目的 探讨血管性血友病因子裂解酶 ADAM 金属肽酶含血小板反应蛋白1型13（ADAMTS13）间隔区结构域在血管性血友病因子（vWF）裂解过程中的生物学功能，阐明ADAMTS13在血栓性血小板减少性紫癜（TTP）发病机制中的作用。 方法 将ADAMTS13间隔区结构域中的氨基酸残基 TEDRLPR 以点突变技术逐个基因突变（突变体M1~M7），将构建的ADAMTS13 与其突变体质粒转染至人胚肾 HEK293 细胞，稳定表达后提纯重组蛋白。观察野生型和突变型 ADAMTS13 在变性条件、剪切应力作用和 ADAMTS13 抗体处理后裂解 vWF 的能力。 结果 荧光共振能量转移（FRET）实验， 与野生型 ADAMTS13 比较， ADAMTS13 突变体M4（R635A）和突变体M7（R638A）对FRET-vWF73剪切能力降低（P<0.05）。变性条件下，野生型ADAMTS13可以将vWF多聚体裂解， 与野生型ADAMTS13比较， ADAMTS13突变体M4（R635A）和突变体M7（R638A）的裂解活性明显降低（P<0.01）。在体外剪切应力作用下，与野生型 ADAMTS13 比较， ADAMTS13 突变体 M4（R635A）和突变体M7（R638A）裂解 vWF 多聚体的能力明显降低（P<0.01）。 与野生型 ADAMTS13 比较， ADAMTS13突变体M4（R635A）和突变体M7（R638A）与vWF之间的结合力差异无统计学意义（P>0.05），表明ADAMTS13的C末端与vWF之间存在多个结合位点。ADAMTS13抗体处理可在一定程度上抑制野生型和突变型ADAMTS13裂解vWF的能力。 结论 间隔区突变后ADAMTS13的活性降低。ADAMTS13突变体M4（R635A）和突变体M7（R638A）可能是ADAMTS13在底物识别时的重要作用位点。

Objective To discuss the biological function of the spacer domain of ADAM metalloproteinase with thrombospondin type 1 motifs 13 （ADAMTS13） in the cleaving process of von Willebrand factor （vWF），and to clarify the role of ADAMTS13 in the pathogenesis of thrombotic thrombocytopenic purpura （TTP）. Methods The point mutation method was introduced sequentially into the amino acid residues TEDRLPR of the ADAMTS13 spacer domain （mutants M1－M7） by site-directed mutagenesis. The constructed ADAMTS13 and its mutants plasmids were transfected into the human embryonic kidney HEK293 cells， and the recombinant proteins were purified after stable expression. The cleavage capabilities of both wild type and mutant ADAMTS13 were observed under denaturation conditions， shear stress， and after treatment with ADAMTS13 antibodies. Results The fluorescence resonance energy transfer （FRET） assay results showed that compared with wild type ADAMTS13，the cleavage abilities of ADAMTS13 mutant M4 （R635A） and mutant M7 （R638A） on the FRET-vWF73 were decreased （P<0.05）. Under denaturation conditions， the wild-type ADAMTS13 could cleave the vWF multimers； compared with wild-type ADAMTS13， the cleavage activities of ADAMTS13 mutant M4 （R635A） and mutant M7 （R638A） were significantly decreased （P<0.01）. Under in vitro shear stress， compared with wild type ADAMTS13， the abilities of ADAMTS13 mutant M4 （R635A） and mutant M7 （R638A） to cleave vWF multimers were significantly decreased （P<0.01）. Compared with wild type ADAMTS13， the binding affinity between vWF and ADAMTS13 mutant M4 （R635A） and mutant M7 （R638A） had no significant difference （P>0.05）， indicating there were multiple binding sites between C-terminal of ADAMTS13 and vWF. The ADAMTS13 antibodies were able to inhibit the cleavage ability of both wild-type and mutant ADAMTS13 to some extent. Conclusion The activity of ADAMTS13 after spacer domain mutation is decreased. The ADAMTS13 mutant M4 （R635A） and mutant M7 （R638A） may be the important action sites for ADAMTS13 in substrate recognition.