The in vivo effect in humans of pyridoxal-5′-phosphate on platelet function and blood coagulation

Vitamin B₆ has an antithrombotic effect. This, based on the results of in vitro studies, has been attributed to an antiplatelet effect. We assessed the in vivo effect of vitamin B₆ by measuring the effect of long-term administration of vitamin B₆ on platelet function and blood coagulation. Vitamin B₆ (pyridoxine hydrochloride), 100mg twice daily p.o. for fifteen days, was administered to 10 healthy volunteers. The bleeding time was measured before the first dose and 15 days after. A baseline value, the acute effect, chronic effect, and the acute-on-chronic effect of vitamin B₆ was estimated by measuring platelet function. The following tests were performed: platelet aggregation induced by collagen, ADP and epinephrine; thromboxane A₂ (TxA₂)-production and prostacyclin inhibition of ADP-induced aggregation. The effects on the coagulation system were monitored by measuring: the prothrombin time, activated partial thromboplastin time and levels of coagulation factor. Vitamin B₆ significantly prolonged the bleeding time from 4.1 ± 1.1 minutes to 6.8 ± 1.0 minutes (p = 0.0063). Aggregation of platelets with collagen was slightly but not significantly inhibited. Platelet aggregation induced with the agonists ADP or epinephrine was significantly inhibited by vitamin B₆, and the platelets tended to aggregate at a slightly decreased rate. The mean TxA₂-production was slightly, but not significantly, decreased. Vitamin B₆ had no effect on the sensitivity of platelets to prostacyclin, or on the coagulation system. Our results indicate that the antithrombotic effects of vitamin B₆ is limited to inhibition of platelet function; there was no measurable influence on coagulation. The results of this in vivo study are however such that clinical trials are warranted to further assess the efficacy of vitamin B₆ as an antiplatelet drug.     

Effect of cobra venom phospholipase A2 on platelet aggregation in comparison with those produced by arachidonic acid and lysophophatidylcholine

Cobra venom phospholipase A₂ induced a biphasic effect on washed rabbit platelets. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The aggregation and thromboxane B₂ formation were inhibited by indomethacin, mepacrine, tetracaine and imipramine, while PGE₁ and sodium nitroprusside inhibited only the aggregation, but not the thromboxane B₂ formation. The second phase was an inhibitory effect on platelet aggregation induced by arachidonic acid, PAF, ADP or collagen but not that by thrombin or ionophore A23187. The longer the incubation time of cobra venom phospholipase A₂ with platelets, the more the inhibitory effect. The aggregating and anti-aggregating effects could be overcome by bovine serum albumin. Lysophosphatidylcholine (Lyso-PC) and arachidonic acid showed synergistic inhibition in platelet aggregation. Lyso-PC decreased thromboxane B₂ formation in platelets formed by collagen. The inhibitory effect of Lyso-PC on platelet aggregation was more marked at lower calcium concentrations. It is concluded that the aggregating effect of exogenous addition of venom phospholipase A₂ is due to thromboxane formation and the antiplatelet effect is similar to those produced by arachidonic acid and lysophosphatidylcholine.     

effects of picotamide on human platelet aggregation, the release reaction and thromboxane B2 production

We studied the effects of picotamide (N,N′ bis 3 picolyl-4-methoxy-isophthalamide) on human platelet aggregation, the release reaction and the production of thromboxane B₂ (TxB₂) induced by several platelet agonists. The effects of picotamide were compared to those of acetylsalicylic acid (ASA). Picotamide (0.5 mmol/1) inhibited platelet aggregation, the release of ATP and TxB₂ production induced by ADP, arachidonic acid (AA), collagen or the prostaglandin endoperoxide (PE) analogue U46619. ASA (0.5 mmol/1) did not affect platelet aggregation and the release of ATP induced by U46619. Picotamide and ASA inhibited the AA-induced platelet TxB₂ production both under stirring and non-stirring conditions, whereas the pure thromboxane A₂ receptor antagonist BM13177 (0.5 mmol/1) was inhibitory only under stirring conditions. Since under non-stirring conditions platelet aggregation does not occur, picotamide directly inhibits TxB₂ production, whereas BM13177 inhibits the potentiation of TxB₂ production due to TxA₂/PE-dependent platelet aggregation. Malondialdehyde (MDA) production by unstirred platelets stimulated with AA was not significantly inhibited by picotamide. In conclusion, picotamide inhibits the TxA2/PE-dependent platelet responses to agonists by a double mechanism: (i), TxA₂/PE antagonism; (ii) inhibition of thromboxane synthase.     

