The heterotrimeric G protein Gβ1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein-coupled receptor signaling in platelets.

PubMed

Pradhan, Subhashree; Khatlani, Tanvir; Nairn, Angus C; Vijayan, K Vinod

2017-08-11

Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gβγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gβ 1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gβ 1 revealed that Gβ 1 interacts with the PP1c α, β, and γ1 isoforms. Purified PP1c bound to recombinant Gβ 1 -GST protein, and PP1c co-immunoprecipitated with Gβ 1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gβ 1 complex, which correlated with an association of PP1c with phospholipase C β3 (PLCβ3), along with a concomitant dephosphorylation of the inhibitory Ser 1105 residue in PLCβ3. siRNA-mediated depletion of GNB1 (encoding Gβ 1 ) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα -/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gβγ. Finally, disruption of PP1c-Gβ 1 complexes with myristoylated Gβ 1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gβ 1 protein enlists PP1c to modulate GPCR signaling in platelets.

Antiplatelet Aggregation Activity of Walnut Hull Extract via Suppression of Reactive Oxygen Species Generation and Caspase Activation.

PubMed

Meshkini, Azadeh; Tahmasbi, Masoumeh

2017-06-01

Walnut hull (wal hull) is an agricultural by-product that is widely used in traditional medicine for alleviating pain and treating skin diseases, however, recently it has gained much attention in modern pharmacology due to its antioxidant properties. The current study was aimed to determine the total phenolic, flavonoid, and tannin content of Persian wal hull extract and evaluate its biological effects on platelet function. Experimental data showed that acetone extract of wal hulls has a high content of polyphenolic compounds and antioxidant properties. The analytical study of crude extract by gas chromatography-mass spectrometry demonstrated different types of high- and low-molecular-weight compounds that are basically and biologically important. Moreover, an in vitro study revealed that wal hull extract at a concentration of 50 μg/mL inhibited thrombin-induced platelet aggregation and protein secretion by 50%, without any cytotoxic effects on platelets. The examined extract suppressed reactive oxygen species generation and also caspase activation in thrombin-stimulated platelets. Identically, N-acetylcysteine inhibited the increase of reactive oxygen species level induced by thrombin in platelets, and supported a link between cellular redox status and caspase activation in activated platelets. Presumably, the antiplatelet activity of wal hull extract is related to its polyphenolic compounds and their antioxidant properties. Therefore, wal hulls can be considered as a candidate for thrombotic disorders. Copyright © 2017. Published by Elsevier B.V.

Platelet-Rich Plasma with Basic Fibroblast Growth Factor for Treatment of Wrinkles and Depressed Areas of the Skin.

PubMed

Kamakura, Tatsuro; Kataoka, Jiro; Maeda, Kazuhiko; Teramachi, Hideaki; Mihara, Hisayuki; Miyata, Kazuhiro; Ooi, Kouichi; Sasaki, Naomi; Kobayashi, Miyuki; Ito, Kouhei

2015-11-01

There are several treatments for wrinkles and depressed areas of the face, hands, and body. Hyaluronic acid is effective, but only for 6 months to 1 year. Autologous fat grafting may cause damage during tissue harvest. In this study, patients were injected with platelet-rich plasma plus basic fibroblast growth factor (bFGF). Platelet-rich plasma was prepared by collecting blood and extracting platelets using double centrifugation. Basic fibroblast growth factor diluted with normal saline was added to platelet-rich plasma. There were 2005 patients who received platelet-rich plasma plus bFGF therapy. Of the 2005 patients treated, 1889 were female and 116 were male patients; patients had a mean age of 48.2 years. Treated areas inlcuded 1461 nasolabial folds, 437 marionette lines, 1413 nasojugal grooves, 148 supraorbital grooves, 253 midcheek grooves, 304 foreheads, 49 temples, and 282 glabellae. Results on the Global Aesthetic Improvement Scale indicated that the level of patient satisfaction was 97.3 percent and the level of investigator satisfaction was 98.4 percent. The period for the therapy's effectiveness to become apparent was an average of 65.4 days. Platelet-rich plasma plus bFGF therapy resulted in an improved grade on the Wrinkle Severity Rating Scale. Improvement was 0.55 for a Wrinkle Severity Rating Scale grade of 2, 1.13 for a Wrinkle Severity Rating Scale grade of 3, 1.82 for a Wrinkle Severity Rating Scale grade of 4, and 2.23 for a Wrinkle Severity Rating Scale grade of 5. Platelet-rich plasma plus bFGF is effective in treating wrinkles and depressed areas of the skin of the face and body. The study revealed that platelet-rich plasma plus bFGF is an innovative therapy that causes minimal complications. Therapeutic, IV.

The nitrative and oxidative stress in blood platelets isolated from breast cancer patients: the protectory action of aronia melanocarpa extract.

PubMed

Kedzierska, Magdalena; Olas, Beata; Wachowicz, Barbara; Stochmal, Anna; Oleszek, Wieslaw; Jeziorski, Arkadiusz; Piekarski, Janusz

2010-01-01

Since mechanisms involved in the relationship between oxidative stress and breast cancer are still unclear, the aim of our present study was to evaluate oxidative/nitrative modifications of blood platelet proteins by measuring the level of biomarkers of oxidative/nitrative stress such as carbonyl groups, thiol groups and 3-nitrotyrosine in proteins in patients with benign breast diseases and in patients with invasive breast cancer, and compare with the control group. Levels of carbonyl groups and 3-nitrotyrosine residues in platelet proteins were measured by ELISA and a competition ELISA, respectively. The method with 5,5′-dithio-bis(2-nitro-benzoic acid) has been used to analyse free thiol groups in platelet proteins. Patients were hospitalized in the Department of Oncological Surgery, Medical University of Lodz, Poland. Exogenous antioxidants reduce oxidative stress, therefore we also investigated in a model system in vitro the effects of a polyphenol rich extract of Aronia melanocarpa (Rosaceae, final concentration of 50 µg/ml, 5 min, 37°C) on modified blood platelet proteins as well from patients with breast cancer and from the healthy group. We demonstrated in platelet proteins from patients with invasive breast cancer a higher level of carbonyl groups than in the control healthy group (p < 0.02). The level of 3-nitrotyrosine in platelet proteins from patients with invasive breast cancer was also significantly higher than in the healthy subject group (p < 0.001). In contrast, the amount of thiol groups in platelet proteins from patients was significantly lower (about < 50%) than in control blood platelets. In a model system in vitro we also observed that the extract from berries of A. melanocarpa (50 µg/ml, 5 min, 37°C) due to antioxidant action, significantly reduced the oxidative/nitrative stress (measured by thiol groups and 3-nitrotyrosine) in platelets, not only from the healthy group, but also from patients with benign breast diseases and in patients with invasive breast cancer.

Platelet activation and aggregation by the opportunistic pathogen Cutibacterium (Propionibacterium) acnes

PubMed Central

Petersson, Frida; Kilsgård, Ola; Shannon, Oonagh

2018-01-01

Cutibacterium (Propionibacterium) acnes, considered a part of the skin microbiota, is one of the most commonly isolated anaerobic bacteria from medical implants in contact with plasma. However, the precise interaction of C. acnes with blood cells and plasma proteins has not been fully elucidated. Herein, we have investigated the molecular interaction of C. acnes with platelets and plasma proteins. We report that the ability of C. acnes to aggregate platelets is dependent on phylotype, with a significantly lower ability amongst type IB isolates, and the interaction of specific donor-dependent plasma proteins (or concentrations thereof) with C. acnes. Pretreatment of C. acnes with plasma reduces the lag time before aggregation demonstrating that pre-deposition of plasma proteins on C. acnes is an important step in platelet aggregation. Using mass spectrometry we identified several plasma proteins deposited on C. acnes, including IgG, fibrinogen and complement factors. Inhibition of IgG, fibrinogen or complement decreased C. acnes-mediated platelet aggregation, demonstrating the importance of these plasma proteins for aggregation. The interaction of C. acnes and platelets was visualized using fluorescence microscopy, verifying the presence of IgG and fibrinogen as components of the aggregates, and co-localization of C. acnes and platelets in the aggregates. Here, we have demonstrated the ability of C. acnes to activate and aggregate platelets in a bacterium and donor-specific fashion, as well as added mechanistic insights into this interaction. PMID:29385206

Platelet activation and aggregation by the opportunistic pathogen Cutibacterium (Propionibacterium) acnes.

PubMed

Petersson, Frida; Kilsgård, Ola; Shannon, Oonagh; Lood, Rolf

2018-01-01

Cutibacterium (Propionibacterium) acnes, considered a part of the skin microbiota, is one of the most commonly isolated anaerobic bacteria from medical implants in contact with plasma. However, the precise interaction of C. acnes with blood cells and plasma proteins has not been fully elucidated. Herein, we have investigated the molecular interaction of C. acnes with platelets and plasma proteins. We report that the ability of C. acnes to aggregate platelets is dependent on phylotype, with a significantly lower ability amongst type IB isolates, and the interaction of specific donor-dependent plasma proteins (or concentrations thereof) with C. acnes. Pretreatment of C. acnes with plasma reduces the lag time before aggregation demonstrating that pre-deposition of plasma proteins on C. acnes is an important step in platelet aggregation. Using mass spectrometry we identified several plasma proteins deposited on C. acnes, including IgG, fibrinogen and complement factors. Inhibition of IgG, fibrinogen or complement decreased C. acnes-mediated platelet aggregation, demonstrating the importance of these plasma proteins for aggregation. The interaction of C. acnes and platelets was visualized using fluorescence microscopy, verifying the presence of IgG and fibrinogen as components of the aggregates, and co-localization of C. acnes and platelets in the aggregates. Here, we have demonstrated the ability of C. acnes to activate and aggregate platelets in a bacterium and donor-specific fashion, as well as added mechanistic insights into this interaction.

The use of autologous blood-derived growth factors in bone regeneration

PubMed Central

Civinini, Roberto; Macera, Armando; Nistri, Lorenzo; Redl, Birgit; Innocenti, Massimo

2011-01-01

Platelet-rich plasma (PRP) is defined as a portion of the plasma fraction of autologous blood having platelet concentrations above baseline. When activated the platelets release growth factors that play an essential role in bone healing such as Platelet-derived Growth Factor, Transforming Growth Factor-β, Vascular Endothelial Growth Factor and others. Multiple basic science and in vivo animal studies agree that PRP has a role in the stimulation of the healing cascade in ligament, tendon, muscle cartilage and in bone regeneration in the last years PRP had a widespread diffusion in the treatment of soft tissue and bone healing. The purpose of this review is to describe the biological properties of platelets and its factors, the methods used for producing PRP, to provide a background on the underlying basic science and an overview of evidence based medicine on clinical application of PRP in bone healing. PMID:22461800

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins.

PubMed

O'Connor, Marie N; Salles, Isabelle I; Cvejic, Ana; Watkins, Nicholas A; Walker, Adam; Garner, Stephen F; Jones, Chris I; Macaulay, Iain C; Steward, Michael; Zwaginga, Jaap-Jan; Bray, Sarah L; Dudbridge, Frank; de Bono, Bernard; Goodall, Alison H; Deckmyn, Hans; Stemple, Derek L; Ouwehand, Willem H

2009-05-07

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins

PubMed Central

O'Connor, Marie N.; Salles, Isabelle I.; Cvejic, Ana; Watkins, Nicholas A.; Walker, Adam; Garner, Stephen F.; Jones, Chris I.; Macaulay, Iain C.; Steward, Michael; Zwaginga, Jaap-Jan; Bray, Sarah L.; Dudbridge, Frank; de Bono, Bernard; Goodall, Alison H.; Stemple, Derek L.; Ouwehand, Willem H.

2009-01-01

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis. PMID:19109564

Early storage lesions in apheresis platelets are induced by the activation of the integrin αIIbβ₃ and focal adhesion signaling pathways.

PubMed

Thiele, Thomas; Iuga, Cristina; Janetzky, Susann; Schwertz, Hansjorg; Gesell Salazar, Manuela; Fürll, Birgit; Völker, Uwe; Greinacher, Andreas; Steil, Leif

2012-12-05

Production and storage of platelet concentrates (PC) induce protein changes in platelets leading to impaired platelet function. This study aimed to identify signaling pathways involved in the development of early platelet storage lesions in apheresis-PCs stored in plasma or additive solution (PAS). Apheresis-PCs from four donors were stored in plasma or in PAS at 22°C (n=4 each). Platelets were analyzed at day 0 (production day) and after 1, 6 and 9 days of storage. Platelet response to agonists (TRAP, collagen, ADP) and to hypotonic shock decreased, CD62P expression increased in both storage media over time. Using DIGE 1550 protein spots were monitored and compared to baseline values at day 0. Platelets in plasma displayed changes in 352 spots (166/day 1, 263/day 6 and 201/day 9); in PAS 325 spots changed (202/day 1, 221/day 6, 200/day 9). LC-ESI-MS/MS analysis of 405 platelet proteins revealed 32 proteins changed during storage in plasma (9/day 1, 15/day 6 and 26/day 9) and 28 in PAS (5/day 1, 20/day 6, 26/day 9). Ingenuity pathway analysis found integrin-αII(b)β(3) and focal adhesion signaling pathways involved in early alterations, being confirmed by Western blotting. Corresponding mRNAs in platelets were identified by next generation sequencing for 84 changed proteins. Integrin-αII(b)β(3) and focal adhesion signaling cause irreversible early storage lesions in apheresis platelets. This article is part of a Special Issue entitled: Integrated omics. Copyright © 2012 Elsevier B.V. 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.

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.

Delineating the roles of the GPIIb/IIIa and GP-Ib-IX-V platelet receptors in mediating platelet adhesion to adsorbed fibrinogen and albumin.

PubMed

Sivaraman, Balakrishnan; Latour, Robert A

2011-08-01

Platelet adhesion to adsorbed plasma proteins, such as fibrinogen (Fg), has been conventionally thought to be mediated by the GPIIb/IIIa receptor binding to Arg-Gly-Asp (RGD)-like motifs in the adsorbed protein. In previous studies, we showed that platelet adhesion response to adsorbed Fg and Alb was strongly influenced by the degree of adsorption-induced protein unfolding and that platelet adhesion was only partially blocked by soluble RGD, with RGD-blocked platelets adhering without activation. Based on these results, we hypothesized that in addition to the RGD-specific GPIIb/IIIa receptor, which mediates both adhesion and activation, a non-RGD-specific receptor set likely also plays a role in platelet adhesion (but not activation) to both Fg and albumin (Alb). To identify and elucidate the role of these receptors, in addition to GPIIb/IIIa, we also examined the GPIb-IX-V receptor complex, which has been shown to mediate platelet adhesion (but not activation) in studies by other groups. The platelet suspension was pretreated with either a GPIIb/IIIa-antagonist drug Aggrastat(®) or monoclonal antibodies 6B4 or 24G10 against GPIb-IX-V prior to adhesion on Fg- and Alb-coated OH- and CH(3)-functionalized alkanethiol self-assembled monolayer surfaces. The results revealed that GPIIb/IIIa is the primary receptor set involved in platelet adhesion to adsorbed Fg and Alb irrespective of their degree of adsorption-induced unfolding, while the GPIb-IX-V receptor complex plays an insignificant role. Overall, these studies provide novel insights into the molecular-level mechanisms mediating platelet interactions with adsorbed plasma proteins, thereby assisting the biomaterials field develop potent strategies for inhibiting platelet-protein interactions in the design of more hemocompatible cardiovascular biomaterials and effective anti-thrombotic therapies. Copyright © 2011 Elsevier Ltd. All rights reserved.

mTOR-dependent synthesis of Bcl-3 controls the retraction of fibrin clots by activated human platelets

PubMed Central

Weyrich, Andrew S.; Denis, Melvin M.; Schwertz, Hansjorg; Tolley, Neal D.; Foulks, Jason; Spencer, Eliott; Kraiss, Larry W.; Albertine, Kurt H.; McIntyre, Thomas M.

2007-01-01

New activities of human platelets continue to emerge. One unexpected response is new synthesis of proteins from previously transcribed RNAs in response to activating signals. We previously reported that activated human platelets synthesize B-cell lymphoma-3 (Bcl-3) under translational control by mammalian target of rapamycin (mTOR). Characterization of the ontogeny and distribution of the mTOR signaling pathway in CD34+ stem cell–derived megakaryocytes now demonstrates that they transfer this regulatory system to developing proplatelets. We also found that Bcl-3 is required for condensation of fibrin by activated platelets, demonstrating functional significance for mTOR-regulated synthesis of the protein. Inhibition of mTOR by rapamycin blocks clot retraction by human platelets. Platelets from wild-type mice synthesize Bcl-3 in response to activation, as do human platelets, and platelets from mice with targeted deletion of Bcl-3 have defective retraction of fibrin in platelet-fibrin clots mimicking treatment of human platelets with rapamycin. In contrast, overexpression of Bcl-3 in a surrogate cell line enhanced clot retraction. These studies identify new features of post-transcriptional gene regulation and signal-dependant protein synthesis in activated platelets that may contribute to thrombus and wound remodeling and suggest that posttranscriptional pathways are targets for molecular intervention in thrombotic disorders. PMID:17110454

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.

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.

Increased expression of urokinase plasminogen activator in Quebec platelet disorder is linked to megakaryocyte differentiation

PubMed Central

Veljkovic, D. Kika; Rivard, Georges E.; Diamandis, Maria; Blavignac, Jessica; Cramer-Bordé, Elisabeth M.

2009-01-01

Quebec platelet disorder (QPD) is an inherited bleeding disorder associated with increased urokinase plasminogen activator (uPA) in platelets but not in plasma, intraplatelet plasmin generation, and α-granule protein degradation. These abnormalities led us to investigate uPA expression by QPD CD34+ progenitors, cultured megakaryocytes, and platelets, and whether uPA was stored in QPD α-granules. Although QPD CD34+ progenitors expressed normal amounts of uPA, their differentiation into megakaryocytes abnormally increased expression of the uPA gene but not the flanking genes for vinculin or calcium/calmodulin-dependent protein kinase IIγ on chromosome 10. The increased uPA production by cultured QPD megakaryocytes mirrored their production of α-granule proteins, which was normal. uPA was localized to QPD α-granules and it showed extensive colocalization with α-granule proteins in both cultured QPD megakaryocytes and platelets, and with plasminogen in QPD platelets. In QPD megakaryocytes, cultured without or with plasma as a source of plasminogen, α-granule proteins were stored undegraded and this was associated with much less uPA-plasminogen colocalization than in QPD platelets. Our studies indicate that the overexpression of uPA in QPD emerges with megakaryocyte differentiation, without altering the expression of flanking genes, and that uPA is costored with α-granule proteins prior to their proteolysis in QPD. PMID:19029443

Increased expression of urokinase plasminogen activator in Quebec platelet disorder is linked to megakaryocyte differentiation.

PubMed

Veljkovic, D Kika; Rivard, Georges E; Diamandis, Maria; Blavignac, Jessica; Cramer-Bordé, Elisabeth M; Hayward, Catherine P M

2009-02-12

Quebec platelet disorder (QPD) is an inherited bleeding disorder associated with increased urokinase plasminogen activator (uPA) in platelets but not in plasma, intraplatelet plasmin generation, and alpha-granule protein degradation. These abnormalities led us to investigate uPA expression by QPD CD34(+) progenitors, cultured megakaryocytes, and platelets, and whether uPA was stored in QPD alpha-granules. Although QPD CD34(+) progenitors expressed normal amounts of uPA, their differentiation into megakaryocytes abnormally increased expression of the uPA gene but not the flanking genes for vinculin or calcium/calmodulin-dependent protein kinase IIgamma on chromosome 10. The increased uPA production by cultured QPD megakaryocytes mirrored their production of alpha-granule proteins, which was normal. uPA was localized to QPD alpha-granules and it showed extensive colocalization with alpha-granule proteins in both cultured QPD megakaryocytes and platelets, and with plasminogen in QPD platelets. In QPD megakaryocytes, cultured without or with plasma as a source of plasminogen, alpha-granule proteins were stored undegraded and this was associated with much less uPA-plasminogen colocalization than in QPD platelets. Our studies indicate that the overexpression of uPA in QPD emerges with megakaryocyte differentiation, without altering the expression of flanking genes, and that uPA is costored with alpha-granule proteins prior to their proteolysis in QPD.

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

Studies on antioxidant properties of polyphenol-rich extract from berries of Aronia melanocarpa in blood platelets.

PubMed

Olas, B; Wachowicz, B; Nowak, P; Kedzierska, M; Tomczak, A; Stochmal, A; Oleszek, W; Jeziorski, A; Piekarski, J

2008-12-01

The antioxidant properties of extract from berries of Aronia melanocarpa (chokeberry) containing: anthocyanidines, phenolic acids and quercetine glycosides on oxidative/nitrative stress induced by peroxynitrite (ONOO(-), a powerful physiological oxidant, nitrating species and inflammatory mediator) in human blood platelets were studied in vitro. The extract from A. melanocarpa (5 - 50 microg/mL) significantly inhibited platelet protein carbonylation (measured by ELISA method) and thiol oxidation estimated with 5,5'-dithio-bis(2-nitro-benzoic acid) (DTNB) induced by peroxynitrite (0.1 mM) (IC(50)--35 microg/mL for protein carbonylation, and IC(50)--33 microg/mL for protein thiol oxidation). The tested extract only slightly reduced platelet protein nitration (measured by C- ELISA method). The extract also caused a distinct reduction of platelet lipid peroxidation induced by peroxynitrite. Moreover, in our preliminary experiments we observed that the extract (50 microg/mL) reduced oxidative/nitrative stress in blood platelets from patients with breast cancer. The obtained results indicate that in vitro the extract from A. melanocarpa has the protective effects against peroxynitrite-induced oxidative/nitrative damage to the human platelet proteins and lipids. The extract from A. melanocarpa seems to be also useful as an antioxidant in patients with breast cancer.

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.

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.

Application of 2-dimensional difference gel electrophoresis (2D-DIGE) to the study of thrombin-activated human platelet secretome.

PubMed

Della Corte, Anna; Maugeri, Norma; Pampuch, Agnieszka; Cerletti, Chiara; de Gaetano, Giovanni; Rotilio, Domenico

2008-02-01

Thrombin is an agonist inducing platelet activation. We combined two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MALDI-TOF MS) to analyse differentially expressed proteins secreted from thrombin-stimulated platelets. Human washed platelets, from healthy volunteers, were stimulated with thrombin 0.5 U/ml at 37 degrees C without stirring and the secreted proteins were resolved by 2D-DIGE. By image analysis, 1094 spots were detected in the 2D gel. The spots whose mean intensity showed at least a five-fold change intensity increase or decrease in the thrombin-activated platelet gel in comparison with unstimulated control were digested by trypsin and subjected to MALDI-TOF MS analysis. Peptides from mass spectra of in-gel digest samples were matched against available databases, using the Mascot search engine (Matrix Science) for peptide mass fingerprint. In the activated platelet secretome, transferrin, glutathione-transferase, WD repeat protein, ER-60, thrombospondin-1 precursor and thrombospondin were the most abundant. Also lamin A, a nuclear protein, not previously identified in platelets, appeared to be released. The novel strategy to combine 2D-DIGE with MALDI-TOF MS is a useful approach for a quantitative analysis of the effect of thrombin on the secretome profile of human platelets.

Essential role of protein kinase C delta in platelet signaling, alpha IIb beta 3 activation, and thromboxane A2 release.

PubMed

Yacoub, Daniel; Théorêt, Jean-François; Villeneuve, Louis; Abou-Saleh, Haissam; Mourad, Walid; Allen, Bruce G; Merhi, Yahye

2006-10-06

The protein kinase C (PKC) family is an essential signaling mediator in platelet activation and aggregation. However, the relative importance of the major platelet PKC isoforms and their downstream effectors in platelet signaling and function remain unclear. Using isolated human platelets, we report that PKCdelta, but not PKCalpha or PKCbeta, is required for collagen-induced phospholipase C-dependent signaling, activation of alpha(IIb)beta(3), and platelet aggregation. Analysis of PKCdelta phosphorylation and translocation to the membrane following activation by both collagen and thrombin indicates that it is positively regulated by alpha(IIb)beta(3) outside-in signaling. Moreover, PKCdelta triggers activation of the mitogen-activated protein kinase-kinase (MEK)/extracellular-signal regulated kinase (ERK) and the p38 MAPK signaling. This leads to the subsequent release of thromboxane A(2), which is essential for collagen-induced but not thrombin-induced platelet activation and aggregation. This study adds new insight to the role of PKCs in platelet function, where PKCdelta signaling, via the MEK/ERK and p38 MAPK pathways, is required for the secretion of thromboxane A(2).

Platelet geometry sensing spatially regulates α-granule secretion to enable matrix self-deposition

PubMed Central

Sakurai, Yumiko; Fitch-Tewfik, Jennifer L.; Qiu, Yongzhi; Ahn, Byungwook; Myers, David R.; Tran, Reginald; Fay, Meredith E.; Ding, Lingmei; Spearman, Paul W.; Michelson, Alan D.; Flaumenhaft, Robert

2015-01-01

Although the biology of platelet adhesion on subendothelial matrix after vascular injury is well characterized, how the matrix biophysical properties affect platelet physiology is unknown. Here we demonstrate that geometric orientation of the matrix itself regulates platelet α-granule secretion, a key component of platelet activation. Using protein microcontact printing, we show that platelets spread beyond the geometric constraints of fibrinogen or collagen micropatterns with <5-µm features. Interestingly, α-granule exocytosis and deposition of the α-granule contents such as fibrinogen and fibronectin were primarily observed in those areas of platelet extension beyond the matrix protein micropatterns. This enables platelets to “self-deposit” additional matrix, provide more cellular membrane to extend spreading, and reinforce platelet-platelet connections. Mechanistically, this phenomenon is mediated by actin polymerization, Rac1 activation, and αIIbβ3 integrin redistribution and activation, and is attenuated in gray platelet syndrome platelets, which lack α-granules, and Wiskott-Aldrich syndrome platelets, which have cytoskeletal defects. Overall, these studies demonstrate how platelets transduce geometric cues of the underlying matrix geometry into intracellular signals to extend spreading, which endows platelets spatial flexibility when spreading onto small sites of exposed subendothelium. PMID:25964667

Tetraspanin Tspan9 regulates platelet collagen receptor GPVI lateral diffusion and activation

PubMed Central

Haining, Elizabeth J.; Matthews, Alexandra L.; Noy, Peter J.; Romanska, Hanna M.; Harris, Helen J.; Pike, Jeremy; Morowski, Martina; Gavin, Rebecca L.; Yang, Jing; Milhiet, Pierre-Emmanuel; Berditchevski, Fedor; Nieswandt, Bernhard; Poulter, Natalie S.; Watson, Steve P.; Tomlinson, Michael G.

2017-01-01

Abstract The tetraspanins are a superfamily of four-transmembrane proteins, which regulate the trafficking, lateral diffusion and clustering of the transmembrane proteins with which they interact. We have previously shown that tetraspanin Tspan9 is expressed on platelets. Here we have characterised gene-trap mice lacking Tspan9. The mice were viable with normal platelet numbers and size. Tspan9-deficient platelets were specifically defective in aggregation and secretion induced by the platelet collagen receptor GPVI, despite normal surface GPVI expression levels. A GPVI activation defect was suggested by partially impaired GPVI-induced protein tyrosine phosphorylation. In mechanistic experiments, Tspan9 and GPVI co-immunoprecipitated and co-localised, but super-resolution imaging revealed no defects in collagen-induced GPVI clustering on Tspan9-deficient platelets. However, single particle tracking using total internal reflection fluorescence microscopy showed that GPVI lateral diffusion was reduced by approximately 50% in the absence of Tspan9. Therefore, Tspan9 plays a fine-tuning role in platelet activation by regulating GPVI membrane dynamics. PMID:28032533

Blood platelet adhesion to protein studied by on-line acoustic wave sensor.

PubMed

Cavic, B A; Freedman, J; Morel, Z; Mody, M; Rand, M L; Stone, D C; Thompson, M

2001-03-01

The attachment of blood platelets to the surface of bare and protein-coated thickness-shear mode acoustic wave devices operating in a flow-through configuration has been studied. Platelets in washed from bind to the gold electrodes of such sensors, but the resulting frequency shifts are far less than predicted by the conventional mass-based model of device operation. Adherence to albumin and various types of collagen can be produced by on-line introduction of protein or by a pre-coating strategy. Differences in attachment of platelets to collagen types I and IV and the Horm variety can be detected. Platelets attached to collagen yield an interesting delayed, but reversible signal on exposure to a flowing medium of low pH. Scanning electron microscopy of sensor surfaces at various time points in this experiment reveals that originally intact platelets are eventually destroyed by the high acidity of the medium. The reversible frequency is attributed to the presence of removable platelet granular components at the sensor-liquid interface.

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.

Lactodifucotetraose, a human milk oligosaccharide, attenuates platelet function and inflammatory cytokine release.

PubMed

Newburg, David S; Tanritanir, Ayse C; Chakrabarti, Subrata

2016-07-01

Human milk strongly quenches inflammatory processes in vitro, and breastfed infants have lower incidence of inflammatory diseases than those fed artificially. Platelets from neonates, in contrast to those from adults, are less responsive to platelet agonists such as collagen, thrombin, ADP, and epinephrine. Breastfed infants absorb oligosaccharides intact from the human milk in their gut to the circulation. This study was to determine whether these oligosaccharides can attenuate platelet function and platelet secretion of pro-inflammatory proteins, and to identify the active component. The natural mixture of oligosaccharides from human milk and pure individual human milk oligosaccharides were tested for their ability to modulate responses of platelets isolated from human blood following exposure to thrombin, ADP, and collagen. Human milk and the natural mixture of human milk oligosaccharides inhibited platelet release of inflammatory proteins. Of the purified human milk oligosaccharides tested, only lactodifucotetraose (LDFT) significantly inhibited thrombin induced release of the pro-inflammatory proteins RANTES and sCD40L. LDFT also inhibited platelet adhesion to a collagen-coated surface, as well as platelet aggregation induced by ADP or collagen. These data indicate that LDFT may help modulate hemostasis by suppressing platelet-induced inflammatory processes in breastfed infants. This activity suggests further study of LDFT for its potential as a therapeutic agent in infants and adults.

Mechanism of inhibition of cyclo-oxygenase in human blood platelets by carbamate insecticides.

PubMed Central

Krug, H F; Hamm, U; Berndt, J

1988-01-01

Carbamates are a widely used class of insecticides and herbicides. They were tested for their ability to affect human blood platelet aggregation and arachidonic acid metabolism in platelets. (1) The herbicides of the carbamate type have no, or only little, influence up to a concentration of 100 microM; the carbamate insecticides, however, inhibit both aggregation and arachidonic acid metabolism in a dose- and time-dependent manner. (2) Carbaryl, the most effective compound, inhibits platelet aggregation and cyclo-oxygenase activity completely at 10 microM. The liberation of arachidonic acid from phospholipids and the lipoxygenase pathway are not affected, whereas the products of the cyclo-oxygenase pathway are drastically decreased. (3) By using [14C]carbaryl labelled in the carbamyl or in the ring moiety, it could be proved that the carbamyl residue binds covalently to platelet proteins. In contrast with acetylsalicylic acid, which acetylates only one protein, carbaryl carbamylates a multitude of platelet proteins. (4) One of the carbamylated proteins was found to be the platelet cyclo-oxygenase, indicating that carbaryl resembles in this respect acetylsalicylic acid, which is known to inhibit this enzyme specifically by acetylation. Images Fig. 4. PMID:3128272

Incorporation of a circulating protein into megakaryocyte and platelet granules

NASA Technical Reports Server (NTRS)

Handagama, P. J.; George, J. N.; Shuman, M. A.; McEver, R. P.; Bainton, D. F.

1987-01-01

To determine whether or not proteins circulating in plasma can be incorporated into megakaryocytes and platelets, horseradish peroxidase (HRP) was injected intravenously into guinea pigs and these cells were examined for its uptake by electron microscopy and cytochemistry. Enriched samples of megakaryocytes enabled ultrastructural analysis of large numbers of these rare cells. In megakaryocytes, 50% of alpha granules contained HRP between 75 min and 7 hr after injection. At 24 hr, 25% of the megakaryocyte granules were peroxidase-positive, less were positive by 48 hr, and there were none at 4 days. Thus, the findings demonstrate that a circulating protein can be endocytosed by megakaryocytes and rapidly packaged into alpha granules. Platelet granules also contain HRP by 7 hr after injection, and they can secrete it in response to thrombin. Unfortunately, our present studies do not allow us to distinguish between direct endocytosis by the platelet and/or shedding of new platelets from recently labeled megakaryocytes. It is concluded that while some alpha granule proteins are synthesized by megakaryocytes, others may be acquired from plasma by endocytosis. In addition to providing evidence that some of the proteins of alpha granules may be of exogenous origin, this study has allowed the definition of a pathway whereby plasma proteins may be temporarily sequestered in megakaryocytes before reentering the circulation in platelets.

Discovery and identification of potential biomarkers of pediatric Acute Lymphoblastic Leukemia

PubMed Central

Shi, Linan; Zhang, Jun; Wu, Peng; Feng, Kai; Li, Jing; Xie, Zhensheng; Xue, Peng; Cai, Tanxi; Cui, Ziyou; Chen, Xiulan; Hou, Junjie; Zhang, Jianzhong; Yang, Fuquan

2009-01-01

Background Acute lymphoblastic leukemia (ALL) is a common form of cancer in children. Currently, bone marrow biopsy is used for diagnosis. Noninvasive biomarkers for the early diagnosis of pediatric ALL are urgently needed. The aim of this study was to discover potential protein biomarkers for pediatric ALL. Methods Ninety-four pediatric ALL patients and 84 controls were randomly divided into a "training" set (45 ALL patients, 34 healthy controls) and a test set (49 ALL patients, 30 healthy controls and 30 pediatric acute myeloid leukemia (AML) patients). Serum proteomic profiles were measured using surface-enhanced laser desorption/ionization-time-of-flight mass spectroscopy (SELDI-TOF-MS). A classification model was established by Biomarker Pattern Software (BPS). Candidate protein biomarkers were purified by HPLC, identified by LC-MS/MS and validated using ProteinChip immunoassays. Results A total of 7 protein peaks (9290 m/z, 7769 m/z, 15110 m/z, 7564 m/z, 4469 m/z, 8937 m/z, 8137 m/z) were found with differential expression levels in the sera of pediatric ALL patients and controls using SELDI-TOF-MS and then analyzed by BPS to construct a classification model in the "training" set. The sensitivity and specificity of the model were found to be 91.8%, and 90.0%, respectively, in the test set. Two candidate protein peaks (7769 and 9290 m/z) were found to be down-regulated in ALL patients, where these were identified as platelet factor 4 (PF4) and pro-platelet basic protein precursor (PBP). Two other candidate protein peaks (8137 and 8937 m/z) were found up-regulated in the sera of ALL patients, and these were identified as fragments of the complement component 3a (C3a). Conclusion Platelet factor (PF4), connective tissue activating peptide III (CTAP-III) and two fragments of C3a may be potential protein biomarkers of pediatric ALL and used to distinguish pediatric ALL patients from healthy controls and pediatric AML patients. Further studies with additional populations or using pre-diagnostic sera are needed to confirm the importance of these findings as diagnostic markers of pediatric ALL. PMID:19291297

Platelet Adhesion and Activation on Chiral Surfaces: The Influence of Protein Adsorption.

PubMed

Fan, Yonghong; Luo, Rifang; Han, Honghong; Weng, Yajun; Wang, Hong; Li, Jing'an; Yang, Ping; Wang, Yunbing; Huang, Nan

2017-10-03

Adsorbed proteins and their conformational change on blood-contacting biomaterials will determine their final hemocompatibility. It has frequently been reported that surface chirality of biomaterials may highly influence their protein adsorption behavior. Here, lysine and tartaric acid with different chirality were immobilized onto TiO 2 films respectively, and the influence of surface chirality on protein adsorption, platelet adhesion, and activation was also investigated. It showed that the l- and d-molecule grafted samples had almost the same grafting density, surface topography, chemical components, and hydrophilicity in this study. However, biological behaviors such as protein adsorption, platelet adhesion, and activation were quite different. The d-lysine grafted surface had a greater ability to inhibit both bovine serum albumin and fibrinogen adsorption, along with less degeneration of fibrinogen compared to the l-lysine anchored surface. However, the d-tartaric acid grafted surface adsorbed more protein but with less denatured fibrinogen compared to the l-tartaric acid grafted one. Further studies showed that the secondary structural change of the adsorbed albumin and fibrinogen on all surfaces with deduction of the α-helix content and increase of disordered structure, while the changing degree was apparently varied. As a result, the d-lysine immobilized surface absorbed less platelets and red blood cells and achieved slightly increased platelet activation. For tartaric acid anchored surfaces, a larger number of platelets adhered to the D-surface but were less activated compared to the L-surface. In conclusion, the surface chirality significantly influenced the adsorption and conformational change of blood plasma protein, which in turn influenced both platelet adhesion and activation.

Dual mechanism of integrin αIIbβ3 closure in procoagulant platelets.

PubMed

Mattheij, Nadine J A; Gilio, Karen; van Kruchten, Roger; Jobe, Shawn M; Wieschhaus, Adam J; Chishti, Athar H; Collins, Peter; Heemskerk, Johan W M; Cosemans, Judith M E M

2013-05-10

Inactivation of integrin αIIbβ3 reverses platelet aggregate formation upon coagulation. Platelets from patient (Scott) and mouse (Capn1(-/-) and Ppif(-/-)) blood reveal a dual mechanism of αIIbβ3 inactivation: by calpain-2 cleavage of integrin-associated proteins and by cyclophilin D/TMEM16F-dependent phospholipid scrambling. These data provide novel insight into the switch mechanisms from aggregating to procoagulant platelets. Aggregation of platelets via activated integrin αIIbβ3 is a prerequisite for thrombus formation. Phosphatidylserine-exposing platelets with a key role in the coagulation process disconnect from a thrombus by integrin inactivation via an unknown mechanism. Here we show that αIIbβ3 inactivation in procoagulant platelets relies on a sustained high intracellular Ca(2+), stimulating intracellular cleavage of the β3 chain, talin, and Src kinase. Inhibition of calpain activity abolished protein cleavage, but only partly suppressed αIIbβ3 inactivation. Integrin αIIbβ3 inactivation was unchanged in platelets from Capn1(-/-) mice, suggesting a role of the calpain-2 isoform. Scott syndrome platelets, lacking the transmembrane protein TMEM16F and having low phosphatidylserine exposure, displayed reduced αIIbβ3 inactivation with the remaining activity fully dependent on calpain. In platelets from Ppif(-/-) mice, lacking mitochondrial permeability transition pore (mPTP) formation, agonist-induced phosphatidylserine exposure and αIIbβ3 inactivation were reduced. Treatment of human platelets with cyclosporin A gave a similar phenotype. Together, these data point to a dual mechanism of αIIbβ3 inactivation via calpain(-2) cleavage of integrin-associated proteins and via TMEM16F-dependent phospholipid scrambling with an assistant role of mPTP formation.

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.

Platelets enhance tissue factor protein and metastasis initiating cell markers, and act as chemoattractants increasing the migration of ovarian cancer cells.

PubMed

Orellana, Renan; Kato, Sumie; Erices, Rafaela; Bravo, María Loreto; Gonzalez, Pamela; Oliva, Bárbara; Cubillos, Sofía; Valdivia, Andrés; Ibañez, Carolina; Brañes, Jorge; Barriga, María Isabel; Bravo, Erasmo; Alonso, Catalina; Bustamente, Eva; Castellon, Enrique; Hidalgo, Patricia; Trigo, Cesar; Panes, Olga; Pereira, Jaime; Mezzano, Diego; Cuello, Mauricio A; Owen, Gareth I

2015-04-15

An increase in circulating platelets, or thrombocytosis, is recognized as an independent risk factor of bad prognosis and metastasis in patients with ovarian cancer; however the complex role of platelets in tumor progression has not been fully elucidated. Platelet activation has been associated with an epithelial to mesenchymal transition (EMT), while Tissue Factor (TF) protein expression by cancer cells has been shown to correlate with hypercoagulable state and metastasis. The aim of this work was to determine the effect of platelet-cancer cell interaction on TF and "Metastasis Initiating Cell (MIC)" marker levels and migration in ovarian cancer cell lines and cancer cells isolated from the ascetic fluid of ovarian cancer patients. With informed patient consent, ascitic fluid isolated ovarian cancer cells, cell lines and ovarian cancer spheres were co-cultivated with human platelets. TF, EMT and stem cell marker levels were determined by Western blotting, flow cytometry and RT-PCR. Cancer cell migration was determined by Boyden chambers and the scratch assay. The co-culture of patient-derived ovarian cancer cells with platelets causes: 1) a phenotypic change in cancer cells, 2) chemoattraction and cancer cell migration, 3) induced MIC markers (EMT/stemness), 3) increased sphere formation and 4) increased TF protein levels and activity. We present the first evidence that platelets act as chemoattractants to cancer cells. Furthermore, platelets promote the formation of ovarian cancer spheres that express MIC markers and the metastatic protein TF. Our results suggest that platelet-cancer cell interaction plays a role in the formation of metastatic foci.

Posttransfusion purpura associated with an autoantibody directed against a previously undefined platelet antigen.

PubMed

Stricker, R B; Lewis, B H; Corash, L; Shuman, M A

1987-05-01

Although alloantibody against the PLA1 platelet antigen is usually found in patients with posttransfusion purpura (PTP), the mechanism of destruction of the patient's own PLA1-negative platelets is unexplained. We used a sensitive immunoblot technique to detect antiplatelet antibodies in a patient with classic PTP. The patient's acute-phase serum contained antibodies against three proteins present in control (PLA1-positive) platelets: an antibody that bound to a previously unrecognized platelet protein of mol wt 120,000 [glycoprotein (GP) 120], antibodies that bound to PLA1 (mol wt 90,000), and an epitope of GP IIb (mol wt 140,000). The antibodies against PLA1 and GP IIb did not react with the patient's own PLA1-negative platelets, control PLA1-negative platelets, or thrombasthenic platelets. In contrast, the antibody against GP 120 recognized this protein in all three platelet preparations, but not in Bernard-Soulier or Leka (Baka)-negative platelets. Antibody against GP 120 was not detected in the patient's recovery serum, although the antibodies against PLA1 and GP IIb persisted. F(ab)2 prepared from the patient's acute-phase serum also bound to GP 120. These results suggest that in PTP, transient autoantibody production may be responsible for autologous (PLA1-negative) platelet destruction. In addition, alloantibodies against more than one platelet alloantigen may be found in this disease. The nature of the GP 120 autoantigen and the GP IIb-related alloantigen defined by our patient's serum remains to be determined.

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

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.

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.

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.

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.

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.

An In Vitro Investigation of Platelet-Rich Plasma-Gel as a Cell and Growth Factor Delivery Vehicle for Tissue Engineering

PubMed Central

Jalowiec, Jagoda M.; D'Este, Matteo; Bara, Jennifer Jane; Denom, Jessica; Menzel, Ursula; Alini, Mauro; Herrmann, Marietta

2016-01-01

Platelet-rich plasma (PRP) has been used for different applications in human and veterinary medicine. Many studies have shown promising therapeutic effects of PRP; however, there are still many controversies regarding its composition, properties, and clinical efficacy. The aim of this study was to evaluate the influence of different platelet concentrations on the rheological properties and growth factor (GF) release profile of PRP-gels. In addition, the viability of incorporated bone marrow-derived human mesenchymal stem cells (MSCs) was investigated. PRP (containing 1000 × 103, 2000 × 103, and 10,000 × 103 platelets/μL) was prepared from human platelet concentrates. Platelet activation and gelification were achieved by addition of human thrombin. Viscoelastic properties of PRP-gels were evaluated by rheological studies. The release of GFs and inflammatory proteins was measured using a membrane-based protein array and enzyme-linked immunosorbent assay. MSC viability and proliferation in PRP-gels were assessed over 7 days by cell viability staining. Cell proliferation was examined using DNA quantification. Regardless of the platelet content, all tested PRP-gels showed effective cross-linking. A positive correlation between protein release and the platelet concentration was observed at all time points. Among the detected proteins, the chemokine CCL5 was the most abundant. The greatest release appeared within the first 4 h after gelification. MSCs could be successfully cultured in PRP-gels over 7 days, with the highest cell viability and DNA content found in PRP-gels with 1000 × 103 platelets/μL. The results of this study suggest that PRP-gels represent a suitable carrier for both cell and GF delivery for tissue engineering. Notably, a platelet concentration of 1000 × 103 platelets/μL appeared to provide the most favorable environment for MSCs. Thus, the platelet concentration is an important consideration for the clinical application of PRP-gels. PMID:26467221

Acetylsalicylic acid is compounding to antiplatelet effect of C-reactive protein.

PubMed

Boncler, Magdalena; Luzak, Boguslawa; Rozalski, Marcin; Golanski, Jacek; Rychlik, Blazej; Watala, Cezary

2007-01-01

The contribution of inflammatory process to the modulation of platelet response to acetylsalicylic acid (ASA) remains obscure. In our study, we examined the in vitro effect of C-reactive protein (CRP) on the ASA-mediated inhibition of collagen-stimulated platelet reactivity. Influence of CRP on platelet responsiveness to ASA was analysed using classical turbidimetric aggregation and flow cytometry. When acting alone, both C-reactive protein and ASA inhibited collagen-dependent platelet aggregation and reduced the expressions of two platelet surface membrane activation markers: P-selectin and activated GPIIbIIIa complex. Compared to the effects observed for ASA alone, the simultaneous action of both agents lead to further reductions in platelet aggregation (by 56.7+/-1.0% vs. 14.9+/-0.6%, p<0.0001) and lowered the expressions of platelet surface membrane P-selectin (by 72.1+/-5.3% vs. 65.0+/-6.0%, p<0.01) and activated GPIIbIIIa (by 67.0+/-5.6% vs. 47.7+/-8.3%, p<0.01). In general, our findings showed for the first time the augmenting effect of native C-reactive protein in the antiplatelet action of acetylsalicylic acid. Thus, we conclude that the effectiveness of aspirin therapy may strongly depend upon the presence of native CRP in circulation.

Impact of ticagrelor on P2Y1 and P2Y12 localization and on cholesterol levels in platelet plasma membrane.

PubMed

Rabani, Vahideh; Montange, Damien; Meneveau, Nicolas; Davani, Siamak

2017-10-11

Ticagrelor is an antiplatelet agent that inhibits platelet activation via P2Y12 antagonism. There are several studies showing that P2Y12 needs lipid rafts to be activated, but there are few data about how ticagrelor impacts lipid raft organization. Therefore, we aimed to investigate how ticagrelor could impact the distribution of cholesterol and consequently alter the organization of lipid rafts on platelet plasma membranes. We identified cholesterol-enriched raft fractions in platelet membranes by quantification of their cholesterol levels. Modifications in cholesterol and protein profiles (Flotillin 1, Flotillin 2, CD36, P2Y1, and P2Y12) were studied in platelets stimulated by ADP, treated by ticagrelor, or both. In ADP-stimulated and ticagrelor-treated groups, we found a decreased level of cholesterol in raft fractions of platelet plasma membrane compared to the control group. In addition, the peak of cholesterol in different experimental groups changed its localization on membrane fractions. In the control group, it was situated on fraction 2, while in ADP-stimulated platelets, it was located in fractions 3 to 5, and in fraction 4 in ticagrelor-treated group. The proteins studied also showed changes in their level of expression and localization in fractions of plasma membrane. Cholesterol levels of plasma membranes have a direct role in the organization of platelet membranes and could be modified by stimulation or drug treatment. Since ticagrelor and ADP both changed lipid composition and protein profile, investigating the lipid and protein composition of platelet membranes is of considerable importance as a focus for further research in anti-platelet management.

Platelet adhesion and plasma protein adsorption control of collagen surfaces by He + ion implantation

NASA Astrophysics Data System (ADS)

Kurotobi, K.; Suzuki, Y.; Nakajima, H.; Suzuki, H.; Iwaki, M.

2003-05-01

He + ion implanted collagen-coated tubes with a fluence of 1 × 10 14 ions/cm 2 were exhibited antithrombogenicity. To investigate the mechanisms of antithrombogenicity of these samples, plasma protein adsorption assay and platelet adhesion experiments were performed. The adsorption of fibrinogen (Fg) and von Willebrand factor (vWf) was minimum on the He + ion implanted collagen with a fluence of 1 × 10 14 ions/cm 2. Platelet adhesion (using platelet rich plasma) was inhibited on the He + ion implanted collagen with a fluence of 1 × 10 14 ions/cm 2 and was accelerated on the untreated collagen and ion implanted collagen with fluences of 1 × 10 13, 1 × 10 15 and 1 × 10 16 ions/cm 2. Platelet activation with washed platelets was observed on untreated collagen and He + ion implanted collagen with a fluence of 1 × 10 14 ions/cm 2 and was inhibited with fluences of 1 × 10 13, 1 × 10 15 and 1 × 10 16 ions/cm 2. Generally, platelets can react with a specific ligand inside the collagen (GFOGER sequence). The results of platelets adhesion experiments using washed platelets indicated that there were no ligands such as GFOGER on the He + ion implanted collagen over a fluence of 1 × 10 13 ions/cm 2. On the 1 × 10 14 ions/cm 2 implanted collagen, no platelet activation was observed due to the influence of plasma proteins. From the above, it is concluded that the decrease of adsorbed Fg and vWf caused the antithrombogenicity of He + ion implanted collagen with a fluence of 1 × 10 14 ions/cm 2 and that plasma protein adsorption took an important role repairing the graft surface.

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

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

An inhibitor of collagen-stimulated platelet activation from the salivary glands of the Haementeria officinalis leech. I. Identification, isolation, and characterization.

PubMed

Connolly, T M; Jacobs, J W; Condra, C

1992-04-05

A protein that blocks collagen-stimulated platelet aggregation has been identified and isolated from the soluble fraction of salivary glands from Haementeria officinalis leeches. We have named this protein leech antiplatelet protein (LAPP). LAPP was isolated from soluble crude salivary gland extract by heparin-agarose, size exclusion, and C18 reverse phase high-performance chromatography. Its molecular weight is approximately 16,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reduced and nonreduced conditions. The sequences of peptides generated by V8 digestion of LAPP as well as its amino acid composition suggested no homology to other known proteins. The IC50 for LAPP to inhibit platelet aggregation was approximately 60 nM. This inhibitory activity is specific for collagen-induced aggregation. Platelet aggregation in response to ADP, arachidonic acid, U46619, thrombin, and ionophore A23187 was not inhibited by LAPP at a concentration that blocked platelet aggregation to collagen by 100%. In contrast, crude salivary gland-soluble extract contained activity(ies) which inhibited aggregation to all these agonists except thrombin at 1 unit/ml and 2 microM A23187. Thus, the H. officinalis leech has evolved multiple mechanisms to prevent hemostasis, including an inhibitor of collagen-stimulated platelet aggregation. The identification and isolation of LAPP demonstrates the existence of a new type of platelet inhibitor that should be useful to better understand the mechanism of collagen stimulation of platelets.

Proteome changes in platelets after pathogen inactivation--an interlaboratory consensus.

PubMed

Prudent, Michel; D'Alessandro, Angelo; Cazenave, Jean-Pierre; Devine, Dana V; Gachet, Christian; Greinacher, Andreas; Lion, Niels; Schubert, Peter; Steil, Leif; Thiele, Thomas; Tissot, Jean-Daniel; Völker, Uwe; Zolla, Lello

2014-04-01

Pathogen inactivation (PI) of platelet concentrates (PCs) reduces the proliferation/replication of a large range of bacteria, viruses, and parasites as well as residual leucocytes. Pathogen-inactivated PCs were evaluated in various clinical trials showing their efficacy and safety. Today, there is some debate over the hemostatic activity of treated PCs as the overall survival of PI platelets seems to be somewhat reduced, and in vitro measurements have identified some alterations in platelet function. Although the specific lesions resulting from PI of PCs are still not fully understood, proteomic studies have revealed potential damages at the protein level. This review merges the key findings of the proteomic analyses of PCs treated by the Mirasol Pathogen Reduction Technology, the Intercept Blood System, and the Theraflex UV-C system, respectively, and discusses the potential impact on the biological functions of platelets. The complementarities of the applied proteomic approaches allow the coverage of a wide range of proteins and provide a comprehensive overview of PI-mediated protein damage. It emerges that there is a relatively weak impact of PI on the overall proteome of platelets. However, some data show that the different PI treatments lead to an acceleration of platelet storage lesions, which is in agreement with the current model of platelet storage lesion in pathogen-inactivated PCs. Overall, the impact of the PI treatment on the proteome appears to be different among the PI systems. Mirasol impacts adhesion and platelet shape change, whereas Intercept seems to impact proteins of intracellular platelet activation pathways. Theraflex influences platelet shape change and aggregation, but the data reported to date are limited. This information provides the basis to understand the impact of different PI on the molecular mechanisms of platelet function. Moreover, these data may serve as basis for future developments of PI technologies for PCs. Further studies should address the impact of both the PI and the storage duration on platelets in PCs because PI may enable the extension of the shelf life of PCs by reducing the bacterial contamination risk. Copyright © 2014 Elsevier Inc. All rights reserved.

Protein levels of ADAM10, BACE1, and PSEN1 in platelets and leukocytes of Alzheimer's disease patients.

PubMed

Bram, Jessyka Maria de França; Talib, Leda Leme; Joaquim, Helena Passarelli Giroud; Sarno, Tamires Alves; Gattaz, Wagner Farid; Forlenza, Orestes Vicente

2018-05-29

The clinical diagnosis of Alzheimer's disease (AD) is a probabilistic formulation that may lack accuracy particularly at early stages of the dementing process. Abnormalities in amyloid-beta precursor protein (APP) metabolism and in the level of APP secretases have been demonstrated in platelets, and to a lesser extent in leukocytes, of AD patients, with conflicting results. The aim of the present study was to compare the protein level of the APP secretases A-disintegrin and metalloprotease 10 (ADAM10), Beta-site APP-cleaving enzyme 1 (BACE1), and presenilin-1 (PSEN1) in platelets and leukocytes from 20 non-medicated older adults with AD and 20 healthy elders, and to determine the potential use of these biomarkers to discriminate cases of AD from controls. The protein levels of all APP secretases were significantly higher in platelets compared to leukocytes. We found statistically a significant decrease in ADAM10 (52.5%, p < 0.0001) and PSEN1 (32%, p = 0.02) in platelets from AD patients compared to controls, but not in leukocytes. Combining all three secretases to generate receiver-operating characteristic (ROC) curves, we found a good discriminatory effect (AD vs. controls) when using platelets (the area under the curve-AUC-0.90, sensitivity 88.9%, specificity 66.7%, p = 0.003), but not in leukocytes (AUC 0.65, sensitivity 77.8%, specificity 50.0%, p = 0.2). Our findings indicate that platelets represent a better biological matrix than leukocytes to address the peripheral level of APP secretases. In addition, combining the protein level of ADAM10, BACE1, and PSEN1 in platelets, yielded a good accuracy to discriminate AD from controls.

c-erbA and v-erbA modulate growth and gene expression of a mouse glial precursor cell line.

PubMed

Iglesias, T; Llanos, S; López-Barahona, M; Pérez-Aranda, A; Rodríguez-Peña, A; Bernal, J; Höhne, A; Seliger, B; Muñoz, A

1994-07-01

The c-erbA alpha protooncogene coding for the thyroid hormone (T3) receptor (TR alpha 1) and the viral, mutated v-erbA oncogene were expressed in an immortal mouse glial cell line (B3.1) using retroviral vectors. c-erbA alpha expression led to a decrease in cell proliferation in high and low serum conditions, both in the presence and in the absence of T3. In serum-free medium, c-erbA-expressing cells (B3.1 + TR alpha 1) were completely arrested, whereas cells expressing v-erbA (B3.1 + v-erbA) showed a higher DNA synthesis rate than normal B3.1 cells. Although proliferation of all three cell types was stimulated by platelet-derived growth factor and basic fibroblast growth factor, differences were also observed in the response to these agents. B3.1 + TR alpha 1 cells were more sensitive to platelet-derived growth factor than B3.1 and B3.1 + v-erbA cells. In contrast, B3.1 cells responded to basic fibroblast growth factor better than B3.1 + TR alpha 1 or B3.1 + v-erbA cells. Insulin-like growth factor I potentiated the action of platelet-derived growth factor and basic fibroblast growth factor. Again, different responses to treatment with insulin-like growth factor I alone were observed; B3.1 + TR alpha 1 cells did not respond to it, whereas B3.1 + v-erbA cells showed a dramatic stimulation by this agent. Interestingly, in the presence of T3, the blockade in B3.1 + TR alpha 1 cell proliferation was accompanied by the down-regulation of the typical astrocytic genes, glial fibrillary acidic protein and vimentin. These hormone effects were not found in v-erbA-expressing cells. In addition, v-erbA inhibited the basal expression of the cyclic nucleotide phosphodiesterase gene, an oligodendrocytic marker.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the. alpha. subunit of the stimulatory guanine nucleotide-binding regulatory protein

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

Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G.

1988-08-01

ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the {alpha} subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost,more » a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5{prime}-({alpha}-{sup 32}P)triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an {alpha} subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera.« less

Evaluation of the Effects of Platelet-Rich Plasma (PRP) Therapy Involved in the Healing of Sports-Related Soft Tissue Injuries

PubMed Central

Middleton, Kellie K.; Barro, Victor; Muller, Bart; Terada, Satosha; Fu, Freddie H.

2012-01-01

Abstract Musculoskeletal injuries are the most common cause of severe long-term pain and physical disability, and affect hundreds of millions of people around the world. One of the most popular methods used to biologically enhance healing in the fields of orthopaedic surgery and sports medicine includes the use of autologous blood products, namely, platelet rich plasma (PRP). PRP is an autologous concentration of human platelets to supra-physiologic levels. At baseline levels, platelets function as a natural reservoir for growth factors including platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor-beta 1 (TGF-β1), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and insulin-like growth factor (IGF-I). PRP is commonly used in orthopaedic practice to augment healing in sports-related injuries of skeletal muscle, tendons, and ligaments. Despite its pervasive use, the clinical efficacy of PrP therapy and varying mechanisms of action have yet to be established. Basic science research has revealed that PRP exerts is effects through many downstream events secondary to release of growth factors and other bioactive factors from its alpha granules. These effects may vary depending on the location of injury and the concentration of important growth factors involved in various soft tissue healing responses. This review focuses on the effects of PrP and its associated bioactive factors as elucidated in basic science research. Current findings in PRP basic science research, which have shed light on its proposed mechanisms of action, have opened doors for future areas of PrP research. PMID:23576936

The saliva proteome of the blood-feeding insect Triatoma infestans is rich in platelet-aggregation inhibitors

NASA Astrophysics Data System (ADS)

Charneau, Sébastien; Junqueira, Magno; Costa, Camila M.; Pires, Daniele L.; Fernandes, Ellen S.; Bussacos, Ana C.; Sousa, Marcelo V.; Ricart, Carlos André O.; Shevchenko, Andrej; Teixeira, Antonio R. L.

2007-12-01

The saliva of the bloodsucking bug Triatoma infestans vector of Chagas disease contains an anti-hemostatic molecular cocktail that prevents coagulation, vasoconstriction and platelet aggregation in a vertebrate prey. In order to characterize T. infestans saliva proteome, we separated the secreted saliva by two-dimensional gel electrophoresis (2-DE). More than 200 salivary proteins were detected on the 2-DE map, mainly in the alkaline region. By nanoLC-MS/MS analysis using a LTQ-Orbitrap equipment followed by a combination of conventional and sequence-similarity searches, we identified 58 main protein spots. Most of such proteins possess potential blood-feeding associated functions, particularly anti-platelet aggregation proteins belonging to lipocalin and apyrase families. The saliva protein composition indicates a highly specific molecular mechanism of early response to platelet aggregation. This first proteome analysis of the T. infestans secreted saliva provides a basis for a better understanding of this fluid protein composition highly directed to counterpart hemostasis of the prey, thus promoting the bug's blood-feeding.

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.

Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting

NASA Astrophysics Data System (ADS)

Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

2016-07-01

Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency.

Inhibiting platelets aggregation could aggravate the acute infection caused by Staphylococcus aureus.

PubMed

Zhang, Xin; Liu, Yu; Gao, Yaping; Dong, Jie; Mu, Chunhua; Lu, Qiang; Shao, Ningsheng; Yang, Guang

2011-01-01

Several fibrinogen binding proteins (Fibs) play important roles in the pathogenesis of Staphylococcus aureus (S. aureus). Most Fibs can promote the aggregation of platelets during infection, but the extracellular fibrinogen-binding protein (Efb) is an exception. It is reported that Efb can specifically bind fibrinogen and inhibit the aggregation of platelet with its N terminal. However, the biological significance of platelet aggregation inhibition in the infection caused by S. aureus is unclear until now. Here, we demonstrated that the persistence and aggregation of platelets were important for killing S. aureus in whole blood. It was found that the N terminal of Efb (EfbN) and platelets inhibitors could increase the survival of S. aureus in whole blood. The study in vivo also showed that EfbN and platelets inhibitors could reduce the killing of S. aureus and increase the lethality rate of S. aureus in the acute infection mouse model.

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 Proteomic Analysis Revealed Differential Pattern of Cytoskeletal- and Immune-Related Proteins at Early Stages of Alzheimer's Disease.

PubMed

González-Sánchez, Marta; Díaz, Teresa; Pascual, Consuelo; Antequera, Desiree; Herrero-San Martín, Alejandro; Llamas-Velasco, Sara; Villarejo-Galende, Alberto; Bartolome, Fernando; Carro, Eva

2018-03-30

Platelets are considered a good model system to study a number of elements associated with neuronal pathways as they share biochemical similarities. Platelets represent the major source of amyloid-β (Aβ) in blood contributing to the Aβ accumulation in the brain parenchyma and vasculature. Peripheral blood platelet alterations including cytoskeletal abnormalities, abnormal cytoplasmic calcium fluxes or increased oxidative stress levels have been related to Alzheimer's disease (AD) pathology. Therefore, platelets can be considered a peripheral model to study metabolic mechanisms occurring in AD. To investigate peripheral molecular alterations, we examined platelet protein expression in a cohort of 164 subjects, including mild cognitive impairment (MCI), and AD patients, and healthy aged-matched controls. A two-dimensional difference gel electrophoresis (2D-DIGE) discovery phase revealed significant differences between patients and controls in five proteins: talin, vinculin, moesin, complement C3b and Rho GDP, which are known to be involved in cytoskeletal regulation including focal adhesions, inflammation and immune functions. Western blot analysis verified that talin was found to be increased in mild and moderate AD groups versus control, while the other three were found to be decreased. We also analysed amyloid precursor protein (APP), amyloid-β 1-40 (Aβ 40 ) and 1-42 (Aβ 42 ) levels in platelets from the same groups of subjects. Upregulation of platelet APP and Aβ peptides was found in AD patients compared to controls. These findings complement and expand previous reports concerning the morphological and functional alterations in AD platelets, and provide more insights into possible mechanisms that participate in the multifactorial and systemic damage in AD.

C-reactive protein enhances IgG-mediated phagocyte responses and thrombocytopenia.

PubMed

Kapur, Rick; Heitink-Pollé, Katja M J; Porcelijn, Leendert; Bentlage, Arthur E H; Bruin, Marrie C A; Visser, Remco; Roos, Dirk; Schasfoort, Richard B M; de Haas, Masja; van der Schoot, C Ellen; Vidarsson, Gestur

2015-03-12

Immune-mediated platelet destruction is most frequently caused by allo- or autoantibodies via Fcγ receptor-dependent phagocytosis. Disease severity can be predicted neither by antibody isotype nor by titer, indicating that other factors play a role. Here we show that the acute phase protein C-reactive protein (CRP), a ligand for Fc receptors on phagocytes, enhances antibody-mediated platelet destruction by human phagocytes in vitro and in vivo in mice. Without antiplatelet antibodies, CRP was found to be inert toward platelets, but it bound to phosphorylcholine exposed after oxidation triggered by antiplatelet antibodies, thereby enhancing platelet phagocytosis. CRP levels were significantly elevated in patients with allo- and autoantibody-mediated thrombocytopenias compared with healthy controls. Within a week, intravenous immunoglobulin treatment in children with newly diagnosed immune thrombocytopenia led to significant decrease of CRP levels, increased platelet numbers, and clinically decreased bleeding severity. Furthermore, the higher the level of CRP at diagnosis, the longer it took before stable platelet counts were reached. These data suggest that CRP amplifies antibody-mediated platelet destruction and may in part explain the aggravation of thrombocytopenia on infections. Hence, targeting CRP could offer new therapeutic opportunities for these patients. © 2015 by The American Society of Hematology.

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.

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.

Thrombin-receptor agonist peptides, in contrast to thrombin itself, are not full agonists for activation and signal transduction in human platelets in the absence of platelet-derived secondary mediators.

