Human platelet electrorotation changed induced by activation: inducer specificity and correlation to serotonin release

Electrorotation of single platelets was compared with [¹⁴C]serotonin release, aggregation and electron microscopy. Activation of washed and degranulated platelets was induced by thrombin, arachidonic acid, collagen, adrenaline, platelet activation factor (PAF (PA), ADP and A23187. A strong correlation between electrorotation decrease and serotonin release was found. Electrorotation did not correlate with aggregation. It was concluded that an increase of the specific conductivity of the platelet membrane by three orders of magnitude (approx. 1.0–10⁻⁷ S · m⁻¹ to 1.0·10⁻⁴ S · m⁻¹) upon activation was responsible for the observed decrease of anti-field rotation and the shift of the first characteristic frequency towards higher values. Electrorotation allowed for time-dependent measurements of activation. Characteristic activation times in the order of minutes were found. There was the following sequence of activators classified by increasing activation time constants: A23187 was the fastest followed by thrombin, collagen, PAF, arachidonic acid, adrenaline, and ADP.     

Antiplatelet effects of chelerythrine chloride isolated from Zanthoxylum simulans

Chelerythrine chloride is an antiplatelet agent isolated from Zanthoxylum simulans. Aggregation and ATP release of washed rabbit platelets caused by ADP, arachidonic acid, PAF, collagen, ionophore A23187 and thrombin were inhibited by chelerythrine chloride. Less inhibition was observed in platelet-rich plasma. The thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, ionophore A23187 and thrombin was decreased by chelerythrine chloride. Phosphoinositides breakdown caused by collagen and PAF was completely inhibited by chelerythrine chloride, while that of thrombin was only partially suppressed. Chelerythrine chloride inhibited the intracellular calcium increase caused by arachidonic acid, PAF, collagen and thrombin in quin-2/AM-loaded platelets. The cyclic AMP level of washed platelets did not elevated by chelerythrine chloride. The antiplatelet effect of chelerythrine chloride was not dependent on the incubation time and the aggregability of platelets inhibited by chelerythrine chloride was easily recovered after sedimenting the platelets by centrifugation and then the platelet pellets were resuspended. Chelerythrine chloride did not cause any platelet lysis, since lactate dehydrogenase activity was not found in the supernatant. These data indicate that the inhibitory effect of chelerythrine chloride on rabbit platelet aggregation and release reaction is due to the inhibition on thromboxane formation and phosphoinositides breakdown.     

Characterization of the normal bovine platelet aggregation response

1. Bovine platelets are more sensitive to stimulation by platelet activating factor (PAF) than adenosine-di-phosphate (ADP) or thrombin. 2. While epinephrine, arachidonic acid and serotonin are ineffective by themselves as aggregatory stimulants of bovine platelets they enhance the aggregation response of other platelet agonists. 3. There is no correlation between thromboxane A2 production and release and the extent of platelet aggregation in bovine platelets. 4. The dependence of bovine platelet aggregation on a phospholipid pathway and calcium mobilization is indicated.     

Antiplatelet effect of butylidenephthalide

Butylidenephthalide inhibited, in a dose-dependent manner, the aggregation and release reaction of washed rabbit platelets induced by collagen and arachidonic acid. Butylidenephthalide also inhibited slightly the platelet aggregation induced by PAF and ADP, but not that by thrombin or ionophore A23187. Thromboxane B2 formation caused by collagen, arachidonic acid, thrombin and ionophore A23187 was in each case markedly inhibited by butylidenephthalide. Butylidenephthalide inhibited the aggregation of ADP-refractory platelets, thrombin-degranulated platelets, chymotrypsin-treated platelets and platelets in the presence of creatine phosphate/creatine phosphokinase. Its inhibition of collagen-induced aggregation was more marked at lower Ca2+ concentrations in the medium. The aggregability of platelets inhibited by butylidenephthalide could be recovered after the washing of platelets. In human platelet-rich plasma, butylidenephthalide and indomethacin prevented the secondary aggregation and blocked ATP release from platelets induced by epinephrine. Prostaglandin E2 formed by the incubation of guinea-pig lung homogenate with arachidonic acid could be inhibited by butylidenephthalide, indomethacin and aspirin. It is concluded that the antiplatelet effect of butylidenephthalide is mainly due to an inhibitory effect on cyclo-oxygenase and may be due partly to interference with calcium mobilization.     