Effect of op 1206, a prostaglandin E1 derivative, on guinea-pig platelet functions

OP 1206, 17(S)-methyl-ω-homo-trans-Δ²-prostaglandin E₁, inhibited guinea-pig platelet aggregation induced by ADP, collagen, A 23187, arachidonic acid, labile aggregation stimulating substances (LASS) and thromboxane A₂ (TXA₂)-like substance, and platelet adhesiveness to a glass bead column. The potency of inhibition was 10–16 times stronger than that of prostaglandin E₁ (PGE₁). ADP-induced platelet aggregation was notably disaggregated by the addition of OP 1206 after induction of aggregation. The release of ATP and ADP from guinea-pig platelets induced by collagen was suppressed by OP 1206, of which potency was 9–10 times stronger than that of PGE₁. OP 1206 and PGE₁ increased guinea-pig platelet cyclic AMP levels, and the increased levels were augmented by the pretreatment with theophylline. OP 1206 and PGE₁ inhibited synthesis of guinea-pig platelet malondialdehyde (MDA) induced by thrombin but not by arachidonic acid. This inhibition was released by exogenous calcium. OP 1206 and PGE₁ showed no influence on synthesis of radioactive TXA₂ (measured as a stable form, TXB₂) from [¹⁴C] arachidonic acid. From these results, increased levels of platelet cyclic AMP by OP 1206 as well as PGE₁ may exert their action on platelet functions.     

The effect of submaximal exercise on platelet aggregation during late follicular and midluteal phases in women

INTRODUCTION: The key role of platelets in the pathogenesis of atherosclerosis prompted considerable interest on the effect of physical exercise on platelets. Due to probable menstrual cycle variations, only a limited number of investigations have studied the effect of exercise on platelets in women. The study was undertaken to determine the effect of acute submaximal exercise on platelet aggregation and thromboxane A(2) (TxA(2)) formation in females during their late follicular and midluteal phases. MATERIALS AND METHODS: Twelve healthy, sedentary, female volunteers performed 15 min of cycling exercise at a workload that increased their heart rate to 75% of maximal in two phases of the menstrual cycle. The maximal rate of ADP and collagen-induced platelet aggregation was evaluated on citrated whole blood using the impedance technique. Thrombin-induced thromboxane A(2) formation was evaluated by the measurement of thromboxane B(2) (TxB(2)) level by enzyme-linked immunoassay. RESULTS AND CONCLUSION: No significant difference was found between maximal rates of platelet aggregation measured in the different phases of menstrual cycle. Collagen-induced platelet aggregation and platelet count increased significantly after the exercise in both late follicular and midluteal phases (p<0.05). ADP-induced platelet aggregation did not change due to the exercise during the two phases of menstrual cycle. The thromboxane B(2) level measured in the midluteal phase was significantly higher than that measured in late follicular phase at rest. It was significantly increased after the exercise in late follicular phase while no significant difference was found between pre-exercise and postexercise levels in the midluteal phase. The differences in thromboxane A(2) formation were pointed out in the changes in platelet reactivity status. The inhibitory systems for platelets need further investigations. Our findings support the idea that menstrual variations do not have pronounced and acute effects on both platelet aggregation and response of platelets to acute exercise.     

Involvement of calmodulin in platelet reaction

Penothiazines, known as selective inhibitors of calmodulin, completely inhibited platelet aggregation and secretion induced by ADP, collagen, epinephrine, thrombin or calcium ionophore. They also completely inhibited aggregation induced by exogenous arachidonate (AA) or a mixture of thromboxane A₂ and prostaglandin endoperoxides (TxA₂/PG G₂,H₂). Also, in the presence of these calmodulin inhibitors, the release of AA from platelet phospholipids (PL) was dosedependently inhibited in stimulated platelets. These observations suggest that in platelet reaction, calmodulin is involved in at least two different steps of the reaction: activation of phospholipases and contraction of platelet actomyosin after the formation of TxA₂.     