PubMed Central

Lau, L F; Pumiglia, K; Côté, Y P; Feinstein, M B

1994-01-01

Synthetic thrombin receptor peptides (TRPs), comprising the first 6-14 amino acids of the new N-terminus tethered ligand of the thrombin receptor that is generated by thrombin's proteolytic activity, were reported to activate platelets equally with thrombin itself and are considered to be full agonists [Vu et al. (1991) Cell 64, 1057-1068]. Using aspirin plus ADP-scavengers or the ADP-receptor antagonist adenosine 5'-[alpha-thio]triphosphate to prevent the secondary effects of the potent agonists that are normally released from stimulated platelets (i.e. ADP and thromboxane A2), we assessed the direct actions of thrombin and TRPs (i.e. TRP42-47 and TRP42-55). Compared with thrombin, under these conditions, TRPs: (1) failed to aggregate platelets completely; (2) produced less activation of glycoprotein (GP)IIb-IIIa; (3) did not cause association of GPIIb and pp60c-src with the cytoskeleton; and (4) caused less alpha-granule secretion, phosphorylation of cytoplasmic phospholipase A2, arachidonic acid release and phosphatidyl inositol (PtdOH) production. Furthermore, TRPs induced transient increases in protein phosphorylation mediated by protein kinase C and protein tyrosine phosphorylation, whereas these same responses to thrombin were greater and more sustained. Hirudin added after thrombin accelerated protein dephosphorylation, thereby mimicking the rate of spontaneous dephosphorylation seen after stimulation by TRPs. Platelets totally desensitized to very high concentrations of TRPs, by prior exposure to maximally effective concentrations of the peptides, remained responsive to alpha- and gamma-thrombins. Thrombin-stimulated PtdOH production in permeabilized platelets desensitized to TRPs was abolished by guanosine 5'-[beta-thio]diphosphate (GDP[beta S]), as in normal platelets. These results are discussed in terms of the allosteric Ternary Complex Model for G-protein linked receptors [Samama et al. (1993) J. Biol. Chem. 268, 4625-4636]. We conclude that: (1) TRPs are partial agonists for the thrombin receptor and produce incomplete receptor desensitization in keeping with their lower intrinsic activity; (2) thrombin's effects in platelets, even in TRP-desensitized platelets, are entirely mediated through the recently cloned G-protein linked receptor, and (3) thrombin's ability to produce sustained signals, compared with TRPs, may require the continued progressive proteolytic activation of naive thrombin receptors. Images Figure 3 PMID:7526841

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

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.

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

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.

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.

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

Survival protein anoctamin-6 controls multiple platelet responses including phospholipid scrambling, swelling, and protein cleavage.

PubMed

Mattheij, Nadine J A; Braun, Attila; van Kruchten, Roger; Castoldi, Elisabetta; Pircher, Joachim; Baaten, Constance C F M J; Wülling, Manuela; Kuijpers, Marijke J E; Köhler, Ralf; Poole, Alastair W; Schreiber, Rainer; Vortkamp, Andrea; Collins, Peter W; Nieswandt, Bernhard; Kunzelmann, Karl; Cosemans, Judith M E M; Heemskerk, Johan W M

2016-02-01

Scott syndrome is a rare bleeding disorder, characterized by altered Ca(2+)-dependent platelet signaling with defective phosphatidylserine (PS) exposure and microparticle formation, and is linked to mutations in the ANO6 gene, encoding anoctamin (Ano)6. We investigated how the complex platelet phenotype of this syndrome is linked to defective expression of Anos or other ion channels. Mice were generated with heterozygous of homozygous deficiency in Ano6, Ano1, or Ca(2+)-dependent KCa3.1 Gardos channel. Platelets from these mice were extensively analyzed on molecular functions and compared with platelets from a patient with Scott syndrome. Deficiency in Ano1 or Gardos channel did not reduce platelet responses compared with control mice (P > 0.1). In 2 mouse strains, deficiency in Ano6 resulted in reduced viability with increased bleeding time to 28.6 min (control 6.4 min, P < 0.05). Platelets from the surviving Ano6-deficient mice resembled platelets from patients with Scott syndrome in: 1) normal collagen-induced aggregate formation (P > 0.05) with reduced PS exposure (-65 to 90%); 2) lowered Ca(2+)-dependent swelling (-80%) and membrane blebbing (-90%); 3) reduced calpain-dependent protein cleavage (-60%); and 4) moderately affected apoptosis-dependent PS exposure. In conclusion, mouse deficiency of Ano6 but not of other channels affects viability and phenocopies the complex changes in platelets from hemostatically impaired patients with Scott syndrome. © FASEB.

Identification of a specific intronic PEAR1 gene variant associated with greater platelet aggregability and protein expression

PubMed Central

Yanek, Lisa R.; Yang, Xiao Ping; Mathias, Rasika; Herrera-Galeano, J. Enrique; Suktitipat, Bhoom; Qayyum, Rehan; Johnson, Andrew D.; Chen, Ming-Huei; Tofler, Geoffrey H.; Ruczinski, Ingo; Friedman, Alan D.; Gylfason, Arnaldur; Thorsteinsdottir, Unnur; Bray, Paul F.; O'Donnell, Christopher J.; Becker, Diane M.; Becker, Lewis C.

2011-01-01

Genetic variation is thought to contribute to variability in platelet function; however, the specific variants and mechanisms that contribute to altered platelet function are poorly defined. With the use of a combination of fine mapping and sequencing of the platelet endothelial aggregation receptor 1 (PEAR1) gene we identified a common variant (rs12041331) in intron 1 that accounts for ≤ 15% of total phenotypic variation in platelet function. Association findings were robust in 1241 persons of European ancestry (P = 2.22 × 10−8) and were replicated down to the variant and nucleotide level in 835 persons of African ancestry (P = 2.31 × 10−27) and in an independent sample of 2755 persons of European descent (P = 1.64 × 10−5). Sequencing confirmed that variation at rs12041331 accounted most strongly (P = 2.07 × 10−6) for the relation between the PEAR1 gene and platelet function phenotype. A dose-response relation between the number of G alleles at rs12041331 and expression of PEAR1 protein in human platelets was confirmed by Western blotting and ELISA. Similarly, the G allele was associated with greater protein expression in a luciferase reporter assay. These experiments identify the precise genetic variant in PEAR1 associated with altered platelet function and provide a plausible biologic mechanism to explain the association between variation in the PEAR1 gene and platelet function phenotype. PMID:21791418

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

Application of Platelet-Rich Plasma to Disorders of the Knee Joint

PubMed Central

Mandelbaum, Bert R.; McIlwraith, C. Wayne

2013-01-01

Importance. The promising therapeutic potential and regenerative properties of platelet-rich plasma (PRP) have rapidly led to its widespread clinical use in musculoskeletal injury and disease. Although the basic scientific rationale surrounding PRP products is compelling, the clinical application has outpaced the research. Objective. The purpose of this article is to examine the current concepts around the basic science of PRP application, different preparation systems, and clinical application of PRP in disorders in the knee. Evidence Acquisition. A systematic search of PubMed for studies that evaluated the basic science, preparation and clinical application of platelet concentrates was performed. The search used terms, including platelet-rich plasma or PRP preparation, activation, use in the knee, cartilage, ligament, and meniscus. Studies found in the initial search and related studies were reviewed. Results. A comprehensive review of the literature supports the potential use of PRP both nonoperatively and intraoperatively, but highlights the absence of large clinical studies and the lack of standardization between method, product, and clinical efficacy. Conclusions and Relevance. In addition to the call for more randomized, controlled clinical studies to assess the clinical effect of PRP, at this point, it is necessary to investigate PRP product composition and eventually have the ability to tailor the therapeutic product for specific indications. PMID:26069674

Role of G protein signaling in the formation of the fibrin(ogen)-integrin αIIbβ3-actin cytoskeleton complex in platelets.

PubMed

Budnik, Ivan; Shenkman, Boris; Savion, Naphtali

2016-09-01

Effective platelet function requires formation of a physical link between fibrin(ogen), integrin αIIbβ3, and cytoplasmic actin filaments. We investigated the role of the Gαq, Gαi, and Gα12/13 families of heterotrimeric GTP-binding proteins (G proteins) in the assembly of a ligand-αIIbβ3-actin cytoskeleton complex. Selective and combined activation of the G proteins was achieved by using combinations of various platelet agonists and inhibitors. Formation and stability of fibrinogen-αIIbβ3 interaction were evaluated by the extent of platelet aggregation and the rate of eptifibatide-induced platelet disaggregation; association of αIIbβ3 with the cytoskeleton was analyzed by western blot. Formation of the fibrin-αIIbβ3-actin cytoskeleton complex was evaluated by rotational thromboelastometry assay in which clot formation was induced by the mixture of reptilase and factor XIIIa. We demonstrated that involvement of heterotrimeric G proteins in the formation of the ligand-αIIbβ3-cytoskeleton complex depends on whether fibrinogen or fibrin serves as the integrin ligand. Formation of the fibrinogen-αIIbβ3-cytoskeleton complex requires combined activation of at least two G protein pathways while the maximal αIIbβ3-cytoskeleton association and the strongest αIIbβ3-fibrinogen binding supporting irreversible platelet aggregation require combined activation of all three-Gαq, Gαi, and Gα12/13-G protein families. In contrast, formation of the fibrin-αIIbβ3-cytoskeleton complex mediating clot retraction is critically dependent on the activation of the Gαi family, especially on the activation of Gαz.

Influence of dynamic flow conditions on adsorbed plasma protein corona and surface-induced thrombus generation on antifouling brushes.

PubMed

Yu, Kai; Andruschak, Paula; Yeh, Han Hung; Grecov, Dana; Kizhakkedathu, Jayachandran N

2018-06-01

The information regarding the nature of protein corona (and its changes) and cell binding on biomaterial surface under dynamic conditions is critical to dissect the mechanism of surface-induced thrombosis. In this manuscript, we investigated the nature of protein corona and blood cell binding in heparinized recalcified human plasma, platelet rich plasma and whole blood on three highly hydrophilic antifouling polymer brushes, (poly(N, N-dimethylacrylamide) (PDMA), poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and poly[N-(2-hydroxypropyl) methacrylamide] (PHPMA) using an in vitro blood loop model at comparable arterial and venous flow, and static conditions. A fluid dynamics model was used initially to better understand the resulting flow patterns in a vertical channel containing the substrates to arrive at the placement of the substrates within the blood loop. The protein binding on the brush modified substrates was determined using ellipsometry, fluorescence microscopy and the nature of the protein corona was investigated using mass spectrometry based proteomics. The flow elevated fouling on brush coated surface from blood. The extent of plasma protein adsorption and platelet adhesion onto PDMA brush was lower than other surfaces in both static and flow conditions. The profiles of adsorbed protein corona showed strong dependence on the test conditions (static vs. flow), and the chemistry of the polymer brushes. Specially, the PDMA brush under flow conditions was more enriched with coagulation proteins, complement proteins, vitronectin and fibronectin but was less enriched with serum albumin. Apolipoprotein B-100 and complement proteins were the most abundant proteins seen on PMPC and PHPMA surfaces under both flow and static conditions, respectively. Unlike PDMA brush, the flow conditions did not affect the composition of protein corona on PMPC and PHPMA brushes. The nature of the protein corona formed in flow conditions influenced the platelet and red blood cell binding. The dependence of shear stress on platelet adhesion from platelet rich plasma and whole blood highlights the contribution of red blood cells in enhancing platelet adhesion on the surface under high shear condition. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

Identification and Characterization of Anti-Platelet Antibodies in Idiopathic Thrombocytopenic Purpura Patients

PubMed Central

Aghabeigi, N; Lindsey, N; Zamani, A; Shishaeyan, B

2012-01-01

Background: The autoimmune disease known as Idiopathic (immune thrombocytopenic purpura thrombocytopenic purpura (ITP) is clinically defined by a low numbers of platelets in the circulation blood. This study aimed to isolate autoantibodies made against the platelet glycoproteins using platelets from healthy volunteers, to determine their specificity and further elucidate their effects on platelet function. Methods: This study used a phage display system to recognize Fab anti-platelet antibodies. Anti-platelet After isolation, the anti-platelet Fab-expressing phage was characterized by ELISA and Western blotting. The Fab-bearing phage pool obtained from five rounds of panning was analysed in order to determine its anti-platelet reactivity. Of the phage colonies obtained, 100 colonies of different sizes were randomly selected for reaction with whole platelets, using M13 phage as a negative control. Results: Twelve colonies of them had strong reactions against the whole platelet preparation, but only four colonies showed substantial reactivity against the lysed platelet preparation (lysate). Three of the four colonies showed three bands representing proteins with different molecular weights. The fourth colony showed only a single band. The final experiment to characterise the protein isolated from the phage library was a DNA gel agarose test. Conclusion: Each colony showed a DNA band that corresponded with the molecular size marker for 5.4 kbase pairs, and this suggested the presence of heavy and light antibody chains in the phage. PMID:23113135

Protein A Sepharose immunoadsorption can restore the efficacy of platelet concentrates in patients with Glanzmann's thrombasthenia and anti-glycoprotein IIb-IIIa antibodies.

PubMed

Martin, Isabelle; Kriaa, Fayçal; Proulle, Valérie; Guillet, Benoît; Kaplan, Cécile; D'Oiron, Roseline; Debré, Marianne; Fressinaud, Edith; Laurian, Yyes; Tchernia, Gil; Charpentier, Bernard; Lambert, Thierry; Dreyfus, Marie

2002-12-01

Type I Glanzmann's thrombasthenia is a rare congenital platelet function disorder, characterized by undetectable platelet membrane glycoprotein IIb-IIIa (GPIIb-IIIa). Severe bleeding is controlled by transfusion of normal platelets, leading in some cases to the occurrence of anti-GPIIb-IIIa isoantibodies, which induces a loss of transfused platelet efficacy. We used immunoadsorption on protein A Sepharose (IA-PA), which has been shown to be efficient in decreasing the titre of antibodies in several immune diseases, in three patients with Glanzmann's thrombasthenia and anti-GPIIb-IIIa isoantibodies on five different occasions. IA-PA was well tolerated with no deleterious side-effects reported. It induced a dramatic decrease of total immunoglobulin (Ig)G, including anti-GPIIb-IIIa isoantibody levels, as assessed by the monoclonal antibody-specific immobilization of platelet antigens test and the ex vivo inhibition of normal platelet aggregation induced by the patient's platelet-rich or platelet-poor plasma. Elimination of the antibody was associated with a correction of the bleeding time following platelet transfusion. IA-PA combined with platelet transfusion made it possible to control two life-threatening haemorrhages, and allowed two surgical procedures and one bone marrow transplantation to be performed safely. Our experience suggests that IA-PA, which restores the haemostatic efficacy of platelet transfusion, is a valuable therapeutic strategy in patients with Glanzmann's thrombasthenia and anti-GPIIb-IIIa isoantibodies.

Several adaptor proteins promote intracellular localisation of the transporter MRP4/ABCC4 in platelets and haematopoietic cells.

PubMed

Schaletzki, Yvonne; Kromrey, Marie-Luise; Bröderdorf, Susanne; Hammer, Elke; Grube, Markus; Hagen, Paul; Sucic, Sonja; Freissmuth, Michael; Völker, Uwe; Greinacher, Andreas; Rauch, Bernhard H; Kroemer, Heyo K; Jedlitschky, Gabriele

2017-01-05

The multidrug resistance protein 4 (MRP4/ABCC4) has been identified as an important transporter for signalling molecules including cyclic nucleotides and several lipid mediators in platelets and may thus represent a novel target to interfere with platelet function. Besides its localisation in the plasma membrane, MRP4 has been also detected in the membrane of dense granules in resting platelets. In polarised cells it is localised at the basolateral or apical plasma membrane. To date, the mechanism of MRP4 trafficking has not been elucidated; protein interactions may regulate both the localisation and function of this transporter. We approached this issue by searching for interacting proteins by in vitro binding assays, followed by immunoblotting and mass spectrometry, and by visualising their co-localisation in platelets and haematopoietic cells. We identified the PDZ domain containing scaffold proteins ezrin-binding protein 50 (EBP50/NHERF1), postsynaptic density protein 95 (PSD95), and sorting nexin 27 (SNX27), but also the adaptor protein complex 3 subunit β3A (AP3B1) and the heat shock protein HSP90 as putative interaction partners of MRP4. The knock-down of SNX27, PSD95, and AP3B1 by siRNA in megakaryoblastic leukaemia cells led to a redistribution of MRP4 from intracellular structures to the plasma membrane. Inhibition of HSP90 led to a diminished expression and retention of MRP4 in the endoplasmic reticulum. These results indicate that MRP4 localisation and function are regulated by multiple protein interactions. Changes in the adaptor proteins can hence lead to altered localisation and function of the transporter.

Identification of functional VEGF receptors on human platelets.

PubMed

Selheim, Frode; Holmsen, Holm; Vassbotn, Flemming S

2002-02-13

Platelets secrete platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) upon stimulation. We have demonstrated that platelets have functionally active PDGF alpha-receptors, a transmembrane tyrosine kinase involved in negative feedback regulation. Here we demonstrate the presence of the related VEGF receptors fms-like tyrosine kinase-1 and kinase-insert domain region on human platelets. VEGF itself did not cause platelet aggregation. However, addition of exogenous VEGF to SFRLLN or thrombin-stimulated platelets potentiated platelet aggregation. Moreover, thrombin-induced phosphoinositide 3-kinase and mitogen-activated protein kinase activity were enhanced in the presence of VEGF.

Platelets as delivery systems for disease treatments

PubMed Central

Shi, Qizhen; Montgomery, Robert R.

2010-01-01

Platelets are small, anucleate, discoid shaped blood cells that play a fundamental role in hemostasis. Platelets contain a large number of biologically active molecules within cytoplasmic granules that are critical to normal platelet function. Because platelets circulate in blood through out the body, release biological molecules and mediators on demand, and participate in hemostasis as well as many other pathophysiologic processes, targeting expression of proteins of interest to platelets and utilizing platelets as delivery systems for disease treatment would be a logical approach. This paper reviews the genetic therapy for inherited bleeding disorders utilizing platelets as delivery system, with a particular focus on platelet-derived FVIII for hemophilia A treatment. PMID:20619307

New and Emerging Strategies in Platelet-Rich Plasma Application in Musculoskeletal Regenerative Procedures: General Overview on Still Open Questions and Outlook

PubMed Central

Veronesi, Francesca; Maglio, Melania; Sartori, Maria; Fini, Milena

2015-01-01

Despite its pervasive use, the clinical efficacy of platelet-rich plasma (PRP) therapy and the different mechanisms of action have yet to be established. This overview of the literature is focused on the role of PRP in bone, tendon, cartilage, and ligament tissue regeneration considering basic science literature deriving from in vitro and in vivo studies. Although this work provides evidence that numerous preclinical studies published within the last 10 years showed promising results concerning the application of PRP, many key questions remain unanswered and controversial results have arisen. Additional preclinical studies are needed to define the dosing, timing, and frequency of PRP injections, different techniques for delivery and location of delivery, optimal physiologic conditions for injections, and the concomitant use of recombinant proteins, cytokines, additional growth factors, biological scaffolds, and stems cells to develop optimal treatment protocols that can effectively treat various musculoskeletal conditions. PMID:26075269

Dynamin-related protein-1 controls fusion pore dynamics during platelet granule exocytosis.

PubMed

Koseoglu, Secil; Dilks, James R; Peters, Christian G; Fitch-Tewfik, Jennifer L; Fadel, Nathalie A; Jasuja, Reema; Italiano, Joseph E; Haynes, Christy L; Flaumenhaft, Robert

2013-03-01

Platelet granule exocytosis serves a central role in hemostasis and thrombosis. Recently, single-cell amperometry has shown that platelet membrane fusion during granule exocytosis results in the formation of a fusion pore that subsequently expands to enable the extrusion of granule contents. However, the molecular mechanisms that control platelet fusion pore expansion and collapse are not known. We identified dynamin-related protein-1 (Drp1) in platelets and found that an inhibitor of Drp1, mdivi-1, blocked exocytosis of both platelet dense and α-granules. We used single-cell amperometry to monitor serotonin release from individual dense granules and, thereby, measured the effect of Drp1 inhibition on fusion pore dynamics. Inhibition of Drp1 increased spike width and decreased prespike foot events, indicating that Drp1 influences fusion pore formation and expansion. Platelet-mediated thrombus formation in vivo after laser-induced injury of mouse cremaster arterioles was impaired after infusion of mdivi-1. These results demonstrate that inhibition of Drp1 disrupts platelet fusion pore dynamics and indicate that Drp1 can be targeted to control thrombus formation in vivo.

Selective protein adsorption modulates platelet adhesion and activation to oligo(ethylene glycol)-terminated self-assembled monolayers with C18 ligands.

PubMed

Gonçalves, Inês C; Martins, M Cristina L; Barbosa, Mário A; Naeemi, Esmaeel; Ratner, Buddy D

2009-06-01

This study focuses on the selective binding of albumin to a nanostructured surfaces to inhibit other blood proteins from adsorbing thereby reducing platelet adhesion and activation. Tetra (ethylene-glycol)-terminated self-assembled monolayers (EG4 SAMs) with different percentages of C18 ligands on the surface were characterized by contact angle measurements, X-ray photoelectron microscopy, infrared reflection-absorption spectroscopy, and ellipsometry. A specific surface (2.5% C18 SAM) was found to be selective for human serum albumin (HSA) in the presence of both albumin and fibrinogen (HFG). The importance of this concentration of C18 ligands was stressed in reversibility studies since that surface exchanged almost all the preadsorbed HSA by HSA in solution, but not by HFG. The effect of protein adsorption in the subsequent adhesion and activation of platelets was studied by pre-immersing the surfaces in albumin and plasma before contact with platelets. Scanning electron microscopy and glutaraldehyde induced fluorescence technique images showed that as surfaces got more hydrophobic due to the immobilization of C18 ligands, the number of adherent platelets increased and their morphology changed from round to fully spread. Pre-immersion in HSA led to an 80% decrease in platelet adhesion and reduction of activation. Pre-immersion in 1% plasma was only relevant in 2.5% C18 SAMs since this was the only surface that demonstrated less adhesion of platelets comparing with buffer pre-immersion. However, they still adsorb more platelets then when HSA was preadsorbed. This was confirmed in competition studies between HSA and plasma that suggested that other plasma proteins were also adsorbing to this surface. 2008 Wiley Periodicals, Inc.

Functional display of platelet-binding VWF fragments on filamentous bacteriophage.

PubMed

Yee, Andrew; Tan, Fen-Lai; Ginsburg, David

2013-01-01

von Willebrand factor (VWF) tethers platelets to sites of vascular injury via interaction with the platelet surface receptor, GPIb. To further define the VWF sequences required for VWF-platelet interaction, a phage library displaying random VWF protein fragments was screened against formalin-fixed platelets. After 3 rounds of affinity selection, DNA sequencing of platelet-bound clones identified VWF peptides mapping exclusively to the A1 domain. Aligning these sequences defined a minimal, overlapping segment spanning P1254-A1461, which encompasses the C1272-C1458 cystine loop. Analysis of phage carrying a mutated A1 segment (C1272/1458A) confirmed the requirement of the cystine loop for optimal binding. Four rounds of affinity maturation of a randomly mutagenized A1 phage library identified 10 and 14 unique mutants associated with enhanced platelet binding in the presence and absence of botrocetin, respectively, with 2 mutants (S1370G and I1372V) common to both conditions. These results demonstrate the utility of filamentous phage for studying VWF protein structure-function and identify a minimal, contiguous peptide that bind to formalin-fixed platelets, confirming the importance of the VWF A1 domain with no evidence for another independently platelet-binding segment within VWF. These findings also point to key structural elements within the A1 domain that regulate VWF-platelet adhesion.

Growth Arrest-Specific 6 (Gas6) and TAM Receptors in Mouse Platelets.

PubMed

Uras, Fikriye; Küçük, Burhanettin; Bingöl Özakpınar, Özlem; Demir, Ahmet Muzaffer

2015-03-05

Growth arrest-specific 6 (Gas6) is a newly discovered vitamin K-dependent protein, which is a ligand for TAM receptors [Tyro3 (Sky), Axl, and Mer] from the tyrosine kinase family. Gas6 knockout mice were resistant to venous and arterial thrombosis. There are contradictory reports on the presence of Gas6 and its receptors in mouse platelets. The objective of this study was to investigate whether Gas6 and its receptors were present in mouse platelets or not. Specific pathogen-free BALB/c male and female mice of 8-10 weeks old and 25-30 g in weight were anesthetized under light ether anesthesia and blood samples were taken from their hearts. RNAs were isolated from isolated platelets, and then mRNAs encoding Gas6 and TAM receptors were detected by reverse transcription-polymerase chain reaction (RT-PCR). Protein concentrations of Gas6 and TAM receptors in platelets were measured by ELISA, but not those of Mer, because of the absence of any commercial ELISA kit for mouse specimens. RT-PCR results indicated the presence of mRNAs encoding Gas6 and Mer in mouse platelets. However, although RT-PCR reactions were performed at various temperatures and cycles, we could not detect the presence of mRNAs encoding Axl and Tyro3 (Sky). Receptor protein levels of Axl and Tyro3 were below the detection limits of the ELISA method. We found the presence of mRNAs encoding Gas6 and the receptor Mer in mouse platelets, but not Axl and Tyro3. Gas6, Axl, and Tyro3 protein levels were below the detection limits of the ELISA. The presence of mRNA is not obvious evidence of protein expression in platelets that have no nucleus or DNA. Further studies are required to clarify the presence of Gas6/TAM receptors in platelets using real-time PCR and more sensitive immunological methods, and future studies on mechanisms will indicate whether the Gas6/TAM pathway is a strategy for treatment of disorders.

Blood platelet counts, morphology and morphometry in lions, Panthera leo.

PubMed

Du Plessis, L

2009-09-01

Due to logistical problems in obtaining sufficient blood samples from apparently healthy animals in the wild in order to establish normal haematological reference values, only limited information regarding the blood platelet count and morphology of free-living lions (Panthera leo) is available. This study provides information on platelet counts and describes their morphology with particular reference to size in two normal, healthy and free-ranging lion populations. Blood samples were collected from a total of 16 lions. Platelet counts, determined manually, ranged between 218 and 358 x 10(9)/l. Light microscopy showed mostly activated platelets of various sizes with prominent granules. At the ultrastructural level the platelets revealed typical mammalian platelet morphology. However, morphometric analysis revealed a significant difference (P < 0.001) in platelet size between the two groups of animals. Basic haematological information obtained in this study may be helpful in future comparative studies between animals of the same species as well as in other felids.

Influence of platelet aspect ratio on the mechanical behaviour of bio-inspired nanocomposites using molecular dynamics.

PubMed

Mathiazhagan, S; Anup, S

2016-06-01

Superior mechanical properties of biocomposites such as nacre and bone are attributed to their basic building blocks. These basic building blocks have nanoscale features and play a major role in achieving combined stiffening, strengthening and toughening mechanisms. Bioinspired nanocomposites based on these basic building blocks, regularly and stairwise staggered arrangements of hard platelets in soft matrix, have huge potential for developing advanced materials. The study of applicability of mechanical principles of biological materials to engineered materials will guide designing advanced materials. To probe the generic mechanical characteristics of these bioinspired nanocomposites, the model material concept in molecular dynamics (MD) is used. In this paper, the effect of platelets aspect ratio (AR) on the mechanical behaviour of bioinspired nanocomposites is investigated. The obtained Young׳s moduli of both the models and the strengths of the regularly staggered models agree with the available theories. However, the strengths of the stairwise staggered models show significant difference. For the stairwise staggered model, we demonstrate the existence of two critical ARs, a smaller critical AR above which platelet fracture occurs and a higher critical AR above which composite strength remains constant. Our MD study also shows the existence of mechanisms of platelet pull-out and breakage for lower and higher ARs. Pullout mechanism acts as a major source of plasticity. Further, we find that the regularly staggered model can achieve an optimal combination of high Young׳s modulus, flow strength and toughness, and the stairwise staggered model is efficient in obtaining high Young׳s modulus and tensile strength. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

Nitric oxide decreases coagulation protein function in rabbits as assessed by thromboelastography.

PubMed

Nielsen, V G

2001-02-01

Nitric oxide (NO) is administered via infusion of donors such as nitroglycerin or in inhaled form for treatment of ischemia and pulmonary hypertension, respectively. In rabbits, the NO donor, DETANONOate, decreases whole blood clotting function as assessed by thromboelastographic variables (R, reaction time; alpha, angle; and G, a measure of clot strength). I hypothesized that DETANONOate-derived NO would adversely affect coagulation protein and platelet function. Blood obtained from ear arteries of conscious rabbits (n = 8) anticoagulated with sodium citrate. The blood was then incubated with 0 or 10mM DETANONOate for 30 min. After incubation and recalcification, thromboelastography was performed for 60 min under four conditions: 1) 0mM DETANONOate, 2) 0mM DETANONOate with platelet inhibition with cytochalasin D, 3) 10mM DETANONOate, and 4) 10mM DETANONOate with platelet inhibition. DETANONOate significantly (P < 0.05) increased R and decreased alpha and G in samples with or without platelet inhibition, compared with samples not exposed to DETANONOate. Lastly, the percentage of total G (G(T)) attributable to platelet function (G(P)) was significantly more in the absence of DETANONOate (G(P) = 92.3% +/- 1.6%; mean +/- SD) than after exposure to DETANONOate (G(P) = 90.2% +/- 2.3%). DETANONOate-derived NO significantly decreased coagulation protein function and platelet function. Coagulation protein function may be similarly affected in clinical situations involving the administration of NO or NO donors.

Plasma-deposited tetraglyme surfaces greatly reduce total blood protein adsorption, contact activation, platelet adhesion, platelet procoagulant activity, and in vitro thrombus deposition.

PubMed

Cao, Lan; Chang, Mark; Lee, Chi-Ying; Castner, David G; Sukavaneshvar, Sivaprasad; Ratner, Buddy D; Horbett, Thomas A

2007-06-15

The ability of tetraethylene glycol dimethyl ether (tetraglyme) plasma deposited coatings exhibiting ultralow fibrinogen adsorption to reduce blood activation was studied with six in vitro methods, namely fibrinogen and von Willebrand's factor adsorption, total protein adsorption, clotting time in recalcified plasma, platelet adhesion and procoagulant activity, and whole blood thrombosis in a disturbed flow catheter model. Surface plasmon resonance results showed that tetraglyme surfaces strongly resisted the adsorption of all proteins from human plasma. The clotting time in the presence of tetraglyme surfaces was lengthened compared with controls, indicating a lower activation of the intrinsic coagulation cascade. Platelet adhesion and thrombin generation by adherent platelets were greatly reduced on tetraglyme-coated materials, compared with uncoated and Biospan-coated glass slides. In the in vitro disturbed blood flow model, tetraglyme plasma coated catheters had 50% less thrombus than did the uncoated catheters. Tetraglyme-coated materials thus had greatly reduced blood interactions as measured with all six methods. The improved blood compatibility of plasma-deposited tetraglyme is thus not only due to their reduced platelet adhesion and activation, but also to a generalized reduction in blood interactions. (c) 2007 Wiley Periodicals, Inc.

α-Synuclein Aggregated with Tau and β-Amyloid in Human Platelets from Healthy Subjects: Correlation with Physical Exercise

PubMed Central

Daniele, Simona; Pietrobono, Deborah; Fusi, Jonathan; Lo Gerfo, Annalisa; Cerri, Eugenio; Chico, Lucia; Iofrida, Caterina; Petrozzi, Lucia; Baldacci, Filippo; Giacomelli, Chiara; Galetta, Fabio; Siciliano, Gabriele; Bonuccelli, Ubaldo; Trincavelli, Maria L.; Franzoni, Ferdinando; Martini, Claudia

2018-01-01

The loss of protein homeostasis that has been associated with aging leads to altered levels and conformational instability of proteins, which tend to form toxic aggregates. In particular, brain aging presents characteristic patterns of misfolded oligomers, primarily constituted of β-amyloid (Aβ), tau, and α-synuclein (α-syn), which can accumulate in neuronal membranes or extracellular compartments. Such aging-related proteins can also reach peripheral compartments, thus suggesting the possibility to monitor their accumulation in more accessible fluids. In this respect, we have demonstrated that α-syn forms detectable hetero-aggregates with Aβ or tau in red blood cells (RBCs) of healthy subjects. In particular, α-syn levels and its heteromeric interactions are modulated by plasma antioxidant capability (AOC), which increases in turn with physical activity. In order to understand if a specific distribution of misfolded proteins can occur in other blood cells, a cohort of human subjects was enrolled to establish a correlation among AOC, the level of physical exercise and the concentrations of aging-related proteins in platelets. The healthy subjects were divided depending on their level of physical exercise (i.e., athletes and sedentary subjects) and their age (young and older subjects). Herein, aging-related proteins (i.e., α-syn, tau and Aβ) were confirmed to be present in human platelets. Among such proteins, platelet tau concentration was demonstrated to decrease in athletes, while α-syn and Aβ did not correlate with physical exercise. For the first time, α-syn was shown to directly interact with Aβ and tau in platelets, forming detectable hetero-complexes. Interestingly, α-syn interaction with tau was inversely related to plasma AOC and to the level of physical activity. These results suggested that α-syn heterocomplexes, particularly with tau, could represent novel indicators to monitor aging-related proteins in platelets. PMID:29441013

Single-step isolation of extracellular vesicles by size-exclusion chromatography

PubMed Central

Böing, Anita N.; van der Pol, Edwin; Grootemaat, Anita E.; Coumans, Frank A. W.; Sturk, Auguste; Nieuwland, Rienk

2014-01-01

Background Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively. Aim To develop a single-step protocol to isolate vesicles from human body fluids. Methods Platelet-free supernatant, derived from platelet concentrates, was loaded on a sepharose CL-2B column to perform size-exclusion chromatography (SEC; n=3). Fractions were collected and analysed by nanoparticle tracking analysis, resistive pulse sensing, flow cytometry and transmission electron microscopy. The concentrations of high-density lipoprotein cholesterol (HDL) and protein were measured in each fraction. Results Fractions 9–12 contained the highest concentrations of particles larger than 70 nm and platelet-derived vesicles (46%±6 and 61%±2 of totals present in all collected fractions, respectively), but less than 5% of HDL and less than 1% of protein (4.8%±1 and 0.65%±0.3, respectively). HDL was present mainly in fractions 18–20 (32%±2 of total), and protein in fractions 19–21 (36%±2 of total). Compared to the starting material, recovery of platelet-derived vesicles was 43%±23 in fractions 9–12, with an 8-fold and 70-fold enrichment compared to HDL and protein. Conclusions SEC efficiently isolates extracellular vesicles with a diameter larger than 70 nm from platelet-free supernatant of platelet concentrates. Application SEC will improve studies on the dimensional, structural and functional properties of extracellular vesicles. PMID:25279113

R1: Platelets and Megakaryocytes contain functional NF-κB

PubMed Central

Spinelli, Sherry L.; Casey, Ann E.; Pollock, Stephen J.; Gertz, Jacqueline M.; McMillan, David H.; Narasipura, Srinivasa D.; Mody, Nipa A.; King, Michael R.; Maggirwar, Sanjay B.; Francis, Charles W.; Taubman, Mark B.; Blumberg, Neil; Phipps, Richard P.

2010-01-01

The Nuclear Factor (NF)-κB transcription factor family is well-known for their role in eliciting inflammation and promoting cell survival. We discovered that human megakaryocytes and platelets express the majority of NF-κB family members including the regulatory Inhibitor (I)-κB and Inhibitor Kappa Kinase (IKK) molecules. Objective Investigate the presence and role of NF-κB proteins in megakaryocytes and platelets. Methods and Results Anucleate platelets exposed to NF-κB inhibitors demonstrated impaired fundamental functions involved in repairing vascular injury and thrombus formation. Specifically, NF-κB inhibition diminished lamellapodia formation, decreased clot retraction times and reduced thrombus stability. Moreover, inhibition of I-κB-α phosphorylation (BAY-11-7082) reverts fully spread platelets back to a spheroid morphology. Addition of recombinant IKK-β or I-κB-α protein to BAY inhibitor-treated platelets partially restore platelet spreading in I-κB-α inhibited platelets, and addition of active IKK-β increased endogenous I-κB-α phosphorylation levels. Conclusions These novel findings support a crucial and non-classical role for the NF-κB family in modulating platelet function and reveal that platelets are sensitive to NF-κB inhibitors. As NF-κB inhibitors are being developed as anti-inflammatory and anti-cancer agents, they may have unintended effects on platelets. Based on these data, NF-κB is also identified as a new target to dampen unwanted platelet activation. PMID:20042710

Comparative lipidomics and proteomics analysis of platelet lipid rafts using different detergents.

PubMed

Rabani, Vahideh; Davani, Siamak; Gambert-Nicot, Ségolène; Meneveau, Nicolas; Montange, Damien

2016-11-01

Lipid rafts play a pivotal role in physiological functions of platelets. Their isolation using nonionic mild detergents is considered as the gold standard method, but there is no consensual detergent for lipid raft studies. We aimed to investigate which detergent is the most suitable for lipid raft isolation from platelet membrane, based on lipidomics and proteomics analysis. Platelets were obtained from healthy donors. Twelve sucrose fractions were extracted by three different detergents, namely Brij 35, Lubrol WX, and Triton X100, at 0.05% and 1%. After lipidomics analysis and determination of fractions enriched in cholesterol (Ch) and sphingomyelin (SM), proteomics analysis was performed. Lipid rafts were mainly observed in 1-4 fractions, and non-rafts were distributed on 5-12 fractions. Considering the concentration of Ch and SM, Lubrol WX 1% and Triton X100 1% were more suitable detergents as they were able to isolate lipid raft fractions that were more enriched than non-raft fractions. By proteomics analysis, overall, 822 proteins were identified in platelet membrane. Lipid raft fractions isolated with Lubrol WX 0.05% and Triton X100 1% contained mainly plasma membrane proteins. However, only Lubrol WX 0.05 and 1% and Triton X100 1% were able to extract non-denaturing proteins with more than 10 transmembrane domains. Our results suggest that Triton X100 1% is the most suitable detergent for global lipid and protein studies on platelet plasma membrane. However, the detergent should be adapted if investigation of an association between specific proteins and lipid rafts is planned.

Molecular mimicry by Helicobacter pylori CagA protein may be involved in the pathogenesis of H. pylori-associated chronic idiopathic thrombocytopenic purpura.

PubMed

Takahashi, Toru; Yujiri, Toshiaki; Shinohara, Kenji; Inoue, Yusuke; Sato, Yutaka; Fujii, Yasuhiko; Okubo, Masashi; Zaitsu, Yuzuru; Ariyoshi, Koichi; Nakamura, Yukinori; Nawata, Ryouhei; Oka, Yoshitomo; Shirai, Mutsunori; Tanizawa, Yukio

2004-01-01

The eradication of Helicobacter pylori often leads to platelet recovery in patients with chronic idiopathic thrombocytopenic purpura (cITP). Although this clinical observation suggests the involvement of H. pylori, little is known about the pathogenesis of cITP. We initially examined the effect of H. pylori eradication on platelet counts in 20 adult Japanese cITP patients. Then, using platelet eluates as the probe in immunoblot analyses, we examined the role of molecular mimicry in the pathogenesis of cITP. Helicobacter pylori infection was detected in 75% (15 of 20) of cITP patients. Eradication was achieved in 13 (87%) of the H. pylori-positive patients, seven (54%) of which showed increased platelet counts within the 4 months following treatment. Completely responsive patients also showed significant declines in platelet-associated immunoglobulin G (PAIgG) levels. Platelet eluates from 12 (nine H. pylori-positive and three H. pylori-negative) patients recognized H. pylori cytotoxin-associated gene A (CagA) protein, and in three completely responsive patients, levels of anti-CagA antibody in platelet eluates declined after eradication therapy. Cross-reactivity between PAIgG and H. pylori CagA protein suggests that molecular mimicry by CagA plays a key role in the pathogenesis of a subset of cITP patients.

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.

Carboxyl‐terminal Heparin‐binding Fragments of Platelet Factor 4 Retain the Blocking Effect on the Receptor Binding of Basic Fibroblast Growth Factor

PubMed Central

Waki, Michinori; Ohno, Motonori; Kuwano, Michihiko; Sakata, Toshiie

1993-01-01

Platelet factor 4 (PF‐4) blocks the binding of basic fibroblast growth factor (bFGF) to its receptor. In the present study, we constructed carboxyl‐terminal fragments, which represent the heparin‐binding region of the PF‐4 molecule, and examined whether these synthetic peptides retain the blocking effects on the receptor binding of bFGF. Synthetic peptides inhibited the receptor binding of bFGF. Furthermore, they inhibited the migration and tube formation of bovine capillary endothelial cells in culture (these phenomena are dependent on endogenous bFGF). PMID:8320164

Procoagulant Platelets Form an α-Granule Protein-covered “Cap” on Their Surface That Promotes Their Attachment to Aggregates*

PubMed Central

Abaeva, Anastasia A.; Canault, Matthias; Kotova, Yana N.; Obydennyy, Sergey I.; Yakimenko, Alena O.; Podoplelova, Nadezhda A.; Kolyadko, Vladimir N.; Chambost, Herve; Mazurov, Aleksei V.; Ataullakhanov, Fazoil I.; Nurden, Alan T.; Alessi, Marie-Christine; Panteleev, Mikhail A.

2013-01-01

Strongly activated “coated” platelets are characterized by increased phosphatidylserine (PS) surface expression, α-granule protein retention, and lack of active integrin αIIbβ3. To study how they are incorporated into thrombi despite a lack of free activated integrin, we investigated the structure, function, and formation of the α-granule protein “coat.” Confocal microscopy revealed that fibrin(ogen) and thrombospondin colocalized as “cap,” a single patch on the PS-positive platelet surface. In aggregates, the cap was located at the point of attachment of the PS-positive platelets. Without fibrin(ogen) retention, their ability to be incorporated in aggregates was drastically reduced. The surface fibrin(ogen) was strongly decreased in the presence of a fibrin polymerization inhibitor GPRP and also in platelets from a patient with dysfibrinogenemia and a fibrinogen polymerization defect. In contrast, a fibrinogen-clotting protease ancistron increased the amount of fibrin(ogen) and thrombospondin on the surface of the PS-positive platelets stimulated with collagen-related peptide. Transglutaminases are also involved in fibrin(ogen) retention. However, platelets from patients with factor XIII deficiency had normal retention, and a pan-transglutaminase inhibitor T101 had only a modest inhibitory effect. Fibrin(ogen) retention was normal in Bernard-Soulier syndrome and kindlin-3 deficiency, but not in Glanzmann thrombasthenia lacking the platelet pool of fibrinogen and αIIbβ3. These data show that the fibrin(ogen)-covered cap, predominantly formed as a result of fibrin polymerization, is a critical mechanism that allows coated (or rather “capped”) platelets to become incorporated into thrombi despite their lack of active integrins. PMID:23995838

Estriol-induced fibrinolysis due to the activation of plasminogen to plasmin by nitric oxide synthesis in platelets.

PubMed

Jana, Pradipta; Maiti, Smarajit; Kahn, Nighat N; Sinha, Asru K

2015-04-01

Estriol, an oestrogen, at 0.6 nmol/l was reported to inhibit ADP-induced platelet aggregation through nitric oxide synthesis. As nitric oxide has been reported to cause fibrinolysis due to the activation of plasminogen to plasmin, the role of estriol as a fibrinolytic agent was investigated. Also, the mechanism of estriol-induced nitric oxide synthesis in anucleated platelets was investigated. The estriol-induced lysis of platelet-rich plasma (PRP) clot was determined by photography of the clot lysis and by the assay of fibrin degradation products in the lysate and was obtained by SDS-PAGE. Nitric oxide was determined by methemoglobin method. The platelet membrane protein was isolated from the platelets by using Triton X-100 (0.05% v/v). The binding of estriol to the protein was determined by Scatchard plot by using an ELISA for estriol. Estriol at 0.6 nmol/l was found to lyse the clotted PRP due to fibrinolysis that produced fibrin degradation products in the lysate. The amino acid analysis of the platelet membrane protein, which resembles with nitric oxide synthase (NOS) activity, was activated nearly 10-fold over the control in the presence of estriol and was identified to be a human serum albumin precursor (Mr. 69 kDa) that binds to estriol with Kd1 of 6.0 × 10 mol/l and 39 ± 2 molecules of estriol bound the NOS molecule. The estriol-induced nitric oxide is capable of inducing fibrinolysis of the clotted PRP. The binding of estriol to platelet membrane NOS activated the enzyme in the absence of DNA in the platelet.

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.

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.

Concerted functions of Streptococcus gordonii surface proteins PadA and Hsa mediate activation of human platelets and interactions with extracellular matrix.

PubMed

Haworth, Jennifer A; Jenkinson, Howard F; Petersen, Helen J; Back, Catherine R; Brittan, Jane L; Kerrigan, Steve W; Nobbs, Angela H

2017-01-01

A range of Streptococcus bacteria are able to interact with blood platelets to form a thrombus (clot). Streptococcus gordonii is ubiquitous within the human oral cavity and amongst the common pathogens isolated from subjects with infective endocarditis. Two cell surface proteins, Hsa and Platelet adherence protein A (PadA), in S. gordonii mediate adherence and activation of platelets. In this study, we demonstrate that PadA binds activated platelets and that an NGR (Asparagine-Glycine-Arginine) motif within a 657 amino acid residue N-terminal fragment of PadA is responsible for this, together with two other integrin-like recognition motifs RGT and AGD. PadA also acts in concert with Hsa to mediate binding of S. gordonii to cellular fibronectin and vitronectin, and to promote formation of biofilms. Evidence is presented that PadA and Hsa are each reliant on the other's active presentation on the bacterial cell surface, suggesting cooperativity in functions impacting both colonization and pathogenesis. © 2016 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.

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

Rho GTPases and their downstream effectors in megakaryocyte biology.

PubMed

Pleines, Irina; Cherpokova, Deya; Bender, Markus

2018-06-18

Megakaryocytes differentiate from hematopoietic stem cells in the bone marrow. The transition of megakaryocytes to platelets is a complex process. Thereby, megakaryocytes extend proplatelets into sinusoidal blood vessels, where the proplatelets undergo fission to release platelets. Defects in platelet production can lead to a low platelet count (thrombocytopenia) with increased bleeding risk. Rho GTPases comprise a family of small signaling G proteins that have been shown to be master regulators of the cytoskeleton controlling many aspects of intracellular processes. The generation of Pf4-Cre transgenic mice was a major breakthrough that enabled studies in megakaryocyte-/platelet-specific knockout mouse lines and provided new insights into the central regulatory role of Rho GTPases in megakaryocyte maturation and platelet production. In this review, we will summarize major findings on the role of Rho GTPases in megakaryocyte biology with a focus on mouse lines in which knockout strategies have been applied to study the function of the best-characterized members Rac1, Cdc42 and RhoA and their downstream effector proteins.

Coagulation parameters and platelet function analysis in patients with acromegaly.

PubMed

Colak, A; Yılmaz, H; Temel, Y; Demirpence, M; Simsek, N; Karademirci, İ; Bozkurt, U; Yasar, E

2016-01-01

Acromegaly is associated with increased cardiovascular morbidity and mortality. The data about the evaluation of coagulation and fibrinolysis in acromegalic patients are very limited and to our knowledge, platelet function analysis has never been investigated. So, we aimed to investigate the levels of protein C, protein S, fibrinogen, antithrombin 3 and platelet function analysis in patients with acromegaly. Thirty-nine patients with active acromegaly and 35 healthy subjects were included in the study. Plasma glucose and lipid profile, fibrinogen levels, GH and IGF-1 levels and protein C, protein S and antithrombin III activities were measured in all study subjects. Also, platelet function analysis was evaluated with collagen/ADP and collagen-epinephrine-closure times. Demographic characteristics of the patient and the control were similar. As expected, fasting blood glucose levels and serum GH and IGF-1 levels were significantly higher in the patient group compared with the control group (pglc: 0.002, pGH: 0.006, pIGF-1: 0.001, respectively). But lipid parameters were similar between the two groups. While serum fibrinogen and antithrombin III levels were found to be significantly higher in acromegaly group (p fibrinogen: 0.005 and pantithrombin III: 0.001), protein S and protein C activity values were significantly lower in the patient group (p protein S: 0.001, p protein C: 0.001). Also significantly enhanced platelet function (measured by collagen/ADP- and collagen/epinephrine-closure times) was demonstrated in acromegaly (p col-ADP: 0.002, p col-epinephrine: 0.002). The results did not change, when we excluded six patients with type 2 diabetes in the acromegaly group. There was a negative correlation between serum GH levels and protein S (r: -0.25, p: 0.04)) and protein C (r: -0.26, p: 0.04) values. Likewise, there was a negative correlation between IGF-1 levels and protein C values (r: -0.39, p: 0.002), protein S values (r: -0.39, p: 0.001), collagen/ADP-closure times (r: -0.28, p: 0.02) and collagen/epinephrine-closure times (r:-0.26, p: 0.04). Also, we observed a positive correlation between IGF-1 levels and fibrinogen levels (r: 0.31, p: 0.01). Acromegaly was found to be associated with increased tendency to coagulation and enhanced platelet activity. This hypercoagulable state might increase the risk for cardiovascular and cerebrovascular events in acromegaly.

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.

Serotoninergic antidepressants positively affect platelet ADAM10 expression in patients with Alzheimer's disease.

PubMed

Bianco, Otávio Augusto Fernandes Marques; Manzine, Patrícia Regina; Nascimento, Carla Manuela Crispim; Vale, Francisco Assis Carvalho; Pavarini, Sofia Cristina Iost; Cominetti, Márcia Regina

2016-06-01

Studies have demonstrated a decreased platelet ADAM10 expression in patients with Alzheimer's Disease (AD), classifying this protein as a blood-based AD biomarker. About 50% of the patients with AD are diagnosed with depression, which is commonly treated with tricyclic and tetracyclic antidepressants, monoaminoxidade (MAO) inhibitors and, more preferably, with selective serotonin reuptake inhibitors (SSRIs). Considering that a large proportion of patients with AD takes antidepressant medications during the course of the disease we investigated the influence of this medication on the expression of platelet ADAM10, which is considered the main α-secretase preventing beta-amyloid (βA) formation. Blood was collected for protein extraction from platelets. ADAM10 was analyzed by using western blotting and reactive bands were measured using β-actin as endogenous control. Platelet ADAM10 protein expression in patients with AD was positively influenced by serotoninergic medication. More studies on the positive effects of serotonergic antidepressants on ADAM10 platelet expression should be performed in order to understand its biological mechanisms and to verify whether these effects are reflected in the central nervous system. This work represents an important advance for the study of AD biomarkers, as well as for more effective pharmacological treatment of patients with AD and associated depression.

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

A role for SNAP-25 but not VAMPs in store-mediated Ca2+ entry in human platelets

PubMed Central

Redondo, Pedro C; Harper, Alan G S; Salido, Ginés M; Pariente, Jose A; Sage, Stewart O; Rosado, Juan A

2004-01-01

Store-mediated Ca2+ entry (SMCE) is a major mechanism for Ca2+ influx in non-excitable cells. Recently, a conformational coupling mechanism allowing coupling between transient receptor potential channels (TRPCs) and IP3 receptors has been proposed to activate SMCE. Here we have investigated the role of two soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs), which are involved in membrane trafficking and docking, in SMCE in human platelets. We found that the synaptosome-associated protein (SNAP-25) and the vesicle-associated membrane proteins (VAMP) coimmunoprecipitate with hTRPC1 in platelets. Treatment with botulinum toxin (BoNT) E or with tetanus toxin (TeTx), induced cleavage and inactivation of SNAP-25 and VAMPs, respectively. BoNTs significantly reduced thapsigargin- (TG) and agonist-evoked SMCE. Treatment with BoNTs once SMCE had been activated decreased Ca2+ entry, indicating that SNAP-25 is required for the activation and maintenance of SMCE. In contrast, treatment with TeTx had no effect on either the activation or the maintenance of SMCE in platelets. Finally, treatment with BoNT E impaired the coupling between naturally expressed hTRPC1 and IP3 receptor type II in platelets. From these findings we suggest SNAP-25 has a role in SMCE in human platelets. PMID:15121806

Autonomous role of Wiskott-Aldrich syndrome platelet deficiency in inducing autoimmunity and inflammation.

PubMed

Sereni, Lucia; Castiello, Maria Carmina; Marangoni, Francesco; Anselmo, Achille; di Silvestre, Dario; Motta, Sara; Draghici, Elena; Mantero, Stefano; Thrasher, Adrian J; Giliani, Silvia; Aiuti, Alessandro; Mauri, Pierluigi; Notarangelo, Luigi D; Bosticardo, Marita; Villa, Anna

2018-02-06

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by eczema, infections, and susceptibility to autoimmunity and malignancies. Thrombocytopenia is a constant finding, but its pathogenesis remains elusive. To dissect the basis of the WAS platelet defect, we used a novel conditional mouse model (CoWas) lacking Wiskott-Aldrich syndrome protein (WASp) only in the megakaryocytic lineage in the presence of a normal immunologic environment, and in parallel we analyzed samples obtained from patients with WAS. Phenotypic and functional characterization of megakaryocytes and platelets in mutant CoWas mice and patients with WAS with and without autoantibodies was performed. Platelet antigen expression was examined through a protein expression profile and cluster proteomic interaction network. Platelet immunogenicity was tested by using ELISAs and B-cell and platelet cocultures. CoWas mice showed increased megakaryocyte numbers and normal thrombopoiesis in vitro, but WASp-deficient platelets had short lifespan and high expression of activation markers. Proteomic analysis identified signatures compatible with defects in cytoskeletal reorganization and metabolism yet surprisingly increased antigen-processing capabilities. In addition, WASp-deficient platelets expressed high levels of surface and soluble CD40 ligand and were capable of inducing B-cell activation in vitro. WASp-deficient platelets were highly immunostimulatory in mice and triggered the generation of antibodies specific for WASp-deficient platelets, even in the context of a normal immune system. Patients with WAS also showed platelet hyperactivation and increased plasma soluble CD40 ligand levels correlating with the presence of autoantibodies. Overall, these findings suggest that intrinsic defects in WASp-deficient platelets decrease their lifespan and dysregulate immune responses, corroborating the role of platelets as modulators of inflammation and immunity. Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

ADAP deficiency impairs megakaryocyte polarization with ectopic proplatelet release and causes microthrombocytopenia.

PubMed

Spindler, Markus; van Eeuwijk, Judith M M; Schurr, Yvonne; Nurden, Paquita; Nieswandt, Bernhard; Stegner, David; Reinhold, Annegret; Bender, Markus

2018-06-27

Bone marrow megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in thrombopoiesis can lead to thrombocytopenia associated with increased bleeding tendency. Recently, the platelet disorder congenital autosomal recessive small-platelet thrombocytopenia (CARST) was described which is caused by mutations in the ADAP (Adhesion and degranulation promoting adaptor protein; synonym: FYB, SLAP130/120) gene, and characterized by microthrombocytopenia and bleeding symptoms. In this study we used constitutive ADAP-deficient mice (Adap -/- ) as a model to investigate mechanisms underlying the microthrombocytopenia in CARST. We show that Adap -/- mice display several characteristics of human CARST, with moderate thrombocytopenia and smaller-sized platelets. Adap -/- platelets had a shorter life span than control platelets, and macrophage depletion, but not splenectomy, increased platelet counts in mutant mice to control levels. Whole sternum 3D confocal imaging and intravital two-photon microscopy revealed altered morphology of ADAP-deficient MKs with signs of fragmentation and ectopic release of (pro)platelet-like particles into the bone marrow compartment. In addition, cultured bone marrow-derived MKs lacking ADAP showed reduced spreading on extracellular matrix proteins as well as activation of β1 integrins, impaired podosome formation, and displayed defective polarization of the demarcation membrane system in vitro. MK-/platelet-specific ADAP deficient mice (PF4-cre) also produced less and smaller-sized platelets and released platelets ectopically. These data demonstrate that the abnormal platelet production in the mutant mice is a MK-intrinsic defect. Taken together, these results point to a so far unidentified role of ADAP in the process of MK polarization and platelet biogenesis. Copyright © 2018 American Society of Hematology.

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.

Photoaffinity labelling of cyclic GMP-inhibited phosphodiesterase (PDE III) in human and rat platelets and rat tissues: effects of phosphodiesterase inhibitors.

PubMed

Tang, K M; Jang, E K; Haslam, R J

1994-06-15

Ultraviolet irradiation of human platelet cytosol in the presence of 32P-labelled cyclic GMP (cGMP) can specifically label 110, 80, 55, 49 and 38 kDa proteins; the 110 kDa species is the subunit of cGMP-inhibited phosphodiesterase (PDE III) and the 80 kDa species that of cGMP-dependent protein kinase (Tang et al., 1993, Biochem. J. 294, 329). We have now shown that although photolabelling of platelet PDE III was inhibited by unlabelled cGMP, 8-bromo-cGMP and cyclic AMP (cAMP), it was not affected by phosphorothioate analogues of these cyclic nucleotides. Specific concentration-dependent inhibitions of the photolabelling of PDE III were observed with the following PDE inhibitors: trequinsin (IC50 = 13 +/- 2 nM), lixazinone (IC50 = 22 +/- 4 nM), milrinone (IC50 = 56 +/- 12 nM), cilostamide (IC50 = 70 +/- 9 nM), siguazodan (IC50 = 117 +/- 29 nM) and 3-isobutyl 1-methylxanthine (IBMX) (IC50 = 3950 +/- 22 nM). Thus, measurements of the inhibitory effects of compounds on the photolabelling of platelet PDE III provide a simple quantitative means of investigating their actions at a molecular level that avoids the need to purify the enzyme. Photolabelling of rat platelet lysate or rat heart homogenate by [32P]cGMP showed that the 110 kDa PDE III present in human material was replaced by a 115 kDa protein, labelling of which was also blocked by PDE III inhibitors. Heart and other rat tissues contained much less of this putative 115 kDa PDE III than rat platelets. In contrast, the 80 kDa protein was labelled much less in platelets than in many other rat tissue homogenates (e.g., heart, aorta, uterus and lung). Thus, comparison of the relative amounts of specific photolabelled proteins in different cells may provide an indication of different patterns of cyclic nucleotide action. We compared the abilities of phosphodiesterase inhibitors to block the photolabelling of PDE III in human platelet cytosol and to increase the iloprost-stimulated accumulation of cAMP in intact platelets. Whereas trequinsin (EC50 = 19 +/- 3 nM), lixazinone (EC50 = 122 +/- 8 nM), milrinone (EC50 = 5320 +/- 970 nM) and siguazodan (EC50 = 18880 +/- 3110 nM) all increased platelet cAMP to the same maximum extent, cilostamide and IBMX increased cAMP further, indicating that they inhibited a PDE isozyme in addition to PDE III.

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.

A New Poly(Methyl Methacrylate) Membrane Dialyzer, NF, with Adsorptive and Antithrombotic Properties.

PubMed

Oshihara, Wataru; Fujieda, Hiroaki; Ueno, Yoshiyuki

2017-01-01

Poly(methyl methacrylate) (PMMA) membranes adsorb several kinds of proteins and can remove high-molecular-weight proteins, including uremic toxins, which are not removed efficiently by hemodialysis or hemodiafiltration. However, the antithrombogenicity of PMMA membranes is insufficient due to their adsorptive properties. Coagulation during hemodialysis occurs because proteins that are adsorbed to the PMMA membrane undergo structural changes and are recognized by platelets, which are then activated by adhesion to the membrane surface. In developing a new PMMA membrane dialyzer, NF, we intended to inhibit platelet adhesion to the membrane surface by suppressing the structural change in the proteins adsorbed on the membrane. In addition, we give examples of clinical trials of the NF in Japan and describe its advantages. Key Message: PMMA membrane dialyzers have been used for 40 years. The PMMA dialyzer NF can suppress the adhesion of platelets to the membrane while maintaining protein adsorption. © 2017 S. Karger AG, Basel.

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.

Functional Divergence of Platelet Protein Kinase C (PKC) Isoforms in Thrombus Formation on Collagen*

PubMed Central

Gilio, Karen; Harper, Matthew T.; Cosemans, Judith M. E. M.; Konopatskaya, Olga; Munnix, Imke C. A.; Prinzen, Lenneke; Leitges, Michael; Liu, Qinghang; Molkentin, Jeffery D.; Heemskerk, Johan W. M.; Poole, Alastair W.

2010-01-01

Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen. PMID:20479008

Functional divergence of platelet protein kinase C (PKC) isoforms in thrombus formation on collagen.

PubMed

Gilio, Karen; Harper, Matthew T; Cosemans, Judith M E M; Konopatskaya, Olga; Munnix, Imke C A; Prinzen, Lenneke; Leitges, Michael; Liu, Qinghang; Molkentin, Jeffery D; Heemskerk, Johan W M; Poole, Alastair W

2010-07-23

Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent alpha-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCalpha and PKCbeta, whereas the novel isoform, PKC, negatively regulates these events. PKCdelta also negatively regulates thrombus formation but not alpha-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCalpha or PKCbeta showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKC. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.

Tyrosine phosphorylated c-Cbl regulates platelet functional responses mediated by outside-in signaling

PubMed Central

Buitrago, Lorena; Langdon, Wallace Y.

2011-01-01

c-Cbl protein functions as an E3 ligase and scaffolding protein, where 3 residues, Y700, Y731, and Y774, upon phosphorylation, have been shown to initiate several signaling cascades. In this study, we investigated the role of these phospho-tyrosine residues in the platelet functional responses after integrin engagement. We observed that c-Cbl Y700, Y731 and Y774 undergo phosphorylation upon platelet adhesion to immobilized fibrinogen, which was inhibited in the presence of PP2, a pan-src family kinase (SFK) inhibitor, suggesting that c-Cbl is phosphorylated downstream of SFKs. However, OXSI-2, a Syk inhibitor, significantly reduced c-Cbl phosphorylation at residues Y774 and Y700, without affecting Y731 phosphorylation. Interestingly, PP2 inhibited both platelet-spreading on fibrinogen as well as clot retraction, whereas OXSI-2 blocked only platelet-spreading, suggesting a differential role of these tyrosine residues. The physiologic role of c-Cbl and Y731 was studied using platelets from c-Cbl KO and c-CblYF/YF knock-in mice. c-Cbl KO and c-CblYF/YF platelets had a significantly reduced spreading over immobilized fibrinogen. Furthermore, clot retraction with c-Cbl KO and c-CblYF/YF platelets was drastically delayed. These results indicate that c-Cbl and particularly its phosphorylated residue Y731 plays an important role in platelet outside-in signaling contributing to platelet-spreading and clot retraction. PMID:21967979

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.

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.

Regulation of platelet granule exocytosis by S-nitrosylation

PubMed Central

Morrell, Craig N.; Matsushita, Kenji; Chiles, Kelly; Scharpf, Robert B.; Yamakuchi, Munekazu; Mason, Rebecca J. A.; Bergmeier, Wolfgang; Mankowski, Joseph L.; Baldwin, William M.; Faraday, Nauder; Lowenstein, Charles J.

2005-01-01

Nitric oxide (NO) regulates platelet activation by cGMP-dependent mechanisms and by mechanisms that are not completely defined. Platelet activation includes exocytosis of platelet granules, releasing mediators that regulate interactions between platelets, leukocytes, and endothelial cells. Exocytosis is mediated in part by N-ethylmaleimide-sensitive factor (NSF), an ATPase that disassembles complexes of soluble NSF attachment protein receptors. We now demonstrate that NO inhibits exocytosis of dense granules, lysosomal granules, and α-granules from human platelets by S-nitrosylation of NSF. Platelets lacking endothelial NO synthase show increased rolling on venules, increased thrombosis in arterioles, and increased exocytosis in vivo. Regulation of exocytosis is thus a mechanism by which NO regulates thrombosis. PMID:15738422

Platelets promote cartilage repair and chondrocyte proliferation via ADP in a rodent model of osteoarthritis.

PubMed

Zhou, Qi; Xu, Chunhua; Cheng, Xingyao; Liu, Yangyang; Yue, Ming; Hu, Mengjiao; Luo, Dongjiao; Niu, Yuxi; Ouyang, Hongwei; Ji, Jiansong; Hu, Hu

2016-01-01

Osteoarthritis (OA) is the most common age-related degenerative joint disease and platelet-rich plasma (PRP) has been shown to be beneficial in OA. Therefore, in this study, we aimed to investigate the effects of platelets on chondrocytes and the underlying mechanisms. Anabolic and catabolic activity and the proliferation rate of chondrocytes were evaluated after co-culture with platelets. Chondrocyte gene expression was measured by real-time PCR. Chondrocyte protein expression and phosphorylation were measured by western blot. Chondrocytes treated with or without platelets were transplanted into a rat model of OA induced by intra-articular injection of monosodium iodoacetate and the repair of articular cartilage was evaluated macroscopically and histologically. Platelets significantly promoted the proliferation of chondrocytes, while mildly influencing anabolic and catabolic activity. Chondrocytes co-cultured with platelets showed significantly increased production of bone morphogenetic protein 7 (BMP7). The autocrine/paracrine effect of BMP7 was responsible for the increased proliferation of chondrocytes, via the ERK/CDK1/cyclin B1 signaling pathway. Transplantation of platelet-treated chondrocytes showed better cartilage repair in the OA model. Platelet-derived ADP was identified as the major mediator to promote the production of BMP7 and the proliferation of chondrocytes, through the ADP receptor P2Y1. Finally, direct injection of α,β-methyleneadenosine-5'-diphosphate into OA joints also enhanced cartilage repair. This study has identified that platelet-derived ADP, but not ATP, is the key mediator for platelet-promoted chondrocyte proliferation and cartilage repair in osteoarthritis. This finding may provide a key explanation for the therapeutic effect of platelets in OA and help shaping a strategy to improve OA therapy.

