The physical exchange of factor VIII (FVIII) between von Willebrand factor and activated platelets and the effect of the FVIII B-domain on platelet binding

Normal hemostasis proceeds through the assembly of coagulant complexes on a lipid surface derived from activated platelets. The activation complex assembly is governed by multiple factors including the binding constants (Kd) of the coagulant factors for the lipid surface. The formation of the tenase complex requires delivery of factor VIII (FVIII) to the activated lipid surface by von Willebrand factor (vWF). Using electrophoretic quasi-elastic light scattering (ELS), we have examined the interaction of FVIII in the presence and absence of vWF with both resting and activated gel-filtered human platelets. Resting platelets do not bind FVIII. Platelets activated by thrombin, epinephrine, or SFLLRN, but not ADP or collagen, bind unactivated FVIII if vWF is not present. In the absence of vWF, unactivated FVIII binds to activated platelets with a Kd of 10.4 nM. B-domain deleted FVIII binds to activated platelets with a Kd of 5.1 nM. Thrombin -activated FVIII (FVIIIa) binds to activated platelets with a Kd of 1.7 nM. The activation of FVIII while bound to the platelet surface can be monitored as a function of time. In the presence of vWF, binding of unactivated FVIII to activated platelets was inhibited, but not the binding of FVIIIa. Displacement of bound unactivated FVIII from the platelet surface occurs when vWF is added to the FVIII-platelet complex. The binding of FVIII to activated platelets is affected by the B-domain, the state of FVIII activation, and the presence of soluble vWF and proceeds as a multistep process. FVIII binding by activated platelets is not affected by platelet gpIIb/IIIa or by platelet vWF.     

Activation and adherence of lyophilized human platelets on canine vessel strips in the Baumgartner perfusion chamber

The Baumgartner perfusion technique was used to test the ability of rehydrated lyophilized human platelets to adhere to the thrombogenic surface of a denuded arterial vessel segment and to undergo platelet activation under conditions of high shear. Twenty preparations of washed platelets were stabilized by 1-hour or 2-hour exposure to paraformaldehyde before freeze-drying in a Virtis 600 lyophilizer. The response of these fixed-dried preparations was compared with that of non-fixed platelets in fresh citrated whole blood. The outcome of each perfusion experiment was quantified in photomicrographs by morphometric estimation of the percent area of the vessel segment covered by adherent platelets after immunofluorescent staining with monoclonal antibodies to glycoprotein Ib (CD42) or glycoprotein IIbIIIa (CD41a). Evidence of activation in nonadherent platelets was examined by flow cytometry for CD62 and GP53 expression. In addition, thromboxane B2 was measured by radioimmunoassay as an index of platelet responsiveness to activation conditions during perfusion. The percent vessel coverage observed with lyophilized platelets in recombined whole blood was somewhat less than that of platelets in fresh whole blood (39% vs 73%, respectively). In other perfusion experiments performed with non-denuded vessel segments, the percent coverage was reduced by half or more for both types of platelet preparation. Scanning electron microscopy confirmed that the lyophilized platelets did not adhere to areas of intact endothelium. Furthermore, the lyophilized platelets showed a small-but-significant rise in CD62 or CD63 activation antigen expression and generated thromboxane B2 at about one third the rate of fresh platelets in these perfusion experiments. The thromboxane generation during perfusion was inhibited in fresh or lyophilized platelet preparations by pretreatment with indomethacin or PGE-1. We interpret these data as evidence of the ability of our lyophiilized platelet preparations to respond at least partially to physiologic stimuli and to adhere to appropriate thrombogenic sites to support hemostasis.     

