Inhibition of platelet thromboxane formation and phosphoinositides breakdown by osthole from Angelica pubescens

Osthole, isolated from Chinese herb Angelica pubescens, inhibited platelet aggregation and ATP release induced by ADP, arachidonic acid, PAF, collagen, ionophore A23187 and thrombin in washed rabbit platelets. It showed a weak activity in platelet-rich plasma. Osthole inhibited the thromboxane B2 formation caused by arachidonic acid, collagen, ionophore A23187 and thrombin in washed platelets, and also the thromboxane B2 formation caused by the incubation of lysed platelet homogenate with arachidonic acid. The generation of inositol phosphates in washed platelets caused by collagen, PAF and thrombin was suppressed by osthole. These data indicate that the inhibitory effect of osthole on platelet aggregation and release reaction was due to the inhibition of thromboxane formation and phosphoinositides breakdown.     

Inhibitory mechanisms of dantrolene on platelet aggregation

The antiplatelet effect of dantrolene and possible inhibitory mechanisms were studied in rabbit platelets. Preincubation of rabbit washed platelets with dantrolene (50-300 microM) inhibited the platelet aggregation and adenosine triphosphate release induced by arachidonic acid (100 microM), collagen (10 microg/mL), or thrombin (0.1 U/mL) in a dose-dependent manner. The thromboxane B2 formation caused by collagen or thrombin was inhibited by dantrolene, while formation of thromboxane B2 and prostaglandin D2 induced by arachidonic acid were not inhibited. In addition, the formation of phosphoinositide breakdown and the rise of intracellular calcium level induced by collagen or thrombin were also inhibited in a dose-dependent manner by dantrolene in the presence of indomethacin (2 microM). However, the platelets cyclic AMP level was not affected by dantrolene. In conclusion, the present study demonstrates that dantrolene inhibits platelet activation mainly due to suppression of phosphoinositide breakdown.     

Investigation on a selective non-prostanoic thromboxane antagonist, BM 13.177, in human platelets

The mode of action of BM 13.177 (4-[2-(benzenesulfonamido)-ethyl] phenoxyacetic acid), a new anti-aggregating and anti-thrombotic agent, was studied in human washed platelets and citrated PRP. With ASA-treated platelets, BM 13.177 (0.1 - 100 microM) did not inhibit the shape change and the aggregation induced by ADP, serotonin, adrenaline, thrombin, or collagen. Therefore, BM 13.177 is neither an antagonist of ADP, serotonin, adrenaline, thrombin, or collagen nor a common pathway inhibitor like PGE1, or an inhibitor of the platelet interactions during aggregation. However, BM 13.177 (greater than or equal to 0.1 microM) produced a dose-dependent reduction of shape change, aggregation and release of [3H]serotonin induced by the stable PGH2 analogues U 46619 and U 44069 in ASA-treated platelets or ASA-treated citrated PRP. In untreated platelets, BM 13.177 inhibited platelet activation by U 46619 or U 44069 and by exogenous arachidonic acid or by endogenous arachidonic acid mobilized by hydrogen peroxide. Consequently, the ADP- and adrenaline-induced secondary aggregation and [3H]serotonin release in citrated PRP and the major effects of collagen were also inhibited. In washed platelets treated with 10 microM arachidonic acid or 100 microM hydrogen peroxide, the formation of TXB2 was not inhibited by 10 microM BM 13.177. However, the TXB2 formation after stimulation with 1,200 microM hydrogen peroxide was partially reduced by BM 13.177 to the same extent as by PGE1. This reduction may be due to the absence of a secondary release of arachidonic acid from phospholipids if the platelets were prevented from activation by BM 13.177 or PGE1. Arachidonic acid and hydrogen peroxide also induced the shape change, aggregation and release of washed platelets when thromboxane formation was inhibited by dazoxiben. Under these conditions, BM 13.177 was able to abolish the platelet response which was due to accumulating prostaglandin endoperoxides. These results show that BM 13.177 acts as a selective antagonist of TXA2 and prostaglandin endoperoxides. Its inhibitory effect on platelet function does not depend on an inhibition of either the primary release of arachidonic acid or the activities of cyclooxygenase or thromboxane synthetase.     

