Mechanism of Action of p-Chlorobiphenyl on the Inhibition of Platelet Aggregation

p-Chlorobiphenyl (1–50 μm ) concentration-dependently inhibited the aggregation and release reaction of rabbit washed platelets induced by arachidonic acid and collagen, but not those induced by platelet-activating factor (PAF), U46619 and thrombin. The IC50 values of p-chlorobiphenyl on the arachidonic acid and collagen-induced platelet aggregation were 2.9 ± 0.5 and 12.8 ± 2.3 μm , respectively. The formation of both platelet thromboxane B₂ and prostaglandin D₂ caused by arachidonic acid was inhibited by p-chlorobiphenyl concentration-dependently. In myo-[³H]inositol-labeled and fura-2-loaded platelets, [³H]inositol monophosphate generation and the rise in intracellular Ca²⁺ stimulated by arachidonic acid were inhibited by p-chlorobiphenyl. In human platelet-rich plasma, p-chlorobiphenyl and indomethacin prevented the secondary aggregation and blocked ATP release from platelets induced by adenosine 5′-diphosphate and adrenaline without affecting the primary aggregation. It is concluded that p-chlorobiphenyl may be a cyclo-oxygenase inhibitor and its antiplatelet action is mainly due to the inhibition of thromboxane formation.     

Effect of anethole dithiolthione on human platelet aggregation.

Anethole dithiolthione (ADT) (10 mumol/l) inhibited platelet aggregation and the formation of thromboxane (Tx)B2 in plasma in response to adenosine diphosphate (ADP), epinephrine and arachidonic acid (AA). ADT partially inhibited platelet aggregation and TxB2 formation in plasma induced by thrombin, phorbol myristate acetate and calcium ionophore A23187 and increased the lag time of collagen-induced aggregation at concentrations in the range 10-40 mumol/l. ADT (100 mumol/l) completely inhibited the aggregation of washed platelets challenged with thrombin. ADT had no additive effect on the inhibition of thrombin-induced platelet aggregation by acetylsalicylic acid. ADT was a more effective inhibitor of AA-induced platelet aggregation than butylated hydroxytoluene. ADT inhibited the release of 3H-AA from platelet phospholipids in response to ADP and collagen. It is suggested that ADT inhibits platelet aggregation by inhibiting thromboxane synthesis and preventing AA release.     

Antiplatelet activity of [5-(2-methoxy-5-chlorophenyl)furan-2-ylcarbonyl]guanidine (KR-32570), a novel sodium/hydrogen exchanger-1 and its mechanism of action

The antiplatelet effects of a novel guanidine derivative, KR-32570 ([5-(2-methoxy-5-chlorophenyl) furan-2-ylcarbonyl]guanidine), were investigated with an emphasis on the mechanisms underlying its inhibition of collagen-induced platelet aggregation. KR-32570 significantly inhibited the aggregation of washed rabbit platelets induced by collagen (10 microg/mL), thrombin (0.05 U/mL), arachidonic acid (100 microM), a thromboxane (TX) A2 mimetic agent U46619 (9,11-dideoxy-9,11-methanoepoxy-prostaglandin F2, 1 microM) and a Ca2+ ATPase inhibitor thapsigargin (0.5 microM) (IC50 values: 13.8 +/- 1.8, 26.3 +/- 1.2, 8.5 +/- 0.9, 4.3 +/- 1.7 and 49.8 +/- 1.4 microM, respectively). KR-32570 inhibited the collagen-induced liberation of [3H]arachidonic acid from the platelets in a concentration dependent manner with complete inhibition being observed at 50 microM. The TXA2 synthase assay showed that KR-32570 also inhibited the conversion of the substrate PGH2 to TXB2 at all concentrations. Furthermore, KR-32570 significantly inhibited the [Ca2+]i mobilization induced by collagen at 50 microM, which is the concentration that completely inhibits platelet aggregation. KR-32570 also decreased the level of collagen (10 microg/mL)-induced secretion of serotonin from the dense-granule contents of platelets, and inhibited the NHE-1-mediated rabbit platelet swelling induced by intracellular acidification. These results suggest that the antiplatelet activity of KR-32570 against collagen-induced platelet aggregation is mediated mainly by inhibiting the release of arachidonic acid, TXA2 synthase, the mobilization of cytosolic Ca2+ and NHE-1.     