Detection of platelets activated during acetylcholine-induced coronary vasospasm

Although platelet activation may play a role in coronary artery spasm, platelets activated following coronary vasospasm have not been clinically detected. We performed flow cytometric analysis of activation-dependent granular proteins, CD62P (P-selectin), CD63, PAC-1 (activated glycoprotein [GP] IIb/IIIa) and thrombospondin on the platelet plasma membrane in patients who exhibited acetylcholine-induced coronary vasospasm and compared findings with those in control patients without vasospasm. We simultaneously investigated the plasma levels of thrombin anti-thrombin III complex (TAT), plasmin alpha2-plasmin inhibitor complex (PIC), and thrombomodulin. In patients with vasospasm, the expression of CD62P, CD63 and PAC-1 on the platelet membrane surface increased in coronary sinus blood samples following coronary vasospasm, although the expression in aortic samples did not change. The TAT level also increased in the coronary sinus after vasospasm. Platelets might be activated by coronary vasospasm within the coronary circulation. The platelet activation process may be modulated by thrombin generation.     

Hyporeactivity of neonatal platelets is not caused by preactivation during birth

Little information exists concerning platelet function in neonates due to the small blood volume. Most studies using conventional aggregation methods have shown a diminished response to various agonists. This is in contrast to the lack of a bleeding tendency and to a short bleeding time in healthy neonates. In previous work we have shown that in healthy term neonates even after an uncomplicated delivery signs of thrombin generation can be demonstrated. This activation of the clotting system may also lead to platelet activation in the neonate. We investigated by means of flow cytometry the expression of activation markers GMP 140 and GP 53 on the surface of neonatal platelets and GP 53 intracellularly by means of a newly developed assay. The expression of GMP 140 and of GP 53 after thrombin stimulation was significantly higher on adult rather than neonatal platelets, while there was no difference between neonatal and adult platelets using GP 53 as intracellular marker. In none of the healthy term neonates after an uncomplicated delivery were activated platelets demonstrated. CONCLUSION: Our data show that the decreased reactivity of neonatal platelets is not caused by preactivation during birth but rather represents a developmental phenomenon. Possibly the observed hyporeactivity of neonatal platelets to thrombin helps to prevent harmful effects of birth stress on the clotting system of neonates.     

Comparison of inotropic and chronotropic effects of vasoactive intestinal peptide in isolated dog atria

In an in vitro study the effect of various thrombin inhibitors (argatroban, efegatran, DuP 714, recombinant hirudin and PEG-hirudin) on platelet activation in whole blood was investigated. Blood was drawn from normal human volunteers using the double syringe technique without use of a tourniquet to avoid autoaggregation of platelets. Blood was anticoagulated with either argatroban, efegatran, DuP 714, hirudin or PEG-hirudin at final concentrations of 10 micrograms/ml. Blood samples were then incubated at 37 degrees C either with saline, r-tissue factor, arachidonic acid, adenosine diphosphate or collagen. At definite times (1, 2.5, 5, 10 min) aliquots were taken and after various steps of fixative procedure the percentage of platelet activation was measured using fluorescent monoclonal antibodies to platelet surface receptors GPIIIa (CD-61) and P-selectin (CD-62). Flow cytometric analysis showed a platelet activation after all agonists used. All thrombin inhibitors studied caused a nearly complete inhibition of r-tissue factor-mediated platelet activation. In contrast, after activation with the other agonists an increased percent CD-62 expression was found with a maximum after 2.5 to 5 min. The results show that in whole blood thrombin inhibitors are effective in preventing platelet activation induced by r-tissue factor. The formation of active serine proteases including thrombin may be effectively inhibited by these agents. The observations further suggest that while thrombin inhibitors may control serine proteases, these agents do not inhibit the activation of platelets mediated by other agonists.     

Phospholipase A2: its role in ADP- and thrombin-induced platelet activation mechanisms

ADP and thrombin are two of the most important agonists of platelet aggregation--a cellular response that is critical for maintaining normal hemostasis. However, aberrant platelet aggregation induced by these agonists plays a central role in the pathogenesis of cardiovascular and cerebrovascular diseases. Agonist-induced primary or secondary activation of phospholipases leads to generation of the second messengers that participate in biochemical reactions essential to a number of platelet responses elicited by ADP and thrombin. Phospholipase A2 (PLA2) has been linked to cardiovascular diseases. However, the mechanism(s) of activation of PLA2 in platelets stimulated by ADP and thrombin has remained less well defined and much less appreciated. The purpose of this review is to examine and compare the molecular mechanisms of activation of PLA2 in platelets stimulated by ADP and thrombin.     

