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.     

N-ethylmaleimide inhibits Ca2+ influx induced by collagen or arachidonate on rabbit platelets

N-Ethylmaleimide dose dependently inhibited platelet aggregation induced by collagen or arachidonate but did not inhibit the aggregation by thrombin or ionophore A23187 within the concentrations tested. [3H]Arachidonate release from membrane phospholipids of the collagen-stimulated platelets was inhibited by N-ethylmaleimide in parallel with the inhibition of aggregation, but not in response to A23187. N-Ethylmaleimide prevented 45Ca2+ influx into platelet cells from outer medium induced by collagen, and also inhibited the increase in the concentration of cytoplasmic free Ca2+, which probably results from Ca2+ influx, as monitored by quin2 fluorescence, under stimulation with arachidonate. The concentration of N-ethylmaleimide giving a complete inhibition of Ca2+ influx was consistent with that required to inhibit collagen- or arachidonate-induced aggregation. Prostaglandin metabolism from arachidonate to thromboxane A2 was not disturbed by N-ethylmaleimide, while phosphatidate formation induced by arachidonate was slightly inhibited by it at concentrations at which aggregation was completely inhibited. These data suggest that N-ethylmaleimide preferentially suppresses increase in cytoplasmic free Ca2+ which is linked to thromboxane A2-receptor occupation in collagen- or arachidonate-stimulated platelets, probably due to blockage of Ca2+ influx through Ca2+-channel protein, thereby inhibiting aggregation induced by these agonists.     

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.     

Some unique features of the metabolic conversion of platelet-activating factor (AGEPC) to alkyl acyl PC by washed rabbit platelets

Recently several synthetic analogs of 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC; platelet-activating factor) were characterized as selective inhibitors of this agonist's effects on rabbit platelets (Tokumura, A., Homma, H., and Hanahan, D. J. (1985) J. Biol. Chem. 260, 12710-12714). In this current investigation, these studies have been extended to include a further inquiry into the biochemical nature of the metabolic inactivation of AGEPC in rabbit platelets, and the effect of these analogs on this process. Two of the latter components (U66985 and CV3988), which blocked AGEPC biological activity on rabbit platelets, also blocked the metabolism of this agonist. The metabolic conversion of AGEPC to alkyl acyl PC was inhibited nearly sevenfold by the most potent analog, U66985. Those analogs with low (U68043) or no biological inhibitory activity (lysoGEPC) had marginal effects on the metabolism of AGEPC. The effects of these compounds on the metabolism of AGEPC was not simply due to competitive inhibition. In platelets which had been pretreated with AGEPC in absence of extracellular Ca2+ (desensitized) and washed, the metabolic conversion of AGEPC to alkyl acyl PC was actually enhanced. This enhanced metabolic inactivation of AGEPC was also observed upon the treatment of the cells with thrombin, collagen, or ionophore A23187, indicating that the metabolism of AGEPC in platelets was enhanced not only by AGEPC itself but by other agonists as well. Nearly 85% of the fatty acyl residues was arachidonate in the alkyl acyl PC derived from AGEPC. This specific acylation with arachidonate was observed in the presence and absence of the inhibitor and in desensitized cells, indicating that selectivity for arachidonate is not dependent on the enhancement of the metabolism of AGEPC. The alkyl acyl PC found in the cells treated with thrombin, collagen, or A23187 was also predominantly alkyl arachidonoyl PC. Thus it has been shown that the inactivation of AGEPC by its conversion to alkyl acyl PC by rabbit platelets is enhanced by this agonist itself and that excess amounts of AGEPC could be further inactivated by the enhanced capacity of the metabolism process.     

