血小板膜糖蛋白Ⅱb/Ⅲa受体复合物的分离和纯化

Isolation and Purification of the Human Platelet Glycoprotein Ⅱb/Ⅲa Receptor Complex

从机采浓缩血小板中 ,应用凝胶柱刀豆素A—琼脂糖、肝素—琼脂糖亲和层析与分子筛层析法 ,分离、纯化并得到血小板膜糖蛋白Ⅱb/Ⅲa受体复合物。血小板膜糖蛋白Ⅱb/Ⅲa受体复合物由α、β两个亚单位组成 ,糖蛋白Ⅱb可还原为糖蛋白Ⅱbα(分子量为 12 5kDa)和糖蛋白Ⅱbβ(分子量为 2 3kDa) ,而糖蛋白Ⅲa为一条多肽链 ,分子量为 95kDa ,还原后为 10 8kDa。经不同凝胶亲和层析、凝胶电泳分析 ,得到血小板膜糖蛋白Ⅱb/Ⅲa受体复合物 ,为进一步筛选单克隆抗体和进行药物研发打下了基础。

Aim A method has been developed for the rapid isolation of platelet membrane glycoproteins (GP) Ⅱb and Ⅲa. Methods The GP Ⅱb/Ⅲa complex was purified with concanavalin A-sepharose, heparin-sepharose and sephacryl S-300HR chromatography. Results The GP Ⅱb/Ⅲa complex, GP Ⅱb is composed of two disulfide-linked chains, a heavy chain of 125 kDa, called GPⅡbα, and a light chain of 23 kDa, called GP Ⅱbβ, in reduced conditions. The GP Ⅲa is a single polypeptide of 108 kDa in reduced conditions, or 95 kDa in nonreduced conditions. Conclusions Concanavalin A affinity chromatography was used to purify a platelet glycoprotein fraction. The concanavalin A-retained glycoproteins were eluted and adsorbed with a heparin-sepharose column to remove a major contaminant, thrombospondin. Sephacryl S-300 gel filtration was used as the final purification step to remove most fibrinogen and low-molecular-weight contaminants. The GP Ⅱb/Ⅲa complex can be used for the development of its biological products and further study.