Increased platelet sensitivity toward platelet inhibitors during physical exercise in patients with coronary artery disease

Generalized atherosclerosis and coronary artery disease (CAD) are associated with endothelial dysfunction and during acute myocardial ischemia platelet activation has been reported. Activated platelets exert activated fibrinogen receptors (GP IIb/IIIa) and express CD 62p being regarded as reliable marker for platelet activation. Patients with angiographically proven CAD performed a bicycle exercise test until the onset of angina or ST-segment depression. We studied the ischemia-induced alterations in fibrinogen binding to activated platelet GP IIb/IIIa receptors and CD 62p expression. Therefore, the basal fibrinogen binding to GP IIb/IIIa and CD 62p expression and the thrombin-concentration for half-maximal platelet activation before and after exercise testing were determined. Additionally, inhibition of thrombin-induced platelet activation by increasing concentrations of the prostacyclin-analog iloprost and the NO-donor SIN-1 was examined. In patients with CAD, a significantly reduced basal activation and a highly significant reduction in sensitivity towards thrombin was measured. The thrombin-induced expression of GP IIb/IIIa and CD 62p was significantly diminished in patients with CAD after physical exercise and their platelets were significantly more sensitive towards the inhibitory effects of iloprost and SIN-1. These data demonstrate a significant reduction in platelet activation in response to physical exercise in patients with CAD and advanced atherosclerosis. Despite exercise induced myocardial ischemia as evidenced by angina and ECG-changes, the platelets are not generally activated, as it could be expected. Thus, patients with myocardial ischemia experienced a reduced platelet activity and enhanced sensitivity towards prostacyclin (PGI2) and nitric oxide, probably due to an augmented release of endogenous platelet inhibitory mediators.     

A dual thrombin receptor system for platelet activation

Platelet-dependent arterial thrombosis triggers most heart attacks and strokes. Because the coagulation protease thrombin is the most potent activator of platelets, identification of the platelet receptors for thrombin is critical for understanding thrombosis and haemostasis. Protease-activated receptor-1 (PAR1) is important for activation of human platelets by thrombin, but plays no apparent role in mouse platelet activation. PAR3 is a thrombin receptor that is expressed in mouse megakaryocytes. Here we report that thrombin responses in platelets from PAR3-deficient mice were markedly delayed and diminished but not absent. We have also identified PAR4, a new thrombin-activated receptor. PAR4 messenger RNA was detected in mouse megakaryocytes and a PAR4-activating peptide caused secretion and aggregation of PAR3-deficient mouse platelets. Thus PAR3 is necessary for normal thrombin responses in mouse platelets, but a second PAR4-mediated mechanism for thrombin signalling exists. Studies with PAR-activating peptides suggest that PAR4 also functions in human platelets, which implies that an analogous dual-receptor system also operates in humans. The identification of a two-receptor system for platelet activation by thrombin has important implications for the development of antithrombotic therapies.     

Inhibition of platelet-mediated, tissue factor-induced thrombin generation by the mouse/human chimeric 7E3 antibody. Potential implications for the effect of c7E3 Fab treatment on acute thrombosis and "clinical restenosis"