Leupeptin selectively inhibits human platelet responses induced by thrombin and trypsin; a role for proteolytic activation of phospholipase C

Thrombin and trypsin induce serotonin release and aggregation in human platelets. Both proteases induce activation of phospholipase C as reflected by formation of inositol phosphates and phosphorylation of the resultant 1,2-diacylglycerol to phosphatidic acid. Also, thrombin and trypsin activate protein kinase C and myosin light chain kinase as indicated, respectively, by phosphorylation of the 40,000 and 20,000 dalton proteins. Leupeptin, a known inhibitor of serine proteases, blocks all the observed responses of human platelets to trypsin and thrombin. Leupeptin does not inhibit serotonin release and aggregation induced by other platelet stimuli such as collagen, platelet-activating factor, ionophore A23187, and arachidonic acid. The implication of a proteolytic-mediated pathway in the transmembrane signalling involved in platelet activation is discussed.     

Effects of C-reactive protein on platelet function. III. The role of cAMP, contractile elements, and prostaglandin metabolism in CRP-induced inhibition of platelet aggregation and secretion.

It was previously demonstrated that C-reactive protein (CRP) inhibits platelet aggregation and release reactions, activation of platelet factor 3, and platelet-dependent clot retraction. Multiple considerations including selective inhibition of secondary wave aggregation suggested that CRP exerted its inhibitory effects by interfering with the release of endogenous ADP. In the present investigation, CRP was found by direct assay to inhibit the release of endogenous ADP and/or serotonin concomitant with inhibition of platelet aggregation stimulated by ADP, epinephrine, thrombin, and AHGG. CRP did not induce an increase in the basal level of platelet cAMP, suggesting independence of a direct effect upon this mediator system. Furthermore, CRP did not inhibit the aggregation and secretion induced by the antibiotic ionophore A23187, suggesting the absence of a direct effect upon the activation of platelet contractile elements. By contrast, CRP did inhibit both thrombin-induced release of malondialdehyde, a prostaglandin endoperoxide nonprostanoate endproduct, and platelet aggregation induced by the prostaglandin endoperoxide precursor arachidonic acid. These data, therefore, raise the possibility that CRP inhibits platelet reactivities by interfering with an aspect of porstaglandin metabolism, and that this occurs subsequent to the hydrolytic accumulation of arachidonic acid and prior to the movement of calcium from the platelet dense tubules. These studies support the concept that CRP serves to modulate platelet reactivities during acute inflammatory reactions.     

Spiramine N-6,一种新的抗血小板和抗血小板-中性粒细胞相互作用的物质

Spiramine N-6属粉花秀线菊植物中提取分离的二十碳二萜生物碱。本实验采用Born,Shen和Hamburger等方法分别观察了spiramine N-6在体外和体内对兔血小板聚集功能的影响。应用荧光分光光度法测定其对血小板5-羟色胺释放反应的作用,同时评价spiramine N-6对激活的血小板与中性粒细胞之间粘附反应的影响。结果表明：spiramine N-6在体外选择性抑制血小板活化因子(PAF)诱导的血小板聚集,并呈量效关系,其IC50=26μmol／L,对花生四烯酸(AA)或腺苷二磷酸(ADP)引起的血小板聚集无明显作用;spiramine N-6静注后明显抑制PAF、AA和ADP诱导的血小板聚集。Spiramine N-6呈浓度依赖性减少AA和PAF引起血小板5-羟色胺的释放,其IC50分别为64．7和33．5μmol／L。Spiramine N-6明显阻抑激活的血小板与中性粒细胞间的粘附率,其IC50为78．6μmol／L。结果提示spiramine N-6作为二十碳二萜生物碱具有较强的抗血小板和阻抑血小板一中性粒细胞相互作用的生物活性。     

Membrane microenvironmental changes during activation of human blood platelets by thrombin. A study with a fluorescent probe.

