Structure and Function of ADAMTS13 Metalloprotease

描述（由申请人提供）：血浆金属蛋白酶（ADAMTS13）缺乏引起的灾难性并发症突出了该蛋白酶在调节血栓性血小板减少性紫癜（TTP）综合征中发生的止血和预防微血管循环血栓形成中的生物学重要性。ADAMTS13金属蛋白酶编码基因的鉴定和编码蛋白结构元件的确定为了解锌金属蛋白酶在止血和血栓形成调控中的作用开辟了新时代，并为解决ADAMTS13的生物合成、激活、底物识别、细胞内分选和自身抗体相互作用等基本（病理）生物学问题提供了必要的工具。拟建研究的目标是：为了确定ADAMTS13的底物识别，1)分析野生型ADAMTS13和ADAMTS13的各种突变体对血管性血液病因子及其类似物VWF73的蛋白水解活性；2)通过放射性配体结合和表面等离子体共振测定VWF或VWF73与野生型或突变型ADAMTS13的结合动力学。具体目标2。通过测定MDCK细胞中全长型和突变型ADAMTS13分泌的极性来确定ADAMTS13的细胞内分选；2)确定ADAMTS13的分选信号和分选机制；3)确定先天性TTP患者ADAMTS13基因点突变或截短对ADAMTS13分泌极性的影响。具体目标3。确定抗ADAMTS13自身抗体结合的ADATMTS13结构域（或位点）以及ADAMTS13金属蛋白酶与抗ADAMTS13自身抗体相互作用的动力学参数（亲和性和特异性），并将TTP患者的临床过程和结果与不同类型的抗ADAMTS13自身抗体相关联。

DESCRIPTION (provided by applicant): The catastrophic complications due to deficiency in plasma metalloprotease (ADAMTS13) highlight the biological importance of this protease in regulation of hemostasis and prevention of thrombosis in microvascular circulation that occurs in the thrombotic thrombocytopenic purpura (TTP) syndrome. Identification of the gene encoding ADAMTS13 metalloprotease and determination of the structure elements in the encoded protein have opened a new era in understanding of the roles of a zinc metalloprotease in regulation of hemostasis and thrombosis, and provided the essential tools to address the fundamental (patho)biological questions concerning ADAMTS13 including its biosynthesis, activation, substrate recognition, intracellular sorting and autoantibody interaction. The goals of the proposed research are: Specific Aim 1. To determine ADAMTS13 substrate recognition by 1) analyzing the proteolytic activity of wild type ADAMTS13 and various mutants of ADAMTS13 toward von Willebrand factor and its analog, VWF73; 2) determining the binding kinetics between VWF or VWF73 and wild type or mutant ADAMTS13 by radioligand binding and surface plasmon resonance assays. Specific Aim 2. To determine intracellular sorting of ADAMTS13 by 1) determining the polarity of full-length and mutant ADAMTS13 secretion in MDCK cells; 2) determining the sorting signal and mechanisms by which ADAMTS13 is sorted; 3) determining the effect of point mutations or truncations of ADAMTS13 gene found in patients with congenital TTP on the polarity of ADAMTS13 secretion. Specific Aim 3. To determine the domains (or sites) of ADATMTS13 to which anti-ADAMTS13 autoantibodies bind and the kinetic parameters (affinity and specificity) of interaction between ADAMTS13 metalloprotease and anti-ADAMTS13 autoantibodies, and to correlate the clinical course and outcome to the distinct type of anti-ADAMTS autoantibodies identified in patients with TTP.