Antiplatelet and antithrombotic effects of the diterpene spiramine Q from Spiraea japonica var. incisa

Spiramine Q, a diterpene, was isolated from a Chinese herbal plant Spiraea japonica var. incisa Yu. Born's and Wan HY's methods were used to investigate effects of spiramine Q on rabbit platelet aggregation and serotonin release, respectively. Its antithrombotic effect in mice was also evaluated by Myers' method. Spiramine Q selectively inhibited arachidonic acid-induced platelet aggregation in vitro or ex vivo, and decreased serotonin secretion from rabbit platelets. Spiramine Q (5 mg/kg) decreased the mouse mortality caused by injection of 80 mg/kg arachidonic acid in the tail vein. The results suggested that spiramine Q showed potent antiplatelet and antithrombotic activites.     

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.     

Antiplatelet activity of carnosic acid, a phenolic diterpene from Rosmarinus officinalis

Carnosic acid is a major phenolic diterpene derived from Rosmarinus officinalis and has been reported to have antioxidant, antibacterial, anticancer, antiobese and photoprotective activities. This study investigated the antiplatelet activity of carnosic acid. carnosic acid significantly inhibited collagen-, arachidonic acid-, U46619- and thrombin-induced washed rabbit platelet aggregation in a concentration-dependent manner, with IC50 values of 39+/-0.3, 34+/-1.8, 29+/-0.8 and 48+/-2.9 microM, respectively, while it failed to inhibit PMA-(a direct PKC activator) and ADP-induced platelet aggregation. In agreement with its antiplatelet activity, carnosic acid blocked collagen-, arachidonic acid-, U46619- and thrombin-mediated cytosolic calcium mobilization. accordingly, serotonin secretion and arachidonic acid liberation were also inhibited in a similar concentration-dependent manner. However, in contrast to the inhibition of arachidonic acid-induced platelet aggregation, carnosic acid had no effect on the formation of arachidonic acid-mediated thromboxane A2 and prostaglandin D2, thus indicating that carnosic acid has no effect on the cyclooxygenase and thromboxane A2 synthase activity. Overall, these results suggest that the antiplatelet activity of carnosic acid is mediated by the inhibition of cytosolic calcium mobilization and that carnosic acid has the potential of being developed as a novel antiplatelet agent.     

The chemistry and biochemistry of Spiraea japonica complex

The characteristic components of Spiraea japonica complex, which consists of seven varieties, are the hetisine- and atisine-type diterpene alkaloids, and the atisane-type diterpenes. From this complex, 20 hetisines, 37 atisines and 7 diterpenes were isolated during 1964-2001, including the observations of the isomerization of spiraea diterpene alkaloids having the oxazolidine ring and the interconversion of the oxazolidine rings, the configuration of the oxygen substitution at C-15 of the atisine-type alkaloids, and the interconversion relationship between the two main subtypes of the hetisine-type alkaloids. The chemotaxonomy based on structures of the reported diterpene alkaloids were suggested. The studies on the anti-inflammation, anti-platelet aggregation and neuroprotective bioactivities of the alkaloids from S. japonica var. acuta were reviewed as well.     

Antiplatelet activity of carnosol is mediated by the inhibition of TXA2 receptor and cytosolic calcium mobilization

Carnosol, a naturally occurring phenolic diterpene found in rosemary, has been reported to exhibit antioxidant, anticancer and hepatoprotective effects. In the present study, the antiplatelet activity of carnosol was investigated. Carnosol concentration-dependently inhibited washed rabbit platelet aggregation induced by collagen and arachidonic acid (AA), with IC(50) values of 5.5+/-0.3 and 42.5+/-0.9 microM, respectively, while failed to inhibit that induced by, ADP and thrombin. Consist with inhibition of collagen-induced platelet aggregation, carnosol revealed blocking of collagen-mediated cytosolic calcium mobilization, serotonin secretion and arachidonic acid liberation. However, contrary to the inhibition of AA-induced platelet aggregation, carnosol has no effect on AA-mediated TXA(2) and PGD(2) formation, indicating carnosol may directly inhibit TXA(2) receptor, which was supported by the finding that carnosol potently inhibited U46619 (a TXA(2) mimic)-induced platelet aggregation, with an IC(50) value of 22.0+/-2.5 microM. In addition, the U46619-induced concentration-response curve was downward shifted by the application of carnosol at concentrations of 22 and 50 microM, indicating a typical non-competitive antagonism on TXA(2) receptor. Taken together, these results suggest that antiplatelet activity of carnosol may be mediated by the inhibition of TXA(2) receptor and cytosolic calcium mobilization, and carnosol has a potential to be developed as a novel-antiplatelet agent.     

