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Sample records for activation platelet activation

  1. Shiga toxin binds to activated platelets.

    PubMed

    Ghosh, S A; Polanowska-Grabowska, R K; Fujii, J; Obrig, T; Gear, A R L

    2004-03-01

    Hemolytic uremic syndrome (HUS) is associated with acute renal failure in children and can be caused by Shiga toxin (Stx)-producing Escherichia coli. Thrombocytopenia and formation of renal thrombi are characteristic of HUS, suggesting that platelet activation is involved in its pathogenesis. However, whether Shiga toxin directly activates platelets is controversial. The present study evaluates if potential platelet sensitization during isolation by different procedures influences platelet interaction with Shiga toxin. Platelets isolated from sodium citrate anticoagulated blood were exposed during washing to EDTA and higher g forces than platelets prepared from acid-citrate-dextrose (ACD) plasma. Platelet binding of Stx was significantly higher in EDTA-washed preparations relative to ACD-derived platelets. Binding of Stx was also increased with ACD-derived platelets when activated with thrombin (1 U mL-1) and exposure of the Gb3 Stx receptor was detected only on platelets subjected to EDTA, higher g forces or thrombin. EDTA-exposed platelets lost their normal discoid shape and were larger. P-selectin (CD62P) exposure was significantly increased in EDTA-washed preparations relative to ACD-derived platelets, suggesting platelet activation. Taken together, these results suggest that direct binding of Stx occurs only on 'activated' platelets rather than on resting platelets. The ability of Stx to interact with previously activated platelets may be an important element in understanding the pathogenesis of HUS. PMID:15009469

  2. Platelet Activation: The Mechanisms and Potential Biomarkers

    PubMed Central

    Yun, Seong-Hoon; Sim, Eun-Hye; Goh, Ri-Young; Park, Joo-In

    2016-01-01

    Beyond hemostasis and thrombosis, an increasing number of studies indicate that platelets play an integral role in intercellular communication, mediating inflammatory and immunomodulatory activities. Our knowledge about how platelets modulate inflammatory and immunity has greatly improved in recent years. In this review, we discuss recent advances in the pathways of platelet activation and potential application of platelet activation biomarkers to diagnosis and prediction of disease states. PMID:27403440

  3. Signaling during platelet adhesion and activation

    PubMed Central

    Li, Zhenyu; Delaney, M. Keegan; O’Brien, Kelly A.; Du, Xiaoping

    2011-01-01

    Upon vascular injury, platelets are activated by adhesion to adhesive proteins like von Willebrand factor and collagen, or by soluble platelet agonists like ADP, thrombin, and thromboxane A2. These adhesive proteins and soluble agonists induce signal transduction via their respective receptors. The various receptor-specific platelet activation signaling pathways converge into common signaling events, which stimulate platelet shape change, granule secretion, and ultimately induce the “inside-out” signaling process leading to activation of the ligand binding function of integrin αIIbβ3. Ligand binding to integrin αIIbβ3 mediates platelet adhesion and aggregation and triggers “outside-in” signaling, resulting in platelet spreading, additional granule secretion, stabilization of platelet adhesion and aggregation, and clot retraction. It has become increasingly evident that agonist-induced platelet activation signals also crosstalk with integrin “outside-in” signals to regulate platelet responses. Platelet activation involves a series of rapid positive feedback loops that greatly amplify initial activation signals, and enable robust platelet recruitment and thrombus stabilization. Recent studies have provided novel insight into the molecular mechanisms of these processes. PMID:21071698

  4. Relationship between potential platelet activation and LCS

    NASA Astrophysics Data System (ADS)

    Shadden, Shawn

    2010-11-01

    In the study of blood flow, emphasis is often directed at understanding shear stress at the vessel wall due to its potentially disruptive influence on the endothelium. However, it is also known that shear stress has a potent effect on platelet activation. Platelet activation is a precursor for blood clotting, which in turn is the cause of most forms of death. Since most platelets are contained in the flow domain, it is important to consider stresses acting on the platelet as they are convected. Locations of high stress can correspond to boundaries between different dynamic regions and locations of hyperbolic points in the Eulerian sense. In the computation of LCS, strain in typically considered in the Lagrangian sense. In this talk we discuss the relationship between locations of potential platelet activation due to increased stress and locations of LCS marking increase Lagrangian deformation.

  5. Effects of platelet inhibitors on propyl gallate-induced platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activation.

    PubMed

    Xiao, Hongyan; Kovics, Richard; Jackson, Van; Remick, Daniel G

    2004-04-01

    Propyl gallate (PG) is a platelet agonist characterized by inducing platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activity. The mechanisms of platelet activation following PG stimulation were examined by pre-incubating platelets with well-defined platelet inhibitors using platelet aggregation, protein tyrosine phosphorylation, activated plasma clotting time, and annexin V binding by flow cytometry. PG-induced platelet aggregation and tyrosine phosphorylation of multiple proteins were substantially abolished by aspirin, apyrase, and abciximab (c7E3), suggesting that PG is associated with activation of platelet cyclooxygenase 1, adenosine phosphate receptors, and glycoprotein IIb/IIIa, respectively. The phosphorylation of the cytoskeletal enzyme pp60(c-src) increased following PG stimulation, but was blunted by pre-incubation of platelets with aspirin, apyrase, and c7E3, suggesting that tyrosine kinase is important for the signal transduction of platelet aggregation. Propyl gallate also activates platelet factor 3 by decreasing the platelet coagulation time and increasing platelet annexin V binding. Platelet incubation with aspirin, apyrase, and c7E3 did not alter PG-induced platelet coagulation and annexin V binding. The results suggest that platelet factor 3 activation and membrane phosphotidylserine expression were not involved with activation of platelet cyclooxygenase, adenosine phosphate receptors, and glycoprotein IIb/IIIa. PG is unique in its ability to stimulate platelet aggregation and coagulation simultaneously, and platelet inhibitors in this study affect only platelet aggregation but not platelet coagulation. PMID:15060414

  6. Titanium surface hydrophilicity enhances platelet activation.

    PubMed

    Alfarsi, Mohammed A; Hamlet, Stephen M; Ivanovski, Saso

    2014-01-01

    Titanium implant surface modification is a key strategy used to enhance osseointegration. Platelets are the first cells that interact with the implant surface whereupon they release a wide array of proteins that influence the subsequent healing process. This study therefore investigated the effect of titanium surface modification on the attachment and activation of human platelets. The surface characteristics of three titanium surfaces: smooth (SMO), micro-rough (SLA) and hydrophilic micro-rough (SLActive) and the subsequent attachment and activation of platelets following exposure to these surfaces were determined. The SLActive surface showed the presence of significant nanoscale topographical features. While attached platelets appeared to be morphologically similar, significantly fewer platelets attached to the SLActive surface compared to both the SMO and SLA surfaces. The SLActive surface however induced the release of the higher levels of chemokines β-thromboglobulin and platelet factor 4 from platelets. This study shows that titanium surface topography and chemistry have a significant effect on platelet activation and chemokine release. PMID:25311339

  7. Equid herpesvirus type 1 activates platelets.

    PubMed

    Stokol, Tracy; Yeo, Wee Ming; Burnett, Deborah; DeAngelis, Nicole; Huang, Teng; Osterrieder, Nikolaus; Catalfamo, James

    2015-01-01

    Equid herpesvirus type 1 (EHV-1) causes outbreaks of abortion and neurological disease in horses. One of the main causes of these clinical syndromes is thrombosis in placental and spinal cord vessels, however the mechanism for thrombus formation is unknown. Platelets form part of the thrombus and amplify and propagate thrombin generation. Here, we tested the hypothesis that EHV-1 activates platelets. We found that two EHV-1 strains, RacL11 and Ab4 at 0.5 or higher plaque forming unit/cell, activate platelets within 10 minutes, causing α-granule secretion (surface P-selectin expression) and platelet microvesiculation (increased small events double positive for CD41 and Annexin V). Microvesiculation was more pronounced with the RacL11 strain. Virus-induced P-selectin expression required plasma and 1.0 mM exogenous calcium. P-selectin expression was abolished and microvesiculation was significantly reduced in factor VII- or X-deficient human plasma. Both P-selectin expression and microvesiculation were re-established in factor VII-deficient human plasma with added purified human factor VIIa (1 nM). A glycoprotein C-deficient mutant of the Ab4 strain activated platelets as effectively as non-mutated Ab4. P-selectin expression was abolished and microvesiculation was significantly reduced by preincubation of virus with a goat polyclonal anti-rabbit tissue factor antibody. Infectious virus could be retrieved from washed EHV-1-exposed platelets, suggesting a direct platelet-virus interaction. Our results indicate that EHV-1 activates equine platelets and that α-granule secretion is a consequence of virus-associated tissue factor triggering factor X activation and thrombin generation. Microvesiculation was only partly tissue factor and thrombin-dependent, suggesting the virus causes microvesiculation through other mechanisms, potentially through direct binding. These findings suggest that EHV-1-induced platelet activation could contribute to the thrombosis that occurs in

  8. Pneumolysin Mediates Platelet Activation In Vitro.

    PubMed

    Nel, Jan Gert; Durandt, Chrisna; Mitchell, Timothy J; Feldman, Charles; Anderson, Ronald; Tintinger, Gregory R

    2016-08-01

    This study has explored the role of the pneumococcal toxin, pneumolysin (Ply), in activating human platelets. Following exposure to Ply (10-80 ng/ml), platelet activation and cytosolic Ca(2+) concentrations were measured flow cytometrically according to the level of expression of CD62P (P-selectin) and spectrofluorimetrically, respectively. Exposure to Ply resulted in marked upregulation of expression of platelet CD62P, achieving statistical significance at concentrations of 40 ng/ml and higher (P < 0.05), in the setting of increased influx of Ca(2+). These potentially pro-thrombotic actions of Ply were attenuated by depletion of Ca(2+) from the extracellular medium or by exposure of the cells to a pneumolysoid devoid of pore-forming activity. These findings are consistent with a mechanism of Ply-mediated platelet activation involving sub-lytic pore formation, Ca(2+) influx, and mobilization of CD62P-expressing α-granules, which, if operative in vivo, may contribute to the pathogenesis of associated acute lung and myocardial injury during invasive pneumococcal disease. PMID:27192991

  9. Platelet activity in the pathophysiology of inflammatory bowel diseases.

    PubMed

    Chen, Chunqiu; Li, Yongyu; Yu, Zhen; Liu, Zhanju; Shi, Yanhong; Lewandowska, Urszula; Sobczak, Marta; Fichna, Jakub; Kreis, Martin

    2015-01-01

    Platelets play a crucial role in immune responses. Impaired platelet activation may cause persistent mucosal inflammation through P-selectin, CD40-CD40L and other systems influencing granulocytes, macrophages or endothelial cells. Pharmacological regulation of platelet activation may reduce thromboembolism and limit the interaction of platelets with endothelial and inflammatory cells, in turn weakening the inflammatory responses. In this review we focus on pathophysiological activities of platelets in inflammatory bowel diseases and discuss the studies on currently available anti-platelet therapies in the treatment of gastrointestinal inflammation. Finally, we provide a prospective view to new anti-platelet agents currently under development. PMID:25585124

  10. Platelet activating factor activity in the phospholipids of bovine spermatozoa

    SciTech Connect

    Parks, J.E.; Hough, S.; Elrod, C. )

    1990-11-01

    Platelet activating factor (PAF) has been detected in sperm from several mammalian species and can affect sperm motility and fertilization. Because bovine sperm contain a high percentage of ether-linked phospholipid precursors required for PAF synthesis, a study was undertaken to determine the PAF activity of bovine sperm phospholipids. Total lipids of washed, ejaculated bull sperm were extracted, and phospholipids were fractionated by thin-layer chromatography. Individual phospholipid fractions were assayed for PAF activity on the basis of (3H)serotonin release from equine platelets. PAF activity was detected in the PAF fraction (1.84 pmol/mumol total phospholipid) and in serine/inositol (PS/PI), choline (CP), and ethanolamine phosphoglyceride (EP) and cardiolipin (CA) fractions. Activity was highest in the CP fraction (8.05 pmol/mumol total phospholipid). Incomplete resolution of PAF and neutral lipids may have contributed to the activity in the PS/PI and CA fractions, respectively. Phospholipids from nonsperm sources did not stimulate serotonin release. Platelet activation by purified PAF and by sperm phospholipid fractions was inhibited by the receptor antagonist SRI 63-675. These results indicate that bovine sperm contain PAF and that other sperm phospholipids, especially CP and EP, which are high in glycerylether components, are capable of receptor-mediated platelet activation.

  11. Epithelial sodium channel modulates platelet collagen activation.

    PubMed

    Cerecedo, Doris; Martínez-Vieyra, Ivette; Alonso-Rangel, Lea; Benítez-Cardoza, Claudia; Ortega, Arturo

    2014-03-01

    Activated platelets adhere to the exposed subendothelial extracellular matrix and undergo a rapid cytoskeletal rearrangement resulting in shape change and release of their intracellular dense and alpha granule contents to avoid hemorrhage. A central step in this process is the elevation of the intracellular Ca(2+) concentration through its release from intracellular stores and on throughout its influx from the extracellular space. The Epithelial sodium channel (ENaC) is a highly selective Na(+) channel involved in mechanosensation, nociception, fluid volume homeostasis, and control of arterial blood pressure. The present study describes the expression, distribution, and participation of ENaC in platelet migration and granule secretion using pharmacological inhibition with amiloride. Our biochemical and confocal analysis in suspended and adhered platelets suggests that ENaC is associated with Intermediate filaments (IF) and with Dystrophin-associated proteins (DAP) via α-syntrophin and β-dystroglycan. Migration assays, quantification of soluble P-selectin, and serotonin release suggest that ENaC is dispensable for migration and alpha and dense granule secretion, whereas Na(+) influx through this channel is fundamental for platelet collagen activation. PMID:24679405

  12. Geometric complexity identifies platelet activation in familial hypercholesterolemic patients.

    PubMed

    Bianciardi, Giorgio; Aglianò, Margherita; Volpi, Nila; Stefanutti, Claudia

    2015-06-01

    Familial hypercholesterolemia (FH), a genetic disease, is associated with a severe incidence of athero-thrombotic events, related, also, to platelet hyperreactivity. A plethora of methods have been proposed to identify those activated circulating platelets, none of these has proved really effective. We need efficient methods to identify the circulating platelet status in order to follow the patients after therapeutic procedures. We propose the use of computerized fractal analysis for an objective characterization of the complexity of circulating platelet shapes observed by means of transmission electron microscopy in order to characterize the in vivo hyperactivated platelets of familial hypercholesterolemic patients, distinguishing them from the in vivo resting platelets of healthy individuals. Platelet boundaries were extracted by means of automatically image analysis. Geometric complexity (fractal dimension, D) by box counting was automatically calculated. The platelet boundary observed by electron microscopy is fractal, the shape of the circulating platelets is more complex in FH (n = 6) than healthy subjects (n = 5, P < 0.01), with 100% correct classification in selected individuals. In vitro activated platelets from healthy subjects show an analogous increase of D. The observed high D in the platelet boundary in FH originates from the in vivo platelet activation. Computerized fractal analysis of platelet shape observed by transmission electron microscopy can provide accurate, quantitative data to study platelet activation in familial hypercholesterolemia and after administration of drugs or other therapeutic procedures. PMID:25877374

  13. Procoagulant activity on platelets adhered to collagen or plasma clot.

    PubMed

    Ilveskero, S; Siljander, P; Lassila, R

    2001-04-01

    In a new 2-stage assay of platelet procoagulant activity (PCA), we first subjected gel-filtered platelets to adhesion on collagen (as a model of primary hemostasis) or plasma clots (as a model of preformed thrombus) for 30 minutes, and then the adherent platelets were supplemented with pooled, reptilase-treated, diluted plasma. Defibrinated plasma provided coagulation factors for assembly on platelet membranes without uncontrolled binding of thrombin to fibrin(ogen). Platelet adhesion to both surfaces showed modest individual variation, which increased at platelet densities that allowed aggregation. However, adhesion-induced PCA varied individually and surface-independently >3-fold, suggesting a uniform platelet procoagulant mechanism. Permanently adhered platelets showed markedly enhanced PCA when compared with the platelet pool in suspension, even after strong activation. The rate of thrombin generation induced by clot-adherent platelets was markedly faster than on collagen-adherent platelets during the initial phase of coagulation, whereas collagen-induced PCA proceeded slowly, strongly promoted by tissue thromboplastin. Therefore at 10 minutes, after adjustment for adhered platelets, collagen supported soluble thrombin formation as much as 5 times that of the thrombin-retaining clots. Activation of platelets by their firm adhesion was accompanied by formation of microparticles, representing about one third of the total soluble PCA. Collagen-adhered platelets provide soluble thrombin and microparticles, whereas the preformed clot serves to localize and accelerate hemostasis at the injury site, with the contribution of retained thrombin and microparticles. PMID:11304482

  14. In vivo effects of eltrombopag on platelet function in immune thrombocytopenia: no evidence of platelet activation

    PubMed Central

    Psaila, Bethan; Bussel, James B.; Linden, Matthew D.; Babula, Bracken; Li, Youfu; Barnard, Marc R.; Tate, Chinara; Mathur, Kanika; Frelinger, Andrew L.

    2012-01-01

    The effects of eltrombopag, a thrombopoietin-receptor agonist, on platelet function in immune thrombocytopenia (ITP) are not fully characterized. This study used whole blood flow cytometry to examine platelet function in 20 patients receiving eltrombopag treatment at days 0, 7, and 28. Platelet surface expression of activated GPIIb/IIIa, P-selectin, and GPIb was measured with and without low and high adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) concentrations. Before eltrombopag treatment with no ex vivo agonist, platelet activation was higher in ITP patients than controls. Platelet GPIb and activated GPIIb/IIIa expression without added agonist was unchanged following eltrombopag treatment, whereas a slight increase in P-selectin was observed. Expression of P-selectin and activated GPIIb/IIIa in response to high-dose ADP was lower during eltrombopag treatment than at baseline. Eltrombopag led to a slight increase in platelet reactivity to TRAP only in responders to eltrombopag but not to levels above those in controls; whole blood experiments demonstrated that this increase was probably because of higher platelet counts rather than higher platelet reactivity. In conclusion, although thrombocytopenic ITP patients have higher baseline platelet activation than controls, eltrombopag did not cause platelet activation or hyper-reactivity, irrespective of whether the platelet count increased. PMID:22294727

  15. Effects of Physical (In)activity on Platelet Function

    PubMed Central

    Heber, Stefan; Volf, Ivo

    2015-01-01

    As platelet activation is closely related to the liberation of growth factors and inflammatory mediators, platelets play a central role in the development of CVD. Virtually all cardiovascular risk factors favor platelet hyperreactivity and, accordingly, also physical (in)activity affects platelet function. Within this paper, we will summarize and discuss the current knowledge on the impact of acute and habitual exercise on platelet function. Although there are apparent discrepancies regarding the reported effects of acute, strenuous exercise on platelet activation, a deeper analysis of the available literature reveals that the applied exercise intensity and the subjects' cardiorespiratory fitness represent critical determinants for the observed effects. Consideration of these factors leads to the summary that (i) acute, strenuous exercise can lead to platelet activation, (ii) regular physical activity and/or physical fitness diminish or prevent platelet activation in response to acute exercise, and (iii) habitual physical activity and/or physical fitness also favorably modulate platelet function at physical rest. Notably, these effects of exercise on platelet function show obvious similarities to the well-recognized relation between exercise and the risk for cardiovascular events where vigorous exercise transiently increases the risk for myocardial infarction and a physically active lifestyle dramatically reduces cardiovascular mortality. PMID:26557653

  16. Platelet activation during angiotensin II infusion in healthy volunteers.

    PubMed

    Larsson, P T; Schwieler, J H; Wallén, N H

    2000-01-01

    The present study was undertaken to evaluate the effects of an intravenous infusion of angiotensin II (10 ng/kg per min) on platelet function and coagulation in vivo in 18 healthy males. The infusion increased mean arterial pressure by 23+/-2 mm Hg. Plasma beta-thromboglobulin levels, reflecting platelet secretion, increased by 66+/-24% during the infusion, as did also platelet surface expression of P-selectin as measured by flow cytometry. Platelet fibrinogen binding increased, whereas platelet aggregability, assessed by ex vivo filtragometry, was unaltered. Angiotensin II caused mild activation of the coagulation cascade with increases in plasma levels of thrombin-antithrombin complex and prothrombin fragment F1 + 2. In conclusion, angiotensin II has mild platelet-activating effects in vivo and also enhances coagulation. Markers of platelet secretion are significantly increased, whereas markers of platelet aggregability are less affected. The clinical relevance of these findings remains to be clarified. PMID:10691100

  17. Minimal regulation of platelet activity by PECAM-1.

    PubMed

    Dhanjal, Tarvinder S; Ross, Ewan A; Auger, Jocelyn M; McCarty, Owen J T; Hughes, Craig E; Senis, Yotis A; Buckley, Chris D; Watson, Steve P

    2007-02-01

    PECAM-1 is a member of the superfamily of immunoglobulins (Ig) and is expressed on platelets at moderate level. PECAM-1 has been reported to have contrasting effects on platelet activation by the collagen receptor GPVI and the integrin, alphaIIbbeta3, even though both receptors signal through Src-kinase regulation of PLCgamma2. The present study compares the role of PECAM-1 on platelet activation by these two receptors and by the lectin receptor, CLEC-2, which also signals via PLCgamma2. Studies using PECAM-1 knockout-mice and cross-linking of PECAM-1 using specific antibodies demonstrated a minor inhibitory role on platelet responses to the above three receptors and also under some conditions to the G-protein agonist thrombin. The degree of inhibition was considerably less than that produced by PGI2, which elevates cAMP. There was no significant difference in thrombus formation on collagen in PECAM-1-/- platelets relative to litter-matched controls. The very weak inhibitory effect of PECAM-1 on platelet activation relative to that of PGI2 indicate that the Ig-receptor is not a major regulator of platelet activation. PECAM-1 has been reported to have contrasting effects on platelet activation. The present study demonstrates a very mild or negligible effect on platelet activation in response to stimulation by a variety of agonists, thereby questioning the physiological role of the immunoglobulin receptor as a major regulator of platelet activation. PMID:17365855

  18. Busulfan Triggers Intrinsic Mitochondrial-Dependent Platelet Apoptosis Independent of Platelet Activation.

    PubMed

    Qiao, Jianlin; Wu, Yulu; Liu, Yun; Li, Xiaoqian; Wu, Xiaoqing; Liu, Na; Zhu, Feng; Qi, Kunming; Cheng, Hai; Li, Depeng; Li, Hongchun; Li, Zhenyu; Zeng, Lingyu; Ma, Ping; Xu, Kailin

    2016-09-01

    As a nonspecific alkylating antineoplastic agent, busulfan has been widely used in the treatment of patients with chronic myeloid leukemia. In vitro and in vivo studies demonstrated busulfan-induced cell apoptosis. Whether busulfan triggers platelet apoptosis remains unclear. This study aimed to evaluate the role of busulfan in platelet apoptosis. Isolated human platelets were incubated with busulfan followed by analysis of platelet apoptosis by flow cytometry or western blot, including mitochondrial depolarization, expression of Bcl-2, and Bax and caspase 3 activation. Meanwhile, platelet activation, expression of glycoprotein Ibα (GPIbα), glycoprotein VI (GPVI), and IIb3 and platelet aggregation in response to collagen and adenosine diphosphate (ADP) were measured. Additionally, busulfan was injected into mice with or without administration of caspase inhibitor QVD-Oph to investigate its effect on platelet lifespan. Our results showed that busulfan-treated platelets displayed increased mitochondrial membrane depolarization, decreased expression of Bcl-2, increased expression of Bax and caspase 3 activation in dose-dependent manner, which were inhibited by QVD-Oph. Platelet activation was not observed in busulfan-treated platelets as showed by no increased P-selectin expression and PAC-1 binding. However, busulfan reduced collagen- or ADP-induced platelet aggregation without affecting expression of GPIbα, GPVI, and IIb3. Furthermore, busulfan reduced circulating platelet lifespan which was ameliorated by QVD-Oph in mice. In conclusion, busulfan triggers mitochondrial-dependent platelet apoptosis and reduces platelet lifespan in mice. These data suggest targeting caspase activation might be beneficial in the prophylaxis of platelet apoptosis-associated thrombocytopenia after administration of busulfan. PMID:27292166

  19. Platelet activation risk index as a prognostic thrombosis indicator.

    PubMed

    Zlobina, K E; Guria, G Th

    2016-01-01

    Platelet activation in blood flow under high, overcritical shear rates is initiated by Von Willebrand factor. Despite the large amount of experimental data that have been obtained, the value of the critical shear rate, above which von Willebrand factor starts to activate platelets, is still controversial. Here, we recommend a theoretical approach to elucidate how the critical blood shear rate is dependent on von Willebrand factor size. We derived a diagram of platelet activation according to the shear rate and von Willebrand factor multimer size. We succeeded in deriving an explicit formula for the dependence of the critical shear rate on von Willebrand factor molecule size. The platelet activation risk index was introduced. This index is dependent on the flow conditions, number of monomers in von Willebrand factor, and platelet sensitivity. Probable medical applications of the platelet activation risk index as a universal prognostic index are discussed. PMID:27461235

  20. Platelet activation risk index as a prognostic thrombosis indicator

    PubMed Central

    Zlobina, K. E.; Guria, G. Th.

    2016-01-01

    Platelet activation in blood flow under high, overcritical shear rates is initiated by Von Willebrand factor. Despite the large amount of experimental data that have been obtained, the value of the critical shear rate, above which von Willebrand factor starts to activate platelets, is still controversial. Here, we recommend a theoretical approach to elucidate how the critical blood shear rate is dependent on von Willebrand factor size. We derived a diagram of platelet activation according to the shear rate and von Willebrand factor multimer size. We succeeded in deriving an explicit formula for the dependence of the critical shear rate on von Willebrand factor molecule size. The platelet activation risk index was introduced. This index is dependent on the flow conditions, number of monomers in von Willebrand factor, and platelet sensitivity. Probable medical applications of the platelet activation risk index as a universal prognostic index are discussed. PMID:27461235

  1. Complement Activation in Trauma Patients Alters Platelet Function.

    PubMed

    Atefi, Gelareh; Aisiku, Omozuanvbo; Shapiro, Nathan; Hauser, Carl; Dalle Lucca, Jurandir; Flaumenhaft, Robert; Tsokos, George C

    2016-09-01

    Trauma remains the main cause of death for both civilians and those in uniform. Trauma-associated coagulopathy is a complex process involving inflammation, coagulation, and platelet dysfunction. It is unknown whether activation of complement, which occurs invariably in trauma patients, is involved in the expression of trauma-associated coagulopathy. We designed a prospective study in which we enrolled 40 trauma patients and 30 healthy donors upon arrival to the emergency department of BIDMC. Platelets from healthy individuals were incubated with sera from trauma patients and their responsiveness to a thrombin receptor-activating peptide was measured using aggregometry. Complement deposition on platelets from trauma patients was measured by flow cytometry. Normal platelets displayed hypoactivity after incubation with trauma sera even though exposure to trauma sera resulted in increased agonist-induced calcium flux. Depletion of complement from sera further blocked activation of hypoactive platelets. Conversely, complement activation increased aggregation of platelets. Platelets from trauma patients were found to have significantly higher amounts of C3a and C4d on their surface compared with platelets from controls. Depletion of complement (C4d, C3a) reversed the ability of trauma sera to augment agonist-induced calcium flux in donor platelets. Our data indicate that complement enhances platelet aggregation. Despite its complement content, trauma sera render platelets hypoactive and complement depletion further blocks activation of hypoactive platelets. The defect in platelet activation induced by trauma sera is distal to receptor activation since agonist-induced Ca2+ flux is elevated in the presence of trauma sera owing to complement deposition. PMID:27355402

  2. Geometric complexity is increased in in vitro activated platelets.

    PubMed

    Bianciardi, Giorgio

    2015-06-01

    This article investigates the use of computerized fractal analysis for objective characterization of the complexity of platelets in vitro stimulated by low level thrombin (0.02 U mL(-1) ), collected from healthy individuals and observed by means of transmission electron microscopy. Platelet boundaries were extracted by means of automatically image analysis. Local fractal dimension was evaluated by the box-counting technique (measure of geometric complexity of the platelet outline). The results showed that the platelet boundary is fractal when observed by transmission electron microscopy and that, after an in vitro platelet activation test, the shape of platelets present increased geometric complexity in comparison to the no stimulated platelets (P < 0.001), with 100% correct classification. Computerized fractal analysis of platelet shape by transmission electron microscopy can provide accurate, quantitative, data to study platelet activation. The results may play important roles in the evaluation of the platelets status in pathological conditions, like as atherosclerosis and diabetes mellitus, where in in vivo activated platelets have been described. PMID:25808036

  3. Protease-induced immunoregulatory activity of platelet factor 4.

    PubMed Central

    Katz, I R; Thorbecke, G J; Bell, M K; Yin, J Z; Clarke, D; Zucker, M B

    1986-01-01

    Intravenous injection of human or mouse serum or platelet material secreted from appropriately stimulated platelets ("releasate") together with antigen alleviates the immunosuppression in SJL/J mice induced by injection of irradiated lymphoma cells or in (CB6)F1 mice induced by injection of concanavalin A. We now report that injection of releasate from 10(6) human platelets restores plaque-forming cells to the unsuppressed number; greater amounts increase responses further. Immunoregulatory activity is released from platelets exposed to thrombin in parallel with other alpha-granule components. Heparin-agarose absorbs activity. Purified platelet factor 4 (PF4) has activity; beta-thromboglobulin and platelet-derived growth factor have little or none. Activity in serum is neutralized by goat anti-human PF4. An enzymatic step is necessary for production of immunoregulatory activity. Releasates boiled immediately after platelet aggregation with 250 nM A23187 or those produced by adding A23187 in the presence of 100 microM serine protease inhibitor (p-amidinophenyl)methanesulfonyl fluoride (APMSF) are ineffective, whereas releasates boiled or mixed with APMSF after incubation for 60 min are active. Activity is generated by incubating a mixture of heparin-absorbed releasate (as enzyme source) and heparin-agarose eluate of releasate made in the presence of APMSF (as substrate source). The enzymatic step does not alter the heparin-neutralizing activity of PF4. Apparently a secreted platelet protease converts PF4 to a form with immunoregulatory activity. PMID:3517862

  4. Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow

    PubMed Central

    De Angelis, Valentina; Koekman, Arnold C.; Weeterings, Cees; Roest, Mark; de Groot, Philip G.; Herczenik, Eszter; Maas, Coen

    2014-01-01

    Background The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells. Additionally, it has been reported that endocannabinoid receptors are present on circulating platelets, but there has been conflicting evidence on their contribution to platelet function. Objectives Our aim was to examine the role of endocannabinoids in platelet function in vitro and in vivo. Methods and Results We studied the effects of the well-characterized endogenous endocannabinoid anandamide on platelet aggregation in suspension, α-granule release, calcium mobilization, Syk phosphorylation, as well as platelet spreading and aggregate formation under flow. Anandamide inhibits platelet aggregation and α-granule release by collagen, collagen-derived peptide CRP-XL, ADP, arachidonic acid and thromboxane A2 analogue U46619. However, activation via thrombin receptor PAR-1 stays largely unaffected. Calcium mobilization is significantly impaired when platelets are stimulated with collagen or CRP-XL, but remains normal in the presence of the other agonists. In line with this finding, we found that anandamide prevents collagen-induced Syk phosphorylation. Furthermore, anandamide-treated platelets exhibit reduced spreading on immobilized fibrinogen, have a decreased capacity for binding fibrinogen in solution and show perturbed platelet aggregate formation under flow over collagen. Finally, we investigated the influence of Cannabis sativa consumption by human volunteers on platelet activation. Similar to our in vitro findings with anandamide, ex vivo collagen-induced platelet aggregation and aggregate formation on immobilized collagen under flow were impaired in whole blood of donors that had consumed Cannabis sativa. Conclusions Endocannabinoid receptor agonists reduce platelet activation and aggregate formation both in vitro and ex vivo after Cannabis sativa consumption. Further elucidation of this novel regulatory mechanism for platelet function

  5. CLEC-2 expression is maintained on activated platelets and on platelet microparticles.

    PubMed

    Gitz, Eelo; Pollitt, Alice Y; Gitz-Francois, Jerney J; Alshehri, Osama; Mori, Jun; Montague, Samantha; Nash, Gerard B; Douglas, Michael R; Gardiner, Elizabeth E; Andrews, Robert K; Buckley, Christopher D; Harrison, Paul; Watson, Steve P

    2014-10-01

    The C-type lectin-like receptor CLEC-2 mediates platelet activation through a hem-immunoreceptor tyrosine-based activation motif (hemITAM). CLEC-2 initiates a Src- and Syk-dependent signaling cascade that is closely related to that of the 2 platelet ITAM receptors: glycoprotein (GP)VI and FcγRIIa. Activation of either of the ITAM receptors induces shedding of GPVI and proteolysis of the ITAM domain in FcγRIIa. In the present study, we generated monoclonal antibodies against human CLEC-2 and used these to measure CLEC-2 expression on resting and stimulated platelets and on other hematopoietic cells. We show that CLEC-2 is restricted to platelets with an average copy number of ∼2000 per cell and that activation of CLEC-2 induces proteolytic cleavage of GPVI and FcγRIIa but not of itself. We further show that CLEC-2 and GPVI are expressed on CD41+ microparticles in megakaryocyte cultures and in platelet-rich plasma, which are predominantly derived from megakaryocytes in healthy donors, whereas microparticles derived from activated platelets only express CLEC-2. Patients with rheumatoid arthritis, an inflammatory disease associated with increased microparticle production, had raised plasma levels of microparticles that expressed CLEC-2 but not GPVI. Thus, CLEC-2, unlike platelet ITAM receptors, is not regulated by proteolysis and can be used to monitor platelet-derived microparticles. PMID:25150298

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

    PubMed Central

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

    2012-01-01

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

  7. Thrombin-Mediated Platelet Activation of Lysed Whole Blood and Platelet-Rich Plasma: A Comparison Between Platelet Activation Markers and Ultrastructural Alterations.

    PubMed

    Augustine, Tanya N; van der Spuy, Wendy J; Kaberry, Lindsay L; Shayi, Millicent

    2016-06-01

    Platelet ultrastructural alterations representing spurious activation have been identified in pathological conditions. A limitation of platelet studies is that sample preparation may lead to artifactual activation processes which may confound results, impacting the use of scanning electron microscopy as a supplemental diagnostic tool. We used scanning electron microscopy and flow cytometry to analyze platelet activation in platelet-rich plasma (PRP) and whole blood (WB) samples. PRP generated using a single high g force centrifugation, and WB samples treated with a red blood cell lysis buffer, were exposed to increasing concentrations of the agonist thrombin. Platelets in lysed WB samples responded to thrombin by elevating the activation marker CD62p definitively, with corresponding ultrastructural changes indicating activation. Conversely, CD62p expression in PRP preparations remained static. Ultrastructural analysis revealed fully activated platelets even under low concentration thrombin stimulation, with considerable fibrin deposition. It is proposed that the method for PRP production induced premature platelet activation, preventable by using an inhibitor of platelet aggregation and fibrin polymerization. Nevertheless, our results show a definitive correspondence between flow cytometry and scanning electron microscopy in platelet activation studies, highlighting the potential of the latter technique as a supplemental diagnostic tool. PMID:27329313

  8. Rupture Forces among Human Blood Platelets at different Degrees of Activation.

    PubMed

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

    2016-01-01

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

  9. Rupture Forces among Human Blood Platelets at different Degrees of Activation

    PubMed Central

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

    2016-01-01

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

  10. Reduced serum inhibition of platelet-activating factor activity in preeclampsia.

    PubMed

    Benedetto, C; Massobrio, M; Bertini, E; Abbondanza, M; Enrieu, N; Tetta, C

    1989-01-01

    We determined in normal nonpregnant (group 1) women, normal pregnant (group 2) women, and patients with preeclampsia (group 3) the serum inhibition of platelet-activating factor activity, the presence of detectable amounts of platelet-activating factor in the blood, and platelet responsiveness in vitro to platelet-activating factor, and to other agonists (adenosine diphosphate, collagen, and ristocetin), and prostacyclin (prostaglandin I2). In patients with preeclampsia (group 3) the serum inhibition of platelet-activating factor activity was significantly lower than that in groups 1 and 2. However, no detectable amounts of platelet-activating factor were observed. The mean values of platelet aggregation induced by platelet-activating factor, adenosine diphosphate, collagen and ristocetin, and the prostaglandin I2-inhibitory concentration of 50% which is inversely correlated with platelet sensitivity to prostaglandin I2, were not significantly different between groups 2 and 3. It is suggested that in preeclampsia the defect in serum inhibitory potential of platelet-activating factor--induced platelet aggregation may contribute to the disturbance in the homeostatic balance between proaggregant and antiaggregant substances. PMID:2912073

  11. Platelet Serotonin Transporter Function Predicts Default-Mode Network Activity

    PubMed Central

    Kasess, Christian H.; Meyer, Bernhard M.; Hofmaier, Tina; Diers, Kersten; Bartova, Lucie; Pail, Gerald; Huf, Wolfgang; Uzelac, Zeljko; Hartinger, Beate; Kalcher, Klaudius; Perkmann, Thomas; Haslacher, Helmuth; Meyer-Lindenberg, Andreas; Kasper, Siegfried; Freissmuth, Michael; Windischberger, Christian; Willeit, Matthäus; Lanzenberger, Rupert; Esterbauer, Harald; Brocke, Burkhard; Moser, Ewald; Sitte, Harald H.; Pezawas, Lukas

    2014-01-01

    Background The serotonin transporter (5-HTT) is abundantly expressed in humans by the serotonin transporter gene SLC6A4 and removes serotonin (5-HT) from extracellular space. A blood-brain relationship between platelet and synaptosomal 5-HT reuptake has been suggested, but it is unknown today, if platelet 5-HT uptake can predict neural activation of human brain networks that are known to be under serotonergic influence. Methods A functional magnetic resonance study was performed in 48 healthy subjects and maximal 5-HT uptake velocity (Vmax) was assessed in blood platelets. We used a mixed-effects multilevel analysis technique (MEMA) to test for linear relationships between whole-brain, blood-oxygen-level dependent (BOLD) activity and platelet Vmax. Results The present study demonstrates that increases in platelet Vmax significantly predict default-mode network (DMN) suppression in healthy subjects independent of genetic variation within SLC6A4. Furthermore, functional connectivity analyses indicate that platelet Vmax is related to global DMN activation and not intrinsic DMN connectivity. Conclusion This study provides evidence that platelet Vmax predicts global DMN activation changes in healthy subjects. Given previous reports on platelet-synaptosomal Vmax coupling, results further suggest an important role of neuronal 5-HT reuptake in DMN regulation. PMID:24667541

  12. p38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo

    PubMed Central

    Canault, Matthias; Duerschmied, Daniel; Brill, Alexander; Stefanini, Lucia; Schatzberg, Daphne; Cifuni, Stephen M.

    2010-01-01

    Platelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-α and GPV. We recently demonstrated that tumor necrosis factor-α converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37°C or 22°C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets. PMID:19965619

  13. Xanthohumol, a Prenylated Flavonoid from Hops (Humulus lupulus), Prevents Platelet Activation in Human Platelets

    PubMed Central

    Lee, Ye-Ming; Hsieh, Kuo-Hsien; Lu, Wan-Jung; Chou, Hsiu-Chu; Chou, Duen-Suey; Lien, Li-Ming; Sheu, Joen-Rong; Lin, Kuan-Hung

    2012-01-01

    Xanthohumol is the principal prenylated flavonoid in the hop plant (Humulus lupulus L.). Xanthohumol was found to be a very potent cancer chemopreventive agent through regulation of diverse mechanisms. However, no data are available concerning the effects of xanthohumol on platelet activation. The aim of this paper was to examine the antiplatelet effect of xanthohumol in washed human platelets. In the present paper, xanthohumol exhibited more-potent activity in inhibiting platelet aggregation stimulated by collagen. Xanthohumol inhibited platelet activation accompanied by relative [Ca2+]i mobilization, thromboxane A2 formation, hydroxyl radical (OH●) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and Akt phosphorylation. Neither SQ22536, an inhibitor of adenylate cyclase, nor ODQ, an inhibitor of guanylate cyclase, reversed the xanthohumol-mediated inhibitory effect on platelet aggregation. Furthermore, xanthohumol did not significantly increase nitrate formation in platelets. This study demonstrates for the first time that xanthohumol possesses potent antiplatelet activity which may initially inhibit the PI3-kinase/Akt, p38 MAPK, and PLCγ2-PKC cascades, followed by inhibition of the thromboxane A2 formation, thereby leading to inhibition of [Ca2+]i and finally inhibition of platelet aggregation. Therefore, this novel role of xanthohumol may represent a high therapeutic potential for treatment or prevention of cardiovascular diseases. PMID:22611436

  14. Use of mean platelet component to measure platelet activation on the ADVIA 120 haematology system.

    PubMed

    Macey, M G; Carty, E; Webb, L; Chapman, E S; Zelmanovic, D; Okrongly, D; Rampton, D S; Newland, A C

    1999-10-15

    Platelet activation results in changes in a number of cell surface molecules including an increase in P-Selectin (CD62P) that may be rapidly and conveniently measured by immunofluorescent flow cytometry. The ADVIA 120 (Bayer) is a new system that facilitates more accurate measurement of platelet volume and in addition provides an approximate measure of the mean refractive index (RI) of the platelets reported as mean platelet component (MPC) concentration. We were interested to determine whether changes in MPC might reflect changes in platelet activation status. To investigate this, the platelet CD62P expression, determined by flow cytometry, and change in MPC, measured on the ADVIA 120 system, was first examined in vitro after stimulation of EDTA anticoagulated whole blood with submaximal concentrations of bovine thrombin in the presence or absence of the thromboxane synthase inhibitor, Ridogrel. Thrombin produced a dose-dependent increase in platelet CD62P expression and a decrease in MPC that could be inhibited by Ridogrel at physiological concentrations. In the second set of experiments, blood from 20 normal controls was collected into both EDTA and sodium citrate (SC) anticoagulants. Within 30 min of venesection and again at 3 h post-venesection after storage at room temperature, the platelet MPC and CD62P expression were determined. Platelets in all samples with both anticoagulants showed very low levels of CD62P expression when first analysed. At 3 h there was a small increase in CD62P expression on platelets in whole blood anticoagulated with SC, but a significant (P < 0.001) increase was observed on platelets anti-coagulated with EDTA. A negative correlation was found between the change in MPC of the platelets and the increase in the mean fluorescence intensity (MFI) (r = -0.69, P < 0.001, n = 20) and the percentage (r = -0.72, P < 0.001, n = 20) of CD62P positive platelets at 3 h in blood anticoagulated with EDTA. We conclude that a reduction in MPC as

  15. Platelet activation and apoptosis modulate monocyte inflammatory responses in dengue

    PubMed Central

    Hottz, Eugenio D.; Medeiros-de-Moraes, Isabel M.; Vieira-de-Abreu, Adriana; de Assis, Edson F.; Vals-de-Souza, Rogério; Castro-Faria-Neto, Hugo C.; Weyrich, Andrew S.; Zimmerman, Guy A.; Bozza, Fernando A.; Bozza, Patrícia T.

    2014-01-01

    Background Dengue is the most prevalent human arbovirus disease in the world. Dengue infection has a large spectrum of clinical manifestations from self-limited febrile illness to severe syndromes accompanied by bleeding and shock. Thrombocytopenia and vascular leak with altered cytokine profiles in plasma are features of severe dengue. Although monocytes have been recognized as important sources of cytokines in dengue, the contributions of platelet-monocyte interactions to inflammatory responses in dengue have not been addressed. Patients/Methods Patients with dengue were investigated for platelet-monocyte aggregate formation and markers of monocyte activation. Platelet-induced cytokine responses by monocytes and underlying mechanisms were also investigated in vitro. Results We observed increased levels of platelet-monocyte aggregates in blood samples from patients with dengue, especially patients with thrombocytopenia and increased vascular permeability. Moreover, the exposure of monocytes from healthy volunteers to platelets from patients with dengue induced the secretion of the cytokines IL-1β, IL-8, IL-10 and MCP-1, while the exposure to platelets from healthy volunteers only induced the secretion of MCP-1. In addition to the well-established modulation of monocyte cytokine responses by activated platelets through P-selectin binding, we found that interaction of monocytes with apoptotic platelets mediate IL-10 secretion through phosphatidylserine recognition in platelet-monocyte aggregates. Moreover, IL-10 secretion required platelet-monocyte contact but not phagocytosis. Conclusions Together, our results demonstrate that activated and apoptotic platelets aggregate with monocytes during dengue infection and signal specific cytokine responses that may contribute to the pathogenesis of dengue. PMID:25015827

  16. ANALYSIS OF ARACHIDONIC ACID METABOLITE AND PLATELET ACTIVATING FACTOR PRODUCTION

    EPA Science Inventory

    Metabolites of arachidonic acid ("eicosanoids") and platelet activating factor are important bioactive lipids that may be involved in the pathobiological alterations in animals induced by pollutant exposure. nalysis of these substances in biological tissue and fluids is important...

  17. Invasive pneumococcal disease leads to activation and hyperreactivity of platelets.

    PubMed

    Tunjungputri, Rahajeng N; de Jonge, Marien I; de Greeff, Astrid; van Selm, Saskia; Buys, Herma; Harders-Westerveen, Jose F; Stockhofe-Zurwieden, Norbert; Urbanus, Rolf T; de Groot, Phillip G; Smith, Hilde E; van der Ven, Andre J; de Mast, Quirijn

    2016-08-01

    Using a novel porcine model of intravenous Streptococcus pneumoniae infection, we showed that invasive pneumococcal infections induce marked platelet activation and hyperreactivity. This may contribute to the vascular complications seen in pneumococcal infection. PMID:27322088

  18. Wdr1-Dependent Actin Reorganization in Platelet Activation.

    PubMed

    Dasgupta, Swapan K; Le, Anhquyen; Da, Qi; Cruz, Miguel; Rumbaut, Rolando E; Thiagarajan, Perumal

    2016-01-01

    In resting platelets, the integrin αIIbβ3 is present in a low-affinity "bent" state. During platelet aggregation, intracytoplasmic signals induce conformational changes (inside-out signaling) that result in a "swung-out" conformation competent to bind ligands such as fibrinogen. The cytoskeleton plays an essential role in αIIbβ3 activation. We investigated the role of the actin interacting protein Wdr1 in αIIbβ3 activation. Wdr1-hypomorphic mice had a prolonged bleeding time (> 10 minutes) compared to that of wild-type mice (2.1 ± 0.7 minutes). Their platelets had impaired aggregation to collagen and thrombin. In a FeCl3 induced carotid artery thrombosis model, vessel occlusion in Wdr1-hypomorphic mice was prolonged significantly compared to wild-type mice (9.0 ± 10.5 minutes versus 5.8 ± 12.6 minutes (p = 0.041). Activation-induced binding of JON/A (a conformation-specific antibody to activated αIIbβ3) was significantly less in Wdr1-hypomorphic platelets at various concentrations of collagen, indicating impaired inside-out activation of αIIbβ3, despite a normal calcium response. Actin turnover, assessed by measuring F-actin and G-actin ratios during collagen- and thrombin-induced platelet aggregation, was highly impaired in Wdr1-hypomorphic platelets. Furthermore, talin failed to redistribute and translocate to the cytoskeleton following activation in Wdr1-hypomorphic platelets. These studies show that Wdr1 is essential for talin-induced activation of αIIbβ3 during platelet activation. PMID:27627652

  19. Activated platelets rescue apoptotic cells via paracrine activation of EGFR and DNA-dependent protein kinase

    PubMed Central

    Au, A E-L; Sashindranath, M; Borg, R J; Kleifeld, O; Andrews, R K; Gardiner, E E; Medcalf, R L; Samson, A L

    2014-01-01

    Platelet activation is a frontline response to injury, not only essential for clot formation but also important for tissue repair. Indeed, the reparative influence of platelets has long been exploited therapeutically where application of platelet concentrates expedites wound recovery. Despite this, the mechanisms of platelet-triggered cytoprotection are poorly understood. Here, we show that activated platelets accumulate in the brain to exceptionally high levels following injury and release factors that potently protect neurons from apoptosis. Kinomic microarray and subsequent kinase inhibitor studies showed that platelet-based neuroprotection relies upon paracrine activation of the epidermal growth factor receptor (EGFR) and downstream DNA-dependent protein kinase (DNA-PK). This same anti-apoptotic cascade stimulated by activated platelets also provided chemo-resistance to several cancer cell types. Surprisingly, deep proteomic profiling of the platelet releasate failed to identify any known EGFR ligand, indicating that activated platelets release an atypical activator of the EGFR. This study is the first to formally associate platelet activation to EGFR/DNA-PK – an endogenous cytoprotective cascade. PMID:25210793

  20. Membrane Changes Associated with Platelet Activation

    PubMed Central

    George, James N.; Lyons, Roger M.; Morgan, Rebecca K.

    1980-01-01

    The effect of aggregation and secretion on membrane proteins was studied in washed human platelets. Reversible aggregation without secretion was stimulated by ADP and secretion without aggregation was stimulated by thrombin in the presence of EDTA. No loss of platelet surface glycoproteins occurred during reversible ADP-induced platelet aggregation, as measured by quantitative polyacrylamide gel electrophoresis analysis of platelets that were labeled with 125I-diazotized diiodosulfanilic acid (DD125ISA) before ADP stimulation. Also, no new proteins became exposed on the platelet surface after ADP aggregation, as determined by DD125ISA labeling after stimulation. Thrombin-induced platelet secretion also caused no loss of platelet surface glycoproteins. However, after platelet secretion two new proteins were labeled by DD125ISA: (a) actin and (b) the 149,000-mol wt glycoprotein (termed GP-G), which is contained in platelet granules and secreted in response to thrombin. The identity of DD125ISA-labeled actin was confirmed by four criteria: (a) comigration with actin in three different sodium dodecyl sulfate-polyacrylamide gel electrophoresis systems, (b) elution from a particulate fraction in low ionic strength buffer, (c) co-migration with actin in isoelectric focusing, and (d) binding to DNase I. The identity of actin and its appearance on the platelet surface after thrombin-induced secretion was also demonstrated by the greater binding of an anti-actin antibody to thrombin-treated platelets, measured with 125I-staphylococcal protein A. Therefore, major platelet membrane changes occur after secretion but not after reversible aggregation. The platelet surface changes occurring with secretion may be important in the formation of irreversible platelet aggregates and in the final retraction of the blood clot. Images PMID:6772667

  1. Exposure to acrolein by inhalation causes platelet activation

    SciTech Connect

    Sithu, Srinivas D.; Srivastava, Sanjay; Siddiqui, Maqsood A.; Vladykovskaya, Elena; Riggs, Daniel W.; Conklin, Daniel J.; Haberzettl, Petra; O'Toole, Timothy E.; Bhatnagar, Aruni; D'Souza, Stanley E.

    2010-10-15

    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 cause 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.

  2. EXPOSURE TO ACROLEIN BY INHALATION CAUSES PLATELET ACTIVATION

    PubMed Central

    Sithu, Srinivas D; Srivastava, Sanjay; Siddiqui, Maqsood A; Vladykovskaya, Elena; Riggs, Daniel W; Conklin, Daniel J; Haberzettl, Petra; O’Toole, Timothy E; Bhatnagar, Aruni; D’Souza, Stanley E

    2010-01-01

    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, 6h/day for 4 days) acrolein inhalation exposures. The acute exposure to acrolein did not cause 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. PMID:20678513

  3. The systemic activation of platelets by Dacron grafts.

    PubMed

    Shoenfeld, N A; Connolly, R; Ramberg, K; Valeri, C R; Eldrup-Jorgensen, J; Callow, A D

    1988-05-01

    Dacron (polyester fiber), a stimulus to platelet aggregation in vitro, accumulates platelets to a greater extent in vivo than autogenous artery, polytetrafluoroethylene (PTFE) or human umbilical vein (HUV). We conducted a series of experiments using the ex vivo shunt in the baboon to determine whether or not systemic activation of platelet function was produced by a Dacron graft. Two 5 centimeter segments of 4 millimeter internal diameter graft materials were placed in series in the ex vivo shunt perfused at 25 milliliters per minute flow rate for two and one-half hours. Deposition of autologous Indium 111 labeled platelets was monitored. The ex vivo shunt procedures were divided into two groups, both with PTFE as the proximal graft: one with a distal Dacron graft (n = 21), the second with PTFE or HUV distally (n = 17). In this study, an increase in platelet deposition on the proximal PTFE graft represents systemic platelet activation caused by the distal graft. Increased platelet deposition on PTFE was noted at all time points in the presence of a Dacron graft (p less than 0.05). This property of Dacron has important clinical implications, potentially accelerating the progression of vascular disease, increasing the failure rate of composite grafts and subsequent arterial reconstruction. PMID:2966442

  4. Effects of dimethylformamide (DMF) on coagulation and platelet activity

    SciTech Connect

    Imbriani, M.; Ghittori, S.; Prestinoni, A.; Longoni, P.; Cascone, G.; Gamba, G.

    1986-03-01

    The effects of dimethylformamide (DMF) on hemostatic functions, especially on platelet activity, were examined both in vitro and in vivo in 15 workers exposed to DMF (27 mg/m3, median value). Twenty-eight control subjects who were not exposed to DMF, but comparable for age, anthropometric data, and smoking habits, were also studied. Workers exposed to DMF showed a decrease in the number of platelets and had longer coagulation times, probably due to a change caused by DMF on the membrane receptor of platelets and on the phospholipid components of the clotting system.

  5. Platelet Activation Test in Unprocessed Blood (Pac-t-UB) to Monitor Platelet Concentrates and Whole Blood of Thrombocytopenic Patients

    PubMed Central

    Roest, Mark; van Holten, Thijs C.; Fleurke, Ger-Jan; Remijn, Jasper A.

    2013-01-01

    Summary Background Platelet concentrate transfusion is the standard treatment for hemato-oncology patients to compensate for thrombocytopenia. We have developed a novel platelet activation test in anticoagulated unprocessed blood (pac-t-UB) to determine platelet function in platelet concentrates and in blood of thrombocytopenic patients. Methods We have measured platelet activity in a platelet concentrate and in anticoagulated unprocessed blood of a post-transfusion thrombocytopenic patient. Results Our data show time-dependent platelet activation by GPVI agonist (collagen related peptide; CRP), PAR-1 agonist (SFLLRN), P2Y12 agonist (ADP), and thromboxane receptor agonist (U46619) in a platelet concentrate. Furthermore, pac-t-UB showed time-dependent platelet activation in unprocessed blood of a post-transfusion patient with thrombocytopenia. Testing platelet function by different agonists in relation to storage show that 3-day-old platelet concentrates are still reactive to the studied agonists. This reactivity rapidly drops for each agonists during longer storage. Discussion Pac-t-UB is a novel tool to estimate platelet function by different agonists in platelet concentrates and in unprocessed blood of thrombocytopenic patients. In the near future, we will validate whether pac-t-UB is an adequate test to monitor the quality of platelet concentrates and whether pac-t-UB predicts the bleeding risk of transfused thrombocytopenic patients. PMID:23652405

  6. Exosomes: novel effectors of human platelet lysate activity.

    PubMed

    Torreggiani, E; Perut, F; Roncuzzi, L; Zini, N; Baglìo, S R; Baldini, N

    2014-01-01

    Despite the popularity of platelet-rich plasma (PRP) and platelet lysate (PL) in orthopaedic practice, the mechanism of action and the effectiveness of these therapeutic tools are still controversial. So far, the activity of PRP and PL has been associated with different growth factors (GF) released during platelet degranulation. This study, for the first time, identifies exosomes, nanosized vesicles released in the extracellular compartment by a number of elements, including platelets, as one of the effectors of PL activity. Exosomes were isolated from human PL by differential ultracentrifugation, and analysed by electron microscopy and Western blotting. Bone marrow stromal cells (MSC) treated with three different exosome concentrations (0.6 μg, 5 μg and 50 μg) showed a significant, dose-dependent increase in cell proliferation and migration compared to the control. In addition, osteogenic differentiation assays demonstrated that exosome concentration differently affected the ability of MSC to deposit mineralised matrix. Finally, the analysis of exosome protein content revealed a higher amount of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-BB) and transforming growth factor beta 1 (TGF-β1) as compared to PL. In regards to RNA content, an enrichment of small RNAs in exosomes as compared to donor platelets has been found. These results suggest that exosomes consistently contribute to PL activity and could represent an advantageous nanodelivery system for cell-free regeneration therapies. PMID:25241964

  7. Chlorogenic Acid Inhibits Human Platelet Activation and Thrombus Formation

    PubMed Central

    Fuentes, Eduardo; Caballero, Julio; Alarcón, Marcelo; Rojas, Armando; Palomo, Iván

    2014-01-01

    Background Chlorogenic acid is a potent phenolic antioxidant. However, its effect on platelet aggregation, a critical factor in arterial thrombosis, remains unclear. Consequently, chlorogenic acid-action mechanisms in preventing platelet activation and thrombus formation were examined. Methods and Results Chlorogenic acid in a dose-dependent manner (0.1 to 1 mmol/L) inhibited platelet secretion and aggregation induced by ADP, collagen, arachidonic acid and TRAP-6, and diminished platelet firm adhesion/aggregation and platelet-leukocyte interactions under flow conditions. At these concentrations chlorogenic acid significantly decreased platelet inflammatory mediators (sP-selectin, sCD40L, CCL5 and IL-1β) and increased intraplatelet cAMP levels/PKA activation. Interestingly, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent A2A receptor antagonist) attenuated the antiplatelet effect of chlorogenic acid. Chlorogenic acid is compatible to the active site of the adenosine A2A receptor as revealed through molecular modeling. In addition, chlorogenic acid had a significantly lower effect on mouse bleeding time when compared to the same dose of aspirin. Conclusions Antiplatelet and antithrombotic effects of chlorogenic acid are associated with the A2A receptor/adenylate cyclase/cAMP/PKA signaling pathway. PMID:24598787

  8. Influence of gold nanoparticles on platelets functional activity in vitro

    NASA Astrophysics Data System (ADS)

    Akchurin, Garif G.; Akchurin, George G.; Ivanov, Alexey N.; Kirichuk, Vyacheslav F.; Terentyuk, George S.; Khlebtsov, Boris N.; Khlebtsov, Nikolay G.

    2008-02-01

    Now in the leading biomedical centers of the world approved new technology of laser photothermal destruction of cancer cells using plasmon gold nanoparticles. Investigations of influence of gold nanoparticles on white rat platelets aggregative activity in vitro have been made. Platelet aggregation was investigated in platelet rich plasma (PRP) with help of laser analyzer 230 LA <>, Russia). Aggregation inductor was ADP solution in terminal concentration 2.5 micromole (<>, Russia). Gold nanoshells soluted in salt solution were used for experiments. Samples of PRP were incubated with 50 or 100 μl gold nanoshells solution in 5 minute, after that we made definition ADP induced platelet aggregation. We found out increase platelet function activity after incubation with nanoparticles solution which shown in maximum ADP-induced aggregation degree increase. Increase platelet function activity during intravenous nanoshells injection can be cause of thrombosis on patients. That's why before clinical application of cancer cell destruction based on laser photothermal used with plasmon gold nanoparticles careful investigations of thrombosis process and detail analyze of physiological blood parameters are very necessary.

  9. Bryostatins activate protein kinase C in intact human platelets

    SciTech Connect

    Smith, J.B.; Tallant, E.A.; Pettit, G.R.; Wallace, R.W.

    1986-05-01

    Bryostatins, macrocyclic lactones isolated from a marine bryozoan, have antineoplastic activity in the P388 lymphocytic leukemia system. These compounds also stimulate growth in Swiss 3T3 cells, induce secretion in leukocytes, inhibit phorbol dibutyrate binding to a high affinity receptor, and activate the C-kinase in vitro. In human platelets, phorbol esters induce aggregation and activate protein kinase C, resulting in phosphorylation of a 47K protein and the 20K myosin light chain. The authors now show that bryostatin 7 (B-7) triggers platelet aggregation to the same rate and extent as phorbol 12-myristate 13-acetate (PMA). B-7 also causes the in vivo activation of the C-kinase, resulting in phosphorylation of both the 47K and the 20K proteins; the time courses and dose-responses of these B-7-induced phosphorylations were similar to those found with PMA. In addition, B-7 increases the level of /sup 32/P-incorporation into the platelet polyphosphoinositides, which also occurs in response to PMA. Bryostatin 3 (B-3), which has been shown to be much less potent than B-7 in mimicking other PMA effects, was much less effective than PMA or B-7 in inducing platelet aggregation and in stimulating /sup 32/P-incorporation into both proteins and the phosphoinositides. These results demonstrate that, intact human platelets, bryostatins mimic the phorbol esters tumor promoters and directly activate protein kinase C.

  10. Metabolic Plasticity in Resting and Thrombin Activated Platelets

    PubMed Central

    Ravi, Saranya; Chacko, Balu; Sawada, Hirotaka; Kramer, Philip A.; Johnson, Michelle S.; Benavides, Gloria A.; O’Donnell, Valerie; Marques, Marisa B.; Darley-Usmar, Victor M.

    2015-01-01

    Platelet thrombus formation includes several integrated processes involving aggregation, secretion of granules, release of arachidonic acid and clot retraction, but it is not clear which metabolic fuels are required to support these events. We hypothesized that there is flexibility in the fuels that can be utilized to serve the energetic and metabolic needs for resting and thrombin-dependent platelet aggregation. Using platelets from healthy human donors, we found that there was a rapid thrombin-dependent increase in oxidative phosphorylation which required both glutamine and fatty acids but not glucose. Inhibition of fatty acid oxidation or glutamine utilization could be compensated for by increased glycolytic flux. No evidence for significant mitochondrial dysfunction was found, and ATP/ADP ratios were maintained following the addition of thrombin, indicating the presence of functional and active mitochondrial oxidative phosphorylation during the early stages of aggregation. Interestingly, inhibition of fatty acid oxidation and glutaminolysis alone or in combination is not sufficient to prevent platelet aggregation, due to compensation from glycolysis, whereas inhibitors of glycolysis inhibited aggregation approximately 50%. The combined effects of inhibitors of glycolysis and oxidative phosphorylation were synergistic in the inhibition of platelet aggregation. In summary, both glycolysis and oxidative phosphorylation contribute to platelet metabolism in the resting and activated state, with fatty acid oxidation and to a smaller extent glutaminolysis contributing to the increased energy demand. PMID:25875958

  11. Platelet activation and platelet-leukocyte interaction in dogs naturally infected with Babesia rossi.

    PubMed

    Goddard, Amelia; Leisewitz, Andrew L; Kristensen, Annemarie T; Schoeman, Johan P

    2015-09-01

    Using flow cytometry, platelet-leukocyte aggregate (PLA) formation has previously been documented in dogs with a variety of systemic inflammatory disorders and immune-mediated haemolytic anaemia. Platelet activation and subsequent interaction between platelets and leukocytes are important for regulating innate immunity and systemic inflammation. The objective of this study was to investigate PLA formation in canine babesiosis and to determine whether it was associated with outcome. Blood was collected from 36 client-owned dogs diagnosed with Babesia rossi infection and 15 healthy controls using EDTA as anticoagulant. Activated platelets and PLA formation were detected by measuring surface expression of P-selectin (CD62P) on platelets, monocytes and neutrophils. Of the Babesia-infected dogs, 29 survived and seven died. The percentage of CD62P-positive monocytes was significantly higher (P = 0.036) in the Babesia-infected dogs (54%) than in healthy control dogs (35.3%). However, there were no significant differences between the Babesia-infected and control groups for CD62P-positive platelets (4.9% and 1.2%, respectively) and CD62P-positive neutrophils (28.3% and 17.9%, respectively). The percentage of CD62P-positive monocytes was significantly higher (P = 0.019) in the survivors (58.9%) than in healthy control dogs; however, there were no significant differences between the non-survivors (39.2%) and the controls or between survivors and non-survivors. There were no significant differences between groups for the percentage of CD62P-positive platelets (survivors 4.8%; non-survivors 5.3%; controls 1.2%) or CD62P-positive neutrophils (survivors 31.6%; non-survivors 5.6%; controls 17.9%). In conclusion, Babesia-infected dogs, specifically dogs that survived, had a significantly increased percentage of platelet-monocyte aggregates compared to healthy control dogs. PMID:26088270

  12. Accuracy of platelet counting by automated hematologic analyzers in acute leukemia and disseminated intravascular coagulation: potential effects of platelet activation.

    PubMed

    Kim, Seon Young; Kim, Ji-Eun; Kim, Hyun Kyung; Han, Kyou-Sup; Toh, Cheng Hock

    2010-10-01

    Platelet counting in patients with acute leukemia or disseminated intravascular coagulation (DIC) may have a risk for erroneous counts owing to the presence of nonplatelet particles or platelet activation. We evaluated automated platelet counting methods using the Abbott Cell-Dyn Sapphire (Abbott Diagnostics, Santa Clara, CA), Sysmex XE-2100 (Sysmex, Kobe, Japan), ADVIA 2120 (Siemens Diagnostics, Tarrytown, NY), and Beckman Coulter LH 750 (Beckman Coulter, Miami, FL) compared with the international reference method (IRM). Automated platelet counting methods were inaccurate compared with the IRM, without evidence of interfering nonplatelet particles. It is interesting that platelet activation markers were associated with DIC severity and erroneous platelet counting, suggesting that platelet activation is a potential source of inaccuracy. Furthermore, the artifactual in vitro platelet activation induced a high degree of intermethod variation in platelet counts. The inaccuracy of automated platelet counts increased the risk for misdiagnosis of DIC. More attention needs to be given to the accuracy of platelet counts, especially in clinical conditions with florid platelet activation. PMID:20855645

  13. Resolvin E1 Regulates ADP Activation of Human Platelets

    PubMed Central

    Fredman, Gabrielle; Van Dyke, Thomas E.; Serhan, Charles N.

    2010-01-01

    Objective Resolvin E1 (RvE1) is an eicosapentaenoic acid (EPA)-derived specialized pro-resolving mediator generated during resolution of acute inflammation. RvE1 exhibits potent organ-protective actions in vivo and acts on specific cell types including platelets. Here, we investigated the ability of RvE1 to regulate adenosine diphosphate (ADP) activation of platelets via specific receptors because RvE1 reduces platelet aggregation with certain agonists including ADP. Methods and Results RvE1 (0.1nM–100nM) incubated with platelets gave reduced ADP-stimulated P-selectin mobilization (IC50 ~1.6×10−12 M) and polymerized actin content compared to control platelets. RvE1 (1–100nM) did not stimulate or block intracellular calcium mobilization. Using a new P2Y12-β-arrestin-coupled cell system, ADP-activated P2Y12 with an EC50 of 5×10−6 M and RvE1 did not directly stimulate P2Y12 or block ADP-P2Y12 signals. In this system, another eicosanoid LTE4 (EC50 1.3×10−11 M) dose dependently activated P2Y12. When recombinant P2Y12-expressing cells were transiently transfected with an RvE1 receptor, human ChemR23 (present on human platelets), addition of RvE1 (0.1nM-10.0nM) blocked ADP signals (IC50 ~1.6×10−11 M) in P2Y12-ChemR23-expressing cells compared to mock transfections. Conclusions These results demonstrate that RvE1’s regulatory actions (i.e reducing ADP-stimulated P-selectin mobilization and actin polymerization) are hChemR23-dependent. Moreover, they document specific platelet actions of RvE1 selectively engaged with ADP-activated platelets that illuminate a new cellular mechanism and impact of omega-3 EPA that may contribute to both resolution of vascular inflammation and ADP-dependent platelet activation relevant in pathologic cardiovascular events. PMID:20702811

  14. Decreased platelet function in aortic valve stenosis: high shear platelet activation then inactivation.

    PubMed Central

    O'Brien, J. R.; Etherington, M. D.; Brant, J.; Watkins, J.

    1995-01-01

    OBJECTIVE--To elucidate the mechanism of the bleeding tendency observed in patients with aortic valve stenosis. DESIGN--A prospective study of high and low shear platelet function tests in vitro in normal controls compared with that in patients with severe aortic valve stenosis with a mean (SD) systolic gradient by Doppler of 75 (18) mm Hg before and at least 4 months after aortic valve replacement. SETTING--District general hospital. RESULTS--The patients showed reduced retention in the high shear platelet function tests. (a) Platelet retention in the filter test was 53.6 (12.6)% in patients with aortic valve stenosis and 84.8 (9.6)% in the controls (P < 0.001). (b) Retention in the glass bead column test was 49.8 (19.2) in the patients and 87.4 (8.7) in the controls (P < 0.001). (c) The standard bleeding time was longer in the patients (P < 0.06). Results of the high shear tests (a, b, and c) after aortic valve replacement were within the normal range. The platelet count was low but within the normal range before surgery and increased postoperatively (P < 0.01). There were no differences in the results of standard clotting tests, plasma and intraplatelet von Willebrand's factor, or in 15 platelet aggregation tests using five agonists between patients with aortic valve stenosis and controls. CONCLUSIONS--The high shear haemodynamics of aortic valve stenosis modify platelet function in vivo predisposing to a bleeding tendency. This abnormality of platelet function is detectable only in vitro using high shear tests. The abnormal function is reversed by aortic valve replacement. High shear forces in vitro activate and then inactivate platelets. By the same mechanisms aortic valve stenosis seems to lead to high shear damage in vivo, resulting in a clinically important bleeding tendency in some patients. PMID:8541170

  15. Immunoregulatory activity of peptides related to platelet factor 4.

    PubMed Central

    Zucker, M B; Katz, I R; Thorbecke, G J; Milot, D C; Holt, J

    1989-01-01

    Platelet factor 4 (PF4), a secreted platelet protein, alleviates concanavalin A-induced immunosuppression in mice. We now find that activity also resides in (i) the C-terminal tridecapeptide of PF4 (P13S), (ii) an analog of this in which arginine replaces the lysine residues and in which the last two amino acids are absent, (iii) the C-terminal 18 amino acids of low-affinity platelet factor 4, which is very similar to P13S, and (iv) peptide fragments of P13S that contain only 5-9 amino acids. P13S treated with fluorescamine to derivatize the free amino groups retained immunoregulatory activity but did not bind to heparin-agarose. The N-terminal and middle portions of PF4, polylysine, protamine, and three unrelated peptides were inactive in this assay. PMID:2678107

  16. The novel platelet activation receptor CLEC-2.

    PubMed

    Suzuki-Inoue, Katsue; Inoue, Osamu; Ozaki, Yukio

    2011-01-01

    The c-type lectin-like receptor 2 (CLEC-2) was first identified from a bio-informatic screen for c-type lectin-like receptors. However, neither its function nor its ligand(s) had been elucidated for several years. In 2006, we reported that the receptor is expressed on the surface of platelets and serves as a receptor for the snake venom rhodocytin, which potently stimulates platelet aggregation. Since then CLEC-2 has been intensively investigated, and its endogenous/exogenous ligands and several physiological/pathological roles have been clarified. In this article and its accompanying poster, we outline the structure, distribution, signal transduction mechanism and functions of CLEC-2. PMID:21714702

  17. gamma. -hexachlorocyclohexane (. gamma. -HCH) activates washed rabbit platelets

    SciTech Connect

    Lalau-Keraly, C.; Delautier, D.; Benveniste, J.; Puiseux-Dao, S.

    1986-03-01

    In guinea-pig macrophages, ..gamma..-HCH triggers activation of the phosphatidylinositol cycle and Ca/sup 2 +/ mobilization. Since these two biochemical events are also involved in platelet activation, the authors examined the effects of ..gamma..-HCH on washed rabbit platelets. Release of /sup 14/C-serotonin (/sup 14/C-5HT) and ATP from platelets prelabelled with /sup 14/C-5HT was measured simultaneously with aggregation. ..gamma..-HCH induced shape-change, aggregation and release reaction of platelets. Maximal aggregation (89 arbitrary units, AU), was observed using 170 ..mu..M ..gamma..-HCH, and was associated with 38.1 +/- 6.9% and 161 +/- 48 nM for /sup 14/C-5HT and ATP release respectively (mean +/- 1 SD, n=3). Using 80 ..mu..M ..gamma..-HCH yielded 18 AU, 12.8 +/- 1.0% and 27 +/- 14 nM for aggregation, C-5HT and ATP release respectively (n=3). No effect was observed with 40 ..mu.. M ..gamma..-HCH. Aspirin (ASA), a cyclooxygenase blocker, did not affect ..gamma..-HCH-induced platelet activation. Apyrase (APY), an ADP scavenger, inhibited by 90% aggregation induced by 170 ..mu..M ..gamma..-HCH and slightly inhibited (15%) the /sup 14/C-5HT release. In the presence of both ASA and APY, 96% inhibition of aggregation and 48% inhibition of /sup 14/C-5HT release were observed. Thus, ..gamma..-HCH induced platelet activation in a dose-dependent manner ADP, but not cyclooxygenase-dependent arachidonate metabolites, is involved in ..gamma..-HCH-induced aggregation, whereas, both appear to play a role in ..gamma..-HCH-induced release reaction.

  18. Flow cytometric analysis of platelet activation under calcium ion-chelating conditions.

    PubMed

    Nishioka, T; Yokota, M; Tsuda, I; Tatsumi, N

    2002-04-01

    Platelet activation and aggregation results in factitious counting and sizing in routine haematology testing. In this study, the possibility of platelet activation in anticoagulated solutions was examined. Whole blood was examined using an automated counter and a flow cytometer before and after strong vortex agitation. Blood treated with ethylenediaminetetraacetic acid (EDTA) exhibited platelet activation both pre- and postagitation but activated platelets did not cause platelet aggregation. With sodium citrate, platelets were only minimally activated both pre- and postagitation. Heparin-treated blood exhibited minimal platelet activation preagitation, but agitation resulted in strong platelet activation and aggregation. Platelet size was increased by agitation in blood with EDTA and with sodium citrate, in association with significant increases in mean platelet volume (MPV) and platelet distribution width (PDW), but MPV and PDW were significantly higher in EDTA solution than in sodium citrate solution. Change in platelet size was observed even in the presence of EDTA, indicating that careful sampling and processing are needed in the collection of specimens. Specimens obtained from patients with EDTA-dependent pseudothrombocytopenia exhibited the same level of activation as controls, although platelets exhibited aggregation in such specimens. In conclusion, platelet activation involving platelet size change can occur in the absence of calcium ions in blood treated with EDTA. PMID:11985558

  19. Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis

    PubMed Central

    Takagi, Satoshi; Takemoto, Ai; Takami, Miho; Oh-hara, Tomoko; Fujita, Naoya

    2014-01-01

    The interactions of tumor cells with platelets contribute to the progression of tumor malignancy, and the expression levels of platelet aggregation-inducing factors positively correlate with the metastatic potential of osteosarcoma cells. However, it is unclear how tumor-platelet interaction contributes to the proliferation of osteosarcomas. We report here that osteosarcoma-platelet interactions induce the release of platelet-derived growth factor (PDGF) from platelets, which promotes the proliferation of osteosarcomas. Co-culture of platelets with MG63 or HOS osteosarcoma cells, which could induce platelet aggregation, enhanced the proliferation of each cell line in vitro. Analysis of phospho-antibody arrays revealed that co-culture of MG63 cells with platelets induced the phosphorylation of platelet derived growth factor receptor (PDGFR) and Akt. The addition of supernatants of osteosarcoma-platelet reactants also increased the growth of MG63 and HOS cells as well as the level of phosphorylated-PDGFR and -Akt. Sunitinib or LY294002, but not erlotinib, significantly inhibited the platelet-induced proliferation of osteosarcoma cells, indicating that PDGF released from platelets plays an important role in the proliferation of osteosarcomas by activating the PDGFR and then Akt. Our results suggest that inhibitors that specifically target osteosarcoma-platelet interactions may eradicate osteosarcomas. PMID:24974736

  20. Unaltered Angiogenesis-Regulating Activities of Platelets in Mild Type 2 Diabetes Mellitus despite a Marked Platelet Hyperreactivity.