Inhibition of Platelet Thromboxane Formation and Phosphoinositides Breakdown by Diisoeugenol

Abstract— Diisoeugenol inhibited the platelet aggregation and ATP release of rabbit platelets caused by ADP, arachidonic acid, platelet-activating factor (PAF), collagen and thrombin. Prolongation of the incubation time of platelets with diisoeugenol did not cause further inhibition and the aggregability of platelets could not be restored after washing. In human platelet-rich plasma, diisoeugenol inhibited the biphasic aggregation and ATP release induced by adrenaline and ADP in a concentration-dependent manner. Thromboxane B₂ formation caused by arachidonic acid, collagen and thrombin was markedly inhibited by diisoeugenol in a concentration-dependent manner. Diisoeugenol also inhibited the formation of inositol monophosphate caused by collagen, PAF and thrombin. The cAMP level of washed platelets was not changed by diisoeugenol. It is concluded that the antiplatelet effect of diisoeugenol is due to the inhibition of thromboxane formation and phosphoinositides breakdown.     

Triphenyltin fluoride in vitro inhibition of rabbit platelet collagen-induced aggregation and ATP secretion and blockade of arachidonic acid mobilization from membrane phospholipids

Recent studies have demonstrated that triphenyltin fluoride (TPTF) inhibits collagen-induced aggregation and ATP secretion of rabbit platelets in vivo [S. Manabe and O. Wada, J. Toxic. Sci. 6, 236 (1981)]. The aim of the present investigation was to test the effects in vitro of TPTF on platelet aggregation and to elucidate the mechanism of the inhibitory action by studying the release and metabolism of arachidonic acid and the cyclic AMP contents of rabbit platelets treated in vitro with TPTF. Although no inhibitory effect of TPTF was found on sodium arachidonate-induced platelet aggregation and ATP secretion, TPTF inhibited both reactions induced by collagen. Triphenylarsine and triphenylantimony did not inhibit, even at a concentration of 10(-3) M. The anti-aggregating concentration (IC50) of TPTF was 6.0 x 10(-6) M against collagen. TPTF had no inhibitory effect on the conversion of exogenous arachidonic acid to malondialdehyde (MDA) by platelets, while the collagen-induced production of arachidonate metabolites [MDA, 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and thromboxane B2] was remarkably inhibited by TPTF. Furthermore, TPTF apparently inhibited the collagen-induced release of arachidonic acid from platelets, although the formation of phosphatidic acid was not inhibited. Total cyclic AMP content after TPTF exposure was not changed significantly. These results indicate that TPTF inhibited the collagen-induced arachidonic acid release from platelet phospholipids, presumably by acting on phospholipase A2. Furthermore, it seems unlikely that the inhibition of arachidonic acid release by TPTF can be explained by the level of cyclic AMP in platelets.     

Antiplatelet Effects of Some Aporphine and Phenanthrene Alkaloids in Rabbits and Man

Two aporphines (boldine and laurolitsine) and five phenanthrene alkaloids (litebamine, secoboldine, N-cyanosecoboldine, N-methylsecoglaucine and N-methylsecopredicentrine) were evaluated in-vitro for their ability to inhibit platelet aggregation. All seven alkaloids inhibited aggregation of rabbit platelets and inhibited the release of ATP induced by arachidonic acid and collagen in rabbit platelets. Those aggregations induced by platelet-activating factor (PAF), thrombin, U46619 and ADP were inhibited by the three N-substituted secoboldine derivatives only. Thromboxane B₂ formation caused by arachidonic acid was also suppressed by these compounds. They did not affect the generation of [³H]inositol monophosphate caused by collagen, PAF and thrombin in the presence of indomethacin. Platelet cyclic AMP level was unaffected by litebamine, but was increased by N-methyl-secoglaucine. Litebamine suppressed the secondary aggregation, but not the primary aggregation, induced by ADP and adrenaline in platelet-rich plasma from man, whereas N-methylsecoglaucine inhibited both primary and secondary aggregation. It is concluded that the antiplatelet effect of these seven aporphine and phenanthrene alkaloids is mainly a result of inhibition of thromboxane A₂ formation; N-methylsecoglaucine has additional antiplatelet activity as a result of increasing the levels of platelet cyclic AMP.     