XANTHINES INHIBIT HUMAN PLATELET AGGREGATION INDUCED BY PLATELET-ACTIVATING FACTOR

1. Platelet-activating factor (PAF) may be involved in the pathogenesis of asthma, and therefore the effects of the anti-asthma drugs theophylline and enprofylline on human platelet aggregation and adenosine triphosphate (ATP) release induced by PAF and adenosine diphosphate (ADP) were studied. 2. Enprofylline (50% inhibitory concentration [IC50] = 94.8 +/- 13.2 mumol/L) was more potent than theophylline (IC50 = 934.1 +/- 40.1 mumol/L) as an inhibitor of PAF-induced aggregation, and the xanthines were twice as potent as inhibitors of PAF-induced aggregation when compared with ADP-induced aggregation. ATP release was 1.4 times more sensitive to inhibition by the xanthines than aggregation. 3. Although high concentrations of xanthines inhibited platelet aggregation and ATP release induced by PAF, therapeutic concentrations are unlikely to inhibit PAF-induced effects.     

Platelet aggregation and anti-inflammatory effects of garden pea, Desi chickpea and Kabuli chickpea

Inflammation is the natural body defense mechanism for the removal of injurious agents, necrosed cells and tissues from the body. This study was aimed to evaluate the anti-inflammatory and platelet aggregation effects of three medicinal plants of Pakistan. Methanolic extract of garden pea inhibited arachidonic acid (AA)-induced platelet aggregation (IC50 = 35 microg/mL) and platelet activating factor (PAF)-induced platelet aggregation (IC50 = 38 microg/mL) in a dose dependent fashion. Methanolic extract of Desi chickpea inhibited arachidonic acid (AA) induced platelet aggregation (IC50 value = AA = 46 microg/mL) in dose dependent fashion while was found not active against PAF-induced platelet aggregation. Methanolic extract of Kabuli chickpea was found not active against both arachidonic acid (AA)-induced platelet aggregation and PAF-induced platelet aggregation. The best potential to inhibit in vitro COX-2 activity showed garden pea (Pisum sativum: the synthesis of PGE2 reduced by 92% in comparison with untreated control wells) followed by Desi chickpea (Cicer arietinum var; 87% inhibition) and Kabuli chickpea extracts (Cicer arietinum var: 65% inhibition). All extracts were tested at concentration 20 microg/mL. in COX-2 assay. The results indicate that if the same were happening in vito, Garden pea, Desi chickpea and Kabuli chickpea could be useful as natural antithrombotic anti-inflammatory materials.     

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.     

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.     

Effect of isoquinoline alkaloids of different structural types on antiplatelet aggregation in vitro

Forty-one isoquinoline alkaloids were tested for antiplatelet aggregation effects. Among them, (-)-discretamine (6), protopine (7), ochotensimine (18), O-methylarmepavinemethine (23), lindoldhamine (25), isotetrandrine (26), thalicarpine (27), papaverine (28), and D-(+)- N-norarmepavine (32) exhibited significant inhibitory activity towards adenosine 5'-diphosphate (ADP)-, arachidonic acid (AA)-, collagen-, and/or platelet-activating factor (PAF)-induced platelet aggregation. The results are discussed on the basis of structure-activity relationships.     

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.     

