Fucosyltransferase VI Induces Platelet Activation: A Novel Property of a Plasma Glycosyltransferase.

Abstract Abstract 4016 Poster Board III-952 A number of glycosyltransferases are present in human plasma with the α(1→3) fucosyltransferase, Fucosyltransferase VI (FTVI), having the highest plasma concentration. Notably, elevated plasma levels of FTVI are associated with a variety of cancers and correlate with tumor load/progression. The well-known association of neoplasia with thromboembolic complications prompted us to examine whether FTVI has direct effect(s) on platelet function. We obtained human platelets from blood of healthy donors and separated from platelet-rich plasma by differential centrifugation. Freshly isolated platelets (x108/ml) were stirred and exposed at 37°C to varying concentrations (20, 40, 60 and 80 mU/mL) of glycosyltransferases FTVI, β-1-4-galactosyltransferase-I (βGalT-I), or α,2-3-N-sialyltransferase (α2,3-N-ST), or to 1 U/mL thrombin. Platelet aggregation and activation was assessed by aggregometry (light transmission) or by flow cytometry of FSC/SSC characteristics and of surface expression of P-Selectin, respectively. FT-VI reproducibly induced platelet aggregation and activation, whereas other glycosyltransferases (β4GalT-I and α2,3-N-ST) had no effect on platelets. FTVI activation of platelets was concentration-dependent, and the aggregation curve for FTVI was one wave, similar to that for thrombin. FTVI-induced platelet activation was independent of catalytic conversion of surface glycans, but was inhibited by FTVI denaturation, indicating that FTVI-induced platelet activation is a lectin-mediated process. To determine the membrane target(s) mediating FTVI-induced platelet activation, biochemical studies were performed after catalytic exofucosylation of the platelet surface. Flow cytometry after platelet exofucosylation showed formation of the carbohydrate structure sLex, detected by the mAb Heca452, but no formation of Lex (CD15). Western blot showed that enforced fucosylation induced sLex on a single platelet surface protein, and further biochemical studies revealed that this protein is GPIbα. These findings unveil a previously unrecognized property of FTVI as an activator of platelets, mediated via a specific lectin/carbohydrate interaction on GP1ba, and offer novel perspectives on the pathobiology of tumor-associated thrombogenesis. Disclosures: No relevant conflicts of interest to declare.

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Platelets as Predictors of Vascular Risk: Is There a Practical Index of Platelet Activity?

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/107602960300900301 ◽

2003 ◽

Vol 9 (3) ◽

pp. 177-190 ◽

Cited By ~ 169

Author(s):

Stavroula Tsiara ◽

Moses Elisaf ◽

I. Anita Jagroop ◽

Dimitri P. Mikhailidis

Keyword(s):

Flow Cytometry ◽

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Rich Plasma ◽

Vascular Risk ◽

Platelet Activity ◽

Vascular Events ◽

Bioactive Substances ◽

Platelet Surface ◽

Soluble P

Activated platelets play a role in the pathogenesis of coronary heart disease (CHD). Following activation, platelets change shape, aggregate, and release several bioactive substances. The aim of this review is to identify if there is a simple and cost-effective method that indicates platelet activation and predicts the risk of CHD and vascular events. The rationale for identifying high-risk patients is to reduce their risk of vascular events by administering appropriate and effective antiplatelet treatment, like aspirin, clopidogrel, or combination regimens. Many laboratory tests estimating platelet activity have been described. Some are relatively simple, such as spontaneous or agonist-induced platelet aggregation. Other tests include measuring the mean platelet volume (MPV) or plasma soluble P-selectin levels. Some more complex tests include flow cytometry to determine platelet GP Ilb/Illa receptors, platelet surface P-selectin, plateletmonocyte aggregates, and microparticles. Only few prospective studies assessed the predictive value of platelet activation in healthy individuals. Although the MPV seems an 'easy method, there are insufficient data supporting its ability to predict the risk of a vascular event in healthy adults. Platelet aggregation, in whole blood or in platelet-rich plasma was not consistently predictive of vascular risk. Soluble P-selectin measurement is a promising method but it needs further evaluation. Flow cytometry methods are costly, time-consuming, and need specialized equipment. Thus, they are unlikely to be useful in estimating the risk in large numbers of patients. There is as yet no ideal test for the detection of platelet activation. Each currently available test has merits and disadvantages. Simple methods such as the MPV and the determination of platelet release products need further evaluation.