HA-29: an inhibitor of thromboxane A2 formation with antagonism of thromboxane A2/prostaglandin endoperoxide receptor in rabbit platelets.

HA-29, 2-[(3-methoxyphenyl)methyl]-pyrano[2,3-c]pyrazol-6(1H)-one, was investigated for its inhibitory mechanism of action in washed rabbit platelets. This compound inhibited the aggregation and ATP release of rabbit platelets induced by arachidonic acid and collagen in a concentration-dependent manner, without affecting those induced by ADP, PAF and thrombin. Prolongation of the incubation time of HA-29 with platelets did not cause further inhibition and the aggregability of the agent-treated platelets could be restored after washing of platelets. The concentration-response curve of U-46619-induced platelet aggregation was shifted to the right by HA-29 in a concentration-dependent manner, but the maximal aggregation was suppressed by HA-29. The pA2 and pA10 values of HA-29 on U-46619-induced platelet aggregation were 4.26 and 3.58, respectively, with a slope value of -1.4. The U-46619-induced aggregation was markedly disaggregated by HA-29 even it was added 5 min after U-46619. HA-29 inhibited the secondary aggregation and ATP release, but not the primary aggregation of human platelet-rich plasma induced by ADP and epinephrine. Thromboxane B2 formation caused by arachidonic acid, collagen and thrombin was markedly suppressed by HA-29. HA-29 also inhibited the formation of prostaglandin D2 caused by arachidonic acid. HA-29 inhibited almost completely the formation of inositol monophosphate caused by U-46619, but not that by collagen or thrombin. HA-29 did not affect U-46619-induced contraction of rat aorta. It is concluded that the antiplatelet effect of HA-29 is due to the inhibition of thromboxane A2 formation and blockade of thromboxane A2/prostaglandin endoperoxide receptor.     

Specific inhibition of 40 K protein phosphorylation in platelet by a new antithrombotic agent, d-indobufen

Indobufen, (+)2[p-oxo-2-isoindolinyl)-phenyl] butyric acid exhibited unique inhibitory pattern of platelet function with suppression of protein phosphorylation. In collagen-induced activation, d-indobufen potently inhibited aggregation and release reaction in a dose-dependent manner (IC50:0.49 microM). This compound delayed the onset of aggregation and release reaction induced by arachidonic acid without reducing the magnitude of final reactions. When platelets were stimulated with thrombin, indobufen did not inhibit the aggregation significantly up to 100 microM. d-Indobufen suppressed platelet 40 kDa protein phosphorylation in collagen-stimulated platelets and delayed the onset of the phosphorylation in arachidonic acid-stimulated platelets. However, d-indobufen did not directly inhibited protein kinases in platelets, in vitro. These findings suggest that the inhibitory actions of d-indobufen may be due to its effects on the initiation process of 40 kDa protein phosphorylation.     

Antiplatelet effects of protopine isolated from Corydalis tubers

Protopine inhibited the aggregation and ATP release of rabbit platelets induced by ADP, arachidonic acid, PAF, collagen and ionophore A23187. Although the platelet aggregation caused by thrombin was not inhibited by protopine (100 micrograms/ml), the release reaction was partially suppressed. In rabbit platelet-rich plasma, protopine also inhibited the platelet aggregation caused by ADP, arachidonic acid, PAF and collagen. The thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, ionophore A23187 and thrombin was suppressed by protopine. Protopine inhibited the intracellular calcium increase caused by arachidonic acid in quin-2/AM loaded rabbit platelets. In the presence of indomethacin, the intracellular calcium increase caused by collagen and PAF was completely suppressed by protopine, and the intracellular calcium increase caused by thrombin was partially inhibited. The phosphoinositides breakdown caused by collagen and PAF was inhibited by protopine, but that by thrombin was not affected significantly. Protopine did not cause the elevation of cyclic AMP level of platelets. It is concluded that the antiplatelet effects of protopine is due to inhibition on thromboxane formation and phosphoinositides breakdown and then lead to the decrease of intracellular calcium concentration.     

12-HETE inhibits the binding of PGH2/TXA2 receptor ligands in human platelets.

