Oral administration of the thrombin receptor antagonist E5555 (atopaxar) attenuates intimal thickening following balloon injury in rats

Thrombin is a powerful agonist for a variety of cellular responses including platelet aggregation and vascular smooth muscle cell (SMC) proliferation. These actions are mediated by a thrombin receptor known as protease-activated receptor-1 (PAR-1). Recently we discovered that 1-(3-tert-butyl-4-methoxy-5-morpholinophenyl)-2-(5,6-diethoxy-7-fluoro-1-imino-1,3-dihydro-2H-isoindol-2-yl)ethanone hydrobromide (E5555, atopaxar) is a potent and selective thrombin receptor antagonist. This study characterized the pharmacological effects of E5555 on SMC proliferation in vitro and in a rat model of intimal thickening after balloon injury in vivo. E5555 selectively inhibited rat aortic SMC proliferation induced by thrombin and thrombin receptor-activating peptide (TRAP) with half maximal inhibitory concentration (IC(50)) values of 0.16 and 0.038 μM, respectively. E5555 did not inhibit rat SMC proliferation induced by basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) at concentrations up to 1μM. In addition, E5555 inhibited human aortic SMC proliferation induced by thrombin at concentrations of 0.3 and 3units/ml with IC(50) values of 0.028 and 0.079 μM, respectively, whereas it did not affect bFGF-induced proliferation at concentrations up to 1μM. Repeated oral administration of 30 mg/kg E5555 (once daily for 16 days) significantly reduced neointimal formation in the balloon-injured rat arterial model. These results suggested that a PAR-1 antagonist could be effective for treating restenosis following vascular intervention in addition to preventing thrombus formation. E5555 could thus have therapeutic potential for restenosis and chronic atherothrombotic disease.     

Quinones. 4. Novel eicosanoid antagonists: synthesis and pharmacological evaluation

A new series of omega-phenyl-omega-quinonylalkanoic acids and related compounds was synthesized. The compounds were tested for their inhibitory effects on U-44069-induced contraction of the rabbit aorta. (+/- )-7-(3,5,6-Trimethyl-1,4-benzoquinon-2-yl)-7-phenylheptanoic acid (4d) (AA-2414) with pA2 value of 8.28 was one of the most potent compounds. Compound 4d inhibited U-46619-induced contraction of the guinea pig lung (pA2 = 8.29) and U-44069-induced aggregation of the guinea pig platelet (IC50 = 3.5 x 10(-7) M). Compound 4d displaced the binding of [3H]U-46619 to guinea pig platelets (IC50 = 7.4 x 10(-9) M). Compound 4d also showed very potent inhibitory effects with an MED of 0.3 mg/kg (po) on U-46619-, LTD4-, PAF-, or IgG1-induced bronchoconstriction in guinea pigs. The enantiomers of 4d were prepared. The R-(+) isomer 8a was active in both in vitro and in vivo tests, but the S-(-) isomer 8b was much less active. We concluded that the antiasthmatic effects of 4d were based mainly on the TXA2 receptor antagonistic action. In addition, compound 4d showed potent inhibitory effects on PGD2-, PGF2 alpha-, and 11-epi-PGF2 alpha-induced contraction of the guinea pig tracheal strips. The diverse inhibitory effects might be expressed in terms of eicosanoid-antagonistic activity.     

Antiplatelet and antithrombotic effects of a novel selective phosphodiesterase 3 inhibitor, NSP-513, in mice and rats