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Comparison of platelet aggregation parameters and expression of platelet granule markers

Тромбоз, гемостаз и реология ◽

10.25555/thr.2019.4.0896 ◽

2019 ◽

Author(s):

В.В. Малышева ◽

О.А. Шустова ◽

С.Г. Хаспекова ◽

Я.А. Наймушин ◽

А.В. Мазуров

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Light Transmission ◽

Platelet Rich Plasma ◽

Thrombin Receptor ◽

Platelet Surface ◽

Dense Granules ◽

Turbidimetric Method ◽

Healthy Donors ◽

Aggregation Parameters

Введение. Активация тромбоцитов стимулирует их агрегацию и ассоциированный с секрецией экзоцитоз внутриклеточных гранул. Агрегацию чаще всего изучают турбидиметрическим методом, а экзоцитоз гранул методом проточной цитофлуориметрии, определяя экспрессию их маркеров на поверхности тромбоцитов. Цель исследования: сравнение чувствительности двух методов оценки активации тромбоцитов, исследования их агрегации и экспрессии маркеров внутриклеточных гранул. Материалы и методы. Тромбоциты здоровых доноров активировали АДФ и пептидом, активирующим рецептор тромбина (thrombin receptor activating peptide, TRAP). Агрегацию тромбоцитов изучали турбидиметрическим методом в обогащенной тромбоцитами плазме, регистрируя максимальный уровень светопропускания (Т макс). Экспрессию маркеров гранул изучали в цельной крови с помощью проточной цитофлуориметрии, регистрируя процент тромбоцитов, окрашенных антителами против маркеров альфагранул (CD62P) и плотных гранул (CD63). Результаты. У всех доноров 20 мкМ АДФ и 10 мкМ TRAP стимулировали мощную, необратимую агрегацию тромбоцитов (62,1 10,3 и 65,1 5,1 T макс, соответственно). Средние уровни агрегации были ниже при ее стимуляции 2,5 мкМ АДФ (30,9 23,2 T макс) и 1 мкМ TRAP (24,4 29,1 T макс). Экспрессия маркеров гранул была максимальной при активации тромбоцитов 10 мкМ TRAP (77,6 13,7 CD62P и 73,9 13,3 CD63), ниже при активации 1 мкМ TRAP (46,3 25,1 CD62P и 38,3 23,8 CD63), еще ниже при активации 20 мкМ АДФ (19,8 8,5 CD62P и 13,6 5,2 CD63) и минимальной при активации 2,5 мкМ АДФ (8,0 4,2 CD62P и 8,3 3,3 CD63). Адреналин (20 мкМ) не стимулировал экспрессию маркеров гранул, но при совместном добавлении с 20 мкМ АДФ повышал ее в 1,9 раза для обоих маркеров CD62P и CD63. Заключение. При активации тромбоцитов АДФ исследование агрегации является более чувствительным методом, чем определение экспрессии маркеров гранул. При активации тромбоцитов TRAP чувствительность обоих методов приблизительно одинакова. Экспрессия маркеров гранул увеличивается при совместном добавлении к тромбоцитам АДФ и адреналина, хотя адреналин сам по себе не стимулирует их экспрессию. Introduction. Platelet activation stimulates their aggregation and secretion associated exocytosis of intracellular granules. Aggregation is usually studied by turbidimetric method and granule exocytosis by detecting expression of their markers on platelet surface using flow cytofluorimetry. Aim: сomparison of the sensitivity of two methods for evaluation of platelet activation, studies of their aggregation and expression of intracellular granule markers. Materials and methods. Healthy donors platelets were activated by ADP and thrombin receptor activating peptide (TRAP). Platelet aggregation was investigated in platelet rich plasma by turbidimetric method, assessed by the maximal level of light transmission (T max). Expression of granule markers was detected in whole blood by fl ow cytofl uorimetry registering the percent of platelets stained with antibodies against the markers of alphagranules (CD62P) and dense granules (CD63). Results. In all donors 20 M ADP and 10 M TRAP stimulated strong irreversible platelet aggregation (62.1 10.3 and 65.1 5.1 T max). Mean aggregation levels were lower when it was stimulated by 2.5 M ADP (30.9 23.2 T max) and 1 M TRAP (24.4 29.1 T max). Expression of granule markers was maximal at platelet activation by 10 M TRAP (77.6 13.7 CD62P and 73.9 13.3 CD63), lower at activation by 1 M TRAP (46.3 25.1 CD62P and 38.3 23.8 CD63), more lower at activation by 20 M ADP (19.8 8.5 CD62P and 13.6 5.4 CD63), and minimal at activation by 2.5 M ADP (8.0 4.2 CD62P and 8.3 3.3 CD63). Epinephrine (20 M) did not stimulate expression of granule markers but at combined addition with 20 M ADP increased its level by 1.9 for both markers, CD62P and CD63. Conclusion. Platelet aggregation is more sensitive method than detection of expression of granule markers when platelets are activated by ADP. Both methods demonstrate about the same sensitivity when platelets are activated by TRAP. Expression of granule markers is increased at combined addition of ADP and epinephrine although epinephrine by itself fails to stimulate their expression.