Effects of Ethanol on Platelet Responses Associated with Adhesion to Collagen

Adhesion of platelets to collagen in damaged blood vessels or ruptured atherosclerotic plaques is important in hemostasis and arterial thrombosis. Adhesion to collagen results in secretion of granule contents and formation of thromboxane A₂; thromboxane A₂ and released ADP synergistically promote aggregation around platelets adherent to collagen. Ethanol inhibits collagen-induced platelet aggregation, secretion, arachidonate mobilization, and thromboxane A₂ formation but does not inhibit platelet adhesion to de-endothelialized rabbit aortae. We investigated whether ethanol affects the initial signalling events and responses of platelets adherent to collagen, independent of the actions of secondary agonists. Suspensions of washed human platelets, labelled by incorporation of [³H]oleate into phospholipids, were used to measure platelet adhesion to collagen by a filtration method; studies were done in the presence of an ADP-removing system and blockers of receptors for thromboxane A₂, platelet-activating factor, serotonin, and fibrinogen. Ethanol (87 mM) did not affect the rate or extent of platelet adhesion to collagen or secretion of [¹⁴C]serotonin from prelabelled platelets adherent to collagen, but ethanol did inhibit thromboxane A₂ formation. Previous studies showed that ethanol does not affect platelet stimulation by arachidonate, leading to the suggestion that reduced mobilization of arachidonate, rather than inhibition of its conversion to thromboxane A₂, is responsible for inhibition by ethanol of thromboxane A₂ formation. Here, we show by a gel mobility shift assay and immunoblotting, that ethanol delays the collagen-induced increase in the phosphorylation of cytosolic phospholipase A₂, the enzyme responsible for arachidonate mobilization. However, ethanol has no effect on collagen-induced tyrosine phosphorylation of phospholipase Cγ2, determined by immunoprecipitation and immunoblotting. Thus, ethanol's effect on signal transduction in collagen-adherent platelets occurs distal to phosphorylation of phospholipase Cγ2 but proximal to phosphorylation of cytosolic phospholipase A₂.     

Purification and characterisation of a snake venom phospholipase A2: A potent inhibitor of platelet aggregation

An inhibitor of human platelet aggregation was identified from the venom of an Australian Copperhead snake, Austrelaps superba, as a novel phospholipase A₂. The inhibitor was purified to homogeneity by chromatography on Q-Sepharose, S-Sepharose and C8 reverse phase HPLC. The purified phospholipase A₂ has a molecular weight of 15 kDa as assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal sequence analysis of the platelet inhibitor revealed 70–80% sequence identity to other previously described secretory phospholipase A₂. Phospholipase activity of the purified protein was confirmed by the ability of the enzyme to hydrolyse lecithin. Pretreatment of the purified protein with the specific phospholipase A₂ inhibitor p-bromophenacyl bromide, resulted in abrogation of both its enzyme and platelet inhibitory activity. The phospholipase A₂ inhibited platelet aggregation and serotonin release, induced by a variety of platelet agonists, in a time and dose dependent manner.     

Effects of trifluoperazine on platelet activation

Previous reports of the inhibitory effects of trifluoperazine on platelet responses to different aggregating agents have been conflicting, and the mechanism of action remains unclear. We have found that aggregation by minimum concentrations of collagen and arachidonic acid, and second phase aggregation by minimum concentrations of ADP, thrombin, epinephrine and the calcium ionophore A23187 were inhibited by 40–60μM trifluoperazine. The first phase of aggregation by a minimum concentration of epinephrine was completely inhibited by 100μM trifluoperazine, and the first phase of aggregation induced by ADP, thrombin or A23187 was decreased by 300μM trifluoperazine. The platelet shape change caused by collagen, but by no other aggregating agent examined, was inhibited by 300μM trifluoperazine. Secretion of ³H-5 hydroxytryptamine by minimum concentrations of ADP, collagen, epinephrine and arachidonic acid was completely suppressed by 50μM trifluoperazine. Secretion by thrombin and A23187 was incompletely inhibited by 300μM trifluoperazine. Thromboxane B₂ formation caused by all aggregating agents, except epinephrine, was incompletely suppressed by 50μM trifluoperazine, and 300μM trifluoperazine only caused complete inhibition of thromboxane B₂ formation by ADP, collagen and epinephrine. The phorbol ester, TPA, which mimics diacylglycerol by activating protein kinase C, caused aggregation and secretion. Aggregation, but not secretion, by low concentrations of TPA was inhibited by concentrations of trifluoperazine as low as 50μM. However, aggregation by a combination of TPA and A23187 was only inhibited by concentrations of trifluoperazine in excess of 100 μM. Secretion by TPA was inhibited by concentrations of trifluoperazine in excess of 200μM. Our findings suggest that low concentrations of trifluoperazine inhibit platelet activation by inhibiting phospholipase A₂, and that higher concentrations inhibit platelet responses by interfering with protein kinase C.     

