Inhibitory mechanisms of dihydroginsenoside Rg3 in platelet aggregation: critical roles of ERK2 and cAMP

Ginsenoside Rg3, a single ginseng saponin, is known to be a major anti-platelet component of protopanaxadiol that is isolated from Korean red ginseng. In this study, we investigated whether dihydroginsenoside Rg3, a stable chemical derivative of ginsenoside Rg3, also demonstrated anti-platelet activity. Dihydroginsenoside Rg3 inhibited thrombin-induced platelet aggregation in a concentration-dependent manner with an IC50 (concentration producing 50% inhibition) of 18.8 +/- 0.4 microM. Ginsenoside Rg3 inhibited platelet aggregation which was induced by thrombin (0.1 U mL(-1)) with an IC50 of 40.2 +/- 0.9 microM. We next determined whether dihydroginsenoside Rg3 affected different types of ligand-induced platelet aggregation. We found that dihydroginsenoside Rg3 inhibited collagen-induced platelet aggregation with an IC50 of 20.0 +/- 0.9 microM. To elucidate the inhibitory mechanism of dihydroginsenoside Rg3 on aggregation, we analysed its downstream signalling pathway. It was interesting to note that dihydroginsenoside Rg3 elevated cyclic AMP production in resting platelets, but did not affect cyclic GMP production. In addition, we found that dihydroginsenoside Rg3 potently suppressed phosphorylation of extracellular signal-regulated kinase 2 (ERK2), which was stimulated by collagen (2.5 microg mL(-1)), but not of p38 mitogen-activated protein kinase. Taken together, our results indicate that dihydroginsenoside Rg3 potently inhibited platelet aggregation via the modulation of downstream signalling components such as cAMP and ERK2.     

Resveratrol attenuates thromboxane A2 receptor agonist-induced platelet activation by reducing phospholipase C activity

Resveratrol (3,5,4'-trihydroxystilbene) is a naturally occurring compound shown to decrease the incidence of thromboembolic disease. Although considerable data are available as to the inhibitory effect of resveratrol on the platelet aggregation and thrombopoiesis in human, its underlying mechanism, at the cellular level, has not been rigorously studied. In this experiment, we studied the effect of resveratrol and 1-[6-[[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione, a phospholipase C inhibitor (U-73122) on the thromboxane A2 receptor agonist (9,11-dideoxy-11 alpha,9 alpha-epoxymethanoprostaglandin F(2 alpha), U46619)-induced platelet aggregation, platelet P-selectin expression, and the activity of phospho-phospholipase C beta 3 (P-PLC beta 3) and total-phospholipase C beta 3 (T-PLC beta 3), which play key roles in the signal transduction system of platelet in human. It was found that resveratrol blocked platelet aggregation and platelet P-selectin expression induced by U46619 in a concentration-dependent manner. U-73122 and resveratrol had additive effect in inhibiting platelet aggregation and platelet P-selectin expression. Resveratrol (final concentration was 50 microM) could reduce the ratio of P-PLC beta 3 to T-PLC beta 3. Taken together, these results show that resveratrol suppresses U46619-induced platelet aggregation and P-selectin expression partly through the decrease of the activity of phospholipase C beta of platelets.     

Antiplatelet and antithrombotic activities of Korean Red Ginseng

The antiplatelet and antithrombotic activities of Korean Red Ginseng (KRG) were examined on rat carotid artery thrombosis in vivo, and platelet aggregation in vitro and ex vivo. Administration of KRG to rats not only prevented carotid artery thrombosis in vivo in a dose-dependent manner, but also significantly inhibited ADP- and collagen-induced platelet aggregation ex vivo, while failed to prolong coagulation times such as activated partial thromboplastin time (APTT) and prothrombin time (PT), indicating the antithrombotic effect of KRG might be due to its antiplatelet aggregation rather than anticoagulation effect. In line with the above observations, KRG inhibited U46619-, arachidonic acid-, collagen- and thrombin-induced rabbit platelet aggregation in vitro in a concentration-dependent manner, with IC50 values of 620 +/- 12, 823 +/- 22, 722 + 21 and 650 +/- 14 microg/mL, respectively. Accordingly, KRG also inhibited various agonists-induced platelet serotonin secretions as it suppressed platelet aggregation. These results suggest that KRG has a potent antithrombotic effect in vivo, which may be due to antiplatelet rather than anticoagulation activity, and KRG intake may be beneficial to the individuals with high risks of thrombotic and cardiovascular diseases.     