Effects of a polyoxyethylene detergent (Brij 58) on platelet aggregation, release and clotting activity

Low concentrations of a polyoxyethylene detergent, Brij 58, inhibited the secondary phase of platelet aggregation induced by ADP in human citrated platelet-rich plasma but had no effect on primary aggregation. Thrombin-induced aggregation of washed human platelets suspended in Tyrode's buffer was inhibited after incubation of cells with 4.10(-6) M detergent. Efflux of [14C]serotonin, 45Ca2+ and labile aorta contracting substance (thromboxane A2) and development of prothrombin-converting activity (platelet factor 3) were abolished concomitantly. Aggregation of washed platelets either by sodium arachidonate or by collagen was also inhibited by the same concentration of Brij 58 which inhibited thrombin aggregation. This concentration did not itself produce any release of a cytoplasmic marker, lactate dehydrogenase, from platelets. Higher concentrations of Brij 58, exceeding 4.10(-5) M, lysed the cells liberating lactate dehydrogenase, serotonin and Ca2+. When albumin was included as a platelet stabilizer in the suspending medium the concentration of detergent required for the inhibitory effects was increased ten-fold. This could be attributed to competitive binding of the detergent to albumin, demonstrated with [14C]acetylated Brij 58. A variety of other polyoxyethylene detergents, at concentrations from 8.10(-4) to 5.10(-3) M, also inhibited platelet aggregation induced by thrombin. It is concluded that low concentrations of Brij 58 stabilize the platelets against the action of aggregating agents, while higher concentrations produce membrane destabilization and cell lysis.     

The influence of lysophosphatidic acid on platelet protein phosphorylation

Lysophosphatidic acid (LPA) is a lysophospholipid that is produced during thrombin stimulation of platelets, which can promote platelet aggregation. The mechanism of the effect of LPA was explored in normal platelets and in platelets from a patient with a storage pool deficiency (SPD). A comparison with other lysophospholipids showed that only LPA exerted significant effects to cause or potentiate platelet aggregation. Aspirin, an inhibitor of prostaglandin endoperoxide synthetase, had little effect on LPA-induced aggregation, but completely blocked LPA-induced serotonin secretion. LPA also promoted phosphorylation of myosin light chain (MLC), a 47 kilodalton (kDa) protein, and actin-binding protein. Aspirin significantly inhibited the phosphorylation of the 47-kDa and actin-binding proteins at 3-8 min after the addition of LPA, but had no effect on protein phosphorylation within the 1st min and had no significant effect on MLC phosphorylation. In SPD platelets, aspirin partially inhibited both aggregation and phosphorylation of the 47-kDa protein (less than 30% inhibition) and MLC (less than 40% inhibition) at time points of 1 min or less. The addition of ADP to SPD platelets enhanced the LPA response in platelets either pretreated or not pretreated with aspirin. Studies with SPD platelets indicate that thromboxane and secreted ADP contribute to, but are not necessary for, LPA-induced aggregation and phosphorylation. A23187 (a calcium ionophore) and LPA showed some selectivity to promote MLC as opposed to the 47-kDa protein phosphorylation, particularly at low concentrations of agonists and at earlier time points. The protein phosphorylation changes seen are consistent with a role for MLC phosphorylation in the granule centralization promoted with LPA.     

Inhibition of platelet aggregation by primary amines. Evidence for a possible role of membrane-associated calcium

The aggregation of human blood platelets by thrombin, adenosine diphosphate, wheat germ agglutinin or ristocetin was inhibited by primary amines. In general, thrombin-induced platelet aggregation was strongly affected by the amines while the effect was weak on cell aggregation by ristocetin. Usually, the diamines were stronger inhibitors of aggregation than the monoamines with cadaverine as the strongest and ethylamine as the weakest inhibitor. At concentration where platelet aggregation was inhibited, the amines neither displaced serotonin from serotinin-loaded platelets nor caused lysis of human red cells. The lectin activity of wheat germ agglutinin on human red cells was not affected by the amines indicating that the amines probably acted on platelets and not on the agglutinin. The clotting activity of thrombin on fibrinogen was partially inhibited by the amines while its esterolytic activity remained unaltered. The inhibitory action of the amines on platelet aggregation could be overcome with small amounts of calcium while other divalent cations tested had little effect. It is suggested that the amines affect platelet aggregation by interfering with the actions of membrane-associated calcium.     