The murine/human chimeric monoclonal antibody fragment (c7E3 Fab) blocks GPIIb/IIIa and alpha v beta 3 receptors, inhibits platelet aggregation, and decreases the frequency of ischemic events after coronary artery angioplasty in patients at high risk of suffering such events. Although inhibition of platelet aggregation is likely to be the major mechanism of c7E3 Fab's effects, since activated platelets facilitate thrombin generation, it is possible that c7E3 Fab also decreases thrombin generation. To test this hypothesis, the effects of c7E3 Fab and other antiplatelet agents were tested in a thrombin generation assay triggered by tissue factor. c7E3 Fab produced dose-dependent inhibition of thrombin generation, reaching a plateau of 45-50% inhibition at concentrations > or = 15 micrograms/ml. It also inhibited thrombin-antithrombin complex formation, prothrombin fragment F1-2 generation, platelet-derived growth factor and platelet factor 4 release, incorporation of thrombin into clots, and microparticle formation. Antibody 6D1, which blocks platelet GPIb binding of von Willebrand factor, had no effect on thrombin generation, whereas antibody 10E5, which blocks GPIIb/IIIa but not alpha v beta 3 receptors decreased thrombin generation by approximately 25%. Combining antibody LM609, which blocks alpha v beta 3 receptors, with 10E5 increased the inhibition of thrombin generation to approximately 32-41%. The platelets from three patients with Glanzmann thrombasthenia, who lacked GPIIb/IIIa receptors but had normal or increased alpha v beta 3 receptors, supported approximately 21% less thrombin generation than normal platelets. We conclude that thrombin generation initiated by tissue factor in the presence of platelets is significantly inhibited by c7E3 Fab, most likely in part through both GPIIb/IIIa and alpha v beta 3 blockade, and that this effect may contribute to its antithrombotic properties.     

Thrombin activates factor XI on activated platelets in the absence of factor XII

Thrombin can activate factor XI in the presence of dextran sulfate or sulfatides. However, a physiological cofactor for thrombin activation of factor XI has not been identified. We examined this question in a cell-based, tissue factor-initiated model system. In the absence of factor XII, factor XI enhanced thrombin generation in this model. The effect on thrombin generation was reproduced by 2 to 5 pmol/L factor XIa. A specific inhibitor of factor XIIa did not diminish the effect of factor XI. Thus, factor XI can be activated in a model system that does not contain factor XIIa or nonphysiological cofactors. Preincubation of factor XI with activated platelets and thrombin or factor Xa enhanced subsequent thrombin generation in the model system. Preincubation of factor XI with thrombin or factor Xa, but without platelets, did not enhance thrombin generation, suggesting that these proteases might activate factor XI on platelet surfaces. Thrombin and factor Xa were then directly tested for their ability to activate factor XI. In the presence of dextran sulfate, thrombin or factor Xa activated factor XI. Thrombin, but not factor Xa, also cleaved detectable amounts of factor XI in the presence of activated platelets. Thus, thrombin activates enough factor XI to enhance subsequent thrombin generation in a model system. Platelet surfaces might provide the site for thrombin activation of functionally significant amounts of factor XI in vivo.     

Thrombin receptor activating peptide does not stimulate platelet procoagulant activity

Platelets after challenge with alpha-thrombin alone, collagen alone or thrombin/collagen mixture were observed to increase the rate of activation of prothrombin by factor Xa in the presence of factor Va and calcium ion (platelet procoagulant activity) by a maximum of 25, 45 and 110 fold, respectively. The increase in platelet procoagulant activity due to these agonists has been described previously and arises from increased expression of phosphatidylserine on the platelet surface. When platelets were treated with the thrombin receptor activating peptide (TRAP) (SFLLRNPNDKYEPK), alone or in the presence of collagen or thrombin, no change in platelet procoagulant activity was observed at concentrations of TRAP sufficient to cause increased intracellular calcium levels and protein phosphorylation in a manner similar to that of thrombin. In addition, no increase in platelet procoagulant activity was seen upon treatment with TRAP in the presence of inactivated thrombin (PPACK-thrombin). These results suggest that the thrombin-mediated increase in procoagulant activity may be due to activation of a thrombin receptor distinct from the recently cloned G-protein-coupled receptor, or to other proteolytic events on the platelet surface.     

