, this subpopulation is demonstrated to be the result of the interaction of an aggregatory platelet and a procoagulant platelet rather than indicative of a novel intracellular mechanism regulating platelet phosphatidylserine externalization. Using electron microscopy, similar interactions between aggregatory and procoagulant platelets are demonstrated in vitro and in vivo within a mesenteric vein hemostatic thrombus. Platelet phosphatidylserine externalization is closely associated with the mitochondrial event of IMM disruption identifying a common pathway in phosphatidylserine-externalizing platelets. The limited interaction of procoagulant platelets and integrin-active aggregatory platelets identifies a potential mechanism for procoagulant platelet retention within the hemostatic thrombus. © 2017 American Heart Association, Inc.

Differential Dynamics of Platelet Contact and Spreading

PubMed Central

Lee, Dooyoung; Fong, Karen P.; King, Michael R.; Brass, Lawrence F.; Hammer, Daniel A.

2012-01-01

Platelet spreading is critical for hemostatic plug formation and thrombosis. However, the detailed dynamics of platelet spreading as a function of receptor-ligand adhesive interactions has not been thoroughly investigated. Using reflection interference contrast microscopy, we found that both adhesive interactions and PAR4 activation affect the dynamics of platelet membrane contact formation during spreading. The initial growth of close contact area during spreading was controlled by the combination of different immobilized ligands or PAR4 activation on fibrinogen, whereas the growth of the total area of spreading was independent of adhesion type and PAR4 signaling. We found that filopodia extend to their maximal length and then contract over time; and that filopodial protrusion and expansion were affected by PAR4 signaling. Upon PAR4 activation, the integrin αIIbβ3 mediated close contact to fibrinogen substrata and led to the formation of ringlike patterns in the platelet contact zone. A systematic study of platelet spreading of GPVI-, α2-, or β3-deficient platelets on collagen or fibrinogen suggests the integrin α2 is indispensable for spreading on collagen. The platelet collagen receptors GPVI and α2 regulate integrin αIIbβ3-mediated platelet spreading on fibrinogen. This work elucidates quantitatively how receptor-ligand adhesion and biochemical signals synergistically control platelet spreading. PMID:22325269

Antiplatelet activity of L-sulforaphane by regulation of platelet activation factors, glycoprotein IIb/IIIa and thromboxane A2.

PubMed

Oh, Chung-Hun; Shin, Jang-In; Mo, Sang Joon; Yun, Sung-Jo; Kim, Sung-Hoon; Rhee, Yun-Hee

2013-07-01

L-sulforaphane was identified as an anticarcinogen that could produce quinine reductase and a phase II detoxification enzyme. In recent decades, multi-effects of L-sulforaphane may have been investigated, but, to the authors' knowledge, the antiplatelet activation of L-sulforaphane has not been studied yet.In this study, 2 μg/ml of collagen, 50 μg/ml of ADP and 5 μg/ml of thrombin were used for platelet aggregations with or without L-sulforaphane. L-sulforaphane inhibited the platelet aggregation dose-dependently. Among these platelet activators, collagen was most inhibited by L-sulforaphane, which markedly decreased collagen-induced glycoprotein IIb/IIIa activation and thromboxane A2 (TxA2) formation in vitro. L-sulforaphane also reduced the collagen and epinephrine-induced pulmonary embolism, but did not affect prothrombin time (PT) in vivo. This finding demonstrated that L-sulforaphane inhibited the platelet activation through an intrinsic pathway.L-sulforaphane had a beneficial effect on various pathophysiological pathways of the collagen-induced platelet aggregation and thrombus formation as a selective inhibition of cyclooxygenase and glycoprotein IIb/IIIa antagonist. Thus, we recommend L-sulforaphane as a potential antithrombotic drug.

Necrotic platelets provide a procoagulant surface during thrombosis

PubMed Central

Hua, Vu Minh; Abeynaike, Latasha; Glaros, Elias; Campbell, Heather; Pasalic, Leonardo; Chen, Vivien M. Y.

2015-01-01

A subpopulation of platelets fulfills a procoagulant role in hemostasis and thrombosis by enabling the thrombin burst required for fibrin formation and clot stability at the site of vascular injury. Excess procoagulant activity is linked with pathological thrombosis. The identity of the procoagulant platelet has been elusive. The cell death marker 4-[N-(S-glutathionylacetyl)amino]phenylarsonous acid (GSAO) rapidly enters a subpopulation of agonist-stimulated platelets via an organic anion-transporting polypeptide and is retained in the cytosol through covalent reaction with protein dithiols. Labeling with GSAO, together with exposure of P-selectin, distinguishes necrotic from apoptotic platelets and correlates with procoagulant potential. GSAO+ platelets form in occluding murine thrombi after ferric chloride injury and are attenuated with megakaryocyte-directed deletion of the cyclophilin D gene. These platelets form a procoagulant surface, supporting fibrin formation, and reduction in GSAO+ platelets is associated with reduction in platelet thrombus size and fibrin formation. Analysis of platelets from human subjects receiving aspirin therapy indicates that these procoagulant platelets form despite aspirin therapy, but are attenuated by inhibition of the necrosis pathway. These findings indicate that the major subpopulation of platelets involved in fibrin formation are formed via regulated necrosis involving cyclophilin D, and that they may be targeted independent of platelet activation. PMID:26474813

The mechanisms how heparin affects the tumor cell induced VEGF and chemokine release from platelets to attenuate the early metastatic niche formation

PubMed Central

Ponert, Jan Moritz; Schwarz, Svenja; Haschemi, Reza; Müller, Jens; Pötzsch, Bernd; Bendas, Gerd

2018-01-01

Metastasis is responsible for the majority of cancer associated fatalities. Tumor cells leaving the primary tumor and entering the blood flow immediately interact with platelets. Activated platelets contribute in different ways to cancer cell survival and proliferation, e.g. in formation of the early metastatic niche by release of different growth factors and chemokines. Here we show that a direct interaction between platelets and MV3 melanoma or MCF7 breast cancer cells induces platelet activation and a VEGF release in citrated plasma that cannot be further elevated by the coagulation cascade and generated thrombin. In contrast, the release of platelet-derived chemokines CXCL5 and CXCL7 depends on both, a thrombin-mediated platelet activation and a direct interaction between tumor cells and platelets. Preincubation of platelets with therapeutic concentrations of unfractionated heparin reduces the tumor cell initiated VEGF release from platelets. In contrast, tumor cell induced CXCL5 and CXCL7 release from platelets was not impacted by heparin pretreatment in citrated plasma. In defibrinated, recalcified plasma, on the contrary, heparin is able to reduce CXCL5 and CXCL7 release from platelets by thrombin inhibition. Our data indicate that different chemokines and growth factors in diverse platelet granules are released in tightly regulated processes by various trigger mechanisms. We show for the first time that heparin is able to reduce the mediator release induced by different tumor cells both in a contact and coagulation dependent manner. PMID:29346400

Platelet lipidomics: a modern day perspective on lipid discovery and characterization in platelets

PubMed Central

O’Donnell, Valerie B; Murphy, Robert C.; Watson, Steve P

2014-01-01

Lipids are diverse families of biomolecules that perform essential structural and signaling roles in platelets. Their formation and metabolism is tightly controlled by enzymes and signal transduction pathways, and their dysregulation leads to significant defects in platelet function and disease. Platelet activation is associated with significant changes to membrane lipids, and formation of diverse bioactive lipids that play essential roles in hemostasis. In recent years, new generation mass spectrometry analysis of lipids (termed “lipidomics”) has begun to alter our understanding of how these molecules participate in key cellular processes. While, the application of lipidomics to platelet biology is still in its infancy, seminal earlier studies have shaped our knowledge of how lipids regulate key aspects of platelet biology, including aggregation, shape change, coagulation and degranulation, as well as how lipids generated by platelets influence other cells, such as leukocytes and the vascular wall, and thus how they regulate hemostasis, vascular integrity and inflammation, as well as contribute to pathologies including arterial/deep vein thrombosis and atherosclerosis. This review will provide a brief historical perspective on the characterization of lipids in platelets, then an overview of the new generation lipidomic approaches, their recent application to platelet biology, and future perspectives for research in this area. The major platelet-regulatory lipid families, their formation, metabolism, and their role in health and disease, will be summarized. PMID:24677238

SLAP/SLAP2 prevent excessive platelet (hem)ITAM signaling in thrombosis and ischemic stroke in mice.

PubMed

Cherpokova, Deya; Bender, Markus; Morowski, Martina; Kraft, Peter; Schuhmann, Michael K; Akbar, Sarah M; Sultan, Cheryl S; Hughes, Craig E; Kleinschnitz, Christoph; Stoll, Guido; Dragone, Leonard L; Watson, Steve P; Tomlinson, Michael G; Nieswandt, Bernhard

2015-01-01

Glycoprotein VI and C-type lectin-like receptor 2 are essential platelet activating receptors in hemostasis and thrombo-inflammatory disease, which signal through a (hem)immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway. The adapter molecules Src-like adapter proteins (SLAP and SLAP2) are involved in the regulation of immune cell surface expression and signaling, but their function in platelets is unknown. In this study, we show that platelets expressed both SLAP isoforms and that overexpression of either protein in a heterologous cell line almost completely inhibited glycoprotein VI and C-type lectin-like receptor 2 signaling. In mice, single deficiency of SLAP or SLAP2 had only moderate effects on platelet function, whereas double deficiency of both adapters resulted in markedly increased signal transduction, integrin activation, granule release, aggregation, procoagulant activity, and thrombin generation in response to (hem)ITAM-coupled, but not G protein-coupled, receptor activation. In vivo, constitutive SLAP/SLAP2 knockout mice displayed accelerated occlusive arterial thrombus formation and a dramatically worsened outcome after focal cerebral ischemia. This was attributed to the absence of both adapter proteins in platelets, as demonstrated by adoptive transfer of Slap(-/-)/Slap2(-/-) platelets into wild-type mice. Our results establish SLAP and SLAP2 as critical inhibitors of platelet (hem)ITAM signaling in the setting of arterial thrombosis and ischemic stroke. © 2015 by The American Society of Hematology.

Effects of a single bout of strenuous exercise on platelet activation in female ApoE/LDLR-/- mice.

PubMed

Przyborowski, K; Kassassir, H; Wojewoda, M; Kmiecik, K; Sitek, B; Siewiera, K; Zakrzewska, A; Rudolf, A M; Kostogrys, R; Watala, C; Zoladz, J A; Chlopicki, S

2017-11-01

Strenuous physical exercise leads to platelet activation that is normally counterbalanced by the production of endothelium-derived anti-platelet mediators, including prostacyclin (PGI 2 ) and nitric oxide (NO). However, in the case of endothelial dysfunction, e.g. in atherosclerosis, there exists an increased risk for intravascular thrombosis during exercise that might be due to an impairment in endothelial anti-platelet mechanisms. In the present work, we evaluated platelet activation at rest and following a single bout of strenuous treadmill exercise in female ApoE/LDLR - /- mice with early (3-month-old) and advanced (7-month-old) atherosclerosis compared to female age-matched WT mice. In sedentary and post-exercise groups of animals, we analyzed TXB 2 generation and the expression of platelet activation markers in the whole blood ex vivo assay. We also measured pre- and post-exercise plasma concentration of 6-keto-PGF 1α , nitrite/nitrate, lipid profile, and blood cell count. Sedentary 3- and 7-month-old ApoE/LDLR - /- mice displayed significantly higher activation of platelets compared to age-matched wild-type (WT) mice, as evidenced by increased TXB 2 production, expression of P-selectin, and activation of GPIIb/IIIa receptors, as well as increased fibrinogen and von Willebrand factor (vWf) binding. Interestingly, in ApoE/LDLR - /- but not in WT mice, strenuous exercise partially inhibited TXB 2 production, the expression of activated GPIIb/IIIa receptors, and fibrinogen binding, with no effect on the P-selectin expression and vWf binding. Post-exercise down-regulation of the activated GPIIb/IIIa receptor expression and fibrinogen binding was not significantly different between 3- and 7-month-old ApoE/LDLR - /- mice; however, only 7-month-old ApoE/LDLR - /- mice showed lower TXB 2 production after exercise. In female 4-6-month-old ApoE/LDLR - /- but not in WT mice, an elevated pre- and post-exercise plasma concentration of 6-keto-PGF 1α was observed. In turn, the pre- and post-exercise plasma concentrations of nitrite (NO 2 - ) and nitrate (NO 3 - ) were decreased in ApoE/LDLR - /- as compared to that in age-matched WT mice. In conclusion, we demonstrated overactivation of platelets in ApoE/LDLR - /- as compared to WT mice. However, platelet activation in ApoE/LDLR - /- mice was not further increased by strenuous exercise, but was instead attenuated, a phenomenon not observed in WT mice. This phenomenon could be linked to compensatory up-regulation of PGI 2 -dependent anti-platelet mechanisms in ApoE/LDLR - /- mice.

Effect of cigarette smoke on monocyte procoagulant activity: Focus on platelet-derived brain-derived neurotrophic factor (BDNF).

PubMed

Amadio, Patrizia; Baldassarre, Damiano; Sandrini, Leonardo; Weksler, Babette B; Tremoli, Elena; Barbieri, Silvia S

2017-01-01

Cigarette smoke (CS) activates platelets, promotes vascular dysfunction, and enhances Tissue Factor (TF) expression in blood monocytes favoring pro-thrombotic states. Brain-derived neurotrophic factor (BDNF), a member of the family of neurotrophins involved in survival, growth, and maturation of neurons, is released by activated platelets (APLTs) and plays a role in the cardiovascular system. The effect of CS on circulating levels of BDNF is controversial and the function of circulating BDNF in atherothrombosis is not fully understood. Here, we have shown that human platelets, treated with an aqueous extract of CS (CSE), released BDNF in a dose-dependent manner. In addition, incubation of human monocytes with BDNF or with the supernatant of platelets activated with CSE increased TF activity by a Tropomyosin receptor kinase B (TrkB)-dependent mechanism. Finally, comparing serum and plasma samples of 12 male never smokers (NS) and 29 male active smokers (AS) we observed a significant increase in microparticle-associated TF activity (MP-TF) as well as BDNF in AS, while in serum, BDNF behaved oppositely. Taken together these findings suggest that platelet-derived BDNF is involved in the regulation of TF activity and that CS plays a role in this pathway by favoring a pro-atherothrombotic state.

Does patchouli oil change blood platelet monoamine oxidase-A activity of adult mammals?

PubMed

Karim, Md Fazlul; Banerjee, Soumyabrata; Poddar, Mrinal K

2018-05-01

Patchouli oil, an essential aroma oil extracted from patchouli leaf during short-term exposure with five and ten drops either inhibited (at 1 or 2 h) or stimulated (at 4 h) the platelet MAO-A activity depending on the dosages of the aroma oil mainly due to inhibition or stimulation of its K m . The long-term 15 consecutive days exposure (with two or five drops) of patchouli oil, on the other hand, maximally stimulated the platelet MAO-A activity with five drops patchouli oil for 1 h exposure, but further continuation of its exposure with same doses (two or five drops) for 30 consecutive days significantly stimulated (with two drops) and inhibited (with five drops) the platelet MAO-A activity due to stimulation and inhibition respectively of its corresponding both K m and V max . These results thus suggest that this aroma oil exposure may modulate the blood platelet serotonergic regulation depending on the dose, duration, and conditions of exposure.

Thrombin-induced activation of RhoA in platelet shape change.

PubMed

Bodie, S L; Ford, I; Greaves, M; Nixon, G F

2001-09-14

Thrombin-induced activation of RhoA and its involvement in the regulation of myosin II light chain(20) phosphorylation (MLC-P) in alpha-toxin permeabilized platelets was investigated. Permeabilized platelets, expressing normal levels of P-selectin, displayed a Ca(2+)-dependent increase in shape change and MLC-P. Thrombin activated RhoA as measured by a rhotekin-binding assay within 30 s of stimulation under conditions of constant [Ca(2+)](i). Under the same conditions and timecourse, thrombin or GTPgammaS induced an increase in MLC-P and platelet shape change which was not dependent on an increase in [Ca(2+)](i). The thrombin- and GTPgammaS-induced MLC-P in constant [Ca(2+)](i) was inhibited by the addition of Y27632, a Rho-kinase inhibitor. This study directly demonstrates that thrombin can activate RhoA in platelets in a timecourse compatible with a role in increasing MLC-P and shape change (not involving an increase in [Ca(2+)](i)). This is also Rho-kinase-dependent. Copyright 2001 Academic Press.

Nitric oxide activity in platelets of dengue haemorrhagic fever patients: the apparent paradoxical role of ADMA and l-NMMA.

PubMed

Matsuura, Cristiane; Moraes, Thalyta L; Barbosa, Julia B; Moss, Monique B; Siqueira, Mariana A S; Mann, Giovanni E; Neto, Miguel Lemos; Brunini, Tatiana M C; Mendes-Ribeiro, Antonio Claudio

2012-03-01

Dengue haemorrhagic fever (DHF) is a prevalent acute disease that occurs in patients infected by an arbovirus in tropical and subtropical regions. We have previously shown increased intraplatelet nitric oxide (NO) production in patients with dengue fever associated with reduced platelet aggregation. In this study, l-arginine transport as well as expression and activity of nitric oxide synthase (NOS) isoforms in the presence or absence of l-arginine analogues were examined in 23 DHF patients. l-arginine transport and NOS activity in platelets were increased in patients with DHF compared with controls. However, platelet endothelial NOS (eNOS) and inducible (iNOS) protein levels did not differ between healthy controls and DHF patients. Endogenous or exogenous analogues did not inhibit platelet NOS activity from DHF patients. In contrast, endogenous l-arginine analogues [N(G)-monomethyl-l-arginine (l-NMMA) and asymmetric dimethylarginine (ADMA)] inhibited NOS activity in platelets from healthy subjects. These results show the first evidence that the intraplatelet l-arginine-NO pathway is activated in DHF patients. The lack of inhibition of NO formation in vitro by all l-arginine analogues tested in DHF platelets may suggest another mechanism by which NOS activity can be regulated. Copyright © 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Platelets and cancer: a casual or causal relationship: revisited

PubMed Central

Menter, David G.; Tucker, Stephanie C.; Kopetz, Scott; Sood, Anil K.; Crissman, John D.; Honn, Kenneth V.

2014-01-01

Human platelets arise as subcellular fragments of megakaryocytes in bone marrow. The physiologic demand, presence of disease such as cancer, or drug effects can regulate the production circulating platelets. Platelet biology is essential to hemostasis, vascular integrity, angiogenesis, inflammation, innate immunity, wound healing, and cancer biology. The most critical biological platelet response is serving as “First Responders” during the wounding process. The exposure of extracellular matrix proteins and intracellular components occurs after wounding. Numerous platelet receptors recognize matrix proteins that trigger platelet activation, adhesion, aggregation, and stabilization. Once activated, platelets change shape and degranulate to release growth factors and bioactive lipids into the blood stream. This cyclic process recruits and aggregates platelets along with thrombogenesis. This process facilitates wound closure or can recognize circulating pathologic bodies. Cancer cell entry into the blood stream triggers platelet-mediated recognition and is amplified by cell surface receptors, cellular products, extracellular factors, and immune cells. In some cases, these interactions suppress immune recognition and elimination of cancer cells or promote arrest at the endothelium, or entrapment in the microvasculature, and survival. This supports survival and spread of cancer cells and the establishment of secondary lesions to serve as important targets for prevention and therapy. PMID:24696047

Investigations of human platelet-type 12-lipoxygenase: role of lipoxygenase products in platelet activation1[S

PubMed Central

Ikei, Kenneth N.; Yeung, Jennifer; Apopa, Patrick L.; Ceja, Jesús; Vesci, Joanne; Holinstat, Michael

2012-01-01

Human platelet-type 12-lipoxygenase (12-LOX) has recently been shown to play an important role in regulation of human platelet function by reacting with arachidonic acid (AA). However, a number of other fatty acids are present on the platelet surface that, when cleaved from the phospholipid, can be oxidized by 12-LOX. We sought to characterize the substrate specificity of 12-LOX against six essential fatty acids: AA, dihomo-γ-linolenic acid (DGLA), eicosapentaenoic acid (EPA), α-linolenic acid (ALA), eicosadienoic acid (EDA), and linoleic acid (LA). Three fatty acids were comparable substrates (AA, DGLA, and EPA), one was 5-fold slower (ALA), and two showed no reactivity with 12-LOX (EDA and LA). The bioactive lipid products resulting from 12-LOX oxidation of DGLA, 12-(S)-hydroperoxy-8Z,10E,14Z-eicosatrienoic acid [12(S)-HPETrE], and its reduced product, 12(S)-HETrE, resulted in significant attenuation of agonist-mediated platelet aggregation, granule secretion, αIIbβ3 activation, Rap1 activation, and clot retraction. Treatment with DGLA similarly inhibited PAR1-mediated platelet activation as well as platelet clot retraction. These observations are in surprising contrast to our recent work showing 12(S)-HETE is a prothrombotic bioactive lipid and support our hypothesis that the overall effect of 12-LOX oxidation of fatty acids in the platelet is dependent on the fatty acid substrates available at the platelet membrane. PMID:22984144

Antiplatelet Activity of Morus alba Leaves Extract, Mediated via Inhibiting Granule Secretion and Blocking the Phosphorylation of Extracellular-Signal-Regulated Kinase and Akt

PubMed Central

Rhee, Man Hee; Sung, Yoon-Young; Yang, Won-Kyung; Kim, Seung Hyung; Kim, Ho-Kyoung

2014-01-01

Ethnopharmacological Relevance. Morus alba L. leaves (MAE) have been used in fork medicine for the treatment of beriberi, edema, diabetes, hypertension, and atherosclerosis. However, underlying mechanism of MAE on cardiovascular protection remains to be elucidated. Therefore, we investigated whether MAE affect platelet aggregation and thrombosis. Materials and Methods. The anti-platelet activity of MAE was studied using rat platelets. The extent of anti-platelet activity of MAE was assayed in collagen-induced platelet aggregation. ATP and serotonin release was carried out. The activation of integrin α IIb β 3 and phosphorylation of signaling molecules, including MAPK and Akt, were investigated with cytofluorometer and immunoblotting, respectively. The thrombus formation in vivo was also evaluated in arteriovenous shunt model of rats. Results. HPLC chromatographic analysis revealed that MAE contained rutin and isoquercetin. MAE dose-dependently inhibited collagen-induced platelet aggregation. MAE also attenuated serotonin secretion and thromboxane A2 formation. In addition, the extract in vivo activity showed that MAE at 100, 200, and 400 mg/kg significantly and dose-dependently attenuated thrombus formation in rat arterio-venous shunt model by 52.3% (P < 0.001), 28.3% (P < 0.01), and 19.1% (P < 0.05), respectively. Conclusions. MAE inhibit platelet activation, TXB2 formation, serotonin secretion, aggregation, and thrombus formation. The plant extract could be considered as a candidate to anti-platelet and antithrombotic agent. PMID:24701244

Tangeretin regulates platelet function through inhibition of phosphoinositide 3-kinase and cyclic nucleotide signaling.

PubMed

Vaiyapuri, Sakthivel; Ali, Marfoua S; Moraes, Leonardo A; Sage, Tanya; Lewis, Kirsty R; Jones, Chris I; Gibbins, Jonathan M

2013-12-01

Dietary flavonoids have long been appreciated in reducing cardiovascular disease risk factors, but their mechanisms of action are complex in nature. In this study, the effects of tangeretin, a dietary flavonoid, were explored on platelet function, signaling, and hemostasis. Tangeretin inhibited agonist-induced human platelet activation in a concentration-dependent manner. It inhibited agonist-induced integrin αIIbβ3 inside-out and outside-in signaling, intracellular calcium mobilization, and granule secretion. Tangeretin also inhibited human platelet adhesion and subsequent thrombus formation on collagen-coated surfaces under arterial flow conditions in vitro and reduced hemostasis in mice. Further characterization to explore the mechanism by which tangeretin inhibits platelet function revealed distinctive effects of platelet signaling. Tangeretin was found to inhibit phosphoinositide 3-kinase-mediated signaling and increase cGMP levels in platelets, although phosphodiesterase activity was unaffected. Consistent with increased cGMP levels, tangeretin increased the phosphorylation of vasodilator-stimulated phosphoprotein at S239. This study provides support for the ability and mechanisms of action of dietary flavonoids to modulate platelet signaling and function, which may affect the risk of thrombotic disease.

Type I Interferon-Mediated Skewing of the Serotonin Synthesis Is Associated with Severe Disease in Systemic Lupus Erythematosus

PubMed Central

Lood, Christian; Tydén, Helena; Gullstrand, Birgitta; Klint, Cecilia; Wenglén, Christina; Nielsen, Christoffer T.; Heegaard, Niels H. H.; Jönsen, Andreas; Kahn, Robin; Bengtsson, Anders A.

2015-01-01

Serotonin, a highly pro-inflammatory molecule released by activated platelets, is formed by tryptophan. Tryptophan is also needed in the production of kynurenine, a process mediated by the type I interferon (IFN)-regulated rate-limiting enzyme indoleamine 2,3-dioxygenase (IDO). The aim of this study was to investigate levels of serotonin in patients with the autoimmune disease systemic lupus erythematosus (SLE), association to clinical phenotype and possible involvement of IDO in regulation of serotonin synthesis. Serotonin levels were measured in serum and plasma from patients with SLE (n=148) and healthy volunteers (n=79) by liquid chromatography and ELISA, as well as intracellularly in platelets by flow cytometry. We found that SLE patients had decreased serotonin levels in serum (p=0.01) and platelets (p<0.0001) as compared to healthy individuals. SLE patients with ongoing type I IFN activity, as determined by an in-house reporter assay, had decreased serum levels of serotonin (p=0.0008) as well as increased IDO activity (p<0.0001), as determined by the kynurenine/tryptophan ratio measured by liquid chromatography. Furthermore, SLE sera induced IDO expression in WISH cells in a type I IFN-dependent manner (p=0.008). Also platelet activation contributed to reduce overall availability of serotonin levels in platelets and serum (p<0.05). Decreased serum serotonin levels were associated with severe SLE with presence of anti-dsDNA antibodies and nephritis. In all, reduced serum serotonin levels in SLE patients were related to severe disease phenotype, including nephritis, suggesting involvement of important immunopathological processes. Further, our data suggest that type I IFNs, present in SLE sera, are able to up-regulate IDO expression, which may lead to decreased serum serotonin levels. PMID:25897671

A Critical Role for the Transient Receptor Potential Channel Type 6 in Human Platelet Activation

PubMed Central

Conlon, Christine; Khasawneh, Fadi T.

2015-01-01

While calcium signaling is known to play vital roles in platelet function, the mechanisms underlying its receptor-operated calcium entry component (ROCE) remain poorly understood. It has been proposed, but never proven in platelets, that the canonical transient receptor potential channel-6 (TRPC6) mediates ROCE. Nonetheless, we have previously shown that the mouse TRPC6 regulates hemostasis, thrombogenesis by regulating platelet aggregation. In the present studies, we used a pharmacological approach to characterize the role of TRPC6 in human platelet biology. Thus, interestingly, we observed that a TRPC6 inhibitor exerted significant inhibitory effects on human platelet aggregation in a thromboxane receptor (TPR)-selective manner; no additional inhibition was observed in the presence of the calcium chelator BAPTA. This inhibitor also significantly inhibited human platelet secretion (dense and alpha granules), integrin IIb-IIIa, Akt and ERK phosphorylation, again, in a TPR-selective manner; no effects were observed in response to ADP receptor stimulation. Furthermore, there was a causal relationship between these inhibitory effects, and the capacity of the TRPC6 inhibitor to abrogate elevation in intracellular calcium, that was again found to be TPR-specific. This effect was not found to be due to antagonism of TPR, as the TRPC6 inhibitor did not displace the radiolabeled antagonist [3H]SQ29,548 from its binding sites. Finally, our studies also revealed that TRPC6 regulates human clot retraction, as well as physiological hemostasis and thrombus formation, in mice. Taken together, our findings demonstrate, for the first time, that TRPC6 directly regulates TPR-dependent ROCE and platelet function. Moreover, these data highlight TRPC6 as a novel promising therapeutic strategy for managing thrombotic disorders. PMID:25928636

Identification of Tspan9 as a novel platelet tetraspanin and the collagen receptor GPVI as a component of tetraspanin microdomains

PubMed Central

Protty, Majd B.; Watkins, Nicholas A.; Colombo, Dario; Thomas, Steven G.; Heath, Victoria L.; Herbert, John M. J.; Bicknell, Roy; Senis, Yotis A.; Ashman, Leonie K.; Berditchevski, Fedor; Ouwehand, Willem H.; Watson, Steve P.; Tomlinson, Michael G.

2008-01-01

Platelets are essential for wound healing and inflammatory processes, but can also play a deleterious role by causing heart attack and stroke. Normal platelet activation is dependent on tetraspanins, a superfamily of glycoproteins that function as ‘organisers’ of cell membranes by recruiting other receptors and signalling proteins into tetraspanin-enriched microdomains. However, our understanding of how tetraspanin microdomains regulate platelets is hindered by the fact that only four of the 33 mammalian tetraspanins have been identified in platelets. This is because of a lack of antibodies to most tetraspanins and difficulties in measuring mRNA, due to low levels in this anucleate cell. To identify potentially platelet-expressed tetraspanins, mRNA was measured in their nucleated progenitor cell, the megakaryocyte, using serial analysis of gene expression and DNA microarrays. Amongst 19 tetraspanins identified in megakaryocytes, Tspan9, a previously uncharacterized tetraspanin, was relatively specific to these cells. Through generating the first Tspan9 antibodies, Tspan9 expression was found to be tightly regulated in platelets. The relative levels of CD9, CD151, Tspan9 and CD63 were 100, 14, 6 and 2 respectively. Since CD9 was expressed at 49000 cell surface copies per platelet, this suggested a copy number of 2800 Tspan9 molecules. Finally, Tspan9 was shown to be a component of tetraspanin microdomains that included the collagen receptor GPVI (glycoprotein VI) and integrin α6β1, but not the von Willebrand receptor GPIbα or the integrins αIIbβ3 or α2β1. These findings suggest a role for Tspan9 in regulating platelet function in concert with other platelet tetraspanins and their associated proteins. PMID:18795891

Differential role of glycolipid-enriched membrane domains in glycoprotein VI- and integrin-mediated phospholipase Cgamma2 regulation in platelets.

PubMed Central

Wonerow, Peter; Obergfell, Achim; Wilde, Jonathan I; Bobe, Régis; Asazuma, Naoki; Brdicka, Tomás; Leo, Albrecht; Schraven, Burkhart; Horejsí, Václav; Shattil, Sanford J; Watson, Steve P

2002-01-01

The platelet collagen receptor glycoprotein VI (GPVI) and the fibrinogen receptor integrin alphaIIbbeta3 trigger intracellular signalling cascades involving the tyrosine kinase Syk, the adapter SLP-76 and phospholipase Cgamma2 (PLCgamma2). Similar pathways are activated downstream of immune receptors in lymphocytes, where they have been localized in part to glycolipid-enriched membrane domains (GEMs). Here we provide several lines of evidence that GPVI-mediated tyrosine phosphorylation of PLCgamma2 in platelets is dependent on GEM-organized signalling and utilizes the GEM resident adapter protein LAT (linker for activation of T cells). In sharp contrast, although fibrinogen binding to platelets stimulates alphaIIbbeta3-dependent activation of Syk and tyrosine phosphorylation of SLP-76 and PLCgamma2, it does not utilize GEMs to promote these responses or to support platelet aggregation. These results establish that GPVI and alphaIIbbeta3 trigger distinct patterns of receptor signalling in platelets, leading to tyrosine phosphorylation of PLCgamma2, and they highlight the role of GEMs in compartmentalizing signalling reactions involved in haemostasis. PMID:12049640

A novel mutation in the P2Y12 receptor and a function-reducing polymorphism in protease-activated receptor 1 in a patient with chronic bleeding.

PubMed

Patel, Y M; Lordkipanidzé, M; Lowe, G C; Nisar, S P; Garner, K; Stockley, J; Daly, M E; Mitchell, M; Watson, S P; Austin, S K; Mundell, S J

2014-05-01

The study of patients with bleeding problems is a powerful approach in determining the function and regulation of important proteins in human platelets. We have identified a patient with a chronic bleeding disorder expressing a homozygous P2RY(12) mutation, predicting an arginine to cysteine (R122C) substitution in the G-protein-coupled P2Y(12) receptor. This mutation is found within the DRY motif, which is a highly conserved region in G-protein-coupled receptors (GPCRs) that is speculated to play a critical role in regulating receptor conformational states. To determine the functional consequences of the R122C substitution for P2Y(12) function. We performed a detailed phenotypic analysis of an index case and affected family members. An analysis of the variant R122C P2Y(12) stably expressed in cells was also performed. ADP-stimulated platelet aggregation was reduced as a result of a significant impairment of P2Y(12) activity in the patient and family members. Cell surface R122C P2Y(12) expression was reduced both in cell lines and in platelets; in cell lines, this was as a consequence of agonist-independent internalization followed by subsequent receptor trafficking to lysosomes. Strikingly, members of this family also showed reduced thrombin-induced platelet activation, owing to an intronic polymorphism in the F2R gene, which encodes protease-activated receptor 1 (PAR-1), that has been shown to be associated with reduced PAR-1 receptor activity. Our study is the first to demonstrate a patient with deficits in two stimulatory GPCR pathways that regulate platelet activity, further indicating that bleeding disorders constitute a complex trait. © 2014 International Society on Thrombosis and Haemostasis.

Opposing Effects of Platelet-Activating Factor and Lyso-Platelet-Activating Factor on Neutrophil and Platelet ActivationS⃞

PubMed Central

Welch, Emily J.; Naikawadi, Ram P.; Li, Zhenyu; Lin, Phoebe; Ishii, Satoshi; Shimizu, Takao; Tiruppathi, Chinnaswamy; Du, Xiaoping; Subbaiah, Papasani V.; Ye, Richard D.

2009-01-01

Platelet-activating factor (PAF) is a potent, bioactive phospholipid that acts on multiple cells and tissues through its G protein-coupled receptor (GPCR). PAF is not stored but is rapidly generated via enzymatic acetylation of the precursor 1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (lysoPAF). The bioactivity of PAF is effectively and tightly regulated by PAF acetylhydrolases, which convert PAF back to lysoPAF. Previous studies report that lysoPAF is an inactive precursor and metabolite of PAF. However, lysoPAF has not been carefully studied in its own context. Here we report that lysoPAF has an opposing effect of PAF in the activation of neutrophils and platelets. Whereas PAF potentiates neutrophil NADPH oxidase activation, lysoPAF dose-dependently inhibits this function. Inhibition by lysoPAF is not affected by the use of a PAF receptor antagonist or genetic deletion of the PAF receptor gene. The mechanism of lysoPAF-mediated inhibition of neutrophils involves an elevation in the intracellular cAMP level, and pharmacological blockade of adenylyl cyclase completely reverses the inhibitory effect of lysoPAF. In addition, lysoPAF increases intracellular cAMP levels in platelets and inhibits thrombin-induced platelet aggregation, which can be reversed by inhibition of protein kinase A. These findings identify lysoPAF as a bioactive lipid with opposing functions of PAF and suggest a novel and intrinsic regulatory mechanism for balance of the potent activity of PAF. PMID:18931035

Platelet Activating Factor: A Growth Factor for Breast Cancer

DTIC Science & Technology

2006-09-01

synthase (ADS) increases ether lipid content, growth and PAF synthesis in MCF-7 cells. 4. Eicosapentaenoic acid (EPA) inhibits the synthesis of PAF...Schmitt, J. D., Bullock, B. C. Wykle, R. L. Reacylation of platelet activating factor with eicosapentaenoic acid in fish-oil-enriched monkey...breast cancer. Recent studies have shown that the ratio of two families of essential fatty acids is important in regulating many cellular processes

IGF-1 facilitates thrombopoiesis primarily through Akt activation.

PubMed

Chen, Shilei; Hu, Mengjia; Shen, Mingqiang; Wang, Song; Wang, Cheng; Chen, Fang; Tang, Yong; Wang, Xinmiao; Zeng, Hao; Chen, Mo; Gao, Jining; Wang, Fengchao; Su, Yongping; Xu, Yang; Wang, Junping

2018-05-25

It is known that insulin-like growth factor-1 (IGF-1) also functions as a hematopoietic factor, while its direct effect on thrombopoiesis remains unclear. In this study, we show that IGF-1 is able to promote CD34+ cell differentiation toward megakaryocytes (MKs), as well as the facilitation of proplatelet formation (PPF) and platelet production from cultured MKs. The in vivo study demonstrates that IGF-1 administration accelerates platelet recovery in mice after 6.0Gy of irradiation and in mice that received bone marrow transplantation (BMT) following 10.0Gy of lethal irradiation. Subsequent investigations reveal that ERK1/2 and Akt activation mediate the effect of IGF-1 on thrombopoiesis. Notably, Akt activation induced by IGF-1 is more apparent than that of ERK1/2, compared with that of thrombopoietin (TPO) treatment. Moreover, the effect of IGF-1 on thrombopoiesis is independent of TPO signaling, because IGF-1 treatment can also lead to a significant increase of platelet counts in homozygous TPO receptor mutant mice. Further analysis indicates that the activation of Akt triggered by IGF-1 requires the assistance of steroid receptor coactivator-3 (SRC-3). Therefore, our data reveal a distinct role of IGF-1 in regulating thrombopoiesis, providing new insights into TPO-independent regulation of platelet generation. Copyright © 2018 American Society of Hematology.

Gap junctions and connexin hemichannels in the regulation of haemostasis and thrombosis.

PubMed

Vaiyapuri, Sakthivel; Flora, Gagan D; Gibbins, Jonathan M

2015-06-01

Platelets are involved in the maintenance of haemostasis but their inappropriate activation leads to thrombosis, a principal trigger for heart attack and ischaemic stroke. Although platelets circulate in isolation, upon activation they accumulate or aggregate together to form a thrombus, where they function in a co-ordinated manner to prevent loss of blood and control wound repair. Previous report (1) indicates that the stability and functions of a thrombus are maintained through sustained, contact-dependent signalling between platelets. Given the role of gap junctions in the co-ordination of tissue responses, it was hypothesized that gap junctions may be present within a thrombus and mediate intercellular communication between platelets. Therefore studies were performed to explore the presence and functions of connexins in platelets. In this brief review, the roles of hemichannels and gap junctions in the control of thrombosis and haemostasis and the future directions for this research will be discussed.

Thrombin Receptor-Activating Protein (TRAP)-Activated Akt Is Involved in the Release of Phosphorylated-HSP27 (HSPB1) from Platelets in DM Patients

PubMed Central

Tokuda, Haruhiko; Kuroyanagi, Gen; Tsujimoto, Masanori; Matsushima-Nishiwaki, Rie; Akamatsu, Shigeru; Enomoto, Yukiko; Iida, Hiroki; Otsuka, Takanobu; Ogura, Shinji; Iwama, Toru; Kojima, Kumi; Kozawa, Osamu

2016-01-01

It is generally known that heat shock protein 27 (HSP27) is phosphorylated through p38 mitogen-activated protein (MAP) kinase. We have previously reported that HSP27 is released from human platelets associated with collagen-induced phosphorylation. In the present study, we conducted an investigation into the effect of thrombin receptor-activating protein (TRAP) on the release of HSP27 in platelets in type 2 diabetes mellitus (DM) patients. The phosphorylated-HSP27 levels induced by TRAP were directly proportional to the aggregation of platelets. The levels of phosphorylated-HSP27 (Ser-78) were correlated with the levels of phosphorylated-p38 MAP kinase and phosphorylated-Akt in the platelets stimulated by 10 µM TRAP but not with those of phosphorylated-p44/p42 MAP kinase. The levels of HSP27 released from the TRAP (10 µM)-stimulated platelets were correlated with the levels of phosphorylated-HSP27 in the platelets. The released platelet-derived growth factor-AB (PDGF-AB) levels were in parallel with the HSP27 levels released from the platelets stimulated by 10 µM TRAP. Although the area under the curve (AUC) of small aggregates (9–25 µm) induced by 10 µM TRAP showed no significant correlation with the released HSP27 levels, AUC of medium aggregates (25–50 µm), large aggregates (50–70 µm) and light transmittance were significantly correlated with the released HSP27 levels. TRAP-induced phosphorylation of HSP27 was truly suppressed by deguelin, an inhibitor of Akt, in the platelets from a healthy subject. These results strongly suggest that TRAP-induced activation of Akt in addition to p38 MAP kinase positively regulates the release of phosphorylated-HSP27 from human platelets, which is closely related to the platelet hyper-aggregation in type 2 DM patients. PMID:27187380

Thrombin Receptor-Activating Protein (TRAP)-Activated Akt Is Involved in the Release of Phosphorylated-HSP27 (HSPB1) from Platelets in DM Patients.

PubMed

Tokuda, Haruhiko; Kuroyanagi, Gen; Tsujimoto, Masanori; Matsushima-Nishiwaki, Rie; Akamatsu, Shigeru; Enomoto, Yukiko; Iida, Hiroki; Otsuka, Takanobu; Ogura, Shinji; Iwama, Toru; Kojima, Kumi; Kozawa, Osamu

2016-05-14

It is generally known that heat shock protein 27 (HSP27) is phosphorylated through p38 mitogen-activated protein (MAP) kinase. We have previously reported that HSP27 is released from human platelets associated with collagen-induced phosphorylation. In the present study, we conducted an investigation into the effect of thrombin receptor-activating protein (TRAP) on the release of HSP27 in platelets in type 2 diabetes mellitus (DM) patients. The phosphorylated-HSP27 levels induced by TRAP were directly proportional to the aggregation of platelets. The levels of phosphorylated-HSP27 (Ser-78) were correlated with the levels of phosphorylated-p38 MAP kinase and phosphorylated-Akt in the platelets stimulated by 10 µM TRAP but not with those of phosphorylated-p44/p42 MAP kinase. The levels of HSP27 released from the TRAP (10 µM)-stimulated platelets were correlated with the levels of phosphorylated-HSP27 in the platelets. The released platelet-derived growth factor-AB (PDGF-AB) levels were in parallel with the HSP27 levels released from the platelets stimulated by 10 µM TRAP. Although the area under the curve (AUC) of small aggregates (9-25 µm) induced by 10 µM TRAP showed no significant correlation with the released HSP27 levels, AUC of medium aggregates (25-50 µm), large aggregates (50-70 µm) and light transmittance were significantly correlated with the released HSP27 levels. TRAP-induced phosphorylation of HSP27 was truly suppressed by deguelin, an inhibitor of Akt, in the platelets from a healthy subject. These results strongly suggest that TRAP-induced activation of Akt in addition to p38 MAP kinase positively regulates the release of phosphorylated-HSP27 from human platelets, which is closely related to the platelet hyper-aggregation in type 2 DM patients.

Characterization of the platelet transcriptome by RNA sequencing in patients with acute myocardial infarction

PubMed Central

Eicher, John D.; Wakabayashi, Yoshiyuki; Vitseva, Olga; Esa, Nada; Yang, Yanqin; Zhu, Jun; Freedman, Jane E.; McManus, David D.; Johnson, Andrew D.

2016-01-01

Transcripts in platelets are largely produced in precursor megakaryocytes but remain physiologically-active as platelets translate RNAs and regulate protein/RNA levels. Recent studies using transcriptome sequencing (RNA-seq) characterized the platelet transcriptome in limited numbers of non-diseased individuals. Here, we expand upon these RNA-seq studies by completing RNA-seq in platelets from 32 patients with acute myocardial infarction (MI). Our goals were to characterize the platelet transcriptome using a population of patients with acute MI and relate gene expression to platelet aggregation measures and ST-segment elevation MI (STEMI) (n=16) versus non-STEMI (NSTEMI) (n=16) subtypes. Similar to other studies, we detected 9,565 expressed transcripts, including several known platelet-enriched markers (e.g., PPBP, OST4). Our RNA-seq data strongly correlated with independently ascertained platelet expression data and showed enrichment for platelet-related pathways (e.g., wound response, hemostasis, and platelet activation), as well as actin-related and post-transcriptional processes. Several transcripts displayed suggestively higher (FBXL4, ECHDC3, KCNE1, TAOK2, AURKB, ERG, and FKBP5) and lower (MIAT, PVRL3and PZP) expression in STEMI platelets compared to NSTEMI. We also identified transcripts correlated with platelet aggregation to TRAP (ATP6V1G2, SLC2A3), collagen (CEACAM1, ITGA2), and ADP (PDGFB, PDGFC, ST3GAL6). Our study adds to current platelet gene expression resources by providing transcriptome-wide analyses in platelets isolated from patients with acute MI. In concert with prior studies, we identify various genes for further study in regards to platelet function and acute MI. Future platelet RNA-seq studies examining more diverse sets of healthy and diseased samples will add to our understanding of platelet thrombotic and non-thrombotic functions. PMID:26367242

Effect of diazepam and clonazepam on the function of isolated rat platelet and neutrophil.

PubMed

Rajtar, Grazyna; Zółkowska, Dorota; Kleinrok, Zdzisław

2002-04-01

Benzodiazepine binding sites distinct from the GABA-receptor-chloride-complex in the central nervous system have been recognized in many peripheral tissues, but their physiological role remains unexplained. Our study was undertaken to examine the effects of diazepam, clonazepam, and PK 11195, a peripheral benzodiazepine receptor antagonist, on the functional and biochemical responses of platelets and neutrophils stimulated by different physiological agonists. The experiments were conducted on isolated washed rat platelets activated by arachidonic acid (AA), adenosine 5'-diphosphate (ADP), or thrombin and on isolated rat neutrophils activated by a chemotactic peptide, formyl methionyl leucyl phenylalanine (fMLP). The results showed that neither diazepam nor clonazepam nor PK 11195 alone augmented the response of resting platelets or modified neutrophil response, but diazepam and clonazepam in a concentration-dependent manner inhibited thrombin, ADP or AA-stimulated platelet aggregation and the thrombin-induced increase in free intracellular Ca2+. Both drugs also exerted an inhibitory effect on reactive oxygen species (ROS) produced by fMLP-stimulated neutrophils. However, diazepam was about 10 times more effective than clonazepam. PK11195 did not influence platelet and neutrophil function stimulated by agonists, but reversed the inhibitory action of both benzodiazepines on platelet activation and ROS production. The results indicated that in vitro diazepam, and in a much smaller degree clonazepam, may down-regulate platelet activation and release of some proinflammatory mediators by stimulated neutrophils. These effects are probably exerted by a specific benzodiazepine binding sites.

Hydroxysafflor yellow A of Carthamus tinctorius attenuates lung injury of aged rats exposed to gasoline engine exhaust by down-regulating platelet activation.

PubMed

Wang, Chaoyun; Wang, Chunhua; Ma, Chunlei; Huang, Qingxian; Sun, Hongliu; Zhang, Xiaomin; Bai, Xianyong

2014-02-15

Long-term inhalation of gasoline engine exhaust (GEE) increases the risk of respiratory disease. Studies have suggested involvement of platelets in the development of some lung diseases. Hydroxysafflor yellow A (HSYA), a flavonoid compound, prevents hemostasis. Therefore, we investigated its effects on GEE-induced lung injury, and role of platelets in injury. Sixty-week-old male Sprague-Dawley rats were exposed to GEE for 4h/day for 6 weeks, and then grouped as follows: control, GEE, GEE+HSYA, GEE+HSYA+GW9662, and GEE+GW9662. Arterial oxygen tension (PaO2), carbon dioxide tension (PaCO2), pH, and the PaO2/fraction of inspired oxygen ratio (PaO2/FiO2) in the blood were detected using a blood gas analyzer. Wet/dry lung weight ratio, total protein in bronchoalveolar lavage fluid (BALF), and cytokine concentrations in serum and BALF were determined. Furthermore, cyclic adenosine monophosphate (cAMP) level and expression levels of target proteins were analyzed. Platelets were counted and their state was evaluated. HSYA attenuated GEE-mediated decreases in PaO2, PaO2/FiO2, platelet cAMP level, protein kinase A (PKA) activity, and peroxisome proliferator-activated receptor γ (PPARγ) expression. HSYA also attenuated GEE-mediated increases in lung permeability, cytokine levels in serum and BALF, plasma platelet count, and ADP-mediated platelet aggregation. Moreover, it suppressed GEE-induced increases in the expression of adhesion molecules and proinflammatory cytokines in platelets and lung tissue. Therefore, HSYA is therapeutically effective for GEE-mediated lung injury and acts by enhancing PKA activity and inhibiting platelet activation. Copyright © 2013 Elsevier GmbH. All rights reserved.

Pathophysiological consequences of receptor mistraffic: Tales from the platelet P2Y12 receptor.

PubMed

Cunningham, Margaret R; Aungraheeta, Riyaad; Mundell, Stuart J

2017-07-05

Genetic variations in G protein-coupled receptor (GPCR) genes can disrupt receptor function in a wide variety of human genetic diseases, including platelet bleeding disorders. Platelets are critical for haemostasis with inappropriate platelet activation leading to the development of arterial thrombosis, which can result in heart attack and stroke whilst decreased platelet activity is associated with an increased risk of bleeding. GPCRs expressed on the surface of platelets play key roles in regulating platelet activity and therefore function. Receptors include purinergic receptors (P2Y 1 and P2Y 12 ), proteinase-activated receptor (PAR1 and PAR4) and thromboxane receptors (TPα), among others. Pharmacological blockade of these receptors forms a powerful therapeutic tool in the treatment and prevention of arterial thrombosis. With the advance of genomic technologies, there has been a substantial increase in the identification of naturally occurring rare and common GPCR variants. These variants include single-nucleotide polymorphisms (SNPs) and insertion or deletions that have the potential to alter GPCR expression or function. A number of defects in platelet GPCRs that disrupt receptor function have now been characterized in patients with mild bleeding disorders. This review will focus on rare, function-disrupting variants of platelet GPCRs with particular emphasis upon mutations in the P2Y 12 receptor gene that affect receptor traffic to modulate platelet function. Further this review will outline how the identification and characterization of function-disrupting GPCR mutations provides an essential link in translating our detailed understanding of receptor traffic and function in cell line studies into relevant human biological systems. Copyright © 2017. Published by Elsevier B.V.

P2Y12 regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries

PubMed Central

André, Patrick; Delaney, Suzanne M.; LaRocca, Thomas; Vincent, Diana; DeGuzman, Francis; Jurek, Marzena; Koller, Beverley; Phillips, David R.; Conley, Pamela B.

2003-01-01

The critical role for ADP in arterial thrombogenesis was established by the clinical success of P2Y12 antagonists, currently used at doses that block 40–50% of the P2Y12 on platelets. This study was designed to determine the role of P2Y12 in platelet thrombosis and how its complete absence affects the thrombotic process. P2Y12-null mice were generated by a gene-targeting strategy. Using an in vivo mesenteric artery injury model and real-time continuous analysis of the thrombotic process, we observed that the time for appearance of first thrombus was delayed and that only small, unstable thrombi formed in P2Y12–/– mice without reaching occlusive size, in the absence of aspirin. Platelet adhesion to vWF was impaired in P2Y12–/– platelets. While adhesion to fibrinogen and collagen appeared normal, the platelets in thrombi from P2Y12–/– mice on collagen were less dense and less activated than their WT counterparts. P2Y12–/– platelet activation was also reduced in response to ADP or a PAR-4–activating peptide. Thus, P2Y12 is involved in several key steps of thrombosis: platelet adhesion/activation, thrombus growth, and stability. The data suggest that more aggressive strategies of P2Y12 antagonism will be antithrombotic without the requirement of aspirin cotherapy and may provide benefits even to the aspirin-nonresponder population. PMID:12897207

Regulating billions of blood platelets: glycans and beyond

PubMed Central

Grozovsky, Renata; Giannini, Silvia; Falet, Hervé

2015-01-01

The human body produces and removes 1011 platelets daily to maintain a normal steady state platelet count. Platelet production must be regulated to avoid spontaneous bleeding or arterial occlusion and organ damage. Multifaceted and complex mechanisms control platelet production and removal in physiological and pathological conditions. This review will focus on different mechanisms of platelet senescence and clearance with specific emphasis on the role of posttranslational modifications. It will also briefly address platelet transfusion and the role of glycans in the clearance of stored platelets. PMID:26330242

Activated platelet-derived growth factor β receptor and Ras-mitogen-activated protein kinase pathway in natural bovine urinary bladder carcinomas.

PubMed

Corteggio, Annunziata; Di Geronimo, Ornella; Roperto, Sante; Roperto, Franco; Borzacchiello, Giuseppe

2012-03-01

Bovine papillomavirus types 1 or 2 (BPV-1/2) are involved in the aetiopathogenesis of bovine urinary bladder cancer. BPV-1/2 E5 activates the platelet-derived growth factor β receptor (PDGFβR). The aim of this study was to analyse the Ras/mitogen-activated protein kinase (MAPK) pathway in relation to activation of PDGFβR in natural bovine urinary bladder carcinomas. Co-immunoprecipitation and Western blot analysis demonstrated that recruitment of growth factor receptor bound protein 2 (GRB-2) and Sos-1 to the activated PDGFβR was increased in carcinomas compared to normal tissues. Higher grade bovine urinary bladder carcinomas were associated with activation of Ras, but not with activation of downstream mitogen-activated protein kinase/extracellular signal-regulated kinase (Mek 1/2) or extracellular signal-regulated kinase (Erk 1/2). Copyright © 2011 Elsevier Ltd. All rights reserved.

ROS-mediated platelet generation: a microenvironment-dependent manner for megakaryocyte proliferation, differentiation, and maturation

PubMed Central

Chen, S; Su, Y; Wang, J

2013-01-01

Platelets have an important role in the body because of their manifold functions in haemostasis, thrombosis, and inflammation. Platelets are produced by megakaryocytes (MKs) that are differentiated from haematopoietic stem cells via several consecutive stages, including MK lineage commitment, MK progenitor proliferation, MK differentiation and maturation, cell apoptosis, and platelet release. During differentiation, the cells migrate from the osteoblastic niche to the vascular niche in the bone marrow, which is accompanied by reactive oxygen species (ROS)-dependent oxidation state changes in the microenvironment, suggesting that ROS can distinctly influence platelet generation and function in a microenvironment-dependent manner. The objective of this review is to reveal the role of ROS in regulating MK proliferation, differentiation, maturation, and platelet activation, thereby providing new insight into the mechanism of platelet generation, which may lead to the development of new therapeutic agents for thrombocytopenia and/or thrombosis. PMID:23846224

Mechanism of platelet functional changes and effects of anti-platelet agents on in vivo hemostasis under different gravity conditions.

PubMed

Li, Suping; Shi, Quanwei; Liu, Guanglei; Zhang, Weilin; Wang, Zhicheng; Wang, Yuedan; Dai, Kesheng

2010-05-01

Serious thrombotic and hemorrhagic problems or even fatalities evoked by either microgravity or hypergravity occur commonly in the world. We recently reported that platelet functions are inhibited in microgravity environments and activated under high-G conditions, which reveals the pathogenesis for gravity change-related hemorrhagic and thrombotic diseases. However, the mechanisms of platelet functional variations under different gravity conditions remain unclear. In this study we show that the amount of filamin A coimmunoprecipitated with GPIbalpha was enhanced in platelets exposed to modeled microgravity and, in contrast, was reduced in 8 G-exposed platelets. Hypergravity induced actin filament formation and redistribution, whereas actin filaments were reduced in platelets treated with modeled microgravity. Furthermore, intracellular Ca2+ levels were elevated by hypergravity. Pretreatment of platelets with the cell-permeable Ca2+ chelator BAPTA-AM had no effect on cytoskeleton reorganization induced by hypergravity but significantly reduced platelet aggregation induced by ristocetin/hypergravity. Two anti-platelet agents, aspirin and tirofiban, effectively reversed the shortened tail bleeding time and reduced the death rate of mice exposed to hypergravity. Furthermore, the increased P-selectin surface expression was obviously reduced in platelets from mice treated with aspirin/hypergravity compared with those from mice treated with hypergravity alone. These data suggest that the actin cytoskeleton reorganization and intracellular Ca2+ level play key roles in the regulation of platelet functions in different gravitational environments. The results with anti-platelet agents not only further confirm the activation of platelets in vivo but also suggest a therapeutic potential for hypergravity-induced thrombotic diseases.

Aspirin Inhibits Platelet-Derived Sphingosine-1-Phosphate Induced Endothelial Cell Migration.

PubMed

Polzin, Amin; Knoop, Betül; Böhm, Andreas; Dannenberg, Lisa; Zurek, Mark; Zeus, Tobias; Kelm, Malte; Levkau, Bodo; Rauch, Bernhard H

2018-01-01

Aspirin plays a crucial role in the prevention of cardiovascular diseases. We previously described that aspirin has effects beyond inhibition of platelet aggregation, as it inhibited thrombin-mediated release of sphingosine-1-phosphate (S1P) from human platelets. S1P is a bioactive lipid with important functions on inflammation and apoptosis. In endothelial cells (EC), S1P is a key regulator of cell migration. In this study, we aimed to analyze the effects of aspirin on platelet-induced EC migration. Human umbilical EC migration was measured by Boyden chamber assay. EC migration was induced by platelet supernatants of thrombin receptor-activating peptide-1 (AP1) stimulated platelets. To investigate the S1P receptor subtype that promotes EC migration, specific inhibitors of S1P receptor subtypes were applied. S1P induced EC migration in a concentration-dependent manner. EC migration induced by AP1-stimulated platelet supernatants was reduced by aspirin. S1P1 receptor inhibition almost completely abolished EC migration induced by activated platelets. The inhibition of S1P2 or S1P3 receptor had no effect. Aspirin inhibits EC migration induced by activated platelets that is in part due to S1P and mediated by the endothelial S1P1 receptor. The clinical significance of this novel mechanism of aspirin action has to be investigated in future studies. © 2017 S. Karger AG, Basel.

Reciprocal regulation of platelet responses to P2Y and thromboxane receptor activation.

PubMed

Barton, J F; Hardy, A R; Poole, A W; Mundell, S J

2008-03-01

Thromboxane A(2) and ADP are two major platelet agonists that stimulate two sets of G protein-coupled receptors to activate platelets. Although aggregation responses to ADP and thromboxane desensitize, there are no reports currently addressing whether activation by one agonist may heterologously desensitize responses to the other. To demonstrate whether responses to ADP or U46619 may be modulated by prior treatment of platelets with the alternate agonist, revealing a level of cross-desensitization between receptor systems. Here we show that pretreatment of platelets with either agonist substantially desensitizes aggregation responses to the other agonist. Calcium responses to thromboxane receptor activation are desensitized by preactivation of P2Y(1) but not P2Y(12) receptors. This heterologous desensitization is mediated by a protein kinase C (PKC)-independent mechanism. Reciprocally, calcium responses to ADP are desensitized by pretreatment of platelets with the thromboxane analogue, U46619, and P2Y(12)-mediated inhibition of adenylate cyclase is also desensitized by pretreatment with U46619. In this direction, desensitization is comprised of two components, a true heterologous component that is PKC-independent, and a homologous component that is mediated through stimulated release of dense granule ADP. This study reveals cross-desensitization between ADP and thromboxane receptor signaling in human platelets. Cross-desensitization is mediated by protein kinases, involving PKC-dependent and independent pathways, and indicates that alterations in the activation state of one receptor may have effects upon the sensitivity of the other receptor system.

Intraplatelet reactive oxygen species (ROS) correlate with the shedding of adhesive receptors, microvesiculation and platelet adhesion to collagen during storage: Does endogenous ROS generation downregulate platelet adhesive function?

PubMed

Ghasemzadeh, Mehran; Hosseini, Ehteramolsadat; Roudsari, Zahra Oushyani; Zadkhak, Parvin

2018-03-01

Platelets storage lesion is mainly orchestrated by platelet activating signals during storage. Reactive oxygen species (ROS) are being considered as important signaling molecules modulating platelet function while their production has also been shown to be augmented by platelet activation. This study investigated to what extent endogenous ROS generation during platelet storage could be correlated with platelet receptor shedding, microvesiculation and adhesive function. 10 PRP-platelet concentrates were subjected to flow cytometry analysis to examine the generation of intraplatelet ROS on days 1, 5 and 7 after storage. In 5 day-stored platelets considering 40% of ROS generation as a cutoff point, samples were divided into two groups of those with higher or lower levels of ROS. The expression of adhesion receptors (GPVI, GPIbα), the amount of microparticles and phosphatidylserine exposure in each group were then examined by flow cytometry. Platelet receptor shedding and adhesion to collagen matrix were respectively measured by western blotting and microscopic assays. Our data showed lowered expression of GPIbα (p < 0.05) and GPVI in samples with ROS > 40% than those with ROS ≤ 40%, whereas receptors shedding and microvesiculation were (p < 0.05) elevated in platelets with higher levels of ROS. Functionally, we observed significantly (p < 0.05) lower levels of platelet adhesion to collagen matrix in samples with ROS generation more than 40%. Taken together, we showed correlations between intraplatelet ROS generation and either platelet receptors or microparticle shedding as well as platelet adhesive capacity to collagen. These findings suggest that augmented ROS generation during storage might be relevant to down-regulation of platelet adhesive function. Copyright © 2018 Elsevier Ltd. All rights reserved.

Platelets Express Activated P2Y12 Receptor in Patients With Diabetes Mellitus.

PubMed

Hu, Liang; Chang, Lin; Zhang, Yan; Zhai, Lili; Zhang, Shenghui; Qi, Zhiyong; Yan, Hongmei; Yan, Yan; Luo, Xinping; Zhang, Si; Wang, Yiping; Kunapuli, Satya P; Ye, Hongying; Ding, Zhongren

2017-08-29

Platelets from patients with diabetes mellitus are hyperactive. Hyperactivated platelets may contribute to cardiovascular complications and inadequate responses to antiplatelet agents in the setting of diabetes mellitus. However, the underlying mechanism of hyperactivated platelets is not completely understood. We measured P2Y 12 expression on platelets from patients with type 2 diabetes mellitus and on platelets from rats with diabetes mellitus. We also assayed platelet P2Y 12 activation by measuring cAMP and VASP phosphorylation. The antiplatelet and antithrombotic effects of AR-C78511 and cangrelor were compared in rats. Finally, we explored the role of the nuclear factor-κB pathway in regulating P2Y 12 receptor expression in megakaryocytes. Platelet P2Y 12 levels are 4-fold higher in patients with type 2 diabetes mellitus compared with healthy subjects. P2Y 12 expression correlates with ADP-induced platelet aggregation (r=0.89, P <0.01). P2Y 12 in platelets from patients with diabetes mellitus is constitutively activated. Although both AR-C78511, a potent P2Y 12 inverse agonist, and cangrelor have similar antiplatelet efficacy on platelets from healthy subjects, AR-C78511 exhibits more powerful antiplatelet effects on diabetic platelets than cangrelor (aggregation ratio 36±3% versus 49±5%, respectively, P <0.05). Using a FeCl 3 -injury mesenteric arteriole thrombosis model in rats and an arteriovenous shunt thrombosis model in rats, we found that the inverse agonist AR-C78511 has greater antithrombotic effects on GK rats with diabetes mellitus than cangrelor (thrombus weight 4.9±0.3 mg versus 8.3±0.4 mg, respectively, P <0.01). We also found that a pathway involving high glucose-reactive oxygen species-nuclear factor-κB increases platelet P2Y 12 receptor expression in diabetes mellitus. Platelet P2Y 12 receptor expression is significantly increased and the receptor is constitutively activated in patients with type 2 diabetes mellitus, which contributes to platelet hyperactivity and limits antiplatelet drug efficacy in type 2 diabetes mellitus. © 2017 American Heart Association, Inc.

NF-E2 p45 Is Important for Establishing Normal Function of Platelets

PubMed Central

Fujita, Rie; Takayama-Tsujimoto, Mariko; Satoh, Hironori; Gutiérrez, Laura; Aburatani, Hiroyuki; Fujii, Satoshi; Sarai, Akinori; Bresnick, Emery H.

2013-01-01

NF-E2 is a heterodimeric transcription factor consisting of p45 and small Maf subunits. Since p45−/− mice display severe thrombocytopenia, p45 is recognized as a critical regulator of platelet production from megakaryocytes. To identify direct p45 target genes in megakaryocytes, we used chromatin immunoprecipitation (ChIP) sequencing to analyze the genome-wide chromatin occupancy of p45 in primary megakaryocytes. p45 target gene candidates obtained from the analysis are implicated in the production and function of platelets. Two of these genes, Selp and Myl9, were verified as direct p45 targets through multiple approaches. Since P-selectin, encoded by Selp, plays a critical role in platelet function during thrombogenesis, we tested whether p45 determines the intrinsic reactivity and potency of platelets generated from megakaryocytes. Mice expressing a hypomorphic p45 mutant instead of wild-type p45 in megakaryocytes (p45−/−:ΔNTD-Tg mice) displayed platelet hypofunction accompanied by mild thrombocytopenia. Furthermore, lung metastasis of melanoma cells, which requires platelet activation, was repressed in p45−/−:ΔNTD-Tg mice compared to control mice, validating the impaired function of platelets produced from p45−/−:ΔNTD-Tg megakaryocytes. By activating genes in megakaryocytes that mediate platelet production and function, p45 determines the quantity and quality of platelets. PMID:23648484

Endothelial- and Platelet-Derived Microparticles Are Generated During Liver Resection in Humans.

PubMed

Banz, Yara; Item, Gian-Marco; Vogt, Andreas; Rieben, Robert; Candinas, Daniel; Beldi, Guido

2016-01-01

Cell-derived plasma microparticles (<1.5 μm) originating from various cell types have the potential to regulate thrombogenesis and inflammatory responses. The aim of this study was to test the hypothesis that microparticles generated during hepatic surgery co-regulate postoperative procoagulant and proinflammatory events. In 30 patients undergoing liver resection, plasma microparticles were isolated, quantitated, and characterized as endothelial (CD31+, CD41-), platelet (CD41+), or leukocyte (CD11b+) origin by flow cytometry and their procoagulant and proinflammatory activity was measured by immunoassays. During liver resection, the total numbers of microparticles increased with significantly more Annexin V-positive, endothelial and platelet-derived microparticles following extended hepatectomy compared to standard and minor liver resections. After liver resection, microparticle tissue factor and procoagulant activity increased along with overall coagulation as assessed by thrombelastography. Levels of leukocyte-derived microparticles specifically increased in patients with systemic inflammation as assessed by C-reactive protein but are independent of the extent of liver resection. Endothelial and platelet-derived microparticles are specifically elevated during liver resection, accompanied by increased procoagulant activity. Leukocyte-derived microparticles are a potential marker for systemic inflammation. Plasma microparticles may represent a specific response to surgical stress and may be an important mediator of postoperative coagulation and inflammation.

Quercetin changes purinergic enzyme activities and oxidative profile in platelets of rats with hypothyroidism.

PubMed

Baldissarelli, Jucimara; Santi, Adriana; Schmatz, Roberta; Zanini, Daniela; Cardoso, Andréia M; Abadalla, Fátima H; Thomé, Gustavo R; Murussi, Camila; Polachini, Carla R N; Delenogare, Diéssica P; Loro, Vania L; Morsch, Vera M; Schetinger, Maria R C

2016-12-01

Diseases related to thyroid hormones have been extensively studied because affect a large number of individuals, and these hormones participate in the regulation of the whole organism homeostasis. However, little is known about the involvement of purinergic signaling related to oxidative stress in hypothyroidism and possible therapeutic adjuncts for treatment of this disorder. Thus, the present study investigates the effects of quercetin on NTPDase, 5'-nucleotidase and adenosine deaminase activities, platelet aggregation and oxidative profile in platelets of rats with methimazole (MMI)-induced hypothyroidism. Methimazole at a concentration of 20mg/100mL was administered for 90days. From the second month the animals received quercetin 10 or 25mg/kg for 60days. Results showed that: Ecto-5'-nucleotidase activity decreased in methimazole/water group and the treatment with quercetin 25mg/kg decreased NTPDase, 5'-nucleotidase and adenosine deaminase activities. Moreover, platelet aggregation increased in methimazole/water group. Lipid peroxidation increased while superoxide dismutase and catalase activities decreased, but, interestingly, the treatment with quercetin reversed these changes. These results demonstrated that quercetin modulates adenine nucleotide hydrolysis decreasing the ADP formation and adenosine deamination. At the same time quercetin improves the oxidative profile, as well as reduces platelet aggregation, which together with the modulation in the nucleotides levels can contribute to the prevention of platelet disorders. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation

PubMed Central

Metcalfe, Clive; Ramasubramoni, Anjana; Pula, Giordano; Harper, Matthew T.; Mundell, Stuart J.; Coxon, Carmen H.

2016-01-01

Thioredoxin (Trx) is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12) to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase). In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb). This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents. PMID:27716777

Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation.

PubMed

Metcalfe, Clive; Ramasubramoni, Anjana; Pula, Giordano; Harper, Matthew T; Mundell, Stuart J; Coxon, Carmen H

2016-01-01

Thioredoxin (Trx) is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12) to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase). In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb). This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents.

Novel iridium (III)‑derived organometallic compound for the inhibition of human platelet activation.

PubMed

Shyu, Kou-Gi; Velusamy, Marappan; Hsia, Chih-Wei; Yang, Chih-Hao; Hsia, Chih-Hsuan; Chou, Duen-Suey; Jayakumar, Thanasekaran; Sheu, Joen-Rong; Li, Jiun-Yi

2018-05-01

Since cisplatin achieved clinical success, transition metal platinum (Pt) drugs have been effectively used for the treatment of cancer. Iridium (Ir) compounds are considered to be potential alternatives to Pt compounds, as they possess promising anticancer effects with minor side effects. Platelet activation is associated with the metastasis and progression of cancer, and also with arterial thrombosis. Therefore, it is necessary to develop novel, effective antithrombotic agents. An Ir (III)‑derived complex, [Ir (Cp*) 1‑(2‑pyridyl)‑3‑(3‑methoxyphenyl)imidazo[1,5‑a]pyridine Cl]BF4 (Ir‑3), was developed as a novel antiplatelet drug. Ir‑3 exerted more potent inhibitory activity on platelet aggregation stimulated by collagen compared with other agonists, including thrombin. In collagen‑activated platelets, Ir‑3 also inhibited adenosine trisphosphate release, intracellular Ca+2 mobilization and surface P‑selectin expression, as well as the phosphorylation of phospholipase Cγ2 (PLCγ2), protein kinase C (PKC), protein kinase B (Akt) and c‑Jun N‑terminal kinase (JNK) 1, but not p38 mitogen‑activated protein kinase or extracellular signal‑regulated kinases. Ir‑3 did not markedly affect phorbol 12, 13‑dibutyrate‑stimulated platelet aggregation. Neither the adenylate cyclase inhibitor SQ22536 nor the guanylate cyclase inhibitor 1H‑[1, 2, 4] oxadiazolo [4,3‑a]quinoxalin‑1‑one significantly reversed the Ir‑3‑mediated inhibition of platelet aggregation. Furthermore, Ir‑3 had no considerable diminishing effects on OH radical signals in collagen‑stimulated platelets or Fenton reaction solution. In conclusion, Ir‑3 serves a novel function in the inhibition of platelet aggregation through inhibiting the PLCγ2‑PKC cascade, and the subsequent suppression of Akt and JNK1 activation. Therefore, Ir‑3 may be a potential novel therapeutic agent for the treatment of thromboembolic disorders, or the interplay between platelets and tumor cells which contributes to tumor cell proliferation and progression.

Cbl-b is a novel physiologic regulator of glycoprotein VI-dependent platelet activation.

PubMed

Daniel, James L; Dangelmaier, Carol A; Mada, Sripal; Buitrago, Lorena; Jin, Jianguo; Langdon, Wallace Y; Tsygankov, Alexander Y; Kunapuli, Satya P; Sanjay, Archana

2010-06-04

Cbl-b, a member of the Cbl family of E3 ubiquitin ligases, plays an important role in the activation of lymphocytes. However, its function in platelets remains unknown. We show that Cbl-b is expressed in human platelets along with c-Cbl, but in contrast to c-Cbl, it is not tyrosine-phosphorylated upon glycoprotein VI (GPVI) stimulation. Cbl-b, unlike c-Cbl, is not required for Syk ubiquitylation downstream of GPVI activation. Phospholipase Cgamma2 (PLCgamma2) and Bruton's tyrosine kinase (BTK) are constituently associated with Cbl-b. Cbl-b-deficient (Cbl-b(-/-)) platelets display an inhibition in the concentration-response curve for GPVI-specific agonist-induced aggregation, secretion, and Ca(2+) mobilization. A parallel inhibition is found for activation of PLCgamma2 and BTK. However, Syk activation is not affected by the absence of Cbl-b, indicating that Cbl-b acts downstream of Syk but upstream of BTK and PLCgamma2. When Cbl-b(-/-) mice were tested in the ferric chloride thrombosis model, occlusion time was increased and clot stability was reduced compared with wild type controls. These data indicate that Cbl-b plays a positive modulatory role in GPVI-dependent platelet signaling, which translates to an important regulatory role in hemostasis and thrombosis in vivo.

Endothelial progenitor cells bind and inhibit platelet function and thrombus formation.

PubMed

Abou-Saleh, Haissam; Yacoub, Daniel; Théorêt, Jean-François; Gillis, Marc-Antoine; Neagoe, Paul-Eduard; Labarthe, Benoit; Théroux, Pierre; Sirois, Martin G; Tabrizian, Maryam; Thorin, Eric; Merhi, Yahye

2009-12-01

Interactions of endothelial progenitor cells (EPCs) with vascular and blood cells contribute to vascular homeostasis. Although platelets promote the homing of EPCs to sites of vascular injury and their differentiation into endothelial cells, the functional consequences of such interactions on platelets remain unknown. Herein, we addressed the interactions between EPCs and platelets and their impact on platelet function and thrombus formation. Cultured on fibronectin in conditioned media, human peripheral blood mononuclear cells differentiated, within 10 days of culture, into EPCs, which uptake acetylated low-density lipoprotein, bind ulex-lectin, lack monocyte/leukocyte markers (CD14, P-selectin glycoprotein ligand-1, L-selectin), express progenitor/endothelial markers (CD34, vascular endothelial growth factor receptor-2, von Willebrand factor, and vascular endothelial cadherin), and proliferate in culture. These EPCs bound activated platelets via CD62P and inhibited its translocation, glycoprotein IIb/IIIa activation, aggregation, and adhesion to collagen, mainly via prostacyclin secretion. Indeed, this was associated with upregulation of cyclooxygenase-2 and inducible nitric oxide synthase. However, the effects on platelets in vitro were reversed by cyclooxygenase and cyclooxygenase-2 inhibition but not by nitric oxide or inducible nitric oxide synthase inhibition. Moreover, in a ferric chloride-induced murine arterial thrombosis model, injection of EPCs led to their incorporation into sites of injury and impaired thrombus formation, leading to an incomplete occlusion with 50% residual flow. Peripheral blood mononuclear cell-derived EPCs bind platelets via CD62P and inhibit platelet activation, aggregation, adhesion to collagen, and thrombus formation, predominantly via upregulation of cyclooxygenase-2 and secretion of prostacyclin. These findings add new insights into the biology of EPCs and define their potential roles in regulating platelet function and thrombosis.

Endothelial Progenitor Cells Bind and Inhibit Platelet Function and Thrombus Formation

PubMed Central

Abou-Saleh, Haissam; Yacoub, Daniel; Théorêt, Jean-François; Gillis, Marc-Antoine; Neagoe, Paul-Eduard; Labarthe, Benoit; Théroux, Pierre; Sirois, Martin G.; Tabrizian, Maryam; Thorin, Eric; Merhi, Yahye

2013-01-01

Background Interactions of endothelial progenitor cells (EPCs) with vascular and blood cells contribute to vascular homeostasis. Although platelets promote the homing of EPCs to sites of vascular injury and their differentiation into endothelial cells, the functional consequences of such interactions on platelets remain unknown. Herein, we addressed the interactions between EPCs and platelets and their impact on platelet function and thrombus formation. Methods and Results Cultured on fibronectin in conditioned media, human peripheral blood mononuclear cells differentiated, within 10 days of culture, into EPCs, which uptake acetylated low-density lipoprotein, bind ulex-lectin, lack monocyte/leukocyte markers (CD14, P-selectin glycoprotein ligand-1, L-selectin), express progenitor/endothelial markers (CD34, vascular endothelial growth factor receptor-2, von Willebrand factor, and vascular endothelial cadherin), and proliferate in culture. These EPCs bound activated platelets via CD62P and inhibited its translocation, glycoprotein IIb/IIIa activation, aggregation, and adhesion to collagen, mainly via prostacyclin secretion. Indeed, this was associated with upregulation of cyclooxygenase-2 and inducible nitric oxide synthase. However, the effects on platelets in vitro were reversed by cyclooxygenase and cyclooxygenase-2 inhibition but not by nitric oxide or inducible nitric oxide synthase inhibition. Moreover, in a ferric chloride–induced murine arterial thrombosis model, injection of EPCs led to their incorporation into sites of injury and impaired thrombus formation, leading to an incomplete occlusion with 50% residual flow. Conclusions Peripheral blood mononuclear cell– derived EPCs bind platelets via CD62P and inhibit platelet activation, aggregation, adhesion to collagen, and thrombus formation, predominantly via upregulation of cyclooxygenase-2 and secretion of prostacyclin. These findings add new insights into the biology of EPCs and define their potential roles in regulating platelet function and thrombosis. PMID:19917882

Dual-specificity phosphatase 3 deficiency or inhibition limits platelet activation and arterial thrombosis.

PubMed

Musumeci, Lucia; Kuijpers, Marijke J; Gilio, Karen; Hego, Alexandre; Théâtre, Emilie; Maurissen, Lisbeth; Vandereyken, Maud; Diogo, Catia V; Lecut, Christelle; Guilmain, William; Bobkova, Ekaterina V; Eble, Johannes A; Dahl, Russell; Drion, Pierre; Rascon, Justin; Mostofi, Yalda; Yuan, Hongbin; Sergienko, Eduard; Chung, Thomas D Y; Thiry, Marc; Senis, Yotis; Moutschen, Michel; Mustelin, Tomas; Lancellotti, Patrizio; Heemskerk, Johan W M; Tautz, Lutz; Oury, Cécile; Rahmouni, Souad

2015-02-17

A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. A better understanding of the molecular mechanisms leading to platelet activation is important for the development of improved therapies. Recently, protein tyrosine phosphatases have emerged as critical regulators of platelet function. This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated by the collagen receptor glycoprotein VI and the C-type lectin-like receptor 2. DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism compared with wild-type mice and showed severely impaired thrombus formation on ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of phospholipase Cγ2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen- and C-type lectin-like receptor 2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. DUSP3 plays a selective and essential role in collagen- and C-type lectin-like receptor 2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a protein tyrosine phosphatase, implicated in platelet signaling, has been targeted with a small-molecule drug. © 2014 American Heart Association, Inc.

DUSP3 Phosphatase Deficiency or Inhibition Limit Platelet Activation and Arterial Thrombosis

PubMed Central

Musumeci, Lucia; Kuijpers, Marijke J; Gilio, Karen; Hego, Alexandre; Théâtre, Emilie; Maurissen, Lisbeth; Vandereyken, Maud; Diogo, Catia V; Lecut, Christelle; Guilmain, William; Bobkova, Ekaterina V; Eble, Johannes A.; Dahl, Russell; Drion, Pierre; Rascon, Justin; Mostofi, Yalda; Yuan, Hongbin; Sergienko, Eduard; Chung, Thomas DY; Thiry, Marc; Senis, Yotis; Moutschen, Michel; Mustelin, Tomas; Lancellotti, Patrizio; Heemskerk, Johan WM; Tautz, Lutz; Oury, Cécile; Rahmouni, Souad

2015-01-01

Background A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. Better understanding of the molecular mechanisms leading to platelet activation is of importance for the development of improved therapies. Recently, protein tyrosine phosphatases (PTPs) have emerged as critical regulators of platelet function. Methods and Results This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated through the collagen receptor glycoprotein VI (GPVI) and the C-type lectin-like receptor 2 (CLEC-2). DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism, compared to wild-type mice, and showed severely impaired thrombus formation upon ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of PLCγ2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen and CLEC-2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. Conclusions DUSP3 plays a selective and essential role in collagen- and CLEC-2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a PTP, implicated in platelet signaling, has been targeted with a small-molecule drug. PMID:25520375

Alternative RNA splicing in the endothelium mediated in part by Rbfox2 regulates the arterial response to low flow

PubMed Central

Butty, Vincent L; Boutz, Paul L; Begum, Shahinoor; Kimble, Amy L; Sharp, Phillip A; Burge, Christopher B

2018-01-01

Low and disturbed blood flow drives the progression of arterial diseases including atherosclerosis and aneurysms. The endothelial response to flow and its interactions with recruited platelets and leukocytes determine disease progression. Here, we report widespread changes in alternative splicing of pre-mRNA in the flow-activated murine arterial endothelium in vivo. Alternative splicing was suppressed by depletion of platelets and macrophages recruited to the arterial endothelium under low and disturbed flow. Binding motifs for the Rbfox-family are enriched adjacent to many of the regulated exons. Endothelial deletion of Rbfox2, the only family member expressed in arterial endothelium, suppresses a subset of the changes in transcription and RNA splicing induced by low flow. Our data reveal an alternative splicing program activated by Rbfox2 in the endothelium on recruitment of platelets and macrophages and demonstrate its relevance in transcriptional responses during flow-driven vascular inflammation. PMID:29293084

Deficiency of Src homology 2 domain–containing inositol 5-phosphatase 1 affects platelet responses and thrombus growth

PubMed Central

Séverin, Sonia; Gratacap, Marie-Pierre; Lenain, Nadège; Alvarez, Laetitia; Hollande, Etienne; Penninger, Josef M.; Gachet, Christian; Plantavid, Monique; Payrastre, Bernard

2007-01-01

Platelets are critical for normal hemostasis. Their deregulation can lead to bleeding or to arterial thrombosis, a primary cause of heart attack and ischemic stroke. Src homology 2 domain–containing inositol 5-phosphatase 1 (SHIP1) is a 5-phosphatase capable of dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate second messenger into phosphatidylinositol 3,4-bisphosphate. SHIP1 plays a critical role in regulating the level of these 2 lipids in platelets. Using SHIP1-deficient mice, we found that its loss affects platelet aggregation in response to several agonists with minor effects on fibrinogen binding and β3 integrin tyrosine phosphorylation. Accordingly, SHIP1-null mice showed defects in arterial thrombus formation in response to a localized laser-induced injury. Moreover, these mice had a prolonged tail bleeding time. Upon stimulation, SHIP1-deficient platelets showed large membrane extensions, abnormalities in the open canalicular system, and a dramatic decrease in close cell-cell contacts. Interestingly, SHIP1 appeared to be required for platelet contractility, thrombus organization, and fibrin clot retraction. These data indicate that SHIP1 is an important element of the platelet signaling machinery to support normal hemostasis. To our knowledge, this is the first report unraveling an important function of SHIP1 in the activation of hematopoietic cells, in contrast to its well-documented role in the negative regulation of lymphocytes. PMID:17347685

Platelet hyperaggregability in obesity: is there a role for nitric oxide impairment and oxidative stress?