We investigated the effects of NSP-513, (R)-4,5-dihydro-5-methyl-6-[4-(2-propyl-3-oxo-1-cyclohexenyl)amino] phenyl-3(2H)-pyridazinone, on phosphodiesterase (PDE) isozyme activities, in vitro platelet aggregation and in vivo thrombus formation. NSP-513 selectively inhibited human platelet PDE 3 isozyme with an IC50 value of 0.039 microM. In an in vitro human platelet aggregation assay, the IC50 values (microM) of NSP-513 for platelet aggregation induced by collagen, U-46619, arachidonic acid, adenosine diphosphate (ADP), epinephrine and thrombin were 0.31, 0.25, 0.082, 0.66, 0.23 and 0.73, respectively. In a mouse pulmonary thromboembolism model, orally administered NSP-513 showed in vivo antithrombotic effects that were 320 to 470 times more potent than those of cilostazol. In a rat carotid arterial thrombosis model, intraduodenally administered NSP-513 (0.1 mg/kg), cilostazol (30 mg/kg) and aspirin (30 mg/kg) reduced thrombus formation by 75%, 66% and 48%, respectively. However, intravenously administered dipyridamole (10 mg/kg) did not significantly prevent thrombus formation. These results demonstrate that NSP-513 has the potential to prevent not only in vitro platelet aggregation but also in vivo thrombus formation and indicate that the highly selective PDE 3 inhibitory effect of NSP-513 may make this compound useful for assessing the physiological role of PDE 3.     

Studies on antiplatelet effect of OP-41483, a prostaglandin I2 analog, in experimental animals. I. Effect on platelet function and thrombosis

Antiplatelet and antithrombotic effects of OP-41483, a PGl2 analog, were studied in experimental animals, and the following results were obtained: 1) With 10 min-intravenous infusion to guinea pigs, OP-41483 inhibited platelet adhesiveness and platelet aggregation at 300-1000 ng/kg/min and 1000 ng/kg/min, respectively. In these effects, OP-41483 was 1-3 times more potent than carbacyclin and 3 times less potent than PGl2. 2) With oral administration to guinea pigs, OP-41483 given as its alpha-cyclodextrin clathrate (OP-41483 alpha-CD) inhibited platelet adhesiveness at doses higher than 1.0 mg/kg (expressed in terms of OP-41483), whereas PGl2 and carbacyclin did not at 10 mg/kg. OP-41483 alpha-CD also inhibited platelet aggregation after a single dose of 3 mg/kg and repeated doses of 3 mg/kg/day for 7 days. 3) In the electrically induced thrombosis model of guinea pig mesenteric artery, OP-41483 (300-1000 ng/kg/min, i.v.-infusion) and OP-41483 alpha-CD (1.0-3.0 mg/kg, p.o.) inhibited thrombus formation, but heparin (1.0-10 U/kg/min, i.v.-infusion) did not. 4) In the rabbit extracorporeal circulation thrombosis model, OP-41483 (100 and 300 ng/kg/min, i.v.-infusion) inhibited thrombus formation in the extracorporeal shunt and prevented the decrease in platelet count, hematocrit and fibrinogen level in circulating blood. Heparin (1.0-3.0 U/kg/min, i.v.-infusion) also inhibited the thrombus formation and the decrease in fibrinogen level, but did not inhibit the decrease in hematocrit and platelet count.     

Antithrombotic activity of NSP-513, a novel selective phosphodiesterase 3 inhibitor, on femoral arterial thrombosis induced by physical stenosis and electrical current: comparison of antithrombotic and hemodynamic effects

NSP-513, a novel potent and selective phosphodiesterase 3 (PDE 3) inhibitor, and cilostazol, a previously developed PDE 3 inhibitor, were compared with respect to antiplatelet, antithrombotic, and hemodynamic effects. In the in vitro antiplatelet aggregation studies, NSP-513 and cilostazol inhibited collagen-induced canine platelet aggregation with median inhibitory concentration (IC50) values of 0.093 and 3.1 miccroM, respectively, and inhibited adenosine diphosphate (ADP)-induced canine platelet aggregation with IC50 values of 0.15 and 12 microM, respectively. For ADP-induced platelet aggregation, the presence of prostaglandin E1 (PGE1; 3 and 10 nM) further decreased the IC50 values for NSP-513 to 0.11 and 0.032 microM, respectively. In ex vivo antiplatelet aggregation studies, orally administered NSP-513 (0.03-1 mg/kg) and cilostazol (50 mg/kg) inhibited collagen-induced canine platelet aggregation. In an in vivo canine femoral arterial thrombosis model, intraduodenally administered NSP-513 (0.01-0.03 mg/ kg) dose-dependently prevented thrombus formation without any changes in blood pressure, heart rate, or bleeding time. In conscious dogs, NSP-513 at oral doses of > or =0.3 mg/kg produced hemodynamic changes such as decreased blood pressure and increased heart rate and LVdP/dt(max). Thus the minimal hemodynamically effective dose of NSP-513 was 0.3 mg/kg, and the hemodynamic effects of this dose were comparable to those of 50 mg/kg of cilostazol. In conclusion, these data suggest that NSP-513 has in vivo selectivity for antiplatelet and antithrombotic activities over hemodynamic activity, and that the selectivity of NSP-513 is higher than that of cilostazol in dogs.     