Inhibitory effects of sulphated flavonoids isolated from Flaveria bidentis on platelet aggregation

Flaveria bidentis is a plant species that has as major constituents sulphated flavonoids in the highest degree of sulphatation. Among them, quercetin 3,7,3′,4′-tetrasulphate (QTS) and quercetin 3-acetyl-7,3′,4′-trisulphate (ATS) are the most important constituents. Both showed anticoagulant properties. The objective of the present study was to evaluate the effects of these flavonoids on human platelet aggregation in comparison with the well-known inhibitor quercetin (Qc) by using several agonists. Platelet-rich plasma (PRP) or washed human platelets (WP) were incubated with different concentrations of the flavonoids to be tested (1 to 1000 μM, final concentration), and the platelet aggregation was induced by using adenosine 5′-diphosphate (ADP), epinephrine (EP), collagen, arachidonic acid (AA) and ristocetin as agonists. QTS (500 μM) and Qc (250 μM) markedly inhibited platelet aggregation with all the aggregant agents, except ristocetin, whereas ATS (1000 μM) showed only slight antiplatelet effects. In addition, QTS and Qc antagonized the aggregation of PRP or WP induced by U-46619, a mimetic thromboxane A2 (TxA₂) receptor agonist. Challenged with collagen or arachidonic acid, the thromboxane B₂ (TxB₂) formation was also inhibited by the flavonoids, mainly by QTS and Qc, in WP. These results demonstrate that QTS and in minor extension ATS induce a deleterious effect on the production of TxA₂, as judged by TxB₂ formation, in stimulated WP and a marked interference on the TxA₂ receptor according to the profile of inhibition of the agonist-induced platelet aggregation when using ADP, EP, AA and collagen and confirmed with U-46619.     

Chloroquine inhibits stimulated platelets at the arachidonic acid pathway

Chloroquine inhibited arachidonic acid liberation from membrane phospholipids of thrombin and A23187- stimulated platelets. In addition, it dose-dependently inhibited stimulated malondialdehyde formation and thromboxane B₂ generation in the same platelets. The linear correlation between the inhibition of arachidonic acid liberation and malondialdehyde formation indicated that chloroquine inhibited activated phospholipase A₂ in thrombin-stimulated platelets, similarly as it does in different cells and tissues. Yet, the nonlinear relationship between arachidonic acid liberation along with malondialdehyde formation and thromboxane generation as well as aggregation suggest that phospholipase A₂ does not seem to be the only site of chloroquine action. Rather, it may affect platelets either at other levels of the arachidonic acid cascade too, or at some different stimulatory pathways, like intraplatelet calcium mobilisation, phosphoinositide cycle, calmodulin and protein kinase C activation.     

Effect of sodium selenite treatment on platelet aggregation of streptozotocin-induced diabetic rats

Introduction: There is a well-known association between diabetes and atherosclerosis. Platelets are involved in the development of atherosclerotic vascular diseases and play a key role in atherosclerotic complications. Diabetes mellitus is related to alteration in the homeostasis of selenium and the protective role of selenium against lipid peroxidation in diabetes is reported. In the present study, thrombin-induced platelet aggregation and thromboxane A₂ (TxA2) formation in diabetes and the effect of sodium selenite were evaluated. Materials and methods: Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) in Wistar rats (n=21). Thirty of them were used as control rats. A week after streptozotocin injection, 11 of the control rats and 12 of the diabetics were injected with 5 μmol/kg/day of sodium selenite for 4 weeks. Thrombin-induced aggregation of the platelets was evaluated by optical technique. Thromboxane B₂ (TxB2), TxA2 metabolite, was measured by enzyme-linked immunoassay (EIA) in thrombin-induced platelets. Results and conclusion: The platelet aggregation and TxB2 level increased in diabetic rats. Sodium selenite reversed the increase in platelet aggregation and TxB2 and caused a small but significant (p<0.05) decrease in the glucose level. The hyperaggregability of platelets in STZ-induced diabetic rats was thought to be related to the enhanced TxA2 formation of platelets. Increase in TxA2 formation implies lipid peroxidation. Sodium selenite decreased the TxA2 formation. Besides its antioxidative effect, further studies are needed to establish the insulin-like effect of selenite because of a small decrease in blood glucose.     