Novel inhibitory actions on platelet thromboxane and inositolphosphate formation by xanthones and their glycosides

Xanthones and their glycosides were tested for their antiplatelet activities in washed rabbit platelets. Tripteroside acetate and norathyriol acetate were the most potent inhibitors. Tripteroside acetate inhibited platelet aggregation and ATP release induced by ADP, arachidonic acid, platelet-activating factor (PAF), collagen, ionophore A23187 and thrombin. The IC50 values of tripteroside acetate toward arachidonic acid- (100 microM) and collagen- (10 micrograms/ml) induced platelet aggregation were 10 and 30 micrograms/ml respectively. It inhibited thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, thrombin and ionophore A23187 and also that caused by the incubation of lysed platelet homogenate with arachidonic acid. Tripteroside acetate decreased the formation of inositolphosphate caused by thrombin, collagen and PAF, whereas it had no direct effect on fibrinogen-platelet interaction. It is concluded that xanthone derivatives inhibited platelet aggregation and release reaction by diminishing thromboxane formation and phosphoinositide breakdown.     

Antiplatelet Mechanism of 2‐Chloro‐3‐(4‐hexylphenyl)‐amino‐1,4‐naphthoquinone (NQ304), an Antithrombotic Agent

Abstract: The effects of 2‐chloro‐3‐(4‐hexylphenyl)‐amino‐1,4‐naphthoquinone (NQ304), an antithrombotic agent, on aggregation, binding of fibrinogen to glycoprotein IIb/IIIa and intracellular signals were investigated using human platelets. NQ304 inhibited thrombin‐, arachidonic acid‐ and thapsigargin‐induced aggregation of washed human platelets with the IC₅₀ values of 22.2±0.7, 6.5±0.2, and 7.6±0.1 μM, respectively. NQ304 significantly inhibited fluorescein isothiocyanate‐conjugated fibrinogen binding to human platelet surface glycoprotein IIb/IIIa receptor by 75%, but failed to inhibit the fibrinogen binding to purified glycoprotein IIb/IIIa receptor. This result suggests that NQ304 inhibit platelet aggregation by suppression of an intracellular pathway that involves exposure of the glycoprotein IIb/IIIa receptor, rather than by direct inhibition of fibrinogen‐glycoprotein IIb/IIIa binding. NQ304 significantly inhibited thrombin‐induced increase in intracellular Ca²⁺ mobilization at the dose of 30 μM and ATP secretion in a dose‐dependent manner. It also inhibited thrombin‐ and arachidonic acid‐induced thromboxane A₂ formation in human platelet dose‐dependently. In conclusion, the antiplatelet mechanism of NQ304 may be due to the reduction of the thromboxane A₂ formation, inhibition of adenosine triphosphate release and intracellular calcium mobilization.     

Mechanisms of antiplatelet activity of PC-09, a newly synthesized pyridazinone derivative

In this study, we examined whether PC-09, a new pyridazinone derivative, has antiplatelet activity in vitro and further investigated the possible mechanisms involved. Pretreatment with PC-09 resulted in an inhibition on rabbit platelet aggregation and ATP release induced by arachidonic acid, collagen or thrombin, with the IC(50) values of 5.4 to 76.8 muM. The thromboxane B(2) formation caused by collagen or thrombin was markedly inhibited by PC-09, but there was no alteration in that caused by arachidonic acid. The rise of platelet intracellular calcium level stimulated by aggregation agonists and collagen-induced platelet membrane surface glycoprotein IIb/IIIa expression was also reduced by PC-09. In addition, PC-09 itself significantly increased the cyclic AMP level through inhibiting cyclic AMP phosphodiesterase activity. These findings demonstrate that PC-09 is an inhibitor of platelet aggregation, which may be associated with mechanisms including inhibition of thromboxane A(2) formation, intracellular calcium mobilization and platelet surface GPIIb/IIIa expression accompanied by increasing cyclic AMP level.     