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von Willebrand factor bound to glycoprotein Ib is cleared from the platelet surface after platelet activation by thrombin

Blood ◽

10.1182/blood.v79.8.2011.2011 ◽

1992 ◽

Vol 79 (8) ◽

pp. 2011-2021 ◽

Cited By ~ 30

Author(s):

P Hourdille ◽

HR Gralnick ◽

E Heilmann ◽

A Derlon ◽

AM Ferrer ◽

...

Keyword(s):

Flow Cytometry ◽

Platelet Activation ◽

Von Willebrand Factor ◽

Platelet Rich Plasma ◽

Surface Expression ◽

Von Willebrand Disease ◽

Platelet Surface ◽

Immunogold Staining ◽

Von Willebrand ◽

Willebrand Factor

Abstract We recently reported that after activation of human platelets by thrombin, glycoprotein (GP) Ib-IX complexes are translocated to the surface-connected canalicular system (SCCS) (Blood 76:1503, 1990). As GPIb is a major receptor for von Willebrand factor (vWF) in platelet adhesion, we have now examined the consequences of thrombin activation on the organization of vWF bound to GPIb on the platelet surface. Studies were performed using monoclonal or polyclonal antibodies in either immunogold staining and electron microscopy (Au-EM) or in flow cytometry. When unstirred platelet-rich plasma was incubated with ristocetin, bound vWF was located by Au-EM as discrete masses regularly distributed over the cell surface. Platelets from a patient with Glanzmann's thrombasthenia, lacking GPIIb-IIIa complexes, gave a similar pattern, confirming that this represented binding to GPIb. That ristocetin was not precipitating vWF before their binding to the platelets was shown by the detection of similar masses on the surface of platelets of a patient with type IIB von Willebrand disease. Experiments were continued using washed normal platelets incubated in Tyrode-EDTA, the purpose of the EDTA being to limit the surface expression of endogenous vWF after platelet stimulation. Under these conditions, platelets were treated with ristocetin for 5 minutes at 37 degrees C in the presence of increasing amounts of purified vWF. This was followed by incubation with thrombin (0.5 U/mL) for periods of up to 10 minutes. Flow cytometry showed a time-dependent loss in the surface expression of vWF bound to GPIb and these changes were confirmed by Au-EM. In particular, immunogold staining performed on ultrathin sections showed that the bulk of the vWF was being cleared to internal membrane systems. Surface clearance of vWF during thrombin- induced platelet activation is a potential mechanism for regulating platelet adhesivity.