Core aldehydes of alkyl glycerophosphocholines in atheroma induce platelet aggregation and inhibit endothelium-dependent arterial relaxation.

Plaque disruption with superimposed thrombosis is considered to be responsible for precipitating acute coronary syndrome. We identified sn-1-alkyl- and sn-1-acyl-type glycerophosphocholine (GroPCho) core aldehydes from human atheromas and demonstrated their activities on platelets and arteries. The naturally occurring core aldehydes were identified and quantified in relation to synthetic standards by high performance liquid chromatography with on-line electrospray mass spectrometry. 1-O-Hexadecyl-2-(5-oxovaleroyl)-sn-GroPCho (C(5) alkyl GroPCho core aldehyde), occurring in atheroma at less than 0.1% of total phosphatide, induced aggregation of washed rabbit platelets (50% effective dose was approximately 50 nM). Aggregations induced by C(5) alkyl GroPCho core aldehydes were completely inhibited by two different platelet-activating factor receptor antagonists. 1-Palmitoyl-2-(5-oxovaleroyl)-sn-GroPCho (C(5) acyl GroPCho core aldehyde) induced platelet shape change, but not aggregation. By contrast, 10 microM C(5) alkyl and C(5) acyl GroPCho core aldehydes both inhibited endothelium-dependent relaxation of rabbit artery by 50% (endothelium-independent relaxation was not affected). The present demonstration of platelet aggregation by physiologically relevant concentrations of alkyl GroPCho core aldehydes suggests that alkyl GroPCho core aldehyde generated in atheroma could be involved in precipitating acute coronary events, in which thrombus formation following lipid-rich plaque disruption plays an important role.     

Lipid peroxide makes rabbit platelet hyperaggregable to agonists through phospholipase A2 activation

Treatment of rabbit platelets with tert-butyl hydroperoxide and Fe2+ caused increasing arachidonic acid release, lysophosphatidylcholine formation, and aggregation with increasing concentrations of Fe2+. A combination of tert-butyl hydroperoxide and a low concentration of Fe2+, which by itself causes slight or no such activation, elicited synergistic release of arachidonic acid and aggregation under stimulation with a suboptimal concentration of collagen or arachidonic acid as an agonist. These responses were inhibited by pretreatment of the platelets with vitamin E or mepacrine in a concentration-dependent manner, but not by uric acid. The arachidonic acid release was dependent on the presence of Ca2+ in the medium. Synergistic formation of lysophosphatidylcholine, but not diacylglycerol, was also observed under this condition. The aggregation was also inhibited by indomethacin, a cyclooxygenase inhibitor. Cyclooxygenase activity was not affected by the oxidative treatment. These results suggest that lipid peroxide formed in membranes causes phospholipase A2 to become hypersusceptible to the agonist used, making the platelets hyperaggregable.     

Inhibition of epinephrine-induced platelet aggregation by a derivative of wheat germ agglutinin

A nonagglutinating derivative of wheat germ agglutinin has been prepared and used as a probe to explore the initial events in platelet activation. The lectin derivative had no effect on platelet aggregation by adenosine diphosphate, collagen, ristocetin, wheat germ agglutinin or trypsin but aggregation induced by epinephrine or thrombin was inhibited. Unlike thrombin, the inhibition of aggregation by the derivative could not be overcome by increasing the concentration of epinephrine. The derivative did not affect the binding of [³H]dihydroergocryptine to platelets. A 74,000 dalton protein isolated from platelet membranes by lectin affinity chromatography strongly inhibited platelet activation by thrombin but not by epinephrine. The receptors for thrombin and for epinephrine on platelets are different but they are closely linked.     

