Mechanisms of the mAb ALB6(CD9) induced human platelet activation: comparison with thrombin

A CD9 monoclonal antibody described to aggregate human platelets was studied on different platelet functions in order to determine its mechanism of action. After a lag phase of 35 sec the mAb ALB6 induced a transient decrease in 32P-polyphosphoinositides, synthesis of 32P-phosphatidate (PA), phosphorylation of myosin light chain (P20) and of 43 KDa protein (P43) and the release reaction. Final biological and metabolic effects of ALB6 thus appear similar to that of thrombin but three differences bring additional information: (i) the lag phase, (ii) the kinetic of ALB6-induced release is identical for all granules whereas the release of dense granules is faster when induced by thrombin. (iii) no external Ca++ is required for ALB6 induced-activation.     

Are polyphosphoinositides involved in platelet activation?

In human platelets, the amounts of triphosphoinositides (TPI) and diphosphoinositides (DPI) increase after 30 sec and level off after 120 sec of thrombin stimulation. After 180 sec of thrombin challenge, TPI and DPI increase accounts for 66 and 80%, respectively. Polyphosphoinositide changes roughly parallel the release of N-acetyl-beta-D-glucosaminidase and appear as a later event compared to aggregation and serotonin secretion. It is concluded that an increased phosphorylation of polyphosphoinositides might participate in platelets to the process of stimulus-activation coupling and might be linked to thrombin receptor occupancy. A role of DPI in platelet activation is suggested by the observation that DPI promote platelet aggregation, the mechanism of which is discussed.     

Ethanol stimulates shape change in human platelets by activation of phosphoinositide-specific phospholipase C

Administration of ethanol to human platelets resulted in a rapid shape change which was maximal within 30 s. Ethanol did not cause aggregation or secretion of ATP at any time and inhibited aggregation induced by collagen. In platelets that were loaded with the intracellular calcium indicator fura2, ethanol induced a rapid mobilization of calcium from internal, thrombin-sensitive pools. Cytosolic calcium increased to a maximum within 5 s and decreased slowly over the ensuing 5 min to near basal levels. The mobilization of calcium by ethanol coincided with the rapid formation of phosphatidic acid and a decrease in the level of phosphatidylinositol 4,5-bisphosphate, as measured in 32P-labeled platelets. In platelets labeled with myo-[2-3H]inositol, ethanol caused a 20-30% increase in the levels of inositol (1,4,5)-trisphosphate and inositol bisphosphate within 10 s. Ethanol also induced the transient phosphorylation of myosin light chain (20 kDa) and a 40 kDa protein, a known substrate for protein kinase C. The results indicate that ethanol activates phosphoinositide-specific phospholipase C in human platelets. The subsequent mobilization of intracellular calcium and activation of protein kinase C can account for the shape change induced by ethanol.     

Thrombin-mediated feedback activation of factor XI on the activated platelet surface is preferred over contact activation by factor XIIa or factor XIa

To study the pathways for initiation of intrinsic blood coagulation, activated human platelets were compared with dextran sulfate as surfaces for factor XI activation by factor XIIa, factor XIa, or thrombin. Activated gel-filtered platelets promoted the activation of factor XI (60 nm) by thrombin (0.02-10 nm, EC(50) approximately 100 pm, threshold concentration approximately 10 pm) at initial rates 2- to 3-fold greater than those obtained with dextran sulfate in the presence of either high molecular weight kininogen (45 nm) and ZnCl(2) (25 micrometer) or prothrombin (1.2 micrometer) and CaCl(2) (2 mm). The maximum rates of factor XI activation achieved in the presence of activated gel-filtered platelets were 30 nm.min(-1) with thrombin, 6 nm.min(-1) with factor XIIa and 2 nm.min(-1) with factor XIa. Values of turnover number calculated at various enzyme concentrations (0.05-1 nm) were 24-167 (mean = 86) min(-1) for thrombin, 4.6-50 (mean = 21) min(-1) for factor XIIa, and 1.3-14 (mean = 8) min(-1) for factor XIa. A physiological concentration of fibrinogen (9.0 micrometer) inhibited factor XI activation by thrombin (but not by factor XIIa) in the presence of dextran sulfate but not in the presence of gel-filtered platelets. Compared with factors XIIa and XIa, thrombin is the preferred factor XI activator, and activated platelets are a relevant physiological surface for thrombin-mediated initiation of intrinsic coagulation in vivo.     