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

Thrombosis and Haemostasis ◽

10.1160/th07-01-0010 ◽

2007 ◽

Vol 98 (07) ◽

pp. 192-200 ◽

Cited By ~ 33

Author(s):

Joseph Jakubowski ◽

You FuLi ◽

Marc Barnard ◽

Marsha Fox ◽

Matthew Linden ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Whole Blood ◽

Blood Cells ◽

Inflammatory Markers ◽

Active Metabolite ◽

Platelet Rich Plasma ◽

Shape Change ◽

Flow Cytometric Analysis ◽

Platelet Surface

SummaryThe novel thienopyridine prodrug prasugrel, a platelet P2Y12 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 in-hibition of these ADP-stimulated thrombo-inflammatory markers.These rapid kinetics required physiological calcium levels, but were largely unaffected by aspirin. Lower IC50 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 P2Y12-mediated ADP-induced shape change, even at concentrations that completely inhibited platelet aggregation, confirming the specificity of R-138727 for P2Y12. In conclusion, R-138727, the active metabolite of prasugrel, results in rapid, potent, consistent, and selective inhibition of P2Y12-mediated up-regulation of thromboinflammatory markers of platelet activation.This inhibition is enhanced in the presence other blood cells and calcium,but not aspirin.

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Sialic Acid Deficiency Impairs GPVI-Mediated Platelet Activation in ST3Gal-IV−/− Mice.

Blood ◽

10.1182/blood.v114.22.3005.3005 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3005-3005

Author(s):

Viktoria Rumjantseva ◽

Anne Louise Sørensen ◽

Karin M Hoffmeister ◽

Hervé Falet

Keyword(s):

Flow Cytometry ◽

Sialic Acid ◽

Platelet Activation ◽

Conflicts Of Interest ◽

Receptor Expression ◽

Platelet Surface ◽

Lectin Domain ◽

Terminal Sialic Acid ◽

Fibrinogen Binding ◽

Surface Receptor

Abstract Abstract 3005 Poster Board II-972 Lack of terminal sialic acid residues on platelet surface glycoproteins results in rapid platelet clearance. Using null mice for the ST3Gal-IV sialyltransferase gene (ST3Gal-IV−/− mice), we have recently identified galactose residues on the N-terminus of the platelet Von Willebrand Factor receptor GPIbαa as a major counter receptor for the lectin domain of the asialoglycoprotein receptor on both hepatocytes and liver Kupffer cells (Sørensen et al., Blood 2009). ST3Gal-IV−/− mice have increased tail bleeding time. However, the role of terminal sialic acid residues on platelet activation is unclear. We investigated here whether loss of sialylation affects platelet activation mediated through the collagen receptor GPVI or by thrombin. Platelets were isolated from ST3Gal-IV−/− and ST3Gal-IV+/+ mouse littermates, stimulated with collagen-related peptide (CRP), convulxin (CVX), or thrombin, and platelet activation was evaluated by flow cytometry using P-selectin expression, as a marker for αa-granule secretion, and fibrinogen binding, as a marker for integrin αaIIbβ3 activation. Stimulation of ST3Gal-IV−/− platelets with CRP and CVX revealed a profound activation defect, compared to ST3Gal-IV+/+ platelets. The defect was not due to loss of surface receptor expression since ST3Gal-IV−/− and ST3Gal-IV+/+ platelets had comparable GPVI expression, as evidenced by flow cytometry. By contrast, activation of ST3Gal-IV−/− platelets with thrombin was normal. The data show that terminal sialic acid residues on GPVI are required for maximal platelet activation by CRP and CVX. Disclosures: No relevant conflicts of interest to declare.

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The GPIIbIIIa Antagonist Drugs Eptifibatide and Tirofiban Inhibit Caspase-3 Activation in Thrombin- and Calcium Ionophore-Stimulated Human Platelets.

Blood ◽

10.1182/blood.v114.22.2998.2998 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2998-2998

Author(s):

Valery Leytin ◽

Asuman Mutlu ◽

Sergiy Mykhaylov ◽

David J. Allen ◽

Armen V. Gyulkhandanyan ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Caspase 3 ◽