2-萘-3-(3,4-二甲氧基)苯丙烯酸抗血小板聚集作用及其机理

研究发现,NMPA在体外可抑制花生四烯酸、胶原及凝血酶诱导的家兔血小板聚集,IC_(50)分别为189±9,105±11.0,49.8±16.6μM。小鼠ig NMPA(25mg/kg)后,以ADP诱导的血小板聚集明显受到抑制。大鼠ig NMPA后(52mg/kg一次或10mg/(kg·d)连续7d,出血时间延长、胶原诱导的血小板聚集率下降、丙二醛生成减少、但对动脉壁PGI_2样物质影响较小。说明MNPA的作用机理可能在于改变体内TXA_2/PGI_2比值,而达到抑制血小板聚集的效应。     

Effects of the new antiplatelet agent 2-methyl-3-(1,4,5,6-tetrahydronicotinoyl)pyrazolo[1,5-a]pyridine on platelet aggregation and thrombosis in experimental animals.

Antiplatelet and antithrombotic effects of KC-764 (2-methyl-3-(1,4,5,6-tetrahydronicotinoyl)pyrazolo[1,5-a]pyridine, CAS 94457-09-7) were studied. KC-764 inhibited arachidonic acid (AA)- and collagen-induced platelet aggregation with IC50s of 1.0 x 10(-8)-2.8 x 10(-7) mol/l for humans, rabbits, guinea pigs and dogs, and IC50s of 3.9 x 10(-6)-3.7 x 10(-5) mol/l for mice and rats in vitro. KC-764 inhibited AA- and collagen-induced aggregation with ID50s of 0.04-0.09 mg/kg p.o. in rabbits and dogs, and ID50 of 13.0 mg/kg p.o. in rats. These antiaggregatory activities of KC-764 were stronger than those of acetyl-salicylic acid (ASA), indometacin, cilostazol and ticlopidine. KC-764 inhibited the production of thromboxane B2 (TXB2) in rabbit platelet microsomes, washed platelets and reconstituted platelet rich plasma (RPRP) with IC50s of 2.9 x 10(-6) mol/l, 2.8 x 10(-7) mol/l and 4.3 x 10(-8) mol/l, respectively. The in vitro inhibitory activity of KC-764 on AA-induced platelet aggregation was more potent when RPRP was used rather than washed platelet suspension containing 30% rabbit plasma. ASA did not show such an augmentation. KC-764 prevented collagen- and AA-induced thrombosis at more than 1 mg/kg p.o. and more than 0.1 mg/kg i.v. in mice and rabbits. KC-764 showed the wider margin of dose between antiplatelet action and prolongation of bleeding time in rabbits than ASA and indometacin. These results indicated that KC-764 was a potent antithrombotic drug to prevent TXB2 production and less possible to induce untoward actions as compared with ASA or indometacin.     

Inhibition of platelet adhesion and aggregation by E4021, a type V phosphodiesterase inhibitor, in guinea pigs

