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.     

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.     

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 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.     

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.     

Inhibition of platelet aggregation by some flavonoids.

The inhibitory effects of five flavonoids on the aggregation and secretion of platelets were studied. These flavonoids inhibited markedly platelet aggregation and ATP release of rabbit platelets induced by arachidonic acid or collagen, and slightly those by platelet-activating factor. ADP-induced platelet aggregation was also suppressed by myricetin, fisetin and quercetin. The IC50 on arachidonic acid-induced platelet aggregation was: fisetin, 22 microM; kaempferol, 20 microM; quercetin, 13 microM; morin, 150 microM less than IC50 less than 300 microM. The thromboxane B2 formations were also inhibited by flavonoids in platelets challenged with arachidonic acid. Fisetin, kaempferol, morin and quercetin antagonized the aggregation of washed platelets induced by U46619, a thromboxane A2/prostaglandin endoperoxides mimetic receptor agonist. In human platelet-rich plasma, quercetin prevented the secondary aggregation and blocked ATP release from platelets induced by epinephrine or ADP. These results demonstrate that the major antiplatelet effect of flavonoids tested may be due to both the inhibition of thromboxane formation and thromboxane receptor antagonism.     

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.     

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.     

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.     

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.     

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 ethanol on pathways of platelet aggregation in vitro

Ethanol, at physiologically tolerable concentrations, did not affect the primary phase of ADP-induced aggregation of human or rabbit platelets, which is not associated with the secretion of granule contents. Potentiation by epinephrine of the primary phase of ADP-induced aggregation of rabbit platelets was also not inhibited by ethanol. However, ethanol did inhibit the secondary phase of ADP-induced aggregation which occurs with human platelets in citrated platelet-rich plasma and is dependent on the formation of thromboxane A2. Inhibition by ethanol of thromboxane production by stimulated platelets is likely due to inhibition of the mobilization of arachidonic acid from membrane phospholipids, as ethanol had little or no effect on aggregation and secretion induced by arachidonic acid or the thromboxane mimetic U46619. Rabbit platelet aggregation and secretion in response to low concentrations of collagen, thrombin, or PAF were inhibited by ethanol. Inhibition of the effects of thrombin and PAF was also observed with aspirin-treated platelets. Thus, in addition to inhibiting the mobilization of arachidonate for thromboxane formation that occurs with most agonists, ethanol can also inhibit aggregation and secretion through other effects on platelet responses.     

Two antiplatelet agents from Magnolia officinalis

Magnolol and honokiol are two position isomers isolated from the bark of Magnolia officinalis. Both inhibited the aggregation and ATP release of rabbit platelet-rich plasma induced by collagen and arachidonic acid without affecting that induced by ADP, PAF or thrombin. Aggregation of washed platelets was more markedly inhibited than that of platelet-rich plasma, while the aggregation of whole blood was least affected by both inhibitors. Thromboxane B2 formation caused by collagen, arachidonic acid or thrombin was in each case inhibited by magnolol and honokiol. The rise of intracellular calcium caused by arachidonic acid or collagen was also suppressed by both agents. Collagen-induced intracellular calcium increase in the presence of indomethacin was suppressed by magnolol. It is concluded that the antiplatelet effect of magnolol and honokiol is due to an inhibitory effect on thromboxane formation and also an inhibition of intracellular calcium mobilization.     

XC 386: A new antiplatelet agent

XC386 was a new antiplatelet compound, which inhibited the aggregation and release reaction of rat platelet-rich plasma induced by collagen. This inhibition was dose-dependent and the IC₅₀was calculated to be 1 mM on collagen-induced aggregation. In washed platelets, the aggregations induced by ADP and collagen were much more markedly inhibited by XC386 than those induced by thrombin, A23187 and arachidonate. High calcium (4 to 8 mM) could not antagonize the inhibition. XC386 did not alter the malondialdehyde (MDA) and thromboxane B (TXB₂) levels of resting platelets. But MDA formation induce by collagen, thrombin and A23187, and TXB₂ formation induced by collagen and thrombin were significantly inhibited, while both formations induced by arachidonate were not changed. Combination of indomethacin or CP/CPK and XC386 enhanced markedly the inhibitory effect of XC386 on collagen- or A23187-induced aggregation. It was concluded that XC386 might inhibit platelet aggregation before the step of arachidonic acid release by phospholipase A₂.     

