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.     

Comparison of the actions of some platelet-activating factor antagonists on platelets and aortic smooth muscles.

The pharmacological actions of five platelet-activating factor (PAF) antagonists were compared in rabbit platelets and rat thoracic aorta. In PAF (2 ng/ml)-induced aggregation of washed rabbit platelets, WEB 2086 and WEB 2170 much were more potent inhibitors than BN 52021, kadsurenone and denudatin B, and the IC50 values were calculated to be 0.1, 0.3, 5, 8 and 10 micrograms/ml, respectively. WEB 2086, WEB 2170 and BN 52021 did not affect the platelet aggregation caused by collagen (10 micrograms/ml), ADP (20 microM), arachidonic acid (100 microM) or thrombin (0.1 U/ml). Kadsurenone and denudatin B suppressed ATP release, thromboxane B2 formation and the rise in intracellular calcium of washed rabbit platelets caused by collagen and thrombin, while WEB 2086, WEB 2170 and BN 52021 did not have an effect. Norepinephrine (3 microM) induced a sustained contraction in rat thoracic aorta. Pretreatment with these PAF antagonists (20-100 micrograms/ml) caused inhibition of the aortic contraction in the following order: kadsurenone greater than denudatin B greater than WEB 2086 greater than BN 52021 greater than WEB 2170. In high potassium (60 mM)-induced contraction of rat aorta, kadsurenone and denudatin B caused marked relaxation, while WEB 2086, WEB 2170 and BN 52021 had only a slight effect. It is concluded that WEB 2086, WEB 2170 and BN 52021 are specific PAF antagonists in rabbit platelets, and weak relaxants in rat aorta. Two other PAF antagonists, kadsurenone and denudatin B, may inhibit some aspects of signal transduction, e.g., thromboxane formation or intracellular Ca2+ mobilization in rabbit platelets, and cause vasorelaxation in rat aorta by inhibiting calcium influx.     

Dicentrine, a novel antiplatelet agent inhibiting thromboxane formation and increasing the cyclic AMP level of rabbit platelets.

Dicentrine is an antiplatelet agent isolated from the Chinese herb Lindera megaphylla. We examined the in vitro effects of dicentrine on various aspects of platelet reactivity. Dicentrine inhibited the aggregation and ATP release of washed rabbit platelets induced by arachidonic acid (AA), collagen, ADP, platelet-activating factor (PAF), thrombin and U46619. Dicentrine also inhibited the thromboxane B2 formation caused by AA, collagen and thrombin in washed intact platelets or that induced by AA in lysed platelet homogenate, while prostaglandin D2 formation caused by AA was not increased. The generation of inositol monophosphates (in the presence of indomethacin) caused by thrombin, collagen and PAF was not suppressed significantly, nor did dicentrine suppress fibrinogen-induced aggregation of elastase-treated platelets. Dicentrine inhibited the intracellular Ca2+ increase in quin-2/AM-loaded platelets caused by thrombin, PAF, collagen and AA. The cyclic AMP level was elevated by dicentrine in a concentration-dependent manner. These data indicate that the inhibitory effect of dicentrine on platelet aggregation and ATP release was due to the inhibition of thromboxane formation and the elevation of the level of cyclic AMP.     