12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), the end-lipoxygenase product of arachidonic acid in platelets has been previously shown to prevent PGH2/TxA2-induced aggregation. From the present study, we show that 12-HETE inhibits the binding of [125I]-PTA-OH, a thromboxane antagonist, to platelet membranes with an IC50 of 8 microM. This value is in accordance with previously reported 12-HETE concentrations required to prevent the aggregation induced by TxA2 mimetics, the methano analogues of PGH2, U44069 and U46619. When [3H]-U44069 was used as a thromboxane agonist to label intact platelets, 12-HETE also inhibited its binding. We conclude that part of the inhibitory effect of 12-HETE on PGH2/TxA2-induced aggregation might be the result of interacting with PGH2/TxA2 receptor sites.     

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.     

Antiplatelet actions of some coumarin compounds isolated from plant sources.

Xanthyletin, xanthoxyletin, suberosin (all from Citrus grandis), aurapten (from Severinia huxifolia) and poncitrin (from Poncirus trifoliata) were isolated and their chemical structures were characterized to be coumarin compounds. All these coumarin compounds except xanthyletin inhibited the aggregation and ATP release of rabbit platelets induced by arachidonic acid, collagen, ADP, platelet-activating factor (PAF) or U46619 (a thromboxane A2 analog). Thrombin-induced ATP release, but not the aggregation, was also inhibited by these compounds. Xanthyletin inhibited only platelet aggregation induced by arachidonic acid and collagen, while poncitrin inhibited that caused by PAF more markedly than other coumarin compounds. The thromboxane B2 formation in washed platelets caused by arachidonic acid and collagen was suppressed by these coumarin compounds. The phosphoinositide breakdown caused by collagen and PAF was also inhibited by these compounds. They did not affect fibrinogen-induced aggregation of elastase-treated platelets. These antiplatelet actions were immediate, reversible by washout and independent on the incubation time (except suberosin). Antiaggregating effect was also studied by an electrical impedance method and the inhibitory effect of coumarin compounds on the whole blood and platelet-rich plasma was much less than that of platelet suspension in the aggregation induced by collagen, PAF and ADP. It is concluded that the antiplatelet actions of these coumarin compounds are due to the inhibition on thromboxane A2 formation and phosphoinositides breakdown.     

Benzodiazepines inhibit human platelet activation: comparison of the mechanism of antiplatelet actions of flurazepam and diazepam

These studies were undertaken to examine the effects and the mechanism of action of flurazepam and diazepam on human platelet activation. One minute preincubation with flurazepam (3-300 microM) or diazepam (3-300 microM) inhibited platelet aggregation, serotonin secretion and prostaglandin synthesis induced by ADP (1-5 microM), epinephrine (1-5 microM), and arachidonic acid (600-1000 microM). However, 357% higher concentration of diazepam (265 microM) as compared to flurazepam (58 microM), was required to inhibit arachidonic acid induced production of malondialdehyde (MDA) by 50%. In addition, flurazepam and not diazepam inhibited the release of arachidonic acid from platelet phospholipids in a concentration dependent manner. In other experiments flurazepam but not diazepam also blocked aggregation and secretion induced by U46619 (2 microM), a stable analog of prostaglandin H2. Platelet aggregation and serotonin secretion induced by collagen (40-300 micrograms/ml) was inhibited by flurazepam with an IC-50 of 153 microM and 136 microM respectively, whereas higher than 300 microM diazepam was required to inhibit collagen-induced aggregation and secretion by 50%. Flurazepam and diazepam both exhibited their most potent antiplatelet effects against phospholipase C-induced aggregation which is mediated by prostaglandin-independent mechanisms. Only 15 microM and 11 microM flurazepam and 31 microM and 27 microM diazepam were needed to inhibit PLC-induced aggregation and secretion of serotonin by 50% respectively. Effects of these benzodiazepines on platelet cyclic AMP and cyclic GMP were also examined. Neither flurazepam nor diazepam caused any significant change in cyclic AMP or cyclic GMP levels in platelets. These findings suggest that: (a) flurazepam, as compared to diazepam, is 106% - 357% more effective in inhibiting platelet aggregation and serotonin secretion induced by arachidonic acid, collagen and phospholipase C; (b) flurazepam inhibits platelet activation by inhibiting the release of arachidonic acid, its conversion into prostaglandins and by blocking the action of prostaglandins on platelets; (c) diazepam does not inhibit thrombin-induced release of arachidonic acid, conversion of exogenously added arachidonic acid into MDA, or the action of prostaglandins; (d) both flurazepam and diazepam inhibit PLC-mediated activation of platelets; and (e) neither diazepam nor flurazepam achieve their antiplatelet actions by affecting platelet cyclic nucleotide levels.     