Membrane microenvironmental changes associated with thrombin-induced platelet activation were followed by fluorescence intensity and polarization studies of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled human platelets. The labeling of washed platelets with DPH did not alter platelet intactness and morphology. In response to thrombin, DPH-labeled platelets exhibited reduced serotonin release, yet aggregation was barely inhibited. Shape change induced by thrombin or ADP was indistinguishable in control and in DPH-labeled platelets. During platelet aggregation induced by thrombin, fluorescence intensity increased by about 14%, which may indicate a more hydrophobic exposure of the probe. However, no change in fluorescence was detected during platelet shape change, induced either by thrombin in presence of EDTA or by ADP. Thrombin-activated platelets exhibited an increase in values of fluorescence polarization (P) during the stages of shape change and secretion, which further increased during aggregation. A similar pattern of increase in P values characterized platelet shape changes, caused either by thrombin in the presence of EDTA or by ADP. Changes in individual platelets are discernible from the alterations of the aggregating cells. These results may indicate that platelet activation is accompanied by an increase in rigidity of the membrane lipids. Functionally, the elevated "microviscosity" may reflect a primary role of membrane lipids in modulating the process of platelet activation or secondary transitions in lipids due to membrane events mediated by proteins.     

Adenosine diphosphate receptors on blood platelets: potential new targets for antiplatelet therapy

Platelets play a key role not only in physiological haemostasis, but also under pathological conditions such as thrombosis. Platelet activation may be initiated by a variety of agonists including thrombin, collagen, thromboxane or adenosine diphosphate (ADP). Although ADP is regarded as a weak agonist of blood platelets, it remains an important mediator of platelet activation evoked by other agonists, which induce massive ADP release from dense granules, where it occurs in molar concentrations. Thus, ADP action underlies a positive feedback that facilitates further platelet aggregation and leads to platelet plug formation. Additionally, ADP acts synergistically to other, even weak, agonists such as serotonin, adrenaline or chemokines. Blood platelets express two types of P2Y ADP receptors: P2Y(1) and P2Y(12). ADP-dependent platelet aggregation is initiated by the P2Y1 receptor, whereas P2Y(12) receptor augments the activating signal and promotes platelet release reaction. Stimulation of P2Y(12) is also essential for ADP-mediated complete activation of GPIIb-IIIa and GPIa-IIa, and further stabilization of platelet aggregates. The crucial role in blood platelet biology makes P2(Y12) an ideal candidate for pharmacological approaches for anti-platelet therapy.     

Ca2+ ionophore A23187 and thrombin inhibit the pertussis-toxin-induced ADP-ribosylation of the alpha-subunit of the inhibitory guanine-nucleotide-binding protein and other proteins in human platelets

Inhibitory guanine-nucleotide-binding proteins (Gi proteins) are substrates for pertussis toxin and the decreased pertussis-toxin-dependent ADP ribosylation of Gi proteins upon prior specific hormonal stimulation of cells is thought to reflect the receptor-mediated activation of Gi proteins, leading to their subsequent dissociation into alpha i and beta/gamma subunits. In the present study, the effect of various platelet stimuli on the subsequent pertussis-toxin-dependent ADP ribosylation of the alpha subunit of Gi (Gi alpha) in saponized platelets and platelet membranes were studied. Stimulation of intact platelets with the Ca(2+)-ionophore A23187 or thrombin, but not phorbol 12,13-dibutyrate, decreased the subsequent pertussis-toxin-dependent ADP ribosylation of Gi alpha in saponin-permeabilized platelets in a time-dependent and dose-dependent manner. Thrombin was more effective than A23187. Parallel measurements of Ca2+ mobilization and pertussis-toxin-dependent ADP ribosylation of Gi alpha in platelets showed that Ca2+ mobilization could only partly account for the decrease in pertussis-toxin-dependent ADP ribosylation in platelets stimulated by thrombin. When the ADP-ribosylation reaction was carried out in platelet membranes, a decrease in ADP ribosylation was still observed after stimulation of platelets with thrombin, but not with A23187. In addition to Gi alpha, two other proteins were found to be ADP ribosylated by pertussis toxin; their ADP ribosylation was also decreased after A23187 and thrombin stimulation of platelets. The results indicate that Ca2+ mobilization can decrease the pertussis-toxin-dependent ADP ribosylation of Gi alpha in saponized platelets; the decrease of pertussis-toxin-dependent ADP ribosylation of Gi alpha after thrombin stimulation of platelets can only, in part, be explained by Ca2+ mobilization and involves additional mechanisms; the decrease in pertussis-toxin-dependent ADP ribosylation after A23187 and thrombin stimulation is not confined to G1 alpha and involves other proteins. We conclude that the decrease in pertussis-toxin-dependent ADP ribosylation of Gi in thrombin-stimulated platelets might not be solely caused by a specific structural change, such as dissociation of Gi. It is likely that A23187 and thrombin stimulation of platelets generates substances which interfere with the ADP-ribosylating activity of pertussis toxin.     