Stimulated Glanzmann's thrombasthenia platelets produced microvesicles. Microvesiculation correlates better to exposure of procoagulant surface than to activation of GPIIb-IIIa

The mechanism of formation of platelet-derived microvesicles remains controversial. The aim of the present work was to study the formation of microvesicles in view of a possible involvement of the GPIIb-IIIa complex, and of exposure of negatively charged phospholipids as procoagulant material on the platelet surface. This was studied in blood from three Glanzmann's thrombasthenia patients lacking GPIIb-IIIa and healthy blood donors. MAb FN52 against CD9 which activates the complement system and produces microvesicles due to a membrane permeabilization, ADP (9.37 microM), and the thrombin receptor agonist peptide SFLLRN (100 microM) that activates platelets via G-proteins were used as inducers. In a series of experiments platelets were also preincubated with PGE1 (20 microM). The number of liberated microvesicles, as per cent of the total number of particles (including platelets), was measured using flow cytometry with FITC conjugated antibodies against GPIIIa or GPIb. Activation of GPIIb-IIIa was detected as binding of PAC-1, and exposure of aminophospholipids as binding of annexin V. With normal donors, activation of the complement system induced a reversible PAC-1 binding during shape change. A massive binding of annexin V was seen during shape change as an irreversible process, as well as formation of large numbers of microvesicles (60.6 +/- 2.7%) which continued after reversal of the PAC-1 binding. Preincubation with PGE1 did not prevent binding of annexin V, nor formation of microvesicles (49.5 +/- 2.7%), but abolished shape change and PAC-1 binding after complement activation. Thrombasthenic platelets behaved like normal platelets after activation of complement except for lack of PAC-1 binding (also with regard to the effect of PGE1 and microvesicle formation). Stimulation of normal platelets with 100 microM SFLLRN gave 16.3 +/- 1.2% microvesicles, and strong PAC-1 and annexin V binding. After preincubation with PGE1 neither PAC-1 nor annexin V binding, nor any significant amount of microvesicles could be detected. SFLLRN activation of the thrombasthenic platelets produced a small but significant number of microvesicles (6.4 +/- 0.8%). Incubation of thrombasthenic platelets with SFLLRN after preincubation with PGE1, gave results identical to those of normal platelets. ADP activation of normal platelets gave PAC-1 binding, but no significant annexin V labelling, nor production of microvesicles. Thus, different inducers of the shedding of microvesicles seem to act by different mechanisms. For all inducers there was a strong correlation between the exposure of procoagulant surface and formation of microvesicles, suggesting that the mechanism of microvesicle formation is linked to the exposure of aminophospholipids. The results also show that the GPIIb-IIIa complex is not required for formation of microvesicles after activation of the complement system, but seems to be of importance, but not absolutely required, after stimulation with SFLLRN.     

P-Selectin and platelet clearance

P-selectin is an adhesion receptor for leukocytes expressed by activated platelets and endothelial cells. To assess a possible role of P-selectin in platelet clearance, we adapted an in vivo biotinylation technique in mice. Wild-type and P-selectin-deficient mice were infused with N-hydroxysuccinimido biotin. The survival of biotinylated platelets was followed by flow cytometry after labeling with fluorescent streptavidin. Both wild-type and P-selectin-deficient platelets presented identical life spans of about 4.7 days, suggesting that P-selectin does not play a role in platelet turnover. When biotinylated platelets were isolated, activated with thrombin, and reinjected into mice, the rate of platelet clearance was unchanged. In contrast, storage of platelets at 4 degreesC caused a significant reduction in their life span in vivo but again no significant differences were observed between the two genotypes. The infused thrombin-activated platelets rapidly lost their surface P-selectin in circulation, and this loss was accompanied by the simultaneous appearance of a 100-kD P-selectin fragment in the plasma. This observation suggests that the platelet membrane P-selectin was shed by cleavage. In conclusion, this study shows that P-selectin, despite its binding to leukocytes, does not mediate platelet clearance. However, the generation of a soluble form of P-selectin on platelet activation may have biological implications in modulating leukocyte recruitment or thrombus growth.     