C-reactive protein, platelets, and patent ductus arteriosus.

PubMed

Meinarde, Leonardo; Hillman, Macarena; Rizzotti, Alina; Basquiera, Ana Lisa; Tabares, Aldo; Cuestas, Eduardo

2016-12-01

The association between inflammation, platelets, and patent ductus arteriosus (PDA) has not been studied so far. The purpose of this study was to evaluate whether C-reactive protein (CRP) is related to low platelet count and PDA. This was a retrospective study of 88 infants with a birth weight ≤1500 g and a gestational age ≤30 weeks. Platelet count, CRP, and an echocardiogram were assessed in all infants. The subjects were matched by sex, gestational age, and birth weight. Differences were compared using the χ 2 , t-test, or Mann-Whitney U-test, as appropriate. Significant variables were entered into a logistic regression model. The association between CRP and platelets was evaluated by correlation and regression analysis. Platelet count (167 000 vs. 213 000 µl -1 , p = 0.015) was lower and the CRP (0.45 vs. 0.20 mg/dl, p = 0.002) was higher, and the platelet count correlated inversely with CRP (r = -0.145, p = 0.049) in the infants with vs. without PDA. Only CRP was independently associated with PDA in a logistic regression model (OR 64.1, 95% confidence interval 1.4-2941, p = 0.033).

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.

Platelet-Rich Blood Derivatives for Stem Cell-Based Tissue Engineering and Regeneration

PubMed Central

Kaushik, Gaurav; Leijten, Jeroen; Khademhosseini, Ali

2016-01-01

Platelet rich blood derivatives have been widely used in different fields of medicine and stem cell based tissue engineering. They represent natural cocktails of autologous growth factor, which could provide an alternative for recombinant protein based approaches. Platelet rich blood derivatives, such as platelet rich plasma, have consistently shown to potentiate stem cell proliferation, migration, and differentiation. Here, we review the spectrum of platelet rich blood derivatives, discuss their current applications in tissue engineering and regenerative medicine, reflect on their effect on stem cells, and highlight current translational challenges. PMID:27047733

Interaction between the Staphylococcus aureus extracellular adherence protein Eap and its subdomains with platelets.

PubMed

Palankar, Raghavendra; Binsker, Ulrike; Haracska, Bianca; Wesche, Jan; Greinacher, Andreas; Hammerschmidt, Sven

2018-04-18

S. aureus associated bacteremia can lead to severe infections with high risk of mortality (e.g. sepsis, infective endocarditis). Many virulence factors and adhesins of S. aureus are known to directly interact with platelets. Extracellular adherence protein, Eap, one of the most important virulence factors in S. aureus mediated infections is a multi-tandem domain protein and has been shown to interact with almost all cell types in the human circulatory system. By using amine reactive fluorescent N-hydroxysuccinimidyl (NHS)-ester dyes and by direct detection with primary fluorescently conjugated anti-histidine (His-tag) antibodies against detect N-terminal His6, we show Eap subdomain Eap D 3 D 4 specifically interacts and rapidly activates human platelets. Furthermore, we validate our finding by using site directed directional immobilization of Eap D 3 D 4 through N-terminal His 6 on nickel (II)-nitrilotriacetic acid (Ni-NTA) functionalized bacteriomimetic microbead arrays to visualize real-time platelet activation through calcium release assay. These methods offer an easily adoptable protocols for screening of S.aureus derived virulence factors and adhesins with platelets. Copyright © 2018 Elsevier GmbH. All rights reserved.

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

Potential Role of Protein Disulfide Isomerase in Metabolic Syndrome-Derived Platelet Hyperactivity

PubMed Central

Gaspar, Renato Simões

2016-01-01

Metabolic Syndrome (MetS) has become a worldwide epidemic, alongside with a high socioeconomic cost, and its diagnostic criteria must include at least three out of the five features: visceral obesity, hypertension, dyslipidemia, insulin resistance, and high fasting glucose levels. MetS shows an increased oxidative stress associated with platelet hyperactivation, an essential component for thrombus formation and ischemic events in MetS patients. Platelet aggregation is governed by the peroxide tone and the activity of Protein Disulfide Isomerase (PDI) at the cell membrane. PDI redox active sites present active cysteine residues that can be susceptible to changes in plasma oxidative state, as observed in MetS. However, there is a lack of knowledge about the relationship between PDI and platelet hyperactivation under MetS and its metabolic features, in spite of PDI being a mediator of important pathways implicated in MetS-induced platelet hyperactivation, such as insulin resistance and nitric oxide dysfunction. Thus, the aim of this review is to analyze data available in the literature as an attempt to support a possible role for PDI in MetS-induced platelet hyperactivation. PMID:28053690

Potential Role of Protein Disulfide Isomerase in Metabolic Syndrome-Derived Platelet Hyperactivity.

PubMed

Gaspar, Renato Simões; Trostchansky, Andrés; Paes, Antonio Marcus de Andrade

2016-01-01

Metabolic Syndrome (MetS) has become a worldwide epidemic, alongside with a high socioeconomic cost, and its diagnostic criteria must include at least three out of the five features: visceral obesity, hypertension, dyslipidemia, insulin resistance, and high fasting glucose levels. MetS shows an increased oxidative stress associated with platelet hyperactivation, an essential component for thrombus formation and ischemic events in MetS patients. Platelet aggregation is governed by the peroxide tone and the activity of Protein Disulfide Isomerase (PDI) at the cell membrane. PDI redox active sites present active cysteine residues that can be susceptible to changes in plasma oxidative state, as observed in MetS. However, there is a lack of knowledge about the relationship between PDI and platelet hyperactivation under MetS and its metabolic features, in spite of PDI being a mediator of important pathways implicated in MetS-induced platelet hyperactivation, such as insulin resistance and nitric oxide dysfunction. Thus, the aim of this review is to analyze data available in the literature as an attempt to support a possible role for PDI in MetS-induced platelet hyperactivation.

Peripheral Blood Mononuclear Cells Enhance the Anabolic Effects of Platelet-Rich Plasma on Anterior Cruciate Ligament Fibroblasts

PubMed Central

Yoshida, Ryu; Murray, Martha M.

2012-01-01

Use of platelet-rich plasma (PRP) has shown promise in various orthopaedic applications, including treatment of anterior cruciate ligament (ACL) injuries. However, various components of blood, including peripheral blood mononuclear cells (PBMCs), are removed in the process of making PRP. It is yet unknown whether these PBMCs have a positive or negative effect on fibroblast behavior. To begin to define the effect of PBMCs on ACL fibroblasts, ACL fibroblasts were cultured on three-dimensional collagen scaffolds for 14 days with and without PBMCs. ACL fibroblasts exposed to PBMCs showed increased type I and type III procollagen gene expression, collagen protein expression, and cell proliferation when the cells were cultured in the presence of platelets and plasma. However, addition of PBMCs to cells cultured without the presence of platelets had no effect. The increase in collagen gene and protein expression was accompanied by an increase in IL-6 expression by the PBMCs with exposure to the platelets. Our results suggest that the interaction between platelets and PBMCs leads to an IL-6 mediated increase in collagen expression by ACL fibroblasts. PMID:22767425

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.

Hypothyroidism coordinately and transiently affects myelin protein gene expression in most rat brain regions during postnatal development.

PubMed

Ibarrola, N; Rodríguez-Peña, A

1997-03-28

To assess the role of thyroid hormone on myelin gene expression, we have studied the effect of hypothyroidism on the mRNA steady state levels for the major myelin protein genes: myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG) and 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in different rat brain regions, during the first postnatal month. We found that hypothyroidism reduces the levels of every myelin protein transcript, with striking differences between the different brain regions. Thus, in the more caudal regions, the effect of hypothyroidism was extremely modest, being only evident at the earlier stages of myelination. In contrast, in the striatum and the cerebral cortex the important decrease in the myelin protein transcripts is maintained beyond the first postnatal month. Therefore, thyroid hormone modulates in a synchronous fashion the expression of the myelin genes and the length of its effect depends on the brain region. On the other hand, hyperthyroidism leads to an increase of the major myelin protein transcripts above control values. Finally, lack of thyroid hormone does not change the expression of the oligodendrocyte progenitor-specific gene, the platelet derived growth factor receptor alpha.

The P2Y12 Antagonists, 2-Methylthioadenosine 5′-Monophosphate Triethylammonium Salt and Cangrelor (ARC69931MX), Can Inhibit Human Platelet Aggregation through a Gi-independent Increase in cAMP Levels*

PubMed Central

Srinivasan, Subhashini; Mir, Fozia; Huang, Jin-Sheng; Khasawneh, Fadi T.; Lam, Stephen C.-T.; Le Breton, Guy C.

2009-01-01

ADP plays an integral role in the process of hemostasis by signaling through two platelet G-protein-coupled receptors, P2Y1 and P2Y12. The recent use of antagonists against these two receptors has contributed a substantial body of data characterizing the ADP signaling pathways in human platelets. Specifically, the results have indicated that although P2Y1 receptors are involved in the initiation of platelet aggregation, P2Y12 receptor activation appears to account for the bulk of the ADP-mediated effects. Based on this consideration, emphasis has been placed on the development of a new class of P2Y12 antagonists (separate from clopidogrel and ticlopidine) as an approach to the treatment of thromboembolic disorders. The present work examined the molecular mechanisms by which two of these widely used adenosine-based P2Y12 antagonists (2-methylthioadenosine 5′-monophosphate triethylammonium salt (2MeSAMP) and ARC69931MX), inhibit human platelet activation. It was found that both of these compounds raise platelet cAMP to levels that substantially inhibit platelet aggregation. Furthermore, the results demonstrated that this elevation of cAMP did not require Gi signaling or functional P2Y12 receptors but was mediated through activation of a separate G protein-coupled pathway, presumably involving Gs. However, additional experiments revealed that neither 2MeSAMP nor ARC69931MX (cangrelor) increased cAMP through activation of A2a, IP, DP, or EP2 receptors, which are known to couple to Gs. Collectively, these findings indicate that 2MeSAMP and ARC69931MX interact with an unidentified platelet G protein-coupled receptor that stimulates cAMP-mediated inhibition of platelet function. This inhibition is in addition to that derived from antagonism of P2Y12 receptors. PMID:19346255

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.

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.

Phorbol ester stimulates calcium sequestration in saponized human platelets

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

Yoshida, K.; Nachmias, V.T.

1987-11-25

When platelets are activated by agonists, calcium (Ca2+) is released from an intracellular storage site. Recent studies using fura-2 show that, after thrombin stimulation, the rise in free calcium is transient and returns to base-line levels in 2-3 min, while the transient following ADP stimulation lasts only 15-20 s. We reported previously that the phorbol ester 12,13-phorbol myristate acetate (PMA), added at nanomolar levels after thrombin, immediately accelerated the rate of return of calcium to the base line severalfold. In the present study, we used both intact and saponized platelets to determine whether this is due to stimulation of calciummore » sequestration. Using fura-2 and intact platelets, we found 1) that PMA stimulated the restoration of free Ca2+ levels after ADP as well as after thrombin, and 2) that H-7, an inhibitor of protein kinase C (Ca2+/phospholipid-dependent enzyme), slowed the return of Ca2+ to baseline levels. Using saponized platelets, we also found 3) that pretreatment of platelets with PMA before saponin treatment increased the ATP-dependent /sup 45/Ca2+ uptake 2-fold, with a half-maximal effect at 5 nm; 4) that most of the Ca2+ released by ionomycin or by myoinositol 1,4,5-trisphosphate; and 5) that a GTP-binding protein inhibitor, guanosine 5'-O-(2-thiodiphosphate), decreased basal or PMA-stimulated /sup 45/Ca2+ uptake in saponin-treated platelets. Our data suggest that activation of protein kinase C stimulates the sequestration of Ca2+ independently of cAMP or myoinositol 1,4,5-trisphosphate.« less

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.

Oxidized LDL activates blood platelets through CD36/NOX2–mediated inhibition of the cGMP/protein kinase G signaling cascade

PubMed Central

Magwenzi, Simbarashe; Woodward, Casey; Wraith, Katie S.; Aburima, Ahmed; Raslan, Zaher; Jones, Huw; McNeil, Catriona; Wheatcroft, Stephen; Yuldasheva, Nadira; Febbriao, Maria; Kearney, Mark

2015-01-01

Oxidized low-density lipoprotein (oxLDL) promotes unregulated platelet activation in dyslipidemic disorders. Although oxLDL stimulates activatory signaling, it is unclear how these events drive accelerated thrombosis. Here, we describe a mechanism for oxLDL-mediated platelet hyperactivity that requires generation of reactive oxygen species (ROS). Under arterial flow, oxLDL triggered sustained generation of platelet intracellular ROS, which was blocked by CD36 inhibitors, mimicked by CD36-specific oxidized phospholipids, and ablated in CD36−/− murine platelets. oxLDL-induced ROS generation was blocked by the reduced NAD phosphate oxidase 2 (NOX2) inhibitor, gp91ds-tat, and absent in NOX2−/− mice. The synthesis of ROS by oxLDL/CD36 required Src-family kinases and protein kinase C (PKC)-dependent phosphorylation and activation of NOX2. In functional assays, oxLDL abolished guanosine 3′,5′-cyclic monophosphate (cGMP)-mediated signaling and inhibited platelet aggregation and arrest under flow. This was prevented by either pharmacologic inhibition of NOX2 in human platelets or genetic ablation of NOX2 in murine platelets. Platelets from hyperlipidemic mice were also found to have a diminished sensitivity to cGMP when tested ex vivo, a phenotype that was corrected by infusion of gp91ds-tat into the mice. This study demonstrates that oxLDL and hyperlipidemia stimulate the generation of NOX2-derived ROS through a CD36-PKC pathway and may promote platelet hyperactivity through modulation of cGMP signaling. PMID:25710879

Peroxisome proliferator-activated receptor (PPAR)-gamma expression in human vascular smooth muscle cells: inhibition of growth, migration, and c-fos expression by the peroxisome proliferator-activated receptor (PPAR)-gamma activator troglitazone.

PubMed

Benson, S; Wu, J; Padmanabhan, S; Kurtz, T W; Pershadsingh, H A

2000-01-01

This study was conducted to determine whether cultured human coronary artery and aorta vascular smooth muscle (VSM) cells express the nuclear transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma); whether the thiazolidinedione troglitazone, a ligand for PPARgamma, would inhibit c-fos expression by these cells; and whether troglitazone would inhibit proliferation and migration induced in these cells by mitogenic growth factors. Using immunoblotting and reverse-transcriptase polymerase chain reaction (RT-PCR) techniques, we show that both human aorta and coronary artery VSM cell lines expressed PPARgamma protein and mRNA for both PPARgamma isoforms, PPARgamma1 and PPARgamma2. Immunocytochemical staining localized the PPARgamma protein primarily within the nucleus. Troglitazone inhibited basic fibroblast growth factor and platelet-derived growth factor-BB induced DNA synthesis in a dose-dependent manner and downregulated the growth-factor-induced expression of c-fos. Troglitazone also inhibited the migration of coronary artery VSM cells along a platelet-derived growth factor-BB concentration gradient. These findings demonstrate for the first time the expression and nuclear localization of PPARgamma in human coronary artery and aorta VSM cells. The data also suggest that the downregulation of c-fos expression, growth-factor-induced proliferation, and migration by VSM may, in part, be mediated by activation of the PPARgamma receptor.

Preclinical and clinical studies on the use of growth factors for bone repair: a systematic review.

PubMed

Fisher, Daniel Mark; Wong, James Min-Leong; Crowley, Conor; Khan, Wasim S

2013-05-01

Bone healing is a complex process. Whilst the majority of fractures heal with conventional treatment, open fractures, large bone defects and non unions still provide great challenges to Orthopaedic Surgeons. Whilst autologous bone graft is seen as the gold standard, the use of growth factors is a growing area of research to find an effective alternative with lower side effects such as donor site morbidity and the finite amount available. This systematic review aims to summarize the pre clinical in-vivo studies and examine the clinical studies on the use of growth factors in bone healing. Databases: PubMed, Medline, OVID, and Cochrane library. The following key words and search terms were used: Growth Factors, Bone Healing, Bone Morphogenic Protein, Transforming Growth Factor Beta, Insulin Like Growth Factor, Platelet Derived Growth Factor, Fracture. All articles were screened based on title with abstracts and full text articles reviewed as appropriate. Reference lists were reviewed from relevant articles to ensure comprehensive and systematic review. Three tables of studies were constructed focussing on Bone Morphogenic Proteins, Platelet Rich Plasma and Growth Factors and Tissue Engineering. Bone Morphogenic Proteins and Platelet Rich Plasma, which contains multiple growth factors, have been shown in preclinical and clinical trials to be an effective alternative to autologous bone graft. Bone Morphogenic Proteins have been shown to be effective in fracture non union, and in open tibial fractures. Platelet Rich Plasma has shown promise in preclinical trials and some small clinical trials, however numbers are limited. Bone Morphogenic Proteins have been shown to be superior to Platelet Rich Protein in one trial. Combining these growth factors with tissue engineering techniques is the focus of ongoing research, and through further clinical trials the most effective techniques for enhancing bone healing will be revealed.

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.

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

HMGB1: a novel protein that induced platelets active and aggregation via Toll-like receptor-4, NF-κB and cGMP dependent mechanisms.

PubMed

Yang, Xinyu; Wang, Haichao; Zhang, Menmen; Liu, Jin; Lv, Ben; Chen, Fangping

2015-08-06

Thrombotic diseases are a group of prevalent and life-threatening diseases. Selective inhibition of pathological thrombosis holds the key to treat variety of thrombotic diseases. The pathological thrombosis can be induced by either tissue necrosis and deregulated inflammation. HMGB1, as an important proinflammatory cytokine and a late mediator, also involves on thrombosis disease. However, the underlying mechanisms are not fully understood. Immunofluorescence, ELISA assay, Platelet Aggregation, Thromboelastogram (TEG) analyzes. Flow cytometric analysis and Western blot analysis were used to investigated the role of HMGB1 in platelet aggregation and obtained following observations. By doing so, we obtained the following observations: i) Highly purified HMGB1 recombinant protein induces platelet aggregation and secretion in a dose-dependent manner in the presence of serum. ii) Low concentration of extracellular HMGB1 could synergistically promote subthreshold concentration of collagen or thrombin induced platelet aggregation. iii) Extracellular HMGB1 promoted platelet aggregation in a platelet-expressed GPIIb/IIIa-dependent manner. iv) We proposed that extracellular HMGB1 seems to promote the phosphorylation of GPIIb/IIIa and subsequent platelet aggregation via TLR4/NF-κB and cGMP pathway. In this study, we provide evidence for the hypothesis that HMGB1 interact with platelet might play an important role in the haemostasis and thrombotic diseases. Our research might be provide an interesting avenue for the treatment of thrombotic diseases in the future.

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.

Characterization of a potent platelet aggregation inducer from Cerastes cerastes (Egyptian sand viper) venom.

PubMed

Basheer, A R; el-Asmar, M F; Soslau, G

1995-07-03

A potent, proteinaceous inducer of platelet aggregation designated as IVa, has been purified to homogeneity from Cerastes cerastes venom by molecular sieve and ion exchange chromatography. It is composed of 2 subunits with total M(r) of 62,000 as shown by native gel chromatography and chemical cross-linking with disuccinimidyl suberate. It is not clear at the present time whether both subunits are identical gene products, however, both have identical N-terminal sequences for the first 15 amino acids. The protein has a pI above 9.6. IVa (0.1 micrograms/ml) could aggregate platelets up to 80% and was inhibited by p-APMSF, leupeptin, iodoacetamide, protein kinase C inhibitor, phosphatase inhibitor, ATP and PGE1, while it was insensitive to acetylsalicylic acid, ADP scavenger system, protein kinase A inhibitor and hirudin. Protein IVa is a serine proteinase with thrombin-like activity as it hydrolysed thrombin chromogenic substrate CBS 34.47, its aggregatory activity was partially inhibited by monoclonal antibodies against GPIb and the thrombin receptor, as was the thrombin, and its ability to induce intracellular Ca2+ release was blocked by pretreating platelets with thrombin. Unlike thrombin, the IVa protein showed very weak coagulant activity as indicated by plasma recalcification time and fibrinogen clotting time although it could hydrolyse fibrinogen alpha-chains.

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.

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

How Blood Clots

MedlinePlus

... with other blood proteins to form fibrin. Fibrin strands form a net that entraps more platelets and ... that is normally dissolved in blood, into long strands of fibrin that radiate from the clumped platelets ...

Global Proteome Analysis Identifies Active Immunoproteasome Subunits in Human Platelets*

PubMed Central

Klockenbusch, Cordula; Walsh, Geraldine M.; Brown, Lyda M.; Hoffman, Michael D.; Ignatchenko, Vladimir; Kislinger, Thomas; Kast, Juergen

2014-01-01

The discovery of new functions for platelets, particularly in inflammation and immunity, has expanded the role of these anucleate cell fragments beyond their primary hemostatic function. Here, four in-depth human platelet proteomic data sets were generated to explore potential new functions for platelets based on their protein content and this led to the identification of 2559 high confidence proteins. During a more detailed analysis, consistently high expression of the proteasome was discovered, and the composition and function of this complex, whose role in platelets has not been thoroughly investigated, was examined. Data set mining resulted in identification of nearly all members of the 26S proteasome in one or more data sets, except the β5 subunit. However, β5i, a component of the immunoproteasome, was identified. Biochemical analyses confirmed the presence of all catalytically active subunits of the standard 20S proteasome and immunoproteasome in human platelets, including β5, which was predominantly found in its precursor form. It was demonstrated that these components were assembled into the proteasome complex and that standard proteasome as well as immunoproteasome subunits were constitutively active in platelets. These findings suggest potential new roles for platelets in the immune system. For example, the immunoproteasome may be involved in major histocompatibility complex I (MHC I) peptide generation, as the MHC I machinery was also identified in our data sets. PMID:25146974

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.

The Distinction of Amyloid-β Protein Precursor (AβPP) Ratio in Platelet Between Alzheimer's Disease Patients and Controls: A Systematic Review and Meta-Analysis.

PubMed

Shi, Yachen; Gu, Lihua; Alsharif, Abdul Azeez; Zhang, Zhijun

2017-01-01

To systematically assess the clinical significance of platelet amyloid-β protein precursor (AβPP) ratio between Alzheimer's disease (AD) patients and controls. 14 articles were selected in this analysis by search of databases including PubMed and Web of Science up to December 2016. Random effects models were used to calculate the standardized mean difference (SMD). Subgroup analyses were used to detect the cause of heterogeneity. The result showed a significant drop in platelet AβPP ratio in AD patients compared to controls [SMD: -1.871; 95% CI: (-2.33, -1.41); p < 0.001; I2 = 88.0% ]. Subgroup analysis revealed races or the quality of studies may be the cause of high heterogeneity. This meta-analysis concluded that there is a close association between platelet AβPP ratio and AD. It is necessary to design a sizable sample study to further support that platelet AβPP ratio can be a biomarker of AD.

Protein A immunoadsorption therapy in HIV-related immune thrombocytopenia: a preliminary report.

PubMed

Bertram, J H; Snyder, H W; Gill, P S; Shulman, I; Henry, D H; Jenkins, D; Kiprov, D D

1988-12-01

Nine homosexual patients with immune thrombocytopenia were treated with autologous plasma that had been perfused over silica-immobilized Staphylococcus aureus protein A (SpA). Pretreatment platelet counts ranged from 10,000 to 98,000 cells/mm3 (mean: 54,000 cells/mm3). Six patients responded to therapy. Platelets increased by a mean of 95,000 cells/mm3 (p less than 0.007) and reached normal levels (greater than 150,000 cells/mm3) in four patients. Increased platelet counts are presently sustained in these four individuals after 5 months of follow-up. Increases in platelet counts significantly correlated with decreases in platelet-associated IgG (PAIgG), platelet-directed IgG (PDIgG), and immune complexes (CIC). PAIgG and PDIgG declined by a mean of 67% (p less than 0.003) and 58% (p less than 0.007), respectively. CIC decreased by a mean of 37% (p = 0.02). Complement was concomitantly activated in all four examined patients. C3a and C5a increased 23-fold and 2.6-fold, respectively, while total hemolytic complement decreased by 50%. Activated complement components and removal of CIC and IgG thus may contribute to the platelet-enhancing activity of SpA immunoadsorption therapy.

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

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.

Serum protein adsorption and platelet adhesion on aspartic-acid-immobilized polysulfone membranes.

PubMed

Higuchi, Akon; Hashiba, Hirokazu; Hayashi, Rika; Yoon, Boo Ok; Sakurai, Masaru; Hara, Mariko

2004-01-01

Polysulfone (PSf) membranes that covalently conjugated with aspartic acid (ASP-PSf) were prepared and analyzed for hemocompatability. Compared to PSf or other types of surface-modified PSf membranes, the ASP-PSf membranes had a reduced ability to adsorb protein from either a plasma solution or a mixed solution of albumin, globulin and fibrinogen. This appears to be due to the creation of a hydrophilic surface by the aspartic acid zwitterion immobilized on the ASP-PSf membranes. Furthermore, the analyses of membrane protein adsorption showed that a mixed protein solution recapitulates the cooperative adsorption of proteins that occurs in plasma. We also found that the number of adhering platelets was the lowest on the ASP-PSf membranes and, in general, that platelet adhesion decreased in parallel with fibrinogen adsorption. In summary, aspartic acid immobilized on the ASP-PSf membranes, which have zwitterions with a net zero charge, effectively contributes to the hydrophilic and hemocompatible sites on the surface of the hydrophobic PSf membranes.

Enhanced generation of megakaryocytes from umbilical cord blood-derived CD34(+) cells expanded in the presence of two nutraceuticals, docosahexanoic acid and arachidonic acid, as supplements to the cytokine-containing medium.

PubMed

Siddiqui, Nikhat Firdaus A; Shabrani, Namrata C; Kale, Vaijayanti P; Limaye, Lalita S

2011-01-01

Ex vivo generation of megakaryocytes (MK) from hematopoietic stem cells (HSC) is important for both basic research, to understand the mechanism of platelet biogenesis, and clinical infusions, for rapid platelet recovery in thrombocytopenic patients. We investigated the role of two nutraceuticals, docosahexanoic acid (DHA) and arachidonic acid (AA), in the in vitro generation of MK. Umbilical cord blood (UCB)-derived CD34+cells were cultured with stem cell factor (SCF) and thrombopoietin (TPO) in the presence (test) or absence (control) of the two additives. On day 10, MK and platelets generated were quantitated by morphologic, phenotypic and functional assays. The cell yield of MK and platelet numbers were significantly higher in test compared with control cells. Phenotypic analyzes and gene expression profiles confirmed these findings. Functional properties, such as colony-forming unit (CFU)-MK formation, chemotaxis and platelet activation, were found to be enhanced in cells cultured with nutraceuticals. The engraftment potential of ex vivo-expanded cells was studied in NOD/SCID mice. Mice that received MK cultured in the presence of DHA/AA engrafted better. There was a reduction in apoptosis and total reactive oxygen species (ROS) levels in the CD41(+) compartment of the test compared with control sets. The data suggest that these compounds probably exert their beneficial effect by modulating apoptotic and redox pathways. Use of nutraceuticals like DHA and AA may prove to be a useful strategy for efficient generation of MK and platelets from cord blood cells, for future use in clinics and basic research.

Modeling HIV-1 Induced Neuroinflammation in Mice: Role of Platelets in Mediating Blood-Brain Barrier Dysfunction

PubMed Central

Jones, Letitia D.; Jackson, Joseph W.; Maggirwar, Sanjay B.

2016-01-01

The number of HIV-1 positive individuals developing some form of HIV-associated neurocognitive disorder (HAND) is increasing. In these individuals, the integrity of the blood-brain barrier (BBB) is compromised due to an increase in exposure to pro-inflammatory mediators, viral proteins, and virus released from infected cells. It has been shown that soluble CD40L (sCD40L) is released upon platelet activation and is an important mediator of the pathogenesis of HAND but the underlying mechanisms are unclear, emphasizing the need of an effective animal model. Here, we have utilized a novel animal model in which wild-type (WT) mice were infected with EcoHIV; a derivative of HIV-1 that contains a substitution of envelope protein gp120 with that of gp80 derived from murine leukemia virus-1 (MuLV-1). As early as two-weeks post-infection, EcoHIV led to increased permeability of the BBB associated with decreased expression of tight junction protein claudin-5, in CD40L and platelet activation-dependent manner. Treatment with an antiplatelet drug, eptifibatide, in EcoHIV-infected mice normalized BBB function, sCD40L release and platelet activity, thus implicating platelet activation and platelet-derived CD40L in virally induced BBB dysfunction. Our results also validate and underscore the importance of EcoHIV infection mouse model as a tool to explore therapeutic targets for HAND. PMID:26986758

Tyrosine kinases activate store-mediated Ca2+ entry in human platelets through the reorganization of the actin cytoskeleton.

PubMed Central

Rosado, J A; Graves, D; Sage, S O

2000-01-01

We have recently reported that store-mediated Ca(2+) entry in platelets is likely to be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, a model termed 'secretion-like coupling'. In this model the actin cytoskeleton plays a key regulatory role. Since tyrosine kinases have been shown to be important for Ca(2+) entry in platelets and other cells, we have now investigated the possible involvement of tyrosine kinases in the secretion-like-coupling model. Treatment of platelets with thrombin or thapsigargin induced actin polymerization by a calcium-independent pathway. Methyl 2,5-dihydroxycinnamate, a tyrosine kinase inhibitor, prevented thrombin- or thapsigargin-induced actin polymerization. The effects of tyrosine kinases in store-mediated Ca(2+) entry were found to be entirely dependent on the actin cytoskeleton. PP1, an inhibitor of the Src family of proteins, partially inhibited store-mediated Ca(2+) entry. In addition, depletion of intracellular Ca(2+) stores stimulated cytoskeletal association of the cytoplasmic tyrosine kinase pp60(src), a process that was sensitive to treatment with cytochalasin D and PP1, but not to inhibition of Ras proteins using prenylcysteine analogues. Finally, combined inhibition of both Ras proteins and tyrosine kinases resulted in complete inhibition of Ca(2+) entry, suggesting that these two families of proteins have independent effects in the activation of store-mediated Ca(2+) entry in human platelets. PMID:11023829

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

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.

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.

Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

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

Morrison, W.J.

1988-01-01

The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. ({sup 3}H)PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 {mu}M. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF ormore » thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRP{gamma}S and GDP{beta}S, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA).« less

Association of Factor V Secretion with Protein Kinase B Signaling in Platelets from Horses with Atypical Equine Thrombasthenia.

PubMed

Norris, J W; Pombo, M; Shirley, E; Blevins, G; Tablin, F

2015-01-01

Two congenital bleeding diatheses have been identified in Thoroughbred horses: Glanzmann thrombasthenia (GT) and a second, novel diathesis associated with abnormal platelet function in response to collagen and thrombin stimulation. Platelet dysfunction in horses with this second thrombasthenia results from a secretory defect. Two affected and 6 clinically normal horses. Ex vivo study. Washed platelets were examined for (1) expression of the αIIb-β3 integrin; (2) fibrinogen binding capacity in response to ADP and thrombin; (3) secretion of dense and α-granules; (4) activation of the mammalian target of rapamycin (mTOR)-protein kinase B (AKT) signaling pathway; and (5) cellular distribution of phosphatidylinositol-4-phosphate-3-kinase, class 2B (PIK3C2B) and SH2 containing inositol-5'-phosphatase 1 (SHIP1). Platelets from affected horses expressed normal amounts of αIIb-β3 integrin and bound fibrinogen normally in response to ADP, but bound 80% less fibrinogen in response to thrombin. α-granules only released 50% as much Factor V as control platelets, but dense granules released their contents normally. Protein kinase B (AKT) phosphorylation was reduced after thrombin activation, but mTOR Complex 2 (mTORC2) and phosphoinositide-dependent kinase 1 (PDK1) signaling were normal. SH2-containing inositol-5'-phosphatase 1 (SHIP1) did not localize to the cytoskeleton of affected platelets and was decreased overall consistent with reduced AKT phosphorylation. Defects in fibrinogen binding, granule secretion, and signal transduction are unique to this thrombasthenia, which we designate as atypical equine thrombasthenia. Copyright © The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of American College of Veterinary Internal Medicine.

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.

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.

[Investigation of mechanisms of action of growth factors of autologous platelet-rich plasma used to treat erectile dysfunction].

PubMed

Epifanova, M V; Chalyi, M E; Krasnov, A O

2017-09-01

To determine the quantitative and qualitative composition of growth factors (PDGF-AA, PDGF-BB, VEGF, VEGF-D, FGF-acid, FGF-basic) and platelets in various modifications of APRP. Blood of 12 male volunteers (control group) and 12 patients with ED was used to prepare APRP and the subsequently determine the concentration of growth factors. The growth factor concentrations (FGF acid, FGF basic, PDGF-AA, PDGF-BB, VEGF, VEGF-D) was determined using a flow cytometry-based xMAP Luminex (Gen-Probe) system. Concentration of platelets in APRP obtained by two stage centrifugation, reached 1480 (1120-1644) in the control group and 1232 (956-1502) in patients with ED. The concentration of growth factors in the samples prepared without preliminary freezing was: PDGF-AA 842 (22-3700), PDGF-BB 2837 (1460-4100), FGF-basic 7.9 (0.28-127), FGF-acid 3, 4 (0.14-11), VEGF 19 (4.6-46), VEGF-D 21 (14-38). After thawing, the concentration of all growth factors in the samples increased. The study findings suggest that the mechanism of erectile function recovery following the use of APRP is through the active substances detected in APRP, i.e. FGF-basic, PDGF-AA, PDGF-BB, VEGF, VEGF-D and FGF-acid. Also, the study showed that the content of growth factors in APRP after of freezing/thawing is higher than in APRP that has not been frozen. This is due to the cell membrane destruction at extremely low temperatures during freezing.

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.

Chemoproteomic Discovery of AADACL1 as a Novel Regulator of Human Platelet Activation

PubMed Central

Holly, Stephen P.; Chang, Jae Won; Li, Weiwei; Niessen, Sherry; Phillips, Ryan M.; Piatt, Raymond; Black, Justin L.; Smith, Matthew C.; Boulaftali, Yacine; Weyrich, Andrew S.; Bergmeier, Wolfgang; Cravatt, Benjamin F.; Parise, Leslie V.

2013-01-01

A comprehensive knowledge of the platelet proteome is necessary for understanding thrombosis and for conceiving novel antiplatelet therapies. To discover new biochemical pathways in human platelets, we screened platelets with a carbamate library designed to interrogate the serine hydrolase subproteome and used competitive activity-based protein profiling to map the targets of active carbamates. We identified an inhibitor that targets arylacetamide deacetylase-like 1 (AADACL1), a lipid deacetylase originally identified in invasive cancers. Using this compound, along with highly selective second-generation inhibitors of AADACL1, metabolomics and RNA interference, we show that AADACL1 regulates platelet aggregation, thrombus growth, RAP1 and PKC activation, lipid metabolism and fibrinogen binding to platelets and megakaryocytes. These data provide the first evidence that AADACL1 regulates platelet and megakaryocyte activation and highlight the value of this chemoproteomic strategy for target discovery in platelets. PMID:23993462

Human platelet lysate as a promising growth-stimulating additive for culturing of stem cells and other cell types.

PubMed

Shanskii, Ya D; Sergeeva, N S; Sviridova, I K; Kirakozov, M S; Kirsanova, V A; Akhmedova, S A; Antokhin, A I; Chissov, V I

2013-11-01

We compared the composition and biological activity of fetal calf serum and platelet lysate from donor platelet concentrate. In platelet lysate, the concentrations of alkaline phosphatase, lactate dehydrogenase, creatinine, and mineral metabolism parameters were lower, while parameters of lipid and protein metabolism were higher than in fetal calf serum. The concentrations of growth factors (platelet-derived (AA, AB, BB), vascular endothelial, insulin-like, and transforming growth factor β) in platelet lysate 1.7-148.7-fold surpassed the corresponding parameters in fetal calf serum. After replacement of fetal calf serum with platelet lysate in the culture medium (0, 25, 50, 75, and 100%), the count of multipotent mesenchymal stromal cells on day 7 (in comparison with day 1) increased by 154.8, 206.6, 228.2, 367.7, and 396.5%, respectively. Thus, platelet lysate can be an adequate non-xenogenic alternative for fetal calf serum.

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

Platelet Storage Lesions: What More Do We Know Now?

PubMed

Ng, Monica Suet Ying; Tung, John-Paul; Fraser, John Francis

2018-04-17

Platelet concentrate (PC) transfusions are a lifesaving adjunct to control and prevent bleeding in cancer, hematologic, surgical, and trauma patients. Platelet concentrate availability and safety are limited by the development of platelet storage lesions (PSLs) and risk of bacterial contamination. Platelet storage lesions are a series of biochemical, structural, and functional changes that occur from blood collection to transfusion. Understanding of PSLs is key for devising interventions that prolong PC shelf life to improve PC access and wastage. This article will review advancements in clinical and mechanistic PSL research. In brief, exposure to artificial surfaces and high centrifugation forces during PC preparation initiate PSLs by causing platelet activation, fragmentation, and biochemical release. During room temperature storage, enhanced glycolysis and reduced mitochondrial function lead to glucose depletion, lactate accumulation, and product acidification. Impaired adenosine triphosphate generation reduces platelet capacity to perform energetically demanding processes such as hypotonic stress responses and activation/aggregation. Storage-induced alterations in platelet surface proteins such as thrombin receptors and glycoproteins decrease platelet aggregation. During storage, there is an accumulation of immunoactive proteins such as leukocyte-derive cytokines (tumor necrosis factor α, interleukin (IL) 1α, IL-6, IL-8) and soluble CD40 ligand which can participate in transfusion-related acute lung injury and nonhemolytic transfusion reactions. Storage-induced microparticles have been linked to enhanced platelet aggregation and immune system modulation. Clinically, stored PCs have been correlated with reduced corrected count increment, posttransfusion platelet recovery, and survival across multiple meta-analyses. Fresh PC transfusions have been associated with superior platelet function in vivo; however, these differences were abrogated after a period of circulation. There is currently insufficient evidence to discern the effect of PSLs on transfusion safety. Various bag and storage media changes have been proposed to reduce glycolysis and platelet activation during room temperature storage. Moreover, cryopreservation and cold storage have been proposed as potential methods to prolong PC shelf life by reducing platelet metabolism and bacterial proliferation. However, further work is required to elucidate and manage the PSLs specific to these storage protocols before its implementation in blood banks. Copyright © 2018 Elsevier Inc. All rights reserved.

Depressed reticuloendothelial clearance of platelets in rats after trauma.

PubMed

Kaplan, J E; Moon, D G; Minnear, F L; Saba, T M

1984-02-01

Platelet microembolization may contribute to microcirculatory and organ damage following trauma and shock. It is hypothesized that posttraumatic reticuloendothelial depression predisposes to such microembolization by failure to clear altered platelets from the circulation. The present study evaluated the short-term (1 h) clearance and organ localization of radiolabeled homologous damaged platelets in normal rats and in rats following sublethal Noble-Collip drum trauma. Platelets were collected in citrated platelet-rich plasma from normal rats and labeled with 51Cr in citrated saline. Platelets were altered by repeated centrifugation in protein-free medium. These platelets differed functionally and morphologically from normal platelets. Disappearance of iv injected damaged platelets conformed to a two-compartment exponential clearance. Velocity of clearance in the rapid compartment correlated with hepatic platelet localization, whereas velocity of clearance in the second compartment correlated with splenic platelet localization. Clearance rate of the rapid compartment was depressed at 1 h after trauma and elevated at 24 h. These changes were associated with a decrease in hepatic platelet localization at 1 h and an increase above normal at 24 h. Splenic platelet localization was decreased by 3 h following trauma. Pulmonary platelet localization was increased at all times following trauma. It is concluded that the posttrauma state is associated with a defect in the reticuloendothelial system clearance of altered platelets, which may augment embolization of platelets in the lung.

Platelet RNA as a circulating biomarker trove for cancer diagnostics.

PubMed

Best, M G; Vancura, A; Wurdinger, T

2017-07-01

Platelets are multifunctional cell fragments, circulating in blood in high abundance. Platelets assist in thrombus formation, sensing of pathogens entering the blood stream, signaling to immune cells, releasing vascular remodeling factors, and, negatively, enhancing cancer metastasis. Platelets are 'educated' by their environment, including in patients with cancer. Cancer cells appear to initiate intraplatelet signaling, resulting in splicing of platelet pre-mRNAs, and enhance secretion of cytokines. Platelets can induce leukocyte and endothelial cell modeling factors, for example, through adenine nucleotides (ATP), thereby facilitating extravasation of cancer cells. Besides releasing factors, platelets can also sequester RNAs and proteins released by cancer cells. Thus, platelets actively respond to queues from local and systemic conditions, thereby altering their transcriptome and molecular content. Platelets contain a rich repertoire of RNA species, including mRNAs, small non-coding RNAs and circular RNAs; although studies regarding the functionality of the various platelet RNA species require more attention. Recent advances in high-throughput characterization of platelet mRNAs revealed 10 to > 1000 altered mRNAs in platelets in the presence of disease. Hence, platelet RNA appears to be dynamically affected by pathological conditions, thus possibly providing opportunities to use platelet RNA as diagnostic, prognostic, predictive, or monitoring biomarkers. In this review, we cover the literature regarding the platelet RNA families, processing of platelet RNAs, and the potential application of platelet RNA as disease biomarkers. © 2017 International Society on Thrombosis and Haemostasis.

Enhancing the Accuracy of Platelet to Lymphocyte Ratio after Adjustment for Large Platelet Count: A Pilot Study in Breast Cancer Patients

PubMed Central

Seretis, Charalampos; Seretis, Fotios; Lagoudianakis, Emmanuel; Politou, Marianna; Gemenetzis, George; Salemis, Nikolaos S.

2012-01-01

Background. The objective of our study is to investigate the potential effect of adjusting preoperative platelet to lymphocyte ratio, an emerging biomarker of survival in cancer patients, for the fraction of large platelets. Methods. A total of 79 patients with breast neoplasias, 44 with fibroadenomas, and 35 with invasive ductal carcinoma were included in the study. Both conventional platelet to lymphocyte ratio (PLR) and the adjusted marker, large platelet to lymphocyte ratio (LPLR), were correlated with laboratory and histopathological parameters of the study sample. Results. LPLR elevation was significantly correlated with the presence of malignancy, advanced tumor stage, metastatic spread in the axillary nodes and HER2/neu overexpression, while PLR was only correlated with the number of infiltrated lymph nodes. Conclusions. This is the first study evaluating the effect of adjustment for large platelet count on improving PLR accuracy, when correlated with the basic independent markers of survival in a sample of breast cancer patients. Further studies are needed in order to assess the possibility of applying our adjustment as standard in terms of predicting survival rates in cancer. PMID:23304480

Enhancing the accuracy of platelet to lymphocyte ratio after adjustment for large platelet count: a pilot study in breast cancer patients.

PubMed

Seretis, Charalampos; Seretis, Fotios; Lagoudianakis, Emmanuel; Politou, Marianna; Gemenetzis, George; Salemis, Nikolaos S

2012-01-01

Background. The objective of our study is to investigate the potential effect of adjusting preoperative platelet to lymphocyte ratio, an emerging biomarker of survival in cancer patients, for the fraction of large platelets. Methods. A total of 79 patients with breast neoplasias, 44 with fibroadenomas, and 35 with invasive ductal carcinoma were included in the study. Both conventional platelet to lymphocyte ratio (PLR) and the adjusted marker, large platelet to lymphocyte ratio (LPLR), were correlated with laboratory and histopathological parameters of the study sample. Results. LPLR elevation was significantly correlated with the presence of malignancy, advanced tumor stage, metastatic spread in the axillary nodes and HER2/neu overexpression, while PLR was only correlated with the number of infiltrated lymph nodes. Conclusions. This is the first study evaluating the effect of adjustment for large platelet count on improving PLR accuracy, when correlated with the basic independent markers of survival in a sample of breast cancer patients. Further studies are needed in order to assess the possibility of applying our adjustment as standard in terms of predicting survival rates in cancer.

The C-terminal CGHC motif of protein disulfide isomerase supports thrombosis

PubMed Central

Zhou, Junsong; Wu, Yi; Wang, Lu; Rauova, Lubica; Hayes, Vincent M.; Poncz, Mortimer; Essex, David W.

2015-01-01

Protein disulfide isomerase (PDI) has two distinct CGHC redox-active sites; however, the contribution of these sites during different physiologic reactions, including thrombosis, is unknown. Here, we evaluated the role of PDI and redox-active sites of PDI in thrombosis by generating mice with blood cells and vessel wall cells lacking PDI (Mx1-Cre Pdifl/fl mice) and transgenic mice harboring PDI that lacks a functional C-terminal CGHC motif [PDI(ss-oo) mice]. Both mouse models showed decreased fibrin deposition and platelet accumulation in laser-induced cremaster arteriole injury, and PDI(ss-oo) mice had attenuated platelet accumulation in FeCl3-induced mesenteric arterial injury. These defects were rescued by infusion of recombinant PDI containing only a functional C-terminal CGHC motif [PDI(oo-ss)]. PDI infusion restored fibrin formation, but not platelet accumulation, in eptifibatide-treated wild-type mice, suggesting a direct role of PDI in coagulation. In vitro aggregation of platelets from PDI(ss-oo) mice and PDI-null platelets was reduced; however, this defect was rescued by recombinant PDI(oo-ss). In human platelets, recombinant PDI(ss-oo) inhibited aggregation, while recombinant PDI(oo-ss) potentiated aggregation. Platelet secretion assays demonstrated that the C-terminal CGHC motif of PDI is important for P-selectin expression and ATP secretion through a non-αIIbβ3 substrate. In summary, our results indicate that the C-terminal CGHC motif of PDI is important for platelet function and coagulation. PMID:26529254

Metformin reduces hyper-reactivity of platelets from patients with polycystic ovary syndrome by improving mitochondrial integrity.

PubMed

Randriamboavonjy, Voahanginirina; Mann, W Alexander; Elgheznawy, Amro; Popp, Rüdiger; Rogowski, Paul; Dornauf, Imke; Dröse, Stefan; Fleming, Ingrid

2015-08-31

Polycystic ovary syndrome (PCOS) is associated with decreased fertility, insulin resistance and an increased risk of developing cardiovascular disease. Treating PCOS patients with metformin improves fertility and decreases cardiovascular complications. Given that platelet activation contributes to both infertility and cardiovascular disease development, we assessed platelet reactivity in PCOS patients and the consequences of metformin treatment. Compared to washed platelets from healthy donors, platelets from PCOS patients demonstrated enhanced reactivity and impaired activation of the AMP-activated kinase (AMPK). PCOS platelets also demonstrated enhanced expression of mitochondrial proteins such as the cytochrome c reductase, ATP synthase and the voltage-dependent anion channel-1. However, mitochondrial function was impaired as demonstrated by a decreased respiration rate. In parallel, the phosphorylation of dynamin-related protein-1 (Drp-1) on Ser616 was increased while that on Ser637 decreased. The latter changes were accompanied by decreased mitochondrial size. In insulin-resistant PCOS patients (HOMA-IR> 2) metformin treatment (1.7 g per day for 4 weeks to 6 months) improved insulin sensitivity, restored mitochondrial integrity and function and normalised platelet aggregation. Treatment was without effect in PCOS patients with HOMA-IR< 2. Moreover, treatment of megakaryocytes with metformin enhanced mitochondrial content and in the same cells metformin enhanced the phosphorylation of the Drp-1 on Ser637 via an AMPKα1-dependent mechanism. In conclusion, the improvement of mitochondrial integrity and platelet reactivity may contribute to the beneficial effects of metformin on cardiovascular disease.

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

Global proteome analysis identifies active immunoproteasome subunits in human platelets.

PubMed

Klockenbusch, Cordula; Walsh, Geraldine M; Brown, Lyda M; Hoffman, Michael D; Ignatchenko, Vladimir; Kislinger, Thomas; Kast, Juergen

2014-12-01

The discovery of new functions for platelets, particularly in inflammation and immunity, has expanded the role of these anucleate cell fragments beyond their primary hemostatic function. Here, four in-depth human platelet proteomic data sets were generated to explore potential new functions for platelets based on their protein content and this led to the identification of 2559 high confidence proteins. During a more detailed analysis, consistently high expression of the proteasome was discovered, and the composition and function of this complex, whose role in platelets has not been thoroughly investigated, was examined. Data set mining resulted in identification of nearly all members of the 26S proteasome in one or more data sets, except the β5 subunit. However, β5i, a component of the immunoproteasome, was identified. Biochemical analyses confirmed the presence of all catalytically active subunits of the standard 20S proteasome and immunoproteasome in human platelets, including β5, which was predominantly found in its precursor form. It was demonstrated that these components were assembled into the proteasome complex and that standard proteasome as well as immunoproteasome subunits were constitutively active in platelets. These findings suggest potential new roles for platelets in the immune system. For example, the immunoproteasome may be involved in major histocompatibility complex I (MHC I) peptide generation, as the MHC I machinery was also identified in our data sets. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

G-protein-coupled receptors signaling pathways in new antiplatelet drug development.

PubMed

Gurbel, Paul A; Kuliopulos, Athan; Tantry, Udaya S

2015-03-01

Platelet G-protein-coupled receptors influence platelet function by mediating the response to various agonists, including ADP, thromboxane A2, and thrombin. Blockade of the ADP receptor, P2Y12, in combination with cyclooxygenase-1 inhibition by aspirin has been among the most widely used pharmacological strategies to reduce cardiovascular event occurrence in high-risk patients. The latter dual pathway blockade strategy is one of the greatest advances in the field of cardiovascular medicine. In addition to P2Y12, the platelet thrombin receptor, protease activated receptor-1, has also been recently targeted for inhibition. Blockade of protease activated receptor-1 has been associated with reduced thrombotic event occurrence when added to a strategy using P2Y12 and cyclooxygenase-1 inhibition. At this time, the relative contributions of these G-protein-coupled receptor signaling pathways to in vivo thrombosis remain incompletely defined. The observation of treatment failure in ≈10% of high-risk patients treated with aspirin and potent P2Y12 inhibitors provides the rationale for targeting novel pathways mediating platelet function. Targeting intracellular signaling downstream from G-protein-coupled receptor receptors with phosphotidylionisitol 3-kinase and Gq inhibitors are among the novel strategies under investigation to prevent arterial ischemic event occurrence. Greater understanding of the mechanisms of G-protein-coupled receptor-mediated signaling may allow the tailoring of antiplatelet therapy. © 2015 American Heart Association, Inc.

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.

Plasma-derived Extracellular Vesicles Contain Predictive Biomarkers and Potential Therapeutic Targets for Myocardial Ischemic (MI) Injury*

PubMed Central

Cheow, Esther Sok Hwee; Cheng, Woo Chin; Lee, Chuen Neng; de Kleijn, Dominique; Sorokin, Vitaly; Sze, Siu Kwan

2016-01-01

Myocardial infarction (MI) triggers a potent inflammatory response via the release of circulatory mediators, including extracellular vesicles (EVs) by damaged cardiac cells, necessary for myocardial healing. Timely repression of inflammatory response are critical to prevent and minimize cardiac tissue injuries, nonetheless, progression in this aspect remains challenging. The ability of EVs to trigger a functional response upon delivery of carried bioactive cargos, have made them clinically attractive diagnostic biomarkers and vectors for therapeutic interventions. Using label-free quantitative proteomics approach, we compared the protein cargo of plasma EVs between patients with MI and from patients with stable angina (NMI). We report, for the first time, the proteomics profiling on 252 EV proteins that were modulated with >1.2-fold after MI. We identified six up-regulated biomarkers with potential for clinical applications; these reflected post-infarct pathways of complement activation (Complement C1q subcomponent subunit A (C1QA), 3.23-fold change, p = 0.012; Complement C5 (C5), 1.27-fold change, p = 0.087), lipoprotein metabolism (Apoliporotein D (APOD), 1.86-fold change, p = 0.033; Apolipoprotein C-III (APOCC3), 2.63-fold change, p = 0.029) and platelet activation (Platelet glycoprotein Ib alpha chain (GP1BA), 9.18-fold change, p < 0.0001; Platelet basic protein (PPBP), 4.72-fold change, p = 0.027). The data have been deposited to the ProteomeXchange with identifier PXD002950. This novel biomarker panel was validated in 43 patients using antibody-based assays (C1QA (p = 0.005); C5 (p = 0.0047), APOD (p = 0.0267); APOC3 (p = 0.0064); GP1BA (p = 0.0031); PPBP (p = 0.0465)). We further present that EV-derived fibrinogen components were paradoxically down-regulated in MI, suggesting that a compensatory mechanism may suppress post-infarct coagulation pathways, indicating potential for therapeutic targeting of this mechanism in MI. Taken together, these data demonstrated that plasma EVs contain novel diagnostic biomarkers and therapeutic targets that can be further developed for clinical use to benefit patients with coronary artery diseases (CADs). PMID:27234505

Comparative Analysis of Cellular and Growth Factor Composition in Bone Marrow Aspirate Concentrate and Platelet-Rich Plasma.

PubMed

Sugaya, Hisashi; Yoshioka, Tomokazu; Kato, Toshiki; Taniguchi, Yu; Kumagai, Hiroshi; Hyodo, Kojiro; Ohneda, Osamu; Yamazaki, Masashi; Mishima, Hajime

2018-01-01

The purpose of this study was to quantify the stem cell and growth factor (GF) contents in the bone marrow aspirate concentrate (BMAC) and platelet-rich plasma (PRP) prepared from whole blood using a protocol established in our laboratory. We examined 10 patients with osteonecrosis of the femoral head who were treated by autologous BMAC transplantation at our hospital between January 2015 and June 2015. We quantified CD34+ and CD31-CD45-CD90+CD105+ cells in BMAC and PRP by flow cytometry. Additionally, we measured various GFs, that is, basic fibroblast growth factor (b-FGF), platelet-derived growth factor-BB (PDGF-BB), vascular endothelial growth factor (VEGF), transforming growth factor- β 1 (TGF- β 1), and bone morphogenetic protein-2 (BMP-2) in BMAC and PRP using enzyme-linked immunosorbent assays and statistical analyses. CD34+ and CD31-45-90+105+ cells accounted for approximately 1.9% and 0.03% of cells in BMAC and no cells in PRP. The concentration of b-FGF was higher in BMAC than in PRP ( P < 0.001), whereas no significant differences in the levels of PDGF-BB, VEGF, TGF- β 1, and BMP-2 were observed between the two types of sample. BMAC had an average of 1.9% CD34+ and 0.03% CD31-45-90+105+ cells and higher levels of b-FGF than those of PRP.

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 rich plasma for the management of hair loss: Better alone or in combination?

PubMed

Anitua, Eduardo; Pino, Ander; Jaén, Pedro; Navarro, Mª Rogelia

2018-06-14

Platelet-rich plasma (PRP) and autologous protein-based treatments have recently emerged as a potential therapeutic approach for hair loss-related disorders including androgenetic alopecia and alopecia areata. The safety and efficacy of repeated intradermal injections of PRP has proved to promote hair growth in a number of randomized clinical trials. Biologically active proteins and cytokines released upon platelet activation have shown to induce folliculogenesis and activate the anagen growing phase of dormant bulbs. Interestingly, further studies have revealed that combining PRP with other hair loss-related products may enhance the final performance of the treatment. These synergistic approaches include Food and Drug Administration (FDA) approved drugs such as finasteride or minoxidil, bioactive macromolecules and cell-based therapies. Here, recent research involving alone or combined therapy with platelet-rich plasma for the management of hair loss-related disorders are outlined and future prospects are discussed. © 2018 Wiley Periodicals, Inc.

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.

Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation*

PubMed Central

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

2010-01-01

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca2+ entry (SOCE) with Orai1 as principal Ca2+ 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 Ca2+ 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 Ca2+ 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, Ca2+ 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) Ca2+ entry, inhibited Ca2+ 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 Ca2+ entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca2+ entry and PS exposure, only one relying on STIM1-Orai1 interaction. PMID:20519511

Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies

PubMed Central

Di Buduo, Christian A.; Wray, Lindsay S.; Tozzi, Lorenzo; Malara, Alessandro; Chen, Ying; Ghezzi, Chiara E.; Smoot, Daniel; Sfara, Carla; Antonelli, Antonella; Spedden, Elise; Bruni, Giovanna; Staii, Cristian; De Marco, Luigi; Magnani, Mauro; Kaplan, David L.

2015-01-01

We present a programmable bioengineered 3-dimensional silk-based bone marrow niche tissue system that successfully mimics the physiology of human bone marrow environment allowing us to manufacture functional human platelets ex vivo. Using stem/progenitor cells, megakaryocyte function and platelet generation were recorded in response to variations in extracellular matrix components, surface topography, stiffness, coculture with endothelial cells, and shear forces. Millions of human platelets were produced and showed to be functional based on multiple activation tests. Using adult hematopoietic progenitor cells our system demonstrated the ability to reproduce key steps of thrombopoiesis, including alterations observed in diseased states. A critical feature of the system is the use of natural silk protein biomaterial allowing us to leverage its biocompatibility, nonthrombogenic features, programmable mechanical properties, and surface binding of cytokines, extracellular matrix components, and endothelial-derived proteins. This in turn offers new opportunities for the study of blood component production ex vivo and provides a superior tissue system for the study of pathologic mechanisms of human platelet production. PMID:25575540

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.

Exogenous modification of platelet membranes with the omega-3 fatty acids EPA and DHA reduces platelet procoagulant activity and thrombus formation.

PubMed

Larson, Mark K; Tormoen, Garth W; Weaver, Lucinda J; Luepke, Kristen J; Patel, Ishan A; Hjelmen, Carl E; Ensz, Nicole M; McComas, Leah S; McCarty, Owen J T

2013-02-01

Several studies have implicated the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in inhibition of normal platelet function, suggesting a role for platelets in EPA- and DHA-mediated cardioprotection. However, it is unclear whether the cardioprotective mechanisms arise from alterations to platelet-platelet, platelet-matrix, or platelet-coagulation factor interactions. Our previous results led us to hypothesize that EPA and DHA alter the ability of platelets to catalyze the generation of thrombin. We tested this hypothesis by exogenously modifying platelet membranes with EPA and DHA, which resulted in compositional changes analogous to increased dietary EPA and DHA intake. Platelets treated with EPA and DHA showed reductions in the rate of thrombin generation and exposure of platelet phosphatidylserine. In addition, treatment of platelets with EPA and DHA decreased thrombus formation and altered the processing of thrombin precursor proteins. Furthermore, treatment of whole blood with EPA and DHA resulted in increased occlusion time and a sharply reduced accumulation of fibrin under flow conditions. These results demonstrate that EPA and DHA inhibit, but do not eliminate, the ability of platelets to catalyze thrombin generation in vitro. The ability of EPA and DHA to reduce the procoagulant function of platelets provides a possible mechanism behind the cardioprotective phenotype in individuals consuming high levels of EPA and DHA.

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

Ethanol inhibits thrombin-induced secretion by human platelets at a site distinct from phospholipase C or protein kinase C.

PubMed Central

Benistant, C; Rubin, R

1990-01-01

Ethanol is known to inhibit the activation of platelets in response to several physiological agonists, but the mechanism of this action is unclear. The addition of physiologically relevant concentrations of ethanol (25-150 mM) to suspensions of washed human platelets resulted in the inhibition of thrombin-induced secretion of 5-hydroxy[14C]tryptamine. Indomethacin was included in the incubation buffer to prevent feedback amplification by arachidonic acid metabolites. Ethanol had no effect on the activation of phospholipase C by thrombin, as determined by the formation of inositol phosphates and the mobilization of intracellular Ca2+. Moreover, ethanol did not interfere with the thrombin-induced formation of diacylglycerol or phosphatidic acid. Stimulation of platelets with phorbol ester (5-50 nM) resulted in 5-hydroxy[14C]tryptamine release comparable with those with threshold doses of thrombin. However, ethanol did not inhibit phorbol-ester-induced secretion. Ethanol also did not interfere with thrombin- or phorbol-ester-induced phosphorylation of myosin light chain (20 kDa) or a 47 kDa protein, a known substrate for protein kinase C. By electron microscopy, ethanol had no effect on thrombin-induced shape change and pseudopod formation, but prevented granule centralization and fusion. The results indicate that ethanol does not inhibit platelet secretion by interfering with the activation of phosphoinositide-specific phospholipase C or protein kinase C by thrombin. Rather, the data demonstrate an inhibition of a Ca2(+)-mediated event such as granule centralization. Images p495-a PMID:2117442

Role of molecular mimicry to HIV-1 peptides in HIV-1–related immunologic thrombocytopenia

PubMed Central

Li, Zongdong; Nardi, Michael A.; Karpatkin, Simon

2005-01-01

Patients with early HIV-1 infection develop an autoimmune thrombocytopenia in which antibody is directed against an immunodominant epitope of the β3 (glycoprotein IIIa [GPIIIa]) integrin, GPIIIa49-66. This antibody induces thrombocytopenia by a novel complement-independent mechanism in which platelets are fragmented by antibody-induced generation of H2O2 derived from the interaction of platelet nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and 12-lipoxygenase. To examine whether sharing of epitope between host and parasite may be responsible for this immunodominant epitope, we screened for antibody-reactive peptides capable of inhibiting platelet lysis and oxidation in vitro, using a filamentous phage display 7-mer peptide library. Fourteen of these phage-peptide clones were identified. Five shared close sequence similarity with GPIIIa49-66, as expected. Ten were molecular mimics with close sequence similarity to HIV-1 proteins nef, gag, env, and pol. Seven were synthesized as 10-mers from their known HIV-1 sequence and found to inhibit anti–GPIIIa49-66–induced platelet oxidation/fragmentation in vitro. Three rabbit antibodies raised against these peptides induced platelet oxidation/fragmentation in vitro and thrombocytopenia in vivo when passively transferred into mice. One of the peptides shared a known epitope region with HIV-1 protein nef and was derived from a variant region of the protein. These data provide strong support for molecular mimicry in HIV-1-immunologic thrombocytopenia within polymorphic regions of HIV-1 proteins. A known epitope of nef is particularly incriminated. PMID:15774614

Genetic Analysis of the Role of Protein Kinase Cθ in Platelet Function and Thrombus Formation

PubMed Central

Hall, Kellie J.; Harper, Matthew T.; Gilio, Karen; Cosemans, Judith M.; Heemskerk, Johan W. M.; Poole, Alastair W.

2008-01-01

Background PKCθ is a novel protein kinase C isozyme, predominately expressed in T cells and platelets. PKCθ−/− T cells exhibit reduced activation and PKCθ−/− mice are resistant to autoimmune disease, making PKCθ an attractive therapeutic target for immune modulation. Collagen is a major agonist for platelets, operating through an immunoreceptor-like signalling pathway from its receptor GPVI. Although it has recently been shown that PKCθ positively regulates outside-in signalling through integrin αIIbβ3 in platelets, the role of PKCθ in GPVI-dependent signalling and functional activation of platelets has not been assessed. Methodology/Principal Findings In the present study we assessed static adhesion, cell spreading, granule secretion, integrin αIIbβ3 activation and platelet aggregation in washed mouse platelets lacking PKCθ. Thrombus formation on a collagen-coated surface was assessed in vitro under flow. PKCθ−/− platelets exhibited reduced static adhesion and filopodia generation on fibrinogen, suggesting that PKCθ positively regulates outside-in signalling, in agreement with a previous report. In contrast, PKCθ−/− platelets also exhibited markedly enhanced GPVI-dependent α-granule secretion, although dense granule secretion was unaffected, suggesting that PKCθ differentially regulates these two granules. Inside-out regulation of αIIbβ3 activation was also enhanced downstream of GPVI stimulation. Although this did not result in increased aggregation, importantly thrombus formation on collagen under high shear (1000 s−1) was enhanced. Conclusions/Significance These data suggest that PKCθ is an important negative regulator of thrombus formation on collagen, potentially mediated by α-granule secretion and αIIbβ3 activation. PKCθ therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCθ inhibitors. PMID:18815612

Can tissue adhesives and platelet-rich plasma prevent pharyngocutaneous fistula formation?