PubMed

Leite, Natália Rodrigues Pereira; Siqueira de Medeiros, Mariana; Mury, Wanda Vianna; Matsuura, Cristiane; Perszel, Monique Bandeira Moss; Noronha Filho, Gerson; Brunini, Tatiana Mc; Mendes-Ribeiro, Antônio Claúdio

2016-08-01

Epidemiological evidence has shown that platelet activation markers are consistently elevated in obesity, contributing to its prothrombotic state. In order to improve the understanding of the regulation of platelet function in obesity, the aim of this study was to investigate the l-arginine-nitric oxide (NO) pathway in obese adults without other cardiovascular risk factor. Seventeen obese (body mass index [BMI] 35.9±1.0 kg/m(2) ) and eighteen age-matched normal weight subjects (BMI 22.0±0.6 kg/m(2) ) were included in this study. l-arginine influx was measured with incubation of l-[(3) H]-arginine. NO synthase (NOS) and arginase activities were determined by the citrulline assay and the conversion of l-[(14) C]-arginine to [(14) C]-urea, respectively. Cyclic guanosine monophosphate (cGMP) content was evaluated by enzyme-linked immunosorbent assay. In addition, the study analyzed: platelet aggregation; intraplatelet antioxidant enzymes, via superoxide dismutase (SOD) and catalase activities; and systemic levels of l-arginine, fibrinogen, and C-reactive protein (CRP). Obese patients presented a significant decrease of platelet l-arginine influx, NOS activity, and cGMP levels, along with platelet hyperaggregability. On the presence of NO donor, platelet aggregation was similar between the groups. The fibrinogen and CRP systemic levels were significantly higher and SOD activity was reduced in obesity. No significant differences were observed in plasma levels of l-arginine and intraplatelet arginase and catalase activities between groups. The diminished NO bioavailability associated with inflammatory status and impaired enzymatic antioxidant defence may contribute to future cardiovascular complications in obesity. © 2016 John Wiley & Sons Australia, Ltd.

Disruption of the Mouse μ-Calpain Gene Reveals an Essential Role in Platelet Function

PubMed Central

Azam, Mohammad; Andrabi, Shaida S.; Sahr, Kenneth E.; Kamath, Lakshmi; Kuliopulos, Athan; Chishti, Athar H.

2001-01-01

Conventional calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. There are two forms of conventional calpains: the μ-calpain, or calpain I, which requires micromolar calcium for half-maximal activation, and the m-calpain, or calpain II, which functions at millimolar calcium concentrations. We evaluated the functional role of the 80-kDa catalytic subunit of μ-calpain by genetic inactivation using homologous recombination in embryonic stem cells. The μ-calpain-deficient mice are viable and fertile. The complete deficiency of μ-calpain causes significant reduction in platelet aggregation and clot retraction but surprisingly the mutant mice display normal bleeding times. No detectable differences were observed in the cleavage pattern and kinetics of calpain substrates such as the β3 subunit of αIIbβ3 integrin, talin, and ABP-280 (filamin). However, μ-calpain null platelets exhibit impaired tyrosine phosphorylation of several proteins including the β3 subunit of αIIbβ3 integrin, correlating with the agonist-induced reduction in platelet aggregation. These results provide the first direct evidence that μ-calpain is essential for normal platelet function, not by affecting the cleavage of cytoskeletal proteins but by potentially regulating the state of tyrosine phosphorylation of the platelet proteins. PMID:11238954

Role of serotonin in the regulation of renal proximal tubular epithelial cells.

PubMed

Erikci, Acelya; Ucar, Gulberk; Yabanoglu-Ciftci, Samiye

2016-08-01

In various renal injuries, tissue damage occurs and platelet activation is observed. Recent studies suggest that some factors, such as serotonin, are released into microenvironment upon platelet activation following renal injury. In the present study, we aimed to investigate whether platelets and platelet-released serotonin are involved in the functional regulation of renal proximal tubular epithelial cells (PTECs). PTECs were obtained by primary cell culture and treated with platelet lysate (PL) (2 × 10(6)/mL, 4 × 10(6)/mL, 8 × 10(6)/mL) or serotonin (1 μM or 5 μM) for 12 or 24 h. Phenotypic transdifferentiation of epithelial cells into myofibroblasts were demonstrated under light microscope and confirmed by the determination of α-smooth muscle actin gene expression. Serotonin and PL were shown to induce epithelial-mesenchymal transdifferentiation of PTECs. After stimulation of PTECs with serotonin or PL, matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-1, and collagen-α1 gene expressions, which were reported to be elevated in renal injury, were determined by real-time PCR and found to be upregulated. Expressions of some inflammatory cytokines such as tumor necrosis factor-α, interleukin-6, and transforming growth factor-β1 were found to be increased in both protein and gene levels. Recently there is no published report on the effect of serotonin on renal PTECs. Results obtained in this study have lightened the role of serotonin and platelet-mediated effects of serotonin on fibrotic and inflammatory processes in PTECs.

PI 3-kinase signalling in platelets: the significance of synergistic, autocrine stimulation.

PubMed

Selheim, F; Holmsen, H; Vassbotn, F S

2000-03-01

Phosphoinositide 3-kinases (PI 3Ks) play a key role in regulation of intracellular signalling and cellular function, including cell proliferation, apoptosis, chemotaxis, membrane trafficking and platelet activation. The PI 3Ks are grouped into three classes on the basis on their structure and in vitro substrate specificity. Class I are activated by a variety of agonists which mediate their effect through tyrosine kinase-linked or G-protein-linked receptors. In vivo class I PI 3Ks seem to preferentially phosphorylate the D3 hydroxyls of the inositol moiety of PtdIns(4,5)P2 to produce PtdIns(3,4,5)P3. However, class II PI 3Ks preferentially phosphorylate the D3 hydroxyl of PtdIns and PtdIns(4)P to produce PtdIns(3)P and PtdIns(3,4)P2, respectively. The late accumulation of PtdIns(3,4)P2 has been suggested to play an important role in irreversible platelet aggregation. In human platelets the class II PI 3K isoform HsC2-PI 3K is activated in an integrin alpha IIb beta 3 + fibrinogen-dependent manner. Class III PI 3Ks phosphorylate PtdIns to produce PtdIns(3)P, which play a crucial role in vesicular trafficking. Recent work has suggested that crosstalk between individual receptors and their downstream signal pathways play a central role in PI 3K signalling responses. In this review, we will concentrate on recent advances regarding the regulation of platelet PI 3Ks.

Binding of thrombin-activated platelets to a fibrin scaffold through α(IIb)β₃ evokes phosphatidylserine exposure on their cell surface.

PubMed

Brzoska, Tomasz; Suzuki, Yuko; Mogami, Hideo; Sano, Hideto; Urano, Tetsumei

2013-01-01

Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an α(IIb)β₃ antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment.

Binding of Thrombin-Activated Platelets to a Fibrin Scaffold through αIIbβ3 Evokes Phosphatidylserine Exposure on Their Cell Surface

PubMed Central

Brzoska, Tomasz; Suzuki, Yuko; Mogami, Hideo; Sano, Hideto; Urano, Tetsumei

2013-01-01

Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an αIIbβ3 antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment. PMID:23383331

Inhibitory mechanisms of CME-1, a novel polysaccharide from the mycelia of Cordyceps sinensis, in platelet activation.

PubMed

Chang, Yi; Hsu, Wen-Hsien; Lu, Wan-Jung; Jayakumar, Thanasekaran; Liao, Jiun-Cheng; Lin, Mei-Jiun; Wang, Shwu-Huey; Geraldine, Pitchairaj; Lin, Kuan-Hung; Sheu, Joen-Rong

2015-01-01

CME-1 is a polysaccharide purified from the mycelia of medicinal mushroom Cordyceps sinensis, its molecular weight was determined to be 27.6 kDa by using nuclear magnetic resonance and gas chromatography-mass spectrometry. The initiation of arterial thromboses is relevant to various cardiovascular diseases (CVDs) and is believed to involve platelet activation. Our recent study exhibited that CME-1 has potent antiplatelet activity via the activation of adenylate cyclase/cyclic AMP ex vivo and in vivo. The aggregometry, and immunoblotting were used in this study. In this study, the mechanisms of CME-1 in platelet activation is further investigated and found that CME-1 inhibited platelet aggregation as well as the ATP-release reaction, relative intracellular [Ca(+2)] mobilization, and the phosphorylation of phospholipase C (PLC)γ2 and protein kinase C (PKC) stimulated by collagen. CME-1 has no effects on inhibiting either convulxin, an agonist of glycoprotein VI, or aggretin, an agonist of integrin α2β1 stimulated platelet aggregation. Moreover, this compound markedly diminished thrombin and arachidonic acid (AA) induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 2, c-Jun N-terminal kinase 1, and Akt. Treatment with SQ22536, an inhibitor of adenylate cyclase, markedly diminished the CME-1-mediated increasing of cyclic AMP level and reversed prostaglandin E1- or CME-1-mediated inhibition of platelet aggregation and p38 MAPK and Akt phosphorylation stimulated by thrombin or AA. Furthermore, phosphodiesterase activity of human platelets was not altered by CME-1. The crucial finding of this study is that the antiplatelet activity of CME-1 may initially inhibit the PLCγ2-PKC-p47 cascade, and inhibit PI3-kinase/Akt and MAPK phosphorylation through adenylate cyclase/ cyclic AMP activation, then inhibit intracellular [Ca(+2)] mobilization, and, ultimately, inhibit platelet activation. The novel role of CME-1 in antiplatelet activity indicates that this compound exhibits high therapeutic potential for treating or preventing CVDs.

Selective Inhibition of ADAM17 Efficiently Mediates Glycoprotein Ibα Retention During Ex Vivo Generation of Human Induced Pluripotent Stem Cell‐Derived Platelets

PubMed Central

Hirata, Shinji; Murata, Takahiko; Suzuki, Daisuke; Nakamura, Sou; Jono‐Ohnishi, Ryoko; Hirose, Hidenori; Sawaguchi, Akira; Nishimura, Satoshi; Sugimoto, Naoshi

2016-01-01

Abstract Donor‐independent platelet concentrates for transfusion can be produced in vitro from induced pluripotent stem cells (iPSCs). However, culture at 37°C induces ectodomain shedding on platelets of glycoprotein Ibα (GPIbα), the von Willebrand factor receptor critical for adhesive function and platelet lifetime in vivo, through temperature‐dependent activation of a disintegrin and metalloproteinase 17 (ADAM17). The shedding can be suppressed by using inhibitors of panmetalloproteinases and possibly of the upstream regulator p38 mitogen‐activated protein kinase (p38 MAPK), but residues of these inhibitors in the final platelet products may be accompanied by harmful risks that prevent clinical application. Here, we optimized the culture conditions for generating human iPSC‐derived GPIbα+ platelets, focusing on culture temperature and additives, by comparing a new and safe selective ADAM17 inhibitor, KP‐457, with previous inhibitors. Because cultivation at 24°C (at which conventional platelet concentrates are stored) markedly diminished the yield of platelets with high expression of platelet receptors, 37°C was requisite for normal platelet production from iPSCs. KP‐457 blocked GPIbα shedding from iPSC platelets at a lower half‐maximal inhibitory concentration than panmetalloproteinase inhibitor GM‐6001, whereas p38 MAPK inhibitors did not. iPSC platelets generated in the presence of KP‐457 exhibited improved GPIbα‐dependent aggregation not inferior to human fresh platelets. A thrombus formation model using immunodeficient mice after platelet transfusion revealed that iPSC platelets generated with KP‐457 exerted better hemostatic function in vivo. Our findings suggest that KP‐457, unlike GM‐6001 or p38 MAPK inhibitors, effectively enhances the production of functional human iPSC‐derived platelets at 37°C, which is an important step toward their clinical application. Stem Cells Translational Medicine 2017;6:720–730 PMID:28297575

Platelet storage in Fresenius/NPBI polyolefin and BTHC-PVC bags: a direct comparison.

PubMed

Hornsey, V S; McColl, K; Drummond, O; Macgregor, I R; Prowse, C V

2008-08-01

New platelet storage systems, such as changes in the plastic of the storage bags, require validation. In this study, pooled buffy coat platelets stored in Fresenius/NPBI polyolefin bags were compared with those stored in Fresenius/NPBI butyryl-trihexyl citrate (BTHC) plasticized polyvinyl chloride (PVC). The CompoSelect thrombocyte polishing filter system (1000 mL polyolefin bag) and the CompoStop F730 system (1300 mL BTHC-PVC bag) were used to prepare paired, plasma-suspended, buffy coat platelet concentrates. Samples were taken up to day 7 for in vitro analysis. In a separate experiment, 12 units were prepared using the CompoStop F730 system and samples taken after leucofiltration for FXIIa assay. By day 7, platelet concentrates stored in BTHC-PVC demonstrated significantly higher pH levels (7.32 +/- 0.05 vs. 7.26 +/- 0.05) and a greater degree of cell lysis as shown by increased lactate dehydrogenase levels (497 +/- 107 vs. 392 +/- 81 U L(-1)). The supernatants contained higher concentrations of soluble P-selectin and the chemokine 'regulated on activation, normal T-cell expressed and presumably secreted', which are released from the alpha-granules during activation. The ATP concentrations were significantly lower in BTHC-PVC. Platelet counts, mean platelet volume and hypotonic shock response were similar for both bags. FXIIa antigen concentrations were 0.6 +/- 0.2 ng mL(-1) indicating that activation of the contact factor pathway had not occurred. Although the CompoStop F730 leucoreduction filter did not activate the contact system, platelets stored in 100% plasma in BTHC-PVC bags demonstrated different in vitro characteristics from those stored in polyolefin. Further work is required to demonstrate whether these differences will affect in vivo recovery and survival.

A novel antithrombotic effect of sulforaphane via activation of platelet adenylate cyclase: ex vivo and in vivo studies.

PubMed

Jayakumar, Thanasekaran; Chen, Wei-Fan; Lu, Wan-Jung; Chou, Duen-Suey; Hsiao, George; Hsu, Chung-Yi; Sheu, Joen-Rong; Hsieh, Cheng-Ying

2013-06-01

Sulforaphane is a naturally occurring isothiocyanate, which can be found in cruciferous vegetables such as broccoli and cabbage. Sulforaphane was found to have very potent inhibitory effects on tumor growth through regulation of diverse mechanisms. However, no data are available concerning the effects of sulforaphane on platelet activation and its relative issues. Activation of platelets caused by arterial thrombosis is relevant to a variety of cardiovascular diseases. Hence, the aim of this study was to examine the in vivo antithrombotic effects of sulforaphane and its possible mechanisms in platelet activation. Sulforaphane (0.125 and 0.25 mg/kg) was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice. Other in vivo studies also revealed that sulforaphane (0.25 mg/kg) significantly prolonged platelet plug formation in mice. In addition, sulforaphane (15-75 μM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen. Sulforaphane inhibited platelet activation accompanied by inhibiting relative Ca(2+) mobilization; phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs) and Akt; and hydroxyl radical (OH(●)) formation. Sulforaphane markedly increased cyclic (c)AMP, but not cyclic (c)GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxal in-1-one), an inhibitor of guanylate cyclase, obviously reversed the sulforaphane-mediated effects on platelet aggregation; PKC activation, p38 MAPK, Akt and VASP phosphorylation; and OH(●) formation. Furthermore, a PI3-kinase inhibitor (LY294002) and a p38 MAPK inhibitor (SB203580) both significantly diminished PKC activation and p38 MAPK and Akt phosphorylation; in contrast, a PKC inhibitor (RO318220) did not diminish p38 MAPK or Akt phosphorylation stimulated by collagen. This study demonstrates for the first time that in addition to it originally being considered as an agent for prevention of tumor growth, sulforaphane possesses potent antiplatelet activity which may initially activate adenylate cyclase/cAMP, followed by inhibiting intracellular signals (such as the PI3-kinase/Akt and PLCγ2-PKC-p47 cascades) and ultimately inhibiting platelet activation. Therefore, this novel role of sulforaphane may represent a high therapeutic potential for treatment or prevention of cardiovascular diseases. Copyright © 2013 Elsevier Inc. All rights reserved.

The neuropeptide substance P stimulates the effector functions of platelets.

PubMed Central

Damonneville, M; Monté, D; Auriault, C; Capron, A

1990-01-01

Sensory neuropeptides, such as substance P, appear as potent mediators of various immunological reactions, and inhibit or stimulate a wide range of functions of immune inflammatory cells. Platelets were recently shown to participate as effector cells in an IgE or lymphokine-dependent killing of parasites. Substance P and its carboxy-terminal fragment SP (4-11) induce the cytotoxic activity of platelets towards the larvae of Schistosoma mansoni, respectively, by 90% and 40%, whereas the modified C terminal SP, the SP-free acid, exhibits no effect on the platelets. The neuropeptide effects occur at low doses (10(-8) M), are specific as shown by inhibition studies with a substance P antagonist, the D-SP. Binding data obtained after flow cytofluorometry with FITC-SP lead to the conclusion that SP binds specifically to about 20% of the homogenous population of platelets. Moreover, IgE could modulate the SP-dependent functions of platelets since the pre-incubation with myeloma human IgE or with AP2 monoclonal antibodies--known to inhibit the IgE-dependent killing of these cells-leads to a dramatic decrease of the SP dependent cytotoxic activity of platelets towards the larvae. These findings identify a potent mechanism for nervous system regulation of host defence responses. PMID:1696868

Arf6 controls platelet spreading and clot retraction via integrin αIIbβ3 trafficking

PubMed Central

Huang, Yunjie; Joshi, Smita; Xiang, Binggang; Kanaho, Yasunori; Li, Zhenyu; Bouchard, Beth A.; Moncman, Carole L.

2016-01-01

Platelet and megakaryocyte endocytosis is important for loading certain granule cargo (ie, fibrinogen [Fg] and vascular endothelial growth factor); however, the mechanisms of platelet endocytosis and its functional acute effects are understudied. Adenosine 5'-diphosphate–ribosylation factor 6 (Arf6) is a small guanosine triphosphate–binding protein that regulates endocytic trafficking, especially of integrins. To study platelet endocytosis, we generated platelet-specific Arf6 knockout (KO) mice. Arf6 KO platelets had less associated Fg suggesting that Arf6 affects αIIbβ3-mediated Fg uptake and/or storage. Other cargo was unaffected. To measure Fg uptake, mice were injected with biotinylated- or fluorescein isothiocyanate (FITC)–labeled Fg. Platelets from the injected Arf6 KO mice showed lower accumulation of tagged Fg, suggesting an uptake defect. Ex vivo, Arf6 KO platelets were also defective in FITC-Fg uptake and storage. Immunofluorescence analysis showed initial trafficking of FITC-Fg to a Rab4-positive compartment followed by colocalization with Rab11-positive structures, suggesting that platelets contain and use both early and recycling endosomes. Resting and activated αIIbβ3 levels, as measured by flow cytometry, were unchanged; yet, Arf6 KO platelets exhibited enhanced spreading on Fg and faster clot retraction. This was not the result of alterations in αIIbβ3 signaling, because myosin light-chain phosphorylation and Rac1/RhoA activation were unaffected. Consistent with the enhanced clot retraction and spreading, Arf6 KO mice showed no deficits in tail bleeding or FeCl3-induced carotid injury assays. Our studies present the first mouse model for defining the functions of platelet endocytosis and suggest that altered integrin trafficking may affect the efficacy of platelet function. PMID:26738539

Arf6 controls platelet spreading and clot retraction via integrin αIIbβ3 trafficking.

PubMed

Huang, Yunjie; Joshi, Smita; Xiang, Binggang; Kanaho, Yasunori; Li, Zhenyu; Bouchard, Beth A; Moncman, Carole L; Whiteheart, Sidney W

2016-03-17

Platelet and megakaryocyte endocytosis is important for loading certain granule cargo (ie, fibrinogen [Fg] and vascular endothelial growth factor); however, the mechanisms of platelet endocytosis and its functional acute effects are understudied. Adenosine 5'-diphosphate-ribosylation factor 6 (Arf6) is a small guanosine triphosphate-binding protein that regulates endocytic trafficking, especially of integrins. To study platelet endocytosis, we generated platelet-specific Arf6 knockout (KO) mice. Arf6 KO platelets had less associated Fg suggesting that Arf6 affects αIIbβ3-mediated Fg uptake and/or storage. Other cargo was unaffected. To measure Fg uptake, mice were injected with biotinylated- or fluorescein isothiocyanate (FITC)-labeled Fg. Platelets from the injected Arf6 KO mice showed lower accumulation of tagged Fg, suggesting an uptake defect. Ex vivo, Arf6 KO platelets were also defective in FITC-Fg uptake and storage. Immunofluorescence analysis showed initial trafficking of FITC-Fg to a Rab4-positive compartment followed by colocalization with Rab11-positive structures, suggesting that platelets contain and use both early and recycling endosomes. Resting and activated αIIbβ3 levels, as measured by flow cytometry, were unchanged; yet, Arf6 KO platelets exhibited enhanced spreading on Fg and faster clot retraction. This was not the result of alterations in αIIbβ3 signaling, because myosin light-chain phosphorylation and Rac1/RhoA activation were unaffected. Consistent with the enhanced clot retraction and spreading, Arf6 KO mice showed no deficits in tail bleeding or FeCl3-induced carotid injury assays. Our studies present the first mouse model for defining the functions of platelet endocytosis and suggest that altered integrin trafficking may affect the efficacy of platelet function. © 2016 by The American Society of Hematology.

Platelet proteomics: from discovery to diagnosis.

PubMed

Looße, Christina; Swieringa, Frauke; Heemskerk, Johan W M; Sickmann, Albert; Lorenz, Christin

2018-05-22

Platelets are the smallest cells within the circulating blood with key roles in physiological haemostasis and pathological thrombosis regulated by the onset of activating/inhibiting processes via receptor responses and signalling cascades. Areas covered: Proteomics as well as genomic approaches have been fundamental in identifying and quantifying potential targets for future diagnostic strategies in the prevention of bleeding and thrombosis, and uncovering the complexity of platelet functions in health and disease. In this article, we provide a critical overview on current functional tests used in diagnostics and the future perspectives for platelet proteomics in clinical applications. Expert commentary: Proteomics represents a valuable tool for the identification of patients with diverse platelet associated defects. In-depth validation of identified biomarkers, e.g. receptors, signalling proteins, post-translational modifications, in large cohorts is decisive for translation into routine clinical diagnostics.

Native thrombopoietin: structure and function.

PubMed

Kato, T; Matsumoto, A; Ogami, K; Tahara, T; Morita, H; Miyazaki, H

1998-01-01

Thrombopoietin (TPO), the c-Mpl ligand, is produced constitutively in liver and other organs, circulates in the bloodstream, and is delivered to bone marrow, where it stimulates the early development of multiple hematopoietic lineages and megakaryocytopoiesis. The concentration of TPO in blood is regulated by c-Mpl mass on platelets and megakaryocytes. In addition to regulation by the number of TPO molecules, including the possible modulation of TPO mRNA abundance in bone marrow, megakaryocytopoiesis and platelet production may be regulated as a result of modulation of TPO activity by proteolytic processing that generates truncated forms of the molecule. Characterization of TPO partially purified from human plasma, however, revealed that the full-length molecule was the predominant form in the blood of both normal individuals and thrombocytopenic patients, although small amounts of truncated species were detected. Thus, truncation of TPO, at least that in the circulation examined, does not appear to contribute to the direct regulation of platelet production in response to increased demand. Given that native TPO isolated from the plasma of thrombocytopenic animals comprises truncated forms, the truncation of TPO is likely of physiological importance in the life history of this molecule.

Activated platelets are the source of elevated levels of soluble CD40 ligand in the circulation of inflammatory bowel disease patients.

PubMed

Danese, S; Katz, J A; Saibeni, S; Papa, A; Gasbarrini, A; Vecchi, M; Fiocchi, C

2003-10-01

The CD40/CD40L system, a key regulator and amplifier of immune reactivity, is activated in inflammatory bowel disease (IBD) mucosa. To determine whether plasma levels of sCD40L are elevated in Crohn's disease (CD) and ulcerative colitis (UC) patients compared with normal controls, to investigate the cellular source of sCD40L, and to explore CD40L induction mechanisms. CD, UC, and normal control subjects were studied. The concentration of sCD40L in plasma and supernatants of freshly isolated platelets and autologous peripheral blood T cells (PBT) was measured by ELISA. Surface CD40L expression level was measured by flow cytometry in resting and thrombin activated platelets, and unstimulated and CD3/CD28 stimulated PBT before and after coculture with human intestinal microvascular endothelial cells (HIMEC). Compared with normal controls, plasma sCD40L levels were significantly higher in both CD and UC patients and proportional to the extent of mucosal inflammation. Platelets from IBD patients displayed a significantly higher surface CD40L expression than those from control subjects, and released greater amounts of sCD40L than autologous PBT. Contact with IL-1beta activated HIMEC induced significant upregulation of CD40L surface expression and release by platelets. Elevated levels of sCD40L in the circulation of IBD patients reflect enhanced surface expression and release of CD40L by platelets. This phenomenon translates to an increased platelet activation state apparently induced by passage through an inflamed mucosal microvascular bed, a conclusion supported by the positive correlation of plasma sCD40L levels with the extent of anatomical involvement by IBD. These results suggest that platelet-endothelial interactions critically contribute to activation of the CD40 pathway in IBD.

von Willebrand factor (VWF) propeptide binding to VWF D'D3 domain attenuates platelet activation and adhesion.

PubMed

Madabhushi, Sri R; Shang, Chengwei; Dayananda, Kannayakanahalli M; Rittenhouse-Olson, Kate; Murphy, Mary; Ryan, Thomas E; Montgomery, Robert R; Neelamegham, Sriram

2012-05-17

Noncovalent association between the von Willebrand factor (VWF) propeptide (VWFpp) and mature VWF aids N-terminal multimerization and protein compartmentalization in storage granules. This association is currently thought to dissipate after secretion into blood. In the present study, we examined this proposition by quantifying the affinity and kinetics of VWFpp binding to mature VWF using surface plasmon resonance and by developing novel anti-VWF D'D3 mAbs. Our results show that the only binding site for VWFpp in mature VWF is in its D'D3 domain. At pH 6.2 and 10mM Ca(2+), conditions mimicking intracellular compartments, VWFpp-VWF binding occurs with high affinity (K(D) = 0.2nM, k(off) = 8 × 10(-5) s(-1)). Significant, albeit weaker, binding (K(D) = 25nM, k(off) = 4 × 10(-3) s(-1)) occurs under physiologic conditions of pH 7.4 and 2.5mM Ca(2+). This interaction was also observed in human plasma (K(D) = 50nM). The addition of recombinant VWFpp in both flow-chamber-based platelet adhesion assays and viscometer-based shear-induced platelet aggregation and activation studies reduced platelet adhesion and activation partially. Anti-D'D3 mAb DD3.1, which blocks VWFpp binding to VWF-D'D3, also abrogated platelet adhesion, as shown by shear-induced platelet aggregation and activation studies. Our data demonstrate that VWFpp binding to mature VWF occurs in the circulation, which can regulate the hemostatic potential of VWF by reducing VWF binding to platelet GpIbα.

Rapid resensitization of purinergic receptor function in human platelets.

PubMed

Mundell, S J; Barton, J F; Mayo-Martin, M B; Hardy, A R; Poole, A W

2008-08-01

Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), the P2Y(1) and P2Y(12) purinergic receptors. Recently, we demonstrated that both receptors desensitize and internalize in human platelets by differential kinase-dependent mechanisms. To demonstrate whether responses to P2Y(1) and P2Y(12) purinergic receptors resensitize in human platelets and determine the role of receptor traffic in this process. These studies were undertaken either in human platelets or in cells stably expressing epitope-tagged P2Y(1) and P2Y(12) purinergic receptor constructs. In this study we show for the first time that responses to both of these receptors can rapidly resensitize following agonist-dependent desensitization in human platelets. Further, we show that in human platelets or in 1321N1 cells stably expressing receptor constructs, the disruption of receptor internalization, dephosphorylation or subsequent receptor recycling is sufficient to block resensitization of purinergic receptor responses. We also show that, in platelets, internalization of both these receptors is dependent upon dynamin, and that this process is required for resensitization of responses. This study is therefore the first to show that both P2Y(1) and P2Y(12) receptor activities are rapidly and reversibly modulated in human platelets, and it reveals that the underlying mechanism requires receptor trafficking as an essential part of this process.

The Small GTPase Rif Is Dispensable for Platelet Filopodia Generation in Mice

PubMed Central

Goggs, Robert; Savage, Joshua S.; Mellor, Harry; Poole, Alastair W.

2013-01-01

Background Formation of filopodia and other shape change events are vital for platelet hemostatic function. The mechanisms regulating filopodia formation by platelets are incompletely understood however. In particular the small GTPase responsible for initiating filopodia formation by platelets remains elusive. The canonical pathway involving Cdc42 is not essential for filopodia formation in mouse platelets. The small GTPase Rif (RhoF) provides an alternative route to filopodia generation in other cell types and is expressed in both human and mouse platelets. Hypothesis/Objective We hypothesized that Rif might be responsible for generating filopodia by platelets and generated a novel knockout mouse model to investigate the functional role of Rif in platelets. Methodology/Principal Findings Constitutive RhoF−/− mice are viable and have normal platelet, leukocyte and erythrocyte counts and indices. RhoF−/− platelets form filopodia and spread normally on various agonist surfaces in static conditions and under arterial shear. In addition, RhoF−/− platelets have normal actin dynamics, are able to activate and aggregate normally and secrete from alpha and dense granules in response to collagen related peptide and thrombin stimulation. Conclusions The small GTPase Rif does not appear to be critical for platelet function in mice. Functional overlap between Rif and other small GTPases may be responsible for the non-essential role of Rif in platelets. PMID:23359340

Partially Defective Store Operated Calcium Entry and Hem(ITAM) Signaling in Platelets of Serotonin Transporter Deficient Mice

PubMed Central

Wolf, Karen; Braun, Attila; Haining, Elizabeth J.; Tseng, Yu-Lun; Kraft, Peter; Schuhmann, Michael K.; Gotru, Sanjeev K.; Chen, Wenchun; Hermanns, Heike M.; Stoll, Guido; Lesch, Klaus-Peter; Nieswandt, Bernhard

2016-01-01

Background Serotonin (5-hydroxytryptamin, 5-HT) is an indolamine platelet agonist, biochemically derived from tryptophan. 5-HT is secreted from the enterochromaffin cells into the gastrointestinal tract and blood. Blood 5-HT has been proposed to regulate hemostasis by acting as a vasoconstrictor and by triggering platelet signaling through 5-HT receptor 2A (5HTR2A). Although platelets do not synthetize 5-HT, they take 5-HT up from the blood and store it in their dense granules which are secreted upon platelet activation. Objective To identify the molecular composite of the 5-HT uptake system in platelets and elucidate the role of platelet released 5-HT in thrombosis and ischemic stroke. Methods: 5-HT transporter knockout mice (5Htt-/-) were analyzed in different in vitro and in vivo assays and in a model of ischemic stroke. Results In 5Htt-/- platelets, 5-HT uptake from the blood was completely abolished and agonist-induced Ca2+ influx through store operated Ca2+ entry (SOCE), integrin activation, degranulation and aggregation responses to glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2) were reduced. These observed in vitro defects in 5Htt-/- platelets could be normalized by the addition of exogenous 5-HT. Moreover, reduced 5-HT levels in the plasma, an increased bleeding time and the formation of unstable thrombi were observed ex vivo under flow and in vivo in the abdominal aorta and carotid artery of 5Htt-/- mice. Surprisingly, in the transient middle cerebral artery occlusion (tMCAO) model of ischemic stroke 5Htt-/- mice showed nearly normal infarct volume and the neurological outcome was comparable to control mice. Conclusion Although secreted platelet 5-HT does not appear to play a crucial role in the development of reperfusion injury after stroke, it is essential to amplify the second phase of platelet activation through SOCE and plays an important role in thrombus stabilization. PMID:26800051

Analysis of aggregation of platelets in thrombosis

NASA Astrophysics Data System (ADS)

Ahuja, Suresh

Platelets are key players in thrombus formation by first rolling over collagen bound von Willebrand factor followed by formation of a stable interaction with collagen. The first adhered platelets bind additional platelets until the whole injury is sealed off by a platelet aggregate. The coagulation system stabilizes the formed platelet plug by creating a tight fibrin network, and then wound contraction takes place because of morphological changes in platelets. Coagulation takes place by platelet activation and aggregation mainly through fibrinogen polymerization into fibrin fibers. The process includes multiple factors, such as thrombin, plasmin, and local shear-rate which regulate and control the process. Coagulation can be divided into two pathways: the intrinsic pathway and the extrinsic pathway. The intrinsic pathway is initiated by the exposure of a negatively charged. It is able to activate factor XII, using a complex reaction that includes prekallikrein and high-molecular-weight kininogen as cofactors.. Thrombin is the final enzyme that is needed to convert fibrinogen into fibrin. The extrinsic pathway starts with the exposure of tissue factor to the circulating blood, which is the major initiator of coagulation. There are several feedback loops that reinforce the coagulation cascade, resulting in large amounts of thrombin. It is dependent on the presence of pro-coagulant surfaces of cells expressing negatively charged phospholipids--which include phosphatidylserine (PS)--on their outer membrane. PS-bearing surfaces are able to increase the efficiency of the reactions by concentrating and co-localizing coagulation factors.. Aggregation of platelets are analyzed and compared to adhesion of platelet to erythrocyte and to endothelial cells. This abstract is replacing MAR16-2015-020003.

Aspirin influences megakaryocytic gene expression leading to up-regulation of multidrug resistance protein-4 in human platelets

PubMed Central

Massimi, Isabella; Guerriero, Raffaella; Lotti, Lavinia Vittoria; Lulli, Valentina; Borgognone, Alessandra; Romani, Federico; Barillà, Francesco; Gaudio, Carlo; Gabbianelli, Marco; Frati, Luigi; Pulcinelli, Fabio M

2014-01-01

Aim The aim of the study was to investigate whether human megakaryocytic cells have an adaptive response to aspirin treatment, leading to an enhancement of multidrug resistance protein-4 (MRP4) expression in circulating platelets responsible for a reduced aspirin action. We recently found that platelet MRP4 overexpression has a role in reducing aspirin action in patients after by-pass surgery. Aspirin enhances MRP4-mRNA levels in rat liver and drug administration transcriptionally regulates MRP4 gene expression through peroxisome proliferator-activated receptor-α (PPARα). Methods The effects induced by aspirin or PPARα agonist (WY14643) on MRP4 modulation were evaluated in vitro in a human megakaryoblastic DAMI cell line, in megakaryocytes (MKs) and in platelets obtained from human haematopoietic progenitor cell (HPC) cultures, and in vivo platelets obtained from aspirin treated healthy volunteers (HV). Results In DAMI cells, aspirin and WY14643 treatment induced a significant increase in MRP4 and PPARα expression. In human MKs grown in the presence of either aspirin or WY14643, MRP4 and PPARα-mRNA were higher than in control cultures and derived platelets showed an enhancement in MRP4 protein expression. The ability of aspirin to modulate MRP4 expression in MKs and to transfer it to platelets was also confirmed in vivo. In fact, we found the highest MRP4 mRNA and protein expression in platelets obtained from HV after 15 days' aspirin treatment. Conclusions The present study provides evidence, for the first time, that aspirin treatment affects the platelet protein pattern through MK genomic modulation. This work represents an innovative and attractive approach, useful both to identify patients less sensitive to aspirin and to improve pharmacological treatment in cardiovascular high-risk patients. PMID:24902864

Ginsenoside-Rp3 inhibits platelet activation and thrombus formation by regulating MAPK and cyclic nucleotide signaling.

PubMed

Irfan, Muhammad; Jeong, Da Hye; Kwon, Hyuk-Woo; Shin, Jung-Hae; Park, Sang-Joon; Kwak, Dongmi; Kim, Tae-Hwan; Lee, Dong-Ha; Park, Hwa-Jin; Rhee, Man Hee

2018-06-08

Ginseng (Panax ginseng C.A. Mayer) contains saponin fractions called ginsenosides, which are thought to be the main components responsible for its various pharmacological activities. Ginsenosides have cardioprotective and antiplatelet effects. In the present study, we evaluated the effects of ginsenoside Rp3 (G-Rp3) on platelet function. The in vitro effects of G-Rp3 were evaluated on agonist-induced human and rat platelet aggregation, while [Ca 2+ ] i mobilization, granule secretion, integrin α IIb β 3 activation, and clot retraction were assessed in rat platelets. Its effects on vasodilator-stimulated phosphoprotein (VASP) expression, phosphorylation of MAPK signaling molecules, and PI3K/Akt activation were also studied. Moreover, the tyrosine phosphorylation of components of the P 2 Y 12 receptor downstream signaling pathway was also examined. The in vivo effects of G-Rp3 were studied using an acute pulmonary thromboembolism model and lung histopathology. G-Rp3 significantly inhibited collagen, ADP, and thrombin-induced platelet aggregation. G-Rp3 elevated cAMP levels and VASP phosphorylation and suppressed agonist-induced [Ca 2+ ] i mobilization, ATP release, and P-selectin expression along with fibrinogen binding to integrin α IIb β 3 , fibronectin adhesion, and clot retraction. G-Rp3 also attenuated the phosphorylation of MAPK, Src, and PLCγ2 as well as PI3K/Akt activation. Furthermore, it inhibited tyrosine phosphorylation of the Src family kinases (Src, Fyn, and Lyn) and PLCγ2 and protected mice from thrombosis. G-Rp3 modulates agonist-induced platelet activation and thrombus formation by inhibiting granule secretion, integrin α IIb β 3 activation, MAPK signaling, and Src, PLCγ2, and PI3K/Akt activation, and VASP stimulation. Our data suggest that G-Rp3 has therapeutic potential as a treatment for platelet-related cardiovascular disorders. Copyright © 2017. Published by Elsevier Inc.

Uncoupling of the ITIM receptor G6b-B from the tyrosine phosphatases Shp1 and Shp2 disrupts platelet homeostasis in mice.

PubMed

Geer, Mitchell J; van Geffen, Johanna P; Gopalasingam, Piraveen; Vögtle, Timo; Smith, Christopher W; Heising, Silke; Kuijpers, Marijke J E; Tullemans, Bibian M E; Jarvis, Gavin E; Eble, Johannes A; Jeeves, Mark; Overduin, Michael; Heemskerk, Johan W M; Mazharian, Alexandra; Senis, Yotis A

2018-06-11

The immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B has emerged as a key regulator of platelet homeostasis. However, it remains unclear how it mediates its effects. Tyrosine phosphorylation of the ITIM and immunoreceptor tyrosine-based switch motif (ITSM) within the cytoplasmic tail of G6b-B provides a docking site for SH2 domain-containing protein-tyrosine phosphatases Shp1 and Shp2, which are also critical regulators of platelet production and function. In this study, we investigate the physiological consequences of uncoupling G6b-B from Shp1 and Shp2. To address this, we generated a transgenic mouse model expressing a mutant form of G6b-B in which tyrosine (Y) residues 212 and 238 within the ITIM and ITSM were mutated to phenylalanine (F), respectively. Mice homozygous for the mutation (G6b-B diY/F) were macrothrombocytopenic, due to a reduction in platelet production, had large clusters of megakaryocytes and myelofibrosis at sites of hematopoiesis, similar to that observed in G6b knockout (G6b KO) mice. Platelets from G6b-B diY/F mice were hypo-responsive to collagen, due to a significant reduction in expression of the immunoreceptor tyrosine-based activation motif (ITAM)-containing collagen receptor complex GPVI-FcR γ-chain, and thrombin, that could be partially rescued by co-stimulating the platelets with ADP. In contrast, platelets from G6b-B diY/F, G6b KO and megakaryocyte-specific Shp2 KO mice were hyper-responsive to antibody-mediated cross-linking of the hemi-ITAM-containing podoplanin receptor CLEC-2, suggesting that G6b-B inhibits CLEC-2-mediated platelet activation through Shp2. Findings from this study demonstrate that G6b-B must engage with Shp1 and Shp2 in order to mediate its regulatory effects on platelet homeostasis. Copyright © 2018 American Society of Hematology.

ADAP interactions with talin and kindlin promote platelet integrin αIIbβ3 activation and stable fibrinogen binding

PubMed Central

Kang, Jian; Kahner, Bryan; Ye, Feng; Ginsberg, Mark H.; Shattil, Sanford J.

2014-01-01

ADAP is a hematopoietic-restricted adapter protein that promotes integrin activation and is a carrier for other adapter proteins, Src kinase–associated phosphoprotein 1 (SKAP1) and SKAP2. In T lymphocytes, SKAP1 is the ADAP-associated molecule that activates integrins through direct linkages with Rap1 effectors (regulator of cell adhesion and polarization enriched in lymphoid tissues; Rap1-interacting adapter molecule). ADAP also promotes integrin αIIbβ3 activation in platelets, which lack SKAP1, suggesting an ADAP integrin–regulatory pathway different from those in lymphocytes. Here we characterized a novel association between ADAP and 2 essential integrin-β cytoplasmic tail-binding proteins involved in αIIbβ3 activation, talin and kindlin-3. Glutathione S-transferase pull-downs identified distinct regions in ADAP necessary for association with kindlin or talin. ADAP was physically proximal to talin and kindlin-3 in human platelets, as assessed biochemically, and by immunofluorescence microscopy and proximity ligation. Relative to wild-type mouse platelets, ADAP-deficient platelets exhibited reduced co-localization of talin with αIIbβ3, and reduced irreversible fibrinogen binding in response to a protease activated receptor 4 (PAR4) thrombin receptor agonist. When ADAP was heterologously expressed in Chinese hamster ovary cells co-expressing αIIbβ3, talin, PAR1, and kindlin-3, it associated with an αIIbβ3/talin complex and enabled kindlin-3 to promote agonist-dependent ligand binding to αIIbβ3. Thus, ADAP uniquely promotes activation of and irreversible fibrinogen binding to platelet αIIbβ3 through interactions with talin and kindlin-3. PMID:24523237

Shiga Toxin 2 and Lipopolysaccharide Induce Human Microvascular Endothelial Cells To Release Chemokines and Factors That Stimulate Platelet Function

PubMed Central

Guessous, Fadila; Marcinkiewicz, Marek; Polanowska-Grabowska, Renata; Kongkhum, Sudawadee; Heatherly, Daniel; Obrig, Tom; Gear, Adrian R. L.

2005-01-01

Shiga toxins (Stxs) produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli are the most common cause of hemolytic-uremic syndrome (HUS). It is well established that vascular endothelial cells, mainly those located in the renal microvasculature, are targets for Stxs. The aim of the present research was to evaluate whether E. coli-derived Shiga toxin 2 (Stx2) incubated with human microvascular endothelial cells (HMEC-1) induces release of chemokines and other factors that might stimulate platelet function. HMEC-1 were exposed for 24 h in vitro to Stx2, lipopolysaccharide (LPS), or the Stx2-LPS combination, and chemokine production was assessed by immunoassay. More interleukin-8 was released than stromal cell-derived factor 1α (SDF-1α) or SDF-1β and RANTES. The Stx2-LPS combination potentiated chemokine release, but Stx2 alone caused more release of SDF-1α at 24 h than LPS or Stx2-LPS did. In the presence of low ADP levels, HMEC-1 supernatants activated platelet function assessed by classical aggregometry, single-particle counting, granule secretion, P-selectin exposure, and the formation of platelet-monocyte aggregates. Supernatants from HMEC-1 exposed only to Stx2 exhibited enhanced exposure of platelet P-selectin and platelet-THP-1 cell interactions. Blockade of platelet cyclooxygenase by indomethacin prevented functional activation. The chemokine RANTES enhanced platelet aggregation induced by SDF-1α, macrophage-derived chemokine, or thymus and activation-regulated chemokine in the presence of very low ADP levels. These data support the hypothesis that microvascular endothelial cells exposed to E. coli O157:H7-derived Stx2 and LPS release chemokines and other factors, which when combined with low levels of primary agonists, such as ADP, cause platelet activation and promote the renal thrombosis associated with HUS. PMID:16299328

Expression and functionality of Toll-like receptor 3 in the megakaryocytic lineage

PubMed Central

D’Atri, L. P.; Etulain, J.; Rivadeneyra, L.; Lapponi, M. J.; Centurion, M.; Cheng, K.; Yin, H.; Schattner, M.

2015-01-01

Summary Background In addition to their key role in hemostasis, platelets and megakaryocytes also regulate immune and inflammatory responses, in part through their expression of Toll-like receptors (TLRs). Among the TLRs, TLR3 recognizes double-stranded (ds) RNA associated with viral infection. Thrombocytopenia is a frequent complication of viral infection. However, the expression and functionality of TLR3 in megakaryocytes and platelets is not yet well understood. Objective To study the expression and functionality of TLR3 in the megakaryocytic lineage. Methods and Results RT-PCR, flow cytometric, and immunofluorescence assays showed that TLR3 is expressed in CD34+ cells, megakaryocytes, and platelets. Immunoblotting assays showed that stimulation of megakaryocytes with two synthetic agonists of TLR3, Poly(I:C) and Poly(A:U), activated the NF-κB, PI3K/Akt, ERK1/2, and p38 pathways. TLR3-megakaryocyte activation resulted in reduced platelet production in vitro and IFN-β release through the PI3K/Akt and NF-κB signaling pathways. TLR3 ligands potentiated the aggregation mediated by classical platelet agonists. This effect was also observed for ATP release, but not for P-selectin or CD40L membrane exposure, indicating that TLR3 activation was not involved in alpha granule release. In addition, TLR3 agonists induced activation of the NF-κB, PI3K/Akt, and ERK1/2 pathways in platelets. Reduction of platelet production and platelet fibrinogen binding mediated by Poly(I:C) or Poly(A:U) were prevented by the presence of an inhibitor of TLR3/dsRNA complex. Conclusions Our findings indicate that functional TLR3 is expressed in CD34+ cells, megakaryocytes, and platelets, and suggest a potential role for this receptor in the megakaryo/thrombopoiesis alterations that occur in viral infections. PMID:25594115

The selective phosphorylation of a guanine nucleotide-binding regulatory protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carlson, K.E.

1989-01-01

Receptor-activated signal transduction pathways regulate the responsiveness of cells to external stimuli. These transduction pathways themselves are subject to regulation, most commonly by phosphorylation. Guanine nucleotide-binding regulatory proteins (G Proteins), as requisite signal transducing elements for many plasma membrane receptors, are considered likely targets for regulation by phosphorylation. Protein kinase C (PKC) has been shown to phosphorylate the {alpha} subunit of G{sub i} and other G proteins in solution. However, the occurrence of the phosphorylation of G{sub 1} within intact cells in response to activation of PKC has not been rigorously demonstrated. In this thesis, the extent to which themore » {alpha} subunits of G{sub i} undergo phosphorylation within human platelets in response to activation of PKC was examined by means of radiolabeling and immunoprecipitation. Incubation of platelets with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, promoted the phosphorylation of several proteins within saponin-permeabilized and intact platelets incubated with ({gamma}{sup 32}P)ATP and ({sup 32}P)H{sub 3}PO{sub 4}, respectively. None of the phosphoproteins, however, were precipitated by either of two antisera containing antibodies differing in specificities for epitopes within G{sub i{alpha}}-despite precipitation of a substantial fraction of the subunit itself. In contrast, other antisera, containing antibodies specific for the recently describe G{sub z{alpha}}, or antibodies for both G{sub z{alpha}} and G{sub i{alpha}}, precipitated a 40-kDa phosphoprotein.« less

Protein kinase C (PKC) phosphorylates human platelet inositol trisphosphate 5/sup +/-/-phosphomonoesterase (IP/sub 3/ 5'-p'tase) increasing phosphatase activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Connolly, T.M.; Majerus, P.W.

1986-05-01

Phosphoinositide breakdown in response to thrombin stimulation of human platelets generates messenger molecules that activate PKC (diglyceride) and mobilize Ca/sup + +/ (inositol tris-phosphates). The water soluble products of phospholipase C-mediated metabolism of phosphatidylinositol 4,5-diphosphate are inositol 1,4,5 P/sub 3/ (IP/sub 3/) and inositol 1:2-cyclic 4,5 P/sub 3/ (cIP/sub 3/). A specific phosphatase, IP/sub 3/ 5'-p'tase, cleaves the 5 phosphate from IP/sub 3/ or cIP/sub 3/ to form IP/sub 2/ or cIP/sub 2/ and P/sub i/, none of which mobilizes Ca/sup + +/. Thus, the IP/sub 3/ 5'-p'tase may regulate cellular responses to IP/sub 3/ or cIP/sub 3/. The authorsmore » find that IP/sub 3/ 5'-p'tase isolated from human platelets is phosphorylated by rat brain PKC, resulting in a 4-fold increase in IP/sub 3/ 5'-p'tase activity. The authors phosphorylated IP/sub 3/ 5'-p'tase using ..gamma.. /sup 32/P-ATP and found that the labeled enzyme comigrated on SDS-PAGE with the previously described 40K protein phosphorylated in response to thrombin stimulation of platelets. The similarity of the PKC-phosphorylated IP/sub 3/ 5'-p'tase observed in vitro and the thrombin-stimulated phosphorylated 40K protein known to be phosphorylated by PKC in vivo, suggests that these proteins may be the same. These results suggest that platelet Ca/sup + +/ mobilization maybe regulated by PKC phosphorylation of the IP/sub 3/ 5'-p'tase and can explain the observation that phorbol ester treatment of intact human platelets results in decreased production of IP/sub 3/ and decreased Ca/sup + +/ mobilization upon subsequent thrombin addition.« less

A molecular signaling model of platelet phosphoinositide and calcium regulation during homeostasis and P2Y1 activation.

PubMed

Purvis, Jeremy E; Chatterjee, Manash S; Brass, Lawrence F; Diamond, Scott L

2008-11-15

To quantify how various molecular mechanisms are integrated to maintain platelet homeostasis and allow responsiveness to adenosine diphosphate (ADP), we developed a computational model of the human platelet. Existing kinetic information for 77 reactions, 132 fixed kinetic rate constants, and 70 species was combined with electrochemical calculations, measurements of platelet ultrastructure, novel experimental results, and published single-cell data. The model accurately predicted: (1) steady-state resting concentrations for intracellular calcium, inositol 1,4,5-trisphosphate, diacylglycerol, phosphatidic acid, phosphatidylinositol, phosphatidylinositol phosphate, and phosphatidylinositol 4,5-bisphosphate; (2) transient increases in intracellular calcium, inositol 1,4,5-trisphosphate, and G(q)-GTP in response to ADP; and (3) the volume of the platelet dense tubular system. A more stringent test of the model involved stochastic simulation of individual platelets, which display an asynchronous calcium spiking behavior in response to ADP. Simulations accurately reproduced the broad frequency distribution of measured spiking events and demonstrated that asynchronous spiking was a consequence of stochastic fluctuations resulting from the small volume of the platelet. The model also provided insights into possible mechanisms of negative-feedback signaling, the relative potency of platelet agonists, and cell-to-cell variation across platelet populations. This integrative approach to platelet biology offers a novel and complementary strategy to traditional reductionist methods.

Platelets release CXCL4L1, a nonallelic variant of the chemokine platelet factor-4/CXCL4 and potent inhibitor of angiogenesis.

PubMed

Struyf, Sofie; Burdick, Marie D; Proost, Paul; Van Damme, Jo; Strieter, Robert M

2004-10-29

Platelet factor-4 (PF-4)/CXCL4 was the first chemokine described to inhibit neovascularization. Here, the product of the nonallelic variant gene of CXCL4, PF-4var1/PF-4alt, designated CXCL4L1, was isolated for the first time from thrombin-stimulated human platelets and purified to homogeneity. Although secreted CXCL4 and CXCL4L1 differ in only three amino acids, CXCL4L1 was more potent in inhibiting chemotaxis of human microvascular endothelial cells toward interleukin-8 (IL-8)/CXCL8 or basic fibroblast growth factor (bFGF). In vivo, CXCL4L1 was also more effective than CXCL4 in inhibiting bFGF-induced angiogenesis in rat corneas. Thus, activated platelets release CXCL4L1, a potent regulator of endothelial cell biology, which affects angiogenesis and vascular diseases.

Lnk regulates integrin αIIbβ3 outside-in signaling in mouse platelets, leading to stabilization of thrombus development in vivo

PubMed Central

Takizawa, Hitoshi; Nishimura, Satoshi; Takayama, Naoya; Oda, Atsushi; Nishikii, Hidekazu; Morita, Yohei; Kakinuma, Sei; Yamazaki, Satoshi; Okamura, Satoshi; Tamura, Noriko; Goto, Shinya; Sawaguchi, Akira; Manabe, Ichiro; Takatsu, Kiyoshi; Nakauchi, Hiromitsu; Takaki, Satoshi; Eto, Koji

2009-01-01

The nature of the in vivo cellular events underlying thrombus formation mediated by platelet activation remains unclear because of the absence of a modality for analysis. Lymphocyte adaptor protein (Lnk; also known as Sh2b3) is an adaptor protein that inhibits thrombopoietin-mediated signaling, and as a result, megakaryocyte and platelet counts are elevated in Lnk–/– mice. Here we describe an unanticipated role for Lnk in stabilizing thrombus formation and clarify the activities of Lnk in platelets transduced through integrin αIIbβ3–mediated outside-in signaling. We equalized platelet counts in wild-type and Lnk–/– mice by using genetic depletion of Lnk and BM transplantation. Using FeCl3- or laser-induced injury and in vivo imaging that enabled observation of single platelet behavior and the multiple steps in thrombus formation, we determined that Lnk is an essential contributor to the stabilization of developing thrombi within vessels. Lnk–/– platelets exhibited a reduced ability to fully spread on fibrinogen and mediate clot retraction, reduced tyrosine phosphorylation of the β3 integrin subunit, and reduced binding of Fyn to integrin αIIbβ3. These results provide new insight into the mechanism of αIIbβ3-based outside-in signaling, which appears to be coordinated in platelets by Lnk, Fyn, and integrins. Outside-in signaling modulators could represent new therapeutic targets for the prevention of cardiovascular events. PMID:20038804

Protease-Activated Receptor 4 Variant p.Tyr157Cys Reduces Platelet Functional Responses and Alters Receptor Trafficking.

PubMed

Norman, Jane E; Cunningham, Margaret R; Jones, Matthew L; Walker, Mary E; Westbury, Sarah K; Sessions, Richard B; Mundell, Stuart J; Mumford, Andrew D

2016-05-01

Protease-activated receptor 4 (PAR4) is a key regulator of platelet reactivity and is encoded by F2RL3, which has abundant rare missense variants. We aimed to provide proof of principle that rare F2LR3 variants potentially affect platelet reactivity and responsiveness to PAR1 antagonist drugs and to explore underlying molecular mechanisms. We identified 6 rare F2RL3 missense variants in 236 cardiac patients, of which the variant causing a tyrosine 157 to cysteine substitution (Y157C) was predicted computationally to have the greatest effect on PAR4 structure. Y157C platelets from 3 cases showed reduced responses to PAR4-activating peptide and to α-thrombin compared with controls, but no reduction in responses to PAR1-activating peptide. Pretreatment with the PAR1 antagonist vorapaxar caused lower residual α-thrombin responses in Y157C platelets than in controls, indicating greater platelet inhibition. HEK293 cells transfected with a PAR4 Y157C expression construct had reduced PAR4 functional responses, unchanged total PAR4 expression but reduced surface expression. PAR4 Y157C was partially retained in the endoplasmic reticulum and displayed an expression pattern consistent with defective N-glycosylation. Mutagenesis of Y322, which is the putative hydrogen bond partner of Y157, also reduced PAR4 surface expression in HEK293 cells. Reduced PAR4 responses associated with Y157C result from aberrant anterograde surface receptor trafficking, in part, because of disrupted intramolecular hydrogen bonding. Characterization of PAR4 Y157C establishes that rare F2RL3 variants have the potential to markedly alter platelet PAR4 reactivity particularly after exposure to therapeutic PAR1 antagonists. © 2016 American Heart Association, Inc.

Ultraviolet-Based Pathogen Inactivation Systems: Untangling the Molecular Targets Activated in Platelets

PubMed Central

Schubert, Peter; Johnson, Lacey; Marks, Denese C.; Devine, Dana V.

2018-01-01

Transfusions of platelets are an important cornerstone of medicine; however, recipients may be subject to risk of adverse events associated with the potential transmission of pathogens, especially bacteria. Pathogen inactivation (PI) technologies based on ultraviolet illumination have been developed in the last decades to mitigate this risk. This review discusses studies of platelet concentrates treated with the current generation of PI technologies to assess their impact on quality, PI capacity, safety, and clinical efficacy. Improved safety seems to come with the cost of reduced platelet functionality, and hence transfusion efficacy. In order to understand these negative impacts in more detail, several molecular analyses have identified signaling pathways linked to platelet function that are altered by PI. Because some of these biochemical alterations are similar to those seen arising in the context of routine platelet storage lesion development occurring during blood bank storage, we lack a complete picture of the contribution of PI treatment to impaired platelet functionality. A model generated using data from currently available publications places the signaling protein kinase p38 as a central player regulating a variety of mechanisms triggered in platelets by PI systems. PMID:29868586

Acidosis downregulates platelet haemostatic functions and promotes neutrophil proinflammatory responses mediated by platelets.

PubMed

Etulain, Julia; Negrotto, Soledad; Carestia, Agostina; Pozner, Roberto Gabriel; Romaniuk, María Albertina; D'Atri, Lina Paola; Klement, Giannoula Lakka; Schattner, Mirta

2012-01-01

Acidosis is one of the hallmarks of tissue injury such as trauma, infection, inflammation, and tumour growth. Although platelets participate in the pathophysiology of all these processes, the impact of acidosis on platelet biology has not been studied outside of the quality control of laboratory aggregation assays or platelet transfusion optimization. Herein, we evaluate the effect of physiologically relevant changes in extracellular acidosis on the biological function of platelets, placing particular emphasis on haemostatic and secretory functions. Platelet haemostatic responses such as adhesion, spreading, activation of αIIbβ3 integrin, ATP release, aggregation, thromboxane B2 generation, clot retraction and procoagulant activity including phosphatidilserine exposure and microparticle formation, showed a statistically significant inhibition of thrombin-induced changes at pH of 7.0 and 6.5 compared to the physiological pH (7.4). The release of alpha granule content was differentially regulated by acidosis. At low pH, thrombin or collagen-induced secretion of vascular endothelial growth factor and endostatin were dramatically reduced. The release of von Willebrand factor and stromal derived factor-1α followed a similar, albeit less dramatic pattern. In contrast, the induction of CD40L was not changed by low pH, and P-selectin exposure was significantly increased. While the generation of mixed platelet-leukocyte aggregates and the increased chemotaxis of neutrophils mediated by platelets were further augmented under acidic conditions in a P-selectin dependent manner, the increased neutrophil survival was independent of P-selectin expression. In conclusion, our results indicate that extracellular acidosis downregulates most of the haemostatic platelet functions, and promotes those involved in amplifying the neutrophil-mediated inflammatory response.

The disulfide isomerase ERp57 is required for fibrin deposition in vivo.

PubMed

Zhou, J; Wu, Y; Wang, L; Rauova, L; Hayes, V M; Poncz, M; Essex, D W

2014-11-01

ERp57 is required for platelet function; however, whether ERp57 contributes to fibrin generation is unknown. Using an inhibitory anti-ERp57 antibody (mAb1), Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice, and mutants of ERp57, we analyzed the function of ERp57 in laser-induced thrombosis. Fibrin deposition was decreased in Pf4-Cre/ERp57(fl/fl) mice, consistent with a role for platelet ERp57 in fibrin generation. Fibrin deposition was further decreased with infusion of mAb1 and in Tie2-Cre/ERp57(fl/fl) mice, consistent with endothelial cells also contributing to fibrin deposition. Infusion of eptibifatide inhibited platelet and fibrin deposition, confirming a role for platelets in fibrin deposition. Infusion of recombinant ERp57 corrected the defect in fibrin deposition but not platelet accumulation, suggesting a direct effect of ERp57 on coagulation. mAb1 inhibited thrombin generation in vitro, consistent with a requirement for ERp57 in coagulation. Platelet accumulation was decreased to similar extents in Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice and normal mice infused with mAb1. Infusion of completely inactivated ERp57 or ERp57 with a non-functional second active site inhibited fibrin deposition and platelet accumulation, indicating that the isomerase activity of the second active site is required for these processes. ERp57 regulates thrombosis via multiple targets. © 2014 International Society on Thrombosis and Haemostasis.

Role of platelet-released growth factors in detoxification of reactive oxygen species in osteoblasts.

PubMed

Tohidnezhad, Mersedeh; Wruck, Christoph-Jan; Slowik, Alexander; Kweider, Nisreen; Beckmann, Rainer; Bayer, Andreas; Houben, Astrid; Brandenburg, Lars-Ove; Varoga, Deike; Sönmez, Tolga-Taha; Stoffel, Marcus; Jahr, Holger; Lippross, Sebastian; Pufe, Thomas

2014-08-01

Oxidative stress can impair fracture healing. To protect against oxidative damage, a system of detoxifying and antioxidative enzymes works to reduce the cellular stress. The transcription of these enzymes is regulated by antioxidant response element (ARE). The nuclear factor (erythroid-derived 2)-like2 (Nrf2) plays a major role in transcriptional activation of ARE-driven genes. Recently it has been shown that vascular endothelial growth factor (VEGF) prevents oxidative damage via activation of the Nrf2 pathway in vitro. Platelet-released growth factor (PRGF) is a mixture of autologous proteins and growth factors, prepared from a determined volume of platelet-rich plasma (PRP). It has already used to enhance fracture healing in vitro. The aim of the present study was to elucidate if platelets can lead to upregulation of VEGF and if platelets can regulate the activity of Nrf2-ARE system in primary human osteoblast (hOB) and in osteoblast-like cell line (SAOS-2). Platelets and PRGF were obtained from healthy human donors. HOB and SAOS-2 osteosarcoma cell line were used. The ARE activity was analysed using a dual luciferase reporter assay system. We used Western blot to detect the nuclear accumulation of Nrf2 and the amount of cytosolic antioxidant Thioredoxin Reductase-1 (TXNRD-1), Heme Oxygenase-1 (HO-1) and NAD(P)H quinine oxidoreductase-1 (NQO1). Gene expression analysis was performed by real-time RT PCR. ELISA was used for the quantification of growth factors. The activity of ARE was increased in the presence of PRGF up to 50%. Western blotting demonstrated enhanced nuclear accumulation of Nrf2. This was followed by an increase in the protein expression of the aforementioned downstream targets of Nrf2. Real-time RT PCR data showed an upregulation in the gene expression of the VEGF after PRGF treatment. This was confirmed by ELISA, where the treatment with PRGF induced the protein level of VEGF in both cells. These results provide a new insight into PRGF's mode of action in osteoblasts. PRGF not only leads to increase the endogenous VEGF, but also it may be involved in preventing oxidative damage through the Nrf2-ARE signalling. Nrf2 activation via PRGF may have great potential as an effective therapeutic drug target in fracture healing. Copyright © 2014 Elsevier Inc. All rights reserved.

Phosphoinositide 3-Kinase p110β Regulates Integrin αIIbβ3 Avidity and the Cellular Transmission of Contractile Forces*

PubMed Central

Schoenwaelder, Simone M.; Ono, Akiko; Nesbitt, Warwick S.; Lim, Joanna; Jarman, Kate; Jackson, Shaun P.

2010-01-01

Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin αIIbβ3, necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin αIIbβ3 activation (affinity modulation) primarily occurs downstream of Gi-coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin αIIbβ3 bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110β isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin αIIbβ3 activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin αIIbβ3 association with the contractile cytoskeleton. Analysis of integrin αIIbβ3 adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin αIIbβ3 bonds. These studies demonstrate an important role for PI3K p110β in regulating the avidity of high affinity integrin αIIbβ3 receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110β inhibitors. PMID:19940148

Platelet monoamine oxidase type B, MAOB intron 13 and MAOA-uVNTR polymorphism and symptoms of post-traumatic stress disorder.

PubMed

Svob Strac, Dubravka; Kovacic Petrovic, Zrnka; Nikolac Perkovic, Matea; Umolac, Danica; Nedic Erjavec, Gordana; Pivac, Nela

2016-07-01

Post-traumatic stress disorder (PTSD), a disorder that develops following exposure to traumatic experience(s), is frequently associated with agitation, aggressive behavior and psychotic symptoms. Monoamine oxidase (MAO) degrades different biogenic amines and regulates mood, emotions and behavior, and has a role in the pathophysiology of various neuropsychiatric disorders. The aim of the study was to investigate the association between different symptoms occurring in PTSD [PTSD symptom severity assessed by the Clinician Administered PTSD Scale (CAPS), agitation and selected psychotic symptoms assessed by the Positive and Negative Syndrome Scale (PANSS)] and platelet MAO-B activity and/or genetic variants of MAOB rs1799836 and MAOA-uVNTR polymorphisms in 249 Croatian male veterans with PTSD. Our study revealed slightly higher platelet MAO-B activity in veterans with PTSD with more severe PTSD symptoms and in veterans with agitation, and significantly higher platelet MAO-B activity in veterans with more pronounced psychotic symptoms compared to veterans with less pronounced psychotic symptoms. Platelet MAO-B activity was associated with smoking but not with age. Genetic variants of MAOB rs1799836 and MAOA-uVNTR were not associated with agitation and selected psychotic symptoms in veterans with PTSD. A marginally significant association was found between MAOB rs1799836 polymorphism and severity of PTSD symptoms, but it was not confirmed since carriers of G or A allele of MAOB rs1799836 did not differ in their total CAPS scores. These findings suggest an association of platelet MAO-B activity, but a lack of association of MAOB rs1799836 and MAOA-uVNTR, with selected psychotic symptoms in ethnically homogenous veterans with PTSD.

Rupture Forces among Human Blood Platelets at different Degrees of Activation

PubMed Central

Nguyen, Thi-Huong; Palankar, Raghavendra; Bui, Van-Chien; Medvedev, Nikolay; Greinacher, Andreas; Delcea, Mihaela

2016-01-01

Little is known about mechanics underlying the interaction among platelets during activation and aggregation. Although the strength of a blood thrombus has likely major biological importance, no previous study has measured directly the adhesion forces of single platelet-platelet interaction at different activation states. Here, we filled this void first, by minimizing surface mediated platelet-activation and second, by generating a strong adhesion force between a single platelet and an AFM cantilever, preventing early platelet detachment. We applied our setup to measure rupture forces between two platelets using different platelet activation states, and blockade of platelet receptors. The rupture force was found to increase proportionally to the degree of platelet activation, but reduced with blockade of specific platelet receptors. Quantification of single platelet-platelet interaction provides major perspectives for testing and improving biocompatibility of new materials; quantifying the effect of drugs on platelet function; and assessing the mechanical characteristics of acquired/inherited platelet defects. PMID:27146004

Phorbol 12,13-dibutyrate and 1-oleyl-2-acetyldiacylglycerol stimulate inositol trisphosphate dephosphorylation in human platelets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Molina y Vedia, L.M.; Lapetina, E.G.

1986-08-15

Inositol trisphosphate (IP3) is formed in response to specific agonists that cause activation of phospholipase C and degradation of phosphatidylinositol bisphosphate. IP3 is a second messenger that releases Ca/sup 2 +/ from the dense tubular system to the cytosol in stimulated platelets. Our present information indicates that (/sup 3/H)IP3 is dephosphorylated to (/sup 3/H)inositol bisphosphate (IP2) and (/sup 3/H)inositol monophosphate (IP) by human platelets treated with 0.05-0.10% Triton X-100. This dephosphorylation of (/sup 3/H)IP3 to (/sup 3/H)IP2 and (/sup 3/H)IP is also observed when platelets are permeabilized by electrical stimulation or by 20 micrograms/ml saponin. These detergents or electropermeabilization allowmore » IP3 to access cytosolic IP3 phosphatase. Pretreatment of intact platelets with phorbol dibutyrate and 1-oleyl-2-acetyldiacylglycerol for 30 s, at concentrations that maximally activate protein kinase C, stimulates the conversion of IP3 to IP2 and IP. This suggests a role for protein kinase C in the regulation of IP3 degradation.« less

Protein kinase C and P2Y12 take center stage in thrombin-mediated activation of mammalian target of rapamycin complex 1 in human platelets.

PubMed

Moore, S F; Hunter, R W; Hers, I

2014-05-01

Rapamycin, an inhibitor of mammalian target of rapamycin complex-1 (mTORC1), reduces platelet spreading, thrombus stability, and clot retraction. Despite an important role of mTORC1 in platelet function, little is known about how it is regulated. The objective of this study was to determine the signaling pathways that regulate mTORC1 in human platelets. Mammalian target of rapamycin complex-1 activation was assessed by measuring the phosphorylation of its downstream substrate ribosomal S6 kinase 1 (p70S6K). Thrombin or the protein kinase C (PKC) activator phorbal 12-myristate 13-acetate stimulated activation of mTORC1 in a PKC-dependent, Akt-independent manner that correlated with phosphorylation of tuberin/tuberous sclerosis 2 (TSC2) (Ser939 and Thr1462). In contrast, insulin-like growth factor 1 (IGF-1)-stimulated TSC2 phosphorylation was completely dependent on phosphoinositide 3 kinase (PI3 kinase)/Akt but did not result in any detectable mTORC1 activation. Early (Ser939 and Thr1462) and late (Thr1462) TSC2 phosphorylation in response to thrombin were directly PKC dependent, whereas later TSC2 (Ser939) and p70S6K phosphorylation were largely dependent on paracrine signaling through P2Y(12). PKC-mediated adenosine diphosphate (ADP) secretion was essential for thrombin-stimulated mTORC1 activation, as (i) ADP rescued p70S6K phosphorylation in the presence of a PKC inhibitor and (ii) P2Y(12) antagonism prevented thrombin-mediated mTORC1 activation. Rescue of mTORC1 activation with exogenous ADP was completely dependent on the Src family kinases but independent of PI3 kinase/Akt. Interestingly, although inhibition of Src blocked the ADP rescue, it had little effect on thrombin-stimulated p70S6K phosphorylation under conditions where PKC was not inhibited. These results demonstrate that thrombin activates the mTORC1 pathway in human platelets through PKC-mediated ADP secretion and subsequent activation of P2Y(12), in a manner largely independent of the canonical PI3 kinase/Akt pathway. © 2014 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

Protein kinase C and P2Y12 take center stage in thrombin-mediated activation of mammalian target of rapamycin complex 1 in human platelets

PubMed Central

Moore, S F; Hunter, R W; Hers, I

2014-01-01

Background Rapamycin, an inhibitor of mammalian target of rapamycin complex-1 (mTORC1), reduces platelet spreading, thrombus stability, and clot retraction. Despite an important role of mTORC1 in platelet function, little is known about how it is regulated. The objective of this study was to determine the signaling pathways that regulate mTORC1 in human platelets. Methods Mammalian target of rapamycin complex-1 activation was assessed by measuring the phosphorylation of its downstream substrate ribosomal S6 kinase 1 (p70S6K). Results Thrombin or the protein kinase C (PKC) activator phorbal 12-myristate 13-acetate stimulated activation of mTORC1 in a PKC-dependent, Akt-independent manner that correlated with phosphorylation of tuberin/tuberous sclerosis 2 (TSC2) (Ser939 and Thr1462). In contrast, insulin-like growth factor 1 (IGF-1)–stimulated TSC2 phosphorylation was completely dependent on phosphoinositide 3 kinase (PI3 kinase)/Akt but did not result in any detectable mTORC1 activation. Early (Ser939 and Thr1462) and late (Thr1462) TSC2 phosphorylation in response to thrombin were directly PKC dependent, whereas later TSC2 (Ser939) and p70S6K phosphorylation were largely dependent on paracrine signaling through P2Y12. PKC-mediated adenosine diphosphate (ADP) secretion was essential for thrombin-stimulated mTORC1 activation, as (i) ADP rescued p70S6K phosphorylation in the presence of a PKC inhibitor and (ii) P2Y12 antagonism prevented thrombin-mediated mTORC1 activation. Rescue of mTORC1 activation with exogenous ADP was completely dependent on the Src family kinases but independent of PI3 kinase/Akt. Interestingly, although inhibition of Src blocked the ADP rescue, it had little effect on thrombin-stimulated p70S6K phosphorylation under conditions where PKC was not inhibited. Conclusion These results demonstrate that thrombin activates the mTORC1 pathway in human platelets through PKC-mediated ADP secretion and subsequent activation of P2Y12, in a manner largely independent of the canonical PI3 kinase/Akt pathway. PMID:24612393

Platelet activation by extracellular matrix proteins in haemostasis and thrombosis.

PubMed

Watson, Steve P

2009-01-01

The prevention of excessive blood loss to avoid fatal haemorrhage is a pivotal process for all organisms possessing a circulatory system. Increased circulating blood volume and pressure, as required in larger animals, make this process all the more important and challenging. It is essential to have a powerful and rapid system to detect damage and generate an effective seal, and which is also exquisitely regulated to prevent unwanted, excessive or systemic activation so as to avoid blockage of vessels. Thus, a highly specialised and efficient haemostatic system has evolved that consists of cellular (platelets) and protein (coagulation factors) components. Importantly, this is able to support haemostasis in both the low shear environment of the venous system and the high shear environment of the arterial system. Endothelial cells, lining the entire circulation system, play a crucial role in the delicate balance between activation and inhibition of the haemostatic system. An intact and healthy endothelium supports blood flow by preventing attachment of cells and proteins which is required for initiation of coagulation and platelet activation. Endothelial cells produce and release the two powerful soluble inhibitors of platelet activation, nitric oxide and prostacyclin, and express high levels of CD39 which rapidly metabolises the major platelet feedback agonist, ADP. This antithrombotic environment however can rapidly change following activation or removal of endothelial cells through injury or rupture of atherosclerotic plaques. Loss of endothelial cells exposes the subendothelial extracellular matrix which creates strong signals for activation of the haemostatic system including powerful platelet adhesion and activation. Quantitative and qualitative changes in the composition of the subendothelial extracellular matrix influence these prothrombotic characteristics with life threatening thrombotic and bleeding complications, as illustrated by formation of atherosclerotic plaques or the disorder Ehler-Danlos syndrome, which is caused by a defect in collagen synthesis and is associated with fragile blood vessels. This review will focus on the role of the subendothelial matrix in haemostasis and thrombosis, highlighting its potential as a target for novel antithrombotics.

Clinical Applications of Platelet-Rich Plasma in Patellar Tendinopathy

PubMed Central

Jeong, D. U.; Lee, C.-R.; Lee, J. H.; Pak, J.; Kang, L.-W.; Jeong, B. C.

2014-01-01

Platelet-rich plasma (PRP), a blood derivative with high concentrations of platelets, has been found to have high levels of autologous growth factors (GFs), such as transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), fibroblastic growth factor (FGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF). These GFs and other biological active proteins of PRP can promote tissue healing through the regulation of fibrosis and angiogenesis. Moreover, PRP is considered to be safe due to its autologous nature and long-term usage without any reported major complications. Therefore, PRP therapy could be an option in treating overused tendon damage such as chronic tendinopathy. Here, we present a systematic review highlighting the clinical effectiveness of PRP injection therapy in patellar tendinopathy, which is a major cause of athletes to retire from their respective careers. PMID:25136568

Polyunsaturated fatty acids balance affects platelet NOX2 activity in patients with liver cirrhosis.

PubMed

Basili, Stefania; Raparelli, Valeria; Napoleone, Laura; Del Ben, Maria; Merli, Manuela; Riggio, Oliviero; Nocella, Cristina; Carnevale, Roberto; Pignatelli, Pasquale; Violi, Francesco

2014-07-01

NADPH-oxidase-2 up-regulation has been suggested in liver damage perpetuation via an oxidative stress-mediated mechanism. n-6/n-3 polyunsaturated fatty acids ratio derangement has been reported in liver disease. To explore polyunsaturated fatty acids balance and its interplay with platelet oxidative stress in liver cirrhosis. A cross-sectional study in 51 cirrhotic patients and sex- and age-matched controls was performed. Serum polyunsaturated fatty acids and oxidative stress markers (urinary isoprostanes and serum soluble NADPH-oxidase-2-derived peptide) were measured. The effect on platelet oxidative stress of n-6/n-3 polyunsaturated fatty acids ratio in vitro and in vivo (1-week supplementation with 3g/daily n-3-polyunsaturated fatty acids) was tested. Compared to controls, cirrhotic patients had significantly higher n-6/n-3 polyunsaturated fatty acids ratio. n-6/n-3 polyunsaturated fatty acids ratio correlated significantly with disease severity and oxidative stress markers. In vitro experiments showed that in Child-Pugh C patients' platelets incubation with low n-6/n-3 polyunsaturated fatty acids ratio resulted in dose-dependent decrease of radical oxigen species (-39%), isoprostanes (-25%) and NADPH-oxidase-2 regulation (-51%). n-3 polyunsaturated fatty acids supplemented patients showed significant oxidative stress indexes reduction. In cirrhosis, n-6/n-3 polyunsaturated fatty acids imbalance up-regulates platelet NADPH-oxidase-2 with ensuing oxidative stress. Further study to evaluate if n-3 supplementation may reduce disease progression is warranted. Copyright © 2014 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

The role of platelets during reproduction.

PubMed

Isermann, Berend; Nawroth, Peter P

2006-01-01

The availability of mice with defined defects within the hemostatic system enabled researchers to identify a role the coagulation system for embryonic and placental development. However, the role of platelets during development has only recently been experimentally addressed, giving some insight into potential functions of platelets during development. Thus, a quantitative embryonic platelet defect (severe thrombopenia secondary to NF-E2 deficiency) is associated with an embryonic growth retardation and reduced vascularisation of the placenta. Maternal platelet deficiency is associated with placental hemorrhage, which, however, does not impair embryonic or maternal survival. In vitro studies established that platelets or platelet conditioned medium regulate the invasive properties of human extravillous trophoblast cells and induce a phenotypical switch of trophoblast cells. These data imply that platelets are of relevance during placentation. Conversely, platelets and the formation of platelet-fibrin aggregates are dispensable for the development of the embryo proper, establishing that the lethal phenotypes observed in some embryo slacking coagulation regulators does not result from an inability to form platelet-fibrin aggregates, but likely reflects altered protease dependent signaling during vascular development.

Functional expression of cysteinyl leukotriene receptors on human platelets.

PubMed

Hasegawa, Shunji; Ichiyama, Takashi; Hashimoto, Kunio; Suzuki, Yasuo; Hirano, Reiji; Fukano, Reiji; Furukawa, Susumu

2010-01-01

Normal peripheral blood leukocytes, such as basophils, eosinophils, B lymphocytes and monocytes/macrophages, have a cysteinyl leukotriene 1 (CysLT1) receptor, while the cysteinyl leukotriene 2 (CysLT2) receptor is expressed in cardiac Purkinje cells, endothelium, brain and leukocytes. However, it is unknown whether or not platelets express the CysLT1 or CysLT2 receptor. In this study we identify and characterize the biological function of the CysLT receptor of human platelets. We determined the CysLT1 or CysLT2 receptor mRNA expression in normal human platelets by RT-PCR and determined protein expression by Western blotting and flow cytometry. Moreover, we examined the effect of cysteinyl leukotrienes (CysLTs) in platelets on the induction of RANTES (Regulated on Activation, Normal T Expressed, and presumably Secreted). We also investigated whether the CysLT1 receptor antagonist pranlukast inhibits CysLT-induced RANTES release. In conclusion, we showed the functional expression of CysLT receptors on human platelets and demonstrated that CysLTs induced the release of significant amounts of RANTES, which suggests a novel role for human platelets in CysLT-mediated allergic inflammation.

Aspirin influences megakaryocytic gene expression leading to up-regulation of multidrug resistance protein-4 in human platelets.

PubMed

Massimi, Isabella; Guerriero, Raffaella; Lotti, Lavinia Vittoria; Lulli, Valentina; Borgognone, Alessandra; Romani, Federico; Barillà, Francesco; Gaudio, Carlo; Gabbianelli, Marco; Frati, Luigi; Pulcinelli, Fabio M

2014-12-01

The aim of the study was to investigate whether human megakaryocytic cells have an adaptive response to aspirin treatment, leading to an enhancement of multidrug resistance protein-4 (MRP4) expression in circulating platelets responsible for a reduced aspirin action. We recently found that platelet MRP4 overexpression has a role in reducing aspirin action in patients after by-pass surgery. Aspirin enhances MRP4-mRNA levels in rat liver and drug administration transcriptionally regulates MRP4 gene expression through peroxisome proliferator-activated receptor-α (PPARα). The effects induced by aspirin or PPARα agonist (WY14643) on MRP4 modulation were evaluated in vitro in a human megakaryoblastic DAMI cell line, in megakaryocytes (MKs) and in platelets obtained from human haematopoietic progenitor cell (HPC) cultures, and in vivo platelets obtained from aspirin treated healthy volunteers (HV). In DAMI cells, aspirin and WY14643 treatment induced a significant increase in MRP4 and PPARα expression. In human MKs grown in the presence of either aspirin or WY14643, MRP4 and PPARα-mRNA were higher than in control cultures and derived platelets showed an enhancement in MRP4 protein expression. The ability of aspirin to modulate MRP4 expression in MKs and to transfer it to platelets was also confirmed in vivo. In fact, we found the highest MRP4 mRNA and protein expression in platelets obtained from HV after 15 days' aspirin treatment. The present study provides evidence, for the first time, that aspirin treatment affects the platelet protein pattern through MK genomic modulation. This work represents an innovative and attractive approach, useful both to identify patients less sensitive to aspirin and to improve pharmacological treatment in cardiovascular high-risk patients. © 2014 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.

Platelet-derived growth factor regulates K-Cl cotransport in vascular smooth muscle cells.

PubMed

Zhang, Jing; Lauf, Peter K; Adragna, Norma C

2003-03-01

Platelet-derived growth factor (PDGF), a potent serum mitogen for vascular smooth muscle cells (VSMCs), plays an important role in membrane transport regulation and in atherosclerosis. K-Cl cotransport (K-Cl COT/KCC), the coupled-movement of K and Cl, is involved in ion homeostasis. VSMCs possess K-Cl COT activity and the KCC1 and KCC3 isoforms. Here, we report on the effect of PDGF on K-Cl COT activity and mRNA expression in primary cultures of rat VSMCs. K-Cl COT was determined as the Cl-dependent Rb influx and mRNA expression by semiquantitative RT-PCR. Twenty four-hour serum deprivation inhibited basal K-Cl COT activity. Addition of PDGF increased total protein content and K-Cl COT activity in a time-dependent manner. PDGF activated K-Cl COT in a dose-dependent manner, both acutely (10 min) and chronically (12 h). AG-1296, a selective inhibitor of the PDGF receptor tyrosine kinase, abolished these effects. Actinomycin D and cycloheximide had no effect on the acute PDGF activation of K-Cl COT, suggesting posttranslational regulation by the drug. Furthermore, PDGF increased KCC1 and decreased KCC3 mRNA expression in a time-dependent manner. These results indicate that chronic activation of K-Cl COT activity by PDGF may involve regulation of the two KCC mRNA isoforms, with KCC1 playing a dominant role in the mechanism of PDGF-mediated activation.

Platelet-derived chemokines CXC chemokine ligand (CXCL)7, connective tissue-activating peptide III, and CXCL4 differentially affect and cross-regulate neutrophil adhesion and transendothelial migration.