YD-3, a novel inhibitor of protease-induced platelet activation

1. In the present study, the antiplatelet effects and mechanisms of a new synthetic compound YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole] were examined. 2. YD-3 inhibited the aggregation of washed rabbit platelets caused by thrombin (IC(50)=28.3 microM), but had no or little inhibitory effect on that induced by arachidonic acid, collagen, platelet-activating factor (PAF) or U46619. YD-3 also suppressed generation of inositol phosphates caused by thrombin. On the other hand, thrombin-induced fibrin formation was not affected by YD-3, indicating YD-3 does not inhibit the proteolytic activity of thrombin. 3. In washed human platelets, however, YD-3 had only mild inhibitory effect on the low concentration (0.05 u ml(-1)) of thrombin-induced human platelet aggregation, and did not affect that induced by higher concentrations (> or =0.1 u ml(-1)) of thrombin or SFLLRN, the protease-activated receptor 1 (PAR1) agonist peptide. By contrast, YD-3 inhibited both human and rabbit platelet aggregation elicited by trypsin with IC(50) values of 38.1 microM and 5.7 microM, respectively. 4. YD-3, at 100 microM, had no effect on ristocetin-induced glycoprotein Ib (GPIb)-dependent aggregation of human platelets. In addition, platelets treated with chymotrypsin, which cleaves GPIb, enhanced rather than attenuated the inhibition of YD-3 on thrombin-induced human platelet aggregation. These data indicate that GPIb plays no role in the antiplatelet effect of YD-3. 5. In SFLLRN-desensitized human platelets, high concentration of thrombin (1 u ml(-1)) could still elicit intracellular Ca(2+) mobilization, and the rise of [Ca(2+)](i) was prevented by either leupeptin or YD-3. 6. Our results suggest that YD-3 inhibits a non-PAR1 thrombin receptor which mediates the major effect of thrombin in rabbit platelets, but in human platelets, this receptor function becomes significant only when the function of PAR1 has been blocked or attenuated.     

Antiplatelet effect of marchantinquinone, isolated from Reboulia hemisphaerica, in rabbit washed platelets

Platelet activation is involved in serious pathological situations, including atherosclerosis and restenosis. It is important to find efficient antiplatelet medicines to prevent fatal thrombous formation during the course of these diseases. Marchantinquinone, a natural compound isolated from Reboulia hemisphaerica, inhibited platelet aggregation and ATP release stimulated by thrombin (0.1 units mL(-1)), platelet-activating factor (PAF; 2 ng mL(-1)), collagen (10 microg mL(-1)), arachidonic acid (100 microM), or U46619 (1 microM) in rabbit washed platelets. The IC50 values of marchantinquinone on the inhibition of platelet aggregation induced by these five agonists were 62.0 +/- 9.0, 86.0 +/- 7.8, 13.6 +/- 4.7, 20.9 +/- 3.1 and 13.4 +/- 5.3 microM, respectively. Marchantinquinone inhibited thromboxane B2 (TxB2) formation induced by thrombin, PAF or collagen. However, marchantinquinone did not inhibit TxB2 formation induced by arachidonic acid, indicating that marchantinquinone did not affect the activity of cyclooxygenase and thromboxane synthase. Marchantinquinone did inhibit the rising intracellular Ca2+ concentration stimulated by the five platelet-aggregation inducers. The formation of inositol monophosphate induced by thrombin was inhibited by marchantinquinone. Platelet cAMP and cGMP levels were unchanged by marchantinquinone. The results indicate that marchantinquinone exerts antiplatelet effects by inhibiting phosphoinositide turnover.     

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.     

An antagonistic activity of etizolam on platelet-activating factor (PAF). In vitro effects on platelet aggregation and PAF receptor binding

The antagonistic effect of etizolam, an anti-anxiety drug, on platelet-activating factor (PAF) was investigated in rabbit platelets in vitro. Etizolam inhibited PAF-induced aggregation in a dose-dependent manner, with an IC50 of 3.8 microM, about one tenth that of triazolam (IC50 = 30 microM). At 300 microM, it inhibited both ADP and arachidonic acid-induced aggregation only slightly, while the other anti-anxiety drugs tested had no effect on PAF-induced aggregation even at this concentration. Etizolam and triazolam inhibited the specific binding of 3H-PAF to PAF receptor sites on washed rabbit platelets with IC50 values of 22 nM and 320 nM, respectively. Diazepam and estazolam were inactive even at 1 microM. These results indicate that etizolam is a specific antagonist of PAF.     

丹酚酸B镁盐对兔洗涤血小板聚集和5-HT释放反应的影响(英文)