PubMed

Eryılmaz, Aylin; Demirci, Buket; Gunel, Ceren; Kacar Doger, Firuzan; Yukselen, Ozden; Kurt Omurlu, Imran; Basal, Yesim; Agdas, Fatih; Basak, Sema

2016-02-01

One of the frequently encountered disorders of wound healing following laryngectomy is pharyngocutaneous fistula. However, although studies have been performed with the aim of prevention of pharyngocutaneous fistulae, there are very few studies with tissue adhesives and platelet-rich plasma. In this study, our aim was to investigate the histopathologic changes in wound healing caused by various tissue adhesives and platelet-rich plasma, together with their effects on prevention of pharyngocutaneous fistula. 40 male rats were randomly divided into five groups: control, platelet-rich plasma, fibrin tissue adhesive, protein-based albumin glutaraldehyde and synthetic tissue adhesive groups. The pharyngotomy procedure was performed and was sutured. Except the control group, tissue adhesives and platelet-rich plasma were applied. Then, the skin was sutured. On the seventh day, the rats were sacrificed. The skin was opened and pharyngotomy site was assessed in terms of fistulae. The pharyngeal suture line was evaluated histopathologically by using Ehrlich Hunt scale. Inflammatory infiltration was found to be higher in "platelet-rich plasma" group than "fibrin tissue adhesive" and "synthetic tissue adhesive" groups. The fibroblastic activity of "platelet-rich plasma", "fibrin tissue adhesive" and "protein-based albumin glutaraldehyde" groups was higher than the control group. The positive changes created by platelet-rich plasma and fibrin tissue adhesive at the histopathologic level were found together with no detected fistula. Among the study groups, there was no statistical difference for pharyngeal fistula development. This result may be obtained by the small number of animal experiments. These results shed light on the suggestion that platelet-rich plasma and fibrin tissue adhesive can be used in clinical studies to prevent pharyngocutaneous fistula. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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.

Sialylation on O-glycans protects platelets from clearance by liver Kupffer cells.

PubMed

Li, Yun; Fu, Jianxin; Ling, Yun; Yago, Tadayuki; McDaniel, J Michael; Song, Jianhua; Bai, Xia; Kondo, Yuji; Qin, Yannan; Hoover, Christopher; McGee, Samuel; Shao, Bojing; Liu, Zhenghui; Sonon, Roberto; Azadi, Parastoo; Marth, Jamey D; McEver, Rodger P; Ruan, Changgeng; Xia, Lijun

2017-08-01

Most platelet membrane proteins are modified by mucin-type core 1-derived glycans (O-glycans). However, the biological importance of O-glycans in platelet clearance is unclear. Here, we generated mice with a hematopoietic cell-specific loss of O-glycans (HC C1galt1 -/- ). These mice lack O-glycans on platelets and exhibit reduced peripheral platelet numbers. Platelets from HC C1galt1 -/- mice show reduced levels of α-2,3-linked sialic acids and increased accumulation in the liver relative to wild-type platelets. The preferential accumulation of HC C1galt1 -/- platelets in the liver was reduced in mice lacking the hepatic asialoglycoprotein receptor [Ashwell-Morell receptor (AMR)]. However, we found that Kupffer cells are the primary cells phagocytosing HC C1galt1 -/- platelets in the liver. Our results demonstrate that hepatic AMR promotes preferential adherence to and phagocytosis of desialylated and/or HC C1galt1 -/- platelets by the Kupffer cell through its C-type lectin receptor CLEC4F. These findings provide insights into an essential role for core 1 O-glycosylation of platelets in their clearance in the liver.

Sialylation on O-glycans protects platelets from clearance by liver Kupffer cells

PubMed Central

Li, Yun; Fu, Jianxin; Ling, Yun; Yago, Tadayuki; McDaniel, J. Michael; Song, Jianhua; Bai, Xia; Kondo, Yuji; Qin, Yannan; Hoover, Christopher; McGee, Samuel; Shao, Bojing; Liu, Zhenghui; Sonon, Roberto; Azadi, Parastoo; Marth, Jamey D.; McEver, Rodger P.; Ruan, Changgeng; Xia, Lijun

2017-01-01

Most platelet membrane proteins are modified by mucin-type core 1-derived glycans (O-glycans). However, the biological importance of O-glycans in platelet clearance is unclear. Here, we generated mice with a hematopoietic cell-specific loss of O-glycans (HC C1galt1−/−). These mice lack O-glycans on platelets and exhibit reduced peripheral platelet numbers. Platelets from HC C1galt1−/− mice show reduced levels of α-2,3-linked sialic acids and increased accumulation in the liver relative to wild-type platelets. The preferential accumulation of HC C1galt1−/− platelets in the liver was reduced in mice lacking the hepatic asialoglycoprotein receptor [Ashwell–Morell receptor (AMR)]. However, we found that Kupffer cells are the primary cells phagocytosing HC C1galt1−/− platelets in the liver. Our results demonstrate that hepatic AMR promotes preferential adherence to and phagocytosis of desialylated and/or HC C1galt1−/− platelets by the Kupffer cell through its C-type lectin receptor CLEC4F. These findings provide insights into an essential role for core 1 O-glycosylation of platelets in their clearance in the liver. PMID:28716912

A Novel Type of Macrothrombocytopenia Associated with a Defect in α2,3-Sialylation

PubMed Central

Jones, Claire; Denecke, Jonas; Sträter, Ronald; Stölting, Torsten; Schunicht, Yvonne; Zeuschner, Dagmar; Klumperman, Judith; Lefeber, Dirk J.; Spelten, Oliver; Zarbock, Alexander; Kelm, Sørge; Strenge, Karen; Haslam, Stuart M.; Lühn, Kerstin; Stahl, Dorothea; Gentile, Luca; Schreiter, Thomas; Hilgard, Philip; Beck-Sickinger, Annette G.; Marquardt, Thorsten; Wild, Martin K.

2011-01-01

We describe a novel type of human thrombocytopenia characterized by the appearance of giant platelets and variable neutropenia. Searching for the molecular defect, we found that neutrophils had strongly reduced sialyl-Lewis X and increased Lewis X surface expression, pointing to a deficiency in sialylation. We show that the glycosylation defect is restricted to α2,3-sialylation and can be detected in platelets, neutrophils, and monocytes. Platelets exhibited a distorted structure of the open canalicular system, indicating defective platelet generation. Importantly, patient platelets, but not normal platelets, bound to the asialoglycoprotein receptor (ASGP-R), a liver cell-surface protein that removes desialylated thrombocytes from the circulation in mice. Taken together, this is the first type of human thrombocytopenia in which a specific defect of α2,3-sialylation and an induction of platelet binding to the liver ASGP-R could be detected. PMID:21864493

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.

Modifying murine von Willebrand factor A1 domain for in vivo assessment of human platelet therapies.

PubMed

Chen, Jianchun; Tan, Kui; Zhou, Hairu; Lo, Hsuan-Fu; Tronik-Le Roux, Diana; Liddington, Robert C; Diacovo, Thomas G

2008-01-01

The A1 domain of von Willebrand factor (VWF-A1) plays a crucial role in hemostasis and thrombosis by initiating platelet adhesion at sites of arterial injury through interactions with the platelet receptor glycoprotein Ib alpha (GPIbalpha). Here we report that murine VWF-A1 supports limited binding of human platelets. However, atomic models of GPIbalpha-VWF-A1 complexes identified an electrostatic 'hot-spot' that, when mutated in murine VWF-A1, switches its binding specificity from mouse to human GPIbalpha. Furthermore, mice expressing this mutant VWF-A1 display a bleeding phenotype that can be corrected by infusion of human platelets. Mechanistically, human platelets correct the phenotype by forming occlusive thrombi, an event that can be abrogated by blockade of GPIbalpha or by the preadministration of inhibitors of platelet activation or adhesion (clopidogrel (Plavix) and abciximab (ReoPro), respectively). Thus, by modifying a protein interface, we have generated a potential biological platform for preclinical screening of antithrombotics that specifically target human platelets.

Negative feedback regulation of human platelets via autocrine activation of the platelet-derived growth factor alpha-receptor.

PubMed

Vassbotn, F S; Havnen, O K; Heldin, C H; Holmsen, H

1994-05-13

Human platelets contain platelet-derived growth factor (PDGF) in their alpha-granules which is released during platelet exocytosis. We show by immunoprecipitation and 125I-PDGF binding experiments that human platelets have functionally active PDGF alpha-receptors, but not beta-receptors. The PDGF alpha-receptor (PDGFR-alpha) was identified as a 170-kDa glycosylated protein-tyrosine kinase as found in other cell types. Stimulation of platelets with 0.1 unit/ml thrombin resulted in a significant increase (2-5-fold) of the tyrosine phosphorylation of the PDGFR-alpha, as determined by immunoprecipitation with phosphotyrosine antiserum as well as with PDGFR-alpha antiserum. The observed thrombin-induced autophosphorylation of the PDGFR-alpha was inhibited by the addition of a neutralizing monoclonal PDGF antibody. Thus, our results suggest that the platelet PDGFR-alpha is stimulated in an autocrine manner by PDGF secreted during platelet activation. Preincubation of platelets with PDGF inhibited thrombin-induced platelet aggregation and secretion of ATP + ADP and beta-hexosaminidase. Thrombin-induced platelet aggregation was also reversed when PDGF was added 30 s after thrombin stimulation. Inhibition of the autocrine PDGF pathway during platelet activation by the PDGF antibody led to a potentiation of thrombin-induced beta-hexosaminidase secretion. Thus, the PDGFR-alpha takes part in a negative feedback regulation during platelet activation. Our demonstration of PDGF alpha-receptors on human platelets and its inhibitory function during platelet activation identifies a new possible role of PDGF in the regulation of thrombosis.

Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens.

PubMed

Lynch, D M; Howe, S E

1985-11-01

Sera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.

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

Increasing platelet concentration in platelet-rich plasma inhibits anterior cruciate ligament cell function in three-dimensional culture.

PubMed

Yoshida, Ryu; Cheng, Mingyu; Murray, Martha M

2014-02-01

Tissue engineering is one new strategy being developed to treat ACL ruptures. One such approach is bio-enhanced ACL repair, where a suture repair is supplemented with a bio-active scaffold containing platelets. However, the optimal concentration of platelets to stimulate ACL healing is not known. We hypothesized that increasing platelet concentrations in the scaffold would enhance critical cell behaviors. Porcine ACL fibroblasts were obtained from explant culture and suspended in platelet poor plasma (PPP), 1× platelet-rich plasma (PRP), 3× PRP, 5× PRP, or phosphate buffered saline (PBS). The cell suspensions were cultured in a 3D collagen scaffold. Cellular metabolism (MTT assay), apoptosis (TUNEL assay), and gene expression for type I and type III collagen were measured. 1× PRP significantly outperformed 5× PRP in all parameters studied: Type I and III collagen gene expression, apoptosis prevention, and cell metabolism stimulation. ACL fibroblasts cultured with 1× PRP had the highest type I and type III collagen gene expression. 1× PRP and PPP groups had the highest cell metabolism and lowest apoptosis rates. Concentration of platelets had significant effects on the behavior of ACL fibroblasts; thus, it is an important parameter that should be specified in clinical or basic science studies. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Tyrosine phosphorylation of P-selectin in intact platelets and in a disulphide-linked complex with immunoprecipitated pp60c-src.

PubMed Central

Modderman, P W; von dem Borne, A E; Sonnenberg, A

1994-01-01

P-selectin is a 140 kDa membrane glycoprotein found in secretory granules of platelets and endothelial cells where it is rapidly translocated to the plasma membrane upon cell activation. It then functions as a receptor for various types of leucocytes. Metabolic labelling of resting platelets with 32Pi showed that P-selectin is primarily phosphorylated on serine residues, although some tyrosine phosphorylation was observed as well. However, tyrosine phosphorylation of P-selectin was greatly stimulated by treatment with the permeating phosphatase inhibitor, pervanadate. When P-selectin immunoprecipitates were incubated with [gamma-32P]ATP (in vitro kinase assay), a fraction of P-selectin was phosphorylated on its tyrosine residues by a co-precipitated kinase. P-selectin phosphorylated in vitro co-migrated with 140 kDa surface-labelled 125I-P-selectin during SDS/PAGE under reducing conditions. Under non-reducing conditions, however, phosphorylated P-selectin was disulphide-linked to unknown protein(s) in a 205 kDa complex. In vitro kinase assays of the most abundant platelet tyrosine kinase, pp60c-src, demonstrated the presence of similar 140 and 205 kDa phosphorylated proteins in SDS/PAGE under reducing and non-reducing conditions respectively. Extraction and reprecipitation studies with proteins phosphorylated in vitro indicated that P-selectin and pp60c-src form a 205 kDa 1:1 disulphide-linked complex. In the complex, pp60c-src autophosphorylation is inhibited and P-selectin is phosphorylated on tyrosine residues. As protein disulphides in the cytoplasm of intact cells are extremely rare, our results suggest that P-selectin and pp60c-src, which co-localize in platelet dense granules, may be non-covalently associated and spontaneously form disulphide bridges during lysis. In addition, the observed tyrosine phosphorylation of P-selectin in intact platelets suggests that its function might be regulated by phosphorylation by pp60c-src. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7514867

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

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-Derived Growth Factor-BB Stimulates Fibronectin Gene Expression in Fibroblasts Isolated from Rat Thoracic Aorta

DTIC Science & Technology

1994-06-13

MARYLAND 20814-4799 TEACHING HOSPITALS WALTER REED ARMY MEDtCA L CENTER APPROVAL SHEET NAVAL HOSPITAL. BETHESDA MALCOLM GROW AIR FORCE MEDICAL ...CENTER WILFORD HALL "IR FORCE MEDICAL CENTER Title of Dissertation: "Platelet-derived growth factor-BB stimulates fibronectin gene expression in...fascinating world of basic medical science. His dedication and pursuit of excellence in all facets of his work are standards by which I will guide my own

Platelets in thrombosis and hemostasis: old topic with new mechanisms.

PubMed

Wang, Yiming; Andrews, Marc; Yang, Yan; Lang, Sean; Jin, Joseph W; Cameron-Vendrig, Alison; Zhu, Guangheng; Reheman, Adili; Ni, Heyu

2012-12-01

Platelets are small anucleate cells generated from megakaryocytes in the bone marrow. After being released into the circulation, platelets play key roles in the surveillance of vascular injury, and can quickly adhere and aggregate at the site of injury, which are critical events for vascular repair and hemostasis. However, the same biological processes of platelet adhesion and aggregation may also cause thrombotic disorders. The formation of a platelet plug at sites of atherosclerotic lesion rupture is the most common mechanism leading to myocardial or cerebral infarction. Platelet-related deep vein thrombosis is also one of the leading causes of mortality worldwide. The contribution of several platelet receptors and their ligands has been highlighted in these processes. In platelet adhesion, particularly at high shear stress, GPIbα-von Willebrand factor (VWF) interaction may initiate this event, which is followed by GPVI signalling and firm platelet adhesion mediated by members of the integrin family, such as β3 (αIIbβ3) and β1 (α2β1, α5β1) integrins. In platelet aggregation, although GPIbα-VWF, P selectin-sulfatides, and other molecules, may be involved, the process is mainly mediated by β3 (αIIbβ3) integrin and its ligands, such as fibrinogen and VWF. It is intriguing that platelet adhesion and aggregation still occur in mice lacking both fibrinogen and VWF, suggesting that other unforeseen molecule(s) may also be important in these processes. Identification and characterization of these molecules will enrich our knowledge in the basic science of hemostasis and thrombosis, and may lead to the development of new therapies against bleeding disorders and thrombotic diseases.

Platelet receptors for the Streptococcus sanguis adhesin and aggregation-associated antigens are distinguished by anti-idiotypical monoclonal antibodies.

PubMed Central

Gong, K; Wen, D Y; Ouyang, T; Rao, A T; Herzberg, M C

1995-01-01

Platelets aggregate in response to an adhesin and the platelet aggregation-associated protein (PAAP) expressed on the cell surfaces of certain strains of Streptococcus sanguis. We sought to identify the corresponding PAAP receptor and accessory adhesin binding sites on platelets. Since the adhesion(s) of S. sanguis for platelets has not been characterized, an anti-idiotype (anti-id) murine monoclonal antibody (MAb2) strategy was developed. First, MAb1s that distinguished the adhesin and PAAP antigens on the surface of S. sanguis I 133-79 were selected. Fab fragments of MAb1.2 (immunoglobulin G2b [IgG2b]; 70 pmol) reacted with 5 x 10(7) cells of S. sanguis to completely inhibit the aggregation of human platelets in plasma. Under similar conditions, MAb1.1 (IgG1) inhibited the adhesion of S. sanguis cells to platelets by a maximum of 34%, with a comparatively small effect on platelet aggregation. Together, these two MAb1s inhibited S. sanguis-platelet adhesion by 63%. In Western immunoblots, both MAb1s reacted with S. sanguis 133-79 87- and 150-kDa surface proteins and MAb1.2 also reacted with purified type I collagen. The hybridomas producing MAb1.1 and MAb1.2 were then injected into BALB/c mice. Enlarged spleens were harvested, and a panel of MAb2 hybridomas was prepared. To identify anti-ids against the specific MAb1s, the MAb2 panel was screened by enzyme-linked immunosorbent assay for reaction with rabbit polyclonal IgG antibodies against the 87- and 150-kDa antigens. The reactions between the specific rabbit antibodies and anti-ids were inhibited by the 87- and 150-kDa antigens. When preincubated with platelets, MAb2.1 (counterpart of MAb1.1) inhibited adhesion to platelets maximally by 46% and MAb2.2 (anti-MAb1.2) inhibited adhesion to platelets maximally by 35%. Together, both MAb2s inhibited the adhesion of S. sanguis to platelets by 81%. MAb2.2 also inhibited induction of platelet aggregation. MAb2.2 immunoprecipitated a biotinylated platelet membrane antigen of 170 kDa (unreduced); MAb2.1 precipitated membrane antigens of 175- and 230-kDa (unreduced). Therefore, platelet binding sites and the receptor for the S. sanguis adhesin and PAAP, respectively, are distinguished by the anti-id MAb2s. PMID:7642300

Hyperreactivity of junctional adhesion molecule A-deficient platelets accelerates atherosclerosis in hyperlipidemic mice.

PubMed

Karshovska, Ela; Zhao, Zhen; Blanchet, Xavier; Schmitt, Martin M N; Bidzhekov, Kiril; Soehnlein, Oliver; von Hundelshausen, Philipp; Mattheij, Nadine J; Cosemans, Judith M E M; Megens, Remco T A; Koeppel, Thomas A; Schober, Andreas; Hackeng, Tilman M; Weber, Christian; Koenen, Rory R

2015-02-13

Besides their essential role in hemostasis, platelets also have functions in inflammation. In platelets, junctional adhesion molecule (JAM)-A was previously identified as an inhibitor of integrin αIIbβ3-mediated outside-in signaling and its genetic knockdown resulted in hyperreactivity. This gain-of-function was specifically exploited to investigate the role of platelet hyperreactivity in plaque development. JAM-A-deficient platelets showed increased aggregation and cellular and sarcoma tyrosine-protein kinase activation. On αIIbβ3 ligation, JAM-A was shown to be dephosphorylated, which could be prevented by protein tyrosine phosphatase nonreceptor type 1 inhibition. Mice with or without platelet-specific (tr)JAM-A-deficiency in an apolipoprotein e (apoe(-/-)) background were fed a high-fat diet. After ≤12 weeks of diet, trJAM-A(-/-)apoe-/- mice showed increased aortic plaque formation when compared with trJAM-A(+/+) apoe(-/-) controls, and these differences were most evident at early time points. At 2 weeks, the plaques of the trJAM-A(-/-) apoe(-/-) animals revealed increased macrophage, T cell, and smooth muscle cell content. Interestingly, plasma levels of chemokines CC chemokine ligand 5 and CXC-chemokine ligand 4 were increased in the trJAM-A(-/-) apoe(-/-)mice, and JAM-A-deficient platelets showed increased binding to monocytes and neutrophils. Whole-blood perfusion experiments and intravital microscopy revealed increased recruitment of platelets and monocytes to the inflamed endothelium in blood of trJAM-A(-/-) apoe(-/-)mice. Notably, these proinflammatory effects of JAM-A-deficient platelets could be abolished by the inhibition of αIIbβ3 signaling in vitro. Deletion of JAM-A causes a gain-of-function in platelets, with lower activation thresholds and increased inflammatory activities. This leads to an increase of plaque formation, particularly in early stages of the disease. © 2014 American Heart Association, Inc.

Tailor-made purified human platelet lysate concentrated in neurotrophins for treatment of Parkinson's disease.

PubMed

Chou, Ming-Li; Wu, Joe-Wei; Gouel, Flore; Jonneaux, Aurélie; Timmerman, Kelly; Renn, Ting-Yi; Laloux, Charlotte; Chang, Hung-Ming; Lin, Liang-Tzung; Devedjian, Jean-Christophe; Devos, David; Burnouf, Thierry

2017-10-01

Human platelet lysates (PLs), which contain multiple neurotrophins, have been proposed for treating neurodegenerative disorders, including Parkinson's disease (PD). However, current PLs suspended in plasma have high protein content and contain fibrinogen/fibrin and, following activation, also proteolytic and thrombogenic enzymes. Upon brain administration, such PLs may saturate the cerebrospinal fluid and exert neurotoxicity. We assessed whether purified PLs, concentrated in neurotrophins, protected dopaminergic neurons in PD models. Platelet concentrates were collected by apheresis and centrifuged to eliminate plasma and recover the platelets. Platelets were lysed by freeze-thaw cycles, and the 10-fold concentrated platelet pellet lysates (PPLs) were heat-treated (at 56 °C for 30 min). The heat-treated PPLs were low in total proteins, depleted in both plasma and platelet fibrinogen, and devoid of thrombogenic and proteolytic activities. They exerted very high neuroprotective activity when non-oncogenic, Lund human mesencephalic (LUHMES) cells that had differentiated into dopaminergic neurons were exposed to the MPP + neurotoxin. Heat treatment improved the neuroprotection and inactivated the neurotoxic blood-borne hepatitis C virus. PPL did not induce inflammation in BV2 microglial cells and inhibited COX-2 expression upon lipopolysaccharide exposure. Intranasal administration in mice revealed (a) diffusion of neurotrophins in the striatum and cortex, and (b) MPTP intoxication neuroprotection in the substantia nigra and striatum and the absence of neuroinflammation. These dedicated heat-treated PPLs can be a safe and valuable candidate for a therapeutic strategy for PD. Copyright © 2017 Elsevier Ltd. All rights reserved.

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-Specific Chemokines Contribute to the Pathogenesis of Acute Lung Injury

PubMed Central

Bdeir, Khalil; Gollomp, Kandace; Stasiak, Marta; Mei, Junjie; Papiewska-Pajak, Izabela; Zhao, Guohua; Worthen, G. Scott; Cines, Douglas B.; Poncz, Mortimer

2017-01-01

Platelets and neutrophils contribute to the development of acute lung injury (ALI). However, the mechanism by which platelets make this contribution is incompletely understood. We investigated whether the two most abundant platelet chemokines, CXCL7, which induces neutrophil chemotaxis and activation, and CXCL4, which does neither, mediate ALI through complementary pathogenic pathways. To examine the role of platelet-derived chemokines in the pathogenesis of ALI using Cxcl7−/− and Cxcl4−/− knockout mice and mice that express human CXCL7 or CXCL4, we measured levels of chemokines in these mice. ALI was then induced by acid aspiration, and the severity of injury was evaluated by histology and by the presence of neutrophils and protein in the bronchoalveolar lavage fluid. Pulmonary vascular permeability was studied in vivo by measuring extravasation of fluorescently labeled dextran. Murine CXCL7, both recombinant and native protein released from platelets, can be N-terminally processed by cathepsin G to yield a biologically active CXCL7 fragment. Although Cxcl7−/− mice are protected from lung injury through the preservation of endothelial/epithelial barrier function combined with impaired neutrophils transmigration, Cxcl4−/− mice are protected through improved barrier function without affecting neutrophils transmigration to the airways. Sensitivity to ALI is restored by transgenic expression of CXCL7 or CXCL4. Platelet-derived CXCL7 and CXCL4 contribute to the pathogenesis of ALI through complementary effects on neutrophil chemotaxis and through activation and vascular permeability. PMID:27755915

Platelet-Specific Chemokines Contribute to the Pathogenesis of Acute Lung Injury.

PubMed

Bdeir, Khalil; Gollomp, Kandace; Stasiak, Marta; Mei, Junjie; Papiewska-Pajak, Izabela; Zhao, Guohua; Worthen, G Scott; Cines, Douglas B; Poncz, Mortimer; Kowalska, M Anna

2017-02-01

Platelets and neutrophils contribute to the development of acute lung injury (ALI). However, the mechanism by which platelets make this contribution is incompletely understood. We investigated whether the two most abundant platelet chemokines, CXCL7, which induces neutrophil chemotaxis and activation, and CXCL4, which does neither, mediate ALI through complementary pathogenic pathways. To examine the role of platelet-derived chemokines in the pathogenesis of ALI using Cxcl7 -/- and Cxcl4 -/- knockout mice and mice that express human CXCL7 or CXCL4, we measured levels of chemokines in these mice. ALI was then induced by acid aspiration, and the severity of injury was evaluated by histology and by the presence of neutrophils and protein in the bronchoalveolar lavage fluid. Pulmonary vascular permeability was studied in vivo by measuring extravasation of fluorescently labeled dextran. Murine CXCL7, both recombinant and native protein released from platelets, can be N-terminally processed by cathepsin G to yield a biologically active CXCL7 fragment. Although Cxcl7 -/- mice are protected from lung injury through the preservation of endothelial/epithelial barrier function combined with impaired neutrophils transmigration, Cxcl4 -/- mice are protected through improved barrier function without affecting neutrophils transmigration to the airways. Sensitivity to ALI is restored by transgenic expression of CXCL7 or CXCL4. Platelet-derived CXCL7 and CXCL4 contribute to the pathogenesis of ALI through complementary effects on neutrophil chemotaxis and through activation and vascular permeability.

A novel ruthenium (II)-derived organometallic compound, TQ-6, potently inhibits platelet aggregation: Ex vivo and in vivo studies.

PubMed

Hsia, Chih-Hsuan; Velusamy, Marappan; Sheu, Joen-Rong; Khamrang, Themmila; Jayakumar, Thanasekaran; Lu, Wan-Jung; Lin, Kuan-Hung; Chang, Chao-Chien

2017-08-25

Arterial thrombosis plays a key role in cardiovascular diseases. Hence, developing more effective antithrombotic agents is necessary. We designed a ruthenium (II)-derived complex, [Ru(η 6 -cymene)2-(1H-benzoimidazol-2-yl)-quinoline Cl]BF 4 (TQ-6), as a new antiplatelet drug. TQ-6 (0.3 µM) exhibited extremely strong inhibitory activity against platelet aggregation, Src, and Syk phosphorylation stimulated by agonists in human platelets. In collagen-activated platelets, TQ-6 also inhibited ATP-release, [Ca +2 ]i, P-selectin expression, FITC-PAC-1 binding, and hydroxyl radical formation, as well as the phosphorylation of phospholipase Cγ2, protein kinase C, mitogen-activated protein kinases, and Akt. Neither FITC-JAQ1 nor FITC-triflavin binding or integrin β 3 phosphorylation stimulated by immobilized fibrinogen were diminished by TQ-6. Furthermore, TQ-6 had no effects in cyclic nucleotide formation. Moreover, TQ-6 substantially prolonged the closure time in whole blood, increased the occlusion time of thrombotic platelet plug formation and bleeding time in mice. In conclusion, TQ-6 has a novel role in inhibiting platelet activation through the inhibition of the agonist receptors-mediated inside-out signaling such as Src-Syk-PLCγ2 cascade and subsequent suppression of granule secretion, leading to disturb integrin α IIb β 3 -mediated outside-in signaling, and ultimately inhibiting platelet aggregation. Therefore, TQ-6 has potential to develop as a therapeutic agent for preventing or treating thromboembolic disorders.

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.

Serum proteomics reveals systemic dysregulation of innate immunity in type 1 diabetes

PubMed Central

Fillmore, Thomas L.; Schepmoes, Athena A.; Clauss, Therese R.W.; Gritsenko, Marina A.; Mueller, Patricia W.; Rewers, Marian; Atkinson, Mark A.; Smith, Richard D.

2013-01-01

Using global liquid chromatography-mass spectrometry (LC-MS)–based proteomics analyses, we identified 24 serum proteins that were significantly variant between those with type 1 diabetes (T1D) and healthy controls. Functionally, these proteins represent innate immune responses, the activation cascade of complement, inflammatory responses, and blood coagulation. Targeted verification analyses were performed on 52 surrogate peptides representing these proteins, with serum samples from an antibody standardization program cohort of 100 healthy control and 50 type 1 diabetic subjects. 16 peptides were verified as having very good discriminating power, with areas under the receiver operating characteristic curve ≥0.8. Further validation with blinded serum samples from an independent cohort (10 healthy control and 10 type 1 diabetics) demonstrated that peptides from platelet basic protein and C1 inhibitor achieved both 100% sensitivity and 100% specificity for classification of samples. The disease specificity of these proteins was assessed using sera from 50 age-matched type 2 diabetic individuals, and a subset of proteins, C1 inhibitor in particular, were exceptionally good discriminators between these two forms of diabetes. The panel of biomarkers distinguishing those with T1D from healthy controls and those with type 2 diabetes suggests that dysregulated innate immune responses may be associated with the development of this disorder. PMID:23277452

Serum proteomics reveals systemic dysregulation of innate immunity in type 1 diabetes

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

Zhang, Qibin; Fillmore, Thomas L.; Schepmoes, Athena A.

Using global liquid chromatography-mass spectrometry (LC-MS)-based proteomics analyses, we identified 24 serum proteins significantly variant between those with type 1 diabetes and healthy controls. Functionally, these proteins represent innate immune responses, the activation cascade of complement, inflammatory responses and blood coagulation. Targeted verification analyses were performed on 52 surrogate peptides representing these proteins with serum samples from an antibody standardization program cohort of 100 healthy control and 50 type 1 diabetic subjects, and 16 peptides were verified having very good discriminating power, with areas under the receiver operator characteristic curve ≥ 0.8. Further validation with blinded serum samples from anmore » independent cohort (10 healthy control and 10 type 1 diabetic) demonstrated that peptides from platelet basic protein and C1 inhibitor achieved both 100% sensitivity and 100% specificity for classification of samples. The disease specificity of these proteins was assessed using serum from 50 age matched type 2 diabetic individuals, and a subset of proteins, particularly C1 inhibitor were exceptionally good discriminators between these two forms of diabetes. The panel of biomarkers distinguishing those with type 1 diabetes from healthy control and type 2 diabetes suggests dysregulated innate immune responses may be associated with the development of this disorder.« 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.

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

Multi-omics Analysis of Serum Samples Demonstrates Reprogramming of Organ Functions Via Systemic Calcium Mobilization and Platelet Activation in Metastatic Melanoma*

PubMed Central

Muqaku, Besnik; Eisinger, Martin; Meier, Samuel M.; Tahir, Ammar; Pukrop, Tobias; Haferkamp, Sebastian; Slany, Astrid; Reichle, Albrecht

2017-01-01

Pathophysiologies of cancer-associated syndromes such as cachexia are poorly understood and no routine biomarkers have been established, yet. Using shotgun proteomics, known marker molecules including PMEL, CRP, SAA, and CSPG4 were found deregulated in patients with metastatic melanoma. Targeted analysis of 58 selected proteins with multiple reaction monitoring was applied for independent data verification. In three patients, two of which suffered from cachexia, a tissue damage signature was determined, consisting of nine proteins, PLTP, CD14, TIMP1, S10A8, S10A9, GP1BA, PTPRJ, CD44, and C4A, as well as increased levels of glycine and asparagine, and decreased levels of polyunsaturated phosphatidylcholine concentrations, as determined by targeted metabolomics. Remarkably, these molecules are known to be involved in key processes of cancer cachexia. Based on these results, we propose a model how metastatic melanoma may lead to reprogramming of organ functions via formation of platelet activating factors from long-chain polyunsaturated phosphatidylcholines under oxidative conditions and via systemic induction of intracellular calcium mobilization. Calcium mobilization in platelets was demonstrated to alter levels of several of these marker molecules. Additionally, platelets from melanoma patients proved to be in a rather exhausted state, and platelet-derived eicosanoids implicated in tumor growth were found massively increased in blood from three melanoma patients. Platelets were thus identified as important source of serum protein and lipid alterations in late stage melanoma patients. As a result, the proposed model describes the crosstalk between lipolysis of fat tissue and muscle wasting mediated by oxidative stress, resulting in the metabolic deregulations characteristic for cachexia. PMID:27879288

A systems approach to hemostasis: 4. How hemostatic thrombi limit the loss of plasma-borne molecules from the microvasculature

PubMed Central

Welsh, John D.; Muthard, Ryan W.; Stalker, Timothy J.; Taliaferro, Joshua P.; Diamond, Scott L.

2016-01-01

Previous studies have shown that hemostatic thrombi formed in response to penetrating injuries have a core of densely packed, fibrin-associated platelets overlaid by a shell of less-activated, loosely packed platelets. Here we asked, first, how the diverse elements of this structure combine to stem the loss of plasma-borne molecules and, second, whether antiplatelet agents and anticoagulants that perturb thrombus structure affect the re-establishment of a tight vascular seal. The studies combined high-resolution intravital microscopy with a photo-activatable fluorescent albumin marker to simultaneously track thrombus formation and protein transport following injuries to mouse cremaster muscle venules. The results show that protein loss persists after red cell loss has ceased. Blocking platelet deposition with an αIIbβ3 antagonist delays vessel sealing and increases extravascular protein accumulation, as does either inhibiting adenosine 5′-diphosphate (ADP) P2Y12 receptors or reducing integrin-dependent signaling and retraction. In contrast, sealing was unaffected by introducing hirudin to block fibrin accumulation or a Gi2α gain-of-function mutation to expand the thrombus shell. Collectively, these observations describe a novel approach for studying vessel sealing after injury in real time in vivo and show that (1) the core/shell architecture previously observed in arterioles also occurs in venules, (2) plasma leakage persists well beyond red cell escape and mature thrombus formation, (3) the most critical events for limiting plasma extravasation are the stable accumulation of platelets, ADP-dependent signaling, and the emergence of a densely packed core, not the accumulation of fibrin, and (4) drugs that affect platelet accumulation and packing can delay vessel sealing, permitting protein escape to continue. PMID:26738537

Platelets Contain Tissue Factor Pathway Inhibitor-2 Derived from Megakaryocytes and Inhibits Fibrinolysis*

PubMed Central

Vadivel, Kanagasabai; Ponnuraj, Sathya-Moorthy; Kumar, Yogesh; Zaiss, Anne K.; Bunce, Matthew W.; Camire, Rodney M.; Wu, Ling; Evseenko, Denis; Herschman, Harvey R.; Bajaj, Madhu S.; Bajaj, S. Paul

2014-01-01

Tissue factor pathway inhibitor-2 (TFPI-2) is a homologue of TFPI-1 and contains three Kunitz-type domains and a basic C terminus region. The N-terminal domain of TFPI-2 is the only inhibitory domain, and it inhibits plasma kallikrein, factor XIa, and plasmin. However, plasma TFPI-2 levels are negligible (≤20 pm) in the context of influencing clotting or fibrinolysis. Here, we report that platelets contain significant amounts of TFPI-2 derived from megakaryocytes. We employed RT-PCR, Western blotting, immunohistochemistry, and confocal microscopy to determine that platelets, MEG-01 megakaryoblastic cells, and bone marrow megakaryocytes contain TFPI-2. ELISA data reveal that TFPI-2 binds factor V (FV) and partially B-domain-deleted FV (FV-1033) with Kd ∼9 nm and binds FVa with Kd ∼100 nm. Steady state analysis of surface plasmon resonance data reveal that TFPI-2 and TFPI-1 bind FV-1033 with Kd ∼36–48 nm and bind FVa with Kd ∼252–456 nm. Further, TFPI-1 (but not TFPI-1161) competes with TFPI-2 in binding to FV. These data indicate that the C-terminal basic region of TFPI-2 is similar to that of TFPI-1 and plays a role in binding to the FV B-domain acidic region. Using pull-down assays and Western blots, we show that TFPI-2 is associated with platelet FV/FVa. TFPI-2 (∼7 nm) in plasma of women at the onset of labor is also, in part, associated with FV. Importantly, TFPI-2 in platelets and in plasma of pregnant women inhibits FXIa and tissue-type plasminogen activator-induced clot fibrinolysis. In conclusion, TFPI-2 in platelets from normal or pregnant subjects and in plasma from pregnant women binds FV/Va and regulates intrinsic coagulation and fibrinolysis. PMID:25262870

Platelet-Rich Plasma in the Animal Long-Bone Model: An Analysis of Basic Science Evidence.

PubMed

Gianakos, Arianna; Zambrana, Lester; Savage-Elliott, Ian; Lane, Joseph M; Kennedy, John G

2015-12-01

Platelet-rich plasma (PRP) has been suggested as an adjunct to aid in long-bone healing. The purpose of this study was to systematically review the basic science in vivo evidence for the use of PRP in the treatment of bone pathology. The PubMed/MEDLINE and EMBASE databases were screened using the following search criteria: "(Platelet-rich plasma OR PRP OR autologous conditioned plasma OR ACP) AND (bone OR osteocytes OR osteogenesis OR nonunion OR delayed union)." Studies were included if they fulfilled the following criteria: (1) studied the effect of PRP or a similar concentrated platelet product, defined as a blood product with platelet concentration elevated to higher than baseline; (2) established a control with which to compare PRP; (3) were published in a peer-reviewed journal; and (4) looked specifically at animal long-bone models. All review articles and clinical studies, including randomized controlled trials and case series, were excluded from the review. Studies examining the effects of PRP on bones of animals with confounding pathology were excluded. In studies that contained additional treatment variables, only the portion of the experiment that compared PRP directly with the control were evaluated. Data were then extracted with a standardized table. The search yielded 29 articles for inclusion. Seventy-two percent of the studies reported platelet concentrations. Eighty-nine percent of studies reported significant improvement in earlier bone healing on histologic/histomorphometric assessment. One hundred percent showed significant increase in bone formation on radiographs in the PRP group. Eighty percent of studies reported a significant increase in bone area on microcomputed tomography. One hundred percent of studies showed a higher torsional stiffness for the PRP-treated defects. In the in vivo studies evaluated, PRP confers several beneficial effects on animal long-bone models. Proof of concept for PRP as a biologic adjunct in long-bone models has been determined. Copyright 2015, SLACK Incorporated.

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

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

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

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.

NCO-sP(EO-stat-PO) coatings on gold sensors--a QCM study of hemocompatibility.

PubMed

Sinn, Stefan; Eichler, Mirjam; Müller, Lothar; Bünger, Daniel; Groll, Jürgen; Ziemer, Gerhard; Rupp, Frank; Northoff, Hinnak; Geis-Gerstorfer, Jürgen; Gehring, Frank K; Wendel, Hans P

2011-01-01

The reliability of implantable blood sensors is often hampered by unspecific adsorption of plasma proteins and blood cells. This not only leads to a loss of sensor signal over time, but can also result in undesired host vs. graft reactions. Within this study we evaluated the hemocompatibility of isocyanate conjugated star shaped polytheylene oxide-polypropylene oxide co-polymers NCO-sP(EO-stat-PO) when applied to gold surfaces as an auspicious coating material for gold sputtered blood contacting sensors. Quartz crystal microbalance (QCM) sensors were coated with ultrathin NCO-sP(EO-stat-PO) films and compared with uncoated gold sensors. Protein resistance was assessed by QCM measurements with fibrinogen solution and platelet poor plasma (PPP), followed by quantification of fibrinogen adsorption. Hemocompatibility was tested by incubation with human platelet rich plasma (PRP). Thrombin antithrombin-III complex (TAT), β-thromboglobulin (β-TG) and platelet factor 4 (PF4) were used as coagulation activation markers. Furthermore, scanning electron microscopy (SEM) was used to visualize platelet adhesion to the sensor surfaces. Compared to uncoated gold sensors, NCO-sP(EO-stat-PO) coated sensors revealed significant better resistance against protein adsorption, lower TAT generation and a lower amount of adherent platelets. Moreover, coating with ultrathin NCO-sP(EO-stat-PO) films creates a cell resistant hemocompatible surface on gold that increases the chance of prolonged sensor functionality and can easily be modified with specific receptor molecules.

Thiols in the alphaIIbbeta3 integrin are necessary for platelet aggregation.

PubMed

Manickam, Nagaraj; Sun, Xiuhua; Hakala, Kevin W; Weintraub, Susan T; Essex, David W

2008-07-01

Sulfhydryl groups of platelet surface proteins are important in platelet aggregation. While p-chloromercuribenzene sulphonate (pCMBS) has been used in most studies on platelet surface thiols, the specific thiol-proteins that pCMBS reacts with to inhibit aggregation have not been well defined. Since the thiol-containing P2Y(12) ADP receptor is involved in most types of platelet aggregation, we used the ADP scavenger apyrase and the P2Y(12) receptor antagonist 2-MeSAMP to examine thiol-dependent reactions in the absence of contributions from this receptor. We provide evidence for a non-P2Y(12) thiol-dependent reaction near the final alphaIIbbeta3-dependent events of aggregation. We then used 3-(N-maleimidylpropionyl)biocytin (MPB) and pCMBS to study thiols in alphaIIbbeta3. As previously reported, disruption of the receptor was required to obtain labelling of thiols with MPB. Specificity of labelling for thiols in the alphaIIb and beta3 subunits was confirmed by identification of the purified proteins by mass spectrometry and by inhibition of labelling with 5,5'-dithiobis-(2-nitrobenzoic acid). In contrast to MPB, pCMBS preferentially reacted with thiols in alphaIIbbeta3 and blocked aggregation under physiological conditions. Similarly, pCMBS preferentially inhibited signalling-independent activation of alphaIIbbeta3 by Mn(2+). Our results suggest that the thiols in alphaIIbbeta3 that are blocked by pCMBS are important in the activation of this integrin.

NCO-sP(EO-stat-PO) Coatings on Gold Sensors—a QCM Study of Hemocompatibility

PubMed Central

Sinn, Stefan; Eichler, Mirjam; Müller, Lothar; Bünger, Daniel; Groll, Jürgen; Ziemer, Gerhard; Rupp, Frank; Northoff, Hinnak; Geis-Gerstorfer, Jürgen; Gehring, Frank K.; Wendel, Hans P.

2011-01-01

The reliability of implantable blood sensors is often hampered by unspecific adsorption of plasma proteins and blood cells. This not only leads to a loss of sensor signal over time, but can also result in undesired host vs. graft reactions. Within this study we evaluated the hemocompatibility of isocyanate conjugated star shaped polytheylene oxide—polypropylene oxide co-polymers NCO-sP(EO-stat-PO) when applied to gold surfaces as an auspicious coating material for gold sputtered blood contacting sensors. Quartz crystal microbalance (QCM) sensors were coated with ultrathin NCO-sP(EO-stat-PO) films and compared with uncoated gold sensors. Protein resistance was assessed by QCM measurements with fibrinogen solution and platelet poor plasma (PPP), followed by quantification of fibrinogen adsorption. Hemocompatibility was tested by incubation with human platelet rich plasma (PRP). Thrombin antithrombin-III complex (TAT), β-thromboglobulin (β-TG) and platelet factor 4 (PF4) were used as coagulation activation markers. Furthermore, scanning electron microscopy (SEM) was used to visualize platelet adhesion to the sensor surfaces. Compared to uncoated gold sensors, NCO-sP(EO-stat-PO) coated sensors revealed significant better resistance against protein adsorption, lower TAT generation and a lower amount of adherent platelets. Moreover, coating with ultrathin NCO-sP(EO-stat-PO) films creates a cell resistant hemocompatible surface on gold that increases the chance of prolonged sensor functionality and can easily be modified with specific receptor molecules. PMID:22163899

Messenger RNA profiling of human platelets by microarray hybridization.

PubMed

Bugert, Peter; Dugrillon, Alex; Günaydin, Ayse; Eichler, Hermann; Klüter, Harald

2003-10-01

Platelets are generally believed to be inactive in terms of de novo protein synthesis. On the other hand, the presence of ribosomes and mRNA molecules is well established. Many studies have used reverse transcriptase (RT) -PCR for detection of gene transcripts in platelets. As RT-PCR is a very sensitive method, any leukocyte contamination of platelet preparations can lead to false results. We performed three filtration procedures to minimize leukocyte contamination of pooled buffy-coat platelet concentrates prior to RNA isolation. Furthermore, by applying a genomic PCR approach with 50 amplification cycles we demonstrated that nucleated cells were not detectable. Microarray hybridization was used to analyze 9,850 individual human genes in RNA from purified platelets. In total we identified 1,526 (15.5%) positive genes. The data were confirmed in six individual experiments each performed on a PC pooled from four individual blood donations. Genes specific for nucleated blood cells such as CD4, CD83 and others were negative and verified the purity of PC. Overrepresentation of positive genes was found in the functional categories of glycoproteins/integrins (22.6% vs. 15.5%, p=0.029) and receptors (20.7% vs. 15.5%, p<0.001). Gene transcripts encoding RANTES, GRO-alpha, MIP-1alpha, MIP-1beta, and others were found at high levels of signal intensity and confirmed literature data. This work provides a mRNA profile of human platelets and a complete list of results can be downloaded from the website of our institute www.ma.uni-heidelberg.de/inst/iti/plt_array.xls. The knowledge about gene transcripts may have an impact on the characterization of novel proteins and their functions in platelets.

Concentration of platelets and growth factors in platelet-rich plasma from Goettingen minipigs.

PubMed

Jungbluth, Pascal; Grassmann, Jan-Peter; Thelen, Simon; Wild, Michael; Sager, Martin; Windolf, Joachim; Hakimi, Mohssen

2014-01-01

In minipigs little is known about the concentration of growth factors in plasma, despite their major role in several patho-physiological processes such as healing of fractures. This prompted us to study the concentration of platelets and selected growth factors in plasma and platelet-rich plasma (PRP) preparation of sixteen Goettingen minipigs. Platelet concentrations increased significantly in PRP in comparison to native blood plasma. Generally, significant increase in the concentration of all growth factors tested was observed in the PRP in comparison to the corresponding plasma or serum. Five of the plasma samples examined contained detectable levels of bone morphogenic protein 2 (BMP-2) whereas eleven of the plasma or serum samples contained minimal amounts of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF-bb) respectively. On the other hand variable concentrations of bone morphogenic protein 7 (BMP-7) and transforming growth factor β1 (TGF-β1) were measured in all plasma samples. In contrast, all PRP samples contained significantly increased amounts of growth factors. The level of BMP-2, BMP-7, TGF-β1, VEGF and PDGF-bb increased by 17.6, 1.5, 7.1, 7.2 and 103.3 fold, in comparison to the corresponding non-enriched preparations. Moreover significant positive correlations were found between platelet count and the concentrations of BMP-2 (r=0.62, p<0.001), TGF-β1 (r=0.85, p<0.001), VEGF (r=0.46, p<0.01) and PDGF-bb (r=0.9, p<0.001). Our results demonstrate that selected growth factors are present in the platelet-rich plasma of minipigs which might thus serve as a source of autologous growth factors.

Productive interaction between transmembrane mutants of the bovine papillomavirus E5 protein and the platelet-derived growth factor beta receptor.

PubMed

Lai, Char-Chang; Edwards, Anne P B; DiMaio, Daniel

2005-02-01

The bovine papillomavirus E5 protein is a 44-amino-acid transmembrane protein that transforms cells by binding to the transmembrane region of the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in sustained receptor signaling. However, there are published reports that certain mutants with amino acid substitutions in the membrane-spanning segment of the E5 protein transform cells without activating the PDGF beta receptor. We re-examined several of these transmembrane mutants, and here we present five lines of evidence that these mutants do in fact activate the PDGF beta receptor, resulting in cellular signaling and transformation.

A peptide affinity column for the identification of integrin alpha IIb-binding proteins.

PubMed

Daxecker, Heide; Raab, Markus; Bernard, Elise; Devocelle, Marc; Treumann, Achim; Moran, Niamh

2008-03-01

To understand the regulation of integrin alpha(IIb)beta(3), a critical platelet adhesion molecule, we have developed a peptide affinity chromatography method using the known integrin regulatory motif, LAMWKVGFFKR. Using standard Fmoc chemistry, this peptide was synthesized onto a Toyopearl AF-Amino-650 M resin on a 6-aminohexanoic acid (Ahx) linker. Peptide density was controlled by acetylation of 83% of the Ahx amino groups. Four recombinant human proteins (CIB1, PP1, ICln and RN181), previously identified as binding to this integrin regulatory motif, were specifically retained by the column containing the integrin peptide but not by a column presenting an irrelevant peptide. Hemoglobin, creatine kinase, bovine serum albumin, fibrinogen and alpha-tubulin failed to bind under the chosen conditions. Immunodetection methods confirmed the binding of endogenous platelet proteins, including CIB1, PP1, ICln RN181, AUP-1 and beta3-integrin, from a detergent-free platelet lysate. Thus, we describe a reproducible method that facilitates the reliable extraction of specific integrin-binding proteins from complex biological matrices. This methodology may enable the sensitive and specific identification of proteins that interact with linear, membrane-proximal peptide motifs such as the integrin regulatory motif LAMWKVGFFKR.

CLONING, EXPRESSION, AND HEMOSTATIC ACTIVITIES OF A DISINTEGRIN, r-MOJASTIN 1, FROM THE MOHAVE RATTLESNAKE (Crotalus scutulatus scutulatus)

PubMed Central

Sánchez, Elda E.; Lucena, Sara E.; Reyes, Steven; Soto, Julio G.; Cantu, Esteban; Lopez-Johnston, Juan Carlos; Guerrero, Belsy; Salazar, Ana Maria; Rodríguez-Acosta, Alexis; Galán, Jacob A.; Tao, W. Andy; Pérez, John C.

2012-01-01

Interactions with exposed subendothelial extracellular proteins and cellular integrins (endothelial cells, platelets and lymphocytes) can cause alterations in the hemostatic system associated with atherothrombotic processes. Many molecules found in snake venoms induce pathophysiological changes in humans, cause edema, hemorrhage, and necrosis. Disintegrins are low molecular weight, non-enzymatic proteins found in snake venom that mediate changes by binding to integrins of platelets or other cells and prevent binding of the natural ligands such as fibrinogen, fibronectin or vitronectin. Disintegrins are of great biomedical importance due to their binding affinities resulting in the inhibition of platelet aggregation, adhesion of cancer cells, and induction of signal transduction pathways. RT-PCR was used to obtain a 216 bp disintegrin cDNA from a C. s. scutulatus snake venom gland. The cloned recombinant disintegrin called r-mojastin 1 codes for 71 amino acids, including 12 cysteines, and an RGD binding motif. r-Mojastin 1 inhibited platelet adhesion to fibronectin with an IC50 of 58.3 nM and ADP-induced platelet aggregation in whole blood with an IC50 of 46 nM. r-Mojastin 1 was also tested for its ability to inhibit platelet ATP release using PRP resulting with an IC50 of 95.6 nM. MALDI-TOF mass spectrum analysis showed that r-mojastin has a mass of 7.9509 kDa. PMID:20598348

Platelet-derived S100 family member myeloid-related protein-14 regulates thrombosis

PubMed Central

Wang, Yunmei; Fang, Chao; Gao, Huiyun; Bilodeau, Matthew L.; Zhang, Zijie; Croce, Kevin; Liu, Shijian; Morooka, Toshifumi; Sakuma, Masashi; Nakajima, Kohsuke; Yoneda, Shuichi; Shi, Can; Zidar, David; Andre, Patrick; Stephens, Gillian; Silverstein, Roy L.; Hogg, Nancy; Schmaier, Alvin H.; Simon, Daniel I.

2014-01-01

Expression of the gene encoding the S100 calcium–modulated protein family member MRP-14 (also known as S100A9) is elevated in platelets from patients presenting with acute myocardial infarction (MI) compared with those from patients with stable coronary artery disease; however, a causal role for MRP-14 in acute coronary syndromes has not been established. Here, using multiple models of vascular injury, we found that time to arterial thrombotic occlusion was markedly prolonged in Mrp14–/– mice. We observed that MRP-14 and MRP-8/MRP-14 heterodimers (S100A8/A9) are expressed in and secreted by platelets from WT mice and that thrombus formation was reduced in whole blood from Mrp14–/– mice. Infusion of WT platelets, purified MRP-14, or purified MRP-8/MRP-14 heterodimers into Mrp14–/– mice decreased the time to carotid artery occlusion after injury, indicating that platelet-derived MRP-14 directly regulates thrombosis. In contrast, infusion of purified MRP-14 into mice deficient for both MRP-14 and CD36 failed to reduce carotid occlusion times, indicating that CD36 is required for MRP-14–dependent thrombosis. Our data identify a molecular pathway of thrombosis that involves platelet MRP-14 and CD36 and suggest that targeting MRP-14 has potential for treating atherothrombotic disorders, including MI and stroke. PMID:24691441

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.

Improvement in hemocompatibility of chitosan/soy protein composite membranes by heparinization.

PubMed

Wang, Xiaomei; Shi, Na; Chen, Yan; Li, Chen; Du, Xinshen; Jin, Weihua; Chen, Yun; Chang, Peter R

2012-01-01

To improve the hemocompatibility of chitosan/soy protein isolate composite membranes by heparinization. Chitosan/soy protein isolate membranes (ChS-n, n=0, 10 and 30, corresponding to the soy protein isolate content in the membranes) and heparinized ChS-n membranes (HChS-n) were prepared by blending in dilute HAc/NaAc solution. The hemocompatibility of ChS-n and HChS-n membranes were comparatively evaluated by measuring surface heparin density, blood platelet adhesion, plasma recalcification time (PRT), thrombus formation and hemolysis assay. The surface heparin density analysis showed that heparinized chitosan/SPI soy protein isolate membranes have been successfully prepared by blending. The density of heparin on the surface of HChS-n membranes was in the range of 0.67-1.29 μg/cm2. The results of platelet adhesion measurement showed that the platelet adhesion numbers of HChS-n membranes were lower than those of the corresponding ChS-n membranes. The PRT of the HChS-0, HChS-10 and HChS-30 membranes were around 292, 306 and 295 s, respectively, which were longer than the corresponding ChS-0 (152 s), ChS-10 (204 s) and ChS-30 (273 s) membranes. The hemolysis rate of HChS-n membranes was lower than 1%. The hemocompatibility of ChS membranes could be improved by blending with heparin. Compared with ChS membranes, HChS membranes showed lower platelet adhesion, longer PRT, higher BCI, significant thromboresistivity and a lower hemolysis rate due to the heparinization. This widens the application of chitosan and soy protein-based biomaterials that may come in contact with blood.

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

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.

Basic characteristics of plasma rich in growth factors (PRGF): blood cell components and biological effects.

PubMed

Nishiyama, Kazuhiko; Okudera, Toshimitsu; Watanabe, Taisuke; Isobe, Kazushige; Suzuki, Masashi; Masuki, Hideo; Okudera, Hajime; Uematsu, Kohya; Nakata, Koh; Kawase, Tomoyuki

2016-11-01

Platelet-rich plasma (PRP) is widely used in regenerative medicine because of its high concentrations of various growth factors and platelets. However, the distribution of blood cell components has not been investigated in either PRP or other PRP derivatives. In this study, we focused on plasma rich in growth factors (PRGF), a PRP derivative, and analyzed the distributions of platelets and white blood cells (WBCs). Peripheral blood samples were collected from healthy volunteers ( N  = 14) and centrifuged to prepare PRGF and PRP. Blood cells were counted using an automated hematology analyzer. The effects of PRP and PRGF preparations on cell proliferation were determined using human periosteal cells. In the PRGF preparations, both red blood cells and WBCs were almost completely eliminated, and platelets were concentrated by 2.84-fold, whereas in the PRP preparations, both platelets and WBCs were similarly concentrated by 8.79- and 5.51-fold, respectively. Platelet counts in the PRGF preparations were positively correlated with platelet counts in the whole blood samples, while the platelet concentration rate was negatively correlated with red blood cell counts in the whole blood samples. In contrast, platelet counts and concentration rates in the PRP preparations were significantly influenced by WBC counts in whole blood samples. The PRP preparations, but not the PRGF preparations, significantly suppressed cell growth at higher doses in vitro. Therefore, these results suggest that PRGF preparations can clearly be distinguished from PRP preparations by both inclusion of WBCs and dose-dependent stimulation of periosteal cell proliferation in vitro.

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.

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.

Platelet-rich plasma and chronic wounds: remaining fibronectin may influence matrix remodeling and regeneration success.

PubMed

Moroz, Andrei; Deffune, Elenice

2013-11-01

Platelet-rich plasma has been largely used as a therapeutic option for the treatment of chronic wounds of different etiologies. The enhanced regeneration observed after the use of platelet-rich plasma has been systematically attributed to the growth factors that are present inside platelets' granules. We hypothesize that the remaining plasma and platelet-bound fibronectin may act as a further bioactive protein in platelet-rich plasma preparations. Recent reports were analyzed and presented as direct evidences of this hypotheses. Fibronectin may directly influence the extracellular matrix remodeling during wound repair. This effect is probably through matrix metalloproteinase expression, thus exerting an extra effect on chronic wound regeneration. Physicians should be well aware of the possible fibronectin-induced effects in their future endeavors with PRP in chronic wound treatment. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Super liquid-repellent gas membranes for carbon dioxide capture and heart-lung machines.

PubMed

Paven, Maxime; Papadopoulos, Periklis; Schöttler, Susanne; Deng, Xu; Mailänder, Volker; Vollmer, Doris; Butt, Hans-Jürgen

2013-01-01

In a gas membrane, gas is transferred between a liquid and a gas through a microporous membrane. The main challenge is to achieve a high gas transfer while preventing wetting and clogging. With respect to the oxygenation of blood, haemocompatibility is also required. Here we coat macroporous meshes with a superamphiphobic-or liquid repellent-layer to meet this challenge. The superamphiphobic layer consists of a fractal-like network of fluorinated silicon oxide nanospheres; gas trapped between the nanospheres keeps the liquid from contacting the wall of the membrane. We demonstrate the capabilities of the membrane by capturing carbon dioxide gas into a basic aqueous solution and in addition use it to oxygenate blood. Usually, blood tends to clog membranes because of the abundance of blood cells, platelets, proteins and lipids. We show that human blood stored in a superamphiphobic well for 24 h can be poured off without leaving cells or adsorbed protein behind.

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

Inhibition of blood platelet adhesion by phenolics' rich fraction of Hippophae rhamnoides L. fruits.

PubMed

Olas, B; Kontek, B; Szczesna, M; Grabarczyk, L; Stochmal, A; Zuchowski, J

2017-04-01

Beneficial influence of fruits on human health may be their ability to prevent the hyperactivation of blood platelets and cardiovascular disorders. Effects of the phenolic fraction from Hippophae rhamnoides fruit on different stages of blood platelet activation (platelet adhesion and aggregation) were studied in vitro. We also examined effects of the H. rhamnoides fraction on metabolism of thiol groups, which plays an important role in platelet functions. The effects of the H. rhamnoides fraction on adhesion of blood platelets to collagen and fibrinogen were determined with Tuszynski's and Murphy's method. The platelet aggregation was determined with turbidimetry. The action of the H. rhamnoides fraction on the level of thiol groups in platelet proteins and a level of glutathione (GSH) in platelets was estimated with 5,5'-dithio-bis(2-nitro-benzoic acid). The tested fraction of H. rhamnoides (0.5 - 50 μg/ml; 30 min of the incubation time 30 min) inhibited blood platelets adhesion to collagen and fibrinogen. The effect of the tested fraction on blood platelet adhesion depended on concentration of fraction. In presence of the highest tested concentration which was 50 μg/ml, inhibition of platelet adhesion for thrombin-activated platelets was about 55%. On the other hand, tested plant fraction had no anti-aggregatory properties. Our results showed anti-adhesive properties of phenolic fraction from H. rhamnoides fruit and we suggest that it may be beneficial for prevention of cardiovascular diseases.

Trimucrin, an Arg-Gly-Asp containing disintegrin, attenuates myocardial ischemia-reperfusion injury in murine by inhibiting platelet function.

PubMed

Hung, Yu-Chun; Kuo, Yu-Ju; Huang, Shiang-Suo; Huang, Tur-Fu

2017-10-15

Trimucrin, a novel small-mass Arg-Gly-Asp (RGD)-containing disintegrin, has been demonstrated to possess anti-platelet and anti-inflammatory effect through blockade of platelet αIIbβ3 and phagocyte αvβ3 integrin. In this study, we found that the platelet-rich plasma prepared from trimucrin-treated rats platelet aggregation was diminished in response to adenosine diphosphate (ADP). We tried to determine whether trimucrin is cardioprotective in rats subjected to myocardial ischemia-reperfusion (I-R) injury. The left anterior descending coronary artery of anesthetized rats was subjected to 1h occlusion and 3h reperfusion. The animals received intravenous trimucrin or saline, and the severities of I-R-induced arrhythmia and infarction were compared. Trimucrin significantly reduced I-R-induced arrhythmias and reduced mortality, as well as infarct volume, troponin-I levels, creatine kinase, and lactate dehydrogenase activity in carotid blood compared with vehicle-treated animals during the same period. Trimucrin also improved cardiac function and survival rates after I-R injury. In addition, trimucrin concentration-dependently inhibited platelet adhesion on collagen- and fibrinogen-coated surfaces without affecting platelet counts. Trimucrin also significantly reduced neutrophil infiltration into heart tissues after I-R compared with controls. Furthermore, trimucrin treatment caused significant downregulation of Bax, Caspase-3 apoptotic proteins and upregulation of anti-apoptotic Bcl-2 protein. These results demonstrate that trimucrin exerts cardioprotective property against myocardial I-R injury mediated through antiplatele, anti-inflammatory, anti-apoptotic mechanism, as well as improvements in cardiac function. Copyright © 2017. Published by Elsevier B.V.

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.

The Role of Platelets and ε-Aminocaproic Acid in Arthrogryposis, Renal Dysfunction, and Cholestasis (ARC) Syndrome Associated Hemorrhage.

PubMed

Weyand, Angela C; Lombel, Rebecca M; Pipe, Steven W; Shavit, Jordan A

2016-03-01

Arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome is a rare disorder associated with platelet abnormalities resembling gray platelet syndrome. Affected patients have normal platelet numbers but abnormal morphology and function. Bleeding symptomatology ranges from postprocedural to spontaneous life-threatening hemorrhage. We report a patient with ARC syndrome and compound heterozygous mutations in VPS33B (vacuolar protein sorting 33B) who presented with significant bleeding requiring numerous admissions and transfusions. She was treated with prophylactic platelet transfusions and ε-aminocaproic acid. This was well-tolerated and significantly decreased transfusion requirements and admissions for bleeding. Our experience provides support for consideration of prophylactic measures in these patients as well as the possibility of using prophylaxis in related disorders. © 2015 Wiley Periodicals, Inc.

Incorporating Platelet-Rich Plasma into Electrospun Scaffolds for Tissue Engineering Applications

PubMed Central

Wolfe, Patricia S.; Ericksen, Jeffery J.; Simpson, David G.; Bowlin, Gary L.

2011-01-01

Platelet-rich plasma (PRP) therapy has seen a recent spike in clinical interest due to the potential that the highly concentrated platelet solutions hold for stimulating tissue repair and regeneration. The aim of this study was to incorporate PRP into a number of electrospun materials to determine how growth factors are eluted from the structures, and what effect the presence of these factors has on enhancing electrospun scaffold bioactivity. PRP underwent a freeze-thaw-freeze process to lyse platelets, followed by lyophilization to create a powdered preparation rich in growth factors (PRGF), which was subsequently added to the electrospinning process. Release of protein from scaffolds over time was quantified, along with the quantification of human macrophage and adipose-derived stem cell (ADSC) chemotaxis and proliferation. Protein assays demonstrated a sustained release of protein from PRGF-containing scaffolds at up to 35 days in culture. Scaffold bioactivity was enhanced as ADSCs demonstrated increased proliferation in the presence of PRGF, whereas macrophages demonstrated increased chemotaxis to PRGF. In conclusion, the work performed in this study demonstrated that the incorporation of PRGF into electrospun structures has a significant positive influence on the bioactivity of the scaffolds, and may prove beneficial in a number of tissue engineering applications. PMID:21679135

Mice lacking the extracellular matrix protein MAGP1 display delayed thrombotic occlusion following vessel injury

PubMed Central

Werneck, Claudio C.; Vicente, Cristina P.; Weinberg, Justin S.; Shifren, Adrian; Pierce, Richard A.; Broekelmann, Thomas J.; Tollefsen, Douglas M.

2008-01-01

Mice lacking the extracellular matrix protein microfibril-associated glycoprotein-1 (MAGP1) display delayed thrombotic occlusion of the carotid artery following injury as well as prolonged bleeding from a tail vein incision. Normal occlusion times were restored when recombinant MAGP1 was infused into deficient animals prior to vessel wounding. Blood coagulation was normal in these animals as assessed by activated partial thromboplastin time and prothrombin time. Platelet number was lower in MAGP1-deficient mice, but the platelets showed normal aggregation properties in response to various agonists. MAGP1 was not found in normal platelets or in the plasma of wild-type mice. In ligand blot assays, MAGP1 bound to fibronectin, fibrinogen, and von Willebrand factor, but von Willebrand factor was the only protein of the 3 that bound to MAGP1 in surface plasmon resonance studies. These findings show that MAGP1, a component of microfibrils and vascular elastic fibers, plays a role in hemostasis and thrombosis. PMID:18281502

Interaction of human platelets with laminin and identification of the 67 kDa laminin receptor on platelets.