Nicotinic-type unitary currents in Helix neurons

1. The platelet aggregation response to several known platelet agonists was evaluated in four Asian elephants. The platelets were highly responsive to stimulation with platelet-activating factor (PAF) and collagen, less responsive to adenosine diphosphate (ADP) and non-responsive to arachidonic acid, serotonin and epinephrine. 2. Arachidonic acid (1 x 10(-4) M), while inducing no aggregation, caused the release of 1248 +/- 1147 pg/ul (mean +/- SD) of thromboxane B2 (TXB2), the stable metabolite of thromboxane A2 from stimulated platelet. The addition of 1 x 10(-4) M ADP to platelets caused suboptimal aggregation and the release of only 25 +/- 10 pg TXB2/microliters. 3. The calcium channel blocker, verapamil, produced a dose-dependent inhibition of PAF-induced but not collagen-induced aggregation. The cyclooxygenase inhibitor, acetylsalicylic acid, produced no inhibition of either collagen- or PAF-induced aggregation.     

Electrorotation measurements of diamide-induced platelet activation changes.

Electrorotation is a special dielectric spectroscopic technique capable of measuring the polarizability of single platelets. The rotational speed of the particles is recorded as a function of the frequency of the applied rotating electric field. As previously shown, the speed of electrorotation in the range of the first characteristic frequency (anti-field rotation) decreased upon activation and was correlated with [14C]serotonin release and an increase of the TMA-DPH-induced fluorescence. Diamide upon activation and was correlated with [14C]serotonin release and an increase of the TMA-DPH-induced fluorescence. Diamide incubation induced morphological changes in control platelets. These changes were accompanied by a shift of the first characteristic frequency of electrorotation toward higher values and a parallel increase of the anti-field rotation. This was explained by a decrease of membrane conductivity and by the changed polarizability of platelet interior due to the observed internal platelet structure changes. Diamide inhibited activation assessed by both electrorotation and TMA-DPH fluorescence in the case of all activators except the ionophore A 23187. Because diamide largely inhibited the A 23187-induced serotonin release, it was concluded that, despite the diamide treatment, the direct increase of cytoplasmic Ca2+ was still able to induce membrane conductivity changes accessible by electrorotation, but this did not complete the final release step of the activation process.     

Antiplatelet effects of clausine-D isolated from Clausena excavata

Clausine-D inhibited concentration-dependently the aggregation and release of washed rabbit platelets induced by arachidonic acid and collagen, without affecting those induced by U46619, PAF and thrombin. The IC₅₀ values of clausine-D on arachidonic acid-and collagen-induced platelet aggregation were calculated to be 9.0±1.1 and 58.9±0.9 μM, respectively. Thromboxane B₂ and prostaglandin D₂ formation in platelets caused by arachidonic acid were also suppressed. Clausine-D inhibited increased intracellular concentration of calcium in platelets caused by arachidonic acid and collagen, and also abolished the generation of inositol monophosphate caused by arachidonic acid, but not that by collagen U46619, PAF and thrombin. In human citrated platelet-rich plasma, clausine-D inhibited the secondary phase, but not the primary phase, of aggregation induced by epinephrine and ADP. These results indicate that the antiplatelet effect of clausine-D is due to inhibition of the formation of thromboxane A₂.     