AIM: To study the effects and mechanism of magnesium lithospermate B(MLB) on rabbit platelet aggregation and 5-HT release. METHODS: The platelet aggregation was determined by Born's method. Release of serotonin (5-HT) and formation of thromboxane A2 (TXA2) were measured by fluorophotometry and radioimmunoassay (RIA) respectively. Cytoplasmic free Ca2+ concentration ([Ca2+]i) in platelets was measured by Fura 2-AM fluorescence technique. RESULTS: In washed platelets, thrombin (200 U/L) or arachidonic acid (AA) (30 mumol/L)-induced aggregation was inhibited by MLB 50-800 mg/L in a concentration-dependent manner. In addition, MLB had more inhibitory effects on platelet aggregation in the absence of extracellular calcium with IC50 of 102 mg/L than in the presence of CaCl2 1 mmol/L with IC50 of 194 mg/L. MLB concentration-dependently decreased the thrombin-activated release of 5-HT, whereas it did not affect the formation of TXA2 in platelets. Furthermore, MLB not only inhibited the rise of [Ca2+]i in thrombin stimulated platelets, but decreased the [Ca2+]i in resting platelets. CONCLUSION: MLB inhibited the aggregation and 5-HT release in rabbit platelets and it is probably by attenuating intracellular calcium concentration.     

Platelet-activating factor synthesis by peritoneal mast cells and its inhibition by two quinoline-based compounds.

1. Peritoneal mast cells from rat were co-incubated in vitro in a platelet aggregometer cuvette with washed rabbit platelets. In response to stimulation with calcium ionophore (A23187; 1-5 microM), the mast cells released a substance which stimulated the platelets to aggregate. These concentrations of ionophore did not stimulate platelet aggregation in the absence of mast cells, nor affect the responsiveness of the platelets to aggregation induced by thrombin or PAF. Release of a PAF-like substance was also observed in response to stimulation of the mast cells with antigen. 2. This pro-aggregatory activity is attributable to the release of PAF by the mast cells, since the activity could be abolished by preincubating the platelets with a specific PAF receptor antagonist (WEB 2086; 10 microM). Furthermore, the platelet-aggregating factor co-migrated with PAF on thin-layer chromatographs and could be abolished by incubation with phospholipase A2 (20 micrograms ml-1) or a specific antibody directed against PAF. 3. The release of PAF by peritoneal mast cells could be inhibited, in a concentration-dependent manner, by PF-5901 (IC50 of 3.9 microM) or Wy-50,295 (IC50 of 1.2 microM), two structurally similar compounds with inhibitory effects on leukotriene synthesis, as well as leukotriene D4 (LTD4) receptor antagonist properties. 4. Inhibition of PAF synthesis was not observed when the mast cells were incubated with a structurally unrelated 5-lipoxygenase inhibitor (A-64077), a structurally dissimilar inhibitor of 5-lipoxygenase activating protein (MK-886) or with a structurally related LTD4 receptor antagonist (MK-571) which lacks inhibitory effects on leukotriene synthesis, each at concentrations of up to 100 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The dopamine D(1) receptor agonist SKF-82958 serves as a discriminative stimulus in the rat

The antiplatelet effect of the pyridazinone analogue, 4, 5-dihydro-6-[4-[2-hydroxy-3-(3,4 dimethoxybenzylamino)propoxy]naphth-1-yl]-3(2H)-pyridazinone (HCL-31D), was investigated in vitro with rabbit platelets. HCL-31D dose-dependently inhibited the platelet aggregation and ATP release induced by collagen (10 microg/ml), arachidonic acid (100 microM) or thrombin (0.1 U/ml) with an IC(50) of about 0.95-5.41 microM. HCL-31D (0.5-5 microM) increased the platelet cyclic AMP level in a dose-dependent manner. Furthermore, HCL-31D potentiated cyclic AMP formation caused by prostaglandin E(1) but not that caused by 3-isobutyl-1-methylxanthine (IBMX). HCL-31D also attenuated phosphoinositide breakdown and intracellular Ca(2+) elevation induced by collagen, arachidonic acid or thrombin. HCL-31D inhibited the formation of thromboxane B(2) induced by collagen or thrombin but not by arachidonic acid. In addition, HCL-31D did not affect platelet cylooxygenase and thromboxane synthase activity. These data indicate that HCL-31D is an inhibitor of phosphodiesterase and that its antiplatelet effect is mainly mediated by elevation of cyclic AMP levels.     

