流动状态下ADAMTS13羧基端的功能研究

Proximal Carboxyl-terminal Domains of ADAMTS13 under Flow Conditions

目的研究ADAMTS13及其突变体于体外流动状态下裂解血管性血友病因子(VWF),降低血小板粘附形成血栓能力的差异。方法将全长ADAMTS13(FL)及缺失TSP2-8CUB1+2(MDTCS)和缺失CUB1+2(delCUB)cDNA质粒瞬时转染COS7细胞,Western blot检测各瞬时表达细胞分泌的蛋白。利用流体力学装置平板流动小室(FlowChamber)培养人脐静脉内皮细胞,并通过蠕动泵加压流动培养液模拟人体血管内流动状态,荧光显微镜观测加入AD-AMTS13或其突变体蛋白后荧光标记的血小板聚集状态的差异。结果成功在真核表达细胞内瞬时表达ADAMTS13及其突变体蛋白,简单纯化并经Western blot检测各表达蛋白相对分子量与预期一致。Flow Chamber模拟人体血管内剪切力作用下,FL蛋白和MDTCS蛋白可以不同程度地抑制血小板凝集,delCUB蛋白抑制血小板凝集的能力显著下降。结论成功瞬时表达全长ADAMTS13及其突变体蛋白。初步证实流动状态下ADAMTS13羧基端是参与裂解VWF以抑制血小板凝集的主要区域。

Objective To compare the cleavage activity and influence on platelets adhesion between ADAMTS13 and its mutants under flow conditions.Methods ADAMTS13 and its mutants plasmids were transiently transfected into COS7 cells,then the secreted proteins were purified and detected by Western blott with anti-V5 IgG.Human umbilical vein endothelial cells（HUVECs）in the Flow Chamber system were cultured,and the flow conditions were imitated in vessels by peristaltic pump.The cleaving activity of Ultralarge von Willebrand factor（UL-VWF）secreted by damaged HUVECs and the ability of reducing platelets adhesion and staying thrombosis formation were compared between ADAMTS13 and its mutants.Results ADAMTS13 and its mutants were successfully transiently expressed in eukaryotic cells,recombinant ADAMTS13 was purified,and the proteins were verified by Western blot.Imitating the flow conditions in vessels by Flow Chamber system,it was demonstrated that FL-ADAMTS13 and MDTCS-ADAMTS13 might reduce platelets aggregation to varying degrees,but the ability of reducing platelets aggregation by delCUB-ADAMTS13 was significantly reduced.Conclusion Under flow conditions,the proximal carboxyl-terminal domains of ADAMTS13 may be crucial for cleaving VWF and inhibiting platelets aggregation.