Conflicts Of Interest ◽

Calcium Ionophore ◽

Human Platelets ◽

Shear Stresses ◽

Ionophore A23187 ◽

Platelet Surface ◽

Apoptotic Effects

Abstract Abstract 2998 Poster Board II-976 Introduction: The platelet surface receptor glycoprotein (GP) IIbIIIa (integrin αaIIbβ3) mediates platelet aggregation and plays a key role in hemostasis and thrombosis. Numerous GPIIbIIIa antagonists have been designed and tested as inhibitors of platelet aggregation. Two of these antagonists, eptifibatide (Integrilin) and tirofiban (Aggrastat) have been approved by the U.S. Food and Drug Administration (FDA) and widely used for preventing and treating thrombotic complications in patients undergoing percutaneous coronary intervention and in patients with acute coronary syndromes. It has been reported, however, that some GPIIbIIIa antagonists, such as orbofiban and xemilofiban, promote apoptosis in cardiomyocytes by activation of the apoptosis executioner caspase-3, raising the possibility that platelets also may be susceptible to pro-apoptotic effects of eptifibatide and tirofiban. Over the past decade it has been well-documented that apoptosis occurs not only in nucleated cells but also in anucleated platelets stimulated with thrombin, calcium ionophores, very high shear stresses and platelet storage (Leytin et al, J Thromb Haemost 4: 2656, 2006; Mason et al, Cell 128: 1173, 2007). It has been further reported that platelet activation and apoptosis may be induced by different mechanisms and/or require different levels of triggering stumuli (Leytin et al, Br J Haematol 136: 762, 2007; Br J Haematol 142: 494, 2008). Recently, we have shown that injection of anti-GPIIb antibody induced caspase-3 activation in mouse platelets in vivo (Leytin et al, Br J Haematol 133: 78, 2006), suggesting that direct GPIIbIIIa-mediated pro-apoptotic signaling is able to trigger caspase-3 activation within platelets. Study Design and Methods: The current study aimed to examine, for the first time, the effect of eptifibatide and tirofiban on caspase-3 activation in human platelets. We studied the effects of eptifibatide and tirofiban on caspase-3 activation in resting platelets, which express GPIIbIIIa receptors in their non-active (“closed”) conformation, and in platelets stimulated with thrombin or calcium ionophore A23187, which induce transition of GPIIbIIIa receptors into active (“open”) conformation. Resting platelets were treated with control buffer, 0.48 μM eptifibatide or 0.48 μM tirofiban, and stimulated platelets were treated with 1 U/mL thrombin or 10 μM A23187, or preincubated with eptifibatide or tirofiban before treatment with thrombin or A23187. Caspase-3 activation was determined by flow cytometry using the cell-penetrating FAM-DEVD-FMK probe, which covalently binds to active caspase-3. Results and Discussion: We found that treatment of resting platelets with eptifibatide and tirofiban did not affect caspase-3 activation (P>0.05, n=7). In contrast, a 2.3-2.7-fold increase of caspase-3 activation was observed in platelets after thrombin or A23187 stimulation (P<0.01, n=7). However, when platelets were preincubated with eptifibatide and tirofiban before agonist treatment, these drugs significantly inhibited agonist-induced caspase-3 activation by an average of 44-50% (P<0.05, n=7). The fact that eptifibatide and tirofiban do not promote caspase-3 activation in unstimulated platelets suggests that these GPIIbIIIa antagonists do not induce transmission of pro-apoptotic transmembrane signals inside platelets through inactive GPIIbIIIa integrin. The inhibitory effect of eptifibatide and tirofiban on thrombin- and A23187-induced caspase-3 activation suggests a role of GPIIbIIIa integrin in caspase-3 activation induced by these platelet agonists. Conclusions: We have demonstrated a novel platelet-directed activity of two clinically used GPIIbIIIa antagonist drugs, eptifibatide (Integrilin) and tirofiban (Aggrastat), with ability to inhibit apoptosis executioner caspase-3 induced by potent platelet agonists, thrombin and A23187, and the absence of adverse pro-apoptotic effects on resting platelets. Taken together with earlier reported data (Leytin et al, Br J Haematol 133: 78, 2006), the current study indicates that, aside from their well-known participation in platelet activation and aggregation, GPIIbIIIa receptors are involved in the modulation of platelet apoptosis. This GPIIbIIIa-mediated mechanism of apoptosis modulation may be very efficient given the extremely large number of GPIIbIIIa copies (≈80,000) on the platelet surface. Disclosures: No relevant conflicts of interest to declare.