Differential expression of platelet activation markers in aspirin-sensitive asthmatics and normal subjects

BACKGROUND: Activation of platelets and expression of adhesion molecules (e.g. CD62P and CD63) which mediate interactions between platelets and other cells may be important in the pathogenesis of aspirin-sensitive asthma. OBJECTIVE: To determine the expression of CD62P and CD63 on platelets from aspirin-sensitive asthmatic (ASA+), aspirin-tolerant asthmatic (ASA-) and normal subjects and to assess the modulatory effect of aspirin on platelet CD62P and CD63 expression following stimulation with either platelet-activating factor (PAF), arachidonic acid (AA) or collagen (COL). METHODS: Platelet-rich plasma was obtained from 10 ASA+, 10 ASA- and 10 normal control subjects, and expression of CD62P and CD63 was measured by flow cytometry. Platelets were stimulated with PAF (10, 80 nM), AA (0.1, 1 mM) or COL (80, 800 micrograms/mL) with or without aspirin (concentration range 0.4-4 mg/mL). RESULTS: In the absence of aspirin, CD62P expression induced by AA and COL was greater in ASA+ patients compared with control subjects (P < 0.001) while CD62P expression with PAF, AA and COL was reduced in ASA- when compared with ASA+ and control subjects (P < 0.001). CD63 expression with PAF and AA was reduced in both ASA+ and ASA- patients compared with control subjects (P < 0.001). Aspirin inhibited the expression of both CD62P and CD63 after agonist stimulation. Greater inhibition of CD62P expression was observed in ASA+ compared with ASA- patients (P < 0.001) and normal subjects (P < 0.05) while greater inhibition of CD63 expression was observed in normal subjects compared with both ASA+ and ASA- patients (P < 0.05). In ASA+ patients and normal subjects, stimulation with PAF and COL resulted in only one platelet population while in contrast with 1 mM AA two populations were observed. CONCLUSIONS: Enhanced AA- and collagen-induced platelet CD62P expression in ASA+ patients compared with normal subjects and greater inhibition by aspirin of CD62P expression in ASA+ may be relevant to the pathogenesis of this syndrome. Reduced expression of CD62P and CD63 in platelets of ASA- patients following stimulation with PAF and AA may also have implications for the role of platelets and these mediators in the pathogenesis of other forms of asthma.     

Procoagulant nature of fibrin

The main threat from a beginning thrombus is that it tends to grow, and hence become occlusive and/or embolise. Although the progressive nature of thrombi has been recognised since a long time, the mechanisms behind thrombus growth remain only partially resolved. In order to investigate in what ways thrombi can themselves become foci of further thrombin -and hence fibrin-formation, we studied the effect of fibrin clots on thrombin generation in platelet poor--and platelet rich plasma (PPP and PRP). The thrombin always adsorbed on a natural fibrin clot is not inactivated by plasmatic antithrombins and could be shown to retain its ability to enhance further thrombin formation by activation of clotting factors V and VIII as well as of blood platelets. To our surprise, fibrin clots without any active thrombin adsorbed, because they were obtained by a snake-venom enzyme or because thrombin had been inhibited, retained their capacity to activate blood platelets and make them procoagulant. The activation could be shown to be due to a rearrangement of cell-membrane phospholipids, by which the procoagulant species (phosphatidyl serine and phosphatidyl ethanolamine) became available at the outer cell surface. The platelet membrane receptor involved could be recognised as glycoprotein Ib, interacting with fibrin through the plasma protein von Willebrand factor (vWf). In fact it appeared that vWf is indispensable for the generation of thrombin in PRP, with or without added clot. This assigns a new and hitherto unknown role to vWf. Our results also show that fibrin is far from being the inert end-product of coagulation but is a potent activator of blood platelets and by this action may foster thrombin generation and hence further fibrin production. We surmise this mechanism to be instrumental in the progression of thrombotic processes.     