E4021 (sodium 1-[6-chloro-4-(3, 4-methylenedioxybenzyl)-aminoquinazolin-2-yl]piperidine-4-carboxylate sesquihydrate) is a highly selective and potent inhibitor of type V phosphodiesterase(PDE5). The in vitro and in vivo effect of E4021 on platelet function was evaluated, using echistatin, a potent disintegrin, as a positive reference agent. E4021 inhibits aggregatory response to collagen in washed human platelets (IC₅₀ =5μM, vs. 0.14μM with echistatin). In the ex vivoplatelet aggregation assay using whole blood from treated guinea pigs, E4021 (9mg/kg i.v.) showed a moderate inhibition (43%) against collagen (0.125μg/ml), whereas echistatin (250μg/kg i.v.) exerted a 88% inhibition. The absence of endothelium-derived factors (NO) may account for the moderate in vitro and ex vivo antiplatelet activity of E4021. In an in vivo model of reversible platelet aggregation elicited by collagen (100μg/kg i.v.), both E4021 and echistatin attenuated the intrapulmonary platelet accumulation in guinea pigs (-36% and -44%, respectively). In addition, E4021 (9mg/kg i.v.) and echistatin (250μg/kg i.v.) caused a similar inhibition of platelet adhesion at sites of microfilament-induced vascular injury in guinea pigs (52% and 65%, respectively). The two agents in combination did not show additive effect, suggesting that E4021 inhibits platelet activation and impairs interactions of adhesion receptors with matrix proteins. E4021 caused a selective increase in cGMP concentrations in the platelets isolated from treated guinea pigs; cAMP was not affected. It is concluded that the antiplatelet activity of E4021 is mediated through cGMP mechanism by virtue of selective inhibition of PDE5 in the platelets. Received: 16 September 1996 / Accepted: 12 December 1996     

Inhibition of cellular action of thrombin by N3-cyclopropyl-7-[[4-(1-methylethyl)phenyl]methyl]-7H-pyrrolo[3, 2-f]quinazoline-1,3-diamine (SCH 79797), a nonpeptide thrombin receptor antagonist

A growing body of evidence suggests an important contribution of the cellular actions of thrombin to thrombosis and restenosis following angioplasty. Recently we reported on SCH 79797 (N3-cyclopropyl-7-?[4-(1-methylethyl)phenyl]methyl?-7H-pyrrolo[3, 2-f]quinazoline-1,3-diamine) and its analogs as new potent, nonpeptide thrombin receptor antagonists. This study further characterizes the biochemical and pharmacological actions of pyrroloquinazoline inhibitors of protease activated receptor-1 (PAR-1) in human platelets and coronary artery smooth muscle cells (hCASMC). SCH 79797 and its N-methyl analog (SCH 203099) inhibited binding of a high-affinity thrombin receptor-activating peptide ([(3)H]haTRAP, Ala-Phe(p-F)-Arg-ChA-HArg-[(3)H]Tyr-NH(2)) to PAR-1 with IC(50) values of 70 and 45 nM, respectively. SCH 79797 inhibited [(3)H]haTRAP binding in a competitive manner. SCH 79797 and SCH 203099 inhibited alpha-thrombin- and haTRAP-induced aggregation of human platelets, but did not inhibit human platelet aggregation induced by the tethered ligand agonist for protease-activated receptor-4 (PAR-4), gamma-thrombin, ADP, or collagen. SCH 203099 inhibited surface expression of P-selectin induced by haTRAP and thrombin, and it did not increase P-selectin expression or prevent thrombin cleavage of the receptor. Thrombin and TFLLRNPNDK-NH(2) (TK), a PAR-1-selective agonist, produced transient increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in hCASMC. This increase in [Ca(2+)](i) was inhibited effectively by SCH 79797. However, the Ca(2+) transients induced by SLIGKV-NH(2,) a PAR-2-selective agonist, were not inhibited by SCH 79797. Thrombin- and TK-stimulated [(3)H]thymidine incorporation also was inhibited completely by SCH 79797. The results of this study demonstrate that SCH 79797 and SCH 203099 are potent, selective antagonists of PAR-1 in human platelets and hCASMC. These data also suggest that the thrombin stimulation of Ca(2+) transients and mitogenesis in hCASMC is mediated primarily through activation of PAR-1.     