    PubMed

    Miao, Xinyan; Zhang, Wei; Huang, Zhangsen; Li, Nailin

    2016-01-01

    Type 2 diabetes mellitus (T2DM) is associated with platelet dysfunction and impaired angiogenesis. Aim of the study is to investigate if platelet dysfunction might hamper platelet angiogenic activities in T2DM patients. Sixteen T2DM patients and gender/age-matched non-diabetic controls were studied. Flow cytometry and endothelial colony forming cell (ECFC) tube formation on matrigel were used to assess platelet reactivity and angiogenic activity, respectively. Thrombin receptor PAR1-activating peptide (PAR1-AP) induced higher platelet P-selectin expression, and evoked more rapid and intense platelet annexin V binding in T2DM patients, seen as a more rapid increase of annexin V+ platelets (24.3±6.4% vs 12.6±3.8% in control at 2 min) and a higher elevation (30.9±5.1% vs 24.3±3.0% at 8 min). However, PAR1-AP and PAR4-AP induced similar releases of angiogenic regulators from platelets, and both stimuli evoked platelet release of platelet angiogenic regulators to similar extents in T2DM and control subjects. Thus, PAR1-stimulated platelet releasate (PAR1-PR) and PAR4-PR similarly enhanced capillary-like network/tube formation of ECFCs, and the enhancements did not differ between T2DM and control subjects. Direct supplementation of platelets to ECFCs at the ratio of 1:200 enhanced ECFC tube formation even more markedly, leading to approximately 100% increases of the total branch points of ECFC tube formation, for which the enhancements were also similar between patients and controls. In conclusion, platelets from T2DM subjects are hyperreactive. Platelet activation induced by high doses of PAR1-AP, however, results in similar releases of angiogenic regulators in mild T2DM and control subjects. Platelets from T2DM and control subjects also demonstrate similar enhancements on ECFC angiogenic activities. PMID:27612088

  1. Endothelial Activation by Platelets from Sickle Cell Anemia Patients

    PubMed Central

    Proença-Ferreira, Renata; Brugnerotto, Ana Flávia; Garrido, Vanessa Tonin; Dominical, Venina Marcela; Vital, Daiana Morelli; Ribeiro, Marilene de Fátima Reis; dos Santos, Melissa Ercolin; Traina, Fabíola; Olalla-Saad, Sara T.; Costa, Fernando Ferreira; Conran, Nicola

    2014-01-01

    Sickle cell anemia (SCA) is associated with a hypercoagulable state. Increased platelet activation is reported in SCA and SCA platelets may present augmented adhesion to the vascular endothelium, potentially contributing to the vaso-occlusive process. We sought to observe the effects of platelets (PLTs) from healthy control (CON) individuals and SCA individuals on endothelial activation, in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured, in the presence, or not, of washed PLTs from CON or steady-state SCA individuals. Supernatants were reserved for cytokine quantification, and endothelial adhesion molecules (EAM) were analyzed by flow cytometry; gene expressions of ICAM1 and genes of the NF-κB pathway were analyzed by qPCR. SCA PLTs were found to be more inflammatory, displaying increased adhesive properties, an increased production of IL-1β and a tendency towards elevated expressions of P-selectin and activated αIIbβ3. Following culture in the presence of SCA PLTs, HUVEC presented significant augmentations in the expressions of the EAM, ICAM-1 and E-selectin, as well as increased IL-8 production and increased ICAM1 and NFKB1 (encodes p50 subunit of NF-κB) gene expressions. Interestingly, transwell inserts abolished the effects of SCA PLTs on EAM expression. Furthermore, an inhibitor of the NF-κB pathway, BAY 11-7082, also prevented the induction of EAM expression on the HUVEC surface by SCA PLTs. In conclusion, we find further evidence to indicate that platelets circulate in an activated state in sickle cell disease and are capable of stimulating endothelial cell activation. This effect appears to be mediated by direct contact, or even adhesion, between the platelets and endothelial cells and via NFκB-dependent signaling. As such, activated platelets in SCD may contribute to endothelial activation and, therefore, to the vaso-occlusive process. Results provide further evidence to support the use of anti-platelet approaches in association

  2. Endothelial activation by platelets from sickle cell anemia patients.

    PubMed

    Proença-Ferreira, Renata; Brugnerotto, Ana Flávia; Garrido, Vanessa Tonin; Dominical, Venina Marcela; Vital, Daiana Morelli; Ribeiro, Marilene de Fátima Reis; dos Santos, Melissa Ercolin; Traina, Fabíola; Olalla-Saad, Sara T; Costa, Fernando Ferreira; Conran, Nicola

    2014-01-01

    Sickle cell anemia (SCA) is associated with a hypercoagulable state. Increased platelet activation is reported in SCA and SCA platelets may present augmented adhesion to the vascular endothelium, potentially contributing to the vaso-occlusive process. We sought to observe the effects of platelets (PLTs) from healthy control (CON) individuals and SCA individuals on endothelial activation, in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured, in the presence, or not, of washed PLTs from CON or steady-state SCA individuals. Supernatants were reserved for cytokine quantification, and endothelial adhesion molecules (EAM) were analyzed by flow cytometry; gene expressions of ICAM1 and genes of the NF-κB pathway were analyzed by qPCR. SCA PLTs were found to be more inflammatory, displaying increased adhesive properties, an increased production of IL-1β and a tendency towards elevated expressions of P-selectin and activated αIIbβ3. Following culture in the presence of SCA PLTs, HUVEC presented significant augmentations in the expressions of the EAM, ICAM-1 and E-selectin, as well as increased IL-8 production and increased ICAM1 and NFKB1 (encodes p50 subunit of NF-κB) gene expressions. Interestingly, transwell inserts abolished the effects of SCA PLTs on EAM expression. Furthermore, an inhibitor of the NF-κB pathway, BAY 11-7082, also prevented the induction of EAM expression on the HUVEC surface by SCA PLTs. In conclusion, we find further evidence to indicate that platelets circulate in an activated state in sickle cell disease and are capable of stimulating endothelial cell activation. This effect appears to be mediated by direct contact, or even adhesion, between the platelets and endothelial cells and via NFκB-dependent signaling. As such, activated platelets in SCD may contribute to endothelial activation and, therefore, to the vaso-occlusive process. Results provide further evidence to support the use of anti-platelet approaches in association

  3. Fibrin activates GPVI in human and mouse platelets.

    PubMed

    Alshehri, Osama M; Hughes, Craig E; Montague, Samantha; Watson, Stephanie K; Frampton, Jon; Bender, Markus; Watson, Steve P

    2015-09-24

    The glycoprotein VI (GPVI)-Fc receptor γ (FcRγ) chain is the major platelet signaling receptor for collagen. Paradoxically, in a FeCl3 injury model, occlusion, but not initiation of thrombus formation, is delayed in GPVI-deficient and GPVI-depleted mice. In this study, we demonstrate that GPVI is a receptor for fibrin and speculate that this contributes to development of an occlusive thrombus. We observed a marked increase in tyrosine phosphorylation, including the FcRγ chain and Syk, in human and mouse platelets induced by thrombin in the presence of fibrinogen and the αIIbβ3 blocker eptifibatide. This was not seen in platelets stimulated by a protease activated receptor (PAR)-4 peptide, which is unable to generate fibrin from fibrinogen. The pattern of tyrosine phosphorylation was similar to that induced by activation of GPVI. Consistent with this, thrombin did not induce tyrosine phosphorylation of Syk and the FcRγ chain in GPVI-deficient mouse platelets. Mouse platelets underwent full spreading on fibrin but not fibrinogen, which was blocked in the presence of a Src kinase inhibitor or in the absence of GPVI. Spreading on fibrin was associated with phosphatidylserine exposure (procoagulant activity), and this too was blocked in GPVI-deficient platelets. The ectodomain of GPVI was shown to bind to immobilized monomeric and polymerized fibrin. A marked increase in embolization was seen following FeCl3 injury in GPVI-deficient mice, likely contributing to the delay in occlusion in this model. These results demonstrate that GPVI is a receptor for fibrin and provide evidence that this interaction contributes to thrombus growth and stability. PMID:26282541

  4. Fibrin activates GPVI in human and mouse platelets

    PubMed Central

    Alshehri, Osama M.; Montague, Samantha; Watson, Stephanie K.; Frampton, Jon; Bender, Markus; Watson, Steve P.

    2015-01-01

    The glycoprotein VI (GPVI)-Fc receptor γ (FcRγ) chain is the major platelet signaling receptor for collagen. Paradoxically, in a FeCl3 injury model, occlusion, but not initiation of thrombus formation, is delayed in GPVI-deficient and GPVI-depleted mice. In this study, we demonstrate that GPVI is a receptor for fibrin and speculate that this contributes to development of an occlusive thrombus. We observed a marked increase in tyrosine phosphorylation, including the FcRγ chain and Syk, in human and mouse platelets induced by thrombin in the presence of fibrinogen and the αIIbβ3 blocker eptifibatide. This was not seen in platelets stimulated by a protease activated receptor (PAR)-4 peptide, which is unable to generate fibrin from fibrinogen. The pattern of tyrosine phosphorylation was similar to that induced by activation of GPVI. Consistent with this, thrombin did not induce tyrosine phosphorylation of Syk and the FcRγ chain in GPVI-deficient mouse platelets. Mouse platelets underwent full spreading on fibrin but not fibrinogen, which was blocked in the presence of a Src kinase inhibitor or in the absence of GPVI. Spreading on fibrin was associated with phosphatidylserine exposure (procoagulant activity), and this too was blocked in GPVI-deficient platelets. The ectodomain of GPVI was shown to bind to immobilized monomeric and polymerized fibrin. A marked increase in embolization was seen following FeCl3 injury in GPVI-deficient mice, likely contributing to the delay in occlusion in this model. These results demonstrate that GPVI is a receptor for fibrin and provide evidence that this interaction contributes to thrombus growth and stability. PMID:26282541

  5. Antithrombotic activity of Vitis labrusca extract on rat platelet aggregation.

    PubMed

    Kwon, Se-Uk; Lee, Hoon-Yeon; Xin, Mingjie; Ji, Su-Jeong; Cho, Hyoung-Kwon; Kim, Dae-Sung; Kim, Dae-Ki; Lee, Young-Mi

    2016-03-01

    Vitis labrusca is a grapevine that has antioxidant, neuroprotective, hepatoprotective, and anticarcinogenic activity. However, the antithrombotic effect of Vitis labrusca leaves on platelets is yet to be ascertained. We investigated the inhibitory effect of V. labrusca leaf extract (VLE) on platelet aggregation in vitro and ex vivo. The thromboxane B2 (TXB2) and serotonin concentrations were measured by ELISA. The flavonoids content was measured by ultraperformance liquid chromatography (UPLC). The antithrombotic activity of VLE was evaluated using various agonists in vitro. VLE strongly inhibited adenosine diphosphate (ADP)-induced platelet aggregation. In rats, VLE treatment (100 mg/kg) reduced ADP-stimulated platelet aggregation, without affecting tail bleeding and coagulation time. Moreover, VLE significantly suppressed TXB2 and serotonin secretion. UPLC analysis indicated that VLE contains quercetin, isorhamnetin, and rutin. Our results indicate that VLE possesses antiplatelet activity via the suppression of TXB2 and serotonin, without affecting bleeding. Further, we identified the flavonoids present in VLE. Thus, VLE may be a potential agent for the prevention of cardiovascular diseases. PMID:26340455

  6. Platelets aggregation in pathological conditions: role of local shear rates and platelet activation delay time.

    NASA Astrophysics Data System (ADS)

    Li, He; Zarif Khalili Yazdani, Alireza; Karniadakis, George

    2015-11-01

    Platelets play an essential role in the initiation and formation of a thrombus, however their detailed motion in blood vessels with complex geometries, such as in the aneurysmal vessel or stenotic vessel in atherosclerosis, has not been studied systematically. Here, we perform spectral element simulations (NEKTAR code) to obtain the 3D flow field in blood vessel with cavities, and we apply the force coupling method (FCM) to simulate the motion of platelets in blood flow. Specifically, simulations of platelets are performed in a 0.25 mm diameter circular blood vessel with 1 mm length. Corresponding coarse-grained molecular dynamics simulations are employed to provide input to the NEKTAR-FCM code. Simulations are conducted at several different Reynolds numbers (Re). An ellipsoid-shaped cavity is selected to intersect with the middle part of the circular vessel to represent the aneurysmal part of the blood vessel. Based on the simulation results, we quantify how the platelets motion and aggregation in the blood vessel cavities depend on Re, platelet activation delay time, and the geometry of the cavities.

  7. Rapid in vitro biocompatibility assay of endovascular stents by flow cytometry using platelet activation and platelet-leukocyte aggregation.

    PubMed

    Tárnok, A; Mahnke, A; Müller, M; Zotz, R J

    1999-02-15

    Clinical studies suggest that stent design and surface texture are responsible for differences in biocompatibility of metallic endovascular stents. A simple in vitro experimental setup was established to test stent-induced degree of platelet and leukocyte activation and platelet-leukocyte aggregation by flow cytometry. Heparin-coated tantalum stents and gold-coated and uncoated stainless steel stents were tested. Stents were implanted into silicone tubes and exposed to blood from healthy volunteers. Platelet and leukocyte activation and percentage of leukocyte-platelet aggregates were determined in a whole-blood assay by subsequent staining for activation-associated antigens (CD41a, CD42b, CD62p, and fibrinogen binding) and leukocyte antigens (CD14 and CD45) and flow cytometric analysis. Blood taken directly after venous puncture or exposed to the silicone tube alone was used as negative controls. Positive control was in vitro stimulation with thrombin receptor activating peptide (TRAP-6). Low degree of platelet activation and significant increase in monocyte- and neutrophil-platelet aggregation were observed in blood exposed to stents (P < 0.05). In addition, leukocyte activation was induced as measured by increased CD45 and CD14 expression. Heparin coated stents continuously induced less platelet activation and leukocyte-platelet aggregation than uncoated stainless steel stents of the same length and shorter stents of the same structure. Stent surface coating and texture plays a role in platelet and leukocyte activation and leukocyte-platelet aggregation. Using this simple in vitro assay and whole blood and flow cytometry, it seems possible to differentiate stents by their potency to activate platelets and/or leukocytes. This assay could be applied for improving the biocompatibility of coronary stents. PMID:10088974

  8. Baseline Platelet Activation and Reactivity in Patients with Critical Limb Ischemia

    PubMed Central

    de Borst, Gert Jan; Verhaar, Marianne C.; Roest, Mark; Moll, Frans L.

    2015-01-01

    Background Patients with critical limb ischemia (CLI) have a high risk to develop cardiovascular events (CVE). We hypothesized that in CLI patients platelets would display increased baseline activation and reactivity. Objectives We investigated baseline platelet activation and platelet reactivity in patients with CLI. Patients/Methods In this study baseline platelet activation and platelet reactivity in response to stimulation of all major platelet activation pathways were determined in 20 CLI patients (11 using aspirin and 9 using vitamin K-antagonists) included in the Juventas-trial (clinicaltrials.gov NCT00371371) and in 17 healthy controls. Platelet activation was quantified with flow cytometric measurement of platelet P-selectin expression and fibrinogen binding. Results CLI patients not using aspirin showed higher baseline platelet activation compared to healthy controls. Maximal reactivity to stimulation of the collagen and thrombin activation pathway was decreased in CLI patients compared to healthy controls. In line, attenuated platelet reactivity to stimulation of multiple activation pathways was associated with several traditional risk factors for cardiovascular disease. Conclusions Baseline platelet activation was increased in CLI patients, whereas the reactivity of circulating platelets to several stimulatory agents is decreased. Reactivity of platelets was inversely correlated with cardiovascular risk factors. PMID:26148006

  9. Treatment of Platelet Concentrates with the Mirasol Pathogen Inactivation System Modulates Platelet Oxidative Stress and NF-κB Activation

    PubMed Central

    Johnson, Lacey; Marks, Denese

    2015-01-01

    Background Pathogen inactivation (PI) technologies for platelets aim to improve transfusion safety by preventing the replication of contaminating pathogens. However, as a consequence of treatment, aspects of the platelet storage lesion are amplified. Mirasol treatment also affects platelet signal transduction and apoptotic protein expression. The aim of this study was to examine the effect of Mirasol treatment on the generation of reactive oxygen species (ROS) and subsequent oxidative stress. Methods Pooled platelet concentrates were prepared in platelet-additive solution (70% SSP+ / 30% plasma). ABO-matched platelets were pooled and split, and treated with the Mirasol system (TerumoBCT) or left untreated as a control. Platelet samples were tested on day 1, 5, and 7 post-collection. Results Mirasol-treated platelets had increased formation of ROS by day 5 of storage. Oxidative damage, in the form of protein carbonylation, was higher in Mirasol-treated platelets, whilst no effect on nitrotyrosine formation or lipid peroxidation was detected. The NF-κB signaling pathway was also activated in Mirasol-treated platelets, with increased expression and phosphorylation of NF-κB p65 and IκBα. Conclusion These data demonstrate that Mirasol-treated platelets produce more ROS and display protein alterations consistent with oxidative damage. PMID:26195930

  10. Influence of activating hormones on human platelet membrane Ca/sup 2 +/-ATPase activity

    SciTech Connect

    Resink, T.J.; Dimitrov, D.; Stucki, S.; Buehler, F.R.

    1986-07-16

    Intact platelets were pretreated with hormones and thereafter membranes were prepared and Ca/sup 2 +/-ATPase activity determined. Thrombin decreased the V/sub max/ of Ca/sup 2 +/-ATPase after pretreatment of intact platelets. Platelet activating factor, vasopressin and ADP also decreased Ca/sup 2 +/-ATPase activity. 12-O-tetradecanoylphorbol-13-acetate (TPA) or A23187 or ionomycin alone had no effect, while the simultaneous pretreatment with TPA and Ca/sup 2 +/-ionophore decreased Ca/sup 2 +/-ATPase activity. cAMP elevating agents prostaglandin E/sub 1/ (PGE/sub 1/) and forskolin had no influence per se on Ca/sup 2 +/-ATPase, but antagonized the inhibitory effect of thrombin. The data suggest a close connection between phosphoinositide metabolism and the Ca/sup 2 +/-ATPase system.

  11. Platelet receptor recognition and cross-talk in collagen-induced activation of platelets.

    PubMed

    Farndale, R W; Slatter, D A; Siljander, P R-M; Jarvis, G E

    2007-07-01

    Comprehensive mapping of protein-binding sites within human collagen III has allowed the recognition motifs for integrin alpha(2)beta(1) and VWF A3 domain to be identified. Glycoprotein VI-binding sites are understood, although less well defined. This information, together with recent developments in understanding collagen fiber architecture, and crystal structures of the receptor collagen-binding domains, allows a coherent model for the interaction of collagen with the platelet surface to be developed. This complements our understanding of the orchestration of receptor presentation by membrane microdomains, such that the polyvalent collagen surface may stabilize signaling complexes within the heterogeneous receptor composition of the lipid raft. The ensuing interactions lead to the convergence of signals from each of the adhesive receptors, mediated by FcR gamma-chain and/or FcgammaRIIa, leading to concerted and co-operative platelet activation. Each receptor has a shear-dependent role, VWF/GpIb essential at high shear, and alpha(2)beta(1) at low and intermediate shear, whilst GpVI provides core signals that contribute to enhanced integrin affinity, tighter binding to collagen and consequent platelet activation. PMID:17635730

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

    SciTech Connect

    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 or 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).

  13. The involvement of platelet activating factor in endotoxin-induced pulmonary platelet recruitment in the guinea-pig.

    PubMed Central

    Beijer, L.; Botting, J.; Crook, P.; Oyekan, A. O.; Page, C. P.; Rylander, R.

    1987-01-01

    1 Exposure of conscious guinea-pigs to an aerosol of endotoxin (25-100 micrograms ml-1) resulted in a dose-related, progressive accumulation of platelets in the thoracic region. Accumulation of 111indium oxine labelled erythrocytes was not observed following exposure to an aerosol of endotoxin (50 micrograms ml-1). 2 Pretreatment of guinea-pigs with the selective platelet activating factor (Paf)-antagonists. CV-3988 or brotizolam resulted in a dose-related inhibition of endotoxin-induced pulmonary platelet recruitment. Pretreatment of guinea-pigs with the selective Paf-antagonist BN 52021 resulted in significant inhibition of endotoxin-induced pulmonary platelet recruitment, although the effects of BN 52021 were not dose-related. 3 Pretreatment of guinea-pigs with indomethacin at doses known to inhibit cyclo-oxygenase did not inhibit endotoxin-induced pulmonary platelet recruitment, whereas higher doses of indomethacin produced a reduction in platelet recruitment in the lung. 4 Pretreatment of guinea-pigs with the anticoagulant heparin and the prostacyclin analogue ZK 36374 inhibited endotoxin-induced platelet recruitment. 5 These observations suggest that endotoxin-induced pulmonary platelet recruitment in the guinea-pig is secondary to the release of platelet activating factor, but not to cyclo-oxygenase products of arachidonic acid and may also involve activation of the coagulation cascade. PMID:2447993

  14. Involvement of platelet cyclic GMP but not cyclic AMP suppression in leukocyte-dependent platelet adhesion to endothelial cells induced by platelet-activating factor in vitro.

    PubMed Central

    Hirafuji, M.; Nezu, A.; Shinoda, H.; Minami, M.

    1996-01-01

    1. Incubation of endothelial cells with platelets in the absence or the presence of PAF (10 nM) markedly increased platelet cyclic AMP levels, which were significantly decreased by indomethacin (3 microM). Co-incubation of endothelial cells and platelets with polymorphonuclear leukocytes (PMNs) did not change the platelet cyclic AMP levels. 2. Incubation of endothelial cells with platelets in the absence of PAF increased platelet cyclic GMP levels, which were increased 3.5 fold by PAF. These cyclic GMP levels were significantly decreased by NG-nitro-L-arginine (100 microM), and completely by methylene blue (10 microM). When endothelial cells and platelets were co-incubated with PMNs, the cyclic GMP level in the cell mixture was 42.5 and 65.3% lower than that in endothelial cells and platelets without and with PAF stimulation, respectively. 3. PAF induced platelet adhesion to endothelial cells only when PMNs were present. Methylene blue dose-dependently potentiated the PMN-dependent platelet adhesion induced by PAF, although it had no effect in the absence of PMNs. 4. Sodium nitroprusside and 8-bromo cyclic GMP but not dibutyryl cyclic AMP significantly, although partially, inhibited the platelet adhesion. Inhibition of cyclic GMP-specific phosphodiesterase by zaprinast slightly inhibited the PMN-induced platelet adhesion and potentiated the inhibitory effect of 8-bromo cyclic GMP, while these drugs markedly inhibited the adhesion of platelet aggregates induced by PMN sonicates. 5. These results suggest that the impairment by activated PMNs of EDRF-induced platelet cyclic GMP formation is involved in part in the mechanism of PMN-dependent platelet adhesion to endothelial cells induced by PAF in vitro. The precise mechanism still remains to be clarified. PMID:8789382

  15. CD44 sensitivity of platelet activation, membrane scrambling and adhesion under high arterial shear rates.

    PubMed

    Liu, Guilai; Liu, Guoxing; Alzoubi, Kousi; Chatterjee, Madhumita; Walker, Britta; Münzer, Patrick; Luo, Dong; Umbach, Anja T; Elvira, Bernat; Chen, Hong; Voelkl, Jakob; Föller, Michael; Mak, Tak W; Borst, Oliver; Gawaz, Meinrad; Lang, Florian

    2016-01-01

    CD44 is required for signalling of macrophage migration inhibitory factor (MIF), an anti-apoptotic pro-inflammatory cytokine. MIF is expressed and released from blood platelets, key players in the orchestration of occlusive vascular disease. Nothing is known about a role of CD44 in the regulation of platelet function. The present study thus explored whether CD44 modifies degranulation (P-selectin exposure), integrin activation, caspase activity, phosphatidylserine exposure on the platelet surface, platelet volume, Orai1 protein abundance and cytosolic Ca(2+)-activity ([Ca2+]i). Platelets from mice lacking CD44 (cd44(-/-)) were compared to platelets from corresponding wild-type mice (cd44(+/+)). In resting platelets, P-selectin abundance, α(IIb)β3 integrin activation, caspase-3 activity and phosphatidylserine exposure were negligible in both genotypes and Orai1 protein abundance, [Ca2+]i, and volume were similar in cd44(-/-) and cd44(+/+) platelets. Platelet degranulation and α(IIb)β3 integrin activation were significantly increased by thrombin (0.02 U/ml), collagen related peptide (CRP, 2 µg/ml and Ca(2+)-store depletion with thapsigargin (1 µM), effects more pronounced in cd44(-/-) than in cd44(+/+) platelets. Thrombin (0.02 U/ml) increased platelet [Ca2+]i, caspase-3 activity, phosphatidylserine exposure and Orai1 surface abundance, effects again significantly stronger in cd44(-/-) than in cd44(+/+) platelets. Thrombin further decreased forward scatter in cd44(-/-) and cd44(+/+) platelets, an effect which tended to be again more pronounced in cd44(-/-) than in cd44(+/+) platelets. Platelet adhesion and in vitro thrombus formation under high arterial shear rates (1,700 s(-1)) were significantly augmented in cd44(-/-) mice. In conclusion, genetic deficiency of CD44 augments activation, apoptosis and pro-thrombotic potential of platelets. PMID:26355696

  16. Platelet activating factor: regulation by mast cells and aspirin.

    PubMed

    Denburg, J A; Williams, D B; Kinlough-Rathbone, R L; Cazenave, J P; Bienenstock, J

    1984-02-01

    We have investigated some aspects of the regulation of production of rat platelet activating factor (PAF)2 in vitro. Suspensions of unseparated (PLC1), mast cell-depleted (PLC2), or mast cell (MC)-enriched rat peritoneal lavage cells (PLC) were analyzed for PAF content by extraction at alkaline pH. PAF activity extracted from PLC1 varied inversely with viable cell concentration: at 1 X 10(6) cells/ml, 32 +/- 9.3 PAF units, decreasing to 11.2 +/- 9.5 units at 10 X 10(6) cells/ml, and no activity at higher concentrations. Incubation of PLC1 in Tyrode's buffer or acetylsalicylic acid (ASA), but not salicylate, resulted in a time-dependent loss of PAF activity. Mean PAF activity of PLC2 was similar to that in PLC1, while no PAF activity was extractable from MC. Co-incubation with MC extracts inhibited PAF activity of PLC1 extracts in a dose-dependent fashion. Ultracentrifugation of PAF-containing samples led to a loss of all PAF activity in PLC1 extracts, suggesting the association of PAF activity with subcellular components. PAF appears to be derived from a non-MC population of rat PLC, is not extractable from rat PLC in the presence of ASA and is inhibited by MC extracts. These studies suggest that ASA regulates PAF availability unrelated to its effect on cyclooxygenase and that MC membrane products directly inhibit PAF activity from rat PLC. PMID:6711391

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-08-01

    Surface modification of single-walled carbon nanotubes (SWCNTs) such as carboxylation, amidation, hydroxylation and pegylation is used to reduce the nanotube toxicity and render them more suitable for biomedical applications than their pristine counterparts. Toxicity can be manifested in platelet activation as it has been shown for SWCNTs. However, the effect of various surface modifications on the platelet activating potential of SWCNTs has not been tested yet. In vitro platelet activation (CD62P) as well as the platelet-granulocyte complex formation (CD15/CD41 double positivity) in human whole blood were measured by flow cytometry in the presence of 0.1mg/ml of pristine or various surface modified SWCNTs. The effect of various SWCNTs was tested by whole blood impedance aggregometry, too. All tested SWCNTs but the hydroxylated ones activate platelets and promote platelet-granulocyte complex formation in vitro. Carboxylated, pegylated and pristine SWCNTs induce whole blood aggregation as well. Although pegylation is preferred from biomedical point of view, among the samples tested by us pegylated SWCNTs induced far the most prominent activation and a well detectable aggregation of platelets in whole blood. PMID:25956790

  19. Lack of association between serum paraoxonase-1 activity and residual platelet aggregation during dual anti-platelet therapy.

    PubMed

    Ohmori, Tsukasa; Yano, Yuichiro; Sakata, Asuka; Ikemoto, Tomokazu; Shimpo, Masahisa; Madoiwa, Seiji; Katsuki, Takaaki; Mimuro, Jun; Shimada, Kazuyuki; Kario, Kazuomi; Sakata, Yoichi

    2012-04-01

    High residual platelet aggregability during thienopyridine treatment occurs because of low levels of the active drug metabolite, and is associated with an increased rate of major adverse cardiovascular events. Recent findings suggest that paraoxonase-1 (PON1) is a major determinant for clopidogrel efficacy. The aim of this study was to assess the impact of serum PON1 activity on platelet aggregability in thienopyridine-treated patients. In 72 patients receiving treatment with aspirin and ticlopidine after acute coronary syndrome, various laboratory data including the formation of platelet aggregations induced by agonists were compared with serum PON1 activities, measured as paraoxonase and homocysteine thiolactone hydrolase (HTLase). Serum paraoxonase activity was significantly associated with HTLase activity (R=0.4487, P<0.0001). These PON1 activities were not correlated with any parameters for platelet aggregation, hypertension, sleep apnea, and diabetes mellitus. In contrast, serum PON1 activities seemed to be involved in cardiac function, with brain natriuretic peptide and ejection fraction being significantly correlated with serum HTLase activity (R=-0.2767, P=0.0214) and paraoxonase activity (R=0.2558, P=0.0339), respectively. Paraoxonase activity also demonstrated a significant association with increased levels of ankle-brachial index (R=0.267, P=0.0255). Serum PON1 activities did not influence platelet aggregability during treatment with thienopyridine. However, they might modulate cardiac function after acute coronary syndrome and progression of atherosclerosis. PMID:22115701

  20. RASA3 is a critical inhibitor of RAP1-dependent platelet activation

    PubMed Central

    Stefanini, Lucia; Paul, David S.; Robledo, Raymond F.; Chan, E. Ricky; Getz, Todd M.; Campbell, Robert A.; Kechele, Daniel O.; Casari, Caterina; Piatt, Raymond; Caron, Kathleen M.; Mackman, Nigel; Weyrich, Andrew S.; Parrott, Matthew C.; Boulaftali, Yacine; Adams, Mark D.; Peters, Luanne L.; Bergmeier, Wolfgang

    2015-01-01

    The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders. PMID:25705885

  1. Secretion of platelet-activating factor by periovulatory ovine follicles

    SciTech Connect

    Alexander, B.M.; Van Kirk, E.A.; Murdoch, W.J. )

    1990-01-01

    Secretion of platelet-activating factor (PAF) in vitro by ovine follicles and ovarian interstitium obtained at various times before, during and after the endogenous preovulatory surge of luteinizing hormone (LH) and ovulation was quantified by radioimmunoassay. Release of PAF by the preovulatory follicle increased within 2 h after initiation of the surge of LH. Capacity for secretion of PAF was greatest at the time of ovulation, then declined thereafter. Production of PAF by ovarian interstitium throughout the periovulatory period was relatively low and did not change with time. It appears that PAF could act as an intrafollicular mediator in the mechanisms of ovulation and(or) luteinization.

  2. Platelet-Activating Factor-Receptor and Tumor Immunity

    PubMed Central

    Sahu, Ravi P; Konger, Raymond L.; Travers, Jeffrey B.

    2016-01-01

    First described in 1972 by Benveniste and colleagues, platelet-activating factor (PAF) remains one of the potent phospholipid known to date. The role of PAF produced enzymatically in mediating diverse biological and pathophysiological processes including inflammatory and allergic diseases and cancers in response to various stimuli has been extensively studied. However, little is known about the role of non-enzymatically-generated PAF-like lipids produced in response to pro-oxidative stressors, particularly in modulating the host immune responses to tumor immunity, which is the focus of this review.

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

    PubMed Central

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

    2015-01-01

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

  4. Platelet surface-associated activation and secretion-mediated inhibition of coagulation factor XII.

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  6. Suboptimal Activation of Protease-activated Receptors Enhances α2β1 Integrin-mediated Platelet Adhesion to Collagen*

    PubMed Central

    Marjoram, Robin J.; Voss, Bryan; Pan, Yumei; Dickeson, S. Kent; Zutter, Mary M.; Hamm, Heidi E.; Santoro, Samuel A.

    2009-01-01

    Thrombin and fibrillar collagen are potent activators of platelets at sites of vascular injury. Both agonists cause platelet shape change, granule secretion, and aggregation to form the primary hemostatic plug. Human platelets express two thrombin receptors, protease-activated receptors 1 and 4 (PAR1 and PAR4) and two collagen receptors, the α2β1 integrin (α2β1) and the glycoprotein VI (GPVI)/FcRγ chain complex. Although these receptors and their signaling mechanisms have been intensely studied, it is not known whether and how these receptors cooperate in the hemostatic function of platelets. This study examined cooperation between the thrombin and collagen receptors in platelet adhesion by utilizing a collagen-related peptide (α2-CRP) containing the α2β1-specific binding motif, GFOGER, in conjunction with PAR-activating peptides. We demonstrate that platelet adhesion to α2-CRP is substantially enhanced by suboptimal PAR activation (agonist concentrations that do not stimulate platelet aggregation) using the PAR4 agonist peptide and thrombin. The enhanced adhesion induced by suboptimal PAR4 activation was α2β1-dependent and GPVI/FcRγ-independent as revealed in experiments with α2β1- or FcRγ-deficient mouse platelets. We further show that suboptimal activation of other platelet Gq-linked G protein-coupled receptors (GPCRs) produces enhanced platelet adhesion to α2-CRP. The enhanced α2β1-mediated platelet adhesion is controlled by phospholipase C (PLC), but is not dependent on granule secretion, activation of αIIbβ3 integrin, or on phosphoinositol-3 kinase (PI3K) activity. In conclusion, we demonstrate a platelet priming mechanism initiated by suboptimal activation of PAR4 or other platelet Gq-linked GPCRs through a PLC-dependent signaling cascade that promotes enhanced α2β1 binding to collagens containing GFOGER sites. PMID:19815553

  7. Hinokitiol inhibits platelet activation ex vivo and thrombus formation in vivo.

    PubMed

    Lin, Kuan H; Kuo, Jinn R; Lu, Wan J; Chung, Chi L; Chou, Duen S; Huang, Shih Y; Lee, Hsiu C; Sheu, Joen R

    2013-05-15

    Hinokitiol is a tropolone-related bioactive compound that has been used in hair tonics, cosmetics, and food as an antimicrobial agent. Recently, hinokitiol has attracted considerable interest because of its anticancer activities. Platelet activation plays a crucial role in atherothrombotic processes. We examined the effects of hinokitiol treatment on platelet activation using human platelets. In the present study, hinokitiol (1 and 2 μM) inhibited the collagen-induced aggregation of human platelets, but did not inhibit the activation of platelets by other agonists, including thrombin, arachidonic acid, and ADP. Hinokitiol inhibited the phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and Akt in collagen-activated human platelets, and significantly reduced intracellular calcium mobilization and hydroxyl radical (OH·) formation. Hinokitiol also reduced the PKC activation and platelet aggregation stimulated by PDBu. In addition, hinokitiol significantly prolonged thrombogenesis in mice. Hinokitiol did not influence the binding of a fluorescent triflavin probe to the αIIbβ3 integrin on platelet membrane, and neither ODQ nor SQ22536 significantly reversed the hinokitiol-mediated inhibition of platelet aggregation. In conclusion, hinokitiol may inhibit platelet activation by inhibiting the PLCγ2-PKC cascade and hydroxyl radical formation, followed by suppressing the activation of MAPKs and Akt. Our study suggests that hinokitiol may represent a potential therapeutic agent for the prevention or treatment of thromboembolic disorders. PMID:23473801

  8. A-Disintegrin-And-Metalloproteinase (ADAM) 10 Activity on Resting and Activated Platelets.

    PubMed

    Facey, Adam; Pinar, Isaac; Arthur, Jane F; Qiao, Jianlin; Jing, Jing; Mado, Belden; Carberry, Josie; Andrews, Robert K; Gardiner, Elizabeth E

    2016-03-01

    The primary platelet collagen receptor, glycoprotein VI (GPVI), plays an important role in platelet activation and thrombosis. The ectodomain of human GPVI (sGPVI) is proteolytically shed from human platelets by a-disintegrin-and-metalloproteinase 10 (ADAM10). In this study, we used a novel ADAM10-sensitive fluorescence resonance energy transfer sensor to analyze ADAM10-mediated shedding of GPVI from human platelets in response to the exposure of GPVI ligands collagen-related peptide (10 μg/mL), collagen (10 μg/mL), and convulxin (0.1 μg/mL) to shear stress (1000-10000 s(-1), 5 min), or a generic activator of metalloproteinases, N-ethylmaleimide (NEM, 5 mM). Elevated shear, NEM, or ligand engagement of GPVI all induced shedding of GPVI, as detected by release of sGPVI; however, only shear or NEM significantly increased ADAM10 enzyme activity. ADAM10 activity was also detectable on the surface of thrombi formed on a collagen-coated surface under flow conditions. Our findings indicate different mechanisms regulate shear- and ligand-induced shedding and shear forces found within the vasculature can regulate ADAM10 activity. PMID:26840909

  9. Serotonin content of platelets in inflammatory rheumatic diseases. Correlation with clinical activity.

    PubMed

    Zeller, J; Weissbarth, E; Baruth, B; Mielke, H; Deicher, H

    1983-04-01

    Significantly decreased platelet serotonin contents were measured in rheumatoid arthritis, systemic lupus erythematosus (SLE), progressive systemic sclerosis, and mixed connective tissue disease. An inverse relationship between platelet serotonin levels and clinical disease activity was observed in both rheumatoid arthritis and systemic lupus erythematosus. SLE patients with multiple organ involvement showed the lowest platelet serotonin values. No correlation was observed between platelet serotonin contents and nonsteroidal antiinflammatory drug treatment, presence of circulating platelet reactive IgG, or the amount of circulating immune complexes. The results are interpreted as indicating platelet release occurring in vivo during inflammatory episodes of the rheumatic disorders investigated. PMID:6838676

  10. Platelet and growth factor concentrations in activated platelet-rich plasma: a comparison of seven commercial separation systems.

    PubMed

    Kushida, Satoshi; Kakudo, Natsuko; Morimoto, Naoki; Hara, Tomoya; Ogawa, Takeshi; Mitsui, Toshihito; Kusumoto, Kenji

    2014-06-01

    Platelet-rich plasma (PRP) is blood plasma that has been enriched with platelets. It holds promise for clinical use in areas such as wound healing and regenerative medicine, including bone regeneration. This study characterized the composition of PRP produced by seven commercially available separation systems (JP200, GLO PRP, Magellan Autologous Platelet Separator System, KYOCERA Medical PRP Kit, SELPHYL, MyCells, and Dr. Shin's System THROMBO KIT) to evaluate the platelet, white blood cell, red blood cell, and growth factor concentrations, as well as platelet-derived growth factor-AB (PDGF-AB), transforming growth factor beta-1 (TGF-β1), and vascular endothelial growth factor (VEGF) concentrations. PRP prepared using the Magellan Autologous Platelet Separator System and the KYOCERA Medical PRP Kit contained the highest platelet concentrations. The mean PDGF-AB concentration of activated PRP was the highest from JP200, followed by the KYOCERA Medical PRP Kit, Magellan Autologous Platelet Separator System, MyCells, and GLO PRP. TGF-β1 and VEGF concentrations varied greatly among individual samples, and there was almost no significant difference among the different systems, unlike for PDGF. The SELPHYL system produced PRP with low concentrations of both platelets and growth factors. Commercial PRP separation systems vary widely, and familiarity with their individual advantages is important to extend their clinical application to a wide variety of conditions. PMID:24748436

  11. Platelet-rich plasma stimulated by pulse electric fields: Platelet activation, procoagulant markers, growth factor release and cell proliferation.

    PubMed

    Frelinger Iii, 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

    2016-03-01

    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. PMID:26030682

  12. Purification of human plasma platelet-activating factor acetylhydrolase

    SciTech Connect

    Stafforini, D.M.; Prescott, S.M.; McIntyre, T.M.

    1986-05-01

    Platelet-activating factor (PAF;1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine is synthesized by a variety of cells. It induces hypotension, and activates platelets, neutrophils, and macrophages at nanomolar concentrations. Removal of the acetate abolishes biological activity, and is catalyzed by a specific PAF acetylhydrolase present in plasma and tissues. The authors developed a rapid assay, based on separation of (/sup 3/H)acetate from (/sup 3/H-acetyl)PAF by reversed-phase chromatography. In human plasma the enzyme exhibits an apparent Km of 5.7..mu..M, with a Vmax of 0.027..mu..mol/h/mg. Ultracentrifugation in density gradients showed that 30% of the activity is associated with high density lipoproteins (HDL) and 70% with low density lipoproteins (LDL). The enzyme was purified from LDL by precipitation with Na phosphotungstate and MgCl/sub 2/, solubilization with Tween 20, column chromatography and electrophoresis. This procedure resulted in a preparation that was 21,000-fold purified from plasma (spec. act. 575..mu..mol/h/mg) with a recovery of 10%. The purified enzyme has a molecular weight of about 43,000, a broad pH optimum (peak 7.5-8.0), and a pl of 4.6. It has greater activity when PAF is in a micellar, as compared to monomeric, and exhibits surface dilution kinetics, which may be important in vivo. The purification and characterization of this enzyme will allow detailed studies of its role in PAF metabolism.

  13. In vitro model of platelet-endothelial activation due to cigarette smoke under cardiovascular circulation conditions.

    PubMed

    Girdhar, Gaurav; Xu, Sulan; Jesty, Jolyon; Bluestein, Danny

    2008-07-01

    Cigarette smoke has been shown to increase platelet activation and endothelial cell (EC) adhesion molecule expression. In the present study, we utilized a hemodynamic shearing device (HSD) to investigate the above effects in vitro in a combined system of platelets and cultured HUVECs (Human Umblical Vein ECs) under physiological shear stress. We investigated the alteration of E-selectin expression on ECs upon exposure to: (1) platelets and nicotine-free smoke extract (NFE), (2) platelets alone, (3) NFE alone, under physiological shear stress. We additionally confirmed the protective effect of nicotine on platelet activation. We found that: (i) surface expression of E-selectin on ECs was significantly increased upon simultaneous exposure of ECs and platelets to NFE relative to exposure of ECs to either platelets or NFE alone (p < 0.05). (ii) Platelet activation was significantly increased in the presence of NFE (p < 0.05). (iii) Nicotine (200 nM) when added to NFE, significantly reduced platelet activation due to NFE (p < 0.05), an effect additionally confirmed by conventional cigarette extracts which contain nicotine (p < 0.05). We therefore conclude that: (a) NFE and platelets additively increase EC E-selectin surface expression, and (b) nicotine modulates platelet activation regardless of ECs. PMID:18452059

  14. Functional validation of platelet-activating factor receptor sites characterized biochemically by a specific and reproducible ( sup 3 H)platelet-activating factor binding in human platelets

    SciTech Connect

    Tahraoui, L.; Floch, A.; Cavero, I. )

    1990-03-01

    In human platelet membranes, (3H)platelet-activating factor(PAF)-C18 binding sites exhibited high affinity (Kd 0.074 +/- 0.005 nM, n = 28 healthy volunteers), saturability, elevated stereoselectivity, marked pharmacological specificity and small intersubject variability. The maximal binding capacity was 215 +/- 12 fmol/mg protein. Saturation of (3H)PAF binding was obtained with 0.3 nM ligand, and its isotherm was compatible with a single class of binding sites. The stereoselectivity for (3H)PAF was clearly indicated by the low displacing potency of enantio-PAF-C16 (the synthetic enantiomer of PAF) that was 5000-fold less potent than PAF. Specific (3H)PAF binding attained 65% with 0.1 nM ligand and was displaced fully not only by cold PAF but also by RP 59227 (Ki = 6.2 +/- 1.3 nM, n = 7), a novel, potent and specific PAF receptor antagonist in a pure enantiomeric form and several other antagonists such as CV-6209, WEB 2086, L-652,731 and BN 52021. Various classical pharmacological agents did not interfere with the (3H)PAF binding. In intact platelets, (3H)PAF binding shared the same properties as those just described for membrane preparations. A functional role for these binding sites was suggested by the high correlation (r = 0.94, P less than .001) between the Ki values for several known PAF antagonists determined in (3H)PAF binding and the IC50 values obtained against PAF-induced aggregation in whole platelets. Thus, the present (3H)PAF binding in human platelet membranes may be a useful pharmacological tool to study possible changes in (3H)PAF binding parameters induced by pathological states for which PAF may be directly or indirectly responsible.

  15. Impaired platelet activation and cAMP homeostasis in MRP4-deficient mice

    PubMed Central

    Decouture, Benoit; Dreano, Elise; Belleville-Rolland, Tiphaine; Kuci, Orjeta; Dizier, Blandine; Bazaa, Amine; Coqueran, Bérard; Lompre, Anne-Marie; Denis, Cécile V.; Hulot, Jean-Sébastien; Gaussem, Pascale

    2015-01-01

    Molecules that reduce the level of cyclic adenosine 5′-monophosphate (cAMP) in the platelet cytosol, such as adenosine 5′-diphosphate (ADP) secreted from dense granules, trigger platelet activation. Therefore, any change in the distribution and/or availability of cyclic nucleotides or ADP may interfere with platelet reactivity. In this study, we evaluated the role of multidrug resistance protein 4 (MRP4, or ABCC4), a nucleotide transporter, in platelet functions in vivo and in vitro by investigating MRP4-deficient mice. MRP4 deletion resulted in a slight increase in platelet count but had no impact on platelet ultrastructure. In MRP4-deficient mice, the arterial occlusion was delayed and the tail bleeding time was prolonged. In a model of platelet depletion and transfusion mimicking a platelet-specific knockout, mice injected with MRP4−/− platelets also showed a significant increase in blood loss compared with mice injected with wild-type platelets. Defective thrombus formation and platelet activation were confirmed in vitro by studying platelet adhesion to collagen in flow conditions, integrin αIIbβ3 activation, washed platelet secretion, and aggregation induced by low concentrations of proteinase-activated receptor 4–activating peptide, U46619, or ADP. We found no role of MRP4 in ADP dense-granule storage, but MRP4 redistributed cAMP from the cytosol to dense granules, as confirmed by increased vasodilator-stimulated phosphoprotein phosphorylation in MRP4-deficient platelets. These data suggest that MRP4 promotes platelet aggregation by modulating the cAMP–protein kinase A signaling pathway, suggesting that MRP4 might serve as a target for novel antiplatelet agents. PMID:26316625

  16. Thrombospondin 1 expression and angiogenesis in breast carcinoma and their relation with platelet activity

    PubMed Central

    Ersoz, Gulriz; Dilek, Fatma Husniye; Gencer, Ercan; Kosar, Mehmet Nuri; Dilek, Osman Nuri

    2009-01-01

    This study investigates angiogenesis and the expression of thrombospondin 1 in invasive ductal carcinoma of the breast and their possible relation to platelet counts and platelet activity. The study included 20 cases of invasive ductal carcinoma. Platelet activity was evaluated by determining thromboxane B2 and cyclic guanosine monophosphate (cGMP) levels by enzyme immunoassay (EIA).Thrombospondin (TSP) 1 and CD34 expression was studied immunohistochemically. Mean platelet count of the patient group was significantly greater than the mean platelet count of the control group (P < 0.05). The platelet counts were positively correlated with tumour size (r=0.609; P < 0.01). Platelet counts were higher in the patients who had grade 3 microvessel density (P < 0.05). The mean basal platelet cGMP level in the patient group was significantly lower than it was in the control group (P < 0.05). Focal TSP immunoreactivity was detectable in 5 (20%) cases in the tumour cells, and in 9 (45%) cases in the stroma. We did not find any correlation between TSP-1 staining and angiogenesis, platelet counts, platelet activity, and the histological prognostic parameters. Our study confirms the essential role of platelets in tumour growth and angiogenesis. Decreased levels of cGMP in the patient group may cause platelet hyperreactivity. Although thrombospondin 1 may be upregulated in malignant breast tissue, this is not sufficient for tumour growth and dissemination according to our results. PMID:19396698

  17. Platelet activation at the onset of human endotoxemia is undetectable in vivo.

    PubMed

    Schrottmaier, Waltraud Cornelia; Kral, Julia Barbara; Zeitlinger, Markus; Salzmann, Manuel; Jilma, Bernd; Assinger, Alice

    2016-07-01

    Infection induces platelet activation and consumption, which leads to thrombocytopenia, enhances microvascular thrombosis, impairs microcirculation and eventually triggers disseminated intravascular coagulation (DIC). It is well characterized that endotoxemia results in a pro-inflammatory and pro-coagulatory state, which favors platelet activation. However the early, direct effects of endotoxemia on platelets have not been investigated so far. Therefore we aimed to determine the early effects of the endotoxin lipopolysaccharide (LPS) on platelet function in vivo. In a human endotoxemia model, 15 healthy volunteers were stimulated with LPS (2 ng/kg). Blood was drawn before, 10, 30 and 60 min after LPS challenge and platelet activation analyzed by flow cytometry (GPIIb/IIIa activation, surface CD62P and CD40L, intraplatelet reactive oxygen formation and platelet-leukocyte aggregates) and ELISA (sCD40L, sCD62P and CXCL4). In parallel, blood samples and platelets were spiked with LPS (50 pg/ml) in vitro and monitored over 60 min for the same platelet activation markers. In vitro platelet stimulation with LPS activated platelets independent of the presence of leukocytes and enhanced their adhesion to endothelial cells. In contrast, in vivo no increase in GPIIb/IIIa activation or surface expression of CD62P was observed. However, endotoxemia resulted in a significant drop in platelet count and elevated the plasma CXCL4 levels already 10 min after the LPS challenge. These data indicate that LPS rapidly activates platelets, leading to α-granule release and endothelial adhesion. This might explain the drop in platelet count observed at the onset of endotoxemia. PMID:26764560

  18. Platelet anti-aggregation activities of compounds from Cinnamomum cassia.

    PubMed

    Kim, Sun Young; Koo, Yean Kyoung; Koo, Ja Yong; Ngoc, Tran Minh; Kang, Sam Sik; Bae, KiHwan; Kim, Yeong Sik; Yun-Choi, Hye Sook

    2010-10-01

    Cinnamomum cassia is a well-known traditional medicine for improvement of blood circulation. An extract of this plant showed both platelet anti-aggregation and blood anti-coagulation effects in preliminary testing. Among the 13 compounds obtained from this plant, eugenol (2), amygdalactone (4), cinnamic alcohol (5), 2-hydroxycinnamaldehyde (7), 2-methoxycinnamaldehyde (8), and coniferaldehyde (9) showed 1.5-73-fold greater inhibitory effects than acetylsalicylic acid (ASA) on arachidonic acid (AA)-induced aggregation (50% inhibitory concentration [IC₅₀] = 3.8, 5.16, 31.2, 40.0, 16.9, and 0.82 μM, respectively, vs. 60.3 μM) and 6.3-730-fold stronger effect than ASA on U46619 (a thromboxane A₂ mimic)-induced aggregation (IC₅₀ = 3.51, 33.9, 31.0, 51.3, 14.6, and 0.44 μM, respectively, vs. 321 μM). The other compounds, coumarin (3), cinnamaldehyde (6), cinnamic acid (10), icariside DC (11), and dihydrocinnacasside (12), also inhibited (2.5 to four times greater than ASA) U46619-induced aggregation. In addition, compounds 2, 4, 5, 6, 7, 8, and 9 were 1.3-87 times more effective than ASA against epinephrine-induced aggregation (IC₅₀ = 1.86, 1.10, 37.7, 25.0, 16.8, 15.3, and 0.57 μM, respectively, vs. 50.0 μM). However, the 13 compounds were only very mildly effective against blood coagulation, if at all. In conclusion, compounds 2, 4, 8, and 9 showed stronger inhibitory potencies than others on AA-, U46619-, and epinephrine-induced platelet aggregation. Eugenol (2) and coniferaldehyde (9) were the two of the most active anti-platelet constituents of C. cassia. PMID:20828311

  19. Pravastatin and C reactive protein modulate protease- activated receptor-1 expression in vitro blood platelets.

    PubMed

    Chu, L-X; Zhou, S-X; Yang, F; Qin, Y-Q; Liang, Z-S; Mo, C-G; Wang, X-D; Xie, J; He, L-P

    2016-01-01

    Protease-activated receptor-1 (PAR-1) plays an important role in mediating activation of human platelets by thrombin. However, mechanism of statin in ADP-induced platelet PAR-1 expression is also unknown. Aggregometry, flow cytometry, immunoblotting and ELISA were used to determine role of pravastatin participating in ADP-induced platelet activation and PAR-1 expression. ADP stimulation significantly increased PAR-1 expression on platelets. PAR-1 antagonist SCH-79797 inhibited platelet aggregation as well as decreased platelet P-selectin expression induced by ADP. CRP inhibited PAR-1 expression induced by ADP in a concentration-dependent manner. Pravastatin treatment reduced PAR-1 expression in a concentration-dependent manner. Combination treatment of CRP and Pravastatin significantly reduced platelet PAR-1 expression induced by ADP. By western-blot analysis, pravastatin treatment did not influence total PAR-1 after ADP treatment. CRP decreased platelet total PAR-1 expression induced by ADP. Pravastatin and CRP reduced TXB2 formation by ADP significantly. CRP decreased thrombin fragment F1+2 level with ADP treatment. Pravastatin, in contrast, did not influence F1+2 level. Upon treatment with Pravastatin reduced platelet LOX-1 expression induced by ADP. In conclusion, PAR-1 served as a critical mechanism to relay platelet activation process induced by ADP. CRP and pravastatin reduce PAR-1 expression in platelet by ADP pathway. PMID:26950455

  20. Epicatechin and Catechin Modulate Endothelial Activation Induced by Platelets of Patients with Peripheral Artery Disease

    PubMed Central

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

    2014-01-01

    Platelet activation contributes to the alteration of endothelial function, a critical initial step in atherogenesis through the production and release of prooxidant mediators. There is uncertainty about the precise role of polyphenols in interaction between platelets and endothelial cells (ECs). We aimed to investigate whether polyphenols are able to reduce endothelial activation induced by activated platelets. First, we compared platelet activation and flow-mediated dilation (FMD) in 10 healthy subjects (HS) and 10 patients with peripheral artery disease (PAD). Then, we evaluated the effect of epicatechin plus catechin on platelet-HUVEC interaction by measuring soluble cell adhesion molecules (CAMs), NOx production, and eNOS phosphorylation (p-eNOS) in HUVEC. Compared to HS, PAD patients had enhanced platelet activation. Conversely, PAD patients had lower FMD than HS. Supernatant of activated platelets from PAD patients induced an increase of sCAMs release and a decrease of p-eNOS and nitric oxide (NO) bioavailability compared to unstimulated HUVEC. Coincubation of HUVEC, with supernatant of PAD platelets patients, pretreated with a scalar dose of the polyphenols, resulted in a decrease of sCAMs release and in an increase of p-eNOS and NO bioavailability. This study demonstrates that epicatechin plus catechin reduces endothelial activation induced by activated platelets. PMID:25180068

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

    PubMed

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

    2014-01-01

    Platelet activation contributes to the alteration of endothelial function, a critical initial step in atherogenesis through the production and release of prooxidant mediators. There is uncertainty about the precise role of polyphenols in interaction between platelets and endothelial cells (ECs). We aimed to investigate whether polyphenols are able to reduce endothelial activation induced by activated platelets. First, we compared platelet activation and flow-mediated dilation (FMD) in 10 healthy subjects (HS) and 10 patients with peripheral artery disease (PAD). Then, we evaluated the effect of epicatechin plus catechin on platelet-HUVEC interaction by measuring soluble cell adhesion molecules (CAMs), NOx production, and eNOS phosphorylation (p-eNOS) in HUVEC. Compared to HS, PAD patients had enhanced platelet activation. Conversely, PAD patients had lower FMD than HS. Supernatant of activated platelets from PAD patients induced an increase of sCAMs release and a decrease of p-eNOS and nitric oxide (NO) bioavailability compared to unstimulated HUVEC. Coincubation of HUVEC, with supernatant of PAD platelets patients, pretreated with a scalar dose of the polyphenols, resulted in a decrease of sCAMs release and in an increase of p-eNOS and NO bioavailability. This study demonstrates that epicatechin plus catechin reduces endothelial activation induced by activated platelets. PMID:25180068

  2. Phosphoproteomic Analysis of Platelets Activated by Pro-Thrombotic Oxidized Phospholipids and Thrombin

    PubMed Central

    Zimman, Alejandro; Titz, Bjoern; Komisopoulou, Evangelia; Biswas, Sudipta; Graeber, Thomas G.; Podrez, Eugene A.

    2014-01-01

    Specific oxidized phospholipids (oxPCCD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPCCD36 are not well defined. We have employed mass spectrometry-based tyrosine, serine, and threonine phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPCCD36 as well as by the strong physiological agonist thrombin. oxPCCD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide candidates in the study of platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCγ2 was identified in platelets activated by oxPCCD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPCCD36. Our results provide multiple insights into the mechanism of platelet activation and specifically in platelet regulation by oxPCCD36. PMID:24400094

  3. Motor-driven marginal band coiling promotes cell shape change during platelet activation

    PubMed Central

    Diagouraga, Boubou; Grichine, Alexei; Fertin, Arnold; Wang, Jin; Khochbin, Saadi

    2014-01-01

    Platelets float in the blood as discoid particles. Their shape is maintained by microtubules organized in a ring structure, the so-called marginal band (MB), in the periphery of resting platelets. Platelets are activated after vessel injury and undergo a major shape change known as disc to sphere transition. It has been suggested that actomyosin tension induces the contraction of the MB to a smaller ring. In this paper, we show that antagonistic microtubule motors keep the MB in its resting state. During platelet activation, dynein slides microtubules apart, leading to MB extension rather than contraction. The MB then starts to coil, thereby inducing the spherical shape of activating platelets. Newly polymerizing microtubules within the coiled MB will then take a new path to form the smaller microtubule ring, in concerted action with actomyosin tension. These results present a new view of the platelet activation mechanism and reveal principal mechanistic features underlying cellular shape changes. PMID:24421335

  4. [Determination of activated platelets: evaluation of methodology and application for patients with idiopathic thrombocytopenic purpura].

    PubMed

    Hayashi, S; Oshida, M; Kiyokawa, T; Aochi, H; Honda, S; Tomiyama, Y; Kurata, Y

    2001-12-01

    Platelet activation causes a change in surface expression of several endogenous proteins, such as CD62P, CD63 and CD40L. Therefore, it is possible to analyze the functional in vivo status of the circulating platelet population directly by flow cytometry. In this study we developed the method to be suitable for use in clinical studies. We used EDTA-2K as anticoagulant since the sample anticoagulated with EDTA-2K, sodium citrate or ACD-A showed no difference in the data of activated platelets. We determined whether fixation of sample is necessary. The samples stained before or without fixation showed abnormally high level of activated platelets, indicating that fixation is necessary before staining. It is controversial whether activated platelets circulate in patients with idiopathic thrombocytopenic purpura(ITP). We measured activated platelets in patients with ITP using our optimised method. The percentages of CD62P, CD63 and CD40L positive platelets were significantly high in patients with ITP and 24%, 55% and 36% (respectively) of ITP patients showed elevated level of activated platelets. These data indicate that activated platelets circulate in ITP patients. PMID:11797399

  5. Relationship between structure of phenothiazine analogues and their activity on platelet calcium fluxes.

    PubMed Central

    Enouf, J.; Lévy-Toledano, S.

    1984-01-01

    Phenothiazine analogues have been tested for their effect on calcium uptake into platelet membrane vesicles and on ionophore-induced platelet activation, both phenomena being Ca2+-dependent. Both calcium uptake into membrane vesicles and ionophore-induced platelet activation were inhibited by the drugs. Evidence for two inhibitors as potent as chlorpromazine and trifluoperazine was found. These drugs are apparently competitive inhibitors of calcium uptake. A structure-activity relationship has been established. The data suggest that the phenothiazines are able to inhibit calmodulin-insensitive calcium uptake of platelet membrane vesicles and that therefore they cannot be assumed to be selective inhibitors of calmodulin interactions under all circumstances. PMID:6697061

  6. Platelet activating factor as a mediator of equine cell locomotion.

    PubMed

    Dawson, J; Lees, P; Sedgwick, A D

    1988-01-01

    Equine polymorphonuclear (PMN) and mononuclear (MN) leucocytes were separated on Percoll gradients and used to study the chemoattractant properties of the polar ether-linked phospholipid, platelet activating factor (PAF). Six concentrations of PAF ranging from 1 ng/ml to 100 micrograms/ml were studied in each of two in vitro assay systems, the agarose microdroplet and a microfilter technique. Very significant (p less than 0.01) increases in the movement of both PMN and MN cells were obtained with most concentrations of PAF. In two instances there was no apparent concentration-response relationship, although the action of PAF was approximately bell-shaped in two others. The possible significance of these findings for equine inflammatory conditions is discussed. PMID:3188378

  7. Interleukin-6 and asymmetric dimethylarginine are associated with platelet activation after percutaneous angioplasty with stent implantation.

    PubMed

    Gremmel, Thomas; Perkmann, Thomas; Kopp, Christoph W; Seidinger, Daniela; Eichelberger, Beate; Koppensteiner, Renate; Steiner, Sabine; Panzer, Simon

    2015-01-01

    Data linking in vivo platelet activation with inflammation and cardiovascular risk factors are scarce. Moreover, the interrelation between endothelial dysfunction as early marker of atherosclerosis and platelet activation has not been studied, so far. We therefore sought to investigate the associations of inflammation, endothelial dysfunction and cardiovascular risk factors with platelet activation and monocyte-platelet aggregate (MPA) formation in 330 patients undergoing angioplasty with stent implantation for atherosclerotic cardiovascular disease. P-selectin expression, activation of glycoprotein IIb/IIIa and MPA formation were determined by flow cytometry. Interleukin (IL)-6, high sensitivity C-reactive protein and asymmetric dimethylarginine (ADMA) were measured by commercially available assays. IL-6 was the only parameter which was independently associated with platelet P-selectin expression and activated GPIIb/IIIa as well as with leukocyte-platelet interaction in multivariate regression analysis (all p<0.05). ADMA was independently associated with GPIIb/IIIa activation (p<0.05). Patients with high IL-6 exhibited a significantly higher expression of P-selectin than patients with low IL-6 (p=0.001), whereas patients with high ADMA levels showed a more pronounced activation of GPIIb/IIIa than patients with low ADMA (p=0.003). In conclusion, IL-6 and ADMA are associated with platelet activation after percutaneous angioplasty with stent implantation. It remains to be established whether they act prothrombotic and atherogenic themselves or are just surrogate markers for atherosclerosis with concomitant platelet activation. PMID:25807315

  8. Interleukin-6 and Asymmetric Dimethylarginine Are Associated with Platelet Activation after Percutaneous Angioplasty with Stent Implantation

    PubMed Central

    Gremmel, Thomas; Perkmann, Thomas; Kopp, Christoph W.; Seidinger, Daniela; Eichelberger, Beate; Koppensteiner, Renate; Steiner, Sabine; Panzer, Simon

    2015-01-01

    Data linking in vivo platelet activation with inflammation and cardiovascular risk factors are scarce. Moreover, the interrelation between endothelial dysfunction as early marker of atherosclerosis and platelet activation has not been studied, so far. We therefore sought to investigate the associations of inflammation, endothelial dysfunction and cardiovascular risk factors with platelet activation and monocyte-platelet aggregate (MPA) formation in 330 patients undergoing angioplasty with stent implantation for atherosclerotic cardiovascular disease. P-selectin expression, activation of glycoprotein IIb/IIIa and MPA formation were determined by flow cytometry. Interleukin (IL)-6, high sensitivity C-reactive protein and asymmetric dimethylarginine (ADMA) were measured by commercially available assays. IL-6 was the only parameter which was independently associated with platelet P-selectin expression and activated GPIIb/IIIa as well as with leukocyte-platelet interaction in multivariate regression analysis (all p<0.05). ADMA was independently associated with GPIIb/IIIa activation (p<0.05). Patients with high IL-6 exhibited a significantly higher expression of P-selectin than patients with low IL-6 (p=0.001), whereas patients with high ADMA levels showed a more pronounced activation of GPIIb/IIIa than patients with low ADMA (p=0.003). In conclusion, IL-6 and ADMA are associated with platelet activation after percutaneous angioplasty with stent implantation. It remains to be established whether they act prothrombotic and atherogenic themselves or are just surrogate markers for atherosclerosis with concomitant platelet activation. PMID:25807315

  9. Thrombin-initiated platelet activation in vivo is vWF independent during thrombus formation in a laser injury model

    PubMed Central

    Dubois, Christophe; Panicot-Dubois, Laurence; Gainor, Justin F.; Furie, Barbara C.; Furie, Bruce

    2007-01-01

    Adhesion of platelets to an injured vessel wall and platelet activation are critical events in the formation of a thrombus. Of the agonists involved in platelet activation, thrombin, collagen, and vWF are known to induce in vitro calcium mobilization in platelets. Using a calcium-sensitive fluorochrome and digital multichannel intravital microscopy to image unstimulated and stimulated platelets, calcium mobilization was monitored as a reporter of platelet activation (as distinct from platelet accumulation) during thrombus formation in live mice. In the absence of vWF, platelet activation was normal, but platelet adherence and aggregation were attenuated during thrombus formation following laser-induced injury in the cremaster muscle microcirculation. In WT mice treated with lepirudin, platelet activation was blocked, and platelet adherence and aggregation were inhibited. The kinetics of platelet activation and platelet accumulation were similar in FcRγ–/– mice lacking glycoprotein VI (GPVI), GPVI-depleted mice, and WT mice. Our results indicate that the tissue factor–mediated pathway of thrombin generation, but not the collagen-induced GPVI-mediated pathway, is the major pathway leading to platelet activation after laser-induced injury under the conditions employed. In the tissue factor–mediated pathway, vWF plays a role in platelet accumulation during thrombus formation but is not required for platelet activation in vivo. PMID:17380206

  10. SDF-1α is a novel autocrine activator of platelets operating through its receptor CXCR4.

    PubMed

    Walsh, Tony G; Harper, Matthew T; Poole, Alastair W

    2015-01-01

    Platelets store and secrete the chemokine stromal cell-derived factor (SDF)-1α upon platelet activation, but the ability of platelet-derived SDF-1α to signal in an autocrine/paracrine manner mediating functional platelet responses relevant to thrombosis and haemostasis is unknown. We sought to explore the role of platelet-derived SDF-1α and its receptors, CXCR4 and CXCR7 in facilitating platelet activation and determine the mechanism facilitating SDF-1α-mediated regulation of platelet function. Using human washed platelets, CXCR4 inhibition, but not CXCR7 blockade significantly abrogated collagen-mediated platelet aggregation, dense granule secretion and thromboxane (Tx) A2 production. Time-dependent release of SDF-1α from collagen-activated platelets supports a functional role for SDF-1α in this regard. Using an in vitro whole blood perfusion assay, collagen-induced thrombus formation was substantially reduced with CXCR4 inhibition. In washed platelets, recombinant SDF-1α in the range of 20-100 ng/mL(-1) could significantly enhance platelet aggregation responses to a threshold concentration of collagen. These enhancements were completely dependent on CXCR4, but not CXCR7, which triggered TxA2 production and dense granule secretion. Rises in cAMP were significantly blunted by SDF-1α, which could also enhance collagen-mediated Ca2+ mobilisation, both of which were mediated by CXCR4. This potentiating effect of SDF-1α primarily required TxA2 signalling acting upstream of dense granule secretion, whereas blockade of ADP signalling could only partially attenuate SDF-1α-induced platelet activation. Therefore, this study supports a potentially novel autocrine/paracrine role for platelet-derived SDF-1α during thrombosis and haemostasis, through a predominantly TxA2-dependent and ADP-independent pathway. PMID:25283599

  11. Biochemistry of platelet-activating factor: A unique class of biologically active phospholipids

    SciTech Connect

    Snyder, F. )

    1989-01-01

    This brief overview describes the chemical features of this unique bioactive phospholipid that possesses biologic properties identical to platelet-activating factor (PAF) and an antihypertensive polar renal lipid (APRL). The current understanding of PAF metabolism and its regulation are emphasized, particularly in the context of explaining the enzymatic source of PAF in physiologic vs pharmacologic processes. Also included are brief accounts of the biologic properties, structural-functional relationships, antagonists, receptors and mode of action of PAF.

  12. Phospholipase A2 activity in platelets. Immuno-purification and localization of the enzyme in rat platelets.

    PubMed

    Aarsman, A J; Leunissen-Bijvelt, J; Van den Koedijk, C D; Neys, F W; Verkleij, A J; Van den Bosch, H

    1989-01-01

    A comparative study on phospholipase A2 activity in platelet lysates from various species was carried out using identical assay conditions with phosphatidylethanolamine as substrate. Platelet phospholipase A2, both when expressed as activity per ml blood and as specific activity in KCl extracts, was low in human, cow, pig and goat. Moderate activities, in increasing order, were found in sheep, horse and rabbit, while rats showed by far the highest activity. In the latter four species total lysate activity was recovered in 1 M KCl extracts, suggesting that the enzyme occurs either in soluble form or as a peripheral membrane-associated protein. Immune cross-reactivity with monoclonal antibodies against rat liver mitochondrial phospholipase A2 was studied in dot-blot and monoclonal antibody-Sepharose binding experiments. Only sheep and rat platelet extracts contained cross-reactive phospholipase(s) A2. Immuno-affinity chromatography of rat platelet extracts indicated virtually complete binding of total phospholipase A2 activity and yielded pure enzyme in a single purification step. Enzyme visualization by immunogold electron microscopy showed a predominant localization in the matrix of alpha-granules. PMID:2519886

  13. Crystal Structure of Human Plasma Platelet-Activating Factor Acetylhydrolase

    SciTech Connect

    Samanta, U.; Bahnson, B

    2008-01-01

    Human plasma platelet-activating factor (PAF) acetylhydrolase functions by reducing PAF levels as a general anti-inflammatory scavenger and is linked to anaphylactic shock, asthma, and allergic reactions. The enzyme has also been implicated in hydrolytic activities of other pro-inflammatory agents, such as sn-2 oxidatively fragmented phospholipids. This plasma enzyme is tightly bound to low and high density lipoprotein particles and is also referred to as lipoprotein-associated phospholipase A{sub 2}. The crystal structure of this enzyme has been solved from x-ray diffraction data collected to a resolution of 1.5{angstrom}. It has a classic lipase {alpha}/{beta}-hydrolase fold, and it contains a catalytic triad of Ser{sup 273}, His{sup 351}, and Asp{sup 296}. Two clusters of hydrophobic residues define the probable interface-binding region, and a prediction is given of how the enzyme is bound to lipoproteins. Additionally, an acidic patch of 10 carboxylate residues and a neighboring basic patch of three residues are suggested to play a role in high density lipoprotein/low density lipoprotein partitioning. A crystal structure is also presented of PAF acetylhydrolase reacted with the organophosphate compound paraoxon via its active site Ser{sup 273}. The resulting diethyl phosphoryl complex was used to model the tetrahedral intermediate of the substrate PAF to the active site. The model of interface binding begins to explain the known specificity of lipoprotein-bound substrates and how the active site can be both close to the hydrophobic-hydrophilic interface and at the same time be accessible to the aqueous phase.

  14. An Assay of Measuring Platelet Reactivity Using Monoclonal Antibody against Activated Platelet Glycoprotein IIb/IIIa in Patients Taking Clopidogrel

    PubMed Central

    Choi, Joon-Hyouk; Kim, Song-Yi; Kim, Ki-Seok; Kim, Young Ree; Kang, Sung Ha

    2015-01-01

    Background and Objectives Residual platelet reactivity in patients who are taking clopidogrel is commonly measured with VerifyNow assay, which is based on the principle of light transmission aggregometry. However, to evaluate the residual platelet reactivity, it would be more accurate if the reactivity of platelet glycoprotein (GP) IIb/IIIa is directly monitored. In this study, PAC1, a monoclonal antibody against activated platelet GP IIb/IIIa, was used to measure the residual platelet reactivity. Subjects and Methods Twenty seven patients with coronary artery disease taking clopidogrel were enrolled. Platelets in whole blood were stained with fluorescein isothiocyanate (FITC)-conjugated PAC1. Mean fluorescence intensity (MFI) and % positive platelets (PP) were measured with flow cytometry, and the binding index (BI; MFI × %PP/100) was calculated. P2Y12 reaction unit (PRU) and % inhibition of VerifyNow assay were also measured in the usual manner. Results PRU of VerifyNow assay correlated significantly with MFI, %PP, and BI at 10 µM (r=0.59, 0.73, and 0.60, respectively, all p<0.005) and 20 µM of adenosine diphosphate (ADP; r=0.61, 0.75, and 0.63, respectively, all p<0.005). The % inhibition also correlated significantly with MFI, %PP, and BI at 10 µM (r=-0.60, -0.69, and -0.59, respectively, all p<0.005) and 20 µM of ADP (r=-0.63, -0.71, and -0.62, respectively, all p<0.005). Conclusion Direct measurements of the reactivity of platelet GP IIb/IIIa were feasible using PAC1 and flow cytometry in patients taking clopidogrel. Further clinical studies are required to determine the cut-off values which would define high residual platelet reactivity in patients on this treatment protocol. PMID:26413105

  15. Segregation of Platelet Aggregatory and Procoagulant Microdomains in Thrombus Formation Regulation by Transient Integrin Activation

    PubMed Central

    Munnix, Imke C.A.; Kuijpers, Marijke J.E.; Auger, Jocelyn; Thomassen, Christella M.L.G.D.; Panizzi, Peter; van Zandvoort, Marc A.M.; Rosing, Jan; Bock, Paul E.; Watson, Steve P.; Heemskerk, Johan W.M.

    2008-01-01

    Objective Platelets play a dual role in thrombosis by forming aggregates and stimulating coagulation. We investigated the commitment of platelets to these separate functions during collagen-induced thrombus formation in vitro and in vivo. Methods and Results High-resolution 2-photon fluorescence microscopy revealed that in thrombus formation under flow, fibrin(ogen)-binding platelets assembled into separate aggregates, whereas distinct patches of nonaggregated platelets exposed phosphatidylserine. The latter platelet population had inactivated αIibβ3 integrins and displayed increased binding of coagulation factors. Coated platelets, expressing serotonin binding sites, were not identified as a separate population. Thrombin generation and coagulation favored the transformation to phosphatidylserine-exposing platelets with inactivated integrins and reduced adhesion. Prolonged tyrosine phosphorylation in vitro resulted in secondary downregulation of active αIIbβ3. Conclusions These results lead to a new spatial model of thrombus formation, in which aggregated platelets ensure thrombus stability, whereas distinct patches of nonaggregated platelets effectuate procoagulant activity and generate thrombin and fibrin. Herein, the hemostatic activity of a developing thrombus is determined by the balance in formation of proaggregatory and procoagulant platelets. This balance is influenced by antiplatelet and anticoagulant medication. PMID:17761939

  16. Platelets mediate increased endothelium permeability in dengue through NLRP3-inflammasome activation

    PubMed Central

    Hottz, Eugenio D.; Lopes, Juliana F.; Freitas, Carla; Valls-de-Souza, Rogério; Oliveira, Marcus F.; Bozza, Marcelo T.; Da Poian, Andrea T.; Weyrich, Andrew S.; Zimmerman, Guy A.

    2013-01-01

    Dengue is the most frequent hemorrhagic viral disease and re-emergent infection in the world. Although thrombocytopenia is characteristically observed in mild and severe forms of dengue, the role of platelet activation in dengue pathogenesis has not been fully elucidated. We hypothesize that platelets have major roles in inflammatory amplification and increased vascular permeability during severe forms of dengue. Here we investigate interleukin (IL)-1β synthesis, processing, and secretion in platelets during dengue virus (DV) infection and potential contribution of these events to endothelial permeability during infection. We observed increased expression of IL-1β in platelets and platelet-derived microparticles from patients with dengue or after platelet exposure to DV in vitro. We demonstrated that DV infection leads to assembly of nucleotide-binding domain leucine rich repeat containing protein (NLRP3) inflammasomes, activation of caspase-1, and caspase-1–dependent IL-1β secretion. Our findings also indicate that platelet-derived IL-1β is chiefly released in microparticles through mechanisms dependent on mitochondrial reactive oxygen species–triggered NLRP3 inflammasomes. Inflammasome activation and platelet shedding of IL-1β–rich microparticles correlated with signs of increased vascular permeability. Moreover, microparticles from DV-stimulated platelets induced enhanced permeability in vitro in an IL-1–dependent manner. Our findings provide new evidence that platelets contribute to increased vascular permeability in DV infection by inflammasome-dependent release of IL-1β. PMID:24009231

  17. Human group II 14 kDa phospholipase A2 activates human platelets.

    PubMed Central

    Polgár, J; Kramer, R M; Um, S L; Jakubowski, J A; Clemetson, K J

    1997-01-01

    Recombinant human group II phospholipase A2 (sPLA2) added to human platelets in the low microg/ml range induced platelet activation, as demonstrated by measurement of platelet aggregation, thromboxane A2 generation and influx of intracellular free Ca2+ concentration and by detection of time-dependent tyrosine phosphorylation of platelet proteins. The presence of Ca2+ at low millimolar concentrations is a prerequisite for the activation of platelets by sPLA2. Mg2+ cannot replace Ca2+. Mg2+, given in addition to the necessary Ca2+, inhibits sPLA2-induced platelet activation. Pre-exposure to sPLA2 completely blocked the aggregating effect of a second dose of sPLA2. Albumin or indomethacin inhibited sPLA2-induced aggregation, similarly to the inhibition of arachidonic acid-induced aggregation. Platelets pre-treated with heparitinase or phosphatidylinositol-specific phospholipase C lost their ability to aggregate in response to sPLA2, although they still responded to other agonists. This suggests that a glycophosphatidylinositol-anchored platelet-membrane heparan sulphate proteoglycan is the binding site for sPLA2 on platelets. Previous reports have stated that sPLA2 is unable to activate platelets. The inhibitory effect of albumin and Mg2+, frequently used in aggregation studies, and the fact that isolated platelets lose their responsiveness to sPLA2 relatively quickly, may explain why the platelet-activating effects of sPLA2 have not been reported earlier. PMID:9355761

  18. Radiation therapy generates platelet-activating factor agonists

    PubMed Central

    Sahu, Ravi P.; Harrison, Kathleen A.; Weyerbacher, Jonathan; Murphy, Robert C.; Konger, Raymond L.; Garrett, Joy Elizabeth; Chin-Sinex, Helen Jan; Johnston, Michael Edward; Dynlacht, Joseph R.; Mendonca, Marc; McMullen, Kevin; Li, Gengxin; Spandau, Dan F.; Travers, Jeffrey B.