Inhibition of rabbit platelet aggregation by nucleoside 5'-alkylphosphates: correlation with inhibition of agonist-induced calcium influx

We investigated the effects of uridine 5'-alkylphosphates on agonist-induced aggregation, increased intracellular calcium concentration [Ca(2+)](i), and Ca(2+) (Mn(2+)) influx in washed rabbit platelets. Uridine 5'-hexadecylphosphate (UMPC16) and uridine 5'-eicosylphosphate (UMPC20) at a concentration of 1 x 10(-5) M inhibited platelet aggregation induced by platelet-activating factor (PAF), thrombin, arachidonic acid, and ADP. UMPC16 did not cause significant interference in the binding of [(3)H-acetyl]PAF to platelets. The inhibition of PAF-induced platelet aggregation by UMPC16 was dependent upon the addition time; UMPC16 was ineffective at 60 sec when the extracellular calcium uptake reached the maximum level in PAF-stimulated platelets. Furthermore, UMPC16 inhibited guanosine 5'-O-(3-thiotriphosphate)-induced platelet aggregation but did not affect ionophore A23187- and calcium-independent agonist phorbol 12-myristate 13-acetate-induced platelet aggregation. UMPC16 markedly inhibited the Ca(2+) (Mn(2+)) influx induced by PAF and ADP, and partly inhibited the [Ca(2+)](i) increase induced by the receptor-mediated stimulation. On the other hand, UMPC16 did not affect the [Ca(2+)](i) increase and Ca(2+) (Mn(2+)) influx induced by ionomycin. These experiments suggest that inhibition of calcium influx associated with receptor-mediated platelet activation may be involved in the action of UMPC16.     

Effects of plumbagin on platelet aggregation and platelet-neutrophil interactions

The effects of plumbagin were investigated on platelet aggregation in vitro and ex vivo, on the binding of thrombin-stimulated platelets to neutrophils, and platelet aggregation induced by intact neutrophils and N-formyl-methionyl-leucyl-phenylalanine (fMLP) or platelet activating factor (PAF) activated neutrophils, by use of the methods of Hamburger, McEver and Born, respectively. The results showed that plumbagin in vitro significantly inhibited adenosine diphosphate (ADP)-, arachidonic acid (AA)-, or platelet activating factor (PAF)-induced platelet aggregation, in a concentration-dependent manner. The medium inhibitory concentrations (IC 50 ) were 39.4, 82.7 and 38.1 microM, respectively. Intragastric plumbagin at 10 mg/kg markedly suppressed platelet aggregation induced by ADP, AA, or PAF. Plumbagin decreased the binding between thrombin-stimulated platelets and neutrophils with an IC 50 of 62.9 microM. Plumbagin significantly inhibited washed platelet aggregation stimulated by fMLP- or PAF-activated neutrophils. The IC 50 values were 54.3 and 47.6 microM, respectively. On the other hand, plumbagin and aspirin increased the inhibition of intact neutrophils on AA-induced platelet aggregation. It is suggested that plumbagin inhibited platelet aggregation in vitro and ex vivo, suppressed the binding of activated platelets to neutrophils, inhibited platelet aggregation induced by activated neutrophils, and increased inhibition of intact neutrophils on platelet reactivity. Abbreviations. DMSO:dimethyl sulphoxide fMLP: N-formyl-methionyl-leucyl-phenylalanine ADP:adenosine diphosphate AA:arachidonic acid PAF:platelet activating factor     

Inhibitory effect of curcumin, a food spice from turmeric, on platelet-activating factor- and arachidonic acid-mediated platelet aggregation through inhibition of thromboxane formation and Ca2+ signaling.

Curcumin, a dietary spice from turmeric, is known to be anti-inflammatory, anticarcinogenic, and antithrombotic. Here, we studied the mechanism of the antiplatelet action of curcumin. We show that curcumin inhibited platelet aggregation mediated by the platelet agonists epinephrine (200 microM), ADP (4 microM), platelet-activating factor (PAF; 800 nM), collagen (20 microg/mL), and arachidonic acid (AA: 0.75 mM). Curcumin preferentially inhibited PAF- and AA-induced aggregation (IC50; 25-20 microM), whereas much higher concentrations of curcumin were required to inhibit aggregation induced by other platelet agonists. Pretreatment of platelets with curcumin resulted in inhibition of platelet aggregation induced by calcium ionophore A-23187 (IC50; 100 microM), but curcumin up to 250 microM had no inhibitory effect on aggregation induced by the protein kinase C (PKC) activator phorbol myrsitate acetate (1 microM). Curcumin (100 microM) inhibited the A-23187-induced mobilization of intracellular Ca2+ as determined by using fura-2 acetoxymethyl ester. Curcumin also inhibited the formation of thromboxane A2 (TXA2) by platelets (IC50; 70 microM). These results suggest that the curcumin-mediated preferential inhibition of PAF- and AA-induced platelet aggregation involves inhibitory effects on TXA2 synthesis and Ca2+ signaling, but without the involvement of PKC.     