PubMed

Schenk, Birgit I; Petersen, Frank; Flad, Hans-Dieter; Brandt, Ernst

2002-09-01

In this study, we have examined the major platelet-derived CXC chemokines connective tissue-activating peptide III (CTAP-III), its truncation product neutrophil-activating peptide 2 (CXC chemokine ligand 7 (CXCL7)), as well as the structurally related platelet factor 4 (CXCL4) for their impact on neutrophil adhesion to and transmigration through unstimulated vascular endothelium. Using monolayers of cultured HUVEC, we found all three chemokines to promote neutrophil adhesion, while only CXCL7 induced transmigration. Induction of cell adhesion following exposure to CTAP-III, a molecule to date described to lack neutrophil-stimulating capacity, depended on proteolytical conversion of the inactive chemokine into CXCL7 by neutrophils. This was evident from experiments in which inhibition of the CTAP-III-processing protease and simultaneous blockade of the CXCL7 high affinity receptor CXCR-2 led to complete abrogation of CTAP-III-mediated neutrophil adhesion. CXCL4 at substimulatory dosages modulated CTAP-III- as well as CXCL7-induced adhesion. Although cell adhesion following exposure to CTAP-III was drastically reduced, CXCL7-mediated adhesion underwent significant enhancement. Transendothelial migration of neutrophils in response to CXCL7 or IL-8 (CXCL8) was subject to modulation by CTAP-III, but not CXCL4, as seen by drastic desensitization of the migratory response of neutrophils pre-exposed to CTAP-III, which was paralleled by selective down-modulation of CXCR-2. Altogether our results demonstrate that there exist multiple interactions between platelet-derived chemokines in the regulation of neutrophil adhesion and transendothelial migration.

Lysophosphatidic Acid Regulation and Roles in Human Prostate Cancer

DTIC Science & Technology

2006-08-01

phosphatidic acid (PA), regulate pivotal processes related to the pathogenesis of cancer. We characterized a novel lipid kinase, designated...pathways. LPA is produced from phosphatidic acid 5 (PA) in activated platelets and ovarian and prostate cancer cells by phospholipase D and subsequent...lysophosphatidic acid (LPA) and phosphatidic acid (PA), regulate pivotal processes related to the pathogenesis of cancer. Here, we report characterization of a novel

Effects of hormones on platelet aggregation.

PubMed

Farré, Antonio López; Modrego, Javier; Zamorano-León, José J

2014-04-01

Platelets and their activation/inhibition mechanisms play a central role in haemostasis. It is well known agonists and antagonists of platelet activation; however, during the last years novel evidences of hormone effects on platelet activation have been reported. Platelet functionality may be modulated by the interaction between different hormones and their platelet receptors, contributing to sex differences in platelet function and even in platelet-mediated vascular damage. It has suggested aspects that apparently are well established should be reviewed. Hormones effects on platelet activity are included among them. This article tries to review knowledge about the involvement of hormones in platelet biology and activity.

Disruption of melatonin synthesis is associated with impaired 14-3-3 and miR-451 levels in patients with autism spectrum disorders.

PubMed

Pagan, Cécile; Goubran-Botros, Hany; Delorme, Richard; Benabou, Marion; Lemière, Nathalie; Murray, Kerren; Amsellem, Frédérique; Callebert, Jacques; Chaste, Pauline; Jamain, Stéphane; Fauchereau, Fabien; Huguet, Guillaume; Maronde, Erik; Leboyer, Marion; Launay, Jean-Marie; Bourgeron, Thomas

2017-05-18

Autism spectrum disorders (ASD) are characterized by a wide genetic and clinical heterogeneity. However, some biochemical impairments, including decreased melatonin (crucial for circadian regulation) and elevated platelet N-acetylserotonin (the precursor of melatonin) have been reported as very frequent features in individuals with ASD. To address the mechanisms of these dysfunctions, we investigated melatonin synthesis in post-mortem pineal glands - the main source of melatonin (9 patients and 22 controls) - and gut samples - the main source of serotonin (11 patients and 13 controls), and in blood platelets from 239 individuals with ASD, their first-degree relatives and 278 controls. Our results elucidate the enzymatic mechanism for melatonin deficit in ASD, involving a reduction of both enzyme activities contributing to melatonin synthesis (AANAT and ASMT), observed in the pineal gland as well as in gut and platelets of patients. Further investigations suggest new, post-translational (reduced levels of 14-3-3 proteins which regulate AANAT and ASMT activities) and post-transcriptional (increased levels of miR-451, targeting 14-3-3ζ) mechanisms to these impairments. This study thus gives insights into the pathophysiological pathways involved in ASD.

The role of microRNAs in platelet biology during storage.

PubMed

Yan, Yuzhong; Zhang, Jingjun; Zhang, Qi; Chen, Yanping; Zhu, Xinfang; Xia, Rong

2017-04-01

Platelet storage lesions seriously affect the quality of stored platelets, even causing them to be ineffective in vivo after transfusion. Past research have been focused on what mechanism(s) cause the formation of storage lesions. One proposed mechanism is microRNAs (miRNAs)-based molecular regulation of the platelet mRNAs that are relevant to the storage lesion. Platelets continue to translate proteins from mRNA while in a storage environment. A strong correlation exists between the platelet transcriptome and its subsequent proteomic profile, which supports de novo platelet translational capabilities. Thus, miRNA may play a crucial role in platelet biology during storage conditions. Importantly, this suggests the exciting possibility of post-transcriptional regulation of gene expression in platelets that are in storage. Given this, the differential profiling of miRNAs could be a useful tool in identifying changes to ex vivo stored platelets. Any identified miRNAs could then be considered as potential markers to assess the viability of platelet concentrates. The present review summarizes the current experimental and clinical evidence that clarifies the role miRNAs play during platelet ex vivo storage. Copyright © 2016 Elsevier Ltd. All rights reserved.

Platelet “First Responders” in Wound Response, Cancer, and Metastasis

PubMed Central

Menter, David G.; Kopetz, Scott; Hawk, Ernest; Sood, Anil K.; Loree, Jonathan M; Gresele, Paolo; Honn, Kenneth V.

2017-01-01

Platelets serve as “First Responders” during normal wounding and homeostasis. Arising from bone marrow stem cell lineage megakaryocytes, anucleate platelets can influence inflammation and immune regulation. Biophysically, platelets are optimized due to size and discoid morphology to distribute near vessel walls, monitor vascular integrity and initiate quick responses to vascular lesions. Adhesion receptors linked to a highly reactive filopodia-generating cytoskeleton maximizes their vascular surface contact allowing rapid response capabilities. Functionally, platelets normally initiate rapid clotting, vasoconstriction, inflammation and wound biology that leads to sterilization, tissue repair and resolution. Platelets also are among the first to sense, phagocytize, decorate, or react to pathogens in the circulation. These platelet first responder properties are commandeered during chronic inflammation, cancer progression and metastasis. Leaky or inflammatory reaction blood vessel genesis during carcinogenesis provides opportunities for platelet invasion into tumors. Cancer is thought of as a non-healing or chronic wound that can be actively aided by platelet mitogenic properties to stimulate tumor growth. This growth ultimately outstrips circulatory support leads to angiogenesis and intravasation of tumor cells into the blood stream. Circulating tumor cells reengage additional platelets, which facilitates tumor cell adhesion, arrest and extravasation and metastasis. This process, along with the hypercoagulable states associated with malignancy is amplified by IL6 production in tumors that stimulate liver thrombopoietin production and elevates circulating platelet numbers by thrombopoiesis in the bone marrow. These complex interactions and the “First Responder” role of platelets during diverse physiologic stresses provides a useful therapeutic target that deserves further exploration. PMID:28730545

The life cycle of platelet granules.

PubMed

Sharda, Anish; Flaumenhaft, Robert

2018-01-01

Platelet granules are unique among secretory vesicles in both their content and their life cycle. Platelets contain three major granule types-dense granules, α-granules, and lysosomes-although other granule types have been reported. Dense granules and α-granules are the most well-studied and the most physiologically important. Platelet granules are formed in large, multilobulated cells, termed megakaryocytes, prior to transport into platelets. The biogenesis of dense granules and α-granules involves common but also distinct pathways. Both are formed from the trans -Golgi network and early endosomes and mature in multivesicular bodies, but the formation of dense granules requires trafficking machinery different from that of α-granules. Following formation in the megakaryocyte body, both granule types are transported through and mature in long proplatelet extensions prior to the release of nascent platelets into the bloodstream. Granules remain stored in circulating platelets until platelet activation triggers the exocytosis of their contents. Soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, located on both the granules and target membranes, provide the mechanical energy that enables membrane fusion during both granulogenesis and exocytosis. The function of these core fusion engines is controlled by SNARE regulators, which direct the site, timing, and extent to which these SNAREs interact and consequently the resulting membrane fusion. In this review, we assess new developments in the study of platelet granules, from their generation to their exocytosis.

Flow cytometric assessment of activation of peripheral blood platelets in dogs with normal platelet count and asymptomatic thrombocytopenia.

PubMed

Żmigrodzka, M; Guzera, M; Winnicka, A

2016-01-01

Platelets play a crucial role in hemostasis. Their activation has not yet been evaluated in healthy dogs with a normal and low platelet count. The aim of this study was to determine the influence of activators on platelet activation in dogs with a normal platelet count and asymptomatic thrombocytopenia. 72 clinically healthy dogs were enrolled. Patients were allocated into three groups. Group 1 consisted of 30 dogs with a normal platelet count, group 2 included 22 dogs with a platelet count between 100 and 200×109/l and group 3 consisted of 20 dogs with a platelet count lower than 100×109/l. Platelet rich-plasma (PRP) was obtained from peripheral blood samples using tripotassium ethylenediaminetetraacetic acid (K3-EDTA) as anticoagulant. Next, platelets were stimulated using phorbol-12-myristate-13-acetate or thrombin, stabilized using procaine or left unstimulated. The expression of CD51 and CD41/CD61 was evaluated. Co-expression of CD41/CD61 and Annexin V served as a marker of platelet activation. The expression of CD41/CD61 and CD51 did not differ between the 3 groups. Thrombin-stimulated platelets had a significantly higher activity in dogs with a normal platelet count than in dogs with asymptomatic thrombocytopenia. Procaine inhibited platelet activity in all groups. In conclusion, activation of platelets of healthy dogs in vitro varied depending on the platelet count and platelet activator.

Comparison of ultrastructural and nanomechanical signature of platelets from acute myocardial infarction and platelet activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Aiqun; Chen, Jianwei; Liang, Zhi-Hong

Acute myocardial infarction (AMI) initiation and progression follow complex molecular and structural changes in the nanoarchitecture of platelets. However, it remains poorly understood how the transformation from health to AMI alters the ultrastructural and biomechanical properties of platelets within the platelet activation microenvironment. Here, we show using an atomic force microscope (AFM) that platelet samples, including living human platelets from the healthy and AMI patient, activated platelets from collagen-stimulated model, show distinct ultrastructural imaging and stiffness profiles. Correlative morphology obtained on AMI platelets and collagen-activated platelets display distinct pseudopodia structure and nanoclusters on membrane. In contrast to normal platelets, AMImore » platelets have a stiffer distribution resulting from complicated pathogenesis, with a prominent high-stiffness peak representative of platelet activation using AFM-based force spectroscopy. Similar findings are seen in specific stages of platelet activation in collagen-stimulated model. Further evidence obtained from different force measurement region with activated platelets shows that platelet migration is correlated to the more elasticity of pseudopodia while high stiffness at the center region. Overall, ultrastructural and nanomechanical profiling by AFM provides quantitative indicators in the clinical diagnostics of AMI with mechanobiological significance.« less

Characterization of 5' end of human thromboxane receptor gene. Organizational analysis and mapping of protein kinase C--responsive elements regulating expression in platelets.

PubMed

D'Angelo, D D; Davis, M G; Houser, W A; Eubank, J J; Ritchie, M E; Dorn, G W

1995-09-01

Platelet thromboxane receptors are acutely and reversibly upregulated after acute myocardial infarction. To determine if platelet thromboxane receptors are under transcriptional control, we isolated and characterized human genomic DNA clones containing the 5' flanking region of the thromboxane receptor gene. The exon-intron structure of the 5' portion of the thromboxane receptor gene was determined initially by comparing the nucleotide sequence of the 5' flanking genomic clone with that of a novel human uterine thromboxane receptor cDNA that extended the mRNA 141 bp further upstream than the previously identified human placental cDNA. A major transcription initiation site was located in three human tissues approximately 560 bp upstream from the translation initiation codon and 380 bp upstream from any previously identified transcription initiation site. The thromboxane receptor gene has neither a TATA nor a CAAT consensus site. Promoter function of the 5' flanking region of the thromboxane receptor gene was evaluated by transfection of thromboxane receptor gene promoter/chloramphenicol acetyltransferase (CAT) chimera plasmids into platelet-like K562 cells. Thromboxane receptor promoter activity, as assessed by CAT expression, was relatively weak but was significantly enhanced by phorbol ester treatment. Functional analysis of 5' deletion constructs in transfected K562 cells and gel mobility shift localized the major phorbol ester-responsive motifs in the thromboxane receptor gene promoter to a cluster of activator protein-2 (AP-2) binding consensus sites located approximately 1.8 kb 5' from the transcription initiation site. These studies are the first to determine the structure and organization of the 5' end of the thromboxane receptor gene and demonstrate that thromboxane receptor gene expression can be regulated by activation of protein kinase C via induction of an AP-2-like nuclear factor binding to upstream promoter elements. These findings strongly suggest that the mechanism for previously described upregulation of platelet thromboxane receptors after acute myocardial infarction is increased thromboxane receptor gene transcription in platelet-progenitor cells.

Platelet-derived growth factor inhibits platelet activation in heparinized whole blood.

PubMed

Selheim, F; Holmsen, H; Vassbotn, F S

1999-08-15

We previously have demonstrated that human platelets have functionally active platelet-derived growth factor alpha-receptors. Studies with gel-filtered platelets showed that an autocrine inhibition pathway is transduced through this tyrosine kinase receptor during platelet activation. The physiological significance of this inhibitory effect of platelet-derived growth factor on gel-filtered platelets activation is, however, not known. In the present study, we investigated whether platelet-derived growth factor inhibits platelet activation under more physiological conditions in heparinized whole blood, which represents a more physiological condition than gel-filtered platelets. Using flow cytometric assays, we demonstrate here that platelet-derived growth factor inhibits thrombin-, thrombin receptor agonist peptide SFLLRN-, and collagen-induced platelet aggregation and shedding of platelet-derived microparticles from the platelet plasma membrane during platelet aggregation in stirred heparinized whole blood. The inhibitory effect of platelet-derived growth factor was dose dependent. However, under nonaggregating conditions (no stirring), we could not demonstrate any significant effect of platelet-derived growth factor on thrombin- and thrombin receptor agonist peptide-induced platelet surface expression of P-selectin. Our results demonstrate that platelet-derived growth factor appears to be a true antithrombotic agent only under aggregating conditions in heparinized whole blood.

Role of Gas6 receptors in platelet signaling during thrombus stabilization and implications for antithrombotic therapy

PubMed Central

Angelillo-Scherrer, Anne; Burnier, Laurent; Flores, Nathalie; Savi, Pierre; DeMol, Maria; Schaeffer, Paul; Herbert, Jean-Marc; Lemke, Greg; Goff, Stephen P.; Matsushima, Glenn K.; Earp, H. Shelton; Vesin, Christian; Hoylaerts, Marc F.; Plaisance, Stéphane; Collen, Désiré; Conway, Edward M.; Wehrle-Haller, Bernhard; Carmeliet, Peter

2005-01-01

Mechanisms regulating thrombus stabilization remain largely unknown. Here, we report that loss of any 1 of the Gas6 receptors (Gas6-Rs), i.e., Tyro3, Axl, or Mer, or delivery of a soluble extracellular domain of Axl that traps Gas6 protects mice against life-threatening thrombosis. Loss of a Gas6-R does not prevent initial platelet aggregation but impairs subsequent stabilization of platelet aggregates, at least in part by reducing “outside-in” signaling and platelet granule secretion. Gas6, through its receptors, activates PI3K and Akt and stimulates tyrosine phosphorylation of the β3 integrin, thereby amplifying outside-in signaling via αIIbβ3. Blocking the Gas6-R–αIIbβ3 integrin cross-talk might be a novel approach to the reduction of thrombosis. PMID:15650770

Preanalytical requirements for flow cytometric evaluation of platelet activation: choice of anticoagulant.

PubMed

Mody, M; Lazarus, A H; Semple, J W; Freedman, J

1999-06-01

Accurate assessment of in vivo or in vitro platelet activation requires optimal preanalytical conditions to prevent artefactual in vitro activation of the platelets. The choice of anticoagulant is one of the critical preanalytical conditions as anticoagulants exert different effects on the activation of platelets ex vivo. We tested the effectiveness of Diatube-H (also known as CTAD; sodium citrate, theophylline, adenosine and dipyridamole) and citrate vacutainer tubes in preventing artefactual activation of platelets and preserving functional reserve. Platelet surface expression of the CD62P (reflecting alpha granule release), CD63 (reflecting lysosomal release) and modulation of normal platelet membrane glycoproteins CD41a and CD42b, were measured in whole blood and in isolated platelets immediately after collection and at 6, 24 and 48 h after venipuncture. Samples taken into Diatube-H showed less spontaneous platelet activation than did those taken into citrate. To measure in vitro platelet functional reserve, thrombin was added as agonist to blood stored for varying periods up to 48 h. Although Diatube-H suppressed in vitro platelet activation for up to 4 h, in samples kept for 6-24 h before thrombin addition, the inhibitory effect was lost and platelets responded fully to agonist activation. Hence, Diatube-H preserved platelets and allowed for measurement of in vivo platelet activation as well as thrombin-induced in vitro platelet activation after 6-24 h, in both whole blood and isolated platelets.

Acetylsalicylic Acid Produces Different Effects on the Production of Active Oxygen Species by Activated Platelets in Different Inflammatory Diseases.

PubMed

Gabbasov, Z A; Kogan-Yasny, V V; Lakhno, D A; Kagan, L G; Ryzhkova, E V; Vasilieva, E Yu; Shpektor, A V

2017-11-01

We studied the effect of acetylsalicylic acid on ROS generation by platelets in patients after surgical interventions and in patients with bronchial asthma was studied. Platelets stimulated with platelet-activating factor are characterized by weak luminol-enhanced chemiluminescence in healthy people and patients after operations with laparoscopic incisions. Addition of platelet activation factor to platelet samples from patients after open abdominal surgery caused intensive chemiluminescence that was suppressed after platelet incubation with acetylsalicylic acid. At the same time, platelets of patients with aspirin-sensitive asthma did not respond to addition of platelet activating factor, but after incubation with acetylsalicylic acid, an intensive burst of chemiluminescence was detected with a maximum in 5-10 sec after the addition of a platelet-activating factor. In patients with bronchial asthma tolerant to aspirin, platelet activation factor did not induce chemiluminescence irrespective of incubation with acetylsalicylic acid.

Drug-Free Platelets Can Act as Seeds for Aggregate Formation During Antiplatelet Therapy

PubMed Central

Hoefer, Thomas; Armstrong, Paul C.; Finsterbusch, Michaela; Chan, Melissa V.; Kirkby, Nicholas S.

2015-01-01

Objective— Reduced antiplatelet drug efficacy occurs in conditions of increased platelet turnover, associated with increased proportions of drug-free, that is, uninhibited, platelets. Here, we detail mechanisms by which drug-free platelets promote platelet aggregation in the face of standard antiplatelet therapy. Approach and Results— To model standard antiplatelet therapy, platelets were treated in vitro with aspirin, the P2Y12 receptor blocker prasugrel active metabolite, or aspirin plus prasugrel active metabolite. Different proportions of uninhibited platelets were then introduced. Light transmission aggregometry analysis demonstrated clear positive associations between proportions of drug-free platelets and percentage platelet aggregation in response to a range of platelet agonists. Using differential platelet labeling coupled with advanced flow cytometry and confocal imaging we found aggregates formed in mixtures of aspirin-inhibited platelets together with drug-free platelets were characterized by intermingled platelet populations. This distribution is in accordance with the ability of drug-free platelets to generate thromboxane A2 and so drive secondary platelet activation. Conversely, aggregates formed in mixtures of prasugrel active metabolite–inhibited or aspirin plus prasugrel active metabolite–inhibited platelets together with drug-free platelets were characterized by distinct cores of drug-free platelets. This distribution is consistent with the ability of drug-free platelets to respond to the secondary activator ADP. Conclusions— These experiments are the first to image the interactions of inhibited and uninhibited platelets in the formation of platelet aggregates. They demonstrate that a general population of platelets can contain subpopulations that respond strikingly differently to overall stimulation of the population and so act as the seed for platelet aggregation. PMID:26272940

Circulating Myeloid‐Related Protein–8/14 is Related to Thromboxane‐Dependent Platelet Activation in Patients With Acute Coronary Syndrome, With and Without Ongoing Low‐Dose Aspirin Treatment

PubMed Central

Santilli, Francesca; Paloscia, Leonardo; Liani, Rossella; Di Nicola, Marta; Di Marco, Massimo; Lattanzio, Stefano; La Barba, Sara; Pascale, Silvia; Mascellanti, Marco; Davì, Giovanni

2014-01-01

Background Platelet activation is involved in acute coronary syndromes (ACS). Incomplete suppression by low‐dose aspirin treatment of thromboxane (TX) metabolite excretion (urinary 11‐dehydro‐TXB2) is predictive of vascular events in high‐risk patients. Myeloid‐related protein (MRP)‐8/14 is a heterodimer secreted on activation of platelets, monocytes, and neutrophils, regulating inflammation and predicting cardiovascular events. Among platelet transcripts, MRP‐14 has emerged as a powerful predictor of ACS. Methods and Results We enrolled 68 stable ischemic heart disease (IHD) and 63 ACS patients, undergoing coronary angiography, to evaluate whether MRP‐8/14 release in the circulation is related to TX‐dependent platelet activation in ACS and IHD patients and to residual TX biosynthesis in low‐dose aspirin–treated ACS patients. In ACS patients, plasma MRP‐8/14 and urinary 11‐dehydro‐TXB2 levels were linearly correlated (r=0.651, P<0.001) but significantly higher than those in IHD patients (P=0.012, P=0.044) only among subjects not receiving aspirin. In aspirin‐treated ACS patients, MRP‐8/14 and 11‐dehydro‐TXB2 were lower versus those not receiving aspirin (P<0.001) and still significantly correlated (r=0.528, P<0.001). Higher 11‐dehydro‐TXB2 significantly predicted higher MRP‐8/14 in both all ACS patients and ACS receiving aspirin (P<0.001, adj R2=0.463 and adj R2=0.497) after multivariable adjustment. Conversely, plasma MRP‐8/14 (P<0.001) and higher urinary 8‐iso‐prostaglandin F2α (P=0.050) levels were significant predictors of residual, on‐aspirin, TX biosynthesis in ACS (adjusted R2=0.384). Conclusions Circulating MRP‐8/14 is associated with TX‐dependent platelet activation in ACS, even during low‐dose aspirin treatment, suggesting a contribution of residual TX to MRP‐8/14 shedding, which may further amplify platelet activation. Circulating MRP‐8/14 may be a target to test different antiplatelet strategies in ACS. PMID:25037196

Synthesis of sFlt-1 by platelet-monocyte aggregates contributes to the pathogenesis of preeclampsia

PubMed Central

Major, Heather D.; Cambell, Robert A.; Silver, Robert M.; Branch, D. Ware; Weyrich, Andrew S.

2014-01-01

Objective Soluble fms-like tyrosine kinase (sFlt-1) is an important mediator in the pathogenesis of preeclampsia. We sought to determine if platelet-monocyte aggregates (PMAs) produced sFlt-1 and if PMAs contributed to sFlt-1 production in preeclampsia. Study Design Case-control study of sFlt-1 release from PMAs using blood samples from women with preeclampsia matched by gestational age to pregnant controls. A third group of nonpregnant, reproductive-age women comprised an additional control group. Experiments were also performed using blood from non-pregnant women to elucidate if inducing PMAs could stimulate sFlt-1 production, and if so, to determine the necessary receptors and pathways. Results Women with preeclampsia had increased total Flt-1 concentrations in platelets and monocytes at baseline compared to pregnant controls (25 vs. 10 pg/ml, p=0.0003). sFlt-1 production was elicited from monocytes incubated with thrombin-activated platelets from non-pregnant women. sFlt-1 production was regulated at the transcriptional level by p38 and NF-κB dependent pathways. Conclusion Activated platelets in preeclampsia bind monocytes to generate sFlt-1. PMAs are a previously unrecognized source of sFlt-1 that may contribute to endothelial dysfunction and systemic inflammation commonly observed in preeclampsia. PMID:24440566

Release of extracellular DNA influences renal ischemia reperfusion injury by platelet activation and formation of neutrophil extracellular traps.

PubMed

Jansen, Marcel P B; Emal, Diba; Teske, Gwendoline J D; Dessing, Mark C; Florquin, Sandrine; Roelofs, Joris J T H

2017-02-01

Acute kidney injury is often the result of ischemia reperfusion injury, which leads to activation of coagulation and inflammation, resulting in necrosis of renal tubular epithelial cells. Platelets play a central role in coagulation and inflammatory processes, and it has been shown that platelet activation exacerbates acute kidney injury. However, the mechanism of platelet activation during ischemia reperfusion injury and how platelet activation leads to tissue injury are largely unknown. Here we found that renal ischemia reperfusion injury in mice leads to increased platelet activation in immediate proximity of necrotic cell casts. Furthermore, platelet inhibition by clopidogrel decreased cell necrosis and inflammation, indicating a link between platelet activation and renal tissue damage. Necrotic tubular epithelial cells were found to release extracellular DNA, which, in turn, activated platelets, leading to platelet-granulocyte interaction and formation of neutrophil extracellular traps ex vivo. Renal ischemia reperfusion injury resulted in increased DNA-platelet and DNA-platelet-granulocyte colocalization in tissue and elevated levels of circulating extracellular DNA and platelet factor 4 in mice. After renal ischemia reperfusion injury, neutrophil extracellular traps were formed within renal tissue, which decreased when mice were treated with the platelet inhibitor clopidogrel. Thus, during renal ischemia reperfusion injury, necrotic cell-derived DNA leads to platelet activation, platelet-granulocyte interaction, and subsequent neutrophil extracellular trap formation, leading to renal inflammation and further increase in tissue injury. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays.

PubMed

Pieters, Marlien; Barnard, Sunelle A; Loots, Du Toit; Rijken, Dingeman C

2017-01-01

Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g), platelet-containing (352 g) and platelet-rich plasma (200 g) were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation). Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin) showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly through release of latent plasminogen activator inhibitor-1 with limited effects on plasminogen activator inhibitor-1 activity, tissue plasminogen activator/plasminogen activator inhibitor-1 complex or plasma clot lysis time. Platelets may however also have functional effects on plasma fibrinolytic potential in the presence of high platelet counts, such as in platelet-rich plasma.

Developmental endothelial locus-1 modulates platelet-monocyte interactions and instant blood-mediated inflammatory reaction in islet transplantation.

PubMed

Kourtzelis, Ioannis; Kotlabova, Klara; Lim, Jong-Hyung; Mitroulis, Ioannis; Ferreira, Anaisa; Chen, Lan-Sun; Gercken, Bettina; Steffen, Anja; Kemter, Elisabeth; Klotzsche-von Ameln, Anne; Waskow, Claudia; Hosur, Kavita; Chatzigeorgiou, Antonios; Ludwig, Barbara; Wolf, Eckhard; Hajishengallis, George; Chavakis, Triantafyllos

2016-04-01

Platelet-monocyte interactions are strongly implicated in thrombo-inflammatory injury by actively contributing to intravascular inflammation, leukocyte recruitment to inflamed sites, and the amplification of the procoagulant response. Instant blood-mediated inflammatory reaction (IBMIR) represents thrombo-inflammatory injury elicited upon pancreatic islet transplantation (islet-Tx), thereby dramatically affecting transplant survival and function. Developmental endothelial locus-1 (Del-1) is a functionally versatile endothelial cell-derived homeostatic factor with anti-inflammatory properties, but its potential role in IBMIR has not been previously addressed. Here, we establish Del-1 as a novel inhibitor of IBMIR using a whole blood-islet model and a syngeneic murine transplantation model. Indeed, Del-1 pre-treatment of blood before addition of islets diminished coagulation activation and islet damage as assessed by C-peptide release. Consistently, intraportal islet-Tx in transgenic mice with endothelial cell-specific overexpression of Del-1 resulted in a marked decrease of monocytes and platelet-monocyte aggregates in the transplanted tissues, relative to those in wild-type recipients. Mechanistically, Del-1 decreased platelet-monocyte aggregate formation, by specifically blocking the interaction between monocyte Mac-1-integrin and platelet GPIb. Our findings reveal a hitherto unknown role of Del-1 in the regulation of platelet-monocyte interplay and the subsequent heterotypic aggregate formation in the context of IBMIR. Therefore, Del-1 may represent a novel approach to prevent or mitigate the adverse reactions mediated through thrombo-inflammatory pathways in islet-Tx and perhaps other inflammatory disorders involving platelet-leukocyte aggregate formation.

Platelet size and density affect shear-induced thrombus formation in tortuous arterioles

NASA Astrophysics Data System (ADS)

Chesnutt, Jennifer K. W.; Han, Hai-Chao

2013-10-01

Thrombosis accounts for 80% of deaths in patients with diabetes mellitus. Diabetic patients demonstrate tortuous microvessels and larger than normal platelets. Large platelets are associated with increased platelet activation and thrombosis, but the physical effects of large platelets in the microscale processes of thrombus formation are not clear. Therefore, the objective of this study was to determine the physical effects of mean platelet volume (MPV), mean platelet density (MPD) and vessel tortuosity on platelet activation and thrombus formation in tortuous arterioles. A computational model of the transport, shear-induced activation, collision, adhesion and aggregation of individual platelets was used to simulate platelet interactions and thrombus formation in tortuous arterioles. Our results showed that an increase in MPV resulted in a larger number of activated platelets, though MPD and level of tortuosity made little difference on platelet activation. Platelets with normal MPD yielded the lowest amount of mural thrombus. With platelets of normal MPD, the amount of mural thrombus decreased with increasing level of tortuosity but did not have a simple monotonic relationship with MPV. The physical mechanisms associated with MPV, MPD and arteriole tortuosity play important roles in platelet activation and thrombus formation.

Platelet Activation in Human Immunodeficiency Virus Type-1 Patients Is Not Altered with Cocaine Abuse

PubMed Central

Kiebala, Michelle; Singh, Meera V.; Piepenbrink, Michael S.; Qiu, Xing; Kobie, James J.; Maggirwar, Sanjay B.

2015-01-01

Recent work has indicated that platelets, which are anucleate blood cells, significantly contribute to inflammatory disorders. Importantly, platelets also likely contribute to various inflammatory secondary disorders that are increasingly associated with Human Immunodeficiency Virus Type-1 (HIV) infection including neurological impairments and cardiovascular complications. Indeed, HIV infection is often associated with increased levels of platelet activators. Additionally, cocaine, a drug commonly abused by HIV-infected individuals, leads to increased platelet activation in humans. Considering that orchestrated signaling mechanisms are essential for platelet activation, and that nuclear factor-kappa B (NF-κB) inhibitors can alter platelet function, the role of NF-κB signaling in platelet activation during HIV infection warrants further investigation. Here we tested the hypothesis that inhibitory kappa B kinase complex (IKK) activation would be central for platelet activation induced by HIV and cocaine. Whole blood from HIV-positive and HIV-negative individuals, with or without cocaine abuse was used to assess platelet activation via flow cytometry whereas IKK activation was analyzed by performing immunoblotting and in vitro kinase assays. We demonstrate that increased platelet activation in HIV patients, as measured by CD62P expression, is not altered with reported cocaine use. Furthermore, cocaine and HIV do not activate platelets in whole blood when treated ex vivo. Finally, HIV-induced platelet activation does not involve the NF-κB signaling intermediate, IKKβ. Platelet activation in HIV patients is not altered with cocaine abuse. These results support the notion that non-IKK targeting approaches will be better suited for the treatment of HIV-associated inflammatory disorders. PMID:26076359

Platelet-rich fibrin matrix improves wound angiogenesis via inducing endothelial cell proliferation.

PubMed

Roy, Sashwati; Driggs, Jason; Elgharably, Haytham; Biswas, Sabyasachi; Findley, Muna; Khanna, Savita; Gnyawali, Urmila; Bergdall, Valerie K; Sen, Chandan K

2011-11-01

The economic, social, and public health burden of chronic ulcers and other compromised wounds is enormous and rapidly increasing with the aging population. The growth factors derived from platelets play an important role in tissue remodeling including neovascularization. Platelet-rich plasma (PRP) has been utilized and studied for the last four decades. Platelet gel and fibrin sealant, derived from PRP mixed with thrombin and calcium chloride, have been exogenously applied to tissues to promote wound healing, bone growth, hemostasis, and tissue sealing. In this study, we first characterized recovery and viability of as well as growth factor release from platelets in a novel preparation of platelet gel and fibrin matrix, namely platelet-rich fibrin matrix (PRFM). Next, the effect of PRFM application in a delayed model of ischemic wound angiogenesis was investigated. The study, for the first time, shows the kinetics of the viability of platelet-embedded fibrin matrix. A slow and steady release of growth factors from PRFM was observed. The vascular endothelial growth factor released from PRFM was primarily responsible for endothelial mitogenic response via extracellular signal-regulated protein kinase activation pathway. Finally, this preparation of PRFM effectively induced endothelial cell proliferation and improved wound angiogenesis in chronic wounds, providing evidence of probable mechanisms of action of PRFM in healing of chronic ulcers. 2011 by the Wound Healing Society.

Phloretin suppresses thrombin-mediated leukocyte-platelet-endothelial interactions.

PubMed

Kim, Min Soo; Park, Sin-Hye; Han, Seon-Young; Kim, Yun-Ho; Lee, Eun-Jung; Yoon Park, Jung Han; Kang, Young-Hee

2014-04-01

Thrombin playing a pivotal role in coagulation cascade may influence the onset and progression of atherosclerosis as a pro-inflammatory mediator. This study investigated whether phloretin found in apple tree leaves, severed a linkage between thrombosis and atherosclerosis by thrombin. Human endothelial cells were pre-treated with 1-20 μM phloretin and stimulated with 10 U/mL thrombin. Phloretin attenuated adhesion of THP-1 monocytes and platelets to thrombin-inflamed endothelial cells with concurrent inhibition of protease-activated receptor (PAR-1) induction. The thrombin induction of endothelial CD40, endothelial integrin β3 and P-selectin, and monocytic CD40L was dampened by phloretin. Additionally, phloretin inhibited monocyte secretion of MCP-1, IL-6 and IL-8 responsible for pro-inflammatory activity of thrombin inducing endothelial CD40. The monocyte COX-2 induction and PGE2 secretion due to thrombin were down-regulated by phloretin, deterring endothelial CD40 expression. Thrombin promoted production of PAI-1 and tissue factor in monocytes was attenuated by phloretin through blocking PAR-1 and CD40. Thrombin up-regulated the induction of endothelial connective tissue growth factor independent of PAR-1 activation, which was reversed by phloretin. Phloretin disturbed tethering and stable adhesion of monocytes and platelets onto endothelium during increased thrombosis by thrombin. Phloretin would be a potent agent preventing thrombosis and atherosclerosis. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phosphatidylserine-mediated platelet clearance by endothelium decreases platelet aggregates and procoagulant activity in sepsis.

PubMed

Ma, Ruishuang; Xie, Rui; Yu, Chengyuan; Si, Yu; Wu, Xiaoming; Zhao, Lu; Yao, Zhipeng; Fang, Shaohong; Chen, He; Novakovic, Valerie; Gao, Chunyan; Kou, Junjie; Bi, Yayan; Thatte, Hemant S; Yu, Bo; Yang, Shufen; Zhou, Jin; Shi, Jialan

2017-07-10

The mechanisms that eliminate activated platelets in inflammation-induced disseminated intravascular coagulation (DIC) in micro-capillary circulation are poorly understood. This study explored an alternate pathway for platelet disposal mediated by endothelial cells (ECs) through phosphatidylserine (PS) and examined the effect of platelet clearance on procoagulant activity (PCA) in sepsis. Platelets in septic patients demonstrated increased levels of surface activation markers and apoptotic vesicle formation, and also formed aggregates with leukocytes. Activated platelets adhered were and ultimately digested by ECs in vivo and in vitro. Blocking PS on platelets or αvβ3 integrin on ECs attenuated platelet clearance resulting in increased platelet count in a mouse model of sepsis. Furthermore, platelet removal by ECs resulted in a corresponding decrease in platelet-leukocyte complex formation and markedly reduced generation of factor Xa and thrombin on platelets. Pretreatment with lactadherin significantly increased phagocytosis of platelets by approximately 2-fold, diminished PCA by 70%, prolonged coagulation time, and attenuated fibrin formation by 50%. Our results suggest that PS-mediated clearance of activated platelets by the endothelium results in an anti-inflammatory, anticoagulant, and antithrombotic effect that contribute to maintaining platelet homeostasis during acute inflammation. These results suggest a new therapeutic target for impeding the development of DIC.

Platelet Activation by Streptococcus pyogenes Leads to Entrapment in Platelet Aggregates, from Which Bacteria Subsequently Escape

PubMed Central

Svensson, Lisbeth; Baumgarten, Maria; Mörgelin, Matthias

2014-01-01

Platelet activation and aggregation have been reported to occur in response to a number of Gram-positive pathogens. Here, we show that platelet aggregates induced by Streptococcus pyogenes were unstable and that viable bacteria escaped from the aggregates over time. This was not due to differential activation in response to the bacteria compared with physiological activators. All the bacterial isolates induced significant platelet activation, including integrin activation and alpha and dense-granule release, at levels equivalent to those induced by potent physiological platelet activators that induced stable aggregates. The ability to escape the aggregates and to resist the antibacterial effects of platelets was dependent on active protein synthesis by the bacteria within the aggregate. We conclude that S. pyogenes bacteria can temporarily cover themselves with activated platelets, and we propose that this may facilitate survival of the bacteria in the presence of platelets. PMID:25069984

LDL oxidation by platelets propagates platelet activation via an oxidative stress-mediated mechanism.

PubMed

Carnevale, Roberto; Bartimoccia, Simona; Nocella, Cristina; Di Santo, Serena; Loffredo, Lorenzo; Illuminati, Giulio; Lombardi, Elisabetta; Boz, Valentina; Del Ben, Maria; De Marco, Luigi; Pignatelli, Pasquale; Violi, Francesco

2014-11-01

Platelets generate oxidized LDL (ox-LDL) via NOX2-derived oxidative stress. We investigated if once generated by activated platelets ox-LDL can propagate platelet activation. Experiments were performed in platelets from healthy subjects (HS), hyper-cholesterolemic patients and patients with NOX2 hereditary deficiency. Agonist-stimulated platelets from HS added with LDL were associated with a dose-dependent increase of reactive oxidant species and ox-LDL. Agonist-stimulated platelets from HS added with a fixed dose of LDL (57.14 μmol/L) or added with homogenized human atherosclerotic plaque showed enhanced ox-LDL formation (approximately +50% and +30% respectively), which was lowered by a NOX2 inhibitor (approximately -35% and -25% respectively). Compared to HS, ox-LDL production was more pronounced in agonist-stimulated platelet rich plasma (PRP) from hyper-cholesterolemic patients but was almost absent in PRP from NOX2-deficient patients. Platelet aggregation and 8-iso-PGF2α-ΙΙΙ formation increased in LDL-treated washed platelets (+42% and +53% respectively) and PRP (+31% and +53% respectively). Also, LDL enhanced platelet-dependent thrombosis at arterial shear rate (+33%) but did not affect platelet activation in NOX2-deficient patients. Platelet activation by LDL was significantly inhibited by CD36 or LOX1 blocking peptides, two ox-LDL receptor antagonists, or by a NOX2 inhibitor. LDL-added platelets showed increased p38MAPK (+59%) and PKC (+51%) phosphorylation, p47(phox) translocation to platelet membrane (+34%) and NOX2 activation (+30%), which were inhibited by ox-LDL receptor antagonists. Platelets oxidize LDL, which in turn amplify platelet activation via specific ox-LDL receptors; both effects are mediated by NOX2 activation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets.

PubMed

Begonja, Antonija Jurak; Pluthero, Fred G; Suphamungmee, Worawit; Giannini, Silvia; Christensen, Hilary; Leung, Richard; Lo, Richard W; Nakamura, Fumihiko; Lehman, William; Plomann, Markus; Hoffmeister, Karin M; Kahr, Walter H A; Hartwig, John H; Falet, Hervé

2015-07-02

Bin-Amphiphysin-Rvs (BAR) and Fes-CIP4 homology BAR (F-BAR) proteins generate tubular membrane invaginations reminiscent of the megakaryocyte (MK) demarcation membrane system (DMS), which provides membranes necessary for future platelets. The F-BAR protein PACSIN2 is one of the most abundant BAR/F-BAR proteins in platelets and the only one reported to interact with the cytoskeletal and scaffold protein filamin A (FlnA), an essential regulator of platelet formation and function. The FlnA-PACSIN2 interaction was therefore investigated in MKs and platelets. PACSIN2 associated with FlnA in human platelets. The interaction required FlnA immunoglobulin-like repeat 20 and the tip of PACSIN2 F-BAR domain and enhanced PACSIN2 F-BAR domain membrane tubulation in vitro. Most human and wild-type mouse platelets had 1 to 2 distinct PACSIN2 foci associated with cell membrane GPIbα, whereas Flna-null platelets had 0 to 4 or more foci. Endogenous PACSIN2 and transfected enhanced green fluorescent protein-PACSIN2 were concentrated in midstage wild-type mouse MKs in a well-defined invagination of the plasma membrane reminiscent of the initiating DMS and dispersed in the absence of FlnA binding. The DMS appeared less well defined, and platelet territories were not readily visualized in Flna-null MKs. We conclude that the FlnA-PACSIN2 interaction regulates membrane tubulation in MKs and platelets and likely contributes to DMS formation. © 2015 by The American Society of Hematology.

Platelet factor XIII increases the fibrinolytic resistance of platelet-rich clots by accelerating the crosslinking of alpha 2-antiplasmin to fibrin

NASA Technical Reports Server (NTRS)

Reed, G. L.; Matsueda, G. R.; Haber, E.

1992-01-01

Platelet clots resist fibrinolysis by plasminogen activators. We hypothesized that platelet factor XIII may enhance the fibrinolytic resistance of platelet-rich clots by catalyzing the crosslinking of alpha 2-antiplasmin (alpha 2AP) to fibrin. Analysis of plasma clot structure by polyacrylamide gel electrophoresis and immunoblotting revealed accelerated alpha 2AP-fibrin crosslinking in platelet-rich compared with platelet-depleted plasma clots. A similar study of clots formed with purified fibrinogen (depleted of factor XIII activity), isolated platelets, and specific factor XIII inhibitors indicated that this accelerated crosslinking was due to the catalytic activity of platelet factor XIII. Moreover, when washed platelets were aggregated by thrombin, there was evidence of platelet factor XIII-mediated crosslinking between platelet alpha 2AP and platelet fibrin(ogen). Specific inhibition (by a monoclonal antibody) of the alpha 2AP associated with washed platelet aggregates accelerated the fibrinolysis of the platelet aggregate. Thus in platelet-rich plasma clots, and in thrombin-induced platelet aggregates, platelet factor XIII actively formed alpha 2AP-fibrin crosslinks, which appeared to enhance the resistance of platelet-rich clots to fibrinolysis.

Computational modelling of biomaterial surface interactions with blood platelets and osteoblastic cells for the prediction of contact osteogenesis.

PubMed

Amor, N; Geris, L; Vander Sloten, J; Van Oosterwyck, H

2011-02-01

Surface microroughness can induce contact osteogenesis (bone formation initiated at the implant surface) around oral implants, which may result from different mechanisms, such as blood platelet-biomaterial interactions and/or interaction with (pre-)osteoblast cells. We have developed a computational model of implant endosseous healing that takes into account these interactions. We hypothesized that the initial attachment and growth factor release from activated platelets is crucial in achieving contact osteogenesis. In order to investigate this, a computational model was applied to an animal experiment [7] that looked at the effect of surface microroughness on endosseous healing. Surface-specific model parameters were implemented based on in vitro data (Lincks et al. Biomaterials 1998;19:2219-32). The predicted spatio-temporal patterns of bone formation correlated with the histological data. It was found that contact osteogenesis could not be predicted if only the osteogenic response of cells was up-regulated by surface microroughness. This could only be achieved if platelet-biomaterial interactions were sufficiently up-regulated as well. These results confirmed our hypothesis and demonstrate the added value of the computational model to study the importance of surface-mediated events for peri-implant endosseous healing. Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Structure-Antiplatelet Activity Relationships of Novel Ruthenium (II) Complexes: Investigation of Its Molecular Targets.

PubMed

Hsia, Chih-Hsuan; Jayakumar, Thanasekaran; Sheu, Joen-Rong; Tsao, Shin-Yi; Velusamy, Marappan; Hsia, Chih-Wei; Chou, Duen-Suey; Chang, Chao-Chien; Chung, Chi-Li; Khamrang, Themmila; Lin, Kao-Chang

2018-02-22

The regulation of platelet function by pharmacological agents that modulate platelet signaling has proven to be a positive approach to the prevention of thrombosis. Ruthenium complexes are fascinating for the development of new drugs, as they possess numerous chemical and biological properties. The present study aims to evaluate the structure-activity relationship (SAR) of newly synthesized ruthenium (II) complexes, TQ-1, TQ-2 and TQ-3 in agonists-induced washed human platelets. Silica gel column chromatography, aggregometry, immunoblotting, NMR, and X-ray analyses were performed in this study. Of the three tested compounds, TQ-3 showed a concentration (1-5 μM) dependent inhibitory effect on platelet aggregation induced by collagen (1 μg/mL) and thrombin (0.01 U/mL) in washed human platelets; however, TQ-1 and TQ-2 had no response even at 250 μM of collagen and thrombin-induced aggregation. TQ-3 was effective with inhibiting collagen-induced ATP release, calcium mobilization ([Ca 2+ ]i) and P-selectin expression without cytotoxicity. Moreover, TQ-3 significantly abolished collagen-induced Lyn-Fyn-Syk, Akt-JNK and p38 mitogen-activated protein kinases (p38 MAPKs) phosphorylation. The compound TQ-3 containing an electron donating amino group with two phenyl groups of the quinoline core could be accounted for by its hydrophobicity and this nature might be the reason for the noted antiplatelet effects of TQ-3. The present results provide a molecular basis for the inhibition by TQ-3 in collagen-induced platelet aggregation, through the suppression of multiple machineries of the signaling pathway. These results may suggest that TQ-3 can be considered a potential agent for the treatment of vascular diseases.

Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nygaard, Gyrid; Department of Biomedicine, University of Bergen, Bergen; Herfindal, Lars

Highlights: • We investigated the impact of cyclic nucleotide analogues on platelet activation. • Different time dependence were found for inhibition of platelet activation. • Additive effect was found using PKA- and PKG-activating analogues. • Our results may explain some of the discrepancies reported for cNMP signalling. - Abstract: In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigatedmore » whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.« less

Activated Platelets Induce Endothelial Cell Activation via an Interleukin-1β Pathway in Systemic Lupus Erythematosus.

PubMed

Nhek, Sokha; Clancy, Robert; Lee, Kristen A; Allen, Nicole M; Barrett, Tessa J; Marcantoni, Emanuela; Nwaukoni, Janet; Rasmussen, Sara; Rubin, Maya; Newman, Jonathan D; Buyon, Jill P; Berger, Jeffrey S

2017-04-01

Systemic lupus erythematosus (SLE) is associated with the premature development of cardiovascular disease. The platelet-endothelium interaction is important in the pathogenesis of cardiovascular disease. In this study, we investigated the platelet phenotype from patients with SLE and matched controls, and their effect on endothelial cells. Platelet aggregability was measured in 54 SLE subjects off antiplatelet therapy (mean age 40.1±12.8 years; 82% female; 37% white) with age- and sex-matched controls. Platelets were coincubated with human umbilical vein endothelial cells (HUVECs) and changes to gene expression assessed by an RNA array and quantitative reverse transcription polymerase chain reaction. SLE disease activity index ranged from 0 to 22 (mean 5.1±3.9). Compared with controls, patients with SLE had significantly increased monocyte and leukocyte-platelet aggregation and platelet aggregation in response to submaximal agonist stimulation. An agnostic microarray of HUVECs cocultured with SLE platelets found a platelet-mediated effect on endothelial gene pathways involved in cell activation. Sera from SLE versus control subjects significantly increased (1) activation of control platelets; (2) platelet adhesion to HUVECs; (3) platelet-induced HUVEC gene expression of interleukin-8, and intercellular adhesion molecule 1; and (4) proinflammatory gene expression in HUVECs, mediated by interleukin-1β-dependent pathway. Incubation of SLE-activated platelets with an interleukin-1β-neutralizing antibody or HUVECs pretreated with interleukin-1 receptor antibodies attenuated the platelet-mediated activation of endothelial cells. Platelet activity measurements and subsequent interleukin-1β-dependent activation of the endothelium are increased in subjects with SLE. Platelet-endothelial interactions may play a role in the pathogenesis of cardiovascular disease in patients with SLE. © 2017 American Heart Association, Inc.

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue.

PubMed

Trugilho, Monique Ramos de Oliveira; Hottz, Eugenio Damaceno; Brunoro, Giselle Villa Flor; Teixeira-Ferreira, André; Carvalho, Paulo Costa; Salazar, Gustavo Adolfo; Zimmerman, Guy A; Bozza, Fernando A; Bozza, Patrícia T; Perales, Jonas

2017-05-01

Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue, and sheds light on new mechanisms of platelet activation and platelet-mediated immune and inflammatory responses.

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue

PubMed Central

Teixeira-Ferreira, André; Carvalho, Paulo Costa; Salazar, Gustavo Adolfo; Zimmerman, Guy A.; Perales, Jonas

2017-01-01

Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue, and sheds light on new mechanisms of platelet activation and platelet-mediated immune and inflammatory responses. PMID:28542641

Effect of serotonin on platelet function in cocaine exposed blood

PubMed Central

Ziu, Endrit; Hadden, Coedy; Li, Yicong; Lowery, Curtis Lee; Singh, Preeti; Ucer, Serra S.; Mercado, Charles P.; Gu, Howard H.; Kilic, Fusun

2014-01-01

5-hydroxytryptamine (5-HT) reuptake inhibitors counteract the pro-thrombotic effect of elevated plasma 5-HT by down-regulating the 5-HT uptake rates of platelets. Cocaine also down-regulates the platelet 5-HT uptake rates but in contrast, the platelets of cocaine-injected mice show a much higher aggregation rate than the platelets of control mice. To examine the involvement of plasma 5-HT in cocaine-mediated platelet aggregation, we studied the function of platelets isolated from wild-type and transgenic, peripheral 5-HT knock-out (TPH1-KO) mice, and cocaine-insensitive dopamine transporter knock in (DAT-KI) mice. In cocaine-injected mice compared to the control mice, the plasma 5-HT level as well as the surface level of P-selectin was elevated; in vitro platelet aggregation in the presence of type I fibrillar collagen was enhanced. However, cocaine injection lowered the 5-HT uptake rates of platelets and increased the plasma 5-HT levels of the DAT-KI mice but did not change their platelets aggregation rates further which are already hyper-reactive. Furthermore, the in vitro studies supporting these in vivo findings suggest that cocaine mimics the effect of elevated plasma 5-HT level on platelets and in 5-HT receptor- and transporter-dependent pathways in a two-step process propagates platelet aggregation by an additive effect of 5-HT and nonserotonergic catecholamine. PMID:25091505

Platelet activation suppresses HIV-1 infection of T cells

PubMed Central

2013-01-01

Background Platelets, anucleate cell fragments abundant in human blood, can capture HIV-1 and platelet counts have been associated with viral load and disease progression. However, the impact of platelets on HIV-1 infection of T cells is unclear. Results We found that platelets suppress HIV-1 spread in co-cultured T cells in a concentration-dependent manner. Platelets containing granules inhibited HIV-1 spread in T cells more efficiently than degranulated platelets, indicating that the granule content might exert antiviral activity. Indeed, supernatants from activated and thus degranulated platelets suppressed HIV-1 infection. Infection was inhibited at the stage of host cell entry and inhibition was independent of the viral strain or coreceptor tropism. In contrast, blockade of HIV-2 and SIV entry was less efficient. The chemokine CXCL4, a major component of platelet granules, blocked HIV-1 entry and neutralization of CXCL4 in platelet supernatants largely abrogated their anti-HIV-1 activity. Conclusions Release of CXCL4 by activated platelets inhibits HIV-1 infection of adjacent T cells at the stage of virus entry. The inhibitory activity of platelet-derived CXCL4 suggests a role of platelets in the defense against infection by HIV-1 and potentially other pathogens. PMID:23634812

Platelet activation suppresses HIV-1 infection of T cells.

PubMed

Solomon Tsegaye, Theodros; Gnirß, Kerstin; Rahe-Meyer, Niels; Kiene, Miriam; Krämer-Kühl, Annika; Behrens, Georg; Münch, Jan; Pöhlmann, Stefan

2013-05-01

Platelets, anucleate cell fragments abundant in human blood, can capture HIV-1 and platelet counts have been associated with viral load and disease progression. However, the impact of platelets on HIV-1 infection of T cells is unclear. We found that platelets suppress HIV-1 spread in co-cultured T cells in a concentration-dependent manner. Platelets containing granules inhibited HIV-1 spread in T cells more efficiently than degranulated platelets, indicating that the granule content might exert antiviral activity. Indeed, supernatants from activated and thus degranulated platelets suppressed HIV-1 infection. Infection was inhibited at the stage of host cell entry and inhibition was independent of the viral strain or coreceptor tropism. In contrast, blockade of HIV-2 and SIV entry was less efficient. The chemokine CXCL4, a major component of platelet granules, blocked HIV-1 entry and neutralization of CXCL4 in platelet supernatants largely abrogated their anti-HIV-1 activity. Release of CXCL4 by activated platelets inhibits HIV-1 infection of adjacent T cells at the stage of virus entry. The inhibitory activity of platelet-derived CXCL4 suggests a role of platelets in the defense against infection by HIV-1 and potentially other pathogens.

Palladin is involved in platelet activation and arterial thrombosis.

PubMed

Chen, Xuejiao; Fan, Xuemei; Tan, Juan; Shi, Panlai; Wang, Xiyi; Wang, Jinjin; Kuang, Ying; Fei, Jian; Liu, Junling; Dang, Suying; Wang, Zhugang

2017-01-01

The dynamics of actin cytoskeleton have been shown to play a critical role during platelet activation. Palladin is an actin-associated protein, serving as a cytoskeleton scaffold to bundle actin fibers and actin cross linker. The functional role of palladin on platelet activation has not been investigated. Here, we characterized heterozygous palladin knockout (palladin +/- ) mice to elucidate the platelet-related functions of palladin. The results showed that palladin was expressed in platelets and moderate palladin deficiency accelerated hemostasis and arterial thrombosis. The aggregation of palladin +/- platelets was increased in response to low levels of thrombin, U46619, and collagen. We also observed enhanced spreading of palladin +/- platelets on immobilized fibrinogen (Fg) and increased rate of clot retraction in platelet-rich plasma (PRP) containing palladin +/- platelets. Furthermore, the activation of the small GTPase Rac1 and Cdc42, which is associated with cytoskeletal dynamics and platelet activation signalings, was increased in the spreading and aggregating palladin +/- platelets compared to that in wild type platelets. Taken together, these findings indicated that palladin is involved in platelet activation and arterial thrombosis, implying a potent role of palladin in pathophysiology of thrombotic diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

Rare platelet GPCR variants: what can we learn?

PubMed

Nisar, S P; Jones, M L; Cunningham, M R; Mumford, A D; Mundell, S J

2015-07-01

Platelet-expressed GPCRs are critical regulators of platelet function. Pharmacological blockade of these receptors forms a powerful therapeutic tool in the treatment and prevention of arterial thrombosis associated with coronary atherosclerosis and ischaemic stroke. However, anti-thrombotic drug therapy is associated with high inter-patient variability in therapeutic response and adverse bleeding side effects. In order to optimize the use of existing anti-platelet drugs and to develop new therapies, more detailed knowledge is required relating to the molecular mechanisms that regulate GPCR and therefore platelet function. One approach has been to identify rare, function-disrupting mutations within key platelet proteins in patients with bleeding disorders. In this review, we describe how an integrated functional genomics strategy has contributed important structure-function information about platelet GPCRs with specific emphasis upon purinergic and thromboxane A2 receptors. We also discuss the potential implications these findings have for pharmacotherapy and for understanding the molecular basis of mild bleeding disorders. © 2014 The British Pharmacological Society.

Responsiveness of platelets during storage studied with flow cytometry--formation of platelet subpopulations and LAMP-1 as new markers for the platelet storage lesion.

PubMed

Södergren, A L; Tynngård, N; Berlin, G; Ramström, S

2016-02-01

Storage lesions may prevent transfused platelets to respond to agonists and arrest bleeding. The aim of this study was to evaluate and quantify the capacity of platelet activation during storage using flow cytometry and new markers of platelet activation. Activation responses of platelets prepared by apheresis were measured on days 1, 5, 7 and 12. In addition, comparisons were made for platelet concentrates stored until swirling was affected. Lysosome-associated membrane protein-1 (LAMP-1), P-selectin and phosphatidylserine (PS) exposure were assessed by flow cytometry on platelets in different subpopulations in resting state or following stimulation with platelet agonists (cross-linked collagen-related peptide (CRP-XL), PAR1- and PAR4-activating peptides). The ability to form subpopulations upon activation was significantly decreased already at day 5 for some agonist combinations. The agonist-induced exposure of PS and LAMP-1 also gradually decreased with time. Spontaneous exposure of P-selectin and PS increased with time, while spontaneous LAMP-1 exposure was unchanged. In addition, agonist-induced LAMP-1 expression clearly discriminated platelet concentrates with reduced swirling from those with retained swirling. This suggests that LAMP-1 could be a good marker to capture changes in activation capacity in stored platelets. The platelet activation potential seen as LAMP-1 exposure and fragmentation into platelet subpopulations is potential sensitive markers for the platelet storage lesion. © 2015 International Society of Blood Transfusion.

Gap Junctions and Connexin Hemichannels Underpin Haemostasis and Thrombosis

PubMed Central

Vaiyapuri, Sakthivel; Jones, Chris I.; Sasikumar, Parvathy; Moraes, Leonardo A.; Munger, Stephanie J.; Wright, Joy R.; Ali, Marfoua S.; Sage, Tanya; Kaiser, William J.; Tucker, Katherine L.; Stain, Christopher J.; Bye, Alexander P.; Jones, Sarah; Oviedo-Orta, Ernesto; Simon, Alexander M.; Mahaut-Smith, Martyn P.; Gibbins, Jonathan M.

2012-01-01

Background Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have now examined the role of connexins in platelets, blood cells that circulate in isolation, but upon tissue injury adhere to each other and the vessel wall to prevent blood loss and facilitate wound repair. Methods and Results We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses prior to platelet-platelet contact, and reduced laser induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion and clot retraction indicating an important role for Cx37 hemichannels and gap junctions in platelet thrombus function. Conclusions Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of haemostasis and thrombosis and represent potential therapeutic targets. PMID:22528526

Platelet-rich plasma derived growth factors contribute to stem cell differentiation in musculoskeletal regeneration

NASA Astrophysics Data System (ADS)

Qian, Yun; Han, Qixin; Chen, Wei; Song, Jialin; Zhao, Xiaotian; Ouyang, Yuanming; Yuan, Weien; Fan, Cunyi

2017-10-01

Stem cell treatment and platelet-rich plasma (PRP) therapy are two significant issues in regenerative medicine. Stem cells such as bone marrow mesenchymal stem cells, adipose-derived stem cells and periodontal ligament stem cells can be successfully applied in the field of tissue regeneration. PRP, a natural product isolated from whole blood, can secrete multiple growth factors (GFs) for regulating physiological activities. These GFs can stimulate proliferation and differentiation of different stem cells in injury models. Therefore, combination of both agents receives wide expectations in regenerative medicine, especially in bone, cartilage and tendon repair. In this review, we thoroughly discussed the interaction and underlying mechanisms of platelet-rich plasma derived growth factors with stem cells, and assessed their functions in cell differentiation for musculoskeletal regeneration.

Dose response of surfactants to attenuate gas embolism related platelet aggregation

NASA Astrophysics Data System (ADS)

Eckmann, David M.; Eckmann, Yonaton Y.; Tomczyk, Nancy

2014-03-01

Intravascular gas embolism promotes blood clot formation, cellular activation, and adhesion events, particularly with platelets. Populating the interface with surfactants is a chemical-based intervention to reduce injury from gas embolism. We studied platelet activation and platelet aggregation, prominent adverse responses to blood contact with bubbles. We examined dose-response relationships for two chemically distinct surfactants to attenuate the rise in platelet function stimulated by exposure to microbubbles. Significant reduction in platelet aggregation and platelet activation occurred with increasing concentration of the surfactants, indicating presence of a saturable system. A population balance model for platelet aggregation in the presence of embolism bubbles and surfactants was developed. Monte Carlo simulations for platelet aggregation were performed. Results agree qualitatively with experimental findings. Surfactant dose-dependent reductions in platelet activation and aggregation indicate inhibition of the gas/liquid interface's ability to stimulate cellular activation mechanically.

Aerobic exercise training lowers platelet reactivity and improves platelet sensitivity to prostacyclin in pre- and postmenopausal women.

PubMed

Lundberg Slingsby, M H; Nyberg, M; Egelund, J; Mandrup, C M; Frikke-Schmidt, R; Kirkby, N S; Hellsten, Y

2017-12-01

Essentials It is unknown how regular exercise affects platelet function after menopause. We studied the effect of 3-months of high-intensity exercise in pre- and postmenopausal women. Platelet sensitivity to the inhibitory effect of arterially infused prostacyclin was increased. Reduced basal platelet reactivity was seen in the premenopausal women only. Background The risk of atherothrombotic events increases after the menopause. Regular physical activity has been shown to reduce platelet reactivity in younger women, but it is unknown how regular exercise affects platelet function after the menopause. Objectives To examine the effects of regular aerobic exercise in late premenopausal and recent postmenopausal women by testing basal platelet reactivity and platelet sensitivity to prostacyclin and nitric oxide. Methods Twenty-five sedentary, but healthy, late premenopausal and 24 matched recently postmenopausal women, mean (95% confidence interval) 49.1 (48.2-49.9) and 53.7 (52.5-55.0) years old, participated in an intervention study: 3-month high-intensity supervised aerobic spinning-cycle training (1 h, × 3/week). Basal platelet reactivity was analyzed in platelet-rich plasma from venous blood as agonist-induced % aggregation. In a subgroup of 13 premenopausal and 14 postmenopausal women, platelet reactivity was tested ex vivo after femoral arterial infusion of prostacyclin, acetylcholine, a cyclooxygenase inhibitor, and after acute one-leg knee extensor exercise. Results Basal platelet reactivity (%aggregation) to TRAP-6 (1 μm) was higher in the postmenopausal, 59% (50-68), than the premenopausal women, 45% (35-55). Exercise training reduced basal platelet reactivity to collagen (1 μg mL -1 ) in the premenopausal women only: from 63% (55-71%) to 51% (41-62%). After the training intervention, platelet aggregation was more inhibited by the arterial prostacyclin infusion and the acute exercise in both premenopausal and postmenopausal women. Conclusions These results highlight previously unknown cardioprotective aspects of regular aerobic exercise in premenopausal and postmenopausal women, improving their regulation of platelet reactivity through an increased platelet sensitivity to prostacyclin, which may counterbalance the increased atherothrombotic risk associated with the menopause. © 2017 International Society on Thrombosis and Haemostasis.

Cyclophilin B binding to platelets supports calcium-dependent adhesion to collagen.

PubMed

Allain, F; Durieux, S; Denys, A; Carpentier, M; Spik, G

1999-08-01

We have recently reported that cyclophilin B (CyPB), a secreted cyclosporine-binding protein, could bind to T lymphocytes through interactions with two types of binding sites. The first ones, referred to as type I, involve interactions with the conserved domain of CyPB and promote the endocytosis of surface-bound ligand, while the second type of binding sites, termed type II, are represented by glycosaminoglycans (GAG). Here, we further investigated the interactions of CyPB with blood cell populations. In addition to lymphocytes, CyPB was found to interact mainly with platelets. The binding is specific, with a dissociation constant (kd) of 9 +/- 3 nmol/L and the number of sites estimated at 960 +/- 60 per cell. Platelet glycosaminoglycans are not required for the interactions, but the binding is dramatically reduced by active cyclosporine derivatives. We then analyzed the biologic effects of CyPB and found a significant increase in platelet adhesion to collagen. Concurrently, CyPB initiates a transmembranous influx of Ca(2+) and induces the phosphorylation of the P-20 light chains of myosin. Taken together, the present results demonstrate for the first time that extracellular CyPB specifically interacts with platelets through a functional receptor related to the lymphocyte type I binding sites and might act by regulating the activity of a receptor-operated membrane Ca(2+) channel.

The effects of platelet lysate patches on the activity of tendon-derived cells.

PubMed

Costa-Almeida, Raquel; Franco, Albina R; Pesqueira, Tamagno; Oliveira, Mariana B; Babo, Pedro S; Leonor, Isabel B; Mano, João F; Reis, Rui L; Gomes, Manuela E

2018-03-01

Platelet-derived biomaterials are widely explored as cost-effective sources of therapeutic factors, holding a strong potential for endogenous regenerative medicine. Particularly for tendon repair, treatment approaches that shift the injury environment are explored to accelerate tendon regeneration. Herein, genipin-crosslinked platelet lysate (PL) patches are proposed for the delivery of human-derived therapeutic factors in patch augmentation strategies aiming at tendon repair. Developed PL patches exhibited a controlled release profile of PL proteins, including bFGF and PDGF-BB. Additionally, PL patches exhibited an antibacterial effect by preventing the adhesion, proliferation and biofilm formation by S. aureus, a common pathogen in orthopaedic surgical site infections. Furthermore, these patches supported the activity of human tendon-derived cells (hTDCs). Cells were able to proliferate over time and an up-regulation of tenogenic genes (SCX, COL1A1 and TNC) was observed, suggesting that PL patches may modify the behavior of hTDCs. Accordingly, hTDCs deposited tendon-related extracellular matrix proteins, namely collagen type I and tenascin C. In summary, PL patches can act as a reservoir of biomolecules derived from PL and support the activity of native tendon cells, being proposed as bioinstructive patches for tendon regeneration. Platelet-derived biomaterials hold great interest for the delivery of therapeutic factors for applications in endogenous regenerative medicine. In the particular case of tendon repair, patch augmentation strategies aiming at shifting the injury environment are explored to improve tendon regeneration. In this study, PL patches were developed with remarkable features, including the controlled release of growth factors and antibacterial efficacy. Remarkably, PL patches supported the activity of native tendon cells by up-regulating tenogenic genes and enabling the deposition of ECM proteins. This patch holds great potential towards simultaneously reducing post-implantation surgical site infections and promoting tendon regeneration for prospective in vivo applications. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Echicetin Coated Polystyrene Beads: A Novel Tool to Investigate GPIb-Specific Platelet Activation and Aggregation

PubMed Central

Petunin, Alexey; Clemetson, Kenneth J.; Gambaryan, Stepan; Walter, Ulrich

2014-01-01

von Willebrand factor/ristocetin (vWF/R) induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y12 and thromboxane receptor through secreted ADP and TxA2, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways. PMID:24705415

Measurement of adhesion of human platelets in plasma to protein surfaces in microplates.

PubMed

Eriksson, Andreas C; Whiss, Per A

2005-01-01

Platelet adhesion is an initial, crucial and complex event for inhibiting blood loss upon vascular injury. Activation and adhesion of platelets also play a fundamental role in the development of thrombosis. A combination of exposed extracellular matrix proteins in the vascular wall and release of activating compounds from the participating cells activate the platelets. New potent anti-platelet agents are in progress but there is a shortage of methods that measure the concerted action of adhesive surfaces and soluble compounds upon platelet adhesion in vitro. The aim of this work was to develop a method to measure adhesion of platelets in plasma with standard laboratory equipment. Platelet-rich plasma from healthy humans was used in studies to optimise the conditions of the present assay. Different proteins were coated in microplate wells and various soluble platelet activators and inhibitors were added to establish the ability of the current method to detect increased as well as decreased platelet adhesion. The amount of platelet adhesion was measured by the reaction between p-nitrophenyl phosphate and the intracellular enzyme acid phosphatase. Adhesion of platelets in plasma to microplate wells coated with albumin, collagen, fibrinogen and activated plasma showed significant surface dependency. The known soluble platelet activators adenosine diphosphate, adrenaline and ristocetin enhanced the levels of adhesion. Available anti-platelet agents such as prostacyclin, forskolin, acetylsalicylic acid and RGD containing peptides caused dose-dependent inhibition of platelet adhesion. This report describes a further development of a previously described method and offers the advantage to use platelets in plasma to measure platelet adhesion to protein surfaces. The assay is simple and flexible and is suitable in basic research for screening and characterisation of platelet adhesion responsiveness.

Fruitflow®: the first European Food Safety Authority-approved natural cardio-protective functional ingredient.

PubMed

O'Kennedy, Niamh; Raederstorff, Daniel; Duttaroy, Asim K

2017-03-01

Hyperactive platelets, in addition to their roles in thrombosis, are also important mediators of atherogenesis. Antiplatelet drugs are not suitable for use where risk of a cardiovascular event is relatively low. It is therefore important to find alternative safe antiplatelet inhibitors for the vulnerable population who has hyperactive platelets in order to reduce the risk of cardiovascular disease. Potent antiplatelet factors were identified in water-soluble tomato extract (Fruitflow ® ), which significantly inhibited platelet aggregation. Human volunteer studies demonstrated the potency and bioavailability of active compounds in Fruitflow ® . Fruitflow ® became the first product in Europe to obtain an approved, proprietary health claim under Article 13(5) of the European Health Claims Regulation 1924/2006 on nutrition and health claims made on foods. Fruitflow ® is now commercially available in different countries worldwide. In addition to its reduction in platelet reactivity, Fruitflow ® contains anti-angiotensin-converting enzyme and anti-inflammatory factors, making it an effective and natural cardio-protective functional food.

In vivo imaging visualizes discoid platelet aggregations without endothelium disruption and implicates contribution of inflammatory cytokine and integrin signaling.

PubMed

Nishimura, Satoshi; Manabe, Ichiro; Nagasaki, Mika; Kakuta, Shigeru; Iwakura, Yoichiro; Takayama, Naoya; Ooehara, Jun; Otsu, Makoto; Kamiya, Akihide; Petrich, Brian G; Urano, Tetsumei; Kadono, Takafumi; Sato, Shinichi; Aiba, Atsu; Yamashita, Hiroshi; Sugiura, Seiryo; Kadowaki, Takashi; Nakauchi, Hiromitsu; Eto, Koji; Nagai, Ryozo

2012-02-23

The mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize the formation of thrombus, especially at the single-platelet level in real time. Here we demonstrate that rapidly developing thrombi composed of discoid platelets can be induced in the mesenteric capillaries, arterioles, and large-sized arteries of living mice, enabling characterization of the kinetics of thrombosis initiation and the multicellular interrelationships during thrombus development. Platelet aggregation without EC disruption was triggered by reactive oxygen species (ROS) photochemically induced by moderate power laser irradiation. The inflammatory cytokines TNF-α and IL-1 could be key components of the EC response, acting through regulation of VWF mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIbα to endothelial VWF in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talin-dependent activation of alphaIIb-beta3 integrin or Rac1 in platelets was required for late-phase thrombus stability. Our novel imaging technology illustrates the molecular mechanism underlying inflammation-based thrombus formation by discoid platelets on undisrupted ECs and suggests control of ROS could be a useful therapeutic target for the prevention of thrombotic diseases.

Thrombopoietin contributes to enhanced platelet activation in cigarette smokers.

PubMed

Lupia, Enrico; Bosco, Ornella; Goffi, Alberto; Poletto, Cesare; Locatelli, Stefania; Spatola, Tiziana; Cuccurullo, Alessandra; Montrucchio, Giuseppe

2010-05-01

Thrombopoietin (TPO) is a humoral growth factor that primes platelet activation in response to several agonists. We recently showed that TPO enhances platelet activation in unstable angina and sepsis. Aim of this study was to investigate the role of TPO in platelet function abnormalities described in cigarette smokers. In a case-control study we enrolled 20 healthy cigarette smokers and 20 nonsmokers, and measured TPO and C-reactive protein (CRP), as well as platelet-leukocyte binding and P-selectin expression. In vitro we evaluated the priming activity of smoker or control plasma on platelet activation, and the role of TPO in this effect. We then studied the effects of acute smoking and smoking cessation on TPO levels and platelet activation indices. Chronic cigarette smokers had higher circulating TPO levels than nonsmoking controls, as well as increased platelet-leukocyte binding, P-selectin expression, and CRP levels. Serum cotinine concentrations correlated with TPO concentrations, platelet-monocyte aggregates and P-selectin expression. In addition, TPO levels significantly correlated with ex vivo platelet-monocyte aggregation and P-selectin expression. In vitro, the plasma from cigarette smokers, but not from nonsmoking controls, primed platelet-monocyte binding, which was reduced when an inhibitor of TPO was used. We also found that acute smoking slightly increased TPO levels, but did not affect platelet-leukocyte binding, whereas smoking cessation induced a significant decrease in both circulating TPO and platelet-leukocyte aggregation. Elevated TPO contributes to enhance platelet activation and platelet-monocyte cross-talk in cigarette smokers. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

Effects of plasma nitric oxide levels on platelet activation in single donor apheresis and random donor concentrates.

PubMed

Büyükkağnici, Demet Iren; Ilhan, Osman; Kavas, Güzin Ozelçi; Arslan, Onder; Arat, Mutlu; Dalva, Klara; Ayyildiz, Erol

2007-02-01

P-selectin is an useful marker to determine platelet activation and nitric oxide inhibits platelet activation, secretion, adhesion and aggregation. The aim of this study was to investigate the relationship between nitric oxide and P-selectin values in both single donor apheresis and random donor platelet concentrates. According to the results of this study, we found that the best platelet concentrate is freshly prepared single donor apheresis concentrate and it is important to prevent activation at the beginning of the donation. Nitric oxide, which is synthesized from platelets during the storage period, is not sufficient to prevent platelet activation.

Disulfide HMGB1 derived from platelets coordinates venous thrombosis in mice

PubMed Central

Philippi, Vanessa; Stockhausen, Sven; Busse, Johanna; Antonelli, Antonella; Miller, Meike; Schubert, Irene; Hoseinpour, Parandis; Chandraratne, Sue; von Brühl, Marie-Luise; Gaertner, Florian; Lorenz, Michael; Agresti, Alessandra; Coletti, Raffaele; Antoine, Daniel J.; Heermann, Ralf; Jung, Kirsten; Reese, Sven; Laitinen, Iina; Schwaiger, Markus; Walch, Axel; Sperandio, Markus; Nawroth, Peter P.; Reinhardt, Christoph; Jäckel, Sven; Bianchi, Marco E.; Massberg, Steffen

2016-01-01

Deep venous thrombosis (DVT) is one of the most common cardiovascular diseases, but its pathophysiology remains incompletely understood. Although sterile inflammation has recently been shown to boost coagulation during DVT, the underlying molecular mechanisms are not fully resolved, which could potentially identify new anti-inflammatory approaches to prophylaxis and therapy of DVT. Using a mouse model of venous thrombosis induced by flow reduction in the vena cava inferior, we identified blood-derived high-mobility group box 1 protein (HMGB1), a prototypical mediator of sterile inflammation, to be a master regulator of the prothrombotic cascade involving platelets and myeloid leukocytes fostering occlusive DVT formation. Transfer of platelets into Hmgb1−/− chimeras showed that this cell type is the major source of HMGB1, exposing reduced HMGB1 on their surface upon activation thereby enhancing the recruitment of monocytes. Activated leukocytes in turn support oxidation of HMGB1 unleashing its prothrombotic activity and promoting platelet aggregation. This potentiates the amount of HMGB1 and further nurtures the accumulation and activation of monocytes through receptor for advanced glycation end products (RAGE) and Toll-like receptor 2, leading to local delivery of monocyte-derived tissue factor and cytokines. Moreover, disulfide HMGB1 facilitates formation of prothrombotic neutrophil extracellular traps (NETs) mediated by RAGE, exposing additional HMGB1 on their extracellular DNA strands. Eventually, a vicious circle of coagulation and inflammation is set in motion leading to obstructive DVT formation. Therefore, platelet-derived disulfide HMGB1 is a central mediator of the sterile inflammatory process in venous thrombosis and could be an attractive target for an anti-inflammatory approach for DVT prophylaxis. PMID:27574188

Activated Platelets Induce Endothelial Cell Activation via an Interleukin-1β Pathway in Systemic Lupus Erythematosus

PubMed Central

Nhek, Sokha; Clancy, Robert; Lee, Kristen A.; Allen, Nicole M.; Barrett, Tessa J.; Marcantoni, Emanuela; Nwaukoni, Janet; Rasmussen, Sara; Rubin, Maya; Newman, Jonathan D.; Buyon, Jill P.; Berger, Jeffrey S.

2017-01-01

Objective Systemic lupus erythematosus (SLE) is associated with the premature development of cardiovascular disease. The platelet–endothelium interaction is important in the pathogenesis of cardiovascular disease. In this study, we investigated the platelet phenotype from patients with SLE and matched controls, and their effect on endothelial cells. Approach and Results Platelet aggregability was measured in 54 SLE subjects off antiplatelet therapy (mean age 40.1±12.8 years; 82% female; 37% white) with age- and sex-matched controls. Platelets were coincubated with human umbilical vein endothelial cells (HUVECs) and changes to gene expression assessed by an RNA array and quantitative reverse transcription polymerase chain reaction. SLE disease activity index ranged from 0 to 22 (mean 5.1±3.9). Compared with controls, patients with SLE had significantly increased monocyte and leukocyte–platelet aggregation and platelet aggregation in response to submaximal agonist stimulation. An agnostic microarray of HUVECs cocultured with SLE platelets found a platelet-mediated effect on endothelial gene pathways involved in cell activation. Sera from SLE versus control subjects significantly increased (1) activation of control platelets; (2) platelet adhesion to HUVECs; (3) platelet-induced HUVEC gene expression of interleukin-8, and intercellular adhesion molecule 1; and (4) proinflammatory gene expression in HUVECs, mediated by interleukin-1β–dependent pathway. Incubation of SLE-activated platelets with an interleukin-1β–neutralizing antibody or HUVECs pretreated with interleukin-1 receptor antibodies attenuated the platelet-mediated activation of endothelial cells. Conclusions Platelet activity measurements and subsequent interleukin-1β–dependent activation of the endothelium are increased in subjects with SLE. Platelet–endothelial interactions may play a role in the pathogenesis of cardiovascular disease in patients with SLE. PMID:28153882

The combined effect of platelet storage media and intercept pathogen reduction technology on platelet activation/activability and cellular apoptosis/necrosis: Lisbon-RBS experience.

PubMed

Carvalho, Helena; Alguero, Carmen; Santos, Matilde; de Sousa, Gracinda; Trindade, Helder; Seghatchian, Jerard

2006-04-01

Platelets are known to undergo shape change, activation, a release reaction and apoptosis/necrosis during processing and storage, all of which are collectively known as the platelet storage lesion. Any additional processing may have some deleterious impact on platelet activability and functional integrity, which need to be investigated. This preliminary investigation was undertaken to establish the combined effects of standard platelet storage media and the intercept pathogen reduction technology on platelet activation and activability during 7 day storage, using buffy-coat derived platelets in standard storage media containing 35% plasma (N=24). P-selectin (CD62p) expression, a classical marker of platelet activation, and phosphatidylserine (PS) exposure on the platelet surface membrane, a hallmark of cellular necrosis/apoptosis, were both measured by flow cytometry. The results reveal significant increases in activation, from an average of 22.7% on day 1 before treatment to 31.6% on day 2 after treatment and 58.7% at the end of storage. Concomitantly, the basal expression of PS was slightly increased from 1.9% to 2.8% at day 2 after treatment and 7.3% at the end of storage. However, the functional reserve of platelets during storage, which reflects their capability to undergo activation and the release reaction when platelets were challenged with either calcium ionophore or thrombin, was relatively well maintained. These preliminary data confirm the earlier data on the use of intercept, and for the first time, based on the assessment of platelet functional integrity, suggest that platelet functional reserve is relatively well maintained, with little change in the formation of apoptotic cells.

Anti-citrullinated protein antibodies contribute to platelet activation in rheumatoid arthritis.

PubMed

Habets, Kim L L; Trouw, Leendert A; Levarht, E W Nivine; Korporaal, Suzanne J A; Habets, Petra A M; de Groot, Philip; Huizinga, Tom W J; Toes, René E M

2015-08-24

Although the role of platelets in rheumatoid arthritis (RA) is relatively unexplored, recent studies point towards a contribution of platelets in arthritis. We set out to determine platelet phenotype in RA and studied whether this could be influenced by the presence of anti-citrullinated protein antibodies (ACPA). Platelets from healthy controls were incubated in the presence of plasma of patients with RA or age- and sex-matched healthy controls and plasma from ACPA(neg) or ACPA(pos) patients or in the presence of plate-bound ACPA. Characteristics of platelets isolated from patients with RA were correlated to disease activity. Platelets isolated from healthy controls displayed markers of platelet activation in the presence of plasma derived from RA patients, as determined by P-selectin expression, formation of aggregates and secretion of soluble CD40 ligand (sCD40L). Furthermore, levels of P-selectin expression and sCD40L release correlated with high ACPA titres. In accordance with these findings, enhanced platelet activation was observed after incubation with ACPA(pos) plasma versus ACPA(neg) plasma. Pre-incubation of platelets with blocking antibodies directed against low-affinity immunoglobulin G receptor (FcγRIIa) completely inhibited the ACPA-mediated activation. In addition, expression of P-selectin measured as number of platelets correlated with Disease Activity Score in 44 joints, C-reactive protein level, ACPA status and ACPA level. We show for the first time that ACPA can mediate an FcγRIIa-dependent activation of platelets. As ACPA can be detected several years before RA disease onset and activated platelets contribute to vascular permeability, these data implicate a possible role for ACPA-mediated activation of platelets in arthritis onset.

Platelet-Derived S100A8/A9 and Cardiovascular Disease in Systemic Lupus Erythematosus.

PubMed

Lood, Christian; Tydén, Helena; Gullstrand, Birgitta; Jönsen, Andreas; Källberg, Eva; Mörgelin, Matthias; Kahn, Robin; Gunnarsson, Iva; Leanderson, Tomas; Ivars, Fredrik; Svenungsson, Elisabet; Bengtsson, Anders A

2016-08-01

Levels of S100A8/A9, a proinflammatory and prothrombotic protein complex, are increased in several diseases, and high levels predispose to cardiovascular disease (CVD). Recently, platelet S100A8/A9 synthesis was described in mice and humans in relation to CVD. The aim of this study was to investigate the role of platelet S100A8/A9 in systemic lupus erythematosus (SLE), a disease with markedly increased cardiovascular morbidity, as well as the exact platelet distribution of the S100A8/A9 proteins. The occurrence and distribution of platelet S100A8/A9 protein were detected by enzyme-linked immunosorbent assay, electron microscopy, Western blotting, and flow cytometry in healthy controls (n = 79) and in 2 individual cohorts of SLE patients (n = 148 and n = 318, respectively) and related to cardiovascular morbidity. We observed that human platelets expressed S100A8/A9 proteins, and that these were localized in close proximity to intracellular membranes and granules as well as on the cell surface upon activation with physiologic and pathophysiologic stimuli. Interestingly, S100A8/A9 was enriched at sites of membrane interactions, indicating a role of S100A8/A9 in cell-cell communication. S100A8/A9 levels were highly regulated by interferon-α, both in vivo and in vitro. Patients with SLE had increased platelet S100A8/A9 content compared with healthy individuals. Increased levels of platelet S100A8/A9 were associated with CVD, particularly myocardial infarction (odds ratio 4.8, 95% confidence interval 1.5-14.9, P = 0.032 [adjusted for age, sex, and smoking]). Platelets contain S100A8/A9 in membrane-enclosed vesicles, enabling rapid cell surface deposition upon activation. Furthermore, platelet S100A8/A9 protein levels were increased in SLE patients, particularly in those with CVD, and may be a future therapeutic target. © 2016, American College of Rheumatology.