Effects of the lipid peroxidation product 4-hydroxynonenal on the aggregation of human platelets

The stimulation by ADP or arachidonic acid of the aggregation of human platelets in plasma was inhibited by 4-hydroxynonenal (HNE). This reduction of aggregation was time related, and was increased by prolonged preincubation of the platelets with the aldehyde. HNE was more potent than its homologue 4-hydroxypentenal (HPE). HNE was less active in decreasing the aggregation induced by calcium ionophore A23187 or collagen in comparison with ADP. HNE was inactive against aggregation of platelet-rich plasma (PRP) stimulated by thrombin whereas it potently inhibited the aggregation of washed platelets in response to both thrombin and collagen. Platelets were found to degrade HNE, and mechanisms additional to covalent binding to glutathione are indicated by the results obtained. The aldehydes, including HNE, generated by platelets originated principally from arachidonic acid metabolism.     

Interrelation of platelet aggregation, release reaction and thromboxane A2 production

Aggregation of platelets, stimulated by different agonists, was inhibited by omitting sample stirring or by preincubation of platelets with a monoclonal antibody against glycoproteins IIb-IIIa or with a pentapeptide containing the sequence Arg-Gly-Asp-Ser. In platelets stimulated by collagen, ADP and epinephrine, the inhibition of aggregation paralleled a reduction of both release reaction and thromboxane A2 formation. When thrombin was the stimulus, ATP release and thromboxane A2 production were unaffected (or only slightly modified) by the inhibition of platelet aggregation. These data add further evidence to the hypothesis that aggregation supports the activation of platelets stimulated by weak agonists.     

On the susceptibility of human platelets to aggregation by concanavalin A and the effect of this lectin on their response to ADP

Concanavalin A aggregated gel-filtered platetes in 0.9% NaCl solution signifying cross-bridging by the lectin. Aggregation of these platelets by concanavalin A was temperature dependent; it did not occur at 0–4 °C unless the platelets were previously trypsinized. The level of aggregation of trypsinized platelets by concanavalin A at 0–4°C was similar to that of untreated platelets at 37°C. It is suggested that trypsin facilitates platelet aggregation by concanavalin A at 0–4°C by causing a configurational change in membrane glycoproteins which orientates concanavalin A receptor sites into positions that favour lectin cross-bridging. Concanavalin A failed to aggregate platelets in plasma. Radioisotope studies showed that the amount of [³H]concanavalin A which combined with platelets in plasma was extremely low compared with gel-filtered platelets in saline. The aggregation of Ehrlich ascites cells by concanavalin A was considerably reduced when platelet-free plasma was added to the medium suggesting that it was due to the presence of concanavalin A-reactive components in the plasma.Concanavalin A inhibited the ADP-induced aggregation of platelets suspended in plasma or in a salts solution supplemented with calcium and fibrinogen, although the inhibitory effect was more conspicuous in the latter case. The results suggests that concanavalin A produces its inhibitory effect on ADP-induced platelet aggregation by interacting with membrane glycoproteins, and this further suggests their involvement in aggregation.     

Aggregation of platelets by muscle actin. A multivalent interaction model of platelet aggregation by ADP

Filamentous muscle actin (F-actin) aggregated blood platelets while G-actin was ineffective. This aggregation could be blocked by ATP suggesting a possible role of actin-bound ADP in this process. Actin-bound ADP caused platelet aggregation at concentrations significantly lower than equivalent concentrations of free ADP. Thus, actin potentiates the aggregating action of ADP. An actin antibody or DNase I inhibited this aggregation showing the requirement of actin in this process. Like other physiological agents, Ca⁺⁺ was necessary for platelet aggregation by actin. Platelets fixed in formaldehyde were not aggregated by actin showing the need for viable platelets. Since F-actin contains 1 mole of bound ADP/mole protein, it is postulated that actin potentiates ADP-induced aggregation by providing multiple interaction sites for platelets.     

