A1500E突变对血管性血友病因子的ADAMTS13依赖性水解的影响

Increased susceptibility of recombinant type 2A von Willebrand factor mutant A1500E to proteolysis by ADAMTS13

目的研究血管性血友病因子（VWF）A1500E突变体对金属蛋白酶ADAMTS13敏感性的改变，为VWF—A1500E突变导致2A型血管性血友病（VWD）的发病机制提供直接依据。方法将野生型VWF质粒和A1500E突变体VWF质粒分别瞬时转染HeLa细胞，收集并浓缩培养上清，分别用重组人ADAMTS13（rADAMTS13）进行水解，然后通过十二烷基硫酸钠-琼脂糖凝胶电泳进行VWF多聚体分析，观察与野生型VWF相比，A1500E突变体VWF对ADAMTS13的敏感性有无改变。结果体外表达研究结果显示WT—VWF和A1500E突变体VWF的表达上清中VWF：舷的平均含量分别为1．10U／ml和0．78U／ml，突变体细胞裂解液中的VWF：Ag表达量为野生型的90．6％，两者差异均无统计学意义（P〉0．05）。VWF多聚体电泳显示突变体VWF与WT—VWF的多聚体分布亦无明显差别。在无尿素和盐酸胍等变性剂的静态条件下，rADAMTS13即可对A1500E突变体VWF进行有效地水解，VWF多聚体分析显示大中分子量VWF多聚体明显减少和小分子量VWF多聚体明显增多；相反，野生型VWF在非变性条件下则无被rADAMTS13水解的证据。结论A1500E突变导致突变体VWF对ADAMTS13的敏感性异常性增高，符合第二组2A型VWD的突变特点。

Objective To investigate the susceptibility of yon Willebrand factor （VWF） type 2A mutant A1500E to proteolysis by metalloprotease ADAMTS13 and to provide the direct supports for the pathogenesis of VWF mutation A1500E responsible for yon Willebrand disease （VWD） type 2A. Methods Recombinant wild-type VWF （WT-VWF） and A1500E mutant VWF transiently expressed on transfected HeLa cell lines. Expression media were collected and concentrated, then cleaved directly by recombinant AD- AMTS13 （rADAMTS13）. Compared with WT-VWF, the susceptibility of A1500E mutant VWF to proteolysis by ADAMTS13 was analyzed using SDS-agarose gel VWF multimers analysis. Results In vitro the expression of VWF： Ag in the supernatants of WT-VWF and A1500E mutant VWF were 1. l0 U/ml and 0.78 U/ml, respectively, while VWF： Ag in cells lysates of A1500E mutant VWF was 90.6% of that of WT-VWF. The SDS-agarose gel VWF multimers analysis showed that there were no differences between WT-VWF and A1500E mutant VWF. The A1500E mutant VWF could be efficiently cleaved by ADAMTS13 under static condition without denaturants such as urea and guanidine HC1. VWF multimeric analysis showed that high and intermediate molecular weight multimers dramatically decreased while low molecular weight muhimers obvious- ly increased. Conversely, WT-VWF could not be cleaved by ADAMTS13 under the same condition. Conclusion The A1500E mutation resulted in VWF more susceptible to ADAMTS13-dependent proteolysis, which belonged to VWD type 2A group 2 mutation.