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Platelet Glycoprotein VI Dimerisation Is An Active Process and Enables the Receptor to Be Competent

Blood ◽

10.1182/blood.v116.21.3194.3194 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3194-3194 ◽

Cited By ~ 1

Author(s):

Stéphane Loyau ◽

Bénédicte Dumont ◽

Nadine Ajzenberg ◽

Martine Jandrot-Perrus

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Adhesion ◽

Transmembrane Domain ◽

Conflicts Of Interest ◽

Blood Platelets ◽

Matrix Proteins ◽

Platelet Glycoprotein ◽

Active Process ◽

Platelet Surface

Abstract Abstract 3194 In the blood, platelets are normally prevented from activation by endothelial inhibitors (i.e. prostacycline, ectonucleotidase). Dysfunctional endothelial cells loose their protective properties and favor platelet adhesion to matrix proteins, platelet aggregation and thrombus growth. Collagen fibers are highly thrombogenic and the platelet Glycoprotein (GP)VI predominantly mediates collagen-induced platelet responses. GPVI is a platelet specific receptor of the immunoglobulin (Ig) superfamily containing two extracellular Ig domains, a single transmembrane domain and a short cytoplasmic tail. GPVI signals through the immunoreceptor tyrosine-based activation motifs (ITAM) of the non-covalently associated immune receptor adaptor FcRg dimer. There is growing evidence that optimal binding of GPVI to collagen depends on the formation of GPVI dimers at the platelet surface: only dimeric GPVI binds to collagen and inhibits collagen-induced platelet aggregation and not monomeric GPVI. Moreover, crystallographic data showed dimerization of GPVI ectodomains. However, the valence of GPVI on resting and activated platelets is still debated. We have obtained an anti-human GPVI monoclonal antibody (9E18), that binds to dimeric GPVI with a 200 fold higher affinity than to monomeric GPVI. In flow cytometry on whole blood, while the 3J24 antibody labels >95% platelets, 9E18 hardly binds to resting platelets with less than 3% positive platelets. The level of 9E18-positive platelets moderately increased (10-15%) after platelet isolation suggesting it could reflect platelet activation. Binding of 9E18 was indeed significantly increased on ADP- or TRAP-activated washed platelets (25±1.9 % and 36±7% positive platelets respectively). Additionally, increased binding of 9E18 was triggered by the GPVI agonists, collagen, convulxin or the activating 9O12 IgG. At sites of vascular lesion, platelet adhesion is initiated by the shear-dependent interaction of GPIb with vWF, assumed to favor GPVI-collagen interaction. When a platelet rich plasma was submitted to a shear of 4000 s-1 for 5 min, 9E18-positive platelets increased from 3.6±1.6% to 7±2% in the whole platelet population and to 26±7.7% on small aggregates (p<0.05).When a2b1 and aIIbb3 were blocked, the relation between the 9E18 binding to stimulated platelets and platelet binding to collagen was linear (r2 = 0.847, p=0.0012, n=8). Interestingly, the cAMP elevating agent PGE1 further lowered the level of 9E18-binding to resting platelets and dropped it to basal values on ADP- or TRAP-treated platelets. Apyrase reduced by 50% TRAP-induced binding of 9E18 whereas indomethacin had no effect. PMA triggered binding of 9E18 on platelets (p<0.001) while the Tyr-phosphatase inhibitor PAO, strongly inhibited PMA-induced 9E18 binding to platelets (p<0.0019) and GPVI-dependent platelet adhesion to collagen. Altogether, these data indicate that 9E18 permit to quantify GPVI dimers on platelets. They show that (i) GPVI is mainly monomeric on resting platelets, (ii) dimerisation is an active process triggered by shear, soluble agonists and matrix proteins, (iii) the level of GPVI dimers is related to the capacity of platelets to adhere to collagen, (iv) GPVI dimerisation is completely prevented in the presence of agents increasing cAMP or by PAO. These data suggested that the formation of GPVI dimer is strictly controlled on resting platelets and that GPVI dimers could thus represent a new marker of platelet activation and susceptibility to collagen. Indeed, in a population of hospitalized patient, a positive correlation was observed between 9E18 binding and P-selectin exposure on platelets. Disclosures: No relevant conflicts of interest to declare.

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von Willebrand factor bound to glycoprotein Ib is cleared from the platelet surface after platelet activation by thrombin

Blood ◽

10.1182/blood.v79.8.2011.bloodjournal7982011 ◽

1992 ◽

Vol 79 (8) ◽

pp. 2011-2021

Author(s):

P Hourdille ◽

HR Gralnick ◽

E Heilmann ◽

A Derlon ◽

AM Ferrer ◽

...