Effect of a new thromboxane A2 antagonist, S-145, on platelet aggregation

The newly synthesized compound S-145, (+/-)-5(Z)-7-(3-endo-phenylsulfonylamino [2.2.1] bicyclohept-2-exo-yl)heptenoic acid, inhibited arachidonic acid (AA)-, 9,11-methanoepoxy-PGH2 (U46619)-, collagen- and ADP-induced human platelet aggregation in vitro with IC50 values of 0.25, 0.34, 0.22, and 0.08 microM, respectively. The inhibiting potency of this compound to AA- or U46619-induced platelet aggregation was about twice that of ONO-3708 and 1/7-1/14 that of SQ29,548 in human platelets, about 7 times that of ONO-3708 and 1/3-1/7 that of SQ29,548 in guinea pig platelets, and 250-800 times that of ONO-3708 and 1-7 times that of SQ29,548 in rabbit platelets. When S-145 was administered orally to guinea pigs at the dose of 0.1 mg/kg, AA-induced platelet aggregation was completely inhibited at 30 and 60 min after the administration, but not at 3 and 6 hr. The minimum effective doses of S-145 (p.o.) to AA- and collagen-induced platelet aggregation at 60 min after the administration were 0.01 mg/kg and 0.03 mg/kg, respectively. The potency of S-145 (p.o.) to inhibit AA- and collagen-induced guinea pig platelet aggregation was 30-300 times that of ONO-3708 or SQ29,548 and 300-1000 times that of aspirin. These results suggest that S-145 is a thromboxane A2 antagonist showing a potent inhibiting effect on platelet aggregation by oral administration.     

LB30057 inhibits platelet aggregation and vascular relaxation induced by thrombin

Previous study showed that an amidrazonophenylalanine derivative, LB30057, which has high water solubility, inhibited the catalytic activity of thrombin potently by interaction with the active site of thrombin. In the current investigation, we examined whether LB30057 inhibited platelet aggregation and vascular relaxation induced by thrombin. Treatment with LB30057 to platelet-rich plasma (PRP) isolated from human blood resulted in a concentration-dependent inhibition of thrombin-induced aggregation. Values for IC50 and IC100 were 54 +/- 4 nM and 96 +/- 3 nM, respectively. This inhibition was agonist (thrombin) specific, since IC50 values for collagen and ADP were much greater than those for thrombin. In addition, concentration-dependent inhibitory effects were observed on the serotonin secretion induced by thrombin in PRP. Consistent with these findings, thrombin-induced increase in cytosolic calcium levels was inhibited in a concentration-dependent manner. When LB30057 was treated with aortic rings isolated from rats, LB30057 resulted in a concentration-dependent inhibition of thrombin-induced vascular relaxation. All these results suggest that LB30057 is a potent inhibitor of platelet aggregation and blood vessel relaxation induced by thrombin.     

Inhibition of platelet function by KB-2796]

The anti-platelet activity of KB-2796, 1-[bis(4-fluorophenyl)methyl]-4- (2,3,4-trimethoxybenzyl) piperazine dihydrochloride, was studied in guinea pigs and mice. When guinea pig platelet-rich plasma (PRP) was employed, platelet function was inhibited at high doses of KB-2796. The IC50 value for [3H]5-HT release was 940 microM, and the IC50 values for collagen- and ADP-induced platelet aggregation were 210 and 390 microM, respectively. Oral administration of KB-2796 at 10-100 mg/kg dose-dependently inhibited the transient thrombocytopenia induced by collagen, but not that caused by ADP. KB-2796 protected mice from death after intravenous injection of collagen plus epinephrine, with an ED50 value of 9.5 mg/kg, p.o. Oral administration of KB-2796 at 10-100 mg/kg dose-dependently reduced guinea pig platelet retention in glass bead columns and reduced the leakage of ADP and ATP from erythrocytes passing through similar columns. KB-2796, at a concentration of 1-10 microM, produced a stabilizing effect on guinea pig erythrocytes against hypotonic hemolysis. These results suggest that KB-2796 is an inhibitor of platelet function and that its inhibition is related mainly to the inhibition of leakage of ADP and ATP from erythrocytes.     

Discovery of potent orally active thrombin receptor (protease activated receptor 1) antagonists as novel antithrombotic agents

Structurally novel thrombin receptor (protease activated receptor 1, PAR-1) antagonists based on the natural product himbacine are described. The prototypical PAR-1 antagonist 55 showed a Ki of 2.7 nM in the binding assay, making it the most potent PAR-1 antagonist reported. 55 was highly active in several functional assays, showed excellent oral bioavailability in rat and monkey models, and showed complete inhibition of agonist-induced ex vivo platelet aggregation in cynomolgus monkeys after oral administration.     