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.     

Purification and characterization of a platelet aggregation inducer from Calloselasma rhodostoma (Malayan pit viper) snake venom

A potent platelet aggregation inducer was purified from Calloselasma rhodostoma snake venom by Sephadex G-75, CM-Sephadex C-50 and Sephacryl S-300 column chromatography. It was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a molecular weight estimated to be 28,160 +/- 1280. It was devoid of phospholipase A2, fibrino(geno)lytic and thrombin-like activities. The venom inducer elicited platelet aggregation and the serotonin release reaction in rabbit platelet-rich plasma and platelet suspension. Exogenous calcium was required for its platelet activation. Creatine phosphate/creatine phosphokinase and indomethacin did not inhibit the venom inducer-induced aggregation and release reaction. Mepacrine and verapamil preferentially inhibited aggregation, while PGE1 completely blocked both aggregation and release reaction. It is concluded that the venom inducer activates platelets through the activation of endogenous phospholipase A2 or C, leading to intracellular calcium mobilization, but independent of the ADP release reaction or thromboxane A2 formation.     

Lipopolysaccharide-binding and neutralizing activities of surfactin C in experimental models of septic shock

To evaluate the anti-endotoxin activity of surfactin C, we studied its lipopolysaccharide-binding activity in vitro and therapeutic efficacy in experimental models of gram-negative septic shock. The ability of surfactin C to bind LPS from Escherichia coli O111:B4 was determined using a limulus chromogenic assay. Male ICR mice and Sprague-Dawley rats were given intraperitoneal administration of 1x10(9) colony forming units of E. coli ATCC 25922. After bacterial challenge, all animals were randomized to receive intraperitoneally saline, polymyxin B or surfactin C. Surfactin C not only completely bound to the LPS (its median effective concentration being 13.75 microM) but also improved the survival and reduced of the number of inoculated bacteria in the mouse model of septic shock. Surfactin C reduced the plasma endotoxin, tumor necrosis factor-alpha and nitric oxide levels in response to septic shock in rats.     

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.     

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.     

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.     

Comparison of the effects of PAR1 antagonists, PAR4 antagonists, and their combinations on thrombin-induced human platelet activation

Thrombin activates human platelets through proteolytic activation of two protease-activated receptors (PARs), PAR1 and PAR4. In the present study, we show that, RWJ-56110, a potent synthetic PAR1 antagonist, inhibited platelet aggregation caused by a low concentration (0.05 U/ml) of thrombin, but lost its effectiveness when higher concentrations of thrombin were used as stimulators. YD-3, a non-peptide PAR4 antagonist, alone had little or no effect on thrombin-induced platelet aggregation, significantly enhanced the anti-aggregatory activity of PAR1 antagonist. In addition, we demonstrate for the first time that P-selectin expression in thrombin-stimulated platelets can be synergistically prevented by combined treatment of PAR1 antagonist and PAR4 antagonist. These results indicate that thrombin-induced platelet activation cannot be effectively inhibited by just blocking either single thrombin receptor pathway, and suggest a rationale for potential combination therapy in arterial thrombosis.     

Synergistic interaction of adrenaline and histamine in human platelet aggregation is mediated through activation of phospholipase, map kinase and cyclo-oxygenase pathways.

