眼镜蛇毒金属蛋白酶atrase A抗血小板聚集作用及机制

Inhibitory effect and mechanism of cobra venom metalloproteinase atrase A on platelet aggregation

目的研究中华眼镜蛇毒金属蛋白酶atrase A对血小板聚集的影响及其相关的机制。方法测定atrase A对二磷酸腺苷、胶原、血小板活化因子、花生四烯酸、瑞斯托霉素、凝血酶诱导血小板聚集的影响情况,并通过蛋白质免疫印迹检测atrase A对血小板膜糖蛋白和血管假血友病因子的酶切情况。结果中华眼镜蛇毒金属蛋白酶atrase A能明显抑制由瑞斯托霉素和凝血酶诱导的血小板聚集,这种抑制作用呈量效、时效关系。而atrase A和血小板预孵5min后对二磷酸腺苷、胶原、血小板活化因子、花生四烯酸诱导的血小板聚集有微弱的抑制作用,预孵时间延长至30min对血小板聚集有明显的抑制作用。蛋白质免疫印迹结果显示atrase A能特异性酶切血小板膜糖蛋白GPIb,但对vWF几乎没有酶切作用。结论中华眼镜蛇毒金属蛋白酶atrase A对二磷酸腺苷、胶原、血小板活化因子、花生四烯酸、瑞斯托霉素、凝血酶诱导的血小板聚集均有抑制作用,其中对瑞斯托霉素和凝血酶诱导的血小板聚集具有明显的抑制作用,其机制是通过酶切血小板膜糖蛋白GPIb。

Aim To investigate the effect and mechanism of cobra venom metalloproteinase atrase A on inhibiting platelet aggregation. Methods Platelet aggregation induced by collagen, ADP, PAF, AA, ristoeetin and thrombin,respectively,was measured turbimetrieally after platelet incubated with atrase A. Western blot was used to detect the effect of atrase A on cleavage of platelet membrane glycoprotein and yon Willebrand Factor. Results Atrase A significantly inhibited platelet aggregation induced by ristocetin and thrombin in a dose-and time-dependent manner. Meanwhile,atrase A just showed slight inhibitory effect on platelet aggregation induced by PAF, AA, collagen, and ADP after incubated with PRP for 5 min. After incubation time was prolonged to 30 min, significant inhibition was shown on platelet aggregation. Western blot revealed that atrase A cleaved platelet membrane glycoprotein GPIb. Conclusions Cobra venom metalloproteinase atrase A significantly inhibits platelet aggregation induced by ristocetin and thrombin due to the cleavage of platelet membrane glycoprotein Ib. And atrase A also has inhibitory effect on platelet aggregation induced by ADP, PAF, AA, and collagen.