Docking based design of diastereoisomeric MTCA as GPIIb/IIIa receptor inhibitor.

PubMed

Wang, Xiaozhen; Wang, Yuji; Wu, Jianhui; Gui, Lin; Zhang, Xiaoyi; Zheng, Meiqing; Wang, Yaonan; Zhao, Shurui; Li, Ze; Zhao, Ming; Peng, Shiqi

2017-12-01

In GPIIb/IIIa mediated arterial thrombosis platelet activation plays a central role. To discover platelet activation inhibitor the pharmacophores of GPIIb/IIIa receptor inhibitors and anti-thrombotic agents were analyzed. This led to the design of (1R,3S)- and (1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acids as GPIIb/IIIa inhibitors. Comparing to (1S,3S)-isomer (1R,3S)-isomer had lower cdocker interaction energy. AFM image showed that the minimal effective concentration of (1S,3S)-isomer and (1R,3S)-isomer inhibiting platelet activation were 10 -5  M and 10 -6  M, respectively. In vivo 1 μmol/kg of oral (1S,3S)-isomer effectively inhibited the rats to form arterial thrombus and down regulated GPIIb/IIIa expression, but the activities were significantly lower than those of 1 μmol/kg of oral (1R,3S)-isomer. Both (1S,3S)-isomer and (1R,3S)-isomer can be safely used for structural modifications, but (1R,3S)-isomer should be superior to (1S,3S)-isomer. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genetic Alterations of the Thrombopoietin/MPL/JAK2 Axis Impacting Megakaryopoiesis.

PubMed

Plo, Isabelle; Bellanné-Chantelot, Christine; Mosca, Matthieu; Mazzi, Stefania; Marty, Caroline; Vainchenker, William

2017-01-01

Megakaryopoiesis is an original and complex cell process which leads to the formation of platelets. The homeostatic production of platelets is mainly regulated and controlled by thrombopoietin (TPO) and the TPO receptor (MPL)/JAK2 axis. Therefore, any hereditary or acquired abnormality affecting this signaling axis can result in thrombocytosis or thrombocytopenia. Thrombocytosis can be due to genetic alterations that affect either the intrinsic MPL signaling through gain-of-function (GOF) activity ( MPL, JAK2, CALR ) and loss-of-function (LOF) activity of negative regulators ( CBL, LNK ) or the extrinsic MPL signaling by THPO GOF mutations leading to increased TPO synthesis. Alternatively, thrombocytosis may paradoxically result from mutations of MPL leading to an abnormal MPL trafficking, inducing increased TPO levels by alteration of its clearance. In contrast, thrombocytopenia can also result from LOF THPO or MPL mutations, which cause a complete defect in MPL trafficking to the cell membrane, impaired MPL signaling or stability, defects in the TPO/MPL interaction, or an absence of TPO production.

Genetic Alterations of the Thrombopoietin/MPL/JAK2 Axis Impacting Megakaryopoiesis

PubMed Central

Plo, Isabelle; Bellanné-Chantelot, Christine; Mosca, Matthieu; Mazzi, Stefania; Marty, Caroline; Vainchenker, William

2017-01-01

Megakaryopoiesis is an original and complex cell process which leads to the formation of platelets. The homeostatic production of platelets is mainly regulated and controlled by thrombopoietin (TPO) and the TPO receptor (MPL)/JAK2 axis. Therefore, any hereditary or acquired abnormality affecting this signaling axis can result in thrombocytosis or thrombocytopenia. Thrombocytosis can be due to genetic alterations that affect either the intrinsic MPL signaling through gain-of-function (GOF) activity (MPL, JAK2, CALR) and loss-of-function (LOF) activity of negative regulators (CBL, LNK) or the extrinsic MPL signaling by THPO GOF mutations leading to increased TPO synthesis. Alternatively, thrombocytosis may paradoxically result from mutations of MPL leading to an abnormal MPL trafficking, inducing increased TPO levels by alteration of its clearance. In contrast, thrombocytopenia can also result from LOF THPO or MPL mutations, which cause a complete defect in MPL trafficking to the cell membrane, impaired MPL signaling or stability, defects in the TPO/MPL interaction, or an absence of TPO production. PMID:28955303

P-selectin ligation induces platelet activation and enhances microaggregate and thrombus formation.

PubMed

Théorêt, Jean-François; Yacoub, Daniel; Hachem, Ahmed; Gillis, Marc-Antoine; Merhi, Yahye

2011-09-01

Platelet P-selectin is a thrombo-inflammatory molecule involved in platelet activation and aggregation. This may occur via the adhesive function of P-selectin and its potential capacity to trigger intracellular signaling. However, its impact on platelet function remains elusive. This study was therefore designed to investigate the relationship between the signaling potential of platelet P-selectin and its function in platelet physiology. Human and mouse platelets were freshly isolated from whole blood. Platelet activation was assessed using flow cytometry and western blot analysis, while platelet physiological responses were evaluated through aggregation, microaggregate formation and in a thrombosis model in wild-type and P-selectin-deficient (CD62P(-/-)) mice. Interaction of P-selectin with its high-affinity ligand, a recombinant soluble form of P-Selectin Glycoprotein Ligand-1 (rPSGL-1), enhances platelet activation, adhesion and microaggregate formation. This augmented platelet microaggregates requires an intact cytoskeleton, but occurs independently of platelet α(IIb)β(3). Thrombus formation and microaggregate were both enhanced by rPSGL-1 in wild-type, but not in CD62P(-/-) mice. In addition, CD62P(-/-) mice exhibited thrombosis abnormalities without an α(IIb)β(3) activation defect. This study demonstrates that the role of platelet P-selectin is not solely adhesive; its binding to PSGL-1 induces platelet activation that enhances platelet aggregation and thrombus formation. Therefore, targeting platelet P-selectin or its ligand PSGL-1 could provide a potential therapeutic approach in the management of thrombotic disorders. Copyright © 2011 Elsevier Ltd. All rights reserved.

Akt Phosphorylation and PI (3, 4, 5) P3 Binding Coordinately Inhibit the Tumor Suppressive Activity of Merlin

DTIC Science & Technology

2010-02-01

C-terminal F-actin binding site. Recent work using either unphosphorylated or T558 phosphorylated moesin purified from platelets revealed a dual...Pestonjamasp K, Luna EJ, & Furthmayr H (1999) Regulation of F-actin binding to platelet moesin in vitro by both phosphorylation of threonine 558 and... Lysate IB: anti-GFP M.W.(kDa) 100 75 50 37 150 em pt y ve ct or G FP -s um o1 IB: anti-GFP CB IB: anti-myc myc-merlin em pt y ve ct or G FP -s um o1 myc

Passive participation of fixed platelets in aggregation facilitated by covalently bound fibrinogen.

PubMed

Agam, G; Livne, A

1983-01-01

The role of fibrinogen in interplatelet recognition during aggregation was examined by combining two cell types: fresh platelets (in limiting density) activated by thrombin or A23187, and formaldehyde-fixed platelets, bearing cross-linked fibrinogen. The fixed platelets did not aggregate by themselves, nor with resting platelets, but were capable of interacting with activated platelets and of participating passively in aggregation. The participation, expressed by enhanced aggregation, was assayed by the conventional turbidometric traces and by cosedimentation of fixed 3H-platelets with aggregates of fresh platelets. Platelet suspensions, prepared without special means to avert spontaneous activation, retained plasma fibrinogen to the extent of 50 micrograms/ml of a suspension containing 10(8) platelets, and the derived fixed platelets participated in aggregation, independently of added fibrinogen. The capability of such fixed platelets to participate in aggregation was sensitive to proteolytic digestion and to massive acetylation. When platelet separation was aided by apyrase or aspirin, PGE1 and gel filtration, the residual plasma fibrinogen was limited to 0.4 micrograms/ml of 10(8) platelet suspension. The derived fixed platelets were incapable of participating in aggregation unless fibrinogen was added prior to fixation. The affixed fibrinogen could not be replaced by soluble fibrinogen or affixed albumin. It is concluded that fibrinogen, which binds to platelets upon activation or is linked to them covalently, is a recognition site for platelet-platelet interaction during aggregation.

Thrombopoietin contributes to enhanced platelet activation in patients with unstable angina.

PubMed

Lupia, Enrico; Bosco, Ornella; Bergerone, Serena; Dondi, Anna Erna; Goffi, Alberto; Oliaro, Elena; Cordero, Marco; Del Sorbo, Lorenzo; Trevi, Giampaolo; Montrucchio, Giuseppe

2006-12-05

We sought to investigate the potential role of elevated levels of thrombopoietin (TPO) in platelet activation during unstable angina (UA). Thrombopoietin is a humoral growth factor that does not induce platelet aggregation per se, but primes platelet activation in response to several agonists. No data concerning its contribution to platelet function abnormalities described in patients with UA are available. We studied 15 patients with UA and, as controls, 15 patients with stable angina (SA) and 15 healthy subjects. We measured TPO and C-reactive protein (CRP), as well as monocyte-platelet binding and the platelet expression of P-selectin and of the TPO receptor, c-Mpl. The priming activity of patient or control plasma on platelet aggregation and monocyte-platelet binding and the role of TPO in this effect also were studied. Patients with UA showed higher circulating TPO levels, as well as increased monocyte-platelet binding, platelet P-selectin expression, and CRP levels, than those with SA and healthy control subjects. The UA patients also showed reduced platelet expression of the TPO receptor, c-Mpl. In vitro, the plasma from UA patients, but not from SA patients or healthy controls, primed platelet aggregation and monocyte-platelet binding, which were both reduced when an inhibitor of TPO was used. Thrombopoietin may enhance platelet activation in the early phases of UA, potentially participating in the pathogenesis of acute coronary syndromes.

Effects of protopine on blood platelet aggregation. II. Effect on metabolic system of adenosine 3',5'-cyclic monophosphate in platelets.

PubMed

Shiomoto, H; Matsuda, H; Kubo, M

1990-08-01

The mode of action of protopine on rabbit platelet aggregation was investigated in the metabolic system of adenosine 3',5'-cyclic monophosphate (cyclic AMP) in vitro experimental models. The inhibitory activity of protopine on adenosine 5'-diphosphate induced platelet aggregation was increased in the presence of prostaglandin I2 or papaverine in platelets. Protopine elevated content of the basal cyclic AMP accumulation in platelets and enhanced activity of crude adenylate cyclase prepared from platelets, but was ineffective on cyclic AMP phosphodiesterase. It is concluded that protopine has an inhibitory activity on platelet aggregation, activates adenylate cyclase and increases cyclic AMP content in platelets, in addition to other inhibitory actions in the metabolic system of cyclic AMP.

Premature remodeling of fat body and fat mobilization triggered by platelet-derived growth factor/VEGF receptor in Drosophila.

PubMed

Zheng, Huimei; Wang, Xuexiang; Guo, Pengfei; Ge, Wanzhong; Yan, Qinfeng; Gao, Weiqiang; Xi, Yongmei; Yang, Xiaohang

2017-05-01

In Drosophila, fat-body remodeling accompanied with fat mobilization is an ecdysone-induced dynamic process that only occurs during metamorphosis. Here, we show that the activated Drosophila platelet-derived growth factor/VEGF receptor (PVR) is sufficient to induce shape changes in the fat body, from thin layers of tightly conjugated polygonal cells to clusters of disaggregated round-shaped cells. These morphologic changes are reminiscent of those seen during early pupation upon initiation of fat-body remodeling. Activation of PVR also triggers an early onset of lipolysis and mobilization of internal storage, as revealed by the appearance of small lipid droplets and up-regulated lipolysis-related genes. We found that PVR displays a dynamic expression pattern in the fat body and peaks at the larval-prepupal transition under the control of ecdysone signaling. Removal of PVR, although it does not prevent ecdysone-induced fat-body remodeling, causes ecdysone signaling to be up-regulated. Our data reveal that PVR is active in a dual-secured mechanism that involves an ecdysone-induced fat-body remodeling pathway and a reinforced PVR pathway for effective lipid mobilization. Ectopic expression of activated c-kit-the mouse homolog of PVR in the Drosophila fat body-also results in a similar phenotype. This may suggest a novel function of c-kit as it relates to lipid metabolism in mammals.-Zheng, H., Wang, X., Guo, P., Ge, W., Yan, Q., Gao, W., Xi, Y., Yang, X. Premature remodeling of fat body and fat mobilization triggered by platelet-derived growth factor/VEGF receptor in Drosophila . © FASEB.

Platelets and atherogenesis: Platelet anti-aggregation activity and endothelial protection from tomatoes (Solanum lycopersicum L.)

PubMed Central

PALOMO, IVÁN; FUENTES, EDUARDO; PADRÓ, TERESA; BADIMON, LINA

2012-01-01

In recent years, it has been shown that platelets are not only involved in the arterial thrombotic process, but also that they play an active role in the inflammatory process of atherogenesis from the beginning. The interaction between platelets and endothelial cells occurs in two manners: activated platelets unite with intact endothelial cells, or platelets in resting adhere to activated endothelium. In this context, inhibition of the platelet function (adhesion/aggregation) could contribute to the prevention of atherothrombosis, the leading cause of cardiovascular morbidity. This can be achieved with antiplatelet agents. However, at the public health level, the level of primary prevention, a healthy diet has also been shown to exert beneficial effects. Among those elements of a healthy diet, the consumption of tomatoes (Solanum lycopersicum L.) stands out for its effect on platelet anti-aggregation activity and endothelial protection, which may be beneficial for cardiovascular health. This article briefly discusses the involvement of platelets in atherogenesis and the possible mechanisms of action provided by tomatoes for platelet anti-aggregation activity and endothelial protection. PMID:22969932

Volatile anesthetics, not intravenous anesthetic propofol bind to and attenuate the activation of platelet receptor integrin αIIbβ3.

PubMed

Yuki, Koichi; Bu, Weiming; Shimaoka, Motomu; Eckenhoff, Roderic

2013-01-01

In clinical reports, the usage of isoflurane and sevoflurane was associated with more surgical field bleeding in endoscopic sinus surgeries as compared to propofol. The activation of platelet receptor αIIbβ3 is a crucial event for platelet aggregation and clot stability. Here we studied the effect of isoflurane, sevoflurane, and propofol on the activation of αIIbβ3. The effect of anesthetics on the activation of αIIbβ3 was probed using the activation sensitive antibody PAC-1 in both cell-based (platelets and αIIbβ3 transfectants) and cell-free assays. The binding sites of isoflurane on αIIbβ3 were explored using photoactivatable isoflurane (azi-isoflurane). The functional implication of revealed isoflurane binding sites were studied using alanine-scanning mutagenesis. Isoflurane and sevoflurane diminished the binding of PAC-1 to wild-type αIIbβ3 transfectants, but not to the high-affinity mutant, β3-N305T. Both anesthetics also impaired PAC-1 binding in a cell-free assay. In contrast, propofol did not affect the activation of αIIbβ3. Residues adducted by azi-isoflurane were near the calcium binding site (an important regulatory site termed SyMBS) just outside of the ligand binding site. The mutagenesis experiments demonstrated that these adducted residues were important in regulating integrin activation. Isoflurane and sevoflurane, but not propofol, impaired the activation of αIIbβ3. Azi-isoflurane binds to the regulatory site of integrin αIIbβ3, thereby suggesting that isoflurane blocks ligand binding of αIIbβ3 in not a competitive, but an allosteric manner.

Functional factor XIII-A is exposed on the stimulated platelet surface

PubMed Central

Mitchell, Joanne L.; Lionikiene, Ausra S.; Fraser, Steven R.; Whyte, Claire S.; Booth, Nuala A.

2014-01-01

Factor XIII (FXIII) stabilizes thrombi against fibrinolysis by cross-linking α2-antiplasmin (α2AP) to fibrin. Cellular FXIII (FXIII-A) is abundant in platelets, but the extracellular functions of this pool are unclear because it is not released by classical secretion mechanisms. We examined the function of platelet FXIII-A using Chandler model thrombi formed from FXIII-depleted plasma. Platelets stabilized FXIII-depleted thrombi in a transglutaminase-dependent manner. FXIII-A activity on activated platelets was unstable and was rapidly lost over 1 hour. Inhibiting platelet activation abrogated the ability of platelets to stabilize thrombi. Incorporating a neutralizing antibody to α2AP into FXIII-depleted thrombi revealed that the stabilizing effect of platelet FXIII-A on lysis was α2AP dependent. Platelet FXIII-A activity and antigen were associated with the cytoplasm and membrane fraction of unstimulated platelets, and these fractions were functional in stabilizing FXIII-depleted thrombi against lysis. Fluorescence confocal microscopy and flow cytometry revealed exposure of FXIII-A on activated membranes, with maximal signal detected with thrombin and collagen stimulation. FXIII-A was evident in protruding caps on the surface of phosphatidylserine-positive platelets. Our data show a functional role for platelet FXIII-A through exposure on the activated platelet membrane where it exerts antifibrinolytic function by cross-linking α2AP to fibrin. PMID:25331118

Epicatechin and catechin modulate endothelial activation induced by platelets of patients with peripheral artery disease.

PubMed

Carnevale, R; Loffredo, L; Nocella, C; Bartimoccia, S; Bucci, T; De Falco, E; Peruzzi, M; Chimenti, I; Biondi-Zoccai, G; Pignatelli, P; Violi, F; Frati, G

2014-01-01

Platelet activation contributes to the alteration of endothelial function, a critical initial step in atherogenesis through the production and release of prooxidant mediators. There is uncertainty about the precise role of polyphenols in interaction between platelets and endothelial cells (ECs). We aimed to investigate whether polyphenols are able to reduce endothelial activation induced by activated platelets. First, we compared platelet activation and flow-mediated dilation (FMD) in 10 healthy subjects (HS) and 10 patients with peripheral artery disease (PAD). Then, we evaluated the effect of epicatechin plus catechin on platelet-HUVEC interaction by measuring soluble cell adhesion molecules (CAMs), NOx production, and eNOS phosphorylation (p-eNOS) in HUVEC. Compared to HS, PAD patients had enhanced platelet activation. Conversely, PAD patients had lower FMD than HS. Supernatant of activated platelets from PAD patients induced an increase of sCAMs release and a decrease of p-eNOS and nitric oxide (NO) bioavailability compared to unstimulated HUVEC. Coincubation of HUVEC, with supernatant of PAD platelets patients, pretreated with a scalar dose of the polyphenols, resulted in a decrease of sCAMs release and in an increase of p-eNOS and NO bioavailability. This study demonstrates that epicatechin plus catechin reduces endothelial activation induced by activated platelets.

Platelet 12-lipoxygenase activation via glycoprotein VI: involvement of multiple signaling pathways in agonist control of H(P)ETE synthesis.

PubMed

Coffey, Marcus J; Jarvis, Gavin E; Gibbins, Jonathan M; Coles, Barbara; Barrett, Natasha E; Wylie, Oliver R E; O'Donnell, Valerie B

2004-06-25

Lipoxygenases (LOX) contribute to vascular disease and inflammation through generation of bioactive lipids, including 12-hydro(pero)xyeicosatetraenoic acid (12-H(P)ETE). The physiological mechanisms that acutely control LOX product generation in mammalian cells are uncharacterized. Human platelets that contain a 12-LOX isoform (p12-LOX) were used to define pathways that activate H(P)ETE synthesis in the vasculature. Collagen and collagen-related peptide (CRP) (1 to 10 microg/mL) acutely induced platelet 12-H(P)ETE synthesis. This implicated the collagen receptor glycoprotein VI (GPVI), which signals via the immunoreceptor-based activatory motif (ITAM)-containing FcRgamma chain. Conversely, thrombin only activated at high concentrations (> 0.2 U/mL), whereas U46619 and ADP alone were ineffective. Collagen or CRP-stimulated 12-H(P)ETE generation was inhibited by staurosporine, PP2, wortmannin, BAPTA/AM, EGTA, and L-655238, implicating src-tyrosine kinases, PI3-kinase, Ca2+ mobilization, and p12-LOX translocation. In contrast, protein kinase C (PKC) inhibition potentiated 12-H(P)ETE generation. Finally, activation of the immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing platelet endothelial cell adhesion molecule (PECAM-1) inhibited p12-LOX product generation. This study characterizes a receptor-dependent pathway for 12-H(P)ETE synthesis via the collagen receptor GPVI, which is negatively regulated by PECAM-1 and PKC, and demonstrates a novel link between immune receptor signaling and lipid mediator generation in the vasculature.

Microparticles from splenectomized β-thalassemia/HbE patients play roles on procoagulant activities with thrombotic potential.

PubMed

Klaihmon, Phatchanat; Phongpao, Kunwadee; Kheansaard, Wasinee; Noulsri, Egarit; Khuhapinant, Archrob; Fucharoen, Suthat; Morales, Noppawan Phumala; Svasti, Saovaros; Pattanapanyasat, Kovit; Chaichompoo, Pornthip

2017-02-01

Thromboembolic events including cerebral thrombosis, deep vein thrombosis, and pulmonary embolism are major complications in β-thalassemia. Damaged red blood cells and chronic platelet activation in splenectomized β-thalassemia/HbE patients were associated with increased microparticles (MPs) releases into blood circulation. MPs are small membrane vesicles, which play important roles on coagulation. However, the role of MP in thalassemia is poorly understood. In this study, the effects of splenectomized-MPs on platelet activation and aggregation were investigated. The results showed that isolated MPs from fresh platelet-free plasma of patients and normal subjects directly induce platelet activation, platelet aggregation, and platelet-neutrophil aggregation in a dose-dependent manner. Interestingly, MPs obtained from splenectomized patients are more efficient in induction of platelet activation (P-selectin + ) when compared to MPs from normal subjects (P < 0.05), tenfold lower than pathophysiological level, at 1:0.1 platelet MP ratio. Co-incubation of splenectomized-MPs with either normal-, non-splenectomized- or splenectomized-platelets at 1:10 platelet MP ratio increased platelet activation up to 5.1 ± 2.2, 5.6 ± 3.7, and 9.5 ± 3.0%, respectively, when normalized with individual baseline. These findings suggest that splenectomized patients were proned to be activated by MPs, and splenectomized-MPs could play an important role on chronic platelet activation and aggregation, leading to thrombus formation in β-thalassemia/HbE patients.

Clot lysis time in platelet-rich plasma: method assessment, comparison with assays in platelet-free and platelet-poor plasmas, and response to tranexamic acid.

PubMed

Panes, Olga; Padilla, Oslando; Matus, Valeria; Sáez, Claudia G; Berkovits, Alejandro; Pereira, Jaime; Mezzano, Diego

2012-01-01

Fibrinolysis dysfunctions cause bleeding or predisposition to thrombosis. Platelets contain several factors of the fibrinolytic system, which could up or down regulate this process. However, the temporal relationship and relative contributions of plasma and platelet components in clot lysis are mostly unknown. We developed a clot lysis time (CLT) assay in platelet-rich plasma (PRP-CLT, with and without stimulation) and compared it to a similar one in platelet-free plasma (PFP) and to another previously reported test in platelet-poor plasma (PPP). We also studied the differential effects of a single dose of tranexamic acid (TXA) on these tests in healthy subjects. PFP- and PPP-CLT were significantly shorter than PRP-CLT, and the three assays were highly correlated (p < 0.0001). PFP- and PPP-, but more significantly PRP-CLT, were positively correlated with age and plasma PAI-1, von Willebrand factor, fibrinogen, LDL-cholesterol, and triglycerides (p < 0.001). All these CLT assays had no significant correlations with platelet aggregation/secretion, platelet counts, and pro-coagulant tests to explore factor X activation by platelets, PRP clotting time, and thrombin generation in PRP. Among all the studied variables, PFP-CLT was independently associated with plasma PAI-1, LDL-cholesterol, and triglycerides and, additionally, stimulated PRP-CLT was also independently associated with plasma fibrinogen. A single 1 g dose of TXA strikingly prolonged all three CLTs, but in contrast to the results without the drug, the lysis times were substantially shorter in non-stimulated or stimulated PRP than in PFP and PPP. This standardized PRP-CLT may become a useful tool to study the role of platelets in clot resistance and lysis. Our results suggest that initially, the platelets enmeshed in the clot slow down the fibrinolysis process. However, the increased clot resistance to lysis induced by TXA is overcome earlier in platelet-rich clots than in PFP or PPP clots. This is likely explained by the display of platelet pro-fibrinolytic effects. Focused research is needed to disclose the mechanisms for the relationship between CLT and plasma cholesterol and its potential pathophysiologic and clinical relevance.

Novel Antiplatelet Activity of Minocycline Involves Inhibition of MLK3-p38 Mitogen Activated Protein Kinase Axis

PubMed Central

Jackson, Joseph W.; Singh, Meera V.; Singh, Vir B.; Jones, Letitia D.; Davidson, Gregory A.; Ture, Sara; Morrell, Craig N.; Schifitto, Giovanni; Maggirwar, Sanjay B.

2016-01-01

Platelets play an essential role in hemostasis and wound healing by facilitating thrombus formation at sites of injury. Platelets also mediate inflammation and contain several pro-inflammatory molecules including cytokines and chemokines that mediate leukocyte recruitment and activation. Not surprisingly, platelet dysfunction is known to contribute to several inflammatory disorders. Antiplatelet therapies, such as aspirin, adenosine diphosphate (ADP) antagonists, glycoprotein IIb/IIIa (GPIIb/IIIa) inhibitors, and anticoagulants such as warfarin, dampen platelet activity at the risk of unwarranted bleeding. Thus, the development of drugs that reduce platelet-mediated inflammation without interfering with thrombus formation is of importance to combat platelet-associated disorders. We have shown here for the first time that the tetracycline antibiotic, minocycline, administered to HIV-infected individuals reduces plasma levels of soluble CD40L and platelet factor 4 levels, host molecules predominately released by platelets. Minocycline reduced the activation of isolated platelets in the presence of the potent platelet activator, thrombin, as measured by ELISA and flow cytometry. Platelet degranulation was reduced upon exposure to minocycline as shown by mepacrine retention and flow cytometry. However, minocycline had no effect on spreading, aggregation, GPIIb/IIIa activation, or in vivo thrombus formation. Lastly, immunoblot analysis suggests that the antiplatelet activity of minocycline is likely mediated by inhibition of mixed lineage kinase 3 (MLK3)-p38 MAPK signaling axis and loss of p38 activity. Our findings provide a better understanding of platelet biology and a novel repurposing of an established antibiotic, minocycline, to specifically reduce platelet granule release without affecting thrombosis, which may yield insights in generating novel, specific antiplatelet therapies. PMID:27270236

Novel Antiplatelet Activity of Minocycline Involves Inhibition of MLK3-p38 Mitogen Activated Protein Kinase Axis.

PubMed

Jackson, Joseph W; Singh, Meera V; Singh, Vir B; Jones, Letitia D; Davidson, Gregory A; Ture, Sara; Morrell, Craig N; Schifitto, Giovanni; Maggirwar, Sanjay B

2016-01-01

Platelets play an essential role in hemostasis and wound healing by facilitating thrombus formation at sites of injury. Platelets also mediate inflammation and contain several pro-inflammatory molecules including cytokines and chemokines that mediate leukocyte recruitment and activation. Not surprisingly, platelet dysfunction is known to contribute to several inflammatory disorders. Antiplatelet therapies, such as aspirin, adenosine diphosphate (ADP) antagonists, glycoprotein IIb/IIIa (GPIIb/IIIa) inhibitors, and anticoagulants such as warfarin, dampen platelet activity at the risk of unwarranted bleeding. Thus, the development of drugs that reduce platelet-mediated inflammation without interfering with thrombus formation is of importance to combat platelet-associated disorders. We have shown here for the first time that the tetracycline antibiotic, minocycline, administered to HIV-infected individuals reduces plasma levels of soluble CD40L and platelet factor 4 levels, host molecules predominately released by platelets. Minocycline reduced the activation of isolated platelets in the presence of the potent platelet activator, thrombin, as measured by ELISA and flow cytometry. Platelet degranulation was reduced upon exposure to minocycline as shown by mepacrine retention and flow cytometry. However, minocycline had no effect on spreading, aggregation, GPIIb/IIIa activation, or in vivo thrombus formation. Lastly, immunoblot analysis suggests that the antiplatelet activity of minocycline is likely mediated by inhibition of mixed lineage kinase 3 (MLK3)-p38 MAPK signaling axis and loss of p38 activity. Our findings provide a better understanding of platelet biology and a novel repurposing of an established antibiotic, minocycline, to specifically reduce platelet granule release without affecting thrombosis, which may yield insights in generating novel, specific antiplatelet therapies.

The feed gas composition determines the degree of physical plasma-induced platelet activation for blood coagulation

NASA Astrophysics Data System (ADS)

Bekeschus, Sander; Brüggemeier, Janik; Hackbarth, Christine; Weltmann, Klaus-Dieter; von Woedtke, Thomas; Partecke, Lars-Ivo; van der Linde, Julia

2018-03-01

Cold atmospheric (physical) plasma has long been suggested to be a useful tool for blood coagulation. However, the clinical applicability of this approach has not been addressed sufficiently. We have previously demonstrated the ability of a clinically accepted atmospheric pressure argon plasma jet (kINPen® MED) to coagulate liver incisions in mice with similar performance compared to the gold standard electrocauterization. We could show that plasma-mediated blood coagulation was dependent on platelet activation. In the present work, we extended on this by investigating kINPen®-mediated platelet activation in anticoagulated human donor blood ex vivo. With focus on establishing high-throughput, multi-parametric platelet activation assays and performing argon feed gas parameter studies we achieved the following results: (i) plasma activated platelets in heparinized but not in EDTA-anticoagulated blood; (ii) plasma decreased total platelet counts but increased numbers of microparticles; (iii) plasma elevated the expression of several surface activation markers on platelets (CD62P, CD63, CD69, and CD41/61); (iv) in platelet activation, wet and dry argon plasma outperformed feed gas admixtures with oxygen and/or nitrogen; (v) plasma-mediated platelet activation was accompanied by platelet aggregation. Platelet aggregation is a necessary requirement for blood clot formation. These findings are important to further elucidate molecular details and clinical feasibility of cold physical plasma-mediated blood coagulation.

The Effects of Thrombin on Adenyl Cyclase Activity and a Membrane Protein from Human Platelets

PubMed Central

Brodie, G. N.; Baenziger, Nancy Lewis; Chase, Lewis R.; Majerus, Philip W.

1972-01-01

Washed human platelets were incubated with 0.1-1.0 U/ml human thrombin and the effects on adenyl cyclase activity and on a platelet membrane protein (designated thrombin-sensitive protein) were studied. Adenyl cyclase activity was decreased 70-90% when intact platelets were incubated with thrombin. The T½ for loss of adenyl cyclase activity was less than 15 sec at 1 U/ml thrombin. There was no decrease of adenyl cyclase activity when sonicated platelets or isolated membranes were incubated with these concentrations of thrombin. Loss of adenyl cyclase activity was relatively specific since the activities of other platelet membrane enzymes were unaffected by thrombin. Prior incubation of platelets with dibutyryl cyclic adenosine monophosphate (AMP), prostaglandin E1, or theophylline protected adenyl cyclase from inhibition by thrombin. Incubation of intact but not disrupted platelets with thrombin resulted in the release of thrombin-sensitive protein from the platelet membrane. The rapid release of this protein (T½ < 15 sec) at low concentrations of thrombin suggested that removal of thrombin-sensitive protein from the platelet membrane is an integral part of the platelet release reaction. This hypothesis is supported by the parallel effects of thrombin on adenyl cyclase activity and thrombin-sensitive protein release in the presence of dibutyryl cyclic AMP, prostaglandin E1, and theophylline at varying concentrations of thrombin. Images PMID:4331802

Blood clots are rapidly assembled hemodynamic sensors: flow arrest triggers intraluminal thrombus contraction.

PubMed

Muthard, Ryan W; Diamond, Scott L

2012-12-01

Blood clots form under flow during intravascular thrombosis or vessel leakage. Prevailing hemodynamics influence thrombus structure and may regulate contraction processes. A microfluidic device capable of flowing human blood over a side channel plugged with collagen (± tissue factor) was used to measure thrombus permeability (κ) and contraction at controlled transthrombus pressure drops. The collagen (κ(collagen)=1.98 × 10(-11) cm(2)) supported formation of a 20-µm thick platelet layer, which unexpectedly underwent massive platelet retraction on flow arrest. This contraction resulted in a 5.34-fold increase in permeability because of collagen restructuring. Without stopping flow, platelet deposits (no fibrin) had a permeability of κ(platelet)=5.45 × 10(-14) cm(2) and platelet-fibrin thrombi had κ(thrombus)=2.71 × 10(-14) cm(2) for ΔP=20.7 to 23.4 mm Hg, the first ever measurements for clots formed under arterial flow (1130 s(-1) wall shear rate). Platelet sensing of flow cessation triggered a 4.6- to 6.5-fold (n=3, P<0.05) increase in contraction rate, which was also observed in a rigid, impermeable parallel-plate microfluidic device. This triggered contraction was blocked by the myosin IIA inhibitor blebbistatin and by inhibitors of thromboxane A2 (TXA(2)) and ADP signaling. In addition, flow arrest triggered platelet intracellular calcium mobilization, which was blocked by TXA(2)/ADP inhibitors. As clots become occlusive or blood pools following vessel leakage, the flow diminishes, consequently allowing full platelet retraction. Flow dilution of ADP and thromboxane regulates platelet contractility with prevailing hemodynamics, a newly defined flow-sensing mechanism to regulate clot function.

The possible involvement of protein phosphatase 1 in thrombin-induced Ca2+ influx of human platelets.

PubMed

Murata, K; Sakon, M; Kambayashi, J; Yukawa, M; Yano, Y; Fujitani, K; Kawasaki, T; Shiba, E; Mori, T

1993-04-01

Protein phosphatase 1 is considered to be involved in thrombin-induced platelet activation (Murata et al., Biochem Int 26:327-334, 1992). To clarify the mechanism, we examined the effects of protein phosphatase 1 and 2A inhibitors (calyculin A, tautomycin, okadaic acid) on Ca2+ influx. In the presence of 1 mM Ca2+, thrombin- (0.1 U/ml) induced platelet aggregation and ATP release were inhibited by calyculin A, while this inhibitory effect was abolished in the absence of Ca2+ (EGTA 1 mM). Furthermore, thrombin-induced Mn2+ influx but not intracellular Ca2+ mobilization was inhibited by calyculin A in a dose-related manner. Calyculin A also blocked the ongoing Ca2+ influx when added 3 min after thrombin stimulation. Similar inhibitory effects were observed with okadaic acid and tautomycin in the same potency sequence as the reported one for protein phosphatase 1 (calyculin A > tautomycin > okadaic acid). These results suggest that the anti-platelet effects of phosphatase inhibitors are due to the inhibition of Ca2+ influx and that protein phosphatase 1 plays a key role in the regulation of receptor operated Ca2+ channel of human platelets.

Putative role of glycogen as a peripheral biomarker of GSK3β activity.

PubMed

Frizzo, Marcos Emilio

2013-09-01

Glycogen synthase kinase 3-β (GSK3β) has a pivotal role in several intracellular signaling cascades that are involved in gene transcription, cytoskeletal reorganization, energy metabolism, cell cycle regulation, and apoptosis. This kinase has pleiotropic functions, and the importance of its activity has recently been shown in neurons and platelets. In addition to its regulatory function in several physiological events, changes in GSK3β activity have been associated with many psychiatric and neurodegenerative illnesses, such as Alzheimer's disease, schizophrenia and autism-spectrum disorders. Beside the reports of its involvement in several pathologies, it has become increasingly apparent that GSK3β might be a common therapeutic target for different classes of psychiatric drugs, and also that the GSK3β ratio may be a useful parameter to determine the biochemical changes that might occur during antidepressant treatment. Although GSK3β is commonly described as a key enzyme in a plethora of signaling cascades, originally it was identified as playing an important role in the regulation of glycogen synthesis, given its ability to inactivate glycogen synthase (GS) by phosphorylation. Acting as a constitutively active kinase, GSK3β phosphorylates GS, which results in a decrease of glycogen production. GSK3β phosphorylation increases glycogen synthesis and storage, while its dephosphorylation decreases glycogen synthesis. Inactivation of GSK3β leads to dephosphorylation of GS and increase in glycogen synthesis in the adipose tissue, muscle and liver. Glycogen levels are reduced by antidepressant treatment, and this effect seems to be related to an effect of drugs on GSK3β activity. Peripherally, glycogen is also abundantly found in platelets, where it is considered a major energy source, required for a variety of its functions, including the release reaction. Recently, analysis of platelets from patients with late-life major depression showed that active forms of GSK3β expression were upregulated by continuous treatment with sertraline. Here, we hypothesized that the quantification of glycogen in platelets might be used as a peripheral biomarker of GSK3β activity. Since it has been recently demonstrated that the modulation of GSK3β activity causes changes in glycogen stores, the glycogen levels in platelets could be used to assay the effects of drugs that have this kinase as a target, or diseases where its activity is affected. In conclusion, we hypothesized that the determination of glycogen peripherally may be useful to indicate a change in the activity of this enzyme, providing a faster and non-invasive approach to guide the therapeutic procedures for the patient. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effect of steroids on the activation status of platelets in patients with Immune thrombocytopenia (ITP).

PubMed

Bhoria, Preeti; Sharma, Saniya; Varma, Neelam; Malhotra, Pankaj; Varma, Subhash; Luthra-Guptasarma, Manni

2015-01-01

The activation status of platelets in Immune Thrombocytopenia (ITP) patients--which is still somewhat controversial--is of potential interest, because activated platelets tend to aggregate (leading to excessive clotting or thromboembolic events) but cannot do so when platelet numbers are low, as in ITP. Although corticosteroids are the first line of therapy in ITP, the effect of steroids on activation of platelets has not been evaluated so far. We examined the status of platelet activation (with and without stimulation with ADP) in ITP patients, at the start of therapy (pre-steroid treatment, naive) and post-steroid treatment (classified on the basis of steroid responsiveness). We used flow cytometry to evaluate the levels of expression of P-selectin, and PAC-1 binding to platelets of 55 ITP patients and a similar number of healthy controls, treated with and without ADP. We found that platelets in ITP patients exist in an activated state. In patients who are responsive to steroids, the treatment reverses this situation. Also, the fold activation of platelets upon treatment with ADP is more in healthy controls than in ITP patients; treatment with steroids causes platelets in steroid-responsive patients to become more responsive to ADP-activation, similar to healthy controls. Thus steroids may cause changes in the ability of platelets to get activated with an agonist like ADP. Our results provide new insights into how, and why, steroid therapy helps in the treatment of ITP.

Effects of pathogen reduction systems on platelet microRNAs, mRNAs, activation, and function

PubMed Central

Osman, Abdimajid; Hitzler, Walter E.; Meyer, Claudius U.; Landry, Patricia; Corduan, Aurélie; Laffont, Benoit; Boilard, Eric; Hellstern, Peter; Vamvakas, Eleftherios C.

2015-01-01

Pathogen reduction (PR) systems for platelets, based on chemically induced cross-linking and inactivation of nucleic acids, potentially prevent transfusion transmission of infectious agents, but can increase clinically significant bleeding in some clinical studies. Here, we documented the effects of PR systems on microRNA and mRNA levels of platelets stored in the blood bank, and assessed their impact on platelet activation and function. Unlike platelets subjected to gamma irradiation or stored in additive solution, platelets treated with Intercept (amotosalen + ultraviolet-A [UVA] light) exhibited significantly reduced levels of 6 of the 11 microRNAs, and 2 of the 3 anti-apoptotic mRNAs (Bcl-xl and Clusterin) that we monitored, compared with platelets stored in plasma. Mirasol (riboflavin + UVB light) treatment of platelets did not produce these effects. PR neither affected platelet microRNA synthesis or function nor induced cross-linking of microRNA-sized endogenous platelet RNA species. However, the reduction in the platelet microRNA levels induced by Intercept correlated with the platelet activation (p < 0.05) and an impaired platelet aggregation response to ADP (p < 0.05). These results suggest that Intercept treatment may induce platelet activation, resulting in the release of microRNAs and mRNAs from platelets. The clinical implications of this reduction in platelet nucleic acids secondary to Intercept remain to be established. PMID:24749844

Existence of a microRNA pathway in anucleate platelets

PubMed Central

Landry, Patricia; Plante, Isabelle; Ouellet, Dominique L; Perron, Marjorie P; Rousseau, Guy; Provost, Patrick

2010-01-01

Platelets play a critical role in the maintenance of hemostasis as well as in thrombosis and vessel occlusion that underlie stroke and acute coronary syndromes. Anucleate platelets contain messenger RNAs (mRNAs) and are capable of protein synthesis, raising the issue of how these mRNAs are regulated. Here we show that human platelets harbor an abundant and diverse array of microRNAs (miRNAs), which are known as key regulators of mRNA translation. Further analyses revealed that platelets contain Dicer and Argonaute 2 (Ago2) complexes functional in exogenously supplied miRNA precursor (pre-miRNA) processing and the control of specific reporter transcripts, respectively. Detection of the receptor P2Y12 mRNA in Ago2 immunoprecipitates suggests that P2Y12 expression may be subjected to miRNA control in human platelets. Our study lends an additional level of complexity to the control of gene expression in these anucleate elements of the cardiovascular system. PMID:19668211

NOD2 Receptor is Expressed in Platelets and Enhances Platelet Activation and Thrombosis

PubMed Central

Zhang, Si; Zhang, Shenghui; Hu, Liang; Zhai, Lili; Xue, Ruyi; Ye, Jianqin; Chen, Leilei; Cheng, Guanjun; Mruk, Jozef; Kunapuli, Satya P.; Ding, Zhongren

2015-01-01

Background Pattern recognition receptor NOD2 (nucleotide binding oligomerization domain 2) is well investigated in immunity, its expression and function in platelets has never been explored. Method and Results Using RT-PCR and Western blot we show that both human and mouse platelets express NOD2, and its agonist MDP induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates platelet aggregation and secretion induced by low concentration of thrombin or collagen, as well as clot retraction. These potentiating effects of MDP were not seen in platelets from NOD2-deficient mice. Plasma from septic patients also potentiates platelet aggregation induced by thrombin or collagen NOD2-dependently. Using intravital microscopy, we found that MDP administration accelerated in vivo thrombosis in FeCl3-injured mesenteric arteriole thrombosis mouse model. Platelet depletion and transfusion experiments confirmed that NOD2 from platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. We further found that platelets express RIP2 (receptor-interacting protein 2), and provided evidences suggesting that MAPK and NO/sGC/cGMP/PGK pathways downstream of RIP2 mediate the role of NOD2 in platelets. Finally, MDP stimulates proinflammatory cytokine IL-1β maturation and accumulation in human and mouse platelets NOD2-dependently. Conclusions NOD2 is expressed in platelets and functions in platelet activation and arterial thrombosis, possibly during infection. To our knowledge, this is the first study on NOD-like receptors in platelets which links thrombotic events to inflammation. PMID:25825396

Enhanced levels of soluble CD40 ligand exacerbate platelet aggregation and thrombus formation through a CD40-dependent tumor necrosis factor receptor-associated factor-2/Rac1/p38 mitogen-activated protein kinase signaling pathway.

PubMed

Yacoub, Daniel; Hachem, Ahmed; Théorêt, Jean-François; Gillis, Marc-Antoine; Mourad, Walid; Merhi, Yahye

2010-12-01

CD40 ligand is a thromboinflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40 ligand (sCD40L), which has been shown to influence platelet activation, although its exact functional impact on platelets and the underlying mechanisms remain undefined. We aimed to determine the impact and the signaling mechanisms of sCD40L on platelets. sCD40L strongly enhances platelet activation and aggregation. Human platelets treated with a mutated form of sCD40L that does not bind CD40, and CD40(-/-) mouse platelets failed to elicit such responses. Furthermore, sCD40L stimulation induces the association of the tumor necrosis factor receptor-associated factor-2 with platelet CD40. Notably, sCD40L primes platelets through activation of the small GTPase Rac1 and its downstream target p38 mitogen-activated protein kinase, which leads to platelet shape change and actin polymerization. Moreover, sCD40L exacerbates thrombus formation and leukocyte infiltration in wild-type mice but not in CD40(-/-) mice. sCD40L enhances agonist-induced platelet activation and aggregation through a CD40-dependent tumor necrosis factor receptor-associated factor-2/Rac1/p38 mitogen-activated protein kinase signaling pathway. Thus, sCD40L is an important platelet primer predisposing platelets to enhanced thrombus formation in response to vascular injury. This may explain the link between circulating levels of sCD40L and cardiovascular diseases.

Effects of extracts and isolated compounds from safflower on some index of promoting blood circulation and regulating menstruation.

PubMed

Yao, Dong; Wang, Zheng; Miao, Li; Wang, Linyan

2016-09-15

Carthamus tinctorius is used as one of the Traditional Chinese Medicine (TCM) materials in prescriptions and composite to promote blood circulation to remove blood stasis, regulate menstruation and alleviate pain for over 2500 years. Modern pharmacological experiments have demonstrated that safflower has wide-reaching biological activities, including dilating coronary artery, modulating immune system, improving myocardial ischemia, anticoagulation and thromboprophylaxis, antioxidation, antihypoxic, antiaging, antifatigue, antiinflammation, anti-hepatic fibrosis, antitumor, analgesia, etc. Platelet aggregation of safflower extract and main constituents in safflower were determined by PAF-induced or ADP-induced platelet aggregation in vitro. Anticoagulation activity was measured by clotting assay of thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (APTT) according to the methods provided by the biological reagents provider (Sun Biochemical). Antioxidant effects of safflower were assessed using DPPH radical-scavenging activity test, ABTS radical-scavenging activity test and ferric reducing antioxidant power test. In addition, rats ovary granulosa cell proliferation activity was used for the bio-activity index on regulate menstruation of safflower. Safflower extract at the concentration of 0.7g/mL (P<0.001) and 0.5g/mL (P<0.01) had significantly antagonistic effect on PAF-induced platelet aggregation, compared with negative control. And the anti-platelet aggregation of 0.7g/mL safflower extract was significantly stronger than that of positive control (P<0.001). 0.7g/mL of hydroxysafflor yellow A (P<0.01), anhydrosafflor yellow B (P<0.05), 6-hydroxykaempferol-3-O-rutinoside (P<0.05), keampferol-3-O-β-rutinoside (P<0.01) had significant effect on platelet aggregation compared with negative control. Safflower extract at the concentration of 0.5g/mL (P<0.001) and 0.125g/mL (P<0.01) could significantly inhibit ADP-induced platelet aggregation, compared with negative control. And antagonistic effect of safflower extract was significantly stronger than the effect of positive control (P<0.001). Adenosine (P<0.001), anhydrosafflor yellow B (P<0.01) and 6-hydroxykaempferol-3-O-rutinoside (P<0.01) at the concentration of 0.5g/mL had significant effect on ADP-induced platelet aggregation compared with negative control. 0.125g/mL of adenosine (P<0.05) had significant effect on ADP-induced platelet aggregation compared with negative control. The effect of 0.5g/mL adenosine (P<0.01) and 6-hydroxykaempferol-3-O-rutinoside (P<0.05) was significantly stronger than that of positive control. Safflower extract at the concentration of 0.7mg/mL (P<0.001) and 0.5mg/mL (P<0.001) had significantly anticoagulation activity in PT, TT and APTT, compared with negative control. However, the respective compound didn't have significant effect on PT and TT at experiment concentration. At the concentration of 0.7mg/mL, hydroxysafflor yellow A (P<0.01), 6-hydroxykaempferol-3,6,7-tri-O-β-d-glucoside (P<0.05), 6-hydroxyapigenin-6-O-glucoside-7-O-glucuronide (P<0.01), anhydrosafflor yellow B (P<0.001), 6-hydroxykaempferol-3-O-rutinoside (P<0.05) and keampferol-3-O-β-rutinoside (P<0.05) significantly prolonged APTT, compared with negative control. At the concentration of 0.5mg/mL, hydroxysafflor yellow A (P<0.05), 6-hydroxyapigenin-6-O-glucoside-7-O-glucuronide (P<0.05), anhydrosafflor yellow B (P<0.001), 6-hydroxykaempferol-3-O-rutinoside (P<0.05) and keampferol-3-O-β-rutinoside (P<0.05) could significantly prolong APTT, compared with negative control. From the results of DPPH, ABTS radical scavenging activity test and Fe(3+) reduction power test, 5mg/mL, 2.5mg/mL and 1.25mg/mL safflower extract had antioxidant effects. Every compound with each concentration (5mg/mL, 2.5mg/mL and 1.25mg/mL) had significant effect on Fe(3+) reduction power (P<0.001 vs. negative control). Safflower extract, cytidine, 6-hydroxy-kaempferol-3,6-di-O-β-d-glucoside-7-O-β-d-glucuronide, 6-hydroxykaemp-ferol-3,6,7-tri-O-β-D-glucoside and keampferol-3-O-β-rutinoside significantly promoted ovarian granulosa cell proliferation. Based on previous researches, the activities of safflower extract and pure compounds isolated from safflower were studied in this paper. This study found some compounds with the effects of anti-platelet aggregation, anticoagulation, antioxidation and ovarian granulosa cell proliferation, and further revealed the possible pharmacological mechanism of safflower. Copyright © 2016. Published by Elsevier Ireland Ltd.

Antioxidants change platelet responses to various stimulating events

PubMed Central

Sobotková, Alžběta; Mášová-Chrastinová, Leona; Suttnar, Jiří; Štikarová, Jana; Májek, Pavel; Reicheltová, Zuzana; Kotlín, Roman; Weisel, John W.; Malý, Martin; Dyr, Jan E.

2010-01-01

The role of platelets in hemostasis may be influenced by alteration of the platelet redox state—the presence of antioxidants and the formation of reactive oxygen and nitrogen species. We investigated the effects of two antioxidants, resveratrol and trolox, on platelet activation. Trolox and resveratrol inhibited aggregation of washed platelets and platelet-rich plasma activated by ADP, collagen, and thrombin receptor-activating peptide. Resveratrol was a more effective agent in reducing platelet static and dynamic adhesion in comparison with trolox. The antioxidant capacity of resveratrol was, however, the same as that of trolox. After incubation of platelets with antioxidants, the resveratrol intraplatelet concentration was about five times lower than the intracellular concentration of trolox. Although both antioxidants comparably lowered hydroxyl radical and malondialdehyde production in platelets stimulated with collagen, TxB2 levels were decreased by resveratrol much more effectively than by trolox. Cyclooxygenase 1 was inhibited by resveratrol and not by trolox. Our data indicate that antioxidants, apart from nonspecific redox or radical-quenching mechanisms, inhibit platelet activation also by specific interaction with target proteins. The results also show the importance of studying platelet activation under conditions of real blood flow in contact with reactive surfaces, e.g., using dynamic adhesion experiments. PMID:19766712

Antioxidants change platelet responses to various stimulating events.

PubMed

Sobotková, Alzbeta; Másová-Chrastinová, Leona; Suttnar, Jirí; Stikarová, Jana; Májek, Pavel; Reicheltová, Zuzana; Kotlín, Roman; Weisel, John W; Malý, Martin; Dyr, Jan E

2009-12-15

The role of platelets in hemostasis may be influenced by alteration of the platelet redox state-the presence of antioxidants and the formation of reactive oxygen and nitrogen species. We investigated the effects of two antioxidants, resveratrol and trolox, on platelet activation. Trolox and resveratrol inhibited aggregation of washed platelets and platelet-rich plasma activated by ADP, collagen, and thrombin receptor-activating peptide. Resveratrol was a more effective agent in reducing platelet static and dynamic adhesion in comparison with trolox. The antioxidant capacity of resveratrol was, however, the same as that of trolox. After incubation of platelets with antioxidants, the resveratrol intraplatelet concentration was about five times lower than the intracellular concentration of trolox. Although both antioxidants comparably lowered hydroxyl radical and malondialdehyde production in platelets stimulated with collagen, TxB(2) levels were decreased by resveratrol much more effectively than by trolox. Cyclooxygenase 1 was inhibited by resveratrol and not by trolox. Our data indicate that antioxidants, apart from nonspecific redox or radical-quenching mechanisms, inhibit platelet activation also by specific interaction with target proteins. The results also show the importance of studying platelet activation under conditions of real blood flow in contact with reactive surfaces, e.g., using dynamic adhesion experiments.

Binary agonist surface patterns prime platelets for downstream adhesion in flowing whole blood.

PubMed

Eichinger, Colin D; Hlady, Vladimir

2017-04-28

As platelets encounter damaged vessels or biomaterials, they interact with a complex milieu of surface-bound agonists, from exposed subendothelium to adsorbed plasma proteins. It has been shown that an upstream, surface-immobilized agonist is capable of priming platelets for enhanced adhesion downstream. In this study, binary agonists were integrated into the upstream position of flow cells and the platelet priming response was measured by downstream adhesion in flowing whole blood. A nonadditive response was observed in which platelets transiently exposed to two agonists exhibited greater activation and downstream adhesion than that from the sum of either agonist alone. Antibody blocking of one of the two upstream agonists eliminated nonadditive activation and downstream adhesion. Crosstalk between platelet activation pathways likely led to a synergistic effect which created an enhanced activation response in the platelet population. The existence of synergy between platelet priming pathways is a concept that has broad implications for the field of biomaterials hemocompatibility and platelet activity testing.

Inhibition of the biosynthesis of prostaglandin E2 by low dose aspirin: implications for adenocarcinoma metastasis

PubMed Central

Boutaud, Olivier; Sosa, I. Romina; Amin, Taneem; Oram, Denise; Adler, David; Hwang, Hyun S.; Crews, Brenda C.; Milne, Ginger; Harris, Bradford K.; Hoeksema, Megan; Knollmann, Bjorn C.; Lammers, Philip E.; Marnett, Lawrence J.; Massion, Pierre P.; Oates, John A.

2016-01-01

Meta-analyses have demonstrated that low dose aspirin reduces the risk of developing adenocarcinoma metastasis, and when colon cancer is detected during aspirin treatment, there is a remarkable 83% reduction in risk of metastasis. As platelets participate in the metastatic process, the anti-platelet action of low dose aspirin likely contributes to its anti-metastatic effect. Cycloxooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) also contributes to metastasis, and we addressed the hypothesis that low dose aspirin also inhibits PGE2 biosynthesis. We show that low dose aspirin inhibits systemic PGE2 biosynthesis by 45% in healthy volunteers (p <0.0001). Aspirin is found to be more potent in colon adenocarcinoma cells than in the platelet, and in lung adenocarcinoma cells its inhibition is equivalent to that in the platelet. Inhibition of COX by aspirin in colon cancer cells is in the context of the metastasis of colon cancer primarily to the liver, the organ exposed to the same high concentrations of aspirin as the platelet. We find that the interaction of activated platelets with lung adenocarcinoma cells up-regulates COX-2 expression and PGE2 biosynthesis, and inhibition of platelet COX-1 by aspirin inhibits PGE2 production by the platelet-tumor cell aggregates. In conclusion, low dose aspirin has a significant effect on extraplatelet cyclooxygenase, and potently inhibits COX-2 in lung and colon adenocarcinoma cells. This supports a hypothesis that the remarkable prevention of metastasis from adenocarcinomas, and particularly from colon adenocarcinomas, by low dose aspirin results from its effect on platelet COX-1 combined with inhibition of PGE2 biosynthesis in metastasizing tumor cells. PMID:27554763

Phosphorothioate backbone modifications of nucleotide-based drugs are potent platelet activators

PubMed Central

Flierl, Ulrike; Nero, Tracy L.; Lim, Bock; Arthur, Jane F.; Yao, Yu; Jung, Stephanie M.; Gitz, Eelo; Pollitt, Alice Y.; Zaldivia, Maria T.K.; Jandrot-Perrus, Martine; Schäfer, Andreas; Nieswandt, Bernhard; Andrews, Robert K.; Parker, Michael W.; Gardiner, Elizabeth E.

2015-01-01

Nucleotide-based drug candidates such as antisense oligonucleotides, aptamers, immunoreceptor-activating nucleotides, or (anti)microRNAs hold great therapeutic promise for many human diseases. Phosphorothioate (PS) backbone modification of nucleotide-based drugs is common practice to protect these promising drug candidates from rapid degradation by plasma and intracellular nucleases. Effects of the changes in physicochemical properties associated with PS modification on platelets have not been elucidated so far. Here we report the unexpected binding of PS-modified oligonucleotides to platelets eliciting strong platelet activation, signaling, reactive oxygen species generation, adhesion, spreading, aggregation, and thrombus formation in vitro and in vivo. Mechanistically, the platelet-specific receptor glycoprotein VI (GPVI) mediates these platelet-activating effects. Notably, platelets from GPVI function–deficient patients do not exhibit binding of PS-modified oligonucleotides, and platelet activation is fully abolished. Our data demonstrate a novel, unexpected, PS backbone–dependent, platelet-activating effect of nucleotide-based drug candidates mediated by GPVI. This unforeseen effect should be considered in the ongoing development programs for the broad range of upcoming and promising DNA/RNA therapeutics. PMID:25646267

Modification of Pulsed Electric Field Conditions Results in Distinct Activation Profiles of Platelet-Rich Plasma.

PubMed

Frelinger, Andrew L; Gerrits, Anja J; Garner, Allen L; Torres, Andrew S; Caiafa, Antonio; Morton, Christine A; Berny-Lang, Michelle A; Carmichael, Sabrina L; Neculaes, V Bogdan; Michelson, Alan D

2016-01-01

Activated autologous platelet-rich plasma (PRP) used in therapeutic wound healing applications is poorly characterized and standardized. Using pulsed electric fields (PEF) to activate platelets may reduce variability and eliminate complications associated with the use of bovine thrombin. We previously reported that exposing PRP to sub-microsecond duration, high electric field (SMHEF) pulses generates a greater number of platelet-derived microparticles, increased expression of prothrombotic platelet surfaces, and differential release of growth factors compared to thrombin. Moreover, the platelet releasate produced by SMHEF pulses induced greater cell proliferation than plasma. To determine whether sub-microsecond duration, low electric field (SMLEF) bipolar pulses results in differential activation of PRP compared to SMHEF, with respect to profiles of activation markers, growth factor release, and cell proliferation capacity. PRP activation by SMLEF bipolar pulses was compared to SMHEF pulses and bovine thrombin. PRP was prepared using the Harvest SmartPreP2 System from acid citrate dextrose anticoagulated healthy donor blood. PEF activation by either SMHEF or SMLEF pulses was performed using a standard electroporation cuvette preloaded with CaCl2 and a prototype instrument designed to take into account the electrical properties of PRP. Flow cytometry was used to assess platelet surface P-selectin expression, and annexin V binding. Platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF) and platelet factor 4 (PF4), and were measured by ELISA. The ability of supernatants to stimulate proliferation of human epithelial cells in culture was also evaluated. Controls included vehicle-treated, unactivated PRP and PRP with 10 mM CaCl2 activated with 1 U/mL bovine thrombin. PRP activated with SMLEF bipolar pulses or thrombin had similar light scatter profiles, consistent with the presence of platelet-derived microparticles, platelets, and platelet aggregates whereas SMHEF pulses primarily resulted in platelet-derived microparticles. Microparticles and platelets in PRP activated with SMLEF bipolar pulses had significantly lower annexin V-positivity than those following SMHEF activation. In contrast, the % P-selectin positivity and surface P-selectin expression (MFI) for platelets and microparticles in SMLEF bipolar pulse activated PRP was significantly higher than that in SMHEF-activated PRP, but not significantly different from that produced by thrombin activation. Higher levels of EGF were observed following either SMLEF bipolar pulses or SMHEF pulses of PRP than after bovine thrombin activation while VEGF, PDGF, and PF4 levels were similar with all three activating conditions. Cell proliferation was significantly increased by releasates of both SMLEF bipolar pulse and SMHEF pulse activated PRP compared to plasma alone. PEF activation of PRP at bipolar low vs. monopolar high field strength results in differential platelet-derived microparticle production and activation of platelet surface procoagulant markers while inducing similar release of growth factors and similar capacity to induce cell proliferation. Stimulation of PRP with SMLEF bipolar pulses is gentler than SMHEF pulses, resulting in less platelet microparticle generation but with overall activation levels similar to that obtained with thrombin. These results suggest that PEF provides the means to alter, in a controlled fashion, PRP properties thereby enabling evaluation of their effects on wound healing and clinical outcomes.

Ultrastructural characteristics of fibrin clots from canine and feline platelet concentrates activated with calcium gluconate or calcium gluconate plus batroxobin.

PubMed

Silva, Raúl F; Carmona, Jorge U; Rezende, Cleuza M F

2013-04-15

The aim of this study was to use transmission electron microscopy to describe the ultrastructural characteristics of clots obtained from canine and feline platelet concentrates (PC) that had been activated with calcium gluconate (CG) or CG plus batroxobin (CGB). Platelets from fibrin clots were classified according their morphological changes. The area of the intercellular space (μm2), the area of the fibrin fibers (μm2), and the width of the fibrin fibers (μm) were determined for the dog clots. The platelet area (μm2), the area of fibrin fibers (μm2), the ratio of the minor and major axes of platelets, the ratio of the major and minor axes of platelets, and the number of α-granules found within platelets were measured for the cat clots. Cat platelets displayed full activation. Dog platelets displayed lysis with loss of normal architecture. In both species, a statistically significant difference was found (P < 0.01) between the fibrin fiber measurements in the PC clots activated with CG and CGB. The findings suggest that activation with CG caused platelet alpha granules to release their contents. In cats, fibrin production was greater when the PC was activated with CG. In dogs, activation with CG produced thick fibrin fibers.

Associations of MDR1, TBXA2R, PLA2G7, and PEAR1 genetic polymorphisms with the platelet activity in Chinese ischemic stroke patients receiving aspirin therapy.

PubMed

Peng, Ling-Ling; Zhao, Yuan-Qi; Zhou, Zi-Yi; Jin, Jing; Zhao, Min; Chen, Xin-Meng; Chen, Ling-Yan; Cai, Ye-Feng; Li, Jia-Li; Huang, Min

2016-11-01

Aspirin resistance has an incidence of 5%-65% in patients with ischemic stroke, who receive the standard dose of aspirin, but the platelet function is inadequately inhibited, thereby leading to thrombotic events. Numerous evidence shows that thromboxane A 2 receptor (TXA 2 receptor, encoded by TBXA2R), lipoprotein-associated phospholipase A 2 (Lp-PLA 2 , encoded by PLA2G7) and platelet endothelial aggregation receptor-1 (PEAR1, encoded by PEAR1) are crucial in regulating platelet activation, and P-glycoprotein (P-gp, encoded by MDR1) influences the absorption of aspirin in the intestine. In this study we examined the correlation between MDR1, TBXA2R, PLA2G7, PEAR1 genetic polymorphisms and platelet activity in Chinese ischemic stroke patients receiving aspirin therapy. A total of 283 ischemic stroke patients receiving 100 mg aspirin for 7 d were genotyped for polymorphisms in MDR1 C3435T, TBXA2R (rs1131882), PLA2G7 (rs1051931, rs7756935), and PEAR1 (rs12566888, rs12041331). The platelet aggregation response was measured using an automatic platelet aggregation analyzer and a commercially available TXB 2 ELISA kit. Thirty-three patients (11.66%) were insensitive to aspirin treatment. MDR1 3435TT genotype carriers, whose arachidonic acid (AA) or adenosine diphosphate (ADP)-induced platelet aggregation was lower than that of CC+CT genotype carriers, were less likely to suffer from aspirin resistance (odds ratio=0.421, 95% CI: 0.233-0.759). The TBXA2R rs1131882 CC genotype, which was found more frequently in the aspirin-insensitive group (81.8% vs 62.4%) than in the sensitive group, was identified as a risk factor for aspirin resistance (odds ratio=2.712, 95% CI: 1.080-6.810) with a higher level of AA-induced platelet aggregation. Due to the combined effects of PLA2G7 rs1051931 and rs7756935, carriers of the AA-CC haplotype had a higher level of ADP-induced platelet aggregation, and were at considerably higher risk of aspirin resistance than noncarriers (odds ratio=8.233, 95% CI: 1.590-42.638). A considerable portion (11.66%) of Chinese ischemic stroke patients are insensitive to aspirin treatment, which may be correlated with the MDR1 C3435T, TBXA2R (rs1131882), and PLA2G7 (rs1051931-rs7756935) polymorphisms.

Platelet-derived microparticles regulates thrombin generation via phophatidylserine in abdominal sepsis.

PubMed

Wang, Yongzhi; Zhang, Su; Luo, Lingtao; Norström, Eva; Braun, Oscar Ö; Mörgelin, Matthias; Thorlacius, Henrik

2018-02-01

Sepsis is associated with dysfunctional coagulation. Recent data suggest that platelets play a role in sepsis by promoting neutrophil accumulation. Herein, we show that cecal ligation and puncture (CLP) triggered systemic inflammation, which is characterized by formation of IL-6 and CXC chemokines as well as neutrophil accumulation in the lung. Platelet depletion decreased neutrophil accumulation, IL-6, and CXC chemokines formation in septic lungs. Depletion of platelets increased peak thrombin formation and total thrombin generation (TG) in plasma from septic animals. CLP elevated circulating levels of platelet-derived microparticles (PMPs). In vitro generated PMPs were a potent inducer of TG. Interestingly, in vitro wild-type recombinant annexin V abolished PMP-induced thrombin formation whereas a mutant annexin V protein, which does not bind to phosphatidylserine (PS), had no effect. Administration of wild-type, but not mutant annexin V, significantly inhibited thrombin formation in septic animals. Moreover, CLP-induced formation of thrombin-antithrombin complexes were reduced in platelet-depleted mice and in animals pretreated with annexin V. PMP-induced TG attenuated in FXII- and FVII-deficient plasma. These findings suggest that sepsis-induced TG is dependent on platelets. Moreover, PMPs formed in sepsis are a potent inducer of TG via PS exposure, and activation of both the intrinsic and extrinsic pathway of coagulation. In conclusion, these observations suggest that PMPs and PS play an important role in dysfunctional coagulation in abdominal sepsis. © 2017 Wiley Periodicals, Inc.

A genetically-engineered von Willebrand disease type 2B mouse model displays defects in hemostasis and inflammation.

PubMed

Adam, Frédéric; Casari, Caterina; Prévost, Nicolas; Kauskot, Alexandre; Loubière, Cécile; Legendre, Paulette; Repérant, Christelle; Baruch, Dominique; Rosa, Jean-Philippe; Bryckaert, Marijke; de Groot, Philip G; Christophe, Olivier D; Lenting, Peter J; Denis, Cécile V

2016-05-23

von Willebrand disease (VWD)-type 2B is characterized by gain-of-function mutations in the von Willebrand factor (VWF) A1-domain, leading to increased affinity for its platelet-receptor, glycoprotein Ibα. We engineered the first knock-in (KI) murine model for VWD-type 2B by introducing the p.V1316M mutation in murine VWF. Homozygous KI-mice replicated human VWD-type 2B with macrothrombocytopenia (platelet counts reduced by 55%, platelet volume increased by 44%), circulating platelet-aggregates and a severe bleeding tendency. Also, vessel occlusion was deficient in the FeCl3-induced thrombosis model. Platelet aggregation induced by thrombin or collagen was defective for KI-mice at all doses. KI-mice manifested a loss of high molecular weight multimers and increased multimer degradation. In a model of VWF-string formation, the number of platelets/string and string-lifetime were surprisingly enhanced in KI-mice, suggesting that proteolysis of VWF/p.V1316M is differentially regulated in the circulation versus the endothelial surface. Furthermore, we observed increased leukocyte recruitment during an inflammatory response induced by the reverse passive Arthus reaction. This points to an active role of VWF/p.V1316M in the exfiltration of leukocytes under inflammatory conditions. In conclusion, our genetically-engineered VWD-type 2B mice represent an original model to study the consequences of spontaneous VWF-platelet interactions and the physiopathology of this human disease.

PPARbeta/delta agonists modulate platelet function via a mechanism involving PPAR receptors and specific association/repression of PKCalpha--brief report.

PubMed

Ali, Ferhana Y; Hall, Matthew G; Desvergne, Béatrice; Warner, Timothy D; Mitchell, Jane A

2009-11-01

Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) is a nuclear receptor found in platelets. PPARbeta/delta agonists acutely inhibit platelet function within a few minutes of addition. As platelets are anucleated, the effects of PPARbeta/delta agonists on platelets must be nongenomic. Currently, the particular role of PPARbeta/delta receptors and their intracellular signaling pathways in platelets are not known. We have used mice lacking PPARbeta/delta (PPARbeta/delta(-/-)) to show the effects of the PPARbeta/delta agonist GW501516 on platelet adhesion and cAMP levels are mediated specifically by PPARbeta/delta, however GW501516 had no PPARbeta/delta-specific effect on platelet aggregation. Studies in human platelets showed that PKCalpha, which can mediate platelet activation, was bound and repressed by PPARbeta/delta after platelets were treated with GW501516. These data provide evidence of a novel mechanism by which PPAR receptors influence platelet activity and thereby thrombotic risk.

Role of thrombin signalling in platelets in haemostasis and thrombosis

NASA Astrophysics Data System (ADS)

Sambrano, Gilberto R.; Weiss, Ethan J.; Zheng, Yao-Wu; Huang, Wei; Coughlin, Shaun R.

2001-09-01

Platelets are critical in haemostasis and in arterial thrombosis, which causes heart attacks and other events triggered by abnormal clotting. The coagulation protease thrombin is a potent activator of platelets ex vivo. However, because thrombin also mediates fibrin deposition and because multiple agonists can trigger platelet activation, the relative importance of platelet activation by thrombin in haemostasis and thrombosis is unknown. Thrombin triggers cellular responses at least in part through protease-activated receptors (PARs). Mouse platelets express PAR3 and PAR4 (ref. 9). Here we show that platelets from PAR4-deficient mice failed to change shape, mobilize calcium, secrete ATP or aggregate in response to thrombin. This result demonstrates that PAR signalling is necessary for mouse platelet activation by thrombin and supports the model that mouse PAR3 (mPAR3) does not by itself mediate transmembrane signalling but instead acts as a cofactor for thrombin cleavage and activation of mPAR4 (ref. 10). Importantly, PAR4-deficient mice had markedly prolonged bleeding times and were protected in a model of arteriolar thrombosis. Thus platelet activation by thrombin is necessary for normal haemostasis and may be an important target in the treatment of thrombosis.

[Role of nitric oxide as a regulator of cell processes in the formation of multiple organ failure].

PubMed

Riabov, G A; Azisov, Iu M

2001-01-01

Main aspects of functional activity of nitric oxide (NO) are discussed. Physicochemical properties of NO, routes of its formation in man, and mechanism of its effects on physiological processes are described. In human body NO is formed as a result of activity of a specific enzyme, nitric oxide synthase. Three isoforms of the enzyme are known: neuronal, inducible, and endothelial. NO regulates vascular tone, cell adhesion, neurotransmission, bronchodilatation, and platelet aggregation. NO can protect and damage cells under different conditions. The effect of NO can be direct and mediated. Mechanisms of vasodilating effect of NO and of its effect on apoptosis are discussed. The role of NO in regulation of the functional activity of hepatocytes is described. Regulation of NO level in human organism is discussed.

Synergistic effect of signaling from receptors of soluble platelet agonists and outside-in signaling in formation of a stable fibrinogen-integrin αIIbβ3-actin cytoskeleton complex.

PubMed

Budnik, Ivan; Shenkman, Boris; Savion, Naphtali

2015-01-01

Thrombus formation in the injured vessel wall is a highly complex process involving various blood-born components that go through specific temporal and spatial changes as observed by intravital videomicroscopy. Platelets bind transiently to the developing thrombus and may either become stably incorporated into or disengage from the thrombus. The aim of the present study was to reveal the processes involved in the formation of a stable thrombus. Platelet-rich plasma and washed platelets were studied by the aggregometer. The aggregate stability was challenged by eptifibatide. Platelet Triton-insoluble fraction was prepared and the actin and αIIb content in the cytoskeleton was analyzed by western blot. Maximal actin polymerization is achieved 1min after platelet activation while maximal αIIbβ3-actin cytoskeleton association requires 5 to 10min of activation and fibrinogen-mediated platelet-to-platelet bridging. Thus, actin polymerization is dependent on platelet activation and requires neither αIIbβ3 integrin occupation nor platelet aggregation. Formation of a stable aggregate requires platelet activation for more than 1min, complete increase in actin cytoskeleton fraction and partial association of αIIbβ3 with the actin cytoskeleton. However, direct αIIbβ3 activation is not sufficient for cytoskeleton complex formation. Thus, stable αIIbβ3-fibrinogen interaction, representing stable aggregate, is achieved after more than 1min agonist activation, involving inside-out and outside-in signaling but not after direct integrin activation, involving only outside-in signaling. Formation of a stable fibrinogen-αIIbβ3-actin cytoskeleton complex is the result of the combined effect of platelet stimulation by soluble agonists, activation of αIIbβ3, fibrinogen binding and platelet-to-platelet bridging. Copyright © 2014 Elsevier Ltd. All rights reserved.

Two distinct roles of mitogen-activated protein kinases in platelets and a novel Rac1-MAPK–dependent integrin outside-in retractile signaling pathway

PubMed Central

Flevaris, Panagiotis; Li, Zhenyu; Zhang, Guoying; Zheng, Yi; Liu, Junling

2009-01-01

Mitogen-activated protein kinases (MAPK), p38, and extracellular stimuli-responsive kinase (ERK), are acutely but transiently activated in platelets by platelet agonists, and the agonist-induced platelet MAPK activation is inhibited by ligand binding to the integrin αIIbβ3. Here we show that, although the activation of MAPK, as indicated by MAPK phosphorylation, is initially inhibited after ligand binding to integrin αIIbβ3, integrin outside-insignaling results in a late but sustained activation of MAPKs in platelets. Furthermore, we show that the early agonist-induced MAPK activation and the late integrin-mediated MAPK activation play distinct roles in different stages of platelet activation. Agonist-induced MAPK activation primarily plays an important role in stimulating secretion of platelet granules, while integrin-mediated MAPK activation is important in facilitating clot retraction. The stimulatory role of MAPK in clot retraction is mediated by stimulating myosin light chain (MLC) phosphorylation. Importantly, integrin-dependent MAPK activation, MAPK-dependent MLC phosphorylation, and clot retraction are inhibited by a Rac1 inhibitor and in Rac1 knockout platelets, indicating that integrin-induced activation of MAPK and MLC and subsequent clot retraction is Rac1-dependent. Thus, our results reveal 2 different activation mechanisms of MAPKs that are involved in distinct aspects of platelet function and a novel Rac1-MAPK–dependent cell retractile signaling pathway. PMID:18957688

hnRNP L regulates differences in expression of mouse integrin alpha2beta1.

PubMed

Cheli, Yann; Kunicki, Thomas J

2006-06-01

There is a 2-fold variation in platelet integrin alpha2beta1 levels among inbred mouse strains. Decreased alpha2beta1 in 4 strains carrying Itga2 haplotype 2 results from decreased affinity of heterogeneous ribonucleoprotein L (hnRNP L) for a 6 CA repeat sequence (CA6) within intron 1. Seven strains bearing haplotype 1 and a 21 CA repeat sequence at this position (CA21) express twice the level of platelet alpha2beta1 and exhibit an equivalent gain of platelet function in vitro. By UV crosslinking and immunoprecipitation, hnRNP L binds more avidly to CA21, relative to CA6. By cell-free, in vitro mRNA splicing, decreased binding of hnRNP L results in decreased splicing efficiency and an increased proportion of alternatively spliced product. The splicing enhancer activity of CA21 in vivo is abolished by prior treatment with hnRNP L-specific siRNA. Thus, decreased surface alpha2beta1 results from decreased Itga2 pre-mRNA splicing regulated by hnRNP L and depends on CA repeat length at a specific site in intron 1.

hnRNP L regulates differences in expression of mouse integrin α2β1

PubMed Central

Cheli, Yann; Kunicki, Thomas J.

2006-01-01

There is a 2-fold variation in platelet integrin α2β1 levels among inbred mouse strains. Decreased α2β1 in 4 strains carrying Itga2 haplotype 2 results from decreased affinity of heterogeneous ribonucleoprotein L (hnRNP L) for a 6 CA repeat sequence (CA6) within intron 1. Seven strains bearing haplotype 1 and a 21 CA repeat sequence at this position (CA21) express twice the level of platelet α2β1 and exhibit an equivalent gain of platelet function in vitro. By UV crosslinking and immunoprecipitation, hnRNP L binds more avidly to CA21, relative to CA6. By cell-free, in vitro mRNA splicing, decreased binding of hnRNP L results in decreased splicing efficiency and an increased proportion of alternatively spliced product. The splicing enhancer activity of CA21 in vivo is abolished by prior treatment with hnRNP L–specific siRNA. Thus, decreased surface α2β1 results from decreased Itga2 pre-mRNA splicing regulated by hnRNP L and depends on CA repeat length at a specific site in intron 1. PMID:16455949

Generation of thromboxane A2 and aorta-contracting activity from platelets stimulated with modified C-reactive protein.

PubMed Central

Simpson, R M; Prancan, A; Izzi, J M; Fiedel, B A

1982-01-01

The classical acute phase reactant, C-reactive protein (CRP), appears in markedly elevated concentration in the sera of individuals undergoing reactions of acute inflammation and tissue degradation. We previously demonstrated that like IgG, appropriately purified CRP could be thermally modified (H-CRP) such that it enhanced platelet activation in plasma and initiated platelet responses in isolated systems. We now report that this direct platelet activation by modified CRP results in the secretion of both platelet dense body and alpha-granule constituents, and is sensitive to non-steroidal anti-inflammatory drugs as well as the adenosine diphosphate (ADP)-removing enzyme system creatine phosphate/creatine phosphokinase. Thin-layer chromatographic (TLC) analysis of prostanoate endproducts following platelet activation with H-CRP revealed the formation of thromboxane B2 (the hydrated endproduct of thromboxane A2), an important endogenous platelet activator and contractor of vascular tissue; bioassay on rabbit aorta strips of supernatants obtained from platelets undergoing challenge with H-CRP supported the TLC analysis. Complexes formed between CRP and one major ligand, the polycation, were found to share certain platelet activating properties with H-CRP, as does latex-aggregated CRP. These data imply a potential agonist role for this acute phase reactant in platelet physiology and suggest that the interaction of modified forms of CRP with the platelet at sites of vascular damage could have pathological significance. PMID:7118160

Mechanism of platelet activation induced by endocannabinoids in blood and plasma.

PubMed

Brantl, S Annette; Khandoga, Anna L; Siess, Wolfgang

2014-01-01

Platelets play a central role in atherosclerosis and atherothrombosis, and circulating endocannabinoids might modulate platelet function. Previous studies concerning effects of anandamide (N-arachidonylethanolamide) and 2-arachidonoylglycerol (2-AG) on platelets, mainly performed on isolated cells, provided conflicting results. We therefore investigated the action of three main endocannabinoids [anandamide, 2-AG and virodhamine (arachidonoylethanolamine)] on human platelets in blood and platelet-rich plasma (PRP). 2-AG and virodhamine induced platelet aggregation in blood, and shape change, aggregation and adenosine triphosphate (ATP) secretion in PRP. The EC50 of 2-AG and virodhamine for platelet aggregation in blood was 97 and 160 µM, respectively. Lower concentrations of 2-AG (20 µM) and virodhamine (50 µM) synergistically induced aggregation with other platelet stimuli. Platelet activation induced by 2-AG and virodhamine resembled arachidonic acid (AA)-induced aggregation: shape change, the first platelet response, ATP secretion and aggregation induced by 2-AG and virodhamine were all blocked by acetylsalicylic acid (ASA) or the specific thromboxane A2 (TXA2) antagonist daltroban. In addition, platelet activation induced by 2-AG and virodhamine in blood and PRP were inhibited by JZL184, a selective inhibitor of monoacylglycerol lipase (MAGL). In contrast to 2-AG and virodhamine, anandamide, a substrate of fatty acid amidohydrolase, was inactive. Synthetic cannabinoid receptor subtype 1 (CB1) and 2 (CB2) agonists lacked stimulatory as well as inhibitory platelet activity. We conclude that 2-AG and virodhamine stimulate platelets in blood and PRP by a MAGL-triggered mechanism leading to free AA and its metabolism by platelet cyclooxygenase-1/thromboxane synthase to TXA2. CB1, CB2 or non-CB1/CB2 receptors are not involved. Our results imply that ASA and MAGL inhibitors will protect platelets from activation by high endocannabinoid levels, and that pharmacological CB1- and CB2-receptor ligands will not affect platelets and platelet-dependent progression and complications of cardiovascular diseases.

Platelet–neutrophil interactions under thromboinflammatory conditions

PubMed Central

Li, Jing; Kim, Kyungho; Barazia, Andrew; Tseng, Alan

2015-01-01

Platelets primarily mediate hemostasis and thrombosis, whereas leukocytes are responsible for immune responses. Since platelets interact with leukocytes at the site of vascular injury, thrombosis and vascular inflammation are closely intertwined and occur consecutively. Recent studies using real-time imaging technology demonstrated that platelet–neutrophil interactions on the activated endothelium are an important determinant of microvascular occlusion during thromboinflammatory disease in which inflammation is coupled to thrombosis. Although the major receptors and counter receptors have been identified, it remains poorly understood how heterotypic platelet–neutrophil interactions are regulated under disease conditions. This review discusses our current understanding of the regulatory mechanisms of platelet– neutrophil interactions in thromboinflammatory disease. PMID:25650236

Protein kinase C activates non-capacitative calcium entry in human platelets

PubMed Central

Rosado, Juan A; Sage, Stewart O

2000-01-01

In many non-excitable cells Ca2+ influx is mainly controlled by the filling state of the intracellular Ca2+ stores. It has been suggested that this store-mediated or capacitative Ca2+ entry is brought about by a physical and reversible coupling of the endoplasmic reticulum with the plasma membrane. Here we provide evidence for an additional, non-capacitative Ca2+ entry mechanism in human platelets. Changes in cytosolic Ca2+ and Sr2+ were measured in human platelets loaded with the fluorescent indicator fura-2. Depletion of the internal Ca2+ stores with thapsigargin plus a low concentration of ionomycin stimulated store-mediated cation entry, as demonstrated upon Ca2+ or Sr2+ addition. Subsequent treatment with thrombin stimulated further divalent cation entry in a concentration-dependent manner. Direct activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol also stimulated divalent cation entry, without evoking the release of Ca2+ from intracellular stores. Cation entry evoked by thrombin or activators of PKC was abolished by the PKC inhibitor Ro-31-8220. Unlike store-mediated Ca2+ entry, jasplakinolide, which reorganises actin filaments into a tight cortical layer adjacent to the plasma membrane, did not inhibit divalent cation influx evoked by thrombin when applied after Ca2+ store depletion, or by activators of PKC. Thrombin also activated Ca2+ entry in platelets in which the release from intracellular stores and store-mediated Ca2+ entry were blocked by xestospongin C. These results indicate that the non-capacitative divalent cation entry pathway is regulated independently of store-mediated entry and does not require coupling of the endoplasmic reticulum and the plasma membrane. These results support the existence of a mechanism for receptor-evoked Ca2+ entry in human platelets that is independent of Ca2+ store depletion. This Ca2+ entry mechanism may be activated by occupation of G-protein-coupled receptors, which activate PKC, or by direct activation of PKC, thus generating non-capacitative Ca2+ entry alongside that evoked following the release of Ca2+ from the intracellular stores. PMID:11080259

Secreted Immunomodulatory Proteins of Staphylococcus aureus Activate Platelets and Induce Platelet Aggregation.

