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

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.

Download Full-text

Chemical Agonists and High Shear Stress Induce Apoptosis in Human Platelets.

Blood ◽

10.1182/blood.v104.11.3875.3875 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3875-3875

Author(s):

Valery Leytin ◽

Sergiy Mykhaylov ◽

David J. Allen ◽

Lukasz Miz ◽

Elena V. Lyubimov ◽

...

Keyword(s):

Shear Stress ◽

Platelet Activation ◽

Caspase 3 ◽

Calcium Ionophore ◽

Shear Stresses ◽

Ionophore A23187 ◽

High Shear ◽

Platelet Surface ◽

Platelet Apoptosis ◽

Chemical Stimuli

Abstract Apoptosis, or programmed cell death, is appreciated as the main physiologic mechanism that regulates cell life-span and serves for controlled deletion of unwanted cells. Since its discovery in 1972, apoptosis was long attributed exclusively to nucleate cells. It took more than 20 years to recognize apoptosis in enucleated cells cytoplasts and anucleate platelets. During the following years, apoptosis has been demonstrated in platelets treated with natural and artificial agonists, in platelet concentrates aged during storage under standard blood banking conditions, and in animal models of suppressed thrombopoiesis and thrombocytopenia. Other studies documented that mechanical forces (shear stresses) stimulate platelet activation and signaling in the absence of exogenous chemical stimuli. We analysed whether shear stresses can trigger platelet apoptosis, a question that has not yet been studied. Using a cone-and-plate viscometer (CAP-2000, Brookfield Engineering Labs, Inc., Middleboro, MA), we exposed human platelet-rich plasma to different shear stresses, ranging from physiologic arterial and arterioles levels (10–44 dynes/cm2) to pathologic high levels (117–388 dynes/cm2) occurring in stenosed coronary, peripheral or cerebral arteries. We found that pathologic shear stresses induce not only platelet activation (P-selectin upregulation and GPIb-alpha downregulation) but also trigger apoptosis events, including mitochondrial transmembrane potential depolarization, caspase 3 activation, phosphatidylserine exposure, and platelet shrinkage and fragmentation into microparticles, whereas physiologic shear stresses are not effective. Platelets subjected to pathologic shear stresses are characterized by impaired platelet function as shown by the absence of ADP-induced platelet aggregation. Apoptosis changes were also induced by the treatment of platelets with calcium ionophore A23187 (10 μM) and thrombin (1 U/mL). Thus, in the present work, we have demonstrated that platelet apoptosis can be induced by chemical stimuli and by mechanical rheological forces (pathologic high shear stresses). Most of shear-induced activation and apoptosis events occur inside of the platelet, including translocation of CD62 from alpha-granules to the platelet surface, depolarization of mitochondrial inner membrane potential, activation of cytosolic enzyme caspase 3, and translocation of phosphatidylserine from the inner to the outer plasma membrane leaflet. These data suggest that the effects of shear stress on platelet activation and apoptosis are mediated by mechanoreceptor(s) that transmit activation and apoptosis signals to the cell interior. The platelet paradigm of apoptosis induced by chemical agonists and shear stresses suggests that apoptotic cytoplasmic machinery may function without nuclear participation.

Download Full-text

Platelet Apoptosis Events Can Be Triggered by Shear Stresses and Chemical Agonists.

Blood ◽

10.1182/blood.v106.11.3940.3940 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3940-3940

Author(s):

Valery Leytin ◽

Sergiy Mykhaylov ◽

David J. Allen ◽

John J. Freedman

Keyword(s):