Inhibition of rabbit platelet aggregation by 1,4-naphthoquinones

The effects of four 1,4-naphthoquinone derivatives on the aggregation of rabbit platelets were examined. All the four 1,4-naphthoquinone derivatives inhibited the platelet aggregation of washed rabbit platelets induced by thrombin (0.1 U/ml) and the IC50 is: 2-chloro-3-methyl-1,4-naphthoquinone (CMN), 5 micrograms/ml; 3-methyl-5,8-dihydroxy-1,4-naphthoquinone, 13 micrograms/ml; 5,8-dihydroxy-1,4-naphthoquinone, 18 micrograms/ml; 3-methyl-1,4-naphthoquinone (vitamin K3), 53 micrograms/ml. CMN was the most potent in inhibiting the aggregation and release reaction induced by ADP, arachidonic acid, PAF, ionophore A23187, collagen and thrombin in a dose-dependent manner in washed platelets, platelet-rich-plasma and whole blood. The thromboxane B2 formation caused by collagen and ionophore A23187 was inhibited by CMN. However, the thromboxane B2 formation by arachidonic acid was markedly increased. The platelet inhibitory effect of CMN could not be antagonized either by raising the concentrations of extracellular Ca++ or by wash out. The phosphoinositides breakdown induced by thrombin was inhibited by CMN. Phospholipids (PE, PC, PI) could slightly antagonize the antiplatelet effect of CMN. It is concluded that the inhibitory effect of CMN on rabbit platelet aggregation may be due to the inhibition of phosphoinositides breakdown caused by the inducers.     

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.     

Influence of granulocyte elastase-like proteinase (ELP) on platelet functions

The influence of granulocyte elastase-like proteinase (ELP) on platelet functions was investigated. ELP inhibited the platelet aggregations induced by a wide variety of agonists. The inhibition was marked in the case of receptor-mediated agonists such as thrombin, ristocetin, etc. It was moderate with the pervading agonist, arachidonic acid, and mild with the bypassing agonist, Ca²⁺ ionophore A23187. ELP inhibited the release of thromboxane A2 from platelets in the case of the platelet aggregation induced by thrombin. On the other hand, ELP did not inhibit the release of thromboxane A2 from platelets in the platelet aggregation induced by arachidonic acid or Ca²⁺ ionophore A23187. ELP suppressed the release of serotonin from platelets induced by thrombin, while it did not markedly suppress the release of serotonin induced by Ca²⁺-ionophore A23187. Treatment of platelets with ELP resulted in a slight increase of intraplatelet cAMP levels. These results suggest that ELP acts on receptors and inhibits platelet functions. As a results, ELP markedly inhibits the platelet functions such as aggregation or release of serotonin or thromboxane A2 stimulated by receptor-mediated agonists. ELP slightly elevates the CAMP level in the platelets, resulting in the mild inhibition of the platelet functions stimulated by the pervading agonist, arachidonic acid, or the bypassing agonist, Ca²⁺-ionophore A23187.     