This study was conducted to examine the mechanism(s) of synergistic interaction of histamine- and adrenaline-mediated human platelet aggregation. We found that platelet aggregation mediated by subthreshold concentrations of histamine (1-4 microm) plus adrenaline (0.5-2 microm) is inhibited by both an alpha(2)-adrenoceptor blocker (yohimbine) and a histamine (H1) receptor antagonist (diphenhydramine). In examining the role of the downstream signalling pathway, we found that such an interaction is inhibited by the calcium channel blockers verapamil and diltiazem. However, platelet aggregation by adrenaline plus histamine was inhibited by very low concentrations of the phospholipase C (PLC) inhibitor, U73122 (IC(50)= 1.2 microm), the MEK inhibitor, PD98059 (IC(50)= 1.1 microm) and the cyclo-oxygenase (COX) inhibitor, indomethacin (IC(50)= 7 microm). However the inhibition of receptor tyrosine kinase, protein kinase C and phosphatidylinositol 3-kinase by genistien, chelerythrine and wortmannin, respectively, had no significant effect on aggregation. Similarly the nitric oxide donor (SNAP) had no effect on this synergism. These data suggest that the synergistic effect of histamine and adrenaline during human platelet aggregation is receptor mediated and involves activation of PLC, COX and MAP kinase signalling pathways.     

槲皮素单硫酸酯钠盐对凝血酶诱导的猪血小板聚集的抑制作用

研究槲皮素单硫酸酯钠盐对凝血酶诱导的猪血小板聚集的抑制作用。方法：用比浊法测定血小板聚集,Ｆｕｒａ ２－ＡＭ荧光法检测胞浆游离钙浓度（「Ｃａ＾２＋）ｉ」。用组蛋白ⅢＳ,「γ＾３２Ｐ」ＡＴＰ与蛋白激酶Ｃ酶液一起温育的方法测定ＰＫＣ的活性。用ＳＤＳ－ＰＡＧＥ分离骨架蛋白。     

眼镜蛇毒蛋白酶natrahagin水解纤维蛋白原的特性及对人血小板聚集的

AIM: To study the fibrinogenolytic properties of natrahagin and its effect on platelet aggregation. METHOD: SDS-PAGE, fibrinogenolytic activity assay, platelet aggregation. RESULTS: Upon incubation of fibrinogen with natrahagin at the ratio of 50:1 (w/w), A alpha-chains of fibrinogen were almost completely hydrolyzed in 5 min; however, at least 6 h was needed for the complete degradation of gamma-chains. Fibrinogenolytic activity of natrahagin was 0.349 +/- 0.044 g.min-1.g-1 as determined by its ability to reduce the clottable fibrinogen. On the other hand, natrahagin concentration-dependently inhibited platelet aggregation induced by ristocetin in platelet-rich plasma and thrombin (80 U.L-1) in washed platelets with IC50 (95% confidence limit) of 56 (40-79) and 3.3 (1.4-8.0) mg.L-1. No inhibitory effect was found on collagen- and ADP-induced platelet aggregation even when the dose of natrahagin reached 200 mg.L-1. CONCLUSION: Natrahagin is an alpha, gamma-fibrinogenase with an inhibitory effect on platelet membrane glycoprotein Ib (GPIb)-dependent platelet aggregation.     

Antithrombotic and antiplatelet activities of Korean red ginseng extract

The antithrombotic and antiplatelet activities of Korean red ginseng extract (KRGE) were examined on rat carotid artery thrombosis in vivo and platelet aggregation in vitro and ex vivo. The KRGE significantly prevented rat carotid arterial thrombosis in vivo in a dose-dependent manner. Administration of the KRGE to rats significantly inhibited adenosine diphosphate (ADP)- and collagen-induced platelet aggregation ex vivo, although it failed to prolong coagulation times such as activated partial thromboplastin and prothrombin time indicating that the antithrombotic effect of the red ginseng may be due to its antiplatelet aggregation rather than anticoagulation effect. In line with the above observations, the red ginseng inhibited the U46619-, arachidonic acid-, collagen- and thrombin-induced rabbit platelet aggregations in vitro in a concentration-dependent manner, with IC(50) values of 390 +/- 15, 485 +/- 19, 387 +/- 11 and 335 +/- 15 microg/ml, respectively. Consistently, serotonin secretion was also inhibited by ginseng in the same pattern. These results suggest that the red ginseng has a potent antithrombotic effect in vivo, which may be due to the antiplatelet rather than the anticoagulation activity, and the red ginseng intake may be beneficial for individuals with high risks of thrombotic and cardiovascular diseases.     