Potentiation by adrenaline of human platelet activation and the inhibition by the alpha-adrenergic antagonist nicergoline of platelet adhesion, secretion and aggregation

Adrenaline (1 to 10 microM) can induce the aggregation of human platelets suspended in citrated plasma but does not induce the aggregation of washed human platelets at doses as high as 1 mM, although these platelets respond normally to ADP, PAF-acether, collagen, arachidonic acid, thrombin, the endoperoxide analog U-46619 and the Ca2+ ionophore A23187. Adrenaline (0.5 microM) potentiates the aggregation and secretion induced by all the previous agonists in citrated platelet-rich plasma (cPRP) or in washed platelets. The activation by adrenaline of human platelets is mediated by alpha 2-adrenergic receptors, as demonstrated by inhibition with a series of adrenergic antagonists. The alpha-adrenergic antagonist nicergoline inhibits the activation of human platelets by adrenaline in the following situations: nicergoline inhibits the aggregation and secretion caused by adrenaline in cPRP (IC50 0.22 microM and 0.28 microM respectively); nicergoline inhibits the aggregation and secretion induced by the combination of adrenaline and each aggregating agent listed above in cPRP (IC50 ranging from 0.1 to 2.5 microM) or in washed platelets (IC50 ranging from 0.1 to 0.8 microM); nicergoline inhibits the binding of 3H-yohimbine to washed human platelets (IC50 0.26 microM); the intravenous administration of nicergoline (0.5 mg/kg per day) to patients inhibits significantly the ex vivo response of their platelets to adrenaline in cPRP. High concentrations of nicergoline also inhibit the aggregation and secretion induced by the aggregating agents listed above in cPRP (IC50 range 108 to 670 microM) and in washed platelets (IC50 range 27 to 140 microM) and the adhesion of platelets to collagen-coated surfaces. This latter effect is not mediated through blockade of alpha-adrenoceptors. A possible role of adrenaline in platelet activation in vivo could justify the use of nicergoline (Sermion), an alpha-adrenergic antagonist in combination therapy to prevent arterial thrombosis.     

Effect of a potent cyclooxygenase inhibitor, 5-ethyl-4-methoxy-2-phenylquinoline (KTC-5), on human platelets

Because the metabolites of arachidonic acid participate in many physiopathological responses, including inflammation and platelet aggregation, cyclooxygenase inhibitors are important in the treatment of associated diseases. A biologically active compound, 5-ethyl-4-methoxy-2-phenylquinoline (KTC-5), selectively and concentration dependently inhibited aggregation of platelets from man and ATP release caused by arachidonic acid (200 microM) and collagen (10 microg mL(-1)) without affecting the aggregation caused by thrombin (0.1 U mL(-1)) and U46619 (2 microM). The IC50 value (drug concentration inhibiting maximum response by 50%) of KTC-5 for aggregation induced by arachidonic acid and collagen was 0.11+/-0.04 microM and 0.20+/-0.03 microM, respectively. This inhibitory effect of KTC-5 was reversible and time dependent. KTC-5 specifically inhibited intracellular calcium mobilization initiated by arachidonic acid or collagen without affecting that caused by thrombin or U46619 in human platelets. Furthermore, KTC-5 inhibited thromboxane B2 and prostaglandin D2 formation provoked by arachidonic acid. The IC50 value of KTC-5 for arachidonic-acid-induced thromboxane B2 formation was 0.07+/-0.02 microM. Based on these observations, the data indicated that KTC-5 potently inhibited human platelet aggregation and ATP release mainly via the inhibition of the cyclooxygenase-1 activity. Moreover, KTC-5 inhibited lipopolysaccharide-induced prostaglandin E2 formation in RAW264.7 cells in the presence of external arachidonic acid with an IC50 value of 0.17+/-0.06 microM. Immunoblot analysis showed that KTC-5 did not affect the cyclooxygenase-2 expression in the presence of lipopolysaccharide on RAW264.7 cells. This result indicated that KTC-5 affects the activity of cyclooxygenase-2. According to these data, we concluded that KTC-5 is a cyclooxygenase inhibitor for both subtypes.     

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.     

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.     

