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目的:研究人工半合成槲皮素—槲皮素-7,4′-二硫酸酯二钠(disodium quercetin-7,4′-disulfate,DQD)对凝血酶(500 U·L~(-1))诱导猪血小板聚集的抑制作用及其机制。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-~(32)P]ATP与蛋白激酶C(PKC)酶液一起保温的方法测定Ca~(2+)/PL依赖的PKC活性。用SDS-PAGE分离骨架蛋白。结果:DQD对凝血酶诱导的血小板聚集有抑制作用,当DQD的浓度为100,200和400μmol/L时,抑制率分别为77％、86％和82％。DQD(10-80μmol/L)抑制凝血酶诱导的血小板胞外钙内流;DQD对凝血酶诱导的血小板胞内钙动员也有抑制作用。DQD(10-160μmol/L)抑制血小板胞浆Ca~(2+)/PL依赖的PKC,但DQD不影响胞膜PKC。DQD(20-200μmol/L)对凝血酶诱导的血小板肌动蛋白聚合有较强的抑制作用。结论:DQD对凝血酶诱导的猪血小板聚集有抑制作用,其分子作用机制是由于抑制血小板外钙内流、内钙动员、Ca~(2+)/PL依赖的PKC和肌动蛋白聚合。 血小板;;黄酮类;;槲皮素;;血小板聚集;;钙;;蛋白激酶C;;肌动蛋白类
Objective: To investigate the effect of artificial semi-synthetic quercetin-4, 4’-disulfate (DQD) on thrombin (500 U · L -1) ) Induced pig platelet aggregation inhibition and its mechanism. Methods: Platelet aggregation was measured by turbidimetry. Fura 2-AM fluorescence detection of cytoplasmic free calcium concentration ([Ca ~ (2 +)] _i). The Ca2 + / PL dependent PKC activity was measured by incubating protein kinase C (PKC) enzyme with histone III S, [gamma- (32) P] ATP. Skeleton proteins were separated by SDS-PAGE. RESULTS: DQD had an inhibitory effect on thrombin-induced platelet aggregation, with inhibition rates of 77%, 86% and 82%, respectively, at DQD concentrations of 100, 200 and 400 μmol / L. DQD (10-80μmol / L) inhibited thrombin-induced platelet extracellular calcium influx; DQD also inhibited thrombin-induced platelet intracellular calcium mobilization. DQD (10-160 μmol / L) inhibited platelet cytoplasm Ca ~ (2 +) / PL-dependent PKC, but DQD did not affect membrane PKC. DQD (20-200 μmol / L) has a strong inhibitory effect on thrombin-induced platelet actin polymerization. CONCLUSION: DQD can inhibit thrombin induced platelet aggregation in pigs. Its molecular mechanism is due to inhibition of platelet extracellular calcium influx, intracellular calcium mobilization, Ca ~ (2 +) / PL-dependent PKC and actin polymerization. Platelets; flavonoids ;; quercetin; platelet aggregation; calcium; protein kinase C ;; actin