Antiplatelet effect of NQ12: a possible mechanism through the arachidonic acid cascade

NQ12, an antithrombotic agent, has been reported to display a potent antiplatelet activity. This study was undertaken to reveal the effect of NQ12 on rabbit platelet aggregation and signal transduction involved in the arachidonic acid (AA) cascade. NQ12 concentration-dependently suppressed collagen-, AA-, and U46619-induced rabbit platelet aggregation, with IC(50) values of 0.71 +/- 0.2, 0.82 +/- 0.3, and 0.45 +/- 0.1 microM, respectively. In addition, the concentration-response curve of U46619 was shifted to the right after NQ12 treatment, indicating an antagonism on thromboxane (TX) A(2) receptors. The collagen-stimulated AA liberation was inhibited by NQ12 in the same pattern as its inhibition of platelet aggregation. Further study revealed that NQ12 potently suppressed AA-mediated TXA(2) formation, but had no effect on the PGD(2) production, indicating an inhibitory effect on TXA(2) synthase, which was supported by a TXA(2) synthase activity assay indicating that NQ12 concentration-dependently inhibited TXA(2) formation converted from PGH(2). On the other hand, the AA-stimulated 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) formation was also suppressed by NQ12. Taken together, these results suggest that NQ12 has a potential to inhibit TXA(2) synthase activity and TXA(2) receptors, and it can modulate AA liberation as well as 12-HETE formation in platelets. This may be a convincing mechanism for the antithrombotic action of NQ12.     

Stereo-dependent inhibition of human platelet function by the optical isomers of trimetoquinol

The stereoisomers of trimetoquinol [1-(3',4',5'-trimethoxybenzyl)-6–7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; TMQ] were shown to have potent and selective inhibitory effects on human platelet function in vitro. the R(+)-isomer was 12.1-, 12.3-, 39.2-, 82.9- and 36.0-fold more effective than the S(?)-isomer as an inhibitor of aggregation induced by arachidonic acid (AA), collagen, the epoxymethano-PGH2 analogs U44069 and U46619, and thromboxane A; (TxA₂) respectively. the concentrations of the R(+)-isomer that produced 50 percent inhibition (IC₅₀) of platelet aggregation were 4.2, 4.3, 1.4, 0.14 and 0.64 μM using AA, collagen, U44069, U46619, and TxA₂₀ as respective inducers. the graphical approximation of an inhibitory Constant (K_i = 0.13 μM) for the effect of TMQ on U46619-induced aggregation suggested that a competitive-like inhibition was operative. In other experiments, platelet aggregation and serotonin release induced by U46619 were inhibited differentially by the TMQ stereoisomers with nearly identical concentration-response curves. In addition, racemicTMQ blocked the secondary phase of platelet aggregation and serotonin release induced by ADP. These data, together with the ability of the TMQ stereoisomers to selectively inhibit TxA₂-induced aggregation, suggest that TMQ is an inhibitor of endoperoxide or TxA₂ action, e.g. a thromboxane A₂ receptor antagonist.     