Thromboxane synthase inhibition causes re-direction of prostaglandin endoperoxides to prostaglandin D2 during collagen stimulated aggregation of human platelet rich plasma

Prostanoid synthesis and release during collagen-induced aggregation of human platelet rich plasma (PRP) was studied using a novel gas chromatography/mass spectrometry assay technique. Aggregation was associated with the production of mainly thromboxane A₂ (TXA₂), measured as TXB₂, and smaller amounts of the prostaglandins (PGs) D₂, E₂ and F₂. UK 37,248 inhibited T₁XB₂ formation by >95% and increased the production of PGD₂, PGE₂ and PGF₂ twenty-fold. The relative amounts of these three prostanoids were not changed by UK 37,248. Even though high concentrations of PGD₂ were formed, aggregation was not inhibited. In contrast, flurbiprofen inhibited aggregation, demonstrating that platelet aggregation produced by this concentration of collagen is cyclooxygenase dependent. These results support the proposal that the prostaglandin endoperoxides can induce aggregation alone, irrespective of the amount of PGD₂ that is produced.     

Effect of thromboxane synthetase inhibition on platelet function and morphology during ovine pregnancy-induced hypertension

Arterial blood pressure, serum fibrin/fibrinogen degratory products, plasma thromboxane B2, in vitro platelet aggregation, and platelet ultrastructure were studied in ten gravid ewes during fast-triggered ovine pregnancy-induced hypertension and subsequent administration of the thromboxane synthetase inhibitors CGS13080 and CGS12970. During the hypertensive period, blood pressure (p less than 0.005) and plasma thromboxane B2 levels (p less than 0.005) were significantly altered. Collagen-induced in vitro platelet aggregation lag times increased (p less than 0.01), and percent aggregation (p less than 0.05), primary (p less than 0.01), and secondary (p less than 0.005) aggregatory slopes decreased. Collagen also failed to induce aggregation in some ewes. Primary slopes of ADP-induced in vitro platelet aggregation decreased (p less than 0.01) during hypertension. Degranulation and open canalicular tubule system swelling were observed in platelets which produced abnormal or no aggregation response. However, these ultrastructural abnormalities did not necessarily correspond to hypertensive periods. Thromboxane synthetase inhibitor administration lowered blood pressure (p less than 0.005) and plasma thromboxane B2 levels (p less than 0.005). Abnormalities in collagen and ADP-induced platelet aggregation curves were also corrected, and ultrastructural abnormalities were not detected. Marked elevations in plasma thromboxane levels during ovine pregnancy-induced hypertension may have had an "exhaustive" effect on thrombocytes which was reversed by thromboxane synthetase inhibition.     

"In vitro" activity of urokinase on platelet function and on ADP degradation by vascular tissue

The effects of urokinase on ADP breakdown by vessel-wall, platelet aggregation and the related prostaglandin system "in vitro" were investigated. It is confirmed that urokinase does not induce platelet aggregation both in humans and rabbits "in vitro". Conversely, in high concentrations, urokinase inhibits ADP-induced platelet aggregation in human and rabbit platelet-rich plasma. No effects were observed on rabbit platelet thromboxane A2 release and on rat vascular prostacyclin production, both measured by radioimmunoassay of thromboxane B2 and 6-keto-F1 alpha prostaglandin, respectively. Moreover, the incubation of urokinase with vascular endothelium resulted in an increased disappearance rate.     

Low platelet apachidonic acid in young patients with brain infarction

Fatty acid patterns of plasma and platelet lipids, platelet aggregation and thromboxane A₂(TxA₂) production were studied in young patients (n = 12) with brain infarction and in healthy controls (n = 13). Platelet arachidonic acid content was significantly reduced in the stroke patients, but in vitro platelet aggregation was similar in the two groups. A low dose of acetosalicylic acid (ASA) (100 mg) suppressed thromboxane production and normalized the platelet arachidonic acid values. The low arachidonic acid in platelets is probably due to its increased consumption, indicating platelet activation in vivo.     