槲皮素对血小板聚集和胞浆游离钙的影响(英文)

目的:研究槲皮素对凝血酶诱导的血小板聚集和胞浆游离钙浓度的影响及钙对槲皮素的血小板聚集抑制效应的作用。方法:用荧光钙离子指示剂观察槲皮素对血小板胞浆游离钙的影响.结果:槲皮素明显抑制凝血酶诱导的血小板聚集和游离钙的升高.IC_(50)和95％可信区间分别为146.2(92.4～231.3)和78.5(49.5—124.4)μmol·L~(-1).槲皮素对血小板的抑制作用可被钙翻转.槲皮素对凝血酶诱导的钙释放无影响.结论:抑制钙内流是槲皮素抑制血小板聚集和[Ca~(2 )]_i升高的机制.     

Effect of (+)-S-145 calcium salt dihydrate, an orally active antagonist of the thromboxane A2/prostaglandin endoperoxide receptor, on platelet aggregation

The effect of (+)-S-145, (1R, 2S, 3S, 4S)-(5Z)-7-(3-phenylsulfonylaminobicyclo [2.2.1] hept-2-yl) heptenoic acid on human and guinea pig platelet aggregation was examined. (+)-S-145 sodium salt inhibited human platelet aggregation induced by arachidonic acid (AA), 9,11-methanoepoxy-PGH2 (U 46619), collagen, ADP or epinephrine with the IC50 being 0.047-0.146 microM in an in vitro system. When (+)-S-145 calcium salt dihydrate was administered orally to guinea pigs, it inhibited AA-, U-46619- or collagen-induced platelet aggregation dose-dependently with the minimum effective dose being 0.03 mg/kg, and the effective duration being maximally 3 hr. The inhibiting potency and effective duration of (+)-S-145 calcium salt dihydrate after multiple administrations, once a day (0.5 mg/kg) for 7 days, were almost the same as those after a single administration. Although (+)-S-145 sodium salt showed a partial agonist effect (shape change) on platelets in vitro, the effect diminished after pretreatment of the platelets with a lower dose of this compound. These data suggest that (+)-S-145 calcium salt dihydrate is an orally effective potent platelet aggregation inhibitor.     

The in vivo pharmacological profile of CS-747, a novel antiplatelet agent with platelet ADP receptor antagonist properties

1. CS-747 is a novel antiplatelet agent that generates an active metabolite, R-99224, in vivo. CS-747 itself was totally inactive in vitro. This study examined in vivo pharmacological profiles of CS-747 after single oral administration to rats. 2. Orally administered CS-747 (0.3 - 10 mg kg(-1)) partially but significantly decreased [(3)H]-2-methylthio-ADP binding to rat platelets. CS-747 (3 mg kg(-1), p.o.) treatment neutralized ADP-induced decreases of cyclic AMP concentrations induced by prostaglandin E(1), suggesting that metabolites of CS-747 interfere with G(i)-linked P2T receptor. 3. CS-747 (0.3 and 3 mg kg(-1), p.o.) markedly inhibited ex vivo washed platelet aggregation in response to ADP but not to thrombin. CS-747 also exhibited a marked inhibition of ADP-induced ex vivo platelet aggregation in PRP with a rapid onset (<0.5 h) and long duration (>3 days) of action (ED(50) at 4 h=1.2 mg kg(-1)). 4. R-99224 (IC(50)=45 microM) inhibited in vitro PRP aggregation in a concentration-related manner. 5. CS-747 prevented thrombus formation in a dose-related manner with an ED(50) value of 0.68 mg kg(-1). CS-747 was more potent than clopidogrel (6.2 mg kg(-1)) and ticlopidine (>300 mg kg(-1)). 6. CS-747, clopidogrel, and ticlopidine prolonged the bleeding time. The order of potency of these agents in this activity was the same as that in antiaggregatory and antithrombotic activities. 7. These findings indicate that CS-747 is an orally active and a potent antiplatelet and antithrombotic agent with a rapid onset and long duration of action, and warrants clinical evaluations of the agent.     