PubMed

Binsker, Ulrike; Palankar, Raghavendra; Wesche, Jan; Kohler, Thomas P; Prucha, Josephine; Burchhardt, Gerhard; Rohde, Manfred; Schmidt, Frank; Bröker, Barbara M; Mamat, Uwe; Pané-Farré, Jan; Graf, Anica; Ebner, Patrick; Greinacher, Andreas; Hammerschmidt, Sven

2018-04-01

Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection. Schattauer GmbH Stuttgart.

PKCδ Regulates Force Signaling during VEGF/CXCL4 Induced Dissociation of Endothelial Tubes

PubMed Central

Jamison, Joshua; Wang, James H-C.; Wells, Alan

2014-01-01

Wound healing requires the vasculature to re-establish itself from the severed ends; endothelial cells within capillaries must detach from neighboring cells before they can migrate into the nascent wound bed to initiate angiogenesis. The dissociation of these endothelial capillaries is driven partially by platelets' release of growth factors and cytokines, particularly the chemokine CXCL4/platelet factor-4 (PF4) that increases cell-cell de-adherence. As this retraction is partly mediated by increased transcellular contractility, the protein kinase c-δ/myosin light chain-2 (PKCδ/MLC-2) signaling axis becomes a candidate mechanism to drive endothelial dissociation. We hypothesize that PKCδ activation induces contractility through MLC-2 to promote dissociation of endothelial cords after exposure to platelet-released CXCL4 and VEGF. To investigate this mechanism of contractility, endothelial cells were allowed to form cords following CXCL4 addition to perpetuate cord dissociation. In this study, CXCL4-induced dissociation was reduced by a VEGFR inhibitor (sunitinib malate) and/or PKCδ inhibition. During combined CXCL4+VEGF treatment, increased contractility mediated by MLC-2 that is dependent on PKCδ regulation. As cellular force is transmitted to focal adhesions, zyxin, a focal adhesion protein that is mechano-responsive, was upregulated after PKCδ inhibition. This study suggests that growth factor regulation of PKCδ may be involved in CXCL4-mediated dissociation of endothelial cords. PMID:24699667

PKCδ regulates force signaling during VEGF/CXCL4 induced dissociation of endothelial tubes.

PubMed

Jamison, Joshua; Wang, James H-C; Wells, Alan

2014-01-01

Wound healing requires the vasculature to re-establish itself from the severed ends; endothelial cells within capillaries must detach from neighboring cells before they can migrate into the nascent wound bed to initiate angiogenesis. The dissociation of these endothelial capillaries is driven partially by platelets' release of growth factors and cytokines, particularly the chemokine CXCL4/platelet factor-4 (PF4) that increases cell-cell de-adherence. As this retraction is partly mediated by increased transcellular contractility, the protein kinase c-δ/myosin light chain-2 (PKCδ/MLC-2) signaling axis becomes a candidate mechanism to drive endothelial dissociation. We hypothesize that PKCδ activation induces contractility through MLC-2 to promote dissociation of endothelial cords after exposure to platelet-released CXCL4 and VEGF. To investigate this mechanism of contractility, endothelial cells were allowed to form cords following CXCL4 addition to perpetuate cord dissociation. In this study, CXCL4-induced dissociation was reduced by a VEGFR inhibitor (sunitinib malate) and/or PKCδ inhibition. During combined CXCL4+VEGF treatment, increased contractility mediated by MLC-2 that is dependent on PKCδ regulation. As cellular force is transmitted to focal adhesions, zyxin, a focal adhesion protein that is mechano-responsive, was upregulated after PKCδ inhibition. This study suggests that growth factor regulation of PKCδ may be involved in CXCL4-mediated dissociation of endothelial cords.

Platelets from patients with the Quebec platelet disorder contain and secrete abnormal amounts of urokinase-type plasminogen activator.

PubMed

Kahr, W H; Zheng, S; Sheth, P M; Pai, M; Cowie, A; Bouchard, M; Podor, T J; Rivard, G E; Hayward, C P

2001-07-15

The Quebec platelet disorder (QPD) is an autosomal dominant platelet disorder associated with delayed bleeding and alpha-granule protein degradation. The degradation of alpha-granule, but not plasma, fibrinogen in patients with the QPD led to the investigation of their platelets for a protease defect. Unlike normal platelets, QPD platelets contained large amounts of fibrinolytic serine proteases that had properties of plasminogen activators. Western blot analysis, zymography, and immunodepletion experiments indicated this was because QPD platelets contained large amounts of urokinase-type plasminogen activator (u-PA) within a secretory compartment. u-PA antigen was not increased in all QPD plasmas, whereas it was increased more than 100-fold in QPD platelets (P <.00009), which contained increased u-PA messenger RNA. Although QPD platelets contained 2-fold more plasminogen activator inhibitor 1 (PAI-1) (P <.0008) and 100-fold greater u-PA-PAI-1 complexes (P <.0002) than normal platelets, they contained excess u-PA activity, predominantly in the form of two chain (tcu-PA), which required additional PAI-1 for full inhibition. There was associated proteolysis of plasminogen in QPD platelets, to forms that comigrated with plasmin. When similar amounts of tcu-PA were incubated with normal platelet secretory proteins, many alpha-granule proteins were proteolyzed to forms that resembled degraded QPD platelet proteins. These data implicate u-PA in the pathogenesis of alpha-granule protein degradation in the QPD. Although patients with the QPD have normal to increased u-PA levels in their plasma, without evidence of systemic fibrinogenolysis, their increased platelet u-PA could contribute to bleeding by accelerating fibrinolysis within the hemostatic plug. QPD is the only inherited bleeding disorder in humans known to be associated with increased u-PA.

Polyphosphate colocalizes with factor XII on platelet-bound fibrin and augments its plasminogen activator activity

PubMed Central

Lionikiene, Ausra S.; Georgiev, Georgi; Klemmer, Anja; Brain, Chelsea; Kim, Paul Y.

2016-01-01

Activated factor XII (FXIIa) has plasminogen activator capacity but its relative contribution to fibrinolysis is considered marginal compared with urokinase and tissue plasminogen activator. Polyphosphate (polyP) is released from activated platelets and mediates FXII activation. Here, we investigate the contribution of polyP to the plasminogen activator function of αFXIIa. We show that both polyP70, of the chain length found in platelets (60-100 mer), and platelet-derived polyP significantly augment the plasminogen activation capacity of αFXIIa. PolyP70 stimulated the autoactivation of FXII and subsequent plasminogen activation, indicating that once activated, αFXIIa remains bound to polyP70. Indeed, complex formation between polyP70 and αFXIIa provides protection against autodegradation. Plasminogen activation by βFXIIa was minimal and not enhanced by polyP70, highlighting the importance of the anion binding site. PolyP70 did not modulate plasmin activity but stimulated activation of Glu and Lys forms of plasminogen by αFXIIa. Accordingly, polyP70 was found to bind to FXII, αFXIIa, and plasminogen, but not βFXIIa. Fibrin and polyP70 acted synergistically to enhance αFXIIa-mediated plasminogen activation. The plasminogen activator activity of the αFXIIa-polyP70 complex was modulated by C1 inhibitor and histidine-rich glycoprotein, but not plasminogen activator inhibitors 1 and 2. Platelet polyP and FXII were found to colocalize on the activated platelet membrane in a fibrin-dependent manner and decorated fibrin strands extending from platelet aggregates. We show that in the presence of platelet polyP and the downstream substrate fibrin, αFXIIa is a highly efficient and favorable plasminogen activator. Our data are the first to document a profibrinolytic function of platelet polyP. PMID:27694320

Synthetic analogues of flavonoids with improved activity against platelet activation and aggregation as novel prototypes of food supplements.

PubMed

Del Turco, Serena; Sartini, Stefania; Cigni, Giulia; Sentieri, Cassandra; Sbrana, Silverio; Battaglia, Debora; Papa, Angela; Da Settimo, Federico; La Motta, Concettina; Basta, Giuseppina

2015-05-15

We investigated the ability of quercetin and apigenin to modulate platelet activation and aggregation, and compared the observed efficacy with that displayed by their synthetic analogues 2-phenyl-4H-pyrido[1,2-a]pyrimidin-4-ones, 1-4, and 2,3-diphenyl-4H-pyrido[1,2-a]pyrimidin-4-ones, 5-7. Platelet aggregation was explored through a spectrophotometric assay on platelet-rich plasma (PRP) treated with the thromboxane A2 mimetic U46619, collagen and thrombin in presence/absence of various bioisosteres of flavonoids (12.5-25-50-100 μM). The platelet density, (mean platelet component, MPC), was measured by the Advia 120 Hematology System as a marker surrogate of platelet activation. The induced P-selectin expression, which reflects platelet degranulation/activation, was quantified by flow cytometry on PRP. Our synthetic compounds modulated significantly both platelet activation and aggregation, thus turning out to be more effective than the analogues quercetin and apigenin when tested at a concentration fully consistent with their use in vivo. Accordingly, they might be used as food supplements to increase the efficacy of natural flavonoids. Copyright © 2014 Elsevier Ltd. All rights reserved.

In vitro platelet activation, aggregation and platelet-granulocyte complex formation induced by surface modified single-walled carbon nanotubes.

PubMed

Fent, János; Bihari, Péter; Vippola, Minnamari; Sarlin, Essi; Lakatos, Susan

2015-08-01

Surface modification of single-walled carbon nanotubes (SWCNTs) such as carboxylation, amidation, hydroxylation and pegylation is used to reduce the nanotube toxicity and render them more suitable for biomedical applications than their pristine counterparts. Toxicity can be manifested in platelet activation as it has been shown for SWCNTs. However, the effect of various surface modifications on the platelet activating potential of SWCNTs has not been tested yet. In vitro platelet activation (CD62P) as well as the platelet-granulocyte complex formation (CD15/CD41 double positivity) in human whole blood were measured by flow cytometry in the presence of 0.1mg/ml of pristine or various surface modified SWCNTs. The effect of various SWCNTs was tested by whole blood impedance aggregometry, too. All tested SWCNTs but the hydroxylated ones activate platelets and promote platelet-granulocyte complex formation in vitro. Carboxylated, pegylated and pristine SWCNTs induce whole blood aggregation as well. Although pegylation is preferred from biomedical point of view, among the samples tested by us pegylated SWCNTs induced far the most prominent activation and a well detectable aggregation of platelets in whole blood. Copyright © 2015 Elsevier Ltd. All rights reserved.

Platelet CLEC-2 protects against lung injury via effects of its ligand podoplanin on inflammatory alveolar macrophages in the mouse

PubMed Central

Rayes, Julie; Wichaiyo, Surasak; Haining, Elizabeth J.; Lowe, Kate; Grygielska, Beata; Laloo, Ryan; Flodby, Per; Borok, Zea; Crandall, Edward D.; Thickett, David R.; Watson, Steve P.

2017-01-01

There is no therapeutic intervention proven to prevent acute respiratory distress syndrome (ARDS). Novel mechanistic insights into the pathophysiology of ARDS are therefore required. Platelets are implicated in regulating many of the pathogenic processes that occur during ARDS; however, the mechanisms remain elusive. The platelet receptor CLEC-2 has been shown to regulate vascular integrity at sites of acute inflammation. Therefore the purpose of this study was to establish the role of CLEC-2 and its ligand podoplanin in a mouse model of ARDS. Platelet-specific CLEC-2-deficient, as well as alveolar epithelial type I cell (AECI)-specific or hematopoietic-specific podoplanin deficient, mice were established using cre-loxP strategies. Combining these with intratracheal (IT) instillations of lipopolysaccharide (LPS), we demonstrate that arterial oxygen saturation decline in response to IT-LPS in platelet-specific CLEC-2-deficient mice is significantly augmented. An increase in bronchoalveolar lavage (BAL) neutrophils and protein was also observed 48 h post-IT-LPS, with significant increases in pro-inflammatory chemokines detected in BAL of platelet-specific CLEC-2-deficient animals. Deletion of podoplanin from hematopoietic cells but not AECIs also reduces lung function and increases pro-inflammatory chemokine expression following IT-LPS. Furthermore, we demonstrate that following IT-LPS, platelets are present in BAL in aggregates with neutrophils, which allows for CLEC-2 interaction with podoplanin expressed on BAL inflammatory alveolar macrophages. Taken together, these data suggest that the platelet CLEC-2-podoplanin signaling axis regulates the severity of lung inflammation in mice and is a possible novel target for therapeutic intervention in patients at risk of developing ARDS. PMID:28839100

Blood clots are rapidly assembled hemodynamic sensors: Flow arrest triggers intraluminal thrombus contraction

PubMed Central

Muthard, Ryan W.; Diamond, Scott L.

2012-01-01

Objective Blood clots form under flow during intravascular thrombosis or vessel leakage. Prevailing hemodynamics influence thrombus structure and may regulate contraction processes. A microfluidic device capable of flowing human blood over a side channel plugged with collagen (± tissue factor) was used to measure thrombus permeability (κ) and contraction at controlled transthrombus pressure drops. Methods and Results The collagen (κcollagen = 1.98 × 10−11 cm2) supported formation of a 20-μm thick platelet layer, which unexpectedly underwent massive platelet retraction upon flow arrest. This contraction resulted in a 5.34-fold increase in permeability due to collagen restructuring. Without stopping flow, platelet deposits (no fibrin) had a permeability of κplatelet = 5.45 × 10−14 cm2 and platelet-fibrin thrombi had κthrombus = 2.71 × 10−14 cm2 for ΔP = 20.7 to 23.4 mm-Hg, the first ever measurements for clots formed under arterial flow (1130 s−1 wall shear rate). Platelet sensing of flow cessation triggered a 4.6 to 6.5-fold (n=3, P<0.05) increase in contraction rate, which was also observed in a rigid, impermeable parallel-plate microfluidic device. This triggered contraction was blocked by the myosin IIA inhibitor blebbistatin and by inhibitors of thromboxane (TXA2) and ADP signaling. In addition, flow arrest triggered platelet intracellular calcium mobilization, which was blocked by TXA2/ADP inhibitors. As clots become occlusive or vessels rupture, flow around developed clots diminishes facilitating full platelet retraction and hemostasis. Conclusion Flow dilution of ADP and thromboxane regulates platelet contractility with prevailing hemodynamics, a newly defined flow sensing mechanism to regulate clot function. PMID:23087356

Plant Food Delphinidin-3-Glucoside Significantly Inhibits Platelet Activation and Thrombosis: Novel Protective Roles against Cardiovascular Diseases

PubMed Central

Yang, Yan; Shi, Zhenyin; Reheman, Adili; Jin, Joseph W.; Li, Conglei; Wang, Yiming; Andrews, Marc C.; Chen, Pingguo; Zhu, Guangheng; Ling, Wenhua; Ni, Heyu

2012-01-01

Delphinidin-3-glucoside (Dp-3-g) is one of the predominant bioactive compounds of anthocyanins in many plant foods. Although several anthocyanin compounds have been reported to be protective against cardiovascular diseases (CVDs), the direct effect of anthocyanins on platelets, the key players in atherothrombosis, has not been studied. The roles of Dp-3-g in platelet function are completely unknown. The present study investigated the effects of Dp-3-g on platelet activation and several thrombosis models in vitro and in vivo. We found that Dp-3-g significantly inhibited human and murine platelet aggregation in both platelet-rich plasma and purified platelets. It also markedly reduced thrombus growth in human and murine blood in perfusion chambers at both low and high shear rates. Using intravital microscopy, we observed that Dp-3-g decreased platelet deposition, destabilized thrombi, and prolonged the time required for vessel occlusion. Dp-3-g also significantly inhibited thrombus growth in a carotid artery thrombosis model. To elucidate the mechanisms, we examined platelet activation markers via flow cytometry and found that Dp-3-g significantly inhibited the expression of P-selectin, CD63, CD40L, which reflect platelet α- and δ-granule release, and cytosol protein secretion, respectively. We further demonstrated that Dp-3-g downregulated the expression of active integrin αIIbβ3 on platelets, and attenuated fibrinogen binding to platelets following agonist treatment, without interfering with the direct interaction between fibrinogen and integrin αIIbβ3. We found that Dp-3-g reduced phosphorylation of adenosine monophosphate-activated protein kinase, which may contribute to the observed inhibitory effects on platelet activation. Thus, Dp-3-g significantly inhibits platelet activation and attenuates thrombus growth at both arterial and venous shear stresses, which likely contributes to its protective roles against thrombosis and CVDs. PMID:22624015

Platelets Toll-like receptor-4 in Crohns disease.

PubMed

Schmid, Werner; Novacek, Gottfried; Vogelsang, Harald; Papay, Pavol; Primas, Christian; Eser, Alexander; Panzer, Simon

2017-02-01

Platelets are activated in Crohn's disease (CD) and interplay with leukocytes. Engagement of Toll-like receptor-4 (TLR-4), which is expressed in human platelets, may be involved in crosstalks between platelets and leukocytes leading to their mutual activation for host defense. Human neutrophil peptides (HNPs), lipoprotein binding peptides, and sCD14 were determined by enzyme-linked immunosorbent assays in 42 patients with active CD, in 43 patients with CD in remission, and in 30 healthy individuals. Neutrophil-platelet aggregates and binding of the TLR-4 monoclonal antibody to platelets were determined by flow cytometry. Levels of HNPs were higher in patients with CD than in controls (P = 0.0003 vs. active CD and P = 0.01 vs. CD in remission). Likewise, neutrophils with adhering platelets were higher in patients with active CD than in controls (P = 0.004). Binding of the TLR-4 antibody in patients with active CD was similar to that in controls, while patients in remission had significantly higher binding capacities (P = 0.59 and P = 0.003). Incubation of plasma from patients with active disease or patients in remission with platelets from healthy controls confirmed lower binding of the TLR-4 antibody in the presence of plasma from active diseased patients compared to controls (P = 0.039), possibly due to high levels of lipopolysaccharides, as suggested by high levels of sCD14 and lipoprotein binding protein. Our study indicates involvement of platelet TLR-4 in enhancing the secretion of antimicrobial peptides from neutrophils. While platelet aggregation can be due to a variety of mechanisms in inflammatory disease, the mutual activation of platelets and neutrophils may augment host defense. © 2016 Stichting European Society for Clinical Investigation Journal Foundation.

The origin and function of platelet glycosyltransferases

PubMed Central

Rumjantseva, Viktoria; Sørensen, Anne Louise Tølbøll; Patel-Hett, Sunita; Josefsson, Emma C.; Bennett, Eric P.; Italiano, Joseph E.; Clausen, Henrik; Hartwig, John H.; Hoffmeister, Karin M.

2012-01-01

Platelets are megakaryocyte subfragments that participate in hemostatic and host defense reactions and deliver pro- and antiangiogenic factors throughout the vascular system. Although they are anucleated cells that lack a complex secretory apparatus with distinct Golgi/endoplasmic reticulum compartments, past studies have shown that platelets have glycosyltransferase activities. In the present study, we show that members of 3 distinct glycosyltransferase families are found within and on the surface of platelets. Immunocytology and flow cytometry results indicated that megakaryocytes package these Golgi-derived glycosyltransferases into vesicles that are sent via proplatelets to nascent platelets, where they accumulate. These glycosyltransferases are active, and intact platelets glycosylate large exogenous substrates. Furthermore, we show that activation of platelets results in the release of soluble glycosyltransferase activities and that platelets contain sufficient levels of sugar nucleotides for detection of glycosylation of exogenously added substrates. Therefore, the results of the present study show that blood platelets are a rich source of both glycosyltransferases and donor sugar substrates that can be released to function in the extracellular space. This platelet-glycosylation machinery offers a pathway to a simple glycoengineering strategy improving storage of platelets and may serve hitherto unknown biologic functions. PMID:22613794

Use of a Cyclooxygenase-2 Inhibitor Does Not Inhibit Platelet Activation or Growth Factor Release From Platelet-Rich Plasma.

PubMed

Ludwig, Hilary C; Birdwhistell, Kate E; Brainard, Benjamin M; Franklin, Samuel P

2017-12-01

It remains unestablished whether use of cyclooxygenase (COX)-2 inhibitors impairs platelet activation and anabolic growth factor release from platelets in platelet-rich plasma (PRP). The purpose of this study was to assess the effects of a COX-2 inhibitor on platelet activation and anabolic growth factor release from canine PRP when using a clinically applicable PRP activator and to determine whether a 3-day washout would be sufficient to abrogate any COX-2 inhibitor-related impairment on platelet function. Controlled laboratory study. Ten healthy dogs underwent blood collection and PRP preparation. Dogs were then administered a COX-2 inhibitor for 7 days, after which PRP preparation was repeated. The COX-2 inhibitor was continued for 4 more days and PRP preparation performed a third time, 3 days after discontinuation of the COX-2 inhibitor. Immediately after PRP preparation, the PRP was divided into 4 aliquots: 2 unactivated and 2 activated using human γ-thrombin (HGT). One activated and 1 unactivated sample were assessed using flow cytometry for platelet expression of CD62P and platelet-bound fibrinogen using the canine activated platelet-1 (CAP1) antibody. The 2 remaining samples were centrifuged and the supernatant assayed for transforming growth factor-β1 (TGF-β1), platelet-derived growth factor-BB (PDGF-BB), and thromboxane B2 (TXB2) concentrations. Differences in platelet activation and TGF-β1, PDGF-BB, and TXB2 concentrations over the 3 study weeks were evaluated using a 1-way repeated-measures ANOVA, and comparisons between activated and unactivated samples within a study week were assessed with paired t tests. There were no statistically significant ( P > .05) effects of the COX-2 inhibitor on percentage of platelets positive for CD62P or CAP1 or on concentrations of TGF-β1, PDGF-BB, or TXB2. All unactivated samples had low levels of activation or growth factor concentrations and significantly ( P < .05) greater activation and growth factor concentrations in HGT-activated samples. This COX-2 inhibitor did not impair platelet activation, growth factor release, or TXB2 production in this canine PRP when using HGT as an activator. Studies are warranted to determine whether COX-2 inhibitors affect platelet activation and growth factor release from human PRPs. These results suggest that there is no need to withhold a COX-2 inhibitor before PRP preparation, particularly if thrombin is going to be used to activate the PRP. This is clinically relevant information because many patients who are candidates for PRP therapy for treatment of musculoskeletal injury are also using COX-2 inhibitors.

An efficient method for isolation of representative and contamination-free population of blood platelets for proteomic studies.

PubMed

Wrzyszcz, Aneta; Urbaniak, Joanna; Sapa, Agnieszka; Woźniak, Mieczysław

2017-01-01

To date, there has been no ideal method for blood platelet isolation which allows one to obtain a preparation devoid of contaminations, reflecting the activation status and morphological features of circulating platelets. To address these requirements, we have developed a method which combines the continuous density gradient centrifugation with washing from PGI 2 -supplemented platelet-rich plasma (PRP). We have assessed the degree of erythrocyte and leukocyte contamination, recovery of platelets, morphological features, activation status, and reactivity of isolated platelets. Using our protocol, we were able to get a preparation free from contaminations, representing well the platelet population prior to the isolation in terms of size and activity. Besides this, we have obtained approximately 2 times more platelets from the same volume of blood compared to the most widely used method. From 10 ml of whole citrated blood we were able to get on average 2.7 mg of platelet-derived protein. The method of platelet isolation presented in this paper can be successfully applied to tests requiring very pure platelets, reflecting the circulating platelet state, from a small volume of blood.

Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM)-mediated Inhibitory Signaling is Regulated by Sequential Phosphorylation Mediated by Distinct Nonreceptor Tyrosine Kinases: A Case Study Involving PECAM-1

PubMed Central

Tourdot, Benjamin E.; Brenner, Michelle K.; Keough, Kathleen C.; Holyst, Trudy; Newman, Peter J.; Newman, Debra K.

2013-01-01

The activation state of many blood and vascular cells is tightly controlled by a delicate balance between receptors that contain immunoreceptor tyrosine-based activation motifs (ITAMs) and those that contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Precisely how the timing of cellular activation by ITAM-coupled receptors is regulated by ITIM-containing receptors is, however, poorly understood. Using platelet endothelial cell adhesion molecule 1 (PECAM-1) as a prototypical ITIM-bearing receptor, we demonstrate that initiation of inhibitory signaling occurs via a novel, sequential process in which Src family kinases phosphorylate the C-terminal ITIM, thereby enabling phosphorylation of the N-terminal ITIM of PECAM-1 by other Src homology 2 domain-containing non-receptor tyrosine kinases (NRTKs). NRTKs capable of mediating the second phosphorylation event include C-terminal Src kinase (Csk) and Bruton’s tyrosine kinase (Btk). Btk and Csk function downstream of phosphatidylinositol 3-kinase (PI3K) activation during ITAM-dependent platelet activation. In ITAM-activated platelets that were treated with a PI3K inhibitor, PECAM-1 was phosphorylated but did not bind the tandem SH2 domain-containing tyrosine phosphatase SHP-2, indicating that it was not phosphorylated on its N-terminal ITIM. Csk bound to and phosphorylated PECAM-1 more efficiently than did Btk, and required its SH2 domain to perform these functions. Additionally, the phosphorylation of the N-terminal ITIM of Siglec-9 by Csk is enhanced by the prior phosphorylation of its C-terminal ITIM, providing evidence that the ITIMs of other dual ITIM-containing receptors are also sequentially phosphorylated. On the basis of these findings, we propose that sequential ITIM phosphorylation provides a general mechanism for precise temporal control over the recruitment and activation of tandem SH2 domain-containing tyrosine phosphatases that dampen ITAM-dependent signals. PMID:23418871

Coated platelets function in platelet-dependent fibrin formation via integrin αIIbβ3 and transglutaminase factor XIII

PubMed Central

Mattheij, Nadine J.A.; Swieringa, Frauke; Mastenbroek, Tom G.; Berny-Lang, Michelle A.; May, Frauke; Baaten, Constance C.F.M.J.; van der Meijden, Paola E.J.; Henskens, Yvonne M.C.; Beckers, Erik A.M.; Suylen, Dennis P.L.; Nolte, Marc W.; Hackeng, Tilman M.; McCarty, Owen J.T.; Heemskerk, Johan W.M.; Cosemans, Judith M.E.M.

2016-01-01

Coated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin αIIbβ3 activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin αIIbβ3. Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin αIIbβ3. Vice versa, star-like fibrin formation from platelets of a patient with deficiency in αIIbβ3 (Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial αIIbβ3 activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin αIIbβ3. PMID:26721892

Protective mechanisms of adenosine 5'-monophosphate in platelet activation and thrombus formation.

PubMed

Fuentes, E; Badimon, L; Caballero, J; Padró, T; Vilahur, G; Alarcón, M; Pérez, P; Palomo, I

2014-03-03

Platelet activation is relevant to a variety of acute thrombotic events. We sought to examine adenosine 5'-monophosphate (AMP) mechanisms of action in preventing platelet activation, thrombus formation and platelet-related inflammatory response. We assessed the effect of AMP on 1) P-selectin expression and GPIIb/IIIa activation by flow cytometry; 2) Platelet aggregation and ATP secretion induced by ADP, collagen, TRAP-6, convulxin and thrombin; 3) Platelet rolling and firm adhesion, and platelet-leukocyte interactions under flow-controlled conditions; and, 4) Platelet cAMP levels, sP-selectin, sCD40L, IL-1β, TGF-β1 and CCL5 release, PDE3A activity and PKA phosphorylation. The effect of AMP on in vivo thrombus formation was also evaluated in a murine model. The AMP docking with respect to A2 adenosine receptor was determined by homology. AMP concentration-dependently (0.1 to 3 mmol/l) inhibited P-selectin expression and GPIIb/IIIa activation, platelet secretion and aggregation induced by ADP, collagen, TRAP-6 and convulxin, and diminished platelet rolling and firm adhesion. Furthermore, AMP induced a marked increase in the rolling speed of leukocytes retained on the platelet surface. At these concentrations AMP significantly decreased inflammatory mediator from platelet, increased intraplatelet cAMP levels and inhibited PDE3A activity. Interestingly, SQ22536, ZM241385 and SCH58261 attenuated the antiplatelet effect of AMP. Docking experiments revealed that AMP had the same orientation that adenosine inside the A2 adenosine receptor binding pocket. These in vitro antithrombotic properties were further supported in an in vivo model of thrombosis. Considering the successful use of combined antiplatelet therapy, AMP may be further developed as a novel antiplatelet agent.

Glycoprotein Ib activation by thrombin stimulates the energy metabolism in human platelets

PubMed Central

Corona de la Peña, Norma; Gutiérrez-Aguilar, Manuel; Hernández-Reséndiz, Ileana; Marín-Hernández, Álvaro

2017-01-01

Thrombin-induced platelet activation requires substantial amounts of ATP. However, the specific contribution of each ATP-generating pathway i.e., oxidative phosphorylation (OxPhos) versus glycolysis and the biochemical mechanisms involved in the thrombin-induced activation of energy metabolism remain unclear. Here we report an integral analysis on the role of both energy pathways in human platelets activated by several agonists, and the signal transducing mechanisms associated with such activation. We found that thrombin, Trap-6, arachidonic acid, collagen, A23187, epinephrine and ADP significantly increased glycolytic flux (3–38 times vs. non-activated platelets) whereas ristocetin was ineffective. OxPhos (33 times) and mitochondrial transmembrane potential (88%) were increased only by thrombin. OxPhos was the main source of ATP in thrombin-activated platelets, whereas in platelets activated by any of the other agonists, glycolysis was the principal ATP supplier. In order to establish the biochemical mechanisms involved in the thrombin-induced OxPhos activation in platelets, several signaling pathways associated with mitochondrial activation were analyzed. Wortmannin and LY294002 (PI3K/Akt pathway inhibitors), ristocetin and heparin (GPIb inhibitors) as well as resveratrol, ATP (calcium-release inhibitors) and PP1 (Tyr-phosphorylation inhibitor) prevented the thrombin-induced platelet activation. These results suggest that thrombin activates OxPhos and glycolysis through GPIb-dependent signaling involving PI3K and Akt activation, calcium mobilization and protein phosphorylation. PMID:28817667

Endothelial dysfunction is associated with activation of the type I interferon system and platelets in patients with systemic lupus erythematosus

PubMed Central

Tydén, Helena; Lood, Christian; Gullstrand, Birgitta; Nielsen, Christoffer Tandrup; Heegaard, Niels H H; Kahn, Robin; Jönsen, Andreas; Bengtsson, Anders A

2017-01-01

Objectives Endothelial dysfunction may be connected to cardiovascular disease (CVD) in systemic lupus erythematosus (SLE). Type I interferons (IFNs) are central in SLE pathogenesis and are suggested to induce both endothelial dysfunction and platelet activation. In this study, we investigated the interplay between endothelial dysfunction, platelets and type I IFN in SLE. Methods We enrolled 148 patients with SLE and 79 sex-matched and age-matched healthy controls (HCs). Type I IFN activity was assessed with a reporter cell assay and platelet activation by flow cytometry. Endothelial dysfunction was assessed using surrogate markers of endothelial activation, soluble vascular cell adhesion molecule-1 (sVCAM-1) and endothelial microparticles (EMPs), and finger plethysmograph to determine Reactive Hyperaemia Index (RHI). Results In patients with SLE, type I IFN activity was associated with endothelial activation, measured by high sVCAM-1 (OR 1.68, p<0.01) and elevated EMPs (OR 1.40, p=0.03). Patients with SLE with high type I IFN activity had lower RHI than HCs (OR 2.61, p=0.04), indicating endothelial dysfunction. Deposition of complement factors on platelets, a measure of platelet activation, was seen in patients with endothelial dysfunction. High levels of sVCAM-1 were associated with increased deposition of C4d (OR 4.57, p<0.01) and C1q (OR 4.10, p=0.04) on platelets. High levels of EMPs were associated with C4d deposition on platelets (OR 3.64, p=0.03). Conclusions Endothelial dysfunction was associated with activation of platelets and the type I IFN system. We suggest that an interplay between the type I IFN system, injured endothelium and activated platelets may contribute to development of CVD in SLE. PMID:29119007

Thrombin generation by activated factor VII on platelet activated by different agonists. Extending the cell-based model of hemostasis

PubMed Central

Altman, Raul; Scazziota, Alejandra Silvia; Herrera, Maria de Lourdes; Gonzalez, Claudio

2006-01-01

Background Platelet activation is crucial in normal hemostasis. Using a clotting system free of external tissue factor, we investigated whether activated Factor VII in combination with platelet agonists increased thrombin generation (TG) in vitro. Methods and results TG was quantified by time parameters: lag time (LT) and time to peak (TTP), and by amount of TG: peak of TG (PTG) and area under thrombin formation curve after 35 minutes (AUC→35min) in plasma from 29 healthy volunteers using the calibrated automated thrombography (CAT) technique. TG parameters were measured at basal conditions and after platelet stimulation by sodium arachidonate (AA), ADP, and collagen (Col). In addition, the effects of recombinant activated FVII (rFVIIa) alone or combined with the other platelet agonists on TG parameters were investigated. We found that LT and TTP were significantly decreased (p < 0.05) and PTG and AUC→35min were significantly increased (p < 0.05) in platelet rich plasma activated with AA, ADP, Col, and rFVIIa compared to non-activated platelet rich plasma from normal subjects (p = 0.01). Furthermore platelet rich plasma activated by the combined effects of rFVIIa plus AA, ADP or Col had significantly reduced LT and TTP and increased AUC→35min (but not PTG) when compared to platelet rich plasma activated with agonists in the absence of rFVIIa. Conclusion Platelets activated by AA, ADP, Col or rFVIIa triggered TG. This effect was increased by combining rFVIIa with other agonists. Our intrinsic coagulation system produced a burst in TG independent of external tissue factor activity an apparent hemostatic effect with little thrombotic capacity. Thus we suggest a modification in the cell-based model of hemostasis. PMID:16630353

Amyloid precursor protein expression is enhanced in human platelets from subjects with Alzheimer's disease and frontotemporal lobar degeneration: a real-time PCR study.

PubMed

Vignini, Arianna; Morganti, Stefano; Salvolini, Eleonora; Sartini, Davide; Luzzi, Simona; Fiorini, Rosamaria; Provinciali, Leandro; Di Primio, Roberto; Mazzanti, Laura; Emanuelli, Monica

2013-12-01

Frontotemporal lobar degeneration (FTLD) and Alzheimer's disease (AD) represent the most frequent causes of early-onset and late-onset degenerative dementia, respectively. A correct diagnosis entails the choice of appropriate therapies. In this view the present study aimed to identify biomarkers that could improve the differential diagnosis. We recently found an overexpression of platelet amyloid precursor protein (APP) in AD; furthermore, recent studies have suggested the presence of changes in APP processing in FTLD. In this context, we analyzed the mRNA expression level of Total APP (TOT) and APP containing a Kunitz-type serine protease inhibitor domain (KPI) in platelets obtained from AD patients, subjects with FTLD, and healthy subjects. In addition, we evaluated the correlation between platelet APP mRNA expression levels and cognitive impairment.Differential gene expression measurements revealed a significant up-regulation of APP TOT and APP KPI in both AD and FTLD patients compared to the controls (being AD/Controls: 1.67 for APP TOT and 1.47 for APP KPI; FTLD/Controls: 1.62 for APP TOT and 1.51 for APP KPI; p < 0.05), although it is interesting to note that in FTLD patients this expression did not correlate with the severity of cognitive impairment.This could be related to a reduced beta-amyloid (Aβ) formation, caused by an alteration of secretase enzymatic activity, even though a post-transcriptional regulation of APP mRNAs in FTLD cannot be excluded.

Amyloid precursor protein expression is enhanced in human platelets from subjects with Alzheimer's disease and Frontotemporal lobar degeneration: A Real-time PCR study.

PubMed

Vignini, Arianna; Morganti, Stefano; Salvolini, Eleonora; Sartini, Davide; Luzzi, Simona; Fiorini, Rosamaria; Provinciali, Leandro; Di Primio, Roberto; Mazzanti, Laura; Emanuelli, Monica

2013-10-26

Frontotemporal lobar degeneration (FTLD) and Alzheimer's disease (AD) represent the most frequent causes of early-onset and late-onset degenerative dementia, respectively. A correct diagnosis entails the choice of appropriate therapies. In this view the present study aimed to identify biomarkers that could improve the differential diagnosis. We recently found an overexpression of platelet amyloid precursor protein (APP) in AD; furthermore, recent studies have suggested the presence of changes in APP processing in FTLD. In this context, we analyzed the mRNA expression level of Total APP (TOT) and APP containing a Kunitz-type serine protease inhibitor domain (KPI) in platelets obtained from AD patients, subjects with FTLD, and healthy subjects. In addition, we evaluated the correlation between platelet APP mRNA expression levels and cognitive impairment. Differential gene expression measurements revealed a significant up-regulation of APP TOT and APP KPI in both AD and FTLD patients compared to the controls (being AD/Controls: 1.67 for APP TOT and 1.47 for APP KPI; FTLD/Controls: 1.62 for APP TOT and 1.51 for APP KPI; p<0.05) , although it is interesting to note that in FTLD patients this expression did not correlate with the severity of cognitive impairment. This could be related to a reduced beta-amyloid (Aβ) formation, caused by an alteration of secretase enzymatic activity, even though a post-transcriptional regulation of APP mRNAs in FTLD cannot be excluded. © 2013.

Talin does not associate exclusively with alpha 2b beta 3 integrin in activated human platelets.

PubMed

Escolar, G; Diaz-Ricart, M; White, J G

1995-05-01

Talin is a high-molecular-weight protein that may stabilize connections between cytoplasmic actin and the submembrane portion of glycoprotein IIb-IIIa (GPIIb-IIIa) (alpha 2b beta 3 integrin) in thrombin-stimulated human platelets. Using morphologic and electrophoretic techniques, we have examined the association of talin with the cytoskeleton of platelets activated by thrombin in the presence of fibrinogen-coated gold particles (Fgn/Au). Ultrastructural studies confirmed the presence of Fgn/Au firmly bound to the outside membranes of detergent-extracted platelets. Immunoblots of protein bands showed GPIIIa, but not talin, associated with cytoskeletons of activated platelets. Immunogold cytochemical techniques were performed on ultrathin cryosections of whole platelets to localize talin at the ultrastructural level. Studies were performed on normal platelets and platelets defective in GPIIb-IIIa (Glanzmann's thrombasthenia) and GPIb (Bernard-Soulier syndrome). Talin was randomly distributed in the cytoplasm of resting platelets. Activation resulted in binding of Fgn/Au to the surface membrane and redistribution of talin to the submembrane region. However, no definitive colocalization between the two markers was noted. Activated thrombasthenic platelets failed to bind Fgn/Au, but talin was localized to the submembrane location. After activation, talin was confined to the submembrane zone of Bernard-Soulier syndrome platelets. No definitive colocalization was observed between large clusters of Fgn/Au-occupied receptors and talin distributed in the submembrane region. GPIb and GPIIb-IIIa are not necessary for talin to localize in the submembrane region of activated cells. Talin does not redistribute exclusively with GPIIb-IIIa, and it may stabilize connections with other glycoproteins.

Effect of TGFβ on calcium signaling in megakaryocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Jing; Schmid, Evi; Department of Pediatric Surgery and Pediatric Urology, University Children's Hospital Tübingen, Tübingen

2015-05-22

TGFβ is a powerful regulator of megakaryocyte maturation and platelet formation. As previously shown for other cell types, TGFβ may up-regulate the expression of the serum & glucocorticoid inducible kinase SGK1, an effect requiring p38 kinase. SGK1 has in turn recently been shown to participate in the regulation of cytosolic Ca{sup 2+} activity ([Ca{sup 2+}]{sub i}) in megakaryocytes and platelets. SGK1 phosphorylates the IκB kinase (IKKα/β), which in turn phosphorylates the inhibitor protein IκBα resulting in nuclear translocation of nuclear factor NFκB. Genes up-regulated by NFκB include Orai1, the pore forming ion channel subunit accomplishing store operated Ca{sup 2+} entrymore » (SOCE). The present study explored whether TGFβ influences Ca{sup 2+} signaling in megakaryocytes. [Ca{sup 2+}]{sub i} was determined by Fura-2 fluorescence and SOCE from the increase of [Ca{sup 2+}]{sub i} following re-addition of extracellular Ca{sup 2+} after store depletion by removal of extracellular Ca{sup 2+} and inhibition of the sarcoendoplasmatic Ca{sup 2+} ATPase (SERCA) with thapsigargin (1 μM). As a result, TGFβ (60 ng, 24 h) increased SOCE, an effect significantly blunted by p38 kinase inhibitor Skepinone-L (1 μM), SGK1 inhibitor EMD638683 (50 μM) and NFκB inhibitor wogonin (100 μM). In conclusion, TGFβ is a powerful regulator of store operated Ca{sup 2+} entry into megakaryocytes, an effect mediated by a signaling cascade involving p38 kinase, SGK1 and NFκB. - Highlights: • TGFβ up-regulates store operated Ca{sup 2+} entry (SOCE) in megakaryocytes. • The effect of TGFβ on SOCE is blunted by p38 kinase inhibitor Skepinone-L. • The effect of TGFβ on SOCE is virtually abrogated by SGK1 inhibitor EMD638683. • The effect of TGFβ on SOCE is almost abolished by NFκB inhibitor wogonin. • The effect of TGFβ is expected to enhance sensitivity of platelets to activation.« less

Inhibitory effects of ethyl pyruvate on platelet aggregation and phosphatidylserine exposure.

PubMed

Li, Wenjin; Yang, Xinyu; Peng, Minyuan; Li, Can; Mu, Guangfu; Chen, Fangping

2017-06-03

Ethyl pyruvate (EP) is a stable lipophilic pyruvate derivative. Studies demonstrated that EP shows potent anti-oxidation, anti-inflammatory and anti-coagulant effects. Inflammation and coagulation are closely interacted with platelet activation. However, it is unclear whether EP has anti-platelet effects. Therefore, we investigated the anti-platelet effect of EP in this study in vitro. We found that EP inhibited agonists induced platelets aggregation, ATP release and adhesion to collagen. Flow cytometric analysis revealed that EP inhibited agonist induced platelets PAC-1 binding, as well as P-selectin and CD40L expression. The underlying mechanism of action may involve the inhibition of platelet PI3K/Akt and Protein Kinase C (PKC) signaling pathways. Additionally, EP dose dependently inhibited platelet PS exposure induced by high concentration thrombin. Lactate dehydrogenase (LDH) activity assay and mice platelet count implied that EP may have no toxic effect on platelets. Therefore, we are the first to report that EP has potent anti-platelet activity and attenuates platelet PS exposure in vitro, suggesting that the inhibitory effects of EP on platelets may also play important roles in improvement of inflammation and coagulation disorder in related animal models. Copyright © 2017 Elsevier Inc. All rights reserved.

Myeloid-related protein-14 regulates deep vein thrombosis

PubMed Central

Wang, Yunmei; Gao, Huiyun; Kessinger, Chase W.; Schmaier, Alvin; Jaffer, Farouc A.; Simon, Daniel I.

2017-01-01

Using transcriptional profiling of platelets from patients presenting with acute myocardial infarction, we identified myeloid-related protein-14 (MRP-14, also known as S100A9) as an acute myocardial infarction gene and reported that platelet MRP-14 binding to platelet CD36 regulates arterial thrombosis. However, whether MRP-14 plays a role in venous thrombosis is unknown. We subjected WT and Mrp-14–deficient (Mrp-14-/-) mice to experimental models of deep vein thrombosis (DVT) by stasis ligation or partial flow restriction (stenosis) of the inferior vena cava. Thrombus weight in response to stasis ligation or stenosis was reduced significantly in Mrp-14-/- mice compared with WT mice. The adoptive transfer of WT neutrophils or platelets, or the infusion of recombinant MRP-8/14, into Mrp-14-/- mice rescued the venous thrombosis defect in Mrp-14-/- mice, indicating that neutrophil- and platelet-derived MRP-14 directly regulate venous thrombogenesis. Stimulation of neutrophils with MRP-14 induced neutrophil extracellular trap (NET) formation, and NETs were reduced in venous thrombi harvested from Mrp-14-/- mice and in Mrp-14-/- neutrophils stimulated with ionomycin. Given prior evidence that MRP-14 also regulates arterial thrombosis, but not hemostasis (i.e., reduced bleeding risk), MRP-14 appears to be a particularly attractive molecular target for treating thrombotic cardiovascular diseases, including myocardial infarction, stroke, and venous thromboembolism. PMID:28570273

Diversity of Interstitial Lung Fibroblasts Is Regulated by Platelet-Derived Growth Factor Receptor α Kinase Activity.

PubMed

Green, Jenna; Endale, Mehari; Auer, Herbert; Perl, Anne-Karina T

2016-04-01

Epithelial-mesenchymal cell interactions and factors that control normal lung development are key players in lung injury, repair, and fibrosis. A number of studies have investigated the roles and sources of epithelial progenitors during lung regeneration; such information, however, is limited in lung fibroblasts. Thus, understanding the origin, phenotype, and roles of fibroblast progenitors in lung development, repair, and regeneration helps address these limitations. Using a combination of platelet-derived growth factor receptor α-green fluorescent protein (PDGFRα-GFP) reporter mice, microarray, real-time polymerase chain reaction, flow cytometry, and immunofluorescence, we characterized two distinct interstitial resident fibroblasts, myo- and matrix fibroblasts, and identified a role for PDGFRα kinase activity in regulating their activation during lung regeneration. Transcriptional profiling of the two populations revealed a myo- and matrix fibroblast gene signature. Differences in proliferation, smooth muscle actin induction, and lipid content in the two subpopulations of PDGFRα-expressing fibroblasts during alveolar regeneration were observed. Although CD140α(+)CD29(+) cells behaved as myofibroblasts, CD140α(+)CD34(+) appeared as matrix and/or lipofibroblasts. Gain or loss of PDGFRα kinase activity using the inhibitor nilotinib and a dominant-active PDGFRα-D842V mutation revealed that PDGFRα was important for matrix fibroblast differentiation. We demonstrated that PDGFRα signaling promotes alveolar septation by regulating fibroblast activation and matrix fibroblast differentiation, whereas myofibroblast differentiation was largely PDGFRα independent. These studies provide evidence for the phenotypic and functional diversity as well as the extent of specificity of interstitial resident fibroblasts differentiation during regeneration after partial pneumonectomy.

Diversity of Interstitial Lung Fibroblasts Is Regulated by Platelet-Derived Growth Factor Receptor α Kinase Activity

PubMed Central

Green, Jenna; Endale, Mehari; Auer, Herbert

2016-01-01

Epithelial–mesenchymal cell interactions and factors that control normal lung development are key players in lung injury, repair, and fibrosis. A number of studies have investigated the roles and sources of epithelial progenitors during lung regeneration; such information, however, is limited in lung fibroblasts. Thus, understanding the origin, phenotype, and roles of fibroblast progenitors in lung development, repair, and regeneration helps address these limitations. Using a combination of platelet-derived growth factor receptor α–green fluorescent protein (PDGFRα-GFP) reporter mice, microarray, real-time polymerase chain reaction, flow cytometry, and immunofluorescence, we characterized two distinct interstitial resident fibroblasts, myo- and matrix fibroblasts, and identified a role for PDGFRα kinase activity in regulating their activation during lung regeneration. Transcriptional profiling of the two populations revealed a myo- and matrix fibroblast gene signature. Differences in proliferation, smooth muscle actin induction, and lipid content in the two subpopulations of PDGFRα-expressing fibroblasts during alveolar regeneration were observed. Although CD140α+CD29+ cells behaved as myofibroblasts, CD140α+CD34+ appeared as matrix and/or lipofibroblasts. Gain or loss of PDGFRα kinase activity using the inhibitor nilotinib and a dominant-active PDGFRα-D842V mutation revealed that PDGFRα was important for matrix fibroblast differentiation. We demonstrated that PDGFRα signaling promotes alveolar septation by regulating fibroblast activation and matrix fibroblast differentiation, whereas myofibroblast differentiation was largely PDGFRα independent. These studies provide evidence for the phenotypic and functional diversity as well as the extent of specificity of interstitial resident fibroblasts differentiation during regeneration after partial pneumonectomy. PMID:26414960

Study on antithrombotic and antiplatelet activities of low molecular weight fucoidan from Laminaria japonica

NASA Astrophysics Data System (ADS)

Chen, Anjin; Zhang, Fang; Shi, Jie; Zhao, Xue

2012-06-01

The antithrombotic and antiplatelet effects of two fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica were compared in order to examine the influence of chemical character on their antithrombotic activity and the possible mechanism. Both LMW fucoidan fractions exhibited favorable antithrombotic activity in an Fecl3-induced arterial thrombosis. The antithrombotic activity of LMW fucoidan was related with decrease of TXB2 and whole blood viscosity and hematocrit. LMW fucoidan showed a correlation between anticoagulant, antiaggregant and antithrombotic effects in vivo. For LMW fucoidan, antithrombotic activity required high dose of 5-10 nmol kg-1, concomitantly with increase in anticoagulant activity and inhibition of platelet aggregation. Administration of LMW fucoidan significantly promoted the 6-keto-PGF1α content and decreased the TXB2 content, indicating its inhibition of tissue factor pathway and regulation of metabolism of arachidonic acid. By comparison, highly sulfated fucoidan LF2 with Mw 3900 seemed to be a more suitable choice for antithrombotic drug for its antithrombotic activity accompanied with specific inhibitory activity on platelet aggregation, low anticoagulant activity and low hemorrhagic risk in vivo.

Effects of FX06 in vitro on platelet, coagulation, and fibrinolytic biomarkers in volunteers and patients with documented coronary artery disease.

PubMed

Hallén, Jonas; Atar, Dan; Serebruany, Victor

2014-01-01

FX06 is a naturally occurring fibrin-derived peptide demonstrated to confer cytoprotection in the setting of primary percutaneous coronary intervention. Because the effect of FX06 on human platelet, coagulation, and fibrinolysis biomarkers (PCFB) is unknown but is important for further clinical development, we evaluated how FX06 affects PCFB. The in vitro effects of the whole-blood pre-incubation with escalating concentrations of FX06 (4, 25, and 75 μg/mL) were assessed in aspirin-naïve healthy volunteers (n = 10), those with multiple risk factors for vascular disease (n = 10), and patients with documented coronary artery disease (n = 10). The last two groups were treated with aspirin (81 mg/daily). Thirty-two variables of PCFB were measured with the vehicle and for each chosen FX06 dose. Pretreatment of blood samples with FX06 resulted in a moderate but significant and mostly dose-dependent increases of platelet aggregation induced by adenosine diphosphate and collagen. Similarly, the closure time was reduced, suggesting share-induced activation, PECAM-1, GP Ib, GP IIb/IIIa activity, and vitronectin receptors, which were also up-regulated. In contrast, P-selectin and GPIIb antigen expression were reduced after FX06. All other PCFB were predominantly unaffected by FX06, with the exception of the increased plasminogen, decreased protein C activity, and activated von Willebrand factor. We conclude that in the therapeutic range, FX06 in vitro mildly affects hemostasis by way of mostly activating platelets. Applying moderate concomitant antiplatelet strategies should be considered for the adequate protection from vascular thrombotic events in patients treated with FX06. Similar ex vivo study in patients receiving aspirin and clopidogrel is warranted.

Elevated thrombopoietin in plasma of burned patients without and with sepsis enhances platelet activation.

PubMed

Lupia, E; Bosco, O; Mariano, F; Dondi, A E; Goffi, A; Spatola, T; Cuccurullo, A; Tizzani, P; Brondino, G; Stella, M; Montrucchio, G

2009-06-01

Thrombopoietin (TPO) is a humoral growth factor that does not induce platelet aggregation per se, but enhances platelet activation in response to several agonists. Circulating levels of TPO are increased in patients with sepsis and are mainly related to sepsis severity. To investigate the potential contribution of elevated TPO levels in platelet activation during burn injury complicated or not by sepsis. We studied 22 burned patients, 10 without and 12 with sepsis, and 10 healthy subjects. We measured plasma levels of TPO, as well as leukocyte-platelet binding and P-selectin expression. The priming activity of plasma from burned patients or healthy subjects on platelet aggregation and leukocyte-platelet binding, and the role of TPO in these effects were also studied in vitro. Burned patients without and with sepsis showed higher circulating TPO levels and increased monocyte-platelet binding compared with healthy subjects. Moreover, TPO levels, monocyte-platelet binding and P-selectin expression were significantly higher in burned patients with sepsis than in burned patients without sepsis. In vitro, plasma from burned patients without and with sepsis, but not from healthy subjects, primed platelet aggregation, monocyte-platelet binding and platelet P-selectin expression. The effect of plasma from burned patients with sepsis was significantly higher than that of plasma from burned patients without sepsis. An inhibitor of TPO prevented the priming effect of plasma from burned patients. Increased TPO levels may enhance platelet activation during burn injury and sepsis, potentially participating in the pathogenesis of multi-organ failure in these diseases.

Quantitative evaluation of morphological changes in activated platelets in vitro using digital holographic microscopy.

PubMed

Kitamura, Yutaka; Isobe, Kazushige; Kawabata, Hideo; Tsujino, Tetsuhiro; Watanabe, Taisuke; Nakamura, Masayuki; Toyoda, Toshihisa; Okudera, Hajime; Okuda, Kazuhiro; Nakata, Koh; Kawase, Tomoyuki

2018-06-18

Platelet activation and aggregation have been conventionally evaluated using an aggregometer. However, this method is suitable for short-term but not long-term quantitative evaluation of platelet aggregation, morphological changes, and/or adhesion to specific materials. The recently developed digital holographic microscopy (DHM) has enabled the quantitative evaluation of cell size and morphology without labeling or destruction. Thus, we aim to validate its applicability in quantitatively evaluating changes in cell morphology, especially in the aggregation and spreading of activated platelets, thus modifying typical image analysis procedures to suit aggregated platelets. Freshly prepared platelet-rich plasma was washed with phosphate-buffered saline and treated with 0.1% CaCl 2 . Platelets were then fixed and subjected to DHM, scanning electron microscopy (SEM), atomic force microscopy, optical microscopy, and flow cytometry (FCM). Tightly aggregated platelets were identified as single cells. Data obtained from time-course experiments were plotted two-dimensionally according to the average optical thickness versus attachment area and divided into four regions. The majority of the control platelets, which supposedly contained small and round platelets, were distributed in the lower left region. As activation time increased, however, this population dispersed toward the upper right region. The distribution shift demonstrated by DHM was essentially consistent with data obtained from SEM and FCM. Therefore, DHM was validated as a promising device for testing platelet function given that it allows for the quantitative evaluation of activation-dependent morphological changes in platelets. DHM technology will be applicable to the quality assurance of platelet concentrates, as well as diagnosis and drug discovery related to platelet functions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Effects of low temperature on shear-induced platelet aggregation and activation.

PubMed

Zhang, Jian-ning; Wood, Jennifer; Bergeron, Angela L; McBride, Latresha; Ball, Chalmette; Yu, Qinghua; Pusiteri, Anthony E; Holcomb, John B; Dong, Jing-fei

2004-08-01

Hemorrhage is a major complication of trauma and often becomes more severe in hypothermic patients. Although it has been known that platelets are activated in the cold, studies have been focused on platelet behavior at 4 degrees C, which is far below temperatures encountered in hypothermic trauma patients. In contrast, how platelets function at temperatures that are commonly found in hypothermic trauma patients (32-37 degrees C) remains largely unknown, especially when they are exposed to significant changes in fluid shear stress that could occur in trauma patients due to hemorrhage, vascular dilation/constriction, and fluid resuscitation. Using a cone-plate viscometer, we have examined platelet activation and aggregation in response to a wide range of fluid shear stresses at 24, 32, 35, and 37 degrees C. We found that shear-induced platelet aggregation was significantly increased at 24, 32, and 35 degrees C as compared with 37 degrees C and the enhancement was observed in whole blood and platelet-rich plasma. In contrast to observation made at 4 degrees C, the increased shear-induced platelet aggregation at these temperatures was associated with minimal platelet activation as determined by the P-selectin expression on platelet surface. Blood viscosity was also increased at low temperature and the changes in viscosity correlated with levels of plasma total protein and fibrinogen. We found that platelets are hyper-reactive to fluid shear stress at temperatures of 24, 32, and 35 degrees C as compared with at 37 degrees C. The hyperreactivity results in heightened aggregation through a platelet-activation independent mechanism. The enhanced platelet aggregation parallels with increased whole blood viscosity at these temperatures, suggesting that enhanced mechanical cross-linking may be responsible for the enhanced platelet aggregation.

Nonthrombogenic Hydrogel Coatings with Carbene-Cross-Linking Bioadhesives.

PubMed

Nanda, Himansu Sekhar; Shah, Ankur Harish; Wicaksono, Gautama; Pokholenko, Oleksandr; Gao, Feng; Djordjevic, Ivan; Steele, Terry W J

2018-05-14

Bioadhesives are a current unmet clinical need for mending of blood contacting soft tissues without inducing thrombosis. Recent development of carbene precursor bioadhesives with the advantages of on-demand curing, tuneable modulus, and wet adhesion have been synthesized by grafting diazirine onto poly (amidoamine) (PAMAM-G5) dendrimers. Herein, the structure activity relationships of platelet adhesion and activation is evaluated for the first time on the cured PAMAM-g-diazirine bioadhesives. Three strategies were employed to prevent healthy human donor platelets from adhering and activating on light-cured bioadhesive surfaces: (1) Attenuation of cationic surface charge, (2) antifouling composites by incorporating heparin and alginate in uncured formulation, and (3) heparin wash of cured bioadhesive surface. Topographical imaging of cured and ethanol dehydrated bioadhesive surfaces was used to quantify the adhered and activated platelets with scanning electron microscopy, whose resolution allowed identification of round senescent, short dendritic, and long dendritic platelets. Cured surfaces of PAMAM-g-diazirine (15%) had 10300 ± 500 adhered platelets mm -2 with 99.7% activation into short/long dendritic cells. Reduction of primary amines by higher degree of diazirine grafting or capping of free amines by acetylation reduces platelet adherence (2400 ± 200 vs 3000 ± 300, respectively). Physical incorporation of heparin and alginate in the formulations reduced the activated platelet; 1300 ± 300 and 300 ± 50, activated platelets mm -2 , in comparison with additive free adhesive formulation. Similarly, heparin rinse of the surface of additive free bioadhesive reduced the activated platelet to platelets of heparin composites at 600 ± 100 platelets mm -2 . PAMAM-g-diazirine (15%) bioadhesive retained the photocured mechanical properties and lap shear adhesion despite the addition of heparin and alginate additives.

Superior integrin activating capacity and higher adhesion to fibrinogen matrix in buffy coat-derived platelet concentrates (PCs) compared to PRP-PCs.

PubMed

Beshkar, Pezhman; Hosseini, Ehteramolsadat; Ghasemzadeh, Mehran

2018-02-01

Regardless of different sources, methods or devices which are applied for preparation of therapeutic platelets, these products are generally isolated from whole blood by the sedimentation techniques which are based on PRP or buffy coat (BC) separation. As a general fact, platelet preparation and storage are also associated with some deleterious changes that known as platelet storage lesion (PSL). Although these alternations in platelet functional activity are aggravated during storage, whether technical issues within preparation can affect integrin activation and platelet adhesion to fibrinogen were investigated in this study. PRP- and BC-platelet concentrates (PCs) were subjected to flowcytometry analysis to examine the expression of platelet activation marker, P-selectin as well as active confirmation of the GPIIb/IIIa (α IIb β 3 ) on day 0, 1, 3 and 5 post-storage. Platelet adhesion to fibrinogen matrix was evaluated by fluorescence microscopy. Glucose concentration and LDH activity were also measured by colorimetric methods. The increasing P-selectin expression during storage was in a reverse correlation with PAC-1 binding (r = -0.67; p = .001). PRP-PCs showed the higher level of P-selectin expression than BC-PCs, whereas the levels of PAC-1 binding and platelet adhesion to fibrinogen matrix were significantly lower in PRP-PCs. Higher levels of active confirmation of the GPIIb/IIIa in BC-PCs were also associated with greater concentration of glucose in these products. We demonstrated the superior capacities of integrin activation and adhesion to fibrinogen for BC-PCs compared to those of PRP-PCs. These findings may provide more advantages for BC method of platelet preparation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Platelets Drive Thrombus Propagation in a Hematocrit and Glycoprotein VI-Dependent Manner in an In Vitro Venous Thrombosis Model.

PubMed

Lehmann, Marcus; Schoeman, Rogier M; Krohl, Patrick J; Wallbank, Alison M; Samaniuk, Joseph R; Jandrot-Perrus, Martine; Neeves, Keith B

2018-05-01

The objective of this study was to measure the role of platelets and red blood cells on thrombus propagation in an in vitro model of venous valvular stasis. A microfluidic model with dimensional similarity to human venous valves consists of a sinus distal to a sudden expansion, where for sufficiently high Reynolds numbers, 2 countercurrent vortices arise because of flow separation. The primary vortex is defined by the points of flow separation and reattachment. A secondary vortex forms in the deepest recess of the valve pocket characterized by low shear rates. An initial fibrin gel formed within the secondary vortex of a tissue factor-coated valve sinus. Platelets accumulated at the interface of the fibrin gel and the primary vortex. Red blood cells at physiological hematocrits were necessary to provide an adequate flux of platelets to support thrombus growth out of the valve sinus. A subpopulation of platelets that adhered to fibrin expose phosphatidylserine. Platelet-dependent thrombus growth was attenuated by inhibition of glycoprotein VI with a blocking Fab fragment or D-dimer. A 3-step process regulated by hemodynamics was necessary for robust thrombus propagation: First, immobilized tissue factor initiates coagulation and fibrin deposition within a low flow niche defined by a secondary vortex in the pocket of a model venous valve. Second, a primary vortex delivers platelets to the fibrin interface in a red blood cell-dependent manner. Third, platelets adhere to fibrin, activate through glycoprotein VI, express phosphatidylserine, and subsequently promote thrombus growth beyond the valve sinus and into the bulk flow. © 2018 American Heart Association, Inc.

Identification of Aspergillus fumigatus Surface Components That Mediate Interaction of Conidia and Hyphae With Human Platelets.

PubMed

Rambach, Günter; Blum, Gerhard; Latgé, Jean-Paul; Fontaine, Thierry; Heinekamp, Thorsten; Hagleitner, Magdalena; Jeckström, Hanna; Weigel, Günter; Würtinger, Philipp; Pfaller, Kristian; Krappmann, Sven; Löffler, Jürgen; Lass-Flörl, Cornelia; Speth, Cornelia

2015-10-01

Platelets were recently identified as a part of innate immunity. They are activated by contact with Aspergillus fumigatus; putative consequences include antifungal defense but also thrombosis, excessive inflammation, and thrombocytopenia. We aimed to identify those fungal surface structures that mediate interaction with platelets. Human platelets were incubated with Aspergillus conidia and hyphae, isolated wall components, or fungal surface mutants. Interaction was visualized microscopically; activation was quantified by flow cytometry of specific markers. The capacity of A. fumigatus conidia to activate platelets is at least partly due to melanin, because this effect can be mimicked with "melanin ghosts"; a mutant lacking melanin showed reduced platelet stimulating potency. In contrast, conidial hydrophobin masks relevant structures, because an A. fumigatus mutant lacking the hydrophobin protein induced stronger platelet activation than wild-type conidia. A. fumigatus hyphae also contain surface structures that interact with platelets. Wall proteins, galactomannan, chitin, and β-glucan are not the relevant hyphal components; instead, the recently identified fungal polysaccharide galactosaminogalactan potently triggered platelet activation. Conidial melanin and hydrophobin as well as hyphal galactosaminogalactan represent important pathogenicity factors that modulate platelet activity and thus might influence immune responses, inflammation, and thrombosis in infected patients. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

UAP56 is an important mediator of Angiotensin II/platelet derived growth factor induced vascular smooth muscle cell DNA synthesis and proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sahni, Abha; Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555; Wang, Nadan

2013-02-15

Highlights: ► Knockdown of UAP56 inhibits Angiotensin II/PDGF induced vascular smooth muscle cell proliferation. ► UAP56 is a positive regulator of E2F transcriptional activation. ► UAP56 is present in the vessel wall of low flow carotid arteries. -- Abstract: Angiotensin (Ang) II and platelet-derived growth factor (PDGF) are important mediators of pathologic vascular smooth muscle cell (VSMC) proliferation. Identifying downstream mediators of Ang II and PDGF signaling may provide insights for therapies to improve vascular proliferative diseases. We have previously demonstrated that breakpoint cluster region (Bcr) is an important mediator of Ang II/PDGF signaling in VSMC. We have recently reportedmore » that the DExD/H box protein UAP56 is an interacting partner of Bcr in regulating VSMC DNA synthesis. We hypothesized that UAP56 itself is an important regulator of VSMC proliferation. In this report we demonstrate that knockdown of UAP56 inhibits Ang II/PDGF induced VSMC DNA synthesis and proliferation, and inhibits E2F transcriptional activity. In addition, we demonstrate that UAP56 is present in the vessel wall of low-flow carotid arteries. These findings suggest that UAP56 is a regulator of VSMC proliferation and identify UAP56 as a target for preventing vascular proliferative disease.« less

Origin-Specific Adhesive Interactions of Mesenchymal Stem Cells with Platelets Influence Their Behavior After Infusion.

PubMed

Sheriff, Lozan; Alanazi, Asma; Ward, Lewis S C; Ward, Carl; Munir, Hafsa; Rayes, Julie; Alassiri, Mohammed; Watson, Steve P; Newsome, Phil N; Rainger, G E; Kalia, Neena; Frampton, Jon; McGettrick, Helen M; Nash, Gerard B

2018-02-28

We investigated the adhesive behavior of mesenchymal stem cells (MSC) in blood, which might influence their fate when infused as therapy. Isolated human bone marrow MSC (BMMSC) or umbilical cord MSC (UCMSC) adhered efficiently from flow to the matrix proteins, collagen, or fibronectin, but did not adhere to endothelial selectins. However, when suspended in blood, BMMSC no longer adhered to collagen, while UCMSC adhered along with many aggregated platelets. Neither MSC adhered to fibronectin from flowing blood, although the fibronectin surface did become coated with a platelet monolayer. UCMSC induced platelet aggregation in platelet rich plasma, and caused a marked drop in platelet count when mixed with whole human or mouse blood in vitro, or when infused into mice. In contrast, BMMSC did not activate platelets or induce changes in platelet count. Interestingly, isolated UCMSC and BMMSC both adhered to predeposited platelets. The differences in behavior in blood were attributable to expression of podoplanin (an activating ligand for the platelet receptor CLEC-2), which was detected on UCMSC, but not BMMSC. Thus, platelets were activated when bound to UCMSC, but not BMMSC. Platelet aggregation by UCMSC was inhibited by recombinant soluble CLEC-2, and UCMSC did not cause a reduction in platelet count when mixed with blood from mice deficient in CLEC-2. We predict that both MSC would carry platelets in the blood, but their interaction with vascular endothelium would depend on podoplanin-induced activation of the bound platelets. Such interactions with platelets might target MSC to damaged tissue, but could also be thrombotic. Stem Cells 2018. © 2018 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Effects of different shear rates on the attachment and detachment of platelet thrombi.

PubMed

Shi, Xiaofeng; Yang, Jichun; Huang, Jiansong; Long, Zhangbiao; Ruan, Zheng; Xiao, Bing; Xi, Xiaodong

2016-03-01

Thrombosis and hemostasis take place in flowing blood, which generates shear forces. The effect of different shear rates, particularly pathological forces, on platelet thrombus formation remains to be fully elucidated. The present study observed the morphological characteristics and hierarchical structure of thrombi on the collagen surface at a wide range of wall shear rates (WSRs) and examined the underlying mechanisms. Calcein AM‑labeled whole blood was perfused over a collagen‑coated surface at different shear rates set by a Bioflux 200 microfluidic device and the thrombi formed were assessed for area coverage, the height and the hierarchical structure defined by the extent of platelet activation and packing density. The factors that affect thrombus formation were also investigated. Platelet thrombus formation varied under different WSRs, for example, dispersed platelet adhesion mixed with erythrocytes was observed at 125‑250 s(‑1), extensive and thin platelet thrombi were observed at 500‑1,500 s(‑1), and sporadic, thick thrombi were observed at pathological WSRs of 2,500‑5,000 s(‑1), which showed a tendency to be shed. With increasing WSRs, the height of the thrombi showed an increasing linear trend, whereas the total fluorescence intensity and area of the thrombi exhibited a parabolic curve‑like change, with a turning point at a WSR of 2,500 s(‑1). The number of thrombi, the average fluorescence intensity and the area per thrombus showed similar trends, with an initial upwards incline followed by a decline. The thrombi formed at higher WSRs had a thicker shell, which led to a more densely packed core. Platelet thrombus formation under shear‑flow was regulated by the adhesive strength, which was mediated by receptor‑ligand interaction, the platelet deposition induced by shear rates and the detachment by the dynamic force of flow. This resulted in a balance between thrombus attachment, including adhesion and aggregation, and detachment. Collectively, compared with physiological low WSRs, pathological high WSRs caused thicker and more easily shed thrombi with more condensed cores, which was regulated by an attachment‑detachment balance. These results provide novel insights into the properties of thrombus formation on collagen at different WSRs, and offers possible explanations for certain clinical physiopathological phenomena, including physical hemostasis and pathological thrombosis.

Flow cytometric analysis reveals the high levels of platelet activation parameters in circulation of multiple sclerosis patients.

PubMed

Morel, Agnieszka; Rywaniak, Joanna; Bijak, Michał; Miller, Elżbieta; Niwald, Marta; Saluk, Joanna

2017-06-01

The epidemiological studies confirm an increased risk of cardiovascular disease in multiple sclerosis, especially prothrombotic events directly associated with abnormal platelet activity. The aim of our study was to investigate the level of blood platelet activation in the circulation of patients with chronic phase of multiple sclerosis (SP MS) and their reactivity in response to typical platelets' physiological agonists. We examined 85 SP MS patients diagnosed according to the revised McDonald's criteria and 50 healthy volunteers as a control group. The platelet activation and reactivity were assessed using flow cytometry analysis of the following: P-selectin expression (CD62P), activation of GP IIb/IIIa complex (PAC-1 binding), and formation of platelet microparticles (PMPs) and platelet aggregates (PA) in agonist-stimulated (ADP, collagen) and unstimulated whole blood samples. Furthermore, we measured the level of soluble P-selectin (sP-selectin) in plasma using ELISA method, to evaluate the in vivo level of platelet activation, both in healthy and SP MS subjects. We found a statistically significant increase in P-selectin expression, GP IIb/IIIa activation, and formation of PMPs and PA, as well as in unstimulated and agonist-stimulated (ADP, collagen) platelets in whole blood samples from patients with SP MS in comparison to the control group. We also determined the higher sP-selectin level in plasma of SP MS subjects than in the control group. Based on the obtained results, we might conclude that during the course of SP MS platelets are chronically activated and display hyperreactivity to physiological agonists, such as ADP or collagen.

Platelet activation in essential hypertension during exercise: pre- and post-treatment changes with an angiotensin II receptor blocker.

PubMed

Gkaliagkousi, Eugenia; Gavriilaki, Eleni; Yiannaki, Efi; Markala, Dimitra; Papadopoulos, Nikolaos; Triantafyllou, Areti; Anyfanti, Panagiota; Petidis, Konstantinos; Garypidou, Vasileia; Doumas, Michael; Ferro, Albert; Douma, Stella

2014-04-01

Acute exercise may exert deleterious effects on the cardiovascular system through a variety of pathophysiological mechanisms, including increased platelet activation. However, the degree of exercise-induced platelet activation in untreated hypertensive (UH) individuals as compared with normotensive (NT) individuals has yet to be established. Furthermore, the effect of antihypertensive treatment on exercise-induced platelet activation in essential hypertension (EH) remains unknown. Study 1 consisted of 30 UH and 15 NT subjects. UH subjects who received treatment were included in study 2 and were followed-up after a 3-month treatment period with an angiotensin II receptor blocker (ARB; valsartan). Circulating monocyte-platelet aggregates (MPA) and platelet P-selectin were measured as platelet activation markers at baseline, immediately after a treadmill exercise test, and 10, 30, and 90 minutes later. Maximal platelet activation was observed at 10 minutes after peak exercise in both groups. In UH subjects, MPA levels remained increased at 30 minutes after peak exercise, despite BP fall to baseline levels. MPA levels were significantly higher in UH subjects than NT subjects at maximal exercise and at 10 and 30 minutes of recovery. Post-treatment MPA levels increased significantly only at 10 minutes into recovery and were similar to those of NT subjects. Acute high-intensity exercise exaggerates platelet activation in untreated patients with EH compared with NT individuals. Angiotensin II receptor blockade with adequate BP control greatly improves exercise-induced platelet activation in EH. Further studies are needed to clarify whether this phenomenon depends purely on BP lowering or benefits also from the pleiotropic effects of ARBs.

Mechanobiology of Platelets: Techniques to Study the Role of Fluid Flow and Platelet Retraction Forces at the Micro- and Nano-Scale

PubMed Central

Feghhi, Shirin; Sniadecki, Nathan J.

2011-01-01

Coagulation involves a complex set of events that are important in maintaining hemostasis. Biochemical interactions are classically known to regulate the hemostatic process, but recent evidence has revealed that mechanical interactions between platelets and their surroundings can also play a substantial role. Investigations into platelet mechanobiology have been challenging however, due to the small dimensions of platelets and their glycoprotein receptors. Platelet researchers have recently turned to microfabricated devices to control these physical, nanometer-scale interactions with a higher degree of precision. These approaches have enabled exciting, new insights into the molecular and biomechanical factors that affect platelets in clot formation. In this review, we highlight the new tools used to understand platelet mechanobiology and the roles of adhesion, shear flow, and retraction forces in clot formation. PMID:22272117

Platelets release pathogenic serotonin and return to circulation after immune complex-mediated sequestration.

PubMed

Cloutier, Nathalie; Allaeys, Isabelle; Marcoux, Genevieve; Machlus, Kellie R; Mailhot, Benoit; Zufferey, Anne; Levesque, Tania; Becker, Yann; Tessandier, Nicolas; Melki, Imene; Zhi, Huiying; Poirier, Guy; Rondina, Matthew T; Italiano, Joseph E; Flamand, Louis; McKenzie, Steven E; Cote, Francine; Nieswandt, Bernhard; Khan, Waliul I; Flick, Matthew J; Newman, Peter J; Lacroix, Steve; Fortin, Paul R; Boilard, Eric

2018-02-13

There is a growing appreciation for the contribution of platelets to immunity; however, our knowledge mostly relies on platelet functions associated with vascular injury and the prevention of bleeding. Circulating immune complexes (ICs) contribute to both chronic and acute inflammation in a multitude of clinical conditions. Herein, we scrutinized platelet responses to systemic ICs in the absence of tissue and endothelial wall injury. Platelet activation by circulating ICs through a mechanism requiring expression of platelet Fcγ receptor IIA resulted in the induction of systemic shock. IC-driven shock was dependent on release of serotonin from platelet-dense granules secondary to platelet outside-in signaling by αIIbβ3 and its ligand fibrinogen. While activated platelets sequestered in the lungs and leaky vasculature of the blood-brain barrier, platelets also sequestered in the absence of shock in mice lacking peripheral serotonin. Unexpectedly, platelets returned to the blood circulation with emptied granules and were thereby ineffective at promoting subsequent systemic shock, although they still underwent sequestration. We propose that in response to circulating ICs, platelets are a crucial mediator of the inflammatory response highly relevant to sepsis, viremia, and anaphylaxis. In addition, platelets recirculate after degranulation and sequestration, demonstrating that in adaptive immunity implicating antibody responses, activated platelets are longer lived than anticipated and may explain platelet count fluctuations in IC-driven diseases.

Inhibition of the plasma SCUBE1, a novel platelet adhesive protein, protects mice against thrombosis.

PubMed

Wu, Meng-Ying; Lin, Yuh-Charn; Liao, Wei-Ju; Tu, Cheng-Fen; Chen, Ming-Huei; Roffler, Steve R; Yang, Ruey-Bing

2014-07-01

Signal peptide-CUB-EGF domain-containing protein 1 (SCUBE1), a secreted and surface-exposed glycoprotein on activated platelets, promotes platelet-platelet interaction and supports platelet-matrix adhesion. Its plasma level is a biomarker of platelet activation in acute thrombotic diseases. However, the exact roles of plasma SCUBE1 in vivo remain undefined. We generated new mutant (Δ) mice lacking the soluble but retaining the membrane-bound form of SCUBE1. Plasma SCUBE1-depleted Δ/Δ mice showed normal hematologic and coagulant features and expression of major platelet receptors, but Δ/Δ platelet-rich plasma showed impaired platelet aggregation in response to ADP and collagen treatment. The addition of purified recombinant SCUBE1 protein restored the aggregation of platelets in Δ/Δ platelet-rich plasma and further enhanced platelet aggregation in +/+ platelet-rich plasma. Plasma deficiency of SCUBE1 diminished arterial thrombosis in mice and protected against lethal thromboembolism induced by collagen-epinephrine treatment. Last, antibodies directed against the epidermal growth factor-like repeats of SCUBE1, which are involved in trans-homophilic protein-protein interactions, protected mice against fatal thromboembolism without causing bleeding in vivo. We conclude that plasma SCUBE1 participates in platelet aggregation by bridging adjacent activated platelets in thrombosis. Blockade of soluble SCUBE1 might represent a novel antithrombotic strategy. © 2014 American Heart Association, Inc.

In vitro glycoxidized low-density lipoproteins and low-density lipoproteins isolated from type 2 diabetic patients activate platelets via p38 mitogen-activated protein kinase.

PubMed

Calzada, Catherine; Coulon, Laurent; Halimi, Déborah; Le Coquil, Elodie; Pruneta-Deloche, Valérie; Moulin, Philippe; Ponsin, Gabriel; Véricel, Evelyne; Lagarde, Michel

2007-05-01

Platelet hyperactivation contributes to the increased risk for atherothrombosis in type 2 diabetes and is associated with oxidative stress. Plasma low-density lipoproteins (LDLs) are exposed to both hyperglycemia and oxidative stress, and their role in platelet activation remains to be ascertained. The aim of this study was to investigate the effects of LDLs modified by both glycation and oxidation in vitro or in vivo on platelet arachidonic acid signaling cascade. The activation of platelet p38 MAPK, the stress kinase responsible for the activation of cytosolic phospholipase A(2), and the concentration of thromboxane B(2), the stable catabolite of the proaggregatory arachidonic acid metabolite thromboxane A(2), were assessed. First, in vitro-glycoxidized LDLs increased the phosphorylation of platelet p38 MAPK as well as the concentration of thromboxane B(2). Second, LDLs isolated from plasma of poorly controlled type 2 diabetic patients stimulated both platelet p38 MAPK phosphorylation and thromboxane B(2) production and possessed high levels of malondialdehyde but normal alpha-tocopherol concentrations. By contrast, LDLs from sex- and age-matched healthy volunteers had no activating effects on platelets. Our results indicate that LDLs modified by glycoxidation may play an important contributing role in platelet hyperactivation observed in type 2 diabetes via activation of p38 MAPK.

Differential changes in platelet VEGF, Tsp, CXCL12, and CXCL4 in patients with metastatic cancer.

PubMed

Wiesner, Tina; Bugl, Stefanie; Mayer, Frank; Hartmann, Jörg T; Kopp, Hans-Georg

2010-03-01

Data from animal studies indicate that platelets play a key role in tumor dissemination and metastasis. We therefore hypothesized that metastastic cancer patients may display a specific platelet phenotype. Percentage of activated, p-selectin positive platelets as well as platelet contents (i.e., plasma and platelet count-corrected serum levels of VEGF-A, CXCL12, CXCL4, and thrombospondin-1) were analyzed in 43 patients with newly diagnosed metastatic disease prior to treatment. Tumor patients had increased platelet counts and significantly elevated percentages of activated platelets. Moreover, the platelet content of VEGF-A in cancer patients was significantly increased compared to healthy controls, while thrombospondin-1, CXCL12 and CXCL4 were significantly decreased. Our data contain several unexpected results: firstly, CXCL12 was found in minute quantities in the serum as compared with murine studies. Secondly, CXCL4, which was found by mass spectrometry to be the single massively upregulated intraplatelet chemokine in mice after tumor xenotransplantation, was decreased in tumor patient platelets. While increased contents of VEGF-A have been attributed to platelet scavenger activity, the differential decrease of specific platelet contents may be due to differential secretion or altered megakaryopoiesis in metastatic cancer patients.

Intracellular activation of the fibrinolytic cascade in the Quebec Platelet Disorder.

PubMed

Sheth, Prameet M; Kahr, Walter H A; Haq, M Anwar; Veljkovic, Dragoslava Kika; Rivard, Georges E; Hayward, Catherine P M

2003-08-01

The Quebec Platelet Disorder (QPD) is an unusual bleeding disorder associated with increased platelet stores of urokinase-type plasminogen activator (u-PA) and proteolysis of platelet alpha-granule proteins. The increased u-PA and proteolyzed plasminogen in QPD platelets led us to investigate possible contributions of intracellular plasmin generation to QPD alpha-granule proteolysis. ELISA indicated there were normal amounts of plasminogen and plasmin-alpha(2)-antiplasmin (PAP) complexes in QPD plasmas. Like normal platelets, QPD platelets contained only a small proportion of the blood plasminogen, however, they contained an increased amount of PAP complexes compared to normal platelets (P < 0.005). The quantities of plasminogen stored in platelets were important to induce QPD-like proteolysis of normal alpha-granule proteins by two chain u-PA (tcu-PA) in vitro. Moreover, adding supplemental plasminogen to QPD, but not to control, platelet lysates, triggered further alpha-granule protein proteolysis to forms that comigrated with plasmin degraded proteins. These data suggest the generation of increased but limiting amounts of plasmin within platelets is involved in producing the unique phenotypic changes to alpha-granule proteins in QPD platelets. The QPD is the only known bleeding disorder associated with chronic, intracellular activation of the fibrinolytic cascade.

Platelet Factor 4 Mediates Inflammation in Cerebral Malaria

PubMed Central

Srivastava, Kalyan; Cockburn, Ian A.; Swaim, AnneMarie; Thompson, Laura E.; Tripathi, Abhai; Fletcher, Craig A.; Shirk, Erin M.; Sun, Henry; Kowalska, M. Anna; Fox-Talbot, Karen; Sullivan, David; Zavala, Fidel; Morrell, Craig N.

2008-01-01

Summary Cerebral malaria is a major complication of Plasmodium falciparum infection in children. The pathogenesis of cerebral malaria involves vascular inflammation, immune stimulation and obstruction of cerebral capillaries. Platelets have a prominent role in both immune responses and vascular obstruction. We now demonstrate that the platelet derived chemokine, platelet factor 4 (PF4)/CXCL4, promotes the development of experimental cerebral malaria. Plasmodium infected red blood cells (RBC) activated platelets independent of vascular effects, resulting in increased plasma PF4. PF4 or CXCR3 null mice had less ECM, decreased brain T-cell recruitment, and platelet depletion or aspirin treatment reduced the development of ECM. We conclude that Plasmodium infected RBC can activate platelets and platelet derived PF4 then contributes to immune activation and T-cell trafficking as part of the pathogenesis of ECM. PMID:18692777

The duplication mutation of Quebec platelet disorder dysregulates PLAU, but not C10orf55, selectively increasing production of normal PLAU transcripts by megakaryocytes but not granulocytes.