Antiplatelet effects of piplartine,an alkamide isolated from Piper tuberculatum: possible involvement of cyclooxygenase blockade and antioxidant activity

Objectives Piplartine (piperlongumine; 5,6‐dihydro‐1‐[1‐oxo‐3‐(3,4,5‐trimethoxyphenyl]‐2(1H) pyridinone) is an alkaloid amide isolated from Piper species (Piperaceae). It has been reported to show multiple pharmacological activities in vitro and in vivo. Methods We evaluated the in‐vitro antiplatelet effect of piplartine isolated from the roots of P. tuberculatum, on human platelet aggregation induced in platelet‐rich plasma by the agonists collagen, adenosine 5′‐diphosphate (ADP), arachidonic acid (AA) and thrombin. Key findings Piplartine (100μg/ml) caused a 30% inhibition in platelet aggregation when collagen was the agonist. At 200 μg/ml, piplartine significantly inhibited the aggregation induced by arachidonic acid (100%), collagen (59%) or ADP (52%) but not that induced by thrombin. The highest concentration of piplartine (300 μg/ml) inhibited thrombin‐ (37%), ADP‐ (71%) and collagen‐ (98%) induced aggregation. The inhibitory effect of piplartine on ADP‐induced platelet aggregation was not modified by pretreatment with pentoxifylline (a phosphodiesterase inhibitor), l ‐arginine (a substrate for nitric oxide synthase) or ticlopidine (a P2Y₁₂ purinoceptor antagonist). However, aspirin, a well‐known inhibitor of cyclooxygenase, greatly increased the inhibitory effect of piplartine on arachidonic‐acid‐induced platelet aggregation. Conclusions The mechanism underlying the piplartine antiplatelet action is not totally clarified. It could be related to the inhibition of cyclooxgenase activity and a decrease in thromboxane A₂ formation, similar to that occurring with aspirin. This and other possible mechanisms require further study.     

Effects of cryptolepine on collagen-induced aggregation and on the mobilization and metabolism of arachidonic acid by rabbit platelets

1. We examined the effect of cryptolepine on collagen-induced aggregation and on the mobilization, and metabolism of arachidonic acid in rabbit platelets.2. Preincubation of platelets with cryptolepine (50–100 μM) did not affect the primary wave of aggregation but resulted in a dose-dependent, surmountable inhibition of the secondary wave of aggregation induced by collagen (5 μg/ml). The inhibition by cryptolepine was greater when cryptolepine was incubated with the platelets after the peak of the primary wave of aggregation.3. Cryptolepine (50–100 μM) dose-dependently inhibited thrombin (1.5 U/ml) and A23187 (2.5 μM)-induced release of ¹⁴C[AA] from platelet membrane phospholipid pools. The percentage inhibition of A23187-induced ¹⁴C[AA] release was 31.3 ± 4.3% (50 μM) and 79.3 ± 5.4% (100 μM), while thrombin-induced release was inhibited by 39.2 ± 2.4% (50 μM) and 68.2 ± 3.6% (100 μM).4. At near maximal concentration (100 μM) which significantly inhibited secondary aggregatory response and ¹⁴C[AA] release, cryptolepine had no effect on the platelet metabolism of ¹⁴C[AA] to thromboxane B₂, HHT and 12 HETE.5. The present findings suggest that cryptolepine inhibited collagen-induced secondary aggregation through a selective antiphospholipase-like activity. There was not effect on platelet cyclooxygenase and lipoxygenase activities of platelets.     