Platelet Activation ◽

Caspase 3 ◽

Platelet Rich Plasma ◽

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Shear Stresses ◽

Ionophore A23187 ◽

Platelet Surface ◽

Platelet Apoptosis ◽

Chemical Stimuli

Abstract Apoptosis, or programmed cell death, is appreciated as the main physiologic mechanism that regulates cell life-span and serves for controlled deletion of unwanted cells. Since its discovery in 1972, apoptosis was long attributed exclusively to nucleate cells. It took more than 20 years to recognize apoptosis in enucleated cells cytoplasts and anucleate platelets. During the following years, apoptosis has been demonstrated in platelets treated with natural and artificial agonists, in platelet concentrates aged during storage under standard blood banking conditions, and in animal models of suppressed thrombopoiesis and thrombocytopenia. Other studies documented that mechanical forces (shear stresses) stimulate platelet activation and signaling in the absence of exogenous chemical stimuli. We analysed whether shear stresses can trigger platelet apoptosis, a question that has not yet been studied. Using a cone-and-plate viscometer (CAP-2000, Brookfield Engineering Labs, Inc., Middleboro, MA), we exposed human platelet-rich plasma to different shear stresses, ranging from physiologic arterial and arterioles levels (10-44 dynes/cm2) to pathologic high levels (117–388 dynes/cm2) occurring in stenosed coronary, peripheral or cerebral arteries. We found that pathologic shear stresses induce not only platelet activation (P-selectin upregulation and GPIbα downregulation) but also trigger apoptosis events, including mitochondrial transmembrane potential depolarization, caspase 3 activation, phosphatidylserine exposure, and platelet shrinkage and fragmentation into microparticles, whereas physiologic shear stresses are not effective. Platelets subjected to pathologic shear stresses are characterized by impaired platelet function as shown by the absence of ADP-induced platelet aggregation. Apoptosis changes were also induced by the treatment of platelets with calcium ionophore A23187 (10 μM) and thrombin (1 U/mL). Thus, in the present work, we have demonstrated that platelet apoptosis can be induced by chemical stimuli and by mechanical rheological forces (pathologic high shear stresses). Most of shear-induced activation and apoptosis events occur inside of the platelet, including translocation of CD62 from α-granules to the platelet surface, depolarization of mitochondrial inner membrane potential, activation of cytosolic enzyme caspase 3, and translocation of phosphatidylserine from the inner to the outer plasma membrane leaflet. These data suggest that the effects of shear stress on platelet activation and apoptosis are mediated by mechanoreceptor(s) that transmit activation and apoptosis signals to the cell interior. The platelet paradigm of apoptosis induced by chemical agonists and shear stresses suggests that apoptotic cytoplasmic machinery may function without nuclear participation.

Download Full-text

Platelet Apoptosis in Fluid-Phase and Adherent Platelets and Thrombi-Like Platelet Aggregates.

Blood ◽

10.1182/blood.v110.11.2102.2102 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2102-2102

Author(s):

Valery Leytin ◽

David J. Allen ◽

Elena Lyubimov ◽

Anna Chavlovski ◽

Mingyu She ◽

...

Keyword(s):

Caspase 3 ◽

Fluid Phase ◽

Human Platelets ◽

Shear Stresses ◽

Ionophore A23187 ◽

High Shear ◽

Platelet Surface ◽

Platelet Suspension ◽

Platelet Apoptosis ◽

Platelet Aggregates

Abstract Apoptosis, or programmed cell death, is the physiologic mechanism that serves for controlled deletion of unwanted cells. Apoptosis was initially attributed exclusively to nucleated cells but over the past decade it has been recognized that apoptosis also occurs in anucleated cytoplasts and platelets. In this study, using flow cytometry we analyzed in human platelets three critical manifestations of mitochondrial, cytoplasmic and plasma membrane apoptosis, mitochondrial inner transmembrane potential (Δψm) depolarization, caspase-3 activation and phosphatidylserine (PS) externalization, respectively. We found that these hallmarks of apoptosis can be induced in human platelet suspension by diverse stimuli, including human α-thrombin (1, 10, 100 nM), calcium ionophore A23187 (3, 5, 10 μM), high shear stresses generated by cone-and-plate viscometer (120, 200, 390 dyn/cm2) and prolonged storage of platelet concentrates in blood banking conditions at 22°C for 6 and 13 days. We also demonstrated that these apoptotic markers can be induced in mouse platelets in vivo in a murine model of immune thrombocytopenia caused by injection of anti-glycoprotein (GP) IIb (rat anti-mouse GPIIb, MWReg30) antibody. Other manifestations of apoptosis were detected in human platelets, including expression of proapoptotic members of Bcl-2 family proteins (Bax and Bak) induced by thrombin, and platelet shrinkage and shedding of microparticles induced by high shear stresses. In addition to apoptosis in fluid-phase platelets, apoptosis was also revealed by confocal fluorescent microscopy in adherent human platelets and thrombi-like platelet aggregates deposited on thrombogenic immobilized human vascular collagen types I and III, as detected by PS exposure and shedding of PS-exposed microparticles. Taken together, these data suggest that platelet apoptosis is a phenomenon that can be triggered by a wide diversity of chemical and physical stimuli using different mechanisms mediated by thrombin-, collagen- and integrin GPIIbIIIa-receptors, mechanoreceptors and Ca2+-overloading. These stimuli trigger platelet apoptosis by impacting on several intracellular apoptotic targets, including shifting the balance between Bcl-2 regulatory proteins in a proapoptotic direction, depolarizing the inner mitochondrial membrane, activating the executioner caspase-3, stimulating aberrant PS exposure on the platelet surface and, eventually, resulting in ‘terminal’ stages of platelet apoptosis, such as platelet shrinkage and shedding of PS-exposed microparticles resembling apoptotic bodies. Platelet apoptosis can be induced both in fluid-phase and adherent platelets and thrombi-like platelet aggregates. These data also indicate that natural PL agonists thrombin and subendothelial vascular collagens and hemodynamic shear forces, can be involved not only in the processes of hemostasis, thrombosis and blood coagulation but also can trigger platelet death via apoptosis. Platelet apoptosis may contribute to the pathophysiology of thrombocytopenia in diseases associated with enhanced thrombin generation, such as sepsis and disseminated intravascular coagulation, as well as in autoimmune and alloimmune thrombocytopenias.