    2016-01-01

    Pro-oxidative stressors can suppress host immunity due to their ability to generate oxidized lipid agonists of the platelet-activating factor-receptor (PAF-R). As radiation therapy also induces reactive oxygen species, the present studies were designed to define whether ionizing radiation could generate PAF-R agonists and if these lipids could subvert host immunity. We demonstrate that radiation exposure of multiple tumor cell lines in-vitro, tumors in-vivo, and human subjects undergoing radiation therapy for skin tumors all generate PAF-R agonists. Structural characterization of radiation-induced PAF-R agonistic activity revealed PAF and multiple oxidized glycerophosphocholines that are produced non-enzymatically. In a murine melanoma tumor model, irradiation of one tumor augmented the growth of the other (non-treated) tumor in a PAF-R-dependent process blocked by a cyclooxygenase-2 inhibitor. These results indicate a novel pathway by which PAF-R agonists produced as a byproduct of radiation therapy could result in tumor treatment failure, and offer important insights into potential therapeutic strategies that could improve the overall antitumor effectiveness of radiation therapy regimens. PMID:26959112

  19. Radiation therapy generates platelet-activating factor agonists.

    PubMed

    Sahu, Ravi P; Harrison, Kathleen A; Weyerbacher, Jonathan; Murphy, Robert C; Konger, Raymond L; Garrett, Joy Elizabeth; Chin-Sinex, Helen Jan; Johnston, Michael Edward; Dynlacht, Joseph R; Mendonca, Marc; McMullen, Kevin; Li, Gengxin; Spandau, Dan F; Travers, Jeffrey B

    2016-04-12

    Pro-oxidative stressors can suppress host immunity due to their ability to generate oxidized lipid agonists of the platelet-activating factor-receptor (PAF-R). As radiation therapy also induces reactive oxygen species, the present studies were designed to define whether ionizing radiation could generate PAF-R agonists and if these lipids could subvert host immunity. We demonstrate that radiation exposure of multiple tumor cell lines in-vitro, tumors in-vivo, and human subjects undergoing radiation therapy for skin tumors all generate PAF-R agonists. Structural characterization of radiation-induced PAF-R agonistic activity revealed PAF and multiple oxidized glycerophosphocholines that are produced non-enzymatically. In a murine melanoma tumor model, irradiation of one tumor augmented the growth of the other (non-treated) tumor in a PAF-R-dependent process blocked by a cyclooxygenase-2 inhibitor. These results indicate a novel pathway by which PAF-R agonists produced as a byproduct of radiation therapy could result in tumor treatment failure, and offer important insights into potential therapeutic strategies that could improve the overall antitumor effectiveness of radiation therapy regimens. PMID:26959112

  20. Platelet Activation Following Implant of the Levitronix PediVAS in the Ovine Model

    PubMed Central

    Johnson, Carl A.; Shankarraman, Venkat; Wearden, Peter D.; Kocyildirim, Ergin; Maul, Timothy M.; Marks, John D.; Richardson, J. Scott; Gellman, Barry N.; Borovetz, Harvey S.; Dasse, Kurt A.; Wagner, William R.

    2011-01-01

    The Levitronix PediVAS is an extracorporeal magnetically levitated pediatric ventricular assist system with an optimal flow rate range of 0.3 – 1.5 L/min. The system is being tested in preclinical studies to assess hemodynamic performance and biocompatibility. The PediVAS was implanted in 9 ovines for 30 days duration using either commercially available cannulae (N = 3) or customized Levitronix cannulae (N = 6). Blood biocompatibility in terms of circulating activated platelets was measured by flow cytometric assays to detect P-selectin. Platelet activation was further examined following exogenous agonist stimulation. Platelet activation rose following surgery and eventually returned to baseline in animal studies where minimal kidney infarcts were observed. Platelet activation remained elevated for the duration of the study in animals where a moderate number of kidney infarcts with or without thrombotic deposition in the cannulae were observed. When platelet activation did return to baseline, platelets appropriately responded to agonist stimulation signifying conserved platelet function following PediVAS implant. Platelet activation returned to baseline in the majority of studies, representing a promising biocompatibility result for the Levitronix PediVAS. PMID:21989419

  1. The influence of statin therapy on platelet activity markers in hyperlipidemic patients after ischemic stroke

    PubMed Central

    Chmielewski, Henryk; Kaczorowska, Beata; Przybyła, Monika; Baj, Zbigniew

    2015-01-01

    Introduction Low-density lipoprotein cholesterol (LDL-C) has been reported to increase platelet activation. Reducing the level of LDL-C with statins induces important pleiotropic effects such as platelet inhibition. This association between platelet activity and statin therapy may be clinically important in reducing the risk of ischemic stroke. We investigated the effect of simvastatin therapy on platelet activation markers (platelet CD62P, sP-selectin, and platelet-derived microparticles (PDMPs)) in hyperlipidemic patients after ischemic stroke. Material and methods The study group consisted of 21 hyperlipidemic patients after ischemic stroke confirmed by CT, and 20 healthy subjects served as controls. We assessed the CD62P expression on resting and thrombin-activated blood platelets. CD62P and PDMPs were analyzed by the use of monoclonal antibodies anti-CD61 and anti-CD62 on a flow cytometer. The level of sP-selectin in serum was measured by the ELISA (enzyme-linked immunosorbent assay) method. All markers were re-analyzed after 6 months of treatment with simvastatin (20 mg/day). Results Hyperlipidemic patients presented a significantly higher percentage of CD62+ platelets and higher reactivity to thrombin compared to control subjects. After simvastatin therapy hyperlipidemic patients showed a reduction of the percentage of resting CD62P(+) platelets (p = 0.005) and a reduction of expression and percentage of CD62P(+) platelets after activation by thrombin (median p < 0.05; percentage: p = 0.001). A decrease of sP-selectin levels (p = 0.001) and percentage of PDMPs (p < 0.05) in this group was also observed. Conclusions HMG-CoA reductase inhibitor therapy in stroke patients with hyperlipidemia may be useful not only due to the lipid-lowering effect but also because of a significant role in reduction of platelet activation and reactivity. PMID:25861297

  2. Spice active principles as the inhibitors of human platelet aggregation and thromboxane biosynthesis.

    PubMed

    Raghavendra, R H; Naidu, K Akhilender

    2009-07-01

    Spice active principles are reported to have anti-diabetic, anti-hypercholesterolemic, antilithogenic, anti-inflammatory, anti-microbial and anti-cancer properties. In our previous report we have shown that spices and their active principles inhibit 5-lipoxygenase and also formation of leukotriene C4. In this study, we report the modulatory effect of spice active principles viz., eugenol, capsaicin, piperine, quercetin, curcumin, cinnamaldehyde and allyl sulphide on in vitro human platelet aggregation. We have demonstrated that spice active principles inhibit platelet aggregation induced by different agonists, namely ADP (50microM), collagen (500microg/ml), arachidonic acid (AA) (1.0mM) and calcium ionophore A-23187 (20microM). Spice active principles showed preferential inhibition of arachidonic acid-induced platelet aggregation compared to other agonists. Among the spice active principles tested, eugenol and capsaicin are found to be most potent inhibitors of AA-induced platelet aggregation with IC50 values of 0.5 and 14.6microM, respectively. The order of potency of spice principles in inhibiting AA-induced platelet aggregation is eugenol>capsaicin>curcumin>cinnamaldehyde>piperine>allyl sulphide>quercetin. Eugenol is found to be 29-fold more potent than aspirin in inhibiting AA-induced human platelet aggregation. Eugenol and capsaicin inhibited thromboxane B2 (TXB2) formation in platelets in a dose-dependent manner challenged with AA apparently by the inhibition of the cyclooxygenase (COX-1). Eugenol-mediated inhibition of platelet aggregation is further confirmed by dose-dependent decrease in malondialdehyde (MDA) in platelets. Further, eugenol and capsaicin inhibited platelet aggregation induced by agonists-collagen, ADP and calcium ionophore but to a lesser degree compared to AA. These results clearly suggest that spice principles have beneficial effects in modulating human platelet aggregation. PMID:19501497

  3. Activated platelets release sphingosine 1-phosphate and induce hypersensitivity to noxious heat stimuli in vivo

    PubMed Central

    Weth, Daniela; Benetti, Camilla; Rauch, Caroline; Gstraunthaler, Gerhard; Schmidt, Helmut; Geisslinger, Gerd; Sabbadini, Roger; Proia, Richard L.; Kress, Michaela

    2015-01-01

    At the site of injury activated platelets release various mediators, one of which is sphingosine 1-phosphate (S1P). It was the aim of this study to explore whether activated human platelets had a pronociceptive effect in an in vivo mouse model and whether this effect was based on the release of S1P and subsequent activation of neuronal S1P receptors 1 or 3. Human platelets were prepared in different concentrations (105/μl, 106/μl, 107/μl) and assessed in mice with different genetic backgrounds (WT, S1P1fl/fl, SNS-S1P1−/−, S1P3−/−). Intracutaneous injections of activated human platelets induced a significant, dose-dependent hypersensitivity to noxious thermal stimulation. The degree of heat hypersensitivity correlated with the platelet concentration as well as the platelet S1P content and the amount of S1P released upon platelet activation as measured with LC MS/MS. Despite the significant correlations between S1P and platelet count, no difference in paw withdrawal latency (PWL) was observed in mice with a global null mutation of the S1P3 receptor or a conditional deletion of the S1P1 receptor in nociceptive primary afferents. Furthermore, neutralization of S1P with a selective anti-S1P antibody did not abolish platelet induced heat hypersensitivity. Our results suggest that activated platelets release S1P and induce heat hypersensitivity in vivo. However, the platelet induced heat hypersensitivity was caused by mediators other than S1P. PMID:25954148

  4. Human platelet glycoprotein Ia. One component is only expressed on the surface of activated platelets and may be a granule constituent

    SciTech Connect

    Bienz, D.; Clemetson, K.J.

    1989-01-05

    Glycoprotein Ia (GP Ia) is a relatively minor component of human blood platelets thought to be a receptor involved in collagen-induced platelet activation. However, some difficulties exist with the definition of this glycoprotein. The expression of GP Ia on resting (prostacyclin analogue-treated) and thrombin-activated platelets was compared by surface labeling with /sup 125/I-lactoperoxidase. Intact platelets or platelets solubilized in sodium dodecyl sulfate were labeled with periodate/(/sup 3/H)NaBH/sub 4/. Analysis on two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed that GP Ia is very poorly labeled in resting platelets. After activation a new spot (GP Ia*) appears with the same relative molecular mass as GP Ia under reducing conditions. GP Ia and Ia* can be clearly separated by two-dimensional nonreduced/reduced gel electrophoresis. Therefore, two glycoproteins which have been termed GP Ia exist in platelets with similar molecular weight and pI under reducing conditions. One of these (GP Ia*) is only surface-labeled when platelets are activated, indicating that it is only exposed on the surface of activated platelets. Supernatant from activated platelets contains this glycoprotein as well as other granule components. This glycoprotein is missing in platelets from two patients with collagen-response defects.

  5. Complement activation on platelets correlates with a decrease in circulating immature platelets in patients with immune thrombocytopenic purpura.

    PubMed

    Peerschke, Ellinor I B; Andemariam, Biree; Yin, Wei; Bussel, James B

    2010-02-01

    The role of the complement system in immune thrombocytopenic purpura (ITP) is not well defined. We examined plasma from 79 patients with ITP, 50 healthy volunteers, and 25 patients with non-immune mediated thrombocytopenia, to investigate their complement activation/fixation capacity (CAC) on immobilized heterologous platelets. Enhanced CAC was found in 46 plasma samples (59%) from patients with ITP, but no samples from patients with non-immune mediated thrombocytopenia. Plasma from healthy volunteers was used for comparison. In patients with ITP, an enhanced plasma CAC was associated with a decreased circulating absolute immature platelet fraction (A-IPF) (<15 x 10(9)/l) (P = 0.027) and thrombocytopenia (platelet count < 100 x 10(9)/l) (P = 0.024). The positive predictive value of an enhanced CAC for a low A-IPF was 93%, with a specificity of 77%. The specificity and positive predictive values increased to 100% when plasma CAC was defined strictly by enhanced C1q and/or C4d deposition on test platelets. Although no statistically significant correlation emerged between CAC and response to different pharmacological therapies, an enhanced response to splenectomy was noted (P < 0.063). Thus, complement fixation may contribute to the thrombocytopenia of ITP by enhancing clearance of opsonized platelets from the circulation, and/or directly damaging platelets and megakaryocytes. PMID:19925495

  6. Complement Activation on Platelets Correlates with a Decrease in Circulating Immature Platelets in Patients with Immune Thrombocytopenic Purpura

    PubMed Central

    Peerschke, Ellinor I.B.; Andemariam, Biree; Yin, Wei; Bussel, James B.

    2010-01-01

    The role of the complement system in immune thrombocytopenic purpura (ITP) is not well defined. We examined plasma from 79 patients with ITP, 50 healthy volunteers, and 25 patients with non-immune mediated thrombocytopenia, to investigate their complement activation/fixation capacity (CAC) on immobilized heterologous platelets. Enhanced CAC was found in 46 plasma samples (59%) from patients with ITP, but no samples from patients with non-immune mediated thrombocytopenia. Plasma from healthy volunteers was used for comparison. In patients with ITP, an enhanced plasma CAC was associated with a decreased circulating absolute immature platelet fraction (A-IPF) (<15 × 109/L) (p = 0.027) and thrombocytopenia (platelet count less than 100K/μl) (p= 0.024). The positive predictive value of an enhanced CAC for a low A-IPF was 93%, with a specificity of 77%. The specificity and positive predictive values increased to 100% when plasma CAC was defined strictly by enhanced C1q and/or C4d deposition on test platelets. Although no statistically significant correlation emerged between CAC and response to different pharmacologic therapies, an enhanced response to splenectomy was noted (p <0.063). Thus, complement fixation may contribute to the thrombocytopenia of ITP by enhancing clearance of opsonized platelets from the circulation, and/or directly damaging platelets and megakaryocytes. PMID:19925495

  7. Repetitive Hypershear Activates and Sensitizes Platelets in a Dose-Dependent Manner.

    PubMed

    Sheriff, Jawaad; Tran, Phat L; Hutchinson, Marcus; DeCook, Tracy; Slepian, Marvin J; Bluestein, Danny; Jesty, Jolyon

    2016-06-01

    Implantation of mechanical circulatory support (MCS) devices-ventricular assist devices and the total artificial heart-has emerged as a vital therapy for advanced and end-stage heart failure. Unfortunately, MCS patients face the requirement of life-long antiplatelet and anticoagulant therapy to combat thrombotic complications resulting from the dynamic and supraphysiologic shear stress conditions associated with such devices, whose effect on platelet activation is poorly understood. We developed a syringe-capillary viscometer-the "platelet hammer"-that repeatedly exposed platelets to average shear stresses up to 1000 dyne/cm(2) for as short as 25 ms. Platelet activation state was measured using a modified prothrombinase assay, with morphological changes analyzed using scanning electron microscopy. We observed an increase in platelet activation state and post-high shear platelet activation rate, or sensitization, with an increase in stress accumulation (SA), the product of shear stress and exposure time. A significant increase in platelet activation state was observed beyond an SA of 1500 dyne-s/cm(2) , with a marked increase in pseudopod length visible beyond an SA of 1000 dyne-s/cm(2) . Utility of the platelet hammer extends to studies of other shear-dependent pathologies, and may assist development of approaches to enhance the safety and effectiveness of MCS devices and objective antithrombotic pharmacotherapy management. PMID:26527361

  8. Lectin-induced activation of platelets may require only limited phosphorylation of the 47K protein

    SciTech Connect

    Ganguly, C.; Chelladurai, M.; Ganguly, P.

    1986-05-01

    Wheat germ agglutinin (WGA) is an N-acetylglucosamine (Glc-NAc) specific lectin which can activate platelets. Like thrombin, stimulation of platelets by WGA is accompanied by enhanced phosphorylation of two polypeptides of M/sub r/ 47K and 20K. Addition of GlcNAc at different time intervals arrested that aggregation of platelets by WGA and paralleled the modification of phosphorylation of the 47K polypeptide. So, the phosphorylation of the 47K polypeptide may regulate the WGA-receptor mediated stimulation of platelets. However, the ratio of phosphoserine to phosphothreonine in the 47K protein was markedly different in WGA-activated than thrombin-stimulated platelets. Thus, the molecular mechanism of action of thrombin and WGA could be different. To explore this idea, /sup 32/P/sub i/-labeled platelets were stimulated with WGA and the activation arrested with N-acetyl-glucosamine at different times. Two-dimensional gel electrophoresis of total protein at 5s showed only two phosphorylated species of 47K protein. At 60s, maximally four phosphorylated species were noted. In contrast, with thrombin using the same technique, seven to nine phosphorylated components have been reported. These results suggest that the different activators of platelets may act by different mechanisms. In addition, activation of platelets may require only limited levels of phosphorylation of the 47K polypeptide.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-02-01

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

  11. Interaction of disintegrins with the alpha IIb beta 3 receptor on resting and activated human platelets.

    PubMed Central

    McLane, M A; Kowalska, M A; Silver, L; Shattil, S J; Niewiarowski, S

    1994-01-01

    Viper venom disintegrins contain the RGD/KGD motif. They inhibit platelet aggregation and cell adhesion, but show structural and functional heterogeneity. We investigated the interaction of four prototypic disintegrins with alpha IIb beta 3 expressed on the surface of resting and activated platelets. The binding affinity (Kd) of 125I-albolabrin, 125I-echistatin, 125I-bitistatin and 125I-eristostatin toward resting platelets was 294, 153, 48 and 18 nM respectively. The Kd value for albolabrin decreased 3-fold and 6-fold after ADP- or thrombin-induced activation. The Kd values for bitistatin and echistatin also decreased with ADP, but there was no further decrease with thrombin. In contrast, eristostatin bound with the same high affinity to resting and activated platelets. The pattern of fluorescein isothiocyanate (FITC)-eristostatin and FITC-albolabrin binding to resting and activated platelets was consistent with observations using radiolabelled material. Eristostatin showed faster and more irreversible binding to platelets, and greater potency compared with albolabrin in inducing conformational neo-epitopes in beta 3. The anti-alpha IIb beta 3 monoclonal antibody OP-G2 that is RGD-dependent inhibited disintegrin binding to activated platelets more strongly than binding to resting platelets and it inhibited the binding to platelets of albolabrin more strongly than eristostatin. The specificity of disintegrin interaction with alpha IIb beta 3 was confirmed by demonstrating cross-linking of these peptides to alpha IIb beta 3 on normal platelets, but not to thrombasthenic platelets deficient in alpha IIb beta 3. Images Figure 6 PMID:8042985

  12. Polymers for the rapid and effective activation and aggregation of platelets.

    PubMed

    Hansen, Anne; McMillan, Loraine; Morrison, Alex; Petrik, Juraj; Bradley, Mark

    2011-10-01

    Platelets are responsible for plugging sites of vascular injury, where upon activation they spread out and become cross-linked, preventing further blood loss. It is desirable to control the activation process on demand for applications such as the rapid staunching of blood flow following trauma. Polymers are the material of choice in many biological areas, with physical properties that allow control of morphology as well as ease of functionalisation and production. Herein, polymer microarrays were used to screen a complex human fluid (platelet rich plasma) to identify polyacrylates that could be used to modulate platelet activation. Several polymers were identified which rapidly activated platelets as determined by CD61P binding and subsequent confirmation by scanning electron microcopy analysis. This approach enabled a direct comparison between the natural agonist collagen and synthetic polymers with respect to the activation status of the platelets as well as the number of bound platelets. Further investigations under physiological flow demonstrated that the static microarray experiments gave viable candidates for potential medical applications while specific protein binding to the polymers was identified as a possible mode of action. The approach demonstrates the ability of polymer microarrays to identify new polymers for specific biological activation events and in this case allowed the identification of materials that allowed higher levels of platelets to bind in advanced activation states than the natural standard collagen in static and flow studies. PMID:21719101

  13. Platelet-activating factor and laser trauma of the iris

    SciTech Connect

    Verbey, N.L.; Van Delft, J.L.; Van Haeringen, N.J.; Braquet, P.

    1989-06-01

    Local application of platelet-activating factor (PAF) on the rabbit eye caused a dose-dependent significant increase in intraocular pressure (IOP). After laser irradiation of the iris the IOP showed a hypertensive phase of about 3 hr. Prophylactic treatment with the PAF antagonist BN 52021 but not with indomethacin abolished the hypertensive phase. Elevated levels of protein (10.6 +/- 0.9 g/l) and prostaglandin E2 (PGE2, 1.7 +/- 0.2 ng/ml) were measured in the aqueous humor 2 hr after laser irradiation of the iris. Prophylactic treatment with BN 52021 showed lower levels of protein (6.1 +/- 0.7) and PGE2 (1.1 +/- 0.02); with indomethacin pretreatment the level of protein was 3.4 +/- 0.7 g/l and of PGE2 0.10 +/- 0.02 ng/ml. A role of PAF as a mediator in ocular inflammatory response is suggested.

  14. Protein Kinase Cδ mediates the activation of Protein Kinase D2 in Platelets

    PubMed Central

    Bhavanasi, Dheeraj; Kim, Soochong; Goldfinger, Lawrence E.; Kunapuli, Satya P.

    2011-01-01

    Protein Kinase D (PKD) is a subfamily of serine/threonine specific family of kinases, comprised of PKD1, PKD2 and PKD3 (PKCμ, PKD2 and PKCν in humans). It is known that PKCs activate PKD, but the relative expression of isoforms of PKD or the specific PKC isoform/s responsible for its activation in platelets is not known. This study is aimed at investigating the pathway involved in activation of PKD in platelets. We show that PKD2 is the major isoform of PKD that is expressed in human as well as murine platelets but not PKD1 or PKD3. PKD2 activation induced by AYPGKF was abolished with a Gq inhibitor YM-254890, but was not affected by Y-27632, a RhoA/p160ROCK inhibitor, indicating that PKD2 activation is Gq-, but not G12/13-mediated Rho-kinase dependent. Calcium-mediated signals are also required for activation of PKD2 as dimethyl BAPTA inhibited its phosphorylation. GF109203X, a pan PKC inhibitor abolished PKD2 phosphorylation but Go6976, a classical PKC inhibitor had no effect suggesting that novel PKC isoforms are involved in PKD2 activation. Importantly, Rottlerin, a non-selective PKCδ inhibitor, inhibited AYPGKF-induced PKD2 activation in human platelets. Similarly, AYPGKF- and Convulxin-induced PKD2 phosphorylation was dramatically inhibited in PKCδ-deficient platelets, but not in PKCθ– or PKCε–deficient murine platelets compared to that of wild type platelets. Hence, we conclude that PKD2 is a common signaling target downstream of various agonist receptors in platelets and Gq-mediated signals along with calcium and novel PKC isoforms, in particular, PKCδ activate PKD2 in platelets. PMID:21736870

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

    PubMed Central

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

    2016-01-01

    Background Activated autologous platelet-rich plasma (PRP) used in therapeutic wound healing applications is poorly characterized and standardized. Using pulsed electric fields (PEF) to activate platelets may reduce variability and eliminate complications associated with the use of bovine thrombin. We previously reported that exposing PRP to sub-microsecond duration, high electric field (SMHEF) pulses generates a greater number of platelet-derived microparticles, increased expression of prothrombotic platelet surfaces, and differential release of growth factors compared to thrombin. Moreover, the platelet releasate produced by SMHEF pulses induced greater cell proliferation than plasma. Aims To determine whether sub-microsecond duration, low electric field (SMLEF) bipolar pulses results in differential activation of PRP compared to SMHEF, with respect to profiles of activation markers, growth factor release, and cell proliferation capacity. Methods PRP activation by SMLEF bipolar pulses was compared to SMHEF pulses and bovine thrombin. PRP was prepared using the Harvest SmartPreP2 System from acid citrate dextrose anticoagulated healthy donor blood. PEF activation by either SMHEF or SMLEF pulses was performed using a standard electroporation cuvette preloaded with CaCl2 and a prototype instrument designed to take into account the electrical properties of PRP. Flow cytometry was used to assess platelet surface P-selectin expression, and annexin V binding. Platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF) and platelet factor 4 (PF4), and were measured by ELISA. The ability of supernatants to stimulate proliferation of human epithelial cells in culture was also evaluated. Controls included vehicle-treated, unactivated PRP and PRP with 10 mM CaCl2 activated with 1 U/mL bovine thrombin. Results PRP activated with SMLEF bipolar pulses or thrombin had similar light scatter profiles, consistent with the

  16. Arsenic Trioxide Induces Apoptosis in Human Platelets via C-Jun NH2-Terminal Kinase Activation

    PubMed Central

    Wu, Yicun; Dai, Jin; Zhang, Weilin; Yan, Rong; Zhang, Yiwen; Ruan, Changgeng; Dai, Kesheng

    2014-01-01

    Arsenic trioxide (ATO), one of the oldest drugs in both Western and traditional Chinese medicine, has become an effective anticancer drug, especially in the treatment of acute promyelocytic leukemia (APL). However, thrombocytopenia occurred in most of ATO-treated patients with APL or other malignant diseases, and the pathogenesis remains unclear. Here we show that ATO dose-dependently induces depolarization of mitochondrial inner transmembrane potential (ΔΨm), up-regulation of Bax and down-regulation of Bcl-2 and Bcl-XL, caspase-3 activation, and phosphotidylserine (PS) exposure in platelets. ATO did not induce surface expression of P-selectin and PAC-1 binding, whereas, obviously reduced collagen, ADP, and thrombin induced platelet aggregation. ATO dose-dependently induced c-Jun NH2-terminal kinase (JNK) activation, and JNK specific inhibitor dicumarol obviously reduced ATO-induced ΔΨm depolarization in platelets. Clinical therapeutic dosage of ATO was intraperitoneally injected into C57 mice, and the numbers of circulating platelets were significantly reduced after five days of continuous injection. The data demonstrate that ATO induces caspase-dependent apoptosis via JNK activation in platelets. ATO does not incur platelet activation, whereas, it not only impairs platelet function but also reduces circulating platelets in vivo, suggesting the possible pathogenesis of thrombocytopenia in patients treated with ATO. PMID:24466103

  17. Arsenic trioxide induces apoptosis in human platelets via C-Jun NH2-terminal kinase activation.

    PubMed

    Wu, Yicun; Dai, Jin; Zhang, Weilin; Yan, Rong; Zhang, Yiwen; Ruan, Changgeng; Dai, Kesheng

    2014-01-01

    Arsenic trioxide (ATO), one of the oldest drugs in both Western and traditional Chinese medicine, has become an effective anticancer drug, especially in the treatment of acute promyelocytic leukemia (APL). However, thrombocytopenia occurred in most of ATO-treated patients with APL or other malignant diseases, and the pathogenesis remains unclear. Here we show that ATO dose-dependently induces depolarization of mitochondrial inner transmembrane potential (ΔΨm), up-regulation of Bax and down-regulation of Bcl-2 and Bcl-XL, caspase-3 activation, and phosphotidylserine (PS) exposure in platelets. ATO did not induce surface expression of P-selectin and PAC-1 binding, whereas, obviously reduced collagen, ADP, and thrombin induced platelet aggregation. ATO dose-dependently induced c-Jun NH2-terminal kinase (JNK) activation, and JNK specific inhibitor dicumarol obviously reduced ATO-induced ΔΨm depolarization in platelets. Clinical therapeutic dosage of ATO was intraperitoneally injected into C57 mice, and the numbers of circulating platelets were significantly reduced after five days of continuous injection. The data demonstrate that ATO induces caspase-dependent apoptosis via JNK activation in platelets. ATO does not incur platelet activation, whereas, it not only impairs platelet function but also reduces circulating platelets in vivo, suggesting the possible pathogenesis of thrombocytopenia in patients treated with ATO. PMID:24466103

  18. Functional responses and molecular mechanisms involved in histone-mediated platelet activation.

    PubMed

    Carestia, A; Rivadeneyra, L; Romaniuk, M A; Fondevila, C; Negrotto, S; Schattner, M

    2013-11-01

    Histones are highly alkaline proteins found in cell nuclei and they can be released by either dying or inflammatory cells. The recent observations that histones are major components of neutrophil extracellular traps and promote platelet aggregation and platelet-dependent thrombin generation have shown that these proteins are potent prothrombotic molecules. Because the mechanism(s) of platelet activation by histones are not completely understood, we explored the ability of individual recombinant human histones H1, H2A, H2B, H3 and H4 to induce platelet activation as well as the possible molecular mechanisms involved. All histones were substrates for platelet adhesion and spreading and triggered fibrinogen binding, aggregation, von Willebrand factor release, P-selectin and phosphatidylserine (PS) exposure and the formation of platelet-leukocyte aggregates; however, H4 was the most potent. Histone-mediated fibrinogen binding, P-selectin and PS exposure and the formation of mixed aggregates were potentiated by thrombin. Histones induced the activation of ERK, Akt, p38 and NFκB. Accordingly, histone-induced platelet activation was significantly impaired by pretreatment of platelets with inhibitors of ERK (U 0126), PI3K/Akt (Ly 294002), p38 (SB 203580) and NFκB (BAY 11-7082 and Ro 106-9920). Preincubation of platelets with either aspirin or dexamethasone markedly decreased fibrinogen binding and the adhesion mediated by histones without affecting P-selectin exposure. Functional platelet responses induced by H3 and H4, but not H1, H2A and H2B, were partially mediated through interaction with Toll-like receptors -2 and -4. Our data identify histones as important triggers of haemostatic and proinflammatory platelet responses, and only haemostatic responses are partially inhibited by anti-inflammatory drugs. PMID:23965842

  19. Effect of Red Blood Cells on Platelet Activation and Thrombus Formation in Tortuous Arterioles

    PubMed Central

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

    2013-01-01

    Thrombosis is a major contributor to cardiovascular disease, which can lead to myocardial infarction and stroke. Thrombosis may form in tortuous microvessels, which are often seen throughout the human body, but the microscale mechanisms and processes are not well understood. In straight vessels, the presence of red blood cells (RBCs) is known to push platelets toward walls, which may affect platelet aggregation and thrombus formation. However in tortuous vessels, the effects of RBC interactions with platelets in thrombosis are largely unknown. Accordingly, the objective of this work was to determine the physical effects of RBCs, platelet size, and vessel tortuosity on platelet activation and thrombus formation in tortuous arterioles. A discrete element computational model was used to simulate the transport, collision, adhesion, aggregation, and shear-induced platelet activation of hundreds of individual platelets and RBCs in thrombus formation in tortuous arterioles. Results showed that high shear stress near the inner sides of curved arteriole walls activated platelets to initiate thrombosis. RBCs initially promoted platelet activation, but then collisions of RBCs with mural thrombi reduced the amount of mural thrombus and the size of emboli. In the absence of RBCs, mural thrombus mass was smaller in a highly tortuous arteriole compared to a less tortuous arteriole. In the presence of RBCs however, mural thrombus mass was larger in the highly tortuous arteriole compared to the less tortuous arteriole. As well, smaller platelet size yielded less mural thrombus mass and smaller emboli, either with or without RBCs. This study shed light on microscopic interactions of RBCs and platelets in tortuous microvessels, which have implications in various pathologies associated with thrombosis and bleeding. PMID:25022613

  20. Effect of Drotrecogin alfa (activated) on platelet receptor expression in vitro.

    PubMed

    Schuerholz, Tobias; Friedrich, Lars; Marx, Gernot; Kornau, Ines; Sümpelmann, Robert; Scheinichen, Dirk

    2007-08-01

    Thrombocytopenia is a common problem in critically ill patients, which is associated with increased mortality. Recently, Drotrecogin alfa (activated) (recombinant human activated protein C (APC)) was shown to reduce mortality in patients with severe sepsis. Only minimal effect of APC on coagulation markers was demonstrated. Nevertheless, low platelet count was identified as a risk factor for bleeding with use of this drug. We conducted this study to evaluate possible influence of APC on in vitro expression of platelet receptors at therapeutic and supra-therapeutic concentrations. Blood samples of volunteers and patients with severe sepsis were adjusted with APC to final concentrations of 0.045 microg mL(-1) APC (APC-45, therapeutic dose) and 0.225 microg mL(-1) APC (APC-225, five-fold therapeutic dose), respectively. The activation of platelets was mediated by two different agonists. APC had no significant influence on platelet activation, with or without stimulation at both concentrations. In group APC-225, CD62P showed a non-significant decrease. This in vitro study demonstrates that therapeutic plasma concentrations of Drotrecogin alfa (activated) have neither influence on expression of platelet activation markers nor on platelet-granulocyte complexes in blood of volunteers and patients with severe sepsis. Thus, a direct drug-platelet interaction seems unlikely. PMID:17654307

  1. Detection and partial characterization of an inhibitor of plasminogen activator in human platelets.

    PubMed Central

    Erickson, L A; Ginsberg, M H; Loskutoff, D J

    1984-01-01

    In this study, we demonstrate the presence of a previously undescribed fibrinolytic inhibitor in human serum. It has an apparent molecular weight of 50,000 and is not detected in serum derived from platelet-poor plasma, suggesting that it originates from platelets. This conclusion is supported by a number of observations. For example, extracts of washed, gel-filtered human platelets contain an inhibitor of similar activity and size, and physiological concentrations of thrombin induce its release from the platelets. Moreover, the kinetics and dose dependency of this release are similar to those observed for the release of platelet factor 4, and the release of both molecules is blocked by pretreating the platelets with prostaglandin E1 and theophylline. Mixing experiments, which were devised to investigate the specificity of the inhibitor, showed that the fibrinolytic activity initiated by both urokinase and tissue-type plasminogen activator was blocked by platelet releasate in a dose-dependent manner. In both cases, the amount of inhibition increased when the releasates were preincubated with the purified activators, indicating a direct interaction between the activators and an inhibitor(s). The inhibitory activity was removed by preincubating the releasates with antiserum prepared against an antiactivator purified from cultured bovine aortic endothelial cells. These results indicate that platelets contain an inhibitor which is released by thrombin, inhibits both urokinase and tissue-type plasminogen activator, and is immunologically similar to an inhibitor produced by endothelial cells. This molecule may represent a new class of inhibitors, the antiactivators, which function together with alpha 2-antiplasmin to regulate the fibrinolytic system of the blood. Its release from platelets by thrombin may protect the growing thrombus against premature dissolution initiated by plasminogen activators released by the endothelium. Images PMID:6434594

  2. Platelet function and activation in Cavalier King Charles Spaniels with subclinical chronic valvular heart disease.

    PubMed

    Tong, Linda J; Hosgood, Giselle L; French, Anne T; Irwin, Peter J; Shiel, Robert E

    2016-08-01

    OBJECTIVE To assess platelet closure time (CT), mean platelet component (MPC) concentration, and platelet component distribution width (PCDW) in dogs with subclinical chronic valvular heart disease. ANIMALS 89 Cavalier King Charles Spaniels (CKCSs) and 39 control dogs (not CKCSs). PROCEDURES Platelet count, MPC concentration, PCDW, and Hct were measured by use of a hematology analyzer, and CT was measured by use of a platelet function analyzer. Murmur grade and echocardiographic variables (mitral valve regurgitant jet size relative to left atrial area, left atrial-to-aortic diameter ratio, and left ventricular internal dimensions) were recorded. Associations between explanatory variables (sex, age, murmur grade, echocardiographic variables, platelet count, and Hct) and outcomes (CT, MPC concentration, and PCDW) were examined by use of multivariate regression models. RESULTS A model with 5 variables best explained variation in CT (R(2), 0.74), with > 60% of the variance of CT explained by mitral valve regurgitant jet size. The model of best fit to explain variation in MPC concentration included only platelet count (R(2), 0.24). The model of best fit to explain variation in PCDW included platelet count and sex (R(2), 0.25). CONCLUSIONS AND CLINICAL RELEVANCE In this study, a significant effect of mitral valve regurgitant jet size on CT was consistent with platelet dysfunction. However, platelet activation, as assessed on the basis of the MPC concentration and PCDW, was not a feature of subclinical chronic valvular heart disease in CKCSs. PMID:27463549

  3. Platelets: the few, the young, and the active.

    PubMed

    D'Souza, Carol; Briggs, Carol; Machin, Samuel J

    2015-03-01

    Many modern automated cell counters in high-volume clinical hematology laboratories use new, improved technologies for routine platelet analysis. The latest progress includes the use of state-of-the art information technology, specific fluorescent dyes, and monoclonal antibodies to obtain more reliable platelet counts. This information allows the accurate and precise enumeration of platelets even in thrombocytopenic patients and the reporting of novel platelet parameters. In the near future, digital image analysis may permit even better platelet analysis. PMID:25676376

  4. Immunodiagnosis of platelet activation in immune thrombocytopenia through scFv antibodies cognate to activated IIb3 integrins

    PubMed Central

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

    2015-01-01

    Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by low platelet count and presence of IgG autoantibodies to platelet surface glycoproteins, such as αIIbβ3 and GPIb/IX. Our previous work has shown that platelets in ITP patients exist in an activated state. Two different marker-based approaches are used to study the course of platelet activation: (1) binding of PAC-1 antibody, signifying a change in αIIbβ3 conformation, and (2) expression of P-selectin, signifying alpha granule content release from platelets. Here, we describe the development of a new scFv antibody (R38) that, compared with PAC-1, appears to better distinguish between platelets of ITP patients and healthy controls. Notably, R38 was generated using commercially sourced resting-state integrin that was coated on a microtiter plate. Its ability to distinguish between ITP patients and healthy controls thus suggests that inadvertent integrin activation caused by coating involves a conformational change and exposure of a cryptic epitope. This report also describes for the first time the potential use of an scFv antibody in the immunodiagnosis of platelet activation in ITP patients. PMID:26301697

  5. VAMP-7 links granule exocytosis to actin reorganization during platelet activation.

    PubMed

    Koseoglu, Secil; Peters, Christian G; Fitch-Tewfik, Jennifer L; Aisiku, Omozuanvbo; Danglot, Lydia; Galli, Thierry; Flaumenhaft, Robert

    2015-07-30

    Platelet activation results in profound morphologic changes accompanied by release of granule contents. Recent evidence indicates that fusion of granules with the plasma membrane during activation provides auxiliary membrane to cover growing actin structures. Yet little is known about how membrane fusion is coupled with actin reorganization. Vesicle-associated membrane protein (VAMP)-7 is found on platelet vesicles and possesses an N-terminal longin domain capable of linking exocytosis to cytoskeletal remodeling. We have evaluated platelets from VAMP-7(-/-) mice to determine whether this VAMP isoform contributes to granule release and platelet spreading. VAMP-7(-/-) platelets demonstrated a partial defect in dense granule exocytosis and impaired aggregation. α Granule exocytosis from VAMP-7(-/-) platelets was diminished both in vitro and in vivo during thrombus formation. Consistent with a role of VAMP-7 in cytoskeletal remodeling, spreading on matrices was decreased in VAMP-7(-/-) platelets compared to wild-type controls. Immunoprecipitation of VAMP-7 revealed an association with VPS9-domain ankyrin repeat protein (VARP), an adaptor protein that interacts with both membrane-bound and cytoskeleton proteins and with Arp2/3. VAMP-7, VARP, and Arp2/3 localized to the platelet periphery during spreading. These studies demonstrate that VAMP-7 participates in both platelet granule secretion and spreading and suggest a mechanism whereby VAMP-7 links granule exocytosis with actin reorganization. PMID:25999457

  6. Glaucocalyxin A inhibits platelet activation and thrombus formation preferentially via GPVI signaling pathway.

    PubMed

    Li, Wei; Tang, Xiaorong; Yi, Wenxiu; Li, Qiang; Ren, Lijie; Liu, Xiaohui; Chu, Chunjun; Ozaki, Yukio; Zhang, Jian; Zhu, Li

    2013-01-01

    Platelets play a pivotal role in atherothrombosis and the antiplatelet agents have been proved to be useful in preventing onset of acute clinical events including myocardial infarction and stroke. Increasing number of natural compounds has been identified to be potential antiplatelet agents. Here we report the antiplatelet effect of glaucocalyxin A (GLA), an ent-diterpenoid that we isolated and purified from the aerial parts of Rabdosia japonica (Burm. f.) var. glaucocalyx (Maxim.) Hara, and investigate the molecular mechanisms by which GLA inhibits platelet activation and thrombus formation. The effect of GLA on platelet activation was measured using platelets freshly isolated from peripheral blood of healthy donors. Results showed that pretreatment of human platelets with lower concentrations of GLA (0.01 μg/ml, 0.1 μg/ml) significantly inhibited platelet aggregation induced by collagen (P<0.001) and CRP (P<0.01), a synthetic GPVI ligand, but not by ADP and U46619. Accordingly, GLA inhibited collagen-stimulated tyrosine phosphorylation of Syk, LAT, and phospholipase Cγ2, the signaling events in collagen receptor GPⅥ pathway. GLA also inhibited platelet p-selectin secretion and integrin activation by convulxin, a GPVI selective ligand. Additionally, GLA was found to inhibit low-dose thrombin-induced platelet activation. Using a flow chamber device, GLA was found to attenuate platelet adhesion on collagen surfaces in high shear condition. In vivo studies showed that GLA administration increased the time for complete occlusion upon vascular injury in mice, but did not extend tail-bleeding time when mice were administered with relatively lower doses of GLA. Therefore, the present results provide the molecular basis for the inhibition effect of GLA on platelet activation and its in vivo effect on thrombus formation, suggesting that GLA could potentially be developed as an antiplatelet and antithrombotic agent. PMID:24386454

  7. Short fungal fractions of β-1,3 glucans affect platelet activation.

    PubMed

    Vancraeyneste, Hélène; Charlet, Rogatien; Guerardel, Yann; Choteau, Laura; Bauters, Anne; Tardivel, Meryem; François, Nadine; Dubuquoy, Laurent; Soloviev, Dmitry; Poulain, Daniel; Sendid, Boualem; Jawhara, Samir

    2016-09-01

    Platelets are capable of binding, aggregating, and internalizing microorganisms, which enhances the elimination of pathogens from the blood. The yeast Candida albicans is a pathobiont causing life-threatening invasive infections. Its cell wall contains β-1,3 glucans that are known to trigger a wide range of host cell activities and to circulate during infection. We studied the effect of β-1,3 glucan fractions (BGFs) consisting of diglucosides (Glc2), tetraglucosides (Glc4), and pentaglucosides (Glc5) on human platelets, their mechanisms of action, and their possible impact on host defenses. The effect of BGFs on the coagulation process was determined by measuring thrombin generation. Platelets pretreated with BGFs were analyzed in terms of activation, receptor expression, aggregation, and adhesion to neutrophils and to C. albicans The results show that BGFs affected the endogenous thrombin potential in a concentration-dependent manner. For platelet activation, BGFs at a low concentration (2 μmol/l) reduced ATP release and prevented the phosphorylation of protein kinase C. BGFs diminished the expression of P-selectin and the activation of αIIbβ3 BGFs decreased platelet aggregation and the interaction between thrombin-stimulated platelets and neutrophils, fibrinogen, and C. albicans GLc5 decreased ATP release and TGF-β1 production in response to TLR4 upregulation in thrombin-stimulated platelets, but TLR4 blockage abolished the effect of BGFs on platelets. This study provides evidence that fungal pentaglucosides modulate platelet activity mediated via TLR4 stimulation and reduce platelet-neutrophil interaction. PMID:27288438

  8. Activation and shedding of platelet glycoprotein IIb/IIIa under non-physiological shear stress.

    PubMed

    Chen, Zengsheng; Mondal, Nandan K; Ding, Jun; Koenig, Steven C; Slaughter, Mark S; Griffith, Bartley P; Wu, Zhongjun J

    2015-11-01

    The purpose of this study was to investigate the influence of non-physiological high shear stress on activation and shedding of platelet GP IIb/IIIa receptors. The healthy donor blood was exposed to three levels of high shear stresses (25, 75, 125 Pa) from the physiological to non-physiological status with three short exposure time (0.05, 0.5, 1.5 s), created by a specific blood shearing system. The activation and shedding of the platelet GPIIb/IIIa were analyzed using flow cytometry and enzyme-linked immunosorbent assay. In addition, platelet P-selectin expression of sheared blood, which is a marker for activated platelets, was also analyzed. The results from the present study showed that the number of activated platelets, as indicated by the surface GPIIb/IIIa activation and P-selectin expression, increased with increasing the shear stress level and exposure time. However, the mean fluorescence of GPIIb/IIIa on the platelet surface, decreased with increasing the shear stress level and exposure time. The reduction of GPIIb/IIIa on the platelet surface was further proved by the reduction of further activated platelet GPIIb/IIIa surface expression induced by ADP and the increase in GPIIb/IIIa concentration in microparticle-free plasma with increasing the applied shear stress and exposure time. It is clear that non-physiological shear stress induce a paradoxical phenomenon, in which both activation and shedding of the GPIIb/IIIa on the platelet surface occur simultaneously. This study may offer a new perspective to explain the reason of both increased thrombosis and bleeding events in patients implanted with high shear blood-contacting medical devices. PMID:26160282

  9. Platelet immunoreceptor tyrosine-based activation motif (ITAM) signaling and vascular integrity.

    PubMed

    Boulaftali, Yacine; Hess, Paul R; Kahn, Mark L; Bergmeier, Wolfgang

    2014-03-28

    Platelets are well-known for their critical role in hemostasis, that is, the prevention of blood loss at sites of mechanical vessel injury. Inappropriate platelet activation and adhesion, however, can lead to thrombotic complications, such as myocardial infarction and stroke. To fulfill its role in hemostasis, the platelet is equipped with various G protein-coupled receptors that mediate the response to soluble agonists such as thrombin, ADP, and thromboxane A2. In addition to G protein-coupled receptors, platelets express 3 glycoproteins that belong to the family of immunoreceptor tyrosine-based activation motif receptors: Fc receptor γ chain, which is noncovalently associated with the glycoprotein VI collagen receptor, C-type lectin 2, the receptor for podoplanin, and Fc receptor γII A, a low-affinity receptor for immune complexes. Although both genetic and chemical approaches have documented a critical role for platelet G protein-coupled receptors in hemostasis, the contribution of immunoreceptor tyrosine-based activation motif receptors to this process is less defined. Studies performed during the past decade, however, have identified new roles for platelet immunoreceptor tyrosine-based activation motif signaling in vascular integrity in utero and at sites of inflammation. The purpose of this review is to summarize recent findings on how platelet immunoreceptor tyrosine-based activation motif signaling controls vascular integrity, both in the presence and absence of mechanical injury. PMID:24677237

  10. TMEM16F is required for phosphatidylserine exposure and microparticle release in activated mouse platelets

    PubMed Central

    Fujii, Toshihiro; Sakata, Asuka; Nishimura, Satoshi; Eto, Koji; Nagata, Shigekazu

    2015-01-01

    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 Ca2+ ionophore at 20 °C induces PtdSer exposure without compromising plasma membrane integrity. Among five transmembrane protein 16 (TMEM16) members that support Ca2+-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. PMID:26417084

  11. Platelet and monocyte activity markers and mediators of inflammation in Takotsubo cardiomyopathy.

    PubMed

    Pirzer, Rainer; Elmas, Elif; Haghi, Dariusch; Lippert, Christiane; Kralev, Stefan; Lang, Siegfried; Borggrefe, Martin; Kälsch, Thorsten

    2012-03-01

    Patients with Takotsubo cardiomyopathy (TC) often present with symptoms similar to those of myocardial infarction (MI). We analyzed blood concentrations of mediators of inflammation and platelet- and monocyte-activity markers in patients with TC and MI for significant differences. Clinical data of patients with TC (n = 16) and acute MI (n = 16) were obtained. Serial blood samples were taken at the time of hospital admission (t(0)), after 2-4 days (t(1)) and after 4-7 weeks (t(2)), respectively. Plasma concentrations of interleukin (IL)-6, IL-7, soluble CD40 ligand (sCD40L), and monocyte chemotactic protein 1 (MCP-1) were determined with an ELISA. Tissue factor binding on monocytes, platelet-activation marker CD62P, platelet CD40-ligand (CD40L), and platelet-monocyte aggregates were measured using flow cytometry. Expression of CD62P on platelets and IL-6 plasma levels were significantly lower in patients with TC compared to MI at the time of hospital admission. IL-7 plasma levels were significantly elevated in patients with TC compared to patients with MI at 2-4 days after hospital admission. No significant differences were observed concerning sCD40L and MCP-1 plasma levels, tissue factor binding on monocytes, CD40L expression on platelets, and platelet-monocyte aggregates at any point in time. Our results indicate that inflammatory mediators and platelet-activity markers contribute to the differences in the pathogenesis of MI and TC. PMID:21416113

  12. Platelet activating factor receptor blockade enhances recovery after multifocal brain ischemia

    SciTech Connect

    Kochanek, P.M.; Dutka, A.J.; Kumaroo, K.K.; Hallenbech, J.M.

    1987-12-14

    The authors treated four anesthetized dogs with the platelet activating factor (PAF) receptor antagonist kadsurenone prior to 60 min of multifocal ischemia induced by air embolism, and measured neuronal recovery, blood flow and autologous /sup 111/In-labeled platelet accumulation for 4 h after ischemia. Four anesthetized animals with identical ischemia served as controls. Kadsurenone administered 5 min prior to ischemia and continuously throughout ischemia and recovery significantly enhanced recovery of cortical somatosensory evoked response (CSER) amplitude when compared to controls. They estimated platelet accumulation as /sup 111/In activity (cmp/g tissue) in the injured hemisphere minus that in the non-injured hemisphere. Kadsurenone treated animals did not exhibit significantly altered /sup 111/In labeled platelet accumulation when compared to controls. Beneficial effects of PAF receptor blockade other than those on platelet accumulation may be involved. 20 references, 1 figure.

  13. Mobilization of hepatic calcium pools by platelet activating factor

    SciTech Connect

    Lapointe, D.S.; Hanahan, D.J.; Olson, M.S.

    1987-03-24

    In the perfused rat liver, platelet activating factor, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC), infusion produces an extensive but transient glycogenolytic response which at low AGEPC concentrations is markedly dependent upon the perfusate calcium levels. The role of calcium in the glycogenolytic response of the liver to AGEPC was investigated by assessing the effect of AGEPC on various calcium pools in the intact liver. Livers from fed rats were equilibrated with /sup 45/Ca/sup 2 +/, and the kinetics of /sup 45/Ca/sup 2 +/ efflux were determined in control, AGEPC-stimulated, and phenylephrine-stimulated livers during steady-state washout of /sup 45/Ca/sup 2 +/. AGEPC treatment had only a slight if any effect on the pattern of steady-state calcium efflux from the liver, as opposed to major perturbations in the pattern of calcium efflux effected by the ..cap alpha..-adrenergic agonist phenylephrine. Infusion of short pulses of AGEPC during the washout of /sup 45/Ca/sup 2 +/ from labeled livers caused a transient release of /sup 45/Ca/sup 2 +/ which was not abolished at low calcium concentrations in the perfusate. Infusion of latex beads, which are removed by the reticuloendothelial cells, caused the release of hepatic /sup 45/Ca/sup 2 +/ in a fashion similar to the case with AGEPC. The findings indicate that AGEPC does not perturb a major pool of calcium within the liver as occurs upon ..cap alpha..-adrenergic stimulation; it is likely that AGEPC mobilizes calcium from a smaller yet very important pool, very possibly from nonparenchymal cells in the liver.

  14. Biological effects of the orally active platelet activating factor receptor antagonist SDZ 64-412.

    PubMed

    Handley, D A; Van Valen, R G; Melden, M K; Houlihan, W J; Saunders, R N

    1988-11-01

    SDZ 64-412 is a trimethoxyphenylethylphenyl imidazo[2,1-a] isoquinoline molecule that displays marked in vitro inhibition of platelet activating factor (PAF)-induced human platelet aggregation (IC50 = 60 nM) but is without inhibition (at 100 microM) of epinephrine-, ADP- or collagen-induced aggregation. SDZ 64-412 antagonized receptor binding of radiolabeled PAF to human platelet membranes with an IC50 = 60 nM. In the rat, SDZ 64-412 inhibited 100 ng kg-1 PAF-induced hypotension when given i.v. (ED50 = 0.23 mg kg-1) or p.o. (ED50 = 13 mg kg-1). In the guinea pig, SDZ 64-412 inhibited 50 ng kg-1 PAF-induced bronchoconstriction (ED50 = 4.2 mg kg-1 p.o.) and hemoconcentration (ED50 = 5.0 mg kg-1 p.o.). SDZ 64-412 exhibited oral activity in the dog against 1.5 micrograms kg-1 PAF-induced hypotension (ED50 = 5.1 mg kg-1 p.o.) and hemoconcentration (ED50 = 4.9 mg kg-1) and 3.5 micrograms kg-1 PAF-induced hemoconcentration in the cebus primate (ED50 = 12.8 mg kg-1 p.o.). SDZ 64-412 protected in a dose-dependent manner against PAF-induced lethality (LD75 = 75 micrograms kg-1 i.v.) in mice, where 20 mg kg-1 p.o. improved survival from 25 +/- 4% to 77 +/- 8%. SDZ 64-412 afforded complete protection against endotoxin-induced lethality (LD90 = 7.5 mg kg-1 endotoxin i.v.) where the ED50 was 45 mg kg-1 twice predose.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3183958

  15. The class I phosphoinositide 3-kinases α and β control antiphospholipid antibodies-induced platelet activation.

    PubMed

    Terrisse, Anne-Dominique; Laurent, Pierre-Alexandre; Garcia, Cédric; Gratacap, Marie-Pierre; Vanhaesebroeck, Bart; Sié, Pierre; Payrastre, Bernard

    2016-06-01

    Antiphospholipid syndrome (APS) is an autoimmune disease characterised by the presence of antiphospholipid antibodies (aPL) associated with increased thrombotic risk and pregnancy morbidity. Although aPL are heterogeneous auto-antibodies, the major pathogenic target is the plasma protein β2-glycoprotein 1. The molecular mechanisms of platelet activation by aPL remain poorly understood. Here, we explored the role of the class IA phosphoinositide 3-kinase (PI3K) α and β isoforms in platelet activation by aPL. Compared to control IgG from healthy individuals, the IgG fraction isolated from patients with APS potentiates platelet aggregation induced by low dose of thrombin in vitro and increases platelet adhesion and thrombus growth on a collagen matrix under arterial shear rate through a mechanism involving glycoprotein Ib (GPIb) and Toll Like Receptor 2 (TLR-2). Using isoforms-selective pharmacological PI3K inhibitors and mice with megakaryocyte/platelet lineage-specific inactivation of class IA PI3K isoforms, we demonstrate a critical role of the PI3Kβ and PI3Kα isoforms in platelet activation induced by aPL. Our data show that aPL potentiate platelet activation through GPIbα and TLR-2 via a mechanism involving the class IA PI3Kα and β isoforms, which represent new potential therapeutic targets in the prevention or treatment of thrombotic events in patients with APS. PMID:26818901

  16. Inhibitory effect of caffeic acid on ADP-induced thrombus formation and platelet activation involves mitogen-activated protein kinases

    PubMed Central

    Lu, Yu; Li, Quan; Liu, Yu-Ying; Sun, Kai; Fan, Jing-Yu; Wang, Chuan-She; Han, Jing-Yan

    2015-01-01

    Caffeic acid (CA), one of the active constituents of Radix Salvia miltiorrhizae, exhibits antioxidant and anti-inflammatory activities. However, few studies have assessed the ability of CA to inhibit platelet mediated thrombus generation in vivo. In this study, we investigated the antithrombotic effect of CA in mouse cerebral arterioles and venules using intravital microscopy. The antiplatelet activity of CA in ADP stimulated mouse platelets in vitro was also examined in attempt to explore the underlying mechanism. Our results demonstrated that CA (1.25–5 mg/kg) significantly inhibited thrombus formation in vivo. In vitro, CA (25–100 μM) inhibited ADP-induced platelet aggregation, P-selectin expression, ATP release, Ca2+ mobilization, and integrin αIIbβ3 activation. Additionally, CA attenuated p38, ERK, and JNK activation, and enhanced cAMP levels. Taken together, these data provide evidence for the inhibition of CA on platelet-mediated thrombosis in vivo, which is, at least partly, mediated by interference in phosphorylation of ERK, p38, and JNK leading to elevation of cAMP and down-regulation of P-selectin expression and αIIbβ3 activation. These results suggest that CA may have potential for the treatment of aberrant platelet activation-related diseases. PMID:26345207

  17. Protease Activated Receptor-1 (PAR-1) Mediated Platelet Aggregation is Dependant on Clopidogrel Response

    PubMed Central

    Kreutz, Rolf P.; Breall, Jeffrey A.; Kreutz, Yvonne; Owens, Janelle; Lu, Deshun; Bolad, Islam; von der Lohe, Elisabeth; Sinha, Anjan; Flockhart, David A.

    2012-01-01

    Introduction Clopidogrel inhibits ADP mediated platelet aggregation through inhibition of the P2Y12 receptor by its active metabolite. Thrombin induces platelet aggregation by binding to protease activated receptor-1 (PAR-1), and inhibition of PAR-1 has been evaluated in patients treated with clopidogrel to reduce ischemic events after acute coronary syndromes. Residual PAR-1 mediated platelet aggregation may be dependent on extent of clopidogrel response. Material and Methods Platelet aggregation was measured in 55 patients undergoing elective PCI at 16-24 hours after 600mg clopidogrel loading dose by light transmittance aggregometry using ADP 20μM and thrombin receptor agonist peptide (TRAP) at 15 μM and 25 μM as agonists. Genomic DNA was genotyped for common CYP2C19 variants. Results Increasing quartiles of 20 μM ADP induced platelet aggregation after clopidogrel loading were associated with increasing levels of TRAP mediated platelet aggregation. Patients in the highest quartile (clopidogrel non-responders) of post treatment ADP aggregation had significantly higher TRAP mediated aggregation than the patients in the lowest quartile (clopidogrel responders) [TRAP 15 μM: 79.6±5% vs. 69.5±8%, p<0.001]. Conclusions Non-responders to clopidogrel show increased residual platelet aggregation induced by TRAP, whereas clopidogrel responders exhibit attenuated response to TRAP. Addition of PAR-1 antiplatelet drugs may be most effective in patients with reduced clopidogrel response and high residual TRAP mediated platelet aggregation. PMID:22459907

  18. Bacillus anthracis peptidoglycan activates human platelets through FcγRII and complement

    PubMed Central

    Sun, Dawei; Popescu, Narcis I.; Raisley, Brent; Keshari, Ravi S.; Dale, George L.; Lupu, Florea

    2013-01-01

    Platelet activation frequently accompanies sepsis and contributes to the sepsis-associated vascular leakage and coagulation dysfunction. Our previous work has implicated peptidoglycan (PGN) as an agent causing systemic inflammation in gram-positive sepsis. We used flow cytometry and fluorescent microscopy to define the effects of PGN on the activation of human platelets. PGN induced platelet aggregation, expression of the activated form of integrin αIIbβ3, and exposure of phosphatidylserine (PS). These changes were dependent on immunoglobulin G and were attenuated by the Fcγ receptor IIa–blocking antibody IV.3, suggesting they are mediated by PGN–anti-PGN immune complexes signaling through Fcγ receptor IIa. PS exposure was not blocked by IV.3 but was sensitive to inhibitors of complement activation. PGN was a potent activator of the complement cascade in human plasma and caused deposition of C5b-9 on the platelet surface. Platelets with exposed PS had greatly accelerated prothrombinase activity. We conclude that PGN derived from gram-positive bacteria is a potent platelet agonist when complexed with anti-PGN antibody and could contribute to the coagulation dysfunction accompanying gram-positive infections. PMID:23733338

  19. A novel role of sesamol in inhibiting NF-κB-mediated signaling in platelet activation

    PubMed Central

    2011-01-01

    Background Platelet activation is relevant to a variety of coronary heart diseases. Our previous studies revealed that sesamol possesses potent antiplatelet activity through increasing cyclic AMP formation. Although platelets are anucleated cells, they also express the transcription factor, NF-κB, that may exert non-genomic functions in platelet activation. Therefore, we further investigated the inhibitory roles of sesamol in NF-κB-mediated platelet function. Methods Platelet aggregation, Fura 2-AM fluorescence, and immunoblotting analysis were used in this study. Results NF-κB signaling events, including IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation, were markedly activated by collagen (1 μg/ml) in washed human platelets, and these signaling events were attenuated by sesamol (2.5~25 μM). Furthermore, SQ22536 and ODQ, inhibitors of adenylate cyclase and guanylate cyclase, respectively, strongly reversed the sesamol (25 μM)-mediated inhibitory effects of IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation stimulated by collagen. The protein kinase A (PKA) inhibitor, H89, also reversed sesamol-mediated inhibition of IκBα degradation. Moreover, BAY11-7082, an NF-κB inhibitor, abolished IκBα degradation, phospholipase C (PLC)γ2 phosphorylation, protein kinase C (PKC) activation, [Ca2+]i mobilization, and platelet aggregation stimulated by collagen. Preincubation of platelets with the inhibitors, SQ22536 and H89, both strongly reversed sesamol-mediated inhibition of platelet aggregation and [Ca2+]i mobilization. Conclusions Sesamol activates cAMP-PKA signaling, followed by inhibition of the NF-κB-PLC-PKC cascade, thereby leading to inhibition of [Ca2+]i mobilization and platelet aggregation. Because platelet activation is not only linked to hemostasis, but also has a relevant role in inflammation and metastasis, our data demonstrating that inhibition of NF-κB interferes with platelet function may have a great impact when

  20. Matrix metalloproteinase-2 of human carotid atherosclerotic plaques promotes platelet activation. Correlation with ischaemic events.

    PubMed

    Lenti, Massimo; Falcinelli, Emanuela; Pompili, Marcella; de Rango, Paola; Conti, Valentina; Guglielmini, Giuseppe; Momi, Stefania; Corazzi, Teresa; Giordano, Giuseppe; Gresele, Paolo

    2014-06-01

    Purified active matrix metalloproteinase-2 (MMP-2) is able to promote platelet aggregation. We aimed to assess the role of MMP-2 expressed in atherosclerotic plaques in the platelet-activating potential of human carotid plaques and its correlation with ischaemic events. Carotid plaques from 81 patients undergoing endarterectomy were tested for pro-MMP-2 and TIMP-2 content by zymography and ELISA. Plaque extracts were incubated with gel-filtered platelets from healthy volunteers for 2 minutes before the addition of a subthreshold concentration of thrombin receptor activating peptide-6 (TRAP-6) and aggregation was assessed. Moreover, platelet deposition on plaque extracts immobilised on plastic coverslips under high shear-rate flow conditions was measured. Forty-three plaque extracts (53%) potentiated platelet aggregation (+233 ± 26.8%), an effect prevented by three different specific MMP-2 inhibitors (inhibitor II, TIMP-2, moAb anti-MMP-2). The pro-MMP-2/TIMP-2 ratio of plaques potentiating platelet aggregation was significantly higher than that of plaques not potentiating it (3.67 ± 1.21 vs 1.01 ± 0.43, p<0.05). Moreover, the platelet aggregation-potentiating effect, the active-MMP-2 content and the active MMP-2/pro-MMP-2 ratio of plaque extracts were significantly higher in plaques from patients who developed a subsequent major cardiovascular event. In conclusion, atherosclerotic plaques exert a prothrombotic effect by potentiating platelet activation due to their content of MMP-2; an elevated MMP-2 activity in plaques is associated with a higher rate of subsequent ischaemic cerebrovascular events. PMID:24499865

  1. The dimeric platelet collagen receptor GPVI-Fc reduces platelet adhesion to activated endothelium and preserves myocardial function after transient ischemia in mice.

    PubMed

    Schönberger, Tanja; Ziegler, Melanie; Borst, Oliver; Konrad, Ildiko; Nieswandt, Bernhard; Massberg, Steffen; Ochmann, Carmen; Jürgens, Tobias; Seizer, Peter; Langer, Harald; Münch, Götz; Ungerer, Martin; Preissner, Klaus T; Elvers, Margitta; Gawaz, Meinrad

    2012-10-01

    Platelets play a critical role in the pathophysiology of reperfusion, sepsis, and cardiovascular diseases. In a multiple step process, they adhere to activated endothelium and release proinflammatory cytokines thereby promoting the inflammatory process. Glycoprotein VI (GPVI) is the major collagen receptor on the platelet surface and triggers platelet activation and primary hemostasis. Activation of GPVI leads to stable platelet adhesion and degranulation of platelet granules. However, GPVI is critically involved in platelet adhesion to activated endothelium without exposure of subendothelial matrix. Earlier studies show that the soluble GPVI-Fc binds to collagen and protects mice from atherosclerosis and decreases neointima proliferation after arterial injury. Here, we show for the first time that recombinant GPVI-Fc binds to activated endothelium mainly via vitronectin and prevents platelet/endothelial interaction. Administration of GPVI-Fc reduced infarct size and preserved cardiac function in a mouse model of myocardial infarction. This process was associated with reduced GPVI-induced platelet degranulation and release of proinflammatory cytokines in vitro and in vivo. Taken together, administration of GPVI-Fc offers a novel strategy to control platelet-mediated inflammation and to preserve myocardial function following myocardial infarction. PMID:22814400

  2. MALT1-Ubiquitination Triggers Non-Genomic NF-κB/IKK Signaling upon Platelet Activation

    PubMed Central

    Karim, Zubair A.; Vemana, Hari Priya; Khasawneh, Fadi T.

    2015-01-01

    We have recently shown that IKK complex plays an important non-genomic role in platelet function, i.e., regulates SNARE machinery-dependent membrane fusion. In this connection, it is well known that MALT1, whose activity is modulated by proteasome, plays an important role in the regulation of IKK complex. Therefore, the present studies investigated the mechanism by which IKK signaling is regulated in the context of the platelet proteasome. It was found that platelets express a functional proteasome, and form CARMA/MALT1/Bcl10 (CBM) complex when activated. Using a pharmacological inhibitor, the proteasome was found to regulate platelet function (aggregation, integrin activation, secretion, phosphatidylserine exposure and changes in intracellular calcium). It was also found to regulate thrombogenesis and physiologic hemostasis. We also observed, upon platelet activation, that MALT1 is ubiquitinated, and this coincides with the activation of the IKK/NF-κB-signaling pathway. Finally, we observed that the proteasome inhibitor blocks CBM complex formation and the interaction of IKKγ and MALT1; abrogates SNARE formation, and the association of MALT1 with TAK1 and TAB2, which are upstream of the CBM complex. Thus, our data demonstrate that MALT1 ubiquitination is critical for the engagement of CBM and IKK complexes, thereby directing platelet signals to the NF-κB pathway. PMID:25748427

  3. A balance between TFPI and thrombin-mediated platelet activation is required for murine embryonic development

    PubMed Central

    Ellery, Paul E. R.; Maroney, Susan A.; Cooley, Brian C.; Luyendyk, James P.; Zogg, Mark; Weiler, Hartmut

    2015-01-01

    Tissue factor pathway inhibitor (TFPI) is a critical anticoagulant protein present in endothelium and platelets. Mice lacking TFPI (Tfpi−/−) die in utero from disseminated intravascular coagulation. They are rescued by concomitant tissue factor (TF) deficiency, demonstrating that TFPI modulates TF function in vivo. Recent studies have found TFPI inhibits prothrombinase activity during the initiation of coagulation and limits platelet accumulation during thrombus formation, implicating TFPI in modulating platelet procoagulant activity. To examine whether altered platelet function would compensate for the lack of TFPI and rescue TFPI-null embryonic lethality, Tfpi+/− mice lacking the platelet thrombin receptor, protease activated receptor 4 (PAR4; Par4−/−), or its coreceptor, PAR3, were mated. PAR3 deficiency did not rescue Tfpi−/− embryos, but >40% of expected Tfpi−/−:Par4−/− offspring survived to adulthood. Adult Tfpi−/−:Par4−/− mice did not exhibit overt thrombosis. However, they had focal sterile inflammation with fibrin(ogen) deposition in the liver and elevated plasma thrombin-antithrombin complexes, indicating activation of coagulation at baseline. Tfpi−/−:Par4−/− mice have platelet and fibrin accumulation similar to Par4−/− mice following venous electrolytic injury but were more susceptible than Par4−/− mice to TF-induced pulmonary embolism. In addition, ∼30% of the Tfpi−/−:Par4−/− mice were born with short tails. Tfpi−/−:Par4−/− mice are the first adult mice described that lack TFPI with unaltered TF. They demonstrate that TFPI physiologically modulates thrombin-dependent platelet activation in a manner that is required for successful embryonic development and identify a role for TFPI in dampening intravascular procoagulant stimuli that lead to thrombin generation, even in the absence of thrombin-mediated platelet activation. PMID:25954015

  4. Highly electronegative LDL from patients with ST-elevation myocardial infarction triggers platelet activation and aggregation

    PubMed Central

    Chan, Hua-Chen; Ke, Liang-Yin; Chu, Chih-Sheng; Lee, An-Sheng; Shen, Ming-Yi; Cruz, Miguel A.; Hsu, Jing-Fang; Cheng, Kai-Hung; Chan, Hsiu-Chuan Bonnie; Lu, Jonathan; Lai, Wen-Ter; Sawamura, Tatsuya; Sheu, Sheng-Hsiung

    2013-01-01

    Platelet activation and aggregation underlie acute thrombosis that leads to ST-elevation myocardial infarction (STEMI). L5—highly electronegative low-density lipoprotein (LDL)—is significantly elevated in patients with STEMI. Thus, we examined the role of L5 in thrombogenesis. Plasma LDL from patients with STEMI (n = 30) was chromatographically resolved into 5 subfractions (L1-L5) with increasing electronegativity. In vitro, L5 enhanced adenosine diphosphate–stimulated platelet aggregation twofold more than did L1 and induced platelet-endothelial cell (EC) adhesion. L5 also increased P-selectin expression and glycoprotein (GP)IIb/IIIa activation and decreased cyclic adenosine monophosphate levels (n = 6, P < .01) in platelets. In vivo, injection of L5 (5 mg/kg) into C57BL/6 mice twice weekly for 6 weeks shortened tail bleeding time by 43% (n = 3; P < .01 vs L1-injected mice) and increased P-selectin expression and GPIIb/IIIa activation in platelets. Pharmacologic blockade experiments revealed that L5 signals through platelet-activating factor receptor and lectin-like oxidized LDL receptor-1 to attenuate Akt activation and trigger granule release and GPIIb/IIIa activation via protein kinase C-α. L5 but not L1 induced tissue factor and P-selectin expression in human aortic ECs (P < .01), thereby triggering platelet activation and aggregation with activated ECs. These findings indicate that elevated plasma levels of L5 may promote thrombosis that leads to STEMI. PMID:24030386

  5. Platelet adhesion and activation on polyethylene glycol modified polyurethane surfaces. Measurement of cytoplasmic calcium.

    PubMed

    Park, K D; Suzuki, K; Lee, W K; Lee, J E; Kim, Y H; Sakurai, Y; Okano, T

    1996-01-01

    Polyurethane (PU) surfaces were modified by coupling polyethylene glycol (PEG; molecular weight, 1,000) chains carrying different terminal groups (PU-PEG1K-OH, PU-PEG1K-NH2, PU-PEG1K-SO3) and longer PEG chains (MW, 3,350; PU-PEG3.4K-OH). The modified PU surfaces have the same PEG (1K) chain density. Surface induced platelet activation was evaluated by measuring cytoplasmic free calcium concentration in platelets contacting modified surfaces, and platelet adhesion onto modified surfaces was investigated in vitro. Cytoplasmic free calcium levels in platelets contacting PU-PEG-SO3 remained relatively constant, in contrast to the significant increase observed for PU-PEG-NH2, PU-PEG-OH, and control PU surfaces. The degree of platelet adhesion clearly demonstrates that all PEG graft surfaces prevented platelet adhesion. Among PEG1K surfaces, PU-PEG-SO3 shows the lowest platelet adhesion. In the case of relatively longer PEG grafted surfaces (PU-PEG3.4K-OH and PU-PEG3.4K-Hep), both surfaces were found to prevent the increase in both cytoplasmic free calcium and platelet adhesion. These results suggest that longer PEG chain grafting is more effective than shorter grafting in preventing platelet activation and adhesion because of the highly dynamic movement of hydrated PEG chains at the interface. In addition, in vitro platelet interaction is dependent upon terminal groups of PEG chains on PEG1K series surfaces. PMID:8945010

  6. The nature of interactions between tissue-type plasminogen activator and platelets

    SciTech Connect

    Torr, S.R.; Winters, K.J.; Santoro, S.A.; Sobel, B.E. )

    1990-07-15

    To elucidate interactions responsible for inhibition of aggregation of platelets in platelet-rich plasma (PRP) harvested from whole blood preincubated with t-PA, experiments were performed with PRP and washed platelets under diverse conditions of preincubation. Both ADP and collagen induced aggregation were inhibited in PRP unless aprotinin had been added to the preincubated whole blood concomitantly with t-PA. However, in washed platelets prepared after the same exposure aggregation was intact. When washed platelets were supplemented with fibrinogen degradation products (FDPs) in concentrations simulating those in whole blood preincubated with t-PA, aggregation induced with either ADP or collagen was inhibited. Thus, the inhibition in PRP depended on generation of FDPs by activated plasminogen. The functional integrity of surface glycoprotein (GP) IIb/IIIa receptors in washed platelets was documented by autoradiography after SDS-PAGE of surface labeled GPs and by fibrinogen binding despite preincubation of the whole blood or washed platelets themselves with t-PA and plasminogen as long as exogenous calcium (greater than or equal to 0.1 microM) was present. In contrast, when calcium was absent, the platelet GP IIb/IIIa receptor was rendered susceptible to degradation by plasmin, and aggregation was inhibited by preincubation at 37 degrees C even if aprotinin was present when aggregation was being assayed. These observations reconcile disparate results in the literature from studies in vivo and in vitro by demonstrating that inhibition of aggregation of platelets in PRP and in whole blood reflects indirect effects of plasminogen activation rather than direct effects of t-PA or plasmin on the platelets themselves.

  7. Hypergravity and hypobaric hypoxic conditions promote endothelial cell and platelet activation.

    PubMed

    Rubenstein, David A; Yin, Wei

    2014-09-01

    Cardiovascular disease risk is heightened during exposure to altered gravity and/or altered barometric conditions. Previous work has suggested that this heightened cardiovascular risk is due to enhancements of endothelial cell inflammatory and/or thrombogenic responses. In recent work, the role of platelets on instigating or inhibiting endothelial cell responses associated with cardiovascular disease has been found to be dependent on both biochemical and biophysical factors. In this work, we aimed to determine how two biophysical forces, gravity and atmospheric pressure, alter endothelial cell and platelet functions and their interactions to instigate or inhibit cardiovascular disease responses. To address this aim, endothelial cells and platelets were subjected to a force 8 times greater than the normal gravitational force, for up to 30 minutes. In separate experiments, endothelial cells and platelets were subjected to 50% of normal atmospheric pressure. Endothelial cell and platelet responses, associated with cardiovascular diseases, were measured as a time course during exposure. In general, the exposure of endothelial cells to either hypergravity or hypobaric conditions enhanced cardiovascular disease responses. However, the presence of platelets generally inhibited endothelial cell responses. Platelet activation was, however, somewhat enhanced under both hypergravity and hypobaric conditions. Our data suggest that altered biophysical forces can modulate endothelial cell and platelet responses that are salient for cardiovascular disease progression. However, the interaction of these two cells tends to restrain the progression of the pro-cardiovascular disease responses. PMID:25211651

  8. Net Increase of platelet membrane tyrosine specific-protein kinase activity by phorbol myristate acetate

    SciTech Connect

    Ishihara, Noriko; Sakamoto, Hikaru; Iwama, Minako; Kobayashi, Bonro )

    1990-01-01

    Tyrosine protein kinase (TPK) activity in rabbit platelets after stimulation by phorbol myristate acetate (PMA) or thrombin was directly estimated by {sup 32}P incorporation from ({gamma}-{sup 32})ATP into synthetic peptide angiotensin II. By PMA-treatment a net increase of TPK activity was obtained, while thrombin acted on the TPK quickly but stimulation was limited within the range attained by the control after lengthy incubation. The responsive TPK to these stimulators was localized mainly in membrane but much less in cytosol. The specific activity of the particulate TPK was low in the sonicate of control ice cold platelets but increased about 6-fold when the platelets were incubated at 37{degree}C. On a brief contact of platelets with PMA at 37{degrees}C the TPK was fully activated and reached a maximum value about 130% of the control. Determination of phosphotyrosine phosphatase in the stimulated platelet sonicate revealed that its participation in the above described increase of {sup 32}P-incorporation was meagre. The quick response suggested a possible role of TPK in the signal transduction through the platelet cell membrane.

