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目的:研究补体激活剂眼镜蛇毒因子(CVF)对大鼠血小板的作用及其细胞机制。方法:比浊法测富血小板血浆内血小板聚集并同步记录ATP释放;生色底物法测血小板表面凝血酶原酶活性;用钙离子荧光指示剂Fura-2 AM负载血小板测细胞内游离钙;放免法测细胞内cAMP含量。结果:CVF在19.5-617nmol·L~(-1)范围内浓度依赖性地诱导血小板聚集和ATP释放,195nmol·L~(-1)诱导的ATP释放不依赖于聚集,且明显弱于凝血酶1U/ml的作用。CVF195nmol·L~(-1)时间依赖性地增加血小板表面凝血酶原酶活性。抗血小板膜糖蛋白Ⅰb/Ⅸ、Ⅲa、Ⅱb的单克隆抗体SZ-1、SZ-21、SZ-22抑制CVF诱导的血小板聚集。CVF 195nmol·L~(-1)使血小板内游离钙从静息态的(141±46)nmol·L~(-1)升高到(240±64)nmol·L~(-1),在61.7-617nmol·L~(-1)范围内轻度降低血小板内的cAMP。结论:补体激活剂CVF能诱导大鼠血小板聚集、ATP释放,增加血小板表面凝血酶原酶活性,其激活血小板的作用与血小板表面纤维蛋白原受体及血小板内游离钙升高、cAMP下降有关。
Objective: To investigate the effect of complement activator cobra venom factor (CVF) on rat platelets and its cellular mechanism. Methods: Platelet aggregation in plasma was detected by turbidimetric method and ATP release was recorded synchronously. The activity of prothrombinase on platelet surface was assayed by chromogenic substrate method. Intracellular free calcium was detected by Fura-2 AM loaded with calcium ion fluorescent indicator. Radioimmunoassay intracellular cAMP content. Results: CVF induced platelet aggregation and ATP release in a concentration range of 19.5-617 nmol·L -1. The release of ATP induced by 195 nmol·L -1 did not depend on aggregation and was significantly weaker than that of thrombin 1U / ml role. CVF195nmol·L -1 increased the platelet surface prothrombinase activity in a time-dependent manner. Monoclonal antibodies SZ-1, SZ-21 and SZ-22 against platelet membrane glycoproteins Ib / IX, IIIa and IIb inhibited CVF-induced platelet aggregation. CVF 195nmol·L -1 increased platelet intracellular free calcium from resting (141 ± 46) nmol·L -1 to (240 ± 64) nmol·L -1 61.7-617nmol·L ~ (-1) within a mild reduction of platelet cAMP. CONCLUSION: Complement activator CVF can induce platelet aggregation and ATP release in rats and increase the prothrombinase activity on platelet surface. The effect of its activation on platelets is related to the increase of platelet fibrinogen receptor and platelet intracellular free calcium and the decrease of cAMP.