PubMed Central

Tandon, N N; Holland, E A; Kralisz, U; Kleinman, H K; Robey, F A; Jamieson, G A

1991-01-01

A microtitre adhesion assay has been developed to define parameters affecting the adherence of washed platelets to laminin. Adherence was optimally supported by Mg2+ and was inhibited by Ca2+ and by anti-laminin Fab fragments, but significant adhesion (75-90% of control) was found both in heparinized plasma containing physiological levels of bivalent cations and in plasma anti-coagulated with EGTA. Adherence was unaffected by platelet activation with ADP but was decreased by 50% by treatment with alpha-thrombin (1 unit/ml, 5 min). Adherence was unaffected by monospecific polyclonal antibodies to glycoprotein (GP) Ib and GPIV, and was normal with platelets from two patients with Glanzmann's thrombasthaenia, indicating that GPIb, the GPIIb/IIIa complex and GPIV are not involved in platelet-laminin interaction. Affinity chromatography of Triton-solubilized membranes on laminin-Sepharose followed by elution with 0.2 M-glycine/HCl (pH 2.85) identified a major band with a molecular mass of 67 kDa in the reduced and of 53 kDa in the unreduced form. This protein gave a positive reaction on Western blotting with a monospecific polyclonal antibody raised against the high-affinity laminin receptor isolated from human breast carcinoma tissue. The adhesion of platelets to laminin was inhibited by two monoclonal IgM antibodies specific to the LR-1 domain of the 67 kDa receptor. The binding protein was surface-oriented, as shown by flow cytofluorimetry and by the fact that it could be iodinated in intact platelets, but it was not labelled by the periodate-borotritide procedure, suggesting that it did not contain terminal sialic acid. The laminin-derived peptides Tyr-Ile-Gly-Ser-Arg and Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg-NH2, which constitute a complementary binding domain in laminin for the 67 kDa receptor, themselves supported platelet adhesion, bound to the receptor and inhibited the adhesion of platelets to laminin. In addition, Fab fragments of anti-Tyr-Ile-Gly-Ser-Arg antibody inhibited platelet adhesion to laminin. These results demonstrate that the high-affinity 67 kDa laminin receptor previously identified in a range of normal and transformed cells and its complementary Tyr-Ile-Gly-Ser-Arg binding domain play an important role in the interaction of platelets with laminin. Images Fig. 4. Fig. 8. PMID:1826081

Platelet antiheparin activity. The isolation and characterisation of platelet factor 4 released from thrombin-aggregated washed human platelets and its dissociation into subunits and the isolation of membrane-bound antiheparin activity.

PubMed

Moore, S; Pepper, D S; Cash, J D

1975-02-27

Platelet factor 4 was isolated by gel filtration from the soluble release products of thrombin-aggregated washed human platelets as a proteoglycan-platelet factor 4 complex of molecular weight 358 000, Stokes radius (r-s) of 14.0 nm, sedimentation coefficient (s) of 7.1 S and frictional ratio (f/f-o) of 3.04. The complex was dissociated at high ionic strength (I equals 0.75) and the proteoglycan separated from platelet factor 4 by gel filtration. Platelet factor 4 had a molecular weight of 27 100, r-s of 2.52 nm, s of 2.4 S and f/f-o of 1.26, was insoluble under physiological conditions but readily soluble at pH 3. Under these conditions platelet factor 4 dissociated into four subunits with a molecular weight of 6900, r-s of 1.92 nm, s of 0.8 S, and f/f-o of 1.52. Qualitative N-terminal amino acid analysis showed the presence of glutamic acid or glutamine as the major end group. Platelet factor 4 was compared with protamine sulphate, which has similar biological properties, by electrophoresis at pH 2.2, in which both migrated as single bands but with differing mobility, and by amino acid analysis which showed a more normal distribution of residues than occurred in protamine sulphate. Of the basic amino acids platelet factor 4 (molecular weight 27 100) contained 5.97% arginine, 3.18% histidine, and 12.31% lysine compared to protamine sulphate with 64.2% arginine, 0.6% lysine and no histidine. A partial specific volume (v) of 0.747 was calculated for platelet factor 4 from its amino acid analysis. A membrane fraction with antiheparin activity, an isopycnic density of 1.090-1.110 and r-s of 15-35 nm, was also isolated by sucrose density gradient centrifugation from the ultrasonicated insoluble platelet residue remaining after thrombin-induced aggregation of washed human platelets. Trypsin treatment of the membrane fraction neither solubilised nor destroyed the activity.

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 glycoproteins associated with aspirin-treatment upon platelet activation

PubMed Central

Shah, Punit; Yang, Weiming; Sun, Shisheng; Pasay, Jered; Faraday, Nauder; Zhang, Hui

2017-01-01

Platelet glycoproteins are known to play central roles in hemostasis and vascular integrity and have pathologic roles in vascular occlusive diseases such as myocardial infarction and stroke. Characterizing glycoproteins within and secreted by platelets can provide insight into the mechanisms that underlie vascular pathologies and the therapeutic benefits or failure of anti-platelet agents. To study the impact of aspirin, which is commonly prescribed for primary and secondary cardiovascular prevention, on the platelet glycoproteome, we evaluated washed platelets from ten donors. The platelet glycoproteome, was studied using an iTRAQ in resting and stimulated states and with and without aspirin treatment. Using solid phase extraction of glycosite-containing peptides (SPEG), we were able to identify 799 unique N-linked glycosylation sites (glycosites) in platelets, representing the largest and the most comprehensive analysis to date. We were able to identity a number of glycoproteins impacted by aspirin treatment, which we validated using global proteomics analysis of platelets and their secreted proteins. In our analyses, metallopeptidase inhibitor 1 (TIMP1) was the single most significantly affected glycoprotein by aspirin treatment. ELISA assays confirmed proteomic results and validated our strategy. Functional analysis demonstrated that TIMP1 levels were highly correlated with platelet reactivity in vitro, with a correlation coefficient of −0.5. The release of TIMP1 from platelets, which was previously unknown to be affected by aspirin treatment, may play important roles in hemostasis and/or vascular integrity. If validated, our findings may be useful for developing assays that assess platelet response to aspirin or other anti-platelet therapies. PMID:27452734

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 Kainate Receptor Signaling Promotes Thrombosis by Stimulating Cyclooxygenase Activation

PubMed Central

Sun, Henry; Swaim, AnneMarie; Herrera, Jesus Enrique; Becker, Diane; Becker, Lewis; Srivastava, Kalyan; Thompson, Laura E.; Shero, Michelle R.; Perez-Tamayo, Alita; Suktitpat, Bhoom; Mathias, Rasika; Contractor, Anis; Faraday, Nauder; Morrell, Craig N.

2009-01-01

Rationale Glutamate is a major signaling molecule that binds to glutamate receptors including the ionotropic glutamate receptors; kainate (KA) receptor (KAR), the N-methyl-D-aspartate (NMDA) receptor (NMDAR), and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR). Each is well characterized in the central nervous system (CNS), but glutamate has important signaling roles in peripheral tissues as well, including a role in regulating platelet function. Objective Our previous work has demonstrated that glutamate is released by platelets in high concentrations within a developing thrombus and increases platelet activation and thrombosis. We now show that platelets express a functional KAR that drives increased agonist induced platelet activation. Methods and Results KAR induced increase in platelet activation is in part the result of activation of platelet cyclooxygenase (COX) in a Mitogen Activated Protein Kinase (MAPK) dependent manner. Platelets derived from KA receptor subunit knockout mice (GluR6−/−) are resistant to KA effects and have a prolonged time to thrombosis in vivo. Importantly, we have also identified polymorphisms in KA receptor subunits that are associated with phenotypic changes in platelet function in a large group of Caucasians and African Americans. Conclusion Our data demonstrate that glutamate regulation of platelet activation is in part COX dependent, and suggest that the KA receptor is a novel anti-thrombotic target. PMID:19679838

FlnA-null megakaryocytes prematurely release large and fragile platelets that circulate poorly

PubMed Central

Jurak Begonja, Antonija; Hoffmeister, Karin M.; Hartwig, John H.

2011-01-01

Filamin A (FlnA) is a large cytoplasmic protein that crosslinks actin filaments and anchors membrane receptors and signaling intermediates. FlnAloxP PF4-Cre mice that lack FlnA in the megakaryocyte (MK) lineage have a severe macrothrombocytopenia because of accelerated platelet clearance. Macrophage ablation by injection of clodronate-encapsulated liposomes increases blood platelet counts in FlnAloxP PF4-Cre mice and reveals the desintegration of FlnA-null platelets into microvesicles, a process that occurs spontaneously during storage. FlnAloxP PF4-Cre bone marrows and spleens have a 2.5- to 5-fold increase in MK numbers, indicating increased thrombopoiesis in vivo. Analysis of platelet production in vitro reveals that FlnA-null MKs prematurely convert their cytoplasm into large CD61+ platelet-sized particles, reminiscent of the large platelets observed in vivo. FlnA stabilizes the platelet von Willebrand factor receptor, as surface expression of von Willebrand factor receptor components is normal on FlnA-null MKs but decreased on FlnA-null platelets. Further, FlnA-null platelets contain multiple GPIbα degradation products and have increased expression of the ADAM17 and MMP9 metalloproteinases. Together, the findings indicate that FlnA-null MKs prematurely release large and fragile platelets that are removed rapidly from the circulation by macrophages. PMID:21652675

[Glutamate-binding membrane proteins from human platelets].

PubMed

Gurevich, V S; Popov, Iu G; Gorodinskiĭ, A I; Dambinova, S A

1991-09-01

Solubilization of the total membrane fraction of human platelets in a 2% solution of sodium deoxycholate and subsequent affinity chromatography on glutamate agarose resulted in two protein fractions possessing a glutamate-binding activity. As can be evidenced from radioligand binding data, the first fraction contains two types of binding sites (Kd1 = 1 microM, Bmax 1 = 100 pmol/mg of protein; Kd2 = 9.3 microMm Bmax2 = 395 pmol/mg of protein). The second fraction has only one type of binding sites (Kd = 1 microM, Bmax = = 110 pmol/mg of protein). SDS-PAAG electrophoresis revealed the presence in the first fraction of proteins with Mr of 14, 24, 56 and 155 kDa, whereas the second fraction was found to contain 14, 46, 71 and 155 kDa proteins. Solid phase immunoenzymatic analysis using poly- and monoclonal specific antibodies against mammalian brain glutamate-binding proteins revealed a marked immunochemical similarity of the isolated protein fractions with human brain synaptic membrane glutamate-binding proteins.

Molecular cloning and characterization of rhesus monkey platelet glycoprotein Ibα, a major ligand-binding subunit of GPIb-IX-V complex.

PubMed

Qiao, Jianlin; Shen, Yang; Shi, Meimei; Lu, Yanrong; Cheng, Jingqiu; Chen, Younan

2014-05-01

Through binding to von Willebrand factor (VWF), platelet glycoprotein (GP) Ibα, the major ligand-binding subunit of the GPIb-IX-V complex, initiates platelet adhesion and aggregation in response to exposed VWF or elevated fluid-shear stress. There is little data regarding non-human primate platelet GPIbα. This study cloned and characterized rhesus monkey (Macaca Mullatta) platelet GPIbα. DNAMAN software was used for sequence analysis and alignment. N/O-glycosylation sites and 3-D structure modelling were predicted by online OGPET v1.0, NetOGlyc 1.0 Server and SWISS-MODEL, respectively. Platelet function was evaluated by ADP- or ristocetin-induced platelet aggregation. Rhesus monkey GPIbα contains 2,268 nucleotides with an open reading frame encoding 755 amino acids. Rhesus monkey GPIbα nucleotide and protein sequences share 93.27% and 89.20% homology respectively, with human. Sequences encoding the leucine-rich repeats of rhesus monkey GPIbα share strong similarity with human, whereas PEST sequences and N/O-glycosylated residues vary. The GPIbα-binding residues for thrombin, filamin A and 14-3-3ζ are highly conserved between rhesus monkey and human. Platelet function analysis revealed monkey and human platelets respond similarly to ADP, but rhesus monkey platelets failed to respond to low doses of ristocetin where human platelets achieved 76% aggregation. However, monkey platelets aggregated in response to higher ristocetin doses. Monkey GPIbα shares strong homology with human GPIbα, however there are some differences in rhesus monkey platelet activation through GPIbα engagement, which need to be considered when using rhesus monkey platelet to investigate platelet GPIbα function. Copyright © 2014 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

Effects of short-term streptozotocin-induced diabetes and vitamin C on platelet non-enzymatic glycation.

PubMed

Batırel, Saime; Yarat, Ayşen; Emekli, Nesrin

2010-01-01

Diabetes mellitus is one of the most prevalent metabolic syndromes worldwide. Glycation, a chemical modification of proteins with reducing sugars, indicates a possible explanation for the association between hyperglycemia and the wide variety of tissue pathologies. Non-enzymatic glycation (NEG) of platelet proteins is one of the key mechanisms in the pathogenesis of diabetic complications and may be significant in diabetic atherothrombosis. The aim of this study was to investigate the effects of streptozotocin (STZ)-induced short-term experimental diabetes on the glycation of platelets and to find out if vitamin C affected this glycation. A total of 40 male Wistar albino rats, 200-250 g, were randomly divided into 4 groups (2 diabetic and 2 control groups). The diabetic groups were made diabetic by intraperitoneal injection of STZ (65 mg/kg, citrate buffer pH 4.5). By daily intraperitoneal injection, 80 mg/kg vitamin C (Roche, Turkey) was administered until the end of the experiment. Blood glucose levels of the diabetic groups were significantly higher than those at day 0 and also higher than those of the non-diabetic control groups. The changes in total protein, NEG and vitamin C levels were not statistically significant. Although the differences among the groups were not statistically significant, vitamin C administration increased NEG levels in the diabetic group. The results of this study demonstrate that 8 days of STZ-induced short-term diabetes did not cause a significant increase in NEG of platelets. However, the effect of vitamin C on platelet NEG needs to be further investigated. Copyright © 2011 S. Karger AG, Basel.

Picomolar platelet-activating factor mobilizes Ca to change platelet shape without activating phospholipase C or protein kinase C; simultaneous fluorometric measurement of intracellular free Ca concentration and aggregation.

PubMed

James-Kracke, M R; Sexe, R B; Shukla, S D

1994-11-01

The purpose of this study was to investigate signal transduction mechanisms activated by low and high concentrations of platelet-activating factor (PAF) in rabbit platelets and to contrast the responses to those induced by thrombin. We measured changes in intracellular free calcium ([Ca++]i) with fura2, while monitoring light scatter simultaneously as a measure of shape change and aggregation in a dual-excitation dual-emission spectrofluorometer. An abrupt 20% fall in light scatter, coincident with the peak of the [Ca++]i, indicated shape change in Ca-containing or Ca-free medium and was blocked by BAPTA loading and 10 microM cytochalasin B. A secondary decline in light scatter, indicating aggregation, occurred only in Ca-containing medium and only under conditions favoring protein kinase C (PKC) activation. PAF at 10(-12) M did not increase 1,4,5-inositol triphosphate content, which suggested PKC would not be activated. However, PAF at 10(-12) rapidly increased [Ca++]i to 900 nM in 7 sec seemingly by Ca influx through receptor-operated channels inducing shape change. PAF at 10(-9) and 10(-8) M increased [Ca++]i to 2 microM in 12 sec and induced both shape change and aggregation. However, in platelets pretreated with 100 nM staurosporine to inhibit protein kinases, 10(-9) M PAF did not cause aggregation even though [Ca++]i still rose to 2 microM, which indicated that PKC plays a role in aggregation but not in Ca++ mobilization.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Platelet-Poor and Platelet-Rich Plasma Stimulate Bone Lineage Differentiation in Periodontal Ligament Stem Cells.

PubMed

Martínez, Constanza E; González, Sergio A; Palma, Verónica; Smith, Patricio C

2016-02-01

Plasma-derived fractions have been used as an autologous source of growth factors; however, limited knowledge concerning their biologic effects has hampered their clinical application. In this study, the authors analyze the content and specific effect of both platelet-rich plasma (PRP) and platelet-poor plasma (PPP) on osteoblastic differentiation using primary cultures of human periodontal ligament stem cells (HPLSCs). The authors evaluated the growth factor content of PRP and PPP using a proteome profiler array and enzyme-linked immunosorbent assay. HPLSCs were characterized by flow cytometry and differentiation assays. The effect of PRP and PPP on HPLSC bone differentiation was analyzed by quantifying calcium deposition after 14 and 21 days of treatment. Albeit at different concentrations, the two fractions had similar profiles of growth factors, the most representative being platelet-derived growth factor (PDGF) isoforms (PDGF-AA, -BB, and -AB), insulin-like growth factor binding protein (IGFBP)-2, and IGFBP-6. Both formulations exerted a comparable stimulus on osteoblastic differentiation even at low doses (2.5%), increasing calcium deposits in HPLSCs. PRP and PPP showed a similar protein profile and exerted comparable effects on bone differentiation. Further studies are needed to characterize and compare the effects of PPP and PRP on bone healing in vivo.

Preventive and therapeutic effect of brozopine on stroke in Dahl Salt-sensitive hypertensive rats.

PubMed

Gao, Yuan; Wang, Yan; Li, Miao; Liu, Yali; Chang, Junbiao; Qiao, Hailing

2017-10-01

Our aim was to explore the preventive and therapeutic effects of sodium (±)-5-bromo-2-(α-hydroxypentyl) benzoate (brand name: brozopine, BZP) on stroke in Dahl Salt-sensitive (Dahl-SS) hypertensive rats. Dahl-SS rats were fed a high-salt diet to observe the effect of BZP on blood pressure, and brain, heart, and kidney tissues. Additionally, the incidence of stroke was recorded according to the neurological score. The relative mechanisms investigated included anti-oxidative effects and anti-platelet aggregation. BZP reduced the incidence of stroke, neuronal necrosis in the brain, and cell swelling and inflammatory infiltration in the kidney. Its mechanisms were related to the increased activities of gluthatione peroxidase and catalase and the decreased level of plasma nitric oxide. BZP inhibited arachidonic acid (AA) - induced platelet aggregation (IC 50 : 12µM) rather than that of adenosine diphosphate (ADP) - and/or thrombin-induced platelet aggregation in vitro. Interestingly, BZP inhibited ADP-, thrombin-, or AA-induced platelet aggregation and elevated the level of AMP-activated protein kinase, cyclic guanosine monophosphate, and vasodilator-stimulated-phosphoprotein, and attenuated ATP contents and mitogen-activated protein kinase levels in platelet and inhibited thrombus formation in a carotid artery thrombosis model, dose-dependently, in Dahl-SS hypertensive-induced stroke rats. In conclusion, BZP can have therapeutic and preventive effects on stroke in Dahl-SS hypertensive rats, the mechanisms of which may be related to anti-oxidant, anti-platelet aggregation and anti-thrombus formation. Copyright © 2017 Elsevier B.V. All rights reserved.

Mechanism underlying bioinertness of self-assembled monolayers of oligo(ethyleneglycol)-terminated alkanethiols on gold: protein adsorption, platelet adhesion, and surface forces.

PubMed

Hayashi, Tomohiro; Tanaka, Yusaku; Koide, Yuki; Tanaka, Masaru; Hara, Masahiko

2012-08-07

The mechanism underlying the bioinertness of the self-assembled monolayers of oligo(ethylene glycol)-terminated alkanethiol (OEG-SAM) was investigated with protein adsorption experiments, platelet adhesion tests, and surface force measurements with an atomic force microscope (AFM). In this work, we performed systematic analysis with SAMs having various terminal groups (-OEG, -OH, -COOH, -NH(2), and -CH(3)). The results of the protein adsorption experiment by the quartz crystal microbalance (QCM) method suggested that having one EG unit and the neutrality of total charges of the terminal groups are essential for protein-resistance. In particular, QCM with energy dissipation analyses indicated that proteins absorb onto the OEG-SAM via a very weak interaction compared with other SAMs. Contrary to the protein resistance, at least three EG units as well as the charge neutrality of the SAM are found to be required for anti-platelet adhesion. When the identical SAMs were formed on both AFM probe and substrate, our force measurements revealed that only the OEG-SAMs possessing more than two EG units showed strong repulsion in the range of 4 to 6 nm. In addition, we found that the SAMs with other terminal groups did not exhibit such repulsion. The repulsion between OEG-SAMs was always observed independent of solution conditions [NaCl concentration (between 0 and 1 M) and pH (between 3 and 11)] and was not observed in solution mixed with ethanol, which disrupts the three-dimensional network of the water molecules. We therefore concluded that the repulsion originated from structured interfacial water molecules. Considering the correlation between the above results, we propose that the layer of the structured interfacial water with a thickness of 2 to 3 nm (half of the range of the repulsion observed in the surface force measurements) plays an important role in deterring proteins and platelets from adsorption or adhesion.

Growth factor and proteinase profile of Vivostat® platelet-rich fibrin linked to tissue repair.

PubMed

Agren, M S; Rasmussen, K; Pakkenberg, B; Jørgensen, B

2014-07-01

Autologous platelet-rich fibrin (PRF(®)) is prepared by the automatic Vivostat(®) system. Conflicting results with Vivostat PRF in acute wound healing prompted us to examine its cellular and biomolecular composition. Specifically, platelets, selected growth factors and matrix metalloproteinase (MMP)-9 were quantified using novel analytical methods. Ten healthy non-thrombocytopenic volunteers donated blood for generation of intermediate fibrin-I and final PRF. Anticoagulated whole blood and serum procured in parallel served as baseline controls. Leucocyte, erythrocyte and platelet counts in whole blood and fibrin-I were determined by automated haematology analyser. Platelet concentration in PRF was quantified manually by stereologic analysis of Giemsa-stained tissue sections, and the total content of five growth factors and MMP-9 by enzyme-linked immunosorbent assays. The number of leucocytes and erythrocytes was reduced (P < 0·001), whereas platelets increased (P < 0·001) in fibrin-I versus whole blood. PRF contained 982 ± 206 × 10(9) platelets/l representing 3·9-fold (P < 0·001) enrichment relative to whole blood. Growth factor abundance in Vivostat PRF and serum was in descending order: transforming growth factor-β1 [5·1-fold higher in PRF than serum, P < 0·001] > platelet-derived growth factor (PDGF)-AB [2·5-fold, P < 0·01] > PDGF-BB [1·6-fold, P < 0·05] > vascular endothelial growth factor > basic fibroblast growth factor [75-fold, P < 0·001]. MMP-9 was reduced 139-fold (P < 0·001) compared with serum, reflecting leucocyte depletion in PRF. The gained knowledge on platelet enrichment and biomolecular constituents may guide clinicians in their optimal use of Vivostat PRF for tissue regenerative applications. © 2013 International Society of Blood Transfusion.

Specific binding of magnetic nanoparticle probes to platelets in whole blood detected by magnetorelaxometry

NASA Astrophysics Data System (ADS)

Eberbeck, Dietmar; Wiekhorst, Frank; Steinhoff, Uwe; Schwarz, Kay Oliver; Kummrow, Andreas; Kammel, Martin; Neukammer, Jörg; Trahms, Lutz

2009-05-01

The binding of monoclonal antibodies labelled with magnetic nanoparticles to CD61 surface proteins expressed by platelets in whole blood samples was measured by magnetorelaxometry. This technique is sensitive to immobilization of the magnetic labels upon binding. Control experiments with previous saturation of the epitopes on the platelet surfaces demonstrated the specificity of the binding. The kinetics of the antibody antigen reaction is accessible with a temporal resolution of 12 s. The minimal detectable platelet concentration is about 2000 μL -1 (sample volume 150 μL). The proportionality of the magnetic relaxation amplitude to the number of bound labels allows a quantification of the antibody binding capacity.

Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.

PubMed

Moreau, Thomas; Evans, Amanda L; Vasquez, Louella; Tijssen, Marloes R; Yan, Ying; Trotter, Matthew W; Howard, Daniel; Colzani, Maria; Arumugam, Meera; Wu, Wing Han; Dalby, Amanda; Lampela, Riina; Bouet, Guenaelle; Hobbs, Catherine M; Pask, Dean C; Payne, Holly; Ponomaryov, Tatyana; Brill, Alexander; Soranzo, Nicole; Ouwehand, Willem H; Pedersen, Roger A; Ghevaert, Cedric

2016-04-07

The production of megakaryocytes (MKs)--the precursors of blood platelets--from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology.

Mutant botrocetin-2 inhibits von Willebrand factor-induced platelet agglutination.

PubMed

Matsui, T; Hori, A; Hamako, J; Matsushita, F; Ozeki, Y; Sakurai, Y; Hayakawa, M; Matsumoto, M; Fujimura, Y

2017-03-01

Essentials Botrocetin-2 (Bot2) binds to von Willebrand factor (VWF) and induces platelet agglutination. We identified Bot2 residues that are required for binding to VWF and glycoprotein (GP) Ib. We produced a mutant Bot2 that binds to VWF but inhibits platelet agglutination. Mutant Bot2 could be used as a potential anti-thrombotic reagent to block VWF-GPIb interaction. Background Botrocetin-2 (Bot2) is a botrocetin-like protein composed of α and β subunits that have been cloned from the snake Bothrops jararaca. Bot2 binds specifically to von Willebrand factor (VWF), and the complex induces glycoprotein (GP) Ib-dependent platelet agglutination. Objectives To exploit Bot2's VWF-binding capacity in order to attempt to create a mutant Bot2 that binds to VWF but inhibits platelet agglutination. Methods and Results Several point mutations were introduced into Bot2 cDNA, and the recombinant protein (recombinant Bot2 [rBot2]) was purified on an anti-botrocetin column. The mutant rBot2 with either Ala at Asp70 in the β subunit (Aspβ70Ala), or Argβ115Ala and Lysβ117Ala, showed reduced platelet agglutination-inducing activity. rBot2 with Aspβ70Ala showed little binding activity towards immobilized VWF on an ELISA plate, whereas rBot2 with Argβ115Ala/Lysβ117Ala showed reduced binding activity towards GPIb (glycocalicin) after forming a complex with VWF. rBot2 point-mutated to oppositely charged Glu at both Argβ115 and Lysβ117 showed normal binding activity towards VWF but no platelet-agglutinating activity. Furthermore, this doubly mutated protein inhibited ristocetin-induced or high shear stress-induced platelet aggregation, and restrained thrombus formation under flow conditions. Conclusions Asp70 in the β subunit of botrocetin is important for VWF binding, and Arg115 and Lys117 in the β subunit are essential for interaction with GPIb. Doubly mutated rBot2, with Argβ115Glu and Lysβ117Glu, repels GPIb and might have potential as an antithrombotic reagent that specifically blocks VWF function. This is the first report on an artificial botrocetin that can inhibit the VWF-GPIb interaction. © 2017 International Society on Thrombosis and Haemostasis.

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.

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

Higher platelet reactivity and platelet-monocyte complex formation in Gram-positive sepsis compared to Gram-negative sepsis.

PubMed

Tunjungputri, Rahajeng N; van de Heijden, Wouter; Urbanus, Rolf T; de Groot, Philip G; van der Ven, Andre; de Mast, Quirijn

2017-09-01

Platelets may play a role in the high risk for vascular complications in Gram-positive sepsis. We compared the platelet reactivity of 15 patients with Gram-positive sepsis, 17 with Gram-negative sepsis and 20 healthy controls using a whole blood flow cytometry-based assay. Patients with Gram-positive sepsis had the highest median fluorescence intensity (MFI) of the platelet membrane expression of P-selectin upon stimulation with high dose adenosine diphosphate (ADP; P = 0.002 vs. Gram-negative and P = 0.005 vs. control groups) and cross-linked collagen-related peptide (CRP-XL; P = 0.02 vs. Gram-negative and P = 0.0001 vs. control groups). The Gram-positive group also demonstrated significantly higher ADP-induced fibrinogen binding (P = 0.001), as wll as platelet-monocyte complex formation (P = 0.02), compared to the Gram-negative group and had the highest plasma levels of platelet factor 4, β-thromboglobulin and soluble P-selectin. In contrast, thrombin-antithrombin complex and C-reactive protein levels were comparable in both patient groups. In conclusion, common Gram-positive pathogens induce platelet hyperreactivity, which may contribute to a higher risk for vascular complications.

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

Phenotypic approaches to gene mapping in platelet function disorders - identification of new variant of P2Y12, TxA2 and GPVI receptors.

PubMed

Watson, S; Daly, M; Dawood, B; Gissen, P; Makris, M; Mundell, S; Wilde, J; Mumford, A

2010-01-01

Platelet number or function disorders cause a range of bleeding symptoms from mild to severe. Patients with platelet dysfunction but normal platelet number are the most prevalent and typically have mild bleeding symptoms. The study of this group of patients is particularly difficult because of the lack of a gold-standard test of platelet function and the variable penetrance of the bleeding phenotype among affected individuals. The purpose of this short review is to discuss the way in which this group of patients can be investigated through platelet phenotyping in combination with targeted gene sequencing. This approach has been used recently to identify patients with mutations in key platelet activation receptors, namely those for ADP, collagen and thromboxane A2 (TxA2). One interesting finding from this work is that for some patients, mild bleeding is associated with heterozygous mutations in platelet proteins that are co-inherited with other genetic disorders of haemostasis such as type 1 von Willebrand's disease. Thus, the phenotype of mild bleeding may be multifactorial in some patients and may be considered to be a complex trait.

Basic characteristics of plasma rich in growth factors (PRGF): blood cell components and biological effects

PubMed Central

Nishiyama, Kazuhiko; Okudera, Toshimitsu; Watanabe, Taisuke; Isobe, Kazushige; Suzuki, Masashi; Masuki, Hideo; Okudera, Hajime; Uematsu, Kohya; Nakata, Koh

2016-01-01

Abstract Platelet‐rich plasma (PRP) is widely used in regenerative medicine because of its high concentrations of various growth factors and platelets. However, the distribution of blood cell components has not been investigated in either PRP or other PRP derivatives. In this study, we focused on plasma rich in growth factors (PRGF), a PRP derivative, and analyzed the distributions of platelets and white blood cells (WBCs). Peripheral blood samples were collected from healthy volunteers (N = 14) and centrifuged to prepare PRGF and PRP. Blood cells were counted using an automated hematology analyzer. The effects of PRP and PRGF preparations on cell proliferation were determined using human periosteal cells. In the PRGF preparations, both red blood cells and WBCs were almost completely eliminated, and platelets were concentrated by 2.84‐fold, whereas in the PRP preparations, both platelets and WBCs were similarly concentrated by 8.79‐ and 5.51‐fold, respectively. Platelet counts in the PRGF preparations were positively correlated with platelet counts in the whole blood samples, while the platelet concentration rate was negatively correlated with red blood cell counts in the whole blood samples. In contrast, platelet counts and concentration rates in the PRP preparations were significantly influenced by WBC counts in whole blood samples. The PRP preparations, but not the PRGF preparations, significantly suppressed cell growth at higher doses in vitro. Therefore, these results suggest that PRGF preparations can clearly be distinguished from PRP preparations by both inclusion of WBCs and dose‐dependent stimulation of periosteal cell proliferation in vitro. PMID:29744155

Activity of blood coagulation and fibrinolysis during and after hydroxyethyl starch (HES) colloidal volume replacement.

PubMed

Omar, M N; Shouk, T A; Khaleq, M A

1999-06-01

To examine the effect of medium molecular weight hydroxyethyl starch on protein C levels and the changes in the activation state of blood platelets, coagulation and fibrinolyis during and after 5 day of its infusion. Fifty male patients (mean age: 47 years, range 45-50 years) who required prostatectomy for benign prostatic hyperplasia were divided into two equal groups. One group was given 15 mL/kg body weight (mean volume 1000 mL +/- 100 mL) of 6% hydroxyethyl starch (HES) 200/0.5, the other received an equal volume of 5% human albumin during the operation. Blood samples were collected immediately before infusion (baseline values) and at 20, 40, 60, 90, 240, and 480 min after the infusion started then daily for the next 5 days postoperatively. Hematocrit, factor VIII:C, thrombin-antithrombin III complex; the anticoagulant protein C levels; the fibrinolytic parameters tissue type plasminogen activator (t-PA), and the fibrinolytic product D-Dimer and the platelet aggregation activity were measured. The data obtained did not detect any significant differences between HES and human albumin in the plasma levels of thrombin-antithrombin III complex, protein C, tissue-type plasminogen activator and the fibrin split products D-Dimer. Factor VIII:C and platelet aggregation were significantly lower in the hydroxyethyl starch group in comparison with albumin. Baseline values were attained postoperatively for factor VIII:C and platelet aggregation by the first and fifth days, respectively. The lowering effect of medium molecular weight hydroxyethyl starch on factor VIII:C would not be attributed to increased proteolytic activity of protein C on this coagulation cofactor because there is a nonsignificant change in protein C levels.

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.

Comparison of platelet serotonin transporter activity in subjects with severe sleep bruxism and control.

PubMed

Minakuchi, Hajime; Sogawa, Chiharu; Hara, Emilio Satoshi; Miki, Haruna; Maekawa, Kenji; Sogawa, Norio; Kitayama, Shigeo; Matsuka, Yoshizo; Clark, Glenn T; Kuboki, Takuo

2014-10-01

The aim of this study was to evaluate the correlation between sleep bruxism (SB) frequency and serotonin transporter (SERT)-driven serotonin (5-HT)-uptake in platelets. Subjects were dental trainee residents and faculty members of Okayama University Hospital who were aware of having severe or no SB. SB frequency was assessed for 3-consecutive nights by a self-contained electromyographic detector/analyzer, which indicated individual SB levels as one of four grades (score 0, 1, 2 and 3). Subjects were classified as normal control (NC) when SB scores indicated only 0 or 1 during the 3 nights, or as severe SB for scores 2 or 3. Those subjects whose scores fluctuated from 0 to 3 during the 3 nights were omitted from further analysis. Fasting peripheral venous blood samples were collected in the morning following the final SB assessment. Amounts of SERTs proteins collected from peripheral platelets were quantified using ELISA, and SERTs transport activity was assessed by uptake assay using [3H]-5-HT. Thirteen severe SB subjects and 7 NC subjects were eligible. Gender distribution, mean age, 5-HT concentration and total amounts of SERT protein in platelets showed no significant differences between NC and severe SB (p=0.85: Chi-squared test; p=0.64, 0.26, 0.46: t-test). However, [3H]-5-HT uptake by platelets was significantly greater in NC compared to severe SB subjects (12.79±1.97, 8.27±1.91 fmol/10(5) platelets/min, p<0.001, t-test). The results of this pilot study suggest a possible correlation between peripheral platelet serotonin transporter uptake ability and SB severity. Copyright © 2014 Japan Prosthodontic Society. Published by Elsevier Ltd. All rights reserved.

Evaluation of blood neutrophil-lymphocyte ratio and platelet distribution width as inflammatory markers in patients with fibromyalgia.

PubMed

Aktürk, Semra; Büyükavcı, Raikan

2017-08-01

Fibromyalgia syndrome (FMS) is characterized by chronic widespread pain and systemic symptoms. The aetiology and pathogenesis of fibromyalgia are not yet fully understood. Blood neutrophil/lymphocyte ratio (NLR) is a marker of systemic inflammatory response. Platelet distribution width (PDW) and mean platelet volume (MPV) are the determinants of platelet activation and studied as markers in inflammatory diseases. The aim of the present study was to evaluate levels of NLR,PDW and MPV in patients with fibromyalgia. A total of 197 FMS patients and 53 healthy controls are included in the study. Demographic characteristics, erythrocyte sedimentation rate, C-reactive protein, neutrophil, lymphocyte and platelet counts, platelet distribution width and mean platelet volume levels were recorded. In the patient group, the blood NLR and MPV were significantly higher and the PDW was significantly lower compared to the control group. In the roc curve analysis, blood PDW ≥had 90.4% sensitivity and 90% specificity in predicting fibromyalgia. The results of this study suggest NLR and PDW as promising inflammatory markers indicating fibromyalgia and may be beneficial in facilitating the diagnosis of FMS patients.

On-pump Cardiac Surgery Enhances Platelet Renewal and Impairs Aspirin Pharmacodynamics: Effects of Improved Dosing Regimens.

PubMed

Cavalca, V; Rocca, B; Veglia, F; Petrucci, G; Porro, B; Myasoedova, V; De Cristofaro, R; Turnu, L; Bonomi, A; Songia, P; Cavallotti, L; Zanobini, M; Camera, M; Alamanni, F; Parolari, A; Patrono, C; Tremoli, E

2017-11-01

On-pump cardiac surgery may trigger inflammation and accelerate platelet cyclooxygenase-1 renewal, thereby modifying low-dose aspirin pharmacodynamics. Thirty-seven patients on standard aspirin 100 mg once-daily were studied before surgery and randomized within 36 hours postsurgery to 100 mg once-daily, 100 mg twice-daily, or 200 mg once-daily for 90 days. On day 7 postsurgery, immature and mature platelets, platelet mass, thrombopoietin, glycocalicin, leukocytes, C-reactive protein, and interleukin-6 significantly increased. Interleukin-6 significantly correlated with immature platelets. At day 7, patients randomized to 100 mg once-daily showed a significant increase in serum thromboxane (TX)B 2 within the 24-hour dosing interval and urinary TXA 2 metabolite (TXM) excretion. Aspirin 100 mg twice-daily lowered serum TXB 2 and prevented postsurgery TXM increase (P < 0.01), without affecting prostacyclin metabolite excretion. After cardiac surgery, shortening the dosing interval, but not doubling the once-daily dose, rescues the impaired antiplatelet effect of low-dose aspirin and prevents platelet activation associated with acute inflammation and enhanced platelet turnover. © 2017 American Society for Clinical Pharmacology and Therapeutics.

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.

Defective PDI release from platelets and endothelial cells impairs thrombus formation in Hermansky-Pudlak syndrome

PubMed Central

Sharda, Anish; Kim, Sarah H.; Jasuja, Reema; Gopal, Srila; Flaumenhaft, Robert; Furie, Barbara C.

2015-01-01

Protein disulfide isomerase (PDI), secreted from platelets and endothelial cells after injury, is required for thrombus formation. The effect of platelet and endothelial cell granule contents on PDI-mediated thrombus formation was studied by intravital microscopy using a mouse model of Hermansky-Pudlak syndrome in which platelet dense granules are absent. Platelet deposition and fibrin generation were nearly absent, and extracellular PDI was significantly reduced in HPS6−/− mice after vascular injury. HPS6−/− platelets displayed impaired PDI secretion and impaired exocytosis of α granules, lysosomes, and T granules due to decreased sensitivity to thrombin, but these defects could be corrected by addition of subthreshold amounts of adenosine 5′-diphosphate (ADP). Human Hermansky-Pudlak syndrome platelets demonstrated similar characteristics. Infusion of wild-type platelets rescued thrombus formation in HPS6−/− mice. Human umbilical vein endothelial cells in which the HPS6 gene was silenced displayed impaired PDI secretion and exocytosis of Weibel-Palade bodies. Defective thrombus formation in Hermansky-Pudlak syndrome, associated with impaired exocytosis of residual granules in endothelial cells and platelets, the latter due to deficiency of ADP, is characterized by a defect in T granule secretion, a deficiency in extracellular PDI secretion, and impaired fibrin generation and platelet aggregation. Hermansky-Pudlak syndrome is an example of a hereditary disease whereby impaired PDI secretion contributes to a bleeding phenotype. PMID:25593336

Clot retraction is mediated by factor XIII-dependent fibrin-αIIbβ3-myosin axis in platelet sphingomyelin-rich membrane rafts.

PubMed

Kasahara, Kohji; Kaneda, Mizuho; Miki, Toshiaki; Iida, Kazuko; Sekino-Suzuki, Naoko; Kawashima, Ikuo; Suzuki, Hidenori; Shimonaka, Motoyuki; Arai, Morio; Ohno-Iwashita, Yoshiko; Kojima, Soichi; Abe, Mitsuhiro; Kobayashi, Toshihide; Okazaki, Toshiro; Souri, Masayoshi; Ichinose, Akitada; Yamamoto, Naomasa

2013-11-07

Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen γ-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunit-deficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-β-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-αIIbβ3-myosin complex is formed as a primary axis to promote platelet contraction.

Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow

PubMed Central

Whyte, Claire S.; Swieringa, Frauke; Mastenbroek, Tom G.; Lionikiene, Ausra S.; Lancé, Marcus D.; van der Meijden, Paola E. J.; Heemskerk, Johan W. M.

2015-01-01

The interaction of plasminogen with platelets and their localization during thrombus formation and fibrinolysis under flow are not defined. Using a novel model of whole blood thrombi, formed under flow, we examine dose-dependent fibrinolysis using fluorescence microscopy. Fibrinolysis was dependent upon flow and the balance between fibrin formation and plasminogen activation, with tissue plasminogen activator-mediated lysis being more efficient than urokinase plasminogen activator-mediated lysis. Fluorescently labeled plasminogen radiates from platelet aggregates at the base of thrombi, primarily in association with fibrin. Hirudin attenuates, but does not abolish plasminogen binding, denoting the importance of fibrin. Flow cytometry revealed that stimulation of platelets with thrombin/convulxin significantly increased the plasminogen signal associated with phosphatidylserine (PS)-exposing platelets. Binding was attenuated by tirofiban and Gly-Pro-Arg-Pro amide, confirming a role for fibrin in amplifying plasminogen binding to PS-exposing platelets. Confocal microscopy revealed direct binding of plasminogen and fibrinogen to different platelet subpopulations. Binding of plasminogen and fibrinogen co-localized with PAC-1 in the center of spread platelets. In contrast, PS-exposing platelets were PAC-1 negative, and bound plasminogen and fibrinogen in a protruding “cap.” These data show that different subpopulations of platelets harbor plasminogen by diverse mechanisms and provide an essential scaffold for the accumulation of fibrinolytic proteins that mediate fibrinolysis under flow. PMID:25712989

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.

Platelets are responsible for the accumulation of β-amyloid in blood clots inside and around blood vessels in mouse brain after thrombosis.

PubMed

Kucheryavykh, Lilia Y; Dávila-Rodríguez, Josué; Rivera-Aponte, David E; Zueva, Lidia V; Washington, A Valance; Sanabria, Priscilla; Inyushin, Mikhail Y

2017-01-01

Platelets contain beta-amyloid precursor protein (APP) as well as Aβ peptide (Aβ) that can be released upon activation. During thrombosis, platelets are concentrated in clots and activated. We used in vivo fluorescent analysis and electron microscopy in mice to determine to what degree platelets are concentrated in clots. We used immunostaining to visualize Aβ after photothrombosis in mouse brains. Both in vivo results and electron microscopy revealed that platelets were 300-500 times more concentrated in clots than in non-clotted blood. After thrombosis in control mice, but not in thrombocytopenic animals, Aβ immunofluorescence was present inside blood vessels in the visual cortex and around capillaries in the entorhinal cortex. The increased concentration of platelets allows enhanced release of Aβ during thrombosis, suggesting an additional source of Aβ in the brains of Alzheimer's patients that may arise if frequent micro-thrombosis events occur in their brains. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

[Heparin and antiheparin in childhood. 2. Clinical relevance of the phenomenon of heparin resistance].

PubMed

Domula, M; Weissbach, G

1982-01-01

Investigations of the content of platelet factor 4 in different thrombocyte lysates and platelet-rich plasma after induced release reaction were aimed at checking the efficiency of the own antiheparin measuring system. In this connection, the age dependent dynamics of platelet factor 4 could be first discovered. In platelet-poor plasma of healthy grown-up test persons there was an evidence of antiheparin titres which were five times higher as compared with those persons born maturely. All patients with disseminated intravascular coagulation processes of different aetiology, however, will have significantly increased values. As demonstrated in two children with hyperpyretic toxicosis, the liberated platelet factor 4 will only show a short plasma half decay period. From investigations made for refinding heparin in the plasma after in vitro addition the conclusion can be drawn that, in addition to platelet factor 4, even unspecific adhesions of heparin to certain plasma proteins may be responsible for increasing heparin resistance.

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

A method for the automated processing and analysis of images of ULVWF-platelet strings.

PubMed

Reeve, Scott R; Abbitt, Katherine B; Cruise, Thomas D; Hose, D Rodney; Lawford, Patricia V

2013-01-01

We present a method for identifying and analysing unusually large von Willebrand factor (ULVWF)-platelet strings in noisy low-quality images. The method requires relatively inexpensive, non-specialist equipment and allows multiple users to be employed in the capture of images. Images are subsequently enhanced and analysed, using custom-written software to perform the processing tasks. The formation and properties of ULVWF-platelet strings released in in vitro flow-based assays have recently become a popular research area. Endothelial cells are incorporated into a flow chamber, chemically stimulated to induce ULVWF release and perfused with isolated platelets which are able to bind to the ULVWF to form strings. The numbers and lengths of the strings released are related to characteristics of the flow. ULVWF-platelet strings are routinely identified by eye from video recordings captured during experiments and analysed manually using basic NIH image software to determine the number of strings and their lengths. This is a laborious, time-consuming task and a single experiment, often consisting of data from four to six dishes of endothelial cells, can take 2 or more days to analyse. The method described here allows analysis of the strings to provide data such as the number and length of strings, number of platelets per string and the distance between each platelet to be found. The software reduces analysis time, and more importantly removes user subjectivity, producing highly reproducible results with an error of less than 2% when compared with detailed manual analysis.

Novel mutations in RASGRP2, which encodes CalDAG-GEFI, abrogate Rap1 activation, causing platelet dysfunction

PubMed Central

Lozano, María Luisa; Cook, Aaron; Bastida, José María; Paul, David S.; Iruin, Gemma; Cid, Ana Rosa; Adan-Pedroso, Rosa; Ramón González-Porras, José; Hernández-Rivas, Jesús María; Fletcher, Sarah J.; Johnson, Ben; Morgan, Neil; Ferrer-Marin, Francisca; Vicente, Vicente; Sondek, John; Watson, Steve P.; Bergmeier, Wolfgang

2016-01-01

In addition to mutations in ITG2B or ITGB3 genes that cause defective αIIbβ3 expression and/or function in Glanzmann’s thrombasthenia patients, platelet dysfunction can be a result of genetic variability in proteins that mediate inside-out activation of αIIbβ3. The RASGRP2 gene is strongly expressed in platelets and neutrophils, where its encoded protein CalDAG-GEFI facilitates the activation of Rap1 and subsequent activation of integrins. We used next-generation sequencing (NGS) and whole-exome sequencing (WES) to identify 2 novel function-disrupting mutations in RASGRP2 that account for bleeding diathesis and platelet dysfunction in 2 unrelated families. By using a panel of 71 genes, we identified a homozygous change (c.1142C>T) in exon 10 of RASGRP2 in a 9-year-old child of Chinese origin (family 1). This variant led to a p.Ser381Phe substitution in the CDC25 catalytic domain of CalDAG-GEFI. In 2 Spanish siblings from family 2, WES identified a nonsense homozygous variation (c.337C>T) (p.Arg113X) in exon 5 of RASGRP2. CalDAG-GEFI expression was markedly reduced in platelets from all patients, and by using a novel in vitro assay, we found that the nucleotide exchange activity was dramatically reduced in CalDAG-GEFI p.Ser381Phe. Platelets from homozygous patients exhibited agonist-specific defects in αIIbβ3 integrin activation and aggregation. In contrast, α- and δ-granule secretion, platelet spreading, and clot retraction were not markedly affected. Integrin activation in the patients’ neutrophils was also impaired. These patients are the first cases of a CalDAG-GEFI deficiency due to homozygous RASGRP2 mutations that are linked to defects in both leukocyte and platelet integrin activation. PMID:27235135

Exome-chip meta-analysis identifies association between variation in ANKRD26 and platelet aggregation.

PubMed

Chen, Ming-Huei; Yanek, Lisa R; Backman, Joshua D; Eicher, John D; Huffman, Jennifer E; Ben-Shlomo, Yoav; Beswick, Andrew D; Yerges-Armstrong, Laura M; Shuldiner, Alan R; O'Connell, Jeffrey R; Mathias, Rasika A; Becker, Diane M; Becker, Lewis C; Lewis, Joshua P; Johnson, Andrew D; Faraday, Nauder

2017-11-29

Previous genome-wide association studies (GWAS) have identified several variants associated with platelet function phenotypes; however, the proportion of variance explained by the identified variants is mostly small. Rare coding variants, particularly those with high potential for impact on protein structure/function, may have substantial impact on phenotype but are difficult to detect by GWAS. The main purpose of this study was to identify low frequency or rare variants associated with platelet function using genotype data from the Illumina HumanExome Bead Chip. Three family-based cohorts of European ancestry, including ~4,000 total subjects, comprised the discovery cohort and two independent cohorts, one of European and one of African American ancestry, were used for replication. Optical aggregometry in platelet-rich plasma was performed in all the discovery cohorts in response to adenosine diphosphate (ADP), epinephrine, and collagen. Meta-analyses were performed using both gene-based and single nucleotide variant association methods. The gene-based meta-analysis identified a significant association (P = 7.13 × 10 -7 ) between rare genetic variants in ANKRD26 and ADP-induced platelet aggregation. One of the ANKRD26 SNVs - rs191015656, encoding a threonine to isoleucine substitution predicted to alter protein structure/function, was replicated in Europeans. Aggregation increases of ~20-50% were observed in heterozygotes in all cohorts. Novel genetic signals in ABCG1 and HCP5 were also associated with platelet aggregation to ADP in meta-analyses, although only results for HCP5 could be replicated. The SNV in HCP5 intersects epigenetic signatures in CD41+ megakaryocytes suggesting a new functional role in platelet biology for HCP5. This is the first study to use gene-based association methods from SNV array genotypes to identify rare variants related to platelet function. The molecular mechanisms and pathophysiological relevance for the identified genetic associations requires further study.

Surface morphology of platelet adhesion influenced by activators, inhibitors and shear stress

NASA Astrophysics Data System (ADS)

Watson, Melanie Groan

Platelet activation involves multiple events, one of which is the generation and release of nitric oxide (NO), a platelet aggregation inhibitor. Platelets simultaneously send and receive various agents that promote a positive and negative feedback control system during hemostasis. Although the purpose of platelet-derived NO is not fully understood, NO is known to inhibit platelet recruitment. NO's relatively large diffusion coefficient allows it to diffuse more rapidly than platelet agonists. It may thus be able to inhibit recruitment of platelets near the periphery of a growing thrombus before agonists have substantially accumulated in those regions. Results from two studies in our laboratory differed in the extent to which platelet-derived NO decreased platelet adhesion. Frilot studied the effect of L-arginine (L-A) and NG-Methyl-L-arginine acetate salt (L-NMMA) on platelet adhesion to collagen under static conditions in a Petri dish. Eshaq examined the percent coverage on collagen-coated and fibrinogen-coated microchannels under shear conditions with different levels of L-A and Adenosine Diphosphate (ADP). Frilot's results showed no effect of either L-A or L-NMMA on surface coverage, thrombus size or serotonin release, while Eshaq's results showed a decrease in surface coverage with increased levels of L-A. A possible explanation for these contrasting results is that platelet-derived NO may be more important under flow conditions than under static conditions. For this project, the effects of L-A. ADP and L-NMMA on platelet adhesion were studied at varying shear stresses on protein-coated glass slides. The surface exposed to platelet-rich-plasma in combination with each chemical solution was observed under AFM, FE-SEM and fluorescence microscopy. Quantitative and qualitative comparisons of images obtained with these techniques confirmed the presence of platelets on the protein coatings. AFM images of fibrinogen and collagen-coated slides presented characteristic differences. Adhered platelets were identified, particularly with the AFM. The effects of chemical additives were examined under the same microscopy techniques. The resulting fluorescent microscopy data suggests statistical differences between the percent surface coverage of different shear regions on the glass slides. No statistically significant change in surface coverage was found with the addition of ADP on fibrinogen-coated slides, but showed differences on collagen with all chemicals. However, in high shear regions. L-A produced a significant decrease in platelet adhesion and L-NMMA produced a statistically significant increase in platelet adhesion on fibrinogen and collagen-coated slides. The AFM images of the chemical additives provided no differences between one another except with ADP. The no shear and low shear conditions provided no variations between AFM images via visual confirmation and statistical significance. The only AFM image shear region differences were obtained from low to high shear regions and static to high shear regions comparisons. The objective of this project was to determine whether the static conditions used by Frilot and the dynamic conditions used by Eshaq could explain the different effects of L-A observed in those studies. In addition, the ability of the fluorescent imaging technique to quantify platelet adhesion was examined by comparison of fluorescent imaging to AFM and FE-SEM. The results of this study were consistent with both the lack of an effect of chemical additives under static conditions reported by Frilot and the reduction of platelet adhesion in response to L-A reported by Eshaq.

Transcriptomic analysis of the ion channelome of human platelets and megakaryocytic cell lines.

PubMed

Wright, Joy R; Amisten, Stefan; Goodall, Alison H; Mahaut-Smith, Martyn P

2016-08-01

Ion channels have crucial roles in all cell types and represent important therapeutic targets. Approximately 20 ion channels have been reported in human platelets; however, no systematic study has been undertaken to define the platelet channelome. These membrane proteins need only be expressed at low copy number to influence function and may not be detected using proteomic or transcriptomic microarray approaches. In our recent work, quantitative real-time PCR (qPCR) provided key evidence that Kv1.3 is responsible for the voltage-dependent K+ conductance of platelets and megakaryocytes. The present study has expanded this approach to assess relative expression of 402 ion channels and channel regulatory genes in human platelets and three megakaryoblastic/erythroleukaemic cell lines. mRNA levels in platelets are low compared to other blood cells, therefore an improved method of isolating platelets was developed. This used a cocktail of inhibitors to prevent formation of leukocyte-platelet aggregates, and a combination of positive and negative immunomagnetic cell separation, followed by rapid extraction of mRNA. Expression of 34 channel-related transcripts was quantified in platelets, including 24 with unknown roles in platelet function, but that were detected at levels comparable to ion channels with established roles in haemostasis or thrombosis. Trace expression of a further 50 ion channel genes was also detected. More extensive channelomes were detected in MEG-01, CHRF-288-11 and HEL cells (195, 185 and 197 transcripts, respectively), but lacked several channels observed in the platelet. These "channelome" datasets provide an important resource for further studies of ion channel function in the platelet and megakaryocyte.

Nobiletin, a citrus flavonoid, activates vasodilator-stimulated phosphoprotein in human platelets through non-cyclic nucleotide-related mechanisms.

PubMed

Jayakumar, Thanasekaran; Lin, Kao-Chang; Lu, Wan-Jung; Lin, Chia-Ying; Pitchairaj, Geraldine; Li, Jiun-Yi; Sheu, Joen-Rong

2017-01-01

Nobiletin, a bioactive polymethoxylated flavone, has been described to possess a diversity of biological effects through its antioxidant and anti-inflammatory properties. Vasodilator-stimulated phosphoprotein (VASP) is a common substrate for cyclic AMP and cyclic GMP-regulated protein kinases [i.e., cyclic AMP-dependent protein kinase (PKA; also known as protein kinase A) and cyclic GMP-dependent protein kinase (PKG; also known as protein kinase G)] and it has been shown to be directly phosphorylated by protein kinase C (PKC). In the present study, we demonstrate that VASP is phosphorylated by nobiletin in human platelets via a non-cyclic nucleotide-related mechanism. This was confirmed by the use of inhibitors of adenylate cyclase (SQ22536) and guanylate cyclase [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)], since they prevented VASP phosphorylation induced by nobiletin. Furthormore, this event was also not affected by specific inhibitors of PKA (H-89), PKG (KT5823) and PKC (Ro318220), representing cyclic nucleotide-dependent pathways upon nobiletin-induced VASP phosphorylation. Similarly, inhibitors of p38 mitogen-activated protein kinase (MAPK; SB203580), extracellular signal-regulated kinase 2 (ERK2; PD98059), c-Jun N-terminal kinase 1 (JNK1; SP600125), Akt (LY294002) and nuclear factor-κB (NF-κB; Bay11-7082) did not affect nobiletin‑induced VASP phosphorylation. Moreover, electron spin resonance, dichlorofluorescein fluorescence and western blotting techniques revealed that nobiletin did not affect hydroxyl radicals (OH•), intracellular reactive oxygen species (ROS) and on protein carbonylation, respectively. Furthermore, the nobiletin‑induced VASP phosphorylation was surprisingly reversed by the intracellular antioxidant, N-acetylcysteine (NAC), but not by the inhibitor of NADPH oxidase, diphenyleneiodonium chloride (DPI). It was surprising to observe the differential effects of nobiletin and NAC on VASP phosphorylation in human platelets, since they both have been reported to have antioxidant properties. The likely explanation for this discrepancy is that NAC may bind to allosteric sites on the receptor different from those that nobiletin binds to in human platelets. Taken together, our findings suggest that nobiletin induces VASP phosphorylation in human platelets through non-cyclic nucleotide-related mechanisms. Nevertheless, the exact mechanisms responsible for these effects need to be further confirmed in future studies.

Sequential expression of cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor in rat hippocampal neurons after fluid percussion injury

PubMed Central

Li, Zhiqiang; Shu, Qingming; Li, Lingzhi; Ge, Maolin; Zhang, Yongliang

2014-01-01

Traumatic brain injury causes gene expression changes in different brain regions. Occurrence and development of traumatic brain injury are closely related, involving expression of three factors, namely cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. However, little is known about the correlation of these three factors and brain neuronal injury. In this study, primary cultured rat hippocampal neurons were subjected to fluid percussion injury according to Scott's method, with some modifications. RT-PCR and semi-quantitative immunocytochemical staining was used to measure the expression levels of cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. Our results found that cyclooxygenase-2 expression were firstly increased post-injury, and then decreased. Both mRNA and protein expression levels reached peaks at 8 and 12 hours post-injury, respectively. Similar sequential changes in glutamate receptor 2 were observed, with highest levels mRNA and protein expression at 8 and 12 hours post-injury respectively. On the contrary, the expressions of platelet activating factor receptor were firstly decreased post-injury, and then increased. Both mRNA and protein expression levels reached the lowest levels at 8 and 12 hours post-injury, respectively. Totally, our findings suggest that these three factors are involved in occurrence and development of hippocampal neuronal injury. PMID:25206921

Platelets, lymphocytes and erythrocytes from Alzheimer's disease patients: the quest for blood cell-based biomarkers.

PubMed

Pluta, Ryszard; Ułamek-Kozioł, Marzena; Januszewski, Sławomir; Czuczwar, Stanisław J

2018-01-01

In elderly population, Alzheimer's disease is a common neurodegenerative disorder and accounts for about 70% of all cases of dementia. The neurodegenerative processes of this disease start presumably 20 years ahead of the clinical beginning of the disorder. The postmortem histopathological examination, brains from Alzheimer's disease patients with characteristic features like amyloid plaques and neurofibrillary tangles, neuronal and synaptic disintegration confirm the final diagnosis of Alzheimer's disease. Senile plaques are composed of -amyloid peptide, deriving from the amyloid protein precursor, which is present not only in the brain tissue, but also in other non-neuronal tissues. Some investigations reported that platelets possess amyloid protein precursor and all the enzymatic activities required for the metabolism of this protein throughout the same pathways present in the brain. Thus, platelets may be a good peripheral blood cell-based biomarker to study the onset of Alzheimer's disease. Another line of research indicated molecular and cellular aberrations in blood lymphocytes and erythrocytes from Alzheimer's disease patients and emphasizes the systemic nature of the disease. In this review, we will summarize the recent knowledge on the involvement and/or response of platelets, lymphocytes and red blood cells in the circulation during Alzheimer's disease development. The facts will be reviewed with the special possibility for applying the above blood cells as Alzheimer's disease preclinical and antemortem blood cell-based biomarkers.

Formation of mixed-layer structures in smectites intercalated with tryptone

NASA Astrophysics Data System (ADS)

Block, K. A.; Trusiak, A.; Steiner, J. C.; Katz, A.; Gottlieb, P.; Alimova, A.

2012-12-01

Stable clay-protein complexes are fundamental to studies of the critical zone, terrestrial ecosystems, pharmacology, and industrial applications such as bioremediation. Two sets of montmorillonite clays were purified and made homoionic for Na and Mg. Mg-montmorillonite and Na-montmorillonite were mixed with tryptone (casein digest) in a 9:1 and 18:1 clay:tryptone ratio, resulting in the formation of reversible intercalated structures. X-ray diffraction analysis of the protein-clay complexes produced profiles consisting of two peaks associated with the smectite 001 reflection and a related tryptone-packet peak similar to that produced by a mixed layer clay structure. Shifts in the 002, 003, and 004 diffraction maxima are attributed to disorder caused by the interaction with the protein. Line broadening in the smectite-tryptone XRD spectra is interpreted to be the result of interlayer absorption. Adsorption produces coherent crystalline packets of regularly interbedded tryptone and smectite platelets. SEM images reveal clay platelets with upwardly rolled edges that tend toward cylindrical structures with the production of occasional tubes in the smaller platelet size range as noted for organic compound-kaolinite intercalation reported by Fenoll Hach-Ali and Weiss (1969). Reference: Fenoll Hach-Ali, P.F., Weiss, A., 1969. Estudio de la reaccion de caolinita y N-metilform- amida. Quimica LXV, 769-790. Scanning electron micrograph of tryptone-intercalated clay platelets exhibiting rolled edge structure.

Poly-2-methoxyethylacrylate-coated cardiopulmonary bypass circuit can reduce transfusion of platelet products compared to heparin-coated circuit during aortic arch surgery.

PubMed

Hosoyama, Katsuhiro; Ito, Koki; Kawamoto, Shunsuke; Kumagai, Kiichiro; Akiyama, Masatoshi; Adachi, Osamu; Kawatsu, Satoshi; Sasaki, Konosuke; Suzuki, Marina; Sugawara, Yumi; Shimizu, Yuya; Saiki, Yoshikatsu

2016-09-01

Several coating techniques for extracorporeal circulation have been developed to reduce the systemic inflammatory response during cardiopulmonary bypass (CPB). We compared the clinical effectiveness and biocompatibility of poly-2-methoxyethylacrylate (PMEA)- and heparin-coated CPB circuits in total aortic arch replacement (TAR) with the prolonged use of the bypass technique. Twenty patients who underwent elective TAR were divided randomly into two equal groups: group P (n = 10) to use PMEA-coated circuits and group H (n = 10) to use heparin-coated circuits. Clinical outcomes, hematological variables, and acute phase inflammatory response were analyzed perioperatively. Demographic, CPB, and clinical outcome data were similar for both groups. Hemoglobin and platelet count showed similar time-course curves. However, the amount of platelet products transfused intraoperatively was significantly larger in group H (group P 26.0 ± 7.0 units; group H 33.0 ± 6.7 units, p = 0.04). Total protein, and albumin levels were significantly higher in group P during and after the operation (total protein, p = 0.04; albumin, p = 0.02). The use of PMEA-coated circuit is associated with retainment of perioperative plasma proteins levels and may help to reduce transfusion of platelet products in TAR in comparison with the heparin-coated circuit.

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

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.

Localization and characterization of an alpha-thrombin-binding site on platelet glycoprotein Ib alpha.

PubMed

De Marco, L; Mazzucato, M; Masotti, A; Ruggeri, Z M

1994-03-04

Glycoprotein (GP) Ib alpha is required for expression of the highest affinity alpha-thrombin-binding site on platelets, possibly contributing to platelet activation through a pathway involving cleavage of a specific receptor. This function may be important for the initiation of hemostasis and may also play a role in the development of pathological vascular occlusion. We have now identified a discrete sequence in the extracytoplasmic domain of GP Ib alpha, including residues 271-284 of the mature protein, which appears to be part of the high affinity alpha-thrombin-binding site. Synthetic peptidyl mimetics of this sequence inhibit alpha-thrombin binding to GP Ib as well as platelet activation and aggregation induced by subnanomolar concentrations of the agonist; they also inhibit alpha-thrombin binding to purified glycocalicin, the isolated extracytoplasmic portion of GP Ib alpha. The inhibitory peptides interfere with the clotting of fibrinogen by alpha-thrombin but not with the amidolytic activity of the enzyme on a small synthetic substrate, a finding compatible with the concept that the identified GP Ib alpha sequence interacts with the anion-binding exosite of alpha-thrombin but not with its active proteolytic site. The crucial structural elements of this sequence necessary for thrombin binding appear to be a cluster of negatively charged residues as well as three tyrosine residues that, in the native protein, may be sulfated. GP Ib alpha has no significant overall sequence homology with the thrombin inhibitor, hirudin, nor with the specific thrombin receptor on platelets; all three molecules, however, possess a distinct region rich in negatively charged residues that appear to be involved in thrombin binding. This may represent a case of convergent evolution of unrelated proteins for high affinity interaction with the same ligand.

Prealbumin, platelet factor 4 and S100A12 combination at baseline predicts good response to TNF alpha inhibitors in Rheumatoid Arthritis.

