VW Factor Cleaving Prostease and Thrombotic Diseases

描述（申请人提供）：血栓性血小板减少性紫癜（UP）是一种血栓性微血管病，与血浆中解聚血管性血友病因子的蛋白酶活性缺乏有关。家族性TIP是由这种蛋白酶的遗传缺陷引起的，而获得性UP是由蛋白酶自身抗体的存在引起的。这种蛋白酶，已被命名为vWF切割蛋白酶（VWFCP），是一种金属蛋白酶，并且特异性地切割vWF亚基的P2结构域中的Tyrl 605-MetI 606键。已将VWFCP纯化至均一，部分测序，并克隆了其cDNA。VWFCP是金属蛋白酶ADAMTS家族的成员，包含结构基序，如解整合素、富含Cys和血小板反应蛋白-1基序，这些是该家族的定义特征。在本申请中，我们建议（1）研究VWFCP的结构和功能关系，并将VWFCP的生物化学性质与其天然存在的变体形式进行比较，这些变体形式是作为选择性mRNA剪接的结果而合成的;（2）开发替代底物，改进用于定量测量VWFCP活性、血浆中VWFCP抗原水平和VWFCP自身抗体滴度的测定方法。这些研究将提供一个更好的理解这种金属蛋白酶如何调节vWF的功能，并保持血栓形成和原发性止血之间的微妙平衡。这种理解将为设计治疗家族性和获得性UP的替代方法提供基础。

DESCRIPTION (provided by applicant): Thrombotic thrombocytopenic purpura (UP) is a thrombotic microangiopathic disorder that is associated with a deficiency of a protease activity that depolymerizes von Willebrand factor in plasma. Familial TIP is caused by a genetic deficiency of this protease, whereas acquired UP is caused by the presence of autoantibodies to the protease. This protease, which has been named vWF cleaving protease (VWFCP), is a metalloprotease and specifically cleaves the Tyrl 605-MetI 606 bond in the P2 domain of the vWF subunit. VWFCP has been purified to homogeneity, partially sequenced, and its cDNA has been cloned. VWFCP is a member of the ADAMTS family of metalloproteases and contains structural motifs such as disintegrin, Cys-rich, and thrombospondin-1 motifs, which are the defining characteristics of this family. In this application, we propose to (1) study the structure and function relationship of VWFCP, and compare the biochemical properties of VWFCP to its naturally occurring variant forms, which are synthesized as a result of alternative mRNA splicing; (2) develop alternative substrates, improved assays for the quantitative measurement of VWFCP activity, the level of VWFCP antigen in plasma, and the titer of autoantibodies to VWFCP. These studies would provide a better understanding of how this metalloprotease regulates the function of vWF and keeps a delicate balance between thrombosis and primary hemostasis. This understanding would provide a basis for devising alternative methods for treating familial and acquired UP.