Agents that elevate cAMP levels in platelets decrease thrombin binding

The effect of high intracellular levels of cAMP on the ability of rabbit and human platelets to bind and respond to thrombin was examined. Control rabbit platelets differed from human platelets in two interesting respects: they showed thrombin-dependent up-regulation of thrombin binding, but also a 3- to 5-fold lower thrombin-binding capacity. Nevertheless, treatment with prostaglandin E1 + theophylline or with forskolin decreased thrombin binding to both rabbit and human platelets by 60 to 70%. This effect was associated with a marked increase in the level of cAMP and seemed to depend on a decrease in number rather than affinity of thrombin-binding sites. Changes in thrombin binding correlated closely with changes in thrombin-stimulated incorporation of 32Pi into phosphatidic acid and a 40-kDa protein. However, regardless of the amount of thrombin that bound to treated platelets, thrombin-stimulated phosphorylation of a 20-kDa protein and serotonin secretion were severely inhibited. Thus, increased levels of platelet cAMP are associated with a reduced ability to bind and respond to thrombin. However, thrombin binding to platelets correlates more closely with some responses than others, presumably because cAMP inhibits additional platelet reactions.     

Adenine nucleotide metabolism of blood platelets. IX. Time course of secretion and changes in energy metabolism in thrombin-treated platelets.

Changes in the energy metabolism of washed human platelets were compared with the kinetics of secretion induced by thrombin (5 units/ml). A 50% decrease in the level of metabolic ATP (3H-labelled), which was essentially complete in 30s, was matched in rate by adenine nucleotide secretion from storage in dense granules. Incubation of platelets with antimycin before thrombin addition increased the rate of fall in metabolic ATP, but did not affect the rate of adenine nucleotide secretion. beta-N-Acetylglucosaminidase secretion, which was slower than adenine nucleotide secretion in control platelets, was noticeably inhibited by antimycin, confirming previous reports that different regulatory mechanisms exist for dense and alpha-granule secretion. The rates of rephosphorylation of metabolic ADP to ATP via glycolysis and oxidative phosphorylation were estimated by measuring lactate production and O2 consumption in resting and thrombin-stimulated platelets and compared to the level of metabolic ATP (9-10 nmol/mg of platelet protein in the resting state). The rate of ATP production was stimulated at least two fold from 12 nmol to 24 nmol/min/mg within seconds of thrombin addition. This increased rate was maintained over the observed period of 5 min although the level of metabolic ATP had decreased to 4-5 nmol/mg within 30 s; the turnover of the remaining metabolic ATP thus increased four fold over the resting state although the actual stimulation of energy production was only two fold.     

Stimulation of human platelets with low concentrations of thrombin: evidence for equimolar accumulation of inositol trisphosphates and phosphatidic acid

1. Gel-filtered human platelets prelabeled with [32P]Pi or [3H]glycerol were exposed to 0-0.3 U/ml of thrombin and analyzed for radioactivities and masses in the phosphoinositides, inositol trisphosphates (IP3), phosphatidic acid (PA) and diacylglycerol (DAG) at 15 and 180 sec of stimulation. 2. At thrombin concentrations below 0.1 U/ml, PA and IP3 accumulated in equimolar amounts. 3. The production and disappearance of the metabolites of the polyphosphoinositide cycle was balanced during 180 sec of stimulation with 0.03-0.1 U/ml of thrombin. 4. Under these conditions no increase in [3H]DAG or [3H]monoacylglycerol could be detected. 5. The data indicate that all DAG is converted to PA and support our conclusion that phosphatidylinositol 4,5-bisphosphate represents the major source for production of DAG upon stimulation of human platelets with low concentrations of thrombin.     

Factors affecting dense and alpha-granule secretion from electropermeabilized human platelets: Ca(2+)-independent actions of phorbol ester and GTP gamma S.