Regulation of human platelet activation--analysis of cyclooxygenase and cyclic AMP-dependent pathways

We have studied the regulation of human platelet activation by cyclic AMP (cAMP), and the cyclooxygenase products by examining the effect of prostacyclin (PGI2) and indomethacin on platelet aggregation, release reaction and thromboxane B2 (TxB2) generation induced by the full dose range of common platelet agonists in both platelet-rich plasma and washed platelets. Platelet aggregation, [14C]-5HT and TxB2 release induced by "threshold" and "supramaximal" concentrations of ADP, adrenaline, platelet-activating factor (PAF) and U46619 were totally abolished by low concentrations of PGI2 (3-6 nM). In contrast, platelet activation induced by submaximal concentrations of collagen, thrombin and the calcium ionophore A23187 was only partially inhibited by PGI2 (3-3000 nM). PAF-induced release reaction like that induced by ADP and adrenaline was totally dependent on the cyclooxygenase products and aggregation, while U46619-induced release reaction was only partially dependent on aggregation and the cyclooxygenase products. While both PGI2 (18-3000 nM) and indomethacin (10 microM) abolished collagen-induced aggregation and the aggregation-mediated release reaction, neither inhibitor significantly inhibited platelet adhesion or the adhesion-mediated release reaction. Maximal thrombin-induced aggregation and release reaction was also not significantly inhibited by PGI2 (300 nM) or indomethacin (10 microM). Thromboxane (TxB2) generation induced by sub-maximal to maximal concentrations of collagen, thrombin and A23187 was, although significantly inhibited, not abolished by PGI2. These results demonstrate that PAF is a "weak" agonist similar to ADP and adrenaline, U46619 is an agonist intermediate between weak and strong which induces a release reaction that is only partially dependent on aggregation, but unlike the strong agonists, is totally susceptible to inhibition by PGI2, PGI2 is an indirect inhibitor of phospholipase activation, which does not significantly inhibit non-aggregation-mediated arachidonate mobilization, induced by the strong agonists, and the so-called third pathway in the collagen and thrombin-induced release reaction, which is insensitive to indomethacin, is also insensitive to elevators of cAMP such as PGI2.     

At low extracellular calcium concentration platelet activating factor induces beta-thromboglobulin release from human platelets through thromboxane-endoperoxides formation

The aim of this study was to investigate the mechanism involved in beta-thromboglobulin (BTG) release induced by platelet activating factor (PAF) in human platelet-rich plasma (PRP) and washed platelets (WP) during aggregation. PAF was used in PRP at increasing concentrations starting at its threshold concentration for irreversible aggregation (TAC: 90-150 nM). In citrated PRP, PAF induced release of BTG (80-95% of total content) and thromboxane B2 (TXB2) formation (30-40 pmol/ml). At low PAF concentrations aggregation and BTG release were blocked by apyrase (a scavenger of ADP), by ASA (an inhibitor of cyclooxygenase) and by BM 13177 (a thromboxane receptor antagonist). Higher concentrations of PAF overcame the effect of apyrase, but only induced reversible aggregation and minor release in the presence of ASA or BM 13177. In heparinized PRP, PAF induced full irreversible aggregation, but only very low BTG release (about 25% of total content) and thromboxane synthesis (2-3 pmol/ml). WP resuspended in the presence of 2 mmol/l Ca2+ seldom responded to PAF alone, as previously shown by others, but full aggregation could be induced by concomitant addition of subthreshold concentrations of PAF (25-50 nM) and epinephrine (1 microM). In these conditions average BTG release from WP was less than 20% of the amount releasable by thrombin. In contrast, when WP were resuspended in the absence of Ca2+, stimulation by PAF + EPI induced sustained BTG release (40-50% of total content) and TXB2 synthesis (15-20 pmol/ml). We conclude that at low Ca2+ concentration PAF induces BTG release mainly through thromboxane-endoperoxides formation. In contrast, when [Ca2+] is normal, PAF does not or weakly induces thromboxane formation and BTG release.     

Characterization of the L-arginine:nitric oxide pathway in human platelets.