Action mechanism of the platelet aggregation inducer and inhibitor from Echis carinatus snake venom

Platelet aggregation inducer and inhibitor were isolated from Echis carinatus snake venom. The venom inducer caused aggregation of washed rabbit platelets which could be inhibited completely by heparin or hirudin. The venom inducer also inhibit both the reversibility of platelet aggregation induced by ADP and the disaggregating effect of prostaglandin E₁ on the aggregation induced by collagen in the presence of heparin. The venom inhibitor decreased the platelet aggregation induced by collagen, thrombin, ionophore A23187, arachidonate, ADP and platelet-activating factor (PAF) with an IC₅₀ of around 10 μg/ml. It did not inhibit the agglutination of formaldehyde-treated platelets induced by polylysine. In the presence of indomethacin or in ADP-refractory platelets or thrombin-degranulated platelets, the venom inhibitor further inhibited the collagen-induced aggregation. Fibrinogen antagonized competitively the inhibitory action of the venom inhibitor in collagen-induced aggregation. In chymotrypsin-treated platelets, the venom inhibitor abolished the aggregation induced by fibrinogen. It was concluded that the venom inducer caused platelet aggregation indirectly by the conversion of prothrombin to thrombin, while the venom inhibitor inhibited platelet aggregation by interfering with the interaction between fibrinogen and platelets.     

The inflammatory and tumor-promoting sesquiterpene lactone, thapsigargin, activates platelets by selective mobilization of calcium as shown by protein phosphorylations

We have studied the activation of human blood platelets by the inflammatory and tumor-promoting sesquiterpene lactone, thapsigargin. The effect of thapsigargin was compared with other common agonists (calcium ionophore A23187, phorbol ester TPA and thrombin). Platelet aggregation, serotonin release, raised cytoplasmic free calcium level and phosphorylation of platelet proteins was examined in platelet-rich plasma and washed platelet suspension. In contrast to A23187 and thrombin, the platelet activation induced by thapsigargin developed slowly, with maximal response obtained after 2-3 min. Both the thapsigargin- and the A23187-induced serotonin releases were synergistically increased by TPA. Studies of the phosphorylation of platelet proteins revealed that thapsigargin and A23187 equally well induced a selective phosphorylation of two proteins with apparent molecular masses of 20 kDa and 47 kDa. These proteins, which are substrates of myosin light-chain kinase and protein kinase C respectively, are known to be involved in platelet activation. The thapsigargin-induced platelet aggregation and serotonin release was completely inhibited by class I (nimodipine), class II (verapamil) and class III (diltiazem) calcium-channel blockers. The inhibitory activity of nimodipine was abolished by the corresponding 1,4-dihydropyridine calcium-channel agonist, BAY K 8644. These results shows that the thapsigargin-induced platelet activation is mediated by an increase in the cytoplasmic free calcium level, presumably obtained by stimulation of the passive calcium transport through specific channels. These thapsigargin-sensitive channels should predominantly be located in the membranes of intracellular calcium stores rather than in the plasma membrane, because removal of extracellular calcium by EGTA had only an insignificant effect on the thapsigargin-induced rise in cytoplasmic free calcium level.     

Effects of acrolein on human platelet aggregation

Acrolein, a component of tobacco smoke, potentiated platelet aggregation and increased thromboxane A2 (TXA2) formation caused by thrombin and arachidonic acid (AA). Acrolein produced these effects at concentrations in the range 50-5000 microM. Acrolein had no effect on platelet responses to ADP, epinephrine, collagen or the ionophore A23187. Acrolein increased the mobilization of [3H]arachidonic acid from prelabelled platelets in response to thrombin and arachidonic acid. The increased availability of substrate could partly explain the enhanced production of TXA2 and increased aggregation observed in the presence of acrolein. These findings could provide an explanation for the increased incidence of vascular disease in cigarette smokers.     

Nitridergic platelet pathway activation by hementerin, a metalloprotease from the leech Haementeria depressa

Hementerin (HT) is an 80 kDa fibrino(geno)lytic metalloprotease, purified from saliva of the leech Haementeria depressa. In the present report, the effect of HT on several functional parameters of human platelets was assessed. HT inhibited platelet aggregation and ATP release induced by different agonists such as ADP, adrenaline, collagen, thrombin, and arachidonic acid. HT did neither modify the expression of platelet glycoproteins (Ib, IIb-IIIa, Ia-IIa, IV) nor intraplatelet fibrinogen levels, whereas it markedly decreased CD62P and CD63 levels after the stimulation with thrombin. HT significantly increased thrombin-induced platelet Ca2+ intracellular levels, cGMP content and nitric oxide synthase (NOS) activity. The effect of HT on platelet aggregation was reversed by two NOS inhibitors, N(omega)-Nitro-L-arginine methyl ester and 2 N(G)-Nitro-L-arginine. In summary, these results indicate that HT is an effective inhibitor of human platelet aggregation, presumably through activation of the platelet's nitridergic pathway.     