  9. Amyloid β peptide stimulates platelet activation through RhoA-dependent modulation of actomyosin organization.

    PubMed

    Sonkar, Vijay K; Kulkarni, Paresh P; Dash, Debabrata

    2014-04-01

    Platelets contribute to 95% of circulating amyloid precursor protein in the body and have widely been employed as a "peripheral" model of neurons in Alzheimer's disease. We sought to analyze the effects of amyloid β (Aβ) on platelets and to understand the underlying molecular mechanism. The Aβ active fragment containing amino acid sequence 25-35 (Aβ(25-35); 10-20 μM) was found to induce strong aggregation of human platelets, granule release, and integrin activation, similar to that elicited by physiological agonists. Platelets exposed to Aβ(25-35) retracted fibrin clot and displayed augmented adhesion to collagen under arterial shear, reflective of a switch to prothrombotic phenotype. Exposure of platelets to Aβ peptide (20 μM) resulted in a 4.2- and 2.3-fold increase in phosphorylation of myosin light chain (MLC) and MLC phosphatase, respectively, which was reversed by Y27632, an inhibitor of Rho-associated coiled-coil protein kinase (ROCK). Aβ(25-35)-induced platelet aggregation and clot retraction were also significantly attenuated by Y27632. Consistent with these findings, Aβ(25-35) elicited a significant rise in the level of RhoA-GTP in platelets. Platelets pretreated with reverse-sequenced Aβ fragment (Aβ(35-25)) and untreated resting platelets served as controls. We conclude that Aβ induces cellular activation through RhoA-dependent modulation of actomyosin, and hence, RhoA could be a potential therapeutic target in Alzheimer's disease and cerebral amyloid angiopathy. PMID:24421399

  10. Activated platelet chemiluminescence and presence of CD45+ platelets in patients with acute myocardial infarction.

    PubMed

    Gabbasov, Zufar; Ivanova, Oxana; Kogan-Yasny, Victor; Ryzhkova, Evgeniya; Saburova, Olga; Vorobyeva, Inna; Vasilieva, Elena

    2014-01-01

    It has been found that in 15% of acute myocardial infarction patients' platelets generate reactive oxygen species that can be detected with luminol-enhanced chemiluminescence of platelet-rich plasma within 8-10 days after acute myocardial infarction. This increase in generate reactive oxygen species production coincides with the emergence of CD45(+) platelets. The ability of platelets to carry surface leukocyte antigen implies their participation in exchange of specific proteins in the course of acute myocardial infarction. Future studies of CD45(+) platelets in peripheral blood of acute myocardial infarction patients in association with generate reactive oxygen species production may provide a new insight into the complex mechanisms of cell-cell interactions associated with acute myocardial infarction. PMID:24102264

  11. Are the changes in the peripheral brain-derived neurotrophic factor levels due to platelet activation?

    PubMed

    Serra-Millàs, Montserrat

    2016-03-22

    Brain-derived neurotrophic factor (BDNF) plays an important role in central nervous system development, neurogenesis and neuronal plasticity. BDNF is also expressed in several non-neuronal tissues, and it could play an important role in other processes, such as cancer, angiogenesis, etc. Platelets are the major source of peripheral BDNF. However, platelets also contain high amounts of serotonin; they express specific surface receptors during activation, and a multitude of pro-inflammatory and immunomodulatory bioactive compounds are secreted from the granules. Until recently, there was insufficient knowledge regarding the relationship between BDNF and platelets. Recent studies showed that BDNF is present in two distinct pools in platelets, in α-granules and in the cytoplasm, and only the BDNF in the granules is secreted following stimulation, representing 30% of the total BDNF in platelets. BDNF has an important role in the pathophysiology of depression. Low levels of serum BDNF have been described in patients with major depressive disorder, and BDNF levels increased with chronic antidepressant treatment. Interestingly, there is an association between depression and platelet function. This review analyzed studies that evaluated the relationship between BDNF and platelet activation and the effect of treatments on both parameters. Only a few studies consider this possible confounding factor, and it could be very important in diseases such as depression, which show changes in both parameters. PMID:27014600

  12. Glucose and collagen regulate human platelet activity through aldose reductase induction of thromboxane

    PubMed Central

    Tang, Wai Ho; Stitham, Jeremiah; Gleim, Scott; Di Febbo, Concetta; Porreca, Ettore; Fava, Cristiano; Tacconelli, Stefania; Capone, Marta; Evangelista, Virgilio; Levantesi, Giacomo; Wen, Li; Martin, Kathleen; Minuz, Pietro; Rade, Jeffrey; Patrignani, Paola; Hwa, John

    2011-01-01

    Diabetes mellitus is associated with platelet hyperactivity, which leads to increased morbidity and mortality from cardiovascular disease. This is coupled with enhanced levels of thromboxane (TX), an eicosanoid that facilitates platelet aggregation. Although intensely studied, the mechanism underlying the relationship among hyperglycemia, TX generation, and platelet hyperactivity remains unclear. We sought to identify key signaling components that connect high levels of glucose to TX generation and to examine their clinical relevance. In human platelets, aldose reductase synergistically modulated platelet response to both hyperglycemia and collagen exposure through a pathway involving ROS/PLCγ2/PKC/p38α MAPK. In clinical patients with platelet activation (deep vein thrombosis; saphenous vein graft occlusion after coronary bypass surgery), and particularly those with diabetes, urinary levels of a major enzymatic metabolite of TX (11-dehydro-TXB2 [TX-M]) were substantially increased. Elevated TX-M persisted in diabetic patients taking low-dose aspirin (acetylsalicylic acid, ASA), suggesting that such patients may have underlying endothelial damage, collagen exposure, and thrombovascular disease. Thus, our study has identified multiple potential signaling targets for designing combination chemotherapies that could inhibit the synergistic activation of platelets by hyperglycemia and collagen exposure. PMID:22005299

  13. Glucose and collagen regulate human platelet activity through aldose reductase induction of thromboxane.

    PubMed

    Tang, Wai Ho; Stitham, Jeremiah; Gleim, Scott; Di Febbo, Concetta; Porreca, Ettore; Fava, Cristiano; Tacconelli, Stefania; Capone, Marta; Evangelista, Virgilio; Levantesi, Giacomo; Wen, Li; Martin, Kathleen; Minuz, Pietro; Rade, Jeffrey; Patrignani, Paola; Hwa, John

    2011-11-01

    Diabetes mellitus is associated with platelet hyperactivity, which leads to increased morbidity and mortality from cardiovascular disease. This is coupled with enhanced levels of thromboxane (TX), an eicosanoid that facilitates platelet aggregation. Although intensely studied, the mechanism underlying the relationship among hyperglycemia, TX generation, and platelet hyperactivity remains unclear. We sought to identify key signaling components that connect high levels of glucose to TX generation and to examine their clinical relevance. In human platelets, aldose reductase synergistically modulated platelet response to both hyperglycemia and collagen exposure through a pathway involving ROS/PLCγ2/PKC/p38α MAPK. In clinical patients with platelet activation (deep vein thrombosis; saphenous vein graft occlusion after coronary bypass surgery), and particularly those with diabetes, urinary levels of a major enzymatic metabolite of TX (11-dehydro-TXB2 [TX-M]) were substantially increased. Elevated TX-M persisted in diabetic patients taking low-dose aspirin (acetylsalicylic acid, ASA), suggesting that such patients may have underlying endothelial damage, collagen exposure, and thrombovascular disease. Thus, our study has identified multiple potential signaling targets for designing combination chemotherapies that could inhibit the synergistic activation of platelets by hyperglycemia and collagen exposure. PMID:22005299

  14. Are the changes in the peripheral brain-derived neurotrophic factor levels due to platelet activation?

    PubMed Central

    Serra-Millàs, Montserrat

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) plays an important role in central nervous system development, neurogenesis and neuronal plasticity. BDNF is also expressed in several non-neuronal tissues, and it could play an important role in other processes, such as cancer, angiogenesis, etc. Platelets are the major source of peripheral BDNF. However, platelets also contain high amounts of serotonin; they express specific surface receptors during activation, and a multitude of pro-inflammatory and immunomodulatory bioactive compounds are secreted from the granules. Until recently, there was insufficient knowledge regarding the relationship between BDNF and platelets. Recent studies showed that BDNF is present in two distinct pools in platelets, in α-granules and in the cytoplasm, and only the BDNF in the granules is secreted following stimulation, representing 30% of the total BDNF in platelets. BDNF has an important role in the pathophysiology of depression. Low levels of serum BDNF have been described in patients with major depressive disorder, and BDNF levels increased with chronic antidepressant treatment. Interestingly, there is an association between depression and platelet function. This review analyzed studies that evaluated the relationship between BDNF and platelet activation and the effect of treatments on both parameters. Only a few studies consider this possible confounding factor, and it could be very important in diseases such as depression, which show changes in both parameters. PMID:27014600

  15. Fractions of aqueous and methanolic extracts from tomato (Solanum lycopersicum L.) present platelet antiaggregant activity.

    PubMed

    Fuentes, Eduado J; Astudillo, Luis A; Gutiérrez, Margarita I; Contreras, Samuel O; Bustamante, Luis O; Rubio, Pia I; Moore-Carrasco, Rodrigo; Alarcón, Marcelo A; Fuentes, Jaime A; González, Daniel E; Palomo, Iván F

    2012-03-01

    Cardiovascular disease (CVD) is the leading cause of death worldwide. Its prevention emphasizes three aspects: not smoking, physical activity and a healthy diet. Recently, we screened the antithrombotic activity of a selected group of fruits and vegetables. Among them, tomato showed an important effect. The aim of this study was to evaluate and characterize the platelet antiaggregatory activity of tomato (Solanum lycopersicum L.). For this, we obtained aqueous and methanolic tomato extracts and evaluated the effect of pH (2 and 10) and temperature (22, 60 and 100°C) on this activity. Furthermore, in order to isolate the antiaggregant principle, we separated tomato extracts into several fractions (A-D) by size exclusion chromatography. In addition, we evaluated the platelet antiaggregating activity ex vivo in Wistar rats. Aqueous and methanolic extracts of tomato treated at 22, 60 and 100°C and pH 2 and 10 still inhibited platelet aggregation (in vitro). Moreover, it was noted that one of the fractions (fraction C), from both aqueous and methanolic extracts, presented the highest activity (∼70% inhibition of platelet aggregation) and concentration dependently inhibited platelet aggregation significantly compared with control (P < 0.05). These fractions did not contain lycopene but presented two peaks of absorption, at 210 and 261 nm, compatible with the presence of nucleosides. In rats treated with tomato macerates, a mild platelet antiaggregating effect ex vivo was observed. Further studies are required to identify the molecules with platelet antiaggregating activity and antiplatelet mechanisms of action. PMID:22185934

  16. An antagonistic activity of etizolam on platelet-activating factor (PAF). In vitro effects on platelet aggregation and PAF receptor binding.

    PubMed

    Mikashima, H; Takehara, S; Muramoto, Y; Khomaru, T; Terasawa, M; Tahara, T; Maruyama, Y

    1987-08-01

    The antagonistic effect of etizolam, an anti-anxiety drug, on platelet-activating factor (PAF) was investigated in rabbit platelets in vitro. Etizolam inhibited PAF-induced aggregation in a dose-dependent manner, with an IC50 of 3.8 microM, about one tenth that of triazolam (IC50 = 30 microM). At 300 microM, it inhibited both ADP and arachidonic acid-induced aggregation only slightly, while the other anti-anxiety drugs tested had no effect on PAF-induced aggregation even at this concentration. Etizolam and triazolam inhibited the specific binding of 3H-PAF to PAF receptor sites on washed rabbit platelets with IC50 values of 22 nM and 320 nM, respectively. Diazepam and estazolam were inactive even at 1 microM. These results indicate that etizolam is a specific antagonist of PAF. PMID:2890779

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

    PubMed Central

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

    2015-01-01

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

  18. Ultraviolet irradiation of platelet concentrate abrogates lymphocyte activation without affecting platelet function in vitro

    SciTech Connect

    Kahn, R.A.; Duffy, B.F.; Rodey, G.G.

    1985-11-01

    We studied the effect of ultraviolet (UV) radiation on platelet concentrates. Samples irradiated at 310 mm for 30 minutes at a dose of 1782 J per m2 showed no loss of platelet function in vitro as determined by adenosine diphosphate, collagen, or ristocetin-induced aggregation. Lymphocytes isolated from irradiated units were unable to act as responders or stimulators in a mixed-lymphocyte reaction. These data suggest that UV radiation of platelet concentrates may result in a cell suspension that is unable to evoke an immunological response.

  19. Plasma Components and Platelet Activation Are Essential for the Antimicrobial Properties of Autologous Platelet-Rich Plasma: An In Vitro Study

    PubMed Central

    Drago, Lorenzo; Bortolin, Monica; Vassena, Christian; Romanò, Carlo L.; Taschieri, Silvio; Fabbro, Massimo Del

    2014-01-01

    Autologous platelet concentrates are successfully adopted in a variety of medical fields to stimulate bone and soft tissue regeneration. The rationale for their use consists in the delivery of a wide range of platelet-derived bioactive molecules that promotes wound healing. In addition, antimicrobial properties of platelet concentrates have been pointed out. In this study, the effect of the platelet concentration, of the activation step and of the presence of plasmatic components on the antimicrobial activity of pure platelet-rich plasma was investigated against gram positive bacteria isolated from oral cavity. The antibacterial activity, evaluated as the minimum inhibitory concentration, was determined through the microdilution two-fold serial method. Results seem to suggest that the antimicrobial activity of platelet-rich plasma against Enterococcus faecalis, Streptococcus agalactiae, Streptococcus oralis and Staphylococcus aureus is sustained by a co-operation between plasma components and platelet-derived factors and that the activation of coagulation is a fundamental step. The findings of this study may have practical implications in the modality of application of platelet concentrates. PMID:25232963

  20. Arf6 plays an early role in platelet activation by collagen and convulxin.

    PubMed

    Choi, Wangsun; Karim, Zubair A; Whiteheart, Sidney W

    2006-04-15

    Small GTPases play critical roles in hemostasis, though the roster of such molecules in platelets is not complete. In this study, we report the presence of Ras-related GTPases of the ADP-ribosylation factor (Arf) family. Platelets contain Arf1 or 3 and Arf6, with the latter being predominantly membrane associated. Using effector domain pull-down assays, we show, counter to other GTPases, that Arf6-GTP is present in resting platelets and decreases rapidly upon activation with collagen or convulxin. This decrease does not completely rely on secondary agonists (ADP and thromboxane A2) or require integrin signaling. The decrease in free Arf6-GTP temporally precedes activation of Rho family GTPases (RhoA, Cdc42, and Rac1). Using a membrane-permeant, myristoylated peptide, which mimics the N-terminus of Arf6, we show that the Arf6-GTP decrease is essential for collagen- and convulxin-induced aggregation, platelet adherence, and spreading on collagen-coated glass. Treatment with this peptide also affects the activation of Rho family GTPases, but has little effect on RalA and Rap1 or on agonist-induced calcium mobilization. These data show that Arf6 is a key element in activation through GPVI, and is required for activation of the Rho family GTPases and the subsequent cytoskeletal rearrangements needed for full platelet function. PMID:16352809

  1. Exploration of the antiplatelet activity profile of betulinic acid on human platelets

    PubMed Central

    Tzakos, Andreas G.; Kontogianni, Vassiliki G.; Tsoumani, Maria; Kyriakou, Eleni; Hwa, John; Rodrigues, Francisco A.; Tselepis, Alexandros D.

    2013-01-01

    Betulinic acid, a natural pentacyclic triterpene acid, presents a diverse mode of biological actions including anti-retroviral, antibacterial, antimalarial and anti-inflammatory activities. The potency of betulinic acid as an inhibitor of human platelet activation was evaluated and its antiplatelet profile against in vitro platelet aggregation, induced by several platelet agonists (Adenosine Diphosphate, Thrombin Receptor Activator Peptide-14 and Arachidonic Acid), was explored. Flow cytometric analysis was performed to examine the effect of betulinic acid on P-selectin membrane expression and PAC-1 binding to activated platelets. Betulinic acid potently inhibits platelet aggregation and also reduced PAC-1 binding and the membrane expression of P-selectin. Principal component analysis was used to screen, on the chemical property space, for potential common pharmacophores of betulinic acid with approved antithrombotic drugs. A common pharmacophore was defined between the NMR derived structure of betulinic acid and prostacyclin agonists (PGI2) and the importance of its carboxylate group in its antiplatelet activity was determined. The present results indicate that betulinic acid has potential use as an antithrombotic compound and suggest that the mechanism underlying the antiplatelet effects of betulinic acid is similar to that of the PGI2 receptor agonists, a hypothesis that reserves further investigation. PMID:22720759

  2. 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

  3. Platelet aggregation and serum adenosine deaminase (ADA) activity in pregnancy associated with diabetes, hypertension and HIV.

    PubMed

    Leal, Claudio A M; Leal, Daniela B R; Adefegha, Stephen A; Morsch, Vera M; da Silva, José E P; Rezer, João F P; Schrekker, Clarissa M L; Abdalla, Faida H; Schetinger, Maria R C

    2016-07-01

    Platelet aggregation and adenosine deaminase (ADA) activity were evaluated in pregnant women living with some disease conditions including hypertension, diabetes mellitus and human immunodeficiency virus infection. The subject population is consisted of 15 non-pregnant healthy women [control group (CG)], 15 women with normal pregnancy (NP), 7 women with hypertensive pregnancy (HP), 10 women with gestational diabetes mellitus (GDM) and 12 women with human immunodeficiency virus-infected pregnancy (HIP) groups. The aggregation of platelets was checked using an optical aggregometer, and serum ADA activity was determined using the colorimetric method. After the addition of 5 µM of agonist adenosine diphosphate, the percentage of platelet aggregation was significantly (p < 0·05) increased in NP, HP, GDM and HIP groups when compared with the CG, while the addition of 10 µM of the same agonist caused significant (p < 0·05) elevations in HP, GDM and HIP groups when compared with CG. Furthermore, ADA activity was significantly (p < 0·05) enhanced in NP, HP, GDM and HIP groups when compared with CG. In this study, the increased platelet aggregation and ADA activity in pregnancy and pregnancy-associated diseases suggest that platelet aggregation and ADA activity could serve as peripheral markers for the development of effective therapy in the maintenance of homeostasis and some inflammatory process in these pathophysiological conditions. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27273565

  4. Exploration of the antiplatelet activity profile of betulinic acid on human platelets.

    PubMed

    Tzakos, Andreas G; Kontogianni, Vassiliki G; Tsoumani, Maria; Kyriakou, Eleni; Hwa, John; Rodrigues, Francisco A; Tselepis, Alexandros D

    2012-07-18

    Betulinic acid, a natural pentacyclic triterpene acid, presents a diverse mode of biological actions including antiretroviral, antibacterial, antimalarial, and anti-inflammatory activities. The potency of betulinic acid as an inhibitor of human platelet activation was evaluated, and its antiplatelet profile against in vitro platelet aggregation, induced by several platelet agonists (adenosine diphosphate, thrombin receptor activator peptide-14, and arachidonic acid), was explored. Flow cytometric analysis was performed to examine the effect of betulinic acid on P-selectin membrane expression and PAC-1 binding to activated platelets. Betulinic acid potently inhibits platelet aggregation and also reduced PAC-1 binding and the membrane expression of P-selectin. Principal component analysis was used to screen, on the chemical property space, for potential common pharmacophores of betulinic acid with approved antithrombotic drugs. A common pharmacophore was defined between the NMR-derived structure of betulinic acid and prostacyclin agonists (PGI2), and the importance of its carboxylate group in its antiplatelet activity was determined. The present results indicate that betulinic acid has potential use as an antithrombotic compound and suggest that the mechanism underlying the antiplatelet effects of betulinic acid is similar to that of the PGI2 receptor agonists, a hypothesis that deserves further investigation. PMID:22720759

  5. Studies of activated GPIIb/IIIa receptors on the luminal surface of adherent platelets. Paradoxical loss of luminal receptors when platelets adhere to high density fibrinogen.

    PubMed Central

    Coller, B S; Kutok, J L; Scudder, L E; Galanakis, D K; West, S M; Rudomen, G S; Springer, K T

    1993-01-01

    The accessibility of activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to damaged blood vessels or atherosclerotic plaques is likely to play a crucial role in subsequent platelet recruitment. To define better the factors involved in this process, we developed a functional assay to assess the presence of activated, luminal GPIIb/IIIa receptors, based on their ability to bind erythrocytes containing a high density of covalently coupled RGD-containing peptides (thromboerythrocytes). Platelets readily adhered to wells coated with purified type I rat skin collagen and the adherent platelets bound a dense lawn of thromboerythrocytes. With fibrinogen-coated wells, platelet adhesion increased as the fibrinogen-coating concentration increased, reaching a plateau at about 11 micrograms/ml. Thromboerythrocyte binding to the platelets adherent to fibrinogen showed a paradoxical response, increasing at fibrinogen coating concentrations up to approximately 4-6 micrograms/ml and then dramatically decreasing at higher fibrinogen-coating concentrations. Scanning electron microscopy demonstrated that the morphology of platelets adherent to collagen was similar to that of platelets adherent to low density fibrinogen, with extensive filopodia formation and ruffling. In contrast, platelets adherent to high density fibrinogen showed a bland, flattened appearance. Immunogold staining of GPIIb/IIIa receptors demonstrated concentration of the receptors on the filopodia, and depletion of receptors on the flattened portion of the platelets. Thus, there is a paradoxical loss of accessible, activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to high density fibrinogen. Two factors may contribute to this result: engagement of GPIIb/IIIa receptors with fibrinogen on the abluminal surface leading to the loss of luminal receptors, and loss of luminal filopodia that interact with thromboerythrocytes. These data provide insight into the differences

  6. Inhibition of platelet-aggregating activity in thrombotic thrombocytopenic purpura plasma by normal adult immunoglobulin G.

    PubMed Central

    Lian, E C; Mui, P T; Siddiqui, F A; Chiu, A Y; Chiu, L L

    1984-01-01

    Plasma from patients with thrombotic thrombocytopenic purpura (TTP) caused the aggregation of autologous and homologous platelets, and effect which was inhibited by normal plasma. IgG purified from seven normal adults at a concentration of 0.7 mg/ml completely inhibited the platelet aggregation induced by plasma obtained from two TTP patients with active disease. The inhibition of platelet aggregation by human adult IgG was concentration dependent, and the inhibitory activity of human IgG was neutralized by rabbit antihuman IgG. Fab fragments inhibited the TTP plasma-induced platelet aggregation as well as intact IgG, whereas Fc fragments had no effect. Platelet aggregation caused by ADP, collagen, epinephrine, or thrombin was not affected by purified human IgG. The prior incubation of IgG with TTP plasma caused a significantly greater reduction of platelet aggregation by TTP plasma than that of IgG and platelet suspension, suggesting that the IgG inhibits TTP plasma-induced platelet aggregation through direct interaction with platelet aggregating factor in TTP plasma. IgG obtained initially from five infants and young children under the age of 4 yr did not possess any inhibitory activity. When one of the children reached 3 yr of age, his IgG inhibited the aggregation induced by one TTP plasma, but not that caused by another plasma. The IgG procured from the same boy at 4 yr of age inhibited the aggregation induced by both TTP plasmas. The IgG purified from the TTP plasma during active disease failed to inhibit the aggregation caused by the same plasma. After recovery, however, the IgG effectively inhibited aggregation. These observations suggest that platelet-aggregating factors present in the TTP plasma are heterogeneous in nature and that the IgG present in the normal adult plasma, which inhibits the TTP plasma-induced platelet aggregation, may be partially responsible for the success of plasma infusion therapy in TTP. Images PMID:6538207

  7. Soluble CD40 ligand induces β3 integrin tyrosine phosphorylation and triggers platelet activation by outside-in signaling

    PubMed Central

    Prasad, K. S. Srinivasa; Andre, Patrick; He, Ming; Bao, Ming; Manganello, Jeanne; Phillips, David R.

    2003-01-01

    We earlier reported that the soluble form of the CD40 ligand (sCD40L), is involved in thrombosis by stabilizing platelet thrombi. In this article, we have determined the mechanism by which this protein affects platelet biology. Addition of sCD40L to washed platelets was found to activate the receptor function of αIIbβ3 as measured by the induction of fibrinogen binding and the formation of platelet microparticles. Mutation in the KGD sequence (D117E) of sCD40L, the αIIbβ3-binding domain in the N terminus of the protein resulted in a loss of the platelet-stimulatory activity of this protein. Integrilin, a αIIbβ3 antagonist, but not an antibody to CD40 that blocked the ligand-binding activity, inhibited these platelet-stimulatory events. CD40-/- platelets bound fibrinogen and formed microparticles similar to WT platelets, again indicating that CD40 is not involved in sCD40L-induced platelet activation. Exposure of platelets to sCD40L, but not D117E-sCD40L-coated surfaces, induced platelet thrombi formation under arterial shear rate. sCD40L-induced platelet stimulation resulted in the phosphorylation of tyrosine-759 in the cytoplasmic domain of β3. Platelets from the diYF mouse strain, expressing β3 in which both cytoplasmic tyrosines are mutated to phenylalanine, were defective in sCD40L-induced platelet stimulation. These data indicate that sCD40L is a primary platelet agonist and that platelet stimulation is induced by the binding of the KGD domain of sCD40L to αIIbβ3, triggering outside-in signaling by tyrosine phosphorylation of β3. PMID:14519852

  8. P-selectin increases angiotensin II-induced cardiac inflammation and fibrosis via platelet activation

    PubMed Central

    LIU, GAIZHEN; LIANG, BIN; SONG, XIAOSU; BAI, RUI; QIN, WEIWEI; SUN, XU; LU, YAN; BIAN, YUNFEI; XIAO, CHUANSHI

    2016-01-01

    Platelet activation is important in hypertension-induced cardiac inflammation and fibrosis. P-selectin expression significantly (P<0.05) increases when platelets are activated during hypertension. Although P-selectin recruits leukocytes to sites of inflammation, the role of P-selectin in cardiac inflammation and fibrosis remains to be elucidated. The present study aimed to investigate whether platelet-derived P-selectin promotes hypertensive cardiac inflammation and fibrosis. P-selectin knockout (P-sel KO) mice and wild-type (WT) C57BL/6 littermates were infused with angiotensin II (Ang II) at 1,500 ng/kg/min for 7 days and then cross-transplanted with platelets originating from either WT or P-sel KO mice. P-selectin expression was increased in the myocardium and plasma of hypertensive mice, and the P-sel KO mice exhibited significantly (P<0.05) reduced cardiac fibrosis. The fibrotic areas were markedly smaller in the hearts of P-sel KO mice compared with WT mice, as assessed by Masson's trichrome staining. In addition, α-smooth muscle actin and transforming growth factor β1 (TGF-β1) expression levels were decreased in the P-sel KO mice, as assessed by immunohistochemistry. Following platelet transplantation into P-sel KO mice, the number of Mac-2 (galectin-3)- and TGF-β1-positive cells was increased in mice that received WT platelets compared with those that received P-sel KO platelets, and the mRNA expression levels of collagen I and TGF-β1 were also increased. The results from the present study suggest that activated platelets secrete P-selectin to promote cardiac inflammation and fibrosis in Ang II-induced hypertension. PMID:27121797

  9. Thrombin-induced conversion of fibrinogen to fibrin results in rapid platelet trapping which is not dependent on platelet activation or GPIb

    PubMed Central

    Jarvis, Gavin E; Atkinson, Ben T; Frampton, Jon; Watson, Steve P

    2003-01-01

    Activation of human platelets by thrombin is mediated by the proteolytic cleavage of two G-protein coupled protease-activated receptors, PAR-1 and PAR-4. However, thrombin also binds specifically to the platelet surface glycoprotein GPIb. It has been claimed that thrombin can induce aggregation of platelets via a novel GPIb-mediated pathway, which is independent of PAR activation and fibrinogen binding to αIIbβ3 integrin, but dependent upon polymerizing fibrin and the generation of intracellular signals. In the presence of both fibrinogen and the αIIbβ3 receptor antagonist lotrafiban, thrombin induced a biphasic platelet aggregation response. The initial primary response was small but consistent and associated with the release of platelet granules. The delayed secondary response was more substantial and was abolished by the fibrin polymerization blocking peptide GPRP. Cleavage of the extracellular portion of GPIb by mocarhagin partially inhibited thrombin-induced αIIbβ3-dependent aggregation and release, but had no effect on the secondary fibrin-dependent response. Fixing of the platelets abolished αIIbβ3-dependent aggregation and release of adenine nucleotides, whereas the fibrin-dependent response remained, indicating that platelet activation and intracellular signalling are not necessary for this secondary ‘aggregation'. In conclusion, the secondary fibrin-dependent ‘aggregation' response observed in the presence of fibrinogen and lotrafiban is a platelet trapping phenomenon dependent primarily on the conversion of soluble fibrinogen to polymerizing fibrin by thrombin. PMID:12598411

  10. Albumin inhibits platelet-activating factor (PAF)-induced responses in platelets and macrophages: implications for the biologically active form of PAF.

    PubMed Central

    Grigoriadis, G.; Stewart, A. G.

    1992-01-01

    1. Platelet-activating factor (PAF) binds with high affinity to albumin leading Clay et al. (1990) to suggest that the active form of PAF is the albumin-PAF complex. 2. In the present study the proposal that albumin-bound, rather than monomeric PAF, is the active form of PAF at PAF receptors was critically evaluated by examining the effect of albumin on the potency of PAF in isolated platelets and macrophages. 3. Bovine serum albumin inhibited concentration-dependently PAF-induced responses in platelets and macrophages. The most probable explanation of this finding is that BSA reduced the concentration of free PAF. 4. Thus, we conclude that free PAF, rather than the albumin-PAF complex is the active form. Consequently, local concentrations of albumin will influence profoundly the potency of endogenously released PAF. Moreover, estimates of the affinity of PAF for PAF receptors made in buffers containing BSA, underestimate the true affinity of PAF for its receptors by approximately 3 orders of magnitude. PMID:1330167

  11. Isoform-specific roles of the GTPase activating protein Nadrin in cytoskeletal reorganization of platelets.

    PubMed

    Beck, S; Fotinos, A; Lang, F; Gawaz, M; Elvers, M

    2013-01-01

    Cytoskeletal reorganization of activated platelets plays a crucial role in hemostasis and thrombosis and implies activation of Rho GTPases. Rho GTPases are important regulators of cytoskeletal dynamics and function as molecular switches that cycle between an inactive and an active state. They are regulated by GTPase activating proteins (GAPs) that stimulate GTP hydrolysis to terminate Rho signaling. The regulation of Rho GTPases in platelets is not explored. A detailed characterization of Rho regulation is necessary to understand activation and inactivation of Rho GTPases critical for platelet activation and aggregation. Nadrin is a RhoGAP regulating cytoplasmic protein explored in the central nervous system. Five Nadrin isoforms are known that share a unique GAP domain, a serine/threonine/proline-rich domain, a SH3-binding motif and an N-terminal BAR domain but differ in their C-terminus. Here we identified Nadrin in platelets where it co-localizes to actin-rich regions and Rho GTPases. Different Nadrin isoforms selectively regulate Rho GTPases (RhoA, Cdc42 and Rac1) and cytoskeletal reorganization suggesting that - beside the GAP domain - the C-terminus of Nadrin determines Rho specificity and influences cell physiology. Furthermore, Nadrin controls RhoA-mediated stress fibre and focal adhesion formation. Spreading experiments on fibrinogen revealed strongly reduced cell adhesion upon Nadrin overexpression. Unexpectedly, the Nadrin BAR domain controls Nadrin-GAP activity and acts as a guidance domain to direct this GAP to its substrate at the plasma membrane. Our results suggest a critical role for Nadrin in the regulation of RhoA, Cdc42 and Rac1 in platelets and thus for platelet adhesion and aggregation. PMID:22975681

  12. 11,12-Epoxyeicosatrienoic acid activates the L-arginine/nitric oxide pathway in human platelets.

    PubMed

    Zhang, Like; Cui, Yuying; Geng, Bing; Zeng, Xiangjun; Tang, Chaoshu

    2008-01-01

    The present study was to test the hypothesis that 11,12-epoxyeicosatrienoic acid (11,12-EET), a metabolic product of arachidonic acid by cytochrome P450 epoxygenase, regulates nitric oxide (NO) generation of the L-arginine/NO synthase (NOS) pathway in human platelets. Human platelets were incubated in the presence or absence of different concentrations of 11,12-EET for 2 h at 37 degrees C, followed by measurements of activities of the L-arginine/NOS pathway. Incubation with 11,12-EET increased the platelet NOS activity, nitrite production, cGMP content, and the platelet uptake of L-[(3)H]arginine in a concentration-dependent manner. In addition, 11,12-EET attenuated intracellular free Ca(2+) accumulation stimulated by collagen, which was at least partly mediated by EET-activated L-arginine/NOS pathway. It is suggested that 11,12-EET regulates platelet function through up-regulating the activity of the L-arginine/NOS/NO pathway. PMID:17932624

  13. Fluorine doping into diamond-like carbon coatings inhibits protein adsorption and platelet activation.

    PubMed

    Hasebe, Terumitsu; Yohena, Satoshi; Kamijo, Aki; Okazaki, Yuko; Hotta, Atsushi; Takahashi, Koki; Suzuki, Tetsuya

    2007-12-15

    The first major event when a medical device comes in contact with blood is the adsorption of plasma proteins. Protein adsorption on the material surface leads to the activation of the blood coagulation cascade and the inflammatory process, which impair the lifetime of the material. Various efforts have been made to minimize protein adsorption and platelet adhesion. Recently, diamond-like carbon (DLC) has received much attention because of their antithrombogenicity. We recently reported that coating silicon substrates with fluorine-doped diamond-like carbon (F-DLC) drastically suppresses platelet adhesion and activation. Here, we evaluated the protein adsorption on the material surfaces and clarified the relationship between protein adsorption and platelet behaviors, using polycarbonate and DLC- or F-DLC-coated polycarbonate. The adsorption of albumin and fibrinogen were assessed using a colorimetric protein assay, and platelet adhesion and activation were examined using a differential interference contrast microscope. A higher ratio of albumin to fibrinogen adsorption was observed on F-DLC than on DLC and polycarbonate films, indicating that the F-DLC film should prevent thrombus formation. Platelet adhesion and activation on the F-DLC films were more strongly suppressed as the amount of fluorine doping was increased. These results show that the F-DLC coating may be useful for blood-contacting devices. PMID:17600326

  14. Signaling-mediated cooperativity between glycoprotein Ib-IX and protease-activated receptors in thrombin-induced platelet activation.

    PubMed

    Estevez, Brian; Kim, Kyungho; Delaney, M Keegan; Stojanovic-Terpo, Aleksandra; Shen, Bo; Ruan, Changgeng; Cho, Jaehyung; Ruggeri, Zaverio M; Du, Xiaoping

    2016-02-01

    Thrombin-induced cellular response in platelets not only requires protease-activated receptors (PARs), but also involves another thrombin receptor, the glycoprotein Ib-IX complex (GPIb-IX). It remains controversial how thrombin binding to GPIb-IX stimulates platelet responses. It was proposed that GPIb-IX serves as a dock that facilitates thrombin cleavage of protease-activated receptors, but there are also reports suggesting that thrombin binding to GPIb-IX induces platelet activation independent of PARs. Here we show that GPIb is neither a passive thrombin dock nor a PAR-independent signaling receptor. We demonstrate a novel signaling-mediated cooperativity between PARs and GPIb-IX. Low-dose thrombin-induced PAR-dependent cell responses require the cooperativity of GPIb-IX signaling, and conversely, thrombin-induced GPIb-IX signaling requires cooperativity of PARs. This mutually dependent cooperativity requires a GPIb-IX-specific 14-3-3-Rac1-LIMK1 signaling pathway, and activation of this pathway also requires PAR signaling. The cooperativity between GPIb-IX signaling and PAR signaling thus drives platelet activation at low concentrations of thrombin, which are important for in vivo thrombosis. PMID:26585954

  15. Effects of Rivaroxaban on Platelet Activation and Platelet–Coagulation Pathway Interaction

    PubMed Central

    Heitmeier, Stefan; Laux, Volker

    2015-01-01

    Introduction: Activation of coagulation and platelets is closely linked, and arterial thrombosis involves coagulation activation as well as platelet activation and aggregation. In these studies, we investigated the possible synergistic effects of rivaroxaban in combination with antiplatelet agents on thrombin generation and platelet aggregation in vitro and on arterial thrombosis and hemostasis in rat models. Materials and Methods: Thrombin generation was measured by the Calibrated Automated Thrombogram method (0.5 pmol/L tissue factor) using human platelet-rich plasma (PRP) spiked with rivaroxaban (15, 30, or 60 ng/mL), ticagrelor (1.0 µg/mL), and acetylsalicylic acid (ASA; 100 µg/mL). Tissue factor-induced platelet aggregation was measured in PRP spiked with rivaroxaban (15 or 30 ng/mL), ticagrelor (1 or 3 µg/mL), or a combination of these. An arteriovenous (AV) shunt model in rats was used to determine the effects of rivaroxaban (0.01, 0.03, or 0.1 mg/kg), clopidogrel (1 mg/kg), ASA (3 mg/kg), and combinations on arterial thrombosis. Results: Rivaroxaban inhibited thrombin generation in a concentration-dependent manner and the effect was enhanced with ticagrelor and ticagrelor plus ASA. Rivaroxaban and ticagrelor also concentration-dependently inhibited tissue factor-induced platelet aggregation, and their combination increased the inhibition synergistically. In the AV shunt model, rivaroxaban dose-dependently reduced thrombus formation. Combining subefficacious or weakly efficacious doses of rivaroxaban with ASA or ASA plus clopidogrel increased the antithrombotic effect. Conclusion: These data indicate that the combination of rivaroxaban with single or dual antiplatelet agents works synergistically to reduce platelet activation, which may in turn lead to the delayed/reduced formation of coagulation complexes and vice versa, thereby enhancing antithrombotic potency. PMID:25848131

  16. DMSO inhibits human platelet activation through cyclooxygenase-1 inhibition. A novel agent for drug eluting stents?

    SciTech Connect

    Asmis, Lars; Tanner, Felix C.; Sudano, Isabella; Luescher, Thomas F.; Camici, Giovanni G.

    2010-01-22

    Background: DMSO is routinely infused together with hematopoietic cells in patients undergoing myeloablative therapy and was recently found to inhibit smooth muscle cells proliferation and arterial thrombus formation in the mouse by preventing tissue factor (TF), a key activator of the coagulation cascade. This study was designed to investigate whether DMSO prevents platelet activation and thus, whether it may represent an interesting agent to be used on drug eluting stents. Methods and results: Human venous blood from healthy volunteers was collected in citrated tubes and platelet activation was studied by cone and platelet analyzer (CPA) and rapid-platelet-function-assay (RPFA). CPA analysis showed that DMSO-treated platelets exhibit a lower adherence in response to shear stress (-15.54 {+-} 0.9427%, n = 5, P < 0.0001 versus control). Additionally, aggregometry studies revealed that DMSO-treated, arachidonate-stimulated platelets had an increased lag phase (18.0% {+-} 4.031, n = 9, P = 0.0004 versus control) as well as a decreased maximal aggregation (-6.388 {+-} 2.212%, n = 6, P = 0.0162 versus control). Inhibitory action of DMSO could be rescued by exogenous thromboxane A2 and was mediated, at least in part, by COX-1 inhibition. Conclusions: Clinically relevant concentrations of DMSO impair platelet activation by a thromboxane A2-dependent, COX-1-mediated effect. This finding may be crucial for the previously reported anti-thrombotic property displayed by DMSO. Our findings support a role for DMSO as a novel drug to prevent not only proliferation, but also thrombotic complications of drug eluting stents.

  17. 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

  18. A novel mathematical model of activation and sensitization of platelets subjected to dynamic stress histories

    PubMed Central

    Soares, João S.; Sheriff, Jawaad

    2013-01-01

    Blood recirculating devices, such as ventricular assist devices and prosthetic heart valves, are burdened by thromboembolic complications requiring complex and lifelong anticoagulant therapy with its inherent hemorrhagic risks. Pathologic flow patterns occurring in such devices chronically activate platelets, and the optimization of their thrombogenic performance requires the development of flow-induced platelet activation models. However, existing models are based on empirical correlations using the well-established power law paradigm of constant levels of shear stress during certain exposure times as factors for mechanical platelet activation. These models are limited by their range of application and do not account for other relevant phenomena, such as loading rate dependence and platelet sensitization to high stress conditions, which characterize the dynamic flow conditions in devices. These limitations were addressed by developing a new class of phenomenological stress-induced platelet activation models that specifies the rate of platelet activation as a function of the entire stress history and results in a differential equation that can be directly integrated to calculate the cumulative levels of activation. The proposed model reverts to the power law under constant shear stress conditions and is able to describe experimental results in response to a diverse range of highly dynamic stress conditions found in blood recirculating devices. The model was tested in vitro under emulated device flow conditions and correlates well with experimental results. This new model provides a reliable and robust mathematical tool that can be incorporated into computational fluid dynamic studies in order to optimize design, with the goal of improving the thrombogenic performance of blood recirculating devices. PMID:23359062

  19. Human Platelets Utilize Cycloxygenase-1 to Generate Dioxolane A3, a Neutrophil-activating Eicosanoid*

    PubMed Central

    Hinz, Christine; Aldrovandi, Maceler; Uhlson, Charis; Marnett, Lawrence J.; Longhurst, Hilary J.; Warner, Timothy D.; Alam, Saydul; Slatter, David A.; Lauder, Sarah N.; Allen-Redpath, Keith; Collins, Peter W.; Murphy, Robert C.; Thomas, Christopher P.; O'Donnell, Valerie B.

    2016-01-01

    Eicosanoids are important mediators of fever, pain, and inflammation that modulate cell signaling during acute and chronic disease. We show by using lipidomics that thrombin-activated human platelets generate a new type of eicosanoid that both stimulates and primes human neutrophil integrin (Mac-1) expression, in response to formylmethionylleucylphenylalanine. Detailed characterization proposes a dioxolane structure, 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (dioxolane A3, DXA3). The lipid is generated in nanogram amounts by platelets from endogenous arachidonate during physiological activation, with inhibition by aspirin in vitro or in vivo, implicating cyclooxygenase-1 (COX). Pharmacological and genetic studies on human/murine platelets revealed that DXA3 formation requires protease-activated receptors 1 and 4, cytosolic phospholipase A2 (cPLA2), Src tyrosine kinases, p38 MAPK, phospholipase C, and intracellular calcium. From data generated by purified COX isoforms and chemical oxidation, we propose that DXA3 is generated by release of an intermediate from the active site followed by oxygenation at C8. In summary, a new neutrophil-activating platelet-derived lipid generated by COX-1 is presented that can activate or prime human neutrophils, suggesting a role in innate immunity and acute inflammation. PMID:27129261

  20. Human Platelets Utilize Cycloxygenase-1 to Generate Dioxolane A3, a Neutrophil-activating Eicosanoid.

    PubMed

    Hinz, Christine; Aldrovandi, Maceler; Uhlson, Charis; Marnett, Lawrence J; Longhurst, Hilary J; Warner, Timothy D; Alam, Saydul; Slatter, David A; Lauder, Sarah N; Allen-Redpath, Keith; Collins, Peter W; Murphy, Robert C; Thomas, Christopher P; O'Donnell, Valerie B

    2016-06-24

    Eicosanoids are important mediators of fever, pain, and inflammation that modulate cell signaling during acute and chronic disease. We show by using lipidomics that thrombin-activated human platelets generate a new type of eicosanoid that both stimulates and primes human neutrophil integrin (Mac-1) expression, in response to formylmethionylleucylphenylalanine. Detailed characterization proposes a dioxolane structure, 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (dioxolane A3, DXA3). The lipid is generated in nanogram amounts by platelets from endogenous arachidonate during physiological activation, with inhibition by aspirin in vitro or in vivo, implicating cyclooxygenase-1 (COX). Pharmacological and genetic studies on human/murine platelets revealed that DXA3 formation requires protease-activated receptors 1 and 4, cytosolic phospholipase A2 (cPLA2), Src tyrosine kinases, p38 MAPK, phospholipase C, and intracellular calcium. From data generated by purified COX isoforms and chemical oxidation, we propose that DXA3 is generated by release of an intermediate from the active site followed by oxygenation at C8. In summary, a new neutrophil-activating platelet-derived lipid generated by COX-1 is presented that can activate or prime human neutrophils, suggesting a role in innate immunity and acute inflammation. PMID:27129261

  1. Antiplatelet aggregation and platelet activating factor (PAF) receptor antagonistic activities of the essential oils of five Goniothalamus species.

    PubMed

    Moharam, Bushra Abdulkarim; Jantan, Ibrahim; Ahmad, Fasihuddin bin; Jalil, Juriyati

    2010-08-01

    Nine essential oils, hydrodistilled from different parts of five Goniothalamus species (G. velutinus Airy-Shaw, G. woodii Merr., G. clemensii Ban, G. tapis Miq. and G. tapisoides Mat Salleh) were evaluated for their ability to inhibit platelet aggregation in human whole blood using an electrical impedance method and their inhibitory effects on platelet activating factor (PAF) receptor binding with rabbit platelets using 3H-PAF as a ligand. The chemical composition of the oils was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The bark oil of G. velutinus was the most effective sample as it inhibited both arachidonic acid (AA) and ADP-induced platelet aggregation with IC(50) values of 93.6 and 87.7 microg/mL, respectively. Among the studied oils, the bark oils of G. clemensii, G. woodii, G. velutinus and the root oil of G. tapis showed significant inhibitory effects on PAF receptor binding, with IC(50 )values ranging from 3.5 to 10.5 microg/mL. The strong PAF antagonistic activity of the active oils is related to their high contents of sesquiterpenes and sesquiterpenoids, and the individual components in the oils could possibly produce a synergistic effect in the overall antiplatelet activity of the oils. PMID:20714290

  2. Point of care platelet activity measurement in primary PCI [PINPOINT-PPCI]: a protocol paper

    PubMed Central

    2014-01-01

    Background Optimal treatment of acute ST-elevation myocardial infarction (STEMI) involves rapid diagnosis, and transfer to a cardiac centre capable of percutaneous coronary intervention (PCI) for immediate mechanical revascularisation. Successful treatment requires rapid return of perfusion to the myocardium achieved by thromboaspiration, passivation of the culprit lesion with stent scaffolding and systemic inhibition of thrombosis and platelet activation. A delicate balance exists between thrombosis and bleeding and consequently anti-thrombotic and antiplatelet treatment regimens continue to evolve. The desire to achieve reperfusion as soon as possible, in the setting of high platelet reactivity, requires potent and fast-acting anti-thrombotic/anti-platelet therapies. The associated bleeding risk may be minimised by use of short-acting anti-thrombotic intravenous agents. However, effective oral platelet inhibition is required to prevent recurrent thrombosis. The interaction between baseline platelet reactivity, timing of revascularisation and effective inhibition of thrombosis is yet to be formally investigated. Methods/Design We present a protocol for a prospective observational study in patients presenting with acute STEMI treated with primary PCI (PPCI) and receiving bolus/infusion bivalirudin and prasugrel therapy. The objective of this study is to describe variation in platelet reactivity, as measured by the multiplate platelet function analyser, at presentation, the end of the PPCI procedure and 1, 2, & 24 hours post-procedure. We intend to assess the prevalence of high residual platelet reactivity within 24 hours of PPCI in acute STEMI patients receiving prasugrel and bivalirudin. Additionally, we will investigate the association between high platelet reactivity before and after PPCI and the door-to-procedure completion time. This is a single centre study with a target sample size of 108 participants. Discussion The baseline platelet reactivity on

  3. Arachidonate metabolism, 5-hydroxytryptamine release and aggregation in human platelets activated by palmitaldehyde acetal phosphatidic acid.

    PubMed Central

    Brammer, J. P.; Maguire, M. H.

    1984-01-01

    Palmitaldehyde acetal phosphatidic acid ( PGAP ) caused dose-dependent aggregation of human platelets resuspended in modified Tyrode medium, with a threshold concentration of 0.5-1 microM and an EC50 of 4 microM. Concentrations of PGAP which elicited biphasic irreversible aggregation concomitantly induced formation of 1.02 +/- 0.029 nmol (mean +/- s.e. mean) of malondialdehyde (MDA) per 10(9) platelets and caused release of 58 +/- 2.8% of platelet [14C]-5-hydroxytryptamine ([14C]-5-HT) from prelabelled platelets; no MDA formation or [14C]-5-HT release occurred at lower doses of PGAP which elicited only monophasic reversible aggregation. Adenosine 5'-pyrophosphate (ADP)-induced platelet activation resulted in formation of 0.344 +/- 0.004 nmol of MDA per 10(9) platelets in association with irreversible aggregation and 49.1 +/- 1% release of [14C]-5-HT. Mepacrine, a phospholipase A2 inhibitor, at 2.5 microM reduced PGAP -induced MDA formation and [14C]-5-HT release by the resuspended platelets without affecting irreversible aggregation; higher concentrations of mepacrine abolished all three responses. Chlorpromazine, a calmodulin antagonist, similarly inhibited PGAP -induced MDA formation and irreversible aggregation, and at 100 microM abolished monophasic aggregation. The cyclo-oxygenase inhibitor indomethacin caused a concentration-dependent reduction of PGAP -induced MDA formation by resuspended human platelets without significantly inhibiting [14C]-5-HT release or irreversible aggregation; concentrations (greater than or equal to 1.75 microM) which inhibited MDA formation by more than 94% abolished [14C]-5-HT release, and converted second phase irreversible aggregation to an extensive reversible response. 2-Methylthioadenosine 5'-phosphate (2 methylthio-AMP), an ADP antagonist, inhibited PGAP -induced MDA formation, [14C]-5-HT release and second phase aggregation in the human platelet suspensions in a parallel, concentration-dependent manner; at 9.4 microM 2

  4. Diminished adhesion and activation of platelets and neutrophils with CD47 functionalized blood contacting surfaces.

    PubMed

    Finley, Matthew J; Rauova, Lubica; Alferiev, Ivan S; Weisel, John W; Levy, Robert J; Stachelek, Stanley J

    2012-08-01

    CD47 is a ubiquitously expressed transmembrane protein that, through signaling mechanisms mediated by signal regulatory protein alpha (SIRPα1), functions as a biological marker of 'self-recognition'. We showed previously that inflammatory cell attachment to polymeric surfaces is inhibited by the attachment of biotinylated recombinant CD47 (CD47B). We test herein the hypothesis that CD47 modified blood conduits can reduce platelet and neutrophil activation under clinically relevant conditions. We appended a poly-lysine tag to the C-terminus of recombinant CD47 (CD47L) allowing for covalent linkage to the polymer. SIRPα1 expression was confirmed in isolated platelets. We then compared biocompatibility between CD47B and CD47L functionalized polyvinyl chloride (PVC) surfaces and unmodified control PVC surfaces. Quantitative and Qualitative analysis of blood cell attachment to CD47B and CD47L surfaces, via scanning electron microscopy, showed strikingly fewer platelets attached to CD47 modified surfaces compared to control. Flow cytometry analysis showed that activation markers for neutrophils (CD62L) and platelets (CD62P) exposed to CD47 modified PVC were equivalent to freshly acquired control blood, while significantly elevated in the unmodified PVC tubing. In addition, ethylene oxide gas sterilization did not inhibit the efficacy of the CD47 modification. In conclusion, CD47 modified PVC inhibits both the adhesion and activation of platelets and neutrophils. PMID:22613135

  5. P‑selectin increases angiotensin II‑induced cardiac inflammation and fibrosis via platelet activation.

    PubMed

    Liu, Gaizhen; Liang, Bin; Song, Xiaosu; Bai, Rui; Qin, Weiwei; Sun, Xu; Lu, Yan; Bian, Yunfei; Xiao, Chuanshi

    2016-06-01

    Platelet activation is important in hypertension‑induced cardiac inflammation and fibrosis. P-selectin expression significantly (P<0.05) increases when platelets are activated during hypertension. Although P‑selectin recruits leukocytes to sites of inflammation, the role of P‑selectin in cardiac inflammation and fibrosis remains to be elucidated. The present study aimed to investigate whether platelet‑derived P‑selectin promotes hypertensive cardiac inflammation and fibrosis. P‑selectin knockout (P‑sel KO) mice and wild‑type (WT) C57BL/6 littermates were infused with angiotensin II (Ang II) at 1,500 ng/kg/min for 7 days and then cross‑transplanted with platelets originating from either WT or P‑sel KO mice. P‑selectin expression was increased in the myocardium and plasma of hypertensive mice, and the P‑sel KO mice exhibited significantly (P<0.05) reduced cardiac fibrosis. The fibrotic areas were markedly smaller in the hearts of P‑sel KO mice compared with WT mice, as assessed by Masson's trichrome staining. In addition, α‑smooth muscle actin and transforming growth factor β1 (TGF‑β1) expression levels were decreased in the P‑sel KO mice, as assessed by immunohistochemistry. Following platelet transplantation into P‑sel KO mice, the number of Mac‑2 (galectin‑3)‑ and TGF‑β1‑positive cells was increased in mice that received WT platelets compared with those that received P‑sel KO platelets, and the mRNA expression levels of collagen I and TGF‑β1 were also increased. The results from the present study suggest that activated platelets secrete P‑selectin to promote cardiac inflammation and fibrosis in Ang II‑induced hypertension. PMID:27121797

  6. Angiotensin peptides attenuate platelet-activating factor-induced inflammatory activity in rats.

    PubMed

    Sato, Akira; Yokoyama, Izumi; Ebina, Keiichi

    2015-11-01

    Angiotensin (Ang)--a peptide that is part of the renin-angiotensin system-induces vasoconstriction and a subsequent increase in blood pressure; Ang peptides, especially AngII, can also act as potent pro-inflammatory mediators. Platelet-activating factor (PAF) is a potent phospholipid mediator that is implicated in many inflammatory diseases. In this study, we investigated the effects of Ang peptides (AngII, AngIII, and AngIV) on PAF-induced inflammatory activity. In experiments using a rat hind-paw oedema model, AngII markedly and dose-dependently attenuated the paw oedema induced by PAF. The inhibitory effects of AngIII and AngIV on PAF-induced paw oedema were lower than that of AngII. Two Ang receptors, the AT1 and AT2 receptors, did not affect the AngII-mediated attenuation of PAF-induced paw oedema. Moreover, intrinsic tyrosine fluorescence studies demonstrated that AngII, AngIII, and AngIV interact with PAF, and that their affinities were closely correlated with their inhibitory effects on PAF-induced rat paw oedema. Also, AngII interacted with metabolite/precursor of PAF (lyso-PAF), and an oxidized phospholipid, 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC), which bears a marked structural resemblance to PAF. Furthermore, POVPC dose-dependently inhibited AngII-mediated attenuation of PAF-induced paw oedema. These results suggest that Ang peptides can attenuate PAF-induced inflammatory activity through binding to PAF and lyso-PAF in rats. Therefore, Ang peptides may be closely involved in the regulation of many inflammatory diseases caused by PAF. PMID:26348270

  7. Assessment of platelet activation in several different anticoagulants by the Advia 120 Hematology System, fluorescence flow cytometry, and electron microscopy.

    PubMed

    Ahnadi, Charaf E; Sabrinah Chapman, E; Lépine, Mariette; Okrongly, David; Pujol-Moix, Nuria; Hernández, Angel; Boughrassa, Faiza; Grant, Andrew M

    2003-11-01

    In vivo platelet activation results are often confounded by activation induced in vitro during the preparative procedures. We measured ex vivo (basal) and in vitro (thrombin-induced) platelet activation in sodium citrate, ethylenediaminetetraacetic acid (EDTA), and Citrate Theophylline Dipyridamole Adenosine (CTAD) whole blood specimens. Determinations were made by measurements of platelet density (mean platelet component: MPC concentration) on the Advia 120 Hematology System. The MPC has been previously shown to correlate with a fluorescence flow cytometric method, also determined in this study, using the surface expression of CD62P. Moreover, platelet shape and structure changes in EDTA and CTAD anticoagulated whole blood specimens were characterized by transmission electron microscopy (TEM). Observations made using the Advia 120 Hematology System platelet density parameter, MPC, in the absence of thrombin were 25.7 +/- 0.9 g/dl, 27.9 +/- 0.9 g/dl and 24.8 +/- 1.2 g/dl in sodium citrate, EDTA and CTAD whole blood specimens, respectively. Addition of thrombin induced a significant change in platelet MPC for sodium citrate (21.9 +/- 1.9 g/dl; p<0.0001) and EDTA (23.2 +/- 0.9 g/dl; p<0.0001) whole blood specimens. In contrast, thrombin had no effect on MPC measured in whole blood taken into CTAD tubes. In vitro fluorescence flow cytometric platelet activation experiments measuring the percentage of platelets expressing anti-CD62P showed increase in sodium citrate specimens from 9.2 +/- 7.0 to 55.5 +/- 23.1 % (p<0.0001) and in EDTA specimens from 1.9 +/- 1.7 to 64.6 +/- 12.4 % (p<0.0001) after addition of thrombin. However, in blood taken into CTAD tubes, there was no significant change. Studies on platelets isolated from whole blood in CTAD showed activation by thrombin indicating that platelets in CTAD, while protected in its presence remained functional upon its removal. When observed by TEM over time, platelets in EDTA appear more activated and contain fewer

  8. Mechanism of activation and functional role of protein kinase Ceta in human platelets.

    PubMed

    Bynagari, Yamini S; Nagy, Bela; Tuluc, Florin; Bhavaraju, Kamala; Kim, Soochong; Vijayan, K Vinod; Kunapuli, Satya P

    2009-05-15

    The novel class of protein kinase C (nPKC) isoform eta is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCeta using pharmacological and gene knock-out approaches. nPKCeta was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y1 receptor antagonist, or YM-254890, a G(q) blocker, abolished 2MeSADP-induced phosphorylation of nPKCeta. Similarly, ADP failed to activate nPKCeta in platelets isolated from P2Y1 and G(q) knock-out mice. However, pretreatment of platelets with P2Y12 receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCeta phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCeta was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin alpha(IIb)beta3 mediated outside-in signaling. Moreover, in the presence of SC-57101, a alpha(IIb)beta3 receptor antagonist, nPKCeta dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cgamma, a catalytic subunit of serine/threonine phosphatase, alpha(IIb)beta3 failed to dephosphorylate nPKCeta. Thus, we conclude that ADP activates nPKCeta via P2Y1 receptor and is subsequently dephosphorylated by PP1gamma phosphatase activated by alpha(IIb)beta3 integrin. In addition, pretreatment of platelets with eta-RACK antagonistic peptides, a specific inhibitor of nPKCeta, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCeta positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation. PMID:19286657

  9. A contrast agent recognizing activated platelets reveals murine cerebral malaria pathology undetectable by conventional MRI.

    PubMed

    von Zur Muhlen, Constantin; Sibson, Nicola R; Peter, Karlheinz; Campbell, Sandra J; Wilainam, Panop; Grau, Georges E; Bode, Christoph; Choudhury, Robin P; Anthony, Daniel C

    2008-03-01

    Human and murine cerebral malaria are associated with elevated levels of cytokines in the brain and adherence of platelets to the microvasculature. Here we demonstrated that the accumulation of platelets in the brain microvasculature can be detected with MRI, using what we believe to be a novel contrast agent, at a time when the pathology is undetectable by conventional MRI. Ligand-induced binding sites (LIBS) on activated platelet glycoprotein IIb/IIIa receptors were detected in the brains of malaria-infected mice 6 days after inoculation with Plasmodium berghei using microparticles of iron oxide (MPIOs) conjugated to a single-chain antibody specific for the LIBS (LIBS-MPIO). No binding of the LIBS-MPIO contrast agent was detected in uninfected animals. A combination of LIBS-MPIO MRI, confocal microscopy, and transmission electron microscopy revealed that the proinflammatory cytokine TNF-alpha, but not IL-1beta or lymphotoxin-alpha (LT-alpha), induced adherence of platelets to cerebrovascular endothelium. Peak platelet adhesion was found 12 h after TNF-alpha injection and was readily detected with LIBS-MPIO contrast-enhanced MRI. Temporal studies revealed that the level of MPIO-induced contrast was proportional to the number of platelets bound. Thus, the LIBS-MPIO contrast agent enabled noninvasive detection of otherwise undetectable cerebral pathology by in vivo MRI before the appearance of clinical disease, highlighting the potential of targeted contrast agents for diagnostic, mechanistic, and therapeutic studies. PMID:18274670

  10. A contrast agent recognizing activated platelets reveals murine cerebral malaria pathology undetectable by conventional MRI

    PubMed Central

    von zur Muhlen, Constantin; Sibson, Nicola R.; Peter, Karlheinz; Campbell, Sandra J.; Wilainam, Panop; Grau, Georges E.; Bode, Christoph; Choudhury, Robin P.; Anthony, Daniel C.

    2008-01-01

    Human and murine cerebral malaria are associated with elevated levels of cytokines in the brain and adherence of platelets to the microvasculature. Here we demonstrated that the accumulation of platelets in the brain microvasculature can be detected with MRI, using what we believe to be a novel contrast agent, at a time when the pathology is undetectable by conventional MRI. Ligand-induced binding sites (LIBS) on activated platelet glycoprotein IIb/IIIa receptors were detected in the brains of malaria-infected mice 6 days after inoculation with Plasmodium berghei using microparticles of iron oxide (MPIOs) conjugated to a single-chain antibody specific for the LIBS (LIBS-MPIO). No binding of the LIBS-MPIO contrast agent was detected in uninfected animals. A combination of LIBS-MPIO MRI, confocal microscopy, and transmission electron microscopy revealed that the proinflammatory cytokine TNF-α, but not IL-1β or lymphotoxin-α (LT-α), induced adherence of platelets to cerebrovascular endothelium. Peak platelet adhesion was found 12 h after TNF-α injection and was readily detected with LIBS-MPIO contrast-enhanced MRI. Temporal studies revealed that the level of MPIO-induced contrast was proportional to the number of platelets bound. Thus, the LIBS-MPIO contrast agent enabled noninvasive detection of otherwise undetectable cerebral pathology by in vivo MRI before the appearance of clinical disease, highlighting the potential of targeted contrast agents for diagnostic, mechanistic, and therapeutic studies. PMID:18274670

  11. Antagonistic activity of etizolam on platelet-activating factor in vivo experiments.

    PubMed

    Terasawa, M; Mikashima, H; Tahara, T; Maruyama, Y

    1987-08-01

    The ability of etizolam, 6-(o-chlorophenyl)-8-ethyl-1-methyl-4H-s-triazolo[3,4-c]thieno[2,3-e] [1,4]diazepine (Y-7131), an anti-anxiety drug, to inhibit platelet-activating factor (PAF)-induced reactions was investigated in experimental animals in vivo. Etizolam (0.01-0.3 mg/kg, i.v.) dose dependently inhibited PAF (0.3 microgram/kg, i.v.)-induced bronchoconstriction (Konzett and Rössler's method) in guinea pigs, but even at doses as large as 3 mg/kg, i.v., it had no effect on bronchoconstriction induced by histamine, serotonin, acetylcholine, arachidonic acid, bradykinin, angiotensin l or leukotriene D4. Etizolam (0.1-1 mg/kg, i.v.) also dose-dependently reversed PAF (1 microgram/kg, i.v.)-induced hypotension in anesthetized rats. Injection of PAF into the tail veins of mice produced lethal shock within 10-30 min. Etizolam (0.1-3 mg/kg, i.v. and 1-10 mg/kg, p.o.) protected against the lethal effect of PAF (75 micrograms/kg, i.v.) in a dose-dependent manner. These results indicate that etizolam specifically inhibits the action of PAF in vivo. PMID:3682404

  12. Coming safely to a stop: a review of platelet activity after cessation of antiplatelet drugs

    PubMed Central

    2015-01-01

    The platelet P2Y12 antagonists are widely used, usually in combination with aspirin, to prevent atherothrombotic events in patients with acute coronary syndromes during percutaneous coronary intervention and after placement of arterial stents. Inhibition by clopidogrel or prasugrel lasts for the lifetime of the affected platelets and platelet haemostatic function gradually recovers after stopping the drug, as new unaffected platelets are formed. The optimal durations for dual antiplatelet therapy are prescribed by clinical guidelines. Continuation beyond the recommended duration is associated with an increased mortality, mainly associated with major bleeding. Fear of a ‘rebound’ of prothrombotic platelet activity on stopping the drug has provoked much discussion and many studies. However, review of the available literature reveals no evidence for production of hyper-reactive platelets after cessation of clopidogrel in patients who are stable. Any increase in acute coronary and other vascular events after stopping seems most likely therefore to be due to premature discontinuation or disruption of treatment while thrombotic risk is still high. No difference in rebound was found with the newer P2Y12 inhibitors, although ticagrelor and prasugrel are more potent platelet inhibitors than clopidogrel. Recent randomized controlled trials confirm it is safe to stop the thienopyridine and continue with aspirin alone in most patients after the duration of treatment recommended by the guidelines. Decisions on when to stop therapy in individuals, however, remain challenging and there is a growing rationale for platelet testing to assist clinical judgement in certain situations such as patients stopping dual antiplatelet therapy before surgery or in individuals at highest bleeding or thrombotic risk. PMID:26301068

  13. Aggregation efficiency of activated normal or fixed platelets in a simple shear field: effect of shear and fibrinogen occupancy.

    PubMed Central

    Xia, Z; Frojmovic, M M

    1994-01-01

    Shear rate can affect protein adsorption and platelet aggregation by regulating both the collision frequency and the capture efficiency (alpha). These effects were evaluated in well defined shear field in a micro-couette for shear rate G = 10 - 1000 s-1. The rate of protein binding was independent of G, shown for adsorption of albumin to latex beads and PAC1 to activated platelets. The initial aggregation rate for ADP-activated platelets in citrated platelet-rich plasma followed second order kinetics at the initial platelet concentrations between 20,000 and 60,000/microliters. alpha values, which dropped nearly fivefold for a 10-fold increase in G, were approximately proportional to G-1, contrary to a minor drop predicted by the theory that includes protein cross-bridging. Varying ADP concentration did not change alpha of maximally activated platelet subpopulations, suggesting that aggregation between unactivated and activated platelets is negligible. Directly blocking the unoccupied but activated GPIIb-IIIa receptors without affecting pre-bound Fg on "RGD"-activated, fixed platelets (AFP) by GRGDSP or Ro 43-5054 eliminated aggregation, suggesting that cross-bridging of GPIIb-IIIa on adjacent platelets by fibrinogen mediates aggregation. Alpha for AFP remained maximal (approximately 0.24) over 25-75% Fg occupancy, otherwise decreasing rapidly, with a half-maximum occurring at around 2% occupancy, suggesting that very few bound Fg were required to cause significant aggregation. Images FIGURE 1 PMID:8075353

  14. Hemocompatibility of Polyvinyl Alcohol-Gelatin Core-Shell Electrospun Nanofibers: A Novel Scaffold for Modulating Platelet Deposition and Activation

    PubMed Central

    Merkle, Valerie M.; Martin, Daniel; Hutchinson, Marcus; Tran, Phat L.; Behrens, Alana; Hossainy, Samir; Bluestein, Danny; Wu, Xiaoyi; Slepian, Marvin J.

    2015-01-01

    In this study, we evaluate coaxial electrospun nanofibers with gelatin in the shell and polyvinyl (PVA) in the core as a potential vascular material by determining fiber surface roughness, as well as human platelet deposition and activation under varying conditions. PVA scaffolds had the highest surface roughness (Ra = 65.5 ± 6.8 nm) but the lowest platelet deposition (34.2 ± 5.8 platelets) in comparison to gelatin nanofibers (Ra = 36.8 ± 3.0 nm & 168.9 ± 29.8 platelets) and coaxial nanofibers (1 Gel: 1 PVA coaxial – Ra = 24.0 ± 1.5 nm & 150.2 ± 17.4 platelets; 3 Gel: 1 PVA coaxial – Ra = 37.1 ± 2.8 nm & 167.8 ± 15.4 platelets). Therefore, the chemical structure of the gelatin nanofibers dominated surface roughness in platelet deposition. Due to their increased stiffness, the coaxial nanofibers had the highest platelet activation rate – rate of thrombin formation, in comparison to gelatin and PVA fibers. Our studies indicate that mechanical stiffness is a dominating factor for platelet deposition and activation, followed by biochemical moieties, and lastly surface roughness. Overall, these coaxial nanofibers are an appealing material for vascular applications by supporting cellular growth while minimizing platelet deposition and activation. PMID:25815434

  15. mTORC1 promotes aging-related venous thrombosis in mice via elevation of platelet volume and activation.

    PubMed

    Yang, Jun; Zhou, Xuan; Fan, Xiaorong; Xiao, Min; Yang, Dinghua; Liang, Bo; Dai, Meng; Shan, Lanlan; Lu, Jingbo; Lin, Zhiqi; Liu, Rong; Liu, Jun; Wang, Liping; Zhong, Mei; Jiang, Yu; Bai, Xiaochun

    2016-08-01

    Aging is associated with an increased incidence of venous thromboembolism (VTE), resulting in significant morbidity and mortality in the elderly. Platelet hyperactivation is linked to aging-related VTE. However, the mechanisms through which aging enhances platelet activation and susceptibility to VTE are poorly understood. In this study, we demonstrated that mechanistic target of rapamycin complex 1 (mTORC1) signaling is essential for aging-related platelet hyperactivation and VTE. mTORC1 was hyperactivated in platelets and megakaryocytes (MKs) from aged mice, accompanied by elevated mean platelet volume (MPV) and platelet activation. Inhibition of mTORC1 with rapamycin led to a significant reduction in susceptibility to experimental deep vein thrombosis (DVT) in aged mice (P < .01). To ascertain the specific role of platelet mTORC1 activation in DVT, we generated mice with conditional ablation of the mTORC1-specific component gene Raptor in MKs and platelets (Raptor knockout). These mice developed markedly smaller and lighter thrombi, compared with wild-type littermates (P < .01) in experimental DVT. Mechanistically, increased reactive oxygen species (ROS) production with aging induced activation of mTORC1 in MKs and platelets, which, in turn, enhanced bone marrow MK size, MPV, and platelet activation to promote aging-related VTE. ROS scavenger administration induced a significant decrease (P < .05) in MK size, MPV, and platelet activation in aged mice. Our findings collectively demonstrate that mTORC1 contributes to enhanced venous thrombotic susceptibility in aged mice via elevation of platelet size and activation. PMID:27288518

  16. Thromboxane-insensitive dog platelets have impaired activation of phospholipase C due to receptor-linked G protein dysfunction.

    PubMed Central

    Johnson, G J; Leis, L A; Dunlop, P C

    1993-01-01

    Human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors are linked to phosphoinositide-specific phospholipase C (PI-PLC) via a G protein tentatively identified as a member of the Gq class. In contrast, platelet thrombin receptors appear to activate PI-PLC via other unidentified G proteins. Platelets from most dogs are TXA2 insensitive (TXA2-); i.e., they do not aggregate irreversibly or secrete although they bind TXA2, but they respond normally to thrombin. In contrast, a minority of dogs have TXA2-sensitive (TXA2+) platelets that are responsive to TXA2. To determine the mechanism responsible for TXA2- platelets, we evaluated receptor activation of PI-PLC. Equilibrium binding of TXA2/PGH2 receptor agonists, [125I]BOP and [3H]U46619, and antagonist, [3H]SQ29,548, revealed comparable high-affinity binding to TXA2-, TXA2+, and human platelets. U46619-induced PI-PLC activation was impaired in TXA2- platelets as evidenced by reduced (a) phosphorylation of the 47-kD substrate of protein kinase C, (b) phosphatidic acid (PA) formation, (c) rise in cytosolic calcium concentration, and (d) inositol 1,4,5 trisphosphate (IP3) formation, while thrombin-induced PI-PLC activation was not impaired. GTPase activity stimulated by U46619, but not by thrombin, was markedly reduced in TXA2- platelets. Antisera to Gq class alpha subunits abolished U46619-induced GTPase activity in TXA2-, TXA2+, and human platelets. Direct G protein stimulation by GTP gamma S yielded significantly less PA and IP3 in TXA2- platelets. Immunotransfer blotting revealed comparable quantities of Gq class alpha-subunits in all three platelet types. Thus, TXA2- dog platelets have impaired PI-PLC activation in response to TXA2/PGH2 receptor agonists secondary to G protein dysfunction, presumably involving a member of the Gq class. Images PMID:8227362

  17. Hepatic sinusoidal endothelium avidly binds platelets in an integrin-dependent manner, leading to platelet and endothelial activation and leukocyte recruitment.

    PubMed

    Lalor, Patricia F; Herbert, John; Bicknell, Roy; Adams, David H

    2013-03-01

    Platelets have recently been shown to drive liver injury in murine models of viral hepatitis and promote liver regeneration through the release of serotonin. Despite their emerging role in inflammatory liver disease, little is known about the mechanisms by which platelets bind to the hepatic vasculature. Therefore, we referenced public expression data to determine the profile of potential adhesive receptors expressed by hepatic endothelium. We then used a combination of tissue-binding and flow-based endothelial-binding adhesion assays to show that resting platelets bind to human hepatic sinusoidal endothelial cells and that the magnitude of adhesion is greatly enhanced by thrombin-induced platelet activation. Adhesion was mediated by the integrins Gp1b, αIIbβIII, and αvβ3, as well as immobilized fibrinogen. Platelet binding to hepatic endothelial cells resulted in NF-κB activation and increased chemokine secretion. The functional relevance of platelet binding was confirmed by experiments that showed markedly increased binding of neutrophils and lymphocytes to hepatic endothelial cells under shear conditions replicating those found in the hepatic sinusoid, which was in part dependent on P-selectin expression. Thus the ability of platelets to activate endothelium and promote leukocyte adhesion may reflect an additional mechanism through which they promote liver injury. PMID:23257923

  18. Cbl-b Is a Novel Physiologic Regulator of Glycoprotein VI-dependent Platelet Activation*

    PubMed Central

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

    2010-01-01

    Cbl-b, a member of the Cbl family of E3 ubiquitin ligases, plays an important role in the activation of lymphocytes. However, its function in platelets remains unknown. We show that Cbl-b is expressed in human platelets along with c-Cbl, but in contrast to c-Cbl, it is not tyrosine-phosphorylated upon glycoprotein VI (GPVI) stimulation. Cbl-b, unlike c-Cbl, is not required for Syk ubiquitylation downstream of GPVI activation. Phospholipase Cγ2 (PLCγ2) and Bruton's tyrosine kinase (BTK) are constituently associated with Cbl-b. Cbl-b-deficient (Cbl-b−/−) platelets display an inhibition in the concentration-response curve for GPVI-specific agonist-induced aggregation, secretion, and Ca2+ mobilization. A parallel inhibition is found for activation of PLCγ2 and BTK. However, Syk activation is not affected by the absence of Cbl-b, indicating that Cbl-b acts downstream of Syk but upstream of BTK and PLCγ2. When Cbl-b−/− mice were tested in the ferric chloride thrombosis model, occlusion time was increased and clot stability was reduced compared with wild type controls. These data indicate that Cbl-b plays a positive modulatory role in GPVI-dependent platelet signaling, which translates to an important regulatory role in hemostasis and thrombosis in vivo. PMID:20400514

  19. Three-dimentional simulation of flow-induced platelet activation in artificial heart valves

    NASA Astrophysics Data System (ADS)

    Hedayat, Mohammadali; Asgharzadeh, Hafez; Borazjani, Iman

    2015-11-01

    Since the advent of heart valve, several valve types such as mechanical and bio-prosthetic valves have been designed. Mechanical Heart Valves (MHV) are durable but suffer from thromboembolic complications that caused by shear-induced platelet activation near the valve region. Bio-prosthetic Heart Valves (BHV) are known for better hemodynamics. However, they usually have a short average life time. Realistic simulations of heart valves in combination with platelet activation models can lead to a better understanding of the potential risk of thrombus formation in such devices. In this study, an Eulerian approach is developed to calculate the platelet activation in three-dimensional simulations of flow through MHV and BHV using a parallel overset-curvilinear immersed boundary technique. A curvilinear body-fitted grid is used for the flow simulation through the anatomic aorta, while the sharp-interface immersed boundary method is used for simulation of the Left Ventricle (LV) with prescribed motion. In addition, dynamics of valves were calculated numerically using under-relaxed strong-coupling algorithm. Finally, the platelet activation results for BMV and MHV are compared with each other.

  20. EFFECT OF PHOTOPERIOD ON PLATELET-ACTIVATING FACTOR CONCENTRATION IN BOAR SPERMATOZOA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Platelet-activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is an important phospholipid mediator shown to be involved in fertilization. We recently reported that boars with a 70% or higher fertility history have a higher concentration of PAF in their spermatozoa. In addition,...