PubMed

Nguyen, Minh Vu Chuong; Baillet, Athan; Romand, Xavier; Trocmé, Candice; Courtier, Anaïs; Marotte, Hubert; Thomas, Thierry; Soubrier, Martin; Miossec, Pierre; Tébib, Jacques; Grange, Laurent; Toussaint, Bertrand; Lequerré, Thierry; Vittecoq, Olivier; Gaudin, Philippe

2018-06-06

Tumour necrosis factor-alpha inhibitors (TNFi) are effective treatments for Rheumatoid Arthritis (RA). Responses to treatment are barely predictable. As these treatments are costly and may induce a number of side effects, we aimed at identifying a panel of protein biomarkers that could be used to predict clinical response to TNFi for RA patients. Baseline blood levels of C-reactive protein, platelet factor 4, apolipoprotein A1, prealbumin, α1-antitrypsin, haptoglobin, S100A8/A9 and S100A12 proteins in bDMARD naive patients at the time of TNFi treatment initiation were assessed in a multicentric prospective French cohort. Patients fulfilling good EULAR response at 6 months were considered as responders. Logistic regression was used to determine best biomarker set that could predict good clinical response to TNFi. A combination of biomarkers (prealbumin, platelet factor 4 and S100A12) was identified and could predict response to TNFi in RA with sensitivity of 78%, specificity of 77%, positive predictive values (PPV) of 72%, negative predictive values (NPV) of 82%, positive likelihood ratio (LR+) of 3.35 and negative likelihood ratio (LR-) of 0.28. Lower levels of prealbumin and S100A12 and higher level of platelet factor 4 than the determined cutoff at baseline in RA patients are good predictors for response to TNFi treatment globally as well as to Infliximab, Etanercept and Adalimumab individually. A multivariate model combining 3 biomarkers (prealbumin, platelet factor 4 and S100A12) accurately predicted response of RA patients to TNFi and has potential in a daily practice personalized treatment. Copyright © 2018. Published by Elsevier Masson SAS.

A virally inactivated functional growth factor preparation from human platelet concentrates.

PubMed

Su, C-Y; Kuo, Y P; Lin, Y C; Huang, C-T; Tseng, Y H; Burnouf, T

2009-08-01

Human platelet growth factors (HPGF) are essential for tissue regeneration and may replace fetal bovine serum (FBS) in cell therapy. No method for the manufacture of standardized virally inactivated HPGF has been developed yet. Platelet concentrates (PC) were subjected to solvent/detergent (S/D) treatment (1% TnBP/1% Triton X-45), oil extraction, hydrophobic interaction chromatography and sterile filtration. Platelet-derived growth factor (PDGF)-AB, -BB and -AA, transforming growth factor-beta1 (TGF-beta1), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1) and vascular endothelium growth factor (VEGF) were measured by ELISA. Composition in proteins and lipids was determined, protein profiles were obtained by SDS-PAGE, and TnBP and Triton X-45 were assessed by gas chromatography and high-performance liquid chromatography, respectively. Cell growth promoting activity of HPGF was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay using human embryonic kidney (HEK293A) fibroblast and Statens Seruminstitute rabbit corneal (SIRC) epithelial cell lines. The GF preparation contained a mean of 16.66, 2.04, 1.53, 72.19, 0.33, 48.59 and 0.44 ng/ml of PDGF-AB, -BB, -AA, TGF-beta1, EGF, IGF-1 and VEGF, respectively. The protein profile was typical of platelet releasates and had less than 2 p.p.m. of residual S/D agents. MTS assay of HEK293A and SIRC cultures showed that the GF preparation at 10% and 0.1% (v/v), respectively, could successfully replace 10% FBS for cell proliferation. Cell-stimulating activity of HPGF on HEK293A was over twice that of PC releasates. STANDARDIZED and functional virally inactivated HPGF can be prepared from human PC for possible applications in cell therapy and regenerative medicine.

Calcium supplementation during pregnancy for preventing hypertensive disorders is not associated with changes in platelet count, urate, and urinary protein: a randomized control trial.

PubMed

Hofmeyr, G J; Mlokoti, Z; Nikodem, V C; Mangesi, L; Ferreira, S; Singata, M; Jafta, Z; Merialdi, M; Hazelden, C; Villar, J

2008-01-01

To test the hypothesis that calcium supplementation inhibits the underlying pathological processes in women with preeclampsia. Seven hundred and eight nulliparous women were enrolled in a WHO randomized double-blind trial, who received 1.5 g of calcium or placebo from 20 weeks of pregnancy or earlier. Platelet count, serum urate, and urinary protein/creatinine ratio were measured at or near 35 gestational weeks. No difference was detected in rates of abnormal platelet count (relative risk [RR] 1.18; 95% confidence interval [CI], 0.63 to 2.18), serum urate level (1.0; 0.64 to 1.57) or urine protein/creatinine ratio (1.01; 0.76 to 1.34). This was consistent with the main trial finding of no difference in the incidence of 'dipstick' proteinuria between women receiving calcium and those receiving placebo (8312 women; RR, 1.01; 95% CI, 0.88 to 1.15). An effect of calcium supplementation in the second half of pregnancy on the rate of abnormal laboratory measures associated with preeclampsia was not demonstrated.

Hemostatic potential of natural/synthetic polymer based hydrogels crosslinked by gamma radiation

NASA Astrophysics Data System (ADS)

Barba, Bin Jeremiah D.; Tranquilan-Aranilla, Charito; Abad, Lucille V.

2016-01-01

Various raw materials and hydrogels prepared from their combination were assessed for hemostatic capability using swine whole blood clotting analysis. Initial screening showed efficient coagulative properties from κ-carrageenan and its carboxymethylated form, and α-chitosan, even compared to commercial products like QuikClot Zeolite Powder. Blending natural and synthetic polymers formed into hydrogels using gamma radiation produced materials with improved properties. KC and CMKC hydrogels were found to have the lowest blood clotting index in granulated form and had the higher capacity for platelet adhesion in foamed form compared to GelFoam. Possible mechanisms involved in the evident thrombogenicity of the materials include adsorption of platelets and related proteins that aid in platelet activation (primary hemostasis), absorption of water to concentrate protein factors that control the coagulation cascade, contact activation by its negatively charged surface and the formation of gel-blood clots.

Platelet Rich Plasma and Knee Surgery

PubMed Central

Sánchez, Mikel; Sánchez, Pello; Orive, Gorka; Anitua, Eduardo; Padilla, Sabino

2014-01-01

In orthopaedic surgery and sports medicine, the knee joint has traditionally been considered the workhorse. The reconstruction of every damaged element in this joint is crucial in achieving the surgeon's goal to restore the knee function and prevent degeneration towards osteoarthritis. In the last fifteen years, the field of regenerative medicine is witnessing a boost of autologous blood-derived platelet rich plasma products (PRPs) application to effectively mimic and accelerate the tissue healing process. The scientific rationale behind PRPs is the delivery of growth factors, cytokines, and adhesive proteins present in platelets and plasma, as well as other biologically active proteins conveyed by the plasma such as fibrinogen, prothrombin, and fibronectin; with this biological engineering approach, new perspectives in knee surgery were opened. This work describes the use of PRP to construct and repair every single anatomical structure involved in knee surgery, detailing the process conducted in ligament, meniscal, and chondral surgery. PMID:25302310

High glucose inhibits the aspirin-induced activation of the nitric oxide/cGMP/cGMP-dependent protein kinase pathway and does not affect the aspirin-induced inhibition of thromboxane synthesis in human platelets.

PubMed

Russo, Isabella; Viretto, Michela; Barale, Cristina; Mattiello, Luigi; Doronzo, Gabriella; Pagliarino, Andrea; Cavalot, Franco; Trovati, Mariella; Anfossi, Giovanni

2012-11-01

Since hyperglycemia is involved in the "aspirin resistance" occurring in diabetes, we aimed at evaluating whether high glucose interferes with the aspirin-induced inhibition of thromboxane synthesis and/or activation of the nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (PKG) pathway in platelets. For this purpose, in platelets from 60 healthy volunteers incubated for 60 min with 5-25 mmol/L d-glucose or iso-osmolar mannitol, we evaluated the influence of a 30-min incubation with lysine acetylsalicylate (L-ASA; 1-300 μmol/L) on 1) platelet function under shear stress; 2) aggregation induced by sodium arachidonate or ADP; 3) agonist-induced thromboxane production; and 4) NO production, cGMP synthesis, and PKG-induced vasodilator-stimulated phosphoprotein phosphorylation. Experiments were repeated in the presence of the antioxidant agent amifostine. We observed that platelet exposure to 25 mmol/L d-glucose, but not to iso-osmolar mannitol, 1) reduced the ability of L-ASA to inhibit platelet responses to agonists; 2) did not modify the L-ASA-induced inhibition of thromboxane synthesis; and 3) prevented the L-ASA-induced activation of the NO/cGMP/PKG pathway. Preincubation with amifostine reversed the high-glucose effects. Thus, high glucose acutely reduces the antiaggregating effect of aspirin, does not modify the aspirin-induced inhibition of thromboxane synthesis, and inhibits the aspirin-induced activation of the NO/cGMP/PKG pathway. These results identify a mechanism by which high glucose interferes with the aspirin action.

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.

Hippophae rhamnoides L. Fruits Reduce the Oxidative Stress in Human Blood Platelets and Plasma.

PubMed

Olas, Beata; Kontek, Bogdan; Malinowska, Paulina; Żuchowski, Jerzy; Stochmal, Anna

2016-01-01

Effects of the phenolic fraction from Hippophae rhamnoides fruits on the production of thiobarbituric acid reactive substances (TBARS, a marker of lipid peroxidation) and the generation of superoxide anion (O2 (-∙)) in human blood platelets (resting platelets and platelets stimulated by a strong physiological agonist, thrombin) were studied in vitro. We also examined antioxidant properties of this fraction against human plasma lipid peroxidation and protein carbonylation induced by a strong biological oxidant, hydrogen peroxide (H2O2) or H2O2/Fe (a donor of hydroxyl radicals). The tested fraction of H. rhamnoides (0.5- 50 µg/mL; the incubation time: 15 and 60 min) inhibited lipid peroxidation induced by H2O2 or H2O2/Fe. The H. rhamnoides phenolic fraction inhibited not only plasma lipid peroxidation, but also plasma protein carbonylation stimulated by H2O2 or H2O2/Fe. Moreover, the level of O2 (-∙) in platelets significantly decreased. In comparative experiments, the H. rhamnoides fraction was a more effective antioxidant than aronia extract or grape seed extract (at the highest tested concentration, 50 µg/mL). The obtained results suggest that H. rhamnoides fruits may be a new, promising source of natural compounds with antioxidant and antiplatelet activity beneficial not only for healthy people, but also for those with oxidative stress-associated diseases.

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

Platelet binding sites for factor VIII in relation to fibrin and phosphatidylserine

PubMed Central

Novakovic, Valerie A.; Shi, Jialan; Rasmussen, Jan; Pipe, Steven W.

2015-01-01

Thrombin-stimulated platelets expose very little phosphatidylserine (PS) but express binding sites for factor VIII (fVIII), casting doubt on the role of exposed PS as the determinant of binding sites. We previously reported that fVIII binding sites are increased three- to sixfold when soluble fibrin (SF) binds the αIIbβ3 integrin. This study focuses on the hypothesis that platelet-bound SF is the major source of fVIII binding sites. Less than 10% of fVIII was displaced from thrombin-stimulated platelets by lactadherin, a PS-binding protein, and an fVIII mutant defective in PS-dependent binding retained platelet affinity. Therefore, PS is not the determinant of most binding sites. FVIII bound immobilized SF and paralleled platelet binding in affinity, dependence on separation from von Willebrand factor, and mediation by the C2 domain. SF also enhanced activity of fVIII in the factor Xase complex by two- to fourfold. Monoclonal antibody (mAb) ESH8, against the fVIII C2 domain, inhibited binding of fVIII to SF and platelets but not to PS-containing vesicles. Similarly, mAb ESH4 against the C2 domain, inhibited >90% of platelet-dependent fVIII activity vs 35% of vesicle-supported activity. These results imply that platelet-bound SF is a component of functional fVIII binding sites. PMID:26162408

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.

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.

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.

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.

Inverse relationship between erythrocyte size and platelet reactivity in elderly.

PubMed

Milovanovic, M; Nilsson, S; Winblad, B; Jelic, V; Behbahani, H; Shahnaz, T; Oweling, M; Järemo, P

2017-03-01

Previous work indicates that erythrocytes (RBCs) accumulate β-amyloid X-40 (Aβ 40 ) in individuals with Alzheimer disease (AD) and to a lesser extent in healthy elderly. The toxin damages RBCs and increases their mean corpuscular volume (MCV). Furthermore, AD platelets demonstrate lower reactivity. This study investigated interactions between RBCs and platelets. Older individuals with moderate hypertension (n = 57) were classified into two groups, depending on MCV in whole blood: The MCV high group comprised individuals with higher MCV (n = 27; 97 ± 3(SD) fl) and MCV low group had relatively lower MCV (n = 30; 90 ± 3(SD) fl). Flow cytometry was used to determine platelet reactivity, i.e., the surface binding of fibrinogen after provocation. Adenosine diphosphate (ADP) and a thrombin receptor-activating protein (TRAP-6) were used as agonists. Subsequently, blood cells were divided according to density into 17 subfractions. Intra-RBC Aβ 40 content was analyzed and in all platelet populations surface-bound fibrinogen was determined to estimate platelet in vivo activity. We found Aβ 40 inside RBCs of approximately 50% of participants, but the toxin did not affect MCV and platelet reactivity. In contrast, MCV associated inversely with platelet reactivity as judged from surface-attached fibrinogen after ADP (1.7 μmol/L) (p < 0.05) and TRAP-6 provocation (57 μmol/L (p = 0.01) and 74 μmol/L (p < 0.05)). In several density fractions (nos. 3, 4, 8, 11-13 (p < 0.05) and nos. 5-7 (p < 0.01)) MCV linked inversely with platelet-attached fibrinogen. In our community-dwelling sample, enhanced MCV associated with decreased platelet reactivity and lower in vivo platelet activity. It resembles RBCs and platelet behavior in AD-type dementia.

A Novel Variant in the Platelet Endothelial Aggregation Receptor-1 Gene is Associated with Increased Platelet Aggregabili

PubMed Central

Herrera-Galeano, J. Enrique; Becker, Diane M.; Wilson, Alexander F.; Yanek, Lisa R.; Bray, Paul; Vaidya, Dhananjay; Faraday, Nauder; Becker, Lewis C

2009-01-01

Objective: Platelet endothelial aggregation receptor-1 (PEAR1) is a recently identified platelet transmembrane protein that becomes activated by platelet contact. We looked for novel genetic variants in PEAR1 and studied their association with agonist-induced native platelet aggregation and with aspirin's inhibitory effect on platelets. Methods and Results: We genotyped PEAR1 for 10 single nucleotide polymorphisms (SNPs), selected for optimal gene coverage at a density of 4kb, in 1486 apparently healthy individuals from two generations of families with premature CAD. Subjects had a mean age of 45 years; 62% were white and 38% African American. Platelet aggregation to collagen, epinephrine, and ADP was measured in platelet rich plasma, at baseline and after 2 weeks of aspirin (ASA, 81 mg/day), and genotype-phenotype associations were examined separately by ethnicity using multivariable generalized linear models adjusted for covariates. The C allele of SNP rs2768759 [A/C], located in the promoter region of the gene, was common in whites and uncommon in African Americans (allele frequency 70.2% vs 17.7%). The C allele was generally associated in both ethnic groups with increased aggregation of native platelets to each agonist. Following ASA, the associations were stronger and more consistent, and remained significant when post ASA aggregation was adjusted for baseline aggregation, consistent with a relationship between the C allele and reduced platelet responsiveness to ASA. The PEAR1 SNP explained up to 6.9% of the locus specific genetic variance in African Americans and up to 2.5% of the genetic variance in whites following ASA. Conclusion: PEAR1 appears to play an important role in agonist-induced platelet aggregation and in the response to ASA in both whites and African Americans. PMID:18511696

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.

5-HT receptor probe (/sup 3/H)8-OH-DPAT labels the 5-HT transporter in human platelets

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

Ieni, J.R.; Meyerson, L.R.

1988-01-01

The present study characterizes a serotonin (5-HT) binding site on human platelet membranes, using (/sup 3/H)8-OH-DPAT as the radioligand. (/sup 3/H)8-OH-DPAT binds specifically and saturably to a site on human platelet membranes with an average K/sub D/ of 43 nM and B/sub max/ of 1078 fmol/mg protein. Determinations of IC/sub 50/ values for various serotonergic characterizing agents in platelets for displacement of (/sup 3/H)8-OH-DPAT were performed. The pharmacological inhibitory profile of the platelet 8-OH-DPAT site is not consistent with profiles reported for brain. 8-OH-DPAT does not inhibit (/sup 3/H) imipramine binding, however, it does inhibit (/sup 3/H)5-HT uptake in humanmore » platelets near 5-HT's K/sub m/ value (IC/sub 50/ = 2-4 ..mu..M). These results suggest that the human platelet site labelled by (/sub 3/H)8-OH-DPAT is pharmocologically different from the neuronal site and probably is a component of the 5-HT transporter. 32 references, 1 figure, 4 tables.« less

Initial blood storage experiment

NASA Technical Reports Server (NTRS)

Surgenor, Douglas MACN.

1988-01-01

The possibility of conducting experiments with the formed elements of the blood under conditions of microgravity opens up important opportunities to improve the understanding of basic formed element physiology, as well as, contribution to improved preservation of the formed elements for use in transfusion. The physiological, biochemical, and physical changes of the membrane of the erythrocyte, platelet, and leukocyte was studied during storage under two specific conditions: standard blood bank conditions and microgravity, utilizing three FDA approved plastic bags. Storage lesions; red cell storage on Earth; platelet storage on Earth; and leukocyte storage Earth were examined. The interaction of biomaterials and blood cells was studied during storage.

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

Platelets derived from the bone marrow of diabetic animals show dysregulated endoplasmic reticulum stress proteins that contribute to increased thrombosis.

PubMed

Hernández Vera, Rodrigo; Vilahur, Gemma; Ferrer-Lorente, Raquel; Peña, Esther; Badimon, Lina

2012-09-01

Patients with diabetes mellitus have an increased risk of suffering atherothrombotic syndromes and are prone to clustering cardiovascular risk factors. However, despite their dysregulated glucose metabolism, intensive glycemic control has proven insufficient to reduce thrombotic complications. Therefore, we aimed to elucidate the determinants of thrombosis in a model of type 2 diabetes mellitus with cardiovascular risk factors clustering. Intravital microscopy was used to analyze thrombosis in vivo in Zucker diabetic fatty rats (ZD) and lean normoglycemic controls. Bone marrow (BM) transplants were performed to test the contribution of each compartment (blood or vessel wall) to thrombogenicity. ZD showed significantly increased thrombosis compared with lean normoglycemic controls. BM transplants demonstrated the key contribution of the hematopoietic compartment to increased thrombogenicity. Indeed, lean normoglycemic controls transplanted with ZD-BM showed increased thrombosis with normal glucose levels, whereas ZD transplanted with lean normoglycemic controls-BM showed reduced thrombosis despite presenting hyperglycemia. Significant alterations in megakaryopoiesis and platelet-endoplasmic reticulum stress proteins, protein disulfide isomerase and 78-kDa glucose-regulated protein, were detected in ZD, and increased tissue factor procoagulant activity was detected in plasma and whole blood of ZD. Our results indicate that diabetes mellitus with cardiovascular risk factor clustering favors BM production of hyperreactive platelets with altered protein disulfide isomerase and 78-kDa glucose-regulated protein expression that can contribute to increase thrombotic risk independently of blood glucose levels.

Covalent complexes of albumin with serotonin, ketanserin and lysergic acid antagonize the activity of serotonin in human platelets

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

VanderBerg, S.R.; Gonias, S.L.

1989-01-01

Covalent conjugates of bovine serum albumin (BSA) and 5-HT, ketanserin or d-lysergic acid were synthesized and characterized by polyacrylamide gel electrophoresis, whole blood clearance experiments in mice and aggregation studies with human platelets. Using the standard synthesis procedure, each mol of BSA bound 13.4 mol of (/sup 3/H)5-HT. Derivatization did not cause significant protein aggregation as determined by electrophoresis. All three conjugates antagonized the ability of 5-HT to amplify aggregation caused by low concentrations of ADP. The antagonist activity of each conjugate was concentration dependent; 2.6 ..mu..M 5-HT-BSA completely inhibited the aggregation caused by 13 ..mu..M 5-HT. None of themore » BSA drug conjugates, including 5-HT-BSA, amplified platelet aggregation caused by ADP in the absence of 5-HT. Aggregation by ristocetin, collagen, epinephrine or ADP alone was not significantly affected by the conjugates. Whole blood elimination experiments in mice demonstrated that the three conjugates and underivatized BSA are equally stable in the circulation. These prototypic 5-HT drug-protein conjugates may be useful for probing 5-HT/sub 2/ receptor-ligand interactions in human platelets.« less

HGF Mediates the Anti-inflammatory Effects of PRP on Injured Tendons

PubMed Central

Zhang, Jianying; Middleton, Kellie K.; Fu, Freddie H.; Im, Hee-Jeong; Wang, James H-C.

2013-01-01

Platelet-rich plasma (PRP) containing hepatocyte growth factor (HGF) and other growth factors are widely used in orthopaedic/sports medicine to repair injured tendons. While PRP treatment is reported to decrease pain in patients with tendon injury, the mechanism of this effect is not clear. Tendon pain is often associated with tendon inflammation, and HGF is known to protect tissues from inflammatory damages. Therefore, we hypothesized that HGF in PRP causes the anti-inflammatory effects. To test this hypothesis, we performed in vitro experiments on rabbit tendon cells and in vivo experiments on a mouse Achilles tendon injury model. We found that addition of PRP or HGF decreased gene expression of COX-1, COX-2, and mPGES-1, induced by the treatment of tendon cells in vitro with IL-1β. Further, the treatment of tendon cell cultures with HGF antibodies reduced the suppressive effects of PRP or HGF on IL-1β-induced COX-1, COX-2, and mPGES-1 gene expressions. Treatment with PRP or HGF almost completely blocked the cellular production of PGE2 and the expression of COX proteins. Finally, injection of PRP or HGF into wounded mouse Achilles tendons in vivo decreased PGE2 production in the tendinous tissues. Injection of platelet-poor plasma (PPP) however, did not reduce PGE2 levels in the wounded tendons, but the injection of HGF antibody inhibited the effects of PRP and HGF. Further, injection of PRP or HGF also decreased COX-1 and COX-2 proteins. These results indicate that PRP exerts anti-inflammatory effects on injured tendons through HGF. This study provides basic scientific evidence to support the use of PRP to treat injured tendons because PRP can reduce inflammation and thereby reduce the associated pain caused by high levels of PGE2. PMID:23840657

Hydrolysis of platelet-activating factor by human serum paraoxonase.

PubMed Central

Rodrigo, L; Mackness, B; Durrington, P N; Hernandez, A; Mackness, M I

2001-01-01

Human serum paraoxonase (human PON1) has been shown to be important in the metabolism of phospholipid and cholesteryl ester hydroperoxides, thereby preventing the oxidation of low-density lipoprotein (LDL) and retarding atherogenesis. However, the exact substrate specificity of PON1 has not been established. In the present study we show that purified PON1 hydrolyses platelet-activating factor (PAF). We could find no evidence for contamination of our preparation with authentic platelet-activating-factor acetylhydrolase (PAFAH) by immunoblotting with a PAFAH monoclonal antibody or by sequencing the purified protein. In addition the specific PAFAH inhibitor SB-222657 did not affect the ability of PON1 to hydrolyse PAF (30.1+/-2.8 micromol/min per mg of protein with no inhibitor; 31.4+/-2.2 micromol/min per mg of protein with 100 nM inhibitor) or phenyl acetate (242.6+/-30.8 versus 240.8+/-31.5 micromol/min per mg of protein with and without inhibitor respectively). SB-222657 was also unable to inhibit PAF hydrolysis by isolated human high-density lipoprotein (HDL), but completely abolished the activity of human LDL. Ostrich (Struthio camelus) HDL, which does not contain PON1, was unable to hydrolyse PAF. These data provide evidence that PON1 may limit the action of this bioactive pro-inflammatory phospholipid. PMID:11171072

Lea blood group antigen on human platelets.

PubMed

Dunstan, R A; Simpson, M B; Rosse, W F

1985-01-01

One- and two-stage radioligand assays were used to determine if human platelets possess the Lea antigen. Goat IgG anti-Lea antibody was purified by multiple adsorptions with Le(a-b-) human red blood cells, followed by affinity chromatography with synthetic Lea substance and labeling with 125I. Human IgG anti-Lea antibody was used either in a two stage radioassay with 125I-labeled mouse monoclonal IgG anti-human IgG as the second antibody or, alternatively, purified by Staph protein A chromatography, labeled with 125I, and used in a one-stage radioassay. Platelets from donors of appropriate red blood cell phenotypes were incubated with the antisera, centrifuged through phthalate esters, and assayed in a gamma scintillation counter. Dose response and saturation curve analysis demonstrate the presence of Lewis a antigen on platelets from Lea+ donors. Furthermore, platelets from an Le(a-b-) donor incubated in Le (a+b-) plasma adsorb Lea antigen in a similar manner to red blood cells. The clinical significance of these antigens in platelet transfusion remains undefined.

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

Antibody-mediated platelet phagocytosis by human macrophages is inhibited by siRNA specific for sequences in the SH2 tyrosine kinase, Syk.

PubMed

Lu, Ying; Wang, Weiming; Mao, Huiming; Hu, Hai; Wu, Yanling; Chen, Bing-Guan; Liu, Zhongmin

2011-01-01

Immune thrombocytopenia depends upon Fc receptor-mediated phagocytosis that involves signaling through the SH2 tyrosine kinase, Syk. We designed small interfering (siRNA) sequences complementary to Syk coding regions to decrease the expression of Syk in the human macrophage cell line, THP-1. To evaluate the functional effect of siRNA on phagocytosis, we developed a new in vitro assay for antibody-mediated platelet ingestion by THP-1 cells. Incubation of THP-1 cells at 37°C with fluorescence-labeled platelets and anti-platelet antibody promoted ingestion of platelets that could be quantitated by flow cytometry. Transfection of THP-1 cells with Syk-specific siRNA resulted in a reduction in the amount of FcγRII-associated Syk protein. Coincident with decreased Syk expression, we observed inhibition of antibody-mediated platelet ingestion. These results confirm a key role for Syk in antibody-mediated phagocytosis and suggest Syk-specific siRNA as a possible therapeutic candidate for immune thrombocytopenia. Copyright © 2011 Elsevier Inc. All rights reserved.

Distinct effects of calorie restriction on adipose tissue cytokine and angiogenesis profiles in obese and lean mice

PubMed Central

2012-01-01

Background Obesity associates with low-grade inflammation and adipose tissue remodeling. Using sensitive high-throughput protein arrays we here investigated adipose tissue cytokine and angiogenesis-related protein profiles from obese and lean mice, and in particular, the influence of calorie restriction (CR). Methods Tissue samples from visceral fat were harvested from obese mice fed with a high-fat diet (60% of energy), lean controls receiving low-fat control diet as well as from obese and lean mice kept under CR (energy intake 70% of ad libitum intake) for 50 days. Protein profiles were analyzed using mouse cytokine and angiogenesis protein array kits. Results In obese and lean mice, CR was associated with 11.3% and 15.6% reductions in body weight, as well as with 4.0% and 4.6% reductions in body fat percentage, respectively. Obesity induced adipose tissue cytokine expressions, the most highly upregulated cytokines being IL-1ra, IL-2, IL-16, MCP-1, MIG, RANTES, C5a, sICAM-1 and TIMP-1. CR increased sICAM-1 and TIMP-1 expression both in obese and lean mice. Overall, CR showed distinct effects on cytokine expressions; in obese mice CR largely decreased but in lean mice increased adipose tissue cytokine expressions. Obesity was also associated with increased expressions of angiogenesis-related proteins, in particular, angiogenin, endoglin, endostatin, endothelin-1, IGFBP-3, leptin, MMP-3, PAI-1, TIMP-4, CXCL16, platelet factor 4, DPPIV and coagulation factor III. CR increased endoglin, endostatin and platelet factor 4 expressions, and decreased IGFBP-3, NOV, MMP-9, CXCL16 and osteopontin expressions both in obese and lean mice. Interestingly, in obese mice, CR decreased leptin and TIMP-4 expressions, whereas in lean mice their expressions were increased. CR decreased MMP-3 and PAI-1 only in obese mice, whereas CR decreased FGF acidic, FGF basic and coagulation factor III, and increased angiogenin and DPPIV expression only in lean mice. Conclusions CR exerts distinct effects on adipocyte cytokine and angiogenesis profiles in obese and lean mice. Our study also underscores the importance of angiogenesis-related proteins and cytokines in adipose tissue remodeling and development of obesity. PMID:22748184

A novel method for automated assessment of megakaryocyte differentiation and proplatelet formation.

PubMed

Salzmann, M; Hoesel, B; Haase, M; Mussbacher, M; Schrottmaier, W C; Kral-Pointner, J B; Finsterbusch, M; Mazharian, A; Assinger, A; Schmid, J A

2018-06-01

Transfusion of platelet concentrates represents an important treatment for various bleeding complications. However, the short half-life and frequent contaminations with bacteria restrict the availability of platelet concentrates and raise a clear demand for platelets generated ex vivo. Therefore, in vitro platelet generation from megakaryocytes represents an important research topic. A vital step for this process represents accurate analysis of thrombopoiesis and proplatelet formation, which is usually conducted manually. We aimed to develop a novel method for automated classification and analysis of proplatelet-forming megakaryocytes in vitro. After fluorescent labelling of surface and nucleus, MKs were automatically categorized and analysed with a novel pipeline of the open source software CellProfiler. Our new workflow is able to detect and quantify four subtypes of megakaryocytes undergoing thrombopoiesis: proplatelet-forming, spreading, pseudopodia-forming and terminally differentiated, anucleated megakaryocytes. Furthermore, we were able to characterize the inhibitory effect of dasatinib on thrombopoiesis in more detail. Our new workflow enabled rapid, unbiased, quantitative and qualitative in-depth analysis of proplatelet formation based on morphological characteristics. Clinicians and basic researchers alike will benefit from this novel technique that allows reliable and unbiased quantification of proplatelet formation. It thereby provides a valuable tool for the development of methods to generate platelets ex vivo and to detect effects of drugs on megakaryocyte differentiation.

Application of platelet-rich plasma and platelet-rich fibrin in fat grafting: basic science and literature review.

PubMed

Liao, Han-Tsung; Marra, Kacey G; Rubin, J Peter

2014-08-01

Due to the natural properties of fat, fat grafting remains a popular procedure for soft tissue volume augmentation and reconstruction. However, clinical outcome varies and is technique dependent. Platelet-rich plasma (PRP) contains α-granules, from which multiple growth factors such as platelet-derived growth factor, transforming growth factor-β, vascular endothelial growth factor, and epidermal growth factor can be released after activation. In recent years, the scope of PRP therapies has extended from bone regeneration, wound healing, and healing of musculoskeletal injuries, to enhancement of fat graft survival. In this review, we focus on the definition of PRP, the different PRP preparation and activation methods, and growth factor concentrations. In addition, we discuss possible mechanisms for the role of PRP in fat grafting by reviewing in vitro studies with adipose-derived stem cells, preadipocytes, and adipocytes, and preclinical and clinical research. We also review platelet-rich fibrin, a so-called second generation PRP, and its slow-releasing biology and effects on fat grafts compared to PRP in both animal and clinical research. Finally, we provide a general foundation on which to critically evaluate earlier studies, discuss the limitations of previous research, and direct plans for future experiments to improve the optimal effects of PRP in fat grafting.

Platelet lysate from whole blood-derived pooled platelet concentrates and apheresis-derived platelet concentrates for the isolation and expansion of human bone marrow mesenchymal stromal cells: production process, content and identification of active components

PubMed Central

Fekete, Natalie; Gadelorge, Mélanie; Fürst, Daniel; Maurer, Caroline; Dausend, Julia; Fleury-Cappellesso, Sandrine; Mailänder, Volker; Lotfi, Ramin; Ignatius, Anita; Sensebé, Luc; Bourin, Philippe; Schrezenmeier, Hubert; Rojewski, Markus Thomas

2012-01-01

Background aims The clinical use of human mesenchymal stromal cells (MSC) requires ex vivo expansion in media containing supplements such as fetal bovine serum or, alternatively, human platelet lysate (PL). Methods Platelet concentrates were frozen, quarantine stored, thawed and sterile filtered to obtain PL. PL content and its effect on fibroblast-colony-forming unit (CFU-F) formation, MSC proliferation and large-scale expansion were studied. Results PL contained high levels of basic fibroblast growth factor (bFGF), soluble CD40L (sCD40L), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), platelet-derived growth factor AA (PDGF-AA), platelet-derived growth factor AB/BB (PDGF-AB/BB), chemokine (C-C) ligand 5 (CCL5; RANTES) transforming growth factor-β1 (TGF-β1) and chemokine (C-X-C) ligand 1/2/3 (GRO), with low batch-to-batch variability, and most were stable for up to 14 days. Inhibition of PDGF-BB and bFGF decreased MSC proliferation by about 20% and 50%, respectively. The strongest inhibition (about 75%) was observed with a combination of anti-bFGF + anti-PDGF-BB and anti-bFGF + anti-TGF-β1 + anti-PDGF-BB. Interestingly, various combinations of recombinant PDGF-BB, bFGF and TGF-β1 were not sufficient to promote cell proliferation. PL from whole blood-derived pooled platelet concentrates and apheresis platelet concentrates did not differ significantly in their growth-promoting activity on MSC. Conclusions PL enhances MSC proliferation and can be regarded as a safe tool for MSC expansion for clinical purposes. \\in particular, PDGF-BB and bFGF are essential components for the growth-promoting effect of PL, but are not sufficient for MSC proliferation. PMID:22296115

Double-chimera proteins to enhance recruitment of endothelial cells and their progenitor cells.

PubMed

Behjati, M; Kazemi, M; Hashemi, M; Zarkesh-Esfahanai, S H; Bahrami, E; Hashemi-Beni, B; Ahmadi, R

2013-08-20

Enhanced attraction of selective vascular reparative cells is of great importance in order to increase vascular patency after endovascular treatments. We aimed to evaluate efficient attachment of endothelial cells and their progenitors on surfaces coated with mixture of specific antibodies, L-selectin and VE-cadherin, with prohibited platelet attachment. The most efficient conditions for coating of L-selectin-Fc chimera and VE-cadherin-Fc chimera proteins were first determined by protein coating on ELISA plates. The whole processes were repeated on titanium substrates, which are commonly used to coat stents. Endothelial progenitor cells (EPCs) and human umbilical vein endothelial cells (HUVECs) were isolated and characterized by flow cytometry. Cell attachment, growth, proliferation, viability and surface cytotoxicity were evaluated using nuclear staining and MTT assay. Platelet and cell attachment were evaluated using scanning electron microscopy. Optimal concentration of each protein for surface coating was 50 ng/ml. The efficacy of protein coating was both heat and pH independent. Calcium ions had significant impact on simultaneous dual-protein coating (P<0.05). Coating stability data revealed more than one year stability for these coated proteins at 4°C. L-selectin and VE-cadherin (ratio of 50:50) coated surface showed highest EPC and HUVEC attachment, viability and proliferation compared to single protein coated and non-coated titanium surfaces (P<0.05). This double coated surface did not show any cytotoxic effect. Surfaces coated with L-selectin and VE-cadherin are friendly surface for EPC and endothelial cell attachment with less platelet attachment. These desirable factors make the L-selectin and VE-cadherin coated surfaces perfect candidate endovascular device. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Effects of Non-Collagenous Proteins, TGF-β1, and PDGF-BB on Viability and Proliferation of Dental Pulp Stem Cells

PubMed Central

Tabatabaei, Fahimeh Sadat

2016-01-01

ABSTRACT Objectives The dentin matrix servers as a reservoir of growth factors, sequestered during dentinogenesis. The aim of this study was to assess the viability and proliferation of dental pulp stem cells in the presence of dentin matrix-derived non-collagenous proteins and two growth factors; platelet-derived growth factor BB and transforming growth factor beta 1. Material and Methods The dental pulp cells were isolated and cultured. The dentin proteins were extracted and purified. The MTT assay was performed for assessment of cell viability and proliferation in the presence of different concentrations of dentin proteins and growth factors during 24 - 72 h post-treatment. Results The cells treated with 250 ng/mL dentin proteins had the best viability and proliferation ability in comparison with other concentrations (P < 0.05). The MTT assay demonstrated that cells cultured with 5 ng/mL platelet-derived growth factor BB had the highest viability at each time point as compared to other groups (P < 0.05). However, in presence of platelet-derived growth factor BB alone and in combination with transforming growth factor beta 1 and dentin proteins (10 ng/mL), significant higher viability was seen at all time points (P < 0.05). The least viability and proliferation at each growth factor concentration was seen in cells treated with combination of transforming growth factor beta 1 and dentin proteins at 72 h (P < 0.05). Conclusions The results indicated that the triple combination of growth factors and matrix-derived non-collagenous proteins (especially at 10 ng/mL concentration) has mitogenic effect on dental pulp stem cells. PMID:27099698

Platelet-rich plasma affects bacterial growth in vitro.

PubMed

Mariani, Erminia; Filardo, Giuseppe; Canella, Valentina; Berlingeri, Andrea; Bielli, Alessandra; Cattini, Luca; Landini, Maria Paola; Kon, Elizaveta; Marcacci, Maurilio; Facchini, Andrea

2014-09-01

Platelet-rich plasma (PRP), a blood derivative rich in platelets, is a relatively new technique used in tissue regeneration and engineering. The increased quantity of platelets makes this formulation of considerable value for their role in tissue healing and microbicidal activity. This activity was investigated against five of the most important strains involved in nosocomial infections (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Streptococcus faecalis) to understand the prophylactic role of pure (P)-PRP. Microbicidal proteins released from activated P-PRP platelets were also determined. The microbicidal activity of P-PRP and platelet-poor plasma (PPP) was evaluated on different concentrations of the five bacterial strains incubated for 1, 2, 4 and 18 h and plated on agar for 18-24 h. P-PRP and PPP-released microbicidal proteins were evaluated by means of multiplex bead-based immunoassays. P-PRP and PPP inhibited bacterial growth for up to 2 h of incubation. The effect of P-PRP was significantly higher than that of PPP, mainly at the low seeding concentrations and/or shorter incubation times, depending on the bacterial strain. Chemokine (C-C motif) ligand-3, chemokine (C-C motif) ligand-5 and chemokine (C-X-C motif) ligand-1 were the molecules mostly related to Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus faecalis inhibition. Escherichia coli and Klebsiella pneumoniae were less influenced. The present results show that P-PRP might supply an early protection against bacterial contaminations during surgical interventions because the inhibitory activity is already evident from the first hour of treatment, which suggests that physiological molecules supplied in loco might be important in the time frame needed for the activation of the innate immune response. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Diversity and impact of rare variants in genes encoding the platelet G protein-coupled receptors.

PubMed

Jones, Matthew L; Norman, Jane E; Morgan, Neil V; Mundell, Stuart J; Lordkipanidzé, Marie; Lowe, Gillian C; Daly, Martina E; Simpson, Michael A; Drake, Sian; Watson, Steve P; Mumford, Andrew D

2015-04-01

Platelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70 % had global minor allele frequency (MAF) < 0.05 %. Functional annotation using six computational algorithms, experimental evidence and structural data identified 156/740 (21 %) SNVs as potentially damaging to GPCR function, most commonly in regions encoding the transmembrane and C-terminal intracellular receptor domains. In 31 index cases with IPFDs (Gi-pathway defect n=15; secretion defect n=11; thromboxane pathway defect n=3 and complex defect n=2) there were 256 SNVs in the target regions of 15 stimulatory platelet GPCRs (34 unique; 12 with MAF< 1 % and 22 with MAF≥ 1 %). These included rare variants predicting R122H, P258T and V207A substitutions in the P2Y12 receptor that were annotated as potentially damaging, but only partially explained the platelet function defects in each case. Our data highlight that potentially damaging variants in platelet GPCR genes have low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes.

Platelets in liver disease, cancer and regeneration.

PubMed

Kurokawa, Tomohiro; Ohkohchi, Nobuhiro

2017-05-14

Although viral hepatitis treatments have evolved over the years, the resultant liver cirrhosis still does not completely heal. Platelets contain proteins required for hemostasis, as well as many growth factors required for organ development, tissue regeneration and repair. Thrombocytopenia, which is frequently observed in patients with chronic liver disease (CLD) and cirrhosis, can manifest from decreased thrombopoietin production and accelerated platelet destruction caused by hypersplenism; however, the relationship between thrombocytopenia and hepatic pathogenesis, as well as the role of platelets in CLD, is poorly understood. In this paper, experimental evidence of platelets improving liver fibrosis and accelerating liver regeneration is summarized and addressed based on studies conducted in our laboratory and current progress reports from other investigators. In addition, we describe our current perspective based on the results of these studies. Platelets improve liver fibrosis by inactivating hepatic stellate cells, which decreases collagen production. The regenerative effect of platelets in the liver involves a direct effect on hepatocytes, a cooperative effect with liver sinusoidal endothelial cells, and a collaborative effect with Kupffer cells. Based on these observations, we ascertained the direct effect of platelet transfusion on improving several indicators of liver function in patients with CLD and liver cirrhosis. However, unlike the results of our previous clinical study, the smaller incremental changes in liver function in patients with CLD who received eltrombopag for 6 mo were due to patient selection from a heterogeneous population. We highlight the current knowledge concerning the role of platelets in CLD and cancer and anticipate a novel application of platelet-based clinical therapies to treat liver disease.

Aspirin inhibits surface glycoprotein IIb/IIIa, P-selectin, CD63, and CD107a receptor expression on human platelets.

PubMed

McKenzie, Marcus E; Malinin, Alex I; Bell, Christopher R; Dzhanashvili, Alex; Horowitz, Eric D; Oshrine, Benjamin R; Atar, Dan; Serebruany, Victor L

2003-04-01

Platelet inhibition after aspirin therapy reduces the risk for the development of acute coronary syndromes. However, the mechanism by which aspirin affect platelets other than by prostaglandin blockade is unclear. We sought to determine the in vitro effects of aspirin on the surface expression of nine platelet receptors using whole blood flow cytometry. Blood from 24 healthy volunteers was incubated for 30 min with 1.8 and 7.2 mg/l phosphate-buffered saline-diluted acetylsalicylic acid in the presence or absence of apyrase. Platelet serotonin release, and the surface expression of platelet receptors with or without apyrase were determined using the following monoclonal antibodies: anit-CD41 [glycoprotein (GP)IIb/IIIa], CD42b (GPIb), CD62p (P-selectin), CD51/CD61 (vitronectin receptor), CD31 [platelet/endothelial cellular adhesion molecule-1 (PECAM-1)], CD107a [lysosomal associated membrane protein (LAMP)-1], CD107b (LAMP-2), CD63 (LIMP or LAMP-3), and CD151 (PETA-3). Samples were then immediately fixed with 2% paraformaldehyde, and run on the flow cytometer within 48 h. Aspirin does not affect serotonin release from human platelets. Dose-dependent inhibition of GPIIb/IIIa, P-selectin, CD63, and CD107a receptor expression was observed in the aspirin-treated whole-blood samples. Apyrase potentiates the effects of aspirin, and independently inhibits PECAM-1. In addition to the known effect of irreversibly inhibiting platelet cyclooxygenase-1, thereby blocking thromboxane A(2) synthesis, it appears that aspirin exhibits direct effects on selective major platelet receptors.

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.

Comparison of platelet function and viscoelastic test results between healthy dogs and dogs with naturally occurring chronic kidney disease.

PubMed

Dudley, Alicia; Byron, Julie K; Burkhard, Mary Jo; Warry, Emma; Guillaumin, Julien

2017-05-01

OBJECTIVE To compare platelet function and viscoelastic test results between healthy dogs and dogs with chronic kidney disease (CKD) to assess whether dogs with CKD have platelet dysfunction and altered blood coagulation. ANIMALS 10 healthy control dogs and 11 dogs with naturally occurring CKD. PROCEDURES Blood and urine were collected once from each dog for a CBC, serum biochemical analysis, urinalysis, and determination of the urine protein-to-creatinine ratio, prothrombin time, activated partial thromboplastin time, plasma fibrinogen concentration, and antithrombin activity. Closure time was determined by use of a platelet function analyzer and a collagen-ADP platelet agonist. Thromboelastography (TEG) variables (reaction time, clotting time, α angle, maximum amplitude, and global clot strength [G value]) were determined by use of recalcified nonactivated TEG. Platelet expression of glycoprotein Ib (GPIb; receptor for von Willebrand factor), integrin αIIbβ3 (αIIbβ3; receptor for fibrinogen), and P-selectin (marker for platelet activation) was assessed by flow cytometry. RESULTS Compared with healthy control dogs, the median closure time was prolonged, the median maximum amplitude and G value were increased, and the median clotting time was decreased for dogs with CKD. Platelet expression of both αIIbβ3 and P-selectin was also significantly increased for dogs with CKD, compared with that for control dogs. Platelet expression of GPIb, αIIbβ3, and P-selectin was not correlated with closure time or any TEG variable. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that dogs with CKD frequently had evidence of platelet dysfunction and hypercoagulability that were not totally attributable to alterations in platelet surface expression of GPIb, αIIbβ3, and P-selectin.

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

Developmental Stage-Specific Manifestations of Absent TPO/c-MPL Signalling in Newborn Mice.

PubMed

Lorenz, Viola; Ramsey, Haley; Liu, Zhi-Jian; Italiano, Joseph; Hoffmeister, Karin; Bihorel, Sihem; Mager, Donald; Hu, Zhongbo; Slayton, William B; Kile, Benjamin T; Sola-Visner, Martha; Ferrer-Marin, Francisca

2017-12-01

Congenital amegakaryocytic thrombocytopaenia (CAMT) is a disorder caused by c-MPL mutations that impair thrombopoietin (TPO) signalling, resulting in a near absence of megakaryocytes (MKs). While this phenotype is consistent in adults, neonates with CAMT can present with severe thrombocytopaenia despite normal MK numbers. To investigate this, we characterized MKs and platelets in newborn c-MPL –/– mice. Liver MKs in c-MPL –/– neonates were reduced in number and size compared with wild-type (WT) age-matched MKs, and exhibited ultrastructural abnormalities not found in adult c-MPL –/– MKs. Platelet counts were lower in c-MPL –/– compared with WT mice at birth and did not increase over the first 2 weeks of life. In vivo biotinylation revealed a significant reduction in the platelet half-life of c-MPL –/– newborn mice (P2) compared with age-matched WT pups, which was not associated with ultrastructural abnormalities. Genetic deletion of the pro-apoptotic Bak did not rescue the severely reduced platelet half-life of c-MPL –/– newborn mice, suggesting that it was due to factors other than platelets entering apoptosis early. Indeed, adult GFP+ (green fluorescent protein transgenic) platelets transfused into thrombocytopenic c-MPL –/– P2 pups also had a shortened lifespan, indicating the importance of cell-extrinsic factors. In addition, neonatal platelets from WT and c-MPL –/– mice exhibited reduced P-selectin surface expression following stimulation compared with adult platelets of either genotype, and platelets from c-MPL –/– neonates exhibited reduced glycoprotein IIb/IIIa (GPIIb/IIIa) activation in response to thrombin compared with age-matched WT platelets. Taken together, our findings indicate that c-MPL deficiency is associated with abnormal maturation of neonatal MKs and developmental stage-specific defects in platelet function.

Platelet gene therapy improves hemostatic function for integrin αIIbβ3-deficient dogs

PubMed Central

Fang, Juan; Jensen, Eric S.; Boudreaux, Mary K.; Du, Lily M.; Hawkins, Troy B.; Koukouritaki, Sevasti B.; Cornetta, Kenneth; Wilcox, David A.

2011-01-01

Activated blood platelets mediate the primary response to vascular injury. Although molecular abnormalities of platelet proteins occur infrequently, taken collectively, an inherited platelet defect accounts for a bleeding diathesis in ≈1:20,000 individuals. One rare example of a platelet disorder, Glanzmann thrombasthenia (GT), is characterized by life-long morbidity and mortality due to molecular abnormalities in a major platelet adhesion receptor, integrin αIIbβ3. Transfusion therapy is frequently inadequate because patients often generate antibodies to αIIbβ3, leading to immune-mediated destruction of healthy platelets. In the most severe cases allogeneic bone marrow transplantation has been used, yet because of the risk of the procedure it has been limited to few patients. Thus, hematopoietic stem cell gene transfer was explored as a strategy to improve platelet function within a canine model for GT. Bleeding complications necessitated the use of a mild pretransplant conditioning regimen; therefore, in vivo drug selection was used to improve engraftment of autologously transplanted cells. Approximately 5,000 αIIbβ3 receptors formed on 10% of platelets. These modest levels allowed platelets to adhere to αIIbβ3’s major ligand (fibrinogen), form aggregates, and mediate retraction of a fibrin clot. Remarkably, improved hemostatic function was evident, with ≤135-fold reduced blood loss, and improved buccal bleeding times decreased to 4 min for up to 5 y after transplant. One of four transplanted dogs developed a significant antibody response to αIIbβ3 that was attenuated effectively with transient immune suppression. These results indicate that gene therapy could become a practical approach for treating inherited platelet defects. PMID:21606353

C3G promotes a selective release of angiogenic factors from activated mouse platelets to regulate angiogenesis and tumor metastasis.

PubMed

Martín-Granado, Víctor; Ortiz-Rivero, Sara; Carmona, Rita; Gutiérrez-Herrero, Sara; Barrera, Mario; San-Segundo, Laura; Sequera, Celia; Perdiguero, Pedro; Lozano, Francisco; Martín-Herrero, Francisco; González-Porras, José Ramón; Muñoz-Chápuli, Ramón; Porras, Almudena; Guerrero, Carmen

2017-12-19

Previous observations indicated that C3G (RAPGEF1) promotes α-granule release, evidenced by the increase in P-selectin exposure on the platelet surface following its activation. The goal of the present study is to further characterize the potential function of C3G as a modulator of the platelet releasate and its implication in the regulation of angiogenesis. Proteomic analysis revealed a decreased secretion of anti-angiogenic factors from activated transgenic C3G and C3G∆Cat platelets. Accordingly, the secretome from both transgenic platelets had an overall pro-angiogenic effect as evidenced by an in vitro capillary-tube formation assay with HUVECs (human umbilical vein endothelial cells) and by two in vivo models of heterotopic tumor growth. In addition, transgenic C3G expression in platelets greatly increased mouse melanoma cells metastasis. Moreover, immunofluorescence microscopy showed that the pro-angiogenic factors VEGF and bFGF were partially retained into α-granules in thrombin- and ADP-activated mouse platelets from both, C3G and C3GΔCat transgenic mice. The observed interaction between C3G and Vesicle-associated membrane protein (Vamp)-7 could explain these results. Concomitantly, increased platelet spreading in both transgenic platelets upon thrombin activation supports this novel function of C3G in α-granule exocytosis. Collectively, our data point out to the co-existence of Rap1GEF-dependent and independent mechanisms mediating C3G effects on platelet secretion, which regulates pathological angiogenesis in tumors and other contexts. The results herein support an important role for platelet C3G in angiogenesis and metastasis.

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

Svensson Holm, Ann-Charlotte B., E-mail: ann-charlotte.svensson@liu.se; Experimental Pathology, Department of Clinical and Experimental Medicine, Linkoeping University, SE-581 85 Linkoeping; Bengtsson, Torbjoern

Hyaluronic acid (HA) is one of the main components of the extracellular matrix (ECM) and is expressed throughout the body including the lung and mostly in areas surrounding proliferating and migrating cells. Furthermore, platelets have been implicated as important players in the airway remodelling process, e.g. due to their ability to induce airway smooth muscle cell (ASMC) proliferation. The aim of the present study was to investigate the role of HA, the HA-binding surface receptor CD44 and focal adhesion kinase (FAK) in platelet-induced ASMC proliferation. Proliferation of ASMC was measured using the MTS-assay, and we found that the CD44 blockingmore » antibody and the HA synthase inhibitor 4-Methylumbelliferone (4-MU) significantly inhibited platelet-induced ASMC proliferation. The interaction between ASMC and platelets was studied by fluorescent staining of F-actin. In addition, the ability of ASMC to synthesise HA was investigated by fluorescent staining using biotinylated HA-binding protein and a streptavidin conjugate. We observed that ASMC produced HA and that a CD44 blocking antibody and 4-MU significantly inhibited platelet binding to the area surrounding the ASMC. Furthermore, the FAK-inhibitor PF 573228 inhibited platelet-induced ASMC proliferation. Co-culture of ASMC and platelets also resulted in increased phosphorylation of FAK as detected by Western blot analysis. In addition, 4-MU significantly inhibited the increased FAK-phosphorylation. In conclusion, our findings demonstrate that ECM has the ability to influence platelet-induced ASMC proliferation. Specifically, we propose that HA produced by ASMC is recognised by platelet CD44. The platelet/HA interaction is followed by FAK activation and increased proliferation of co-cultured ASMC. We also suggest that the mitogenic effect of platelets represents a potential important and novel mechanism that may contribute to airway remodelling.« less

Platelet gene therapy improves hemostatic function for integrin alphaIIbbeta3-deficient dogs.

PubMed

Fang, Juan; Jensen, Eric S; Boudreaux, Mary K; Du, Lily M; Hawkins, Troy B; Koukouritaki, Sevasti B; Cornetta, Kenneth; Wilcox, David A

2011-06-07

Activated blood platelets mediate the primary response to vascular injury. Although molecular abnormalities of platelet proteins occur infrequently, taken collectively, an inherited platelet defect accounts for a bleeding diathesis in ≈1:20,000 individuals. One rare example of a platelet disorder, Glanzmann thrombasthenia (GT), is characterized by life-long morbidity and mortality due to molecular abnormalities in a major platelet adhesion receptor, integrin αIIbβ3. Transfusion therapy is frequently inadequate because patients often generate antibodies to αIIbβ3, leading to immune-mediated destruction of healthy platelets. In the most severe cases allogeneic bone marrow transplantation has been used, yet because of the risk of the procedure it has been limited to few patients. Thus, hematopoietic stem cell gene transfer was explored as a strategy to improve platelet function within a canine model for GT. Bleeding complications necessitated the use of a mild pretransplant conditioning regimen; therefore, in vivo drug selection was used to improve engraftment of autologously transplanted cells. Approximately 5,000 αIIbβ3 receptors formed on 10% of platelets. These modest levels allowed platelets to adhere to αIIbβ3's major ligand (fibrinogen), form aggregates, and mediate retraction of a fibrin clot. Remarkably, improved hemostatic function was evident, with ≤135-fold reduced blood loss, and improved buccal bleeding times decreased to 4 min for up to 5 y after transplant. One of four transplanted dogs developed a significant antibody response to αIIbβ3 that was attenuated effectively with transient immune suppression. These results indicate that gene therapy could become a practical approach for treating inherited platelet defects.

Leukocyte Count is Associated with Increased Platelet Reactivity and Diminished Response to Aspirin in Healthy Individuals with a Family History of Coronary Artery Disease

PubMed Central

Faraday, Nauder; Yanek, Lisa R.; Vaidya, Dhananjay; Kral, Brian; Qayyum, Rehan; Herrera-Galeano, J. Enrique; Moy, Taryn F.; Becker, Diane M.; Becker, Lewis C.

2009-01-01

Background Markers of systemic inflammation, including blood leukocyte count, are associated with increased cardiovascular risk, but the mechanisms underlying this association are unclear. Leukocytes may promote platelet reactivity and thrombus formation, providing a basis for increased risk, but a relation between leukocyte count and platelet function has not been studied. Methods We evaluated the relation of blood leukocyte count, C-reactive protein (CRP), and interleukin-6 (IL-6) to platelet aggregation to collagen, ADP and arachidonic acid, and to urinary excretion of 11-dehydro thromboxane B2. Studies were conducted in 1600 individuals (45.0 ± 12.9 years, 42.7% male) at risk for coronary artery disease (CAD) before and after low dose aspirin. Results At baseline, platelet reactivity increased with increasing quartile of leukocyte count (median counts for each quartile were normal) for all measures of platelet function (P<0.0001). These relations were unchanged by aspirin. The relation between leukocyte count and each measure of platelet reactivity remained significant (P<0.05) after multivariable adjustment for CRP, IL-6, cardiac risk factors, hematologic variables, and platelet thromboxane production. CRP and IL-6 were independently associated with few measures of platelet reactivity. Conclusions Increasing quartile of leukocyte count, even within the normal range, is associated with increasing platelet reactivity in individuals at risk for CAD. This relationship is not altered by aspirin and is independent of inflammatory markers and platelet thromboxane production. Additional studies are needed to determine the mechanism(s) for this association and therapies to reduce cardiovascular risk in patients with elevated leukocyte counts. PMID:19185906

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.

Insights into abnormal hemostasis in the Quebec platelet disorder from analyses of clot lysis.

PubMed

Diamandis, M; Adam, F; Kahr, W H A; Wang, P; Chorneyko, K A; Arsenault, A L; Rivard, G E; Hayward, C P M

2006-05-01

The Quebec platelet disorder (QPD) is inherited and characterized by delayed-onset bleeding following hemostatic challenge. Other characteristics include increased expression and storage of active urokinase-type plasminogen activator (u-PA) in platelets in the setting of normal to increased u-PA in plasma. There is also consumption of platelet plasminogen activator inhibitor-1 and increased generation of plasmin in platelets accompanied by proteolysis of stored alpha-granule proteins, including Factor V. Although fibrinolysis has been proposed to contribute to QPD bleeding, the effects of QPD blood and platelets on clot lysis have not been evaluated. We used thromboelastography (TEG), biochemical evaluations of whole blood clot lysis, assessments of clot ultrastructure, and perfusion of blood over preformed fibrin to gain insights into the disturbed hemostasis in the QPD. Thromboelastography was not sensitive to the increased u-PA in QPD blood. However, there was abnormal plasmin generation in QPD whole blood clots, generated at low shear, with biochemical evidence of increased fibrinolysis. The incorporation of QPD platelets into a forming clot led to progressive disruption of fibrin and platelet aggregates unless drugs were added to inhibit plasmin. In whole blood perfusion studies, QPD platelets showed normal adherence to fibrin, but their adhesion was followed by accelerated fibrinolysis. The QPD is associated with "gain-of-function" abnormalities that increase the lysis of forming or preformed clots. These findings suggest accelerated fibrinolysis is an important contributor to QPD bleeding.

Activated platelets can promote tumor cell invasion into healthy tissue | Center for Cancer Research

Cancer.gov

Pre-clinical studies conducted by CCR investigators and colleagues show that platelets, tiny cells that promote blood clotting, when activated by the CD97 protein on the surface of tumor cells, enable the tumor cells to invade healthy tissue and then metastasize. The study, published April 17, 2018, in Cell Reports, was led by Kathleen Kelly, Ph.D., Chief, Laboratory of

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.

Sphingosine 1-phosphate (S1P) suppresses the collagen-induced activation of human platelets via S1P4 receptor.

PubMed

Onuma, Takashi; Tanabe, Kumiko; Kito, Yuko; Tsujimoto, Masanori; Uematsu, Kodai; Enomoto, Yukiko; Matsushima-Nishiwaki, Rie; Doi, Tomoaki; Nagase, Kiyoshi; Akamatsu, Shigeru; Tokuda, Haruhiko; Ogura, Shinji; Iwama, Toru; Kozawa, Osamu; Iida, Hiroki

2017-08-01

Sphingosine 1-phosphate (S1P) is as an extracellular factor that acts as a potent lipid mediator by binding to specific receptors, S1P receptors (S1PRs). However, the precise role of S1P in human platelets that express S1PRs has not yet been fully clarified. We previously reported that heat shock protein 27 (HSP27) is released from human platelets accompanied by its phosphorylation stimulated by collagen. In the present study, we investigated the effect of S1P on the collagen-induced platelet activation. S1P pretreatment markedly attenuated the collagen-induced aggregation. Co-stimulation with S1P and collagen suppressed collagen-induced platelet activation, but the effect was weaker than that of S1P-pretreatment. The collagen-stimulated secretion of platelet-derived growth factor (PDGF)-AB and the soluble CD40 ligand (sCD40L) release were significantly reduced by S1P. In addition, S1P suppressed the collagen-induced release of HSP27 as well as the phosphorylation of HSP27. S1P significantly suppressed the collagen-induced phosphorylation of p38 mitogen-activated protein kinase. S1P increased the levels of GTP-bound Gαi and GTP-bound Gα13 coupled to S1PPR1 and/or S1PR4. CYM50260, a selective S1PR4 agonist, but not SEW2871, a selective S1PR1 agonist, suppressed the collagen-stimulated platelet aggregation, PDGF-AB secretion and sCD40L release. In addition, CYM50260 reduced the release of phosphorylated-HSP27 by collagen as well as the phosphorylation of HSP27. The selective S1PR4 antagonist CYM50358, which failed to affect collagen-induced HSP27 phosphorylation, reversed the S1P-induced attenuation of HSP27 phosphorylation by collagen. These results strongly suggest that S1P inhibits the collagen-induced human platelet activation through S1PR4 but not S1PR1. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

Platelet amyloid precursor protein isoform expression in Alzheimer's disease: evidence for peripheral marker.

PubMed

Vignini, A; Sartini, D; Morganti, S; Nanetti, L; Luzzi, S; Provinciali, L; Mazzanti, L; Emanuelli, M

2011-01-01

Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized by a progressive cognitive and memory decline. Among peripheral markers of AD, great interest has been focused on the amyloid precursor protein (APP). In this regard, platelets represent an important peripheral source of APP since it has been demonstrated that the three major isoforms, that are constituted of 770, 751 and 695 aa residues, are inserted in the membrane of resting platelets. APP 751 and APP 770 contain a Kunitz-type serine protease inhibitor domain (APP KPI) and APP 695 lacks this domain. To address this issue, we first examined the platelet APP isoform mRNAs prospectively as biomarker for the diagnosis of AD by means of real-time quantitative PCR, and then evaluated the correlation between APP mRNA expression levels and cognitive impairment of enrolled subjects. Differential gene expression measurements in the AD patient group (n=18) revealed a significant up-regulation of APP TOT (1.52-fold), APP KPI (1.32-fold), APP 770 (1.33-fold) and APP 751 (1.26-fold) compared to controls (n=22). Moreover, a statistically significant positive correlation was found between APP mRNA levels (TOT, KPI, 770 and 751) and cognitive impairment. Since AD definitive diagnosis still relies on pathological evaluation at autopsy, the present results are consistent with the hypothesis that platelet APP could be considered a potential reliable peripheral marker for studying AD and could contribute to define a signature for the presence of AD pathology.

An Investigation of Hierachical Protein Recruitment to the Inhibitory Platelet Receptor, G6B-b

PubMed Central

Coxon, Carmen H.; Sadler, Amanda J.; Huo, Jiandong; Campbell, R. Duncan

2012-01-01

Platelet activation is regulated by both positive and negative signals. G6B-b is an inhibitory platelet receptor with an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). The molecular basis of inhibition by G6B-b is currently unknown but thought to involve the SH2 domain-containing tyrosine phosphatase SHP-1. Here we show that G6B-b also associates with SHP-2, as well as SHP-1, in human platelets. Using a number of biochemical approaches, we found these interactions to be direct and that the tandem SH2 domains of SHP-2 demonstrated a binding affinity for G6B-b 100-fold higher than that of SHP-1. It was also observed that while SHP-1 has an absolute requirement for phosphorylation at both motifs to bind, SHP-2 can associate with G6B-b when only one motif is phosphorylated, with the N-terminal SH2 domain and the ITIM being most important for the interaction. A number of other previously unreported SH2 domain-containing proteins, including Syk and PLCγ2, also demonstrated specificity for G6B-b phosphomotifs and may serve to explain the observation that G6B-b remains inhibitory in the absence of both SHP-1 and SHP-2. In addition, the presence of dual phosphorylated G6B-b in washed human platelets can reduce the EC50 for both CRP and collagen. PMID:23185356

Sulforaphane prevents human platelet aggregation through inhibiting the phosphatidylinositol 3-kinase/Akt pathway.