PubMed

Hayward, Catherine P M; Liang, Minggao; Tasneem, Subia; Soomro, Asim; Waye, John S; Paterson, Andrew D; Rivard, Georges E; Wilson, Michael D

2017-01-01

Quebec Platelet disorder (QPD) is a unique bleeding disorder that markedly increases urokinase plasminogen activator (uPA) in megakaryocytes and platelets but not in plasma or urine. The cause is tandem duplication of a 78 kb region of chromosome 10 containing PLAU (the uPA gene) and C10orf55, a gene of unknown function. QPD increases uPA in platelets and megakaryocytes >100 fold, far more than expected for a gene duplication. To investigate the tissue-specific effect that PLAU duplication has on gene expression and transcript structure in QPD, we tested if QPD leads to: 1) overexpression of normal or unique PLAU transcripts; 2) increased uPA in leukocytes; 3) altered levels of C10orf55 mRNA and/or protein in megakaryocytes and leukocytes; and 4) global changes in megakaryocyte gene expression. Primary cells and cultured megakaryocytes from donors were prepared for quantitative reverse polymerase chain reaction analyses, RNA-seq and protein expression analyses. Rapidly isolated blood leukocytes from QPD subjects showed only a 3.9 fold increase in PLAU transcript levels, in keeping with the normal to minimally increased uPA in affinity purified, QPD leukocytes. All subjects had more uPA in granulocytes than monocytes and minimal uPA in lymphocytes. QPD leukocytes expressed PLAU alleles in proportions consistent with an extra copy of PLAU on the disease chromosome, unlike QPD megakaryocytes. QPD PLAU transcripts were consistent with reference gene models, with a much higher proportion of reads originating from the disease chromosome in megakaryocytes than granulocytes. QPD and control megakaryocytes contained minimal reads for C10orf55, and C10orf55 protein was not increased in QPD megakaryocytes or platelets. Finally, our QPD megakaryocyte transcriptome analysis revealed a global down regulation of the interferon type 1 pathway. We suggest that the low endogenous levels of uPA in blood are actively regulated, and that the regulatory mechanisms are disrupted in QPD in a megakaryocyte-specific manner.

The duplication mutation of Quebec platelet disorder dysregulates PLAU, but not C10orf55, selectively increasing production of normal PLAU transcripts by megakaryocytes but not granulocytes

PubMed Central

Soomro, Asim; Waye, John S.; Paterson, Andrew D.; Rivard, Georges E.; Wilson, Michael D.

2017-01-01

Quebec Platelet disorder (QPD) is a unique bleeding disorder that markedly increases urokinase plasminogen activator (uPA) in megakaryocytes and platelets but not in plasma or urine. The cause is tandem duplication of a 78 kb region of chromosome 10 containing PLAU (the uPA gene) and C10orf55, a gene of unknown function. QPD increases uPA in platelets and megakaryocytes >100 fold, far more than expected for a gene duplication. To investigate the tissue-specific effect that PLAU duplication has on gene expression and transcript structure in QPD, we tested if QPD leads to: 1) overexpression of normal or unique PLAU transcripts; 2) increased uPA in leukocytes; 3) altered levels of C10orf55 mRNA and/or protein in megakaryocytes and leukocytes; and 4) global changes in megakaryocyte gene expression. Primary cells and cultured megakaryocytes from donors were prepared for quantitative reverse polymerase chain reaction analyses, RNA-seq and protein expression analyses. Rapidly isolated blood leukocytes from QPD subjects showed only a 3.9 fold increase in PLAU transcript levels, in keeping with the normal to minimally increased uPA in affinity purified, QPD leukocytes. All subjects had more uPA in granulocytes than monocytes and minimal uPA in lymphocytes. QPD leukocytes expressed PLAU alleles in proportions consistent with an extra copy of PLAU on the disease chromosome, unlike QPD megakaryocytes. QPD PLAU transcripts were consistent with reference gene models, with a much higher proportion of reads originating from the disease chromosome in megakaryocytes than granulocytes. QPD and control megakaryocytes contained minimal reads for C10orf55, and C10orf55 protein was not increased in QPD megakaryocytes or platelets. Finally, our QPD megakaryocyte transcriptome analysis revealed a global down regulation of the interferon type 1 pathway. We suggest that the low endogenous levels of uPA in blood are actively regulated, and that the regulatory mechanisms are disrupted in QPD in a megakaryocyte-specific manner. PMID:28301587

New gene functions in megakaryopoiesis and platelet formation

PubMed Central

Gieger, Christian; Radhakrishnan, Aparna; Cvejic, Ana; Tang, Weihong; Porcu, Eleonora; Pistis, Giorgio; Serbanovic-Canic, Jovana; Elling, Ulrich; Goodall, Alison H.; Labrune, Yann; Lopez, Lorna M.; Mägi, Reedik; Meacham, Stuart; Okada, Yukinori; Pirastu, Nicola; Sorice, Rossella; Teumer, Alexander; Voss, Katrin; Zhang, Weihua; Ramirez-Solis, Ramiro; Bis, Joshua C.; Ellinghaus, David; Gögele, Martin; Hottenga, Jouke-Jan; Langenberg, Claudia; Kovacs, Peter; O’Reilly, Paul F.; Shin, So-Youn; Esko, Tõnu; Hartiala, Jaana; Kanoni, Stavroula; Murgia, Federico; Parsa, Afshin; Stephens, Jonathan; van der Harst, Pim; van der Schoot, C. Ellen; Allayee, Hooman; Attwood, Antony; Balkau, Beverley; Bastardot, François; Basu, Saonli; Baumeister, Sebastian E.; Biino, Ginevra; Bomba, Lorenzo; Bonnefond, Amélie; Cambien, François; Chambers, John C.; Cucca, Francesco; D’Adamo, Pio; Davies, Gail; de Boer, Rudolf A.; de Geus, Eco J. C.; Döring, Angela; Elliott, Paul; Erdmann, Jeanette; Evans, David M.; Falchi, Mario; Feng, Wei; Folsom, Aaron R.; Frazer, Ian H.; Gibson, Quince D.; Glazer, Nicole L.; Hammond, Chris; Hartikainen, Anna-Liisa; Heckbert, Susan R.; Hengstenberg, Christian; Hersch, Micha; Illig, Thomas; Loos, Ruth J. F.; Jolley, Jennifer; Khaw, Kay Tee; Kühnel, Brigitte; Kyrtsonis, Marie-Christine; Lagou, Vasiliki; Lloyd-Jones, Heather; Lumley, Thomas; Mangino, Massimo; Maschio, Andrea; Leach, Irene Mateo; McKnight, Barbara; Memari, Yasin; Mitchell, Braxton D.; Montgomery, Grant W.; Nakamura, Yusuke; Nauck, Matthias; Navis, Gerjan; Nöthlings, Ute; Nolte, Ilja M.; Porteous, David J.; Pouta, Anneli; Pramstaller, Peter P.; Pullat, Janne; Ring, Susan M.; Rotter, Jerome I.; Ruggiero, Daniela; Ruokonen, Aimo; Sala, Cinzia; Samani, Nilesh J.; Sambrook, Jennifer; Schlessinger, David; Schreiber, Stefan; Schunkert, Heribert; Scott, James; Smith, Nicholas L.; Snieder, Harold; Starr, John M.; Stumvoll, Michael; Takahashi, Atsushi; Tang, W. H. Wilson; Taylor, Kent; Tenesa, Albert; Thein, Swee Lay; Tönjes, Anke; Uda, Manuela; Ulivi, Sheila; van Veldhuisen, Dirk J.; Visscher, Peter M.; Völker, Uwe; Wichmann, H.-Erich; Wiggins, Kerri L.; Willemsen, Gonneke; Yang, Tsun-Po; Zhao, Jing Hua; Zitting, Paavo; Bradley, John R.; Dedoussis, George V.; Gasparini, Paolo; Hazen, Stanley L.; Metspalu, Andres; Pirastu, Mario; Shuldiner, Alan R.; van Pelt, L. Joost; Zwaginga, Jaap-Jan; Boomsma, Dorret I.; Deary, Ian J.; Franke, Andre; Froguel, Philippe; Ganesh, Santhi K.; Jarvelin, Marjo-Riitta; Martin, Nicholas G.; Meisinger, Christa; Psaty, Bruce M.; Spector, Timothy D.; Wareham, Nicholas J.; Akkerman, Jan-Willem N.; Ciullo, Marina; Deloukas, Panos; Greinacher, Andreas; Jupe, Steve; Kamatani, Naoyuki; Khadake, Jyoti; Kooner, Jaspal S.; Penninger, Josef; Prokopenko, Inga; Stemple, Derek; Toniolo, Daniela; Wernisch, Lorenz; Sanna, Serena; Hicks, Andrew A.; Rendon, Augusto; Ferreira, Manuel A.; Ouwehand, Willem H.; Soranzo, Nicole

2012-01-01

Platelets are the second most abundant cell type in blood and are essential for maintaining haemostasis. Their count and volume are tightly controlled within narrow physiological ranges, but there is only limited understanding of the molecular processes controlling both traits. Here we carried out a high-powered meta-analysis of genome-wide association studies (GWAS) in up to 66,867 individuals of European ancestry, followed by extensive biological and functional assessment. We identified 68 genomic loci reliably associated with platelet count and volume mapping to established and putative novel regulators of megakaryopoiesis and platelet formation. These genes show megakaryocyte-specific gene expression patterns and extensive network connectivity. Using gene silencing in Danio rerio and Drosophila melanogaster, we identified 11 of the genes as novel regulators of blood cell formation. Taken together, our findings advance understanding of novel gene functions controlling fate-determining events during megakaryopoiesis and platelet formation, providing a new example of successful translation of GWAS to function. PMID:22139419

Platelets miRNA as a Prediction Marker of Thrombotic Episodes

PubMed Central

Dzieciol, Malgorzata

2016-01-01

The blood platelets are crucial for the coagulation physiology to maintain haemostatic balance and are involved in various pathologies such as atherosclerosis and thrombosis. The studies of recent years have shown that anucleated platelets are able to succeed protein synthesis. Additionally, mRNA translation in blood platelets is regulated by miRNA molecules. Recent works postulate the possibility of using miRNAs as biomarkers of atherosclerosis and ischemic episodes. This review article describes clinical studies that presented blood platelets miRNAs expression profile changes in different thrombotic states, which suggest use of these molecules as predictive biomarkers. PMID:28042196

US definitions, current use, and FDA stance on use of platelet-rich plasma in sports medicine.

PubMed

Beitzel, Knut; Allen, Donald; Apostolakos, John; Russell, Ryan P; McCarthy, Mary Beth; Gallo, Gregory J; Cote, Mark P; Mazzocca, Augustus D

2015-02-01

With increased utilization of platelet-rich plasma (PRP), it is important for clinicians to understand the United States, the Food and Drug Administration (FDA) regulatory role and stance on PRP. Blood products such as PRP fall under the prevue of FDA's Center for Biologics Evaluation and Research (CBER). CBER is responsible for regulating human cells, tissues, and cellular and tissue-based products. The regulatory process for these products is described in the FDA's 21 CFR 1271 of the Code of Regulations. Under these regulations, certain products including blood products such as PRP are exempt and therefore do not follow the FDA's traditional regulatory pathway that includes animal studies and clinical trials. The 510(k) application is the pathway used to bring PRP preparation systems to the market. The 510(k) application allows devices that are "substantially equivalent" to a currently marketed device to come to the market. There are numerous PRP preparation systems on the market today with FDA clearance; however, nearly all of these systems have 510(k) clearance for producing platelet-rich preparations intended to be used to mix with bone graft materials to enhance bone graft handling properties in orthopedic practices. The use of PRP outside this setting, for example, an office injection, would be considered "off label." Clinicians are free to use a product off-label as long as certain responsibilities are met. Per CBER, when the intent is the practice of medicine, clinicians "have the responsibility to be well informed about the product, to base its use on firm scientific rationale and on sound medical evidence, and to maintain records of the product's use and effects." Finally, despite PRP being exempted, the language in 21 CFR 1271 has caused some recent concern over activated PRP; however to date, the FDA has not attempted to regulate activated PRP. Clinicians using activated PRP should be mindful of these concerns and continued to stay informed. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Platelet storage lesion in interim platelet unit concentrates: A comparison with buffy-coat and apheresis concentrates.

PubMed

Singh, Sukhi; Shams Hakimi, Caroline; Jeppsson, Anders; Hesse, Camilla

2017-12-01

Platelet storage lesion is characterized by morphological changes and impaired platelet function. The collection method and storage medium may influence the magnitude of the storage lesion. The aim of this study was to compare the newly introduced interim platelet unit (IPU) platelet concentrates (PCs) (additive solution SSP+, 40% residual plasma content) with the more established buffy-coat PCs (SSP, 20% residual plasma content) and apheresis PCs (autologous plasma) in terms of platelet storage lesions. Thirty PCs (n=10 for each type) were assessed by measuring metabolic parameters (lactate, glucose, and pH), platelet activation markers, and in vitro platelet aggregability on days 1, 4, and 7 after donation. The expression of platelet activation markers CD62p (P-selectin), CD63 (LAMP-3), and phosphatidylserine was measured using flow cytometry and in vitro aggregability was measured with multiple electrode aggregometry. Higher platelet activation and lower in vitro aggregability was observed in IPU than in buffy-coat PCs on day 1 after donation. In contrast, metabolic parameters, expression of platelet activation markers, and in vitro aggregability were better maintained in IPU than in buffy-coat PCs at the end of the storage period. Compared to apheresis PCs, IPU PCs had higher expression of activation markers and lower in vitro aggregability throughout storage. In conclusion, the results indicate that there are significant differences in platelet storage lesions between IPU, buffy-coat, and apheresis PCs. The quality of IPU PCs appears to be at least comparable to buffy-coat preparations. Further studies are required to distinguish the effect of the preparation methods from storage conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

The role of lectins and glycans in platelet clearance

PubMed Central

Hoffmeister, Karin M.

2015-01-01

Summary In recent years, it has become increasingly apparent that the life span of transfused platelets in circulation is regulated, at least in part, by glycan-lectin mediated mechanisms. There is clear evidence that refrigerated platelets are cleared by glycan-lectin mediated clearance mechanisms. Acute platelet cooling clusters glycoprotein (GP) Ibα receptors bearing uncovered N-acetylglucosamine (GlcNAc), and αMβ2 integrins on hepatic macrophages recognise clustered GlcNAc to rapidly clear these platelets from circulation. With prolonged refrigeration GPIbα clustering bearing uncovered galactose increases, which mediates the removal of long-term refrigerated platelets via hepatic Ashwell-Morell receptors (AMR), originally named as asialoglycoprotein receptors. In contrast, little is known about the molecular mechanisms of transfused room temperature platelet clearance. This review examines the role of glycan-lectin mediated clearance of exogenous, i.e. transfused chilled platelet clearance and briefly addresses the current knowledge of stored platelet function, degradation and its relation to platelet clearance. PMID:21781240

Insomnia, platelet serotonin and platelet monoamine oxidase in chronic alcoholism.

PubMed

Nenadic Sviglin, Korona; Nedic, Gordana; Nikolac, Matea; Mustapic, Maja; Muck-Seler, Dorotea; Borovecki, Fran; Pivac, Nela

2011-08-18

Insomnia is a common sleep disorder frequently occurring in chronic alcoholic patients. Neurobiological basis of insomnia, as well as of alcoholism, is associated with disrupted functions of the main neurotransmitter systems, including the serotonin (5-hydroxytryptamine, 5-HT) system. Blood platelets are considered a limited peripheral model for the central 5-HT neurons, since both platelets and central 5-HT synaptosomes have similar dynamics of 5-HT. Platelet 5-HT concentration and platelet monoamine oxidase type B (MAO-B) are assumed to represent biomarkers for particular symptoms and behaviors in psychiatric disorders. The hypothesis of this study was that platelet 5-HT concentration and platelet MAO-B activity will be altered in chronic alcoholic patients with insomnia compared to comparable values in patients without insomnia. The study included 498 subjects: 395 male and 103 female medication-free patients with alcohol dependence and 502 healthy control subjects: 325 men and 177 women. The effects of early, middle and late insomnia (evaluated using the Hamilton Depression Rating Scale), as well as sex, age and smoking on platelet 5-HT concentration and platelet MAO-B activity were evaluated using one-way ANOVA and multiple regression analysis by the stepwise method. Platelet 5-HT concentration, but not platelet MAO-B activity, was significantly reduced in alcoholic patients with insomnia compared to patients without insomnia. Multiple regression analysis revealed that platelet 5-HT concentration was affected by middle insomnia, smoking and sex, while platelet MAO activity was affected only by sex and age. The present and previous data suggest that platelet 5-HT concentration might be used, after controlling for sex and smoking, as a biomarker for insomnia in alcoholism, PTSD and in rotating shift workers. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Novel whole blood assay for phenotyping platelet reactivity in mice identifies ICAM-1 as a mediator of platelet-monocyte interaction

PubMed Central

Kirkby, Nicholas S.; Chan, Melissa V.; Finsterbusch, Michaela; Hogg, Nancy; Nourshargh, Sussan; Warner, Timothy D.

2015-01-01

Testing of platelet function is central to the cardiovascular phenotyping of genetically modified mice. Traditional platelet function tests have been developed primarily for testing human samples and the volumes required make them highly unsuitable for the testing of mouse platelets. This limits research in this area. To address this problem, we have developed a miniaturized whole blood aggregometry assay, based on a readily accessible 96-well plate format coupled with quantification of single platelet depletion by flow cytometric analysis. Using this approach, we observed a concentration-dependent loss of single platelets in blood exposed to arachidonic acid, collagen, U46619 or protease activated receptor 4 activating peptide. This loss was sensitive to well-established antiplatelet agents and genetic manipulation of platelet activation pathways. Observations were more deeply analyzed by flow cytometric imaging, confocal imaging, and measurement of platelet releasates. Phenotypic analysis of the reactivity of platelets taken from mice lacking intercellular adhesion molecule (ICAM)-1 identified a marked decrease in fibrinogen-dependent platelet-monocyte interactions, especially under inflammatory conditions. Such findings exemplify the value of screening platelet phenotypes of genetically modified mice and shed further light upon the roles and interactions of platelets in inflammation. PMID:26215112

Polyphosphate colocalizes with factor XII on platelet-bound fibrin and augments its plasminogen activator activity.

PubMed

Mitchell, Joanne L; Lionikiene, Ausra S; Georgiev, Georgi; Klemmer, Anja; Brain, Chelsea; Kim, Paul Y; Mutch, Nicola J

2016-12-15

Activated factor XII (FXIIa) has plasminogen activator capacity but its relative contribution to fibrinolysis is considered marginal compared with urokinase and tissue plasminogen activator. Polyphosphate (polyP) is released from activated platelets and mediates FXII activation. Here, we investigate the contribution of polyP to the plasminogen activator function of αFXIIa. We show that both polyP 70 , of the chain length found in platelets (60-100 mer), and platelet-derived polyP significantly augment the plasminogen activation capacity of αFXIIa. PolyP 70 stimulated the autoactivation of FXII and subsequent plasminogen activation, indicating that once activated, αFXIIa remains bound to polyP 70 Indeed, complex formation between polyP 70 and αFXIIa provides protection against autodegradation. Plasminogen activation by βFXIIa was minimal and not enhanced by polyP 70 , highlighting the importance of the anion binding site. PolyP 70 did not modulate plasmin activity but stimulated activation of Glu and Lys forms of plasminogen by αFXIIa. Accordingly, polyP 70 was found to bind to FXII, αFXIIa, and plasminogen, but not βFXIIa. Fibrin and polyP 70 acted synergistically to enhance αFXIIa-mediated plasminogen activation. The plasminogen activator activity of the αFXIIa-polyP 70 complex was modulated by C1 inhibitor and histidine-rich glycoprotein, but not plasminogen activator inhibitors 1 and 2. Platelet polyP and FXII were found to colocalize on the activated platelet membrane in a fibrin-dependent manner and decorated fibrin strands extending from platelet aggregates. We show that in the presence of platelet polyP and the downstream substrate fibrin, αFXIIa is a highly efficient and favorable plasminogen activator. Our data are the first to document a profibrinolytic function of platelet polyP. © 2016 by The American Society of Hematology.

Effects of Antimalarial Tafenoquine on Blood Platelet Activity and Survival.

PubMed

Cao, Hang; Bissinger, Rosi; Umbach, Anja T; Al Mamun Bhuyan, A; Lang, Florian; Gawaz, Meinrad

2017-01-01

The 8-aminoquinoline tafenoquine has been shown to be effective against Plasmodia, Leishmania and Trypanosoma. The substance is at least in part effective by triggering apoptosis of the parasites. Moreover, tafenoquine has been shown to trigger eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. The effect of tafenoquine on eryptosis is in part due to stimulation of Ca2+ entry and oxidative stress. Ca2+ entry is a critical event in the activation of blood platelets by thrombin and collagen related peptide (CRP). The present study explored, whether tafenoquine influences Ca2+ entry, activation and apoptosis of blood platelets. Platelets isolated from wild-type mice were exposed for 30 minutes to tafenoquine (2.5 µg/ml) without or with an additional treatment with thrombin (0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+] i ) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from α IIb β 3 integrin abundance, phosphatidylserine abundance from annexin-V-binding, relative platelet volume from forward scatter, reactive oxygen species (ROS) from DCF fluorescence, caspase 3 activity with an active caspase-3 Staining kit, and aggregation utilizing staining with CD9-APC and CD9-PE. Both, thrombin (0.01 U/ml) and CRP (2 µg/ml or 5 µg/ml), significantly increased [Ca2+] i , P-selectin abundance, active α IIb β 3 integrin, and annexin-V-binding, and both significantly decreased platelet volume, activated caspase 3 and stimulated aggregation. Administration of tafenoquine (2.5 µg/ml, 30 min) significantly decreased [Ca2+] i both, in the absence and presence of thrombin and CRP. Tafenoquine significantly blunted the effect of thrombin and CRP on [Ca2+] i , P-selectin abundance, and active α IIb β 3 integrin, but significantly increased ROS and annexin-V-binding, significantly augmented the effect of thrombin on caspase 3 activity and platelet volume and significantly enhanced platelet aggregation. Tafenoquine counteracts thrombin and CRP induced increase of cytosolic Ca2+ activity and platelet activation, but enhances platelet apoptosis and platelet aggregation. © 2017 The Author(s) Published by S. Karger AG, Basel.

[The effect of electromagnetic waves of very high frequency of molecular spectra of radiation and absorption of nitric oxide on the functional activity of platelets].

PubMed

Kirichuk, V F; Maĭborodin, A V; Volin, M V; Krenitskiĭ, A P; Tupikin, V D

2001-01-01

A study was made of the effect of electromagnetic EMI MMD-fluctuation on the frequencies of molecular spectra of radiation, and nitric oxide absorption under in vitro conditions on the functional activity of platelets in patients with unstable angina pectoris, with the help of a specially created generator. At amplitude-modulated and continuous modes of EMI MMD-irradiation of platelet-rich plasma for 5, 15 and 30 min the platelet functional activity decreases, which was shown up in reduction of their activation and fall of aggregative ability. The degree, to which platelet functional activity was inhibited, depended on the mode of irradiation and on duration of EMI MMD effect. The most obvious changes in platelet activation and in their readiness to aggregative response were observed at a continuous mode of irradiation within a 15 min interval.

Quebec platelet disorder: features, pathogenesis and treatment.

PubMed

Diamandis, Maria; Veljkovic, D Kika; Maurer-Spurej, Elisabeth; Rivard, Georges E; Hayward, Catherine P M

2008-03-01

Quebec platelet disorder (QPD) is a rare, autosomal-dominant, inherited bleeding disorder that is associated with unique abnormalities in fibrinolysis. Its hallmark features are delayed-onset bleeding following hemostatic challenges that responds to fibrinolytic inhibitor therapy and increased expression and storage of the fibrinolytic enzyme urokinase plasminogen activator in platelets, without increased plasma urokinase plasminogen activator or systemic fibrinolysis. The increased urokinase plasminogen activator in QPD platelets is only partially inhibited, and, as a result, there is intraplatelet generation of plasmin, and secondary degradation of many platelet alpha-granule proteins. During clot formation, the urokinase plasminogen activator released by QPD platelets leads to platelet-dependent increased fibrinolysis, and this is postulated to be a major contributor to QPD bleeding. The focus of the present review is to summarize the current state of knowledge on QPD, including the history of this disorder, its clinical and laboratory features, and recommended approaches for its diagnosis and treatment.

Comparison of platelet activation through hinge vs bulk flow in mechanical heart valves

NASA Astrophysics Data System (ADS)

Hedayat, Mohammadali; Borazjani, Iman

2017-11-01

Bileaflet mechanical heart valves increase the risk of thrombus formation in patients which is believed to be initiated by platelet activation. Platelets can be activated by the elevated shear stresses in the bulk flow during the systole phase or the flow through the hinge during the diastole. However, the importance of platelet activation by the bulk flow vs the hinge in MHVs has yet to be studied. Here, we investigate the contribution of each of the above mechanisms to the activation of platelets in MHs by performing simulation of the flow through a 25mm St. Jude Medical valve placed in a straight aorta. Two different gap sizes (250 and 150 micrometer) are used in this study. The simulations are done using a sharp interface curvilinear immersed boundary method along with a strong-coupling algorithm for FSI solver on overset grids. The platelet activation through the hinge for different gap sizes is compared to the activation in the bulk flow using two platelet activation models to ensure the consistency of the results. Our results for all gap sizes using different activation models show that the integration of platelet activation caused by the bulk flow is several times higher in comparison to the activation through the hinge. This work is supported by the American Heart Association Grant 13SDG17220022, and the computational resources were partly provided by Center for Computational Research (CCR) at University at Buffalo.

Platelet-rich plasma stimulated by pulse electric fields: Platelet activation, procoagulant markers, growth factor release and cell proliferation.

PubMed

Frelinger, A L; Torres, A S; Caiafa, A; Morton, C A; Berny-Lang, M A; Gerrits, A J; Carmichael, S L; Neculaes, V B; Michelson, A D

2016-01-01

Therapeutic use of activated platelet-rich plasma (PRP) has been explored for wound healing, hemostasis and antimicrobial wound applications. Pulse electric field (PEF) stimulation may provide more consistent platelet activation and avoid complications associated with the addition of bovine thrombin, the current state of the art ex vivo activator of therapeutic PRP. The aim of this study was to compare the ability of PEF, bovine thrombin and thrombin receptor activating peptide (TRAP) to activate human PRP, release growth factors and induce cell proliferation in vitro. Human PRP was prepared in the Harvest SmartPreP2 System and treated with vehicle, PEF, bovine thrombin, TRAP or Triton X-100. Platelet activation and procoagulant markers and microparticle generation were measured by flow cytometry. Released growth factors were measured by ELISA. The releasates were tested for their ability to stimulate proliferation of human epithelial cells in culture. PEF produced more platelet-derived microparticles, P-selectin-positive particles and procoagulant annexin V-positive particles than bovine thrombin or TRAP. These differences were associated with higher levels of released epidermal growth factor after PEF than after bovine thrombin or TRAP but similar levels of platelet-derived, vascular-endothelial, and basic fibroblast growth factors, and platelet factor 4. Supernatant from PEF-treated platelets significantly increased cell proliferation compared to plasma. In conclusion, PEF treatment of fresh PRP results in generation of microparticles, exposure of prothrombotic platelet surfaces, differential release of growth factors compared to bovine thrombin and TRAP and significant cell proliferation. These results, together with PEF's inherent advantages, suggest that PEF may be a superior alternative to bovine thrombin activation of PRP for therapeutic applications.

Effects of clopidogrel and aspirin in combination versus aspirin alone on platelet activation and major receptor expression in diabetic patients: the PLavix Use for Treatment Of Diabetes (PLUTO-Diabetes) trial.

PubMed

Serebruany, Victor L; Malinin, Alex I; Pokov, Alex; Barsness, Gregory; Hanley, Dan F

2008-01-01

Clopidogrel is widely used in diabetic patients after vascular events; however, the ability of this thienopyridine to yield additional antiplatelet protection on top of aspirin has never been explored in a controlled study with comprehensive assessment of platelet activity. The objective of this study was to compare the antiplatelet profiles of clopidogrel + aspirin in combination (C + ASA) versus aspirin alone (ASA) in patients with type 2 diabetes mellitus. Seventy patients with documented diabetes already treated with antecedent aspirin were randomly assigned to receive C + ASA or ASA in the PLUTO-Diabetes trial. Platelet studies included adenosine diphosphate-, collagen-, and arachidonic acid-induced aggregometry; PFA-100 (Dade-Behring, Miami, FL) and Ultegra (Accumetrics, San Diego, CA) analyzers; and expression of 6 major receptors by flow cytometry at baseline and at day 30 after randomization. There were no differences in the baseline clinical and platelet characteristics between the C + ASA and ASA groups, or subsequent significant changes in platelet biomarkers in the ASA group, except for diminished collagen-induced aggregation (P = .02). In contrast, when compared with the ASA group, therapy with C + ASA resulted in significant inhibition of platelet activity assessed by adenosine diphosphate aggregation (P = .0001); closure time prolongation (P = .0003) and reduction of platelet activation units with Ultegra (P = .0001); and expression of platelet/endothelial cell adhesion molecule 1 (P = .002), glycoprotein IIb/IIIa antigen (P = .0002), and activity (P = .0001). Treatment with C + ASA for 1 month provides significantly greater inhibition of platelet activity than ASA alone in diabetic patients in this small randomized trial. However, despite dual antiplatelet regimen, diabetic patients exhibit high residual activity of some platelet biomarkers, including unaffected protease-activated receptor 1 receptor expression.

Role of platelet activating factor in pathogenesis of acute pancreatitis in rats.

PubMed Central

Konturek, S J; Dembinski, A; Konturek, P J; Warzecha, Z; Jaworek, J; Gustaw, P; Tomaszewska, R; Stachura, J

1992-01-01

The importance of platelet activating factor in acute pancreatitis was examined by determining the tissue content of endogenous platelet activating factor and the protective effects of TCV-309, a highly selective platelet activating factor blocker, against caerulein induced pancreatitis in rats. Infusion of caerulein (10 micrograms/kg/h) for five hours resulted in about 70% increase in pancreatic weight, 22% rise in protein content, 50% reduction in tissue blood flow, nine fold increase in tissue level of platelet activating factor and 165% rise in plasma amylase as well as histological evidence of acute pancreatitis. Such infusion of caerulein in chronic pancreatic fistula rats caused a marked increase in protein output from basal secretion of 10 mg/30 minutes to 40 mg/30 minutes in the first hour of infusion followed by a decline in protein output to 15-20 mg/30 minutes in the following hours of the experiment. Exogenous platelet activating factor (50 micrograms/kg) injected ip produced similar alterations in weight, protein content, blood flow, and histology of the pancreas but the increment in serum amylase was significantly smaller and pancreatic secretion was reduced below the basal level. TCV-309 (50 micrograms/kg) given ip before caerulein or platelet activating factor administration significantly reduced the biochemical and morphological alterations caused by caerulein and abolished those induced by exogenous platelet activating factor. These results indicate that platelet activating factor plays an important role in the pathogenesis of acute pancreatitis probably by reducing the blood flow and increasing vascular permeability in the pancreas. PMID:1385272

Piperine Inhibits the Activities of Platelet Cytosolic Phospholipase A2 and Thromboxane A2 Synthase without Affecting Cyclooxygenase-1 Activity: Different Mechanisms of Action Are Involved in the Inhibition of Platelet Aggregation and Macrophage Inflammatory Response

PubMed Central

Son, Dong Ju; Akiba, Satoshi; Hong, Jin Tae; Yun, Yeo Pyo; Hwang, Seock Yeon; Park, Young Hyun; Lee, Sung Eun

2014-01-01

PURPOSE: Piperine, a major alkaloid of black pepper (Piper nigrum) and long pepper (Piper longum), was shown to have anti-inflammatory activity through the suppression of cyclooxygenase (COX)-2 gene expression and enzyme activity. It is also reported to exhibit anti-platelet activity, but the mechanism underlying this action remains unknown. In this study, we investigated a putative anti-platelet aggregation mechanism involving arachidonic acid (AA) metabolism and how this compares with the mechanism by which it inhibits macrophage inflammatory responses; METHODS: Rabbit platelets and murine macrophage RAW264.7 cells were treated with piperine, and the effect of piperine on the activity of AA-metabolizing enzymes, including cytosolic phospholipase A2 (cPLA2), COX-1, COX-2, and thromboxane A2 (TXA2) synthase, as well as its effect on AA liberation from the plasma membrane components, were assessed using isotopic labeling methods and enzyme immunoassay kit; RESULTS: Piperine significantly suppressed AA liberation by attenuating cPLA2 activity in collagen-stimulated platelets. It also significantly inhibited the activity of TXA2 synthase, but not of COX-1, in platelets. These results suggest that piperine inhibits platelet aggregation by attenuating cPLA2 and TXA2 synthase activities, rather than through the inhibition of COX-1 activity. On the other hand, piperine significantly inhibited lipopolysaccharide-induced generation of prostaglandin (PG)E2 and PGD2 in RAW264.7 cells by suppressing the activity of COX-2, without effect on cPLA2; CONCLUSION: Our findings indicate that piperine inhibits platelet aggregation and macrophage inflammatory response by different mechanisms. PMID:25153972

Gallic Acid Attenuates Platelet Activation and Platelet-Leukocyte Aggregation: Involving Pathways of Akt and GSK3β

PubMed Central

Chang, Shih-Sheng; Lee, Viola S. Y.; Tseng, Yu-Lun; Chang, Kuan-Cheng; Chen, Kuen-Bao; Chen, Yuh-Lien; Li, Chi-Yuan

2012-01-01

Platelet activation and its interaction with leukocytes play an important role in atherothrombosis. Cardiovascular diseases resulted from atherothrombosis remain the major causes of death worldwide. Gallic acid, a major constituent of red wine and tea, has been believed to have properties of cardiovascular protection, which is likely to be related to its antioxidant effects. Nonetheless, there were few and inconsistent data regarding the effects of gallic acid on platelet function. Therefore, we designed this in vitro study to determine whether gallic acid could inhibit platelet activation and the possible mechanisms. From our results, gallic acid could concentration-dependently inhibit platelet aggregation, P-selectin expression, and platelet-leukocyte aggregation. Gallic acid prevented the elevation of intracellular calcium and attenuated phosphorylation of PKCα/p38 MAPK and Akt/GSK3β on platelets stimulated by the stimulants ADP or U46619. This is the first mechanistic explanation for the inhibitory effects on platelets from gallic acid. PMID:22811749

Accurate measurement of volume and shape of resting and activated blood platelets from light scattering.

PubMed

Moskalensky, Alexander E; Yurkin, Maxim A; Konokhova, Anastasiya I; Strokotov, Dmitry I; Nekrasov, Vyacheslav M; Chernyshev, Andrei V; Tsvetovskaya, Galina A; Chikova, Elena D; Maltsev, Valeri P

2013-01-01

We introduce a novel approach for determination of volume and shape of individual blood platelets modeled as an oblate spheroid from angle-resolved light scattering with flow-cytometric technique. The light-scattering profiles (LSPs) of individual platelets were measured with the scanning flow cytometer and the platelet characteristics were determined from the solution of the inverse light-scattering problem using the precomputed database of theoretical LSPs. We revealed a phenomenon of parameter compensation, which is partly explained in the framework of anomalous diffraction approximation. To overcome this problem, additional a priori information on the platelet refractive index was used. It allowed us to determine the size of each platelet with subdiffraction precision and independent of the particular value of the platelet aspect ratio. The shape (spheroidal aspect ratio) distributions of platelets showed substantial differences between native and activated by 10 μM adenosine diphosphate samples. We expect that the new approach may find use in hematological analyzers for accurate measurement of platelet volume distribution and for determination of the platelet activation efficiency.

Relationship between platelet and urinary 8-Iso-PGF2α levels in subjects with different degrees of NOX2 regulation.

PubMed

Carnevale, Roberto; Iuliano, Luigi; Nocella, Cristina; Bartimoccia, Simona; Trapè, Stefano; Russo, Roberta; Gentile, Maria Cristina; Cangemi, Roberto; Loffredo, Lorenzo; Pignatelli, Pasquale; Violi, Francesco

2013-06-14

Urinary 8-iso-PGF2α, a marker of oxidative stress, is influenced by the activation of NOX2. It is unclear if platelets 8-iso-PGF2α contribute to urinary 8-iso-PGF2α. In a cross-sectional study, platelet, urinary, and serum 8-iso-PGF2α were determined in subjects with downregulation (X-linked chronic granulomatous disease [X-CGD], n=25) and upregulation (type II diabetic patients [T2D], n=121) of NOX2 and 153 controls matched for sex and age. In diabetic patients (n=18), the above variables were repeated before and after 7 days treatment with 100 mg/day aspirin or 100 mg/day aspirin plus 40 mg/day atorvastatin. In vitro study was performed to see the contribution of blood cells to serum 8-iso-PGF2α. Compared with controls, X-CGD patients had lower platelet, serum, and urinary 8-iso-PGF2α values; conversely, diabetic patients had higher values of 8-iso-PGF2α compared with controls. Urinary 8-iso-PGF2α significantly correlated with both platelet and serum 8-iso-PGF2α in the 2 cohorts. A parallel increase of platelet, serum, and urinary 8-iso-PGF2α by aspirin and a parallel decrease by aspirin plus atorvastatin were detected in the interventional study. In vitro study demonstrated that platelets contribute to 37% of serum 8-iso-PGF2α and that only 13% of it is of extravascular origin. The study suggests that NOX2 contributes to the formation of 8-iso-PGF2α in both platelets and urine. The direct correlation between platelet and urinary 8-iso-PGF2α suggests that, at least partly, urinary 8-iso-PGF2α reflects platelet 8-iso-PGF2α production. Analysis of serum 8-iso-PGF2α may represent a novel tool to investigate the production of 8-iso-PGF2α by blood cells including platelets. URL: ClinicalTrials.gov. Unique Identifier: NCT01250340.

Growth Factor Receptor–Bound Protein 2 Contributes to (Hem)Immunoreceptor Tyrosine-Based Activation Motif–Mediated Signaling in Platelets

PubMed Central

Morowski, Martina; Schiessl, Sarah; Schäfer, Carmen M.; Watson, Stephanie K.; Hughes, Craig E.; Ackermann, Jochen A.; Radtke, Daniel; Hermanns, Heike M.; Watson, Steve P.; Nitschke, Lars; Nieswandt, Bernhard

2015-01-01

Rationale Platelets are anuclear cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity but may also cause pathological vessel occlusion. One major pathway of platelet activation is triggered by 2 receptors that signal through an (hem)immunoreceptor tyrosine-based activation motif (ITAM), the activating collagen receptor glycoprotein (GP) VI and the C-type lectin-like receptor 2 (CLEC-2). Growth factor receptor–bound protein 2 (Grb2) is a ubiquitously expressed adapter molecule involved in signaling processes of numerous receptors in different cell types, but its function in platelets and MKs is unknown. Objective We tested the hypothesis that Grb2 is a crucial adapter protein in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets. Methods and Results Here, we show that genetic ablation of Grb2 in MKs and platelets did not interfere with MK differentiation or platelet production. However, Grb2-deficiency severely impaired glycoprotein VI–mediated platelet activation because of defective stabilization of the linker of activated T-cell (LAT) signalosome and activation of downstream signaling proteins that resulted in reduced adhesion, aggregation, and coagulant activity on collagen in vitro. Similarly, CLEC-2–mediated signaling was impaired in Grb2-deficient platelets, whereas the cells responded normally to stimulation of G protein–coupled receptors. In vivo, this selective (hem) immunoreceptor tyrosine-based activation motif signaling defect resulted in prolonged bleeding times but affected arterial thrombus formation only after concomitant treatment with acetylsalicylic acid, indicating that defective glycoprotein VI signaling in the absence of Grb2 can be compensated through thromboxane A2–induced G protein–coupled receptor signaling pathways. Conclusions These results reveal an important contribution of Grb2 in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets in hemostasis and thrombosis by stabilizing the LAT signalosome. PMID:24265393

The modulation of platelet adhesion and activation by chitosan through plasma and extracellular matrix proteins.

PubMed

Lord, Megan S; Cheng, Bill; McCarthy, Simon J; Jung, MoonSun; Whitelock, John M

2011-10-01

Chitosan has been shown to promote initial wound closure events to prevent blood loss. Platelet adhesion and activation are crucial early events in these processes after traumatic bleeding leading to thrombus formation. Platelet adhesion to chitosan was found to be enhanced in the presence of adsorbed plasma and extracellular matrix proteins and was found to be primarily mediated by α(IIb)β(3) integrins, while α(2)β(1) integrins were found to be involved in platelet adhesion to collagen and perlecan. Platelets were found to be activated by chitosan, as shown by an increase in the expression of α(IIb)β(3) integrins and P-selectin, while the extent of activation was modulated by the presence of proteins including perlecan and fibrinogen. Collagen-coated chitosan was found to activate platelets to the same extent as either chitosan or collagen alone. These data support the role of plasma and extracellular matrix proteins in promoting chitosan mediated platelet adhesion and activation supporting the hypothesis that chitosan promotes wound healing via these interactions. Copyright © 2011 Elsevier Ltd. All rights reserved.

Platelet receptor polymorphisms do not influence Staphylococcus aureus–platelet interactions or infective endocarditis

PubMed Central

Daga, Shruti; Shepherd, James G.; Callaghan, J. Garreth S.; Hung, Rachel K.Y.; Dawson, Dana K.; Padfield, Gareth J.; Hey, Shi Y.; Cartwright, Robyn A.; Newby, David E.; Fitzgerald, J. Ross

2011-01-01

Cardiac vegetations result from bacterium–platelet adherence, activation and aggregation, and are associated with increased morbidity and mortality in infective endocarditis. The GPIIb/IIIa and FcγRIIa platelet receptors play a central role in platelet adhesion, activation and aggregation induced by endocarditis pathogens such as Staphylococcus aureus, but the influence of known polymorphisms of these receptors on the pathogenesis of infective endocarditis is unknown. We determined the GPIIIa platelet antigen PlA1/A2 and FcγRIIa H131R genotype of healthy volunteers (n = 160) and patients with infective endocarditis (n = 40), and investigated the influence of these polymorphisms on clinical outcome in infective endocarditis and S. aureus–platelet interactions in vitro. Platelet receptor genotype did not correlate with development of infective endocarditis, vegetation characteristics on echocardiogram or the composite clinical end-point of embolism, heart failure, need for surgery or mortality (P > 0.05 for all), even though patients with the GPIIIa PlA1/A1 genotype had increased in vivo platelet activation (P = 0.001). Furthermore, neither GPIIIa PlA1/A2 nor FcγRIIa H131R genotype influenced S. aureus-induced platelet adhesion, activation or aggregation in vitro (P > 0.05). Taken together, our data suggest that the GPIIIa and FcγRIIa platelet receptor polymorphisms do not influence S. aureus–platelet interactions in vitro or the clinical course of infective endocarditis. PMID:21044892

Immune complexes formed following the binding of anti-platelet factor 4 (CXCL4) antibodies to CXCL4 stimulate human neutrophil activation and cell adhesion.

PubMed

Xiao, Zhihua; Visentin, Gian P; Dayananda, Kannayakanahalli M; Neelamegham, Sriram

2008-08-15

We tested the possibility that immune complexes formed following platelet factor 4 (PF4/CXCL4) binding to anti-PF4 antibody can stimulate neutrophil activation, similar to previous reports with platelets. Monoclonal Abs against PF4 and IgG from a heparin-induced thrombocytopenia (HIT) patient were applied. We observed that although PF4 or anti-PF4 antibody alone did not alter neutrophil function, costimulation with both reagents resulted in approximately 3-fold increase in cell surface Mac-1 expression, enhanced cell adhesion via L-selectin and CD18 integrins, and degranulation of secondary and tertiary granules. The level of Mac-1 up-regulation peaked at an intermediate PF4 dose, suggesting that functional response varies with antigen-antibody stoichiometry. PF4 binding to neutrophils was blocked by chondroitinase ABC. Cell activation was inhibited by both chondroitinase ABC and anti-CD32/FcgammaRII blocking mAb, IV.3. Confocal microscopy demonstrated that immune complexes colocalize with CD32a. Studies with HIT IgG demonstrated that neutrophils could be activated in the absence of exogenous heparin. These data, together, show that leukocyte surface chondroitin sulfates promote neutrophil activation by enhancing immune-complex binding to CD32a. Studies with recombinant PF4 suggest a role for arginine 49 in stabilizing PF4-chondroitin binding. Neutrophils activated via this mechanism may contribute to thrombosis and inflammation in patients mounting an immune response to PF4-heparin.

Human platelet activation by C3a and C3a des-arg

PubMed Central

1983-01-01

C3a liberated from C3 by treatment with C3 convertase (or by trypsin) induced aggregation of gel-filtered human platelets and stimulated serotonin release. At concentrations of 10(-10) M to 8 X 10(-12) M, C3a induced aggregation when added alone to platelets. However, at lower concentrations (2 X 10(-12) M) C3a did not aggregate platelets directly but exhibited highly significant synergism (two-way analysis of variance P less than 0.0001) with ADP in mediating platelet aggregation and release of serotonin. Removal of the C-terminus arginine from C3a abolished anaphylotoxin activity but did not affect the platelet- stimulating activity of the peptide. C3a and C3a des-arg were equally reactive in mediating platelet aggregation and release of serotonin. Further C3a and C3a des-arg exhibited synergism with ADP of equal significance in both aggregation and the release reaction. The concentrations of C3a required for the platelet-stimulating activity involve relatively small number of molecules per platelet (4,000-10,000 for the synergistic reaction with ADP). These data suggest the possibility of a C3a (C3a des-arg) receptor on human platelets. This premise is strengthened by the demonstration ultrastructurally of C3a on the platelet membrane subsequent to C3a stimulation. PMID:6604123

Genotypic and phenotypic diversity of Lactobacillus rhamnosus clinical isolates, their comparison with strain GG and their recognition by complement system

PubMed Central

Douillard, François P.; Ritari, Jarmo; Paulin, Lars; Järvinen, Hanna M.; Rasinkangas, Pia; Haapasalo, Karita; Meri, Seppo; Jarva, Hanna; de Vos, Willem M.

2017-01-01

Lactobacillus rhamnosus strains are ubiquitous in fermented foods, and in the human body where they are commensals naturally present in the normal microbiota composition of gut, vagina and skin. However, in some cases, Lactobacillus spp. have been implicated in bacteremia. The aim of the study was to examine the genomic and immunological properties of 16 clinical blood isolates of L. rhamnosus and to compare them to the well-studied L. rhamnosus probiotic strain GG. Blood cultures from bacteremic patients were collected at the Helsinki University Hospital laboratory in 2005–2011 and L. rhamnosus strains were isolated and characterized by genomic sequencing. The capacity of the L. rhamnosus strains to activate serum complement was studied using immunological assays for complement factor C3a and the terminal pathway complement complex (TCC). Binding of complement regulators factor H and C4bp was also determined using radioligand assays. Furthermore, the isolated strains were evaluated for their ability to aggregate platelets and to form biofilms in vitro. Genomic comparison between the clinical L. rhamnosus strains showed them to be clearly different from L. rhamnosus GG and to cluster in two distinct lineages. All L. rhamnosus strains activated complement in serum and none of them bound complement regulators. Four out of 16 clinical blood isolates induced platelet aggregation and/or formed more biofilms than L. rhamnosus GG, which did not display platelet aggregation activity nor showed strong biofilm formation. These findings suggest that clinical L. rhamnosus isolates show considerable heterogeneity but are clearly different from L. rhamnosus GG at the genomic level. All L. rhamnosus strains are still normally recognized by the human complement system. PMID:28493885

Differential endosomal sorting of a novel P2Y12 purinoreceptor mutant.

PubMed

Cunningham, Margaret R; Nisar, Shaista P; Cooke, Alexandra E; Emery, Elizabeth D; Mundell, Stuart J

2013-05-01

P2Y12 receptor internalization and recycling play an essential role in ADP-induced platelet activation. Recently, we identified a patient with a mild bleeding disorder carrying a heterozygous mutation of P2Y12 (P341A) whose P2Y12 receptor recycling was significantly compromised. Using human cell line models, we identified key proteins regulating wild-type (WT) P2Y12 recycling and investigated P2Y12 -P341A receptor traffic. Treatment with ADP resulted in delayed Rab5-dependent internalization of P341A when compared with WT P2Y12 . While WT P2Y12 rapidly recycled back to the membrane via Rab4 and Rab11 recycling pathways, limited P341A recycling was observed, which relied upon Rab11 activity. Although minimal receptor degradation was evident, P341A was localized in Rab7-positive endosomes with considerable agonist-dependent accumulation in the trans-Golgi network (TGN). Rab7 activity is known to facilitate recruitment of retromer complex proteins to endosomes to transport cargo to the TGN. Here, we identified that P341A colocalized with Vps26; depletion of which blocked limited recycling and promoted receptor degradation. This study has identified key points of divergence in the endocytic traffic of P341A versus WT-P2Y12 . Given that these pathways are retained in human platelets, this research helps define the molecular mechanisms regulating P2Y12 receptor traffic and explain the compromised receptor function in the platelets of the P2Y12 -P341A-expressing patient. © 2013 John Wiley & Sons A/S.

Differential procoagulant activity of microparticles derived from monocytes, granulocytes, platelets and endothelial cells: impact of active tissue factor.

PubMed

Shustova, Olga N; Antonova, Olga A; Golubeva, Nina V; Khaspekova, Svetlana G; Yakushkin, Vladimir V; Aksuk, Svetlana A; Alchinova, Irina B; Karganov, Mikhail Y; Mazurov, Alexey V

2017-07-01

: Microparticles released by activated/apoptotic cells exhibit coagulation activity as they express phosphatidylserine and some of them - tissue factor. We compared procoagulant properties of microparticles from monocytes, granulocytes, platelets and endothelial cells and assessed the impact of tissue factor in observed differences. Microparticles were sedimented (20 000g, 30 min) from the supernatants of activated monocytes, monocytic THP-1 cells, granulocytes, platelets and endothelial cells. Coagulation activity of microparticles was examined using plasma recalcification assay. The size of microparticles was evaluated by dynamic light scattering. Tissue factor activity was measured by its ability to activate factor X. All microparticles significantly accelerated plasma coagulation with the shortest lag times for microparticles derived from monocytes, intermediate - for microparticles from THP-1 cells and endothelial cells, and the longest - for microparticles from granulocytes and platelets. Average diameters of microparticles ranged within 400-600 nm. The largest microparticles were produced by endothelial cells and granulocytes, smaller - by monocytes, and the smallest - by THP-1 cells and platelets. The highest tissue factor activity was detected in microparticles from monocytes, lower activity - in microparticles from endothelial cells and THP-1 cells, and no activity - in microparticles from platelets and granulocytes. Anti-tissue factor antibodies extended coagulation lag times for microparticles from monocytes, endothelial cells and THP-1 cells and equalized them with those for microparticles from platelets and granulocytes. Higher coagulation activity of microparticles from monocytes, THP-1 cells and endothelial cells in comparison with microparticles from platelets and granulocytes is determined mainly by the presence of active tissue factor.

P2 receptor subtypes in the cardiovascular system.

PubMed Central

Kunapuli, S P; Daniel, J L

1998-01-01

Extracellular nucleotides have been implicated in a number of physiological functions. Nucleotides act on cell-surface receptors known as P2 receptors, of which several subtypes have been cloned. Both ATP and ADP are stored in platelets and are released upon platelet activation. Furthermore, nucleotides are also released from damaged or broken cells. Thus during vascular injury nucleotides play an important role in haemostasis through activation of platelets, modulation of vascular tone, recruitment of neutrophils and monocytes to the site of injury, and facilitation of adhesion of leucocytes to the endothelium. Nucleotides also moderate these functions by generating nitric oxide and prostaglandin I2 through activation of endothelial cells, and by activating different receptor subtypes on vascular smooth muscle cells. In the heart, P2 receptors regulate contractility through modulation of L-type Ca2+ channels, although the molecular mechanisms involved are still under investigation. Classical pharmacological studies have identified several P2 receptor subtypes in the cardiovascular system. Molecular pharmacological studies have clarified the nature of some of these receptors, but have complicated the picture with others. In platelets, the classical P2T receptor has now been resolved into three P2 receptor subtypes: the P2Y1, P2X1 and P2TAC receptors (the last of these, which is coupled to the inhibition of adenylate cyclase, is yet to be cloned). In peripheral blood leucocytes, endothelial cells, vascular smooth muscle cells and cardiomyocytes, the effects of classical P2X, P2Y and P2U receptors have been found to be mediated by more than one P2 receptor subtype. However, the exact functions of these multiple receptor subtypes remain to be understood, as P2-receptor-selective agonists and antagonists are still under development. PMID:9841859

Differential effects of somatostatin on circulating tissue factor procoagulant activity and protein.

PubMed

Boden, Guenther; Vaidyula, Vijender; Homko, Carol; Mozzoli, Maria; Rao, A Koneti

2007-05-01

The tissue factor (TF) pathway is the primary mechanism for initiation of blood coagulation. Circulating blood contains TF, which originates mainly from monocytes and is thrombogenic. The presence of somatostatin (SMS) receptors on monocytes suggests the possibility that SMS may regulate TF synthesis and/or release. Circulating TF procoagulant activity (TF-PCA), factor VIIa activity (FVIIa; clotting assays), TF antigen (TF-Ag; ELISA), prothrombin fragment 1.2 (F1.2), thrombin-antithrombin complexes (ELISAs), CD40 ligand expression on platelets, and monocyte-platelet aggregates (flow cytometry) were determined in blood from normal volunteers undergoing 24 h of basal glucose/basal insulin (BG/BI) clamps and high-glucose/high-insulin (HG/HI) clamps with and without SMS. Infusions of SMS under basal conditions (BG/BI) raised TF-PCA 1.8-fold (P < 0.03), TF-Ag 2.3-fold (P < 0.001), and TF expression on monocytes by 36% (P < 0.001) and decreased plasma levels of FVIIa by 30% (P < 0.001). Infusion of SMS reduced the 8.6-fold HG/HI-induced increase in TF-Ag by 26% and the 8.6-fold increase in TF-PCA by 100%. SMS also prevented the 60% increase in TF expression on monocytes, the 2.2-fold increase in F1.2, the 40% increase in CD40L expression on platelets, and the 17% increase in monocyte-platelet aggregates seen during HG/HI. We conclude that SMS completely prevented HG/HI-induced TF activation in normal volunteers and may be of use to reduce the procoagulant state and acute vascular events in hyperinsulinemic insulin-resistant patients with type 2 diabetes.

Ex vivo and in vivo studies of CME-1, a novel polysaccharide purified from the mycelia of Cordyceps sinensis that inhibits human platelet activation by activating adenylate cyclase/cyclic AMP.

PubMed

Lu, Wan-Jung; Chang, Nen-Chung; Jayakumar, Thanasekaran; Liao, Jiun-Cheng; Lin, Mei-Jiun; Wang, Shwu-Huey; Chou, Duen-Suey; Thomas, Philip Aloysius; Sheu, Joen-Rong

2014-12-01

CME-1, a novel water-soluble polysaccharide, was purified from the mycelia of Cordyceps sinensis, and its chemical structure was characterized to contain mannose and galactose in a ratio of 4:6 (27.6 kDa). CME-1 was originally observed to exert a potent inhibitory effect on tumor migration and a cytoprotective effect against oxidative stress. Activation of platelets caused by arterial thrombosis is relevant to various cardiovascular diseases (CVDs). However, no data are available concerning the effects of CME-1 on platelet activation. Hence, the purpose of this study was to examine the ex vivo and in vivo antithrombotic effects of CME-1 and its possible mechanisms in platelet activation. The aggregometry, immunoblotting, flow cytometric analysis and platelet functional analysis were used in this study. CME-1 (2.3-7.6 μM) exhibited highly potent activity in inhibiting human platelet aggregation when stimulated by collagen, thrombin, and arachidonic acid but not by U46619. CME-1 inhibited platelet activation accompanied by inhibiting Akt, mitogen-activated protein kinases (MAPKs), thromboxane B2 (TxB2) and hydroxyl radical (OH(●)) formation. However, CME-1 interrupted neither FITC-triflavin nor FITC-collagen binding to platelets. CME-1 markedly increased cyclic AMP levels, but not cyclic GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ, an inhibitor of guanylate cyclase, obviously reversed the CME-1-mediated effects on platelet aggregation and vasodilator-stimulated phosphoprotein (VASP), Akt, p38 MAPK phosphorylation, and TxB2 formation. CME-1 substantially prolonged the closure time of whole blood and the occlusion time of platelet plug formation. This study demonstrates for the first time that CME-1 exhibits highly potent antiplatelet activity that may initially activate adenylate cyclase/cyclic AMP and, subsequently, inhibit intracellular signals (such as Akt and MAPKs), ultimately inhibiting platelet activation. This novel role of CME-1 indicates that CME-1 exhibits high potential for application in treating and preventing CVDs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Modulation of platelet aggregation by areca nut and betel leaf ingredients: roles of reactive oxygen species and cyclooxygenase.

PubMed

Jeng, Jiiang-Huei; Chen, Shiao-Yun; Liao, Chang-Hui; Tung, Yuan-Yii; Lin, Bor-Ru; Hahn, Liang-Jiunn; Chang, Mei-Chi

2002-05-01

There are 2 to 6 billion betel quid (BQ) chewers in the world. Areca nut (AN), a BQ component, modulates arachidonic acid (AA) metabolism, which is crucial for platelet function. AN extract (1 and 2 mg/ml) stimulated rabbit platelet aggregation, with induction of thromboxane B2 (TXB2) production. Contrastingly, Piper betle leaf (PBL) extract inhibited AA-, collagen-, and U46619-induced platelet aggregation, and TXB2 and prostaglandin-D2 (PGD2) production. PBL extract also inhibited platelet TXB2 and PGD2 production triggered by thrombin, platelet activating factor (PAF), and adenosine diphosphate (ADP), whereas little effect on platelet aggregation was noted. Moreover, PBL is a scavenger of O2(*-) and *OH, and inhibits xanthine oxidase activity and the (*)OH-induced PUC18 DNA breaks. Deferoxamine, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and neomycin prevented AN-induced platelet aggregation and TXB2 production. Indomethacin, genistein, and PBL extract inhibited only TXB2 production, but not platelet aggregation. Catalase, superoxide dismutase, and dimethylthiourea (DMT) showed little effect on AN-induced platelet aggregation, whereas catalase and DMT inhibited the AN-induced TXB2 production. These results suggest that AN-induced platelet aggregation is associated with iron-mediated reactive oxygen species production, calcium mobilization, phospholipase C activation, and TXB2 production. PBL inhibited platelet aggregation via both its antioxidative effects and effects on TXB2 and PGD2 production. Effects of AN and PBL on platelet aggregation and AA metabolism is crucial for platelet activation in the oral mucosa and cardiovascular system in BQ chewers.

High shear induces platelet dysfunction leading to enhanced thrombotic propensity and diminished hemostatic capacity.

PubMed

Chen, Zengsheng; Mondal, Nandan K; Zheng, Shirong; Koenig, Steven C; Slaughter, Mark S; Griffith, Bartley P; Wu, Zhongjun J

2017-11-28

Thrombosis and bleeding are devastating adverse events in patients supported with blood-contacting medical devices (BCMDs). In this study, we delineated that high non-physiological shear stress (NPSS) caused platelet dysfunction that may contribute to both thrombosis and bleeding. Human blood was subjected to NPSS with short exposure time. Levels of platelet surface GPIbα and GPVI receptors as well as activation level of GPIIb/IIIa in NPSS-sheared blood were examined with flow cytometry. Adhesion of sheared platelets on fibrinogen, von Willibrand factor (VWF), and collagen was quantified with fluorescent microscopy. Ristocetin- and collagen-induced platelet aggregation was characterized by aggregometry. NPSS activated platelets in a shear and exposure time-dependent manner. The number of activated platelets increased with increasing levels of NPSS and exposure time, which corresponded well with increased adhesion of sheared platelets on fibrinogen. Concurrently, NPSS caused shedding of GPIbα and GPVI in a manner dependent on shear and exposure time. The loss of intact GPIbα and GPVI increased with increasing levels of NPSS and exposure time. The number of platelets adhered on VWF and collagen decreased with increasing levels of NPSS and exposure time, respectively. The decrease in the number of platelets adhered on VWF and collagen corresponded well with the loss in GPIbα and GPVI on platelet surface. Both ristocetin- and collagen-induced platelet aggregation in sheared blood decreased with increasing levels of NPSS and exposure time. The study clearly demonstrated that high NPSS causes simultaneous platelet activation and receptor shedding, resulting in a paradoxical effect on platelet function via two distinct mechanisms. The results from the study suggested that the NPSS could induce the concurrent propensity for both thrombosis and bleeding in patients.

Glaucocalyxin A Inhibits Platelet Activation and Thrombus Formation Preferentially via GPVI Signaling Pathway

PubMed Central

Li, Qiang; Ren, Lijie; Liu, Xiaohui; Chu, Chunjun; Ozaki, Yukio; Zhang, Jian; Zhu, Li

2013-01-01

Platelets play a pivotal role in atherothrombosis and the antiplatelet agents have been proved to be useful in preventing onset of acute clinical events including myocardial infarction and stroke. Increasing number of natural compounds has been identified to be potential antiplatelet agents. Here we report the antiplatelet effect of glaucocalyxin A (GLA), an ent-diterpenoid that we isolated and purified from the aerial parts of Rabdosia japonica (Burm. f.) var. glaucocalyx (Maxim.) Hara, and investigate the molecular mechanisms by which GLA inhibits platelet activation and thrombus formation. The effect of GLA on platelet activation was measured using platelets freshly isolated from peripheral blood of healthy donors. Results showed that pretreatment of human platelets with lower concentrations of GLA (0.01μg/ml, 0.1μg/ml) significantly inhibited platelet aggregation induced by collagen (P<0.001) and CRP (P<0.01), a synthetic GPVI ligand, but not by ADP and U46619. Accordingly, GLA inhibited collagen-stimulated tyrosine phosphorylation of Syk, LAT, and phospholipase Cγ2, the signaling events in collagen receptor GPⅥ pathway. GLA also inhibited platelet p-selectin secretion and integrin activation by convulxin, a GPVI selective ligand. Additionally, GLA was found to inhibit low-dose thrombin-induced platelet activation. Using a flow chamber device, GLA was found to attenuate platelet adhesion on collagen surfaces in high shear condition. In vivo studies showed that GLA administration increased the time for complete occlusion upon vascular injury in mice, but did not extend tail-bleeding time when mice were administered with relatively lower doses of GLA. Therefore, the present results provide the molecular basis for the inhibition effect of GLA on platelet activation and its in vivo effect on thrombus formation, suggesting that GLA could potentially be developed as an antiplatelet and antithrombotic agent. PMID:24386454

The influence of Rubus idaeus and Rubus caesius leaf extracts on platelet aggregation in whole blood. Cross-talk of platelets and neutrophils.

PubMed

Dudzinska, Dominika; Bednarska, Katarzyna; Boncler, Magdalena; Luzak, Boguslawa; Watala, Cezary

2016-07-01

Recently, polyphenols have gained attention as potential natural cardioprotective therapeutics, due to their antiplatelet, anti-inflammatory and anticoagulant activity. Species belonging to the genus Rubus sp. have been reported to be a source of polyphenolic compounds with antioxidative proprieties and beneficial biological activities. This study investigates the effects of leaf extracts obtained from red raspberry (Rubus idaeus L.) and European dewberry (Rubus caesius L.) on the reactivity of blood platelets. In ADP-stimulated blood, raspberry and dewberry extracts (15 µg/ml) markedly decreased platelet surface membrane expression of activated GPIIbIIIa receptor by 16% and 21%, respectively (P < 0.01) and significantly inhibited platelet aggregation (by 31-41% for raspberry and by 38-55% for dewberry, P < 0.01). In platelet-rich plasma (PRP), the extracts had no effect on ADP-induced platelet aggregation. The effectiveness of the extracts in whole blood and the lack of their activity in PRP indicate that leukocytes are likely to participate in the platelet response to the extracts. Our experiments show that the extracts significantly reduced the amount of free radicals released by activated neutrophils in whole blood (P < 0.001), as well as in suspensions of isolated neutrophils (P < 0.05). Moreover, the reduced number of neutrophils leads to the decreased efficiency of the extracts in the inhibition of platelet aggregation. In summary, our findings show that the raspberry and dewberry leaf extracts considerably modulated blood platelet reactivity in whole blood: they influenced blood platelet aggregation, possibly via the modulation of the redox status dependent on the oxidative activity of neutrophils.

Effects of clopidogrel and aspirin in combination versus aspirin alone on platelet activation and major receptor expression in patients after recent ischemic stroke: for the Plavix Use for Treatment of Stroke (PLUTO-Stroke) trial.

PubMed

Serebruany, Victor L; Malinin, Alex I; Ziai, Wendy; Pokov, Alex N; Bhatt, Deepak L; Alberts, Mark J; Hanley, Dan F

2005-10-01

Clopidogrel is widely used in patients after recent ischemic stroke; however, its ability to yield additional antiplatelet protection on top of aspirin has never been explored in a controlled study. To determine whether clopidogrel with aspirin (C+ASA) will produce more potent platelet inhibition than aspirin alone (ASA) in patients after ischemic stroke, we conducted the Plavix Use for Treatment of Stroke trial. Seventy patients after ischemic stroke were randomly assigned to C+ASA or ASA groups. Platelet studies included aggregometry; cartridge-based analyzers; expression of PECAM-1, P-selectin, GP IIb/IIIa (antigen and activity), vitronectin receptor, and formation of platelet-leukocyte microparticles by flow cytometry. Platelet tests were performed at baseline and after 30 days after randomization. There were no deaths, hospitalizations, or serious adverse events. There were no differences in the baseline platelet characteristics between C+ASA and ASA groups, or significant changes in platelet parameters in the ASA group, except diminished collagen-induced aggregation (P=0.001). In contrast, therapy with C+ASA resulted in a significant inhibition of platelet activity assessed by ADP- (P=0.00001) and collagen-induced (P=0.02) aggregation; closure time prolongation (P=0.03), and reduction of platelet activation units with Ultegra (P=0.00001); expression of PECAM-1 (P=0.01), and GP IIb/IIIa activity with PAC-1 (P=0.02) when compared with ASA group. Therapy with C+ASA also resulted in the reduced formation of platelet-leukocyte microparticles (P=0.02). Treatment with C+ASA for 1 month provides significantly greater inhibition of platelet activity than ASA alone in patients after recent ischemic stroke in the frame of the small randomized trial.

Hyperglycemia-Induced Platelet Activation in Type 2 Diabetes Is Resistant to Aspirin but Not to a Nitric Oxide–Donating Agent

PubMed Central

Gresele, Paolo; Marzotti, Stefania; Guglielmini, Giuseppe; Momi, Stefania; Giannini, Silvia; Minuz, Pietro; Lucidi, Paola; Bolli, Geremia B.

2010-01-01

OBJECTIVE Acute, short-term hyperglycemia enhances high shear stress–induced platelet activation in type 2 diabetes. Several observations suggest that platelets in type 2 diabetes are resistant to inhibition by aspirin. Our aim was to assess comparatively the effect of aspirin, a nitric oxide–donating agent (NCX 4016), their combination, or placebo on platelet activation induced by acute hyperglycemia in type 2 diabetes. RESEARCH DESIGN AND METHODS In a double-blind, placebo-controlled, randomized trial, 40 type 2 diabetic patients were allocated to 100 mg aspirin once daily, 800 mg NCX 4016 b.i.d., both of them, or placebo for 15 days. On day 15, 1 h after the morning dose, a 4-h hyperglycemic clamp (plasma glucose 13.9 mmol/l) was performed, and blood samples were collected before and immediately after it for platelet activation and cyclooxygenase-1 (COX-1) inhibition studies. RESULTS Acute hyperglycemia enhanced shear stress–induced platelet activation in placebo-treated patients (basal closure time 63 ± 7.1 s, after hyperglycemia 49.5 ± 1.4 s, −13.5 ± 6.3 s, P < 0.048). Pretreatment with aspirin, despite full inhibition of platelet COX-1, did not prevent it (−12.7 ± 6.9 s, NS vs. placebo). On the contrary, pretreatment with the NO donor NCX 4016, alone or in combination with aspirin, suppressed platelet activation induced by acute hyperglycemia (NCX 4016 +10.5 ± 8.3 s; NCX 4016 plus aspirin: +12.0 ± 10.7 s, P < 0.05 vs. placebo for both). Other parameters of shear stress–dependent platelet activation were also more inhibited by NCX 4016 than by aspirin, despite lesser inhibition of COX-1. CONCLUSIONS Acute hyperglycemia-induced enhancement of platelet activation is resistant to aspirin; a NO-donating agent suppresses it. Therapeutic approaches aiming at a wider platelet inhibitory action than that exerted by aspirin may prove useful in patients with type 2 diabetes. PMID:20299485

Potential fluid mechanic pathways of platelet activation.

PubMed

Shadden, Shawn C; Hendabadi, Sahar

2013-06-01

Platelet activation is a precursor for blood clotting, which plays leading roles in many vascular complications and causes of death. Platelets can be activated by chemical or mechanical stimuli. Mechanically, platelet activation has been shown to be a function of elevated shear stress and exposure time. These contributions can be combined by considering the cumulative stress or strain on a platelet as it is transported. Here, we develop a framework for computing a hemodynamic-based activation potential that is derived from a Lagrangian integral of strain rate magnitude. We demonstrate that such a measure is generally maximized along, and near to, distinguished material surfaces in the flow. The connections between activation potential and these structures are illustrated through stenotic flow computations. We uncover two distinct structures that may explain observed thrombus formation at the apex and downstream of stenoses. More broadly, these findings suggest fundamental relationships may exist between potential fluid mechanic pathways for mechanical platelet activation and the mechanisms governing their transport.

Potential fluid mechanic pathways of platelet activation

PubMed Central

Shadden, Shawn C.; Hendabadi, Sahar

2012-01-01

Platelet activation is a precursor for blood clotting, which plays leading roles in many vascular complications and causes of death. Platelets can be activated by chemical or mechanical stimuli. Mechanically, platelet activation has been shown to be a function of elevated shear stress and exposure time. These contributions can be combined by considering the cumulative stress or strain on a platelet as it is transported. Here we develop a framework for computing a hemodynamic-based activation potential that is derived from a Lagrangian integral of strain rate magnitude. We demonstrate that such a measure is generally maximized along, and near to, distinguished material surfaces in the flow. The connections between activation potential and these structures are illustrated through stenotic flow computations. We uncover two distinct structures that may explain observed thrombus formation at the apex and downstream of stenoses. More broadly, these findings suggest fundamental relationships may exist between potential fluid mechanic pathways for mechanical platelet activation and the mechanisms governing their transport. PMID:22782543

Farnesoid X Receptor and Liver X Receptor Ligands Initiate Formation of Coated Platelets

PubMed Central

Unsworth, Amanda J.; Bye, Alexander P.; Tannetta, Dionne S.; Desborough, Michael J.R.; Kriek, Neline; Sage, Tanya; Allan, Harriet E.; Crescente, Marilena; Yaqoob, Parveen; Warner, Timothy D.; Jones, Chris I.

2017-01-01

Objectives— The liver X receptors (LXRs) and farnesoid X receptor (FXR) have been identified in human platelets. Ligands of these receptors have been shown to have nongenomic inhibitory effects on platelet activation by platelet agonists. This, however, seems contradictory with the platelet hyper-reactivity that is associated with several pathological conditions that are associated with increased circulating levels of molecules that are LXR and FXR ligands, such as hyperlipidemia, type 2 diabetes mellitus, and obesity. Approach and Results— We, therefore, investigated whether ligands for the LXR and FXR receptors were capable of priming platelets to the activated state without stimulation by platelet agonists. Treatment of platelets with ligands for LXR and FXR converted platelets to the procoagulant state, with increases in phosphatidylserine exposure, platelet swelling, reduced membrane integrity, depolarization of the mitochondrial membrane, and microparticle release observed. Additionally, platelets also displayed features associated with coated platelets such as P-selectin exposure, fibrinogen binding, fibrin generation that is supported by increased serine protease activity, and inhibition of integrin αIIbβ3. LXR and FXR ligand-induced formation of coated platelets was found to be dependent on both reactive oxygen species and intracellular calcium mobilization, and for FXR ligands, this process was found to be dependent on cyclophilin D. Conclusions— We conclude that treatment with LXR and FXR ligands initiates coated platelet formation, which is thought to support coagulation but results in desensitization to platelet stimuli through inhibition of αIIbβ3 consistent with their ability to inhibit platelet function and stable thrombus formation in vivo. PMID:28619996

Platelet compatibility of magnesium alloys.

PubMed

Yahata, Chie; Mochizuki, Akira

2017-09-01

Lately, Mg alloys have been investigated as a new class of biomaterials owing to their excellent biodegradability and biocompatibility. It has previously been reported that the in vitro compatibility of a Mg alloy containing aluminum and zinc (AZ) alloy with the blood coagulation system is excellent due to Mg 2+ ions eluting from the alloy. In this study, the compatibility of the AZ alloy with platelets was evaluated by scanning electron microscopy (SEM) and flow cytometry. In the flow cytometry analysis, the platelets were stained using PAC-1 and P-selectin antibodies. SEM images and PAC-1 analyses showed no negative effects on the platelets, whereas P-selectin analysis showed marked platelet activation. To understand these contradictory results, the amount of β-thromboglobulin (β-TG) released from the platelets was investigated. From that investigation, it was concluded that platelets are markedly activated by the alloys. In addition to clarifying divergent results depending on the analysis method used, the effects of Mg 2+ ions and pH on platelet activation were studied. These results show that platelet activation is caused by an increase in pH at the alloy surface owing to the erosion of the alloy. Copyright © 2017 Elsevier B.V. All rights reserved.

Intracellular origin and ultrastructure of platelet-derived microparticles.

PubMed

Ponomareva, A A; Nevzorova, T A; Mordakhanova, E R; Andrianova, I A; Rauova, L; Litvinov, R I; Weisel, J W

2017-08-01

Essentials Platelet microparticles play a major role in pathologies, including hemostasis and thrombosis. Platelet microparticles have been analyzed and classified based on their ultrastructure. The structure and intracellular origin of microparticles depend on the cell-activating stimulus. Thrombin-treated platelets fall apart and form microparticles that contain cellular organelles. Background Platelet-derived microparticles comprise the major population of circulating blood microparticles that play an important role in hemostasis and thrombosis. Despite numerous studies on the (patho)physiological roles of platelet-derived microparticles, mechanisms of their formation and structural details remain largely unknown. Objectives Here we studied the formation, ultrastructure and composition of platelet-derived microparticles from isolated human platelets, either quiescent or stimulated with one of the following activators: arachidonic acid, ADP, collagen, thrombin or calcium ionophore A23187. Methods Using flow cytometry, transmission and scanning electron microscopy, we analyzed the intracellular origin, structural diversity and size distributions of the subcellular particles released from platelets. Results The structure, dimensions and intracellular origin of microparticles depend on the cell-activating stimulus. The main structural groups include a vesicle surrounded by one thin membrane or multivesicular structures. Thrombin, unlike other stimuli, induced formation of microparticles not only from the platelet plasma membrane and cytoplasm but also from intracellular structures. A fraction of these vesicular particles having an intracellular origin contained organelles, such as mitochondria, glycogen granules and vacuoles. The size of platelet-derived microparticles depended on the nature of the cell-activating stimulus. Conclusion The results obtained provide a structural basis for the qualitative differences of various platelet activators, for specific physiological and pathological effects of microparticles, and for development of advanced assays. © 2017 International Society on Thrombosis and Haemostasis.

Combined aspirin and cilostazol treatment is associated with reduced platelet aggregation and prevention of exercise-induced platelet activation.

PubMed

Cleanthis, M; Bhattacharya, V; Smout, J; Ashour, H; Stansby, G

2009-05-01

Cilostazol has proven efficacy in increasing walking distance in claudicants, but it has not been demonstrated to be more effective than placebo in secondary cardiovascular prevention. The direct effect of exercise on platelet function remains less well defined. We have investigated the effect of combination treatment with aspirin and cilostazol on platelet activity in claudicants subjected to repeated treadmill exercise. Nineteen claudicants completed a double-blind, randomised, controlled, cross-over trial. Each subject received a 2-week course of aspirin (75mg) and placebo and aspirin and cilostazol (100mg twice daily). Following each 2-week treatment period, patients participated in a standardised treadmill test (3.2kmh(-1), 10 degrees incline) walking to maximal claudication distance. The exercise was repeated thrice in total, and blood was sampled before and after exercise. Platelet activation was measured using free platelet counting aggregation, flow cytometry for surface markers of platelet activation and soluble P-selectin assay. Compared to aspirin and placebo, combination treatment with aspirin and cilostazol was associated with reduced arachidonic-acid-induced platelet aggregation (p<0.01, Wilcoxon signed-rank test). Aspirin and placebo treatment were associated with elevated P-selectin expression, platelet-monocyte aggregation and reduced CD42b expression (p<0.05, Wilcoxon signed-rank test) post-exercise. No difference was seen in spontaneous platelet aggregation whilst soluble P-selectin was reduced post-exercise with combination treatment with aspirin and cilostazol (p<0.05, Wilcoxon signed-rank test). Combination treatment with aspirin and cilostazol results in suppression of platelet activation and reduces the effect of exercise on platelets. The benefit seen may be a result of cilostazol enhancing the inhibitory effect of aspirin on the cyclo-oxygenase pathway.

Platelet aggregation but not activation and degranulation during the acute post-ischemic reperfusion phase in livers with no underlying disease

PubMed Central

van Golen, Rowan F.; Stevens, Katarzyna M.; Colarusso, Pina; Jaeschke, Hartmut; Heger, Michal

2016-01-01

Background Platelets and P-selectin (CD62P) play an unequivocal role in the pathology of hepatic ischemia/reperfusion (I/R) injury. Inhibition or knock-out of P-selectin or immunodepletion of platelets results in amelioration of post-ischemic inflammation, reduced hepatocellular damage, and improved survival. However, P-selectin expression on platelets and endothelial cells, which concurs with platelet activation, has never been clearly demonstrated in I/R-subjected livers. Aims To determine whether platelets become activated and degranulate in the acute phase of liver I/R and whether the platelets interact with neutrophils. Methods Hepatic I/R was induced in male C57BL/6J mice (N = 12) using 37.5-min ischemia time. Platelets, endothelial cells, and neutrophils were fluorescently labeled by systemic administration of non-blocking antibodies. Cell kinetics were monitored by intravital spinning disk confocal microscopy during 90 min of reperfusion. Image analysis and quantification was performed with dedicated software. Results Platelets adhered to sinusoids more extensively in post-ischemic livers compared to livers not subjected to I/R and formed aggregates, which occurred directly after ischemia. Platelets and endothelial cells did not express P-selectin in post-ischemic livers. There was no interaction between platelets and neutrophils. Conclusions Platelets aggregate but do not become activated and do not degranulate in post-ischemic livers. There is no platelet-neutrophil interplay during the early reperfusion phase in a moderate model of hepatic I/R injury. The mechanisms underlying the biological effects of platelets and P-selectin in this setting warrant further investigation. Relevance for patients I/R in surgical liver patients may compromise outcome due to post-ischemic oxidative stress and sterile inflammation. Both processes are mediated in part by platelets. Understanding platelet function during I/R is key to developing effective interventions for I/R injury and improving clinical outcomes. PMID:26925465

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weber, Thomas J.; Markillie, Lye MENG.

The thromboxane A{sub 2} (TXA{sub 2}) receptor (TP) is represented by two alternatively spliced forms, termed the platelet/placental (TP-P) and endothelial (TP-E) type receptors. Experimental evidence suggests that TP isoforms may be regulated by novel ligands termed the isoprostanes, which paradoxically act as TP agonists in smooth muscle and TP antagonists in platelet preparations. Here we have investigated whether prototypical isoprostanes (8-iso-PG{sub 2{sub {alpha}}} and 8-iso-PGE{sub 2}) regulate the activity of TP isoforms expressed in Chinese Hamster Ovary (CHO) cells using activator protein-1 (AP-1)-luciferase activity as a reporter. AP-1-luciferase activity was increased by a TP agonist (U46619) in CHO cellsmore » transfected with the human TP-P and TP-E receptors and this response was fully inhibited by TP antagonists (ISAP, SQ29,548). AP-1-luciferase activity was potently (nM) increased by 8-iso-PGE2 in CHO TP-P and TP-E cells, and this response was partially inhibited by cotreatment of cells with TP antagonists, while 8-iso-PGF{sub 2{sub {alpha}}} was without effect. Cyclooxygenase inhibitors did not abolish 8-iso-PGE{sub 2} mediated AP-1-luciferase activity, indicating that this response is not dependent on de novo TXA2 biosynthesis. Interestingly, 8-iso-PGE{sub 2}-mediated AP-1-luciferase activity was near maximal in naive cells between 1-10 nM concentrations, and this response was not inhibited by TP antagonist or reproduced by agonists for TP or EP1/EP3 receptors. These observations (1) support a role for novel ligands in the regulation of TP-dependent signaling, (2) indicate that TP-P and TP-E couple to AP-1, (3) provide further evidence that isoprostanes function as TP agonists in a cell-type specific fashion, and (4) indicate that additional targets regulated by 8-iso-PGE{sub 2} couple to AP-1.« less

Adhesion of human platelets to albumin is synergistically increased by lysophosphatidic acid and adrenaline in a donor-dependent fashion.

PubMed

Eriksson, Andreas C; Whiss, Per A; Nilsson, Ulrika K

2006-07-01

Lysophosphatidic acid (LPA) and adrenaline are weak platelet activators considered important for thrombus formation, and were previously shown to synergistically increase platelet aggregation. Here we investigate synergistic activation by LPA and adrenaline when measuring platelet adhesion. Platelet-rich plasma from healthy blood donors together with adrenaline and/or LPA were added to protein-coated microplates. Platelets were allowed to adhere and the amount of adhesion detected enzymatically. The LPA and adrenaline combination induced a synergistic increase of platelet adhesion to a normally non-adhesive albumin surface. The degree of synergy varied markedly between individuals; these variations could not be explained by age, gender, blood type or different amounts of platelets, oxidized low-density lipoprotein, insulin or glucose in plasma. There was a trend indicating increased synergistic effect for platelets sensitive to adrenaline stimulation. The synergistic effect was blocked by the alpha2-adrenoceptor antagonist yohimbine and inhibited by the ADP scavenger system creatine phosphate/creatine phosphokinase and antibodies against alphaIIbbeta3. Furthermore, platelets adhering to albumin after adrenaline and LPA treatment expressed P-selectin. In conclusion, LPA and adrenaline act synergistically to increase alphaIIbbeta3-mediated platelet adhesion to albumin, dependent on alpha2-adrenoceptor signalling and platelet secretion. We also confirm that synergistic platelet activation achieved with LPA and adrenaline is highly donor dependent.

Fibrinogen adsorption, platelet adhesion and activation on mixed hydroxyl-/methyl-terminated self-assembled monolayers.