Tyrosine phosphorylation of the novel protein-tyrosine kinase RAFTK during an early phase of platelet activation by an integrin glycoprotein IIb-IIIa-independent mechanism

A key regulatory event controlling platelet activation is mediated through the phosphorylation of several cellular proteins by protein-tyrosine kinases. The related adhesion focal tyrosine kinase (RAFTK) is a novel cytoplasmic tyrosine kinase and a member of the focal adhesion kinase (FAK) gene family. FAK phosphorylation in platelets is integrin-dependent, occurs in a late stage of platelet activation, and is dependent on platelet aggregation. In this study, we have investigated the involvement of RAFTK phosphorylation during different stages of platelet activation. Treatment of platelets with thrombin induced, in as early as 10 s, a rapid tyrosine phosphorylation of RAFTK in a time- and concentration-dependent manner. Treatment of platelets with thrombin in the absence of stirring or pretreatment of platelets with RGDS peptide prevented platelet aggregation, but not RAFTK phosphorylation. Furthermore, phosphorylation of RAFTK did not require integrin engagement since platelets treated with the 7E3 inhibitory antibodies that block fibrinogen binding to glycoprotein IIb-IIIa did not inhibit RAFTK phosphorylation. Similarly, platelets treated with LIBS6 antibodies, which specifically activate glycoprotein IIb-IIIa, did not induce RAFTK phosphorylation. Stimulation of platelets by several agonists such as collagen, ADP, epinephrine, and calcium ionophore A23187 induced RAFTK phosphorylation. Tyrosine phosphorylation of RAFTK in platelets is regulated by calcium and is mediated through the protein kinase C pathway. Phosphorylation of RAFTK is dependent upon the formation of actin cytoskeleton as disruption of actin polymerization by cytochalasin D significantly inhibited this phosphorylation. The RAFTK protein appears to be proteolytically cleaved by calpain in an aggregation dependent manner upon thrombin stimulation. These results demonstrate that RAFTK is tyrosine-phosphorylated during an early phase of platelet activation by an integrin- independent mechanism and is not dependent on platelet aggregation, suggesting different mechanisms of regulation for FAK and RAFTK phosphorylation during platelet activation.     

Ethanol inhibits thrombin-induced secretion of the contents of human platelet dense and alpha-granules and lysosomes

Moderate consumption of alcoholic beverages is associated with a reduction in thromboembolic complications of coronary artery disease, possibly partially attributable to inhibition by ethanol of platelet responses to some aggregating agents. Although ethanol is known to inhibit thrombin-induced secretion of platelet dense granule contents, the effect of ethanol on secretion of alpha-granule and lysosomal contents has not been studied. Using suspensions of washed platelets, and a range of thrombin concentrations (up to 0.1 U/ml), we examined the effect of 87 mM ethanol on secretion of [14C]serotonin from prelabelled platelets as a measure of secretion of dense granule contents. Secretion of alpha-granule and lysosomal contents was examined by flow cytometric measurement of the surface expression of CD62P (P-selectin) and CD63, respectively. Secretion of the lysosomal enzyme, beta-N-acetylglucosaminidase was also quantified. Results were expressed as % of maximum response induced by 1 U/ml thrombin. Ethanol inhibited the thrombin-induced secretion of both dense and alpha-granule contents (P <0.001, 2-way ANOVA), and of lysosomal contents (P <0.005 for CD63 expression and P <0.001 for beta-N-acetylglucosaminidase secretion). When platelets were pretreated with aspirin, thrombin-induced secretion of storage granule and lysosomal contents was slightly inhibited, but secretion was inhibited by ethanol to the same extent as the untreated platelets, indicating that this inhibition was independent of thromboxane A2. Surface expression of CD63 occurred at lower thrombin concentrations than those required for secretion of beta-N-acetylglucosaminidase, possibly due to the presence of some CD63 on granule membranes. Although the role of lysosomal contents in thrombus formation is not established, some constituents of storage granules are known to augment thrombus formation; ethanol's inhibition of their secretion by stimulated platelets may contribute to its beneficial effect on thromboembolism.     