1. The activation of the L-arginine: nitric oxide (NO) pathway during aggregation of human platelets by adenosine 5'-diphosphate (ADP), arachidonic acid, thrombin and the calcium ionophore A23187 and its inhibition by NG-monomethyl-L-arginine (L-NMMA), NG-nitro-L-arginine methyl ester (L-NAME) and N-iminoethyl-L-ornithine (L-NIO) were studied. The inhibition of the cytosolic platelet NO synthase by these compounds was also examined. 2. Platelet aggregation induced by ADP (1-10 microM) and arachidonic acid (0.1-10 microM), but not that induced by thrombin (1-30 mu ml-1) or A23187 (1-10 nM), was inhibited by L-, but not D-arginine (1-30 microM). However, in the presence of a subthreshold concentration of prostacyclin (0.1 nM) or of M & B 22948 (1 microM), a selective inhibitor of guanosine 3':5'-cyclic monophosphate (cyclic GMP) phosphodiesterase, L-arginine caused concentration-dependent inhibition of aggregation induced by all of these aggregating agents. 3. L-NMMA, L-NAME and L-NIO (all at 1-30 microM), but not their D-enantiomers, enhanced to the same extent platelet aggregation induced by ADP, arachidonic acid and thrombin without affecting that induced by A23187. 4. In the presence of 300 microM L-arginine, the NO synthase in platelet cytosol was inhibited by L-NMMA, L-NAME and L-NIO with IC50s of 74 +/- 9, 79 +/- 8 and 8.5 +/- 1.5 microM (n = 3), respectively. 5. These results indicate that the L-arginine: NO pathway in human platelets plays a role in the modulation of platelet aggregation.     

Antiplatelet effect of amlodipine: a possible mechanism through a nitric oxide-mediated process.

The effect of amlodipine, a novel calcium channel blocker of the dihydropyridine type, on rabbit platelet aggregation, and the possible antiaggregatory mechanisms of amlodipine, especially on the nitric oxide (NO) guanosine 3',5'-cyclic monophosphate (cyclic GMP)-mediated pathway, were investigated. Other effects of amlodipine on thromboxane B2 (TXB2) formation in platelets also were examined. Amlodipine concentration-dependently inhibited rabbit platelet aggregation induced by collagen (10 microg/mL) or thrombin (0.1 U/mL) with an IC50 range of 32-69 microM. Along with this inhibition, our results also demonstrated that in the presence of L-arginine (100 IM), amlodipine (50 microM) increased nitric oxide synthetase (NOS) activity (from the resting activity of 2.05+/-0.36 to 7.11+/-0.95 pmol/mg protein/min) and NO release (by 80%), accompanied by an elevation of the cyclic GMP level (from the resting platelet level of 1.27+/-0.12 to 6.21+/-0.55 pmol/10(9) platelets) induced by collagen (10 microg/mL). However, the antiaggregatory effect of amlodipine (50 microM) could be attenuated significantly by oxyhemoglobin (5 microM), a NO scavenger, or N(G)-nitro-L-arginine methyl ester (100 microM), a specific NOS inhibitor. In addition, the TXB2 production in platelets induced by collagen or thrombin was concentration-dependently inhibited by amlodipine. Therefore, we propose that the antiaggregatory mechanisms of amlodipine might be mediated, in part, by a NO-cyclic GMP process accompanied by the inhibition of TXB2 formation in platelets.     

The antiplatelet activity of PMC, a potent alpha-tocopherol analogue, is mediated through inhibition of cyclo-oxygenase.

PMC, a potent alpha-tocopherol derivative, dose-dependently (5-25 microM) inhibited the ATP-release reaction and platelet aggregation in washed human platelets stimulated by agonists (collagen and ADP). PMC also dose-dependently inhibited the intracellular Ca2+ mobilization, whereas it did not inhibit phosphoinositide breakdown in human platelets stimulated by collagen. PMC (10 and 25 microM) significantly inhibited collagen-stimulated thromboxane A2 (TxA2) formation in human platelets. On the other hand, PMC (25 and 100 microM) did not increase the formation of cyclic AMP or cyclic GMP in platelets. Moreover, PMC (25, 100, and 200 microM) did not affect the thromboxane synthetase activity of aspirin-treated platelet microsomes. PMC (10 and 25 microM) markedly inhibited the exogenous arachidonic acid (100 microM)-induced prostaglandin E2 (PGE2) formation in the presence of imidazole (600 microM) in washed human platelets, indicating that PMC inhibits cyclo-oxygenase activity. We conclude that PMC may exert its anti-platelet aggregation activity by inhibiting cyclooxygenase activity, which leads to reduced prostaglandin formation; this, in turn, is followed by a reduction of TxA2 formation, and finally inhibition of [Ca2+]i mobilization and ATP-release.