PubMed

Rodrigues, Sofia N; Gonçalves, Inês C; Martins, M C L; Barbosa, Mário A; Ratner, Buddy D

2006-11-01

The effect of surface wettability on fibrinogen adsorption, platelet adhesion and platelet activation was investigated using self-assembled monolayers (SAMs) containing different ratios of longer chain methyl- and shorter chain hydroxyl-terminated alkanethiols (C15CH3 vs. C11OH) on gold. Protein adsorption studies were performed using radiolabeled human fibrinogen (HFG). Platelet adhesion and activation studies with and without pre-adsorbed fibrinogen, albumin and plasma were assessed using scanning electron microscopy (SEM) and a glutaraldehyde-induced fluorescence technique (GIFT). Results demonstrated a linear decrease of HFG adsorption with the increase of OH groups on the monolayer (increase of the hydrophilicity). Platelet adhesion and activation also decrease with increase of hydrophilicity of surface. Concerning SAMs pre-immersed in proteins, fibrinogen adsorption was related with high platelet adhesion and activation. The passivant effect of albumin on platelet adhesion and activation was only demonstrated on SAMs contained C11OH. When all the blood proteins are present (plasma) platelet adhesion was almost absent on SAMs with 65% and 100% C11OH. This could be explained by the higher albumin affinity of the SAMs with 65% C11OH and the lower total protein adsorption associated with SAMs with 100% C11OH.

Involvement of nuclear factor {kappa}B in platelet CD40 signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hachem, Ahmed; Yacoub, Daniel; Centre Hospitalier Universite de Montreal, 264 boul. Rene-Levesque est, Montreal, Quebec, Canada H2X 1P1

Highlights: Black-Right-Pointing-Pointer sCD40L induces TRAF2 association to CD40 and NF-{kappa}B activation in platelets. Black-Right-Pointing-Pointer I{kappa}B{alpha} phosphorylation downstream of CD40L/CD40 signaling is independent of p38 MAPK phosphorylation. Black-Right-Pointing-Pointer I{kappa}B{alpha} is required for sCD40L-induced platelet activation and potentiation of aggregation. -- Abstract: CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-{kappa}B). Givenmore » that platelets contain NF-{kappa}B, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of I{kappa}B{alpha}, which are abolished by CD40L blockade. Inhibition of I{kappa}B{alpha} phosphorylation reverses sCD40L-induced I{kappa}B{alpha} phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on I{kappa}B{alpha} phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of I{kappa}B{alpha} phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-{kappa}B activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against thrombo-inflammatory disorders.« less

Unfractionated and Low-Molecular-Weight Heparin and the Phosphodiesterase Inhibitors, IBMX and Cilostazol, Block Ex Vivo Equid Herpesvirus Type-1-Induced Platelet Activation.

PubMed

Stokol, Tracy; Serpa, Priscila Beatriz da Silva; Zahid, Muhammad N; Brooks, Marjory B

2016-01-01

Equid herpes virus type-1 (EHV-1) is a major pathogen of horses, causing abortion storms and outbreaks of herpes virus myeloencephalopathy. These clinical syndromes are partly attributed to ischemic injury from thrombosis in placental and spinal vessels. The mechanism of thrombosis in affected horses is unknown. We have previously shown that EHV-1 activates platelets through virus-associated tissue factor-initiated thrombin generation. Activated platelets participate in thrombus formation by providing a surface to localize coagulation factor complexes that amplify and propagate thrombin generation. We hypothesized that coagulation inhibitors that suppress thrombin generation (heparins) or platelet inhibitors that impede post-receptor thrombin signaling [phosphodiesterase (PDE) antagonists] would inhibit EHV-1-induced platelet activation ex vivo . We exposed platelet-rich plasma (PRP) collected from healthy horses to the RacL11 abortigenic and Ab4 neuropathogenic strains of EHV-1 at 1 plaque-forming unit/cell in the presence or absence of unfractionated heparin (UFH), low-molecular-weight heparin (LMWH) or the PDE inhibitors, 3-isobutyl-1methylxanthine (IBMX), and cilostazol. We assessed platelet activation status in flow cytometric assays by measuring P-selectin expression. We found that all of the inhibitors blocked EHV-1- and thrombin-induced platelet activation in a dose-dependent manner. Platelet activation in PRP was maximally inhibited at concentrations of 0.05 U/mL UFH and 2.5 μg/mL LMWH. These concentrations represented 0.1-0.2 U/mL anti-factor Xa activity measured in chromogenic assays. Both IBMX and cilostazol showed maximal inhibition of platelet activation at the highest tested concentration of 50 μM, but inhibition was lower than that seen with UFH and LMWH. Our results indicate that heparin anticoagulants and strong non-selective (IBMX) or isoenzyme-3 selective (cilostazol) PDE antagonists inhibit ex vivo EHV-1-induced platelet activation. These drugs have potential as adjunctive therapy to reduce the serious complications associated with EHV-1-induced thrombosis. Treatment trials are warranted to determine whether these drugs yield clinical benefit when administered to horses infected with EHV-1.

Mice Lacking the SLAM Family Member CD84 Display Unaltered Platelet Function in Hemostasis and Thrombosis

PubMed Central

Hofmann, Sebastian; Braun, Attila; Pozgaj, Rastislav; Morowski, Martina; Vögtle, Timo; Nieswandt, Bernhard

2014-01-01

Background Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity by forming thrombi at sites of vascular injury. Although the early events of thrombus formation—platelet adhesion and aggregation—have been intensively studied, less is known about the mechanisms and receptors that stabilize platelet-platelet interactions once a thrombus has formed. One receptor that has been implicated in this process is the signaling lymphocyte activation molecule (SLAM) family member CD84, which can undergo homophilic interactions and becomes phosphorylated upon platelet aggregation. Objective The role of CD84 in platelet physiology and thrombus formation was investigated in CD84-deficient mice. Methods and Results We generated CD84-deficient mice and analyzed their platelets in vitro and in vivo. Cd84−/− platelets exhibited normal activation and aggregation responses to classical platelet agonists. Furthermore, CD84 deficiency did not affect integrin-mediated clot retraction and spreading of activated platelets on fibrinogen. Notably, also the formation of stable three-dimensional thrombi on collagen-coated surfaces under flow ex vivo was unaltered in the blood of Cd84−/− mice. In vivo, Cd84−/− mice exhibited unaltered hemostatic function and arterial thrombus formation. Conclusion These results show that CD84 is dispensable for thrombus formation and stabilization, indicating that its deficiency may be functionally compensated by other receptors or that it may be important for platelet functions different from platelet-platelet interactions. PMID:25551754

An investigation of the antiplatelet effects of succinobucol (AGI-1067).

PubMed

Houston, Stephanie A; Ugusman, Azizah; Gnanadesikan, Sukanya; Kennedy, Simon

2017-05-01

Succinobucol is a phenolic antioxidant with anti-inflammatory and antiplatelet effects. Given the importance of oxidant stress in modulating platelet-platelet and platelet-vessel wall interactions, the aim of this study was to establish if antioxidant activity was responsible for the antiplatelet activity of succinobucol. Platelet aggregation in response to collagen and adenosine diphosphate (ADP) was studied in rabbit whole blood and platelet-rich plasma using impedance aggregometry. The effect of oxidant stress on aggregation, platelet lipid peroxides, and vascular tone was studied by incubating platelets, washed platelets or preconstricted rabbit iliac artery rings respectively with a combination of xanthine and xanthine oxidase (X/XO). To study the effect of succinobucol in vivo, anaesthetized rats were injected with up to 150 mg/kg succinobucol and aggregation measured in blood removed 15 mins later. Succinobucol (10 -5 -10 -4 M) significantly attenuated platelet aggregation to collagen and ADP in whole blood and platelet-rich plasma. X/XO significantly increased aggregation to collagen and platelet lipid peroxides and this was reversed by succinobucol. Addition of X/XO to denuded rabbit iliac arteries caused a dose-dependent relaxation which was significantly inhibited by succinobucol. In vivo administration up to 150 mg/kg had no effect on heart rate or mean arterial blood pressure but significantly inhibited platelet aggregation to collagen ex vivo. In conclusion, succinobucol displays anti-platelet activity in rabbit and rat blood and reverses the increase in platelet aggregation in response to oxidant stress.

Platelet response heterogeneity in thrombus formation.

PubMed

Munnix, Imke C A; Cosemans, Judith M E M; Auger, Jocelyn M; Heemskerk, Johan W M

2009-12-01

Vascular injury leads to formation of a structured thrombus as a consequence of platelet activation and aggregation, thrombin and fibrin formation, and trapping of leukocytes and red cells. This review summarises current evidence for heterogeneity of platelet responses and functions in the thrombus-forming process. Environmental factors contribute to response heterogeneity, as the platelets in a thrombus adhere to different substrates, and sense specific (ant)agonists and rheological conditions. Contraction of platelets and interaction with fibrin and other blood cells cause further response variation. On the other hand, response heterogeneity can also be due to intrinsic differences between platelets in age and in receptor and signalling proteins. As a result, at least three subpopulations of platelets are formed in a thrombus: aggregating platelets with (reversible) integrin activation, procoagulant (coated) platelets exposing phosphatidylserine and binding coagulation factors, and contracting platelets with cell-cell contacts. This recognition of thrombus heterogeneity has implications for the use and development of antiplatelet medication.

Induction and contribution of beta platelet-derived growth factor signalling by hepatic stellate cells to liver regeneration after partial hepatectomy in mice.

PubMed

Kocabayoglu, Peri; Zhang, David Y; Kojima, Kensuke; Hoshida, Yujin; Friedman, Scott L

2016-06-01

Hepatic stellate cells (HSCs) activate during injury to orchestrate the liver's inflammatory and fibrogenic responses. A critical feature of HSC activation is the rapid induction of beta platelet-derived growth factor (β-PDGFR), which drives cellular fibrogenesis and proliferation; in contrast, normal liver has minimal β-PDGFR expression. While the role of β-PDGFR is well established in liver injury, its expression and contribution during liver regeneration are unknown. The aim of this study was to determine whether β-PDGFR is induced during liver regeneration following partial hepatectomy (pHx), and to define its contribution to the regenerative response. Control mice or animals with HSC-specific β-PDGFR-depletion underwent two-thirds pHx followed by assessment of hepatocyte proliferation and expression of β-PDGFR. RNA-sequencing from whole liver tissue of both groups after pHx was used to uncover pathways regulated by β-PDGFR signalling in HSCs. Beta platelet-derived growth factor expression on HSCs was up-regulated within 24 h following pHx in control mice, whereas absence of β-PDGFR blunted the expansion of HSCs. Mice lacking β-PDGFR displayed prolonged increases of transaminase levels within 72 h following pHx. Hepatocyte proliferation was impaired within the first 24 h based on Ki-67 and PCNA expression in β-PDGFR-deficient mice. This was associated with dysregulated growth in the β-PDGFR-deficient mice based on RNAseq with pathway analysis, and real-time quantitative PCR, which demonstrated reduced expression of Hgf, Igfbp1, Mapk and Il-6. Beta platelet-derived growth factor is induced in HSCs following surgical pHx and its deletion in HSCs leads to prolonged liver injury. However, there is no significant difference in liver regeneration. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The adhesion of blood platelets on fibrinogen surface: comparison of two biochemical microplate assays.

PubMed

Vanícková, Martina; Suttnar, Jirí; Dyr, Jan Evangelista

2006-11-01

The biocompatibility of materials is frequently assessed by blood platelet adhesion, since platelet adhesion plays a considerable role in blood interaction with artificial surfaces. Blood platelets adhesion is an essential event in haemostatic and thrombotic processes. The aim of this study was to simultaneously compare simple biochemical assays widely used for evaluation of platelet static adhesion based on the determination of enzymatic activity of either lactate dehydrogenase (LDH) or acid phosphatase (ACP) in lysates of adhered platelets. Adhesion of platelets from platelet-rich plasma and washed platelets activated by either ADP or thrombin on surfaces covered with fibrinogen and well defined fibrin was studied. The results demonstrated that the amounts of adhered platelets estimated by the LDH method were significantly lower as compared with the amount obtained by ACP method. LDH but not ACP release from platelets during adhesion was shown to take place. It suggests that the LDH method should be used rather as an assay of platelet integrity. The ACP method is much more suitable for quantitative determination of platelet adhesion especially in the development and evaluation of haemocompatibility of new biomaterials.

New Therapeutic Agent against Arterial Thrombosis: An Iridium(III)-Derived Organometallic Compound.

PubMed

Hsia, Chih-Wei; Velusamy, Marappan; Tsao, Jeng-Ting; Hsia, Chih-Hsuan; Chou, Duen-Suey; Jayakumar, Thanasekaran; Lee, Lin-Wen; Li, Jiun-Yi; Sheu, Joen-Rong

2017-12-05

Platelet activation plays a major role in cardio and cerebrovascular diseases, and cancer progression. Disruption of platelet activation represents an attractive therapeutic target for reducing the bidirectional cross talk between platelets and tumor cells. Platinum (Pt) compounds have been used for treating cancer. Hence, replacing Pt with iridium (Ir) is considered a potential alternative. We recently developed an Ir(III)-derived complex, [Ir(Cp*)1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine Cl]BF₄ (Ir-11), which exhibited strong antiplatelet activity; hence, we assessed the therapeutic potential of Ir-11 against arterial thrombosis. In collagen-activated platelets, Ir-11 inhibited platelet aggregation, adenosine triphosphate (ATP) release, intracellular Ca 2+ mobilization, P-selectin expression, and OH · formation, as well as the phosphorylation of phospholipase Cγ2 (PLCγ2), protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and Akt. Neither the adenylate cyclase inhibitor nor the guanylate cyclase inhibitor reversed the Ir-11-mediated antiplatelet effects. In experimental mice, Ir-11 prolonged the bleeding time and reduced mortality associated with acute pulmonary thromboembolism. Ir-11 plays a crucial role by inhibiting platelet activation through the inhibition of the PLCγ2-PKC cascade, and the subsequent suppression of Akt and MAPK activation, ultimately inhibiting platelet aggregation. Therefore, Ir-11 can be considered a new therapeutic agent against either arterial thrombosis or the bidirectional cross talk between platelets and tumor cells.

Identification of anti-Lea by platelet complement fixation.

PubMed

Ando, B; Ibayashi, H

1986-01-01

Two anti-Lea sera which were able to detect Lea antigen on platelets were identified in a screening for anti-platelet antibodies by means of a platelet complement fixation test. These two antisera hemolyzed erythrocytes without enzyme treatment. The anti-Lea activity could be completely absorbed by red cells, platelets and lymphocytes of Le(a+b-) donors but not by cells from Le(a-b+) or Le(a-b-) donors. The antibody activity against red cells was eliminated by treatment of the antisera with dithiothreitol, thereby suggesting that the activity resided in the IgM class of immunoglobulins. As the anti-Lea was more reactive at 37 degrees C than at room temperature against both red cells and platelets, we suggest that transfusion of platelets of Lea-negative donors should be considered for patients with this type of anti-Lea.

Towards optical control of single blood platelet activation

NASA Astrophysics Data System (ADS)

Spiryova, Darya V.; Karmatskih, Oleg Yu.; Vorob'ev, Alexei Yu.; Moskalensky, Alexander E.

2018-04-01

Blood platelets play a pivotal role in blood coagulation and in other normal and pathological processes. The understanding of fundamental mechanisms underlying their functions is very important for diagnostics and treatment. Single-cell experiments are needed for this purpose, which are complicated by insufficient spatiotemporal precision of conventional activation protocols. We present an approach to trigger single platelet activation optically, without the need of reagent mixing. This is achieved using photolabile compound, which rapidly delivers epinephrine upon UV irradiation. We demonstrated the applicability of the technique to rapidly induce platelet activation for studying dynamics of activation. The presented method may give novel fundamental knowledge about platelet functions and facilitate current research of their ability to deliver drugs to tumors or vascular injury sites.

Platelet GpIbα Binding to von Willebrand Factor Under Fluid Shear: Contributions of the D'D3‐Domain, A1‐Domain Flanking Peptide and O‐Linked Glycans

PubMed Central

Madabhushi, Sri R.; Zhang, Changjie; Kelkar, Anju; Dayananda, Kannayakanahalli M.; Neelamegham, Sriram

2014-01-01

Background Von Willebrand Factor (VWF) A1‐domain binding to platelet receptor GpIbα is an important fluid‐shear dependent interaction that regulates both soluble VWF binding to platelets, and platelet tethering onto immobilized VWF. We evaluated the roles of different structural elements at the N‐terminus of the A1‐domain in regulating shear dependent platelet binding. Specifically, the focus was on the VWF D′D3‐domain, A1‐domain N‐terminal flanking peptide (NFP), and O‐glycans on this peptide. Methods and Results Full‐length dimeric VWF (ΔPro‐VWF), dimeric VWF lacking the D′D3 domain (ΔD′D3‐VWF), and ΔD′D3‐VWF variants lacking either the NFP (ΔD′D3NFP─‐VWF) or just O‐glycans on this peptide (ΔD′D3OG─‐VWF) were expressed. Monomeric VWF‐A1 and D′D3‐A1 were also produced. In ELISA, the apparent dissociation constant (KD) of soluble ΔPro‐VWF binding to immobilized GpIbα (KD≈100 nmol/L) was 50‐ to 100‐fold higher than other proteins lacking the D′D3 domain (KD~0.7 to 2.5 nmol/L). Additionally, in surface plasmon resonance studies, the on‐rate of D′D3‐A1 binding to immobilized GpIbα (kon=1.8±0.4×104 (mol/L)−1·s−1; KD=1.7 μmol/L) was reduced compared with the single VWF‐A1 domain (kon=5.1±0.4×104 (mol/L)−1·s−1; KD=1.2 μmol/L). Thus, VWF‐D′D3 primarily controls soluble VWF binding to GpIbα. In contrast, upon VWF immobilization, all molecular features regulated A1‐GpIbα binding. Here, in ELISA, the number of apparent A1‐domain sites available for binding GpIbα on ΔPro‐VWF was ≈50% that of the ΔD′D3‐VWF variants. In microfluidics based platelet adhesion measurements on immobilized VWF and thrombus formation assays on collagen, human platelet recruitment varied as ΔPro‐VWF<ΔD′D3‐VWF<ΔD′D3NFP─‐VWF<ΔD′D3OG─‐VWF. Conclusions Whereas VWF‐D′D3 is the major regulator of soluble VWF binding to platelet GpIbα, both the D′D3‐domain and N‐terminal peptide regulate platelet translocation and thrombus formation. PMID:25341886

Influence of chirality on catalytic generation of nitric oxide and platelet behavior on selenocystine immobilized TiO2 films.

PubMed

Fan, Yonghong; Pan, Xiaxin; Wang, Ke; Wu, Sisi; Han, Honghong; Yang, Ping; Luo, Rifang; Wang, Hong; Huang, Nan; Tan, Wei; Weng, Yajun

2016-09-01

As nitric oxide (NO) plays vital roles in the cardiovascular system, incorporating this molecule into cardiovascular stents is considered as an effective method. In the present study, selenocystine with different chirality (i.e., l- and d-selenocystine) was used as the catalytic molecule immobilized on TiO2 films for decomposing endogenous NO donor. The influences of surface chirality on NO release and platelet behavior were evaluated. Results show that although the amount of immobilized l-selenocystine on the surface was nearly the same as that of immobilized d-selenocystine, in vitro catalytic NO release tests showed that l-selenocystine immobilized surfaces were more capable of catalyzing the decomposition of S-nitrosoglutathione and thus generating more NO. Accordingly, l-selenocystine immobilized surfaces demonstrated significantly increased inhibiting effects on the platelet adhesion and activation, when compared to d-selenocystine immobilized ones. Measurement of the cGMP concentration of platelets further confirmed that surface chirality played an important role in regulating NO generation and platelet behaviors. Additionally, using bovine serum albumin and fibrinogen as model proteins, the protein adsorption determined with quartz crystal microbalance showed that the l-selenocystine immobilized surface enhanced protein adsorption. In conclusion, surface chirality significantly influences protein adsorption and NO release, which may have significant implications in the design of NO-generating cardiovascular stents. Copyright © 2016 Elsevier B.V. All rights reserved.

Modular flow chamber for engineering bone marrow architecture and function.

PubMed

Di Buduo, Christian A; Soprano, Paolo M; Tozzi, Lorenzo; Marconi, Stefania; Auricchio, Ferdinando; Kaplan, David L; Balduini, Alessandra

2017-11-01

The bone marrow is a soft, spongy, gelatinous tissue found in the hollow cavities of flat and long bones that support hematopoiesis in order to maintain the physiologic turnover of all blood cells. Silk fibroin, derived from Bombyx mori silkworm cocoons, is a promising biomaterial for bone marrow engineering, because of its tunable architecture and mechanical properties, the capacity of incorporating labile compounds without loss of bioactivity and demonstrated ability to support blood cell formation. In this study, we developed a bone marrow scaffold consisting of a modular flow chamber made of polydimethylsiloxane, holding a silk sponge, prepared with salt leaching methods and functionalized with extracellular matrix components. The silk sponge was able to support efficient platelet formation when megakaryocytes were seeded in the system. Perfusion of the chamber allowed the recovery of functional platelets based on multiple activation tests. Further, inhibition of AKT signaling molecule, which has been shown to be crucial in regulating physiologic platelet formation, significantly reduced the number of collected platelets, suggesting the applicability of this tissue model for evaluation of the effects of bone marrow exposure to compounds that may affect platelet formation. In conclusion, we have bioengineered a novel modular system that, along with multi-porous silk sponges, can provide a useful technology for reproducing a simplified bone marrow scaffold for blood cell production ex vivo. Copyright © 2017 Elsevier Ltd. All rights reserved.

Potent antiplatelet activity of sesamol in an in vitro and in vivo model: pivotal roles of cyclic AMP and p38 mitogen-activated protein kinase.

PubMed

Chang, Chao C; Lu, Wan J; Chiang, Cheng W; Jayakumar, Thanasekaran; Ong, Eng T; Hsiao, George; Fong, Tsorng H; Chou, Duen S; Sheu, Joen R

2010-12-01

Sesamol is a potent phenolic antioxidant which possesses antimutagenic, antihepatotoxic and antiaging properties. Platelet activation is relevant to a variety of acute thrombotic events and coronary heart diseases. There have been few studies on the effect of sesamol on platelets. Therefore, the aim of this study was to systematically examine the detailed mechanisms of sesamol in preventing platelet activation in vitro and in vivo. Sesamol (2.5-5 μM) exhibited more potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists. Sesamol inhibited collagen-stimulated platelet activation accompanied by [Ca(2+)](i) mobilization, thromboxane A(2) (TxA(2)) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) phosphorylation in washed platelets. Sesamol markedly increased cAMP and cGMP levels, endothelial nitric oxide synthase (eNOS) expression and NO release, as well as vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the sesamol-mediated inhibitory effects on platelet aggregation and p38 MAPK phosphorylation, and sesamol-mediated stimulatory effects on VASP and eNOS phosphorylation, and NO release. Sesamol also reduced hydroxyl radical (OH(●)) formation in platelets. In an in vivo study, sesamol (5 mg/kg) significantly prolonged platelet plug formation in mice. The most important findings of this study demonstrate for the first time that sesamol possesses potent antiplatelet activity, which may involve activation of the cAMP-eNOS/NO-cGMP pathway, resulting in inhibition of the PLCγ2-PKC-p38 MAPK-TxA(2) cascade, and, finally, inhibition of platelet aggregation. Sesamol treatment may represent a novel approach to lowering the risk of or improving function in thromboembolism-related disorders. Copyright © 2010 Elsevier Inc. All rights reserved.

Phosphodiesterase from Daboia russelli russelli venom: purification, partial characterization and inhibition of platelet aggregation.

PubMed

Mitra, Jyotirmoy; Bhattacharyya, Debasish

2014-09-01

Phosphodiesterases (PDEs) belong to a super-family of enzymes that have multiple roles in the metabolism of extracellular nucleotides and regulation of nucleotide-based intercellular signalling. A PDE from Russell's viper (Daboia russelli russelli) venom (DR-PDE) was purified by gel filtration, ion exchange and affinity chromatographies. Homogeneity of the preparation was verified by SDS-PAGE, SE-HPLC and mass spectrometry. It was free from 5'-nucleotidase, alkaline phosphatase and protease activities. Identity of the enzyme was ensured from partial sequence homology with other PDEs. DR-PDE was inactivated by polyvalent anti-venom serum and metal chelators. The enzyme was partially inhibited by the root extracts of four medicinal plants but remained unaffected by inhibitors of intracellular PDEs. DR-PDE hydrolyses ADP and thus, strongly inhibits ADP-induced platelet aggregation in human platelet rich plasma. This study leads to better understanding of a component of Russell's viper venom that affects homoeostatic system of the victim. Copyright © 2014 Elsevier Ltd. All rights reserved.

Estrogen promotes megakaryocyte polyploidization via estrogen receptor beta-mediated transcription of GATA1.

PubMed

Du, C; Xu, Y; Yang, K; Chen, S; Wang, X; Wang, S; Wang, C; Shen, M; Chen, F; Chen, M; Zeng, D; Li, F; Wang, T; Wang, F; Zhao, J; Ai, G; Cheng, T; Su, Y; Wang, J

2017-04-01

Estrogen is reported to be involved in thrombopoiesis and the disruption of its signaling may cause myeloproliferative disease, yet the underlying mechanisms remain largely unknown. GATA-binding factor 1 (GATA1) is a key regulator of megakaryocyte (MK) differentiation and its deficiency will lead to megakaryoblastic leukemia. Here we show that estrogen can dose-dependently promote MK polyploidization and maturation via activation of estrogen receptor beta (ERβ), accompanied by a significant upregulation of GATA1. Chromatin immunoprecipitation and a dual luciferase assay demonstrate that ERβ can directly bind the promoter region of GATA1 and activate its transcription. Steroid receptor coactivator 3 (SRC3) is involved in ERβ-mediated GATA1 transcription. The deficiency of ERβ or SRC3, similar to the inhibition of GATA1, leads to the impediment of estrogen-induced MK polyploidization and platelet production. Further investigations reveal that signal transducer and activator of transcription 1 signaling pathway downstream of GATA1 has a crucial role in estrogen-induced MK polyploidization, and ERβ-mediated GATA1 upregulation subsequently enhances nuclear factor erythroid-derived 2 expression, thereby promoting proplatelet formation and platelet release. Our study provides a deep insight into the molecular mechanisms of estrogen signaling in regulating thrombopoiesis and the pathogenesis of ER deficiency-related leukemia.

Platelet-free shear flow assay facilitates analysis of shear-dependent functions of VWF and ADAMTS13.

PubMed

Kraus, Emma; Kraus, Kristina; Obser, Tobias; Oyen, Florian; Klemm, Ulrike; Schneppenheim, Reinhard; Brehm, Maria A

2014-12-01

The multimeric form of von Willebrand factor (VWF), is the largest soluble protein in mammals and exhibits a multidomain structure resulting in multiple functions. Upon agonist stimulation endothelial cells secrete VWF multimers from Weibel-Palade bodies into the blood stream where VWF plays an essential role in platelet-dependent primary hemostasis. Elongation of VWF strings on the cells' surface leads to accessibility of VWF binding sites for proteins, such as platelet membrane glycoprotein Ib. The prothrombotic strings are size-regulated by the metalloprotease ADAMTS13 by shear force-activated proteolytic cleavage. VWF string formation was induced by histamine stimulation of HUVEC cells under unidirectional shear flow and VWF strings were detected employing the VWF binding peptide of platelet glycoprotein Ib coupled to latex beads. VWF strings were then used as substrate for kinetic studies of recombinant and plasma ADAMTS13. To investigate specific aspects of the shear-dependent functions of VWF and ADAMTS13, we developed a shear flow assay that allows observation of VWF string formation and their degradation by ADAMTS13 without the need for isolated platelets. Our assay specifically detects VWF strings, can be coupled with fluorescent applications and allows semi-automated, quantitative assessment of recombinant and plasma ADAMTS13 activity. Our assay may serve as a valuable research tool to investigate the biochemical characteristics of VWF and ADAMTS13 under shear flow and could complement diagnostics of von Willebrand Disease and Thrombotic Thrombocytopenic Purpura as it allows detection of shear flow-dependent dysfunction of VWD-associated VWF mutants as well as TTP-associated ADAMTS13 mutants. Copyright © 2014 Elsevier Ltd. All rights reserved.

Clopidogrel, a P2Y12 Receptor Antagonist, Potentiates the Inflammatory Response in a Rat Model of Peptidoglycan Polysaccharide-Induced Arthritis

PubMed Central

Rico, Mario C.; Dela Cadena, Raul A.; Kunapuli, Satya P.

2011-01-01

The P2Y12 receptor plays a crucial role in the regulation of platelet activation by several agonists, which is irreversibly antagonized by the active metabolite of clopidogrel, a widely used anti-thrombotic drug. In this study, we investigated whether reduction of platelet reactivity leads to reduced inflammatory responses using a rat model of erosive arthritis. We evaluated the effect of clopidogrel on inflammation in Lewis rats in a peptidoglycan polysaccharide (PG-PS)-induced arthritis model with four groups of rats: 1) untreated, 2) clopidogrel-treated, 3) PG-PS-induced, and 4) PG-PS-induced and clopidogrel-treated. There were significant differences between the PG-PS+clopidogrel group when compared to the PG-PS group including: increased joint diameter and clinical manifestations of inflammation, elevated plasma levels of pro-inflammatory cytokines (IL-1 beta, interferon (IFN) gamma, and IL-6), an elevated neutrophil blood count and an increased circulating platelet count. Plasma levels of IL-10 were significantly lower in the PG-PS+clopidogrel group compared to the PG-PS group. Plasma levels of platelet factor 4 (PF4) were elevated in both the PG-PS and the PG-PS+clopidogrel groups, however PF4 levels showed no difference upon clopidogrel treatment, suggesting that the pro- inflammatory effect of clopidogrel may be due to its action on cells other than platelets. Histology indicated an increase in leukocyte infiltration at the inflammatory area of the joint, increased pannus formation, blood vessel proliferation, subsynovial fibrosis and cartilage erosion upon treatment with clopidogrel in PG-PS-induced arthritis animals. In summary, animals treated with clopidogrel showed a pro-inflammatory effect in the PG-PS-induced arthritis animal model, which might not be mediated by platelets. Elucidation of the mechanism of clopidogrel-induced cell responses is important to understand the role of the P2Y12 receptor in inflammation. PMID:22028806

Supramolecular Chitosan Micro-Platelets Synergistically Enhance Anti-Candida albicans Activity of Amphotericin B Using an Immunocompetent Murine Model.

PubMed

Grisin, Tiphany; Bories, Christian; Bombardi, Martina; Loiseau, Philippe M; Rouffiac, Valérie; Solgadi, Audrey; Mallet, Jean-Maurice; Ponchel, Gilles; Bouchemal, Kawthar

2017-05-01

The aim of this work is to design new chitosan conjugates able to self-organize in aqueous solution in the form of micrometer-size platelets. When mixed with amphotericin B deoxycholate (AmB-DOC), micro-platelets act as a drug booster allowing further improvement in AmB-DOC anti-Candida albicans activity. Micro-platelets were obtained by mixing oleoyl chitosan and α-cyclodextrin in water. The formulation is specifically-engineered for mucosal application by dispersing chitosan micro-platelets into thermosensitive pluronic ® F127 20 wt% hydrogel. The formulation completely cured C. albicans vaginal infection in mice and had a superior activity in comparison with AmB-DOC without addition of chitosan micro-platelets. In vitro studies showed that the platelets significantly enhance AmB-DOC antifungal activity since the IC 50 and the MIC 90 decrease 4.5 and 4.8-times. Calculation of fractional inhibitory concentration index (FICI = 0.198) showed that chitosan micro-platelets act in a synergistic way with AmB-DOC against C. albicans. No synergy is found between spherical nanoparticles composed poly(isobutylcyanoacrylate)/chitosan and AmB-DOC. These results demonstrate for the first time the ability of flattened chitosan micro-platelets to have synergistic activity with AmB-DOC against C. albicans candidiasis and highlight the importance of rheological and mucoadhesive behaviors of hydrogels in the efficacy of the treatment.

TCDD and omeprazole prime platelets through the aryl hydrocarbon receptor (AhR) non-genomic pathway.

PubMed

Pombo, Mónica; Lamé, Michael W; Walker, Naomi J; Huynh, Danh H; Tablin, Fern

2015-05-19

The role of the aryl hydrocarbon receptor (AhR) in hemostasis has recently gained increased attention. Here, we demonstrate, by qRT-PCR and western blot, that human platelets express both AhR mRNA and AhR protein. AhR protein levels increase in a dose dependent manner when incubated with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or omeprazole. Treatment of platelets with puromycin blocks increased AhR protein synthesis in the presence of AhR activators. Additionally, treatment of platelets with either activator results in phosphorylation of p38MAPK and cPLA2, two key signaling molecules in platelet activation pathways. Using the AhR competitive inhibitors alpha naphthoflavone and CH-223191, we show that phosphorylation of p38MAPK is AhR dependent. Further, inhibition of p38MAPK blocks downstream cPLA2 phosphorylation induced by TCDD or omeprazole. Treatment with AhR activators results in platelet priming, as demonstrated by increased platelet aggregation, which is inhibited by AhR antagonists. Our data support a model of the platelet AhR non-genomic pathway in which treatment with AhR activators results in increased expression of the AhR, phosphorylation of p38MAPK and cPLA2, leading to platelet priming in response to agonist. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Platelet and leukocyte activation, atherosclerosis and inflammation in European and South Asian men.

PubMed

Dotsenko, O; Chaturvedi, N; Thom, S A McG; Wright, A R; Mayet, J; Shore, A; Schalkwijk, C; Hughes, A D

2007-10-01

Increased platelet activation occurs in ischemic heart disease (IHD), but increased platelet activation is also seen in cerebrovascular atherosclerosis and peripheral artery disease. It is not clear therefore whether platelet activation is an indicator of IHD or a marker of generalized atherosclerosis and inflammation. South Asian subjects are at high risk of IHD, but little is known regarding differences in platelet and leukocyte function between European and South Asian subjects. Fifty-four male subjects (age 49-79 years) had coronary artery calcification measured by multislice computed tomography (CT), aortic atherosclerosis assessed by measurement of carotid-femoral pulse wave velocity (aortic PWV), and femoral and carotid atherosclerosis measured by B-mode ultrasound. Platelet and leukocyte activation was assessed by flow cytometry of platelet-monocyte complexes (PMC), platelet expression of PAC-1 binding site and CD62P, and expression of L-selectin on leukocytes. Elevated circulating PMC correlated significantly with elevated aortic PWV and PMC were higher in subjects with femoral plaques. In contrast PMC did not differ by increasing coronary artery calcification category or presence of carotid plaques. Higher numbers of PMC were independently related to elevated levels of C-reactive protein (CRP), higher aortic PWV, hypertension and smoking in a multivariate model. Markers of platelet and leukocyte activation did not differ significantly by ethnicity. Increased PMC are related to the extent of aortic and femoral atherosclerosis rather than coronary or carotid atherosclerosis. The association between elevated CRP and increased PMC suggests that inflammation in relation to generalized atherosclerosis may play an important role in PMC activation.

Prothrombin Activation by Platelet-associated Prothrombinase Proceeds through the Prethrombin-2 Pathway via a Concerted Mechanism*

PubMed Central

Haynes, Laura M.; Bouchard, Beth A.; Tracy, Paula B.; Mann, Kenneth G.

2012-01-01

The protease α-thrombin is a key enzyme of the coagulation process as it is at the cross-roads of both the pro- and anti-coagulant pathways. The main source of α-thrombin in vivo is the activation of prothrombin by the prothrombinase complex assembled on either an activated cell membrane or cell fragment, the most relevant of which is the activated platelet surface. When prothrombinase is assembled on synthetic phospholipid vesicles, prothrombin activation proceeds with an initial cleavage at Arg-320 yielding the catalytically active, yet effectively anticoagulant intermediate meizothrombin, which is released from the enzyme complex ∼30–40% of the time. Prothrombinase assembled on the surface of activated platelets has been shown to proceed through the inactive intermediate prethrombin-2 via an initial cleavage at Arg-271 followed by cleavage at Arg-320. The current work tests whether or not platelet-associated prothrombinase proceeds via a concerted mechanism through a study of prothrombinase assembly and function on collagen-adhered, thrombin-activated, washed human platelets in a flow chamber. Prothrombinase assembly was demonstrated through visualization of bound factor Xa by confocal microscopy using a fluorophore-labeled anti-factor Xa antibody, which demonstrated the presence of distinct platelet subpopulations capable of binding factor Xa. When prothrombin activation was monitored at a typical venous shear rate over preassembled platelet-associated prothrombinase neither potential intermediate, meizothrombin or prethrombin-2, was observed in the effluent. Collectively, these findings suggest that platelet-associated prothrombinase activates prothrombin via an efficient concerted mechanism in which neither intermediate is released. PMID:22989889

Differences in levels of platelet-derived microparticles in platelet components prepared using the platelet rich plasma, buffy coat, and apheresis procedures.

PubMed

Noulsri, Egarit; Udomwinijsilp, Prapaporn; Lerdwana, Surada; Chongkolwatana, Viroje; Permpikul, Parichart

2017-04-01

There has been an increased interest in platelet-derived microparticles (PMPs) in transfusion medicine. Little is known about PMP status during the preparation of platelet concentrates for transfusion. The aim of this study is to compare the PMP levels in platelet components prepared using the buffy coat (BC), platelet-rich plasma platelet concentrate (PRP-PC), and apheresis (AP) processes. Platelet components were prepared using the PRP-PC and BC processes. Apheresis platelets were prepared using the Trima Accel and Amicus instruments. The samples were incubated with annexin A5-FITC, CD41-PE, and CD62P-APC. At day 1 after processing, the PMPs and activated platelets were determined using flow cytometry. Both the percentage and number of PMPs were higher in platelet components prepared using the Amicus instrument (2.6±1.8, 32802±19036 particles/μL) than in platelet components prepared using the Trima Accel instrument (0.5±0.4, 7568±5298 particles/μL), BC (1.2±0.6, 12,920±6426 particles/μL), and PRP-PC (0.9±0.6, 10731±5514 particles/μL). Both the percentage and number of activated platelets were higher in platelet components prepared using the Amicus instrument (33.2±13.9, 427553±196965 cells/μL) than in platelet components prepared using the Trima Accel instrument (16.2±6.1, 211209±87706 cells/μL), BC (12.9±3.2, 140624±41003 cells/μL), and PRP-PC (21.1±6.3, 265210±86257 cells/μL). The study suggests high variability of PMPs and activated platelets in platelet components prepared using different processes. This result may be important in validating the instruments involved in platelet blood collection and processing. Copyright © 2016 Elsevier Ltd. All rights reserved.

Multiscale Particle-Based Modeling of Flowing Platelets in Blood Plasma Using Dissipative Particle Dynamics and Coarse Grained Molecular Dynamics

PubMed Central

Zhang, Peng; Gao, Chao; Zhang, Na; Slepian, Marvin J.; Deng, Yuefan; Bluestein, Danny

2014-01-01

We developed a multiscale particle-based model of platelets, to study the transport dynamics of shear stresses between the surrounding fluid and the platelet membrane. This model facilitates a more accurate prediction of the activation potential of platelets by viscous shear stresses - one of the major mechanisms leading to thrombus formation in cardiovascular diseases and in prosthetic cardiovascular devices. The interface of the model couples coarse-grained molecular dynamics (CGMD) with dissipative particle dynamics (DPD). The CGMD handles individual platelets while the DPD models the macroscopic transport of blood plasma in vessels. A hybrid force field is formulated for establishing a functional interface between the platelet membrane and the surrounding fluid, in which the microstructural changes of platelets may respond to the extracellular viscous shear stresses transferred to them. The interaction between the two systems preserves dynamic properties of the flowing platelets, such as the flipping motion. Using this multiscale particle-based approach, we have further studied the effects of the platelet elastic modulus by comparing the action of the flow-induced shear stresses on rigid and deformable platelet models. The results indicate that neglecting the platelet deformability may overestimate the stress on the platelet membrane, which in turn may lead to erroneous predictions of the platelet activation under viscous shear flow conditions. This particle-based fluid-structure interaction multiscale model offers for the first time a computationally feasible approach for simulating deformable platelets interacting with viscous blood flow, aimed at predicting flow induced platelet activation by using a highly resolved mapping of the stress distribution on the platelet membrane under dynamic flow conditions. PMID:25530818

A selective antagonist reveals a potential role of G protein-coupled receptor 55 in platelet and endothelial cell function.

PubMed

Kargl, Julia; Brown, Andrew J; Andersen, Liisa; Dorn, Georg; Schicho, Rudolf; Waldhoer, Maria; Heinemann, Akos

2013-07-01

The G protein-coupled receptor 55 (GPR55) is a lysophosphatidylinositol (LPI) receptor that is also responsive to certain cannabinoids. Although GPR55 has been implicated in several (patho)physiologic functions, its role remains enigmatic owing mainly to the lack of selective GPR55 antagonists. Here we show that the compound CID16020046 ((4-[4-(3-hydroxyphenyl)-3-(4-methylphenyl)-6-oxo-1H,4H,5H,6H-pyrrolo[3,4-c]pyrazol-5-yl] benzoic acid) is a selective GPR55 antagonist. In yeast cells expressing human GPR55, CID16020046 antagonized agonist-induced receptor activation. In human embryonic kidney (HEK293) cells stably expressing human GPR55, the compound behaved as an antagonist on LPI-mediated Ca²⁺ release and extracellular signal-regulated kinases activation, but not in HEK293 cells expressing cannabinoid receptor 1 or 2 (CB₁ or CB₂). CID16020046 concentration dependently inhibited LPI-induced activation of nuclear factor of activated T-cells (NFAT), nuclear factor κ of activated B cells (NF-κB) and serum response element, translocation of NFAT and NF-κB, and GPR55 internalization. It reduced LPI-induced wound healing in primary human lung microvascular endothelial cells and reversed LPI-inhibited platelet aggregation, suggesting a novel role for GPR55 in platelet and endothelial cell function. CID16020046 is therefore a valuable tool to study GPR55-mediated mechanisms in primary cells and tissues.

GDP beta S enhances the activation of phospholipase C caused by thrombin in human platelets: evidence for involvement of an inhibitory GTP-binding protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oberdisse, E.; Lapetina, E.G.

1987-05-14

Guanosine 5'-O-thiotriphosphate (GTP gamma S) and thrombin stimulate the activity of phospholipase C in platelets that have been permeabilized with saponin and whose inositol phospholipids have been prelabeled with (/sup 3/H)inositol. Ca/sup 2 +/ has opposite effects on the formation of (/sup 3/H)inositol phosphates induced by thrombin or GTP gamma S. While the action of GTP gamma S on the formation of (/sup 3/H)inositol phosphates is inhibited by Ca/sup 2 +/, action of thrombin is stimulated by Ca/sup 2 +/. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits the function of GTP-binding proteins, also inhibits the effect of GTP gamma Smore » on phospholipase C stimulation but, surprisingly, increases the effect of thrombin. Ca/sup 2 +/ increases the inhibitory effect of GDP beta S on GTP gamma S activation of phospholipase C, but Ca/sup 2 +/ further enhances the stimulatory effect of GDP beta S on the thrombin activation of phospholipase C. This indicates that two mechanisms are responsible for the activation of phospholipase C in platelets. A GTP-binding protein is responsible for regulation of phospholipase C induced by GTP gamma S, while the effect of thrombin on the stimulation of phospholipase C is independent of GTP-binding proteins. However, the effect of thrombin may be modulated by the action of an inhibitory GTP-binding protein.« less

Protein C Inhibitor (PCI) Binds to Phosphatidylserine Exposing Cells with Implications in the Phagocytosis of Apoptotic Cells and Activated Platelets

PubMed Central

Rieger, Daniela; Assinger, Alice; Einfinger, Katrin; Sokolikova, Barbora; Geiger, Margarethe

2014-01-01

Protein C Inhibitor (PCI) is a secreted serine protease inhibitor, belonging to the family of serpins. In addition to activated protein C PCI inactivates several other proteases of the coagulation and fibrinolytic systems, suggesting a regulatory role in hemostasis. Glycosaminoglycans and certain negatively charged phospholipids, like phosphatidylserine, bind to PCI and modulate its activity. Phosphatidylerine (PS) is exposed on the surface of apoptotic cells and known as a phagocytosis marker. We hypothesized that PCI might bind to PS exposed on apoptotic cells and thereby influence their removal by phagocytosis. Using Jurkat T-lymphocytes and U937 myeloid cells, we show here that PCI binds to apoptotic cells to a similar extent at the same sites as Annexin V, but in a different manner as compared to live cells (defined spots on ∼10–30% of cells). PCI dose dependently decreased phagocytosis of apoptotic Jurkat cells by U937 macrophages. Moreover, the phagocytosis of PS exposing, activated platelets by human blood derived monocytes declined in the presence of PCI. In U937 cells the expression of PCI as well as the surface binding of PCI increased with time of phorbol ester treatment/macrophage differentiation. The results of this study suggest a role of PCI not only for the function and/or maturation of macrophages, but also as a negative regulator of apoptotic cell and activated platelets removal. PMID:25000564

CXC chemokine ligand 4 (CXCL4) down-regulates CC chemokine receptor expression on human monocytes.

PubMed

Schwartzkopff, Franziska; Petersen, Frank; Grimm, Tobias Alexander; Brandt, Ernst

2012-02-01

During acute inflammation, monocytes are essential in abolishing invading micro-organisms and encouraging wound healing. Recruitment by CC chemokines is an important step in targeting monocytes to the inflamed tissue. However, cell surface expression of the corresponding chemokine receptors is subject to regulation by various endogenous stimuli which so far have not been comprehensively identified. We report that the platelet-derived CXC chemokine ligand 4 (CXCL4), a known activator of human monocytes, induces down-regulation of CC chemokine receptors (CCR) 1, -2, and -5, resulting in drastic impairment of monocyte chemotactic migration towards cognate CC chemokine ligands (CCL) for these receptors. Interestingly, CXCL4-mediated down-regulation of CCR1, CCR2 and CCR5 was strongly dependent on the chemokine's ability to stimulate autocrine/paracrine release of TNF-α. In turn, TNF-α induced the secretion CCL3 and CCL4, two chemokines selective for CCR1 and CCR5, while the secretion of CCR2-ligand CCL2 was TNF-α-independent. Culture supernatants of CXCL4-stimulated monocytes as well as chemokine-enriched preparations thereof reproduced CXCL4-induced CCR down-regulation. In conclusion, CXCL4 may act as a selective regulator of monocyte migration by stimulating the release of autocrine, receptor-desensitizing chemokine ligands. Our results stress a co-ordinating role for CXCL4 in the cross-talk between platelets and monocytes during early inflammation.

Functional role of endothelial CXCL16/CXCR6-platelet-leukocyte axis in angiotensin II-associated metabolic disorders.

PubMed

Collado, Aida; Marques, Patrice; Escudero, Paula; Rius, Cristina; Domingo, Elena; Martinez-Hervás, Sergio; Real, José T; Ascaso, Juan F; Piqueras, Laura; Sanz, Maria-Jesus

2018-05-23

Angiotensin-II (Ang-II) is the main effector peptide of the renin-angiotensin system (RAS) and promotes leukocyte adhesion to the stimulated endothelium. Because RAS activation and Ang-II signaling are implicated in metabolic syndrome (MS) and abdominal aortic aneurysm (AAA), we investigated the effect of Ang-II on CXCL16 arterial expression, the underlying mechanisms, and the functional role of the CXCL16/CXCR6 axis in these cardiometabolic disorders. Results from in vitro chamber assays revealed that CXCL16 neutralization significantly inhibited mononuclear leukocyte adhesion to arterial but not to venous endothelial cells. Flow cytometry and immunofluorescence studies confirmed that Ang-II induced enhanced endothelial CXCL16 expression, which was dependent on Nox5 up-regulation and subsequent RhoA/p38-MAPK/NFκB activation. Flow cytometry analysis further showed that MS patients had higher levels of platelet activation and a higher percentage of circulating CXCR6-expressing platelets, CXCR6-expressing-platelet-bound neutrophils, monocytes and CD8+ lymphocytes than age-matched controls, leading to enhanced CXCR6/CXCL16-dependent adhesion to the dysfunctional (Ang-II- and TNFα-stimulated) arterial endothelium. Ang-II-challenged apolipoprotein E-deficient (apoE-/-) mice had a higher incidence of AAA, macrophage, CD3+ and CXCR6+ cell infiltration and neovascularization than unchallenged animals, which was accompanied by greater CCL2, CXCL16 and VEGF mRNA expression within the lesion together with elevated levels of circulating soluble CXCL16. Significant reductions in these parameters were found in animals co-treated with the AT1 receptor antagonist losartan or in apoE-/- mice lacking functional CXCR6 receptor (CXCR6GFP/GFP). CXCR6 expression on platelet-bound monocytes and CD8+ lymphocytes may constitute a new membrane-associated biomarker for adverse cardiovascular events. Moreover, pharmacological modulation of this axis may positively affect cardiovascular outcome in metabolic disorders linked to Ang-II.

Improving platelet transfusion safety: biomedical and technical considerations

PubMed Central

Garraud, Olivier; Cognasse, Fabrice; Tissot, Jean-Daniel; Chavarin, Patricia; Laperche, Syria; Morel, Pascal; Lefrère, Jean-Jacques; Pozzetto, Bruno; Lozano, Miguel; Blumberg, Neil; Osselaer, Jean-Claude

2016-01-01

Platelet concentrates account for near 10% of all labile blood components but are responsible for more than 25% of the reported adverse events. Besides factors related to patients themselves, who may be particularly at risk of side effects because of their underlying illness, there are aspects of platelet collection and storage that predispose to adverse events. Platelets for transfusion are strongly activated by collection through disposal equipment, which can stress the cells, and by preservation at 22 °C with rotation or rocking, which likewise leads to platelet activation, perhaps more so than storage at 4 °C. Lastly, platelets constitutively possess a very large number of bioactive components that may elicit pro-inflammatory reactions when infused into a patient. This review aims to describe approaches that may be crucial to minimising side effects while optimising safety and quality. We suggest that platelet transfusion is complex, in part because of the complexity of the “material” itself: platelets are highly versatile cells and the transfusion process adds a myriad of variables that present many challenges for preserving basal platelet function and preventing dysfunctional activation of the platelets. The review also presents information showing - after years of exhaustive haemovigilance - that whole blood buffy coat pooled platelet components are extremely safe compared to the gold standard (i.e. apheresis platelet components), both in terms of acquired infections and of immunological/inflammatory hazards. PMID:26674828

Influence of the direct NO-donor SIN-1 on the interaction between platelets and stainless steel stents under dynamic conditions.

PubMed

Jung, F; Mrowietz, C; Seyfert, U T; Grewe, R; Franke, R P

2003-01-01

It was investigated whether the NO-donor SIN-1, the active metabolite of molsidomine, influenced the activation of platelets, the formation of circulating platelet aggregates, the spontaneous aggregation of platelets and the activation of the clotting system triggered by a body foreign surface in an in vitro closed-loop perfusion model. With human platelet-rich plasma at micromolar concentrations SIN-1 exerted pronounced effects on the interaction between platelets and an exogenous surface. In the absence of SIN-1, the number of circulating single platelets decreased significantly, which could be due either to the formation of circulating platelet aggregates or to the adhesion of platelets to the stent. Both these processes were blocked by the addition of SIN-1. Moreover, the platelets exhibited hyperaggregability in the absence of SIN-1 whereas the NO-donor was able to completely inhibit spontaneous platelet aggregation. Similar results were obtained in flow cytometry experiments. Without SIN-1, high platelet surface densities of both the GPIb/IX and GPIIb/IIIa receptors were observed. In addition, the density of the fibrinogen receptor increased significantly with the number of perfusion cycles. SIN-1 was able to suppress the augmented GPIIb/IIIa receptor expression significantly. Molsidomine seemed to have the potential to reduce the incidence of thrombotic processes triggered by the exogenous surface of the stent.

Influence of cryopreservation and mechanical stimulation on equine Autologous Conditioned Plasma (ACP®).

PubMed

Mageed, M; Ionita, C; Kissich, C; Brehm, W; Winter, K; Ionita, J-C

2015-01-01

To determine the influence of cryopreservation at two different temperatures on platelet concentration, growth factor (GF) levels and platelet activation parameters in equine ACP®; moreover, to determine if adding mechanical ACP® stimulation to freeze-thaw activation amplifies GF release from platelets. Firstly, blood from five horses was used to prepare ACP®. Platelet, platelet derived growth factor BB (PDGF-BB) and transforming growth factor β1 (TGF-β1) concentrations as well as mean platelet volume (MPV) and mean platelet component (MPC) were determined in fresh and corresponding ACP® samples after 2 months cryopreservation at -20 °C and -80 °C, respectively. Secondly, ACP® was prepared from blood of nine horses. Half of ACP® was activated using one freeze-thaw-cycle at -20 °C, whereas the rest was first vortexed. Their PDGF-BB and TGF-β1 concentrations were subsequently determined. Platelet concentration significantly decreased after -80 °C cryopreservation. PDGF-BB level augmented significantly after both storage methods, whereas TGF-β1 concentration was not significantly altered. MPV significantly increased after -20 °C cryopreservation. Both storage regimens induced a significant MPC decrease. No significant differences in GF concentrations between the vortexed and non-vortexed samples were detected. Both cryopreservation methods induced platelet activation, but storage at -80 °C apparently harmed the platelets without generating higher GF release than -20 °C. The mechanical stimulation process could not enhance GF release in subsequently frozen-thawed ACP®. Storage of ACP® at -20 °C could be useful in equine practice, but, before this procedure can be recommended, further qualitative tests are needed. The mechanical stimulation technique should be adjusted in order to increase platelet activation.

Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase

PubMed Central

Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

2016-01-01

Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase. PMID:26867010

Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase.

PubMed

Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

2016-01-01

Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase.

Paradoxical Effect of Nonphysiological Shear Stress on Platelets and von Willebrand Factor.

PubMed

Chen, Zengsheng; Mondal, Nandan K; Ding, Jun; Koenig, Steven C; Slaughter, Mark S; Wu, Zhongjun J

2016-07-01

Blood can become hypercoagulable by shear-induced platelet activation and generation of microparticles. It has been reported that nonphysiological shear stress (NPSS) could induce shedding of platelet receptor glycoprotein (GP) Ibα, which may result in an opposite effect to hemostasis. The aim of this study was to investigate the influence of the NPSS on platelets and von Willebrand factor (vWF). Human blood was exposed to two levels of NPSS (25 Pa, 125 Pa) with an exposure time of 0.5 s, generated by using a novel blood-shearing device. Platelet activation (P-selectin expression, GPIIb/IIIa activation and generation of microparticles) and shedding of three platelet receptors (GPIbα, GPVI, GPIIb/IIIa) in sheared blood were quantified using flow cytometry. Aggregation capacity of sheared blood induced by ristocetin and collagen was evaluated using an aggregometer. Shear-induced vWF damage was characterized with Western blotting. Consistent with the published data, the NPSS caused significantly more platelets to become activated with increasing NPSS level. Meanwhile, the NPSS induced the shedding of platelet receptors. The loss of the platelet receptors increased with increasing NPSS level. The aggregation capacity of sheared blood induced by ristocetin and collagen decreased. There was a loss of high molecular weight multimers (HMWMs) of vWF in sheared blood. These results suggest that the NPSS induced a paradoxical effect. More activated platelets increase the risk of thrombosis, while the reduction in platelet receptors and the loss of HMWM-vWF increased the propensity of bleeding. The finding might provide a new perspective to understand thrombosis and acquired bleeding disorder in patients supported with blood contacting medical devices. Copyright © 2015 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Paradoxical Effect of Non-Physiological Shear Stress on Platelets and von Willebrand factor

PubMed Central

Chen, Zengsheng; Mondal, Nandan K; Ding, Jun; Koenig, Steven C.; Slaughter, Mark S.; Wu, Zhongjun J.

2016-01-01

Blood can become hypercoagulable by shear-induced platelet activation and generation of microparticles. It has been reported that non-physiological shear stress (NPSS) could induce shedding of platelet receptor glycoprotein (GP) Ibα, which may result in an opposite effect to hemostasis. The aim of this study was to investigate the influence of the NPSS on platelets and von Willebrand factor (vWF). Human blood was exposed to two levels of NPSS (25Pa, 125Pa) with an exposure time of 0.5 sec., generated by using a novel blood shearing device. Platelet activation (P-selectin expression, GPIIb/IIIa activation and generation of microparticles) and shedding of three platelet receptors (GPIbα, GPVI, GPIIb/IIIa) in sheared blood were quantified using flow cytometry. Aggregation capacity of sheared blood induced by ristocetin and collagen was evaluated using an aggregometer. Shear-induced vWF damage was characterized with western blotting. Consistent with the published data, the NPSS caused significantly more platelets to become activated with increasing NPSS level. Meanwhile, the NPSS induced the shedding of platelet receptors. The loss of the platelet receptors increased with increasing NPSS level. The aggregation capacity of sheared blood induced by ristocetin and collagen decreased. There was a loss of high molecular weight multimers (HMWM) of vWF in sheared blood. These results suggest that the NPSS induced a paradoxical effect. More activated platelets increase the risk of thrombosis while the reduction in platelet receptors and the loss of HMWM-vWF increased the propensity of bleeding. The finding might provide a new perspective to understand thrombosis and acquired bleeding disorder in patients supported with blood contacting medical devices. PMID:26582038

Characterization of the aggregation responses of camel platelets.

PubMed

Al Ghumlas, Abeer K; Gader, Abdel Galil M Abdel

2013-09-01

Despite evidence of active hemostasis, camel platelets barely respond to common aggregating agents at standard doses used for human platelet aggregation. The purpose of the study was to find out whether camel platelets can be activated by high doses or combinations of aggregation agonists, and to characterize the receptor that mediates the aggregation response to adenosine diphosphate (ADP), the most potent agonist for camel platelets known so far. Aggregation studies were performed with platelet-rich plasma (PRP) in response to multiple doses or combinations of ADP, epinephrine (EPN), collagen, and arachidonic acid (AA). Aggregation responses to ADP were performed before and after the addition of the ADP receptor (P2Y12) antagonist Clopidogrel. Camel platelets responded to ADP at doses higher than the standard dose for human platelets, and to combinations of EPN and other agonists, while no aggregation was elicited with EPN or AA alone. Clopidogrel blocked the ADP-induced aggregation responses in a dose-dependent fashion in vitro. Camel platelet aggregation can be activated by increasing the dose of some agonists such as ADP, but not AA or EPN. Irreversible aggregation of camel platelets could also be triggered by a combination of EPN and ADP, and collagen and AA. Inhibition with clopidogrel suggests that camel platelets express the ADP receptor, P2Y12. Understanding platelet function in camels will add to the understanding of platelet function in health and disease. © 2013 American Society for Veterinary Clinical Pathology.

Mechanisms of the priming effect of low doses of lipopoly-saccharides on leukocyte-dependent platelet aggregation in whole blood.

PubMed

Montrucchio, Giuseppe; Bosco, Ornella; Del Sorbo, Lorenzo; Fascio Pecetto, Paolo; Lupia, Enrico; Goffi, Alberto; Omedè, Paola; Emanuelli, Giorgio; Camussi, Giovanni

2003-11-01

Several studies focused on the ability of bacterial lipopolysac-charides (LPS) in triggering platelet and/or leukocyte activation. The aim of this study was to investigate the molecular mechanisms involved in the aggregation of platelets and in their interaction with leukocytes in whole blood after stimulation with low doses of LPS. LPS did not directly induce platelet aggregation in whole blood, but they primed the aggregation of platelets induced by epinephrine, adenosine diphosphate and arachidonic acid. As shown by cytofluorimetry, platelets neither bind FITC-LPS, nor express the LPS-receptors CD14 and toll-like receptor 4 (TLR4). On the contrary, LPS primed monocytes and to a lesser extent polymorphonuclear neutrophils to adhere to platelets. Both platelet-leukocyte interaction and platelet aggregation in whole blood were inhibited by blockade of CD14 and TLR4. Moreover, the interaction between platelets and leukocytes was inhibited by P-selectin, and by blockade of PAF and reactive oxygen species, suggesting a role of P-selectin and of leukocyte-derived mediators. In conclusion, these results elucidate the mechanisms leading to platelet activation and interaction with leukocytes triggered by LPS. They suggest that the activation of platelets by LPS is mainly dependent on leukocytes and especially monocytes as a result of CD14 and TLR4 engagement. Moreover, we found that leukocyte-platelet interaction was triggered by the synthesis of PAF and the generation of oxygen radicals that induced upregulation of surface expression of P-selectin.

[Platelet function in acute myeloid leukemia. II. Aggregation of isolated platelets].

PubMed

Zawilska, K; Komarnicki, M; Mańka, B

1978-01-01

In 22 patients with acute myeloid leukaemia (17 cases of myeloblastic leukaemia, 4 cases of myelomonocytic leukaemia and 1 case of undifferentiated-cell leukaemia) platelets were isolated from the plasma by the method of Nicholls and Hampton as modified by Levy-Toledano by centrifugation in albumin gradient. The aim of platelet isolation was their "concentration" in cases of thrombocytopenia to values making possible aggregation tests, and platelet separation from the influence of plasma factors. Then aggregation of isolated platelets caused by ADP was studied. In 16 out of 22 patients a fall of aggregation was observed, with the mean values of aggregation rate and intensity were significantly lower. Parallelly done determinations of aggregating activity released from the platelets by thrombin showed lower values as compared with platelets from healthy subjects. In might be thought, in this connection, that the demonstrated reduction of isolated platelets is associated with a diminution of the nucleotide pool or disturbances of the platelet release reaction. The disturbances of the platelet release reaction. The disturbances of aggregation of isolated platelets and reduction of the aggregating activity were most pronounced in acute myelomonocytic leukaemia.

Effect of platelet lysate on human cells involved in different phases of wound healing.

PubMed

Barsotti, Maria Chiara; Chiara Barsotti, Maria; Losi, Paola; Briganti, Enrica; Sanguinetti, Elena; Magera, Angela; Al Kayal, Tamer; Feriani, Roberto; Di Stefano, Rossella; Soldani, Giorgio

2013-01-01

Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization). Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2) and inflammatory response evaluation (NFκB). Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v). Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (p<0.05 vs. control), comparably to the positive control. Both platelet lysate concentrations activated important inflammatory pathways such as ERK1/2 and NFκB with the same early kinetics, whereas the effect was different for later time-points. These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing.

Effect of Platelet Lysate on Human Cells Involved in Different Phases of Wound Healing

PubMed Central

Briganti, Enrica; Sanguinetti, Elena; Magera, Angela; Al Kayal, Tamer; Feriani, Roberto; Di Stefano, Rossella; Soldani, Giorgio

2013-01-01

Background Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization). Methodology/Principal Findings Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2) and inflammatory response evaluation (NFκB). Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v). Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (p<0.05 vs. control), comparably to the positive control. Both platelet lysate concentrations activated important inflammatory pathways such as ERK1/2 and NFκB with the same early kinetics, whereas the effect was different for later time-points. Conclusion/Significance These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing. PMID:24386412

TMEM16F is required for phosphatidylserine exposure and microparticle release in activated mouse platelets.

PubMed

Fujii, Toshihiro; Sakata, Asuka; Nishimura, Satoshi; Eto, Koji; Nagata, Shigekazu

2015-10-13

Phosphatidylserine (PtdSer) exposure on the surface of activated platelets requires the action of a phospholipid scramblase(s), and serves as a scaffold for the assembly of the tenase and prothrombinase complexes involved in blood coagulation. Here, we found that the activation of mouse platelets with thrombin/collagen or Ca(2+) ionophore at 20 °C induces PtdSer exposure without compromising plasma membrane integrity. Among five transmembrane protein 16 (TMEM16) members that support Ca(2+)-dependent phospholipid scrambling, TMEM16F was the only one that showed high expression in mouse platelets. Platelets from platelet-specific TMEM16F-deficient mice exhibited defects in activation-induced PtdSer exposure and microparticle shedding, although α-granule and dense granule release remained intact. The rate of tissue factor-induced thrombin generation by TMEM16F-deficient platelets was severely reduced, whereas thrombin-induced clot retraction was unaffected. The imaging of laser-induced thrombus formation in whole animals showed that PtdSer exposure on aggregated platelets was TMEM16F-dependent in vivo. The phenotypes of the platelet-specific TMEM16F-null mice resemble those of patients with Scott syndrome, a mild bleeding disorder, indicating that these mice may provide a useful model for human Scott syndrome.

The properties of B-form monoamine oxidase in mitochondria from monkey platelet.

PubMed

Obata, Toshio; Aomine, Masahiro

The present study was examined the effect of the properties of monkey platelet monoamine oxidase (MAO) based on inhibitor sensitivity. Monkey platelet showed a high MAO activity with beta-phenylethylamine (beta-PEA) as substrate and a very low A-form MAO activity with 5 hydroxytryptamine (5-HT) as substrate. Moreover, monkey platelet MAO was sensitive to the drugs deprenyl as B-form MAO inhibitor and less sensitive to clorgyline and harmaline as A form MAO inhibitor with beta-PEA as the B-form MAO substrate. B-form MAO from monkey platelet was more stable against heat treatment at 55 degrees C than B-form MAO in brain. After digestion with trypsin at 37 degrees C for 4 hrs, it was found that MAO from platelet was inhibited about 70% with beta-PEA as substrate with brain. The tricyclic antidepressant imipramine and nortriptyline inhibited B-form MAO activity more potency than B-form MAO in brain. However, when the noncyclic antidepressant nomifensine was used, monkey platelet B-form MAO activities were less potently inhibited. All these reagents were noncompetitive inhibitors of B form MAO in monkey platelet. The present studies demonstrated that monkey platelet MAO is a single of B-form MAO and sensitive to tricyclic antidepressants.

Platelets: versatile effector cells in hemostasis, inflammation, and the immune continuum

PubMed Central

Vieira-de-Abreu, Adriana; Campbell, Robert A.; Weyrich, Andrew S.

2015-01-01

Platelets are chief effector cells in hemostasis. In addition, however, their specializations include activities and intercellular interactions that make them key effectors in inflammation and in the continuum of innate and adaptive immunity. This review focuses on the immune features of human platelets and platelets from experimental animals and on interactions between inflammatory, immune, and hemostatic activities of these anucleate but complex and versatile cells. The experimental findings and evidence for physiologic immune functions include previously unrecognized biologic characteristics of platelets and are paralleled by new evidence for unique roles of platelets in inflammatory, immune, and thrombotic diseases. PMID:21818701

First Selective 12-LOX Inhibitor, ML355, Impairs Thrombus Formation and Vessel Occlusion In Vivo With Minimal Effects on Hemostasis.

PubMed

Adili, Reheman; Tourdot, Benjamin E; Mast, Katherine; Yeung, Jennifer; Freedman, John C; Green, Abigail; Luci, Diane K; Jadhav, Ajit; Simeonov, Anton; Maloney, David J; Holman, Theodore R; Holinstat, Michael

2017-10-01

Adequate platelet reactivity is required for maintaining hemostasis. However, excessive platelet reactivity can also lead to the formation of occlusive thrombi. Platelet 12(S)-lipoxygenase (12-LOX), an oxygenase highly expressed in the platelet, has been demonstrated to regulate platelet function and thrombosis ex vivo, supporting a key role for 12-LOX in the regulation of in vivo thrombosis. However, the ability to pharmacologically target 12-LOX in vivo has not been established to date. Here, we studied the effect of the first highly selective 12-LOX inhibitor, ML355, on in vivo thrombosis and hemostasis. ML355 dose-dependently inhibited human platelet aggregation and 12-LOX oxylipin production, as confirmed by mass spectrometry. Interestingly, the antiplatelet effects of ML355 were reversed after exposure to high concentrations of thrombin in vitro. Ex vivo flow chamber assays confirmed that human platelet adhesion and thrombus formation at arterial shear over collagen were attenuated in whole blood treated with ML355 comparable to aspirin. Oral administration of ML355 in mice showed reasonable plasma drug levels by pharmacokinetic assessment. ML355 treatment impaired thrombus growth and vessel occlusion in FeCl 3 -induced mesenteric and laser-induced cremaster arteriole thrombosis models in mice. Importantly, hemostatic plug formation and bleeding after treatment with ML355 was minimal in mice in response to laser ablation on the saphenous vein or in a cremaster microvasculature laser-induced rupture model. Our data strongly support 12-LOX as a key determinant of platelet reactivity in vivo, and inhibition of platelet 12-LOX with ML355 may represent a new class of antiplatelet therapy. © 2017 American Heart Association, Inc.

Blockade of GpIIb/IIIa inhibits the release of vascular endothelial growth factor (VEGF) from tumor cell-activated platelets and experimental metastasis.

PubMed

Amirkhosravi, A; Amaya, M; Siddiqui, F; Biggerstaff, J P; Meyer, T V; Francis, J L

1999-01-01

Evidence that platelets play a role in tumor metastasis includes the observation of circulating tumor cell-platelet aggregates and the anti-metastatic effect of thrombocytopenia and anti-platelet drugs. Platelets have recently been shown to contain vascular endothelial growth factor (VEGF) which is released during clotting. We therefore studied the effects of (1) tumor cell-platelet adherence and tumor cell TF activity on platelet VEGF release; and (2) the effects of GpIIb/IIIa blockade on tumor cell-induced platelet VEGF release, tumor cell-induced thrombocytopenia and experimental metastasis. Adherent A375 human melanoma cells (TF+) and KG1 myeloid leukemia (TF-) cells were cultured in RPMI containing 10% fetal bovine serum. Platelet-rich plasma was obtained from normal citrated whole blood and the presence of VEGF (34 and 44 kDa isoforms) confirmed by immunoblotting. Platelet-rich plasma with or without anti-GpIIb/IIIa (Abciximab) was added to A375 monolayers and supernatant VEGF measured by ELISA. Tumor cell-induced platelet activation and release were determined by CD62P expression and serotonin release respectively. In vitro, tumor cell-platelet adherence was evaluated by flow cytometry. In vivo, thrombocytopenia and lung seeding were assessed 30 min and 18 days, respectively, after i.v. injection of Lewis Lung carcinoma (LL2) cells into control or murine 7E3 F(ab')(2) (6 mg/ kg) athymic rats. Maximal in vitro platelet activation (72% serotonin release) occurred 30 min after adding platelets to tumor cells. At this time, 87% of the A375 cells had adhered to platelets. Abciximab significantly (P<0.05) reduced platelet adherence to tumor cells as evidenced by flow cytometry. Incubation of A375 cells with platelets induced VEGF release in a time-dependent manner. This release was significantly inhibited by Abciximab (81% at 30 min; P<0.05). In the presence of fibrinogen and FII, VEGF release induced by A375 (TF+) cells was significantly higher than that induced by KG1 (TF-) cells (105.5+/-24 vs. 42+/-7 pg/ml; P<0.001). Omitting fibrinogen or FII from the reaction mixture markedly decreased VEGF release. In vivo, GpIIb/IIIa blockade with murine 7E3 F(ab')(2) reduced LL2 tumor cell-induced thrombocytopenia by 90% (P<0.001) and lung seeding by 82% (P<0.05). We conclude that TF-bearing tumor cells can activate platelets largely via thrombin generation, and that such activation is associated with release of VEGF. This may enhance metastasis, possibly by increasing extravasation at points of adhesion to vascular endothelium.

Involvement of nuclear factor κB in platelet CD40 signaling.

PubMed

Hachem, Ahmed; Yacoub, Daniel; Zaid, Younes; Mourad, Walid; Merhi, Yahye

2012-08-17

CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-κB). Given that platelets contain NF-κB, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of IκBα, which are abolished by CD40L blockade. Inhibition of IκBα phosphorylation reverses sCD40L-induced IκBα phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on IκBα phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of IκBα phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-κB activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against thrombo-inflammatory disorders. Copyright © 2012 Elsevier Inc. All rights reserved.

Meal-induced platelet activation in diabetes mellitus type 1 or type 2 is related to postprandial insulin rather than glucose levels.

PubMed

Spectre, Galia; Stålesen, Ragnhild; Östenson, Claes-Göran; Hjemdahl, Paul

2016-05-01

Postprandial platelet activation was related to postprandial insulin rather than glucose levels in a previous meal insulin study in type 2 diabetes mellitus (T2DM). We therefore compared postprandial platelet activation in type 1 (T1DM) patients without insulin secretion and T2DM patients with high postprandial insulin levels. Patients with T1DM (n=11) and T2DM (n=12) were studied before and 90min after a standardized meal without premeal insulin. Five T1DM patients volunteered for a restudy with their regular premeal insulin. Platelet activation was assessed by flow cytometry, with and without the thromboxane analogue U46619 or ADP, and by whole blood aggregometry (Multiplate®). Effects of insulin (100μU/mL) in vitro were also studied. Before the meal, glucose, insulin and platelet activation markers other than platelet-leukocyte aggregates (PLAs) were similar in T1DM and T2DM; PLAs were higher in T1DM. Postprandial glucose levels increased more markedly in T1DM (to 22.1±1.4 vs. 11.2±0.6mmol/L) while insulin levels increased only in T2DM (from 24.4±4.4 to 68.8±12.3μU/mL). Platelet P-selectin expression, fibrinogen binding and PLA formation stimulated by U46619 were markedly enhanced (approximately doubled) and whole blood aggregation stimulated by U46619 was increased (p<0.05 for all) after the meal in T2DM patients but not in T1DM patients. The pilot study with premeal insulin in T1DM patients showed postprandial platelet activation when postprandial insulin levels increased. In vitro insulin mildly activated platelets in both groups. Postprandial platelet activation via the thromboxane pathway is related to postprandial hyperinsulinemia and not to postprandial hyperglycaemia in patients with diabetes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Non-redundant roles of phosphoinositide 3-kinase isoforms alpha and beta in glycoprotein VI-induced platelet signaling and thrombus formation.

PubMed

Gilio, Karen; Munnix, Imke C A; Mangin, Pierre; Cosemans, Judith M E M; Feijge, Marion A H; van der Meijden, Paola E J; Olieslagers, Servé; Chrzanowska-Wodnicka, Magdalena B; Lillian, Rivka; Schoenwaelder, Simone; Koyasu, Shigeo; Sage, Stewart O; Jackson, Shaun P; Heemskerk, Johan W M

2009-12-04

Platelets are activated by adhesion to vascular collagen via the immunoglobulin receptor, glycoprotein VI (GPVI). This causes potent signaling toward activation of phospholipase Cgamma2, which bears similarity to the signaling pathway evoked by T- and B-cell receptors. Phosphoinositide 3-kinase (PI3K) plays an important role in collagen-induced platelet activation, because this activity modulates the autocrine effects of secreted ADP. Here, we identified the PI3K isoforms directly downstream of GPVI in human and mouse platelets and determined their role in GPVI-dependent thrombus formation. The targeting of platelet PI3Kalpha or -beta strongly and selectively suppressed GPVI-induced Ca(2+) mobilization and inositol 1,4,5-triphosphate production, thus demonstrating enhancement of phospholipase Cgamma2 by PI3Kalpha/beta. That PI3Kalpha and -beta have a non-redundant function in GPVI-induced platelet activation and thrombus formation was concluded from measurements of: (i) serine phosphorylation of Akt, (ii) dense granule secretion, (iii) intracellular Ca(2+) increases and surface expression of phosphatidylserine under flow, and (iv) thrombus formation, under conditions where PI3Kalpha/beta was blocked or p85alpha was deficient. In contrast, GPVI-induced platelet activation was insensitive to inhibition or deficiency of PI3Kdelta or -gamma. Furthermore, PI3Kalpha/beta, but not PI3Kgamma, contributed to GPVI-induced Rap1b activation and, surprisingly, also to Rap1b-independent platelet activation via GPVI. Together, these findings demonstrate that both PI3Kalpha and -beta isoforms are required for full GPVI-dependent platelet Ca(2+) signaling and thrombus formation, partly independently of Rap1b. This provides a new mechanistic explanation for the anti-thrombotic effect of PI3K inhibition and makes PI3Kalpha an interesting new target for anti-platelet therapy.

Equid Herpesvirus Type 1 Activates Platelets

PubMed Central

Stokol, Tracy; Yeo, Wee Ming; Burnett, Deborah; DeAngelis, Nicole; Huang, Teng; Osterrieder, Nikolaus; Catalfamo, James

2015-01-01

Equid herpesvirus type 1 (EHV-1) causes outbreaks of abortion and neurological disease in horses. One of the main causes of these clinical syndromes is thrombosis in placental and spinal cord vessels, however the mechanism for thrombus formation is unknown. Platelets form part of the thrombus and amplify and propagate thrombin generation. Here, we tested the hypothesis that EHV-1 activates platelets. We found that two EHV-1 strains, RacL11 and Ab4 at 0.5 or higher plaque forming unit/cell, activate platelets within 10 minutes, causing α-granule secretion (surface P-selectin expression) and platelet microvesiculation (increased small events double positive for CD41 and Annexin V). Microvesiculation was more pronounced with the RacL11 strain. Virus-induced P-selectin expression required plasma and 1.0 mM exogenous calcium. P-selectin expression was abolished and microvesiculation was significantly reduced in factor VII- or X-deficient human plasma. Both P-selectin expression and microvesiculation were re-established in factor VII-deficient human plasma with added purified human factor VIIa (1 nM). A glycoprotein C-deficient mutant of the Ab4 strain activated platelets as effectively as non-mutated Ab4. P-selectin expression was abolished and microvesiculation was significantly reduced by preincubation of virus with a goat polyclonal anti-rabbit tissue factor antibody. Infectious virus could be retrieved from washed EHV-1-exposed platelets, suggesting a direct platelet-virus interaction. Our results indicate that EHV-1 activates equine platelets and that α-granule secretion is a consequence of virus-associated tissue factor triggering factor X activation and thrombin generation. Microvesiculation was only partly tissue factor and thrombin-dependent, suggesting the virus causes microvesiculation through other mechanisms, potentially through direct binding. These findings suggest that EHV-1-induced platelet activation could contribute to the thrombosis that occurs in clinically infected horses and provides a new mechanism by which viruses activate hemostasis. PMID:25905776

The role of platelet and endothelial GARP in thrombosis and hemostasis.

PubMed

Vermeersch, Elien; Denorme, Frederik; Maes, Wim; De Meyer, Simon F; Vanhoorelbeke, Karen; Edwards, Justin; Shevach, Ethan M; Unutmaz, Derya; Fujii, Hodaka; Deckmyn, Hans; Tersteeg, Claudia

2017-01-01

Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32) is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish. To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice. Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice. Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected. Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice.

The role of platelet and endothelial GARP in thrombosis and hemostasis

PubMed Central

Vermeersch, Elien; Denorme, Frederik; Maes, Wim; De Meyer, Simon F.; Vanhoorelbeke, Karen; Edwards, Justin; Shevach, Ethan M.; Unutmaz, Derya; Fujii, Hodaka; Deckmyn, Hans; Tersteeg, Claudia

2017-01-01

Background Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32) is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish. Objectives To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice. Methods Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice. Results Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected. Conclusions Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice. PMID:28278197

Platelet-rich preparations to improve healing. Part II: platelet activation and enrichment, leukocyte inclusion, and other selection criteria.

PubMed

Davis, Vicki L; Abukabda, Alaeddin B; Radio, Nicholas M; Witt-Enderby, Paula A; Clafshenkel, William P; Cairone, J Vito; Rutkowski, James L

2014-08-01

Multiple platelet-rich preparations have been reported to improve wound and bone healing, such as platelet-rich plasma (PRP) and platelet rich fibrin (PRF). The different methods employed during their preparation are important, as they influence the quality of the product applied to a wound or surgical site. Besides the general protocol for preparing the platelet-rich product (discussed in Part 1 of this review), multiple choices need to be considered during its preparation. For example, activation of the platelets is required for the release and enmeshment of growth factors, but the method of activation may influence the resulting matrix, growth factor availability, and healing. Additionally, some methods enrich leukocytes as well as platelets, but others are designed to be leukocyte-poor. Leukocytes have many important roles in healing and their inclusion in PRP results in increased platelet concentrations. Platelet and growth factor enrichment reported for the different types of platelet-rich preparations are also compared. Generally, TGF-β1 and PDGF levels were higher in preparations that contain leukocytes compared to leukocyte-poor PRP. However, platelet concentration may be the most reliable criterion for comparing different preparations. These and other criteria are described to help guide dental and medical professionals, in large and small practices, in selecting the best procedures for their patients. The healing benefits of platelet-rich preparations along with the low risk and availability of simple preparation procedures should encourage more clinicians to incorporate platelet-rich products in their practice to accelerate healing, reduce adverse events, and improve patient outcomes.

Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity

PubMed Central

Wood, Jeremy P.; Silveira, Jay R.; Maille, Nicole M.; Haynes, Laura M.

2011-01-01

Effective hemostasis relies on the timely formation of α-thrombin via prothrombinase, a Ca2+-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrombin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when prothrombin is processed on the activated platelet surface, the cleavage of prothrombin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin- or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating prethrombin-2, with α-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma- or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC50 = 0.3μM), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagulant activity expressed by activated platelets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin. PMID:21131592

Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity.

PubMed

Wood, Jeremy P; Silveira, Jay R; Maille, Nicole M; Haynes, Laura M; Tracy, Paula B

2011-02-03

Effective hemostasis relies on the timely formation of α-thrombin via prothrombinase, a Ca(2+)-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrombin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when prothrombin is processed on the activated platelet surface, the cleavage of prothrombin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin- or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating prethrombin-2, with α-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma- or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC(50) = 0.3μM), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagulant activity expressed by activated platelets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin.

P-selectin mediates Ca(2+)-dependent adhesion of activated platelets to many different types of leukocytes: detection by flow cytometry.

PubMed

de Bruijne-Admiraal, L G; Modderman, P W; Von dem Borne, A E; Sonnenberg, A

1992-07-01

Previous studies have shown that thrombin-activated platelets interact through the P-selectin with neutrophils and monocytes. To identify other types of leukocytes capable of such an interaction, eosinophils, basophils, and lymphocytes were isolated from whole blood. Binding of these cells to activated platelets was examined in a double immunofluorescence assay and the results show that activated platelets not only bind to neutrophils and monocytes, but also to eosinophils, basophils, and subpopulations of T lymphocytes. Using monoclonal antibodies (MoAbs) specific for subsets of T cells, we could further demonstrate that the T cells which bind activated platelets are natural killer (NK) cells and an undefined subpopulation of CD4+ and CD8+ cells. All these interactions were dependent on divalent cations and were completely inhibited by an MoAb against P-selectin. Thus, P-selectin mediates the binding of activated platelets to many different types of leukocytes. Studies with leukocytes treated with proteases or neuraminidase have shown that the structures recognized by P-selectin are glycoproteins carrying sialic acid residues. Because the loss of binding of activated platelets to neuraminidase-treated neutrophils was almost complete, but only partial to treated eosinophils, basophils, and monocytes, the latter cell types may have different P-selectin ligands in addition to those present on neutrophils. We found that two previously identified ligands for P-selectin, the oligosaccharides Le(x) and sialyl-Le(x), had little or no inhibitory effect on adhesion of activated platelets to leukocytes and that binding was not inhibited by MoAbs against these oligosaccharides. In addition, there was no correlation between the expression of Le(x) on several cell types and their capacity to bind activated platelets. In contrast, the expression of sialyl-Le(x) on cells was almost perfectly correlated with their ability to bind activated platelets. Thus, while Le(x) cannot be a major ligand for P-selectin, a possible role for sialyl-Le(x) in P-selectin-mediated adhesion processes cannot be dismissed. Finally, activated platelets were found to bind normally to monocytes and neutrophils of patients with paroxysmal nocturnal hemoglobulinuria (PNH) and to neutrophils from which phosphatidyl inositol (PI)-linked proteins had been removed by glycosylphosphatidyl inositol-specific phospholipase C (GPI-PLC) digestion. This suggests that at least part of the P-selectin ligands on these cells are not GPI-anchored.

