广西圆斑蝰蛇毒凝血因子X激活剂的分离纯化及其止血作用的研究

Purification and Haemostatic Effect of Factor X activator from venom of Vipera russellii in Guangxi

目的从广西圆斑蝰蛇毒中分离纯化凝血因子X激活剂(factor X activator of Russell’sviper venom,RVV-X)并研究其止血作用。方法应用阴离子交换色谱和凝胶过滤从广西圆斑蝰蛇毒中分离纯化RVV-X,以复钙化时间进行活性鉴定,以SDS-PAGE测定纯度和分子量。观察RVV-X对家兔肝脏创伤出血模型出血时间及出血量,家兔和肝素化小鼠凝血时间的影响。结果广西圆斑蝰粗毒经阴离子交换色谱获得六个蛋白峰,将RVV-X所在第V峰用凝胶过滤获得的组分A为RVV-X纯品,其凝血活性具有钙依赖性。SDS-PAGE非还原性条件下呈单一蛋白条带,分子量约为97.8kDa,还原性条件下呈二蛋白条带,为一条重链(约76.3kDa)和一条轻链(约21.5kDa)。RVV-X可显著减少家兔肝脏切口的失血量,缩短切口出血时间;能显著缩短家兔及肝素化小鼠凝血时间。结论用层析方法从广西圆斑蝰蛇毒中分离得到的凝血因子X激活剂具有较强的止血活性。

Objective To isolate and purify the coagulation factor X activator from venom of Vipera russellii in Guangxi and to investigate its hemostasis effects. Methods RVV-X was purified from Russell’s viper venom by ion-exchange chromatography and by gel filtration. Homogenicity and molecular weight of it were determined in SDS-PAGE. Then we observed effects of intravenous RVV-X on model of hepatic trauma rabbit , on blood coagulation time in rabbit and heparinized mice. Results RVV-X was isolated from the venom of Vipera russellii. It could shorten the coagulation time in the presence of Ca2 ＋ ions. It consisted of a single polypeptide chain,with a mol. wt of 97,800 in nonreducing conditions and composed of one heavy chain （ 76,300Da） and one light chain（ 21,500Da） in reducing conditions. RVV-X could obviously decrease blood loss of rabbits liver wound and shorten bleeding time. It could also shorten the coagulation time of rabbit and heparinized mice. Conclusion RVV-X extracted from the venom of Vipera Russellii in Guangxi possesses considerable hemostatic activities.