Download Full-text

Src Family Kinases Contribute to GI and Gq-Mediated Platelet Activation

Blood ◽

10.1182/blood.v118.21.5263.5263 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5263-5263

Author(s):

Binggang Xiang ◽

Guoying Zhang ◽

Zhenyu Li

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Src Family Kinases ◽

Ionophore A23187 ◽

Akt Phosphorylation ◽

Mrs 2179

Abstract Abstract 5263 The Src family kinases (SFKs) play an essential role in collagen- and von Willebrand factor (vWF)-mediated platelet activation. However, the role of SFKs in G protein-coupled receptor (GPCR)-mediated platelet activation is not fully understood, and little is known about the molecular mechanisms by which SFKs are activated by GPCR stimulation. Here we demonstrate that SFKs are activated by the Gq and Gi pathways, respectively and SFKs play important roles in Gq- and Gi-dependent secretion and activation. ADP induced SFK phosphorylation in wild type and Gq−/− platelets, and ADP-induced SFK phosphorylation was inhibited by the P2Y12 antagonist AR-C69931MX or P2Y12 knockout but was not affected by the P2Y1 antagonist MRS-2179. Lyn and Fyn were co-immunoprecipitated with Gi2 in platelets, and ADP-induced SFK phosphorylation was diminished in Lyn−/− platelets. These results demonstrate that ADP induces SFK activation mainly depending on the Gi pathway activated via its receptor P2Y12. Furthermore, epinephrine also dose-dependently induced SFK phosphorylation in mouse platelets. A selective inhibitor of Src family kinase PP2 inhibited ADP-induced Akt phosphorylation, fibrinogen binding, and platelet aggregation. Thus, activation of Gi is sufficient to induce SFK activation, which plays an important role in Gi-dependent platelet activation. We further show that the thrombin receptor PAR4 peptide AYPGKF elicited SFK phosphorylation in P2Y12−/−, but not in Gq−/− platelets, and AYPGKF-induced SFK phosphorylation was inhibited by the calcium chelator dimethyl-BAPTA, suggesting that Gq-dependent SFK phosphorylation is downstream from the Ca2+ signaling. The calcium ionophore, A23187-induced TXA2 synthesis, platelet aggregation and secretion were inhibited by pre-incubation of platelets with PP2. PAR4-induced TXA2 synthesis was also abolished by PP2. Moreover, PAR4-mediated granule secretion, integrin aIIbb3 activation, and aggregation of P2Y12 deficient platelets were partially inhibited by PP2 or a PKC inhibitor Ro-31-8220, but were completely abolished by Ro-31-8220 plus PP2 or dimethyl-BAPTA, suggesting that Ca2+/SFKs and PKC represent two parallel signaling pathways mediating Gq-dependent platelet activation. In summary, SFKs can be activated by Gq/Ca2+-dependent mechanisms and by Gi-dependent mechanisms, and SFKS play important roles in Gq- and Gi-dependent platelet activation. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C

Blood ◽

10.1182/blood.v82.9.2704.2704 ◽

1993 ◽

Vol 82 (9) ◽

pp. 2704-2713 ◽

Cited By ~ 53

Author(s):

R Vezza ◽

R Roberti ◽

GG Nenci ◽

P Gresele

Keyword(s):

Protein Kinase C ◽

Platelet Aggregation ◽

Protein Kinase ◽

Prostaglandin E2 ◽

Platelet Activation ◽

Human Platelets ◽

Platelet Surface ◽

Kinase C ◽

Activated Platelets ◽

Induced Aggregation

Abstract Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P- labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.