Thyroid hormones inhibit platelet function and myosin light chain kinase

We examined the extranuclear effects of thyroid hormones on human platelets. Pretreatment with DL-thyroxine or DL-triiodothyronine inhibited collagen-induced aggregation, in a dose-dependent manner, but other derivatives of thyroid hormone had no significant effects. In contrast to collagen, 12-O-tetradecanoylphorbol-13-acetate-induced aggregation was not affected by thyroid hormones at the same concentration range. Thyroxine also inhibited the release of [14C] serotonin from collagen-stimulated platelets, with a marked reduction in the phosphorylation of 20,000-dalton protein. Thyroxine and triiodothyronine had inhibitory effects on myosin light chain kinase purified from human platelets and inhibited more markedly the myosin light chain kinase than protein kinase C (Ca2+/phospholipid-dependent enzyme) and cAMP-dependent protein kinase. In addition, L-thyroxine behaved as a competitive inhibitor of myosin light chain kinase toward calmodulin, and the Ki value was calculated to be 2.6 microM. To determine whether or not thyroxine directly binds myosin light chain kinase, we prepared an affinity column, using L-thyroxine as the ligand. Myosin light chain kinase was selectively bound to the column while calmodulin passed through. We also designed a procedure for the purification of myosin light chain kinase from human platelets, using L-thyroxine-affinity chromatography. A markedly increased purification was thus achieved, and DEAE-cellulose and L-thyroxine-affinity chromatography were made feasible. These results suggest that thyroxine can serve as a pharmacological tool for elucidating the biological significance of myosin light chain kinase-mediated reactions and is a pertinent ligand which can be used to purify myosin light chain kinase from platelets as a substitute for calmodulin.     

Structure-activity analysis of the effects of lysophosphatidic acid on platelet aggregation.

Lysophosphatidic acid (1-acyl-sn-glycero-3-phosphate or LPA) is a phospholipid mediator displaying numerous and widespread biological activities and thought to act via G-protein-coupled receptors. Here we have studied the effects on human platelets of a number of LPA analogues, including two enantiomers of both N-palmitoyl-(L)-serine-3-phosphate ((L) and (D)NAPS for N-acyl-phosphoserine) and 2-(R)-N-palmitoyl-norleucinol-1-phosphate ((R) and (S)PNPA), cyclic analogues of 1-acyl-sn-glycero-3-phosphate (cPA) and of 1-O-hexadecyl-sn-glycero-3-phosphate (cAGP), sphingosine-1-phosphate (SPP), as well as two palmitoyl derivatives of dioxazaphosphocanes bearing either a P-H or a P-OH bond (DOXP-H and DOXP-OH, respectively). Nine of these compounds induced platelet aggregation with the following order of potency: SPP < cAGP < DOXP-OH < (L)NAPS = (D)NAPS < (R)PNPA = (S)PNPA < LPA < AGP, EC50 varying between 9.8 nM and 8.3 microM. Two of these compounds (SPP and cAGP) appeared as weak agonists inducing platelet aggregation to only 33% and 41%, respectively, of the maximal response attained with LPA and other analogues. In cross-desensitization experiments, all of these compounds specifically inhibited LPA-induced aggregation, suggesting that they were all acting on the same receptor(s). In contrast, cPA and DOXP-H did not trigger platelet aggregation but instead specifically inhibited the effects of LPA in a concentration-dependent manner. The inhibitory action of cPA did not vary with the acyl chain length or the presence of a double bond and did not involve an increase in cAMP. These data thus confirm the lack of stereospecificity of platelet LPA receptor(s). In addition, since the order of potency of some analogues is different from that described in other cells, our results suggest that platelets contain (a) pharmacologically distinct receptor(s) whose molecular identity still remains to be established. Finally, this unique series of compounds might be used for further characterization of other endogenous or recombinant LPA receptors.     

Neomycin cannot be used as a selective inhibitor of inositol phospholipid hydrolysis in intact or semi-permeabilized human platelets. Aminoglycosides activate semi-permeabilized platelets.