Electropermeabilized human platelets containing 5-hydroxy[14C]tryptamine ([14C]5-HT) were suspended in a glutamate medium containing ATP and incubated for 10 min with (in various combinations) Ca2+ buffers, phorbol 12-myristate 13-acetate (PMA), guanine nucleotides, and thrombin. Release of [14C]5-HT and beta-thromboglobulin (beta TG) were used to measure secretion from dense and alpha-granules, respectively. Ca2+ alone induced secretion from both granule types; half-maximal effects were seen at a -log [Ca2+ free] (pCa) of 5.5 and maximal secretion at a pCa of 4.5, when approximately 80% of 5-HT and approximately 50% of beta TG were released. Addition of PMA, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), GTP, or thrombin shifted the Ca2+ dose-response curves for secretion of both 5-HT and beta TG to the left and caused small increases in the maximum secretion observed. These results suggested that secretion from alpha-granules, like that from dense granules, is a Ca(2+)-dependent process stimulated by the sequential activation of a G-protein, phospholipase C, and protein kinase C (PKC). However, high concentrations of PMA and GTP gamma S had distinct effects in the absence of Ca2+ (pCa greater than 9); 100 nM PMA released approximately 20% of platelet 5-HT but little beta TG, whereas 100 microM GTP gamma S stimulated secretion of approximately 25% of each. Simultaneous addition of PMA greatly enhanced these effects of GTP gamma S. Phosphorylation of pleckstrin in permeabilized platelets incubated with [gamma-32P]ATP was used as an index of the activation of PKC during secretion. In the absence of Ca2+, 100 nM PMA caused maximal phosphorylation of pleckstrin and 100 microM GTP gamma S was approximately 50% as effective as PMA; neither GTP gamma S nor Ca2+ enhanced the phosphorylation of pleckstrin caused by 100 nM PMA. These results indicate that, although activation of PKC promoted secretion, GTP gamma S exerted additional stimulatory effects on secretion from both dense and alpha-granules that were not mediated by PKC. Measurement of [3H]inositol phosphate formation in permeabilized platelets containing [3H]phosphoinositides showed that GTP gamma S did not stimulate phosphoinositide-specific phospholipase C in the absence of Ca2+. It follows that in permeabilized platelets, GTP gamma S can both stimulate PKC and enhance secretion via G-protein-linked effectors other than this phospholipase.     

Cytosolic alkalinization alone is not sufficient for Ca2+ mobilization, phosphatidic acid formation, and protein phosphorylation in human platelets

One of the earliest events following stimulation of human platelets with thrombin is a rise in the cytosolic pH, pHi, mediated by Na+/H+ exchange, and an increase in the cytosolic free Ca2+ concentration, [Ca2+]i. In the present study we investigated whether an increase in pHi alone, induced by the Na+/H+ ionophore monensin, is sufficient for platelet activation. Although monensin (20 microM) raised pHi from 7.10 +/- 0.05 (n = 21) to 7.72 +/- 0.17 (n = 13), neither Ca2+ influx nor mobilization were detectable upon this treatment in fura2-loaded platelets. In contrast, thrombin (0.05 U/ml) raised pHi to 7.31 +/- 0.10 (n = 10) and increased [Ca2+]i by more than 250 nM both in the presence and absence of extracellular Ca2+. Thrombin also caused the formation of phosphatidic acid and phosphorylation of the 20 kDa and 47 kDa proteins in platelets labeled with 32P. Monensin, however, induced none of these responses. It is concluded that an increase in pHi alone is not a sufficient trigger for platelet activation but enhances intracellular signal transduction in platelets stimulated by natural agonists.     

Meizothrombin formation during factor Xa-catalyzed prothrombin activation. Formation in a purified system and in plasma

