CAJ | 학술논문

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:Ag的平均含量分别为1.10 U/ml和0.78 U/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.
목적 연구혈관성혈우병인자(VWF) A1500E돌변체대금속단백매ADAMTS13민감성적개변,위VWF-A1500E돌변도치2A형혈관성혈우병(VWD)적발병궤제제공직접의거.방법 장야생형VWF질립화A1500E돌변체VWF질립분별순시전염HeLa세포,수집병농축배양상청,분별용중조인ADAMTS13( rADAMTS13)진행수해,연후통과십이완기류산납-경지당응효전영진행VWF다취체분석,관찰여야생형VWF상비,A1500E돌변체VWF대ADAMTS13적민감성유무개변.결과 체외표체연구결과현시WT-VWF화A1500E돌변체VWF적표체상청중VWF:Ag적평균함량분별위1.10 U/ml화0.78 U/ml,돌변체세포렬해액중적VWF:Ag표체량위야생형적90.6％,량자차이균무통계학의의(P＞0.05).VWF다취체전영현시돌변체VWF여WT-VWF적다취체분포역무명현차별.재무뇨소화염산고등변성제적정태조건하,rADAMTS13즉가대A1500E돌변체VWF진행유효지수해,VWF다취체분석현시대중분자량VWF다취체명현감소화소분자량VWF다취체명현증다；상반,야생형VWF재비변성조건하칙무피rADAMTS13수해적증거.결론 A1500E돌변도치돌변체VWF대ADAMTS13적민감성이상성증고,부합제이조2A형VWD적돌변특점.
Objective To investigate the susceptibility of von 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 von 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 ADAMTS13 (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 1.10 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 multimers obviously 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.