NP-313, 2-acetylamino-3-chloro-1,4-naphthoquinone, a novel antithrombotic agent with dual inhibition of thromboxane A(2) synthesis and calcium entry

BACKGROUND AND PURPOSE

1,4-Naphthoquinones exhibit antiplatelet activity both in vivo and in vitro. In the present study, we investigated the antiplatelet effect of a novel naphthoquinone derivative NP-313, 2-acetylamino-3-chloro-1,4-naphthoquinone and its mechanism of action.

EXPERIMENTAL APPROACH

We measured platelet aggregation, Ca²⁺ mobilization, thromboxane B2 formation and P-selectin expression and examined several enzymatic activities. Furthermore, we used the irradiated mesenteric venules in fluorescein sodium–treated mice to monitor the antithrombotic effect of NP-313 in vivo.

KEY RESULTS

NP-313 concentration-dependently inhibited human platelet aggregation induced by collagen, arachidonic acid, thapsigargin, thrombin and A23187. NP-313 also inhibited P-selectin expression, thromboxane B₂ formation and [Ca²⁺]_i elevation in platelets stimulated by thrombin and collagen. NP-313 at 10 µM inhibited cyclooxygenase, thromboxane A₂ synthase, and protein kinase Cα, whereas it did not affect phospholipase A₂ or phospholipase C activity. In the presence of indomethacin and an adenosine 5-diphosphate scavenger, NP-313 concentration-dependently inhibited thrombin- and A23187-induced [Ca²⁺]_i increase through its inhibitory effects on Ca²⁺ influx, rather than blocking Ca²⁺ release from intracellular stores. NP-313 also inhibited thapsigargin-mediated Ca²⁺ influx through store-operated calcium channel but had no effect on Ca²⁺ influx through store-independent calcium channel evoked by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol. Nevertheless, it had little effect on cyclic AMP and cyclic GMP levels. Also, intravenously administered NP-313 dose-dependently inhibited the thrombus occlusion of the irradiated mesenteric vessels of fluorescein-pretreated mice.

CONCLUSIONS AND IMPLICATIONS

Taken together, these results indicate that NP-313 exerts its antithrombotic activity through dual inhibition of thromboxane A₂ synthesis and Ca²⁺ influx through SOCC.     

Effects of brazilin on the phospholipase A2 activity and changes of intracellular free calcium concentration in rat platelets

Brazilin {7,11 b-dihydrobenz[b]indeno[1,2-d]pyran-3,6a,9,10(6H)-tetrol} inhibited thrombin-, collagen-and ADP-induced aggregation of washed rat platelets. Thrombin- and collagen-induced ATP release were also inhibited by brazilin in a concentration-dependent manner. Brazilin inhibited the formation of platelet thromboxane A₂ caused by thrombin, whereas it had no effect on the prostaglandin D₂ formation. Brazilin inhibited [³H]-arachidonic acid liberation from membrane phospholipids of thrombin-stimulated platelets. Brazilin inhibited the rise of intracellular free calcium caused by thrombin. These results indicate that the inhibition of phospholipase (PLA₂) activity and [Ca²⁺]_i elevation might be at least a part of antiplatelet mechanism of brazilin.     

Anti-inflammatory and platelet anti-aggregant activity of phospholipase-A2 inhibitors

Mepacrine, papaverine, p-bromophenacyl bromide and 2,3-dibromo(4′-cyclohexyl-3′-chloro)-phenyl-4-oxo-butyric acid (CB 874) inhibit the hydrolysis of phospholipids induced by thrombin in dog platelets. They also exhibit anti-inflammatory and anti-aggregant properties. These biological activities may be explained by a direct or indirect inhibitory action on phospholipase A₂. Phospholipase A₂ inhibitors may block not only the release of arachidonic acid and its subsequent conversion into prostaglandins but also the formation of lysophospholipids involved in inflammation and/or platelet aggregation.     