Meizothrombin and thrombin formation were quantitated during factor Xa-catalyzed activation of human prothrombin in reaction systems containing purified proteins and in plasma. In the purified system considerable amounts of meizothrombin accumulated when prothrombin was activated by factor Xa (with or without accessory components) under initial steady state conditions. The ratio of the rates of meizothrombin and thrombin formation was not influenced by variation of the pH, temperature, or ionic strength of the reaction medium. When 2 microM prothrombin was activated by the complete prothrombinase complex (factor Xa, factor Va, Ca2+, and phospholipid) 80-90% of the initially formed reaction product was meizothrombin. Lowering the prothrombin concentration from 2 to 0.03 microM caused a gradual decrease in the ratio of meizothrombin/thrombin formation from 5 to 0.6. When the phosphatidylserine content of the phospholipid vesicles was varied between 20 and 1 mol % and prothrombin activation was analyzed at 2 microM prothrombin the relative amount of meizothrombin formed decreased from 85 to 55%. With platelets, cephalin, or thromboplastin as procoagulant lipid, thrombin was the major reaction product and only 30-40% of the activation product was meizothrombin. We also analyzed complete time courses of prothrombin activation both with purified proteins and in plasma. In reaction systems with purified proteins substantial amounts of meizothrombin accumulated under a wide variety of experimental conditions. However, little or no meizothrombin was detected in plasma in which coagulation was initiated via the extrinsic pathway with thromboplastin or via the intrinsic pathway with kaolin plus phospholipid (cephalin, platelets, or phosphatidylserine-containing vesicles). Thus, thrombin was the only active prothrombin activation product that accumulated during ex vivo coagulation experiments in plasma.     

Phosphoinositide breakdown as an indirect link between stimulation and aggregation of rat platelets by thrombin and collagen

When washed rat platelets (1.5 x 10(9)/ml) were stimulated by a threshold concentration of thrombin (0.3 unit/ml) or collagen (10 micrograms/ml), a lag period of about 10 or 30 s, respectively, was seen before the start of aggregation. During the lag period, [32P]phosphatidylinositol 4,5-bisphosphate was degraded as the earliest event within 5-10 s of addition of the stimulus. However, though the extent of phosphatidylinositol 4,5-bisphosphate degradation within 10 s of addition of collagen was greater than that within 20 s of addition of thrombin (0.3 unit/ml), a lag of about 20 s remained before the initiation of aggregation by collagen. This casts doubt on the hypothesis that the stimulus-dependent phosphatidylinositol 4,5-bisphosphate breakdown induces the aggregation of platelets. Phosphatidylinositol labeled with 32Pi or [1-14C]arachidonic acid was scarcely degraded during the lag period. As aggregation proceeded, [14C-arachidonic acid]phosphatidylinositol was degraded with generation of diacylglycerol, phosphatidic acid, arachidonic acid and its metabolites. The maximum aggregation by collagen of rat platelets in which arachidonic acid of phospholipids was replaced in vivo with eicosapentaenoic acid was reduced, but that by thrombin was not, though reduction of thromboxane A2 generation was caused by both stimuli. Indomethacin also fully inhibited the aggregation induced by collagen, but not that induced by thrombin. Hence, thromboxane A2 is required for full aggregation by collagen, but not that by thrombin. These results indicate that thrombin-induced phosphoinositide metabolism may proceed independently of aggregation.     

Chemotactic factor causes rapid decreases in phosphatidylinositol,4,5-bisphosphate and phosphatidylinositol 4-monophosphate in rabbit neutrophils

Stimulation of rabbit neutrophils prelabeled with 32P by the synthetic chemotactic peptide f-Met-Leu-Phe induces a rapid decrease in the radioactivity in both phosphatidylinositol, 4,5 bis phosphate and phosphatidylinositol 4-monophosphate. The mean +/- standard error of the mean values of the maximum decrease in phosphatidylinositol, 4,5 bis phosphate occurred at 10 seconds following stimulation and is equal to 19 +/- 3% of the control value. The corresponding value for phosphatidylinositol 4-monophosphate occurred at 60 seconds following stimulation and is equal to 37 +/- 7% of the control value. On the other hand, the radioactivity in phosphatidic acid and lysophospholipids increased continuously with time following stimulation. The relationship of these changes to calcium release and neutrophil activation is discussed.     

Signal transduction in normal and pathological thrombin-stimulated human platelets