Effect of physiologic shear stresses and calcium on agonist-induced platelet aggregation, secretion, and thromboxane A2 formation

INTRODUCTION: Platelets in vivo react under low-shear venous-flow as well as high-shear arterial-flow conditions. Because most studies were carried out at low shear stresses, platelet granule secretion at high shear has not been examined thoroughly. We investigated the secretion of all three types of platelet granules and thromboxane A(2) formation at high shear after stimulation with ADP or thrombin. MATERIALS AND METHODS: Washed human platelets were reacted rapidly (<5 s) in a quenched-flow system simulating high-shear arterial-flow conditions (30 dyn/cm(2)). For comparison, we employed a low-shear stirring system (1-5 dyn/cm(2)). Serotonin release and membrane exposure of P-selectin (alpha), CD63 (dense), and CD107a (lysosomes) were used to assess granule secretion. Aggregation was evaluated by resistive-particle counting of the remaining platelet singlets. RESULTS AND CONCLUSIONS: ADP and thrombin induced similar strong levels of aggregation ( approximately 70%) at high shear by 5 s. Thrombin also caused release of about 40% of all alpha and dense granules within 5 s. However, by 5 s at high shear, ADP failed to induce significant granule secretion or thromboxane A(2) formation (<5%, p>0.05). By 10 min at low shear, ADP caused secretion and thromboxane A(2) formation only at non-physiological, micromolar extracellular Ca(2+) concentrations. These results emphasize the ability of thrombin to initiate multiple aspects of platelet function within seconds, while ADP was only able to induce rapid aggregation.     

Discrimination between platelet prostaglandin receptors with a specific antagonist of bisenoic prostaglandins

Platelet aggregation and secretion induced by PGG₂ and by analogues of PGH₂ and PGE₂ were inhibited by NO164. Inhibition was apparently competitive (K_i 20 μM). Responses to ADP, vasopressin and arachidonic acid were unaffected. Inhibition of ADP-induced platelet aggregation by PGD₂ and PGE₂ (but not by PGE₁) was antagonised by NO164.     

Ethanol stimulates prostacyclin biosynthesis by human neutrophils and potentiates anti-platelet aggregatory effects of prostacyclin

Previous reports on the direct effects of ethanol on human platelet aggregation function have been inconsistent. Ethanol ingestion produces vasodilation and raises intracellular cyclic AMP concentrations, effects similar to those of prostacyclin. We, therefore, hypothesized that ethanol may influence biosynthesis and/or bioactivity of prostacyclin. In our experiments, ethanol in concentrations up to 400 mg% had no consistent inhibitory effect on platelet aggregation in response to epinephrine, ADP, or combination of subthreshold concentrations of epinephrine plus ADP. However, ethanol in concentrations as low as 10 mg% potentiated the platelet aggregation inhibitory effects of prostacyclin. In addition, ethanol (20 mg%) decreased formation of thromboxane A₂ in whole blood by 41% and stimulated formation of prostacyclin by 160% (both P c 0.01). Additional studies using isolated human cells demonstrated synthesis of prostacyclin by neutrophils in the presence of platelets, and this neutrophil prostacyclin formation was enhanced in the presence of ethanol. These effects of alcohol in concentrations achieved after moderate intake may relate to the hemodynamic, biochemical, and cardioprotective effects of ethanol.     

The influence of glutathione and other thiols on human platelet aggregation

The platelet membrane contains sulfhydryl groups which are essential for normal platelet function. Reduced glutathione (GSH) and other thiols such as cysteine and 6-mercaptopurine were found to inhibit human platelet aggregation induced by adenosine diphosphate (ADP), collagen and arachidonic acid. The inhibition of ADP-induced aggregation by GSH (IC₅₀=0.61 ± 0.05 mM) was greater than that by cysteine (IC₅₀=13 ± 1 mM) or 6-mercaptopurine (IC₅₀=5.4 ± 0.2 mM). Two other thiols, dithiothreitol and beta-mercaptoethanol were found to cause platelet aggregation instead of inhibition. The interaction of GSH with the ADP receptor was noncompetitive in nature.