The protective effects of paclitaxel on platelet aggregation through the inhibition of thromboxane A<Subscript>2</Subscript> synthase

Paclitaxel is an anticancer drug used in the treatment of ovarian, breast, head and neck, lung, and prostate cancer. We investigated the anti-platelet activity of paclitaxel in vitro as well as a possible anti-platelet mechanism. Paclitaxel inhibited washed rabbit platelet aggregation induced by collagen in a concentration-dependent manner, with an IC₅₀ of 59.7 ± 3.5. However, it had little effect on platelet aggregation mediated by arachidonic acid, U46619, a thromboxane (TX) A₂ mimic, or thrombin, suggesting that paclitaxel may strongly inhibit collagenmediated signal transduction. In accordance with these findings, paclitaxel blocked collageninduced cytosolic calcium mobilization, arachidonic acid liberation, and serotonin secretion. In addition, it inhibited arachidonic acid-mediated platelet aggregation by about 37% by interfering with TXA₂ synthase as measured by the formation of arachidonic acid-mediated TXA₂ and prostaglandin D₂, as well as cyclooxygenase-1 and TXA₂ synthase activity assays. Taken together, these results point to a cellular mechanism for the anti-platelet activity of paclitaxel through the inhibition of TXA₂ synthase and cytosolic calcium mobilization. This may contribute to the beneficial effects of paclitaxel on the cardiovascular system.     

Inhibition by picotamide of thromboxane production in vitro and ex vivo

Summary The effect of picotamide on platelet function has been studied in vitro and ex vivo.Picotamide at micromolar concentrations inhibited platelet aggregation induced by ADP, arachidonic acid and collagen, and it also inhibited the production of thromboxane A₂ (TxA₂). Unlike aspirin, picotamide did not affect the synthesis of prostacyclin by blood vessels.In eight healthy subjects who took picotamide 1200 mg/d platelet aggregation and TxA₂ production were inhibited.Picotamide appears to be an antiplatelet drug that reduces TxA₂ synthesis without affecting cyclooxygenase activity.     

Alterations in Membrane Transport Function and Cell Viability Induced by ATP Depletion in Primary Cultured Rabbit Renal Proximal Tubular Cells

This study was undertaken to elucidate the underlying mechanisms of ATP depletion-induced membrane transport dysfunction and cell death in renal proximal tubular cells. ATP depletion was induced by incubating cells with 2.5 mM potassium cyanide (KCN)/0.1 mM iodoacetic acid (IAA), and membrane transport function and cell viability were evaluated by measuring Na⁺-dependent phosphate uptake and trypan blue exclusion, respectively. ATP depletion resulted in a decrease in Na⁺-dependent phosphate uptake and cell viability in a time-dependent manner. ATP depletion inhibited Na⁺-dependent phosphate uptake in cells, when treated with 2 mM ouabain, a Na⁺ pump-specific inhibitor, suggesting that ATP depletion impairs membrane transport functional integrity. Alterations in Na⁺-dependent phosphate uptake and cell viability induced by ATP depletion were prevented by the hydrogen peroxide scavenger such as catalase and the hydroxyl radical scavengers (dimethylthiourea and thiourea), and amino acids (glycine and alanine). ATP depletion caused arachidonic acid release and increased mRNA levels of cytosolic phospholipase A₂ (cPLA₂). The ATP depletion-dependent arachidonic acid release was inhibited by cPLA₂ specific inhibitor AACOCF₃. ATP depletion-induced alterations in Na⁺-dependent phosphate uptake and cell viability were prevented by AACOCF₃. Inhibition of Na⁺-dependent phosphate uptake by ATP depletion was prevented by antipain and leupetin, serine/cysteine protease inhibitors, whereas ATP depletion-induced cell death was not altered by these agents. These results indicate that ATP depletion-induced alterations in membrane transport function and cell viability are due to reactive oxygen species generation and cPLA₂ activation in renal proximal tubular cells. In addition, the present data suggest that serine/cysteine proteases play an important role in membrane transport dysfunction, but not cell death, induced by ATP depletion.     