Human blood platelets stimulated by thrombin undergo very rapid morphological changes, the most characteristic of which are pseudopod formation and granule centralization. These early changes in shape are accompanied by a transient decrease (30%) in phosphatidyl inositol 4,5-bisphosphate (PIP2) which occurs in the first 10 s after thrombin addition. Transient decreases in phosphatidyl inositol 4-phosphate (PIP) and phosphatidyl inositol (PI) occur later (20-30 s). These events lead to the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) and hence phosphatidate (PA). Two polypeptides are phosphorylated during the same time span: the myosin light chain (P20) and a 43 kDa protein (P43). Concomitant with these molecular changes, platelet 'release reaction' occurs, i.e., liberation of the different granule constituents into the external medium: the earliest concerns dense bodies which liberate adenine nucleotides, calcium and serotonin; alpha-granules then liberate adhesive and specific proteins and are followed by lysosomes which liberate hydrolases. Pathological platelets from patients with inherited disorders, presenting well-characterized and specific defects of either the platelet membrane (GT) or storage granules (GPS and HPS), have also been studied. The results obtained lead to the following conclusions: (1) the transducing system is normal in platelets unable to aggregate; (2) phosphorylation of P20 and P43 proteins can be complete with impaired release; and (3) when platelets lack alpha-granules the transducing system as well as the release of other granule populations are impaired. These results evidence the relationship between the absence of intraplatelet components and metabolic events.     

Reversible thrombin-induced modification of positional distribution of fatty acids in platelet phospholipids: Involvement of deacylation-reacylation

The positional distribution of fatty acids in phospholipids was analyzed for human platelet activated by thrombin. At 30 sec after thrombin-activation, when the content of phosphatidylinositol(PI) fell transiently, the 2-position of PI underwent a significant decrease in arachidonate (77.6% → 61.5%) with a compensating increase in oleate and stearate. However, accompanying PI resynthesis, its positional distribution tended to revert to that of non-activated platelets. There was no significant change in the fatty acyl positioning in other phospholipids. In addition, evidence is presented that human platelet lysates acylate 1-acyl-sn-glycero-3-phosphorylinositol and the acylation rate for arachidonate was 2.5 times higher than for any other unsaturated fatty acids tested. These data indicate that the thrombin-induced alteration in fatty acid pairing of PI is ameliorated by the deacylation-reacylation follwing PI resynthesis.     

Copper deficiency alters protein kinase C mediation of thrombin-induced dense granule secretion from rat platelets

Experiments were conducted to determine if copper deficiency enhances the rate of thrombin-induced dense granule secretion by modifying the major signal transduction pathways of rat platelets. Platelets were obtained from male, weanling Sprague-Dawley rats fed diets containing either deficient ( < 0.5 μg/g diet) or adequate (5.5 μg/g diet) copper for 5 weeks. Following stimulation with thrombin (0.1 U/mL), the rate of dense granule secretion as measured by ATP release was 160% higher in platelets from copper-deficient than from control rats. Inhibition of the rate of thrombin-induced ATP release by (6-aminohexyl)-1-naphthalene-sulfonamide, a calmodulin antagonist was independent of copper status. However, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, a protein kinase C inhibitor, inhibited the rate of ATP release only in platelets from copper-deficient rats. Aspirin had no effect on ATP release from platelets obtained from either copper-deficient or control rats. This suggests that copper deficiency alters the role of protein kinase C in regulating dense granule secretion. Analysis of autoradiographs showing [³²P]-labeled platelet proteins indicated that the phosphorylation of a 40 kDa protein, a known substrate for protein kinase C in platelets, was significantly less following thrombin stimulation in platelets from copper-deficient than from control rats. When protein kinase C was activated by phorbol 12-myristate 13-acetate prior to thrombin stimulation, ATP release was attenuated regardless of copper status. These findings suggest that protein kinase C can still function as a feedback inhibitor of platelet dense granule secretion in copper deficiency, but impaired activation of this enzyme following thrombin stimulation may prevent it from achieving full regulatory capacity.     

Potentiation of platelet aggregation by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) has binding sites on a variety of tissues, including human platelets. We have used a new, quenched-flow approach coupled to single-particle counting to investigate the effects of ANP (rat, 1-28) on the initial events (within the first several seconds) following human platelet activation. While ANP alone (1 pM-100 nM) had no effect, ANP significantly potentiated thrombin (0.4 units/ml)-, epinephrine (15 microM)- and ADP (2 or 10 microM)-induced aggregation. Maximum stimulation occurred between 10 to 100 pM. ANP had no influence on the thrombin or ADP-induced increase in platelet volume associated with the "shape change." Since ANP receptors are coupled to a particulate guanylate cyclase and some ANP-induced effects may be mediated through cyclic GMP, we studied how another activator of platelet guanylate cyclase, sodium nitroprusside, affected platelet activation and cyclic nucleotide levels. Sodium nitroprusside (1 microM) inhibited ADP, but not thrombin or epinephrine-induced aggregation. Both sodium nitroprusside (1 microM) and ANP (10 nM) increased cyclic GMP levels by 80% and 37%, respectively, within 60 sec in washed platelets. ANP had no effect on platelet cyclic AMP, while sodium nitroprusside induced a 77% increase. These data suggest that the platelet ANP receptor may be coupled to guanylate cyclase and the rise in cyclic GMP may potentiate platelet function.     