Inhibitory effect of hexapeptide (RGRHGD) on platelet aggregation

The B chain of beta-bungarotoxin 1-6 sequence, RGRHGD, presents the highest local average hydrophilicity measured by Kyte and Doolittle modeling analysis. The RGRHGD holds parts of both RGD and KGD peptides, which have been reported as having high binding affinity to GPIIb-IIIa. The present study evaluates whether the synthesized hexapeptide, RGRHGD, has an antiplatelet effect and further elucidates the possible mechanisms of action. RGRHGD dose-dependently inhibited rabbit platelet aggregation and adenosine triphosphate release induced by arachidonic acid, collagen, platelet-activating factor, thrombin, or U46619 with the IC50 range of 82.7 to 510 microg/mL. The platelet thromboxane B2 formation induced by collagen or thrombin was also significantly decreased by RGRHGD, but there was no effect on arachidonic acid-induced thromboxane B2 formation. In addition, RGRHGD also inhibited the rise of intracellular calcium level stimulated by arachidonic acid, collagen, or thrombin in Fura 2-AM-loaded platelets. The adenosine 3',5'-cyclic monophosphate level of washed platelets was not affected by RGRHGD. In conclusion, these data indicate that the inhibitory effect of RGRHGD on platelet aggregation may be due to the attenuation of thromboxane A2 formation and intracellular calcium mobilization. In addition, this study may provide a useful method of finding potential therapeutic agents by using molecular modeling analysis.     

Effects of etamsylate on platelet functions and arachidonic acid metabolism

The effects of etamsylate on human platelet aggregation and ATP release as well as on the arachidonate metabolism by the platelet have been studied. Etamsylate enhanced these platelet functions induced by arachidonic acid (AA), thromboxane A2, collagen and calcium ionophore A23187 but not those induced by ADP and epinephrine. In experiments with cyclooxygenase-inhibited platelets, AA-induced platelet aggregation was completely inhibited and it was not enhanced by etamsylate, while A23187-induced aggregation was partially inhibited and this aggregation was enhanced by etamsylate. Platelet AA metabolism including thrombin-induced AA liberation from phospholipids as well as the lipoxygenase and cyclo-oxygenase pathways was not significantly affected by etamsylate. These results suggested that etamsylate enhanced platelet response to thromboxane A2 and calcium ionophore and that this could be included as a mechanism for its potentiating effect on platelet functions.     

Effects of higenamine and its 1-naphthyl analogs, YS-49 and YS-51, on platelet TXA2 synthesis and aggregation

The effects of higenamine and its 1-naphthyl analogs, YS-49 and YS-51, on thromboxane A(2) (TXA(2)) formation from arachidonic acid (AA) and aggregation in platelets, were investigated. YS-49 and YS-51 (IC(50); 32.8 and 39.4 microM respectively) exhibited much stronger inhibitory effects on TXA(2) formation than higenamine (IC(50); 2.99 mM). The higher inhibitory potencies of YS-49 and YS-51 (IC(50): 3.3 and 5.7 microM respectively) than higenamine (IC(50): 140 microM) on AA induced rat platelet aggregation was presumed to be the result of low inhibitory effect of higenamine than YS-49 and YS-51 on TXA(2) production from AA. Among the present three compounds, the more hydrophobic naphthylmethyl groups were supposed to be more favorable than p-hydroxybenzyl moiety, at 1-position of the tetrahydroisoquinoline ring, to display the inhibitory effects on TXA(2) production and AA induced aggregation of platelets. In addition, higenamine, YS-49 and YS-51 were observed directly antagonistic on TXA(2) receptor (TP receptors) by displaying inhibitory effects to U46619 (TXA(2) mimetic) induced platelet aggregation, however all of the three compounds showed similar order of inhibitory potencies. The present results are suggestive that YS-49 and YS-51 exert their inhibitory effects on AA-induced platelet aggregation partly by inhibiting the production of TXA(2) from AA and partly by directly blocking the TP receptor, in addition to the previously reported effects on alpha(2)-adrenergic receptor. On the other hand, higenamine is supposed to antagonize AA-induced platelet aggregation by mostly directly blocking the TP receptor.     

Inhibition of thrombin- and collagen-induced phosphoinositides breakdown in rabbit platelets by a PAF antagonist--denudatin B, an isomer of kadsurenone