Keyword(s):

Flow Cytometry ◽

Platelet Activation ◽

Von Willebrand Factor ◽

Platelet Rich Plasma ◽

Surface Expression ◽

Von Willebrand Disease ◽

Platelet Surface ◽

Immunogold Staining ◽

Von Willebrand ◽

Willebrand Factor

We recently reported that after activation of human platelets by thrombin, glycoprotein (GP) Ib-IX complexes are translocated to the surface-connected canalicular system (SCCS) (Blood 76:1503, 1990). As GPIb is a major receptor for von Willebrand factor (vWF) in platelet adhesion, we have now examined the consequences of thrombin activation on the organization of vWF bound to GPIb on the platelet surface. Studies were performed using monoclonal or polyclonal antibodies in either immunogold staining and electron microscopy (Au-EM) or in flow cytometry. When unstirred platelet-rich plasma was incubated with ristocetin, bound vWF was located by Au-EM as discrete masses regularly distributed over the cell surface. Platelets from a patient with Glanzmann's thrombasthenia, lacking GPIIb-IIIa complexes, gave a similar pattern, confirming that this represented binding to GPIb. That ristocetin was not precipitating vWF before their binding to the platelets was shown by the detection of similar masses on the surface of platelets of a patient with type IIB von Willebrand disease. Experiments were continued using washed normal platelets incubated in Tyrode-EDTA, the purpose of the EDTA being to limit the surface expression of endogenous vWF after platelet stimulation. Under these conditions, platelets were treated with ristocetin for 5 minutes at 37 degrees C in the presence of increasing amounts of purified vWF. This was followed by incubation with thrombin (0.5 U/mL) for periods of up to 10 minutes. Flow cytometry showed a time-dependent loss in the surface expression of vWF bound to GPIb and these changes were confirmed by Au-EM. In particular, immunogold staining performed on ultrathin sections showed that the bulk of the vWF was being cleared to internal membrane systems. Surface clearance of vWF during thrombin- induced platelet activation is a potential mechanism for regulating platelet adhesivity.

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Platelet Mitochondrial Potential and Function Is Altered During Severe Septic Shock.

Blood ◽

10.1182/blood.v114.22.5074.5074 ◽

2009 ◽

Vol 114 (22) ◽

pp. 5074-5074

Author(s):

Andrea Artoni ◽

Alessandro Protti ◽

Anna Lecchi ◽

Giovanna Motta ◽

Francesca Rosini ◽

...

Keyword(s):