  1. Associations between arterial stiffness and platelet activation in normotensive overweight and obese young adults.

    PubMed

    Cooper, Jennifer N; Evans, Rhobert W; Mori Brooks, Maria; Fried, Linda; Holmes, Chris; Barinas-Mitchell, Emma; Sutton-Tyrrell, Kim

    2014-01-01

    Obese individuals have elevated platelet activation and arterial stiffness, but the strength and temporality of the relationship between these factors remain unclear. We aimed to determine the effect of increased arterial stiffness on circulating platelet activity in overweight/obese young adults. This analysis included 92 participants (mean age 40 years, 60 women) in the Slow Adverse Vascular Effects of excess weight (SAVE) trial, a clinical trial examining the effects of a lifestyle intervention with or without sodium restriction on vascular health in normotensive overweight/obese young adults. Carotid-femoral (cf), brachial-ankle (ba) and femoral-ankle (fa) pulse wave velocity (PWV) served as measures of arterial stiffness and were measured at baseline and 6, 12 and 24 months follow-up. Platelet activity was measured as plasma β-thromboglobulin (β-TG) at 24 months. Higher plasma β-TG was correlated with greater exposure to elevated cfPWV (p = 0.02) and baPWV (p = 0.04) during the preceding two years. After adjustment for serum leptin, greater exposure to elevated baPWV remained significant (p = 0.03) and exposure to elevated cfPWV marginally significant (p = 0.054) in predicting greater plasma β-TG. Greater arterial stiffness, particularly central arterial stiffness, predicts greater platelet activation in overweight/obese individuals. This relationship might partly explain the association between increased arterial stiffness and incident atherothrombotic events. PMID:23654212

  2. Unconjugated Bilirubin exerts Pro-Apoptotic Effect on Platelets via p38-MAPK activation

    PubMed Central

    NaveenKumar, Somanathapura K.; Thushara, Ram M.; Sundaram, Mahalingam S.; Hemshekhar, Mahadevappa; Paul, Manoj; Thirunavukkarasu, Chinnasamy; Basappa; Nagaraju, Ganesh; Raghavan, Sathees C.; Girish, Kesturu S.; Kemparaju, Kempaiah; Rangappa, Kanchugarakoppal S.

    2015-01-01

    Thrombocytopenia is one of the most frequently observed secondary complications in many pathological conditions including liver diseases, where hyperbilirubinemia is very common. The present study sought to find the cause of thrombocytopenia in unconjugated hyperbilirubinemic conditions. Unconjugated bilirubin (UCB), an end-product of heme catabolism, is known to have pro-oxidative and cytotoxic effects at high serum concentration. We investigated the molecular mechanism underlying the pro-apoptotic effect of UCB on human platelets in vitro, and followed it up with studies in phenylhydrazine-induced hyperbilirubinemic rat model and hyperbilirubinemic human subjects. UCB is indeed found to significantly induce platelet apoptotic events including elevated endogenous reactive oxygen species generation, mitochondrial membrane depolarization, increased intracellular calcium levels, cardiolipin peroxidation and phosphatidylserine externalization (p < 0.001) as evident by FACS analysis. The immunoblots show the elevated levels of cytosolic cytochrome c and caspase activation in UCB-treated platelets. Further, UCB is found to induce mitochondrial ROS generation leading to p38 activation, followed by downstream activation of p53, ultimately resulting in altered expression of Bcl-2 and Bax proteins as evident from immunoblotting. All these parameters conclude that elevated unconjugated bilirubin causes thrombocytopenia by stimulating platelet apoptosis via mitochondrial ROS-induced p38 and p53 activation. PMID:26459859

  3. Molecular Imaging of Activated Platelets Allows the Detection of Pulmonary Embolism with Magnetic Resonance Imaging

    PubMed Central

    Heidt, Timo; Ehrismann, Simon; Hövener, Jan-Bernd; Neudorfer, Irene; Hilgendorf, Ingo; Reisert, Marco; Hagemeyer, Christoph E.; Zirlik, Andreas; Reinöhl, Jochen; Bode, Christoph; Peter, Karlheinz; von Elverfeldt, Dominik; von zur Muhlen, Constantin

    2016-01-01

    Early and reliable detection of pulmonary embolism (PE) is critical for improving patient morbidity and mortality. The desire for low-threshold screening for pulmonary embolism is contradicted by unfavorable radiation of currently used computed tomography or nuclear techniques, while standard magnetic resonance imaging still struggles to provide sufficient diagnostic sensitivity in the lung. In this study we evaluate a molecular-targeted contrast agent against activated platelets for non-invasive detection of murine pulmonary thromboembolism using magnetic resonance imaging. By intravenous injection of human thrombin, pulmonary thromboembolism were consistently induced as confirmed by immunohistochemistry of the lung. Magnetic resonance imaging after thrombin injection showed local tissue edema in weighted images which co-localized with the histological presence of pulmonary thromboembolism. Furthermore, injection of a functionalized contrast agent targeting activated platelets provided sensitive evidence of focal accumulation of activated platelets within the edematous area, which, ex vivo, correlated well with the size of the pulmonary embolism. In summary, we here show delivery and specific binding of a functionalized molecular contrast agent against activated platelets for targeting pulmonary thromboembolism. Going forward, molecular imaging may provide new opportunities to increase sensitivity of magnetic resonance imaging for detection of pulmonary embolism. PMID:27138487

  4. Molecular Imaging of Activated Platelets Allows the Detection of Pulmonary Embolism with Magnetic Resonance Imaging.

    PubMed

    Heidt, Timo; Ehrismann, Simon; Hövener, Jan-Bernd; Neudorfer, Irene; Hilgendorf, Ingo; Reisert, Marco; Hagemeyer, Christoph E; Zirlik, Andreas; Reinöhl, Jochen; Bode, Christoph; Peter, Karlheinz; von Elverfeldt, Dominik; von Zur Muhlen, Constantin

    2016-01-01

    Early and reliable detection of pulmonary embolism (PE) is critical for improving patient morbidity and mortality. The desire for low-threshold screening for pulmonary embolism is contradicted by unfavorable radiation of currently used computed tomography or nuclear techniques, while standard magnetic resonance imaging still struggles to provide sufficient diagnostic sensitivity in the lung. In this study we evaluate a molecular-targeted contrast agent against activated platelets for non-invasive detection of murine pulmonary thromboembolism using magnetic resonance imaging. By intravenous injection of human thrombin, pulmonary thromboembolism were consistently induced as confirmed by immunohistochemistry of the lung. Magnetic resonance imaging after thrombin injection showed local tissue edema in weighted images which co-localized with the histological presence of pulmonary thromboembolism. Furthermore, injection of a functionalized contrast agent targeting activated platelets provided sensitive evidence of focal accumulation of activated platelets within the edematous area, which, ex vivo, correlated well with the size of the pulmonary embolism. In summary, we here show delivery and specific binding of a functionalized molecular contrast agent against activated platelets for targeting pulmonary thromboembolism. Going forward, molecular imaging may provide new opportunities to increase sensitivity of magnetic resonance imaging for detection of pulmonary embolism. PMID:27138487

  5. Adhesion and activation of platelets from subjects with coronary artery disease and apparently healthy individuals on biomaterials.

    PubMed

    Braune, S; Groß, M; Walter, M; Zhou, S; Dietze, S; Rutschow, S; Lendlein, A; Tschöpe, C; Jung, F

    2016-01-01

    On the basis of the clinical studies in patients with coronary artery disease (CAD) presenting an increased percentage of activated platelets, we hypothesized that hemocompatibility testing utilizing platelets from healthy individuals may result in an underestimation of the materials' thrombogenicity. Therefore, we investigated the interaction of polymer-based biomaterials with platelets from CAD patients in comparison to platelets from apparently healthy individuals. In vitro static thrombogenicity tests revealed that adherent platelet densities and total platelet covered areas were significantly increased for the low (polydimethylsiloxane, PDMS) and medium (Collagen) thrombogenic surfaces in the CAD group compared to the healthy subjects group. The area per single platelet-indicating the spreading and activation of the platelets-was markedly increased on PDMS treated with PRP from CAD subjects. This could not be observed for collagen or polytetrafluoroethylene (PTFE). For the latter material, platelet adhesion and surface coverage did not differ between the two groups. Irrespective of the substrate, the variability of these parameters was increased for CAD patients compared to healthy subjects. This indicates a higher reactivity of platelets from CAD patients compared to the healthy individuals. Our results revealed, for the first time, that utilizing platelets from apparently healthy donors bears the risk of underestimating the thrombogenicity of polymer-based biomaterials. PMID:25631281

  6. Coagulation-driven platelet activation reduces cholestatic liver injury and fibrosis in mice

    PubMed Central

    Joshi, N.; Kopec, A. K.; O’Brien, K. M.; Towery, K. L.; Cline-Fedewa, H.; Williams, K.J.; Copple, B. L.; Flick, M. J.; Luyendyk, J. P.

    2014-01-01

    Summary Background The coagulation cascade has been shown to participate in chronic liver injury and fibrosis, but the contribution of various thrombin targets, such as protease activated receptors (PARs) and fibrin(ogen), has not been fully described. Emerging evidence suggests that in some experimental settings of chronic liver injury, platelets can promote liver repair and inhibit liver fibrosis. However, the precise mechanisms linking coagulation and platelet function to hepatic tissue changes following injury remain poorly defined. Objectives To determine the role of PAR-4, a key thrombin receptor on mouse platelets, and fibrin(ogen) engagement of the platelet αIIbβ3 integrin in a model of cholestatic liver injury and fibrosis. Methods Biliary and hepatic injury was characterized following 4 week administration of the bile duct toxicant α-naphthylisothiocyanate (ANIT) (0.025%) in PAR-4-deficient mice (PAR-4−/− mice), mice expressing a mutant form of fibrin(ogen) incapable of binding integrin αIIbβ3 (FibγΔ5), and wild-type mice. Results Elevated plasma thrombin-antithrombin and serotonin levels, hepatic fibrin deposition and platelet accumulation in liver accompanied hepatocellular injury and fibrosis in ANIT-treated wild-type mice. PAR-4 deficiency reduced plasma serotonin levels, increased serum bile acid concentration, and exacerbated ANIT-induced hepatocellular injury and peribiliary fibrosis. Compared to PAR-4-deficient mice, ANIT-treated FibγΔ5 mice displayed more widespread hepatocellular necrosis accompanied by marked inflammation, robust fibroblast activation and extensive liver fibrosis. Conclusions Collectively, the results indicate that PAR-4 and fibrin-αIIbβ3 integrin engagement, pathways coupling coagulation to platelet activation, each exert hepatoprotective effects during chronic cholestasis. PMID:25353084

  7. Cytokine-release kinetics of platelet-rich plasma according to various activation protocols

    PubMed Central

    Roh, Y. H.; Kim, W.; Park, K. U.

    2016-01-01

    Objectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocols. Methods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes; double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects. Both preparations were tested for platelet activation by one of three activation protocols: no activation, activation with calcium (Ca) only, or calcium with a low dose (50 IU per 1 ml PRP) of thrombin. Each preparation was divided into four aliquots and incubated for one hour, 24 hours, 72 hours, and seven days. The cytokine-release kinetics were evaluated by assessing PDGF, TGF, VEGF, FGF, IL-1, and MMP-9 concentrations with bead-based sandwich immunoassay. Results The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols. Ca-only activation had a significant effect on the DS PRPs (where the VEGF, FGF, and IL-1 concentrations were sustained) while Ca/thrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short). The IL-1 content showed a significant increase with Ca-only or Ca/thrombin activation while these activations did not increase the MMP-9 concentration. Conclusion The SS and DS methods differed in their effect on cytokine release, and this effect varied among the cytokines analysed. In addition, low dose of thrombin/calcium activation increased the overall cytokine release of the PRP preparations over seven days, relative to that with a calcium-only supplement or non-activation. Cite this article: Professor J. H. Oh. Cytokine-release kinetics of platelet-rich plasma according to various activation protocols. Bone Joint Res 2016;5:37–45. DOI: 10.1302/2046-3758.52.2000540 PMID:26862077

  8. Degranulation of rabbit platelets with PAF-acether: a new procedure for unravelling the mode of action of platelet-activating substances.

    PubMed

    Vargaftig, B B; Joseph, D; Marlas, G; Chevance, L G

    1982-08-24

    Aggregation and secretion of ATP induced by thrombin, collagen, the snake venom component convulxin and platelet-activating factor (PAF-acether) were studied after the exposure of rabbit platelets to 1 microM of PAF-acether. This concentration, which is around 6 orders of magnitude above the concentration needed to induce full aggregation, was required to remove most of the releasable ATP from the platelets. The depleted platelets aggregated to PAF-acether, to thrombin and to convulxin under conditions where only very low amounts of ATP were secreted, confirming that these agents do not require the release of dense body components to trigger aggregation. Furthermore, when exposure to PAF-acether was associated to inactivation of platelet cyclooxygenase with aspirin, aggregation to thrombin persisted, validating the claim that thrombin induces aggregation by a third pathway unrelated to ADP and to thromboxane A2. Aggregation by collagen was markedly reduced by exposure of the platelets to PAF-acether or to aspirin; when both procedures were associated, aggregation was suppressed. Failure to desensitize the rabbit platelets to PAF-acether upon exposure to high amounts of it indicates the absence of irreversible membrane changes due to PAF-acether, and allows its use as a depleting procedure for the dense body materials, which does not affect platelet membrane components as is the case for thrombin. PMID:7135345

  9. 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

  10. New urushiols with platelet aggregation inhibitory activities from resin of Toxicodendron vernicifluum.

    PubMed

    Xie, Ya; Zhang, Jie; Liu, Wenyuan; Xie, Ning; Feng, Feng; Qu, Wei

    2016-07-01

    Eight new urushiol-type compounds (1-7b), along with seven known compounds were isolated from the resin of Toxicodendron vernicifluum Stokes. Their structures were determined by extensive spectroscopic methods, included (1)H NMR, (13)C NMR, HMQC, HMBC, HRESIMS, EI-MS in combination with CD methods. All the compounds except 7a and 7b were evaluated for their anti-platelet aggregation activities in vitro. Among them, compound 5 (IC50=5.12±0.85μmol/L), with a vic-diol moiety in the long alkyl chain showed the most potent inhibitory of platelet aggregation activity induced by ADP. In addition, compound 6 showed the effect of anti-platelet aggregation induced by AA with the IC50 value of 3.09±0.70μmol/L. Thus, these compounds might be the active components to the traditional use of Resina Toxicodendri for breaking up blood stasis, which could be related to the anti-platelet aggregation. PMID:27156871

  11. Platelet Activation in Ovines Undergoing Sham Surgery or Implant of the Second Generation PediaFlow™ Pediatric Ventricular Assist Device

    PubMed Central

    Johnson, Carl A.; Wearden, Peter D.; Kocyildirim, Ergin; Maul, Timothy M.; Woolley, Joshua R.; Ye, Sang-Ho; Strickler, Elise M.; Borovetz, Harvey S.; Wagner, William R.

    2011-01-01

    The PediaFlow™ pediatric ventricular assist device (VAD) is a magnetically levitated turbodynamic pump under development for circulatory support of small children with a targeted flow rate range of 0.3 - 1.5 L/min. As the design of this device is refined, ensuring high levels of blood biocompatibility is essential. In this study we characterized platelet activation during the implantation and operation of a second generation prototype of the PediaFlow VAD (PF2) and also performed a series of surgical sham studies to examine purely surgical effects on platelet activation. In addition, a newly available monoclonal antibody was characterized and shown to be capable of quantifying ovine platelet activation. The PF2 was implanted in 3 chronic ovine experiments of 16, 30, and 70 days, while surgical sham procedures were performed in 5 ovines with 30 d monitoring. Blood biocompatibility in terms of circulating activated platelets was measured by flow cytometric assays with and without exogenous agonist stimulation. Platelet activation following sham surgery returned to baseline in approximately 2 weeks. Platelets in PF2 implanted ovines returned to baseline activation levels in all three animals, and showed an ability to respond to agonist stimulation. Late term platelet activation was observed in one animal corresponding with unexpected pump stoppages related to a manufacturing defect in the percutaneous cable. The results demonstrated encouraging platelet biocompatibility for the PF2 in that basal platelet activation was achieved early in the pump implant period. Furthermore, this first characterization of the effect of a major cardiothoracic procedure on temporal ovine platelet activation provides comparative data for future cardiovascular device evaluation in the ovine model. PMID:21463346

  12. Platelet phospholipase A(2) activity in Alzheimer's disease and mild cognitive impairment.

    PubMed

    Gattaz, W F; Forlenza, O V; Talib, L L; Barbosa, N R; Bottino, C M C

    2004-05-01

    Phospholipase A(2) (PLA(2)) controls the metabolism of phospholipids in cell membranes. In the brain, PLA(2) influences the processing of the amyloid precursor protein (APP) and thus the production of the amyloid-beta peptides (Abeta), which are the major components of the senile plaques in Alzheimer's disease (AD). Reduced PLA(2) activity has been reported in brain and in platelets of AD patients. In the present study we investigated PLA(2) activity in platelets from 21 AD patients as compared to 17 healthy elderly controls and 11 individuals with mild cognitive impairment (MCI). Subjects were cognitively assessed by the Mini-Mental State Examination (MMSE) and the CAMDEX schedule. Platelet PLA(2) activity was determined by radio-enzymatic assay, which mainly detected a calcium-independent form of the enzyme present also in the brain (iPLA(2)). PLA(2) activity was significantly lower in AD than in controls (p < 0.001). Mean PLA(2) activity in MCI individuals was between the values of AD patients and controls, with a subgroup showing PLA as low as the lowest AD patients, but the differences from MCI were not significant from AD and control groups. Lower PLA(2) activity was significantly correlated with a worse cognitive performance both at the MMSE (p = 0.001) and the cognitive sub-scale of the CAMDEX inventory (p = 0.002). Our data replicate previous findings of reduced platelet PLA(2) activity in AD. Both reduced PLA(2) activity and the correlation with impaired cognition were also reported in brain tissue of AD patients, suggesting thus that the present determinations in platelets may be related to a reduction in the brain. In the brain the inhibition of PLA(2) inhibits the physiological secretion of the APP, a mechanism that increases Abeta formation. Further longitudinal studies should investigate whether those MCI individuals with the lowest PLA(2) values in platelets would be at a higher risk to develop AD during a longitudinal follow up. PMID:15088152

  13. Inhibitory effects of kiwifruit extract on human platelet aggregation and plasma angiotensin-converting enzyme activity.

    PubMed

    Dizdarevic, Lili L; Biswas, Dipankar; Uddin, M D Main; Jørgenesen, Aud; Falch, Eva; Bastani, Nasser E; Duttaroy, Asim K

    2014-01-01

    Previous human studies suggest that supplementation with kiwifruits lowers several cardiovascular risk factors such as platelet hyperactivity, blood pressure and plasma lipids. The cardiovascular health benefit of fruit and vegetables is usually attributed to the complex mixture of phytochemicals therein; however, kiwifruit's cardioprotective factors are not well studied. In this study, we investigated the effects of kiwifruit extract on human blood platelet aggregation and plasma angiotensin-converting enzyme (ACE) activity. A sugar-free, heat-stable aqueous extract with molecular mass less than 1000 Da was prepared from kiwifruits. Typically, 100 g kiwifruits produced 66.3 ± 5.8 mg (1.2 ± 0.1 mg CE) of sugar-free kiwifruit extract (KFE). KFE inhibited both human platelet aggregation and plasma ACE activity in a dose-dependent manner. KFE inhibited platelet aggregation in response to ADP, collagen and arachidonic acid, and inhibitory action was mediated in part by reducing TxA2 synthesis. The IC50 for ADP-induced platelet aggregation was 1.6 ± 0.2 mg/ml (29.0 ± 3.0 μg CE/ml), whereas IC50 for serum ACE was 0.6 ± 0.1 mg/ml (11.0 ± 1.2 μg CE/ml). Consuming 500 mg of KFE (9.0 mg CE) in 10 g margarine inhibited ex vivo platelet aggregation by 12.7%, 2 h after consumption by healthy volunteers (n = 9). All these data indicate that kiwifruit contains very potent antiplatelet and anti-ACE components. Consuming kiwifruits might be beneficial as both preventive and therapeutic regime in cardiovascular disease. PMID:24219176

  14. Modulation of heparin cofactor II activity by histidine-rich glycoprotein and platelet factor 4.

    PubMed Central

    Tollefsen, D M; Pestka, C A

    1985-01-01

    Heparin cofactor II is a plasma protein that inhibits thrombin rapidly in the presence of either heparin or dermatan sulfate. We have determined the effects of two glycosaminoglycan-binding proteins, i.e., histidine-rich glycoprotein and platelet factor 4, on these reactions. Inhibition of thrombin by heparin cofactor II and heparin was completely prevented by purified histidine-rich glycoprotein at the ratio of 13 micrograms histidine-rich glycoprotein/microgram heparin. In contrast, histidine-rich glycoprotein had no effect on inhibition of thrombin by heparin cofactor II and dermatan sulfate at ratios of less than or equal to 128 micrograms histidine-rich glycoprotein/microgram dermatan sulfate. Removal of 85-90% of the histidine-rich glycoprotein from plasma resulted in a fourfold reduction in the amount of heparin required to prolong the thrombin clotting time from 14 s to greater than 180 s but had no effect on the amount of dermatan sulfate required for similar anti-coagulant activity. In contrast to histidine-rich glycoprotein, purified platelet factor 4 prevented inhibition of thrombin by heparin cofactor II in the presence of either heparin or dermatan sulfate at the ratio of 2 micrograms platelet factor 4/micrograms glycosaminoglycan. Furthermore, the supernatant medium from platelets treated with arachidonic acid to cause secretion of platelet factor 4 prevented inhibition of thrombin by heparin cofactor II in the presence of heparin or dermatan sulfate. We conclude that histidine-rich glycoprotein and platelet factor 4 can regulate the antithrombin activity of heparin cofactor II. Images PMID:3838317

  15. Blockade of C5a and C5b-9 generation inhibits leukocyte and platelet activation during extracorporeal circulation.

    PubMed Central

    Rinder, C S; Rinder, H M; Smith, B R; Fitch, J C; Smith, M J; Tracey, J B; Matis, L A; Squinto, S P; Rollins, S A

    1995-01-01

    Complement activation contributes to the systemic inflammatory response induced by cardiopulmonary bypass. At the cellular level, cardiopulmonary bypass activates leukocytes and platelets; however the contribution of early (3a) versus late (C5a, soluble C5b-9) complement components to this activation is unclear. We used a model of simulated extracorporeal circulation that activates complement (C3a, C5a, and C5b-9 formation), platelets (increased percentages of P-selectin-positive platelets and leukocyte-platelet conjugates), and neutrophils (upregulated CD11b expression). to specifically target complement activation in this model, we added a blocking mAb directed at the human C5 complement component and assessed its effect on complement and cellular activation. Compared with a control mAB, the anti-human C5 mAb profoundly inhibited C5a and soluble C5b-9 generation and serum complement hemolytic activity but had no effect on C3a generation. Additionally, the anti-human C5 mAb significantly inhibited neutrophil CD11b upregulation and abolished the increase in P-selectin-positive platelets and leukocyte-platelet conjugate formation compared to experiments performed with the control mAb. This suggests that the terminal components C5a and C5b-9, but not C3a, directly contribute to platelet and neutrophil activation during extracorporeal circulation. Furthermore, these data identify the C5 component as a site for therapeutic intervention in cardiopulmonary bypass. PMID:7657827

  16. Thrombus imaging in a primate model with antibodies specific for an external membrane protein of activated platelets

    SciTech Connect

    Palabrica, T.M.; Furie, B.C.; Konstam, M.A.; Aronovitz, M.J.; Connolly, R.; Brockway, B.A.; Ramberg, K.L.; Furie, B.

    1989-02-01

    The activated platelet is a potential target for the localization of thrombi in vivo since, after stimulation and secretion of granule contents, activated platelets are concentrated at sites of blood clot formation. In this study, we used antibodies specific for a membrane protein of activated platelets to detect experimental thrombi in an animal model. PADGEM (platelet activation-dependent granule-external membrane protein), a platelet alpha-granule membrane protein, is translocated to the plasma membrane during platelet activation and granule secretion. Since PADGEM is internal in unstimulated platelets, polyclonal anti-PADGEM and monoclonal KC4 antibodies do not bind to circulating resting platelets but do interact with activated platelets. Dacron graft material incubated with radiolabeled KC4 or anti-PADGEM antibodies in the presence of thrombin-activated platelet-rich plasma bound most of the antibody. Imaging experiments with 123I-labeled anti-PADGEM in baboons with an external arterial-venous Dacron shunt revealed rapid uptake in the thrombus induced by the Dacron graft; control experiments with 123I-labeled nonimmune IgG exhibited minimal uptake. Deep venous thrombi, formed by using percutaneous balloon catheters to stop blood flow in the femoral vein of baboons, were visualized with 123I-labeled anti-PADGEM. Thrombi were discernible against blood pool background activity without subtraction techniques within 1 hr. No target enhancement was seen with 123I-labeled nonimmune IgG. 123I-labeled anti-PADGEM cleared the blood pool with an initial half-disappearance time of 6 min and did not interfere with hemostasis. These results indicate that radioimmunoscintigraphy with anti-PADGEM antibodies can visualize thrombi in baboon models and is a promising technique for clinical thrombus detection in humans.

  17. Response to platelet-activating factor in human platelets stored and aged in plasma. Decrease in aggregation, phosphoinositide turnover, and receptor affinity

    SciTech Connect

    Shukla, S.D.; Morrison, W.J.; Klachko, D.M.

    1989-07-01

    Human platelet concentrates were stored in polyolefin bags at 22 to 24 degrees C on a horizontal shaker for up to 8 days. At different intervals, aliquots of platelet-rich plasma (PRP) were removed aseptically and five variables, i.e., platelet counts, morphology, platelet-activating factor (PAF)-stimulated aggregation, phosphoinositide turnover, and (3H)PAF binding to platelet receptors, were studied. The number of platelets did not change during the 8 days of storage. Scanning electron microscopy of the platelets revealed a gradual morphologic change from biconcave flat discs to irregular, crenated forms. The PAF-induced aggregation of platelets declined with time of storage. A decrease to 50 percent of the Day 1 aggregatory response to PAF was evident on Day 2, and there was a further decline to about 20 percent by Day 6. Similarly, PAF receptor-coupled phosphoinositide turnover, as monitored by 32P incorporation into individual phosphoinositides, decreased dramatically with storage. After 2 to 3 days of storage, the phosphoinositide turnover was reduced to 50 percent of the original response, and it continued to decline to about 25 percent of original response by Day 5 or 6. The binding of (3H)PAF to washed human platelets indicated subtle changes between Days 2 and 4, which became more noticeable by Day 6. These results have raised the possibility of changes in the number of the receptors and/or their affinity for the ligand during storage. We conclude that although the number of platelets was maintained during storage for 8 days, a general deterioration of their responses to PAF occurred at the levels of cell surface receptor, transmembrane signaling (phosphoinositide turnover), and response (aggregation).

  18. Role of platelet-activating factor (PAF) in platelet accumulation in rabbit skin: effect of the novel long-acting PAF antagonist, UK-74,505.

    PubMed Central

    Pons, F.; Rossi, A. G.; Norman, K. E.; Williams, T. J.; Nourshargh, S.

    1993-01-01

    1. The contribution of platelet-activating factor (PAF) to platelet deposition and oedema formation induced by exogenous soluble mediators, zymosan particles and associated with a reversed passive Arthus (RPA) reaction in rabbit skin was investigated by use of a novel long-acting PAF receptor antagonist, UK-74,505. 2. Oedema formation and platelet accumulation were simultaneously measured by i.v. injection of [125I]-albumin and 111In-labelled rabbit platelets. UK-74,505 was either administered i.v. or used to pretreat radiolabelled platelets in vitro before their injection into recipient animals. Platelets pretreated with UK-74,505 were also labelled with the fluorescent calcium indicator, Fura-2, to assess their ex vivo reactivity to PAF at the end of the in vivo experiment. 3. UK-74,505 (0.5 mg kg-1), administered i.v., inhibited PAF-induced oedema formation, but did not affect oedema induced by zymosan particles, bradykinin (BK), histamine, formyl-methionyl-leucylphenylalanine (FMLP), zymosan-activated plasma (ZAP, as a source of C5a des Arg), leukotriene B4 (LTB4) or interleukin-8 (IL-8). 4. UK-74,505, administered i.v. also suppressed the small platelet accumulation induced by exogenous PAF, but had no effect on accumulation induced by IL-8 or ZAP. Although oedema induced by zymosan was not affected by i.v. UK-74,505, zymosan-induced platelet accumulation was significantly attenuated by the antagonist. 5. The RPA reaction in rabbit skin was associated with marked oedema formation and platelet accumulation which were both inhibited by i.v. UK-74,505.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8495241

  19. Inhibitory effect of Andrographis paniculata extract and its active diterpenoids on platelet aggregation.

    PubMed

    Thisoda, Piengpen; Rangkadilok, Nuchanart; Pholphana, Nanthanit; Worasuttayangkurn, Luksamee; Ruchirawat, Somsak; Satayavivad, Jutamaad

    2006-12-28

    Andrographis paniculata has been widely used for the prevention and treatment of common cold especially in Asia and Scandinavia. The three active diterpenoids from this plant, including aqueous plant extracts, were investigated for the inhibitory effect on platelet aggregation in vitro. The results indicated that andrographolide (AP(1)) and 14-deoxy-11,12-didehydroandrographolide (AP(3)) significantly inhibited thrombin-induced platelet aggregation in a concentration-(1-100 microM) and time-dependent manner while neoandrographolide (AP(4)) had little or no activity. AP(3) exhibited higher antiplatelet activity than AP(1) with IC(50) values ranging from 10 to 50 microM. The inhibitory mechanism of AP(1) and AP(3) on platelet aggregation was also evaluated and the results indicated that the inhibition of extracellular signal-regulated kinase1/2 (ERK1/2) pathway may contribute to antiplatelet activity of these two compounds. In addition, standardized aqueous extracts of A. paniculata containing different amounts of AP(3) inhibited thrombin-induced aggregation to different degrees. The extracts significantly decreased platelet aggregation in a concentration-(10-100 microg/ml) and time-dependent manner. However, the extract with high level of AP(3) (Extract B) (IC(50) values=50-75 microg/ml) showed less inhibitory activity against thrombin than the extract with lower level of AP(3) (Extract A) (IC(50) values=25-50 microg/ml). These results indicate that the standardized A. paniculata extract may contain other antiplatelet compounds rather than AP(1) and AP(3), which contribute to high antiplatelet activity. Therefore, the consumption of A. paniculata products may help to prevent or treat some cardiovascular disorders i.e. thrombosis; however, it should be used with caution by patients with bleeding disorders. PMID:17081514

  20. Bilirubin, platelet activation and heart disease: a missing link to cardiovascular protection in Gilbert's syndrome?

    PubMed

    Kundur, Avinash R; Singh, Indu; Bulmer, Andrew C

    2015-03-01

    Gilbert's syndrome (GS) is a relatively common condition, inducing a benign, non-hemolytic, unconjugated hyperbilirubinemia. Gilbert's Syndrome is associated with mutation in the Uridine Glucuronosyl Transferase 1A1 (UGT1A1) gene promoter, reducing UGT1A1 activity, which normally conjugates bilirubin allowing its elimination from the blood. Individuals with GS demonstrate mildly elevated plasma antioxidant capacity caused by elevated levels of unconjugated bilirubin (UCB), reduced thiols and glutathione. Interestingly, the development of, and risk of mortality from, cardiovascular disease is remarkably reduced in GS individuals. An explanation for this protection may be explained by bilirubin's ability to inhibit multiple processes that induce platelet hyper-reactivity and thrombosis, thus far under-appreciated in the literature. Reactive oxygen species are produced continuously via metabolic processes and have the potential to oxidatively modify proteins and lipids within cell membranes, which may encourage the development of thrombosis and CVDs. Oxidative stress induced platelet hyper-reactivity significantly increases the risk of thrombosis, which can potentially lead to tissue infarction. Here, we discuss the possible mechanisms by which increased antioxidant status might influence platelet function and link this to cardiovascular protection in GS. In summary, this is the first article to discuss the possible role of bilirubin as an anti-thrombotic agent, which inhibits platelet activation and potentially, organ infarction, which could contribute to the reduced mortality rate in mildly hyperbilirbinemic individuals. PMID:25576848

  1. The active metabolite of prasugrel inhibits ADP-stimulated thrombo-inflammatory markers of platelet activation: Influence of other blood cells, calcium, and aspirin.

    PubMed

    Frelinger, Andrew L; Jakubowski, Joseph A; Li, Youfu; Barnard, Marc R; Fox, Marsha L; Linden, Matthew D; Sugidachi, Atsuhiro; Winters, Kenneth J; Furman, Mark I; Michelson, Alan D

    2007-07-01

    The novel thienopyridine prodrug prasugrel, a platelet P2Y(12) ADP receptor antagonist, requires in vivo metabolism for activity. Although pharmacological data have been collected on the effects of prasugrel on platelet aggregation, there are few data on the direct effects of the prasugrel's active metabolite, R-138727, on other aspects of platelet function. Here we examined the effects of R-138727 on thrombo-inflammatory markers of platelet activation, and the possible modulatory effects of other blood cells, calcium, and aspirin. Blood (PPACK or citrate anticoagulated) from healthy donors pre- and post-aspirin was incubated with R-138727 and the response to ADP assessed in whole blood or platelet-rich plasma (PRP) by aggregometry and flow cytometric analysis of leukocyte-platelet aggregates, platelet surface P-selectin, and GPIIb-IIIa activation. Low-micromolar concentrations of R-138727 resulted in a rapid and consistent inhibition of these ADP-stimulated thrombo-inflammatory markers. These rapid kinetics required physiological calcium levels, but were largely unaffected by aspirin. Lower IC(50) values in whole blood relative to PRP suggested that other blood cells affect ADP-induced platelet activation and hence the net inhibition by R-138727. R-138727 did not inhibit P2Y(12)-mediated ADP-induced shape change, even at concentrations that completely inhibited platelet aggregation, confirming the specificity of R-138727 for P2Y(12). In conclusion, R-138727, the active metabolite of prasugrel, results in rapid, potent, consistent, and selective inhibition of P2Y(12)-mediated up-regulation of thrombo-inflammatory markers of platelet activation. This inhibition is enhanced in the presence other blood cells and calcium, but not aspirin. PMID:17598013

  2. The role of prostanoids in the production of acute acalculous cholecystitis by platelet-activating factor.

    PubMed Central

    Kaminski, D L; Andrus, C H; German, D; Deshpande, Y G

    1990-01-01

    Gallbladder tissue from patients with acute acalculous cholecystitis contains increased amounts of prostanoids when compared to normal gallbladder tissue. Platelet-activating factor (PAF) is a potent stimulus of eicosanoid formation. It has been implicated as a mediator of acute inflammatory processes and systemic responses to shock. In this study the role of PAF in acute acalculous cholecystitis was evaluated. Anesthetized cats underwent gallbladder perfusion with a physiologic buffer solution containing [14C]polyethylene glycol as a nonabsorbable tracer to quantitate mucosal water absorption. Platelet-activating factor was infused into the hepatic artery for 2 hours. Control experiments were performed when vehicle alone was infused. Experiments also were performed when indomethacin was administered intravenously and when indomethacin and PAF were administered. Gallbladder mucosal absorption/secretion and perfusate and tissue prostaglandin E (PGE) and 6 keto prostaglandin F1 alpha (6-keto PGF1 alpha) levels were evaluated. Gallbladder inflammation was evaluated by beta-glucuronidase and myeloperoxidase tissue concentrations and by a histologic scoring system. Platelet-activating factor eliminated gallbladder absorption and produced net fluid secretion associated with dose-related increases in perfusate PGE concentrations and gallbladder tissue PGE and 6 keto PGF1 alpha levels when compared to control values. Platelet-activating factor produced significant inflammation in the gallbladder with increases in the histologic score of inflammation and tissue lysosomal enzyme activities. Indomethacin significantly decreased the fluid secretion, prostanoid levels, and inflammation produced by PAF. The results suggest that PAF may induce acute gallbladder inflammation associated with systemic stress through a prostanoid-mediated mechanism. Images Fig. 2. PMID:2171443

  3. CV-6209, a highly potent antagonist of platelet activating factor in vitro and in vivo.

    PubMed

    Terashita, Z; Imura, Y; Takatani, M; Tsushima, S; Nishikawa, K

    1987-07-01

    2-[N-acetyl-N-(2-methoxy-3-octadecylcarbamoyloxypropoxycarbonyl) aminomethyl]-1-ethylpyridinium chloride (CV-6209) inhibited aggregation of rabbit and human platelets induced by platelet activating factor (PAF) with the IC50 values of 7.5 X 10(-8) and 1.7 X 10(-7) M, respectively, and had little effects on the aggregation induced by arachidonic acid, ADP and collagen. The inhibitory effect of CV-6209 on the PAF-induced rabbit platelet aggregation was 104, 9, 8 and 3 times more potent than the PAF antagonists CV-3988, ONO-6240, Ginkgolide B and etizolam, respectively. CV-6209 inhibited [3H]serotonin release from rabbit platelets stimulated with PAF (3 X 10(-8) M) with a similar potency as the inhibition on the platelet aggregation. CV-6209 inhibited PAF (0.3 microgram/kg i.v.)-induced hypotension in rats (ED50, 0.009 mg/kg i.v.) with no effect on the hypotension induced by arachidonic acid, histamine, bradykinin and isoproterenol. CV-6209 (1 mg/kg) inhibited slightly the acetylcholine-induced hypotension. In rats, post-treatment with CV-6209 reversed the PAF (1 microgram/kg i.v.)-induced hypotension rapidly (ED50, 0.0046 mg/kg i.v.); CV-6209 was 74, 20, 185 and over 2100 times more potent than CV-3988, ONO-6240, Ginkgolide B and etizolam, respectively. Thus, the relative potency of the anti-PAF action of PAF analog (CV-6209, CV-3988 and ONO-6240) differed little between the inhibition of PAF-induced platelet aggregation and the reversal of PAF-induced hypotension, but that of nonPAF analogs (Ginkgolide B and etizolam) differed greatly with these assay systems, when standardized with CV-6209.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3612533

  4. Low platelet activity predicts 30 days mortality in patients undergoing heart surgery.

    PubMed

    Kuliczkowski, Wiktor; Sliwka, Joanna; Kaczmarski, Jacek; Zysko, Dorota; Zembala, Michal; Steter, Dawid; Zembala, Marian; Gierlotka, Marek; Kim, Moo Hyun; Serebruany, Victor

    2016-03-01

    Despite advanced techniques and improved clinical outcomes, patient survival following coronary artery bypass grafting (CABG) is still a major concern. Therefore, predicting future CABG mortality represents an unmet medical need and should be carefully explored. The objective of this study is to assess whether pre-CABG platelet activity corresponds with 30 days mortality post-CABG. Retrospective analyses of platelet biomarkers and death at 30 days in 478 heart surgery patients withdrawn from aspirin or/and clopidogrel. Platelet activity was assessed prior to CABG for aspirin (ASPI-test) with arachidonic acid and clopidogrel (ADP-test) utilizing Multiplate impedance aggregometer. Most patients (n = 198) underwent conventional CABG, off-pump (n = 162), minimally invasive (n = 30), artificial valve implantation (n = 48) or valves in combination with CABG (n = 40). There were 22 deaths at 30 days, including 10 in-hospital fatalities. With the cut-off value set below 407 area under curve (AUC) for the ASPI-test, the 30-day mortality was 5.90% for the lower cohort and 2.66% for patients with significantly higher platelet reactivity (P = 0.038). For the ADP-test with a cut-off at 400AUC, the 30-day mortality was 9.68% for the lower cohort and 3.66% for patients with higher platelet reactivity, representing a borderline significant difference (P = 0.046). Aside from the platelet indices, patients who received red blood cell (RBC) concentrate had a highly significant (P < 0.0001) risk of death at 30 days. Both aspirin and clopidogrel tests were useful in predicting 30 days mortality following heart surgery, suggesting the danger of diminished platelet activity prior to CABG in such high-risk patients. These preliminary evidence supports early discontinuation of antiplatelet therapy for elective CABG and requires adequately powered randomized trials to test the hypothesis and potentially improve survival. PMID:26366827

  5. Activation of human blood platelets by arginine-vasopressin. Role of bivalent cations

    SciTech Connect

    Pletscher, A.; Erne, P.; Buergisser, E.F.; Ferracin, F.

    1985-12-01

    Arginine-vasopressin caused platelet activation, i.e., a shape change reaction and a rise in intracellular free Ca/sup 2 +/ ((Ca/sup 2 +/)i) only in the presence of certain bivalent cations. The EC50 of arginine-vasopressin (concentration causing half-maximal shape change) decreased with rising concentrations of Mn/sup 2 +/, Mg/sup 2 +/, or Ca/sup 2 +/ in the medium, but was at least an order higher with Ca/sup 2 +/ than with Mn/sup 2 +/ or Mg/sup 2 +/. The EC50 of the active bivalent cations (concentrations enabling 100 nM arginine-vasopressin to exert half-maximal shape change and rise in (Ca/sup 2 +/)i) varied with the individual cations, being by far the highest for Ca/sup 2 +/. The KD of (3H)arginine-vasopressin binding to platelet membranes and intact platelets markedly decreased when extracellular Mg/sup 2 +/ or Mn/sup 2 +/ were present, and the KD values were inversely related to the concentration of the cations. Ca/sup 2 +/ also lowered the KD values; however, the effect was less marked than that of Mg/sup 2 +/ or Mn/sup 2 +/ and, in physiological conditions, significant only in intact platelets. Vasopressin-1 antagonists counteracted arginine-vasopressin binding and the shape change reaction and (Ca/sup 2 +/)i rise induced by arginine-vasopressin. In the presence of Mn/sup 2 +/ in the medium, administration of arginine-vasopressin led to quenching of the intracellular fluorescence of 2-methyl-6-methoxy-8-nitroquinoline-loaded platelets, possibly due to influx of Mn/sup 2 +/. In conclusion, the dependency of the arginine-vasopressin-induced platelet activation on bivalent cations is at least partly due to an enhancement by these cations of the affinity of the vasopressin-1 receptor for arginine-vasopressin. Thereby, under physiological conditions, Mg/sup 2 +/ seems to be of primary importance. Other mechanisms may be involved, too, e.g., an enhancement by arginine-vasopressin of the influx of bivalent cations into the platelets.

  6. Anti-platelet activity of a three-finger toxin (3FTx) from Indian monocled cobra (Naja kaouthia) venom.

    PubMed

    Chanda, Chandrasekhar; Sarkar, Angshuman; Sistla, Srinivas; Chakrabarty, Dibakar

    2013-11-22

    A low molecular weight anti-platelet peptide (6.9 kDa) has been purified from Naja kaouthia venom and was named KT-6.9. MALDI-TOF/TOF mass spectrometry analysis revealed the homology of KT-6.9 peptide sequence with many three finger toxin family members. KT-6.9 inhibited human platelet aggregation process in a dose dependent manner. It has inhibited ADP, thrombin and arachidonic acid induced platelet aggregation process in dose dependent manner, but did not inhibit collagen and ristocetin induced platelet aggregation. Strong inhibition (70%) of the ADP induced platelet aggregation by KT-6.9 suggests competition with ADP for its receptors on platelet surface. Anti-platelet activity of KT-6.9 was found to be 25 times stronger than that of anti-platelet drug clopidogrel. Binding of KT-6.9 to platelet surface was confirmed by surface plasma resonance analysis using BIAcore X100. Binding was also observed by a modified sandwich ELISA method using anti-KT-6.9 antibodies. KT-6.9 is probably the first 3 FTx from Indian monocled cobra venom reported as a platelet aggregation inhibitor. PMID:24183721

  7. Polyurethane blended with polylactides for improved cell adhesion and reduced platelet activation.

    PubMed

    Hsu, S H; Tseng, H J; Fang, Z K

    1999-10-01

    Poly(L-lactide) (PLLA) or 50:50 poly(lactide-co-glycolide) (PLGA) was blended with a commercial polyurethane (PU), Pellethane 2103-80A, using a mutual solvent technique. After preparation by salt casting, the mechanical properties of the porous matrices were measured, and the surface element was characterized by electron spectroscopy for chemical analysis. The attachment and growth of fibroblasts as well as human umbilical vein endothelial cells on the substrates were investigated by the cell culture test. Platelet activation on different substrates was also studied. It was found that blending with these biodegradable polymers, especially PLGA. enhanced the cellular attachment and growth. This enhancement may be correlated with the higher oxygen/carbon (0/C) atomic ratio on the surface of the blends. Blending with either PLLA or PLGA was also found to decrease platelet adhesion and activation in vitro. These characteristics make PU/polylactide blends potential substrates for cardiovascular applications. PMID:10610681

  8. Numerical Investigation of the Effects of Channel Geometry on Platelet Activation and Blood Damage

    PubMed Central

    Wu, Jingshu; Yun, B. Min; Fallon, Anna M.; Hanson, Stephen R.; Aidun, Cyrus K.; Yoganathan, Ajit P.

    2011-01-01

    Thromboembolic complications in Bileaflet mechanical heart valves (BMHVs) are believed to be due to the combination of high shear stresses and large recirculation regions. Relating blood damage to design geometry is therefore essential to ultimately optimize the design of BMHVs. The aim of this research is to quantitatively study the effect of 3D channel geometry on shear-induced platelet activation and aggregation, and to choose an appropriate blood damage index (BDI) model for future numerical simulations. The simulations in this study use a recently developed lattice-Boltzmann with external boundary force (LBM-EBF) method [Wu, J., and C. K. Aidun. Int. J. Numer. Method Fluids 62(7):765–783, 2010; Wu, J., and C. K. Aidun. Int. J. Multiphase flow 36:202–209, 2010]. The channel geometries and flow conditions are re-constructed from recent experiments by Fallon [The Development of a Novel in vitro Flow System to Evaluate Platelet Activation and Procoagulant Potential Induced by Bileaflet Mechanical Heart Valve Leakage Jets in School of Chemical and Biomolecular Engineering. Atlanta: Georgia Institute of Technology] and Fallon et al. [Ann. Biomed. Eng. 36(1):1]. The fluid flow is computed on a fixed regular ‘lattice’ using the LBM, and each platelet is mapped onto a Lagrangian frame moving continuously throughout the fluid domain. The two-way fluid–solid interactions are determined by the EBF method by enforcing a no-slip condition on the platelet surface. The motion and orientation of the platelet are obtained from Newtonian dynamics equations. The numerical results show that sharp corners or sudden shape transitions will increase blood damage. Fallon’s experimental results were used as a basis for choosing the appropriate BDI model for use in future computational simulations of flow through BMHVs. PMID:20976558

  9. Eugenol: a dual inhibitor of platelet-activating factor and arachidonic acid metabolism.

    PubMed

    Saeed, S A; Simjee, R U; Shamim, G; Gilani, A H

    1995-07-01

    Eugenol is an active principal and responsible for several pharmacological activities of clove oil. We studied the effects of eugenol on human platelet aggregation, arachidonic acid (AA) and platelet-activating factor (PAF) metabolism and in vivo effects on AA and PAF-induced shock in rabbits. Eugenol strongly inhibited PAF-induced platelet aggregation with lesser effect against AA and collegen. The IC(50) values were against AA: 31 ± 0.5; collagen: 64 ± 0.7 and PAF 7 ± 0.2 μM (n=9) respectively. In addition, eugenol stimulated PAF-acetylhydrolase activity suggesting that inhibition of PAF could be due to its inactivation to lyso-PAF. Pretreatment of rabbits with eugenol (50-100 mg/kg) prevented the lethal effects of intravenous PAF (11 μgg/kg) or AA (2 mg/kg) in a dose-dependent fashion. The protective effects of eugenol in the rabbits, however, were more pronounced against PAF-induced mortality (100% protection). In addition, eugenol also inhibited AA metabolism via cyclooxygenase and lipoxygenase pathways in human platelets. Both the production of thromboxane-A(2) and 12-hydroxy-eicosatetraenoic acid was inhibited by eugenol in a concentration-related manner (30-120 μM). In vivo, eugenol (50-100 mg/kg; i.p.) inhibited carrageenan-induced rat paw oedema (P < 0.001). In this test, eugenol was 5 times more potent than aspirin. These results provide evidence that eugenol acts as a dual antagonist of AA and PAF. PMID:23196096

  10. Diminished nitric oxide generation from neutrophils suppresses platelet activation in chronic renal failure.

    PubMed

    Abrantes, Daniele C; Brunini, Tatiana M C; Matsuura, Cristiane; Mury, Wanda Vianna; Corrêa, Carolina R; Santos, Sérgio F; Ormonde do Carmo, Monique B O; Mendes-Ribeiro, Antônio Cláudio

    2015-03-01

    Chronic renal failure (CRF) is a complex clinical condition associated with accelerated atherosclerosis and thrombosis leading to cardiovascular events. The aim of this study was to investigate in detail the NO pathway in neutrophils obtained from hemodialysis patients and its association with platelet function and oxidative status. Fifteen CRF patients on hemodialysis and fifteen controls were included in this study. Laboratory and experimental evaluations were performed after hemodialysis in CRF patients. We evaluated L-[³H] arginine transport, NO synthase (NOS) activity, amino acid concentration in neutrophils, and expressions of NOS isoforms and p47(phox) by western blotting. Platelet aggregation was analyzed in the presence or absence of neutrophils. Oxidative status was measured through glutathione peroxidase, catalase activities, protein oxidation, lipid peroxidation, and DNA/RNA oxidation in serum. Basal NOS activity (pmol/10⁶ cells/min) was impaired in CRF patients on hemodialysis (0.33 ± 0.17) compared to controls (0.65 ± 0.12), whereas the expression of NOS isoforms remained unaltered. L-Arginine transport into neutrophils was similar in CRF patients on hemodialysis and controls. In addition, intracellular concentration of L-arginine was increased fourfold in the patient group. Systemic oxidative stress markers were not affected by CRF. On the other hand, NADPH oxidase subunit p47(phox) in neutrophils was overexpressed in CRF. In the presence of neutrophils, there was a reduction time-dependent in platelet aggregation in both groups with no difference between them. This data suggest that reduced basal generation of NO by neutrophils in CRF patients on hemodialysis occurs independently of L-arginine bioavailability and is able to suppress platelet activation. PMID:25524601

  11. Characterization of cutaneous vascular permeability induced by platelet-activating factor in guinea pigs and rats and its inhibition by a platelet-activating factor receptor antagonist

    SciTech Connect

    Hwang, S.B.; Li, C.L.; Lam, M.H.; Shen, T.Y.

    1985-06-01

    Mechanisms of platelet-activating factor (PAF)-induced increases of cutaneous vascular permeability in guinea pigs and in rats were further explored. PAF so far is the most potent vasoactive mediator, being more than 1000-fold more potent than histamine and bradykinin in both species. In guinea pigs, there is a time delay of 5 to 10 minutes before PAF action, whereas, in the rat, the increased vasopermeability occurs immediately following the intradermal PAF injection. Relative vasoactive potencies of PAF and several structure-related analogues in both species correlate very well with their relative inhibition of the binding of /sup 3/H-PAF to specific receptor sites on isolated rabbit platelet plasma membranes and their aggregatory abilities of rabbit platelets. Furthermore, the PAF-induced cutaneous vascular permeability is inhibitable by a competitive specific PAF receptor antagonist, kadsurenone, suggesting that binding of PAF to its specific receptor site is the first step to initiate its action of increased cutaneous vascular permeability. Several pure cyclooxygenase inhibitors, including indomethacin, diflunisal, and flurbiprofen, and the dual cyclooxygenase/lipoxygenase inhibitor, BW755C, but not the histamine antagonists, inhibit the PAF-induced vasopermeability in guinea pigs. The inhibition by indomethacin or BW755C can be fully reversed by coinjection intradermally with PAF and prostaglandin E1 but not leukotriene B4. Also, prostaglandin E1 but not leukotriene B4 enhances the guinea pig in vivo response to PAF in this model. However, in rats, none of the cyclooxygenase inhibitors, histamine antagonists, or BW755C inhibit the PAF effect of cutaneous phenomena.

  12. Effect of an Activated Platelet Concentrate on Differentiated Cells Involved in Tissue Healing.

    PubMed

    Brini, Anna T; Ceci, Caterina; Taschieri, Silvio; Niada, Stefania; Lolato, Alessandra; Giannasi, Chiara; Mortellaro, Carmen; Del Fabbro, Massimo

    2016-05-01

    Tissue healing is a complex process involving several players such as cells and growth factors released from platelets upon activation. Today, platelet concentrates (PCs) are used in many different medical fields including oral, orthopaedic, and reconstructive surgery since they allow growth factors delivery to the injured site, aiming at enhancing tissue regeneration. The purpose of this in vitro study was to evaluate the effect of the acellular plasma of an activated platelet concentrate obtained using a manual protocol, on the proliferation, and biological activity of differentiated cells involved in tissue healing. Human osteoblasts and dermal fibroblasts were grown in serum-free medium supplemented with PC derived from several donors. Human osteoblast and human dermal fibroblast proliferation was assessed by MTT test after 7 days and cells were count up to 12-day incubation. Human osteoblast osteo-differentiation was tested after 7 and 14-day incubation by alkaline phosphatase assay. The addition of PC to the culture medium caused an increased proliferation with respect to cells grown in standard condition. The results of the present study suggest that PC supports the proliferation of terminally differentiated cells involved in wound healing and tissue regeneration, confirming its beneficial clinical application in regenerative therapies. PMID:27054419

  13. Human platelet activation by Escherichia coli: roles for FcγRIIA and integrin αIIbβ3

    PubMed Central

    Watson, Callum N.; Kerrigan, Steven W.; Cox, Dermot; Henderson, Ian R.; Watson, Steve P.; Arman, Mònica

    2016-01-01

    Abstract Gram-negative Escherichia coli cause diseases such as sepsis and hemolytic uremic syndrome in which thrombotic disorders can be found. Direct platelet–bacterium interactions might contribute to some of these conditions; however, mechanisms of human platelet activation by E. coli leading to thrombus formation are poorly understood. While the IgG receptor FcγRIIA has a key role in platelet response to various Gram-positive species, its role in activation to Gram-negative bacteria is poorly defined. This study aimed to investigate the molecular mechanisms of human platelet activation by E. coli, including the potential role of FcγRIIA. Using light-transmission aggregometry, measurements of ATP release and tyrosine-phosphorylation, we investigated the ability of two E. coli clinical isolates to activate platelets in plasma, in the presence or absence of specific receptors and signaling inhibitors. Aggregation assays with washed platelets supplemented with IgGs were performed to evaluate the requirement of this plasma component in activation. We found a critical role for the immune receptor FcγRIIA, αIIbβ3, and Src and Syk tyrosine kinases in platelet activation in response to E. coli. IgG and αIIbβ3 engagement was required for FcγRIIA activation. Moreover, feedback mediators adenosine 5’-diphosphate (ADP) and thromboxane A2 (TxA2) were essential for platelet aggregation. These findings suggest that human platelet responses to E. coli isolates are similar to those induced by Gram-positive organisms. Our observations support the existence of a central FcγRIIA-mediated pathway by which human platelets respond to both Gram-negative and Gram-positive bacteria. PMID:27025455

  14. Hemoglobin interaction with GP1bα induces platelet activation and apoptosis: a novel mechanism associated with intravascular hemolysis.

    PubMed

    Singhal, Rashi; Annarapu, Gowtham K; Pandey, Ankita; Chawla, Sheetal; Ojha, Amrita; Gupta, Avinash; Cruz, Miguel A; Seth, Tulika; Guchhait, Prasenjit

    2015-12-01

    Intravascular hemolysis increases the risk of hypercoagulation and thrombosis in hemolytic disorders. Our study shows a novel mechanism by which extracellular hemoglobin directly affects platelet activation. The binding of Hb to glycoprotein1bα activates platelets. Lower concentrations of Hb (0.37-3 μM) significantly increase the phosphorylation of signaling adapter proteins, such as Lyn, PI3K, AKT, and ERK, and promote platelet aggregation in vitro. Higher concentrations of Hb (3-6 μM) activate the pro-apoptotic proteins Bak, Bax, cytochrome c, caspase-9 and caspase-3, and increase platelet clot formation. Increased plasma Hb activates platelets and promotes their apoptosis, and plays a crucial role in the pathogenesis of aggregation and development of the procoagulant state in hemolytic disorders. Furthermore, we show that in patients with paroxysmal nocturnal hemoglobinuria, a chronic hemolytic disease characterized by recurrent events of intravascular thrombosis and thromboembolism, it is the elevated plasma Hb or platelet surface bound Hb that positively correlates with platelet activation. PMID:26341739

  15. Hemoglobin interaction with GP1bα induces platelet activation and apoptosis: a novel mechanism associated with intravascular hemolysis

    PubMed Central

    Singhal, Rashi; Annarapu, Gowtham K.; Pandey, Ankita; Chawla, Sheetal; Ojha, Amrita; Gupta, Avinash; Cruz, Miguel A.; Seth, Tulika; Guchhait, Prasenjit

    2015-01-01

    Intravascular hemolysis increases the risk of hypercoagulation and thrombosis in hemolytic disorders. Our study shows a novel mechanism by which extracellular hemoglobin directly affects platelet activation. The binding of Hb to glycoprotein1bα activates platelets. Lower concentrations of Hb (0.37–3 μM) significantly increase the phosphorylation of signaling adapter proteins, such as Lyn, PI3K, AKT, and ERK, and promote platelet aggregation in vitro. Higher concentrations of Hb (3–6 μM) activate the pro-apoptotic proteins Bak, Bax, cytochrome c, caspase-9 and caspase-3, and increase platelet clot formation. Increased plasma Hb activates platelets and promotes their apoptosis, and plays a crucial role in the pathogenesis of aggregation and development of the procoagulant state in hemolytic disorders. Furthermore, we show that in patients with paroxysmal nocturnal hemoglobinuria, a chronic hemolytic disease characterized by recurrent events of intravascular thrombosis and thromboembolism, it is the elevated plasma Hb or platelet surface bound Hb that positively correlates with platelet activation. PMID:26341739

  16. Lipid phosphate phosphatases regulate lysophosphatidic acid production and signaling in platelets: studies using chemical inhibitors of lipid phosphate phosphatase activity.

    PubMed

    Smyth, Susan S; Sciorra, Vicki A; Sigal, Yury J; Pamuklar, Zehra; Wang, Zuncai; Xu, Yong; Prestwich, Glenn D; Morris, Andrew J

    2003-10-31

    Blood platelets play an essential role in ischemic heart disease and stroke contributing to acute thrombotic events by release of potent inflammatory agents within the vasculature. Lysophosphatidic acid (LPA) is a bioactive lipid mediator produced by platelets and found in the blood and atherosclerotic plaques. LPA receptors on platelets, leukocytes, endothelial cells, and smooth muscle cells regulate growth, differentiation, survival, motility, and contractile activity. Definition of the opposing pathways of synthesis and degradation that control extracellular LPA levels is critical to understanding how LPA bioactivity is regulated. We show that intact platelets and platelet membranes actively dephosphorylate LPA and identify the major enzyme responsible as lipid phosphate phosphatase 1 (LPP1). Localization of LPP1 to the platelet surface is increased by exposure to LPA. A novel receptor-inactive sn-3-substituted difluoromethylenephosphonate analog of phosphatidic acid that is a potent competitive inhibitor of LPP1 activity potentiates platelet aggregation and shape change responses to LPA and amplifies LPA production by agonist-stimulated platelets. Our results identify LPP1 as a pivotal regulator of LPA signaling in the cardiovascular system. These findings are consistent with genetic and cell biological evidence implicating LPPs as negative regulators of lysophospholipid signaling and suggest that the mechanisms involve both attenuation of lysophospholipid actions at cell surface receptors and opposition of lysophospholipid production. PMID:12909631

  17. Cutting edge: Leukotriene C4 activates mouse platelets in plasma exclusively through the type 2 cysteinyl leukotriene receptor.

    PubMed

    Cummings, Hannah E; Liu, Tao; Feng, Chunli; Laidlaw, Tanya M; Conley, Pamela B; Kanaoka, Yoshihide; Boyce, Joshua A

    2013-12-15

    Leukotriene C4 (LTC4) and its extracellular metabolites, LTD4 and LTE4, mediate airway inflammation. They signal through three specific receptors (type 1 cys-LT receptor [CysLT1R], CysLT2R, and GPR99) with overlapping ligand preferences. In this article, we demonstrate that LTC4, but not LTD4 or LTE4, activates mouse platelets exclusively through CysLT2R. Platelets expressed CysLT1R and CysLT2R proteins. LTC4 induced surface expression of CD62P by wild-type mouse platelets in platelet-rich plasma (PRP) and caused their secretion of thromboxane A2 and CXCL4. LTC4 was fully active on PRP from mice lacking either CysLT1R or GPR99, but completely inactive on PRP from CysLT2R-null (Cysltr2(-/-)) mice. LTC4/CysLT2R signaling required an autocrine ADP-mediated response through P2Y12 receptors. LTC4 potentiated airway inflammation in a platelet- and CysLT2R-dependent manner. Thus, CysLT2R on platelets recognizes LTC4 with unexpected selectivity. Nascent LTC4 may activate platelets at a synapse with granulocytes before it is converted to LTD4, promoting mediator generation and the formation of leukocyte-platelet complexes that facilitate inflammation. PMID:24244016

  18. Early increase in DcR2 expression and late activation of caspases in the platelet storage lesion.

    PubMed

    Plenchette, S; Moutet, M; Benguella, M; N'Gondara, J P; Guigner, F; Coffe, C; Corcos, L; Bettaieb, A; Solary, E

    2001-10-01

    Platelet transfusion is widely used to prevent bleeding in patients with severe thrombocytopenia. The maximal storage duration of platelet concentrates is usually 5 days, due to the platelet storage lesion that impairs their functions when stored for longer times. Some of the morphological and biochemical changes that characterize this storage lesion are reminiscent of cell death by apoptosis. The present study analyzed whether proteins involved in nucleated cell apoptosis could play a role in the platelet storage lesion. Storage of leukocyte-depleted platelets obtained by apheresis is associated with a late and limited activation of caspases, mainly caspase-3. This event correlates with an increased expression of the pro-apoptotic BH3-only protein Bim in the particulate fraction and a slight and late release of the pro-apoptotic mitochondrial protein Diablo/Smac in the cytosol. Platelets do not express the death receptors Fas, DR4 and DR5 on their plasma membrane, while the expression of the decoy receptor DcR2 increases progressively during platelet storage. Addition of low concentrations of the cryoprotector dimethylsulfoxide accelerates platelet caspase activation during storage, an effect that is partially prevented by the caspase inhibitor z-VAD-fmk. Altogether, DcR2 expression on the plasma membrane is an early event while caspase activation is a late event during platelet storage. These observations suggest that caspases are unlikely to account for the platelet storage lesion. As a consequence, addition of caspase inhibitors may not improve the quality of platelet concentrates stored in standard conditions. PMID:11587215

  19. 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

  20. 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

    2016-01-01

    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

  1. An Ultrasound Contrast Agent targeted to P-selectin detects Activated Platelets at Supra-arterial Shear Flow Conditions

    PubMed Central

    Guenther, Felix; von zur Muhlen, Constantin; Ferrante, Elisa A.; Grundmann, Sebastian; Bode, Christoph; Klibanov, Alexander L.

    2012-01-01

    Objectives To evaluate targeting of a microbubble contrast agent to platelets under high shear flow using the natural selectin ligand sialyl Lewisa. Materials and Methods Biotinylated polyacrylamide Sialyl Lewisa or biotinylated carbohydrate-free polymer (used as a control) were attached to biotinylated microbubbles via a streptavidin linker. Activated human platelets were isolated and attached to fibrinogen-coated culture dishes. Fibrinogen-coated dishes without platelets or platelet dishes blocked by an anti-P-selectin antibody served as negative control substrates. Dishes coated by recombinant P-selectin served as a positive control substrate. Microbubble adhesion was assessed by microscopy in an inverted parallel plate flow chamber, with wall shear stress values of 40, 30, 20, 10 and 5 dynes/cm2. The ratio of binding and passing microbubbles was defined as capture efficiency. Results There was no significant difference between the groups regarding the number of microbubbles in the fluid flow at each shear rate. Sialyl Lewisa-targeted microbubbles were binding and slowly rolling on the surface of activated platelets and P-selectin-coated dishes at all the flow conditions including 40 dynes/cm2. Capture efficiency of targeted microbubbles to activated platelets and recombinant P-selectin decreased with increasing shear flow: at 5 dynes/cm2, capture efficiency was 16.11% on activated platelets vs. 21.83 % on P-selectin, and, at 40 dynes/cm2, adhesion efficiency was still 3.4 % in both groups. There was neither significant adhesion of Sialyl Lewisa-targeted microbubbles to control substrates, nor adhesion of control microbubbles to activated platelets or to recombinant P-selectin. Conclusions Microbubble targeting using sialyl Lewisa, a fast-binding ligand to P-selectin, is a promising strategy for the design of ultrasound contrast binding to activated platelets under high shear stress conditions. PMID:20808239

  2. Platelet Mitochondrial Activity and Pesticide Exposure in Early Parkinson’s Disease

    PubMed Central

    Bronstein, Jeff M.; Paul, Kimberly; Yang, Laurice; Haas, Richard H.; Shults, Clifford W.; Le, Thuy; Ritz, Beate

    2015-01-01

    Background Mitochondrial dysfunction has been implicated in the pathogenesis of Parkinson’s disease (PD) but the cause of this dysfunction is unclear. Methods Platelet mitochondrial complex I and I/III (NADH cytochrome c reductase, NCCR) activities were measured in early PD patients and matched controls enrolled in a population based case-control study. Ambient agricultural pesticide exposures were assessed with a geographic information system and California Pesticide Use Registry. Results In contrast to some previous reports, we found no differences in complex I and I/III activities in subjects with PD and controls. We did find that NCCR activity correlated with subjects’ exposure to pesticides known to inhibit mitochondrial activity regardless of their diagnosis. Conclusions ETC activity is not altered in PD in this well-characterized cohort when compared to community-matched controls but appears to be affected by environmental toxins, such as mitochondria-inhibiting pesticides. PMID:25757798

  3. In Vivo Platelet Activation and Aspirin Responsiveness in Type 1 Diabetes.

    PubMed

    Zaccardi, Francesco; Rizzi, Alessandro; Petrucci, Giovanna; Ciaffardini, Flavia; Tanese, Luigi; Pagliaccia, Francesca; Cavalca, Viviana; Ciminello, Angela; Habib, Aida; Squellerio, Isabella; Rizzo, Paola; Tremoli, Elena; Rocca, Bianca; Pitocco, Dario; Patrono, Carlo

    2016-02-01

    Platelet activation is persistently enhanced, and its inhibition by low-dose aspirin is impaired in type 2 diabetes mellitus. We investigated in vivo thromboxane (TX) and prostacyclin (PGI2) biosynthesis and their determinants, as well as aspirin responsiveness, in young adult subjects with type 1 diabetes mellitus (T1DM) without overt cardiovascular disease and stable glycemic control. The biosynthesis of TXA2 was persistently increased in subjects with T1DM versus matched healthy subjects, with females showing higher urinary TX metabolite (TXM) excretion than male subjects with T1DM. Microalbuminuria and urinary 8-iso-PGF2α, an index of in vivo oxidative stress, independently predicted TXM excretion in T1DM. No homeostatic increase in PGI2 biosynthesis was detected. Platelet COX-1 suppression by low-dose aspirin and the kinetics of its recovery after drug withdrawal were similar in patients and control subjects and were unaffected by glucose variability. We conclude that patients with T1DM and stable glycemic control display enhanced platelet activation correlating with female sex and microvascular and oxidative damages. Moreover, aspirin responsiveness is unimpaired in T1DM, suggesting that the metabolic disturbance per se is unrelated to altered pharmacodynamics. The efficacy and safety of low-dose aspirin in T1DM warrant further clinical investigation. PMID:26470782

  4. Activation of signal transduction in platelets by the tyrosine phosphatase inhibitor pervanadate (vanadyl hydroperoxide).

    PubMed Central

    Pumiglia, K M; Lau, L F; Huang, C K; Burroughs, S; Feinstein, M B

    1992-01-01

    The protein tyrosine phosphatase (PTPase) inhibitor pervanadate (vanadyl hydroperoxide) stimulated protein tyrosine phosphorylation 29-fold more than did thrombin in intact and saponin-permeabilized platelets. Increased tyrosine phosphorylation preceded, or was coincident with, a fall in PtdIns(4,5)P2 levels, production of PtdIns(3,4)P2 and phosphatidic acid, mobilization of intracellular Ca2+, stimulation of protein kinase C-dependent protein phosphorylation, secretion of dense and alpha-granules, increased actin polymerization, shape change and aggregation which required fibrinogen and was mediated by increased surface expression of GPIIb-IIIa. The tyrosine kinase inhibitor RG 50864 totally prevented induction of tyrosine phosphorylation by pervanadate, as well as all other responses measured; in contrast, the inactive structural analogue, tyrphostin #1, had no effect. Dense-granule secretion induced by pervanadate required protein kinase C activity; however, aggregation and alpha-granule secretion were independent of protein kinase C. In saponin-permeabilized platelets pervanadate and thrombin stimulated phospholipase C activity by GTP-independent and GTP-dependent mechanisms respectively. We conclude that PTPases are important regulators of signal transduction in platelets. Images Fig. 1. Fig. 2. PMID:1530576

  5. Thrombin-activated human platelets acutely generate oxidized docosahexaenoic-acid-containing phospholipids via 12-lipoxygenase.

    PubMed

    Morgan, Lloyd T; Thomas, Christopher P; Kühn, Hartmut; O'Donnell, Valerie B

    2010-10-01

    Arachidonate-containing oxidized phospholipids are acutely generated by 12-LOX (12-lipoxygenase) in agonist-activated platelets. In the present study, formation of structurally related lipids by oxidation of DHA (docosahexaenoic acid)-containing phospholipids is demonstrated using lipidomic approaches. Precursor scanning reverse-phase LC (liquid chromatography)-MS/MS (tandem MS) identified a new family of lipids that comprise phospholipid-esterified HDOHE (hydroxydocosahexaenoic acid). Two diacyl and two plasmalogen PEs (phosphatidylethanolamines) containing predominantly the 14-HDOHE positional isomer (18:0p/14-HDOHE-PE, 18:0a/14-HDOHE-PE, 16:0a/14-HDOHE-PE and 16:0p/14-HDOHE-PE) were structurally characterized using MS/MS and by comparison with biogenic standards. An involvement of 12-LOX was indicated as purified recombinant human 12-LOX also generated the 14-HDOHE isomer from DHA. Pharmacological studies using inhibitors and recombinant platelet 12-LOX indicate that they form via esterification of newly formed non-esterified HDOHE. HDOHE-PEs formed at significant rates (2-4 ng/4×10(7) cells) within 2-180 min of thrombin stimulation, and their formation was blocked by calcium chelation. In summary, a new family of oxidized phospholipid was identified in thrombin-activated human platelets. PMID:20653566

  6. Platelet-activating factor in bronchoalveolar lavage from patients with sarcoidosis.

    PubMed

    Scappaticci, E; Libertucci, D; Bottomicca, F; Da Col, R; Silvestro, L; Tetta, C; Camussi, G

    1992-08-01

    Platelet-activating factor (PAF), a lipid mediator of inflammation and anaphylaxis, may play a role in several physiopathologic alterations of the lung. A lipid compound with physicochemical and biologic characteristics similar to synthetic PAF was extracted and purified from bronchoalveolar lavage (BAL) fluid of 15 of 34 patients with sarcoidosis. PAF was quantitated by a bioassay on washed rabbit platelets. The specificity of platelet aggregation was assessed by using two different PAF receptor antagonists. The incidence of detectable amounts of PAF in BAL fluid of sarcoid patients was statistically significant (chi 2 = 4.064, p = 0.044) when compared with the 14 normal control subjects. The results demonstrated an increased production of PAF in the lower respiratory tract of patients with sarcoidosis. The presence of PAF in BAL fluid, however, did not correlate with radiologic stage, intensity of alveolitis, gallium scanning positivity, angiotensin-converting enzyme serum level, or lung function tests. Therefore, a direct relationship between presence of PAF in BAL fluid and activity of lung disease in patients with sarcoidosis was not directly established. PMID:1336939

  7. Exosome poly-ubiquitin inhibits platelet activation, downregulates CD36, and inhibits pro-atherothombotic cellular functions

    PubMed Central

    Srikanthan, Sowmya; Li, Wei; Silverstein, Roy L.; McIntyre, Thomas M.

    2014-01-01

    Introduction Activated platelets shed microparticles from plasma membranes, but also release smaller exosomes from internal compartments. While microparticles participate in athero-thrombosis, little is known of exosomes in this process. Materials & Methods Ex vivo biochemical experiments with human platelets and exosomes, and FeCl3-induced murine carotid artery thrombosis. Results Both microparticles and exosomes were abundant in human plasma. Platelet-derived exosomes suppressed ex vivo platelet aggregation and reduced adhesion to collagen-coated microfluidic channels at high shear. Injected exosomes inhibited occlusive thrombosis in FeCl3-damaged murine carotid arteries. Control platelets infused into irradiated, thrombocytopenic mice reconstituted thrombosis in damaged carotid arteries, but failed to do so after prior ex vivo incubation with exosomes. CD36 promotes platelet activation, and exosomes dramatically reduced platelet CD36. CD36 is also expressed by macrophages where it binds and internalizes oxidized LDL and microparticles, supplying lipid to promote foam cell formation. Platelet exosomes inhibited oxidized-LDL binding and cholesterol loading into macrophages. Exosomes were not competitive CD36 ligands, but instead sharply reduced total macrophage CD36 content. Exosomal proteins, in contrast to microparticle or cellular proteins, were highly adducted by ubiquitin. Exosomes enhanced ubiquitination of cellular proteins, including CD36, and blockade of proteosome proteolysis with MG-132 rescued CD36 expression. Recombinant unanchored K48 poly-ubiquitin behaved similarly to exosomes, inhibiting platelet function, macrophage CD36 expression, and macrophage particle uptake. Conclusions Platelet-derived exosomes inhibit athero-thrombotic processes by reducing CD36-dependent lipid loading of macrophages and by suppressing platelet thrombosis. Exosomes increase protein ubiquitination, and enhance proteasome degradation of CD36. PMID:25163645

  8. Full activation of mouse platelets requires ADP secretion regulated by SERCA3 ATPase-dependent calcium stores.

    PubMed

    Elaïb, Ziane; Adam, Frédéric; Berrou, Eliane; Bordet, Jean-Claude; Prévost, Nicolas; Bobe, Régis; Bryckaert, Marijke; Rosa, Jean-Philippe

    2016-08-25

    The role of the sarco-endoplasmic reticulum calcium (Ca(2+)) adenosine triphosphatase (ATPase) 3 (SERCA3) in platelet physiology remains poorly understood. Here, we show that SERCA3 knockout (SERCA3(-/-)) mice exhibit prolonged tail bleeding time and rebleeding. Thrombus formation was delayed both in arteries and venules in an in vivo ferric chloride-induced thrombosis model. Defective platelet adhesion and thrombus growth over collagen was confirmed in vitro. Adenosine 5'-diphosphate (ADP) removal by apyrase diminished adhesion and thrombus growth of control platelets to the level of SERCA3(-/-) platelets. Aggregation, dense granule secretion, and Ca(2+) mobilization of SERCA3(-/-) platelets induced by low collagen or low thrombin concentration were weaker than controls. Accordingly, SERCA3(-/-) platelets exhibited a partial defect in total stored Ca(2+) and in Ca(2+) store reuptake following thrombin stimulation. Importantly ADP, but not serotonin, rescued aggregation, secretion, and Ca(2+) mobilization in SERCA3(-/-) platelets, suggesting specificity. Dense granules appeared normal upon electron microscopy, mepacrine staining, and total serotonin content, ruling out a dense granule defect. ADP induced normal platelet aggregation, excluding a defect in ADP activation pathways. The SERCA3-specific inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone diminished both Ca(2+) mobilization and secretion of control platelets, as opposed to the SERCA2b inhibitor thapsigargin. This confirmed the specific role of catalytically active SERCA3 in ADP secretion. Accordingly, SERCA3-dependent Ca(2+) stores appeared depleted in SERCA3(-/-) platelets. Finally, αIIbβ3 integrin blockade did not affect SERCA3-dependent secretion, therefore proving independent of αIIbβ3 engagement. Altogether, these results show that SERCA3-dependent Ca(2+) stores control a specific ADP secretion pathway required for full platelet secretion induced by agonists at low concentration and independent

  9. Effect of Antrodia camphorata on Inflammatory Arterial Thrombosis-Mediated Platelet Activation: The Pivotal Role of Protein Kinase C

    PubMed Central

    Lu, Wan-Jung; Lan, Chang-Chou; Lee, Tzu-Yin; Hsia, Chih-Hsuan; Lee, Hsiu-Chuan; Sheu, Joen-Rong

    2014-01-01

    Antrodia camphorata is a rare Taiwanese medicinal mushroom. Antrodia camphorata extract has been reported to exhibit antioxidant, anti-inflammation, antimetastasis, and anticancer activities and plays a role in liver fibrosis, vasorelaxation, and immunomodulation. Critical vascular inflammation leads to vascular dysfunction and cardiovascular diseases, including abdominal aortic aneurysms, hypertension, and atherosclerosis. Platelet activation plays a crucial role in intravascular thrombosis, which is involved in a wide variety of cardiovascular diseases. However, the effect of Antrodia camphorata on platelet activation remains unclear. We examined the effects of Antrodia camphorata on platelet activation. In the present study, Antrodia camphorata treatment (56–224 μg/mL) inhibited platelet aggregation induced by collagen, but not U46619, an analogue of thromboxane A2, thrombin, and arachidonic acid. Antrodia camphorata inhibited collagen-induced calcium (Ca2+) mobilization and phosphorylation of protein kinase C (PKC) and Akt. In addition, Antrodia camphorata significantly reduced the aggregation and phosphorylation of PKC in phorbol-12, 13-dibutyrate (PDBu) activated platelets. In conclusion, Antrodia camphorata may inhibit platelet activation by inhibiting of Ca2+ and PKC cascade and the Akt pathway. Our study suggests that Antrodia camphorata may be a potential therapeutic agent for preventing or treating thromboembolic disorders. PMID:25541625

  10. Altered E-NTPDase/E-ADA activities and CD39 expression in platelets of sickle cell anemia patients.

    PubMed

    Castilhos, Lívia G; Doleski, Pedro H; Adefegha, Stephen A; Becker, Lara V; Ruchel, Jader B; Leal, Daniela B R

    2016-04-01

    Sickle cell anemia (SCA) is a hemoglobinopathy characterized by hemolysis and vaso-occlusions caused by rigidly distorted red blood cells. Sickle cell crisis is associated with extracellular release of nucleotides and platelets, which are critical mediators of hemostasis participating actively in purinergic thromboregulatory enzymes system.This study aimed to investigate the activities of purinergic system ecto-enzymes present on the platelet surface as well as CD39 and CD73 expressions on platelets of SCA treated patients. Fifteen SCA treated patients and 30 health subjects (control group) were selected. Ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), ecto-5'-nucleotidase (E-5'-NT) and ecto-adenosine deaminase (E-ADA) activities were measured in platelets isolated from these individuals. Results demonstrated an increase of 41 % in the E-NTPDase for ATP hydrolysis, 52% for ADP hydrolysis and 60 % in the E-ADA activity in SCA patients (P<0.05); however, a two folds decrease in the CD39 expression in platelets was observed in the same group (P<0.01). The increased E-NTPDase activity could be a compensatory mechanism associated with the low expression of CD39 in platelets. Besides, alteration of these enzymes activities suggests that the purinergic system could be involved in the thromboregulatory process in SCA patients. PMID:27044834

  11. Enhanced antimicrobial activity of peptide-cocktails against common bacterial contaminants of ex vivo stored platelets.