High concentrations of neomycin (2-10 mM) inhibited aggregation, but not shape change, of intact platelets by collagen, ADP and the Ca2+ ionophore, A23187, the last two studies being carried out in the presence of the cyclo-oxygenase inhibitor indomethacin. In contrast, over the same range of concentrations neomycin inhibited both aggregation and shape change induced by thrombin. Under these conditions activation of platelets by collagen and by thrombin, but not by A23187 or by ADP, is believed to be dependent on the hydrolysis of membrane inositol phospholipids. These data therefore suggest that the inhibitory action of neomycin on intact platelets is not related to its previously reported inhibitory effect on phosphoinositide metabolism. The selective inhibition of thrombin-induced shape change indicates a second site of action of neomycin on intact platelets. On platelets rendered semi-permeable with saponin, neomycin and a second aminoglycoside antibiotic, streptomycin (each 0.06-2 mM), stimulated secretion and aggregation responses. These effects were inhibited by indomethacin and by EGTA. Activation of semi-permeabilized platelets by neomycin is associated with the formation of inositol phosphates and phosphatidic acid, indicating activation by phospholipase C. This effect is also inhibited by indomethacin, implying that it is secondary to the formation of prostaglandins and endoperoxides. These results are discussed in the context of the use of neomycin as a selective inhibitor of polyphosphoinositide metabolism.     

Aggregation and release reaction of washed platelets stimulated by lanthanum.

Ionized lanthanum caused clumping of washed platelets. This clumping response could be reversed by chelating agents but was not impaired by known inhibitors of platelets aggregation. Aggregation by lanthanum was not restricted to the unique clumping properties of platelets but occurred in fixed platelets and red cells and was most likely based on an electrostatic interaction.Lanthanum was able to stimulate as well as to inhibit serotonin release from platelets.At a concentration of 1 mM, lanthanum evoked a release of serotonin from washed platelets at 37°C. This release reaction was inhibited at 18°C or by prior treatment of platelets with neuraminidase or NEM.At a high concentration (10 mM), lanthanum did not stimulate the platelet release reaction but inhibited that induced by all stimuli investigated, presumably due to a fixation of membrane molecules.The release reaction promoted by thrombin or A 23187, but not that by collagen, was inhibited by a low concentration of lanthanum (0.1 mM). This inhibition is based on an interaction of lanthanum with the stimuli rather than with the platelet surface.     

Clq inhibition of the interaction of collagen with human platelets.

Human Clq, isolated in pure state after affinity chromatography on IgG-Sepharose, inhibited collagen-induced aggregation and release of 14C-Serotonin from prelabeled human platelets. Platelet aggregation induced by ADP or thrombin was not inhibited by Clq. Also, the adherence of platelets to glass surfaces was significantly diminished by Clq. In contrast, aggregated Clq mimicked the effect of collagen in causing platelet aggregation and release of serotonin. It appears that monomeric Clq, which has structural similarities to collagen competes with collagen for specific sites on the platelet surface.     

Complex effects of different green tea catechins on human platelets

Epigallocatechin gallate (EGCG), a major component of green tea, has been previously shown to inhibit platelet aggregation. The effects of other green tea catechins on platelet function are not known. Pre-incubation with EGCG concentration-dependently inhibited thrombin-induced aggregation and phosphorylation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinases-1/2. In contrast EGCG stimulated tyrosine phosphorylation of platelet proteins, including Syk and SLP-76 but inhibited phosphorylation of focal adhesion kinase. Other catechins did not inhibit platelet aggregation. Interestingly, when EGCG was added to stirred platelets, a tyrosine kinase-dependent stimulation of platelet aggregation was observed. The two other catechins containing a galloyl group in the 3' position (catechin gallate, epicatechin gallate) also stimulated platelet aggregation, while catechins without a galloyl group (catechin, epicatechin) or the catechin with a galloyl group in the 2' position (epigallocatechin) did not.