Glycoprotein VI is a major collagen receptor for platelet activation: it recognizes the platelet-activating quaternary structure of collagen, whereas CD36, glycoprotein IIb/IIIa, and von Willebrand factor do not

Simple collagen-related peptides (CRPs) containing a repeat Gly-Pro-Hyp sequence are highly potent platelet agonists. Like collagen, they must exhibit tertiary (triple-helical) and quaternary (polymeric) structure to activate platelets. Platelet signaling events induced by the peptides are the same as most of those induced by collagen. The peptides do not recognize the alpha 2 beta 1 integrin. To identify the signaling receptor involved, we have evaluated the response to the CRP, Gly-Lys-Hyp(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly of platelets with defined functional deficiencies. These studies exclude a primary recognition role for CD36, von Willebrand factor (vWF), or glycoprotein (GP) IIb/IIIa. Thus, both CD36 and vWF-deficient platelets exhibited normal aggregation, normal fibrinogen binding, and normal expression of CD62 and CD63, measured by flow cytometry, in response to the peptide, and there was normal expression of CD62 and CD63 on thrombasthenic platelets. In contrast, GPVI-deficient platelets were totally unresponsive to the peptide, indicating that this receptor recognizes the Gly-Pro-Hyp sequence in collagen. GPVI-deficient platelets showed some fibrinogen binding in response to collagen but failed to aggregate and to express CD62 and CD63. Collagen, but not CRP-XL, contains binding sites for alpha 2 beta 1. Therefore, it is possible that collagen still induces some signaling via alpha 2 beta 1, leading to activation of GPIIb/IIIa. Our findings are consistent with a two-site, two-step model of collagen interaction with platelets involving recognition of specific sequences in collagen by an adhesive receptor such as alpha 2 beta 1 to arrest platelets under flow and subsequent recognition of another specific collagen sequence by an activatory receptor, namely GPVI.     

IgG from patients with antiphospholipid syndrome binds to platelets without induction of platelet activation

The clinical manifestations of the antiphospholipid syndrome (APLS) include arterial and venous thrombosis, thrombocytopenia and fetal loss, but the pathogenic mechanisms remain unclear. It has been hypothesized that platelet activation by autoantibody may be a pathogenic mechanism. We studied IgG binding, microparticle (mp) formation and P-selectin expression by flow cytometry in normal platelets after incubation in serum from 11 patients with antiphospholipid antibodies and that from 10 normal healthy subjects. Levels of platelet-associated IgG were significantly higher after incubation in patient sera (mean 17.2, range 2.0-75.0%) compared with normal sera (mean 2.0, range 1.2-3.7%, P<0.05). Incubation of normal platelets in serum led to increased microparticle formation (P<0.01) and P-selectin expression (P < 0.05), compared with unstimulated platelets. There was no significant difference, however, between microparticle formation nor P-Selectin expression induced by patient serum (mp 3.0 (1.6-5.0)%; P-selectin 8.0 (4.0-16.6)%) versus normal serum (mp 3.2 (2.1-4.5)%; P-selectin 10.1 (4.0-15.6); median (range)). Pre-activation of platelets with sub-threshold ADP concentrations or thrombin receptor activator peptide resulted in a small increase in microparticle formation, but there was still no significant difference between the effects of patient and control sera. Despite the presence of platelet membrane binding IgG in serum from 5/11 patients with antiphospholipid antibodies, there was no evidence for associated enhanced platelet-activating ability. This study supports antiplatelet reactivity in antiphospholipid syndrome, but not a direct platelet-activating role for platelet-directed autoantibodies.