The role of phospholipase C in platelet responses

Degradation of inositides induced by phospholipase C in activated platelets leads to the formation of 1,2-diacylglycerol (1,2-DG) and its phosphorylated product, phosphatidic acid (PA). We have studied the relationship between activation of phospholipase C and the appearance of specific platelet responses, such as phosphorylation of proteins, shape change, release reaction and aggregation induced by different stimuli such as thrombin, platelet-activating factor, collagen, arachidonic acid (AA) and dihomogamma linolenic acid. A low degree of platelet activation induces only shape change which is associated with partial activation of phospholipase C (formation of phosphatidic acid), and phosphorylation of both a 40K molecular weight protein (protein kinase C activation) and a 20K molecular weight protein (myosin light chain). A higher degree of platelet activation induces aggregation, release of serotonin and a higher level of phospholipase C and protein kinase C activities. Metabolism of AA occurs concomitantly to aggregation and serotonin release, but AA metabolites are not related to the shape change of human platelets. Platelet shape change and the initial activation of phospholipase C induced by thrombin or platelet-activating factor is independent of the metabolites derived from cyclo-oxygenase activity. Further activation of phospholipase C which occurs during platelet aggregation and release reaction is, however, partly dependent on cyclo-oxygenase metabolites.     

Secretion, subcellular localization and metabolic status of inorganic pyrophosphate in human platelets. A major constituent of the amine-storing granules.

The platelet content of PPi is 1.90 +/- mumol/10(11) platelets (S.E.M., n = 19) or about 10.5 nmol/mg of protein, several hundred times that found for rat liver. Some 80% of this PPi is secreted by platelets treated with thrombin with a time course and dose-response relationship similar to secretion of ATP, ADP and 5-hydroxytryptamine (serotonin) from the platelet dense granules. During platelet aggregation induced by ADP and adrenaline, substantial amounts of PPi were secreted, but no release of acid hydrolases was observed. Subcellular-fractionation studies showed that the PPi is highly enriched in the same fraction that contains the storage organelles which store ATP, ADP, Ca2+ and 5-hydroxytryptamine. Inorganic pyrophosphatase was present mainly in the soluble fraction and in the mitochondria. Secretion studies done with platelets prelabelled with [32P]Pi showed that the sequestered PPi was relatively metabolically inactive, as is the ATP and ADP in the storage organelles. The possible participation of PPi in the formation of a bivalent-cation-nucleotide complex associated with amine storage is discussed.     

Potential involvement of vicinal sulfhydryls in stimulus-induced rabbit platelet activation

The potential involvement of vicinal dithiols in the expression of platelet-activating factor (AGEPC)- and A23187-induced alterations in rabbit platelets was explored through the use of phenylarsine oxide (PhAsO) and certain analogous derivatives. PhAsO (As3+) but not phenylarsonic acid (As5+) inhibited markedly at 1 microM concentration the release of arachidonic acid initiated by AGEPC and the ionophore A23187. In contrast, AGEPC-induced phosphatidic acid formation, phosphorylation of 40- and 20-kDa proteins, and Ca2+ uptake from external medium were not inhibited substantially by 1 microM PhAsO. However, these latter metabolic responses to AGEPC were inhibited by PhAsO at higher doses (10 microM). AGEPC- and thrombin-induced platelet aggregation and serotonin secretion also were prevented by PhAsO. The IC50 value of PhAsO was 2.7 +/- 1.2 microM toward AGEPC (5 X 10(-10) M)-induced serotonin release. Further, ATP and cAMP levels in PhAsO-treated platelets were not changed from controls. Interestingly, addition of Ca2+ to platelet sonicates (prepared in EDTA) caused diacylglycerol production and free arachidonic acid formation, even in the presence of 133 microM PhAsO. This would suggest that in the intact platelets PhAsO acted indirectly on phospholipase A2 and/or phospholipase C activities. Finally, a dithiol compound, 2,3-dimercaptopropanol, reversed the inhibition of platelet aggregation and arachidonic acid release effected by PhAsO. On the other hand, a monothiol compound, 2-mercaptoethanol, was not effective in preventing or in reversing the action of PhAsO. These observations suggest that vicinal sulfhydryl residues may be involved in stimulus-induced platelet activation.