Studies on the inhibitory effect of etafenone hydrochloride on platelet aggregation

The inhibitory effect of etafenone hydrochloride (etafenone) on platelet aggregation in rabbit platelet rich plasma and the involvement of the arachidonic acid (AA) cascade in the inhibitory mechanism for etafenone on platelet aggregation were studied. 1) Etafenone exhibited a dose-dependent inhibitory effect on collagen (15--20 micrograms/ml)-induced platelet aggregation, and its median inhibitory concentration (IC50) was 1.7 X 10(-5)M. 2) In ADP (20 microM)-induced aggregation, etafenone also exhibited a dose-dependent inhibitory effect, but its IC50 was 2.7 X 10(-4)M and was significantly higher than that in the case of collagen. 3) Etafenone inhibited AA (0.3--0.5mM)-induced platelet aggregation dose-dependently. Its IC50 was 2.8 X 10(-5)M. 4) In thromboxane (TX) A2-induced aggregation, etafenone exhibited a dose-dependent inhibition, and the IC50 was 3.2 X 10(-4)M. 5) Trapidil which was reported to inhibit platelet aggregation via phosphodiesterase (PDE) inhibition had a similar IC50 on ADP- and TXA2-induced platelet aggregation to that of etafenone, but in collagen- and AA-induced aggregation, its IC50 was higher than that of etafenone. 6) Etafenone (3 X 10(-6)--3 X 10(-4)M) dose-dependently inhibited the production of TXB2 in PRP induced by collagen. 7) Etafenone scarcely affected TXA2 synthetase activity in rabbit platelet homogenate. 8) The correlation between the inhibitory effect of etafenone on platelet aggregation and inhibition of AA metabolism activation and PDE inhibition was discussed.     

A potent platelet aggregation inhibitor purified from Agkistrodon halys (mamushi) snake venom

By means of gel filtration on Sephadex G-75, DEAE-Sephadex A-50 column chromatography and three gel filtrations on Sephadex G-75, a potent platelet aggregation inhibitor was purified from Agkistrodon halys snake venom and shown to be a single peptide chain, as judged by SDS-polyacrylamide gel electrophoresis. The purified platelet aggregation inhibitor was an acidic protein with a molecular weight of 14,000 and possessed phospholipase A₂ activity. Its inhibitory activity on platelet aggregation was heat stable (at 96°C, 30 min) in an acidic medium (pH 5.5), while its phospholipase A enzymatic activity was heat labile under the same conditions. Its inhibitory activity on platelet aggregation induced by thrombin, sodium arachidonate, collagen or ionophore A-23187 was non-competitive and dose-dependent with a similar id₅₀ (～ 11 μg/ml). It exerted its inhibitory action without pre-incubation with platelet suspension, however, its inhibitory effect could be moderately increased after longer incubation (30 min).     

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.     

Antiplatelet aggregation activity of diterpene alkaloids from Spiraea japonica

Six diterpene alkaloids with an atisine-type C(20)-skeleton isolated from the Chinese herbal medicines Spiraea japonica var. acuta and S. japonica var. ovalifolia, as well as eight derivatives of spiramine C and spiradine F were evaluated for the ability to inhibit aggregation of rabbit platelets induced by arachidonic acid, ADP, and platelet-activating factor (PAF) in vitro. The results showed that 12 of the 14 atisine-type diterpene alkaloids significantly inhibited PAF-induced platelet aggregation in a concentration-dependent manner, but had no effect on ADP- or arachidonic acid-induced aggregation, exhibiting a selective inhibition. It is the first report that C(20)-diterpene alkaloids inhibit PAF-induced platelet aggregation. However, spiramine C1 concentration-dependently inhibited platelet aggregation induced by PAF, ADP and arachidonic acid with IC(50) values of 30.5+/-2.7, 56.8+/-8.4 and 29.9+/-9.9 microM, respectively, suggesting a non-selective antiplatelet aggregation action. The inhibitory effect of spiramine C1 on arachidonic acid was as potent as that of aspirin. Primary studies of the structure-activity relationships for inhibition of PAF-induced aggregation showed that the oxygen substitution at the C-15 position and the presence of an oxazolidine ring in spiramine alkaloids were essential to their antiplatelet aggregation effects. These results suggest that the atisine-type alkaloids isolated from S. japonica are a class of novel antiplatelet aggregation agents.