Human platelets stimulated by thrombin produce platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) when the degrading enzyme acetyl hydrolase is blocked.

It has been shown [Touqui, Jacquemin & Vargaftig (1983) Thromb. Haemostasis 50, 163; Touqui, Jacquemin & Vargaftig (1983) Biochem. Biophys. Res. Commun. 110, 890-893; Alam, Smith & Melvin (1983) Lipids 18, 534-538; Pieroni & Hanahan (1983) Arch. Biochem. Biophys. 224, 485-493] that rabbit platelets inactivate exogenous PAF (platelet-activating factor, PAF-acether) by a deacetylation-reacylation mechanism. The deacetylation step is catalysed by an acetyl hydrolase sensitive to the serine-hydrolase inhibitor PMSF (phenylmethanesulphonyl fluoride) [Touqui, Jacquemin, Dumarey & Vargaftig (1985) Biochim. Biophys. Acta 833, 111-118]. We report here that human platelets can produce PAF on thrombin stimulation. This production is marginal and transient, reaching a maximum at 10 min and decreasing thereafter. In contrast, 10-12 times more PAF is produced when platelets are treated with PMSF and stimulated with thrombin. Under these conditions, the maximum formation is observed at 30 min and no decline occurs for up to 60 min after stimulation. In addition, these platelets (treated with PMSF and stimulated with thrombin) incorporate exogenous labelled acetate in the 2-position of PAF, probably by an acetyltransferase-dependent mechanism. Production of PAF by human platelets during physiological stimulation can be demonstrated when PAF degradation is suppressed by the acetyl-hydrolase inhibitor PMSF.     

Thrombin and collagen induce rapid phosphorylation of a common set of cellular proteins on tyrosine in human platelets

The ability of thrombin and collagen to induce protein-tyrosine phosphorylation in intact human platelets was assessed by using antibodies to phosphotyrosine in conjugation with immunoblots. Upon stimulation by thrombin there was an increase in the amount of protein-tyrosine phosphorylation of three bands with molecular masses of 135, 124, and 76 kDa in a time-dependent manner. The tyrosine phosphorylation in these three proteins increased in a concurrent fashion and reached a maximum level in 10 s and then a plateau or a slight decrease. Stimulation by collagen was also followed by an increase in tyrosine phosphorylation of 135- and 124-kDa proteins. Unlike stimulation by thrombin, collagen induced no obvious tyrosine phosphorylation of 76-kDa protein. The time courses for thrombin- or collagen-induced protein-tyrosine phosphorylation were similar to that for [14C] serotonin release. These results suggest that 135- and 124-kDa proteins are a common set of proteins that become phosphorylated on tyrosine residue during platelet activation.     

The rapid polyphosphoinositide metabolism may be a triggering event for thrombin-mediated stimulation of human platelets

The metabolism of polyphosphoinositides was examined in human platelets activated by thrombin. The addition of thrombin to [3H]glycerol-labeled platelets induced an initial loss and a subsequent increase of the radioactivity in phosphatidylinositol-4,5-bisphosphate (TPI) without any significant change in phosphatidylinositol-4-phosphate (DPI). A marked enhancement of [32P]Pi incorporation into TPI occurred in parallel with an increase in this lipid content, which was accompanied with a conccurent decrease in phosphatidylinositol (PI). The rate of this subsequent increase in TPI was smaller than that observed in [3H]arachidonic acid-labeled platelets, suggesting that formed TPI in activated platelets may contain much greater amount of arachidonate than preexisting TPI in resting platelets. These data indicate that thrombin causes a rapid change in TPI metabolism (initial degradation of preexisting TPI and subsequent production of arachidonate-rich TPI), which might be a primary candidate to modulate thrombin-induced function in human platelets.