Download Full-text

Junctional Adhesion Molecule a Helps Maintain Integrin αIIbβ3 in Resting State

Blood ◽

10.1182/blood.v112.11.111.111 ◽

2008 ◽

Vol 112 (11) ◽

pp. 111-111 ◽

Cited By ~ 1

Author(s):

Meghna Ulhas Naik ◽

Timothy J. Stalker ◽

Lawrence F. Brass ◽

Ulhas Pandurang Naik

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Adhesion Molecule ◽

Resting State ◽

Human Platelets ◽

Artery Occlusion ◽

In Vivo Model ◽

Platelet Surface ◽

Integrin Αiibβ3

Abstract Under physiological conditions, fibrinogen receptor integrin αIIbβ3 on the circulating platelets is in a low-affinity, or resting state, unable to bind soluble ligands. During platelet activation by agonists, a cascade of signaling events induces a conformational change in the extracellular domain of αIIbβ3, thereby converting it into a high-affinity state capable of binding ligands through a process known as “inside-out signaling”. What maintains this integrin in a low-affinity state is not well understood. We have previously identified JAM-A, junctional adhesion molecule A, on the platelet surface. We have shown that an antibody blockade of JAM-A dose-dependently activates platelets. To understand the molecular mechanism through which JAM-A regulates platelet aggregation, we used Jam-A null mice. Interestingly, the mouse bleeding times were significantly shortened in Jam-A null mice compared to wildtype littermates. Furthermore, the majority of these mice showed a rebleeding phenotype. This phenotype was further confirmed by FeCl3-induced carotid artery occlusion, a well-accepted in vivo model for thrombosis. Platelets derived from Jam-A-null mice were used to evaluate the role of JAM-A in agonist-induced platelet aggregation. We found that Jam-A null platelets showed enhanced aggregation in response to physiological agonists such as PAR4 peptide, collagen, and ADP as compared to platelets from wildtype littermates. JAM-A was found to associate with αIIbβ3 in unactivated human platelets, but this association was disrupted by both agonist-induced platelet aggregation and during outside-in signaling initiated upon platelet spreading on immobilized Fg. We also found that in resting platelets, JAM-A is phosphorylated on a conserved tyrosine 280 in its cytoplasmic domain, which was dephosphorylated upon platelet activation. Furthermore, JAM-A is rapidly and transiently phosphorylated on serine 284 residue during platelet activation by agonists. Interestingly, JAM-A also formed a complex with Csk, a tyrosine kinase known to be inhibitory to Src activation, in resting platelets. This complex was dissociated upon activation of platelets by agonists. These results suggest that tyrosine-phosphorylated JAM-A recruits Csk to αIIbβ3 in resting platelets, thus maintaining a low-affinity state of integrin αIIbβ3. Agonist–induced activation of platelets results in rapid dephosphorylation of JAM-A on Y280 and phosphorylation on S284 residues. This causes dissociation of JAM-A from integrin αIIbβ3 facilitating platelet aggregation.

Download Full-text

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

Blood ◽

10.1182/blood.v114.22.4016.4016 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4016-4016

Author(s):

José-Tomás Navarro ◽

Shwan Tawfiq ◽

Roland Wohlgemuth ◽

Karin M. Hoffmeister ◽

Robert Sackstein

Keyword(s):

Flow Cytometry ◽

Platelet Aggregation ◽

Platelet Activation ◽

Light Transmission ◽

Conflicts Of Interest ◽

Platelet Rich Plasma ◽

Surface Protein ◽

Surface Flow ◽

Platelet Surface ◽

Biochemical Studies

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.

Download Full-text

Evidence that Calcium Regulates Platelet Function

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648653 ◽

1978 ◽

Vol 40 (02) ◽

pp. 207-211 ◽

Cited By ~ 96

Author(s):

Thomas C Detwiler ◽

Israel F Charo ◽

Richard D Feinman

Keyword(s):

Platelet Aggregation ◽

Sarcoplasmic Reticulum ◽

Platelet Activation ◽

Platelet Function ◽

Calcium Ions ◽

Direct Evidence ◽

Calcium Ionophore ◽

Indirect Evidence ◽

Ionophore A23187 ◽

General Role

SummaryIt is generally believed that calcium ions play a key role in regulation of platelet function.This is based on 3 types of evidence.1. Analogies with other cells. Calcium ions are known to trigger secretion and contraction in many cells, possibly reflecting a general role for calcium in all secretion and contraction.2. Indirect evidence. Platelet aggregation and secretion are induced by divalent cation ionophores. The response to the ionophore A23187 is identical to that induced by other potent stimuli.3. Direct evidence. Platelet activation can be blocked by drugs (e. g. certain local anesthetics) that block release of calcium ions from sarcoplasmic reticulum; the inhibition can be overcome by addition of extracellular calcium in the presence of a calcium ionophore. While this does not constitute definitive proof, the central role for calcium ions remains an attractive hypothesis that justifies attempts to further define calcium pools and fluxes in platelets.