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.     

Effect of 4-hydroxy-2,3-trans-nonenal on platelet function

4-Hydroxy-2,3-trans-nonenal (HNE), an aldehyde end-product of lipid peroxidation, potentiated aggregation and increased thromboxane A2 formation in platelets challenged with ADP, thrombin or the ionophore A23187. These effects were observed at HNE concentrations in the range 10-100 microM. Platelet responses to collagen, epinephrine and arachidonic acid were not affected by HNE. Concentrations of HNE in excess of 100 microM inhibited platelet activation. HNE increased the release of 3H-arachidonic acid from prelabelled platelet phospholipids in response to thrombin or ADP. It is proposed that HNE may play an important role in controlling platelet function by regulating the activity of phospholipase A2.     

The anti-aggregation effects of ondansetron on platelets involve IP3 signaling and MAP kinase pathway, but not 5-HT3-dependent pathway

Ondansetron is a 5-HT3 receptor antagonist with potent antiemetic, analgesic, and antiphlogistic effects. Literature concerning 5-HT3 antagonists on platelets is limited. In this report we examined the pharmacological effects of ondansetron on human washed platelets. Platelet aggregation induced by thrombin (0.1 U/mL), collagen (2 μg/mL), arachidonic acid (0.5 mM), ADP (10 μM), or U46619 (2 μM) was observed. The effects of ondansetron on platelet aggregation and ATP release were investigated at different concentrations. Cytosolic Ca²⁺ influx concentration, TXB2, IP3, and the levels of cAMP and cGMP were monitored, and flow cytometric analysis and immunoblotting were performed to investigate downstream signaling components. Our results showed that ondansetron, in a concentration-dependent manner, inhibited agonist-induced platelet aggregation. At 75 μM, ondansetron significantly attenuated intracellular Ca²⁺ mobilization, thromboxane B2 formation, and ATP release by human washed platelets activated by thrombin, collagen, or U46619, whereas it only partially attenuated arachidonic acid-driven platelet activation. Administration of ondansetron resulted in attenuated IP3 production in the washed platelets stimulated by thrombin, as determined by reduced IP1 levels, as well as diminished p38 and ERK2 phosphorylation in response to thrombin. No effect of ondansetron on the levels of either cAMP or cGMP in washed platelets was observed. Furthermore, ondansetron-mediated inhibition of platelet aggregation was not impacted by SR 57227A, the 5-HT3 agonist. Thus, rather than involving the 5-HT3-dependent pathway, the negative effect of ondansetron on platelet aggregation is instead manifested through the attenuation of agonist-induced IP3 production and MAPK (p38 and ERK2) phosphorylation that results in suppressed intracellular Ca²⁺ mobilization, TXB2 formation, and ATP release.     

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.     

Chloroquine: a multipotent inhibitor of human platelets in vitro

Chloroquine inhibited human platelet aggregation in vitro both at receptor- and nonreceptor-operated stimuli. The inhibition was dose-dependent, recorded on isolated platelets as well as in platelet-rich plasma, and followed the rank order of stimuli: adrenaline (second phase)>phorbol 12-myristate 13 acetate>adenosine diphosphate>adrenaline (first phase)>thrombin>calcium ionophore A23187. In thrombin-activated platelets, chloroquine decreased in a dose-dependent manner phospholipase A(2)-induced arachidonic acid liberation from membrane phospholipids, malondialdehyde formation (a marker of membrane phospholipid peroxidation), and thromboxane generation, considered the most potent autoaggregatory agent. Chloroquine only slightly altered the arachidonic acid cascade of platelets stimulated with A23187 and phorbol 12-myristate 13 acetate. Histamine formation and liberation induced with thrombin and A23187 were not affected by chloroquine. On the other hand, thrombin-stimulated serotonin secretion was significantly decreased with chloroquine in the concentration of 10 micromol/L. This indicated that chloroquine might interfere with stimulated secretion from platelets. The results suggest that chloroquine inhibited activated platelets: first, intracellularly; second, in a close relationship to the intraplatelet Ca(2+) mobile pool; and third, most probably at the site of platelet phospholipase A(2) activation.     

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.