    PubMed

    Mohan, K V K; Rao, S Sainath; Gao, Y; Atreya, C D

    2014-01-01

    Bacterial contamination of blood components such as ex vivo-stored platelets is a major safety risk in transfusion medicine. We have recently shown that synthetic antimicrobial peptides named PD1-PD4 derived from the thrombin-induced human platelet-derived antimicrobial proteins, and repeats of Arg-Trp (RW1-RW5) demonstrate microbicidal activity against selected bacteria and viruses. In the present study, we selected PD3, PD4, RW2, RW3 and RW4 and evaluated each individual peptide and their various combinations to see whether the cocktail regimen enhances the antimicrobial activity above and over the individual peptides. Stored platelet or plasma samples spiked with known titres of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Bacillus cereus were treated with either individual peptides or with peptides in various combinations. Analyses revealed that individual peptides show moderate microbicidal activity (10- to 100-fold reduction) against the tested bacteria relative to their combined regimen. The peptide combinations (RW2 + RW4, RW2 + RW3 + RW4 and PD4 + RW3 + RW4) on the other hand enhanced the microbicidal activity (c.10 000-fold reduction) and revealed a minimal inhibitory concentration of 5 μM. Time-kill kinetics indicated that these three peptide combinations exhibited enhanced antimicrobial activity bringing about a 100-fold reduction of bacterial titres within 20 min of incubation. The present study therefore demonstrates the synergistic effect of antimicrobial peptides when used in combinations and provides a proof-of-concept of its potential application as a molecular tool towards pathogen reduction and further extends the possibility of using peptide combinatorial therapeutics as broad-spectrum antibiotics or as alternatives to combat drug-resistant bacteria. PMID:23926880

  12. Interference of anti-inflammatory and anti-asthmatic drugs with neutrophil-mediated platelet activation: singularity of azelastine.

    PubMed Central

    Renesto, P.; Balloy, V.; Vargaftig, B. B.; Chignard, M.

    1991-01-01

    1. The capacity of various drugs (acetylsalicylic acid (ASA), ketoprofen, diclofenac, piroxicam, BW 755C, BW A4C, nedocromil sodium and azelastine) to inhibit human polymorphonuclear neutrophil (PMN)-mediated platelet activation was investigated. In this model, stimulated PMN release cathepsin G (Cat G), a serine proteinase which, in turn, induces platelet activation. 2. Among the different tested drugs, azelastine (100 microM for 1 min) was the only one able to prevent platelet aggregation. The cyclo-oxygenase inhibitors were all inactive, although used at effective concentrations as judged by inhibition of thromboxane B2 (TxB2) formation. Inhibition of platelet aggregation by azelastine was concentration-dependent, the range of active concentrations being of 20-70 microM. Release from platelets of 5-hydroxytryptamine was also inhibited at 30 microM and above, but never reached 100%. 3. The inhibition by azelastine is due to an effect on both cells. Indeed, beta-glucuronidase release from activated PMN and platelet activation by purified Cat G were both affected. 4. However, used at high concentrations (greater than 100 microM) azelastine was toxic since it released significant amounts of lactate dehydrogenase (LDH) from PMN and platelets. 5. These results show the capacity of azelastine, an anti-allergic and anti-asthmatic compound, to inhibit the cell-to-cell communication between PMN and platelets, an effect which may be relevant for its therapeutic efficacy or for a new application in diseases in which PMN and platelets are involved. PMID:1653073

  13. Structural and Functional Analysis of a Platelet-Activating Lysophosphatidylcholine of Trypanosoma cruzi

    PubMed Central

    Gazos-Lopes, Felipe; Oliveira, Mauricio M.; Hoelz, Lucas V. B.; Vieira, Danielle P.; Marques, Alexandre F.; Nakayasu, Ernesto S.; Gomes, Marta T.; Salloum, Nasim G.; Pascutti, Pedro G.; Souto-Padrón, Thaïs; Monteiro, Robson Q.

    2014-01-01

    Background Trypanosoma cruzi is the causative agent of the life-threatening Chagas disease, in which increased platelet aggregation related to myocarditis is observed. Platelet-activating factor (PAF) is a potent intercellular lipid mediator and second messenger that exerts its activity through a PAF-specific receptor (PAFR). Previous data from our group suggested that T. cruzi synthesizes a phospholipid with PAF-like activity. The structure of T. cruzi PAF-like molecule, however, remains elusive. Methodology/Principal findings Here, we have purified and structurally characterized the putative T. cruzi PAF-like molecule by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Our ESI-MS/MS data demonstrated that the T. cruzi PAF-like molecule is actually a lysophosphatidylcholine (LPC), namely sn-1 C18:1(delta 9)-LPC. Similar to PAF, the platelet-aggregating activity of C18:1-LPC was abrogated by the PAFR antagonist, WEB 2086. Other major LPC species, i.e., C16:0-, C18:0-, and C18:2-LPC, were also characterized in all T. cruzi stages. These LPC species, however, failed to induce platelet aggregation. Quantification of T. cruzi LPC species by ESI-MS revealed that intracellular amastigote and trypomastigote forms have much higher levels of C18:1-LPC than epimastigote and metacyclic trypomastigote forms. C18:1-LPC was also found to be secreted by the parasite in extracellular vesicles (EV) and an EV-free fraction. A three-dimensional model of PAFR was constructed and a molecular docking study was performed to predict the interactions between the PAFR model and PAF, and each LPC species. Molecular docking data suggested that, contrary to other LPC species analyzed, C18:1-LPC is predicted to interact with the PAFR model in a fashion similar to PAF. Conclusions/Significance Taken together, our data indicate that T. cruzi synthesizes a bioactive C18:1-LPC, which aggregates platelets via PAFR. We propose that C18:1-LPC might be an important lipid mediator in the

  14. Increased levels of platelet-activating factor in blood following intestinal ischemia in the dog.

    PubMed

    Filep, J; Hermán, F; Braquet, P; Mózes, T

    1989-01-31

    The possible role of platelet-activating factor (PAF) in superior mesenteric artery occlusion induced circulatory collapse was studied in anesthetized dogs. PAF was measured by platelet aggregation assay. Identity of PAF-like product in blood was ascertained by thin layer chromatography, high pressure liquid chromatography and alkaline treatment. Low amount of PAF was detected in the mesenteric blood under normal conditions, during reperfusion PAF levels were significantly higher. Pretreatment of the animals with BN 52021, a specific PAF receptor antagonist abolished the fall in mean arterial pressure and the rise in hematocrit due to ischemia/reperfusion. These findings suggest that PAF may play an important role in mesenteric ischemia-induced circulatory collapse. PMID:2916986

  15. Carotid endarterectomy in patients with heparin-induced platelet activation: comparative efficacy of aspirin and iloprost (ZK36374).

    PubMed

    Kappa, J R; Cottrell, E D; Berkowitz, H D; Fisher, C A; Sobel, M; Ellison, N; Addonizio, V P

    1987-05-01

    Patients with heparin-induced platelet activation who are reexposed to heparin may have recurrent thrombocytopenia, intravascular thrombosis, arterial emboli, or sudden death. To permit carotid endarterectomy in two patients with confirmed heparin-induced platelet activation, we compared the efficacies of aspirin and iloprost, a stable analogue of prostacyclin, in preventing heparin-induced platelet activation. In the first patient, although aspirin prevented both in vitro heparin-induced platelet aggregation (70% without and 7.5% with aspirin) and 14C serotonin release (48% without and 0% with aspirin), intraoperative administration of heparin resulted in an increase in plasma levels of platelet factor 4 from 8 to 260 ng/ml and beta-thromboglobulin levels from 29 to 39 ng/ml. In addition, the circulating platelet count decreased from 221,000 to 174,000 microliters, and 15% spontaneous platelet aggregation was observed. Fortunately, fibrinopeptide A levels remained less than 10 ng/ml intraoperatively, and no thrombotic complications occurred. In the second patient, aspirin did not prevent heparin-induced platelet aggregation in vitro (65% without and 41% with aspirin); however, iloprost (0.01 mumol/L) prevented both in vitro heparin-induced platelet aggregation (59.5% without and 0.0% with iloprost) and 14C serotonin release (56.7% without and 0.0% with iloprost). Therefore, a continuous infusion of iloprost was begun before administration of heparin and was continued until 20 minutes after reversal of heparin with protamine. After intraoperative administration of heparin, plasma levels of platelet factor 4 increased from 19 to 200 ng/ml, and beta-thromboglobulin levels increased from 56 to 76 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2437338

  16. Platelet-Activating Factor Receptors Mediate Excitatory Postsynaptic Hippocampal Injury in Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Geathers, Jasmine S.; Allan, Kevin C.; Gelbard, Harris A.

    2016-01-01

    Gray matter degeneration contributes to progressive disability in multiple sclerosis (MS) and can occur out of proportion to measures of white matter disease. Although white matter pathology, including demyelination and axon injury, can lead to secondary gray matter changes, we hypothesized that neurons can undergo direct excitatory injury within the gray matter independent of these. We tested this using a model of experimental autoimmune encephalomyelitis (EAE) with hippocampal degeneration in C57BL/6 mice, in which immunofluorescent staining showed a 28% loss of PSD95-positive excitatory postsynaptic puncta in hippocampal area CA1 compared with sham-immunized controls, despite preservation of myelin and VGLUT1-positive excitatory axon terminals. Loss of postsynaptic structures was accompanied by appearance of PSD95-positive debris that colocalized with the processes of activated microglia at 25 d after immunization, and clearance of debris was followed by persistently reduced synaptic density at 55 d. In vitro, addition of activated BV2 microglial cells to hippocampal cultures increased neuronal vulnerability to excitotoxic dendritic damage following a burst of synaptic activity in a manner dependent on platelet-activating factor receptor (PAFR) signaling. In vivo treatment with PAFR antagonist BN52021 prevented PSD95-positive synapse loss in hippocampi of mice with EAE but did not affect development of EAE or local microglial activation. These results demonstrate that postsynaptic structures can be a primary target of injury within the gray matter in autoimmune neuroinflammatory disease, and suggest that this may occur via PAFR-mediated modulation of activity-dependent synaptic physiology downstream of microglial activation. SIGNIFICANCE STATEMENT Unraveling gray matter degeneration is critical for developing treatments for progressive disability and cognitive impairment in multiple sclerosis (MS). In a mouse model of MS, we show that neurons can undergo injury

  17. Myosin-II repression favors pre/proplatelets but shear activation generates platelets and fails in macrothrombocytopenia

    PubMed Central

    Spinler, Kyle R.; Shin, Jae-Won; Lambert, Michele P.

    2015-01-01

    Megakaryocyte ploidy and the generation of pre/proplatelets are both increased in culture by pharmacologic inhibition of myosin-II, but nonmuscle myosin-IIA (MIIA) mutations paradoxically cause MYH9-related diseases (MYH9-RD) that adversely affect platelets. In marrow, megakaryocytes extend projections into the microcirculation, where shear facilitates fragmentation to large pre/proplatelets, suggesting that fluid stresses and myosin-II activity somehow couple in platelet biogenesis. Here, in bulk shear, plateletlike particles generated from megakaryocytes are maximized at a shear stress typical of that in the microcirculation and after treatment with a myosin-II inhibitor. MIIA activity in static cells is naturally repressed through phosphorylation at Serine-1943, but shear decreases phosphorylation, consistent with MIIA activation and localization to platelet cortex. Micropipette aspiration of cells shows myosin-II accumulates at stressed sites, but its inhibition prevents such mechanoactivation and facilitates generation of CD41+ fragments similar in size to pre/proplatelets. MYH9-RD mutants phenocopy inhibition, revealing a dominant negative effect. MIIA is diffuse in the large platelets of a MYH9-RD patient with macrothrombocytopenia and is also diffuse in normal pre/proplatelets treated with inhibitor that blocks in vitro division to small platelets. The findings explain the large platelets in MYH9-RD and the near-normal thrombocrit of patients. Myosin-II regulation thus controls platelet size and number. PMID:25395423

  18. A Potential Mechanism of High-Dose Ticagrelor in Modulating Platelet Activity and Atherosclerosis Mediated by Thymic Stromal Lymphopoietin Receptor

    PubMed Central

    Mao, Yi; Peng, Yudong; Zeng, Qiutang; Cheng, Longxian; Wang, Boyuan; Mao, Xiaobo; Meng, Kai; Liu, Yuzhou; Lian, Yitian; Li, Dazhu

    2015-01-01

    Abnormal expression of thymic stromal lymphopoietin (TSLP) and its receptor (TSLPR) was found in patients with acute coronary syndrome. Ticagrelor, an oral platelet ADP P2Y12 receptor antagonist, is widely used in these patients. The aim of this study was to verify whether different doses of ticagrelor regulated plaque progression and platelet activity by modulating TSLP/TSLPR. Seventy-five ApoE-/- mice were randomly divided into five groups: (1) high-cholesterol diet (HCD, n = 15); (2) HCD plus ticagrelor 25 mg/kg/d (T1, n = 15); (3) HCD plus ticagrelor 50 mg/kg/d (T2, n = 15); (4) HCD plus ticagrelor 100 mg/kg/d (T3, n = 15); and (5) a normal diet group (ND, n = 15). At day 0 and at week 16, blood lipids and serum TSLP levels, expression of TSLPR, CD62, and CD63, platelet aggregation, platelet ATP release, PI3K/Akt signaling pathway, and plaque morphology were assessed. HCD increased TSLPR expression and atherosclerosis progression but high-dose ticagrelor (100 mg/kg) moderated this trend. TSLPR was positively correlated with Akt1, platelet aggregation, corrected plaque area, and vulnerability index in the T3 group (P<0.01). In conclusion, low-dose ticagrelor only inhibited platelet activity. Besides this inhibition, high-dose ticagrelor modulated platelet activity and atherosclerosis mediated by TSLPR, potentially through the PI3K/Akt signal pathway. PMID:26517374

  19. Prothrombin Activation by Platelet-associated Prothrombinase Proceeds through the Prethrombin-2 Pathway via a Concerted Mechanism*

    PubMed Central

    Haynes, Laura M.; Bouchard, Beth A.; Tracy, Paula B.; Mann, Kenneth G.

    2012-01-01

    The protease α-thrombin is a key enzyme of the coagulation process as it is at the cross-roads of both the pro- and anti-coagulant pathways. The main source of α-thrombin in vivo is the activation of prothrombin by the prothrombinase complex assembled on either an activated cell membrane or cell fragment, the most relevant of which is the activated platelet surface. When prothrombinase is assembled on synthetic phospholipid vesicles, prothrombin activation proceeds with an initial cleavage at Arg-320 yielding the catalytically active, yet effectively anticoagulant intermediate meizothrombin, which is released from the enzyme complex ∼30–40% of the time. Prothrombinase assembled on the surface of activated platelets has been shown to proceed through the inactive intermediate prethrombin-2 via an initial cleavage at Arg-271 followed by cleavage at Arg-320. The current work tests whether or not platelet-associated prothrombinase proceeds via a concerted mechanism through a study of prothrombinase assembly and function on collagen-adhered, thrombin-activated, washed human platelets in a flow chamber. Prothrombinase assembly was demonstrated through visualization of bound factor Xa by confocal microscopy using a fluorophore-labeled anti-factor Xa antibody, which demonstrated the presence of distinct platelet subpopulations capable of binding factor Xa. When prothrombin activation was monitored at a typical venous shear rate over preassembled platelet-associated prothrombinase neither potential intermediate, meizothrombin or prethrombin-2, was observed in the effluent. Collectively, these findings suggest that platelet-associated prothrombinase activates prothrombin via an efficient concerted mechanism in which neither intermediate is released. PMID:22989889

  20. Novel phosphatidylethanolamine derivatives accumulate in circulation in hyperlipidemic ApoE-/- mice and activate platelets via TLR2.

    PubMed

    Biswas, Sudipta; Xin, Liang; Panigrahi, Soumya; Zimman, Alejandro; Wang, Hua; Yakubenko, Valentin P; Byzova, Tatiana V; Salomon, Robert G; Podrez, Eugene A

    2016-05-26

    A prothrombotic state and increased platelet reactivity are common in dyslipidemia and oxidative stress. Lipid peroxidation, a major consequence of oxidative stress, generates highly reactive products, including hydroxy-ω-oxoalkenoic acids that modify autologous proteins generating biologically active derivatives. Phosphatidylethanolamine, the second most abundant eukaryotic phospholipid, can also be modified by hydroxy-ω-oxoalkenoic acids. However, the conditions leading to accumulation of such derivatives in circulation and their biological activities remain poorly understood. We now show that carboxyalkylpyrrole-phosphatidylethanolamine derivatives (CAP-PEs) are present in the plasma of hyperlipidemic ApoE(-/-) mice. CAP-PEs directly bind to TLR2 and induces platelet integrin αIIbβ3 activation and P-selectin expression in a Toll-like receptor 2 (TLR2)-dependent manner. Platelet activation by CAP-PEs includes assembly of TLR2/TLR1 receptor complex, induction of downstream signaling via MyD88/TIRAP, phosphorylation of IRAK4, and subsequent activation of tumor necrosis factor receptor-associated factor 6. This in turn activates the Src family kinases, spleen tyrosine kinase and PLCγ2, and platelet integrins. Murine intravital thrombosis studies demonstrated that CAP-PEs accelerate thrombosis in TLR2-dependent manner and that TLR2 contributes to accelerate thrombosis in mice in the settings of hyperlipidemia. Our study identified the novel end-products of lipid peroxidation, accumulating in circulation in hyperlipidemia and inducing platelet activation by promoting cross-talk between innate immunity and integrin activation signaling pathways. PMID:27015965

  1. Novel phosphatidylethanolamine derivatives accumulate in circulation in hyperlipidemic ApoE−/− mice and activate platelets via TLR2

    PubMed Central

    Biswas, Sudipta; Xin, Liang; Panigrahi, Soumya; Zimman, Alejandro; Wang, Hua; Yakubenko, Valentin P.; Byzova, Tatiana V.; Salomon, Robert G.

    2016-01-01

    A prothrombotic state and increased platelet reactivity are common in dyslipidemia and oxidative stress. Lipid peroxidation, a major consequence of oxidative stress, generates highly reactive products, including hydroxy-ω-oxoalkenoic acids that modify autologous proteins generating biologically active derivatives. Phosphatidylethanolamine, the second most abundant eukaryotic phospholipid, can also be modified by hydroxy-ω-oxoalkenoic acids. However, the conditions leading to accumulation of such derivatives in circulation and their biological activities remain poorly understood. We now show that carboxyalkylpyrrole-phosphatidylethanolamine derivatives (CAP-PEs) are present in the plasma of hyperlipidemic ApoE−/− mice. CAP-PEs directly bind to TLR2 and induces platelet integrin αIIbβ3 activation and P-selectin expression in a Toll-like receptor 2 (TLR2)-dependent manner. Platelet activation by CAP-PEs includes assembly of TLR2/TLR1 receptor complex, induction of downstream signaling via MyD88/TIRAP, phosphorylation of IRAK4, and subsequent activation of tumor necrosis factor receptor–associated factor 6. This in turn activates the Src family kinases, spleen tyrosine kinase and PLCγ2, and platelet integrins. Murine intravital thrombosis studies demonstrated that CAP-PEs accelerate thrombosis in TLR2-dependent manner and that TLR2 contributes to accelerate thrombosis in mice in the settings of hyperlipidemia. Our study identified the novel end-products of lipid peroxidation, accumulating in circulation in hyperlipidemia and inducing platelet activation by promoting cross-talk between innate immunity and integrin activation signaling pathways. PMID:27015965

  2. Induction and antimicrobial activity of platelet basic protein derivatives in human monocytes.

    PubMed

    Schaffner, Andreas; King, Charles C; Schaer, Dominik; Guiney, Donald G

    2004-11-01

    The antimicrobial activity of a number of chemokines has recently come into focus of research about innate immunity. We have previously shown that platelet basic protein (PBP), which gives rise to several antimicrobial peptides of platelets, is also expressed in human monocytes. In the present studies, we show that exposure of human monocytes to bacteria or microbial components (lipopolysaccharide and zymosan) induces a several-fold greater expression of derivates of PBP. Also, activation of proteinase-activated receptors (PARs) by thrombin or the synthetic peptide ligand SFLLRN of PAR-1 significantly increased PBP expression, presumably on the transcriptional level, as evidenced by higher mRNA levels. Derivates of PBP appeared to reach phago-lysosomes, as higher concentration was found in latex phagosomes isolated by a flotation method. By the gel-overlay technique, two bactericidal derivatives of PBP could be visualized, which were immunoreactive with anti-PBP antibody in Western blots. By matrix-assisted laser desorption/ionization time of flight and surface-enhanced laser desorption and ionization techniques, it was confirmed that the bands corresponded to PBP derivates. After immunofixation with a monoclonal antibody to PBP, the major peptide in zymosan-stimulated monocytes was identified to correspond by molecular weight to connective tissue-activating peptide III, which has been reported to be a major antimicrobial PBP derivate also in platelets. Our observations indicate that PBP and its derivates are constituents of the antimicrobial arsenal of human monocytes. Their increased expression after exposure to microorganisms allows a rapid host response to pathogens. PMID:15316029

  3. Dissociation of SERPINE1 mRNA from the translational repressor proteins Ago2 and TIA-1 upon platelet activation.

    PubMed

    Corduan, Aurélie; Plé, Hélène; Laffont, Benoit; Wallon, Thérèse; Plante, Isabelle; Landry, Patricia; Provost, Patrick

    2015-05-01

    Platelets play an important role in haemostasis, as well as in thrombosis and coagulation processes. They harbour a wide variety of messenger RNAs (mRNAs), that can template de novo protein synthesis, and an abundant array of microRNAs, which are known to mediate mRNA translational repression through proteins of the Argonaute (Ago) family. The relationship between platelet microRNAs and proteins capable of mediating translational repression, however, remains unclear. Here, we report that half of platelet microRNAs is associated to mRNA-regulatory Ago2 protein complexes, in various proportions. Associated to these Ago2 complexes are platelet mRNAs known to support de novo protein synthesis. Reporter gene activity assays confirmed the capacity of the platelet microRNAs, found to be associated to Ago2 complexes, to regulate translation of these platelet mRNAs through their 3'UTR. Neither the microRNA repertoire nor the microRNA composition of Ago2 complexes of human platelets changed upon activation with thrombin. However, under conditions favoring de novo synthesis of Plasminogen Activator Inhibitor-1 (PAI-1) protein, we documented a rapid dissociation of the encoding platelet SERPINE1 mRNA from Ago2 protein complexes as well as from the translational repressor protein T-cell-restricted intracellular antigen-1 (TIA-1). These findings are consistent with a scenario by which lifting of the repressive effects of Ago2 and TIA-1 protein complexes, involving a rearrangement of proteinmRNA complexes rather than disassembly of Ago2microRNA complexes, would allow translation of SERPINE1 mRNA into PAI-1 in response to platelet activation. PMID:25673011

  4. Platelet MAO-B activity and vitamin B12 in old age dementias.

    PubMed

    Parnetti, L; Mecocci, P; Reboldi, G P; Santucci, C; Brunetti, M; Gaiti, A; Cadini, D; Senin, U

    1992-01-01

    Platelet MAO-B activity, serum vitamin B12 levels, and plasma folate were measured in patients suffering from presenile (AD) and senile (SDAT) dementia of Alzheimer-type, and vascular dementia (VD). MAO-B was higher in the SDAT group than in AD and controls. An inverse relationship between MAO-B activity and vit. B12 levels was documented in the whole group and in each category studied; furthermore, MAO-B was positively related to age. All the patients were then divided into two groups, according to vit. B12 levels (Group I: less than 200 pg/mL; Group II: greater than or equal to 200 pg/mL); Group I showed a significantly higher MAO-B activity with respect to Group II. The results indicate the existence of a negative association between platelet MAO-B activity and serum levels of vitamin B12 and confirm the existence of biological differences between presenile and senile dementia of Alzheimer type. PMID:1520404

  5. Mediators and molecular pathways involved in the regulation of neutrophil extracellular trap formation mediated by activated platelets.

    PubMed

    Carestia, Agostina; Kaufman, Tomás; Rivadeneyra, Leonardo; Landoni, Verónica Inés; Pozner, Roberto Gabriel; Negrotto, Soledad; D'Atri, Lina Paola; Gómez, Ricardo Martín; Schattner, Mirta

    2016-01-01

    In addition to being key elements in hemostasis and thrombosis, platelets amplify neutrophil function. We aimed to gain further insight into the stimuli, mediators, molecular pathways, and regulation of neutrophil extracellular trap formation mediated by human platelets. Platelets stimulated by lipopolysaccharide, a wall component of gram-negative bacteria, Pam3-cysteine-serine-lysine 4, a mimetic of lipopeptide from gram-positive bacteria, Escherichia coli, Staphylococcus aureus, or physiologic platelet agonists promoting neutrophil extracellular trap formation and myeloperoxidase-associated DNA activity under static and flow conditions. Although P-selectin or glycoprotein IIb/IIIa were not involved, platelet glycoprotein Ib, neutrophil cluster of differentiation 18, and the release of von Willebrand factor and platelet factor 4 seemed to be critical for the formation of neutrophil extracellular traps. The secretion of these molecules depended on thromboxane A(2) production triggered by lipopolysaccharide or Pam3-cysteine-serine-lysine 4 but not on high concentrations of thrombin. Accordingly, aspirin selectively inhibited platelet-mediated neutrophil extracellular trap generation. Signaling through extracellular signal-regulated kinase, phosphatidylinositol 3-kinase, and Src kinases, but not p38 or reduced nicotinamide adenine dinucleotide phosphate oxidase, was involved in platelet-triggered neutrophil extracellular trap release. Platelet-mediated neutrophil extracellular trap formation was inhibited by prostacyclin. Our results support a role for stimulated platelets in promoting neutrophil extracellular trap formation, reveal that an endothelium-derived molecule contributes to limiting neutrophil extracellular trap formation, and highlight platelet inhibition as a potential target for controlling neutrophil extracellular trap cell death. PMID:26320263

  6. Antimicrobial activity of pure platelet-rich plasma against microorganisms isolated from oral cavity

    PubMed Central

    2013-01-01

    Background Autologous platelet concentrates (PCs) have been extensively used in a variety of medical fields to promote soft and hard tissue regeneration. The significance behind their use lies in the abundance of growth factors in platelets α-granules that promotes wound healing. In addition, antibacterial properties of PCs against various bacteria have been recently pointed out. In this study, the antimicrobial effect of pure platelet-rich plasma (P-PRP) was evaluated against oral cavity microorganisms such as Enterococcus faecalis, Candida albicans, Streptococcus agalactiae, Streptococcus oralis and Pseudomonas aeruginosa. Blood samples were obtained from 17 patients who underwent oral surgery procedures involving the use of P-PRP. The antibacterial activity of P-PRP, evaluated as the minimum inhibitory concentration (MIC), was determined through the microdilution twofold serial method. Results P-PRP inhibited the growth of Enterococcus faecalis, Candida albicans, Streptococcus agalactiae and Streptococcus oralis, but not of Pseudomonas aeruginosa strains. Conclusions P-PRP is a potentially useful substance in the fight against postoperative infections. This might represent a valuable property in adjunct to the enhancement of tissue regeneration. PMID:23442413

  7. Markers of inflammation, activation of blood platelets and coagulation disorders in inflammatory bowel diseases.

    PubMed

    Matowicka-Karna, Joanna

    2016-01-01

    Inflammatory bowel disease (IBD) includes ulcerative colitis and Crohn's disease. It is a group of chronic disorders characterized by inflammation of the gastrointestinal track with unknown etiology. Currently applied biomarkers include CRP, ESR, pANCA, ASCA, and fecal calprotectin. The etiopathogenesis of IBD is multifactorial. In patients with IBD in inflamed alimentary tract mucosa the number of recruited monocytes and activated macrophages which are source of cytokines. In IBD, the exacerbation is accompanied by thrombocytosis. Platelets play a crucial role in the hemostasis and inflammatory response. Selectins, which regulates the hemostasis and inflammatory response, stimulates the secretion of many inflammatory mediators such as β-thromboglobuline, CD40L, fibrinogen, IL-1β, platelet factor-4. In the course of IBD the following changes are observed: an increase in the number of platelets (reactive thrombocytosis), PDW and PCT, reduction in MPV, increased production and excretion of granular content products (P-selectin, GP53, β-TG, PF-4, vWF, fibrinolytic inhibitors). PMID:27117106

  8. Platelet-activating factor induces phospholipid turnover, calcium flux, arachidonic acid liberation, eicosanoid generation, and oncogene expression in a human B cell line

    SciTech Connect

    Schulam, P.G.; Kuruvilla, A.; Putcha, G.; Mangus, L.; Franklin-Johnson, J.; Shearer, W.T. )

    1991-03-01

    Platelet-activating factor is a potent mediator of the inflammatory response. Studies of the actions of platelet-activating factor have centered mainly around neutrophils, monocytes, and platelets. In this report we begin to uncover the influence of platelet-activating factor on B lymphocytes. Employing the EBV-transformed human B cell line SKW6.4, we demonstrate that platelet-activating factor significantly alters membrane phospholipid metabolism indicated by the incorporation of 32P into phosphatidylcholine, phosphatidylinositol, and phosphatidic acid but not significantly into phosphatidylethanolamine at concentrations ranging from 10(-9) to 10(-6) M. The inactive precursor, lyso-platelet-activating factor, at a concentration as high as 10(-7) M had no effect on any of the membrane phospholipids. We also show that platelet-activating factor from 10(-12) to 10(-6) M induced rapid and significant elevation in intracellular calcium levels, whereas lyso-platelet-activating factor was again ineffective. We further demonstrate the impact of platelet-activating factor binding to B cells by measuring platelet-activating factor induced arachidonic acid release and 5-hydroxyeicosatetraenoic acid production. Moreover, platelet-activating factor was capable of inducing transcription of the nuclear proto-oncogenes c-fos and c-jun. Finally we explored the possible role of 5-hydroxyeicosatetraenoic acid as a regulator of arachidonic acid liberation demonstrating that endogenous 5-lipoxygenase activity modulates platelet-activating factor induced arachidonic acid release perhaps acting at the level of phospholipase A2. In summary, platelet-activating factor is shown here to have a direct and profound effect on a pure B cell line.

  9. Involvement of Nitric Oxide on Calcium Mobilization and Arachidonic Acid Pathway Activation during Platelet Aggregation with different aggregating agonists.

    PubMed

    Banerjee, Debipriya; Mazumder, Sahana; Kumar Sinha, Asru

    2016-03-01

    Platelet aggregation by different aggregating agonists is essential in the normal blood coagulation process, the excess of which caused acute coronary syndrome (ACS). In all cases, the activation of arachidonic acid by cycloxygenase was needed for the synthesis of thromboxane A2 (TXA2) but the mechanism of arachidonic acid release in platelets remains obscure. Studies were conducted to determine the role of nitric oxide (NO), if any, on the release of arachidonic acid in platelets. The cytosolic Ca(2+) was visualized and quantitated by fluorescent spectroscopy by using QUIN-2. NO was measured by methemoglobin method. Arachidonic acid was determined by HPLC. TXA2 was measured as ThromboxaneB2 (TXB2) by ELISA. Treatment of platelets in platelet-rich plasma (PRP) with different aggregating agents resulted in the inhibition of nitric oxide synthase (NOS) which inhibited the production of NO synthesis and increased TXA2 synthesis. Furthermore, the treatment of washed PRP with different platelet aggregating agents resulted in the increase of [Ca(2+)] in nM ranges. In contrast, the pre-treatment of washed PRP with aspirin increased platelet NO level and inhibited the Ca(2+) mobilization and TXA2 synthesis. These results indicated that the aggregation of platelets by different aggregating agonists was caused by the cytosolic Ca(2+) mobilization due to the inhibition of NOS. PMID:27127451

  10. The effects of an inhibitor of diglyceride lipase on collagen-induced platelet activation.

    PubMed

    Jackson, Elke C G; Ortar, Giorgio; McNicol, Archie

    2013-12-01

    Human platelet activation by collagen occurs in a dose-dependent manner. High concentrations of collagen bind to a pair of receptors, the α2β1 integrin and glycoprotein (GP)VI/Fc-receptor γ-chain (FcRγ), which stimulate a cascade of events including Syk, LAT, Btk, Gads, and phospholipase Cγ2, leading to calcium release and protein kinase C (PKC) activation. Calcium and PKC are responsible for a range of platelet responses including exocytosis and aggregation, as well as the cytosolic phospholipase A2 (cPLA2)-mediated release of arachidonic acid, which is converted to thromboxane (Tx)A2. In contrast, low concentrations of collagen are acutely aspirin-sensitive, and calcium release and aggregation are TxA2-dependent. Under these conditions, cPLA2 is not involved and it has been suggested that phospholipase C generates 1,2-diacylglycerol (DG) from which arachidonic acid is liberated by diglyceride lipase (DGL). Here a novel DGL blocker (OMDM-188) inhibited collagen-, but not arachidonic acid-induced aggregation and TxA2 synthesis. Furthermore, OMDM-188 inhibited collagen-induced arachidonic acid release. Finally OMDM-188 inhibited collagen-induced p38(MAPK) phosphorylation, but not extracellular signal-regulated kinase (ERK) phosphorylation, with no effect on the phosphorylation of either enzyme in response to arachidonic acid. Taken together, these data suggest a role for a pathway involving phospholipase C liberating DG from membrane phospholipids in response to minimally activating concentrations of collagen. The DG serves as a substrate for DGL, potentially under the regulations of p38(MAPK), to release arachidonic acid, which is subsequently converted to TxA2, which mediates the final platelet response. PMID:24042163

  11. Studies of adhesion-dependent platelet activation: distinct roles for different participating receptors can be dissociated by proteolysis of collagen.

    PubMed

    Siljander, P; Lassila, R

    1999-12-01

    The molecular differences between native-type collagen type I fibrils (NC) and their pepsinated monomers (PC) were used to uncover receptors involved in platelet-collagen interaction along the adhesion-activation axis. The platelet-depositing capacity of NC and PC under blood flow and their adhesive properties and respective morphologies, aggregation, procoagulant capacity, and tyrosine phosphorylation were compared under different cationic milieus, including or excluding the glycoprotein (GP) Ia/IIa. NC was consistently a more preferable and activating substrate than PC during flow (5 minutes) and in platelet aggregation. In PPACK-treated blood, both NC (3.3-fold) and PC (2.7-fold) increased platelet attachment on elevation of the shear rate from 500 to 1640 s(-1), whereas in citrated blood, adhesion and thrombus growth on PC were negligible under the high shear rate, unlike on NC (1.9-fold increase). The complete lack of platelet deposition on PC in citrated blood could be overcome by restoring physiological Mg(2+) concentration, and in contrast to NC, platelets interacting with PC were highly dependent on Mg(2+) during adhesion, aggregation, and procoagulant response. Monoclonal antibody (mAb 131.7) against GP IV inhibited platelet deposition to NC in citrated blood (2 minutes) by 49%, which was not further increased by coincubation with mAb against GP Ia (6F1). These results stress the importance of GP Ia/IIa in shear-resistant platelet deposition on collagen monomers. In native fibers, however, the preserved quaternary structure with telopeptides activates additional platelet receptors capable of substituting GP Ia/IIa and GP IV. PMID:10591685

  12. Preparation and photoelectrocatalytic activity of a nano-structured WO{sub 3} platelet film

    SciTech Connect

    Yagi, Masayuki Maruyama, Syou; Sone, Koji; Nagai, Keiji; Norimatsu, Takayoshi

    2008-01-15

    A tungsten trioxide (WO{sub 3}) film was prepared by calcination from a precursor paste including suspended ammonium tungstate and polyethylene glycol (PEG). The ammonium tungstate suspension was yielded by an acid-base reaction of tungstic acid and an ammonium solution followed by deposition with ethanol addition. Thermogravimetric (TG) analysis showed that the TG profile of PEG is significantly influenced by deposited ammonium tungstate, suggesting that PEG is interacting strongly with deposited ammonium tungstate in the suspension paste. X-ray diffraction (XRD) data indicated that the WO{sub 3} film is crystallized by sintering over 400 deg. C. The scanning electron microscopic (SEM) measurement showed that the film is composed of the nano-structured WO{sub 3} platelets. The semiconductor properties of the film were examined by Mott-Schottky analysis to give flat band potential E{sub FB}=0.30 V vs. saturated calomel reference electrode (SCE) and donor carrier density N{sub D}=2.5x10{sup 22} cm{sup -3}, latter of which is higher than previous WO{sub 3} films by two orders of magnitude. The higher N{sub D} was explained by the large interfacial heterojunction area caused by the nano-platelet structure, which apparently increases capacitance per a unit electrode area. The WO{sub 3} film sintered at 550 deg. C produced 3.7 mA cm{sup -2} of a photoanodic current at 1.2 V vs. SCE under illumination with a 500 W xenon lamp due to catalytic water oxidation. This photocurrent was 4.5-12.8 times higher than those for the other control WO{sub 3} films prepared by similar but different procedures. The high catalytic activity could be explained by the nano-platelet structure. The photocurrent was generated on illumination of UV and visible light below 470 nm, and the maximum incident photon-to-current conversion efficiency (IPCE) was 47% at 320 nm at 1.2 V. Technically important procedures for preparation of nano-structured platelets were discussed. - Graphical abstract: A

  13. The effect of aluminium on NTPDase and 5'-nucleotidase activities from rat synaptosomes and platelets.

    PubMed

    Kaizer, Rosilene Rodrigues; Maldonado, Paula Acosta; Spanevello, Rosélia Maria; Corrêa, Maísa C; Gonçalves, Jamile F; Becker, Lara Vargas; Morsch, Vera Maria; Schetinger, Maria Rosa Chitolina

    2007-10-01

    Aluminium (Al), a neurotoxic compound, has been investigated in a large number of studies both in vivo and in vitro. In this study, we investigated the effect in vivo of long-term exposure to Al on NTPDase (nucleoside triphosphate diphosphohydrolase) and 5'-nucleotidase activities in the synaptosomes (obtained from the cerebral cortex and hippocampus) and platelets of rats. Here, we investigated a possible role of platelets as peripheral markers in rats. Rats were loaded by gavage with AlCl(3) 50 mg/(kg day), 5 days per week, totalizing 60 administrations. The animals were divided into four groups: (1) control (C), (2) 50 mg/kg of citrate solution (Ci), (3) 50 mg/kg of Al plus citrate (Al+Ci) solution and (4) 50 mg/kg of Al (Al). ATP hydrolysis was increased in the synaptosomes from the cerebral cortex by 42.9% for Al+Ci and 39.39% for Al, when compared to their respective control (p<0.05). ADP hydrolysis was increased by 13.15% for both Al and Al+Ci, and AMP hydrolysis increased by 32.7% for Al and 27.25% for Al+Ci (p<0.05). In hippocampal synaptosomes, the hydrolysis of ATP, ADP and AMP, was increased by 58.5%, 28.5% and 25.92%, respectively, for Al (p<0.05) and 36.7%, 22.5% and 37.64% for Al+Ci, both when compared to their respective controls. ATP, ADP and AMP hydrolysis, in platelets, was increased by 172.3%, 188.52% and 92.1%, respectively in Al+Ci, and 317.9%, 342.8% and 177.9%, respectively, for Al, when compared to their respective controls (p<0.05). Together, these results indicate that Al increases NTPDase and 5'-nucleotidase activities, in synaptosomal fractions and platelets. Thus, we suggest that platelets could be sensitive peripheral markers of Al toxicity of the central nervous system. PMID:17686601

  14. Plasma Fibrinogen Is a Natural Deterrent to Amyloid β–Induced Platelet Activation and Neuronal Toxicity

    PubMed Central

    Sonkar, Vijay K; Kulkarni, Paresh P; Chaurasia, Susheel N; Dash, Ayusman; Jauhari, Abhishek; Parmar, Devendra; Yadav, Sanjay; Dash, Debabrata

    2016-01-01

    Alzheimer’s disease (AD) is a devastating neurodegenerative disorder, characterized by extensive loss of neurons and deposition of amyloid β (Aβ) in the form of extracellular plaques. Aβ is considered to have a critical role in synaptic loss and neuronal death underlying cognitive decline. Platelets contribute to 95% of circulating amyloid precursor protein that releases Aβ into circulation. We have recently demonstrated that the Aβ active fragment containing amino acid sequence 25–35 (Aβ25–35) is highly thrombogenic in nature and elicits strong aggregation of washed human platelets in a RhoA-dependent manner. In this study, we evaluated the influence of fibrinogen on Aβ-induced platelet activation. Intriguingly, Aβ failed to induce aggregation of platelets suspended in plasma but not in buffer. Fibrinogen brought about dose-dependent decline in aggregatory response of washed human platelets elicited by Aβ25–35, which could be reversed by increasing doses of Aβ. Fibrinogen also attenuated Aβ-induced platelet responses such as secretion, clot retraction, rise in cytosolic Ca+2 and reactive oxygen species. Fibrinogen prevented intracellular accumulation of full-length Aβ peptide (Aβ42) in platelets as well as neuronal cells. We conclude that fibrinogen serves as a physiological check against the adverse effects of Aβ by preventing its interaction with cells. PMID:27262026

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

    PubMed

    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; Rivera, José

    2016-09-01

    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

  16. Isolation of Bioactive Compounds That Relate to the Anti-Platelet Activity of Cymbopogon ambiguus

    PubMed Central

    Grice, I. Darren; Rogers, Kelly L.; Griffiths, Lyn R.

    2011-01-01

    Infusions and decoctions of Cymbopogon ambiguus have been used traditionally in Australia for the treatment of headache, chest infections and muscle cramps. The aim of the present study was to screen and identify bioactive compounds from C. ambiguus that could explain this plant's anti-headache activity. A dichloromethane extract of C. ambiguus was identified as having activity in adenosine-diphosphate-induced human platelet aggregation and serotonin-release inhibition bioassays. Subsequent fractionation of this extract led to the isolation of four phenylpropenoids, eugenol, elemicin, eugenol methylether and trans-isoelemicin. While both eugenol and elemicin exhibited dose-dependent inhibition of ADP-induced human platelet serotonin release, only eugenol displayed potent inhibitory activity with an IC50 value of 46.6 μM, in comparison to aspirin, with an IC50 value of 46.1 μM. These findings provide evidence to support the therapeutic efficacy of C. ambiguus in the non-conventional treatment of headache and inflammatory conditions. PMID:20047890

  17. Micro-structuring of polycarbonate-urethane surfaces in order to reduce platelet activation and adhesion.

    PubMed

    Clauser, Johanna; Gester, Kathrin; Roggenkamp, Jan; Mager, Ilona; Maas, Judith; Jansen, Sebastian V; Steinseifer, Ulrich

    2014-01-01

    In the development of new hemocompatible biomaterials, surface modification appears to be a suitable method in order to reduce the thrombogenetic potential of such materials. In this study, polycarbonate-urethane (PCU) tubes with different surface microstructures to be used for aortic heart valve models were investigated with regard to the thrombogenicity. The surface structures were produced by using a centrifugal casting process for manufacturing PCU tubes with defined casting mold surfaces which are conferred to the PCU surface during the process. Tubes with different structures defined by altering groove widths were cut into films and investigated under dynamic flow conditions in contact with porcine blood. The analysis was carried out by laser scanning microscopy which allowed for counting various morphological types of platelets with regard to the grade of activation. The comparison between plain and shaped PCU samples showed that the surface topography led to a decline of the activation of the coagulation cascade and thus to the reduction of the fibrin synthesis. Comparing different types of structures revealed that smooth structures with a small groove width (d ~ 3 μm) showed less platelet activation as well as less adhesion in contrast to a distinct wave structure (d ~ 90 μm). These results prove surface modification of polymer biomaterials to be a suitable method for reducing thrombogenicity and hence give reason for further alterations and improvements. PMID:24484511

  18. Leishmania parasites possess a platelet-activating factor acetylhydrolase important for virulence

    PubMed Central

    Pawlowic, Mattie C.; Zhang, Kai

    2012-01-01

    Leishmania parasites are intracellular protozoans capable of salvaging and remodeling lipids from the host. To understand the role of lipid metabolism in Leishmania virulence, it is necessary to characterize the enzymes involved in the uptake and turnover of phospholipids. This study focuses on a putative phospholipase A2 (PLA2)/platelet-activating factor acetylhydrolase (PAF-AH) in L. major. In mammals, PAF-AH is a subgroup of PLA2 catalyzing the hydrolysis/inactivation of platelet-activating factor (PAF), a potent mediator of many leukocyte functions. By immunofluorescence microscopy, L. major PLA2/PAF-AH is predominantly localized in the ER. While wild type L. major parasites are able to hydrolyze PAF, this activity is completely absent in the PLA2/PAF-AH-null mutants. Meanwhile, deletion of PLA2/PAF-AH had no significant effect on the turnover of common glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. PLA2/PAF-AH is not required for the growth of L. major parasites in culture, or the production of GPI-anchored virulence factors. Nonetheless, it does play a key role in the mammalian host as the PLA2/PAF-AH null mutants exhibit attenuated virulence in BALB/c mice. In conclusion, these data suggest that Leishmania parasites possess a functional PAF-AH and the degradation of PAF or PAF-like lipids is an important step in infection. PMID:22954769

  19. The immunological generation of a platelet-activating factor and a platet-lytic factor in the rat.

    PubMed Central

    Valone, F H; Whitmer, D I; Pickett, W C; Austen, K F; Goetzl, E J

    1979-01-01

    Antigen challenge of the rat peritoneal cavity which had been prepared with IgGa-rich antiserum generated activities which released [14C]-serotonin from pre-labelled human platelets. After adsorption of these activities onto Amberlite XAD-8 and elution in 80% ethanol, two factors of differing polarity were resolved by chromatography on diethylaminoethyl cellulose in organic solvents. The activity eluting in the 7:1 chloroform:methanol solvent contained a platelet-lytic factor (PLF) assessed by the parallel release of lactic acid dehydrogenase and [14C]-serotonin; the cytotoxicity of this fraction was confirmed by phase-contrast microscopy examination which demonstrated fragmentation of the exposed platelets. The activity eluting in the 1:1 methanol: aqueous 1.0 M ammonium carbonate solvent was a platelet-activating factor (PAF) as defined by release of [14C]-serotonin without lactic acid dehydrogenase. Both the lytic and the activating principles were separable from slow reacting substance of anaphylaxis and polymorphonuclear leucocyte chemotactic activity, and each presented a single activity peak of differing mobility when chromatographed on silica gel H plates. Human eosinophil phospholipase D inactivated the lytic factor by more than 85% in 2 h at 37 degrees without affecting the activity of the activating factor. The release of [14C]-serotonin induced by the PAF was not affected by the absence of calcium from the medium or by elevations in the platelet concentrations of cyclic AMP or cyclic GMP that resulted from pre-incubation of platelets with prostaglandin D2 or sodium ascorbate, respectively. PMID:227784

  20. Genetic Polymorphism for Human Platelet Thermostable Phenol Sulfotransferase (Ts Pst) Activity

    PubMed Central

    Price, R. A.; Spielman, R. S.; Lucena, A. L.; Van-Loon, J. A.; Maidak, B. L.; Weinshilboum, R. M.

    1989-01-01

    Platelet TS PST basal activity and thermal stability were measured in blood samples from 237 individuals in 50 nuclear families. Significant correlations were found among first degree relatives, confirming the previously reported familial aggregation of TS PST basal activity and thermal stability. Commingling analysis of basal TS PST activity provided evidence for multiple component distributions, and after transformation to remove skewness, segregation analysis supported a major gene hypothesis. For TS PST thermal stability, commingling analysis also provided evidence for multiple component distributions. However, segregation analyses were equivocal with regard to the presence of a major gene for thermal stability, since support for a major gene model depended on skewness. Bivariate commingling analysis, which examined thermal stability by simultaneously considering basal activity and activity after heating, suggested that genotypes, as defined by the inferred component distributions for TS PST activity, differ in thermal stability. A three-allele model is proposed as one hypothesis that may account for the combined results of basal activity and thermal stability. The results of this study indicate that a major gene polymorphism in conjunction with polygenic inheritance plays an important role in the regulation of both level of activity and thermal stability of this important drug-metabolizing enzyme in humans. PMID:2759428

  1. Enhanced nitric oxide and cyclic GMP formation plays a role in the anti-platelet activity of simvastatin

    PubMed Central

    Chou, T-C; Lin, Y-F; Wu, W-C; Chu, K-M

    2008-01-01

    Background and purpose: It has been found that 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exert various vascular protective effects, beyond their cholesterol-lowering property, including inhibition of platelet-dependent thrombus formation. The objective of the present study was to determine whether the nitric oxide (NO)/cyclic GMP-mediated processes in platelets contribute to the anti-aggregatory activity of simvastatin. Experimental approach: After rabbit platelets were incubated with simvastatin for 5 min, aggregation was induced and the platelet aggregation, nitric oxide synthase activity, guanylyl cyclase activity, NO and cyclic GMP formation were measured appropriately. Key results: Treatment with simvastatin concentration-dependently inhibited platelet aggregation induced by collagen or arachidonic acid with an IC50 range of 52–158 μM. We also demonstrated that simvastatin (20–80 μM) concentration-dependently further enhanced collagen-induced NO and cyclic GMP formation through increasing NOS activity (from 2.64±0.12 to 3.52±0.21–5.10±0.14 μmol min−1 mg protein−1) and guanylyl cyclase activity (from 142.9±7.2 to 163.5±17.5–283.8±19.5 pmol min−1 mg protein−1) in the platelets. On the contrary, inhibition of platelet aggregation by simvastatin was markedly attenuated (by about 50%) by addition of a nitric oxide synthase inhibitor, a NO scavenger or a NO-sensitive guanylyl cyclase inhibitor. The anti-aggregatory effects of simvastatin were significantly increased by addition of a selective inhibitor of cyclic GMP phosphodiesterase. Conclusions and implications: Our findings indicate that enhancement of a NO/cyclic GMP-mediated process plays an important role in the anti-aggregatory activity of simvastatin. PMID:18264124

  2. Suppressive effect of exogenous carbon monoxide on endotoxin-stimulated platelet over-activation via the glycoprotein-mediated PI3K-Akt-GSK3β pathway

    PubMed Central

    Liu, Dadong; Wang, Xu; Qin, Weiting; Chen, Jingjia; Wang, Yawei; Zhuang, Mingfeng; Sun, Bingwei

    2016-01-01

    Platelet activation is an important event involved in the pathophysiological processes of the coagulation system. Clinical evidence has shown that platelets undergo distinctive pathological processes during sepsis. Unfortunately, how platelets physiologically respond to inflammation or sepsis is not well understood. In this study, we used a lipopolysaccharide (LPS)-stimulated platelet model to systemically investigate alterations in membrane glycoprotein expression, molecular signaling, morphology and critical functions of platelets. We found that platelet adhesion, aggregation, secretion, and spreading on immobilized fibrinogen and the expression of platelet membrane glycoproteins were significantly increased by LPS stimulation, and these changes were accompanied by a significant decrease in cGMP levels and an abnormal distribution of platelet α-granules. Exogenous CO reversed these alterations. Profound morphological changes in LPS-stimulated platelets were observed using atomic force microscopy and phase microscopy. Furthermore, the elevated activities of PI3Ks, AKt and GSK-3β were effectively suppressed by exogenous CO, leading to the improvement of platelet function. Together, these results provide evidence that platelet over-activation persists under LPS-stimulation and that exogenous CO plays an important role in suppressing platelet activation via the glycoprotein-mediated PI3K-Akt-GSK3β pathway. PMID:27020460

  3. Anti-platelet activity of erythro-(7S,8R)-7-acetoxy-3,4,3',5'-tetramethoxy-8-O-4'-neolignan from Myristica fragrans.

    PubMed

    Kang, Jung Won; Min, Byung-Sun; Lee, Jeong-Hyung

    2013-11-01

    Platelets play a critical role in pathogenesis of cardiovascular disorders and strokes. The inhibition of platelet function is beneficial for the treatment and prevention of these diseases. In this study, we investigated the anti-platelet activity of erythro-(7S,8R)-7-acetoxy-3,4,3',5'-tetramethoxy-8-O-4'-neolignan (EATN), a neolignan isolated from Myristica fragrans, using human platelets. EATN preferentially inhibited thrombin- and platelet-activating factor (PAF)-induced platelet aggregation without affecting platelet damage in a concentration-dependent manner with IC50 values of 3.2 ± 0.4 and 3.4 ± 0.3 μM, respectively. However, much higher concentrations of EATN were required to inhibit platelet aggregation induced by arachidonic acid. EATN also inhibited thrombin-induced serotonin and ATP release, and thromboxane B2 formation in human platelets. Moreover, EATN caused an increase in cyclic AMP (cAMP) levels and attenuated intracellular Ca(2+) mobilization in thrombin-activated human platelets. Therefore, we conclude that the inhibitory mechanism of EATN on platelet aggregation may increase cAMP levels and subsequently inhibit intracellular Ca(2+) mobilization by interfering with a common signaling pathway rather than by directly inhibiting the binding of thrombin or PAF to their receptors. This is the first report of the anti-platelet activity of EATN isolated from M. fragrans. PMID:23296979

  4. Association of platelet activation markers with cancer-associated venous thromboembolism.

    PubMed

    Riedl, Julia; Hell, Lena; Kaider, Alexandra; Koder, Silvia; Marosi, Christine; Zielinski, Christoph; Panzer, Simon; Pabinger, Ingrid; Ay, Cihan

    2016-01-01

    Venous thromboembolism (VTE) is a frequent complication in cancer patients. Platelet activation is thought to be involved in cancer-associated VTE. Here, we determined the association between evolving markers of platelet activation (soluble P-selectin [sP-selectin], soluble CD40 ligand [sCD40L], thrombospondin-1 [TSP-1] and platelet factor-4 [PF-4]) and the development of cancer-associated VTE. A nested matched case-control study was applied within a cohort of 1779 patients with different types of cancer that had been included in the Vienna Cancer and Thrombosis Study (CATS), a prospective, observational study on patients with newly diagnosed or progressive cancer after remission. Primary endpoint is symptomatic VTE during a maximum follow-up of 2 years. Cases (patients who developed VTE during follow-up) were matched in a 1:2 ratio to controls without VTE during follow-up with respect to tumor type, stage and time of observation in the study. In total, 131 VTE cases were compared to 262 controls. In logistic regression analysis, only sP-selectin was associated with risk of VTE. The odds ratios (OR) per double increase of sP-selectin, sCD40L, TSP-1 and PF-4 were 1.66 (95% confidence interval: 1.17-2.35, p = 0.005), 1.04 (0.89-1.21, p = 0.635), 1.09 (0.90-1.32, p = 0.360) and 1.03 (0.87-1.21, p = 0.737), respectively. In conclusion, sP-selectin, but not sCD40L, TSP-1 or PF-4 were associated with risk of VTE in cancer patients in this nested case-control study. PMID:25970326

  5. Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA

    PubMed Central

    Duchez, Anne-Claire; Boudreau, Luc H.; Naika, Gajendra S.; Bollinger, James; Belleannée, Clémence; Cloutier, Nathalie; Laffont, Benoit; Mendoza-Villarroel, Raifish E.; Lévesque, Tania; Rollet-Labelle, Emmanuelle; Rousseau, Matthieu; Allaeys, Isabelle; Tremblay, Jacques J.; Poubelle, Patrice E.; Lambeau, Gérard; Pouliot, Marc; Provost, Patrick; Soulet, Denis; Gelb, Michael H.; Boilard, Eric

    2015-01-01

    Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms. PMID:26106157

  6. Glucosamine suppresses platelet-activating factor-induced activation of microglia through inhibition of store-operated calcium influx.

    PubMed

    Park, Jae-Hyung; Kim, Jeong-Nam; Jang, Byeong-Churl; Im, Seung-Soon; Song, Dae-Kyu; Bae, Jae-Hoon

    2016-03-01

    Microglia activation and subsequent release of inflammatory mediators are implicated in the pathophysiology of neurodegenerative diseases. Platelet-activating factor (PAF), a potent lipid mediator synthesized by microglia, is known to stimulate microglia functional responses. In this study, we determined that endogenous PAF exert autocrine effects on microglia activation, as well as the underlying mechanism involved. We also investigated the effect of D-glucosamine (GlcN) on PAF-induced cellular activation in human HMO6 microglial cells. PAF induced sustained intracellular Ca(2+) ([Ca(2+)]i) increase through store-operated Ca(2+) channels (SOC) and reactive oxygen species (ROS) generation. PAF also induced pro-inflammatory markers through NFκB/COX-2 signaling. GlcN significantly inhibited PAF-induced Ca(2+) influx and ROS generation without significant cytotoxicity. GlcN downregulated excessive expression of pro-inflammatory markers and promoted filopodia formation through NFκB/COX-2 inhibition in PAF-stimulated HMO6 cells. Taken together, these data suggest that GlcN may offer substantial therapeutic potential for treating inflammatory and neurodegenerative diseases accompanied by microglial activation. PMID:26745504

  7. Anti-thrombogenic properties of a nitric oxide-releasing dextran derivative: evaluation of platelet activation and whole blood clotting kinetics

    PubMed Central

    Damodaran, Vinod B.; Leszczak, Victoria; Wold, Kathryn A.; Lantvit, Sarah M.; Popat, Ketul C.; Reynolds, Melissa M.

    2013-01-01

    Controlling platelet activation and clotting initiated by cardiovascular interventions remains a major challenge in clinical practice. In this work, the anti-thrombotic properties of a polysaccharide-based nitric oxide (NO)-releasing dextran derivative are presented. Total platelet adhesion, platelet morphology and whole blood clotting kinetics were used as indicators to evaluate the anti-clotting properties of this material. With a total NO delivery of 0.203±0.003 μmol, the NO-releasing dextran derivative (Dex-SNO) mixed with blood plasma demonstrated a significantly lower amount of platelet adhesion and activation onto a surface and reduced whole blood clotting kinetics. Nearly 75% reduction in platelet adhesion and a significant retention of platelet morphology were observed with blood plasma treated with Dex-SNO, suggesting this to be a potential anti-platelet therapeutic agent for preventing thrombosis that does not have an adverse effect on circulating platelets. PMID:24349705

  8. Fish-Free Diet in Patients with Phenylketonuria Is Not Associated with Early Atherosclerotic Changes and Enhanced Platelet Activation

    PubMed Central

    Htun, Patrik; Nee, Jens; Ploeckinger, Ursula; Eder, Klaus; Geisler, Tobias; Gawaz, Meinrad; Bocksch, Wolfgang; Fateh-Moghadam, Suzanne

    2015-01-01

    Background and Purpose Since patients with phenylketonuria (PKU) have to follow a lifelong restriction of natural protein to lower phenylalanine-intake, they never eat fish. This diet may lead to a chronic deficit of omega-3 and omega-6 fatty acids with the risk of early atherosclerotic changes. The aim of the study was to analyse the fatty acid profile of PKU patients and to correlate the results with surrogate markers of early atherosclerotic changes [enhanced carotid intima media thickness (CIMT) and ß-stiffness index] and platelet activation. Methods In 43 PKU patients and in 58 healthy controls we prospectively examined the fatty acid profile, CIMT, ß-stiffness index and platelet activation (flow cytometric determination of markers of platelet activation). CIMT was measured bilaterally by ultrasound. CIMT mean was defined as the mean value of the sum of CIMT left and CIMT right. Results Despite of lower HDL-cholesterol and higher triglyceride concentrations in the PKU group, there was no significant difference in the omega-6 or omega-3 fatty acid profile, CIMT, ß-stiffness index between both groups. Platelet activation was not enhanced in the PKU group. Conclusions Fish-free diet does not induce early atherosclerotic changes or enhanced platelet activation in PKU patients. PMID:26291823

  9. Platelet-activating factor (PAF-acether) induces high- and low-affinity binding of fibrinogen to human platelets via independent mechanisms.

    PubMed Central

    Kloprogge, E; Akkerman, J W

    1986-01-01

    When human platelets are incubated with 500 nM-PAF-acether (platelet-activating factor. 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) under equilibrium conditions (60 min, 22 degrees C, non-stirred suspensions), two classes of fibrinogen binding sites are exposed: one class with a high affinity [Kd (7.2 +/- 2.1) X 10(-8) M, 2367 +/- 485 sites/platelet, n = 9] and one class with a low affinity [Kd (5.9 +/- 2.4) X 10(-7) M, 26972 +/- 8267 sites/platelet]. Preincubation with inhibitors of cyclo-oxygenase (acetylsalicylic acid, indomethacin) or thromboxane synthetase (UK 38.485) completely abolishes high-affinity binding, leaving low-affinity binding unchanged. In contrast, ADP scavengers (phosphocreatine/creatine kinase or phosphoenol pyruvate/pyruvate kinase) completely prevent low-affinity binding, leaving high-affinity binding unaltered. Initial binding studies (2-10 min incubation) confirm these findings with a major part of the binding being sensitive to ADP scavengers, a minor part sensitive to indomethacin and complete blockade with both inhibitors. Increasing the temperature to 37 degrees C decreases the number of low affinity-binding sites 6-fold without changing high-affinity binding. Aggregation, measured as the rate of single platelet disappearance, then depends on high-affinity binding at 10 nM-fibrinogen or less, whereas at 100 nM-fibrinogen or more low-affinity binding becomes predominant. These findings point at considerable platelet activation during binding experiments. However, arachidonate metabolism [( 3H]arachidonate mobilization and thromboxane synthesis) and secretion [( 14C]serotonin and beta-thromboglobulin) are about 10% or less of the amounts found under optimal conditions (5 units of thrombin/ml 37 degrees C, stirring). We conclude that PAF-acether induces little platelet activation under binding conditions. The amounts of thromboxane A2 and secreted ADP, however, are sufficient for initiating high- and low-affinity fibrinogen binding

  10. Plant extracts inhibit ADP-induced platelet activation in humans: their potential therapeutic role as ADP antagonists.

    PubMed

    Jagroop, Indera Anita

    2014-01-01

    Adenosine diphosphate (ADP) plays a pivotal role in platelet activation. Platelet hyperactivity is associated with vascular disease and also has a key role in haemostasis and thrombosis. ADP activates platelets through three purinoceptor subtypes, the G(q)-coupled P2Y(1) receptor, G(i)-coupled P2Y(12) receptor and P2X(1) ligand-gated cation channel. Platelet ADP purinergic receptors are therefore suitable targets for antiplatelet drugs. Thienopyridines such as clopidogrel and ticlopidine, as well as other ADP receptor antagonists like prasugrel, ticagrelor, cangrelor and elinogrel have demonstrated clinical benefits via the inhibition of the selective purinergic ADP receptor, P2Y(12). However, they still have limitations in their mode of action and efficacy, like increased risk of bleeding. Thus, the ongoing pursuit to develop newer and more effective antiplatelet agents continues. There is a growing interest in the purinergic antiplatelet properties exhibited by plant extracts. This article considers the following: pomolic acid isolated from Licania pittieri, brazilin isolated from the heartwood of Caesalpinia sappan L, phylligenin isolated from the twigs of Muraltia vulpina, bark oil of Gonystylus velutinus, seed and bark extracts from Aesculus hippocastanum L. and red wine phenolics and catechins isolated from green tea. Moreover, the method used to investigate platelet purinergic receptors should be considered, since using a more sensitive, high-resolution platelet sizer can sometimes detect platelet variations when the light transmission method was not able to do so. The exact mechanisms by which these plant extracts work need further investigation. They all however inhibit ADP-induced activation in human platelets. This could explain, at least in part, the protective effect of plant extracts as antiplatelet agents. PMID:24190032

  11. Cognitive Stimulation Modulates Platelet Total Phospholipases A2 Activity in Subjects with Mild Cognitive Impairment

    PubMed Central

    Balietti, Marta; Giuli, Cinzia; Fattoretti, Patrizia; Fabbietti, Paolo; Postacchini, Demetrio; Conti, Fiorenzo

    2016-01-01

    We evaluated the effect of cognitive stimulation (CS) on platelet total phospholipases A2 activity (tPLA2A) in patients with mild cognitive impairment (MCI_P). At baseline, tPLA2A negatively correlated with Mini-Mental State Examination score (MMSE_s): patients with MMSE_s <26 (Subgroup 1) had significantly higher activity than those with MMSE_s ≥26 (Subgroup 2), who had values similar to the healthy elderly. Regarding CS effect, Subgroup 1 had a significant tPLA2A reduction, whereas Subgroup 2 did not significantly changes after training. Our results showed for the first time that tPLA2A correlates with the cognitive conditions of MCI_P, and that CS acts selectively on subjects with a dysregulated tPLA2A. PMID:26836161

  12. Deficiency of platelet-activating factor acetylhydrolase is a severity factor for asthma

    PubMed Central

    Stafforini, Diana M.; Numao, Toshio; Tsodikov, Alexander; Vaitkus, Darius; Fukuda, Takeshi; Watanabe, Naoto; Fueki, Naoto; McIntyre, Thomas M.; Zimmerman, Guy A.; Makino, Sohei; Prescott, Stephen M.

    1999-01-01

    Asthma, a family of airway disorders characterized by airway inflammation, has an increasing incidence worldwide. Platelet-activating factor (PAF) may play a role in the pathophysiology of asthma. Its proinflammatory actions are antagonized by PAF acetylhydrolase. A missense mutation (V279F) in the PAF acetylhydrolase gene results in the complete loss of activity, which occurs in 4% of the Japanese population. We asked if PAF acetylhydrolase deficiency correlates with the incidence and severity of asthma in Japan. We found that the prevalence of PAF acetylhydrolase deficiency is higher in Japanese asthmatics than healthy subjects and that the severity of this syndrome is highest in homozygous-deficient subjects. We conclude that the PAF acetylhydrolase gene is a modulating locus for the severity of asthma. PMID:10194471

  13. Crystallization and Preliminary X-Ray Crystallographic Analysis of Human Plasma Platelet Activating Factor Acetylhydrolase

    SciTech Connect

    Samanta, U.; Wilder, C; Bahnson, B

    2009-01-01

    The plasma form of the human enzyme platelet activating factor acetylhydrolase (PAF-AH) has been crystallized, and X-ray diffraction data were collected at a synchrotron source to a resolution of 1.47 {angstrom}. The crystals belong to space group C2, with unit cell parameters of a = 116.18, b = 83.06, c = 96.71 {angstrom}, and {beta} = 115.09 and two molecules in the asymmetric unit. PAF-AH functions as a general anti-inflammatory scavenger by reducing the levels of the signaling molecule PAF. Additionally, the LDL bound enzyme has been linked to atherosclerosis due to its hydrolytic activities of pro-inflammatory agents, such as sn-2 oxidatively fragmented phospholipids.

  14. Platelet-Activating Factor Induces Epigenetic Modifications in Human Mast Cells.

    PubMed

    Damiani, Elisabetta; Puebla-Osorio, Nahum; Gorbea, Enrique; Ullrich, Stephen E

    2015-12-01

    UV radiation-induced systemic immune suppression is a major risk factor for skin cancer induction. The migration of dermal mast cells from the skin to the draining lymph nodes has a prominent role in activating systemic immune suppression. UV-induced keratinocyte-derived platelet-activating factor (PAF) activates mast cell migration, in part by upregulating the expression of CXCR4 on the surface of mast cells. Others have indicated that epigenetic mechanisms regulate CXCR4 expression; therefore, we asked whether PAF activates epigenetic mechanisms in mast cells. Human mast cells were treated with PAF, and the effect on DNA methylation and/or acetylation was measured. PAF suppressed the expression of DNA methyltransferase (DNMT) 1 and 3b. On the other hand, PAF increased p300 histone acetyltransferase expression, and the acetylation of histone H3, which coincided with a decreased expression of the histone deacetylase HDAC2. Chromatin immunoprecipitation assays indicated that PAF treatment activated the acetylation of the CXCR4 promoter. Finally, inhibiting histone acetylation blocked p300 upregulation and suppressed PAF-induced surface expression of CXCR4. Our findings suggest a novel molecular mechanism for PAF, activation of epigenetic modifications. We suggest that PAF may serve as an endogenous molecular mediator that links the environment (UV radiation) with the epigenome. PMID:26316070

  15. Butyric acid stimulates bovine neutrophil functions and potentiates the effect of platelet activating factor.

    PubMed

    Carretta, M D; Hidalgo, A I; Burgos, J; Opazo, L; Castro, L; Hidalgo, M A; Figueroa, C D; Taubert, A; Hermosilla, C; Burgos, R A

    2016-08-01

    Increased short-chain fatty acid (SCFA) production is associated with subacute ruminal acidosis (SARA) and activation of inflammatory processes. In humans and rodents, SCFAs modulate inflammatory responses in the gut via free fatty acid receptor 2 (FFA2). In bovines, butyric acid is one of the most potent FFA2 agonists. Its expression in bovine neutrophils has recently been demonstrated, suggesting a role in innate immune response in cattle. This study aimed to evaluate if butyric acid modulates oxidative and non-oxidative functions or if it can potentiate other inflammatory mediators in bovine neutrophils. Our results showed that butyric acid can activate bovine neutrophils, inducing calcium (Ca(2+)) influx and mitogen-activated protein kinase (MAPK) phosphorylation, two second messengers involved in FFA2 activation. Ca(2+) influx induced by butyric acid was dependent on the extracellular and intracellular Ca(2+) source and phospholipase C (PLC) activation. Butyric acid alone had no significant effect on reactive oxygen species (ROS) production and chemotaxis; however, a priming effect on platelet-activating factor (PAF), a potent inflammatory mediator, was observed. Butyric acid increased CD63 expression and induced the release of neutrophil granule markers matrix metalloproteinase-9 (MMP-9) and lactoferrin. Finally, we observed that butyric acid induced neutrophil extracellular trap (NET) formation without affecting cellular viability. These findings suggest that butyric acid, a component of the ruminal fermentative process, can modulate the innate immune response of ruminants. PMID:27288853

  16. Platelet-Activating Factor Induces Epigenetic Modifications in Human Mast Cells

    PubMed Central

    Gorbea, Enrique; Ullrich, Stephen E.

    2015-01-01

    Ultraviolet (UV) radiation-induced systemic immune suppression is a major risk factor for skin cancer induction. The migration of dermal mast cells from the skin to the draining lymph nodes plays a prominent role in activating systemic immune suppression. UV-induced keratinocyte-derived platelet-activating factor (PAF) activates mast cell migration, in part by up regulating the expression of CXCR4 on the surface of mast cells. Others have indicated that epigenetic mechanisms regulate CXCR4 expression, so we asked whether PAF activates epigenetic mechanisms in mast cells. Human mast cells were treated with PAF and the effect on DNA methylation and/or acetylation was measured. PAF suppressed the expression of DNA methyltransferase (DNMT) 1 and 3b. On the other hand, PAF increased p300 histone acetyltransferase expression, and the acetylation of histone H3, which coincided with a decreased expression of the histone deacetylase HDAC2. Chromatin immunoprecipitation assays indicated that PAF-treatment activated the acetylation of the CXCR4 promoter. Finally, inhibiting histone acetylation blocked p300 up-regulation and suppressed PAF-induced surface expression of CXCR4. Our findings suggest a novel molecular mechanism for PAF, activation of epigenetic modifications. We suggest that PAF may serve as an endogenous molecular mediator that links the environment (UV radiation) with the epigenome. PMID:26316070

  17. Stimulation of platelet-activating factor synthesis by progesterone and A23187 in human spermatozoa.

    PubMed Central

    Baldi, E; Falsetti, C; Krausz, C; Gervasi, G; Carloni, V; Casano, R; Forti, G

    1993-01-01

    The presence of platelet-activating factor (PAF) has been demonstrated recently in mammalian spermatozoa, together with evidence for a role of this phospholipid in enhancing sperm motility and fertilizing ability. To investigate whether PAF synthesis and release occurs in human spermatozoa following incubation with stimuli that induce acrosome reaction, spermatozoa were incubated with progesterone and A23187, two known inducers of the exocytotic event. PAF synthesis (remodelling pathway) was assessed by [3H]acetate incorporation into PAF. Treatment of spermatozoa with progesterone and A23187 resulted in an increase of [3H]acetate incorporation into PAF. Most of the newly synthesized [3H]PAF formed in response to acrosome reaction was found in the supernatant, suggesting a release of the phospholipid from spermatozoa. PAF-like material extracted from human spermatozoa was able to induce aggregation of rabbit platelets and showed identical retention time and the same ion m/e values as authentic PAF when analysed with g.c.-m.s. Lyso-PAF:acetyl-CoA acetyltransferase (EC 2.3.1.67) activity in human spermatozoa was also studied and showed similar kinetic parameters to those described for other cell systems. Stimulation of spermatozoa with progesterone and A23187 induced an increase of [3H]arachidonic acid release, suggesting an activation of phospholipase A. In conclusion, our results demonstrated increased production and release of PAF in human sperm following stimulation with progesterone and A23187 and suggest a role for this phospholipid in the activation of spermatozoa. Images Figure 2 Figure 4 Figure 7 PMID:8503848

  18. Mathematical analysis of mural thrombogenesis. Concentration profiles of platelet-activating agents and effects of viscous shear flow.

    PubMed Central

    Folie, B J; McIntire, L V

    1989-01-01

    The concentration profiles of adenosine diphosphate (ADP), thromboxane A2 (TxA2), thrombin, and von Willebrand factor (vWF) released extracellularly from the platelet granules or produced metabolically on the platelet membrane during thrombus growth, were estimated using finite element simulation of blood flow over model thrombi of various shapes and dimensions. The wall fluxes of these platelet-activating agents were estimated for each model thrombus at three different wall shear rates (100 s-1, 800 s-1, and 1,500 s-1), employing experimental data on thrombus growth rates and sizes. For that purpose, whole human blood was perfused in a parallel-plate flow chamber coated with type l fibrillar human collagen, and the kinetic data collected and analyzed by an EPl-fluorescence video microscopy system and a digital image processor. It was found that thrombin concentrations were large enough to cause irreversible platelet aggregation. Although heparin significantly accelerated thrombin inhibition by antithrombin lll, the remaining thrombin levels were still significantly above the minimum threshold required for irreversible platelet aggregation. While ADP concentrations were large enough to cause irreversible platelet aggregation at low shear rates and for small aggregate sizes, TxA2 concentrations were only sufficient to induce platelet shape change over the entire range of wall shear rates and thrombi dimensions studied. Our results also indicated that the local concentration of vWF multimers released from the platelet alpha-granules could be sufficient to modulate platelet aggregation at low and intermediate wall shear rates (less than 1,000 s-1). The sizes of standing vortices formed adjacent to a growing aggregate and the embolizing stresses and the torque, acting at the aggregate surface, were also estimated in this simulation. It was found that standing vortices developed on both sides of the thrombus even at low wall shear rates. Their sizes increased with

  19. Visualization of Activated Platelets by Targeted Magnetic Resonance Imaging Utilizing Conformation-Specific Antibodies against Glycoprotein IIb/IIIa

    PubMed Central

    von zur Muhlen, Constantin; Peter, Karlheinz; Ali, Ziad A.; Schneider, Jürgen E.; McAteer, Martina A.; Neubauer, Stefan; Channon, Keith M.; Bode, Christoph; Choudhury, Robin P.