In Vitro and Ex Vivo Approaches to Evaluate Next-Generation Tobacco and Non-Tobacco Products on Human Blood Platelets

PubMed Central

Spinelli, Sherry L.; Lannan, Katie L.; Loelius, Shannon G.

2017-01-01

Abstract Human blood platelets are major hemostatic regulators in the circulation and important in the mediation of chronic inflammation and immunomodulation. They are key elements that promote cardiovascular pathogenesis that leads to atherosclerosis, thrombosis, myocardial infarction, and stroke. New information on tobacco use and platelet dysregulation shows that these highly understudied vascular cells are dysregulated by tobacco smoke. Thus, platelet function studies should be an important consideration for the evaluation of existing and next-generation tobacco and non-tobacco products. Novel in vitro approaches are being sought to investigate these products and their influence on platelet function. Platelets are ideally suited for product assessment, as robust and novel in vitro translational methods are available to assess platelet function. Furthermore, the use of human biological systems has the advantage that risk predictions will better reflect the human condition. PMID:28337466

Influence of calcium salts and bovine thrombin on growth factor release from equine platelet-rich gel supernatants.

PubMed

Giraldo, Carlos E; Álvarez, María E; Carmona, Jorge U

2017-01-16

To compare five activation methods in equine platelet-rich plasma (PRP) by determination of platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta 1 (TGF-β1) concentrations in platelet-rich gel (PRG) supernatants. Platelet-rich plasma from 20 horses was activated by calcium chloride (CC), calcium gluconate (CG), bovine thrombin (BT), and their combinations, BTCC and BTCG. Both growth factor concentrations in PRG supernatants were measured by ELISA and compared with plasma and platelet lysates (PL) over time. Growth factor concentrations were significantly lower in plasma and higher for all PRG supernatants. Platelet lysates contained a significantly lower concentration of PDGF-BB than PRG supernatants and a significantly higher concentration of TGF-β1 than PRG supernatants. Clots from PRP activated with sodium salts were more stable over time and had significant growth factor release, whereas CC produced gross salt deposition. Significant correlations were noticed for platelet with leukocyte concentrations in PRP (r s : 0.76), platelet counts in PRP with TGF-β1 concentrations in PRG supernatants (r s : 0.86), platelet counts in PRP with PDGF-BB concentrations in PRG supernatants (r s : 0.78), leukocyte counts in PRP with TGF-β1 concentrations in PRG supernatants (r s : 0.76), and PDGF-BB concentrations with activating substances (r s : 0.72). Calcium gluconate was the better substance to induce PRP activation. It induced growth factor release free from calcium precipitates in the clots. Use of BT alone or combined with calcium salts was not advantageous for growth factor release.

Erythrocyte and platelet fatty acids in retinitis pigmentosa.

PubMed

Stanzial, A M; Bonomi, L; Cobbe, C; Olivieri, O; Girelli, D; Trevisan, M T; Bassi, A; Ferrari, S; Corrocher, R

1991-05-01

The fatty acid composition and the glutathione-peroxidase activity (GSH-Px) of erythrocytes and platelets, the production of malondialdehyde (MDA) by platelets and the activity of the main systems of transmembrane cation transport in erythrocyte have been studied in 12 patients (5 males and 7 females) affected by retinitis pigmentosa (RP). A remarkable increase of saturated fatty acids (SFA), particularly of stearic acid (C18:0), has been noted in these patients. The reduced unsaturated/saturated fatty acids ratio (PUFA/SFA) observed in both erythrocytes and platelets and the decrease of arachidonic acid in platelets may depend by an active peroxidation process as documented by the increase of MDA. Platelet glutathione-peroxidase (PTL-GSH-PX) and plasma retinol were in the normal range, whereas erythrocyte glutathione-peroxidase (E-GSH-PX), MDA and plasma alfa-toco-pherol were increased in patients with RP. The activities of Na(+)-K+ pump, cotransport and Na(+)-Li+ countertransport were normal in RP erythrocytes.

Heparin-induced thrombocytopenia: real-world issues.

PubMed

Linkins, Lori-Ann; Warkentin, Theodore E

2011-09-01

Heparin-induced thrombocytopenia (HIT) is a prothrombotic drug reaction caused by platelet-activating antibodies. HIT sera often activate platelets without needing heparin-such heparin-"independent" platelet activation can be associated with HIT beginning or worsening despite stopping heparin ("delayed-onset HIT"). We address important issues in HIT diagnosis and therapy, using a recent cohort of HIT patients to illustrate influences of heparin type; triggers for HIT investigation; serological features of heparin-independent platelet activation; and treatment. In our cohort of recent HIT cases ( N = 13), low-molecular-weight heparin (dalteparin) was a common causative agent ( N = 8, 62%); most patients were diagnosed after HIT-thrombosis had occurred; and danaparoid was the most frequently selected treatment. Heparin-independent platelet activation was common (7/13 [54%]) and predicted slower platelet count recovery (>1 week) among evaluable patients (5/5 vs 1/6; P = 0.015). In our experience with argatroban-treated patients, HIT-associated consumptive coagulopathy confounds anticoagulant monitoring. Our observations provide guidance on practical aspects of HIT diagnosis and management. Thieme Medical Publishers.

Modulation of platelet functions by crude rice (Oryza sativa) bran policosanol extract.

PubMed

Wong, Wai-Teng; Ismail, Maznah; Imam, Mustapha Umar; Zhang, Yi-Da

2016-07-28

Rice bran is bioactive-rich and has proven health benefits for humans. Moreover, its source, the brown rice has antioxidant, hypolipidemic and other functional properties that are increasingly making it a nutritional staple especially in Asian countries. This study investigated the antiplatelet aggregation mechanisms of crude hexane/methanolic rice bran extract, in which policosanol was the targeted bioactive. Platelets play a vital role in pathogenesis of atherosclerosis and cardiovascular diseases, and their increased activities could potentially cause arterial thrombus formation or severe bleeding disorders. Thus, in this study, platelet aggregation and adhesion of platelets to major components of basal lamina were examined in vitro. In addition, cellular protein secretion was quantified as a measurement of platelet activation. Adenosine diphosphate (ADP), collagen, and arachidonic acid (AA)-induced aggregation were studied using the microtiter technique. Rat platelets were pre-treated with various concentrations of policosanol extract, and the adhesion of platelets onto collagen- and laminin-coated surface (extracellular matrix) was studied using the acid phosphatase assay. The effect of crude policosanol extract on released proteins from activated platelets was measured using modified Lowry determination method. Rice bran policosanol extract significantly inhibited in vitro platelet aggregation induced by different agonists in a dose dependent manner. The IC50 of ADP-, collagen-, and AA-induced platelet aggregation were 533.37 ± 112.16, 635.94 ± 78.45 and 693.86 ± 70.57 μg/mL, respectively. The present study showed that crude rice bran policosanol extract significantly inhibited platelet adhesion to collagen in a dose dependent manner. Conversely, at a low concentration of 15.625 μg/mL, the extract significantly inhibited platelet adhesion to laminin stimulated by different platelet agonists. In addition to the alteration of cell adhesive properties, cellular protein secretion of the treated platelets towards different stimulants were decreased upon crude extract treatment. Our results showed that crude rice bran policosanol extract could inhibit in vitro platelet adhesion, aggregation and secretion upon activation using agonists. These findings serve as a scientific platform to further explore alternative therapies in cardiovascular diseases related to platelet malfunction.

Platelets and their chemokines in atherosclerosis—clinical applications

PubMed Central

von Hundelshausen, Philipp; Schmitt, Martin M. N.

2014-01-01

The concept of platelets as important players in the process of atherogenesis has become increasingly accepted due to accumulating experimental and clinical evidence. Despite the progress in understanding the molecular details of atherosclerosis, particularly by using animal models, the inflammatory and thrombotic roles of activated platelet s especially in the human system remain difficult to dissect, as often only the complications of atherosclerosis, i.e., stroke and myocardial infarction are definable but not the plaque burden. Platelet indices including platelet count and mean platelet volume (MPV) and soluble mediators released by activated platelets are associated with atherosclerosis. The chemokine CXCL4 has multiple atherogenic activities, e.g., altering the differentiation of T cells and macrophages by inhibiting neutrophil and monocyte apoptosis and by increasing the uptake of oxLDL and synergizing with CCL5. CCL5 is released and deposited on endothelium by activated platelets thereby triggering atherogenic monocyte recruitment, which can be attenuated by blocking the corresponding chemokine receptor CCR5. Atheroprotective and plaque stabilizing properties are attributed to CXCL12, which plays an important role in regenerative processes by attracting progenitor cells. Its release from luminal attached platelets accelerates endothelial healing after injury. Platelet surface molecules GPIIb/IIIa, GP1bα, P-selectin, JAM-A and the CD40/CD40L dyade are crucially involved in the interaction with endothelial cells, leukocytes and matrix molecules affecting atherogenesis. Beyond the effects on the arterial inflammatory infiltrate, platelets affect cholesterol metabolism by binding, modifying and endocytosing LDL particles via their scavenger receptors and contribute to the formation of lipid laden macrophages. Current medical therapies for the prevention of atherosclerotic therapies enable the elucidation of mechanisms linking platelets to inflammation and atherosclerosis. PMID:25152735

Platelet morphology and plasma indices of platelet activation in essential hypertension: effects of amlodipine-based antihypertensive therapy.

PubMed

Nadar, Sunil; Blann, Andrew D; Lip, Gregory Y H

2004-01-01

Platelet abnormalities have been described in hypertension, especially in the presence of target organ damage. Our aim was to study the differences in morphology and indices of platelet activation in treatment-naive patients with essential hypertension as compared to normotensive controls and secondly, to study the effects of amlodipine-based antihypertensive therapy on these indices. We recruited 42 previously untreated, newly diagnosed hypertensive patients (25 men; mean age 53 years) for the cross-sectional study, where data were compared with those from 30 normotensive controls (20 men; mean age 57 years). Of the 42 untreated hypertensive patients who were recruited, 27 patients successfully completed, the six-month treatment phase with amlodipine-based antihypertensive therapy. Platelet morphology (volume and mass) was quantified, and plasma markers of platelet activation (betaTG and sPsel) measured in citrated plasma. The mass of P-selectin in each platelet (pPsel) was determined by lysing a fixed number of platelets and then determining the levels of P-selectin in the lysate. Hypertensive patients had significantly higher platelet volume (P = 0.01) and mass (P = 0.003), plasma betaTG and sPsel, and pPsel levels (all P < 0.001) compared to the controls. After a mean treatment time of 6 months, there was a decrease in platelet volume (P < 0.001) and mass (P = 0.02), with lower pPsel, sPsel and BTG levels (all P < 0.001) compared to the untreated state. Treatment of uncomplicated essential hypertension using amlodipine-based anti-hypertensive therapy results in a reversal of the platelet morphology abnormalities and indices of platelet activation. This may contribute to a reduction in thrombosis-related complications seen in those whose blood pressure lowering is effective.

Pyridoxine improves platelet nitric oxide synthase dysfunction induced by advanced glycation end products in vitro.

PubMed

Han, Yi; Liu, Yuan; Mi, Qiongyu; Xie, Liping; Huang, Yan; Jiang, Qin; Chen, Qi; Ferro, Albert; Liu, Naifeng; Ji, Yong

2010-06-01

Advanced glycation end products (AGEs) increase platelet aggregation and suppress vascular nitric oxide (NO) synthase (NOS) activity, and these effects may contribute to the atherothrombotic disease seen in diabetes. The aims of this study were to determine in vitro whether pyridoxine can abrogate the impairment in platelet NOS activity caused by AGEs, and to determine the mechanism by which it does this. Platelet aggregation was measured by Born aggregometry. Intraplatelet cyclic guanosine-3',5'-monophosphate (cGMP, an index of bioactive NO) was measured by radioimmunoassay. Serine-1177-specific phosphorylation of NOS type 3 (NOS-3) and phosphorylation of protein kinase Akt were determined in platelets by Western blotting. Phosphatidylinositol 3-kinase (PI3K) activity in platelets was ascertained by homogeneous time-resolved fluorescence (HTRF) assay. We found that AGE-modified albumin (AGEs) 200 mg/L increased platelet aggregability and decreased intraplatelet cGMP; these effects were largely attenuated by pyridoxine. Western blotting studies revealed that AGEs decreased NOS-3 phosphorylation on serine-1177, increased NOS-3 O-glycosylation, and decreased serine phosphorylation of protein kinase Akt; all of these changes were abrogated by pyridoxine. Direct measurement of PI3K activity in platelets demonstrated that all of the above effects could be attributed to a suppression by AGEs of PI3K activity, which was prevented by co-incubation with pyridoxine. We conclude that pyridoxine is effective in ameliorating the dysfunction of platelet NO signaling in response to AGEs, through improving PI3K activity, and hence downstream Akt phosphorylation and in turn serine-1177 phosphorylation of NOS-3.

[The influence of nitrates on platelet oxygen metabolism: in vitro studies].

PubMed

Buczyński, A; Dziedziczak-Buczyńska, M; Gnitecki, W; Kocur, J

1999-01-01

Our investigations were carried out on human blood platelets obtained from persons aged 20-23, free from any systemic diseases. Drugs were incubated with blood platelets. Changes of antioxidant enzymes were detected. Glyceryl trinitrate increased the activity of Zn Cu-SOD (4.62%) and GPx (275.91%), concentration of ATP (13.01%) and the blood platelets aggregations (17.88%). Izosorbide dinitrate increased the activity of ZnCu-SOD (19.46%), GPx (150.36%) and Cat (15.62%), increased concentration of ATP (23.73%) and blood platelets aggregation (3.64%). Both preparats decreased concentration of MDA (Sustonit--30.79%, Iso-Mack--35.04%). Gliceryl trinitrate decreased the activity of catalase otherwise izosorbide dinitrate increased the activity of this enzyme.

Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis.

PubMed

Takagi, Satoshi; Takemoto, Ai; Takami, Miho; Oh-Hara, Tomoko; Fujita, Naoya

2014-08-01

The interactions of tumor cells with platelets contribute to the progression of tumor malignancy, and the expression levels of platelet aggregation-inducing factors positively correlate with the metastatic potential of osteosarcoma cells. However, it is unclear how tumor-platelet interaction contributes to the proliferation of osteosarcomas. We report here that osteosarcoma-platelet interactions induce the release of platelet-derived growth factor (PDGF) from platelets, which promotes the proliferation of osteosarcomas. Co-culture of platelets with MG63 or HOS osteosarcoma cells, which could induce platelet aggregation, enhanced the proliferation of each cell line in vitro. Analysis of phospho-antibody arrays revealed that co-culture of MG63 cells with platelets induced the phosphorylation of platelet derived growth factor receptor (PDGFR) and Akt. The addition of supernatants of osteosarcoma-platelet reactants also increased the growth of MG63 and HOS cells as well as the level of phosphorylated-PDGFR and -Akt. Sunitinib or LY294002, but not erlotinib, significantly inhibited the platelet-induced proliferation of osteosarcoma cells, indicating that PDGF released from platelets plays an important role in the proliferation of osteosarcomas by activating the PDGFR and then Akt. Our results suggest that inhibitors that specifically target osteosarcoma-platelet interactions may eradicate osteosarcomas. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Plasma and serum serotonin concentrations and surface-bound platelet serotonin expression in Cavalier King Charles Spaniels with myxomatous mitral valve disease.

PubMed

Cremer, Signe E; Kristensen, Annemarie T; Reimann, Maria J; Eriksen, Nynne B; Petersen, Stine F; Marschner, Clara B; Tarnow, Inge; Oyama, Mark A; Olsen, Lisbeth H

2015-06-01

To investigate serum and plasma serotonin concentrations, percentage of serotonin-positive platelets, level of surface-bound platelet serotonin expression (mean fluorescence intensity [MFI]), and platelet activation (CD62 expression) in platelet-rich plasma from Cavalier King Charles Spaniels with myxomatous mitral valve disease (MMVD). Healthy dogs (n = 15) and dogs with mild MMVD (18), moderate-severe MMVD (19), or severe MMVD with congestive heart failure (CHF; 10). Blood samples were collected from each dog. Serum and plasma serotonin concentrations were measured with an ELISA, and surface-bound platelet serotonin expression and platelet activation were determined by flow cytometry. Dogs with mild MMVD had higher median serum (746 ng/mL) and plasma (33.3 ng/mL) serotonin concentrations, compared with MMVD-affected dogs with CHF (388 ng/mL and 9.9 ng/mL, respectively), but no other group differences were found. Among disease groups, no differences in surface-bound serotonin expression or platelet activation were found. Thrombocytopenic dogs had lower serum serotonin concentration (482 ng/mL) than nonthrombocytopenic dogs (731 ng/mL). In 26 dogs, a flow cytometry scatterplot subpopulation (FSSP) of platelets was identified; dogs with an FSSP had a higher percentage of serotonin-positive platelets (11.0%), higher level of surface-bound serotonin expression (MFI, 32,068), and higher platelet activation (MFI, 2,363) than did dogs without an FSSP (5.7%, 1,230, and 1,165, respectively). An FSSP was present in 93.8% of thrombocytopenic dogs and in 29.5% of nonthrombocytopenic dogs. A substantive influence of circulating serotonin on MMVD stages prior to CHF development in Cavalier King Charles Spaniels was not supported by the study findings. An FSSP of highly activated platelets with pronounced serotonin binding was strongly associated with thrombocytopenia but not MMVD.

Platelet Activation and Clopidogrel Effects on ADP-Induced Platelet Activation in Cats with or without the A31P Mutation in MYBPC3.

PubMed

Li, R H L; Stern, J A; Ho, V; Tablin, F; Harris, S P

2016-09-01

Clopidogrel is commonly prescribed to cats with perceived increased risk of thromboembolic events, but little information exists regarding its antiplatelet effects. To determine effects of clopidogrel on platelet responsiveness in cats with or without the A31P mutation in the MYBPC3 gene. A secondary aim was to characterize variability in feline platelet responses to clopidogrel. Fourteen healthy cats from a Maine Coon/outbred mixed Domestic cat colony: 8 cats homozygous for A31P mutation in the MYPBC3 gene and 6 wild-type cats without the A31P mutation. Ex vivo study. All cats received clopidogrel (18.75 mg PO q24h) for 14 days. Before and after clopidogrel treatment, adenosine diphosphate (ADP)-induced P-selectin expression was evaluated. ADP- and thrombin-induced platelet aggregation was measured by optical aggregometry (OA). Platelet pVASP and ADP receptor response index (ARRI) were measured by Western blot analysis. Platelet activation from cats with the A31P mutation was significantly (P = .0095) increased [35.55% (18.58-48.55) to 58.90% (24.85-69.90)], in response to ADP. Clopidogrel treatment attenuated ADP-induced P-selectin expression and platelet aggregation. ADP- and PGE 1 -treated platelets had a similar level of pVASP as PGE 1 -treated platelets after clopidogrel treatment. Clopidogrel administration resulted in significantly lower ARRI [24.13% (12.46-35.50) to 11.30% (-7.383 to 23.27)] (P = .017). Two of 13 cats were nonresponders based on OA and flow cytometry. Clopidogrel is effective at attenuating platelet activation and aggregation in some cats. Cats with A31P mutation had increased platelet activation relative to the variable response seen in wild-type cats. Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

N-octanoyl-dopamine is a potent inhibitor of platelet function.

PubMed

Ait-Hsiko, Lamia; Kraaij, Tineke; Wedel, Johannes; Theisinger, Bastian; Theisinger, Sonja; Yard, Benito; Bugert, Peter; Schedel, Angelika

2013-01-01

Dopamine (DA) is a co-agonist for platelet activation; yet, donor DA treatment is associated with improved transplantation outcome in renal and heart recipients. Recently, N-octanoyl-dopamine (NOD) was developed which displays superior effects compared to DA in terms of graft protecting properties. Whereas DA is a known platelet co-agonist, the effect of NOD on platelet function is unknown. This is a hypothesis generating study with the aim to assess the effects and molecular mechanisms of NOD and NOD-like compounds on platelet function. The influence of DA, NOD, and NOD-like compounds on platelet responses to classical agonists (adenosine 5'-diphosphate (ADP), U46619) was investigated in six healthy donors by applying whole blood aggregometry (Multiplate®) and flow cytometry for Pac-1, CD62P, and CD63 expression. Changes in platelet cAMP concentrations were assessed by ELISA. While DA showed synergy in platelet activation by ADP and U46619, NOD caused significant inhibition of platelet function both in whole blood aggregometry and flow cytometry. The inhibitory effect of NOD was not mediated via cAMP levels. The nonredox-active NOD-analog N-octanoyl-tyramine had no effects on platelet function. Acetylated NOD conferred to NOD by intracellular esterases showed similar inhibitory effects as NOD. In contrast to DA, NOD is a potent inhibitor of platelet function most likely through intracellular redox-active processes. This adds to the overall protective effect of NOD on pre-transplantation injury and makes NOD an attractive candidate compound for donor or organ conditioning prior to transplantation.

Platelet-activated clotting time does not measure platelet reactivity during cardiac surgery.

PubMed

Shore-Lesserson, L; Ammar, T; DePerio, M; Vela-Cantos, F; Fisher, C; Sarier, K

1999-08-01

Platelet dysfunction is a major contributor to bleeding after cardiopulmonary bypass (CPB), yet it remains difficult to diagnose. A point-of-care monitor, the platelet-activated clotting time (PACT), measures accelerated shortening of the kaolin-activated clotting time by addition of platelet activating factor. The authors sought to evaluate the clinical utility of the PACT by conducting serial measurements of PACT during cardiac surgery and correlating postoperative measurements with blood loss. In 50 cardiac surgical patients, blood was sampled at 10 time points to measure PACT. Simultaneously, platelet reactivity was measured by the thrombin receptor agonist peptide-induced expression of P-selectin, using flow cytometry. These tests were temporally analyzed. PACT values, P-selectin expression, and other coagulation tests were analyzed for correlation with postoperative chest tube drainage. PACT and P-selectin expression were maximally reduced after protamine administration. Changes in PACT did not correlate with changes in P-selectin expression at any time interval. Total 8-h chest tube drainage did not correlate with any coagulation test at any time point except with P-selectin expression after protamine administration (r = -0.4; P = 0.03). The platelet dysfunction associated with CPB may be a result of depressed platelet reactivity, as shown by thrombin receptor activating peptide-induced P-selectin expression. Changes in PACT did not correlate with blood loss or with changes in P-selectin expression suggesting that PACT is not a specific measure of platelet reactivity.

A Study of Platelet Inhibition, Using a 'Point of Care' Platelet Function Test, following Primary Percutaneous Coronary Intervention for ST-Elevation Myocardial Infarction [PINPOINT-PPCI].

PubMed

Johnson, Thomas W; Mumford, Andrew D; Scott, Lauren J; Mundell, Stuart; Butler, Mark; Strange, Julian W; Rogers, Chris A; Reeves, Barnaby C; Baumbach, Andreas

2015-01-01

Rapid coronary recanalization following ST-elevation myocardial infarction (STEMI) requires effective anti-platelet and anti-thrombotic therapies. This study tested the impact of door to end of procedure ('door-to-end') time and baseline platelet activity on platelet inhibition within 24hours post-STEMI. 108 patients, treated with prasugrel and procedural bivalirudin, underwent Multiplate® platelet function testing at baseline, 0, 1, 2 and 24hours post-procedure. Major adverse cardiac events (MACE), bleeding and stent thrombosis (ST) were recorded. Baseline ADP activity was high (88.3U [71.8-109.0]), procedural time and consequently bivalirudin infusion duration were short (median door-to-end time 55minutes [40-70] and infusion duration 30minutes [20-42]). Baseline ADP was observed to influence all subsequent measurements of ADP activity, whereas door-to-end time only influenced ADP immediately post-procedure. High residual platelet reactivity (HRPR ADP>46.8U) was observed in 75% of patients immediately post-procedure and persisted in 24% of patients at 2hours. Five patients suffered in-hospital MACE (4.6%). Acute ST occurred in 4 patients, all were <120mins post-procedure and had HRPR. No significant bleeding was observed. In a post-hoc analysis, pre-procedural morphine use was associated with significantly higher ADP activity following intervention. Baseline platelet function, time to STEMI treatment and opiate use all significantly influence immediate post-procedural platelet activity.

Platelet activation through a Bi-leaflet mechanical heart valve

NASA Astrophysics Data System (ADS)

Hedayat, Mohammadali; Borazjani, Iman

2016-11-01

Platelet activation is one of the major drawbacks of the Mechanical Heart Valves (MHVs) which can increase the risk of thrombus formation in patients. The platelet activation in MHVs can be due to the abnormal shear stress during the systole, the backward leakage flow during the diastole, and the flow through the hinge region. We investigate the contribution of each of the above mechanism to the activation of platelets in MHVs by performing simulations of the flow through the MHV and in the hinge region. The large scale heart valve simulations are performed in a straight aorta using a sharp interface curvilinear immersed boundary method along with a strong-coupling algorithm under physiological flow conditions. In addition, in order to perform the simulation of hinge region the flow field boundary conditions are obtained from the largescale simulations during a whole cardiac cycle. In order to investigate the role of hinge flow on platelet activation in MHVs, a 23mm St. Jude Medical Regent valve hinge with three different gap sizes is tested along with different platelet activation models to ensure the consistency of our results with different activation models. We compare the platelet activation of the hinge region against the bulk of the flow during one cardiac cycle. This work is supported by the American Heart Association Grant 13SDG17220022, and the computational resources were partly provided by Center for Computational Research (CCR) at University at Buffalo.

Force-activatable biosensor enables single platelet force mapping directly by fluorescence imaging.

PubMed

Wang, Yongliang; LeVine, Dana N; Gannon, Margaret; Zhao, Yuanchang; Sarkar, Anwesha; Hoch, Bailey; Wang, Xuefeng

2018-02-15

Integrin-transmitted cellular forces are critical for platelet adhesion, activation, aggregation and contraction during hemostasis and thrombosis. Measuring and mapping single platelet forces are desired in both research and clinical applications. Conventional force-to-strain based cell traction force microscopies have low resolution which is not ideal for cellular force mapping in small platelets. To enable platelet force mapping with submicron resolution, we developed a force-activatable biosensor named integrative tension sensor (ITS) which directly converts molecular tensions to fluorescent signals, therefore enabling cellular force mapping directly by fluorescence imaging. With ITS, we mapped cellular forces in single platelets at 0.4µm resolution. We found that platelet force distribution has strong polarization which is sensitive to treatment with the anti-platelet drug tirofiban, suggesting that the ITS force map can report anti-platelet drug efficacy. The ITS also calibrated integrin molecular tensions in platelets and revealed two distinct tension levels: 12-54 piconewton (nominal values) tensions generated during platelet adhesion and tensions above 54 piconewton generated during platelet contraction. Overall, the ITS is a powerful biosensor for the study of platelet mechanobiology, and holds great potential in antithrombotic drug development and assessing platelet activity in health and disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Coincident regulation of PKCδ in human platelets by phosphorylation of Tyr311 and Tyr565 and phospholipase C signalling

PubMed Central

Hall, Kellie J.; Jones, Matthew L.; Poole, Alastair W.

2007-01-01

PKC (protein kinase C)δ plays a complex role in platelets, having effects on both positive and negative signalling functions. It is phosphorylated on tyrosine residues in response to thrombin and collagen, and it has recently been shown that Tyr311 is phosphorylated in response to PAR (protease-activated receptor) 1 and PAR4 receptor activation. In the present study, we show that Tyr311 and Tyr565 are phosphorylated in response to thrombin, and have examined the interplay between phosphorylation and the classical lipid-mediated activation of PKCδ. Phosphorylation of both Tyr311 and Tyr565 is dependent on Src kinase and PLC (phospholipase C) activity in response to thrombin. Importantly, direct allosteric activation of PKCδ with PMA also induced phosphorylation of Tyr311 and Tyr565, and this was dependent on the activity of Src kinases, but not PLC. Membrane recruitment of PKCδ is essential for phosphorylation of this tyrosine residue, but tyrosine phosphorylation is not required for membrane recruitment of PKCδ. Both thrombin and PMA induce recruitment of PKCδ to the membrane, and for thrombin, this recruitment is a PLC-dependent process. In order to address the functional role of tyrosine residue phosphorylation of PKCδ, we demonstrate that phosphorylation can potentiate the activity of the kinase, although phosphorylation does not play a role in membrane recruitment of the kinase. PKCδ is therefore regulated in a coincident fashion, PLC-dependent signals recruiting it to the plasma membrane and by phosphorylation on tyrosine residues, potentiating its activity. PMID:17570831

Inhibitory effect of hydrophilic polymer brushes on surface-induced platelet activation and adhesion.

PubMed

Zou, Yuquan; Lai, Benjamin F L; Kizhakkedathu, Jayachandran N; Brooks, Donald E

2010-12-08

Poly(N,N-dimethylacrylamide) (PDMA) brushes are successfully grown from unplasticized poly(vinyl chloride) (uPVC) by well-controlled surface-initiated atom transfer radical polymerization (SI-ATRP). Molecular weights of the grafted PDMA brushes vary from ≈ 35,000 to 2,170000 Da, while the graft density ranges from 0.08 to 1.13 chains · nm(-2). The polydispersity of the grafted PDMA brushes is controlled within 1.20 to 1.80. Platelet activation (expression of CD62) and adhesion studies reveal that the graft densities of the PDMA brushes play an important role in controlling interfacial properties. PDMA brushes with graft densities between 0.35 and 0.50 chains · nm(-2) induce a significantly reduced platelet activation compared to unmodified uPVC. Moreover, the surface adhesion of platelets on uPVC is significantly reduced by the densely grafted PDMA brushes. PDMA brushes that have high molecular weights lead to a relatively lower platelet activation compared to low-molecular-weight brushes. However, the graft density of the brush is more important than molecular weight in controlling platelet interactions with PVC. PDMA brushes do not produce any significant platelet consumption in platelet rich plasma. Up to a seven-fold decrease in the number of platelets adhered on high graft density brushes is observed compared to the bare PVC surface. Unlike the bare PVC, platelets do not form pseudopodes or change morphology on PDMA brush-coated surfaces. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sulfation of minoxidil by multiple human cytosolic sulfotransferases.

PubMed

Anderson, R J; Kudlacek, P E; Clemens, D L

1998-02-20

Minoxidil is an antihypertensive agent and hair growth promoter that is metabolized by sulfation to the active compound, minoxidil sulfate. Thermostable phenol sulfotransferase (TS PST or P-PST) was initially thought to catalyze the reaction, and the enzyme was designated minoxidil sulfotransferase (MNX-ST). Information about human ST activities toward minoxidil would be useful in developing the capacity to predict individual responses to minoxidil based on tissue levels of STs. Therefore, human STs were studied from platelet homogenates, partially purified platelets, scalp skin high speed supernatants and COS-1 cell cDNA expressed preparations using a radiochemical enzymatic assay with minoxidil as the substrate. Studies showed the presence of TS PST, TL (thermolabile) PST and MNX-ST activities in human scalp skin. Biochemical properties and correlation studies suggested that in addition to TS PST, the TL PST activity, another ST activity or both were involved in the reaction. Partially purified human platelet TL PST tested with minoxidil and dopamine showed identical thermal stabilities and similar responses to the inhibitors 2,6-dichloro-4-nitrophenol (DCNP) and NaCl. To characterize the activity of TL PST toward minoxidil, several biochemical properties of the enzyme expressed from a human liver cDNA clone were investigated. When assayed with minoxidil and dopamine, thermal stabilities of the expressed enzyme were identical and IC50 values for the inhibitors DCNP and NaCl were similar. It was also demonstrated that cDNA encoded human liver dehydroepiandrosterone sulfotransferase and estrogen sulfotransferase contributed to the sulfation of minoxidil. The results confirm that at least four human STs contribute to minoxidil sulfation. MNX-ST activity represents a combination of ST activities. The data indicate that multiple ST activities should be taken into account in attempts to predict the regulation of minoxidil sulfation and individual responses to minoxidil.

Characteristics of platelet gels combined with silk

PubMed Central

Pallotta, Isabella; Kluge, Jonathan A.; Moreau, Jodie; Calabrese, Rossella

2014-01-01

Platelet gel, a fibrin network containing activated platelets, is widely used in regenerative medicine due the capacity of platelet-derived growth factors to accelerate and direct healing processes. However, limitations to this approach include poor mechanical properties, relatively rapid degradation, and the lack of control of release of growth factors at the site of injection. These issues compromise the ability of platelet gels for sustained function in regenerative medicine. In the present study, a combination of platelet gels with silk fibroin gel was studied to address the above limitations. Mixing sonicated silk gels with platelet gels extended the release of growth factors without inhibiting gel forming ability. The released growth factors were biologically active and their delivery was modified further by manipulation of the charge of the silk protein. Moreover, the silk gel augmented both the rheological properties and compressive stiffness of the platelet gel, tuned by the silk concentration and/or silk/platelet gel ratio. Silk-platelet gel injections in nude rats supported enhanced cell infiltration and blood vessel formation representing a step towards new platelet gel formulations with enhanced therapeutic impact. PMID:24480538

Protein kinase Cι/λ is dispensable for platelet function in thrombosis and hemostasis in mice.

PubMed

Beck, Sarah; Leitges, Michael; Stegner, David

2017-10-01

Platelet activation at sites of vascular injury is crucial for hemostasis, but it may also cause myocardial infarction or ischemic stroke. Upon platelet activation, cytoskeletal reorganization is essential for platelet secretion and thrombus formation. Members of the protein kinase C family, which includes 12 isoforms, are involved in most platelet responses required for thrombus formation. The atypical protein kinase Cι/λ (PKCι/λ) has been implicated as an important mediator of cell polarity, carcinogenesis and immune cell responses. PKCι/λ is known to be associated with the small GTPase Cdc42, an important mediator of multiple platelet functions; however, its exact function in platelets is not known. To study the role of PKCι/λ, we generated platelet- and megakaryocyte-specific PKCι/λ knockout mice (Prkci fl/fl, Pf4-Cre ) and used them to investigate the function of PKCι/λ in platelet activation and aggregation in vitro and in vivo. Surprisingly, lack of PKCι/λ had no detectable effect on platelet spreading and function in vitro and in vivo under all tested conditions. These results indicate that PKCι/λ is dispensable for Cdc42-triggered processes and for thrombosis and hemostasis in mice. Copyright © 2017 Elsevier Inc. All rights reserved.

Low angle light scattering analysis: a novel quantitative method for functional characterization of human and murine platelet receptors.

PubMed

Mindukshev, Igor; Gambaryan, Stepan; Kehrer, Linda; Schuetz, Claudia; Kobsar, Anna; Rukoyatkina, Natalia; Nikolaev, Viacheslav O; Krivchenko, Alexander; Watson, Steve P; Walter, Ulrich; Geiger, Joerg

2012-07-01

Determinations of platelet receptor functions are indispensable diagnostic indicators of cardiovascular and hemostatic diseases including hereditary and acquired receptor defects and receptor responses to drugs. However, presently available techniques for assessing platelet function have some disadvantages, such as low sensitivity and the requirement of large sample sizes and unphysiologically high agonist concentrations. Our goal was to develop and initially characterize a new technique designed to quantitatively analyze platelet receptor activation and platelet function on the basis of measuring changes in low angle light scattering. We developed a novel technique based on low angle light scattering registering changes in light scattering at a range of different angles in platelet suspensions during activation. The method proved to be highly sensitive for simultaneous real time detection of changes in size and shape of platelets during activation. Unlike commonly-used methods, the light scattering method could detect platelet shape change and aggregation in response to nanomolar concentrations of extracellular nucleotides. Furthermore, our results demonstrate that the advantages of the light scattering method make it a choice method for platelet receptor monitoring and for investigation of both murine and human platelets in disease models. Our data demonstrate the suitability and superiority of this new low angle light scattering method for comprehensive analyses of platelet receptors and functions. This highly sensitive, quantitative, and online detection of essential physiological, pathophysiological and pharmacological-response properties of human and mouse platelets is a significant improvement over conventional techniques.

Platelet activation in pregnancy-induced hypertension.

PubMed

Karalis, Ioannis; Nadar, Sunil K; Al Yemeni, Eman; Blann, Andrew D; Lip, Gregory Y H

2005-01-01

Although excess platelet activation, as indicated by increased plasma beta thromboglobulin (beta-TG), has been shown in pregnancy-induced hypertension (PIH), platelet adhesion, platelet morphology and a comparison of platelet and soluble (plasma) levels of the adhesion molecules P-selectin (pPsel and sPsel, respectively) have not been studied. We conducted a cross-sectional study of 35 consecutive women with PIH (age 31+/-6 years), 31 consecutive women with normotensive pregnancies (age 29+/-5 years) and 30 normotensive non pregnant women (age 30+/-5 years). Platelet adhesion was studied in vitro by binding to fibrinogen-coated microwells, platelet morphology [mass and volume by flow cytometry], whole-platelet P-selectin (pPsel) by ELISA of the lysate of 2 x 10(8) cells, and the plasma markers soluble P-selectin (sP-sel) and beta-TG, by ELISA. The women with PIH had significantly raised sPsel, pPsel and (as expected) beta-TG (all p<0.05), when compared to the normotensive pregnant women and controls. However, in PIH platelet adhesion was similar to that in the normotensive pregnancy, but still higher than the normal controls (p<0.001). There was no difference among the three groups with respect to platelet mass and volume. pPsel and platelet adhesion correlated with gestational age and with systolic and diastolic blood pressure (all p<0.05). Increased platelet activation and adhesion develop during normal pregnancy, with some indices being further altered in PIH.

Immune complexes formed following the binding of anti–platelet factor 4 (CXCL4) antibodies to CXCL4 stimulate human neutrophil activation and cell adhesion

PubMed Central

Xiao, Zhihua; Visentin, Gian P.; Dayananda, Kannayakanahalli M.

2008-01-01

We tested the possibility that immune complexes formed following platelet factor 4 (PF4/CXCL4) binding to anti-PF4 antibody can stimulate neutrophil activation, similar to previous reports with platelets. Monoclonal Abs against PF4 and IgG from a heparin-induced thrombocytopenia (HIT) patient were applied. We observed that although PF4 or anti-PF4 antibody alone did not alter neutrophil function, costimulation with both reagents resulted in approximately 3-fold increase in cell surface Mac-1 expression, enhanced cell adhesion via L-selectin and CD18 integrins, and degranulation of secondary and tertiary granules. The level of Mac-1 up-regulation peaked at an intermediate PF4 dose, suggesting that functional response varies with antigen-antibody stoichiometry. PF4 binding to neutrophils was blocked by chondroitinase ABC. Cell activation was inhibited by both chondroitinase ABC and anti-CD32/FcγRII blocking mAb, IV.3. Confocal microscopy demonstrated that immune complexes colocalize with CD32a. Studies with HIT IgG demonstrated that neutrophils could be activated in the absence of exogenous heparin. These data, together, show that leukocyte surface chondroitin sulfates promote neutrophil activation by enhancing immune-complex binding to CD32a. Studies with recombinant PF4 suggest a role for arginine 49 in stabilizing PF4-chondroitin binding. Neutrophils activated via this mechanism may contribute to thrombosis and inflammation in patients mounting an immune response to PF4-heparin. PMID:18539895

Influence of gold nanoparticles on platelets functional activity in vitro

NASA Astrophysics Data System (ADS)

Akchurin, Garif G.; Akchurin, George G.; Ivanov, Alexey N.; Kirichuk, Vyacheslav F.; Terentyuk, George S.; Khlebtsov, Boris N.; Khlebtsov, Nikolay G.

2008-02-01

Now in the leading biomedical centers of the world approved new technology of laser photothermal destruction of cancer cells using plasmon gold nanoparticles. Investigations of influence of gold nanoparticles on white rat platelets aggregative activity in vitro have been made. Platelet aggregation was investigated in platelet rich plasma (PRP) with help of laser analyzer 230 LA <>, Russia). Aggregation inductor was ADP solution in terminal concentration 2.5 micromole (<>, Russia). Gold nanoshells soluted in salt solution were used for experiments. Samples of PRP were incubated with 50 or 100 μl gold nanoshells solution in 5 minute, after that we made definition ADP induced platelet aggregation. We found out increase platelet function activity after incubation with nanoparticles solution which shown in maximum ADP-induced aggregation degree increase. Increase platelet function activity during intravenous nanoshells injection can be cause of thrombosis on patients. That's why before clinical application of cancer cell destruction based on laser photothermal used with plasmon gold nanoparticles careful investigations of thrombosis process and detail analyze of physiological blood parameters are very necessary.

The effect of anthocyanin supplementation in modulating platelet function in sedentary population: a randomised, double-blind, placebo-controlled, cross-over trial.

PubMed

Thompson, Kiara; Hosking, Holly; Pederick, Wayne; Singh, Indu; Santhakumar, Abishek B

2017-09-01

The anti-thrombotic properties of anthocyanin (ACN) supplementation was evaluated in this randomised, double-blind, placebo (PBO) controlled, cross-over design, dietary intervention trial in sedentary population. In all, sixteen participants (three males and thirteen females) consumed ACN (320 mg/d) or PBO capsules for 28 d followed by a 2-week wash-out period. Biomarkers of thrombogenesis and platelet activation induced by ADP; platelet aggregation induced by ADP, collagen and arachidonic acid; biochemical, lipid, inflammatory and coagulation profile were evaluated before and after supplementation. ACN supplementation reduced monocyte-platelet aggregate formation by 39 %; inhibited platelet endothelial cell adhesion molecule-1 expression by 14 %; reduced platelet activation-dependant conformational change and degranulation by reducing procaspase activating compound-1 (PAC-1) (↓10 %) and P-selectin expression (↓14 %), respectively; and reduced ADP-induced whole blood platelet aggregation by 29 %. Arachidonic acid and collagen-induced platelet aggregation; biochemical, lipid, inflammatory and coagulation parameters did not change post-ACN supplementation. PBO treatment did not have an effect on the parameters tested. The findings suggest that dietary ACN supplementation has the potential to alleviate biomarkers of thrombogenesis, platelet hyperactivation and hyper-aggregation in sedentary population.

Development of a New Method for Platelet Function Test and Its Shearing Condition in Microfludic System

NASA Astrophysics Data System (ADS)

Lee, Hoyoon; Kim, Gyehyu; Choi, Seawhan; Shin, Sehyun; Korea University Department of Mechanical Engineering Team

2015-11-01

Platelet is a crucial blood cell on hemostasis. As platelet exposed to high shear stress, it can be activated showing morphological and functional changes to stop bleeding. When platelet is abnormal, there is high risk of cardiovascular diseases. Thus, quick and precise assay for platelet function is important in clinical treatment. In this study, we design a microfluidic system, which can test platelet function exposed with the stimulation of shear and agonists. The microfluidic system consists of three parts: 1) a shear mechanism with rotating stirrer; 2) multiple microchannels to flow samples and to stop; 3) camera-interfaced migration distance(MD) analyzing system. When sheared blood is driven by pressure through the microchannel, shear-activated platelets adhere to a collagen-coated surface, causing blood flow to significantly slow and eventually stop. As the micro-stirrer speed increases, MD decreases exponentially at first, but it increases beyond a critical rpm after all. These results are coincident with data measured by FACS flowcytometry. These results imply that the present system could quantitatively measure the degree of activation, aggregation and adhesion of platelets and that blood MD is potent index for measuring the shear-dependence of platelet function.

Megakaryocyte and polyploidization.

PubMed

Mazzi, Stefania; Lordier, Larissa; Debili, Najet; Raslova, Hana; Vainchenker, William

2018-01-01

In mammals, platelets are produced in the blood by cytoplasmic fragmentation of megakaryocytes (MKs). Platelet production is thus dependent on both the MK number and size. During differentiation, MKs switch from a division by mitosis to polyploidization by endomitosis to increase their size. The endomitotic process includes several successive rounds of DNA replication with an entry in mitosis with a failure in late cytokinesis and a defect in karyokinesis. This leads to a giant cell with a modal ploidy at 16N and one multilobulated nucleus. The entire genome is duplicated several times and all alleles remain functional producing a hypermetabolic cell. A defect in abscission explains the cytokinesis failure and is related to an altered accumulation of actomyosin at the cleavage furrow as a consequence of both a low local RhoA activity and silencing of the MYH10 gene. This mechanism is regulated by transcription factors that govern differentiation explaining the intricacies of both processes. However, the endomitotic cell cycle regulation is still incompletely understood, particularly mitosis entry, escape to the tetraploid checkpoint, and defect in karyokinesis. Polyploidization is regulated during ontogeny, the first embryonic MKs being 2N. The molecular mechanism of this embryo-fetal/adult transition is beginning to be understood. In physiological conditions, MK ploidy is increased by an enhanced platelet demand through the thrombopoietin/myeloproliferative leukemia axis. In numerous hematologic malignancies, MK ploidy decreases, but it is always associated with a defect in MK differentiation. It has been proposed that polyploidization induction could be a treatment for some malignant MK disorders. Copyright © 2018 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Blood Platelets in the Progression of Alzheimer’s Disease

PubMed Central

Gowert, Nina S.; Donner, Lili; Chatterjee, Madhumita; Eisele, Yvonne S.; Towhid, Seyda T.; Münzer, Patrick; Walker, Britta; Ogorek, Isabella; Borst, Oliver; Grandoch, Maria; Schaller, Martin; Fischer, Jens W.; Gawaz, Meinrad; Weggen, Sascha; Lang, Florian; Jucker, Mathias; Elvers, Margitta

2014-01-01

Alzheimer’s disease (AD) is characterized by neurotoxic amyloid-ß plaque formation in brain parenchyma and cerebral blood vessels known as cerebral amyloid angiopathy (CAA). Besides CAA, AD is strongly related to vascular diseases such as stroke and atherosclerosis. Cerebrovascular dysfunction occurs in AD patients leading to alterations in blood flow that might play an important role in AD pathology with neuronal loss and memory deficits. Platelets are the major players in hemostasis and thrombosis, but are also involved in neuroinflammatory diseases like AD. For many years, platelets were accepted as peripheral model to study the pathophysiology of AD because platelets display the enzymatic activities to generate amyloid-ß (Aß) peptides. In addition, platelets are considered to be a biomarker for early diagnosis of AD. Effects of Aß peptides on platelets and the impact of platelets in the progression of AD remained, however, ill-defined. The present study explored the cellular mechanisms triggered by Aß in platelets. Treatment of platelets with Aß led to platelet activation and enhanced generation of reactive oxygen species (ROS) and membrane scrambling, suggesting enhanced platelet apoptosis. More important, platelets modulate soluble Aß into fibrillar structures that were absorbed by apoptotic but not vital platelets. This together with enhanced platelet adhesion under flow ex vivo and in vivo and platelet accumulation at amyloid deposits of cerebral vessels of AD transgenic mice suggested that platelets are major contributors of CAA inducing platelet thrombus formation at vascular amyloid plaques leading to vessel occlusion critical for cerebrovascular events like stroke. PMID:24587388

β3 Integrin Haplotype Influences Gene Regulation and Plasma von Willebrand Factor Activity

PubMed Central

Payne, Katie E; Bray, Paul F; Grant, Peter J; Carter, Angela M

2008-01-01

The Leu33Pro polymorphism of the gene encoding β3 integrin (ITGB3) is associated with acute coronary syndromes and influences platelet aggregation. Three common promoter polymorphisms have also been identified. The aims of this study were to (1) investigate the influence of the ITGB3 −400C/A, −425A/C and −468G/A promoter polymorphisms on reporter gene expression and nuclear protein binding and (2) determine genotype and haplotype associations with platelet αIIbβ3 receptor density. Promoter haplotypes were introduced into an ITGB3 promoter-pGL3 construct by site directed mutagenesis and luciferase reporter gene expression analysed in HEL and HMEC-1 cells. Binding of nuclear proteins was assessed by electrophoretic mobility shift assay. The association of ITGB3 haplotype with platelet αIIbβ3 receptor density was determined in 223 subjects. Species conserved motifs were identified in the ITGB3 promoter in the vicinity of the 3 polymorphisms. The GAA, GCC, AAC, AAA and ACC constructs induced ~50% increased luciferase expression relative to the GAC construct in both cell types. Haplotype analysis including Leu33Pro indicated 5 common haplotypes; no associations between ITGB3 haplotypes and receptor density were found. However, the GCC-Pro33 haplotype was associated with significantly higher vWF activity (128.6 [112.1–145.1]%) compared with all other haplotypes (107.1 [101.2–113.0]%, p=0.02). In conclusion, the GCC-Pro33 haplotype was associated with increased vWF activity but not with platelet αIIbβ3 receptor density, which may indicate ITGB3 haplotype influences endothelial function. PMID:18045606

Platelet morphology, soluble P selectin and platelet P-selectin in acute ischaemic stroke. The West Birmingham Stroke Project.

PubMed

Nadar, Sunil K; Lip, Gregory Y H; Blann, Andrew D

2004-12-01

The pathophysiology of ischaemic stroke involves the platelet. In this study, we hypothesised that abnormalities in platelet morphology, as well as soluble (sPsel) and total platelet P-selectin (pPsel) levels would be present in patients presenting with an acute ischaemic stroke, and that these changes would improve at > or = 3 months' follow-up. We studied 59 hypertensive patients (34 male; mean age 68 +/- 12 years) who presented with an acute ischaemic stroke (ictus < 24 hours), and compared them with 2 groups: (i) age-, sex- and ethnic- origin matched normotensive healthy controls; and (ii) uncomplicated 'high risk' hypertensive patients as 'risk factor control' subjects. Platelet morphology (volume and mass) was quantified, and sPsel (plasma marker of platelet activation) was measured (ELISA) in citrated plasma. The mass of P-selectin in each platelet (pPsel) was determined by lysing a fixed number of platelets and then determining the levels of P-selectin in the lysate. Results show that patients who presented with a stroke had significantly higher levels of sPsel and pPsel (both p < 0.001), compared to the normal controls and the hypertensive patients. Patients with an acute stroke had lower mean platelet mass (MPM) and mean platelet volume (MPV) as compared to the uncomplicated hypertensive patients, who had significantly higher mean MPM and MPV values, as compared to normal controls. On follow-up, the levels of both sPsel (p = 0.011), pPsel (< 0.001) and MPV (p = 0.03) were significantly lower. Mean MPM levels remained unchanged. We conclude that patients presenting with an acute ischaemic stroke have activated platelets, as evident by the increased levels of soluble and platelet P-selectin. Further study of platelet activation and the role of P-selectin is warranted.

Oligomeric state regulated trafficking of human platelet-activating factor acetylhydrolase type-II.

PubMed

Monillas, Elizabeth S; Caplan, Jeffrey L; Thévenin, Anastasia F; Bahnson, Brian J

2015-05-01

The intracellular enzyme platelet-activating factor acetylhydrolase type-II (PAFAH-II) hydrolyzes platelet-activating factor and oxidatively fragmented phospholipids. PAFAH-II in its resting state is mainly cytoplasmic, and it responds to oxidative stress by becoming increasingly bound to endoplasmic reticulum and Golgi membranes. Numerous studies have indicated that this enzyme is essential for protecting cells from oxidative stress induced apoptosis. However, the regulatory mechanism of the oxidative stress response by PAFAH-II has not been fully resolved. Here, changes to the oligomeric state of human PAFAH-II were investigated as a potential regulatory mechanism toward enzyme trafficking. Native PAGE analysis in vitro and photon counting histogram within live cells showed that PAFAH-II is both monomeric and dimeric. A Gly-2-Ala site-directed mutation of PAFAH-II demonstrated that the N-terminal myristoyl group is required for homodimerization. Additionally, the distribution of oligomeric PAFAH-II is distinct within the cell; homodimers of PAFAH-II were localized to the cytoplasm while monomers were associated to the membranes of the endoplasmic reticulum and Golgi. We propose that the oligomeric state of PAFAH-II drives functional protein trafficking. PAFAH-II localization to the membrane is critical for substrate acquisition and effective oxidative stress protection. It is hypothesized that the balance between monomer and dimer serves as a regulatory mechanism of a PAFAH-II oxidative stress response. Copyright © 2015 Elsevier B.V. All rights reserved.

Synthesis of analogues of gingerol and shogaol, the active pungent principles from the rhizomes of Zingiber officinale and evaluation of their anti-platelet aggregation effects.

PubMed

Shih, Hung-Cheng; Chern, Ching-Yuh; Kuo, Ping-Chung; Wu, You-Cheng; Chan, Yu-Yi; Liao, Yu-Ren; Teng, Che-Ming; Wu, Tian-Shung

2014-03-04

The present study was aimed at discovering novel biologically active compounds based on the skeletons of gingerol and shogaol, the pungent principles from the rhizomes of Zingiber officinale. Therefore, eight groups of analogues were synthesized and examined for their inhibitory activities of platelet aggregation induced by arachidonic acid, collagen, platelet activating factor, and thrombin. Among the tested compounds, [6]-paradol (5b) exhibited the most significant anti-platelet aggregation activity. It was the most potent candidate, which could be used in further investigation to explore new drug leads.

Synthesis of Analogues of Gingerol and Shogaol, the Active Pungent Principles from the Rhizomes of Zingiber officinale and Evaluation of Their Anti-Platelet Aggregation Effects

PubMed Central

Shih, Hung-Cheng; Chern, Ching-Yuh; Kuo, Ping-Chung; Wu, You-Cheng; Chan, Yu-Yi; Liao, Yu-Ren; Teng, Che-Ming; Wu, Tian-Shung

2014-01-01

The present study was aimed at discovering novel biologically active compounds based on the skeletons of gingerol and shogaol, the pungent principles from the rhizomes of Zingiber officinale. Therefore, eight groups of analogues were synthesized and examined for their inhibitory activities of platelet aggregation induced by arachidonic acid, collagen, platelet activating factor, and thrombin. Among the tested compounds, [6]-paradol (5b) exhibited the most significant anti-platelet aggregation activity. It was the most potent candidate, which could be used in further investigation to explore new drug leads. PMID:24599082

Space and Time Resolved Detection of Platelet Activation and von Willebrand Factor Conformational Changes in Deep Suspensions.

PubMed

Biasetti, Jacopo; Sampath, Kaushik; Cortez, Angel; Azhir, Alaleh; Gilad, Assaf A; Kickler, Thomas S; Obser, Tobias; Ruggeri, Zaverio M; Katz, Joseph

2017-01-01

Tracking cells and proteins' phenotypic changes in deep suspensions is critical for the direct imaging of blood-related phenomena in in vitro replica of cardiovascular systems and blood-handling devices. This paper introduces fluorescence imaging techniques for space and time resolved detection of platelet activation, von Willebrand factor (VWF) conformational changes, and VWF-platelet interaction in deep suspensions. Labeled VWF, platelets, and VWF-platelet strands are suspended in deep cuvettes, illuminated, and imaged with a high-sensitivity EM-CCD camera, allowing detection using an exposure time of 1 ms. In-house postprocessing algorithms identify and track the moving signals. Recombinant VWF-eGFP (rVWF-eGFP) and VWF labeled with an FITC-conjugated polyclonal antibody are employed. Anti-P-Selectin FITC-conjugated antibodies and the calcium-sensitive probe Indo-1 are used to detect activated platelets. A positive correlation between the mean number of platelets detected per image and the percentage of activated platelets determined through flow cytometry is obtained, validating the technique. An increase in the number of rVWF-eGFP signals upon exposure to shear stress demonstrates the technique's ability to detect breakup of self-aggregates. VWF globular and unfolded conformations and self-aggregation are also observed. The ability to track the size and shape of VWF-platelet strands in space and time provides means to detect pro- and antithrombotic processes.

NPP4 is a procoagulant enzyme on the surface of vascular endothelium

PubMed Central

Albright, Ronald A.; Chang, William C.; Robert, Donna; Ornstein, Deborah L.; Cao, Wenxiang; Liu, Lynn; Redick, Meredith E.; Young, J. Isaac; De La Cruz, Enrique M.

2012-01-01

Ap3A is a platelet-dense granule component released into the extracellular space during the second wave of platelet aggregation on activation. Here, we identify an uncharacterized enzyme, nucleotide pyrophosphatase/phosphodiesterase-4 (NPP4), as a potent hydrolase of Ap3A capable of stimulating platelet aggregation and secretion. We demonstrate that NPP4 is present on the surface of vascular endothelium, where it hydrolyzes Ap3A into AMP and ADP, and Ap4A into AMP and ATP. Platelet aggregation assays with citrated platelet-rich plasma reveal that the primary and secondary waves of aggregation and dense granule release are strongly induced by nanomolar NPP4 in a concentration-dependent manner in the presence of Ap3A, while Ap3A alone initiates a primary wave of aggregation followed by rapid disaggregation. NPP2 and an active site NPP4 mutant, neither of which appreciably hydrolyzes Ap3A, have no effect on platelet aggregation and secretion. Finally, by using ADP receptor blockade we confirm that NPP4 mediates platelet aggregation via release of ADP from Ap3A and activation of ADP receptors. Collectively, these studies define the biologic and enzymatic basis for NPP4 and Ap3A activity in platelet aggregation in vitro and suggest that NPP4 promotes hemostasis in vivo by augmenting ADP-mediated platelet aggregation at the site of vascular injury. PMID:22995898

Alternatives to allogeneic platelet transfusion.

PubMed

Desborough, Michael J R; Smethurst, Peter A; Estcourt, Lise J; Stanworth, Simon J

2016-11-01

Allogeneic platelet transfusions are widely used for the prevention and treatment of bleeding in thrombocytopenia. Recent evidence suggests platelet transfusions have limited efficacy and are associated with uncertain immunomodulatory risks and concerns about viral or bacterial transmission. Alternatives to transfusion are a well-recognised tenet of Patient Blood Management, but there has been less focus on different strategies to reduce bleeding risk by comparison to platelet transfusion. Direct alternatives to platelet transfusion include agents to stimulate endogenous platelet production (thrombopoietin mimetics), optimising platelet adhesion to endothelium by treating anaemia or increasing von Willebrand factor levels (desmopressin), increasing formation of cross-linked fibrinogen (activated recombinant factor VII, fibrinogen concentrate or recombinant factor XIII), decreasing fibrinolysis (tranexamic acid or epsilon aminocaproic acid) or using artificial or modified platelets (cryopreserved platelets, lyophilised platelets, haemostatic particles, liposomes, engineered nanoparticles or infusible platelet membranes). The evidence base to support the use of these alternatives is variable, but an area of active research. Much of the current randomised controlled trial focus is on evaluation of the use of thrombopoietin mimetics and anti-fibrinolytics. It is also recognised that one alternative strategy to platelet transfusion is choosing not to transfuse at all. © 2016 John Wiley & Sons Ltd.

Potentiation by adrenaline of human platelet activation and the inhibition by the alpha-adrenergic antagonist nicergoline of platelet adhesion, secretion and aggregation.

PubMed

Lanza, F; Cazenave, J P; Beretz, A; Sutter-Bay, A; Kretz, J G; Kieny, R

1986-08-01

Adrenaline (1 to 10 microM) can induce the aggregation of human platelets suspended in citrated plasma but does not induce the aggregation of washed human platelets at doses as high as 1 mM, although these platelets respond normally to ADP, PAF-acether, collagen, arachidonic acid, thrombin, the endoperoxide analog U-46619 and the Ca2+ ionophore A23187. Adrenaline (0.5 microM) potentiates the aggregation and secretion induced by all the previous agonists in citrated platelet-rich plasma (cPRP) or in washed platelets. The activation by adrenaline of human platelets is mediated by alpha 2-adrenergic receptors, as demonstrated by inhibition with a series of adrenergic antagonists. The alpha-adrenergic antagonist nicergoline inhibits the activation of human platelets by adrenaline in the following situations: nicergoline inhibits the aggregation and secretion caused by adrenaline in cPRP (IC50 0.22 microM and 0.28 microM respectively); nicergoline inhibits the aggregation and secretion induced by the combination of adrenaline and each aggregating agent listed above in cPRP (IC50 ranging from 0.1 to 2.5 microM) or in washed platelets (IC50 ranging from 0.1 to 0.8 microM); nicergoline inhibits the binding of 3H-yohimbine to washed human platelets (IC50 0.26 microM); the intravenous administration of nicergoline (0.5 mg/kg per day) to patients inhibits significantly the ex vivo response of their platelets to adrenaline in cPRP. High concentrations of nicergoline also inhibit the aggregation and secretion induced by the aggregating agents listed above in cPRP (IC50 range 108 to 670 microM) and in washed platelets (IC50 range 27 to 140 microM) and the adhesion of platelets to collagen-coated surfaces. This latter effect is not mediated through blockade of alpha-adrenoceptors. A possible role of adrenaline in platelet activation in vivo could justify the use of nicergoline (Sermion), an alpha-adrenergic antagonist in combination therapy to prevent arterial thrombosis.

Relationship Between Platelet and Urinary 8‐Iso‐PGF2α Levels in Subjects With Different Degrees of NOX2 Regulation

PubMed Central

Carnevale, Roberto; Iuliano, Luigi; Nocella, Cristina; Bartimoccia, Simona; Trapè, Stefano; Russo, Roberta; Gentile, Maria Cristina; Cangemi, Roberto; Loffredo, Lorenzo; Pignatelli, Pasquale; Violi, Francesco

2013-01-01

Background Urinary 8‐iso‐PGF2α, a marker of oxidative stress, is influenced by the activation of NOX2. It is unclear if platelets 8‐iso‐PGF2α contribute to urinary 8‐iso‐PGF2α. Methods and Results In a cross‐sectional study, platelet, urinary, and serum 8‐iso‐PGF2α were determined in subjects with downregulation (X‐linked chronic granulomatous disease [X‐CGD], n=25) and upregulation (type II diabetic patients [T2D], n=121) of NOX2 and 153 controls matched for sex and age. In diabetic patients (n=18), the above variables were repeated before and after 7 days treatment with 100 mg/day aspirin or 100 mg/day aspirin plus 40 mg/day atorvastatin. In vitro study was performed to see the contribution of blood cells to serum 8‐iso‐PGF2α. Compared with controls, X‐CGD patients had lower platelet, serum, and urinary 8‐iso‐PGF2α values; conversely, diabetic patients had higher values of 8‐iso‐PGF2α compared with controls. Urinary 8‐iso‐PGF2α significantly correlated with both platelet and serum 8‐iso‐PGF2α in the 2 cohorts. A parallel increase of platelet, serum, and urinary 8‐iso‐PGF2α by aspirin and a parallel decrease by aspirin plus atorvastatin were detected in the interventional study. In vitro study demonstrated that platelets contribute to 37% of serum 8‐iso‐PGF2α and that only 13% of it is of extravascular origin. Conclusions The study suggests that NOX2 contributes to the formation of 8‐iso‐PGF2α in both platelets and urine. The direct correlation between platelet and urinary 8‐iso‐PGF2α suggests that, at least partly, urinary 8‐iso‐PGF2α reflects platelet 8‐iso‐PGF2α production. Analysis of serum 8‐iso‐PGF2α may represent a novel tool to investigate the production of 8‐iso‐PGF2α by blood cells including platelets. Clinical Trial Registration URL: ClinicalTrials.gov. Unique Identifier: NCT01250340. PMID:23770972

Key role of integrin α(IIb)β (3) signaling to Syk kinase in tissue factor-induced thrombin generation.

PubMed

van der Meijden, Paola E J; Feijge, Marion A H; Swieringa, Frauke; Gilio, Karen; Nergiz-Unal, Reyhan; Hamulyák, Karly; Heemskerk, Johan W M

2012-10-01

The fibrin(ogen) receptor, integrin α(IIb)β(3), has a well-established role in platelet spreading, aggregation and clot retraction. How α(IIb)β(3) contributes to platelet-dependent coagulation is less well resolved. Here, we demonstrate that the potent suppressing effect of clinically used α(IIb)β(3) blockers on tissue factor-induced thrombin generation is linked to diminished platelet Ca(2+) responses and phosphatidylserine (PS) exposure. The same blockers suppress these responses in platelets stimulated with collagen and thrombin receptor agonists, whereas added fibrinogen potentiates these responses. In platelets spreading on fibrinogen, outside-in α(IIb)β(3) signaling similarly enhances thrombin-induced Ca(2+) rises and PS exposure. These responses are reduced in α(IIb)β(3)-deficient platelets from patients with Glanzmann's thrombasthenia. Furthermore, the contribution of α(IIb)β(3) to tissue factor-induced platelet Ca(2+) rises, PS exposure and thrombin generation in plasma are fully dependent on Syk kinase activity. Tyrosine phosphorylation analysis confirms a key role of Syk activation, which is largely but not exclusively dependent on α(IIb)β(3) activation. It is concluded that the majority of tissue factor-induced procoagulant activity of platelets relies on Syk activation and ensuing Ca(2+) signal generation, and furthermore that a considerable part of Syk activation relies on α(IIb)β(3) signaling. These results hence point to a novel role of Syk in integrin-dependent thrombin generation.

Effects of clopidogrel and aspirin combination versus aspirin alone on platelet aggregation and major receptor expression in patients with heart failure: the Plavix Use for Treatment Of Congestive Heart Failure (PLUTO-CHF) trial.

PubMed

Serebruany, Victor L; Malinin, Alex I; Jerome, Scott D; Lowry, David R; Morgan, Athol W; Sane, David C; Tanguay, Jean-François; Steinhubl, Steven R; O'connor, Christopher M

2003-10-01

Persistent platelet activation may contribute to thrombotic events in patients with congestive heart failure (CHF). Chronic use of mild platelet inhibitors could therefore represent an independent avenue to improve morbidity, mortality, and quality of life in this expanding population. Although clopidogrel is widely used in patients with acute coronary syndromes and ischemic stroke, the ability of this novel ADP-receptor antagonist to inhibit platelet function in patients with CHF is unknown. We assessed antiplatelet properties of clopidogrel with aspirin (C+A) versus aspirin alone (A) in patients with CHF with heightened platelet activity. Patients with left ventricular ejection fraction <40%, or CHF symptoms in the setting of preserved systolic function and New York Heart Association class II-IV were screened. Patients were considered to have platelet activation when 4 of the following 5 parameters were met: ADP-induced platelet aggregation >60%; collagen-induced aggregation >70%; whole blood aggregation >18 ohms; expression of GP IIb/IIIa >220 log MFI; and P-selectin cell positivity >8%. All patients were treated with 325 mg of acetylsalycilic acid (ASA) for at least 1 month. Patients receiving an antithrombotic agent other than ASA were excluded. Patients meeting clinical and laboratory criteria were randomly assigned to C+A (n=25), A (n=25) groups, or represent screen failures (n=38). Platelet studies (conventional and whole blood aggregometry, shear-induced activation, expression of 10 major receptors and formation of platelet-leukocyte microparticles) were performed at baseline and after 30 days of therapy. There were no deaths, hospitalizations, or serious adverse events. There were no changes in platelet parameters in the A group. In contrast, therapy with C+A resulted in a significant inhibition of platelet activity assessed by ADP-induced (P =.00001), and epinephrine-induced (P =.0016) aggregation, closure time (P =.04), expression of PECAM-1 (P =.009), GP Ib (P =.006), GP IIb/IIIa antigen (P =.0001), GP IIb/IIIa activity with PAC-1 (P =.0021), and CD151 (P =.0026) when compared with the A group. Therapy with C+A also resulted in the reduced formation of platelet-leukocyte microparticles (P =.021). Collagen-induced aggregation in plasma and in whole blood, expression of vitronectin receptor, P-selectin, CD63, CD107a, and CD107b did not differ among groups. Treatment with C+A for 1 month provides significantly greater inhibition of platelet activity than ASA alone in patients with CHF. Patients with CHF with heightened platelet activity represent a potential target population in which addition of clopidogrel may decrease mortality rates by reducing the incidence of thrombotic vascular events.

Platelet–Eosinophil Interactions As a Potential Therapeutic Target in Allergic Inflammation and Asthma

PubMed Central

Shah, Sajeel A.; Page, Clive P.; Pitchford, Simon C.

2017-01-01

The importance of platelet activation during hemostasis is well understood. An understanding of these mechanisms has led to the use of several classes of anti-platelet drugs to inhibit aggregation for the prevention of thrombi during cardiovascular disease. It is now also recognized that platelets can function very differently during inflammation, as part of their role in the innate immune response against pathogens. This dichotomy in platelet function occurs through distinct physiological processes and alternative signaling pathways compared to that of hemostasis (leading to platelet aggregation) and is manifested as increased rheological interactions with leukocytes, the ability to undergo chemotaxis, communication with antigen-presenting cells, and direct anti-pathogen responses. Mounting evidence suggests platelets are also critical in the pathogenesis of allergic diseases such as asthma, where they have been associated with antigen presentation, bronchoconstriction, bronchial hyperresponsiveness, airway inflammation, and airway remodeling in both clinical and experimental studies. In particular, platelets have been reported bound to eosinophils in the blood of patients with asthma and the incidence of these events increases after both spontaneous asthma attacks in a biphasic manner, or after allergen challenge in the clinic. Platelet depletion in animal models of allergic airway inflammation causes a profound reduction in eosinophil recruitment to the lung, suggesting that the association of platelets with eosinophils is indeed an important event during eosinophil activation. Furthermore, in cases of severe asthma, and in animal models of allergic airways inflammation, platelet–eosinophil complexes move into the lung through a platelet P-selectin-mediated, eosinophil β1-integrin activation-dependent process, while platelets increase adherence of eosinophils to the vascular endothelium in vitro, demonstrating a clear interaction between these cell types in allergic inflammatory diseases. This review will explore non-thrombotic platelet activation in the context of allergy and the association of platelets with eosinophils, to reveal how these phenomena may lead to the discovery of novel therapeutic targets. PMID:28848732

Origin of platelet-derived growth factor in megakaryocytes in guinea pigs.

PubMed Central

Chernoff, A; Levine, R F; Goodman, D S

1980-01-01

Growth factor activity, as determined by the stimulation of [3H]thymidine incorporation into the DNA of quiescent 3T3 cells in culture, was found in lysates of guinea pig platelets and megakaryocytes. Quantitative dilution studies demonstrated that, of the cells present in the guinea pig bone marrow, only the megakaryocyte possessed quantitatively significant growth factor activity. The amount of activity present in one megakaryocyte was equivalent to that present in 1,000-5,000 platelets, a value approximately comparable to the number of platelets shed from a single megakaryocyte. It is suggested that guinea pig platelet-derived growth factor has its origin in the megakaryocyte. PMID:7358851

Protocatechuic aldehyde inhibits migration and proliferation of vascular smooth muscle cells and intravascular thrombosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moon, Chang Yoon; Endocrinology, Brain Korea 21 Project for Medical Science, Institute of Endocrine Research, and Severance Integrative Research Institute for Cerebral and Cardiovascular Disease, Yonsei University College of Medicine, Seoul; Ku, Cheol Ryong

2012-06-22

Highlights: Black-Right-Pointing-Pointer Protocatechuic aldehyde (PCA) inhibits ROS production in VSMCs. Black-Right-Pointing-Pointer PCA inhibits proliferation and migration in PDGF-induced VSMCs. Black-Right-Pointing-Pointer PCA has anti-platelet effects in ex vivo rat whole blood. Black-Right-Pointing-Pointer We report the potential therapeutic role of PCA in atherosclerosis. -- Abstract: The migration and proliferation of vascular smooth muscle cells (VSMCs) and formation of intravascular thrombosis play crucial roles in the development of atherosclerotic lesions. This study examined the effects of protocatechuic aldehyde (PCA), a compound isolated from the aqueous extract of the root of Salvia miltiorrhiza, an herb used in traditional Chinese medicine to treat a varietymore » of vascular diseases, on the migration and proliferation of VSMCs and platelets due to platelet-derived growth factor (PDGF). DNA 5-bromo-2 Prime -deoxy-uridine (BrdU) incorporation and wound-healing assays indicated that PCA significantly attenuated PDGF-induced proliferation and migration of VSMCs at a pharmacologically relevant concentration (100 {mu}M). On a molecular level, we observed down-regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt and the mitogen-activated protein kinase (MAPK) pathways, both of which regulate key enzymes associated with migration and proliferation. We also found that PCA induced S-phase arrest of the VSMC cell cycle and suppressed cyclin D2 expression. In addition, PCA inhibited PDGF-BB-stimulated reactive oxygen species production in VSMCs, indicating that PCA's antioxidant properties may contribute to its suppression of PDGF-induced migration and proliferation in VSMCs. Finally, PCA exhibited an anti-thrombotic effect related to its inhibition of platelet aggregation, confirmed with an aggregometer. Together, these findings suggest a potential therapeutic role of PCA in the treatment of atherosclerosis and angioplasty-induced vascular restenosis.« less

Mechanics and contraction dynamics of single platelets and implications for clot stiffening

NASA Astrophysics Data System (ADS)

Lam, Wilbur A.; Chaudhuri, Ovijit; Crow, Ailey; Webster, Kevin D.; Li, Tai-De; Kita, Ashley; Huang, James; Fletcher, Daniel A.

2011-01-01

Platelets interact with fibrin polymers to form blood clots at sites of vascular injury. Bulk studies have shown clots to be active materials, with platelet contraction driving the retraction and stiffening of clots. However, neither the dynamics of single-platelet contraction nor the strength and elasticity of individual platelets, both of which are important for understanding clot material properties, have been directly measured. Here we use atomic force microscopy to measure the mechanics and dynamics of single platelets. We find that platelets contract nearly instantaneously when activated by contact with fibrinogen and complete contraction within 15 min. Individual platelets can generate an average maximum contractile force of 29 nN and form adhesions stronger than 70 nN. Our measurements show that when exposed to stiffer microenvironments, platelets generated higher stall forces, which indicates that platelets may be able to contract heterogeneous clots more uniformly. The high elasticity of individual platelets, measured to be 10 kPa after contraction, combined with their high contractile forces, indicates that clots may be stiffened through direct reinforcement by platelets as well as by strain stiffening of fibrin under tension due to platelet contraction. These results show how the mechanosensitivity and mechanics of single cells can be used to dynamically alter the material properties of physiologic systems.

[PLATELET AGGREGATION IN CLINICALLY HEALTHY PERSONS OF THE SECOND COMING OF AGE LIVING IN THE KURSK REGION].

PubMed

Kutafina, N V; Medvedev, I N

2015-01-01

This work is dedicated to the study of the aggregation of platelet activity in healthy persons the second coming of age. The study group included 146 clinically healthy people of the second coming of age, leading a healthy lifestyle and not having metabolic and cardiovascular diseases. In healthy people of 36-45 years noted the lack of reliable antioxidant dynamics of platelets and levels of lipid peroxidation. Platelet aggregation with a range of inductors in people of 36-60 years confirmed the age-dependent increase of aggregation platelet function. After 45 years of age, the activity of platelet aggregative in healthy people increases gradually, leading to an increase in their blood platelet active forms, which inevitably leads to the increase in the number of circulating units of various sizes. This tendency is accompanied by painful with age, increasing negative impact of environmental factors, contributing to the realization of a genetic predisposition to various, primarily cardiovascular, diseases.

In vitro effects of polychlorinated biphenyls on human platelets.

PubMed Central

Raulf, M; König, W

1991-01-01

Incubation of human platelets with polychlorinated biphenyls (PCB) induced and modulated cellular responses to a different degree. 3,3',4,4'-tetrachlorobiphenyl (TCB) was a more potent inducer of platelet aggregation, serotonin release and 12-HETE generation compared to the other PCB [2,2',3,3'-TCB,3,3'-dichlorobiphenyl (DCB),2,2',4,5,5'-pentachlorobiphenyl (PCB)]. 3,3',4,4'-TCB showed synergistic effects, in combination with other PCB, such as an enhanced formation of 12-HETE, when 3,3'-DCB and 2,2',3,3'-TCB were applied simultaneously. The combined incubation of platelets with PCB and sodium fluoride (NaF), an activator of G-proteins, resulted in synergistic 12-HETE generation compared to stimulation with NaF or PCB alone. Furthermore, when platelets were incubated with the PCB the enzymatic steps controlling the metabolism of the platelet-activating factor (PAF) were modulated. A direct relationship between the extent of platelet activation and the chloro-substitution pattern of PCB exists. PMID:1901832

Antimicrobial effect of platelet-rich plasma and platelet-rich fibrin.

PubMed

Badade, Pallavi S; Mahale, Swapna A; Panjwani, Alisha A; Vaidya, Prutha D; Warang, Ayushya D

2016-01-01

Platelet concentrates have been extensively used in a variety of medical fields to promote soft- and hard-tissue regeneration. The significance behind their use lies in the abundance of growth factors (GFs) in platelets α-granules that promote wound healing. Other than releasing a pool of GFs upon activation, platelets also have many features that indicate their role in the anti-infective host defense. The aim of this study is to evaluate the antimicrobial activities of platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) against periodontal disease-associated bacteria. Blood samples were obtained from ten adult male patients. PRP and PRF were procured using centrifugation. The antimicrobial activity of PRP and PRF was evaluated by microbial culturing using bacterial strains of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. P. gingivalis and A. actinomycetemcomitans were inhibited by PRP but not by PRF. PRP is a potentially useful substance in the fight against periodontal pathogens. This might represent a valuable property in adjunct to the enhancement of tissue regeneration.

Patients with metabolic syndrome exhibit higher platelet activity than those with conventional risk factors for vascular disease.

PubMed

Serebruany, Victor L; Malinin, Alex; Ong, Stephen; Atar, Dan

2008-04-01

The metabolic syndrome is a matter of ongoing debate with regard to its existence, classification, clinical meaningfulness, and associated risks for vessel occlusion. Considering that persistent platelet activation is a cornerstone for the development of acute vascular events, and that patients with type 2 diabetes consistently exhibit high platelet activity, these characteristics may be critical for distinguishing and triageing specific features of metabolic syndrome among established risk factors for vascular disease. We assessed the platelet activity by conventional aggregation, expression of major surface receptors by flow cytometry, and quantitatively by rapid bedside analyzers in 20 aspirin-naïve patients with documented metabolic syndrome, and compared these with 20 untreated subjects with multiple cardiovascular risk factors. Closure time by the PFA-100 analyzer was significantly (P = 0.002) shorter in patients with metabolic syndrome indicating platelet inhibition under high shear conditions. Ultegra analyzer readings revealed increased fibrinogen binding (P = 0.0003) what in combination with the increased expression of PAC-1 (P = 0.32) strongly suggest activation of platelet glycoprotein IIb/IIIa receptor. Surface expression of CD107a (P = 0.014), and SPAN-12 (P = 0.003) were also higher in patients with metabolic syndrome. In contrast, platelet aggregation induced by collagen or ADP, CD31, CD41, CD42b, CD51/61, CD62p, CD63, CD154, CD165, so as formation of platelet-monocyte aggregates, PAR-1 thrombin receptor, and thrombospondin did not differ between groups. Patients with metabolic syndrome exhibited a higher degree of platelet activation than subjects with conventional risk factors for vascular disease. Conceptually, applying adequate antiplatelet strategies may reduce the risk of acute thrombotic events in these patients. Further prospective studies exploring this notion are encouraged.

Exercise-induced myocardial ischemia in patients with coronary artery disease: lack of evidence for platelet activation or fibrin formation in peripheral venous blood.

PubMed

Marcella, J J; Nichols, A B; Johnson, L L; Owen, J; Reison, D S; Kaplan, K L; Cannon, P J

1983-05-01

The hypothesis that exercise-induced myocardial ischemia is associated with abnormal platelet activation and fibrin formation or dissolution was tested in patients with coronary artery disease undergoing upright bicycle stress testing. In vivo platelet activation was assessed by radioimmunoassay of platelet factor 4, beta-thrombo-globulin and thromboxane B2. In vivo fibrin formation was assessed by radioimmunoassay of fibrinopeptide A, and fibrinolysis was assessed by radioimmunoassay of thrombin-increasable fibrinopeptide B which reflects plasmin cleavage of fibrin I. Peripheral venous concentrations of these substances were measured in 10 normal subjects and 13 patients with coronary artery disease at rest and during symptom-limited peak exercise. Platelet factor 4, beta-thromboglobulin and thromboxane B2 concentrations were correlated with rest and exercise catecholamine concentrations to determine if exercise-induced elevation of norepinephrine and epinephrine enhances platelet activation. Left ventricular end-diastolic and end-systolic volumes, ejection fraction and segmental wall motion were measured at rest and during peak exercise by first pass radionuclide angiography. All patients with coronary artery disease had documented exercise-induced myocardial ischemia manifested by angina pectoris, ischemic electrocardiographic changes, left ventricular segmental dyssynergy and a reduction in ejection fraction. Rest and peak exercise plasma concentrations were not significantly different for platelet factor 4, beta-thromboglobulin, thromboxane B2, fibrinopeptide A and thrombin-increasable fibrinopeptide B. Peripheral venous concentrations of norepinephrine and epinephrine increased significantly (p less than 0.001) in both groups of patients. The elevated catecholamine levels did not lead to detectable platelet activation. This study demonstrates that enhanced platelet activation, thromboxane release and fibrin formation or dissolution are not detectable in peripheral venous blood of patients with coronary disease during exercise-induced myocardial ischemia.

Critical Role for CD38-mediated Ca2+ Signaling in Thrombin-induced Procoagulant Activity of Mouse Platelets and Hemostasis*

PubMed Central

Mushtaq, Mazhar; Nam, Tae-Sik; Kim, Uh-Hyun

2011-01-01

CD38, a multifunctional enzyme that catalyzes the synthesis of intracellular Ca2+ messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), is known to be expressed on platelets. However, the role of CD38 in platelets remains unclear. Our present results show that treatment of platelets with thrombin results in a rapid and sustained Ca2+ signal, resulting from a coordinated interplay of Ca2+-mobilizing messengers, inositol 1,4,5-trisphosphate, cADPR, and NAADP. By dissecting the signaling pathway using various agents, we delineated that cADPR and NAADP are sequentially produced through CD38 internalization by protein kinase C via myosin heavy chain IIA following phospholipase C activation in thrombin-induced platelets. An inositol 1,4,5-trisphosphate receptor antagonist blocked the thrombin-induced formation of cADPR and NAADP as well as Ca2+ signals. An indispensable response of platelets relying on cytosolic calcium is the surface exposure of phosphatidylserine (PS), which implicates platelet procoagulant activity. Scrutinizing this parameter reveals that CD38+/+ platelets fully express PS on the surface when stimulated with thrombin, whereas this response was decreased on CD38−/− platelets. Similarly, PS exposure and Ca2+ signals were attenuated when platelets were incubated with 8-bromo-cADPR, bafilomycin A1, and a PKC inhibitor. Furthermore, in vivo, CD38-deficient mice exhibited longer bleeding times and unstable formation of thrombus than wild type mice. These results demonstrate that CD38 plays an essential role in thrombin-induced procoagulant activity of platelets and hemostasis via Ca2+ signaling mediated by its products, cADPR and NAADP. PMID:21339289

Roles of platelet STIM1 and Orai1 in glycoprotein VI- and thrombin-dependent procoagulant activity and thrombus formation.

PubMed

Gilio, Karen; van Kruchten, Roger; Braun, Attila; Berna-Erro, Alejandro; Feijge, Marion A H; Stegner, David; van der Meijden, Paola E J; Kuijpers, Marijke J E; Varga-Szabo, David; Heemskerk, Johan W M; Nieswandt, Bernhard

2010-07-30

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca(2+) entry (SOCE) with Orai1 as principal Ca(2+) entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca(2+) entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1(-/-) and Orai1(-/-) platelets had greatly impaired glycoprotein (GP) VI-dependent Ca(2+) signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2(-/-) platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca(2+) signals of Stim1(-/-) and Orai1(-/-) platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1(-/-) and Orai1(-/-) platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca(2+) entry, inhibited Ca(2+) and procoagulant responses even in Stim1(-/-) and Orai1(-/-) platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca(2+) entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca(2+) entry and PS exposure, only one relying on STIM1-Orai1 interaction.