Denudatin B, an isomer of kadsurenone, was isolated from Magnolia fargesii. It inhibited the aggregation and ATP release of washed rabbit platelets caused by platelet-activating factor (PAF) in a concentration-dependent manner. The IC50 on PAF (2 ng/ml)-induced aggregation was about 10 micrograms/ml. High concentration of denudatin B (greater than 50 micrograms/ml) also inhibited the aggregation and ATP release of platelets caused by ADP, collagen, arachidonic acid and thrombin. However, shape change of platelets still existed. Prolongation of the incubation time with platelets could not cause further inhibition, and the aggregability of platelets could be restored after denudatin B was washed out from platelets. Thrombin-induced thromboxane B2 formation was almost completely suppressed. In the absence of extracellular calcium (EGTA 1 mM), ATP release caused by thrombin was inhibited. Thrombin-induced rise of the intracellular calcium concentration was suppressed by denudatin B, but not by BN52021 or kadsurenone. The generation of inositol phosphate in washed platelets caused by collagen, PAF and thrombin was also suppressed. The data indicate that PAF antagonist denudatin B has nonspecific antiplatelet action at high concentration by inhibiting phosphoinositides breakdown induced by collagen and thrombin.     

Suppression by beta-lactam antibiotics of thromboxane A2 generation and arachidonic acid release in rabbit platelets in vitro

High concentrations of latamoxef and some other beta-lactam antibiotics suppressed thromboxane A2 generation as determined from the thromboxane B2 level in the in vitro aggregation of rabbit platelets in agonist-induced stimulations. In aggregation induced with a low concentration of collagen, the suppression of thromboxane B2 generation correlated well with the suppressions of aggregation and serotonin (5-HT) release; collagen produced thromboxane A2-dependent aggregation and 5-HT release as judged from the inhibitory action of indomethacin. Latamoxef also suppressed thromboxane B2 generation when platelet stimulation was induced by thrombin or platelet activating factor at concentrations at which it did not affect either aggregation or 5-HT release. However, latamoxef did not affect platelet responses including thromboxane B2 generation induced by exogenous arachidonic acid or calcium ionophore, A23187. Beta-lactam antibiotics also interfered with arachidonic acid release from the membrane phospholipids of platelets which had been prelabelled with [3H]arachidonic acid and aggregated with collagen. These results suggest that in the in vitro aggregation of platelets, beta-lactam antibiotics interfere with some of the receptor-stimulated processes which lead to arachidonic acid release from the membranes and this, in turn, suppresses thromboxane B2 generation.     

Vinblastine inhibits platelet aggregation by a microtubule-independent mechanism, probably by its perturbing action on the plasma membrane

Inhibitory mechanism of vinblastine on platelet activation was examined with respect to its effect on disassembly of the microtubule system. Vinblastine at 10 microM concentration caused washed platelets to become sphere with disorganized microtubule system, but did not affect aggregation induced by collagen, arachidonic acid or thrombin. Collagen-induced aggregation was inhibited by 50-100 microM of vinblastine and much higher concentration was required to inhibit arachidonic acid- and thrombin-induced aggregation. When the vinblastine (100 microM)-treated platelets were washed with albumin medium, the impaired aggregability was well recovered in response to collagen. In this case, however, both the vinblastine-induced sphered shape and disappeared microtubule system were not recovered to the normal states. Within the concentration ranges that inhibited collagen-induced aggregation, vinblastine also suppressed reversibly Ca2+ influx and arachidonic acid liberation from membrane phospholipids by phospholipase A2. Conversion of added arachidonic acid to thromboxane A2 was not inhibited even by such concentration. These results suggest that vinblastine interacts non-specifically with the cell membrane to cause reversible inhibition of arachidonic acid liberation by phospholipase A2 and Ca2+ influx and thereby aggregation through physical perturbation of membrane lipid bilayer, independent of the activity to disassemble platelet microtubule system.     

Inhibition of the effects of thrombin on guinea pig platelets by the diacylglycerol lipase inhibitor RHC 80267

Phospholipase C (PLC) and diacylglycerol lipase (DGL) activities were found in guinea pig platelet microsome preparations. No phospholipase A2 (PLA2) activity was detected. RHC 80267 (1,6-di (0-(carbamoyl) cyclohexanone oxime)hexane) inhibited DGL activity (IC50 = 4 uM) from guinea pig platelet microsomes but had no effect on PLC. RHC 80267 inhibited platelet aggregation (IC50 = 11 uM), release of arachidonic acid (AA), its metabolites, and ATP (IC50 = 4.5 uM) when guinea pig platelets were challenged with a low concentration of thrombin. We propose that PLC-DGL is an important enzymatic pathway for the release of AA in guinea pig platelets.