Flow Cytometry ◽

Septic Shock ◽

Platelet Aggregation ◽

Platelet Function ◽

Sofa Score ◽

Conflicts Of Interest ◽

Platelet Rich Plasma ◽

Annexin V ◽

Mitochondrial Potential ◽

Serotonin Content

Abstract Abstract 5074 Background Septic shock is a condition characterized by a systemic inflammatory response associated to organ dysfunction. Primary hemostasis is impaired in sepsis, and multiple causative mechanisms have been proposed. Methods In this study we evaluated platelet function in patients with septic shock. In particular platelet mitochondrial potential was studied as a possible cause of platelet function impairment in septic shock. To test the hypothesis we enrolled 21 consecutive patients admitted to our intensive care unit (ICU) with diagnosis of septic shock. All patients were analysed within 24 hours from admission. 30 ml of citrated blood were withdrawn from each patient and the platelet function tests were performed. Platelet mitochondrial potential (Dy) was assessed by flow cytometry through JC1 staining, espressed as the ratio of FL-1 and FL-2 fluorescence. Platelet aggregation was performed on platelet rich plasma in 5 patients not assuming any drug known to influence platelet function with the following aggregating agents: ADP (final concentration 4 μM), collagen (2 μg/ml), U46619 (10 μM) and TRAP (10 μM). Platelet ATP secretion was quantified and intraplatelet δ-granules content dosage was performed. Expression of GpIb, of GpIIb/IIIa, and of annexin V binding were evaluated by flow cytometry. The study was approved by the Hospital Ethical Committee. Results at admission platelet mitochondrial potential was reduced in septic patients in comparison to normal controls (2.6±1.4 vs 3.4±0.3 p=0.06). At day one there was a good correlation between platelet mitochondrial impairment and the Sepsis-related Organ Failure Assessment (SOFA) score, an indicator of sepsis severity (R2=0,39 p<0.001 n=18) with higher values indicating higher degree of severity. Platelets taken from patients with a SOFA score above the group median value (>9, n=8) had Dy values significantly lower than those taken from less severely ill patients (SOFA score ' 9, n=10) and healthy volunteers (1.6±0.6 vs. 3.3±1.4 vs. 3.4±0.3; p<0.01 one way analysis of variance). At day one platelet aggregation was severely impaired, especially with strong aggregating agents as collagen (3/5 of patients with maximum platelet aggregation below 25%). The maximum aggregation caused by collagen seemed to correlate to platelet mitochondrial potential (R2=0,72 n=5 p=0.07). Platelet secretion was equally defective at admission, with pathological secretion to collagen stimulus in 40% of tested patients. Intraplatelet ADP content resulted low in 66% tested (n= 12), while serotonin content was pathological in 58% of patients (n= 12). Platelet GpIIb/IIIa and GpIb expression and annexin V binding were within normal limits. Conclusion we demonstrated in a very well selected group of patients that during the early phase of a septic shock a mitochondrial dysfunction occurs, and this may have an impact on platelets dysfunction observed in this condition. Disclosures No relevant conflicts of interest to declare.

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Parstatin Mediates Platelet Activation That Is Unaffected By PAR Antagonism

Blood ◽

10.1182/blood.v126.23.2234.2234 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2234-2234

Author(s):

Paige Selvy Dunphy ◽

Jayaprakash Kotha ◽

Mason L. A. Dixon ◽

Jay M. Jalenak ◽

Lisa K. Jennings

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Light Transmission ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Shape Change ◽

Antiplatelet Agent ◽

Surface Expression ◽

Thrombin Receptor ◽

Protein Markers

Abstract Thrombin cleaves PAR-1 at amino acids arg41-ser42 to yield the canonical tethered ligand as well as a soluble peptide, parstatin, which has been reported to have divergent physiological functions. In animal models, this peptide demonstrates anti-angiogenic, anti-inflammatory, and cardioprotective properties. In ex vivo studies, parstatin was also demonstrated to affect platelet aggregation and enhance GPIIb/IIIa-mediated adhesion, suggesting this cleaved peptide may participate in pathological thrombosis. With recent approval of a first-in-class PAR-1 antagonist as an antiplatelet agent, it is clinically imperative to fully appreciate the physiologic mechanisms through which thrombin-activation of PAR-1 contributes to platelet aggregation and thrombosis. As such, the effects of PAR antagonism in modulating parstatin-mediated platelet activation requires evaluation. In this study, we characterized parstatin-mediated effects on platelets and investigated the potential involvement of platelet thrombin receptor (PAR-1, PAR-4)-associated signaling in this phenomenon. Light-transmission aggregometry was used to measure aggregation response in washed platelet preparations, and flow cytometry was used to assess expression of protein markers indicative of platelet activation. Consistent with previous reports, we demonstrated parstatin induces P-Selectin surface expression (degranulation), GPIIb/IIIa activation (PAC-1 binding), and aggregation independent of thrombin receptor cleavage (n=10, healthy donors). Interestingly, platelet shape change was not observed following parstatin treatment, even in the presence of PAR-1 activating peptide (PAR-1-AP, SFLLRN), suggesting parstatin-mediated activation does not signal through G12/13-dependent mechanisms, and may override canonical G12/13-associated PAR-1 signaling. Pretreatment with Gq-selective PAR-1 antagonist, ML161 (3 µM), or PAR-4-selective antagonist, ML354 (500 nM) did not inhibit parstatin-mediated platelet activation. These findings are consistent with previous reports suggesting this peptide may signal through a Gi-dependent mechanism. Platelet PAR receptors couple to Gαq and Gα12/13, but direct coupling to Gαi is controversial; therefore, parstatin-mediated activation may occur through a signaling cascade unrelated to canonical PAR-associated mechanisms. Disclosures No relevant conflicts of interest to declare.

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