人von Willebrand因子A1和A3功能结构域嵌合体的表达及生物学功能

vWF介导血小板粘附到细胞外基质 ,在血栓形成过程中发挥重要作用 ,可通过阻断vWF与细胞外基质的结合阻止血小板的粘附 .应用RT PCR方法从人脐静脉内皮细胞中克隆vWF分子A1、A3区基因并在大肠杆菌中表达 ,表达的重组蛋白量占菌体总蛋白 12 6 % ,包涵体经过变性剂溶解、纯化和复性 ,获得重组蛋白 (rvWF A1 A3) .应用流式细胞术检测rvWF A1 A3与血小板膜糖蛋白 (GPⅠb)的结合功能 ;血小板聚集实验观察rvWF A1 A3对瑞斯托霉素 (ristocetin)诱导的血小板聚集 (RIPA)的影响 ;ELISA胶原结合实验及竞争抑制实验分析rvWF A1 A3与胶原的结合活性 .结果显示 :rvWF A1 A3嵌合体与血小板的结合阳性率为 70 4 % .rvWF A1 A3嵌合体不能引起血小板的聚集 ,但rvWF A1 A3嵌合体与血小板温育后可以阻断ristocetin诱导人血浆vWF对血小板的聚集作用 ,而且呈剂量依赖性 ,IC50 的rvWF A1 A3浓度为 0 76 μmol L ,当浓度为 1 17μmol L时抑制率最高达 76 8% .rvWF A1 A3具有良好的胶原结合活性 ,同时它可以竞争性抑制vWF与Ⅲ型胶原的结合 ,抑制率为 76 % .表明rvWF A1 A3可作为阻断剂用于干预vWF介导的血小板粘附过程 ,同时又可以阻断血浆vWF与血小板GPIb结合抑制血小板聚集 ,具有良好的抗栓应用前景 .

Expression of von Willebrand Factor-A1/A3 Chimera in E.coli and Its Biological Activities

Platelets adhere to the subendothelium of damaged blood vessels through a interaction with von Willebrand factor (vWF), which form a bridge between collagens within the damaged vessel wall and the platelet receptor glycoprotein Ib (GPⅠb/Ⅸ/Ⅴ). The interaction between collagen-vWF and glycoprotein Ⅰb axis is the first step in hemostasis and thrombosis especially under high shear condition, thus specific blockade of the interaction between GPⅠb, vWF and collagen could have antithrombotic effects.The mRNA from endothelial cells was extracted, and vWF domain A1 and A3 gene fragments was amplified by RT-PCR. After sequencing, the amplified DNA fragments of vWF domain A1 and A3 were inserted into expression vector pET22b(+) with 6×His tag. The chimeric molecule containing residues 449—728 of mature vWF subunit (vWF-A1 polypeptide) fused in frame to residues 918—1114 of the vWF (vWF-A3 polypeptide) was constructed. The recombinant vector was transformed into E.coli DE3 and induced by IPTG,the recombinant fragment designate rvWF-A1/A3.The protein was expressed in E.coli DE3, accounting for 12.6% of totalbacterial protein. The protein was purified through Ni-NTA resin column and refolded in Tris-HCl buffer containing GSH and GSSG,its purity was over 95%. Flow cytometry, collagen binding test and ristocetin-induced platelet aggregation were used to analyze the biological activities of the refolded rvWF-A1/A3. The protein was identified to have abilities to bind to collagen and to inhibit vWF binding to collagen, with 76% of inhibitory rate. Meanwhile, its binding rates to platelet glycoprotein Ⅰb/Ⅸ/Ⅴ was 70.4%,and IC 50 of 0.76 μmol/L for ability of inhibiting ristocetin-induced platelet aggregation. The inbibitory rate was a dose dependent and the maximal inhibiting rate was 76.8% at 1.17μmol/L. These results indicated that rvWF-A1/A3 fragment might be an effective antithrombotic agent for preventing arterial thrombosis.