    2009-01-01

    Ruptured atherosclerotic plaques, lined with activated platelets, constitute an attractive target for magnetic resonance imaging (MRI). This study evaluated whether microparticles of iron oxide (MPIO) targeting ligand-induced binding sites (LIBS) on the activated conformation of glycoprotein IIb/IIIa could be used to image platelets. MPIO (size: 1 μm) were conjugated to anti-LIBS or control single-chain antibody. Following guidewire injury to mouse femoral artery, platelet adhesion was present after 24 h. Mice were perfused with anti-LIBS-MPIO (or control MPIO) via the left ventricle and 11.7-tesla MRI was performed on femoral arteries ex vivo. A 3D gradient echo sequence attained an isotropic resolution of 25 μm. MPIO binding, quantified by MRI, was 4-fold higher with anti-LIBS-MPIO in comparison to control MPIO (p < 0.01). In histological sections, low signal zones on MRI and MPIO correlated strongly (R2 = 0.72; p < 0.001), indicating accurate MR quantification. In conclusion, anti-LIBS-MPIO bind to activated platelets in mouse arteries, providing a basis for the use of function-specific single-chain antibody-MPIO conjugates for molecular MRI, and represent the first molecular imaging of a conformational change in a surface receptor. This presents an opportunity to specifically image activated platelets involved in acute atherothrombosis with MRI. PMID:18515970

  20. Identification of small peptide analogues having agonist and antagonist activity at the platelet thrombin receptor.

    PubMed

    Ruda, E M; Petty, A; Scrutton, M C; Tuffin, D P; Manley, P W

    1988-06-15

    phosphatidylethanolamine. SC40476 causes no detectable hydrolysis of glycoprotein V as detected by release of the proteolytic product (glycoprotein VFR). The results indicate that SC40476 and SC42619 interact selectively with the platelet thrombin receptor. Both peptide analogues act as effective antagonists for this receptor but also possess weak agonist activity which may also result from interaction with the thrombin receptor. The molecular basis for this latter activity has not been defined. SC42619 non-selectively inhibits Ca2+ influx induced by several agonists but this effect does not appear to contribute to the observed inhibition of the aggregatory and secretory responses. PMID:2839193

  1. Antigen-and ionophore-stimulated synthesis of platelet-activating factor by the cloned mast cell line, MC9

    SciTech Connect

    Musch, M.W.; Billah, M.M.; Siegel, M.I.

    1987-05-14

    MC9 mast cells stimulated by a soluble (calcium ionophore A23187) or by an Fc epsilon-receptor agonist (IgE plus hapten) produce platelet activating factor (PAF). MC9 cells incorporate either exogenous (/sup 3/H)acetic acid or (/sup 3/H)lyso-PAF into PAF. PAF was identified by mobility on thin layer chromatography, platelet aggregatory activity inhibitable by known PAF antagonists, and by enzymatic modification. Quantified by aggregation of rabbit platelets, MC9 cells produce 6 pmoles PAF/10(6) cells. MC9 cells express acetyltransferase activity of 0.19 nmole/5 min-mg protein. Analysis of MC9 phospholipids by HPLC showed that MC9 cells contain large amounts of phosphatidylcholine (82 nmoles/10(7) cells) but contain little ether-linked phosphatidylcholine (4 nmoles/10(7) cells).

  2. New analogues of 13-hydroxyocatdecadienoic acid and 12-hydroxyeicosatetraenoic acid block human blood platelet aggregation and cyclooxygenase-1 activity

    PubMed Central

    2012-01-01

    Background Thromboxane A2 is derived from arachidonic acid through the action of cyclooxygenases and thromboxane synthase. It is mainly formed in blood platelets upon activation and plays an important role in aggregation. Aspirin is effective in reducing the incidence of complications following acute coronary syndrome and stroke. The anti-thrombotic effect of aspirin is obtained through the irreversible inhibition of cyclooxygenases. Analogues of 12-hydroxyeicosatetraenoic acid and 13-hydroxyocatdecadienoic acid were shown previously to modulate platelet activation and to block thromboxane receptors. Results and discussion We synthesized 10 compounds based on the structures of analogues of 12-hydroxyeicosatetraenoic acid and 13-hydroxyocatdecadienoic acid and evaluated their effect on platelet aggregation triggered by arachidonic acid. The structure activity relationship was evaluated. Five compounds showed a significant inhibition of platelet aggregation and highlighted the importance of the lipidic hydrophobic hydrocarbon chain and the phenol group. Their IC50 ranged from 7.5 ± 0.8 to 14.2 ± 5.7 μM (Mean ± S.E.M.). All five compounds decreased platelet aggregation and thromboxane synthesis in response to collagen whereas no modification of platelet aggregation in response to thromboxane receptor agonist, U46619, was observed. Using COS-7 cells overexpressing human cyclooxygenase-1, we showed that these compounds are specific inhibitors of cyclooxygenase-1 with IC50 ranging from 1.3 to 12 μM. Docking observation of human recombinant cyclooxygenase-1 supported a role of the phenol group in the fitting of cyclooxygenase-1, most likely related to hydrogen bonding with the Tyr 355 of cyclooxygenase-1. Conclusions In conclusion, the compounds we synthesized at first based on the structures of analogues of 12 lipoxygenase metabolites showed a role of the phenol group in the anti-platelet and anti-cyclooxygenase-1 activities. These compounds mediate their

  3. Impact of aspirin dose on adenosine diphosphate-mediated platelet activities. Results of an in vitro pilot investigation.

    PubMed

    Tello-Montoliu, Antonio; Thano, Estela; Rollini, Fabiana; Patel, Ronakkumar; Wilson, Ryan E; Muñiz-Lozano, Ana; Franchi, Francesco; Darlington, Andrew; Desai, Bhaloo; Guzman, Luis A; Bass, Theodore A; Angiolillo, Dominick J

    2013-10-01

    Different aspirin dosing regimens have been suggested to impact outcomes when used in combination with adenosine diphosphate (ADP) P2Y12 receptor antagonists. Prior investigations have shown that not only aspirin, but also potent ADP P2Y12 receptor blockade can inhibit thromboxane A2-mediated platelet activation. The impact of aspirin dosing on ADP mediated platelet activities is unknown and represents the aim of this in vitro pilot pharmacodynamic (PD) investigation. Twenty-six patients with stable coronary artery disease on aspirin 81 mg/day and P2Y12 naïve were enrolled. PD assessments were performed at baseline, while patients were on 81 mg/day aspirin and after switching to 325 mg/day for 7 ± 2 days with and without escalating concentrations (vehicle, 1, 3, and 10 μM) of prasugrel's active metabolite (P-AM). PD assays included flow cytometric assessment of VASP to define the platelet reactivity index (PRI) and the Multiplate Analyzer (MEA) using multiple agonists [ADP, ADP + prostaglandin (PGE1), arachidonic acid (AA), and collagen]. Escalating P-AM concentrations showed incremental platelet P2Y12 inhibition measured by VASP-PRI (p<0.001). However, there were no differences according to aspirin dosing regimen at any P-AM concentration (vehicle: p=0.899; 1 μM: p=0.888; 3 μM: p=0.524; 10 μM: p=0.548). Similar findings were observed in purinergic markers assessed by MEA (ADP and ADP+PGE1). P-AM addition significantly reduced AA and collagen induced platelet aggregation (p<0.001 for all measures), irrespective of aspirin dose. In conclusion, aspirin dosing does not appear to affect PD measures of ADP-mediated platelet reactivity irrespective of the degree of P2Y12 receptor blockade. P2Y12 receptor blockade modulates platelet reactivity mediated by alternative activators. PMID:23884248

  4. How do the full-generation poly(amido)amine (PAMAM) dendrimers activate blood platelets? Activation of circulating platelets and formation of "fibrinogen aggregates" in the presence of polycations.

    PubMed

    Watala, Cezary; Karolczak, Kamil; Kassassir, Hassan; Talar, Marcin; Przygodzki, Tomasz; Maczynska, Katarzyna; Labieniec-Watala, Magdalena

    2016-04-30

    Direct use of poly(amido)amine (PAMAM) dendrimers as drugs may be limited, due to uncertain (cyto)toxicity. Peripheral blood components, which constitute the first line of a contact with administered pharmaceuticals, may become vastly affected by PAMAM dendrimers. The aim of this study was to explore how PAMAMs' polycationicity might affect blood platelet activation and reactivity, and thus trigger various haemostatic events. We monitored blood platelet reactivity in rats with experimental diabetes upon a long-term administration of the unmodified PAMAM dendrimers. In parallel, the effects on blood flow in a systemic circulation was recorded intravitally in mice administered with PAMAM G2, G3 or G4. Compounding was the in vitro approach to monitor the impact of PAMAM dendrimers on blood platelet activation and reactivity and on selected haemostatic and protein conformation parameters. We demonstrated the activating effects of polycations on blood platelets. Some diversity of the revealed outcomes considerably depended on the used approach and the particular technique employed to monitor blood platelet function. We discovered undesirable impact of plain PAMAM dendrimers on primary haemostasis and their prothrombotic influence. We emphasize the need of a more profound verifying of all the promising findings collected for PAMAMs with the use of well-designed in vivo preclinical studies. PMID:26319628

  5. Net platelet angiogenic activity (NPAA) correlates with progression and prognosis of non-small cell lung cancer.

    PubMed

    Yao, Lijuan; Dong, Hang; Luo, Yiqin; Du, Jianping; Hu, Wen

    2014-01-01

    Circulating platelets are abundant sources of angiogensis molecules for the tumor vasculature affecting tumor growth and metastasis. The relationship between non-small cell lung cancer (NSCLC) and intra-platelet levels of VEGF, TSP-1 and net platelet angiogenic activity (NPAA) is unclear. The aim of this study was to better understand the role of these factors in the progression of NSCLC cancer and to assess its clinical significance. Platelet VEGF and TSP-1 and NPAA were measured preoperatively in 68 patients with NSCLC by ELISA or Capillary tube formation assay. VEGF, TSP-1 and NPAA distributions in cancer patients and healthy volunteers were compared using the Mann-Whitney U test. The Kaplan-Meier method, univariate and multivariate regression analysis was used to analyze the correlation between these factors and clinicopathological features, overall survival and disease-free survival. Mean intra-platelet TSP-1 level was slightly higher in patients than in healthy subjects (p = 0.092). Intra-platelet TSP-1 levels were significantly higher in patients with involvement greater than T2 or stage III, compared to other patients. Mean intra-platelet VEGF level was 40.8 pg/10⁶ in patients compared to 21.9 ng/10⁶ in healthy subjects (p = 0.041). Median value of NPAA in patients was significantly higher than that in healthy controls (p<0.001). Patients with high NPAA are more likely to exhibit aggressive clinical pathological features. NPAA greater than the median are associated with poor prognosis. The elevated NPAA have better correlation with tumor microvessel density (MVD) than platelet-derived VEGF. The areas under receiver operating curve (AUROC) of NPAA were higher than that of platelet derived VEGF in different groups. A multivariate analysis showed that NPAA are independent prognostic factors. These results indicated that NPAA may be a clinically useful indicator for diagnostic and prognostic evaluation in NSCLC patients. PMID:24788022

  6. Purification and characterization of bovine brain platelet-activating factor acetylhydrolase.

    PubMed

    Hattori, M; Arai, H; Inoue, K

    1993-09-01

    Platelet-activating factor (PAF) acetylhydrolase, which removes the acetyl moiety at the sn-2 position, has been found in plasma and tissue cytosol. PAF acetylhydrolase in bovine brain cytosol was chromatographically separated into three distinct fractions, all of which exhibited pH optima in the neutral to mild alkaline region and were unaffected by EDTA. We have purified the major fraction of the enzyme to near homogeneity. The purified enzyme had a molecular mass of about 100 kDa, as estimated by gel filtration chromatography, and gave three distinct bands of 45, 30, and 29 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These polypeptides exclusively co-migrated with the activity throughout the purification steps. These data suggest that this set of polypeptides corresponds to the subunits of bovine brain PAF acetylhydrolase. Diisopropyl fluorophosphate completely inhibited the activity at 0.1 mM. [3H]Diisopropyl fluorophosphate labeled only the 29-kDa polypeptide, suggesting that this polypeptide possesses an active serine residue(s). The purified enzyme displayed similar activity against PAF and oxidatively modified phosphatidylcholine, but did not hydrolyze phosphatidylcholine or phosphatidylethanolamine with two long chain acyl groups. Thus, the intracellular PAF acetylhydrolase is likely to be a new member of the calcium-independent phospholipases A2 in mammalian tissues. PMID:8360169

  7. Participation of the cysteinyl leukotrienes in the acute bronchoconstrictor response to inhaled platelet activating factor in man.

    PubMed Central

    Spencer, D A; Evans, J M; Green, S E; Piper, P J; Costello, J F

    1991-01-01

    To determine whether the effects of platelet activating factor on the airways may be due to the production of leukotrienes we studied the effects of pretreatment with the selective cysteinyl leukotriene antagonist SK&F 104353-Z2 on the airway and cellular responses to inhaled platelet activating factor. Eight healthy men were studied in a randomised, double blind placebo controlled cross-over study. A single dose of platelet activating factor that caused a fall of at least 35% in specific airways conductance (sGaw) was determined initially for each subject. Challenge with this dose of platelet activating factor was then carried out on two further occasions after pretreatment with a single nebulised dose of SK&F 104353-Z2 or placebo. The % reductions in specific airways conductance and of partial flow at 30% of vital capacity (PVmax30) were less after SK&F 104353-Z2 than after placebo (22 versus 34 for sGaw, 19 versus 31 for PVmax30). The mean (95% confidence limits (CL] differences in the maximum % fall from control values for SK&F 104353-Z2 and placebo were -12.6 (-23.8, -1.4) for sGaw and -12.5, (-20.8 -4.2) for PVmax30. The mean % fall in neutrophil count was similar after SK&F 104353-Z2 (46%) and after placebo (50%) (95% CL of difference 13.6, 6.6). Bronchial responsiveness to methacholine did not increase above baseline values in any subject when measured two weeks after challenge by platelet activating factor. This study suggests that leukotrienes play a part in the response to platelet activating factor in man. Images PMID:1858084

  8. The peptide LSARLAF causes platelet secretion and aggregation by directly activating the integrin alphaIIbbeta3.

    PubMed Central

    Derrick, J M; Taylor, D B; Loudon, R G; Gartner, T K

    1997-01-01

    A novel peptide (designed to bind to alphaIIbbeta3) caused platelet aggregation and aggregation-independent secretion of the contents of alpha-granules in an alphaIIbbeta3-dependent fashion. The agonist peptide induced secretion in the presence of prostaglandin E1. In cell-free assays, alphaIIbbeta3 bound specifically to immobilized agonist peptide and the peptide enhanced the binding of fibrinogen to immobilized alphaIIbbeta3. The agonist peptide apparently activates alphaIIbbeta3 by directly inducing a conformational change in the receptor. This change activates the platelets and causes secretion in a manner independent of fibrinogen binding. PMID:9230107

  9. Activated Platelets in Carotid Artery Thrombosis in Mice Can Be Selectively Targeted with a Radiolabeled Single-Chain Antibody

    PubMed Central

    Goldschmidt, Jürgen; Pethe, Annette; Hagemeyer, Christoph E.; Neudorfer, Irene; Zirlik, Andreas; Weber, Wolfgang A.; Bode, Christoph; Meyer, Philipp T.

    2011-01-01

    Background Activated platelets can be found on the surface of inflamed, rupture-prone and ruptured plaques as well as in intravascular thrombosis. They are key players in thrombosis and atherosclerosis. In this study we describe the construction of a radiolabeled single-chain antibody targeting the LIBS-epitope of activated platelets to selectively depict platelet activation and wall-adherent non-occlusive thrombosis in a mouse model with nuclear imaging using in vitro and ex vivo autoradiography as well as small animal SPECT-CT for in vivo analysis. Methodology/Principal Findings LIBS as well as an unspecific control single-chain antibody were labeled with 111Indium (111In) via bifunctional DTPA ( = 111In-LIBS/111In-control). Autoradiography after incubation with 111In-LIBS on activated platelets in vitro (mean 3866±28 DLU/mm2, 4010±630 DLU/mm2 and 4520±293 DLU/mm2) produced a significantly higher ligand uptake compared to 111In-control (2101±76 DLU/mm2, 1181±96 DLU/mm2 and 1866±246 DLU/mm2) indicating a specific binding to activated platelets; P<0.05. Applying these findings to an ex vivo mouse model of carotid artery thrombosis revealed a significant increase in ligand uptake after injection of 111In-LIBS in the presence of small thrombi compared to the non-injured side, as confirmed by histology (49630±10650 DLU/mm2 vs. 17390±7470 DLU/mm2; P<0.05). These findings could also be reproduced in vivo. SPECT-CT analysis of the injured carotid artery with 111In-LIBS resulted in a significant increase of the target-to-background ratio compared to 111In-control (1.99±0.36 vs. 1.1±0.24; P<0.01). Conclusions/Significance Nuclear imaging with 111In-LIBS allows the detection of platelet activation in vitro and ex vivo with high sensitivity. Using SPECT-CT, wall-adherent activated platelets in carotid arteries could be depicted in vivo. These results encourage further studies elucidating the role of activated platelets in plaque pathology and atherosclerosis

  10. Platelet-activating factor: an endogenous mediator of mesenteric ischemia-reperfusion-induced shock.

    PubMed

    Mózes, T; Braquet, P; Filep, J

    1989-10-01

    The role of platelet-activating factor (PAF) in circulatory shock of intestinal origin was investigated in anesthetized dogs by measuring PAF levels in the superior mesenteric vein during reperfusion after 2-h occlusion of the superior mesenteric artery; by monitoring the effects of BN 52021, a specific PAF receptor antagonist; and by studying the circulatory effects of exogenous PAF injected into the superior mesenteric vein. PAF was measured by a platelet-aggregation assay. Identity of PAF-like bioactivity was ascertained by thin-layer chromatography, high-pressure liquid chromatography, and alkaline treatment. Removal of the superior mesenteric artery occlusion caused an immediate dramatic decrease in mean arterial blood pressure with concomitant increase in mean portal venous pressure and hematocrit values. PAF concentration in the superior mesenteric vein increased from 0.2 +/- 0.1 to 2.8 +/- 0.4 ng/ml (n = 4, P less than 0.05) within the first 5 min of reperfusion. Administration of exogenous PAF (0.1 microgram/kg) injected into the superior mesenteric vein produced similar hemodynamical effects. Pretreatment of the animals with BN 52021 (4 mg/kg), a specific PAF receptor antagonist, prevented the circulatory collapse. The present results suggest that PAF release during intestinal ischemia may play an important role in the development of circulatory collapse caused by mesenteric artery occlusion. PMID:2802004

  11. Regulation of platelet-activating factor synthesis in human neutrophils by MAP kinases.

    PubMed

    Baker, Paul R S; Owen, John S; Nixon, Andrew B; Thomas, Leslie N; Wooten, Rhonda; Daniel, Larry W; O'Flaherty, Joseph T; Wykle, Robert L

    2002-10-21

    Human neutrophils (PMN) are potentially a major source of platelet-activating factor (PAF) produced during inflammatory responses. The stimulated synthesis of PAF in PMN is carried out by a phospholipid remodeling pathway involving three enzymes: acetyl-CoA:lyso-PAF acetyltransferase (acetyltransferase), type IV phospholipase A(2) (cPLA(2)) and CoA-independent transacylase (CoA-IT). However, the coordinated actions and the regulatory mechanisms of these enzymes in PAF synthesis are poorly defined. A23187 has been widely used to activate the remodeling pathway, but it has not been shown how closely its actions mimic those of physiological stimuli. Here we address this important problem and compare responses of the three remodeling enzymes and PAF synthesis by intact cells. In both A23187- and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN, acetyltransferase activation is blocked by SB 203580, a p38 MAP kinase inhibitor, but not by PD 98059, which blocks activation of the ERKs. In contrast, either agent attenuated cPLA(2) activation. Correlating with these results, SB 203580 decreased stimulated PAF formation by 60%, whereas PD 98059 had little effect. However, the combination of both inhibitors decreased PAF formation to control levels. Although a role for CoA-IT in PAF synthesis is recognized, we did not detect activation of the enzyme in stimulated PMN. CoA-IT thus appears to exhibit full activity in resting as well as stimulated cells. We conclude that the calcium ionophore A23187 and the receptor agonist fMLP both act through common pathways to stimulate PAF synthesis, with p38 MAP kinase regulating acetyltransferase and supplementing ERK activation of cPLA(2). PMID:12379481

  12. Lipoteichoic Acid-Induced Nitric Oxide Production Depends on the Activation of Platelet-Activating Factor Receptor and Jak21

    PubMed Central

    Han, Seung Hyun; Kim, Je Hak; Seo, Ho Seong; Martin, Michael H.; Chung, Gook-Hyun; Michalek, Suzanne M.; Nahm, Moon H.

    2006-01-01

    NO production by macrophages in response to lipoteichoic acid (LTA) and a synthetic lipopeptide (Pam3CSK4) was investigated. LTA and Pam3CSK4 induced the production of both TNF-α and NO. Inhibitors of platelet-activating factor receptor (PAFR) blocked LTA- or Pam3CSK4-induced production of NO but not TNF-α. Jak2 tyrosine kinase blocked LTA-induced production of NO but not TNF-α. PAFR inhibition blocked phosphorylation of Jak2 and STAT1, a key factor for expressing inducible NO synthase. In addition, LTA did not induce IFN-β expression, and p38 mitogen-activated protein serine kinase was necessary for LTA-induced NO production but not for TNF-α production. These findings suggest that Gram-positive bacteria induce NO production using a PAFR signaling pathway to activate STAT1 via Jak2. This PAFR/Jak2/STAT1 signaling pathway resembles the IFN-β, type I IFNR/Jak/STAT1 pathway described for LPS. Consequently, Gram-positive and Gram-negative bacteria appear to have different but analogous mechanisms for NO production. PMID:16365452

  13. Epidermal Platelet-activating Factor Receptor Activation and Ultraviolet B Radiation Result in Synergistic Tumor Necrosis Factor-alpha Production

    PubMed Central

    Wolverton, Jay E.; Al-Hassani, Mohammed; Yao, Yongxue; Zhang, Qiwei; Travers, Jeffrey B.

    2010-01-01

    Ultraviolet B radiation (UVB) is a potent stimulator of epidermal cytokine production which has been implicated in photoaggravated dermatoses. In addition to cytokines such as tumor necrosis factor-α (TNF-α), UVB generates bioactive lipids including platelet-activating factor (PAF). Our previous studies have demonstrated that UVB-mediated production of keratinocyte TNF-α is in part due to PAF. The current studies use a human PAF-receptor (PAF-R) negative epithelial cell line transduced with PAF-Rs and PAF–R-deficient mice to demonstrate that activation of the epidermal PAF-R along with UVB irradiation results in a synergistic production of TNF-α. It should be noted that PAF-R effects are mimicked by the protein kinase C (PKC) agonist phorbol myristic acetate, and are inhibited by pharmacological antagonists of the PKC gamma isoenzyme. These studies suggest that concomitant PAF-R activation and UVB irradiation results in a synergistic production of the cytokine TNF-α which is mediated in part via PKC. These studies provide a novel potential mechanism for photosensitivity responses. PMID:19769579

  14. Cystamine immobilization on TiO 2 film surfaces and the influence on inhibition of collagen-induced platelet activation

    NASA Astrophysics Data System (ADS)

    Zhou, Yujuan; Weng, Yajun; Zhang, Liping; Jing, Fengjuan; Huang, Nan; Chen, Junying

    2011-12-01

    Poor haemocompatibility is a main issue of artificial cardiovascular materials in clinical application. Nitric oxide (NO), produced by vascular endothelial cells, is a well known inhibitor of platelet adhesion and activation. Thus, NO-releasing biomaterials are beneficial for improving haemocompatibility of blood-contacting biomedical devices. In this paper, a novel method was developed for enhancement of haemocompatibility by exploiting endogenous NO donors. TiO 2 films were firstly synthesized on Si (1 0 0) wafers via unbalanced magnetron sputtering technology, and then polydopamine was grafted on TiO 2 films and used as a linker for further immobilization of cystamine. The obtained surfaces were characterized by scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analysis. NO generation is evaluated by saville-griess reagents, and it shows that cystamine immobilized samples are able to catalytically generate NO by decomposing endogenous S-nitrosothiols (RSNO). In vitro platelet adhesion results reveal that cystamine modified surfaces can inhibit collagen-induced platelet activation. ELISA analysis reveals that cGMP in platelets obviously increases on cystamine immobilized surface, which suggests the reducing of platelet activation is through NO/cGMP signal channel. It can be concluded that cystamine immobilized surface shows better blood compatibility by catalyzing NO release from the endogenous NO donor. It may be a promising method for improvement of haemocompatibility of blood-contacting implants.

  15. Regulation of platelet-activating factor receptor gene expression in vivo by endotoxin, platelet-activating factor and endogenous tumour necrosis factor.

    PubMed Central

    Wang, H; Tan, X; Chang, H; Gonzalez-Crussi, F; Remick, D G; Hsueh, W

    1997-01-01

    A competitive PCR assay was developed to quantify platelet-activating factor (PAF) receptor (PAF-R) transcripts in rat tissues using a synthetic RNA as a competitor. We found PAF-R mRNA constitutively expressed in the eight organs tested, with the ileum containing the highest concentration [(3.49+/-0.15) x 10(7) molecules/microg of RNA]. Significant but lower levels were also detected in the jejunum, spleen, lungs, kidneys, heart, stomach and liver. Furthermore we defined the regulatory role of inflammatory mediators in ileal PAF-R gene expression using a rat model of intestinal injury induced by PAF or lipopolysaccharide (LPS). Injection of LPS or low-dose PAF resulted in a marked increase in ileal PAF-R mRNA within 30 min. The up-regulation on PAF-R elicited by PAF was biphasic, peaking first at 90 min, then again at 6 h. In contrast, LPS elicited a weak monophasic response. The second phase of PAF-R mRNA increase after PAF administration was completely abolished by WEB 2170, a PAF antagonist, and partially inhibited by antitumour necrosis factor (TNF) antibody. These observations indicate the involvement of endogenous PAF and TNF in this event. In conclusion, we found: (a) preferential PAF-R expression in the ileum, suggesting a role for PAF in intestinal inflammation; (b) induction of PAF-R expression in vivo by its own agonist; (c) a complex regulation of PAR-R gene expression in vivo involving a network of various pro-inflammatory mediators. PMID:9065783

  16. [Genetic variants of platelet ADP receptor P2Y12 associated with changed platelet functional activity and development of cardiovascular diseases].

    PubMed

    Sirotkina, O V; Zabotina, A M; Berkovich, O A; Bazhenova, E A; Vavilova, T V; Shvartsman, A L

    2009-02-01

    The key role in platelet aggregation is played by the platelet ADP receptor P2Y12, which is the target for antiaggregant drugs, clopidogrel and ticlopidine. At present, only sporadic data on genetic variants of platelet ADP receptor P2Y12 are available from literature, and their association with thromboembolic and cardiovascular diseases still remains obscure. Analysis of the group of subjects with high platelet reactivity resulted in identification of two nucleotide substitutions, C18T and G36T, in the coding region of the P2Y12 gene. The frequency of the P2Y12 T1 8 allele was higher in control group than in the group of patients survived from myocardial infarction at the age under 45 years (39% versus 28%, respectively, P = 0.04). Moreover, in the T18 carriers, platelet aggregation activity was lower than in the carriers of the wild-type genotype (0.84 +/- 0.05% versus 1.01 +/- 0.08%, respectively, P = 0.03). In the group of patients with early myocardial infarctions, a tendency towards the increased frequency of 16T allele in comparison with control group (20 and 12%, respectively, P = 0.07) was observed. The rate of ADP-induced platelet aggregation in the carriers of 16T allele from the control group was somewhat higher than in the subjects with the GG36 genotype (1.31 +/- 0.16% versus 1.12 +/- 0.06%, respectively, P = 0.07). The nucleotide substitutions identified were in absolute disequilibrium, i.e., allele T18 conformed to allele G36. On the contrary, allele C18 conformed to allele T36. Haplotype T18G36 was found to be responsible for the decreased risk of myocardial infarction and decreased platelet reactivity. It is suggested that polymorphisms of the P2Y12 gene identified can be used for determination of the risk group for myocardial infarction in the young males. PMID:19334620

  17. Highly active antiretroviral therapy-related mechanisms of endothelial and platelet function alterations.

    PubMed

    Gresele, Paolo; Falcinelli, Emanuela; Momi, Stefania; Francisci, Daniela; Baldelli, Franco

    2014-01-01

    Highly active antiretroviral therapy (HAART) has transformed human immunodeficiency virus (HIV) infection into a chronic condition, which has allowed the infected population to age and become prone to chronic degenerative diseases common to the general population, including atherosclerotic cardiovascular disease, and coronary artery disease (CAD). Possible causative mechanisms of HIV-associated CAD are related to classic cardiovascular risk factors, such as dyslipidemia, insulin resistance, and fat redistribution, which may be due to either HIV infection or to HAART-associated toxicity. However, other mechanisms are emerging as crucial for the cardiovascular complication of HIV and HAART. This article analyzes the effects of HIV and HAART on endothelial function, endothelium-leukocyte interactions, and platelets as possible mechanisms of enhanced cardiovascular risk. PMID:24987863

  18. Platelet-activating factor induces eosinophil peroxidase release from purified human eosinophils.

    PubMed Central

    Kroegel, C; Yukawa, T; Dent, G; Chanez, P; Chung, K F; Barnes, P J

    1988-01-01

    The degranulation response of purified human eosinophils to platelet-activating factor (PAF) has been studied. PAF induced release of eosinophil peroxidase (EPO) and beta-glucuronidase from highly purified human eosinophils with an EC50 of 0.9 nM. The order of release was comparable with that induced by phorbol myristate acetate (PMA). The new specific PAF antagonist 3-[4-(2-chlorophenyl)-9-methyl-H-thieno[3,2-f] [1,2,4]triazolo-[4,3a][1,4]-diazepin-2-yl](4-morpholinyl)- 1-propane-one (WEB 2086) inhibited the PAF-induced enzyme release by human eosinophils in a dose-dependent manner. The viability of eosinophils were unaffected both by PAF and WEB 2086. The results suggest that PAF may amplify allergic and inflammatory reactions by release of preformed proteins from eosinophil granules. PMID:3410498

  19. Influence of experimental Anaplasma marginale infection and splenectomy on NTPDase and 5'nucleotidase activities in platelets of cattle.

    PubMed

    Doyle, Rovaina L; Oliveira, Camila B; França, Raqueli T; Doleski, Pedro H; Souza, Viviane C; Leal, Daniela B R; Martins, João R; Lopes, Sonia T A; Machado, Gustavo; Da Silva, Aleksandro S; Andrade, Cinthia M

    2016-06-01

    The objective of this paper was to evaluate NTPDase and 5'-nucleotidase activities in platelets of bovine with and without spleen and infected by Anaplasma marginale. Our results demonstrate that infection along with splenectomy is able of inducing a profile of cellular protection, which showed an increase in the degradation of the nucleotides ATP and ADP by NTPDase, in addition to AMP by 5'nucleotidase to form the nucleoside adenosine in platelets, i.e., the enzymatic activities of platelets were increased in splenectomized animals when compared to non-splenectomized group. It notes that adenosine is a molecule with anti-inflammatory function. But this profile is related to a deficiency in immune signaling triggered by nucleotide ATP, which may be related to the increase in bacteremia and disability in combating the parasite in splenectomized host. PMID:26945560

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

    PubMed Central

    Fuentes, Eduardo; Palomo, Iván

    2013-01-01

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

  1. Hemocompatibility of Poly(vinyl alcohol)-Gelatin Core-Shell Electrospun Nanofibers: A Scaffold for Modulating Platelet Deposition and Activation.

    PubMed

    Merkle, Valerie M; Martin, Daniel; Hutchinson, Marcus; Tran, Phat L; Behrens, Alana; Hossainy, Samir; Sheriff, Jawaad; Bluestein, Danny; Wu, Xiaoyi; Slepian, Marvin J

    2015-04-22

    In this study, we evaluate coaxial electrospun nanofibers with gelatin in the shell and poly(vinyl alcohol) (PVA) in the core as a potential vascular material by determining fiber surface roughness, as well as human platelet deposition and activation under varying conditions. PVA scaffolds had the highest surface roughness (Ra=65.5±6.8 nm) but the lowest platelet deposition (34.2±5.8 platelets) in comparison to gelatin nanofibers (Ra=36.8±3.0 nm and 168.9±29.8 platelets) and coaxial nanofibers (1 Gel:1 PVA coaxial, Ra=24.0±1.5 nm and 150.2±17.4 platelets. 3 Gel:1 PVA coaxial, Ra=37.1±2.8 nm and 167.8±15.4 platelets). Therefore, the chemical structure of the gelatin nanofibers dominated surface roughness in platelet deposition. Due to their increased stiffness, the coaxial nanofibers had the highest platelet activation rate, rate of thrombin formation, in comparison to gelatin and PVA fibers. Our studies indicate that mechanical stiffness is a dominating factor for platelet deposition and activation, followed by biochemical signals, and lastly surface roughness. Overall, these coaxial nanofibers are an appealing material for vascular applications by supporting cellular growth while minimizing platelet deposition and activation. PMID:25815434

  2. Extracellular matrix is a source of mitogenically active platelet-derived growth factor.

    PubMed

    Field, S L; Khachigian, L M; Sleigh, M J; Yang, G; Vandermark, S E; Hogg, P J; Chesterman, C N

    1996-08-01

    Platelet-derived growth factor (PDGF) is a chemotactic and mitogenic agent for fibroblasts and smooth muscle cells and plays a key role in the development of atherosclerotic lesions. PDGF is produced by a number of normal and transformed cell types and occurs as homo- or heterodimers of A and B polypeptide chains. Using Chinese hamster ovary (CHO) cells transfected with various forms of PDGF, we have previously shown that PDGF A(s) (short splice version) is secreted, PDGF A(l) (long splice version) predominantly extracellular matrix-associated, and PDGF B divided between medium, cells, and matrix. In the present study we have demonstrated the mitogenic activity of matrix-localized PDGF in artificial and more physiologically relevant models by culturing Balb/c-3T3 cells (3T3), human foreskin fibroblasts (HFF), and rabbit aortic smooth muscle cells (SMC) on extracellular matrix (ECM) laid down by PDGF-expressing CHO cells and human umbilical vein endothelial cells (HUVEC). These cells responded to the local growth stimulus of PDGF-containing CHO ECM and HUVEC ECM. We showed that 3T3 cells required proteolytic activity to utilize matrix-localized PDGF, as aprotinin and epsilon-ACA inhibited growth and 3T3 cells were shown to possess plasminogen activator activity. HFF and SMC did not appear to require proteolytic activity (including metalloproteinase and serine protease activity) as a prerequisite for mitogenesis but were able to access immobilized PDGF by contact with the matrix. An understanding of the mechanisms whereby the utilization of stored PDGF is controlled in situations of excessive cellular proliferation will aid in the development of therapy for these conditions. PMID:8707868

  3. Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

    SciTech Connect

    Walker, G.; Bourguignon, L.Y. )

    1990-08-01

    Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation.

  4. Effect of active synthetic 2-substituted quinazolinones on anti-platelet aggregation and the inhibition of superoxide anion generation by neutrophils.

    PubMed

    Chang, Fang-Rong; Wu, Chin-Chung; Hwang, Tsong-Long; Patnam, Ramesh; Kuo, Reen-Yen; Wang, Wei-Ya; Lan, Yu-Hsuan; Wu, Yang-Chang

    2003-07-01

    Quinazolinones, 2-substituted and 3-substituted, mainly synthesized by microwave irradiation, were subjected to anti-platelet aggregation and inhibition of superoxide anion generation assays. Interestingly, 2-phenyl-4-quinazolinone (4) exhibited significant inhibitory activities toward platelet aggregation and neutrophil activation, and it might therefore serve as a prototype lead compound. PMID:12934640

  5. Essential role of platelet-activating factor in male reproduction: a review.

    PubMed

    Ali, Atef; Virirak Kattygnarath, Tiao; Benkhalifa, Moncef; Miron, Pierre

    2007-02-01

    The use of pharmacological adjuvants to enhance sperm function is a real possibility and a very attractive proposition. However, the role of such adjuvants must be well defined to maximize therapeutic outcome for specific patient populations. Platelet-activating factor (PAF) is a potent signalling phospholipid that has been implicated in a number of biological activities. In the field of reproductive biology, PAF has been shown to have an important role in ovulation, sperm function, embryo implantation, fetal lung maturation, and initiation and maintenance of parturition. Although the exact mechanisms are not clear, it has been demonstrated in several species that PAF can influence sperm function by affecting the motility, capacitation, acrosome reaction and fertility of spermatozoa. The reproductive significance of PAF activity in spermatozoa and fertilization and its role in the establishment of pregnancy requires further study. Understanding the role of PAF in a reproductive process, such as in intrauterine insemination, could eventually lead researchers to explore its role in more complex reproductive technologies. This review summarizes the current research on the significant role of PAF in male mammalian reproductive functions, as well as its possible application in some assisted reproductive technologies. PMID:17298732

  6. Chemotherapeutic agents subvert tumor immunity by generating agonists of platelet-activating factor.

    PubMed

    Sahu, Ravi P; Ocana, Jesus A; Harrison, Kathleen A; Ferracini, Matheus; Touloukian, Christopher E; Al-Hassani, Mohammed; Sun, Louis; Loesch, Mathew; Murphy, Robert C; Althouse, Sandra K; Perkins, Susan M; Speicher, Paul J; Tyler, Douglas S; Konger, Raymond L; Travers, Jeffrey B

    2014-12-01

    Oxidative stress suppresses host immunity by generating oxidized lipid agonists of the platelet-activating factor receptor (PAF-R). Because many classical chemotherapeutic drugs induce reactive oxygen species (ROS), we investigated whether these drugs might subvert host immunity by activating PAF-R. Here, we show that PAF-R agonists are produced in melanoma cells by chemotherapy that is administered in vitro, in vivo, or in human subjects. Structural characterization of the PAF-R agonists induced revealed multiple oxidized glycerophosphocholines that are generated nonenzymatically. In a murine model of melanoma, chemotherapeutic administration could augment tumor growth by a PAF-R-dependent process that could be blocked by treatment with antioxidants or COX-2 inhibitors or by depletion of regulatory T cells. Our findings reveal how PAF-R agonists induced by chemotherapy treatment can promote treatment failure. Furthermore, they offer new insights into how to improve the efficacy of chemotherapy by blocking its heretofore unknown impact on PAF-R activation. PMID:25304264

  7. Ligustrazine improves blood circulation by suppressing Platelet activation in a rat model of allergic asthma.

    PubMed

    Wang, Yajuan; Zhu, Huizhi; Tong, Jiabing; Li, Zegeng

    2016-07-01

    Chuan-xiong (Ligusticum wallichii) is a traditional herbal medicine in Eastern Asia, but the effect of its active component ligustrazine remains unclear. We explored its effect and possible mechanism in a well-characterized rat model of allergic asthma. Ligustrazine suppressed bronchial hyper-responsiveness to methacholine, and suppressed lung inflammation in asthmatic rats. Ligustrazine exhibited potent immuno-modulatory and anti-inflammatory effects: it suppressed lymphocyte and eosinophil mobilization, and reduced cytokine IL-5 and IL-13 production significantly in lung tissues from asthmatic rats (P<0.05). Further histological examinations clearly demonstrated that ligustrazine improved blood circulation and ameliorated platelet activation, aggregation and adhesion, which induced sustained infiltration and activation of various inflammatory cells, including lymphocytes and eosinophils, followed by synthesis and release of a variety of pro-inflammatory mediators and cytokines. The present study suggests that ligustrazine is a potent agent for the treatment of allergic asthma due to its strong anti-inflammatory and immuno-modulatory properties. PMID:27362664

  8. Regional Neonatal Associates for cooperative study of platelet-activating factor (PAF). Summary report

    SciTech Connect

    Snyder, F.

    1992-11-01

    Lipid inflammatory mediators are thought to play an important role in the pathogenesis of the respiratory distress syndrome, including neonatal lung injury and bronchopulmonary dysplasia (BPD). One such mediator is platelet-activating factor (PAF), a potent bioactive phospholipid that induces adverse airway, vascular, and microcirculatory responses. To study the role of PAF in neonatal lung disease, we used an {sup 125}I-radioimmunoassay to measure PAF in whole blood and tracheal lavage in very low birthweight infants at 1, 3, 5, 9, 21 and 28 days after birth. PAF was found in the pulmonary lavagate and blood of ventilated infants as early as one day after birth. Lavagate levels of PAF increased with acute injury (pneumothorax, pneumonia) but were not associated with BPD. Our results indicate PAF could be associated with the pathogenesis of BPD. We suggest that as a consequence of the pathophysiologic processes associated with BPD, PAF is released by pulmonary cells. Our preliminary data indicate that low birthweight infants also have lower PAF acetylhydrolase levels in cord blood and tracheal lavagate as compared to adults. Therefore, it is possible the increased levels of PAF in the blood of low birthweight infants might be due to persistent transient increases in PAF alveolar levels coupled with lower blood acetylhydrolase activities and could be important in the development of symptoms associated with BPD. Future plans for this project call for completing the enzymatic study of acetylhydrolase activity in pulmonary lavage of the BPD infants.

  9. Mechanisms of endothelial cell-dependent leukocyte adhesion stimulated by platelet-activating factor.

    PubMed

    Ding, Z; Li, S; Wu, Z

    1992-04-01

    Platelet-activating factor (PAF) stimulates leukocyte-endothelial cell (EC) adhesion through its effects either on leukocytes or on ECs. ECs may be injured, synthesize, or express new adhesive proteins to increase leukocyte adhesion. Intermediary mediators produced by activated ECs are also likely involved in promoting leukocyte adhesion. Our experiments demonstrated that PAF induced no obvious damage to bovine pulmonary artery ECs evaluated by lactic dehydrogenase release rate, angiotensin-converting enzyme activity, and cellular malondialdehyde content. Treatment of EC monolayers with 10(-9) M PAF increased polymorphonuclear leukocyte (PMN) adhesion. Increasing PAF concentration did not induce more PMN adherence. PAF elicited both a rapid and prolonged increment of PMN adherence to EC monolayers. The rapid adherence was greatly attenuated by pretreatment of ECs with PAF receptor antagonist SRI 63-441 but was not affected by pretreatment of PMNs with SRI 63-441, suggesting that PAF increases PMN adherence rapidly through its effects on specific receptors on ECs. Increased PMN adherence lasted if PAF treatment of ECs was sustained for 3 or 6 h. Pretreatment of ECs with actinomycin D, a protein synthesis inhibitor, significantly decreased PAF-induced sustained PMN adherence, but the inhibition is incomplete, suggesting that other mechanisms than protein synthesis also participated in the prolonged PMN adherence. We concluded from the results that PAF may induce both rapid and prolonged PMN adhesion to ECs. The effects are receptor mediated. The prolonged PMN adhesion is partly the result of protein synthesis. PMID:1592489

  10. [Comparison of platelet activity, fibrinolysis and environmental factors in 50 Africans and 50 Europeans. Role of fish consumption].

    PubMed

    Bertrand, E; Cloitre, B; Ticolat, R; Darracq, R; Rain, S F

    1987-01-01

    The authors have studied 50 Ivorians and 50 Europeans people, all living in Abidjan for at least 10 years. Platelet aggregability with increasing ADP concentration (0.6, 1.2, 2.4 mumoles/l), collagen (0.4 mg/l), or ristocetin (1 g/l) was examined. Fibrinolysis and the euglobulin test were also studied before and after anoxia. Other blood parameters measured were: hematocrit, hemoglobin level, platelet count, bleeding time, Howell coagulation test, cephalin tests, prothrombin activity ratio, fibrinogen level. Metabolic tests included: glycemia, cholesterolemia, triglyceridemia, uricemia, A1 and B apoproteins, protidemia, gamma globulinemia. Environmental factors such as physical activity, alcohol and smoking habits, fish consumption, chloroquine prophylaxis were evaluated. The most evident result was lower platelet aggregability in Ivorian people as compared to Europeans. A more precocious and important fibrinolysis activity, either spontaneous or after anoxia was noted in the Ivorian group. Lower platelet number, fibrinogen level, and prothrombin activity were present in the Ivorian group as compared to the European people. The authors eliminated the influence of age, and considered environmental factors as predominant in the genesis of such difference, i.e., hypocholesterolemia, lower smoking and drinking levels. They emphasized the higher fish consumption in Ivorian people. PMID:2827101

  11. Dietary Supplementation of Ginger and Turmeric Rhizomes Modulates Platelets Ectonucleotidase and Adenosine Deaminase Activities in Normotensive and Hypertensive Rats.

    PubMed

    Akinyemi, Ayodele Jacob; Thomé, Gustavo Roberto; Morsch, Vera Maria; Bottari, Nathieli B; Baldissarelli, Jucimara; de Oliveira, Lizielle Souza; Goularte, Jeferson Ferraz; Belló-Klein, Adriane; Oboh, Ganiyu; Schetinger, Maria Rosa Chitolina

    2016-07-01

    Hypertension is associated with platelet alterations that could contribute to the development of cardiovascular complications. Several studies have reported antiplatelet aggregation properties of ginger (Zingiber officinale) and turmeric (Curcuma longa) with limited scientific basis. Hence, this study assessed the effect of dietary supplementation of these rhizomes on platelet ectonucleotidase and adenosine deaminase (ADA) activities in Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) induced hypertensive rats. Animals were divided into seven groups (n = 10): normotensive control rats; induced (l-NAME hypertensive) rats; hypertensive rats treated with atenolol (10 mg/kg/day); normotensive and hypertensive rats treated with 4% supplementation of turmeric or ginger, respectively. After 14 days of pre-treatment, the animals were induced with hypertension by oral administration of l-NAME (40 mg/kg/day). The results revealed a significant (p < 0.05) increase in platelet ADA activity and ATP hydrolysis with a concomitant decrease in ADP and AMP hydrolysis of l-NAME hypertensive rats when compared with the control. However, dietary supplementation with turmeric or ginger efficiently prevented these alterations by modulating the hydrolysis of ATP, ADP and AMP with a concomitant decrease in ADA activity. Thus, these activities could suggest some possible mechanism of the rhizomes against hypertension-derived complications associated to platelet hyperactivity. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27151061

  12. Mapping the Human Platelet Lipidome Reveals Cytosolic Phospholipase A2 as a Regulator of Mitochondrial Bioenergetics during Activation.

    PubMed

    Slatter, David A; Aldrovandi, Maceler; O'Connor, Anne; Allen, Stuart M; Brasher, Christopher J; Murphy, Robert C; Mecklemann, Sven; Ravi, Saranya; Darley-Usmar, Victor; O'Donnell, Valerie B

    2016-05-10

    Human platelets acutely increase mitochondrial energy generation following stimulation. Herein, a lipidomic circuit was uncovered whereby the substrates for this are exclusively provided by cPLA2, including multiple fatty acids and oxidized species that support energy generation via β-oxidation. This indicates that acute lipid membrane remodeling is required to support energetic demands during platelet activation. Phospholipase activity is linked to energy metabolism, revealing cPLA2 as a central regulator of both lipidomics and energy flux. Using a lipidomic approach (LipidArrays), we also estimated the total number of lipids in resting, thrombin-activated, and aspirinized platelets. Significant diversity between genetically unrelated individuals and a wealth of species was revealed. Resting platelets demonstrated ∼5,600 unique species, with only ∼50% being putatively identified. Thrombin elevated ∼900 lipids >2-fold with 86% newly appearing and 45% inhibited by aspirin supplementation, indicating COX-1 is required for major activation-dependent lipidomic fluxes. Many lipids were structurally identified. With ∼50% of the lipids being absent from databases, a major opportunity for mining lipids relevant to human health and disease is presented. PMID:27133131

  13. Nitric oxide mediates the inhibitory action of platelet-activating factor on angiotensin II-induced renal vasoconstriction, in vivo.

    PubMed

    Handa, R K; Strandhoy, J W

    1996-06-01

    The objective of our study was to determine the mechanism(s) involved in the inhibitory effect of platelet-activating factor on renal vascular reactivity, in vivo. Bolus injections of vasoconstrictor agonists were administered into the renal circulation of pentobarbital anesthetized male Wistar rats at a dose to cause a transient 45 to 50% decrease in renal blood flow. Intrarenal infusion of platelet-activating factor (PAF) at 2.5 ng/min/kg attenuated the vasoconstrictor response to angiotensin II by 66%, a significantly smaller reduction of 35% for norepinephrine-mediated vasoconstriction, 22% for vasopressin-mediated vasoconstriction and no alteration of KCl-mediated vasoconstriction. The preferential inhibitory effect of platelet-activating factor on angiotensin II-mediated renal vasoconstriction was mimicked by the intrarenal infusion of either 0.2 to 5 micrograms/min/kg methacholine (endothelium-dependent vasodilator) or 2 micrograms/min/kg sodium nitroprusside (nitric oxide donor). After inhibition of nitric oxide synthesis with NG-monomethyl-L-arginine, intrarenal infusion of PAF or methacholine reduced angiotensin II-mediated renal vasoconstriction significantly less than that observed in the absence of NG-monomethyl-L-arginine. Therefore, this study provides evidence that the shared ability of platelet-activating factor and methacholine to selectively reduce angiotensin II-mediated renal vasoconstriction involves endothelium-derived nitric oxide. PMID:8667214

  14. 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

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

    PubMed Central

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

    2012-01-01

    Noncovalent association between the von Willebrand factor (VWF) propeptide (VWFpp) and mature VWF aids N-terminal multimerization and protein compartmentalization in storage granules. This association is currently thought to dissipate after secretion into blood. In the present study, we examined this proposition by quantifying the affinity and kinetics of VWFpp binding to mature VWF using surface plasmon resonance and by developing novel anti-VWF D′D3 mAbs. Our results show that the only binding site for VWFpp in mature VWF is in its D′D3 domain. At pH 6.2 and 10mM Ca2+, conditions mimicking intracellular compartments, VWFpp-VWF binding occurs with high affinity (KD = 0.2nM, koff = 8 × 10−5 s−1). Significant, albeit weaker, binding (KD = 25nM, koff = 4 × 10−3 s−1) occurs under physiologic conditions of pH 7.4 and 2.5mM Ca2+. This interaction was also observed in human plasma (KD = 50nM). The addition of recombinant VWFpp in both flow-chamber–based platelet adhesion assays and viscometer-based shear-induced platelet aggregation and activation studies reduced platelet adhesion and activation partially. Anti-D′D3 mAb DD3.1, which blocks VWFpp binding to VWF-D′D3, also abrogated platelet adhesion, as shown by shear-induced platelet aggregation and activation studies. Our data demonstrate that VWFpp binding to mature VWF occurs in the circulation, which can regulate the hemostatic potential of VWF by reducing VWF binding to platelet GpIbα. PMID:22452980

  16. Evidence for Lipid Packaging in the Crystal Structure of the GM2-Activator Complex with Platelet Activating Factor

    SciTech Connect

    Wright, Christine S.; Mi, Li-Zhi; Rastinejad, Fraydoon

    2010-11-16

    GM2-activator protein (GM2-AP) is a lipid transfer protein that has the ability to stimulate the enzymatic processing of gangliosides as well as T-cell activation through lipid presentation. Our previous X-ray crystallographic studies of GM2-AP have revealed a large lipid binding pocket as the central overall feature of the structure with non-protein electron density within this pocket suggesting bound lipid. To extend these studies, we present here the 2 {angstrom} crystal structure of GM2-AP complexed with platelet activating factor (PAF). PAF is a potent phosphoacylglycerol whose toxic patho-physiological effects can be inhibited by GM2-AP. The structure shows an ordered arrangement of two bound lipids and a fatty acid molecule. One PAF molecule binds in an extended conformation within the hydrophobic channel that has an open and closed conformation, and was seen to contain bound phospholipid in the low pH apo structure. The second molecule is submerged inside the pocket in a U-shaped conformation with its head group near the single polar residue S141. It was refined as lyso-PAF as it lacks electron density for the sn-2 acetate group. The alkyl chains of PAF interact through van der Waals contacts, while the head groups bind in different environments with their phosphocholine moieties in contact with aromatic rings (Y137, F80). The structure has revealed further insights into the lipid binding properties of GM2-AP, suggesting an unexpected unique mode of lipid packaging that may explain the efficiency of GM2-AP in inhibiting the detrimental biological effects of PAF.

  17. Synthesis, biochemical evaluation and molecular modeling studies of novel rhodium complexes with nanomolar activity against Platelet Activating Factor.

    PubMed

    Tsoupras, Alexandros B; Papakyriakou, Athanasios; Demopoulos, Constantinos A; Philippopoulos, Athanassios I

    2013-03-01

    Two square planar Rh(I) organometallic complexes namely [Rh(L(1))(cod)]Cl (cod = cycloocta-1,5-diene, L(1)=2,2'-pyridylquinoxaline (1-Cl), [Rh(L1)(cod)](NO3) (1-NO(3)) and a series of novel octahedral rhodium(III) complexes of the general formulae mer-[Rh(L(1))Cl(3)(MeOH)] (2) and cis-[Rh(L(2))(2)Cl(2)]Cl (L(2)=4 carboxy 2 (2' pyridyl)quinoline (3), L(3)=2,2' bipyridine 4,4' dicarboxylic acid (4) were synthesized and characterized spectroscopically. All the synthesized compounds including the previously prepared cis-[Rh(L(1))(2)Cl(2)]Cl complex (5) were biologically evaluated as potential inhibitors of the Platelet Activation Factor (PAF) and thrombin induced aggregation. In particular compounds 1-Cl and 1-NO(3) were found to be strong inhibitors of PAF with IC(50) values in the range of 16 nM and 15 nM rendering them good candidates for further investigation. Their potency is comparable to that of the widely used PAF receptor antagonists WEB2170, BN52021, and Rupatadine (IC(50) of 20, 30 and 260 nM respectively). Molecular docking calculations suggest that 1-Cl, 1-NO3 and 2 can be accommodated within the ligand-binding site of PAF receptor and block the activity of PAF. On the other hand, the octahedral rhodium(III) complexes 3-5 that cannot fit the ligand-binding domain, could potentially exhibit their activity at the extracellular domain of the receptor. PMID:23318288

  18. Effect of platelet activating factor (PAF) on the formation of blood vessels in subcutaneous implants in mice.

    PubMed

    Andrade, S P; Vieira, L B; Bakhle, Y S; Piper, P J

    1994-03-01

    Angiogenesis accompanies inflammatory processes and many other pathological conditions. We have studied the effect of platelet-activating factor (PAF) a well-known inflammatory mediator, as a promoter of angiogenesis in the sponge implant model in mice. Development of blood vessels and blood flow were monitored by use of a 133Xe washout technique. The results showed PAF to have angiogenic activity, which was inhibited by WEB 2086, and the PAF-induced vasculature to have normal pharmacological reactivity. PMID:7516807

  19. Platelet-activating factor (PAF) receptor binding antagonist activity of the methanol extracts and isolated flavonoids from Chromolaena odorata (L.) King and Robinson.

    PubMed

    Ling, Sui Kiong; Pisar, Mazura Md; Man, Salbiah

    2007-06-01

    The leaf, stem and root extracts of Chromolaena odorata were evaluated for their effect on platelet-activating factor (PAF) receptor binding on rabbit platelets using 3H-PAF as a ligand. The leaf extract demonstrated high PAF receptor binding inhibitory activity of 79.2+/-2.1% at 18.2 microg/ml. A total of eleven flavonoids were subsequently isolated from the active leaf extract and evaluated for their effects on PAF receptor binding. Eight of the flavonoids exhibited >50% inhibition on the binding activity at 18.2 microg/ml. These flavonoids were identified as eriodictyol 7,4'-dimethyl ether, quercetin 7,4'-methyl ether, naringenin 4'-methyl ether, kaempferol 4'-methyl ether, kaempferol 3-O-rutinoside, taxifolin 4'-methyl ether, taxifolin 7-methyl ether and quercetin 4'-methyl ether. Their IC50 values ranged from 19.5 to 62.1 microM. PMID:17541171

  20. Anti-proliferative lichen compounds with inhibitory activity on 12(S)-HETE production in human platelets.

    PubMed

    Bucar, F; Schneider, I; Ogmundsdóttir, H; Ingólfsdóttir, K

    2004-11-01

    Several lichen compounds, i.e. lobaric acid (1), a beta-orcinol depsidone from Stereocaulon alpinum L., (+)-protolichesterinic acid (2), an aliphatic alpha-methylene-gamma-lactone from Cetraria islandica Laur. (Parmeliaceae), (+)-usnic acid (3), a dibenzofuran from Cladonia arbuscula (Wallr.) Rabenh. (Cladoniaceae), parietin (4), an anthraquinone from Xanthoria elegans (Link) Th. Fr. (Calaplacaceae) and baeomycesic acid (5), a beta-orcinol depside isolated from Thamnolia vermicularis (Sw.) Schaer. var. subuliformis (Ehrh.) Schaer. were tested for inhibitory activity on platelet-type 12(S)-lipoxygenase using a cell-based in vitro system in human platelets. Lobaric acid (1) and (+)-protolichesterinic acid (2) proved to be pronounced inhibitors of platelet-type 12(S)-lipoxygenase, whereas baeomycesic acid (5) showed only weak activity (inhibitory activity at a concentration of 100 microg/ml: (1) 93.4+/-6.62%, (2) 98,5+/-1.19%, 5 14.7+/-2.76%). Usnic acid (3) and parietin (4) were not active at this concentration. 1 and 2 showed a clear dose-response relationship in the range of 3.33-100 microg/ml. According to the calculated IC50 values the highest inhibitory activity was observed for the depsidone 1 (IC50 = 28.5 microM) followed by 2 (IC50 = 77.0 microM). The activity of 1 was comparable to that of the flavone baicalein, which is known as a selective 12(S)-lipoxygenase inhibitor (IC50 = 24.6 microM). PMID:15636173

  1. Metabolism of platelet activating factor at the whole organ and cellular level

    SciTech Connect

    Haroldsen, P.E.

    1987-01-01

    Platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-3-glycerophosphocholine) has been characterized as a phospholipid possessing a myriad of effects from the cellular to whole organism levels. Analytical methods and procedures were developed in order to measure and identify PAF precursors and metabolites. Two quantitative physicochemical methods based on isotope dilution mass spectrometry (MS) were developed to measure lyso-PAF and applied to the calcium ionophore stimulated human neutrophil. Levels of lyso-PAF were found to be significantly increased, 2-3 fold, upon cell activation with a stimulus that concomitantly elicits the production of PAF. Investigation into the metabolism of PAF by the isolated perfused rat lung by intratracheal instillation revealed (/sup 3/H)-PAF to be extensively metabolized over a 15 minute time course. Greater than 96% of the administered dose was retained by the lung and was distributed as: lyso-PAF (3.3%), phosphatidylcholine (GPC, 82.3%), phosphatidylethanolamine (2.5%), and neutral lipid (2.5%), the remainder was intact PAF.

  2. Multidrug-resistance P-glycoprotein (MDR1) secretes platelet-activating factor.

    PubMed Central

    Raggers, R J; Vogels, I; van Meer, G

    2001-01-01

    The human multidrug-resistance (MDR1) P-glycoprotein (Pgp) is an ATP-binding-cassette transporter (ABCB1) that is ubiquitously expressed. Often its concentration is high in the plasma membrane of cancer cells, where it causes multidrug resistance by pumping lipophilic drugs out of the cell. In addition, MDR1 Pgp can transport analogues of membrane lipids with shortened acyl chains across the plasma membrane. We studied a role for MDR1 Pgp in transport to the cell surface of the signal-transduction molecule platelet-activating factor (PAF). PAF is the natural short-chain phospholipid 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine. [(14)C]PAF synthesized intracellularly from exogenous alkylacetylglycerol and [(14)C]choline became accessible to albumin in the extracellular medium of pig kidney epithelial LLC-PK1 cells in the absence of vesicular transport. Its translocation across the apical membrane was greatly stimulated by the expression of MDR1 Pgp, and inhibited by the MDR1 inhibitors PSC833 and cyclosporin A. Basolateral translocation was not stimulated by expression of the basolateral drug transporter MRP1 (ABCC1). It was insensitive to the MRP1 inhibitor indomethacin and to depletion of GSH which is required for MRP1 activity. While efficient transport of PAF across the apical plasma membrane may be physiologically relevant in MDR1-expressing epithelia, PAF secretion in multidrug-resistant tumours may stimulate angiogenesis and thereby tumour growth. PMID:11463358

  3. Systemic chemotherapy is modulated by platelet-activating factor-receptor agonists.

    PubMed

    Sahu, Ravi P; Ferracini, Matheus; Travers, Jeffrey B

    2015-01-01

    Chemotherapy is used to treat numerous cancers including melanoma. However, its effectiveness in clinical settings is often hampered by various mechanisms. Previous studies have demonstrated that prooxidative stressor-mediated generation of oxidized lipids with platelet-activating factor-receptor (PAF-R) agonistic activity induces systemic immunosuppression that augments the growth of experimental melanoma tumors. We have recently shown that treatment of murine B16F10 melanoma cells in vitro or tumors implanted into syngeneic mice and treated intratumorally with various chemotherapeutic agents generated PAF-R agonists in a process blocked by antioxidants. Notably, these intratumoral chemotherapy-generated PAF-R agonists augmented the growth of secondary (untreated) tumors in a PAF-R dependent manner. As both localized and systemic chemotherapies are used based on tumor localization/stage and metastases, the current studies were sought to determine effects of PAF-R agonists on systemic chemotherapy against experimental melanoma. Here, we show that systemic chemotherapy with etoposide (ETOP) attenuates the growth of melanoma tumors when given subsequent to the tumor cell implantation. Importantly, this ETOP-mediated suppression of melanoma tumor growth was blocked by exogenous administration of a PAF-R agonist, CPAF. These findings indicate that PAF-R agonists not only negatively affect the ability of localized chemotherapy but also compromise the efficacy of systemic chemotherapy against murine melanoma. PMID:25922565

  4. Leptin signalling and leptin-mediated activation of human platelets: importance of JAK2 and the phospholipases Cgamma2 and A2.

    PubMed

    Dellas, Claudia; Schäfer, Katrin; Rohm, Ilonka K; Lankeit, Mareike; Leifheit, Maren; Loskutoff, David J; Hasenfuss, Gerd; Konstantinides, Stavros V

    2007-11-01

    Leptin enhances agonist-induced platelet aggregation, and human platelets have been reported to express the leptin receptor. However, the pathways and mediators lying downstream of leptin binding to platelets remain, with few exceptions, unknown. In the present study, we sought to gain further insight into the possible role of leptin as a platelet agonist. Stimulation of platelets with leptin promoted thromboxane generation and activation of alpha(IIb)beta(3), as demonstrated by PAC-1 binding. Furthermore, it increased the adhesion to immobilised fibrinogen (p<0.001) and induced cytoskeletal rearrangement of both platelets and Meg01 cells. Leptin time- and dose-dependently phosphorylated the intracellular signalling molecules JAK2 and STAT3, although the importance of STAT3 for leptin-induced platelet activation remains to be determined. Important intracellular mediators and pathways activated by leptin downstream of JAK2 were found to include phosphatidylinositol-3 kinase, phospholipase Cgamma2 and protein kinase C, as well as the p38 MAP kinase-phospholipase A(2) axis. Accordingly, incubation with the specific inhibitors AG490, Ly294002, U73122, and SB203580 prevented leptin-mediated platelet activation. These results help delineate biologically relevant leptin signalling pathways in platelets and may improve our understanding of the mechanisms linking hyperleptinaemia to the increased thrombosis risk in human obesity. PMID:18000612

  5. In Vivo Detection of Activated Platelets Allows Characterizing Rupture of Atherosclerotic Plaques with Molecular Magnetic Resonance Imaging in Mice

    PubMed Central

    Wiens, Kristina; Neudorfer, Irene; Zirlik, Andreas; Meissner, Mirko; Tilly, Peg; Charles, Anne-Laure; Bode, Christoph; Peter, Karlheinz; Fabre, Jean-Etienne

    2012-01-01

    Background Early and non-invasive detection of platelets on micro atherothrombosis provides a means to identify unstable plaque and thereby allowing prophylactic treatment towards prevention of stroke or myocardial infarction. Molecular magnetic resonance imaging (mMRI) of activated platelets as early markers of plaque rupture using targeted contrast agents is a promising strategy. In this study, we aim to specifically image activated platelets in murine atherothrombosis by in vivo mMRI, using a dedicated animal model of plaque rupture. Methods An antibody targeting ligand-induced binding sites (LIBS) on the glycoprotein IIb/IIIa-receptor of activated platelets was conjugated to microparticles of iron oxide (MPIO) to form the LIBS-MPIO contrast agent causing a signal-extinction in T2*-weighted MRI. ApoE−/− mice (60 weeks-old) were fed a high fat diet for 5 weeks. Using a small needle, the surface of their carotid plaques was scratched under blood flow to induce atherothrombosis. In vivo 9.4 Tesla MRI was performed before and repetitively after intravenous injection of either LIBS-MPIO versus non-targeted-MPIO. Results LIBS-MPIO injected animals showed a significant signal extinction (p<0.05) in MRI, corresponding to the site of plaque rupture and atherothrombosis in histology. The signal attenuation was effective for atherothrombosis occupying ≥2% of the vascular lumen. Histology further confirmed significant binding of LIBS-MPIO compared to control-MPIO on the thrombus developing on the surface of ruptured plaques (p<0.01). Conclusion in vivo mMRI detected activated platelets on mechanically ruptured atherosclerotic plaques in ApoE−/− mice with a high sensititvity. This imaging technology represents a unique opportunity for noninvasive detection of atherothrombosis and the identification of unstable atherosclerotic plaques with the ultimate promise to prevent strokes and myocardial infarctions. PMID:23028736

  6. The attenuation of platelet and monocyte activation in a rabbit model of extracorporeal circulation by a nitric oxide releasing polymer

    PubMed Central

    Major, Terry C; Brant, David O; Reynolds, Melissa M; Bartlett, Robert H; Meyerhoff, Mark E; Handa, Hitesh; Annich, Gail M.

    2010-01-01

    Nitric oxide (NO) has been shown to reduce thrombogenicity by decreasing platelet and monocyte activation by the surface glycoprotein, P-selectin and the integrin, CD11b, respectively. In order to prevent platelet and monocyte activation with exposure to an extracorporeal circulation (ECC), a nitric oxide releasing (NORel) polymeric coating composed of plasticized polyvinyl chloride (PVC) blended with a lipophilic N-diazeniumdiolate was evaluated in a 4 hour rabbit thrombogenicity model using flow cytometry. The NORel polymer significantly reduced ECC thrombus formation compared to polymer control after 4 hours blood exposure (2.8 ± 0.7 NORel vs 6.7 ± 0.4 pixels/cm2 control). Platelet count (3.4 ± 0.3 NORel vs 2.3 ± 0.3 × 108/ml control) and function as measured by aggregometry (71 ± 3 NORel vs 17 ± 6 % control) were preserved after 4 hours exposure in NORel versus control ECC. Plasma fibrinogen levels significantly decreased in both NORel and control groups. Platelet P-selectin mean fluorescence intensity (MFI) as measured by flow cytometry was attenuated after 4 hours on ECC to ex vivo collagen stimulation (27 ± 1 NORel vs 40 ± 2 MFI control). Monocyte CD11b expression was reduced after 4 hours on ECC with NORel polymer (87 ± 14 NORel vs 162 ± 30 MFI control). These results suggest that the NORel polymer coatings attenuate the increase in both platelet P-selectin and monocytic CD11b integrin expression in blood exposure to ECCs. These NO-mediated platelet and monocytic changes were shown to improve thromboresistance of these NORel-polymer-coated ECCs for biomedical devices. PMID:20042236

  7. Synthesis of Analogues of Gingerol and Shogaol, the Active Pungent Principles from the Rhizomes of Zingiber officinale and Evaluation of Their Anti-Platelet Aggregation Effects

    PubMed Central

    Shih, Hung-Cheng; Chern, Ching-Yuh; Kuo, Ping-Chung; Wu, You-Cheng; Chan, Yu-Yi; Liao, Yu-Ren; Teng, Che-Ming; Wu, Tian-Shung

    2014-01-01

    The present study was aimed at discovering novel biologically active compounds based on the skeletons of gingerol and shogaol, the pungent principles from the rhizomes of Zingiber officinale. Therefore, eight groups of analogues were synthesized and examined for their inhibitory activities of platelet aggregation induced by arachidonic acid, collagen, platelet activating factor, and thrombin. Among the tested compounds, [6]-paradol (5b) exhibited the most significant anti-platelet aggregation activity. It was the most potent candidate, which could be used in further investigation to explore new drug leads. PMID:24599082

  8. Platelet activating factor-induced ceramide micro-domains drive endothelial NOS activation and contribute to barrier dysfunction.

    PubMed

    Predescu, Sanda; Knezevic, Ivana; Bardita, Cristina; Neamu, Radu Florin; Brovcovych, Viktor; Predescu, Dan

    2013-01-01

    The spatial and functional relationship between platelet activating factor-receptor (PAF-R) and nitric oxide synthase (eNOS) in the lateral plane of the endothelial plasma membrane is poorly characterized. In this study, we used intact mouse pulmonary endothelial cells (ECs) as well as endothelial plasma membrane patches and subcellular fractions to define a new microdomain of plasmalemma proper where the two proteins colocalize and to demonstrate how PAF-mediated nitric oxide (NO) production fine-tunes ECs function as gatekeepers of vascular permeability. Using fluorescence microscopy and immunogold labeling electron microscopy (EM) on membrane patches we demonstrate that PAF-R is organized as clusters and colocalizes with a subcellular pool of eNOS, outside recognizable vesicular profiles. Moreover, PAF-induced acid sphingomyelinase activation generates a ceramide-based microdomain on the external leaflet of plasma membrane, inside of which a signalosome containing eNOS shapes PAF-stimulated NO production. Real-time measurements of NO after PAF-R ligation indicated a rapid (5 to 15 min) increase in NO production followed by a > 45 min period of reduction to basal levels. Moreover, at the level of this new microdomain, PAF induces a dynamic phosphorylation/dephosphorylation of Ser, Thr and Tyr residues of eNOS that correlates with NO production. Altogether, our findings establish the existence of a functional partnership PAF-R/eNOS on EC plasma membrane, at the level of PAF-induced ceramide plasma membrane microdomains, outside recognized vesicular profiles. PMID:24086643

  9. Platelet Activating Factor-Induced Ceramide Micro-Domains Drive Endothelial NOS Activation and Contribute to Barrier Dysfunction

    PubMed Central

    Predescu, Sanda; Knezevic, Ivana; Bardita, Cristina; Neamu, Radu Florin; Brovcovych, Viktor; Predescu, Dan

    2013-01-01

    The spatial and functional relationship between platelet activating factor-receptor (PAF-R) and nitric oxide synthase (eNOS) in the lateral plane of the endothelial plasma membrane is poorly characterized. In this study, we used intact mouse pulmonary endothelial cells (ECs) as well as endothelial plasma membrane patches and subcellular fractions to define a new microdomain of plasmalemma proper where the two proteins colocalize and to demonstrate how PAF-mediated nitric oxide (NO) production fine-tunes ECs function as gatekeepers of vascular permeability. Using fluorescence microscopy and immunogold labeling electron microscopy (EM) on membrane patches we demonstrate that PAF-R is organized as clusters and colocalizes with a subcellular pool of eNOS, outside recognizable vesicular profiles. Moreover, PAF-induced acid sphingomyelinase activation generates a ceramide-based microdomain on the external leaflet of plasma membrane, inside of which a signalosome containing eNOS shapes PAF-stimulated NO production. Real-time measurements of NO after PAF-R ligation indicated a rapid (5 to 15 min) increase in NO production followed by a > 45 min period of reduction to basal levels. Moreover, at the level of this new microdomain, PAF induces a dynamic phosphorylation/dephosphorylation of Ser, Thr and Tyr residues of eNOS that correlates with NO production. Altogether, our findings establish the existence of a functional partnership PAF-R/eNOS on EC plasma membrane, at the level of PAF-induced ceramide plasma membrane microdomains, outside recognized vesicular profiles. PMID:24086643

  10. Occurrence of platelet-activating factor (PAF) and an endogenous inhibitor of platelet aggregation in diffuse cutaneous mastocytosis.

    PubMed Central

    Macpherson, J L; Kemp, A; Rogers, M; Mallet, A I; Toia, R F; Spur, B; Earl, J W; Chesterman, C N; Krilis, S A

    1989-01-01

    We have identified PAF in the blister fluid from a patient with bullous mastocytosis, a rare form of mast-cell disease. We have found a novel endogenous inhibitor of platelet aggregation which obscured the presence of the PAF in unprocessed blister fluid and in ethanol or lipid extracts. The PAF was characterized by the demonstration of chromatographic, mass spectral and biological properties identical to those of authentic PAF. Thus this is the first demonstration of PAF in biological fluid from a patient with mastocytosis. High levels of immunoreactive prostaglandin D2 (PGD2) and histamine were also present in the blister fluid. The interaction between PAF and the inhibitor of platelet aggregation in patients with systemic mastocytosis may provide an explanation for some of the manifestations of the disease, in particular the episodic hypotension, cutaneous flushing and pallor. PMID:2805409

  11. Protein kinase C promotes arachidonate mobilization through enhancement of CoA-independent transacylase activity in platelets.

    PubMed Central

    Breton, M; Colard, O

    1991-01-01

    A role for protein kinase C in arachidonate mobilization was demonstrated. Treatment of rat platelets with phorbol myristate acetate (PMA) or the diacylglycerol 1-oleoyl-2-acetylglycerol increased the transfer rate of arachidonate (AA) from phosphatidylcholine to phosphatidylethanolamine and stimulated AA release. The transfer dose-dependently induced by PMA was inhibited by staurosporine. Ether phospholipids were the acceptors of AA in these stimulated transfer reactions. Membrane-bound protein kinase C activity was enhanced by PMA, and this increase was inhibited by staurosporine. AA transfer between phospholipids is due to the action of polyunsaturated-fatty-acid-specific transacylases. For this purpose, transacylase activities were assayed in cell-free systems from PMA-treated platelets. We observed that the CoA-independent transacylase activity was modulated in parallel to AA transfer as a function of PMA concentration. Taken together, the data show that protein kinase C activation might promote the mobilization of AA in platelets through the enhancement of CoA-independent transacylase activity. PMID:1741761

  12. Mechanism of arachidonic acid liberation in platelet-activating factor-stimulated human polymorphonuclear neutrophils

    SciTech Connect

    Nakashima, S.; Suganuma, A.; Sato, M.; Tohmatsu, T.; Nozawa, Y. )

    1989-08-15

    Upon stimulation of human polymorphonuclear neutrophils with platelet-activating factor (PAF), arachidonic acid (AA) is released from membrane phospholipids. The mechanism for AA liberation, a key step in the synthesis of biologically active eicosanoids, was investigated. PAF was found to elicit an increase in the cytoplasmic level of free Ca2+ as monitored by fluorescent indicator fura 2. When (3H) AA-labeled neutrophils were exposed to PAF, the enhanced release of AA was observed with a concomitant decrease of radioactivity in phosphatidylinositol and phosphatidylcholine fractions. The inhibitors of phospholipase A2, mepacrine and 2-(p-amylcinnamoyl)-amino-4-chlorobenzoic acid, effectively suppressed the liberation of (3H)AA from phospholipids, indicating that liberation of AA is mainly catalyzed by the action of phospholipase A2. The extracellular Ca2+ is not required for AA release. However, intracellular Ca2+ antagonists, TMB-8 and high dose of quin 2/AM drastically reduced the liberation of AA induced by PAF, indicating that Ca2+ is an essential factor for phospholipase A2 activation. PAF raised the fluorescence of fura 2 at concentrations as low as 8 pM which reached a maximal level about 8 nM, whereas more than nM order concentrations of PAF was required for the detectable release of (3H)AA. Pretreatment of neutrophils with pertussis toxin resulted in complete abolition of AA liberation in response to PAF. However, the fura 2 response to PAF was not effectively inhibited by toxin treatment. In human neutrophil homogenate and membrane preparations, guanosine 5'-O-(thiotriphosphate) stimulated AA release and potentiated the action of PAF. Guanosine 5'-O-(thiodiphosphate) inhibited the effects of guanosine 5'-O-(thiotriphosphate).