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.     

血小板释放的ADP通过稳定细胞内钙浓度而稳定PAF诱导的兔血小板 …

AIM: To examine whether platelet-released adenosine diphosphate (ADP) would contribute to the stabilization of rabbit platelet aggregation induced by platelet activating factor (PAF). METHODS: Rabbit platelet aggregation induced by PAF was measured turbimetrically. ADP release from rabbit platelets stimulated by PAF was determined by HPLC. Intracellular Ca2+ was measured using Ca(2+)-sensitive fluorescent indicator Fura 2-AM. RESULTS: PAF > or = 1 nmol.L-1 induced full platelet aggregation, which did not deaggregate over 5 min after aggregation reached peak. Platelet aggregation was deaggregated in a concentration-dependent manner by subsequent addition of ADP scavenger ATP-diphosphohydrolase (apyrase) at 5-100 mg.L-1. PAF 3 nmol.L-1 stimulated release of ADP (29% vs 6% of control), and elicited a rapid rise in intracellular calcium ([Ca2+]i) which peaked at approximately 15 s. Then the [Ca2+]i gradually decayed from 585 +/- 80 nmol.L-1 within 100 s to a low level (364 +/- 82 nmol.L-1). Apyrase 100 mg.L-1, added 2 min after PAF, reduced [Ca2+]i to a lower level (171 +/- 29 nmol.L-1). CONCLUSION: Platelet-released ADP stabilizes PAF-induced rabbit platelet aggregation by stabilizing [Ca2+]i at elevated level.     

香草酸抗血小板聚集作用的体内外研究

目的香草酸是一种酚酸类化合物,具有抗氧化、抗炎等药理作用,其抗血栓作用尚未有报道。本实验室前期通过筛选发现香草酸具有良好的抗血小板聚集作用,因此本研究通过体内外实验对香草酸抗血小板聚集的作用进行系统评价。方法采用花生四烯酸(arachidonic acid,AA)、二磷酸腺苷(adenosine diphosphate,ADP)、凝血酶(thrombin,THR)诱导体内外血小板聚集模型,结合凝血四项检测评价香草酸抗血小板聚集及抗血栓的作用。结果体外实验中,香草酸对ADP和AA诱导的血小板聚集具有显著抑制作用,并剂量依赖性抑制ADP诱导的血小板聚集;体内试验中,香草酸(10、30和100 mg/kg)能够剂量依赖性抑制ADP和AA诱导的血小板聚集;同时,香草酸(100 mg/kg)能显著降低纤维蛋白原、增加凝血酶原时间,对活化部分凝血活酶时间和血浆凝血酶时间无明显影响。结论本研究首次发现香草酸对ADP和AA诱导的血小板聚集具有抑制作用,为研发具有自主知识产权的抗血小板聚集药物提供了实验依据。     

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.     

花生壳提取物对家兔血小板聚集的影响

目的:研究花生壳提取物体内、外给药对血小板活化因子(PAF)诱导家兔血小板聚集的影响。方法:分别采用体内和体外给药,比浊法测定血小板的聚集率,观察花生壳提取物对PAF诱导的家兔血小板聚集的抑制作用。结果:花生壳提取物在20、10mg·mL-1浓度下体外给药对PAF诱导血小板聚集有明显抑制作用(P<0.05),抑制率分别为47.13%、33.48%;在100mg·kg-1剂量下体内给药对PAF诱导血小板聚集有明显抑制作用(P<0.01),抑制率为21.78%。结论:花生壳提取物对PAF诱导的血小板聚集有抑制作用,其作用机制可能与拮抗PAF受体有关。