PubMed

Chuang, Wen-Ying; Kung, Po-Hsiung; Kuo, Chih-Yun; Wu, Chin-Chung

2013-06-01

Sulforaphane, a dietary isothiocyanate found in cruciferous vegetables, has been shown to exert beneficial effects in animal models of cardiovascular diseases. However, its effect on platelet aggregation, which is a critical factor in arterial thrombosis, is still unclear. In the present study, we show that sulforaphane inhibited human platelet aggregation caused by different receptor agonists, including collagen, U46619 (a thromboxane A2 mimic), protease-activated receptor 1 agonist peptide (PAR1-AP), and an ADP P2Y12 receptor agonist. Moreover, sulforaphane significantly reduced thrombus formation on a collagen-coated surface under whole blood flow conditions. In exploring the underlying mechanism, we found that sulforaphane specifically prevented phosphatidylinositol 3-kinase (PI3K)/Akt signalling, without markedly affecting other signlaling pathways involved in platelet aggregation, such as protein kinase C activation, calcium mobilisation, and protein tyrosine phosphorylation. Although sulforaphane did not directly inhibit the catalytic activity of PI3K, it caused ubiquitination of the regulatory p85 subunit of PI3K, and prevented PI3K translocation to membranes. In addition, sulforaphane caused ubiquitination and degradation of phosphoinositide-dependent kinase 1 (PDK1), which is required for Akt activation. Therefore, sulforaphane is able to inhibit the PI3K/Akt pathway at two distinct sites. In conclusion, we have demonstrated that sulforaphane prevented platelet aggregation and reduced thrombus formation in flow conditions; our data also support that the inhibition of the PI3K/Akt pathway by sulforaphane contributes it antiplatelet effects.

Fetal hemorrhage and platelet dysfunction in SLP-76–deficient mice

PubMed Central

Clements, James L.; Lee, Jong Ran; Gross, Barbara; Yang, Baoli; Olson, John D.; Sandra, Alexander; Watson, Stephen P.; Lentz, Steven R.; Koretzky, Gary A.

1999-01-01

The adapter protein SLP-76 is expressed in T lymphocytes and hematopoietic cells of the myeloid lineage, and is known to be a substrate of the protein tyrosine kinases that are activated after ligation of the T-cell antigen receptor. Transient overexpression of SLP-76 in a T-cell line potentiates transcriptional activation after T-cell receptor ligation, while loss of SLP-76 expression abrogates several T-cell receptor–dependent signaling pathways. Mutant mice that lack SLP-76 manifest a severe block at an early stage of thymocyte development, implicating SLP-76 in signaling events that promote thymocyte maturation. While it is clear that SLP-76 plays a key role in development and activation of T lymphocytes, relatively little is understood regarding its role in transducing signals initiated after receptor ligation in other hematopoietic cell types. In this report, we describe fetal hemorrhage and perinatal mortality in SLP-76–deficient mice. Although megakaryocyte and platelet development proceeds normally in the absence of SLP-76, collagen-induced platelet aggregation and granule release is markedly impaired. Furthermore, treatment of SLP-76–deficient platelets with collagen fails to elicit tyrosine phosphorylation of phospholipase C-γ2 (PLC-γ2), suggesting that SLP-76 functions upstream of PLC-γ2 activation. These data provide one potential mechanism for the fetal hemorrhage observed in SLP-76–deficient mice and reveal that SLP-76 expression is required for optimal receptor-mediated signal transduction in platelets as well as T lymphocytes. PMID:9884330

Hippophae rhamnoides L. Fruits Reduce the Oxidative Stress in Human Blood Platelets and Plasma

PubMed Central

Olas, Beata; Kontek, Bogdan; Malinowska, Paulina; Żuchowski, Jerzy; Stochmal, Anna

2016-01-01

Effects of the phenolic fraction from Hippophae rhamnoides fruits on the production of thiobarbituric acid reactive substances (TBARS, a marker of lipid peroxidation) and the generation of superoxide anion (O2 −∙) in human blood platelets (resting platelets and platelets stimulated by a strong physiological agonist, thrombin) were studied in vitro. We also examined antioxidant properties of this fraction against human plasma lipid peroxidation and protein carbonylation induced by a strong biological oxidant, hydrogen peroxide (H2O2) or H2O2/Fe (a donor of hydroxyl radicals). The tested fraction of H. rhamnoides (0.5– 50 µg/mL; the incubation time: 15 and 60 min) inhibited lipid peroxidation induced by H2O2 or H2O2/Fe. The H. rhamnoides phenolic fraction inhibited not only plasma lipid peroxidation, but also plasma protein carbonylation stimulated by H2O2 or H2O2/Fe. Moreover, the level of O2 −∙ in platelets significantly decreased. In comparative experiments, the H. rhamnoides fraction was a more effective antioxidant than aronia extract or grape seed extract (at the highest tested concentration, 50 µg/mL). The obtained results suggest that H. rhamnoides fruits may be a new, promising source of natural compounds with antioxidant and antiplatelet activity beneficial not only for healthy people, but also for those with oxidative stress-associated diseases. PMID:26933473

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.

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

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.

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 production proceeds independently of the intrinsic and extrinsic apoptosis pathways.

PubMed

Josefsson, Emma C; Burnett, Deborah L; Lebois, Marion; Debrincat, Marlyse A; White, Michael J; Henley, Katya J; Lane, Rachael M; Moujalled, Diane; Preston, Simon P; O'Reilly, Lorraine A; Pellegrini, Marc; Metcalf, Donald; Strasser, Andreas; Kile, Benjamin T

2014-03-17

BH3 mimetic drugs that target BCL-2 family pro-survival proteins to induce tumour cell apoptosis represent a new era in cancer therapy. Clinical trials of navitoclax (ABT-263, which targets BCL-2, BCL-XL and BCL-W) have shown great promise, but encountered dose-limiting thrombocytopenia. Recent work has demonstrated that this is due to the inhibition of BCL-XL, which is essential for platelet survival. These findings raise new questions about the established model of platelet shedding by megakaryocytes, which is thought to be an apoptotic process. Here we generate mice with megakaryocyte-specific deletions of the essential mediators of extrinsic (Caspase-8) and intrinsic (BAK/BAX) apoptosis. We show that megakaryocytes possess a Fas ligand-inducible extrinsic apoptosis pathway. However, Fas activation does not stimulate platelet production, rather, it triggers Caspase-8-mediated killing. Combined loss of Caspase-8/BAK/BAX does not impair thrombopoiesis, but can protect megakaryocytes from death in mice infected with lymphocytic choriomeningitis virus. Thus, apoptosis is dispensable for platelet biogenesis.

Quebec platelet disorder: update on pathogenesis, diagnosis, and treatment.

PubMed

Blavignac, Jessica; Bunimov, Natalia; Rivard, Georges E; Hayward, Catherine P M

2011-09-01

Quebec platelet disorder (QPD) is an autosomal dominant bleeding disorder associated with reduced platelet counts and a unique gain-of-function defect in fibrinolysis due to increased expression and storage of urokinase plasminogen activator (uPA) by megakaryocytes. QPD increases risks for bleeding and its key clinical feature is delayed-onset bleeding, following surgery, dental procedures or trauma, which responds only to treatment with fibrinolytic inhibitors. The genetic cause of the disorder is a tandem duplication mutation of the uPA gene, PLAU, which upregulates uPA expression in megakaryocytes by an unknown mechanism. The increased platelet stores of uPA trigger plasmin-mediated degradation of QPD α-granule proteins. The gain-of-function defect in fibrinolysis is thought to be central to the pathogenesis of QPD bleeding as the activation of QPD platelets leads to release of uPA from α-granules and accelerated clot lysis. The purpose of this review is to summarize current knowledge on QPD pathogenesis and the recommended approaches to QPD diagnosis and treatment. Thieme Medical Publishers.

Two patients with Hermansky Pudlak syndrome type 2 and novel mutations in AP3B1

PubMed Central

Wenham, Matt; Grieve, Samantha; Cummins, Michelle; Jones, Matthew L.; Booth, Sarah; Kilner, Rachel; Ancliff, Philip J.; Griffiths, Gillian M.; Mumford, Andrew D.

2010-01-01

Hermansky Pudlak syndrome type 2 (HPS2) is a rare disorder associated with mutations in the Adaptor Protein 3 (AP-3) complex, which is involved in sorting transmembrane proteins to lysosomes and related organelles. We now report 2 unrelated subjects with HPS2 who show a characteristic clinical phenotype of oculocutaneous albinism, platelet and T-lymphocyte dysfunction and neutropenia. The subjects were homozygous for different deletions within AP3B1 (g.del180242-180866, c.del153-156), which encodes the AP-3β3A subunit, resulting in frame shifts and introduction of nonsense substitutions (p.E693fsX13, p.E52fsX11). In the subject with p.E693fsX13, this resulted in expression of a truncated variant β3A protein. Cytotoxic T-lymphocyte (CTL) clones from both study subjects showed increased cell-surface expression of CD63 and reduced cytotoxicity. Platelets showed impaired aggregation and reduced uptake of 3H-serotonin. These findings are consistent with CTL granule and platelet dense granule defects, respectively. This report extends the clinical and laboratory description of HPS2. PMID:19679886

A Plasma Protein Indistinguishable from Ribosomal Protein S19

PubMed Central

Semba, Umeko; Chen, Jun; Ota, Yoshihiko; Jia, Nan; Arima, Hidetoshi; Nishiura, Hiroshi; Yamamoto, Tetsuro

2010-01-01

A monocyte-chemoattracting factor is generated during blood coagulation and during clotting of platelet-rich plasma. This chemotactic factor attracts monocytes as a ligand of the C5a receptor; however, it inhibits C5a-induced neutrophil chemotaxis as an apparent receptor antagonist. The curious dual function of the serum monocyte chemotactic factor resembles that of the cross-linked homodimer of ribosomal protein S19 (RP S19). Indeed, the inactive precursor of the monocyte chemotactic factor was present in plasma, and the precursor molecule and RP S19, as well as the active form and the RP S19 dimer, were indistinguishable in terms of immunological reactivity and molecular size. Coagulation factor XIIIa, plasma transglutaminase, and membrane phosphatidylserine on the activated platelets were required for conversion of the precursor to the active form. In addition, the precursor molecule in plasma could be replaced by wild-type recombinant RP S19 but not by mutant forms of it. These results indicate that a molecule indistinguishable from RP S19 was present in plasma, and that the RP S19-like molecule was converted to the active form by a transglutaminase-catalyzed reaction on a scaffold that included the phosphatidylserine-exposed platelet membrane. PMID:20093496

The platelet P2Y(12) receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [(3)H]PSB-0413.

PubMed

Ohlmann, Philippe; Lecchi, Anna; El-Tayeb, Ali; Müller, Christa E; Cattaneo, Marco; Gachet, Christian

2013-03-01

Various radioligands have been used to characterize and quantify the platelet P2Y(12) receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y(1) and P2Y(12). We used the [(3)H]PSB-0413 selective P2Y(12) receptor antagonist radioligand to reevaluate the number of P2Y(12) receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [(3)H]PSB-0413 bound to 425 ± 50 sites/platelet (K (D) = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [(3)H]PSB-0413 bound to 1 mg protein of platelet membranes (K (D) = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y(12), with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y(1) ligand MRS2179 and the P2X(1) ligand α,β-Met-ATP did not displace [(3)H]PSB-0413 binding. Patients with severe P2Y(12) deficiency displayed virtually no binding of [(3)H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y(12) receptor had normal binding. Studies in mice showed that: (1) [(3)H]PSB-0413 bound to 634 ± 87 sites/platelet (K (D) = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [(3)H]PSB-0413 bound to 1 mg protein of platelet membranes (K (D) = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [(3)H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y(12) receptors, to identify patients with P2Y(12) deficiencies or quantify the effect of P2Y(12) targeting drugs.

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.

Glucose impairs aspirin inhibition in platelets through a NAD(P)H oxidase signaling pathway.

PubMed

Kobzar, Gennadi; Mardla, Vilja; Samel, Nigulas

2017-07-01

Hyperglycemia has been suggested to play a role in the increased platelet resistance to antiplatelet therapy in patients with diabetes mellitus. Exposure to high glucose impairs platelet inhibition by aspirin. It has been found that antioxidant agents reduce the effect of glucose, confirming the involvement of reactive oxygen species (ROS) in the effect of glucose. The aim of the study was to examine the mechanism of ROS increase by high glucose in aspirin-treated platelets. Platelet aggregation was measured by the optical method, and the production of ROS was detected using luminol-dependent horseradish peroxidase-enhanced chemiluminescence. We found that glucose did not affect ADP-induced platelet aggregation. However, it reduced the effect of aspirin on platelet aggregation, which was accompanied by an increase in ROS generation. The inhibition of NAD(P)H oxidase (NOX) prevented the glucose effect and ROS generation. The same result was recorded after the inhibition of p38 mitogen-activated protein kinases (p38 MAPK), phospholipase A 2 (PLA 2 ) or 12-lipoxygenase (12-LOX). The inhibition of TxA 2 receptor did not decrease the effect of glucose indicating that the effect was not caused by activation of TxA 2 receptors. Copyright © 2017 Elsevier Inc. All rights reserved.

Synthesis and preliminary evaluation of [3H]PSB-0413, a selective antagonist radioligand for platelet P2Y12 receptors.

PubMed

El-Tayeb, Ali; Griessmeier, Kerstin J; Müller, Christa E

2005-12-15

The selective antagonist radioligand [(3)H]2-propylthioadenosine-5'-adenylic acid (1,1-dichloro-1-phosphonomethyl-1-phosphonyl) anhydride ([(3)H]PSB-0413) was prepared by catalytic hydrogenation of its propargyl precursor with a high specific radioactivity of 74Ci/mmol. In preliminary saturation binding studies, [(3)H]PSB-0413 showed high affinity for platelet P2Y(12) receptors with a K(D) value of 4.57nM. Human platelets had a high density of P2Y(12) receptors exhibiting a B(max) value of 7.66pmol/mg of protein.

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

Did dinosaurs have megakaryocytes? New ideas about platelets and their progenitors.

PubMed

Brass, Lawrence F

2005-12-01

Biological evolution has struggled to produce mechanisms that can limit blood loss following injury. In humans and other mammals, control of blood loss (hemostasis) is achieved through a combination of plasma proteins, most of which are made in the liver, and platelets, anucleate blood cells that are produced in the bone marrow by megakaryocytes. Much has been learned about the underlying mechanisms, but much remains to be determined. The articles in this series review current ideas about the production of megakaryocytes from undifferentiated hematopoietic precursors, the steps by which megakaryocytes produce platelets, and the molecular mechanisms within platelets that make hemostasis possible. The underlying theme that connects the articles is the intense investigation of a complex system that keeps humans from bleeding to death, but at the same time exposes us to increased risk of thrombosis and vascular disease.

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

Calcium diacylglycerol guanine nucleotide exchange factor I (CalDAG-GEFI) gene mutations in a thrombopathic Simmental calf.

PubMed

Boudreaux, M K; Schmutz, S M; French, P S

2007-11-01

Simmental thrombopathia is an inherited platelet disorder that closely resembles the platelet disorders described in Basset Hounds and Eskimo Spitz dogs. Recently, two different mutations in the gene encoding calcium diacylglycerol guanine nucleotide exchange factor I (CalDAG-GEFI) were described to be associated with the Basset Hound and Spitz thrombopathia disorders, and a third distinct mutation was identified in CalDAG-GEFI in thrombopathic Landseers of European Continental Type. The gene encoding CalDAG-GEFI was sequenced using DNA obtained from normal cattle and from a thrombopathic calf studied in Canada. The affected calf was found to have a nucleotide change (c.701 T>C), which would result in the substitution of a proline for a leucine within structurally conserved region two (SCR2) of the catalytic domain of the protein. This change is likely responsible for the thrombopathic phenotype observed in Simmental cattle and underscores the critical nature of this signal transduction protein in platelets.

Activated platelets can promote tumor cell invasion into healthy tissue | Center for Cancer Research

Cancer.gov

Pre-clinical studies conducted by CCR investigators and colleagues show that platelets, tiny cells that promote blood clotting, when activated by the CD97 protein on the surface of tumor cells, enable the tumor cells to invade healthy tissue and then metastasize. The study, published April 17, 2018, in Cell Reports, was led by Kathleen Kelly, Ph.D., Chief, Laboratory of Genitourinary Cancer Pathogenesis.

Macromolecular Colloids of Diblock Poly(amino acids) That Bind Insulin.

PubMed

Constancis; Meyrueix; Bryson; Huille; Grosselin; Gulik-Krzywicki; Soula

1999-09-15

The diblock polymer poly(l-leucine-block-l-glutamate), bLE, was synthesized by acid hydrolysis of the ester poly(l-leucine-block-l-methyl glutamate). During the hydrolysis reaction the leucine block precipitates from the reaction mixture, forming nanosized particulate structures. These particles can be purified and further suspended in water or in 0.15 M phosphate saline buffer (PBS) to give stable, colloidal dispersions. TEM analysis shows the predominant particle form to be that of platelets with a diameter of 200 nm. Smaller cylindrical or spherical particles form a relatively minor fraction of the sample. After fractionation, analysis shows the platelets to be compositionally rich in leucine, while the spheres are glutamate-rich. (1)H NMR, CD, and X-ray diffraction indicate that the core of the platelets is composed of crystalline, helical leucine segments. The poly(l-glutamate) polyelectrolyte brush extending out from the two faces of the disk stabilizes individual particles from flocculation. At pH 7.4, the nanoparticles (platelets and cylinders) spontaneously adsorb proteins, such as insulin, directly from solution. Partial desorption of the protein in its native configuration can be induced by simple dilution. The reversibility of the insulin-nanoparticle complex is the basis for a potential new delivery system. Copyright 1999 Academic Press.

Zwitterionic sulfobetaine-grafted poly(vinylidene fluoride) membrane with highly effective blood compatibility via atmospheric plasma-induced surface copolymerization.

PubMed

Chang, Yung; Chang, Wan-Ju; Shih, Yu-Ju; Wei, Ta-Chin; Hsiue, Ging-Ho

2011-04-01

Development of nonfouling membranes to prevent nonspecific protein adsorption and platelet adhesion is critical for many biomedical applications. It is always a challenge to control the surface graft copolymerization of a highly polar monomer from the highly hydrophobic surface of a fluoropolymer membrane. In this work, the blood compatibility of poly(vinylidene fluoride) (PVDF) membranes with surface-grafted electrically neutral zwitterionic poly(sulfobetaine methacrylate) (PSBMA), from atmospheric plasma-induced surface copolymerization, was studied. The effect of surface composition and graft morphology, electrical neutrality, hydrophilicity and hydration capability on blood compatibility of the membranes were determined. Blood compatibility of the zwitterionic PVDF membranes was systematically evaluated by plasma protein adsorption, platelet adhesion, plasma-clotting time, and blood cell hemolysis. It was found that the nonfouling nature and hydration capability of grafted PSBMA polymers can be effectively controlled by regulating the grafting coverage and charge balance of the PSBMA layer on the PVDF membrane surface. Even a slight charge bias in the grafted zwitterionic PSBMA layer can induce electrostatic interactions between proteins and the membrane surfaces, leading to surface protein adsorption, platelet activation, plasma clotting and blood cell hemolysis. Thus, the optimized PSBMA surface graft layer in overall charge neutrality has a high hydration capability and the best antifouling, anticoagulant, and antihemolytic activities when comes into contact with human blood. © 2011 American Chemical Society

Hyaluronic acid influence on platelet-induced airway smooth muscle cell proliferation.

PubMed

Svensson Holm, Ann-Charlotte B; Bengtsson, Torbjörn; Grenegård, Magnus; Lindström, Eva G

2012-03-10

Hyaluronic acid (HA) is one of the main components of the extracellular matrix (ECM) and is expressed throughout the body including the lung and mostly in areas surrounding proliferating and migrating cells. Furthermore, platelets have been implicated as important players in the airway remodelling process, e.g. due to their ability to induce airway smooth muscle cell (ASMC) proliferation. The aim of the present study was to investigate the role of HA, the HA-binding surface receptor CD44 and focal adhesion kinase (FAK) in platelet-induced ASMC proliferation. Proliferation of ASMC was measured using the MTS-assay, and we found that the CD44 blocking antibody and the HA synthase inhibitor 4-Methylumbelliferone (4-MU) significantly inhibited platelet-induced ASMC proliferation. The interaction between ASMC and platelets was studied by fluorescent staining of F-actin. In addition, the ability of ASMC to synthesise HA was investigated by fluorescent staining using biotinylated HA-binding protein and a streptavidin conjugate. We observed that ASMC produced HA and that a CD44 blocking antibody and 4-MU significantly inhibited platelet binding to the area surrounding the ASMC. Furthermore, the FAK-inhibitor PF 573228 inhibited platelet-induced ASMC proliferation. Co-culture of ASMC and platelets also resulted in increased phosphorylation of FAK as detected by Western blot analysis. In addition, 4-MU significantly inhibited the increased FAK-phosphorylation. In conclusion, our findings demonstrate that ECM has the ability to influence platelet-induced ASMC proliferation. Specifically, we propose that HA produced by ASMC is recognised by platelet CD44. The platelet/HA interaction is followed by FAK activation and increased proliferation of co-cultured ASMC. We also suggest that the mitogenic effect of platelets represents a potential important and novel mechanism that may contribute to airway remodelling. Copyright © 2011 Elsevier Inc. All rights reserved.

Inhibitory Effect of Flavonolignans on the P2Y12 Pathway in Blood Platelets.

PubMed

Bijak, Michal; Szelenberger, Rafal; Dziedzic, Angela; Saluk-Bijak, Joanna

2018-02-10

Adenosine diphosphate (ADP) is the major platelet agonist, which is important in the shape changes, stability, and growth of the thrombus. Platelet activation by ADP is associated with the G protein-coupled receptors P2Y1 and P2Y12. The pharmacologic blockade of the P2Y12 receptor significantly reduces the risk of peripheral artery disease, myocardial infarction, ischemic stroke, and vascular death. Recent studies demonstrated the inhibition of ADP-induced blood platelet activation by three major compounds of the flavonolignans group: silybin, silychristin, and silydianin. For this reason, the aim of the current work was to verify the effects of silybin, silychristin, and silydianin on ADP-induced physiological platelets responses, as well as mechanisms of P2Y12-dependent intracellular signal transduction. We evaluated the effect of tested flavonolignans on ADP-induced blood platelets' aggregation in platelet-rich plasma (PRP) (using light transmission aggregometry), adhesion to fibrinogen (using the static method), and the secretion of PF-4 (using the ELISA method). Additionally, using the double labeled flow cytometry method, we estimated platelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation. We demonstrated a dose-dependent reduction of blood platelets' ability to perform ADP-induced aggregation, adhere to fibrinogen, and secrete PF-4 in samples treated with flavonolignans. Additionally, we observed that all of the tested flavonolignans were able to increase VASP phosphorylation in blood platelets samples, which is correlated with P2Y12 receptor inhibition. All of these analyses show that silychristin and silybin have the strongest inhibitory effect on blood platelet activation by ADP, while silydianin also inhibits the ADP pathway, but to a lesser extent. The results obtained in this study clearly demonstrate that silybin, silychristin, and silydianin have inhibitory properties against the P2Y12 receptor and block ADP-induced blood platelet activation.

Identification of a receptor for ADP on blood platelets by photoaffinity labelling.

PubMed Central

Cristalli, G; Mills, D C

1993-01-01

The synthesis of a new analogue of ADP, 2-(p-azidophenyl)-ethythioadenosine 5'-diphosphate (AzPET-ADP), is described. This compound contains a photolabile phenylazide group attached to the ADP molecule by a thioether link at the purine 2 position. It has been prepared in radioactive form with 32P in the beta-phosphate at a specific radioactivity of 100 mCi/mumol. The reagent activated platelets, causing shape change and aggregation, with somewhat lower affinity than ADP. On photolysis the affinity was increased. The reagent also inhibited platelet adenylate cyclase stimulation by prostaglandin E1, with considerably higher affinity than ADP. On photolysis the affinity was decreased. AzPET-ADP competitively inhibited the binding of 2-methylthio[beta-32P]ADP, a ligand for the receptor by which ADP causes inhibition of adenylate cyclase. In the dark, AzPET-[beta-32P]ADP bound reversibly and with high affinity to a single population of sites similar in number to the sites that bind 2-methylthio[beta-32P]ADP. Binding was inhibited by ADP and by ATP and by p-chloromercuribenzenesulphonic acid (pCMBS). On exposure to u.v. light in the presence of platelets, AzPET-[beta-32P]ADP was incorporated covalently but non-specifically into several platelet proteins, although prominent intracellular proteins were not labelled. Specific labelling was confined to a single region of SDS/polyacrylamide gels, overlying but not comigrating with actin. Incorporation of radioactivity into this region was inhibited by ADP and by ATP as well as by ADP beta S, ATP alpha S and pCMBS, but not by adenosine, GDP or AMP. Inhibition of AzPET-[beta-32P]ADP incorporation was closely correlated with inhibition of equilibrium binding of 2-methylthio[beta-32P]ADP. These results suggests that the labelled protein, which migrates with an apparent molecular mass of 43 kDa in reduced gels, is the receptor through which ADP inhibits adenylate cyclase. Images Figure 5 PMID:8387782

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.

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

Release of Phosphorylated HSP27 (HSPB1) from Platelets Is Accompanied with the Acceleration of Aggregation in Diabetic Patients

PubMed Central

Tokuda, Haruhiko; Kuroyanagi, Gen; Tsujimoto, Masanori; Enomoto, Yukiko; Matsushima-Nishiwaki, Rie; Onuma, Takashi; Kojima, Akiko; Doi, Tomoaki; Tanabe, Kumiko; Akamatsu, Shigeru; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Iwama, Toru; Tanikawa, Takahisa; Ishikawa, Kei; Kojima, Kumi; Kozawa, Osamu

2015-01-01

We investigated the relationship between HSP27 phosphorylation and collagen-stimulated activation of platelets in patients with diabetes mellitus (DM). Platelet-rich plasma was prepared from blood of type 2 DM patients. The platelet aggregation was analyzed in size of aggregates by an aggregometer using a laser scattering method. The protein phosphorylation was analyzed by Western blotting. Phosphorylated-HSP27 and PDGF-AB released from platelets were measured by ELISA. The phosphorylated-HSP27 levels at Ser-78 and Ser-82 induced by collagen were directly proportional to the platelet aggregation. Total HSP27 levels in platelets were decreased concomitantly with the phosphorylation. The released HSP27 levels were significantly correlated with the phosphorylated levels of HSP27 in the platelets stimulated by 0.3 μg/ml collagen. The low dose collagen-stimulated release of HSP27 was detected but relatively small in healthy donors. The released levels of PDGF-AB were in parallel with the levels of released HSP27. Area under the curve (AUC) of small aggregation (9-25 μm) induced by 0.3 μg/ml collagen was inversely proportional to the levels of released HSP27. AUC of large aggregation (50-70 μm) was directly proportional to the levels of released HSP27. Exogenous recombinant phosphorylated- HSP27 hardly affected the aggregation or the released levels of PDGF-AB induced by collagen. These results strongly suggest that HSP27 is released from human platelets accompanied with its phosphorylation induced by collagen, which is correlated with the acceleration of platelet aggregation in type 2 DM patients. PMID:26046355

Release of Phosphorylated HSP27 (HSPB1) from Platelets Is Accompanied with the Acceleration of Aggregation in Diabetic Patients.

PubMed

Tokuda, Haruhiko; Kuroyanagi, Gen; Tsujimoto, Masanori; Enomoto, Yukiko; Matsushima-Nishiwaki, Rie; Onuma, Takashi; Kojima, Akiko; Doi, Tomoaki; Tanabe, Kumiko; Akamatsu, Shigeru; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Iwama, Toru; Tanikawa, Takahisa; Ishikawa, Kei; Kojima, Kumi; Kozawa, Osamu

2015-01-01

We investigated the relationship between HSP27 phosphorylation and collagen-stimulated activation of platelets in patients with diabetes mellitus (DM). Platelet-rich plasma was prepared from blood of type 2 DM patients. The platelet aggregation was analyzed in size of aggregates by an aggregometer using a laser scattering method. The protein phosphorylation was analyzed by Western blotting. Phosphorylated-HSP27 and PDGF-AB released from platelets were measured by ELISA. The phosphorylated-HSP27 levels at Ser-78 and Ser-82 induced by collagen were directly proportional to the platelet aggregation. Total HSP27 levels in platelets were decreased concomitantly with the phosphorylation. The released HSP27 levels were significantly correlated with the phosphorylated levels of HSP27 in the platelets stimulated by 0.3 μg/ml collagen. The low dose collagen-stimulated release of HSP27 was detected but relatively small in healthy donors. The released levels of PDGF-AB were in parallel with the levels of released HSP27. Area under the curve (AUC) of small aggregation (9-25 μm) induced by 0.3 μg/ml collagen was inversely proportional to the levels of released HSP27. AUC of large aggregation (50-70 μm) was directly proportional to the levels of released HSP27. Exogenous recombinant phosphorylated- HSP27 hardly affected the aggregation or the released levels of PDGF-AB induced by collagen. These results strongly suggest that HSP27 is released from human platelets accompanied with its phosphorylation induced by collagen, which is correlated with the acceleration of platelet aggregation in type 2 DM patients.

Meal-induced platelet activation in Type 2 diabetes mellitus: effects of treatment with repaglinide and glibenclamide.

PubMed

Yngen, M; Ostenson, C-G; Hjemdahl, P; Wallén, N H

2006-02-01

To compare the effects of treatment with repaglinide and glibenclamide on platelet function and endothelial markers in patients with Type 2 diabetes mellitus, before and after a standardized meal. Fifteen patients with Type 2 diabetes were investigated on three occasions: at baseline without oral hypoglycaemic drug treatment, and after 6 weeks' treatment with repaglinide or glibenclamide, respectively, in an open randomized cross-over study. Agonist-induced platelet P-selectin expression and platelet aggregation, urinary thromboxane, soluble P-selectin, von Willebrand factor (VWF), soluble E-selectin, intercellular adhesion molecule (ICAM-1) and C-reactive protein (CRP) were measured. In addition, pre-meal data were compared with non-diabetic control subjects (n = 15), matched for sex, age and BMI. Adenosine diphosphate (ADP)-induced platelet P-selectin expression increased post-meal in Type 2 diabetic patients both at baseline and after treatment with repaglinide and glibenclamide (P < 0.01 for all; repeated measures anova). Repaglinide treatment reduced fasting ADP-induced P-selectin expression compared with baseline (P = 0.01), but did not influence meal-induced platelet hyper-reactivity (P = 0.32). No significant anti-platelet effects of glibenclamide treatment were found. Plasma concentrations of VWF and ICAM-1 were elevated in patients with Type 2 diabetes compared with control subjects (P < 0.05 for both) and were reduced during treatment with repaglinide (P < 0.01 for both) but did not change during glibenclamide treatment. The post-meal state is associated with enhanced platelet reactivity in patients with Type 2 diabetes mellitus. Pre-meal treatment with repaglinide or glibenclamide does not inhibit postprandial platelet activation, but repaglinide treatment is associated with attenuated platelet and endothelial activity in the fasting state.

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.

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

A Proteomic View at T Cell Costimulation

PubMed Central

Hombach, Andreas A.; Recktenwald, Christian V.; Dressler, Sven P.; Abken, Hinrich; Seliger, Barbara

2012-01-01

The “two-signal paradigm” in T cell activation predicts that the cooperation of “signal 1,” provided by the T cell receptor (TCR) through engagement of major histocompatility complex (MHC)-presented peptide, with “signal 2″ provided by costimulatory molecules, the prototype of which is CD28, is required to induce T cell effector functions. While the individual signalling pathways are well understood, little is known about global changes in the proteome pattern during TCR/CD28-mediated activation. Therefore, comparative 2-DE-based proteome analyses of CD3+ CD69- resting T cells versus cells incubated with (i) the agonistic anti-CD3 antibody OKT3 mimicking signal 1 in absence or presence of IL-2 and/or with (ii) the agonistic antibody 15E8 triggering CD28-mediated signaling were performed. Differentially regulated spots were defined leading to the identification of proteins involved in the regulation of the metabolism, shaping and maintenance of the cytoskeleton and signal transduction. Representative members of the differentially expressed protein families, such as calmodulin (CALM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), L-lactate dehydrogenase (LDH), Rho GDP-dissociation inhibitor 2 (GDIR2), and platelet basic protein (CXCL7), were independently verified by flow cytometry. Data provide a detailed map of individual protein alterations at the global proteome level in response to TCR/CD28-mediated T cell activation. PMID:22539942

Phenotypic modulation of smooth muscle cells during formation of neointimal thickenings following vascular injury.

PubMed

Thyberg, J

1998-07-01

Smooth muscle cells build up the media of mammalian arteries and constitute one of the principal cell types in atherosclerotic and restenotic lesions. Accordingly, they show a high degree of plasticity and are able to shift from a differentiated, contractile phenotype to a less differentiated, synthetic phenotype, and then back again. This modulation occurs as a response to vascular injury and includes a prominent structural reorganization with loss of myofilaments and formation of an extensive endoplasmic reticulum and a large Golgi complex. At the same time, the expression of cytoskeletal proteins and other gene products is altered. As a result, the cells lose their contractility and become able to migrate from the media to the intima, proliferate, and secrete extracellular matrix components, thereby contributing to the formation of intimal thickenings. The mechanisms behind this change in morphology and function of the smooth muscle cells are still incompletely understood. A crucial role has been ascribed to basement membrane proteins such as laminin and collagen type IV and adhesive proteins such as fibronectin. A significant role is also played by mitogenic proteins such as platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF). An improved knowledge of the regulation of smooth muscle differentiated properties represents an important part in the search for new methods of prevention and treatment of vascular disease.

Deposition of Fibrinogen on the Surface of in vitro Thrombi Prevents Platelet Adhesion

PubMed Central

Owaynat, Hadil; Yermolenko, Ivan S.; Turaga, Ramya; Lishko, Valeryi K.; Sheller, Michael R.; Ugarova, Tatiana P.

2015-01-01

The initial accumulation of platelets after vessel injury is followed by thrombin-mediated generation of fibrin which is deposited around the plug. While numerous in vitro studies have shown that fibrin is highly adhesive for platelets, the surface of experimental thrombi in vivo contains very few platelets suggesting the existence of natural anti-adhesive mechanisms protecting stabilized thrombi from platelet accumulation and continuous thrombus propagation. We previously showed that adsorption of fibrinogen on pure fibrin clots results in the formation of a nonadhesive matrix, highlighting a possible role of this process in surface-mediated control of thrombus growth. However, the deposition of fibrinogen on the surface of blood clots has not been examined. In this study, we investigated the presence of intact fibrinogen on the surface of fibrin-rich thrombi generated from flowing blood and determined whether deposited fibrinogen is nonadhesive for platelets. Stabilized fibrin-rich thrombi were generated using a flow chamber and the time that platelets spend on the surface of thrombi was determined by video recording. The presence of fibrinogen and fibrin on the surface of thrombi was analyzed by confocal microscopy using specific antibodies. Examination of the spatial distribution of two proteins revealed the presence of intact fibrinogen on the surface of stabilized thrombi. By manipulating the surface of thrombi to display either fibrin or intact fibrinogen, we found that platelets adhere to fibrin- but not to fibrinogen-coated thrombi. These results indicate that the fibrinogen matrix assembled on the outer layer of stabilized in vitro thrombi protects them from platelet adhesion. PMID:26482763

Deposition of fibrinogen on the surface of in vitro thrombi prevents platelet adhesion.

PubMed

Owaynat, Hadil; Yermolenko, Ivan S; Turaga, Ramya; Lishko, Valeryi K; Sheller, Michael R; Ugarova, Tatiana P

2015-12-01

The initial accumulation of platelets after vessel injury is followed by thrombin-mediated generation of fibrin which is deposited around the plug. While numerous in vitro studies have shown that fibrin is highly adhesive for platelets, the surface of experimental thrombi in vivo contains very few platelets suggesting the existence of natural anti-adhesive mechanisms protecting stabilized thrombi from platelet accumulation and continuous thrombus propagation. We previously showed that adsorption of fibrinogen on pure fibrin clots results in the formation of a nonadhesive matrix, highlighting a possible role of this process in surface-mediated control of thrombus growth. However, the deposition of fibrinogen on the surface of blood clots has not been examined. In this study, we investigated the presence of intact fibrinogen on the surface of fibrin-rich thrombi generated from flowing blood and determined whether deposited fibrinogen is nonadhesive for platelets. Stabilized fibrin-rich thrombi were generated using a flow chamber and the time that platelets spend on the surface of thrombi was determined by video recording. The presence of fibrinogen and fibrin on the surface of thrombi was analyzed by confocal microscopy using specific antibodies. Examination of the spatial distribution of two proteins revealed the presence of intact fibrinogen on the surface of stabilized thrombi. By manipulating the surface of thrombi to display either fibrin or intact fibrinogen, we found that platelets adhere to fibrin- but not to fibrinogen-coated thrombi. These results indicate that the fibrinogen matrix assembled on the outer layer of stabilized in vitro thrombi protects them from platelet adhesion. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

Sithu, Srinivas D.; Diabetes and Obesity Center, University of Louisville, Louisville, KY 40202; Srivastava, Sanjay

Acrolein is a common air pollutant that is present in high concentrations in wood, cotton, and tobacco smoke, automobile exhaust and industrial waste and emissions. Exposure to acrolein containing environmental pollutants such as tobacco smoke and automobile exhaust has been linked to the activation of the coagulation and hemostasis pathways and thereby to the predisposition of thrombotic events in human. To examine the effects of acrolein on platelets, adult male C57Bl/6 mice were subjected acute (5 ppm for 6 h) or sub-chronic (1 ppm, 6 h/day for 4 days) acrolein inhalation exposures. The acute exposure to acrolein did not causemore » pulmonary inflammation and oxidative stress, dyslipidemia or induce liver damage or muscle injury. Platelet GSH levels in acrolein-exposed mice were comparable to controls, but acrolein-exposure increased the abundance of protein-acrolein adducts in platelets. Platelets isolated from mice, exposed to both acute and sub-chronic acrolein levels, showed increased ADP-induced platelet aggregation. Exposure to acrolein also led to an increase in the indices of platelet activation such as the formation of platelet-leukocyte aggregates in the blood, plasma PF4 levels, and increased platelet-fibrinogen binding. The bleeding time was decreased in acrolein exposed mice. Plasma levels of PF4 were also increased in mice exposed to environmental tobacco smoke. Similar to inhalation exposure, acrolein feeding to mice also increased platelet activation and established a pro-thrombotic state in mice. Together, our data suggest that acrolein is an important contributing factor to the pro-thrombotic risk in human exposure to pollutants such as tobacco smoke or automobile exhaust, or through dietary consumption.« less

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.

Primary porcine Kupffer cell phagocytosis of human platelets involves the CD18 receptor.

PubMed

Chihara, Ray K; Paris, Leela L; Reyes, Luz M; Sidner, Richard A; Estrada, Jose L; Downey, Susan M; Wang, Zheng-Yu; Tector, A Joseph; Burlak, Christopher

2011-10-15

Hepatic failure has been treated successfully with clinical extracorporeal perfusions of porcine livers. However, dog-to-pig and pig-to-baboon liver xenotransplant models have resulted in severe bleeding secondary to liver xenograft-induced thrombocytopenia. Kupffer cells (KC) are abundant phagocytic cells in the liver. KC express the CD11b/CD18 receptor, which has been implicated in chilled platelet binding and phagocytosis through interaction with platelet surface proteins and carbohydrates. We sought to identify the role of KC CD18 in liver xenograft-induced thrombocytopenia. Primary pig KC were characterized by flow cytometry, immunoblots, and quantitative polymerase chain reaction. Pig KC were used in inhibition assays with fluorescently labeled human platelets. The CD18 receptor was targeted for siRNA knockdown. Domestic and α1,3-galactosyltransferase double knockout porcine KC cultures were approximately 92% positive for CD18 as detected by quantitative polymerase chain reaction and flow cytometry. Use of CD18 blocking antibodies resulted in reduction of human platelet binding and phagocytosis. Additionally, asialofetuin, not fetuin, inhibited platelet phagocytosis suggesting the involvement of an oligosaccharide-binding site. Furthermore, reduced CD18 expression by siRNA resulted in decreased human platelet binding. Our data suggest that primary pig KC bind and phagocytose human platelets with involvement of CD18. Further understanding and modification of CD18 expression in pigs may result in a liver xenograft with reduced thrombocytopenic effects, which could be used as a bridge to allogeneic liver transplantation.

Structure and hemocompatibility of nanocrystalline titanium nitride produced under glow-discharge conditions

NASA Astrophysics Data System (ADS)

Sowińska, Agnieszka; Czarnowska, Elżbieta; Tarnowski, Michał; Witkowska, Justyna; Wierzchoń, Tadeusz

2018-04-01

Significant efforts are being made towards developing novel antithrombotic materials. The purpose of the presented study was to characterize two variants of nitrided surface layers produced on alloy Ti-6Al-4V in different areas of low-temperature plasma - at the plasma potential (TiNp) or at the cathode potential (TiNc). The layers were characterized in terms of their microstructure, surface topography and wettability, and platelet response to the environment of different pH. The produced layers were of the TiN + Ti2N + αTiN-type, but the layer produced at the plasma potential was thinner, smoother and had lower surface free energy compared with that produced at the cathode potential. Biological evaluation demonstrated more fibrinogen buildup, less platelet adhesion and aggregation, and fewer strongly activated platelets on the TiNp surface compared with those parameters on the TiNc surface and on the titanium alloy in its initial state. Interestingly, both surface types were significantly resistant to fibrinogen adsorption and platelet adhesion in the environment of lower pH. In conclusion, the nitrided surface layer produced at the plasma potential is a promising material and this basic information is critical for further development of hemocompatible materials.

Umbilical Cord Blood Platelet Lysate as Serum Substitute in Expansion of Human Mesenchymal Stem Cells.

PubMed

Shirzad, Negin; Bordbar, Sima; Goodarzi, Alireza; Mohammad, Monire; Khosravani, Pardis; Sayahpour, Froughazam; Baghaban Eslaminejad, Mohamadreza; Ebrahimi, Marzieh

2017-10-01

The diverse clinical applications for human mesenchymal stem cells (hMSCs) in cellular therapy and regenerative medicine warrant increased focus on developing adequate culture supplements devoid of animal-derived products. In the present study, we have investigated the feasibility of umbilical cord blood-platelet lysate (UCB-PL) as a standard substitute for fetal bovine serum (FBS) and human peripheral blood-PL (PB-PL). In this experimental study, platelet concentrates (PC) from UCB and human PB donors were frozen, melted, and sterilized to obtain PL. Quality control included platelet cell counts, sterility testing (viral and microbial), total protein concentrations, growth factor levels, and PL stability. The effects of UCB-PL and PB-PL on hMSCs proliferation and differentiation into osteocytes, chondrocytes, and adipocytes were studied and the results compared with FBS. UCB-PL contained high levels of protein content, platelet-derived growth factor- AB (PDGF-AB), and transforming growth factor (TGF) compared to PB-PL. All growth factors were stable for at least nine months post-storage at -70˚C. hMSCs proliferation enhanced following treatment with UCB-PL. With all three supplements, hMSCs could differentiate into all three lineages. PB-PL and UCB-PL both were potent in hMSCs proliferation. However, PB promoted osteoblastic differentiation and UCB-PL induced chondrogenic differentiation. Because of availability, ease of use and feasible standardization of UCB-PL, we have suggested that UCB-PL be used as an alternative to FBS and PB-PL for the cultivation and expansion of hMSCs in cellular therapy. Copyright© by Royan Institute. All rights reserved.

Cell-free released components of Streptococcus sanguis inhibit human platelet aggregation.

PubMed Central

Herzberg, M C; Brintzenhofe, K L; Clawson, C C

1983-01-01

To study the role of surface components in the selective binding and aggregation of platelet-rich plasma (PRP) by strains of viridans streptococci, we treated the binding, aggregation strain Streptococcus sanguis I 2017-78 by sonication or trypsinization. Morphologically identifiable electron-dense fibrils were released from the cell wall, apparently from an inner electron-dense layer, under conditions that left cells intact. These controlled conditions were determined to cause submaximal loss in adhesion to platelet ghosts and PRP aggregation by treated, washed S. sanguis. Soluble components were recovered from the controlled sonic or L-(tosylamido 2-phenyl)ethyl chloromethyl ketone-trypsin treatments. Each showed dose-response inhibition of aggregation when preincubated with PRP before challenge with fresh, untreated S. sanguis. The time to onset of PRP aggregation was inhibited by 50% with 0.2 mg of TPCK-trypsin peptides or 1.0 mg of the sonicate per ml per 2 X 10(8) platelets. Components of both preparations were immunologically cross-reactive, but lipoteichoic acid was not a major antigen of either. By weight, the TPCK-trypsin peptides were virtually all protein; the sonicate residues identified were about 50% protein and 7% hexose. Each was a complex mixture of components as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 8 TPCK-trypsin peptides and 16 sonicate components were so identified. In contrast, at least four or five components from either preparation were recognized as surface determinants by a rabbit antiserum to whole homologous microbes. Platelet-binding ligands of S. sanguis could be among these determinants. Images PMID:6618669

Trivalent methylated arsenical-induced phosphatidylserine exposure and apoptosis in platelets may lead to increased thrombus formation

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

Bae, Ok-Nam; Lim, Kyung-Min; AMOREPACIFIC CO/R and D Center, Gyeonggi-do 446-729

2009-09-01

Trivalent methylated metabolites of arsenic, monomethylarsonous acid (MMA{sup III}) and dimethylarsinous acid (DMA{sup III}), have been found highly reactive and toxic in various cells and in vivo animal models, suggesting their roles in the arsenic-associated toxicity. However, their effects on cardiovascular system including blood cells, one of the most important targets for arsenic toxicity, remain poorly understood. Here we found that MMA{sup III} and DMA{sup III} could induce procoagulant activity and apoptosis in platelets, which play key roles in the development of various cardiovascular diseases (CVDs) through excessive thrombus formation. In freshly isolated human platelets, treatment of MMA{sup III} resultedmore » in phosphatidylserine (PS) exposure, a hallmark of procoagulant activation, accompanied by distinctive apoptotic features including mitochondrial membrane potential disruption, cytochrome c release, and caspase-3 activation. These procoagulant activation and apoptotic features were found to be mediated by the depletion of protein thiol and intracellular ATP, and flippase inhibition by MMA{sup III}, while the intracellular calcium increase or reactive oxygen species generation was not involved. Importantly, increased platelet procoagulant activity by MMA{sup III} resulted in enhanced blood coagulation and excessive thrombus formation in a rat in vivo venous thrombosis model. DMA{sup III} also induced PS-exposure with apoptotic features mediated by protein thiol depletion, which resulted in enhanced thrombin generation. In summary, we believe that this study provides an important evidence for the role of trivalent methylated arsenic metabolites in arsenic-associated CVDs, giving a novel insight into the role of platelet apoptosis in toxicant-induced cardiovascular toxicity.« less

A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting

PubMed Central

Vallet-Courbin, Amelie; Larivière, Mélusine; Hocquellet, Agnès; Hemadou, Audrey; Parimala, Sarjapura-Nagaraja; Laroche-Traineau, Jeanny; Santarelli, Xavier; Clofent-Sanchez, Gisèle; Jacobin-Valat, Marie-Josée; Noubhani, Abdelmajid

2017-01-01

Cells of the innate and adaptive immune system are key factors in the progression of atherosclerotic plaque, leading to plaque instability and rupture, potentially resulting in acute atherothrombotic events such as coronary artery disease, cerebrovascular disease and peripheral arterial disease. Here, we describe the cloning, expression, purification, and immunoreactivity assessment of a recombinant single-chain variable fragment (scFv) derived from a human anti-αIIbβ3 antibody (HuAb) selected to target atheromatous lesions for the presence of platelets. Indeed, platelets within atheroma plaques have been shown to play a role in inflammation, in platelet-leucocyte aggregates and in thrombi formation and might thus be considered relevant biomarkers of atherosclerotic progression. The DNA sequence that encodes the anti-αIIbβ3 TEG4 scFv previously obtained from a phage-display selection on activated platelets, was inserted into the eukaryote vector (pPICZαA) in fusion with a tag sequence encoding 2 cysteines useable for specific probes grafting experiments. The recombinant protein was expressed at high yields in Pichia pastoris (30 mg/L culture). The advantage of P. pastoris as an expression system is the production and secretion of recombinant proteins in the supernatant, ruling out the difficulties encountered when scFv are produced in the cytoplasm of bacteria (low yield, low solubility and reduced affinity). The improved conditions allowed for the recovery of highly purified and biologically active scFv fragments ready to be grafted in a site-directed way to nanoparticles for the imaging of atherosclerotic plaques involving inflammatory processes and thus at high risk of instability. PMID:28125612

Cell-based Assay System for Predicting Bone Regeneration in Patient Affected by Aseptic Nonunion and Treated with Platelet Rich Fibrin.

PubMed

Perut, Francesca; Dallari, Dante; Rani, Nicola; Baldini, Nicola; Granchi, Donatella

Regenerative strategies based on the use of platelet concentrates as an autologous source of growth factors (GF) has been proposed to promote the healing of long bone nonunions. However, the relatively high failure rate stimulates interest in growing knowledge and developing solutions to obtain the best results from the regenerative approach. In this study we evaluated whether a cell-based assay system could be able to recognize patients who will benefit or not from the use of autologous platelet preparations. The autologous serum was used in culture medium to promote the osteogenic differentiation of normal bone-marrow stromal cells (BMSC). Blood samples were collected from 16 patients affected by aseptic long bone nonunion who were candidates to the treatment with autologous platelet-rich fibrin. The osteoinductive effect was detected by measuring the BMSC proliferation, the mineralization activity, and the expression of bone-related genes. Serum level of basic fibroblast growth factor (bFGF) was considered as a representative marker of the delivery of osteogenic GFs from platelets. Laboratory results were related to the characteristics of the disease before the treatment and to the outcome at 12 months. Serum samples from "good responders" showed significantly higher levels of bFGF and were able to induce a significantly higher proliferation of BMSC, while no significant differences were observed in terms of osteoblast differentiation. BMSC-based assay could be a useful tool to recognize patients who have a low probability to benefit from the use of autologous platelet concentrate to promote the healing of long bone nonunion.

Population Pharmacokinetic and Pharmacodynamic Modeling of Lusutrombopag, a Newly Developed Oral Thrombopoietin Receptor Agonist, in Healthy Subjects.

PubMed

Katsube, Takayuki; Ishibashi, Toru; Kano, Takeshi; Wajima, Toshihiro

2016-11-01

The aim of this study was to develop a population pharmacokinetic (PK)/pharmacodynamic (PD) model for describing plasma lusutrombopag concentrations and platelet response following oral lusutrombopag dosing and for evaluating covariates in the PK/PD profiles. A population PK/PD model was developed using a total of 2539 plasma lusutrombopag concentration data and 1408 platelet count data from 78 healthy adult subjects following oral single and multiple (14-day once-daily) dosing. Covariates in PK and PK/PD models were explored for subject age, body weight, sex, and ethnicity. A three-compartment model with first-order rate and lag time for absorption was selected as a PK model. A three-transit and one-platelet compartment model with a sigmoid E max model for drug effect and feedback of platelet production was selected as the PD model. The PK and PK/PD models well described the plasma lusutrombopag concentrations and the platelet response, respectively. Body weight was a significant covariate in PK. The bioavailability of non-Japanese subjects (White and Black/African American subjects) was 13 % lower than that of Japanese subjects, while the simulated platelet response profiles using the PK/PD model were similar between Japanese and non-Japanese subjects. There were no significant covariates of the tested background data including age, sex, and ethnicity (Japanese or non-Japanese) for the PD sensitivity. A population PK/PD model was developed for lusutrombopag and shown to provide good prediction for the PK/PD profiles. The model could be used as a basic PK/PD model in the drug development of lusutrombopag.

Did dinosaurs have megakaryocytes? New ideas about platelets and their progenitors

PubMed Central

Brass, Lawrence F.

2005-01-01

Biological evolution has struggled to produce mechanisms that can limit blood loss following injury. In humans and other mammals, control of blood loss (hemostasis) is achieved through a combination of plasma proteins, most of which are made in the liver, and platelets, anucleate blood cells that are produced in the bone marrow by megakaryocytes. Much has been learned about the underlying mechanisms, but much remains to be determined. The articles in this series review current ideas about the production of megakaryocytes from undifferentiated hematopoietic precursors, the steps by which megakaryocytes produce platelets, and the molecular mechanisms within platelets that make hemostasis possible. The underlying theme that connects the articles is the intense investigation of a complex system that keeps humans from bleeding to death, but at the same time exposes us to increased risk of thrombosis and vascular disease. PMID:16322776

Thrombolytic along with anti-platelet activity of crinumin, a protein constituent of Crinum asiaticum.

PubMed

Singh, Kunwar Awaneesh; Nayak, Manasa K; Jagannadham, Medicherla V; Dash, Debabrata

2011-08-15

Several anticoagulants, anti-platelet and thrombolytic medications are used for the treatment of thrombotic disorders. Anti-coagulants and anti-platelet agents prevent the formation of blood clots but do not dissolve existing clots, whereas thrombolytic agents are able to dissolve a clot but emboli can form even after successful treatment. Thus, none of them provide a permanent and complete solution. In this regard a single molecule that could both dissolve the clot and prevent the formation of new clots would be useful in the treatment of thrombotic diseases. Crinumin, a stable and active (in many adverse conditions) serine protease, shows plasmin-like fibrinolytic activity and inhibits platelet aggregation and P-selectin exposure, as established by photography, phase contrast microscopy, whole blood optical Lumi-aggregometry and flow cytometry. Crinumin could be an efficient and inexpensive therapeutic agent for the treatment and prevention of thromboembolic diseases. Copyright © 2011 Elsevier Inc. All rights reserved.

Modulation of the pharmacological effects of enzymatically-active PLA2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga

PubMed Central

Oliveira, Simone CB; Fonseca, Fabiana V; Antunes, Edson; Camargo, Enilton A; Morganti, Rafael P; Aparício, Ricardo; Toyama, Daniela O; Beriam, Luís OS; Nunes, Eudismar V; Cavada, Benildo S; Nagano, Celso S; Sampaio, Alexandre H; Nascimento, Kyria S; Toyama, Marcos H

2008-01-01

Background An interaction between lectins from marine algae and PLA2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2), isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA2 isolated from rattlesnake venom (Crotalus durissus cascavella), to better understand the enzymatic and pharmacological mechanisms of the PLA2 and its complex. Results This PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa), its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity. The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24–26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm), but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap). PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm. PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48/80 compound. Conclusion The unexpected results observed for the PLA2-BTL-2 complex strongly suggest that the pharmacological activity of this PLA2 is not solely dependent on the presence of enzymatic activity, and that other pharmacological regions may also be involved. In addition, we describe for the first time an interaction between two different molecules, which form a stable complex with significant changes in their original biological action. This opens new possibilities for understanding the function and action of crude venom, an extremely complex mixture of different molecules. PMID:18534036

Proteins, Platelets, and Blood Coagulation at Biomaterial Interfaces

PubMed Central

Xu, Li-Chong; Bauer, James; Siedlecki, Christopher A.

2015-01-01

Blood coagulation and platelet adhesion remain major impediments to the use of biomaterials in implantable medical devices. There is still significant controversy and question in the field regarding the role that surfaces play in this process. This manuscript addresses this topic area and reports on state of the art in the field. Particular emphasis is placed on the subject of surface engineering and surface measurements that allow for control and observation of surface-mediated biological responses in blood and test solutions. Appropriate use of surface texturing and chemical patterning methodologies allow for reduction of both blood coagulation and platelet adhesion, and new methods of surface interrogation at high resolution allow for measurement of the relevant biological factors. PMID:25448722

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.

Distinct contributions of complement factors to platelet activation and fibrin formation in venous thrombus development

PubMed Central

Jäckel, Sven; Saffarzadeh, Mona; Langer, Florian

2017-01-01

Expanding evidence indicates multiple interactions between the hemostatic system and innate immunity, and the coagulation and complement cascades. Here we show in a tissue factor (TF)–dependent model of flow restriction-induced venous thrombosis that complement factors make distinct contributions to platelet activation and fibrin deposition. Complement factor 3 (C3) deficiency causes prolonged bleeding, reduced thrombus incidence, thrombus size, fibrin and platelet deposition in the ligated inferior vena cava, and diminished platelet activation in vitro. Initial fibrin deposition at the vessel wall over 6 hours in this model was dependent on protein disulfide isomerase (PDI) and TF expression by myeloid cells, but did not require neutrophil extracellular trap formation involving peptidyl arginine deiminase 4. In contrast to C3−/− mice, C5-deficient mice had no apparent defect in platelet activation in vitro, and vessel wall platelet deposition and initial hemostasis in vivo. However, fibrin formation, the exposure of negatively charged phosphatidylserine (PS) on adherent leukocytes, and clot burden after 48 hours were significantly reduced in C5−/− mice compared with wild-type controls. These results delineate that C3 plays specific roles in platelet activation independent of formation of the terminal complement complex and provide in vivo evidence for contributions of complement-dependent membrane perturbations to prothrombotic TF activation on myeloid cells. PMID:28223279

Platelet-rich plasma for long bone healing

PubMed Central

Lenza, Mário; Ferraz, Silvia de Barros; Viola, Dan Carai Maia; dos Santos, Oscar Fernando Pavão; Cendoroglo, Miguel; Ferretti, Mario

2013-01-01

ABSTRACT Objective: To evaluate effectiveness of the use of platelet-rich plasma as coadjuvant for union of long bones. Methods: The search strategy included the Cochrane Library (via Central) and MEDLINE (via PubMed). There were no limits as to language or publication media. The latest search strategy was conducted in December 2011. It included randomized clinical trials that evaluated the use of platelet-rich plasma as coadjuvant medication to accelerate union of long bones (acute fractures, pseudoarthrosis and bone defects). The outcomes of interest for this review include bone regeneration, adverse events, costs, pain, and quality of life. The authors selected eligible studies, evaluated the methodological quality, and extracted the data. It was not possible to perform quantitative analysis of the grouped studies (meta-analyses). Results: Two randomized prospective clinical trials were included, with a total of 148 participants. One of them compared recombinant human morphogenic bone protein-7 versus platelet-rich plasma for the treatment of pseudoarthrosis; the other evaluated the effects of three coadjuvant treatments for union of valgising tibial osteotomies (platelet-rich plasma, platelet-rich plasma plus bone marrow stromal cells, and no coadjuvant treatment). Both had low statistical power and moderate to high risk of bias. Conclusion: There was no conclusive evidence that sustained the use of platelet-rich plasma as a coadjuvant to aid bone regeneration of fractures, pseudoarthrosis, or bone defects. PMID:23579757

Smoking-induced alterations in platelet membrane fluidity and Na(+)/K(+)-ATPase activity in chronic cigarette smokers.

PubMed

Padmavathi, Pannuru; Reddy, Vaddi Damodara; Maturu, Paramahamsa; Varadacharyulu, Nallanchakravarthula

2010-06-30

Cigarette smoking is a recognized risk factor for cardiovascular diseases and has been implicated in the pathogenesis of atherosclerosis. Platelet adhesiveness and aggregation increases as a result of smoking. Cigarette smoking modifies haemostatic parameters via thrombosis with a consequently higher rate of cardiovascular events, but smoking-induced alterations of platelet membrane fluidity and other changes have not been studied. Thirty experimental and control subjects (mean age 35+/-8) were selected for the study. Experimental subjects had smoked 10+/-2 cigarettes per day for 7-10 years. The plasma lipid profile, platelet carbonyls, sulfhydryl groups, Na(+)/k(+)-ATPase activity, fluidity using a fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), total cholesterol and phospholipids as well individual phospholipids were determined. Increases in the platelet membrane cholesterol phospholipid (C/P) ratio, phosphotidylethanolamine, phosphotidylserine with decreased phosphotidylcholine, Na(+)/k(+)-ATPase activity, fluidity and no significant change in phosphotidylinositol and sphingomylein, as well as increases in plasma total cholesterol, LDL-cholesterol, protein carbonyls with decreased HDL-cholesterol and sulfhydryl groups were observed in cigarette smokers. Platelet membrane total phospholipids were positively correlated with plasma LDL-cholesterol (r=0.568) and VLDL-cholesterol (r=0.614) in cigarette smokers. Increased plasma LDL-cholesterol, VLDL-cholesterol and total cholesterol might have resulted in the increased C/P ratio and decreased platelet membrane fluidity of cigarette smokers.

Increasing platelet concentrations in leukocyte-reduced platelet-rich plasma decrease collagen gene synthesis in tendons.

PubMed

Boswell, Stacie G; Schnabel, Lauren V; Mohammed, Hussni O; Sundman, Emily A; Minas, Tom; Fortier, Lisa A

2014-01-01

Platelet-rich plasma (PRP) is used for the treatment of tendinopathy. There are numerous PRP preparations, and the optimal combination of platelets and leukocytes is not known. Within leukocyte-reduced PRP (lrPRP), there is a plateau effect of platelet concentration, with increasing platelet concentrations being detrimental to extracellular matrix synthesis. Controlled laboratory study. Different formulations of lrPRP with respect to the platelet:leukocyte ratio were generated from venous blood of 8 horses. Explants of the superficial digital flexor tendon were cultured in lrPRP products for 96 hours. Platelet-derived growth factor-BB (PDGF-BB), tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), and interleukin-1β (IL-1β) concentrations were determined in the media by enzyme-linked immunosorbent assay. Gene expression in tendon tissue for collagen type I and III (COL1A1 and COL3A1, respectively), matrix metalloproteinase-3 and -13 (MMP-3 and MMP-13, respectively), cartilage oligomeric matrix protein (COMP), and IL-1β was determined. Data were divided into 3 groups of lrPRP based on the ratio of platelets:leukocytes and evaluated to determine the effect of platelet concentration. Complete blood counts verified leukocyte reduction and platelet enrichment in all PRP preparations. In the lrPRP preparation, the anabolic growth factors PDGF-BB and TGF-β1 were increased with increasing platelet concentrations, and the catabolic cytokine IL-1β was decreased with increasing platelet concentrations. Increasing the platelet concentration resulted in a significant reduction in COL1A1 and COL3A1 synthesis in tendons. Increasing the platelet concentration within lrPRP preparations results in the delivery of more anabolic growth factors and less proinflammatory cytokines, but the biological effect on tendons is diminished metabolism as indicated by a decrease in the synthesis of both COL1A1 and COL3A1. Together, this information suggests that minimizing leukocytes in PRP is more important than maximizing platelet numbers with respect to decreasing inflammation and enhancing matrix gene synthesis. This study suggests that reducing leukocytes to minimize catabolic signaling appears to be more important than increasing platelets in an effort to maximize anabolic signaling. Further, a maximum biological threshold of benefit was demonstrated with regard to the number of platelets beyond which further increases in platelet concentration did not result in further anabolic upregulation. In vivo investigations documenting the use of platelets for the treatment of tendinopathy are justified as well as further in vitro characterization of the ideal PRP product for the treatment of tendinopathy and other musculoskeletal applications.

Evidence for shear-mediated Ca2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line.

PubMed

Ilkan, Zeki; Wright, Joy R; Goodall, Alison H; Gibbins, Jonathan M; Jones, Chris I; Mahaut-Smith, Martyn P

2017-06-02

The role of mechanosensitive (MS) Ca 2+ -permeable ion channels in platelets is unclear, despite the importance of shear stress in platelet function and life-threatening thrombus formation. We therefore sought to investigate the expression and functional relevance of MS channels in human platelets. The effect of shear stress on Ca 2+ entry in human platelets and Meg-01 megakaryocytic cells loaded with Fluo-3 was examined by confocal microscopy. Cells were attached to glass coverslips within flow chambers that allowed applications of physiological and pathological shear stress. Arterial shear (1002.6 s -1 ) induced a sustained increase in [Ca 2+ ] i in Meg-01 cells and enhanced the frequency of repetitive Ca 2+ transients by 80% in platelets. These Ca 2+ increases were abrogated by the MS channel inhibitor Grammostola spatulata mechanotoxin 4 (GsMTx-4) or by chelation of extracellular Ca 2+ Thrombus formation was studied on collagen-coated surfaces using DiOC 6 -stained platelets. In addition, [Ca 2+ ] i and functional responses of washed platelet suspensions were studied with Fura-2 and light transmission aggregometry, respectively. Thrombus size was reduced 50% by GsMTx-4, independently of P2X1 receptors. In contrast, GsMTx-4 had no effect on collagen-induced aggregation or on Ca 2+ influx via TRPC6 or Orai1 channels and caused only a minor inhibition of P2X1-dependent Ca 2+ entry. The Piezo1 agonist, Yoda1, potentiated shear-dependent platelet Ca 2+ transients by 170%. Piezo1 mRNA transcripts and protein were detected with quantitative RT-PCR and Western blotting, respectively, in both platelets and Meg-01 cells. We conclude that platelets and Meg-01 cells express the MS cation channel Piezo1, which may contribute to Ca 2+ entry and thrombus formation under arterial shear. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Platelet-rich plasma, the ultimate secret for youthful skin elixir and hair growth triggering.