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.     

Synthesis and Antithrombotic Effect of Xanthone Derivatives

A series of xanthone derivatives was synthesized and tested in-vitro for their ability to inhibit aggregation of rabbit washed platelets and human platelet-rich plasma (PRP) induced by various inducers. 2-Prenyloxyxanthone showed the most potent inhibition of rabbit washed platelet aggregation induced by arachidonic acid (IC50 = 10.2 μM). Of the compounds tested in human PRP, 2-[3 (propylamino)-2-hydroxypropoxy]xanthone (4) hydrochloride salt exhibited the most potent inhibition of platelet aggregation induced by adrenaline (IC50 = 4.4 μM), whereas in evaluation of mouse antithrombotic activity, compound 4 exhibited the most potent protection of mice from thrombotic challenge. Compound 4, 2-[3-(isopropylamino)-2-hydroxypropoxylxanthone hydrochloride salt and 2,5 dihydroxyxanthone suppressed the secondary aggregation induced by adrenaline in human PRP. We conclude that the antiplatelet effects of these compounds are mainly due to an inhibitory effect on thromboxane formation.     

Pharmacology: Effects of Copper-aspirin Complex on Platelet Aggregation and Thrombosis in Rabbits and Mice

The effects of intragastric and intraduodenal copper-aspirin complex on rabbit platelet aggregation were observed by Born's method. Myers's method was used to evaluate the antithrombotic effect of copper-aspirin complex in mice. In-vitro copper-aspirin complex selectively inhibited arachidonic acid-induced platelet aggregation with an IC50 value (concentration resulting in 50% inhibition) of 13.2 μM (95% confidence limits 9.1–16.8 μM). Copper-aspirin complex (10 mg kg^?1 given intragastrically or intraduodenally) was more potent than aspirin in inhibiting arachidonic acid-induced platelet aggregation. Copper-aspirin complex (10 mg kg^?1) had a stronger inhibitory effect and a longer duration of action when given intragastrically than when given intraduodenally. It was shown by radioimmunoassay that copper-aspirin complex significantly reduced the level of thromboxane B₂ in plasma while markedly increasing that of 6-ketoprostaglandin F_1α (6keto-PGF_1α). Copper-aspirin complex (10 mg kg^?1 given intragastrically for 7 days) significantly reduced mouse mortality caused by intravenous injection of arachidonic acid. The results suggest that both in-vitro and in-vivo copper-aspirin complex is more potent in selectively inhibiting arachidonic acid-induced platelet aggregation than aspirin. When given intragastrically the complex has a more potent antiplatelet effect and a longer duration of action than when given intraduodenally. The antithrombotic effect of the complex was more potent than that of aspirin.     

Inhibitory effect of various thrombin inhibitors on shear-induced platelet function and dynamic coagulation

We assessed the effects of active site-directed, fibrinogen recognition exosite (FRE)-directed and bifunctional thrombin inhibitors, on shear-induced platelet reactivity (adhesion/aggregation) and dynamic coagulation (coagulation of flowing blood). An in vitro test for shear-induced haemostatic plug formation and dynamic coagulation (haemostatometry) was employed using non-anticoagulated rat blood. The active site-directed inhibitors (argatroban, P891, P899) caused inhibition of platelet reactivity and coagulation at 1-, 100- and 100-microM concentrations, respectively. Bifunctional inhibitors (P553, P1053) exerted inhibitory effects at 0.1 microM. A dimeric bifunctional inhibitor P824 caused significant inhibition at 1 microM. The FRE-directed inhibitor (P960) inhibited shear-induced platelet reactivity at 10 microM but the dynamic coagulation at 1 microM. Combination of active site-directed argatroban and FRE-directed P960 did not show any synergistic effect. The most potent inhibition was observed in monomeric bifunctional inhibitors. The inhibitory effects were compared with the K(i) values against human thrombin and with the IC(50) values against fibrin clot formation. The minimum effective concentrations on shear-induced platelet reactivity and dynamic coagulation were comparable with the IC(50) values, but not with the K(i) values.