槲皮素-7，4＇-二硫酸酯对凝血酶诱导猪血小板聚集的抑制作用及其机制

摘要

目的：研究人工半合成槲皮素一槲皮素-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([Ca2+]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Ca2+／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400 μmol／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(10-160 μmol／L)抑制血小板胞浆Ca2+／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、Ca2+／PL依赖的PKC和肌动蛋白聚合．%AIM: To study the inhibitory effect of semi-synthesized quercetin derivatives- disodium quercetin-7,4＇-disulfate (DQD) on the platelet aggregation induced by thrombin and its mechanism. METHODS: Platelet aggregation was analysed by mrbidimetry. Cytosolic free calcium concentration ([ Ca2 + ]i) was determined by Fura-2 fluorescence technique. Activity of Ca2 +/PL dependent protein kinase C (PKC) was assayed by incubating PKC with histone Ⅲ S and Ⅲ[γ-32P] ATP. The cytoskeletal proteins were precipitated by Triton and separated by SDS-PAGE.RESULTS: DQD inhibited the platelet aggregation induced by thrombin (500 U/L), when DQD concentrations were 100, 200, and 400 μmol/L, the inhibition rates were 77 %, 86 %, and 82 % respectively. DQD inhibited Ca2 + influx in platelets induced by thrombin (500 U/L ) in the presence of extracellular Ca2+ 1mmol/L in a concentration-dependent manner (10- 80μmol/L); DQD also had inhibitory effect on intracellular Ca2 + mobilization in the absence of extracellular Ca2 +.DQD (10- 160 μmol/L) inhibited the cytosolic Ca2+/PL dependent PKC from platelets in a concentration-de-pendent manner, but had no effect on membrane PKC.DQD (20- 200 μmol/L) inhibited the actin polymerization induced by thrombin (500 U/L) in platelets in a concentration-dependent manner. CONCLUSION:DQD inhibited pig platelet aggregation induced by thrombin and its molecular mechanism was due to its inhibition of Ca2+ influx, intracellular Ca2 + mobilization, Ca2+/PL dependent PKC activity, and actin polymerization.