PubMed

Elghblawi, Ebtisam

2018-06-01

The clinical application of platelet-rich plasma (PRP) is based on the increase in the concentration of growth factors that are released from alpha-granule of the concentrated platelets and in the secretion of proteins which are able to capitalize on the healing process at the cellular level. It has been invented to restore the natural beauty by starting the natural rejuvenation process of the skin and aiming to make it function as a younger one and to keep the skin youthful and maintain it. Besides that, it is also emerged to include hairs as a new injectable procedure to enable stimulating hair growth locally and topically; preventing its fall; improving hair shaft, hair stem, and its caliber; increasing its shine, vitality, and pliability; and declining hair splitting and breakage. Thus, youth is in your blood as it has a magical power imposed in the platelet factors. There is, however, no standardization of the techniques besides insufficient description of the adopted procedures. Not long, autologous platelet-rich plasma (PRP) has surfaced strongly in diverse medical specialties including plastic, wound healing and diabetic ulcers, orthopedic, trauma, ocular surgery, dry eye for eyelid injection, urology for urinary incontinence, sexual wellness, cutaneous surgery, sport medicine, dentistry and dermatology, and aesthetic applications. PRP proved to promote wound healing and aid in facelift, volumetric skin, skin rejuvenation, regeneration, and reconstruction; improve wrinkling; stimulate hair growth; increase hair follicle viability and its survival rate; prevent apoptosis; increase and prolong the anagen hair growth stage; and delay the progression to catagen hair cycle stage with increased density in hair loss and hair transplantation. The aims of this extensive review were to cover all PRP application aspects that are carried out in aesthetic dermatology and to assess the literature on platelet-rich plasma outcomes on main aesthetic practices of general dermatology. A literature review was conducted by searching through PubMed, Biomedical Library database, Google Scholar, and Research Gate for the terms PRP, platelet-rich plasma, platelet-rich fibrin matrix, platelet preparations, platelet application therapy, platelet growth factors, platelet facial, platelet facial rejuvenation, platelet hairs, and platelet wound healing, from inception till 2017, and they were combined using Boolean operators. All those retrieved articles in English language were looked at and explored thoroughly. © 2017 Wiley Periodicals, Inc.

Characterization of Molecules Binding to the 70K N-terminal Region of Fibronectin by IFAST Purification Coupled with Mass Spectrometry

PubMed Central

Moussavi-Harami, S. Farshid; Annis, Douglas S.; Ma, Wenjiang; Berry, Scott M.; Coughlin, Emma E.; Strotman, Lindsay N.; Maurer, Lisa M.; Westphall, Michael S.; Coon, Joshua J.; Mosher, Deane F.; Beebe, David J.

2013-01-01

Fibronectin (Fn) is a large glycoprotein present in plasma and extracellular matrix and is important for many processes. Within Fn the 70kDa N-terminal region (70k-Fn) is involved in cell-mediated Fn assembly, a process that contributes to embryogenesis, development, and platelet thrombus formation. In addition, major human pathogens including Staphlycoccus aureus and Streptococcus pyogenes, bind the 70k-Fn region by a novel form of protein-protein interaction called β-zipper formation, facilitating bacterial spread and colonization. Knowledge of blood plasma and platelet proteins that interact with 70k-Fn by β-zipper formation is incomplete. In the current study, we aimed to characterize these proteins through affinity purification. For this affinity purification, we used a novel purification technique termed immiscible filtration assisted by surface tension (IFAST). The foundation of this technology is immiscible phase filtration, using a magnet to draw paramagnetic particle (PMP)-bound analyte through an immiscible barrier (oil or organic solvent) that separates an aqueous sample from an aqueous eluting buffer. The immiscible barrier functions to remove unbound proteins via exclusion rather than dilutive washing used in traditional isolation methods. We identified 31 interactors from plasma, of which only seven were previously known to interact with Fn. Furthermore, five proteins were identified to interact with 70k-Fn from platelet lysate, of which one was previously known. These results demonstrate that IFAST offers advantages for proteomic studies of interacting molecules in that the technique requires small sample volumes, can be done with high enough throughput to sample multiple interaction conditions, and is amenable to exploratory mass spectrometric and confirmatory immuno-blotting read-outs. PMID:23750785

The aryl hydrocarbon receptor (AHR) transcription factor regulates megakaryocytic polyploidization

PubMed Central

Lindsey, Stephan; T. Papoutsakis, Eleftherios

2012-01-01

Summary We propose that the aryl hydrocarbon receptor (AHR) is a novel transcriptional regulator of megakaryopoietic polyploidization. Functional evidence was obtained that AHR impacts in vivo megakaryocytic differentiation and maturation; compared to wild-type mice, AHR-null mice had lower platelet counts, fewer numbers of newly synthesized platelets, increased bleeding times and lower-ploidy megakaryocytes (Mks). AHR mRNA increased 3·6-fold during ex vivo megakaryocytic differentiation, but reduced or remained constant during parallel isogenic granulocytic or erythroid differentiation. We interrogated the role of AHR in megakaryopoiesis using a validated Mk model of megakaryopoiesis, the human megakaryoblastic leukaemia CHRF cell line. Upon CHRF Mk differentiation, AHR mRNA and protein levels increased, AHR protein shifted from the cytoplasm to the nucleus and AHR binding to its consensus DNA binding sequence increased. Protein and mRNA levels of the AHR transcriptional target HES1 also increased. Mk differentiation of CHRF cells where AHR or HES1 was knocked-down using RNAi resulted in lower ploidy distributions and cells that were incapable of reaching ploidy classes ≥16n. AHR knockdown also resulted in increased DNA synthesis of lower ploidy cells, without impacting apoptosis. Together, these data support a role for AHR in Mk polyploidization and in vivo platelet function, and warrant further detailed investigations. PMID:21226706

The aryl hydrocarbon receptor (AHR) transcription factor regulates megakaryocytic polyploidization.

PubMed

Lindsey, Stephan; Papoutsakis, Eleftherios T

2011-02-01

We propose that the aryl hydrocarbon receptor (AHR) is a novel transcriptional regulator of megakaryopoietic polyploidization. Functional evidence was obtained that AHR impacts in vivo megakaryocytic differentiation and maturation; compared to wild-type mice, AHR-null mice had lower platelet counts, fewer numbers of newly synthesized platelets, increased bleeding times and lower-ploidy megakaryocytes (Mks). AHR mRNA increased 3·6-fold during ex vivo megakaryocytic differentiation, but reduced or remained constant during parallel isogenic granulocytic or erythroid differentiation. We interrogated the role of AHR in megakaryopoiesis using a validated Mk model of megakaryopoiesis, the human megakaryoblastic leukaemia CHRF cell line. Upon CHRF Mk differentiation, AHR mRNA and protein levels increased, AHR protein shifted from the cytoplasm to the nucleus and AHR binding to its consensus DNA binding sequence increased. Protein and mRNA levels of the AHR transcriptional target HES1 also increased. Mk differentiation of CHRF cells where AHR or HES1 was knocked-down using RNAi resulted in lower ploidy distributions and cells that were incapable of reaching ploidy classes ≥16n. AHR knockdown also resulted in increased DNA synthesis of lower ploidy cells, without impacting apoptosis. Together, these data support a role for AHR in Mk polyploidization and in vivo platelet function, and warrant further detailed investigations. © 2011 Blackwell Publishing Ltd.

Drug Target Mining and Analysis of the Chinese Tree Shrew for Pharmacological Testing

PubMed Central

Liu, Jie; Lee, Wen-hui; Zhang, Yun

2014-01-01

The discovery of new drugs requires the development of improved animal models for drug testing. The Chinese tree shrew is considered to be a realistic candidate model. To assess the potential of the Chinese tree shrew for pharmacological testing, we performed drug target prediction and analysis on genomic and transcriptomic scales. Using our pipeline, 3,482 proteins were predicted to be drug targets. Of these predicted targets, 446 and 1,049 proteins with the highest rank and total scores, respectively, included homologs of targets for cancer chemotherapy, depression, age-related decline and cardiovascular disease. Based on comparative analyses, more than half of drug target proteins identified from the tree shrew genome were shown to be higher similarity to human targets than in the mouse. Target validation also demonstrated that the constitutive expression of the proteinase-activated receptors of tree shrew platelets is similar to that of human platelets but differs from that of mouse platelets. We developed an effective pipeline and search strategy for drug target prediction and the evaluation of model-based target identification for drug testing. This work provides useful information for future studies of the Chinese tree shrew as a source of novel targets for drug discovery research. PMID:25105297

[Experimental research on the effects of different activators on the formation of platelet-rich gel and the release of bioactive substances in human platelet-rich plasma].

PubMed

Yang, Y; Zhang, W; Cheng, B

2017-01-20

Objective: To explore the effects of calcium gluconate and thrombin on the formation of platelet-rich gel (PRG) and the release of bioactive substances in human platelet-rich plasma (PRP) and the clinical significance. Methods: Six healthy blood donors who met the inclusion criteria were recruited in our unit from May to August in 2016. Platelet samples of each donor were collected for preparation of PRP. (1) PRP in the volume of 10 mL was collected from each donor and divided into thrombin activation group (TA, added with 0.5 mL thrombin solution in dose of 100 U/mL) and calcium gluconate activation group (CGA, added with 0.5 mL calcium gluconate solution in dose of 100 g/L) according to the random number table, with 5 mL PRP in each group. Then the PRP of the two groups was activated in water bath at 37 ℃ for 1 h. The formation time of PRG was recorded, and the formation situation of PRG was observed within 1 hour of activation. After being activated for 1 h, one part of PRG was collected to observe the distribution of fibrous protein with HE staining, and another part of PRG was collected to observe platelet ultrastructure under transmission electron microscope (TEM). After being activated for 1 h, the supernatant was collected to determine the content of transforming growth factor β(1, )platelet-derived growth factor BB (PDGF-BB), vascular endothelial growth factor, basic fibroblast growth factor (bFGF), epidermal growth factor, and insulin-like growth factorⅠby enzyme-linked immunosorbent assay. (2) Another 10 mL PRP from each donor was collected and grouped as above, and the platelet suspension was obtained after two times of centrifugation and resuspension with phosphate buffered saline, respectively. And then they were treated with corresponding activator for 1 h as that in experiment (1). Nanoparticle tracking analyzer was used to detect the concentrations of microvesicles with different diameters and total microvesicles derived from platelet. Data were processed with t test. Results: (1) The formation time of PRG in group TA was (228±40) s, and the PRG volume reached the maximum at this moment. The PRG volume shrunk to the minimum after 30 minutes of activation. The formation time of PRG in group CGA was (690±71) s, and the PRG volume reached the maximum at this moment. After 55 minutes of activation, the PRG volume shrunk to the minimum. The formation time of PRG in group TA was obviously shorter than that in group CGA ( t =15.17, P <0.01). (2) HE staining showed that after 1 hour of activation, the red-stained area of fibrous protein in PRG of group TA was large and densely distributed, while that of group CGA was small and loosely distributed. TEM revealed that after 1 hour of activation, the platelets in PRG of group TA were fragmented, while lysing platelet structure, lysing α granule structure, intact α granule structure, and intact dense body structure were observed in PRG of group CGA. (3) The content of PDGF-BB released by PRP in group TA was (7.4±0.8) ng/mL, which was obviously higher than that in group CGA [(4.9±0.5) ng/mL, t =5.41, P <0.01]. The content of bFGF released by PRP in group CGA was (960±151) pg/mL, which was significantly higher than that in group TA [(384±56) pg/mL, t =8.75, P <0.01]. The content of the other 4 growth factors released by PRP in the two groups was close (with t values from 0.11 to 1.97, P values above 0.05). (4) The concentrations of total microvesicles, microvesicles with diameter more than 100 nm, and exosomes with diameter less than or equal to 100 nm derived from platelet in group CGA were (165.8±15.1)×10(8)/mL, (142.4±12.3)×10(8)/mL, and (23.4±2.9)×10(8)/mL respectively, which were significantly higher than those in group TA [(24.7±4.6)×10(8)/mL, (22.6±4.0)×10(8)/mL, and (2.1±0.7)×10(8)/mL, with t values from 17.36 to 22.66, P values below 0.01]. Conclusions: Calcium gluconate can slowly activate PRP, resulting in slowly shrunk PRG with high content of bFGF and high concentration of microvesicles, which is suitable for repairing articular cavity and sinus tract wound. Thrombin can rapidly activate PRP, resulting in quickly shrunk PRG with high content of PDGF-BB and a certain concentration of microvesicles, which is suitable for repairing acute trauma.

Platelet Vascular Endothelial Growth Factor is a Potential Mediator of Transfusion-Related Acute Lung Injury.

PubMed

Maloney, James P; Ambruso, Daniel R; Voelkel, Norbert F; Silliman, Christopher C

The occurrence of non-hemolytic transfusion reactions is highest with platelet and plasma administration. Some of these reactions are characterized by endothelial leak, especially transfusion related acute lung injury (TRALI). Elevated concentrations of inflammatory mediators secreted by contaminating leukocytes during blood product storage may contribute to such reactions, but platelet-secreted mediators may also contribute. We hypothesized that platelet storage leads to accumulation of the endothelial permeability mediator vascular endothelial growth factor (VEGF), and that intravascular administration of exogenous VEGF leads to extensive binding to its lung receptors. Single donor, leukocyte-reduced apheresis platelet units were sampled over 5 days of storage. VEGF protein content of the centrifuged supernatant was determined by ELISA, and the potential contribution of VEGF from contaminating leukocytes was quantified. Isolated-perfused rat lungs were used to study the uptake of radiolabeled VEGF administered intravascularly, and the effect of unlabeled VEGF on lung leak. There was a time-dependent release of VEGF into the plasma fraction of the platelet concentrates (62 ± 9 pg/ml on day one, 149 ± 23 pg/ml on day 5; mean ± SEM, p<0.01, n=8) and a contribution by contaminating leukocytes was excluded. Exogenous 125I-VEGF bound avidly and specifically to the lung vasculature, and unlabeled VEGF in the lung perfusate caused vascular leak. Rising concentrations of VEGF occur during storage of single donor platelet concentrates due to platelet secretion or disintegration, but not due to leukocyte contamination. Exogenous VEGF at these concentrations rapidly binds to its receptors in the lung vessels. At higher VEGF concentrations, VEGF causes vascular leak in uninjured lungs. These data provide further evidence that VEGF may contribute to the increased lung permeability seen in TRALI associated with platelet products.

The Mediation of Platelet Quiescence by NO-Releasing Polymers via cGMP-Induced Serine 239 Phosphorylation of Vasodilator-Stimulated Phosphoprotein

PubMed Central

Major, Terry C; Handa, Hitesh; Brisbois, Elizabeth J; Reynolds, Melissa M; Annich, Gail M; Meyerhoff, Mark E; Bartlett, Robert H

2013-01-01

Nitric oxide (NO) releasing (NORel) materials have been shown to create localized increases in NO concentration by the release of NO from a diazeniumdiaolate-containing or S-nitrosothiol-containing polymer coating and the improvement of extracorporeal circulation (ECC) hemocompatibility. However, the mechanism and, in particular, the platelet upregulation of the NO/cGMP signaling protein, vasodilator-stimulated phosphoprotein phosphorylated at serine 239 (P-VASP (ser 239), for the improved ECC hemocompatibility via NO release still needs elucidation. In this work, two NORel polymeric coatings were evaluated in a 4 h rabbit thrombogenicity (RT) model and the anti-thrombotic mechanism investigated for rabbit platelet P-VASP upregulation. Polymer films containing 25 wt% diazeniumdiolated dibutylhexansdiamine (DBHD) or 5 wt% S-nitroso-N-acetylpenicillamine (SNAP) coated on the inner walls of ECC circuits yielded significantly reduced ECC thrombus formation and maintained normal platelet aggregation compared to polymer controls after 4 h of blood exposure. Platelet P-VASP (ser 239), a useful tool to monitor NO/cGMP signaling, was upregulated after 4 h on ECC and markedly increased after ex vivo sodium nitroprusside (SNP) stimulation. Interestingly, in the rabbit platelet, NO did not upregulate the cAMP P-VASP phosphoprotein P-VASP (ser 157) as previously shown in human platelets. These results suggest that NORel polymers preserve rabbit platelet quiescence by sustainng a level of cGMP signaling as monitored by P-VASP (ser 239) upregulation. The upregulation of this NO-mediated platelet signaling mechanism in this RT model indicates the potential for improved thromboresistance of any NORel-coated medical device. PMID:23906514

The trans-sialidase from Trypanosoma cruzi induces thrombocytopenia during acute Chagas' disease by reducing the platelet sialic acid contents.

PubMed

Tribulatti, María Virginia; Mucci, Juan; Van Rooijen, Nico; Leguizamón, María Susana; Campetella, Oscar

2005-01-01

Strong thrombocytopenia is observed during acute infection with Trypanosoma cruzi, the parasitic protozoan agent of American trypanosomiasis or Chagas' disease. The parasite sheds trans-sialidase, an enzyme able to mobilize the sialyl residues on cell surfaces, which is distributed in blood and is a virulence factor. Since the sialic acid content on the platelet surface is crucial for determining the half-life of platelets in blood, we examined the possible involvement of the parasite-derived enzyme in thrombocytopenia induction. We found that a single intravenous injection of trans-sialidase into naive mice reduced the platelet count by 50%, a transient effect that lasted as long as the enzyme remained in the blood. CD43(-/-) mice were affected to a similar extent. When green fluorescent protein-expressing platelets were treated in vitro with trans-sialidase, their sialic acid content was reduced together with their life span, as determined after transfusion into naive animals. No apparent deleterious effect on the bone marrow was observed. A central role for Kupffer cells in the clearance of trans-sialidase-altered platelets was revealed after phagocyte depletion by administration of clodronate-containing liposomes and splenectomy. Consistent with this, parasite strains known to exhibit more trans-sialidase activity induced heavier thrombocytopenia. Finally, the passive transfer of a trans-sialidase-neutralizing monoclonal antibody to infected animals prevented the clearance of transfused platelets. Results reported here strongly support the hypothesis that the trans-sialidase is the virulence factor that, after depleting the sialic acid content of platelets, induces the accelerated clearance of the platelets that leads to the thrombocytopenia observed during acute Chagas' disease.

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.

Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming

PubMed Central

Moreau, Thomas; Evans, Amanda L.; Vasquez, Louella; Tijssen, Marloes R.; Yan, Ying; Trotter, Matthew W.; Howard, Daniel; Colzani, Maria; Arumugam, Meera; Wu, Wing Han; Dalby, Amanda; Lampela, Riina; Bouet, Guenaelle; Hobbs, Catherine M.; Pask, Dean C.; Payne, Holly; Ponomaryov, Tatyana; Brill, Alexander; Soranzo, Nicole; Ouwehand, Willem H.; Pedersen, Roger A.; Ghevaert, Cedric

2016-01-01

The production of megakaryocytes (MKs)—the precursors of blood platelets—from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 105 mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology. PMID:27052461

Off-target effect of the Epac agonist 8-pCPT-2'-O-Me-cAMP on P2Y12 receptors in blood platelets.

PubMed

Herfindal, Lars; Nygaard, Gyrid; Kopperud, Reidun; Krakstad, Camilla; Døskeland, Stein Ove; Selheim, Frode

2013-08-09

The primary target of the cAMP analogue 8-pCPT-2'-O-Me-cAMP is exchange protein directly activated by cAMP (Epac). Here we tested potential off-target effects of the Epac activator on blood platelet activation signalling. We found that the Epac analogue 8-pCPT-2'-O-Me-cAMP inhibits agonist-induced-GPCR-stimulated, but not collagen-stimulated, P-selectin surface expression on Epac1 deficient platelets. In human platelets, 8-pCPT-2'-O-Me-cAMP inhibited P-selectin expression elicited by the PKC activator PMA. This effect was abolished in the presence of the extracellular ADP scavenger system CP/CPK. In silico modelling of 8-pCPT-2'O-Me-cAMP binding into the purinergic platelet receptor P2Y12 revealed that the analogue docks similar to the P2Y12 antagonist 2MeSAMP. The 8-pCPT-2'-O-Me-cAMP analogue per se, did not provoke Rap 1 (Rap 1-GTP) activation or phosphorylation on the vasodilator-stimulated phosphoprotein (VASP) at Ser-157. In addition, the protein kinase A (PKA) antagonists Rp-cAMPS and Rp-8-Br-cAMPS failed to block the inhibitory effect of 8-pCPT-2'-O-Me-cAMP on thrombin- and TRAP-induced Rap 1 activation, thus suggesting that PKA is not involved. We conclude that the 8-pCPT-2'-O-Me-cAMP analogue is able to inhibit agonist-induced-GPCR-stimulated P-selectin independent from Epac1; the off-target effect of the analogue appears to be mediated by antagonistic P2Y12 receptor binding. This has implications when using cAMP analogues on specialised system involving such receptors. We found, however that the Epac agonist 8-Br-2'-O-Me-cAMP did not affect platelet activation at similar concentrations. Copyright © 2013 Elsevier Inc. All rights reserved.

Effects of Pharmacologic Intervention on Oxygenation, Lung Water and Protein Leak in the Pseudomonas ARDS Porcine Model

DTIC Science & Technology

1989-07-01

model indicate that Pseudomonas primes these cells to produce superoxide anion at a much higher rate post-injury than pre-injury, thus implicating...as oxygen free radicals, the neutrophils, platelets, monocytes and lymphocytes can release a number of other factors which have an affect on...mediator in the lung injury seen with endotoxin induced ARDS is the inappropriately named platelet activating factor (PAF). This phospholipid, which is

Potential applications of venom peptides as anti-thrombotic agents in arterial & deep-vein thrombosis.

PubMed

Khan, Samiullah; Gul, Aqsa; Noreen, Rabia; Ashraf, Muhammad; Ahmad, Sohail; Awan, Sattar Bakhsh

2018-06-13

Thrombus is composed of two main substances i.e. red blood cells and aggregated platelets which make a web of inter-connected fibrin proteins. During injury it prevents bleeding, so it is very useful but it can be very dangerous if it is produced in healthy blood vessels and block the blood flow through it. Mural thrombi attaches with the blood vessels but in most cases do not block it completely. Venoms are an incredible source of peptides having amazing bioactivities with varying number of amino acid residues. Anticoagulant venom peptides however inhibit the enzyme taking part in coagulation like factor Xa and thrombin. The anticoagulant potential of venom peptides have also been reported by the degradation of the fibrin or fibrinogen related to serine or metalloproteases. Designing and development of numerous therapeutic agents or lead molecules mostly for cardiovascular diseases have been motivated from toxins/proteins from snake venoms. For example, disintegrins, a large family of platelet aggregation inhibitors found in viperid and crotalid snake venoms were the basis for designing of platelet aggregation inhibitors such as eptifibatide and tirofiban. Ancrod isolated from Malayan pit viper venom can cause reduction in level of blood fibrinogen and has been effectively tried in various ischemic conditions, including stroke. In order to search for novel lead molecules, the emphasis should be on isolation and characterization of pharmacologically active snake venoms proteins affecting blood coagulation and platelet aggregation. In this review an attempt has been made to recapitulates and discuss venoms of different animals and arthropod having anticoagulant peptides for their potential use in therapeutics and diagnostics. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The Effect of Brief Exposure to Sub-Therapeutic Concentrations of Chlorhexidine Digluconate on the Susceptibility of Staphylococci to Platelet Microbicidal Protein.

PubMed

Ivanov, Iuri B; Gritsenko, Viktor A; Kuzmin, Michael D

2015-06-01

Antiseptic agents are widely used in hospitals and are essential when prevention and control of nosocomial infections is required. It is necessary to consider several aspects that affect the biocide activity because they have direct impact on the nosocomial infection rate. Organisms belonging to the Staphylococcus genus are involved in such infections and chlorhexidine digluconate (CHXD) is one of the most used antiseptic agents for human and animal health. In the context of such infections, anti-bacterial peptides have been isolated from platelets and have been termed platelet microbicidal proteins (PMP). Platelet microbicidal proteins have been shown to enhance the bacterial inhibitory activities of sub-therapeutic concentrations of antibiotics. The main objective of this study was to investigate the effect of brief exposure to different sub-therapeutic concentrations of CHXD on the susceptibility of staphylococci to PMP. The influence of brief exposure to three different sub-therapeutic concentrations of CHXD (0.005%, 0.0025%, and 0.00125%) on the subsequent staphylocidal effect of PMP was evaluated. Among all clinical staphylococcal strains studied, all isolates were considered to be resistant to the bactericidal action of PMP. Exposure of staphylococci to CHXD prior to PMP resulted in significantly increased staphylococcal killing compared with the killing achieved with PMP alone. This enhanced effect was most marked for concentrations of CHXD of 0.005%. The combined data indicate that PMP exerts cooperative bactericidal effect with CHXD. The anti-staphylococcal PMP and CHXD synergistic activity in vitro demonstrated in the present study make these molecules potentially useful for preventing endovascular catheter-associated infections. Future research based on animal and human models is needed to elucidate the in vivo efficacies and toxicities and utility in clinical practice.

Biomimetic hybrid porous scaffolds immobilized with platelet derived growth factor-BB promote cellularization and vascularization in tissue engineering.

PubMed

Murali, Ragothaman; Ponrasu, Thangavel; Cheirmadurai, Kalirajan; Thanikaivelan, Palanisamy

2016-02-01

Development of hybrid scaffolds with synergistic combination of growth factor is a promising approach to promote early in vivo wound repair and tissue regeneration. Here, we show the rapid wound healing in Wistar albino rats using biomimetic collagen-poly(dialdehyde) guar gum based hybrid porous scaffolds covalently immobilized with platelet derived growth factor-BB. The immobilized platelet derived growth factor in the hybrid scaffolds not only enhance the total protein, collagen, hexosamine, and uronic acid contents in the granulation tissue but also provide stronger tissues. The wound closure analysis reveal that the complete epithelialization period is 15.4 ± 0.9 days for collagen-poly(dialdehyde) guar gum-platelet derived growth factor hybrid scaffolds, whereas it is significantly higher for control, collagen, collagen- poly(dialdehyde) guar gum and povidine-iodine treated groups. Further, the histological evaluation shows that the immobilized platelet derived growth factor in the hybrid scaffolds induced a more robust cellular and vascular response in the implanted site. Hence, we demonstrate that the collagen-poly(dialdehyde) guar gum hybrid scaffolds loaded with platelet derived growth factor stimulates chemotactic effects in the implanted site to promote rapid tissue regeneration and wound repair without the assistance of antibacterial agents. © 2015 Wiley Periodicals, Inc.

A rare case of bleeding disorder: Glanzmann's thrombasthenia.

PubMed

Swathi, Jami; Gowrishankar, A; Jayakumar, S A; Jain, Karun

2017-01-01

Glanzmann's thrombasthenia (GT) is a rare bleeding disorder, which is characterized by a lack of platelet aggregation. It is characterized by qualitative or quantitative abnormalities of the platelet membrane glycoprotein IIb/IIIa. Physiologically, this platelet receptor normally binds several adhesive plasma proteins, and this facilitates attachment and aggregation of platelets to ensure thrombus formation at sites of vascular injury. The lack of resultant platelet aggregation in GT leads to mucocutaneous bleeding whose manifestation may be clinically variable, ranging from easy bruising to severe and potentially life-threatening hemorrhages. To highlight this rare but potentially life-threating disorder, GT. We report a case of GT that was first detected because of the multiple episodes of gum bleeding. The patient was an 18-year-old girl who presented with a history of repeated episodes of gum bleeding since childhood. Till the first visit to our hospital, she had not been diagnosed with GT despite a history of bleeding tendency, notably purpura in areas of easy bruising, gum bleeding, and prolonged bleeding time after abrasions and insect stings. GT was diagnosed on the basis of prolonged bleeding time, lack of platelet aggregation with adenosine di phosphate, epinephrine and collagen. GT should always be considered as differential diagnosis while evaluating any case of bleeding disorder.

A Rare Case of Bleeding Disorder: Glanzmann's Thrombasthenia

PubMed Central

Swathi, Jami; Gowrishankar, A.; Jayakumar, S. A.; Jain, Karun

2017-01-01

Background: Glanzmann's thrombasthenia (GT) is a rare bleeding disorder, which is characterized by a lack of platelet aggregation. It is characterized by qualitative or quantitative abnormalities of the platelet membrane glycoprotein IIb/IIIa. Physiologically, this platelet receptor normally binds several adhesive plasma proteins, and this facilitates attachment and aggregation of platelets to ensure thrombus formation at sites of vascular injury. The lack of resultant platelet aggregation in GT leads to mucocutaneous bleeding whose manifestation may be clinically variable, ranging from easy bruising to severe and potentially life-threatening hemorrhages. Objective: To highlight this rare but potentially life-threating disorder, GT. Case Report: We report a case of GT that was first detected because of the multiple episodes of gum bleeding. The patient was an 18-year-old girl who presented with a history of repeated episodes of gum bleeding since childhood. Till the first visit to our hospital, she had not been diagnosed with GT despite a history of bleeding tendency, notably purpura in areas of easy bruising, gum bleeding, and prolonged bleeding time after abrasions and insect stings. GT was diagnosed on the basis of prolonged bleeding time, lack of platelet aggregation with adenosine di phosphate, epinephrine and collagen. Conclusion: GT should always be considered as differential diagnosis while evaluating any case of bleeding disorder. PMID:29063905

Identification and Characterization of Novel Variations in Platelet G-Protein Coupled Receptor (GPCR) Genes in Patients Historically Diagnosed with Type 1 von Willebrand Disease.

PubMed

Stockley, Jacqueline; Nisar, Shaista P; Leo, Vincenzo C; Sabi, Essa; Cunningham, Margaret R; Eikenboom, Jeroen C; Lethagen, Stefan; Schneppenheim, Reinhard; Goodeve, Anne C; Watson, Steve P; Mundell, Stuart J; Daly, Martina E

2015-01-01

The clinical expression of type 1 von Willebrand disease may be modified by co-inheritance of other mild bleeding diatheses. We previously showed that mutations in the platelet P2Y12 ADP receptor gene (P2RY12) could contribute to the bleeding phenotype in patients with type 1 von Willebrand disease. Here we investigated whether variations in platelet G protein-coupled receptor genes other than P2RY12 also contributed to the bleeding phenotype. Platelet G protein-coupled receptor genes P2RY1, F2R, F2RL3, TBXA2R and PTGIR were sequenced in 146 index cases with type 1 von Willebrand disease and the potential effects of identified single nucleotide variations were assessed using in silico methods and heterologous expression analysis. Seven heterozygous single nucleotide variations were identified in 8 index cases. Two single nucleotide variations were detected in F2R; a novel c.-67G>C transversion which reduced F2R transcriptional activity and a rare c.1063C>T transition predicting a p.L355F substitution which did not interfere with PAR1 expression or signalling. Two synonymous single nucleotide variations were identified in F2RL3 (c.402C>G, p.A134 =; c.1029 G>C p.V343 =), both of which introduced less commonly used codons and were predicted to be deleterious, though neither of them affected PAR4 receptor expression. A third single nucleotide variation in F2RL3 (c.65 C>A; p.T22N) was co-inherited with a synonymous single nucleotide variation in TBXA2R (c.6680 C>T, p.S218 =). Expression and signalling of the p.T22N PAR4 variant was similar to wild-type, while the TBXA2R variation introduced a cryptic splice site that was predicted to cause premature termination of protein translation. The enrichment of single nucleotide variations in G protein-coupled receptor genes among type 1 von Willebrand disease patients supports the view of type 1 von Willebrand disease as a polygenic disorder.

[Distribution diversity of integrins and calcium channels on major human and mouse host cells of Leptospira species].

PubMed

Li, Cheng-xue; Zhao, Xin; Qian, Jing; Yan, Jie

2012-07-01

To determine the distribution of integrins and calcium channels on major human and mouse host cells of Leptospira species. The expression of β1, β2 and β3 integrins was detected with immunofluorescence assay on the surface of human monocyte line THP-1, mouse mononuclear-macrophage-like cell line J774A.1, human vascular endothelial cell line HUVEC, mouse vascular endothelial cell EOMA, human hepatocyte line L-02, mouse hepatocyte line Hepa1-6, human renal tubular epithelial cell line HEK-293, mouse glomerular membrane epithelial cell line SV40-MES13, mouse collagen blast line NIH/3T3, human and mouse platelets. The distribution of voltage gate control calcium channels Cav3.1, Cav3.2, Cav3.3 and Cav2.3, and receptor gate calcium channels P(2)X(1), P(2)2X(2), P(2)X(3), P(2)X(4), P(2)X(5), P(2)X(6) and P(2)X(7) were determined with Western blot assay. β1 integrin proteins were positively expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, L-02, Hepa1-6 and HEK-239 cells as well as human and mouse platelets. β2 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, and NIH/3T3 cells. β3 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, Hepa1-6, HEK-239 and NIH/3T3 cells as well as human and mouse platelets. P(2)X(1) receptor gate calcium channel was expressed on the membrane surface of human and mouse platelets, while P(2)X(5) receptor gate calcium channel was expressed on the membrane surface of J774A.1, THP-1, L-02, Hepa1-6, HEK-239 and HUVEC cells. However, the other calcium channels were not detected on the tested cell lines or platelets. There is a large distribution diversity of integrins and calcium channel proteins on the major human and mouse host cells of Leptospira species, which may be associated with the differences of leptospira-induced injury in different host cells.

Effects of astaxanthin on blood coagulation, fibrinolysis and platelet aggregation in hyperlipidemic rats.

PubMed

Deng, Zu-Yue; Shan, Wei-Guang; Wang, Shen-Feng; Hu, Meng-Mei; Chen, Yan

2017-12-01

Astaxanthin (ASTX) is a xanthophyll carotenoid that reduces hemostasis in hyperlipidemic organisms. Its antihemostatic mechanisms remain unclear. The effects of ASTX on coagulation, the fibrinolytic system and platelet aggregation were investigated in hyperlipidemic rats. Different doses of ASTX (5, 10 and 30 mg/kg/day, p.o.) were administered for four weeks to high-fat diet-induced hyperlipidemic rats. Serum lipid and lipoprotein levels were measured with an automatic biochemical analyzer. The prothrombin time (PT), activated partial thromboplastin time (APTT) and maximum platelet aggregation rate (MAR) were determined by a coagulation analyzer. The activities of the tissue-type plasminogen activator (t-PA), type-1 plasminogen activator inhibitor (PAI-1) and endothelial nitric oxide synthase (eNOS), as well as the levels of thromboxane B(2) [TXB(2)], 6-keto prostaglandin F(1α) [6-keto-PGF(1α)] and platelet granule membrane protein (GMP-140), were measured with enzyme-linked immunosorbent assay kits. Gene and protein expression levels were analyzed by reverse transcriptase polymerase chain reaction and Western blot, respectively. ASTX (30 mg/kg) treatment in hyperlipidemic rats reduced serum TG (0.58 ± 0.14 versus 1.12 ± 0.24 mmol/L), serum TC (1.77 ± 0.22 versus 2.24 ± 0.21 mmol/L), serum LDL-C (1.13 ± 0.32 versus 2.04 ± 0.48 mmol/L), serum MDA (69%), plasma MAR (55%), serum TXB2/6-keto-PGF1α (34%) and serum GMP-140 levels (25%), plasma PAI-1 activity (48%) and downregulated the mRNA (33%) and protein (23%) expression of aorta eNOS, the mRNA (79%) and protein (72%) expression levels of aorta PAI-1. However, ASTX (30 mg/kg/d) treatment increased serum SOD activity (2.1 fold), serum GPx activity (1.8 fold), plasma PT (1.3 fold), plasma APTT (1.7 fold), serum NO (1.4-fold), serum 6-keto-PGF1α (1.3 fold). ASTX reduced blood coagulation and platelet aggregation and promoted fibrinolytic activity in hyperlipidemic rats. These activities were closely correlated with ASTX, maintaining the balance of t-PA/PAI-1, NO/ROS and TXA2/PGI2 in vivo.

Synergistic effects of FGF-2 and PDGF-BB on angiogenesis and muscle regeneration in rabbit hindlimb ischemia model.

PubMed

Li, Jie; Wei, Yuquan; Liu, Kang; Yuan, Chuang; Tang, Yajuan; Quan, Qingli; Chen, Ping; Wang, Wei; Hu, Huozhen; Yang, Li

2010-07-01

Combinatorial strategy has been used in therapeutic angiogenesis in animal models of peripheral arterial disease (PAD) and coronary artery disease for decades. Previous studies have shown that basic fibroblast growth factor (FGF-2) and platelet-derived growth factor BB (PDGF-BB) proteins together establish functional and stable vascular networks on mouse corneal and also in animal model of hindlimb ischemia. However, the short half life of protein by single injection is not sufficient to achieve effective dosage, repeated and prolonged injection causes systemic toxicity. Here we study the synergistic effects of FGF-2 and PDGF-BB by intramuscular injection of naked plasmid DNA on therapeutic angiogenesis in rabbit model of hindlimb ischemia. We found that transient delivery of FGF-2 and PDGF-BB naked DNA together resulted in greater increases in capillary growth, collateral formation and popliteal blood flow compared with control and single gene delivery. Our data provided novel evidence of beneficial effects of DNA-based FGF-2 and PDFG-BB on muscle repair after ischemic injury. These findings reveal an alternative therapeutic approach in the treatment of ischemic diseases and even in muscular disorders. Copyright 2010. Published by Elsevier Inc.

Platelet Function in Basset Hound Hereditary Thrombopathy.

DTIC Science & Technology

1986-01-01

4021, 1975. 20. DZANDU, J.K., DEH, M.E., BARRETT, D.L., AND WISE, G.E. Detec- tion of erythrocyte membrane proteins, sialoproteins , and lipids in the...Wise GE. Detection of erythrocyte membrane proteins, sialoproteins , and lipids in the same polyacrylamide gel using a double staining technique. Proc

Augmenting tendon and ligament repair with platelet-rich plasma (PRP)

PubMed Central

Yuan, Ting; Zhang, Chang-Qing; Wang, James H-C.

2013-01-01

Summary Tendon and ligament injuries (TLI) commonly occur in athletes and non-athletes alike, and remarkably debilitate patients’ athletic and personal abilities. Current clinical treatments, such as reconstruction surgeries, do not adequately heal these injuries and often result in the formation of scar tissue that is prone to re-injury. Platelet-rich plasma (PRP) is a widely used alternative option that is also safe because of its autologous nature. PRP contains a number of growth factors that are responsible for its potential to heal TLIs effectively. In this review, we provide a comprehensive report on PRP. While basic science studies in general indicate the potential of PRP to treat TLIs effectively, a review of existing literature on the clinical use of PRP for the treatment of TLIs indicates a lack of consensus due to varied treatment outcomes. This suggests that current PRP treatment protocols for TLIs may not be optimal, and that not all TLIs may be effectively treated with PRP. Certainly, additional basic science studies are needed to develop optimal treatment protocols and determine those TLI conditions that can be treated effectively. PMID:24367773

Surface modification of CoCr alloy using varying concentrations of phosphoric and phosphonoacetic acids: albumin and fibrinogen adsorption, platelet adhesion, activation, and aggregation studies.

PubMed

Thiruppathi, Eagappanath; Larson, Mark K; Mani, Gopinath

2015-01-01

CoCr alloy is commonly used in various cardiovascular medical devices for its excellent physical and mechanical properties. However, the formation of blood clots on the alloy surfaces is a serious concern. This research is focused on the surface modification of CoCr alloy using varying concentrations (1, 25, 50, 75, and 100 mM) of phosphoric acid (PA) and phosphonoacetic acid (PAA) to generate various surfaces with different wettability, chemistry, and roughness. Then, the adsorption of blood plasma proteins such as albumin and fibrinogen and the adhesion, activation, and aggregation of platelets with the various surfaces generated were investigated. Contact angle analysis showed PA and PAA coatings on CoCr provided a gradient of hydrophilic surfaces. FTIR showed PA and PAA were covalently bound to CoCr surface and formed different bonding configurations depending on the concentrations of coating solutions used. AFM showed the formation of homogeneous PA and PAA coatings on CoCr. The single and dual protein adsorption studies showed that the amount of albumin and fibrinogen adsorbed on the alloy surfaces strongly depend on the type of PA and PAA coatings prepared by different concentrations of coating solutions. All PA coated CoCr showed reduced platelet adhesion and activation when compared to control CoCr. Also, 75 and 100 mM PA-CoCr showed reduced platelet aggregation. For PAA coated CoCr, no significant difference in platelet adhesion and activation was observed between PAA coated CoCr and control CoCr. Thus, this study demonstrated that CoCr can be surface modified using PA for potentially reducing the formation of blood clots and improving the blood compatibility of the alloy.

Different translocation and calcium dependence of protein kinase C isoforms induced by phorbol ester in human platelets

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

Crabos, M.; Fabbro, D.; Imber, R.

1991-03-11

Protein kinase C (PKC) is an important intraplatelet second messenger which is activated and translocated from cytosol to membrane in response to extracellular stimuli. Molecular cloning revealed that PKC represents a family of closely related subspecies. Immunoblot analysis using monoclonal antibodies specific for {alpha}, {beta}, and {gamma} and polyclonal antibodies specific for the {delta}, {epsilon}, and {zeta} subspecies revealed the presence of {alpha}, {beta}, and {zeta} isoforms in human platelets. The subcellular distribution of {alpha}, {beta} and {zeta} in resting state was in the range of 80% in cytosol and 20% in membrane. After 2 min incubation of platelets withmore » 300 nM TPA there was an increase of 10% of {beta} and {zeta} subspecies in membrane whereas incubation after one hour incubation with TPA about 70% of all isoforms were associated with the membrane. Incubation of platelets with 1mM of CaCl{sub 2} for 10 min prior to stimulation with 100 nM TPA for 30 min resulted in an increase in the membrane of: 31{plus minus}1 for {alpha}, 30{plus minus}1 for {beta} and 36{plus minus}6 for {zeta}, while in the presence of 1mM EDTA the increase was 14{plus minus}2 for {alpha}, 28{plus minus}1 for {beta} and 34{plus minus}1 for {zeta} (mean %{plus minus}sem). These results demonstrate the presence of three different subtypes of PKC in human platelets which display different time courses of translocation and different sensitivity to external calcium with respect to TPA. This suggest that these isoforms can be activated differently with hormones and may be involved in different intracellular pathways.« less

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.

Dual Therapy with Aspirin and Cilostazol May Improve Platelet Aggregation in Noncardioembolic Stroke Patients: A Pilot Study.

PubMed

Ohnuki, Yoichi; Ohnuki, Yuko; Kohara, Saori; Shimizu, Mie; Takizawa, Shunya

2017-01-01

Objective Some previous studies have found clinical benefit of dual antiplatelet therapy with aspirin and cilostazol for prevention of secondary stroke, but the physiological mechanism involved remains unknown. We aimed to clarify the effects of aspirin/cilostazol therapy on the platelet and endothelial functions of patients with acute noncardioembolic ischemic stroke, in comparison to patients who were treated with aspirin alone. Methods The present randomized prospective pilot study enrolled 24 patients within a week after the onset of noncardioembolic ischemic stroke. The patients were randomly allocated to receive aspirin (100 mg/day) (A group; 11 patients) or cilostazol (200 mg/day) plus aspirin (100 mg/day) (CA group; 13 patients). We measured platelet aggregation, platelet activation, and the thrombomodulin (TM), highly sensitive C-reactive protein (hs-CRP), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and von Willebrand (vWF) antigen levels and vWF activity over a 4-week period after enrollment. Results There was no significant difference in the platelet functions of the A and CA groups. However, the platelet aggregation induced by adenosine diphosphate (ADP) was decreased at 2 and 4 weeks (p<0.05) after treatment in comparison to the pre-treatment values in the CA group, but not in the A group. Platelet activation, and the hs-CRP, TM, ICAM-1, VCAM-1 and vWF values did not significantly decrease after treatment in either group. Conclusion Although there were no significant differences in platelet aggregation, platelet activation or the endothelial biomarker levels of the A and CA groups, dual therapy with aspirin and cilostazol inhibited platelet aggregation in comparison to the pre-treatment values, similarly to patients who received aspirin alone. This may suggest the clinical usefulness of dual therapy with aspirin and cilostazol in the treatment of patients with noncardioembolic ischemic stroke.

Global phenotypic characterisation of human platelet lysate expanded MSCs by high-throughput flow cytometry.

PubMed

Reis, Monica; McDonald, David; Nicholson, Lindsay; Godthardt, Kathrin; Knobel, Sebastian; Dickinson, Anne M; Filby, Andrew; Wang, Xiao-Nong

2018-03-02

Mesenchymal stromal cells (MSCs) are a promising cell source to develop cell therapy for many diseases. Human platelet lysate (PLT) is increasingly used as an alternative to foetal calf serum (FCS) for clinical-scale MSC production. To date, the global surface protein expression of PLT-expended MSCs (MSC-PLT) is not known. To investigate this, paired MSC-PLT and MSC-FCS were analysed in parallel using high-throughput flow cytometry for the expression of 356 cell surface proteins. MSC-PLT showed differential surface protein expression compared to their MSC-FCS counterpart. Higher percentage of positive cells was observed in MSC-PLT for 48 surface proteins, of which 13 were significantly enriched on MSC-PLT. This finding was validated using multiparameter flow cytometry and further confirmed by quantitative staining intensity analysis. The enriched surface proteins are relevant to increased proliferation and migration capacity, as well as enhanced chondrogenic and osteogenic differentiation properties. In silico network analysis revealed that these enriched surface proteins are involved in three distinct networks that are associated with inflammatory responses, carbohydrate metabolism and cellular motility. This is the first study reporting differential cell surface protein expression between MSC-PLT and MSC-FSC. Further studies are required to uncover the impact of those enriched proteins on biological functions of MSC-PLT.

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.

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

Improved platelet survival after cold storage by prevention of glycoprotein Ibα clustering in lipid rafts

PubMed Central

Gitz, Eelo; Koekman, Cornelis A; van den Heuvel, Dave J.; Deckmyn, Hans; Akkerman, Jan W.; Gerritsen, Hans C.; Urbanus, Rolf T.

2012-01-01

Background Storing platelets for transfusion at room temperature increases the risk of microbial infection and decreases platelet functionality, leading to out-date discard rates of up to 20%. Cold storage may be a better alternative, but this treatment leads to rapid platelet clearance after transfusion, initiated by changes in glycoprotein Ibα, the receptor for von Willebrand factor. Design and Methods: We examined the change in glycoprotein Ibα distribution using Förster resonance energy transfer by time-gated fluorescence lifetime imaging microscopy. Results Cold storage induced deglycosylation of glycoprotein Ibα ectodomain, exposing N-acetyl-Dglucosamine residues, which sequestered with GM1 gangliosides in lipid rafts. Raft-associated glycoprotein Ibα formed clusters upon binding of 14-3-3ζ adaptor proteins to its cytoplasmic tail, a process accompanied by mitochondrial injury and phosphatidyl serine exposure. Cold storage left glycoprotein Ibα surface expression unchanged and although glycoprotein V decreased, the fall did not affect glycoprotein Ibα clustering. Prevention of glycoprotein Ibα clustering by blockade of deglycosylation and 14-3-3ζ translocation increased the survival of cold-stored platelets to above the levels of platelets stored at room temperature without compromising hemostatic functions. Conclusions We conclude that glycoprotein Ibα translocates to lipid rafts upon cold-induced deglycosylation and forms clusters by associating with 14-3-3ζ. Interference with these steps provides a means to enable cold storage of platelet concentrates in the near future. PMID:22733027

Hierarchical assembly strategy and multiscale structural origin of exceptional mechanical performance in nacre

NASA Astrophysics Data System (ADS)

Huang, Zaiwang

Nacre (mother of pearl) is a self-assembled hierarchical nanocomposite in possession of exquisite multiscale architecture and exceptional mechanical properties. Previous work has shown that the highly-ordered brick-mortar-like structure in nacre is assembled via epitaxial growth and the aragonite platelets are pure single-crystals. Our results challenge this conclusion and propose that nacre's individual aragonite platelets are constructed with highly-aligned aragonite nanoparticles mediated by screw dislocation and amorphous aggregation. The underlying physics mechanism why the aragonite nanoparticles choose highly-oriented attachment as its crystallization pathway is rationalized in terms of thermodynamics. The aragonite nanoparticle order-disorder transformation can be triggered by high temperature and mechanical deformation, which in turn confirms that the aragonite nanoparticles are basic building blocks for aragonite platelets. Particularly fascinating is the fracture toughness enhancement of nacre through exquisitely collecting mechanically inferior calcium carbonate (CaCO3) and biomolecules. The sandwich-like microarchitecture with a geometrically staggered arrangement can induce crack deflection along its biopolymer interface, thus significantly enhancing nacre's fracture toughness. Our new findings ambiguously demonstrate that, aside from crack deflection, the advancing crack can invade aragonite platelet, leaving a zigzag crack propagation pathway. These unexpected experimental observations disclose, for the first time, the inevitable structural role of aragonite platelets in enhancing nacre's fracture toughness. Simultaneously, the findings that the crack propagates in a zigzag manner within individual aragonite platelets overturn the previously well-established wisdom that considers aragonite platelets as brittle single-crystals. Moreover, we investigated the dynamical mechanical response of nacre under unixial compression. Our results show that the high strain rate sensitivity reaching ˜0.1 can be directly related to the localized plastic activation volume. Nacre's hierarchical energy-dissipation mechanism under dynamic compression loading comes from a mechanical optimization derived from its inherently multiscale functional structure design.

Intracellular signaling pathways required for rat vascular smooth muscle cell migration. Interactions between basic fibroblast growth factor and platelet-derived growth factor.

PubMed Central

Bilato, C; Pauly, R R; Melillo, G; Monticone, R; Gorelick-Feldman, D; Gluzband, Y A; Sollott, S J; Ziman, B; Lakatta, E G; Crow, M T

1995-01-01

Intracellular signaling pathways activated by both PDGF and basic fibroblast growth factor (bFGF) have been implicated in the migration of vascular smooth muscle cells (VSMC), a key step in the pathogenesis of many vascular diseases. We demonstrate here that, while bFGF is a weak chemoattractant for VSMCs, it is required for the PDGF-directed migration of VSMCs and the activation of calcium/calmodulin-dependent protein kinase II (CamKinase II), an intracellular event that we have previously shown to be important in the regulation of VSMC migration. Neutralizing antibodies to bFGF caused a dramatic reduction in the size of the intracellular calcium transient normally seen after PDGF stimulation and inhibited both PDGF-directed VSMC migration and CamKinase II activation. Partially restoring the calcium transient with ionomycin restored migration and CamKinase II activation as did the forced expression of a mutant CamKinase II that had been "locked" in the active state by site-directed mutagenesis. These results suggest that bFGF links PDGF receptor stimulation to changes in intracellular calcium and CamKinase II activation, reinforcing the central role played by CamKinase II in regulating VSMC migration. Images PMID:7560082

Structure-Guided Design of a High-Affinity Platelet Integrin αIIbβ3 Receptor Antagonist That Disrupts Mg2+ Binding to the MIDAS | Center for Cancer Research

Cancer.gov

A Better Fit. An improved anticoagulant drug called RUC-2 (ball and stick structure) fits snugly into its binding pocket on integrin (blue), a protein found on the surface of platelets. RUC-2 binds both subunits of integrin, inhibiting the excessive blood coagulation that can lead to strokes and heart attacks. Unlike similar drugs that alter integrin's structure when they bind

Platelet activation is a key event in the pathogenesis of streptococcal infections.

PubMed

Jia, Ming; Xiong, Yuling; Lu, Hua; Li, Ruqing; Wang, Tiantian; Ye, Yanyao; Song, Min; Li, Bing; Jiang, Tianlun; Zhao, Shuming

2015-06-01

Diverse Streptococcus species including Streptococcus Pneumoniae, Sanguis, Gordonii, Mitis and Mutans cause life-threatening conditions including pneumonia, bacteremia and meningitis. These diseases bear a high morbidity and mortality and for this reason, understanding the key events in the pathogenesis of these infections have a great significance in their prevention and/or treatment. Here, we describe as how the activation of the platelets and their affinity to bind to bacterial proteins act as early key events in the pathogenesis of Streptococcal infections.

Kaolinite flocculation induced by smectite addition - a transmission X-ray microscopic study.

PubMed

Zbik, Marek S; Song, Yen-Fang; Frost, Ray L

2010-09-01

The influence of smectite addition on kaolinite suspensions in water was investigated by transmission X-ray microscopy (TXM) and Scanning Electron Microscopy (SEM). Sedimentation test screening was also conducted. Micrographs were processed by the STatistic IMage Analysing (STIMAN) program and structural parameters were calculated. From the results of the sedimentation tests important influences of small smectite additions to about 3wt.% on kaolinite suspension flocculation has been found. In order to determine the reason for this smectite impact on kaolinite suspension, macroscopic behaviour micro-structural examination using Transmission X-ray Microscope (TXM) and SEM has been undertaken. TXM & SEM micrographs of freeze-dried kaolinite-smectite suspensions with up to 20% smectite showed a high degree of orientation of the fabric made of highly oriented particles and greatest density when 3wt.% of smectite was added to the 10wt.% dense kaolinite suspension. In contrast, suspensions containing pure kaolinite do not show such platelet mutual orientation but homogenous network of randomly oriented kaolinite platelets. This suggests that in kaolinite-smectite suspensions, smectite forms highly oriented basic framework into which kaolinite platelets may bond in face to face preferential contacts strengthening structure and allowing them to show plastic behaviour which is cause of platelets orientation. Copyright 2010 Elsevier Inc. All rights reserved.

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

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

Human Neutrophil Peptides Mediate Endothelial-Monocyte Interaction, Foam Cell Formation, and Platelet Activation

PubMed Central

Quinn, Kieran L.; Henriques, Melanie; Tabuchi, Arata; Han, Bing; Yang, Hong; Cheng, Wei-Erh; Tole, Soumitra; Yu, Hanpo; Luo, Alice; Charbonney, Emmanuel; Tullis, Elizabeth; Lazarus, Alan; Robinson, Lisa A.; Ni, Heyu; Peterson, Blake R.; Kuebler, Wolfgang M.; Slutsky, Arthur S.; Zhang, Haibo

2016-01-01

Objective Neutrophils are involved in the inflammatory responses during atherosclerosis. Human neutrophil peptides (HNPs) released from activated neutrophils exert immune modulating properties. We hypothesized that HNPs play an important role in neutrophil-mediated inflammatory cardiovascular responses in atherosclerosis. Methods and Results We examined the role of HNPs in endothelial-leukocyte interaction, platelet activation, and foam cell formation in vitro and in vivo. We demonstrated that stimulation of human coronary artery endothelial cells with clinically relevant concentrations of HNPs resulted in monocyte adhesion and transmigration; induction of oxidative stress in human macrophages, which accelerates foam cell formation; and activation and aggregation of human platelets. The administration of superoxide dismutase or anti-CD36 antibody reduced foam cell formation and cholesterol efflux. Mice deficient in double genes of low-density lipoprotein receptor and low-density lipoprotein receptor–related protein (LRP), and mice deficient in a single gene of LRP8, the only LRP phenotype expressed in platelets, showed reduced leukocyte rolling and decreased platelet aggregation and thrombus formation in response to HNP stimulation. Conclusion HNPs exert proatherosclerotic properties that appear to be mediated through LRP8 signaling pathways, suggesting an important role for HNPs in the development of inflammatory cardiovascular diseases. PMID:21817096

Comparison of sea turtle thrombocyte aggregation to human platelet aggregation in whole blood.

PubMed

Soslau, Gerald; Prest, Phillip J; Class, Reiner; George, Robert; Paladino, Frank; Violetta, Gary

2005-11-01

The endangered sea turtles are living "fossils" that afford us an opportunity to study the hemostatic process as it likely existed millions of years ago. There are essentially no data about turtle thrombocyte aggregation prior to our studies. Thrombocytes are nucleated cells that serve the same hemostatic functions as the anucleated mammalian platelet. Sea turtle thrombocytes aggregate in response to collagen and beta-thrombin. Ristocetin induces an agglutination/aggregation response indicating the presence of a von Willebrand-like receptor, GPIb, found in all mammalian platelets. Samples treated with alpha-thrombin plus gamma-thrombin followed by ristocetin results in a rapid, stronger response than ristocetin alone. These responses are inhibited by the RGDS peptide that blocks fibrinogen cross-linking of mammalian platelets via the fibrinogen receptor, GPIIb/IIIa. Three platelet-like proteins, GPIb, GPIIb/IIIa and P-selection are detected in sea turtle thrombocytes by fluorescence activated cell sorting. Turtle thrombocytes do not respond to ADP, epinephrine, serotonin, thromboxane A2 mimetic, U46619, trypsin, or alpha-thrombin and gamma-thrombin added alone. Comparison of hemostasis in sea turtles to other vertebrates could provide a framework for understanding the structure/function and evolution of these pathways and their individual components.

Clot contraction: compression of erythrocytes into tightly packed polyhedra and redistribution of platelets and fibrin

PubMed Central

Cines, Douglas B.; Lebedeva, Tatiana; Nagaswami, Chandrasekaran; Hayes, Vincent; Massefski, Walter; Litvinov, Rustem I.; Rauova, Lubica; Lowery, Thomas J.

2014-01-01

Contraction of blood clots is necessary for hemostasis and wound healing and to restore flow past obstructive thrombi, but little is known about the structure of contracted clots or the role of erythrocytes in contraction. We found that contracted blood clots develop a remarkable structure, with a meshwork of fibrin and platelet aggregates on the exterior of the clot and a close-packed, tessellated array of compressed polyhedral erythrocytes within. The same results were obtained after initiation of clotting with various activators and also with clots from reconstituted human blood and mouse blood. Such close-packed arrays of polyhedral erythrocytes, or polyhedrocytes, were also observed in human arterial thrombi taken from patients. The mechanical nature of this shape change was confirmed by polyhedrocyte formation from the forces of centrifugation of blood without clotting. Platelets (with their cytoskeletal motility proteins) and fibrin(ogen) (as the substrate bridging platelets for contraction) are required to generate the forces necessary to segregate platelets/fibrin from erythrocytes and to compress erythrocytes into a tightly packed array. These results demonstrate how contracted clots form an impermeable barrier important for hemostasis and wound healing and help explain how fibrinolysis is greatly retarded as clots contract. PMID:24335500

Fabrication of Semiordered Nanopatterned Diamond-like Carbon and Titania Films for Blood Contacting Applications.

PubMed

Nandakumar, Deepika; Bendavid, Avi; Martin, Philip J; Harris, Kenneth D; Ruys, Andrew J; Lord, Megan S

2016-03-23

Biomaterials with the ability to interface with, but not activate, blood components are essential for a multitude of medical devices. Diamond-like carbon (DLC) and titania (TiO2) have shown promise for these applications; however, both support platelet adhesion and activation. This study explored the fabrication of nanostructured DLC and TiO2 thin film coatings using a block copolymer deposition technique that produced semiordered nanopatterns with low surface roughness (5-8 nm Rrms). These surfaces supported fibrinogen and plasma protein adsorption that predominantly adsorbed between the nanofeatures and reduced the overall surface roughness. The conformation of the adsorbed fibrinogen was altered on the nanopatterned surfaces as compared with the planar surfaces to reveal higher levels of the platelet binding region. Planar DLC and TiO2 coatings supported less platelet adhesion than nanopatterned DLC and TiO2. However, platelets on the nanopatterned DLC coatings were less spread indicating a lower level of platelet activation on the nanostructured DLC coatings compared with the planar DLC coatings. These data indicated that nanostructured DLC coatings may find application in blood contacting medical devices in the future.

Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via tissue factor activation.

PubMed

Reinhardt, Christoph; von Brühl, Marie-Luise; Manukyan, Davit; Grahl, Lenka; Lorenz, Michael; Altmann, Berid; Dlugai, Silke; Hess, Sonja; Konrad, Ildiko; Orschiedt, Lena; Mackman, Nigel; Ruddock, Lloyd; Massberg, Steffen; Engelmann, Bernd

2008-03-01

The activation of initiator protein tissue factor (TF) is likely to be a crucial step in the blood coagulation process, which leads to fibrin formation. The stimuli responsible for inducing TF activation are largely undefined. Here we show that the oxidoreductase protein disulfide isomerase (PDI) directly promotes TF-dependent fibrin production during thrombus formation in vivo. After endothelial denudation of mouse carotid arteries, PDI was released at the injury site from adherent platelets and disrupted vessel wall cells. Inhibition of PDI decreased TF-triggered fibrin formation in different in vivo murine models of thrombus formation, as determined by intravital fluorescence microscopy. PDI infusion increased - and, under conditions of decreased platelet adhesion, PDI inhibition reduced - fibrin generation at the injury site, indicating that PDI can directly initiate blood coagulation. In vitro, human platelet-secreted PDI contributed to the activation of cryptic TF on microvesicles (microparticles). Mass spectrometry analyses indicated that part of the extracellular cysteine 209 of TF was constitutively glutathionylated. Mixed disulfide formation contributed to maintaining TF in a state of low functionality. We propose that reduced PDI activates TF by isomerization of a mixed disulfide and a free thiol to an intramolecular disulfide. Our findings suggest that disulfide isomerases can act as injury response signals that trigger the activation of fibrin formation following vessel injury.

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.

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

Erythrocyte sedimentation rate and fibrinogen concentration of whole blood influences the cellular composition of platelet-rich plasma obtained from centrifugation methods.

PubMed

Yin, Wenjing; Xu, Zhengliang; Sheng, Jiagen; Xie, Xuetao; Zhang, Changqing

2017-09-01

Erythrocyte sedimentation rate (ESR), which reflects the sedimentation rate of platelets, leukocytes and erythrocytes in response to centrifugal force, may influence the cellular composition of platelet-rich plasma (PRP) obtained via centrifugation methods. However, no relevant studies have substantiated this. In the present study, blood was collected from 40 healthy volunteers and used to prepare PRP with two plasma-based preparation systems [YinPRP and Plasma Rich in Growth Factor (PRGF) systems] and two buffy coat-based systems (RegenPRP and WEGOPRP systems) in a single-donor model. Volumes of PRP and platelet-poor plasma (PPP) that were removed in the preparation process were recorded. Analyses of ESR, haematocrit, C-reaction protein, coagulation, serum glucose and serum lipid of the whole blood used for PRP preparation were performed to evaluate the levels of ESR and the factors known to influence it. Whole blood analysis was performed to evaluate the cellular composition of PRP. Results demonstrated that there were marked positive correlations between the ESR of the whole blood used for PRP preparation and PPP removal efficiencies, platelet concentrations, platelet capture efficiencies and platelet enrichment factors of PRP formulations obtained from plasma-based systems, and PRP yield efficiency of RegenPRP and PPP removal efficiency of WEGOPRP. Furthermore, there were marked negative correlations between ESR and concentrations and enrichment factors of platelets, leukocytes and erythrocytes of RegenPRP. Fibrinogen concentration of the whole blood, which had a marked positive correlation with ESR, also influenced the cellular composition of PRP. These findings may increase the understanding of PRP preparation and provide substantial evidence for the individualised optimisation of PRP preparation systems used in clinical practice.

Platelet aggregation caused by Carybdea rastonii toxins (CrTX-I, II and III) obtained from a jellyfish, Carybdea rastonii.

PubMed

Azuma, H; Sekizaki, S; Satoh, A; Nakajima, T

1986-05-01

The pharmacological mechanisms of platelet aggregation induced by highly toxic proteins (CrTX-I, CrTX-II, and CrTX-III) obtained from tentacles of a jellyfish, Carybdea rastonii, were investigated. When the partially purified toxin (pCrTX) and CrTXs were added to the citrated platelet-rich plasma (PRP), aggregation was produced in a concentration-dependent manner. The activity of CrTXs was approximately 100 times more potent than pCrTX. The CrTXs-induced aggregation was little affected by indomethacin and quinacrine at concentrations sufficient to inhibit arachidonic acid- and collagen-induced aggregation. The CrTXs-induced aggregation in washed platelets was significantly augmented in the presence of Ca2+. The pretreatment with verapamil failed to modify this augmentation of aggregation. The concentration of cytoplasmic-free calcium ([Ca2+]i) of platelets was increased by CrTXs at the same concentrations that produced aggregation. This effect of CrTXs was again little affected by verapamil. CrTXs at the same concentrations as those that produced aggregation and increased [Ca2+]i caused depolarization of platelets, which was unchanged after pretreatment with sodium or potassium transport inhibitors. CrTX-I significantly increased the 22Na flux into platelets and this effect of CrTX-I was unaffected by tetrodotoxin. The CrTX-I-induced aggregation, depolarization, and increase in [Ca2+]i were all significantly attenuated in the low Na+ medium. These results suggest that CrTXs cause a massive depolarization by increasing cation permeability and this generalized depolarization permits an inward movement of Ca2+ down its electrochemical gradient which, in turn, triggers platelet aggregation.

Can platelet-rich plasma enhance anterior cruciate ligament and meniscal repair?

PubMed

Hutchinson, Ian D; Rodeo, Scott A; Perrone, Gabriel S; Murray, Martha M

2015-02-01

The use of platelet-rich plasma (PRP) to improve clinical outcome following a soft tissue injury, regeneration, and repair has been the subject of intense investigation and discussion. This article endeavors to relate clinical and basic science strategies focused on biological augmentation of the healing response in anterior cruciate ligament (ACL) and meniscus repair and replacement using PRP. Therein, a translational feedback loop is created in the literature and targeted towards the entire multidisciplinary team. Ultimately, it is hoped that the theoretical benefits of PRP on soft-tissue interfacial healing will emerge clinically following a careful, focused characterization at the benchtop, and prospective randomized controlled clinical study. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.