Bakkenolide G,a Natural PAF-receptor Antagonist

Because platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) participates in many physiopathological responses, including inflammatory reaction, endotoxic shock, allergic diseases and platelet aggregation, PAF-receptor antagonists are important in the treatment of these diseases. A biologically active compound, bakkenolide G, extracted from the plant Petasites formosanus selectively and concentration-dependently inhibited PAF-induced platelet aggregation and ATP release. The IC50 of bakkenolide G for PAF (2 ng mL^?1)-induced platelet aggregation was 5.6 ± 0.9 μm . Bakkenolide G also concentration-dependently inhibited PAF-induced intracellular signal transductions, including thromboxane B₂ formation, and increased intra-cellular calcium concentration and phosphoinositide breakdown without affecting those caused by thrombin (01 units mL^?1), collagen (10 μg mL^?1), arachidonic acid (100 μm ) and U46619 (1 μm ). Bakkenolide G shifted the concentration-response curves of PAF-induced platelet aggregation parallel to the right; the Schild plot slope and the pA₂ value were 1.31 ± 0.31 and 6.21 ± 0.75, respectively. Moreover, bakkenolide G concentration-dependently competed with [³H]PAF binding to platelets, with an IC50 value of 2.5 ± 0.4 μm . These data strongly indicate that bakkenolide G is a specific PAF-receptor antagonist as an antiplatelet aggregatory agent.     

Antiplatelet Effect of Gingerol Isolated from Zingiber officinale

The purpose of this investigation was to determine the antiplatelet mechanism of gingerol. Gingerol concentration-dependently (0·5–20 μm ) inhibited the aggregation and release reaction of rabbit washed platelets induced by arachidonic acid and collagen, but not those induced by platelet-activating factor (PAF), U46619 (9,11-dideoxy-9α,11 α-methano-epoxy-PGF_2α) and thrombin. Gingerol also concentration-dependently (0·5–10μ m ) inhibited thromboxane B₂ and prostaglandin D₂ formation caused by arachidonic acid, and completely abolished phosphoinositide breakdown induced by arachidonic acid but had no effect on that of collagen, PAF or thrombin even at concentrations as high as 300 μ m . In human platelet-rich plasma, gingerol and indomethacin prevented the secondary aggregation and blocked ATP release from platelets induced by adenosine 5′-diphosphate (ADP, 5 μ m ) and adrenaline (5 ä m ) but had no influence on the primary aggregation. The maximal antiplatelet effect was obtained when platelets were incubated with gingerol for 30 min and this inhibition was reversible. It is concluded that the antiplatelet action of gingerol is mainly due to the inhibition of thromboxane formation.     

Inhibitory effect of various traditional Chinese medicines on rabbit platelet phospholipase A2 in vitro and suppressive effect of t?ki-syakuyaku-san on increased aggregability in hypercholesterolemic rabbit ex vivo

The inhibitory mechanism of 6 traditional Chinese medicines on rabbit platelet aggregation in vitro, and the suppressive effect of oral administration of T?ki-syakuyakusan on hyper-aggregability of the platelet from rabbit fed high cholesterol diet for 2 months, were investigated. Collagen-induced aggregation was inhibited by Keisi-bukury?gan, Kami-sy?y?-san, Dai-saiko-t?, T?ki-syakuyaku-san, Hatimi-zi?-gan and Sy?-saiko-t? in their lower concentrations than those inhibiting arachidonic acid- and thrombin-induced aggregation. These traditional Chinese medicines inhibited the release of [3H]arachidonic acid from membrane phospholipids by phospholipase A2, in [3H]arachidonic acid-labelled platelets under stimulation with collagen and thrombin in the concentration ranges that inhibited each aggregation. In their higher concentrations to inhibit arachidonic acid-induced aggregation, they suppressed the conversion of arachidonic acid to thromboxane A2 by about 50%. However, they had no effect on diacylglycerol formation induced by thrombin. The oral administration of T?ki-syakuyaku-san depressed the increased aggregability of platelets from rabbit fed high cholesterol diet by 20-40% at the period of 1-2 months of feeding, without affecting plasma and platelet cholesterol level. These results indicate that the traditional Chinese medicines used here have an inhibitory effect on platelet phospholipase A2 activation, rather than on cyclooxygenase, and therefore inhibit platelet activation in vitro and ex vivo.