  13. Platelet-activating factor acetylhydrolase: selective inhibition by potent n-alkyl methylphosphonofluoridates.

    PubMed

    Quistad, Gary B; Fisher, Karl J; Owen, Sarah C; Klintenberg, Rebecka; Casida, John E

    2005-06-01

    Platelet-activating factor (PAF) is a potent endogenous phospholipid modulator of diverse biological activities, including inflammation and shock. PAF levels are primarily regulated by PAF acetylhydrolases (PAF-AHs). These enzymes are candidate secondary targets of organophosphorus (OP) pesticides and related toxicants. Previously known OP inhibitors of other serine hydrolases were tested with PAF-AH from mouse brain and testes of established functional importance compared with the structurally different human plasma enzyme. Several key OP pesticides and their oxon metabolites were very poor inhibitors of mouse brain and human plasma PAF-AH in vitro but moderately active for mouse brain and blood PAF-AH in vivo (e.g., tribufos defoliant and profenofos insecticide, presumably following oxidative bioactivation). OP compounds were then designed for maximum in vitro potency and selectivity for mouse brain PAF-AH vs. acetylcholinesterase (AChE). Lead compounds were found in a series of benzodioxaphosphorin 2-oxides. Ultrahigh potency and selectivity were achieved with n-alkyl methylphosphonofluoridates (long-chain sarin analogs): mouse brain and testes IC50 < or = 5 nM for C(8)-C(18) analogs and 0.1-0.6 nM for C(13) and C(14) compounds; human plasma IC50 < or = 2 nM for C(13)-C(18) analogs. AChE inhibitory potency decreased as chain length increased with maximum brain PAF-AH/AChE selectivity (>3000-fold) for C(13)-C(18) compounds. The toxicity of i.p.-administered PAF (LD50 ca. 0.5 mg/kg) was increased less than 2-fold by pretreatment with tribufos or the C(13)n-alkyl methylphosphonofluoridate. These studies with a mouse model indicate that PAF-AH is not a major secondary target of OP pesticide poisoning. The optimized PAF-AH inhibitors may facilitate investigations on other aspects of PAF metabolism and action. PMID:15893542

  14. Structure-activity relationships of alphaIIb 313-320 derived peptide inhibitors of human platelet aggregation.

    PubMed

    Stanica, Ruxandra Maria; Benaki, Dimitra; Rodis, Foteini I; Mikros, Emmanuel; Tsoukatos, Dimokritos; Tselepis, Alexandros; Tsikaris, Vasilios

    2008-11-01

    The alphaIIbbeta3 receptor, which is the most abundant receptor on the surface of platelets, can interact with a variety of adhesive proteins including fibrinogen, fibronectin and the von Willebrand factor. Fibrinogen binding on alphaIIbbeta3 is an event essential for platelet aggregation and thrombus formation. Mapping of the fibrinogen-binding domains on alphaIIb subunit suggested the sequence 313-332 as a possible binding site. This region was restricted to sequence alphaIIb 313-320 (Y313MESRADR320) using synthetic octapeptides overlapping by six residues. The YMESRADR octapeptide inhibits ADP-stimulated human platelets aggregation and binds to immobilized fibrinogen. In this study, we used the Ala scanning methodology within the sequence 313-320 aiming to evaluate the contribution of each amino acid in inhibiting platelet aggregation. It was found that the substitution of Y313, M314, E315 or S316 by A does not affect the activity of the parent octapeptide. The-RADR-motif seems to be the most essential for the biological activity of the alphaIIb 313-320 site. The conformational analysis of the YAESRADR, YMESAADR and YMESRAAR analogs by using NMR spectroscopy and distance geometry calculations revealed significant differences in their conformational states in DMSO-d6. PMID:18646252

  15. Increased urinary excretion of platelet activating factor in mice with lupus nephritis

    SciTech Connect

    Macconi, D.; Noris, M.; Benfenati, E.; Quaglia, R.; Pagliarino, G. ); Remuzzi, G. Ospedali Riuniti di Bergamo )

    1991-01-01

    Platelet activating factor (PAF) is present in urine from humans and experimental animals in normal conditions. Very little is known about changes in PAF urinary excretion under pathologic conditions and no data are available about the origin of PAF in the urine. In the present study we explored the possibility that immunologic renal disease is associated with an increase in PAF urinary excretion using gas chromatography-mass spectrometry technique. To clarify the renal or extrarenal origin of urinary PAF we evaluated whether exogenously administered PAF (1-(1{prime},2{prime}-{sup 3}H)alkyl) is filtered through the glomerulus and excreted in the urine. The results show that: (1) urine from mice with lupus nephritis in the early phase of the disease contained amounts of PAF comparable to those excreted in normal mouse urine, (2) PAF levels increased when animals started to develop high grade proteinuria, (3) after intravenous injection of ({sup 3}H) PAF In nephritic mice, a negligible amount of ({sup 3}H) ether lipid, corresponding to ({sup 3}H)1-alkyl -2-acyl-3-phosphocholine (alkyl-2-acyl-GPC), was recovered from the 24 h urine extract.

  16. UVB Generates Microvesicle Particle Release in Part Due to Platelet-activating Factor Signaling.

    PubMed

    Bihl, Ji C; Rapp, Christine M; Chen, Yanfang; Travers, Jeffrey B

    2016-05-01

    The lipid mediator platelet-activating factor (PAF) and oxidized glycerophosphocholine PAF agonists produced by ultraviolet B (UVB) have been demonstrated to play a pivotal role in UVB-mediated processes, from acute inflammation to delayed systemic immunosuppression. Recent studies have provided evidence that microvesicle particles (MVPs) are released from cells in response to various signals including stressors. Importantly, these small membrane fragments can interact with various cell types by delivering bioactive molecules. The present studies were designed to test if UVB radiation can generate MVP release from epithelial cells, and the potential role of PAF receptor (PAF-R) signaling in this process. We demonstrate that UVB irradiation of the human keratinocyte-derived cell line HaCaT resulted in the release of MVPs. Similarly, treatment of HaCaT cells with the PAF-R agonist carbamoyl PAF also generated equivalent amounts of MVP release. Of note, pretreatment of HaCaT cells with antioxidants blocked MVP release from UVB but not PAF-R agonist N-methyl carbamyl PAF (CPAF). Importantly, UVB irradiation of the PAF-R-negative human epithelial cell line KB and KB transduced with functional PAF-Rs resulted in MVP release only in PAF-R-positive cells. These studies demonstrate that UVB can generate MVPs in vitro and that PAF-R signaling appears important in this process. PMID:26876152

  17. Nuclear localization of platelet-activating factor receptor controls retinal neovascularization

    PubMed Central

    K Bhosle, Vikrant; Rivera, José Carlos; Zhou, Tianwei (Ellen); Omri, Samy; Sanchez, Melanie; Hamel, David; Zhu, Tang; Rouget, Raphael; Rabea, Areej Al; Hou, Xin; Lahaie, Isabelle; Ribeiro-da-Silva, Alfredo; Chemtob, Sylvain

    2016-01-01

    Platelet-activating factor (PAF) is a pleiotropic phospholipid with proinflammatory, procoagulant and angiogenic actions on the vasculature. We and others have reported the presence of PAF receptor (Ptafr) at intracellular sites such as the nucleus. However, mechanisms of localization and physiologic functions of intracellular Ptafr remain poorly understood. We hereby identify the importance of C-terminal motif of the receptor and uncover novel roles of Rab11a GTPase and importin-5 in nuclear translocation of Ptafr in primary human retinal microvascular endothelial cells. Nuclear localization of Ptafr is independent of exogenous PAF stimulation as well as intracellular PAF biosynthesis. Moreover, nuclear Ptafr is responsible for the upregulation of unique set of growth factors, including vascular endothelial growth factor, in vitro and ex vivo. We further corroborate the intracrine PAF signaling, resulting in angiogenesis in vivo, using Ptafr antagonists with distinct plasma membrane permeability. Collectively, our findings show that nuclear Ptafr translocates in an agonist-independent manner, and distinctive functions of Ptafr based on its cellular localization point to another dimension needed for pharmacologic selectivity of drugs. PMID:27462464

  18. Isotopic exchange during derivatization of platelet activating factor for gas chromatography-mass spectrometry

    SciTech Connect

    Haroldsen, P.E.; Gaskell, S.J.; Weintraub, S.T.; Pinckard, R.N. )

    1991-04-01

    One approach to the quantitative analysis of platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine; also referred to as AGEPC, alkyl glyceryl ether phosphocholine) is hydrolytic removal of the phosphocholine group and conversion to an electron-capturing derivative for gas chromatography-negative ion mass spectrometry. (2H3)Acetyl-AGEPC has been commonly employed as an internal standard. When 1-hexadecyl-2-(2H3)acetyl glycerol (obtained by enzymatic hydrolysis of (2H3)-C16:0 AGEPC) is treated with pentafluorobenzoyl chloride at 120 degrees C, the resulting 3-pentafluorobenzoate derivative shows extensive loss of the deuterium label. This exchange is evidently acid-catalyzed since derivatization of 1-hexadecyl-2-acetyl glycerol under the same conditions in the presence of a trace of 2HCl results in the incorporation of up to three deuterium atoms. Isotope exchange can be avoided if the reaction is carried out at low temperature in the presence of base. Direct derivatization of (2H3)-C16:0 AGEPC by treatment with pentafluorobenzoyl chloride or heptafluorobutyric anhydride also results in loss of the deuterium label. The use of (13C2)-C16:0 AGEPC as an internal standard is recommended for rigorous quantitative analysis.

  19. Inhibition of platelet activation by lachrymatory factor synthase (LFS)-silenced (tearless) onion juice.

    PubMed

    Thomson, Susan J; Rippon, Paula; Butts, Chrissie; Olsen, Sarah; Shaw, Martin; Joyce, Nigel I; Eady, Colin C

    2013-11-01

    Onion and garlic are renowned for their roles as functional foods. The health benefits of garlic are attributed to di-2-propenyl thiosulfinate (allicin), a sulfur compound found in disrupted garlic but not found in disrupted onion. Recently, onions have been grown with repressed lachrymatory factor synthase (LFS) activity, which causes these onions to produce increased amounts of di-1-propenyl thiosulfinate, an isomer of allicin. This investigation into the key health attributes of LFS-silenced (tearless) onions demonstrates that they have some attributes more similar to garlic and that this is likely due to the production of novel thiosulfinate or metabolites. The key finding was that collagen-induced in vitro platelet aggregation was significantly reduced by tearless onion extract over normal onion extract. Thiosulfinate or derived compounds were shown not to be responsible for the observed changes in the inflammatory response of AGS (stomach adenocarcinoma) cells to tumor necrosis factor alpha (TNFα) when pretreated with model onion juices. A preliminary rat feeding trial indicated that the tearless onions may also play a key role in reducing weight gain. PMID:24147811

  20. Platelet-activating factor mediates hemodynamic changes and lung injury in endotoxin-treated rats.

    PubMed Central

    Chang, S W; Feddersen, C O; Henson, P M; Voelkel, N F

    1987-01-01

    Within 20 min after intraperitoneal injection of Salmonella enteritidis endotoxin in rats, blood platelet-activating factor (PAF) increased from 4.3 +/- 1.3 to 13.7 +/- 2.0 ng/ml (P less than 0.01) and lung PAF from 32.3 +/- 4.9 to 312.3 +/- 19.6 ng (P less than 0.01), but not lung lavage PAF. We tested the effect of PAF receptor antagonists, CV 3988 and SRI 63-441, on endotoxin-induced hemodynamic changes and lung vascular injury. Pretreatment with CV 3988 attenuated systemic hypotension, preserved hypoxic pulmonary vasoconstriction, and prolonged survival of awake catheter-implanted endotoxin-treated (20 mg/kg) rats. Pretreatment with SRI 63-441 prevented the depressed hypoxic pulmonary vasoconstriction after low dose (2 mg/kg) endotoxin. Both CV 3988 and SRI 63-441 blocked the increased extravascular accumulation of 125I-albumin and water in perfused lungs isolated from endotoxin-treated rats. We conclude that PAF is produced in the lung during endotoxemia and may be an important mediator of the systemic and pulmonary hemodynamic changes as well as the acute lung vascular injury after endotoxemia. PMID:3553241

  1. Endothelium-dependent vasodilator effects of platelet activating factor on rat resistance vessels.

    PubMed Central

    Kamata, K.; Mori, T.; Shigenobu, K.; Kasuya, Y.

    1989-01-01

    1. To elucidate the mechanisms of the powerful and long-lasting hypotension produced by platelet activating factor (PAF), its effects on perfusion pressure in the perfused mesenteric arterial bed of the rat were examined. 2. Infusion of PAF (10(-11) to 3 x 10(-10) M; EC50 = 4.0 x 10(-11) M; 95%CL = 1.6 x 10(-11) - 9.4 x 10(-11) M) and acetylcholine (ACh) (10(-10) to 10(-6) M; EC50 = 3.0 +/- 0.1 x 10(-9) M) produced marked concentration-dependent vasodilatations which were significantly inhibited by treatment with detergents (0.1% Triton X-100 for 30 s or 0.3% CHAPS for 90 s). 3. Pretreatment with CV-6209, a PAF antagonist, inhibited PAF- but not ACh-induced vasodilation. 4. Treatment with indomethacin (10(-6) M) had no effect on PAF- or ACh-induced vasodilatation. 5. These results demonstrate that extremely low concentrations of PAF produce vasodilatation of resistance vessels through the release of endothelium-derived relaxing factor (EDRF). This may account for the strong hypotension produced by PAF in vivo. PMID:2611496

  2. Platelet-activating factor mediates angiotensin II-induced proteinuria in isolated perfused rat kidney.

    PubMed

    Perico, N; Lapinski, R; Konopka, K; Aiello, S; Noris, M; Remuzzi, G

    1997-09-01

    Isolated kidney preparations (IPK) from male Sprague Dawley rats perfused at constant pressure were used to evaluate the effect of angiotensin II (AII) and platelet-activating factor (PAF) on renal function and urinary protein excretion. Compared with basal, intrarenal infusion of AII at 8 ng/min caused a progressive increase in protein excretion (11 +/- 6 versus 73 +/- 21 micrograms/min) in parallel with a decline in renal perfusate flow (RPF) (29 +/- 3 versus 18 +/- 3 ml/min). Addition to the perfusate of PAF at 50 nM final concentration also induced proteinuria (9 +/- 4 versus 55 +/- 14 micrograms/min) but did not change RPF (29 +/- 3 versus 30 +/- 3 ml/min). Preexposure of isolated kidneys to the PAF receptor antagonist WEB 2086 prevented the increase in urinary protein excretion induced by AII infusion (basal: 13 +/- 6; post-AII: 12 +/- 7 micrograms/min) but failed to prevent the vasoactive effect of AII (RPF, basal: 30 +/- 2; post-AII: 21 +/- 3 ml/min). In additional experiments, dexamethasone reduced the proteinuric effect of PAF remarkably. These results indicate that in isolated kidney preparation: (1) AII infusion induced proteinuria and decreased RPF; and (2) the effect of AII in enhancing urinary protein excretion was completely prevented by a specific PAF receptor antagonist, which, however, did not influence the AII-induced fall in RPF. It is suggested that PAF plays a major role in AII-induced changes in the permselective function of the glomerular capillary barrier. PMID:9294830

  3. Platelet-activating factor modulates pulmonary vasomotor tone in the perinatal lamb.

    PubMed

    Ibe, B O; Hibler, S; Raj, J U

    1998-09-01

    Eight near-term fetal lambs were studied acutely in utero to determine role of platelet-activating factor (PAF) in the regulation of vasomotor tone in systemic and pulmonary circulations in the immediate perinatal period. Four fetal lambs were studied predelivery and 2 h postdelivery to determine circulating PAF levels. Aortic and pulmonary arterial pressures and cardiac output were measured continuously, and systemic and pulmonary vascular resistances were calculated. Left pulmonary arterial blood flow was also measured in four fetal lambs. After delivery and oxygenation, circulating PAF levels fell significantly. When WEB-2170, a specific PAF-receptor antagonist, was infused to block effect of endogenous PAF in the eight near-term fetal lambs, systemic vascular resistance fell 30% but pulmonary vascular resistance fell dramatically by 68%. Specificity of WEB-2170 was tested in juvenile lambs and was found to be very specific in lowering vasomotor tone only when tone was elevated by action of PAF. Our data show that endogenous PAF levels in the fetus contribute to maintain a high basal systemic and pulmonary vasomotor tone and that a normal fall in circulating PAF levels after birth and oxygenation may facilitate fall in pulmonary vascular resistance at birth. PMID:9729586

  4. Platelet activation via the collagen receptor GPVI is not altered in platelets from chronic myeloid leukaemia patients despite the presence of the constitutively phosphorylated adapter protein CrkL.

    PubMed

    Best, D; Pasquet, S; Littlewood, T J; Brunskill, S J; Pallister, C J; Watson, S P

    2001-03-01

    In this study, we show that the adapter proteins CrkL and Cbl undergo increases in tyrosine phosphorylation and form an intracellular complex in platelets stimulated with the snake venom toxin convulxin, a selective agonist at the collagen receptor glycoprotein VI (GPVI). Constitutive tyrosine phosphorylation of CrkL has previously been reported in platelets from chronic myeloid leukaemia (CML) patients. This was confirmed in the present study, and shown to result in a weak constitutive association of CrkL with Cbl and a number of other unidentified tyrosine-phosphorylated proteins. There was no further increase in phosphorylation of CrkL in CML platelets in response to GPVI activation, whereas phosphorylation of Cbl and its association with CrkL were potentiated. In addition, this was accompanied by a small increase in p42/ 44 mapkinase (MAPK) activity in CML platelets. The functional consequence of the presence of constitutively phosphorylated proteins in CML platelets was investigated by measurement of aminophospholipid exposure and alpha-granule secretion. This revealed little alteration in the concentration-response curves for either in CML platelets stimulated via GPVI, although maximal levels of P-selectin were depressed. Despite the minimal effect on platelet activation in CML patients, we cannot exclude a role for CrkL or Cbl in signal transduction pathways stimulated via GPVI. PMID:11260061

  5. Is mean platelet volume a new activity criteria in Behçet's disease?

    PubMed

    Uzkeser, Hulya; Haliloglu, Sema; Cayir, Yasemin; Bilen, Nurhan; Karaaslan, Yasar; Kosar, Ali; Carlioglu, Ayse

    2015-10-01

    The aim of this study was to assess mean platelet volume (MPV) and its relationship with disease activity in patients with Behçet's disease. Thirty-six patients with an age of 38.9 ± 11 (mean ± SD) years and 40 controls aged 36.5 ± 12 (mean ± SD) years were enrolled the study. Demographic data, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), MPV, clinical findings such as oral aphthae, genital aphthae, erythema nodosum, acne, central nervous system involvement, uveitis, arthritis and arthralgia were all recorded. The MPV value in patients with Behçet's disease was 8.06 ± 1.0 (mean ± SD) and the MPV value of the control participants was 7.45 ± 0.6 (mean ± SD). MPV was statistically higher in patients with Behçet's disease than in the controls (P = 0.003). There were also significant differences between patients and controls according to ESR and CRP values (P < 0.001 and P = 0.001, respectively). MPV was positively correlated with arthralgia (P < 0.001, r = 0.438), arthritis (P = 0.008, r = 0.307), erythema nodosum (P = 0.002, r =  0.354), central nervous system involvement (P = 0.002, r = 0.357), acne (P = 0.008, r = 0.312), genital aphthae (P < 0.001, r = 0.401) and oral aphthae (P = 0.001 r = 0.377). MPV can be easily obtained from the patients. It was a cheap and practical method. In the future, MPV may be used as a new marker to detect the activation of BD. PMID:26196194

  6. Comparison of activation process of platelets and neutrophils after coronary stent implantation versus balloon angioplasty for stable angina pectoris.

    PubMed

    Inoue, T; Sohma, R; Miyazaki, T; Iwasaki, Y; Yaguchi, I; Morooka, S

    2000-11-15

    The pathophysiologic features of stent-induced cellular responses of platelets and leukocytes have not been established. This study was designed to clinically investigate the activation of platelets and neutrophils after coronary stenting and to identify its effects on the long-term results of coronary stents. Forty-eight consecutive patients with left anterior descending coronary artery disease indicating coronary intervention were randomly assigned to either a balloon angioplasty group or a coronary stent group. Flow cytometric analysis demonstrated that the transcardiac gradient (the value of coronary sinus blood minus the value of peripheral blood) of platelet surface expression of CD62P (p < 0.001) and CD63 (p < 0.01) increased immediately after coronary stenting, but increased less significantly immediately after balloon angioplasty (CD62P, p < 0.01; CD63, p < 0.05). These increases were persistently observed after coronary stenting but transiently after balloon angioplasty alone during a 48-hour observation period after the procedures. The gradient for neutrophil surface expression of CD11b increased, and that of CD62 L decreased 48 hours after coronary stenting (CD11b, p < 0.001; CD62 L, p < 0.05), but these changes showed less significance 48 hours after balloon angioplasty alone (CD11b, p < 0.05; CD62 L, p = NS). The gradients 48 hours after the procedures for both CD62P (r = 0.39, p < 0.05) and CD11b (r = 0.44, p < 0.01) were independently correlated with the late loss in the stent group, whereas the correlation was seen only for CD11b (r = 0.38, p < 0.05) in the balloon angioplasty group. Both platelet and neutrophil activation was greater after coronary stenting than after balloon angioplasty. Cellular interactions between platelets and neutrophils may be related to the progression of neointimal proliferation leading to restenosis after coronary stent implantation. PMID:11074199

  7. The inflammatory and tumor-promoting sesquiterpene lactone, thapsigargin, activates platelets by selective mobilization of calcium as shown by protein phosphorylations.

    PubMed

    Thastrup, O; Linnebjerg, H; Bjerrum, P J; Knudsen, J B; Christensen, S B

    1987-01-19

    We have studied the activation of human blood platelets by the inflammatory and tumor-promoting sesquiterpene lactone, thapsigargin. The effect of thapsigargin was compared with other common agonists (calcium ionophore A23187, phorbol ester TPA and thrombin). Platelet aggregation, serotonin release, raised cytoplasmic free calcium level and phosphorylation of platelet proteins was examined in platelet-rich plasma and washed platelet suspension. In contrast to A23187 and thrombin, the platelet activation induced by thapsigargin developed slowly, with maximal response obtained after 2-3 min. Both the thapsigargin- and the A23187-induced serotonin releases were synergistically increased by TPA. Studies of the phosphorylation of platelet proteins revealed that thapsigargin and A23187 equally well induced a selective phosphorylation of two proteins with apparent molecular masses of 20 kDa and 47 kDa. These proteins, which are substrates of myosin light-chain kinase and protein kinase C respectively, are known to be involved in platelet activation. The thapsigargin-induced platelet aggregation and serotonin release was completely inhibited by class I (nimodipine), class II (verapamil) and class III (diltiazem) calcium-channel blockers. The inhibitory activity of nimodipine was abolished by the corresponding 1,4-dihydropyridine calcium-channel agonist, BAY K 8644. These results shows that the thapsigargin-induced platelet activation is mediated by an increase in the cytoplasmic free calcium level, presumably obtained by stimulation of the passive calcium transport through specific channels. These thapsigargin-sensitive channels should predominantly be located in the membranes of intracellular calcium stores rather than in the plasma membrane, because removal of extracellular calcium by EGTA had only an insignificant effect on the thapsigargin-induced rise in cytoplasmic free calcium level. PMID:3098302

  8. A Novel Direct Factor Xa Inhibitory Peptide with Anti-Platelet Aggregation Activity from Agkistrodon acutus Venom Hydrolysates

    PubMed Central

    Chen, Meimei; Ye, Xiaohui; Ming, Xin; Chen, Yahui; Wang, Ying; Su, Xingli; Su, Wen; Kong, Yi

    2015-01-01

    Snake venom is a natural substance that contains numerous bioactive proteins and peptides, nearly all of which have been identified over the last several decades. In this study, we subjected snake venom to enzymatic hydrolysis to identify previously unreported bioactive peptides. The novel peptide ACH-11 with the sequence LTFPRIVFVLG was identified with both FXa inhibition and anti-platelet aggregation activities. ACH-11 inhibited the catalytic function of FXa towards its substrate S-2222 via a mixed model with a Ki value of 9.02 μM and inhibited platelet aggregation induced by ADP and U46619 in a dose-dependent manner. Furthermore, ACH-11 exhibited potent antithrombotic activity in vivo. It reduced paralysis and death in an acute pulmonary thrombosis model by 90% and attenuated thrombosis weight in an arterio-venous shunt thrombosis model by 57.91%, both at a dose of 3 mg/kg. Additionally, a tail cutting bleeding time assay revealed that ACH-11 did not prolong bleeding time in mice at a dose of 3 mg/kg. Together, our results reveal that ACH-11 is a novel antithrombotic peptide exhibiting both FXa inhibition and anti-platelet aggregation activities, with a low bleeding risk. We believe that it could be a candidate or lead compound for new antithrombotic drug development. PMID:26035670

  9. Characterization of streptococcal platelet-activating factor acetylhydrolase variants that are involved in innate immune evasion.

    PubMed

    Liu, Guanghui; Liu, Mengyao; Xie, Gang; Lei, Benfang

    2013-09-01

    Human pathogen group A streptococcus (GAS) has developed mechanisms to subvert innate immunity. We recently reported that the secreted esterase produced by serotype M1 GAS (SsE(M1)) reduces neutrophil recruitment by targeting platelet-activating factor (PAF). SsE(M1) and SsE produced by serotype M28 GAS (SsE(M28)) have a 37% sequence difference. This study aims at determining whether SsE(M28) is also a PAF acetylhydrolase and participates in innate immune evasion. We also examined whether SsE evolved to target PAF by characterizing the PAF acetylhydrolase (PAF-AH) activity and substrate specificity of SsE(M1), SsE(M28), SeE, the SsE homologue in Streptococcus equi, and human plasma PAF-AH (hpPAF-AH). PAF incubated with SsE(M28) or SeE was converted into lyso-PAF. SsE(M1) and SsE(M28) had kcat values of 373 s(-1) and 467 s(-1), respectively, that were ≥ 30-fold greater than that of hpPAF-AH (12 s(-1)). The comparison of SsE(M1), SsE(M28), and hpPAF-AH in kcat and Km in hydrolyzing triglycerides, acetyl esters, and PAF indicates that the SsE proteins are more potent hydrolases against PAF and have high affinity for PAF. SsE(M28) possesses much lower esterase activities against triglycerides and other esters than SsE(M1) but have similar potency with SsE(M1) in PAF hydrolysis. Deletion of sse(M28) in a covS deletion mutant of GAS increased neutrophil recruitment and reduced skin infection, whereas in trans expression of SsE(M28) in GAS reduced neutrophil infiltration and increased skin invasion in subcutaneous infection of mice. These results suggest that the SsE proteins evolved to target PAF for enhancing innate immune evasion and skin invasion. PMID:23774595

  10. Characterization of Streptococcal Platelet-Activating Factor Acetylhydrolase Variants That Are Involved in Innate Immune Evasion

    PubMed Central

    Liu, Guanghui; Liu, Mengyao; Xie, Gang

    2013-01-01

    Human pathogen group A streptococcus (GAS) has developed mechanisms to subvert innate immunity. We recently reported that the secreted esterase produced by serotype M1 GAS (SsEM1) reduces neutrophil recruitment by targeting platelet-activating factor (PAF). SsEM1 and SsE produced by serotype M28 GAS (SsEM28) have a 37% sequence difference. This study aims at determining whether SsEM28 is also a PAF acetylhydrolase and participates in innate immune evasion. We also examined whether SsE evolved to target PAF by characterizing the PAF acetylhydrolase (PAF-AH) activity and substrate specificity of SsEM1, SsEM28, SeE, the SsE homologue in Streptococcus equi, and human plasma PAF-AH (hpPAF-AH). PAF incubated with SsEM28 or SeE was converted into lyso-PAF. SsEM1 and SsEM28 had kcat values of 373 s−1 and 467 s−1, respectively, that were ≥30-fold greater than that of hpPAF-AH (12 s−1). The comparison of SsEM1, SsEM28, and hpPAF-AH in kcat and Km in hydrolyzing triglycerides, acetyl esters, and PAF indicates that the SsE proteins are more potent hydrolases against PAF and have high affinity for PAF. SsEM28 possesses much lower esterase activities against triglycerides and other esters than SsEM1 but have similar potency with SsEM1 in PAF hydrolysis. Deletion of sseM28 in a covS deletion mutant of GAS increased neutrophil recruitment and reduced skin infection, whereas in trans expression of SsEM28 in GAS reduced neutrophil infiltration and increased skin invasion in subcutaneous infection of mice. These results suggest that the SsE proteins evolved to target PAF for enhancing innate immune evasion and skin invasion. PMID:23774595

  11. Activity.

    ERIC Educational Resources Information Center

    Clearing: Nature and Learning in the Pacific Northwest, 1984

    1984-01-01

    Presents three activities: (1) investigating succession in a schoolground; (2) investigating oak galls; and (3) making sun prints (photographs made without camera or darkroom). Each activity includes a list of materials needed and procedures used. (JN)

  12. Platelet-activating factor induces collagenase expression in corneal epithelial cells.

    PubMed Central

    Bazan, H E; Tao, Y; Bazan, N G

    1993-01-01

    Platelet-activating factor (PAF), a potent lipid mediator involved in inflammatory and immune responses, accumulates rapidly in response to injury in a variety of tissues, including the corneal epithelium. However, the precise role of this compound in the cascade of events following insult has not been defined. Here we examined the effect of PAF on gene expression in the epithelial cells of rabbit corneas in organ culture. We found that incubation with 100 nM methylcarbamoyl PAF, a nonhydrolyzable analog of PAF, produced rapid transient 2.8- and 3.5-fold increases in the expression of c-fos and c-jun, respectively, at 1 hr, followed by increased expression of the collagenase type I gene beginning at 3 hr and peaking at 14-fold by 8 hr. Addition of the protein-synthesis-inhibitor cycloheximide superinduced c-fos and c-jun, strongly potentiating the PAF effect, but inhibited the induction of collagenase type I expression, suggesting the existence of a transcriptional factor linking the two events. BN-50730, a selective antagonist of intracellular PAF-binding sites, blocked the expression of the immediate-early genes as well as the increase in collagenase type I mRNA. Our results suggest that one of the functions of PAF may be to enhance the breakdown of the extracellular matrix as a part of the remodeling process during corneal wound healing after injury. Pathologically, a PAF-induced overproduction of collagenase may be a factor in the development of corneal ulcers, as well as other pathophysiological conditions such as cartilage destruction in arthritis. If so, inhibitors of this signal-transduction pathway may be useful as tools for further investigation and, eventually, as therapeutic agents to treat such disorders. Images Fig. 1 Fig. 2 PMID:8378347

  13. Characterization of platelet-activating factor-induced cutaneous edema and erythema in dogs.

    PubMed

    Watanabe, Miwa; Osada, Hironari; Shimizu, Sunao; Goto, Shun; Nagai, Makoto; Shirai, Junsuke; Sasaki, Kazuaki; Shimoda, Minoru; Itoh, Hiroshi; Ohmori, Keitaro

    2016-09-01

    OBJECTIVE To characterize platelet-activating factor (PAF)-induced edema and erythema in the skin of dogs and compare those reactions with histamine-induced cutaneous reactions. ANIMALS 6 healthy Beagles. PROCEDURES Experiments were performed at ≥ 2-week intervals. Each dog received ID injections (5 μg/site) of PAF C16, PAF C18, lyso-PAF, and histamine. Edema (mean diameter) and erythema scores (none, mild, moderate, or severe) were assessed 30 minutes after the injections. Dogs received ID injections of PAF and histamine each with various concentrations of WEB 2086 (PAF receptor antagonist) or underwent ID testing with PAF and histamine before and 3 hours after oral administration of cetirizine hydrochloride or prednisolone (at 2 doses each). RESULTS ID injections of PAF C16 and PAF C18, but not lyso-PAF, induced comparable levels of edema and erythema. The PAF-induced edema and erythema peaked at 30 minutes and lasted for 6 hours after the injection; histamine-induced edema and erythema peaked at 30 minutes and lasted for 3 hours after the injection. Edema sizes and erythema scores were significantly smaller and lower, respectively, for PAF than for histamine. The WEB 2086 inhibited PAF-induced but not histamine-induced edema and erythema. Cetirizine slightly, but significantly, repressed PAF-induced edema and erythema as well as histamine-induced cutaneous reactions. Prednisolone suppressed both PAF-induced and histamine-induced edema and erythema. CONCLUSIONS AND CLINICAL RELEVANCE In canine skin, the duration of PAF-induced inflammation was longer than that of histamine-induced inflammation. The PAF- and histamine-induced cutaneous reactions were effectively suppressed by oral administration of prednisolone. The importance of PAF in dogs with anaphylaxis and allergic disorders warrants further investigation. PMID:27580108

  14. Platelet-activating factor causes ventilation-perfusion mismatch in humans.

    PubMed Central

    Rodriguez-Roisin, R; Félez, M A; Chung, K F; Barberà, J A; Wagner, P D; Cobos, A; Barnes, P J; Roca, J

    1994-01-01

    We hypothesized that platelet-activating factor (PAF), a potent inflammatory mediator, could induce gas exchange abnormalities in normal humans. To this end, the effect of aerosolized PAF (2 mg/ml solution; 24 micrograms) on ventilation-perfusion (VA/Q) relationships, hemodynamics, and resistance of the respiratory system was studied in 14 healthy, nonatopic, and nonsmoking individuals (23 +/- 1 [SEM]yr) before and at 2, 4, 6, 8, 15, and 45 min after inhalation, and compared to that of inhaled lyso-PAF in 10 other healthy individuals (24 +/- 2 yr). PAF induced, compared to lyso-PAF, immediate leukopenia (P < 0.001) followed by a rebound leukocytosis (P < 0.002), increased minute ventilation (P < 0.05) and resistance of the respiratory system (P < 0.01), and decreased systemic arterial pressure (P < 0.05). Similarly, compared to lyso-PAF, PaO2 showed a trend to fall (by 12.2 +/- 4.3 mmHg, mean +/- SEM maximum change from baseline), and arterial-alveolar O2 gradient increased (by 16.7 +/- 4.3 mmHg) (P < 0.02) after PAF, because of VA/Q mismatch: the dispersion of pulmonary blood flow and that of ventilation increased by 0.45 +/- 0.1 (P < 0.01) and 0.29 +/- 0.1 (P < 0.04), respectively. We conclude that in normal subjects, inhaled PAF results in considerable immediate VA/Q inequality and gas exchange impairment. These results reinforce the notion that PAF may play a major role as a mediator of inflammation in the human lung. Images PMID:8282786

  15. Effect of platelet activating factor (PAF) on pulmonary circulation in isolated rabbit lung.

    PubMed

    Comellas, A; Tristano, S; Pesce, L; Friedman, E; Marcano, H; Sánchez de León, R

    1999-03-01

    Platelet activating factor (PAF) has been implicated in the pathophysiology of acute lung injury. The aim of this work is to study the effect of PAF on isolated and perfused rabbit lungs with blood and with a blood-free solution. 24 isolated and perfused rabbit lungs have been used: 8 control preparations (CP), 4 vehicles preparations (VP), 8 PAF preparations (PP) to which we administered PAF (1 microg/Kg of rabbit weight) and 4 acellular preparations (AP) with the same dose of PAF as in PP but dissolved in BSA-Krebs buffer solution. In the preparations pulmonary artery pressure (Ppa), airway pressure (Paw), left atria pressure (Pla) and fluid filtration rate (FFR) were registered. Ppa resulted in a significant difference in AP vs PP, with a value of 21 cm of water (CI 95%: 12-26) vs 205.1 cm of water (CI 95%: 141.3 - 271) respectively. A increase in FFR was observed in PP but it did not occur in AP, the difference being statistically significant: 5.515 g/min (CI 95 %: 2.425 - 8.865) vs 0.049 g/min (CI 95%: 0.008 - 0.32) respectively. Paw was statistically different in PP vs AP, with a value of 14.3 cm of water (CI 95%: 11.57 - 16.7) vs 8.5 cm of water (CI 95%: 8-9) respectively. These results suggest that PAF does not have a direct effect on the endothelium or smooth muscle in the production of lung edema. PMID:10494654

  16. Interaction of vasoactive substances released by platelet-activating factor in the rat perfused heart.

    PubMed Central

    Hu, W. M.; Man, R. Y.

    1991-01-01

    1. The coronary vascular effects of platelet-activating factor (PAF) have been intensively studied and it has been proposed that they are mediated by the release of vasoactive substances. In this study, a cascade perfusion model using two rat perfused hearts was developed to investigate the properties of PAF-released vasoactive substances and the interplay of these substances. The properties of the vasoactive substances after an injection of PAF (100 pmol) in the rat perfused heart were examined by collecting the effluent from the first heart for the perfusion of a second (recipient) heart. The presence of vasoconstrictor substances in the effluent was characterized by an increase in the perfusion pressure of the recipient heart. 2. Previous exposure of the recipient heart of PAF (100 pmol) abolished the response of the heart to subsequent administration of PAF, but did not affect the response of the recipient heart to the effluent. This suggested that the coronary vasoconstrictor response of the recipient heart was not due to the presence of PAF in the effluent but to other vasoactive substances. 3. Pretreatment of the recipient heart with the leukotriene receptor antagonist, L-649,923 (5 microM), partially reduced the vasoconstrictor effect of the effluent. Pretreatment of the first heart with indomethacin (2.8 microM) also partially reduced the vasoconstrictor effect of the effluent. The combination of indomethacin pretreatment of the first heart and L-649,923 pretreatment of the recipient heart completely abolished the vasoconstrictor effect of the effluent suggesting that both prostaglandins and leukotrienes are involved in the vasoconstrictor effect of the effluent. 4. Pretreatment of both hearts with L-649,923 or the first heart with the leukotriene synthesis inhibitor (MK-886, 10 microM) completely abolished the vasoconstrictor effect of the effluent.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1810604

  17. Cholesterol synthesis inhibitors protect against platelet-activating factor-induced neuronal damage

    PubMed Central

    Bate, Clive; Rumbold, Louis; Williams, Alun

    2007-01-01

    Background Platelet-activating factor (PAF) is implicated in the neuronal damage that accompanies ischemia, prion disease and Alzheimer's disease (AD). Since some epidemiological studies demonstrate that statins, drugs that reduce cholesterol synthesis, have a beneficial effect on mild AD, we examined the effects of two cholesterol synthesis inhibitors on neuronal responses to PAF. Methods Primary cortical neurons were treated with cholesterol synthesis inhibitors (simvastatin or squalestatin) prior to incubation with different neurotoxins. The effects of these drugs on neuronal cholesterol levels and neuronal survival were measured. Immunoblots were used to determine the effects of simvastatin or squalestatin on the distribution of the PAF receptor and an enzyme linked immunoassay was used to quantify the amounts of PAF receptor. Results PAF killed primary neurons in a dose-dependent manner. Pre-treatment with simvastatin or squalestatin reduced neuronal cholesterol and increased the survival of PAF-treated neurons. Neuronal survival was increased 50% by 100 nM simvastatin, or 20 nM squalestatin. The addition of mevalonate restored cholesterol levels, and reversed the protective effect of simvastatin. Simvastatin or squalestatin did not affect the amounts of the PAF receptor but did cause it to disperse from within lipid rafts. Conclusion Treatment of neurons with cholesterol synthesis inhibitors including simvastatin and squalestatin protected neurons against PAF. Treatment caused a percentage of the PAF receptors to disperse from cholesterol-sensitive domains. These results raise the possibility that the effects of statins on neurodegenerative disease are, at least in part, due to desensitisation of neurons to PAF. PMID:17233902

  18. Platelet activating factors alters calcium homeostasis in cultured vascular endothelial cells

    SciTech Connect

    Brock, T.A.; Gimbrone, M.A. Jr.

    1986-06-01

    Platelet activating factor (1-O-alkyl-2-acetyl-sn-glycerol-3-phosphorylcholine; PAF), a potent in vivo mediator of allergic and inflammatory reactions, induced a rapid (onset less than 30 s), concentration-dependent (threshold approximately 10(-11) M, half-maximal approximately 10(-10) M, maximal approximately 10(-8)-10(-7) M) efflux of /sup 45/Ca/sup 2 +/ from preloaded cultured bovine aortic endothelial cells (BAEC). In contrast, deacetylated and other PAF analogues were essentially ineffective. PAF (10(-7) M) was also shown to increase cytosolic free calcium (49 +/- 5%) in suspensions of quin 2 (calcium-sensitive fluorescent dye)-loaded BAEC. PAF-stimulated /sup 45/Ca/sup 2 +/ efflux was not blocked by aspirin treatment (100 or 500 microM, 30 min). In the absence of external calcium, PAF was still highly effective in stimulating unidirectional /sup 45/Ca/sup 2 +/ efflux, thus suggesting that PAF mobilized a sequestered pool of intracellular calcium. CV-3988, a PAF antagonist, inhibited PAF-stimulated /sup 45/Ca/sup 2 +/ efflux in a dose-dependent manner. Pretreatment of BAEC with PAF (10(-8) M, 15 min), but not with other PAF analogues, resulted in a decrease in subsequent PAF-stimulated /sup 45/Ca/sup 2 +/ efflux, thus suggesting an agonist-specific desensitization. PAF also stimulated a 30% net decrease in the equilibrium /sup 45/Ca/sup 2 +/ content of BAEC within 1 min, which gradually recovered to prestimulus levels in 10-15 min. PAF-stimulated /sup 45/Ca/sup 2 +/ efflux was also observed in endothelial cells cultured from human umbilical vein and baboon cephalic vein but not from cultured human dermal fibroblasts or bovine aortic smooth muscle. These studies provide direct evidence for agonist- and cell-specific effects of PAF on vascular endothelium.

  19. Platelet-activating factor biosynthesis in rat vascular smooth muscle cells.

    PubMed

    Tomlinson, P R; Croft, K; Harris, T; Stewart, A G

    1994-01-01

    The ability of platelet-activating factor (PAF) receptor antagonists to protect rats from the cardiovascular collapse induced by large doses of endothelin 1 led us to examine the capacity of rat cultured vascular smooth muscle cells to produce PAF and also to evaluate its potential functional roles in this cell type. Adenosine triphosphate and the vasoactive peptides, endothelin 1, angiotensin II, and arginine vasopressin, each elicited an increase in the PAF level in extracts of rat cultured vascular smooth muscle cells as determined by bioassay. PAF was not detectable (above 20 fmol/mg protein) in the supernatants of these cells. The identity of the bioactivity as PAF was confirmed by GC/MS which indicated that more than 80% of the PAF was 1-O-hexadecyl-2-acetyl-3-sn-glyceryl-phosphorylcholine. Exogenous PAF (100 nM) elicited increases in intracellular calcium that were inhibited by WEB 2086 (10 microM). Endothelin 1, at a concentration which stimulated PAF synthesis, (1 nM), elicited increases in intracellular calcium levels that were not inhibited by WEB 2086 (10 microM). Thus, endogenous PAF is unlikely to be involved in the endothelin-1-induced calcium increases. Although WEB 2086 (3-100 microM) inhibited concentration dependently fetal calf serum (10% v/v) induced [3H]-thymidine incorporation, reaching a maximum effect at 30 microM of 40-50% reduction, in parallel experiments WEB 2086 had no effect on serum-induced increases in cell numbers. We conclude that PAF is produced and retained by cultured rat vascular smooth muscle and that it is unlikely to contribute to the signaling of increases in intracellular calcium or proliferation. PMID:8148465

  20. Chemical synthesis of echistatin, a potent inhibitor of platelet aggregation from Echis carinatus: synthesis and biological activity of selected analogs.

    PubMed

    Garsky, V M; Lumma, P K; Freidinger, R M; Pitzenberger, S M; Randall, W C; Veber, D F; Gould, R J; Friedman, P A

    1989-06-01

    Echistatin, a polypeptide from the venom of the saw-scaled viper, Echis carinatus, containing 49 amino acids and 4 cystine bridges was synthesized by solid-phase methodology in 4% yield. In the final step, air oxidation of the octahydroderivative was found to be optimal at pH 8. The synthetic product was shown to be physically and biologically indistinguishable from native material. It inhibits fibrinogen-dependent platelet aggregation stimulated by ADP with IC50 = 3.3 x 10(-8) M and also prevents aggregation initiated by thrombin, epinephrine, collagen, or platelet-activating factor. Reduction of purified synthetic echistatin to octahydroechistatin with dithiothreitol followed by air oxidation regenerated homogeneous echistatin in quantitative yield. This highly specific refolding strongly suggests that the linear sequence of octahydroechistatin contains all of the information that is required for the proper folding of the peptide. The sequence Arg24-Gly-Asp of echistatin occurs also in adhesive glycoproteins that bind to the platelet fibrinogen receptor--a heterodimeric complex composed of glycoproteins IIb and IIIa. In an effort to evaluate the role of this putative binding site we have synthesized analogs of echistatin with substitution of Arg-24. Replacement with ornithine-24 (Orn-24) resulted in an analog having a platelet aggregation inhibitory activity with IC50 = 1.05 x 10(-7) M. Substitution with Ala-24 gave IC50 = 6.1 x 10(-7) M. The inhibitory activity of the corresponding short sequence analogs Arg-Gly-Asp-Phe (IC50 = 6 x 10(-6) M), Orn-Gly-Asp-Phe (IC50 = 1.3 x 10(-4) M), and Ala-Gly-Asp-Phe (IC50 = 5.0 x 10(-4) M) was also determined. These results suggest that arginine plays a more important role in the binding of the tetrapeptide than in that of echistatin. PMID:2726764

  1. Effects of Platelet-Rich Plasma Composition on Anabolic and Catabolic Activities in Equine Cartilage and Meniscal Explants

    PubMed Central

    McIlwraith, C. Wayne; Rodkey, William G.; Frisbie, David D.; Steadman, J.Richard

    2012-01-01

    Objective: To evaluate the effects of single- and double-spin preparations of platelet-rich plasma (PRP) on anabolic and catabolic activities of cartilage and meniscal explants in vitro. Methods: Single- and double-spin PRP was prepared using laboratory processing or commercial kits. The cellular contents were quantified, and each PRP was mixed in equal quantities with cell culture medium and added to cartilage or meniscus explant cultures, with or without interleukin 1 β (IL-1β). Extracellular matrix synthesis was quantified over 24 hours via 35S-sulfate and 3H-proline incorporation, while gene expression of catabolic enzymes was evaluated using real-time PCR. Results: The platelet concentration in single-spin laboratory PRP was 59% higher than blood. Platelet and white blood cell concentrations in single-spin laboratory and kit PRP were not significantly different, while the double-spin kit resulted in approximately 2.5-fold higher platelet and approximately 400-fold higher white blood cell concentrations. In cartilage cultures without IL-1β, radiolabel incorporation in single-spin PRP cultures was significantly higher than in double-spin cultures. Similar results were obtained for 35S-sulfate incorporation in meniscus cultures without IL-1β. In IL-1β, radiolabel incorporation was largely similar among all PRPs. After 24 hours of culture, ADAMTS-4 gene expression in cartilage was lowest for single-spin PRP, while expression in the double-spin kit was not significantly different from double-spin laboratory PRP in which platelets were concentrated 6-fold. Conclusions This study suggests that single-spin PRP preparations may be the most advantageous for intra-articular applications and that double-spin systems should be considered with caution. PMID:26069637

  2. Glycoprotein VI/Fc receptor γ chain-independent tyrosine phosphorylation and activation of murine platelets by collagen

    PubMed Central

    2004-01-01

    We have investigated the ability of collagen to induce signalling and functional responses in suspensions of murine platelets deficient in the FcRγ (Fc receptor γ) chain, which lack the collagen receptor GPVI (glycoprotein VI). In the absence of the FcRγ chain, collagen induced a unique pattern of tyrosine phosphorylation which was potentiated by the thromboxane analogue U46619. Immunoprecipitation studies indicated that neither collagen alone nor the combination of collagen plus U46619 induced phosphorylation of the GPVI-regulated proteins Syk and SLP-76 (Src homology 2-containing leucocyte protein of 76 kDa). A low level of tyrosine phosphorylation of phospholipase Cγ2 was observed, which was increased in the presence of U46619, although the degree of phosphorylation remained well below that observed in wild-type platelets (∼10%). By contrast, collagen-induced phosphorylation of the adapter ADAP (adhesion- and degranulation-promoting adapter protein) was substantially potentiated by U46619 to levels equivalent to those observed in wild-type platelets. Collagen plus U46619 also induced significant phosphorylation of FAK (focal adhesion kinase). The functional significance of collagen-induced non-GPVI signals was highlighted by the ability of U46619 and collagen to induce the secretion of ATP in FcRγ chain-deficient platelets, even though neither agonist was effective alone. Protein tyrosine phosphorylation and the release of ATP were abolished by the anti-(α2 integrin) antibodies Ha1/29 and HMα2, but not by blockade of αIIbβ3. These results illustrate a novel mechanism of platelet activation by collagen which is independent of the GPVI–FcRγ chain complex, and is facilitated by binding of collagen to integrin α2β1. PMID:15283702

  3. Indium-111-labeled platelets: effect of heparin on uptake by venous thrombi and relationship to the activated partial thromboplastin time

    SciTech Connect

    Fedullo, P.F.; Moser, K.M.; Moser, K.S.; Konopka, R.; Hartman, M.T.

    1982-09-01

    The goal of heparin thepapy in deep vein thrombosis is to prevent thrombus extension. The relationship between thrombus extension and the results of coagulation tests used to monitor heparin thepapy is unclear. To expose this relationship, we studied the effect of several heparin regimens on the accretion of indium-111-labeled platelets on fresh venous thrombi, as detected by gamma imaging, and monitored the activated partial thromboplastin time (APTT). Six dogs were treated with a 300-U/kg bolus of heparin followed by a 90-U/kg/hour heparin infusion, a dose of heparin sufficient to increase the APTT to levels greater than eight times baseline (APTT ratio); platelet accretion (thrombus imaging) occurred only after the heparin effect was reversed with protamine sulfate. Nineteen dogs were treated with a 150-U/kg bolus of heparin followed by a 4-hour, 45-U/kg/hour heparin infusion; a thrombus was demonstrated only after protamine injection in 12 (mean APTT ratio 1.3 +/- 0.19) and before protamine injection in seven. In thirteen of these 19 dogs, 30 minutes separated the platelet injection from heparin therapy, while in six this duration was less than 30 minutes. In four of these six dogs, thrombi were demonstrated before protamine therapy and at APTT ratios greater than 3.0. Finally, 10 dogs were treated with a 100-U/kg bolus followed by a 3-hour, 50-U/kg/hour heparin infusion, after which the APTT was allowed to return to baseline values spontaneously. In all 10 dogs, a thrombus was demonstrated only after cessation of the heparin infusion, and at a mean APTT ratio of 1.4 +/- 0.15 times baseline. These results suggest that, except with very early platelet injection, platelet accretion by thrombi is consistently inhibited by heparin at APTT ratios greater than 2.5.

  4. Effective components of Panax quinquefolius and Corydalis tuber protect the myocardium by inhibiting platelet activation and improving the hypercoagulable state

    PubMed Central

    XUE, MEI; LIU, MEI-LIN; ZHU, XIN-YUAN; SHI, DA-ZHUO; YIN, HUI-JUN

    2015-01-01

    The aim of the present study was to investigate the effects of extract of Panax quinquefolius and Corydalis tuber (EPC) on platelet activation and the hypercoagulable state in rats with acute myocardial infarction (AMI). The MI model in Wistar rats was induced by coronary artery ligation. Sham surgery was performed as a control. The surviving rats that underwent MI surgery were divided into control (administered normal saline), metoprolol (9 mg/kg) and low-, moderate- and high-dose EPC groups (0.54, 1.08 g/kg and 2.16 g/kg, respectively). Saline, metoprolol and EPC were administered by gastrogavage for two consecutive weeks. The morphological changes of the myocardium were assessed by hematoxylin and eosin and nitroblue tetrazolium staining. Serum von Willebrand factor (vWF), D-dimer (DD), platelet membrane glycoproteins IIb-IIIa (GPIIb-IIIa) and CD62P levels were assessed using enzyme-linked immunosorbent assay. EPC attenuated the pathological changes of the myocardium. High-dose EPC decreased the serum concentration of vWF when compared with control group. Moderate and high doses of EPC decreased the DD and GPIIb-IIIa levels, and the CD62P level was gradually decreased with EPC dose escalation. The results therefore demonstrated that EPC protects the myocardium by inhibiting platelet activation and improving the hypercoagulable state in a rat model of AMI. PMID:25780455

  5. Metabolomic investigation of the anti-platelet aggregation activity of ginsenoside Rk₁ reveals attenuated 12-HETE production.

    PubMed

    Ju, Hyun Kyoung; Lee, Jin Gyun; Park, Mi Kyung; Park, So-Jung; Lee, Chang Hoon; Park, Jeong Hill; Kwon, Sung Won

    2012-10-01

    Comprehensive metabolomics analysis is an effective method of measuring metabolite levels in the body following administration of a pharmaceutical compound and can allow for monitoring of the effects of the compound or assessment of appropriate treatment options for individual patients. In the present metabolomics study, samples pretreated with antiplatelet compounds were extracted and subjected to ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry. The acquired data were processed using peak clustering and evaluated by partial least-squares (PLS) and orthogonal projections to latent structures discriminant analyses (OPLS-DA). As a result, meaningful endogenous metabolites, namely eicosanoids and thromboxane B(2) (TXB(2)), were identified. TXB(2), a key element in platelet aggregation, was decreased upon ginsenoside Rk(1) treatment via inhibition of cyclooxygenase (COX) activity. One of the arachidonic acid (AA) metabolites, 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), was decreased significantly in the ginsenoside Rk(1)-treated platelets compared to the AA-induced group. In the mechanism study of ginsenoside Rk(1), a strong linkage to intracellular calcium levels, which induce platelet activation, was found. Additionally, the translocation of 12-LOX from cytosol to membrane, which is related with the intracellular calcium levels, was determined. Therefore, a decreased 12-HETE level induced by ginsenoside Rk(1) on antiplatelet aggregation is related to 12-LOX translocation resulting from decreased Ca(2+) levels. This study shows that global metabolomic analysis has potential for use in understanding the biological behavior of antiplatelet drugs. PMID:22873173

  6. 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) PMID:7965802

  7. Long-term untreated streptozotocin-diabetes leads to increased expression and elevated activity of prostaglandin H2 synthase in blood platelets.

    PubMed

    Siewiera, Karolina; Kassassir, Hassan; Talar, Marcin; Wieteska, Lukasz; Watala, Cezary

    2016-05-01

    In diabetes-related states of chronic hyperglycaemia elevated concentrations of glucose may alter the functioning of platelet enzymes involved in arachidonic acid metabolism, including prostaglandin H2 synthase (cyclooxygenase) (PGHS, COX). Therefore, the principal aim of this study was to assess the effects of experimental chronic hyperglycaemia on platelet PGHS-1 (COX-1) expression and activity. Blood platelet activation and reactivity were assessed in Sprague-Dawley rats with the 5-month streptozotocin (STZ) diabetes. The PGHS-1 abundance in platelets was evaluated with flow cytometry and Western blotting, while its activity monitored using a high resolution respirometry and the peroxidase fluorescent assay. The production of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) in platelets were assayed immunoenzymatically. Circulating platelets from diabetic were characterised by increased size, elevated 'priming' and altered reactivity, compared to non-diabetic animals. Both Western blot analysis and flow cytometry revealed significantly elevated expressions of platelet PGHS-1 in STZ-diabetic rats (p < 0.05). We also observed significantly elevated platelet PGHS-1-related arachidonic acid metabolism in diabetic vs. non-diabetic animals, with the use of polarographic (p < 0.05) and total activity assay (p < 0.001). Such increases were accompanied by the elevated production of PGE2 (p < 0.001) and TXB2 (p < 0.05) in diabetic animals. The increased PGHS-1-dependent oxygen consumption and the total activity of PGHS-1 in diabetic animals remained very significant (p < 0.001) also upon adjusting for blood platelet PGHS-1 abundance. Therefore, our results further contribute to the explanation of the increased metabolism of arachidonic acid observed in diabetes. PMID:26325148

  8. Low-dose sirolimus-eluting hydroxyapatite coating on stents does not increase platelet activation and adhesion ex vivo.

    PubMed

    Alviar, Carlos L; Tellez, Armando; Wang, Michael; Potts, Pamela; Smith, Doug; Tsui, Manus; Budzynski, Wladyslaw; Raizner, Albert E; Kleiman, Neal S; Lev, Eli I; Granada, Juan F; Kaluza, Greg L

    2012-07-01

    We previously found paclitaxel-eluting polymer-coated stents causing more human platelet-monocyte complex formation than bare metal stents in vitro. Presently, we examined patterns of platelet activation and adhesion after exposure to 6 nanofilm HAp-coated (HAp-nano) stents, 6 HAp-microporous-coated (HAp-micro) stents, 5 HAp sirolimus-eluting microporous-coated (HAp-SES) stents and 5 cobalt-chromium stents (BMS) deployed in an in vitro flow system. Blood obtained from healthy volunteers was circulated and sampled at 0, 10, 30 and 60 min. By flow cytometry, there were no significant differences in P-Selectin expression between the 4 stent types (HAp-nano = 32.5%; HAp-micro = 42.5%, HAp-SES = 10.23%, BMS = 7% change from baseline at 60 min, p = NS); PAC-1 antibody binding (HAp-nano = 11.8%; HAp-micro = 2.9%, HAp-SES = 18%, BMS = 6.4% change from baseline at 60 min, p = NS) or PMC formation (HAp-nano = 21.6%; HAp-micro = 4%, HAp-SES = 6.6%, BMS = 17.4% change from baseline at 60 min, p = NS). The 4 stent types did not differ in the average number of platelet clusters >10 μm in diameter by SEM (HAp-nano = 2.39 ± 5.75; HAp-micro = 2.26 ± 3.43; HAp-SES = 1.93 ± 3.24; BMS = 1.94 ± 2.41, p = NS). The majority of the struts in each stent group were only mildly covered by platelets, (HAp-nano = 80%, HAp-micro = 61%, HAp-SES = 78% and BMS = 52.1%, p = NS). The HAp-microporous-coated stents (ECD) attracted slightly more proteinaceous material than bare metal stents (HAp-micro = 35% struts with complete protein coverage, P < 0.0001 vs. other 3 stent types). In conclusion, biomimetic stent coating with nanofilm or microporous hydroxyapatite, even when eluting low-dose sirolimus, does not increase the platelet activation in circulating human blood, or platelet adhesion to stent surface when compared to bare metal stents in vitro. PMID:22350685

  9. The role of class I, II and III PI 3-kinases in platelet production and activation and their implication in thrombosis.

    PubMed

    Valet, Colin; Severin, Sonia; Chicanne, Gaëtan; Laurent, Pierre Alexandre; Gaits-Iacovoni, Frédérique; Gratacap, Marie-Pierre; Payrastre, Bernard

    2016-05-01

    Blood platelets play a pivotal role in haemostasis and are strongly involved in arterial thrombosis, a leading cause of death worldwide. Besides their critical role in pathophysiology, platelets represent a valuable model to investigate, both in vitro and in vivo, the biological roles of different branches of the phosphoinositide metabolism, which is highly active in platelets. While the phospholipase C (PLC) pathway has a crucial role in platelet activation, it is now well established that at least one class I phosphoinositide 3-kinase (PI3K) is also mandatory for proper platelet functions. Except class II PI3Kγ, all other isoforms of PI3Ks (class I α, β, γ, δ; class II α, β and class III) are expressed in platelets. Class I PI3Ks have been extensively studied in different models over the past few decades and several isoforms are promising drug targets to treat cancer and immune diseases. In platelet activation, it has been shown that while class I PI3Kδ plays a minor role, class I PI3Kβ has an important function particularly in thrombus growth and stability under high shear stress conditions found in stenotic arteries. This class I PI3K is a potentially interesting target for antithrombotic strategies. The role of class I PI3Kα remains ill defined in platelets. Herein, we will discuss our recent data showing the potential impact of inhibitors of this kinase on thrombus formation. The role of class II PI3Kα and β as well as class III PI3K (Vps34) in platelet production and function is just emerging. Based on our data and those very recently published in the literature, we will discuss the impact of these three PI3K isoforms in platelet production and functions and in thrombosis. PMID:26714793

  10. Crosstalk between Protease-activated Receptor 1 and Platelet-activating Factor Receptor Regulates Melanoma Cell Adhesion Molecule (MCAM/MUC18) Expression and Melanoma Metastasis*

    PubMed Central

    Melnikova, Vladislava O.; Balasubramanian, Krishnakumar; Villares, Gabriel J.; Dobroff, Andrey S.; Zigler, Maya; Wang, Hua; Petersson, Frederik; Price, Janet E.; Schroit, Alan; Prieto, Victor G.; Hung, Mien-Chie; Bar-Eli, Menashe

    2009-01-01

    The cellular and molecular pathways that regulate platelet activation, blood coagulation, and inflammation are emerging as critical players in cancer progression and metastasis. Here, we demonstrate a novel signaling mechanism whereby protease-activated receptor 1 (PAR1) mediates expression of melanoma cell adhesion molecule MCAM/MUC18 (MUC18), a critical marker of melanoma metastasis, via activation of platelet-activating factor receptor (PAFR) and cAMP-responsive element-binding protein (CREB). We found that PAR1 silencing with small hairpin RNA inhibits MUC18 expression in metastatic melanoma cells by inhibiting CREB phosphorylation, activity, and binding to the MUC18 promoter. We further demonstrate that the PAF/PAFR pathway mediates MUC18 expression downstream of PAR1. Indeed, PAR1 silencing down-regulates PAFR expression and PAF production, PAFR silencing blocks MUC18 expression, and re-expression of PAFR in PAR1-silenced cells rescues MUC18 expression. We further demonstrate that the PAR1-PAFR-MUC18 pathway mediates melanoma cell adhesion to microvascular endothelial cells, transendothelial migration, and metastatic retention in the lungs. Rescuing PAFR expression in PAR1-silenced cells fully restores metastatic phenotype of melanoma, indicating that PAFR plays critical role in the molecular mechanism of PAR1 action. Our results link the two pro-inflammatory G-protein-coupled receptors, PAR1 and PAFR, with the metastatic dissemination of melanoma and suggest that PAR1, PAFR, and MUC18 are attractive therapeutic targets for preventing melanoma metastasis. PMID:19703903

  11. Crosstalk between protease-activated receptor 1 and platelet-activating factor receptor regulates melanoma cell adhesion molecule (MCAM/MUC18) expression and melanoma metastasis.

    PubMed

    Melnikova, Vladislava O; Balasubramanian, Krishnakumar; Villares, Gabriel J; Dobroff, Andrey S; Zigler, Maya; Wang, Hua; Petersson, Frederik; Price, Janet E; Schroit, Alan; Prieto, Victor G; Hung, Mien-Chie; Bar-Eli, Menashe

    2009-10-16

    The cellular and molecular pathways that regulate platelet activation, blood coagulation, and inflammation are emerging as critical players in cancer progression and metastasis. Here, we demonstrate a novel signaling mechanism whereby protease-activated receptor 1 (PAR1) mediates expression of melanoma cell adhesion molecule MCAM/MUC18 (MUC18), a critical marker of melanoma metastasis, via activation of platelet-activating factor receptor (PAFR) and cAMP-responsive element-binding protein (CREB). We found that PAR1 silencing with small hairpin RNA inhibits MUC18 expression in metastatic melanoma cells by inhibiting CREB phosphorylation, activity, and binding to the MUC18 promoter. We further demonstrate that the PAF/PAFR pathway mediates MUC18 expression downstream of PAR1. Indeed, PAR1 silencing down-regulates PAFR expression and PAF production, PAFR silencing blocks MUC18 expression, and re-expression of PAFR in PAR1-silenced cells rescues MUC18 expression. We further demonstrate that the PAR1-PAFR-MUC18 pathway mediates melanoma cell adhesion to microvascular endothelial cells, transendothelial migration, and metastatic retention in the lungs. Rescuing PAFR expression in PAR1-silenced cells fully restores metastatic phenotype of melanoma, indicating that PAFR plays critical role in the molecular mechanism of PAR1 action. Our results link the two pro-inflammatory G-protein-coupled receptors, PAR1 and PAFR, with the metastatic dissemination of melanoma and suggest that PAR1, PAFR, and MUC18 are attractive therapeutic targets for preventing melanoma metastasis. PMID:19703903

  12. A Human Platelet Receptor Protein Microarray Identifies the High Affinity Immunoglobulin E Receptor Subunit α (FcεR1α) as an Activating Platelet Endothelium Aggregation Receptor 1 (PEAR1) Ligand*

    PubMed Central

    Sun, Yi; Vandenbriele, Christophe; Kauskot, Alexandre; Verhamme, Peter; Hoylaerts, Marc F.; Wright, Gavin J.

    2015-01-01

    Genome-wide association studies to identify loci responsible for platelet function and cardiovascular disease susceptibility have repeatedly identified polymorphisms linked to a gene encoding platelet endothelium aggregation receptor 1 (PEAR1), an “orphan” cell surface receptor that is activated to stabilize platelet aggregates. To investigate how PEAR1 signaling is initiated, we sought to identify its extracellular ligand by creating a protein microarray representing the secretome and receptor repertoire of the human platelet. Using an avid soluble recombinant PEAR1 protein and a systematic screening assay designed to detect extracellular interactions, we identified the high affinity immunoglobulin E (IgE) receptor subunit α (FcεR1α) as a PEAR1 ligand. FcεR1α and PEAR1 directly interacted through their membrane-proximal Ig-like and 13th epidermal growth factor domains with a relatively strong affinity (KD ∼ 30 nm). Precomplexing FcεR1α with IgE potently inhibited the FcεR1α-PEAR1 interaction, and this was relieved by the anti-IgE therapeutic omalizumab. Oligomerized FcεR1α potentiated platelet aggregation and led to PEAR1 phosphorylation, an effect that was also inhibited by IgE. These findings demonstrate how a protein microarray resource can be used to gain important insight into the function of platelet receptors and provide a mechanistic basis for the initiation of PEAR1 signaling in platelet aggregation. PMID:25713122

  13. Activation of monocytes and platelets by monoclonal antibodies or malaria-infected erythrocytes binding to the CD36 surface receptor in vitro.

    PubMed Central

    Ockenhouse, C F; Magowan, C; Chulay, J D

    1989-01-01

    The CD36 leukocyte differentiation antigen, recognized by MAbs OKM5 and OKM8 and found on human monocytes and endothelial cells, has been implicated as a sequestration receptor for erythrocytes infected with the human malaria parasite Plasmodium falciparum (IRBC). CD36 is also expressed on platelets and appears to be identical to platelet glycoprotein IV. We investigated receptor activation of monocytes and platelets by anti-CD36 MAbs and by IRBC. Incubation of human monocytes with anti-CD36 MAbs or IRBC resulted in stimulation of the respiratory burst as measured by reduction of nitroblue tetrazolium and generation of chemiluminescence. Incubation of human platelets with anti-CD36 MAbs resulted in platelet activation as measured by aggregation or ATP secretion. Activation of monocytes and platelets required appropriate intracellular transmembrane signaling and was inhibited by calcium antagonists or by specific inhibitors of protein kinase C or guanine nucleotide binding proteins. Soluble CD36 inhibited binding of IRBC to both monocytes and platelets, suggesting that these interactions are mediated by the CD36 receptor. Using a cytochemical electron microscopic technique, the presence of reactive oxygen intermediates was identified at the interface between human monocytes and IRBC. These data provide support for the hypothesis that reactive oxygen intermediates produced by monocytes when IRBC ligands interact with cell surface receptors may play a role in the pathophysiology of falciparum malaria. Images PMID:2474569

  14. Activities.

    ERIC Educational Resources Information Center

    Moody, Mally

    1992-01-01

    A series of four activities are presented to enhance students' abilities to appreciate and use trigonometry as a tool in problem solving. Activities cover problems applying the law of sines, the law of cosines, and matching equivalent trigonometric expressions. A teacher's guide, worksheets, and answers are provided. (MDH)

  15. Effects of TRA-418, a novel TP-receptor antagonist, and IP-receptor agonist, on human platelet activation and aggregation.

    PubMed

    Miyamoto, Mitsuko; Yamada, Naohiro; Ikezawa, Shiho; Ohno, Michihiro; Otake, Atsushi; Umemura, Kazuo; Matsushita, Teruo

    2003-11-01

    [4-[2-(1,1-Diphenylethylsulfanyl)-ethyl]-3,4-dihydro-2H-benzo[1,4]oxazin-8-yloxy]-acetic acid N-Methyl-d-glucamine salt (TRA-418) has both thromboxane A2 (TP)-receptor antagonist and prostacyclin (IP)-receptor agonist properties. The present study examined the advantageous effects of TRA-418 based on the dual activities, over an agent having either activity alone and also the difference in the effects of TRA-418 and a glycoprotein alphaIIb/beta3 integrin (GPIIb/IIIa) inhibitor. TRA-418 inhibited platelet GPIIb/IIIa activation as well as P-selectin expression induced by adenosine 5'-diphosphate, thrombin receptor agonist peptide 1-6 (Ser-Phe-Leu-Leu-Arg-Asn-NH2), and U-46619 in the presence of epinephrine (U-46619+ epinephrine). TRA-418 also inhibited platelet aggregation induced by those platelet-stimulants in Ca2+ chelating anticoagulant, citrate and in nonchelating anticoagulant, d-phenylalanyl-l-prolyl-l-arginyl-chloromethyl ketone (PPACK). The TP-receptor antagonist SQ-29548 inhibited only U-46619+epinephrine-induced GPIIb/IIIa activation, P-selectin expression, and platelet aggregation. The IP-receptor agonist beraprost sodium inhibited platelet activation. Beraprost also inhibited platelet aggregation induced by platelet stimulants we tested in citrate and in PPACK. The GPIIb/IIIa inhibitor abciximab blocked GPIIb/IIIa activation and platelet aggregation. However, abciximab showed slight inhibitory effects on P-selectin expression. TRA-418 is more advantageous as an antiplatelet agent than TP-receptor antagonists or IP-receptor agonists separately used. TRA-418 showed a different inhibitory profile from abciximab in the effects on P-selectin expression. PMID:14504133

  16. Protein C Inhibitor (PCI) Binds to Phosphatidylserine Exposing Cells with Implications in the Phagocytosis of Apoptotic Cells and Activated Platelets

    PubMed Central

    Rieger, Daniela; Assinger, Alice; Einfinger, Katrin; Sokolikova, Barbora; Geiger, Margarethe

    2014-01-01

    Protein C Inhibitor (PCI) is a secreted serine protease inhibitor, belonging to the family of serpins. In addition to activated protein C PCI inactivates several other proteases of the coagulation and fibrinolytic systems, suggesting a regulatory role in hemostasis. Glycosaminoglycans and certain negatively charged phospholipids, like phosphatidylserine, bind to PCI and modulate its activity. Phosphatidylerine (PS) is exposed on the surface of apoptotic cells and known as a phagocytosis marker. We hypothesized that PCI might bind to PS exposed on apoptotic cells and thereby influence their removal by phagocytosis. Using Jurkat T-lymphocytes and U937 myeloid cells, we show here that PCI binds to apoptotic cells to a similar extent at the same sites as Annexin V, but in a different manner as compared to live cells (defined spots on ∼10–30% of cells). PCI dose dependently decreased phagocytosis of apoptotic Jurkat cells by U937 macrophages. Moreover, the phagocytosis of PS exposing, activated platelets by human blood derived monocytes declined in the presence of PCI. In U937 cells the expression of PCI as well as the surface binding of PCI increased with time of phorbol ester treatment/macrophage differentiation. The results of this study suggest a role of PCI not only for the function and/or maturation of macrophages, but also as a negative regulator of apoptotic cell and activated platelets removal. PMID:25000564

  17. Characterization of the de novo biosynthetic enzyme of platelet activating factor, DDT-insensitive cholinephosphotransferase, of human mesangial cells.

    PubMed

    Tsoupras, Alexandros Basilios; Fragopoulou, Elizabeth; Nomikos, Tzortzis; Iatrou, Christos; Antonopoulou, Smaragdi; Demopoulos, Constantinos Alexandros

    2007-01-01

    Platelet activating factor (PAF), a potent inflammatory mediator, is implicated in several proinflammatory/inflammatory diseases such as glomerulonephritis, glomerulosclerosis, atherosclerosis, cancer, allergy, and diabetes. PAF can be produced by several renal cells under appropriate stimuli and it is thought to be implicated in renal diseases. The aim of this study is the characterization of DTT-insensitive cholinephosphotransferase (PAF-CPT) of human mesangial cell (HMC), the main regulatory enzyme of PAF de novo biosynthetic pathway. Microsomal fractions of mesangial cells were isolated and enzymatic activity and kinetic parameters were determined by TLC and in vitro biological test in rabbit washed platelets. The effect of bovine serum albumin (BSA), dithiothreitol (DTT), divalent cations (Mg2+ and Ca2+), EDTA, and various chemicals on the activity of PAF-CPT of HMC was also studied. Moreover, preliminary in vitro tests have been performed with several anti-inflammatory factors such as drugs (simvastatin, IFNa, rupatadine, tinzaparin, and salicylic acid) and bioactive compounds of Mediterranean diet (resveratrol and lipids of olive oil, olive pomace, sea bass "Dicentrarchus labrax," and gilthead sea bream "Sparus aurata"). The results indicated that the above compounds can influence PAF-CPT activity of HMC. PMID:17710109

  18. Increased active metabolite formation explains the greater platelet inhibition with prasugrel compared to high-dose clopidogrel.

    PubMed

    Payne, Christopher D; Li, Ying Grace; Small, David S; Ernest, C Steven; Farid, Nagy A; Jakubowski, Joseph A; Brandt, John T; Salazar, Daniel E; Winters, Kenneth J

    2007-11-01

    Prasugrel pharmacodynamics and pharmacokinetics after a 60-mg loading dose (LD) and daily 10-mg maintenance doses (MD) were compared in a 3-way crossover study to clopidogrel 600-mg/75-mg and 300-mg/75-mg LD/MD in 41 healthy, aspirin-free subjects. Each LD was followed by 7 days of daily MD and a 14-day washout period. Inhibition of platelet aggregation (IPA) was assessed by turbidometric aggregometry (20 and 5 microM ADP). Prasugrel 60-mg achieved higher mean IPA (54%) 30 minutes post-LD than clopidogrel 300-mg (3%) or 600-mg (6%) (P < 0.001) and greater IPA by 1 hour (82%) and 2 hours (91%) than the 6-hour IPA for clopidogrel 300-mg (51%) or 600-mg (69%) (P < 0.01). During MD, IPA for prasugrel 10-mg (78%) exceeded that of clopidogrel (300-mg/75-mg, 56%; 600-mg/75-mg, 52%; P < 0.001). Active metabolite area under the concentration-time curve (AUC0-tlast) after prasugrel 60-mg (594 ng.hr/mL) was 2.2 times that after clopidogrel 600-mg. Prasugrel active metabolite AUC0-tlast was consistent with dose-proportionality from 10-mg to 60-mg, while clopidogrel active metabolite AUC0-tlast exhibited saturable absorption and/or metabolism. In conclusion, greater exposure to prasugrel's active metabolite results in faster onset, higher levels, and less variability of platelet inhibition compared with high-dose clopidogrel in healthy subjects. PMID:18030066

  19. Direct effects of platelet-activating factor on glomerular capillary permeability.

    PubMed

    Sharma, R; Sharma, M; Li, J Z; McCarthy, E T; Savin, V J

    1997-01-01

    Platelet-activating factor (PAF) is an important mediator of injury in acute renal failure and glomerulonephritis. Intrarenal infusion of PAF reduces glomerular filtration rate and renal plasma flow and increases glomerular permselectivity via its renal hemodynamic and/or immunologic effects. Direct effects of PAF on glomerular capillary permeability are not known. We studied the direct effects of PAF on mesangial contraction (a measure of filtration area), glomerular capillary hydraulic conductivity (L[p]) and capillary albumin permeability (P[albumin]). Glomeruli were isolated from Sprague-Dawley rats and incubated with or without various concentrations of PAF (10[-9], 10[-7] and 10[-5] M) for up to 5 h at 37 degrees C. Mesangial contraction (percent change in glomerular volume) was assessed from the gradual decrease in volume of glomeruli during 20 min of incubation with PAF. L(p) was calculated from the rate of change in glomerular volume during the 0.1 s of capillary expansion in response to a transcapillary oncotic gradient. P(albumin) was calculated from a change in relative volume of glomeruli in response to an oncotic gradient. Mesangial contraction was maximal after 20 min of incubation and was concentration dependent (5.2+/-0.9, 7.9+/-1.0 and 10.0+/-1.0%, respectively, with PAF 10(-9), 10(-7) and 10(-5) M). Incubation of glomeruli with PAF 10(-7) M for 60 min at 37 degrees C caused a significant decrease in L(p) (2.25+/-0.30 vs. control 3.12+/-0.28 microl x min(-1) x mm Hg(-1) x cm(-1), n = 5). P(albumin) of glomeruli incubated with PAF was unchanged up to 2 h but increased significantly with the highest concentration of PAF (10(-5) M) after 3 h of incubation (0.60+/-0.18, n=15, vs. control 0.00+/-0.08, n = 20), whereas lower concentrations of PAF (10[-7] or 10[-9] M) required at least 5 h of incubation with glomeruli to cause a significant increase in P(albumin) (0.45+/-0.09 and 0.48+/-0.07, respectively, n=15, vs. control 0.00+/-0.08, n=15). We

  20. Inhibition by recombinant SLPI and half-SLPI (Asn55-Ala107) of elastase and cathepsin G activities: consequence for neutrophil-platelet cooperation.

    PubMed Central

    Renesto, P.; Balloy, V.; Kamimura, T.; Masuda, K.; Imaizumi, A.; Chignard, M.