Download Full-text

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.

Download Full-text

Platelet Apoptosis in Adult Immune Thrombocytopenia. Relationship with Auto-Antibodies, Platelet Function and Treatment

Blood ◽

10.1182/blood.v124.21.2792.2792 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2792-2792 ◽

Cited By ~ 1

Author(s):

Geraldine Contrufo ◽

Ana C Glembotsky ◽

Nora P Goette ◽

Paola R Lev ◽

Matías Grodzielski ◽

...

Keyword(s):

Platelet Count ◽

Platelet Activation ◽

Immune Thrombocytopenia ◽

Calcium Ionophore ◽

Activation Parameters ◽

Calcium Ionophore A23187 ◽

Ionophore A23187 ◽

Platelet Surface ◽

Platelet Apoptosis ◽

Cell Penetrating

Abstract Similarly to nucleated cells, platelet life span is also controlled by an intrinsic apoptotic program that triggers collapse of the mitochondrial inner membrane potential, activation of caspases-3, -8 and -9, phosphatidylserine (PS) externalization and microparticle shedding. The aim of the present study was to investigate platelet apoptosis in adult patients with immune thrombocytopenia (ITP) under different treatment conditions and to search for its relationship with the type of auto-antibody and the platelet-activation status. Twenty-four patients with chronic ITP, age 42 (21-80) years (median and range) diagnosed according to current criteria (Rodeghiero et al, 2009) were included after written informed consent in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee from Instituto de Investigaciones Medicas Alfredo Lanari. Platelet count was 38x109/L (6-85). Platelet apoptosis was evaluated by phosphatidylserine (PS) exposure on the platelet surface using FITC-conjugated Anexin-V, mitochondrial electrochemical potential changes (ΔΨm) using the cell penetrating lipophilic cationic fluorochrome JC-1, and activated caspase-3 (a-casp3) measured by the cell-penetrating carboxyfluorescein-labelled fluoromethyl ketone tetrapeptide (FAM-DEVD-FMK). These parameters were studied in resting platelets and after stimulation with calcium ionophore (A23187). Platelet activation was evaluated by FITC-PAC-1 binding to activated GPIIb-IIIa and GPIb-IX internalization using PE-CD42b, in resting conditions and after stimulation either with ADP or TRAP. Apoptosis and activation parameters were evaluated by flow cytometry. In resting conditions, platelets from ITP patients showed increased PS expression and a- casp3 and abnormal ΔΨm (table 1). TablePSΔΨma-casp3Patients19.6 (1.9-82.0)31.2 (5.8-92.4)11.3 (1.8-40.9)Controls4.7 (1.9-10.8)10.3 (2.2-27.5)4.3 (1.9-8.2)p (Mann-Whitney)0.002<0.00010.012n212312 After stimulation with A23187, ITP platelets had similar levels of PS expression (p=0.305, n=20) and ΔmΨ (p=0.383, n=25) compared to normal controls. However, an increased sensitivity to the apoptotic stimulus was evidenced by elevated levels of a-casp3 at low and high A23187 concentrations (1-3 mM, p=0.097; 6-10 mM, p=0.002). Platelet apoptosis was not related to platelet activation, as PAC-1 binding was not increased in ITP platelets (basal p=0.847, ADP-induced p=0.059, TRAP-induced p=0.103, n=16). Besides, internalization of GPIb-IX after ADP and TRAP stimulation was also normal (p=NS, n=9 for both agonists). Platelets from ITP patients bearing two of the most frequently found auto-antibodies (5 with anti-GPIIb-IIIa and 1 with anti-GPIb-IX) had similar levels of PS expression and ΔmΨ at resting conditions than those who were negative for these auto-antibodies (n=14) (p=0.265 and 0.148, respectively). There were no differences in apoptosis markers either in resting platelets or after stimulation when comparing untreated patients (n=9) vs patients under any kind of treatment (n=15) (resting conditions, PS p=0.737; ΔmΨ p=0.270; stimulated, PS p=0.966; ΔmΨ p=0.987), although platelet count was similar in both groups. Normal platelets incubated during 1 hour with plasma from ITP had higher a-casp3 than those incubated with normal plasma (n=12 and 9, respectively, p=0.027), suggesting a plasmatic component could be responsible for the apoptotic stimulus. In conclusion, increased platelet apoptosis in ITP patients could be induced by a plasmatic factor, contributing to thrombocytopenia in this entity. Disclosures Riveros: Roche: Speakers Bureau.

Download Full-text