Structure and Function of ADAMTS13 Metalloprotease

描述（由申请方提供）：由于血浆金属蛋白酶（ADAMTS 13）缺乏引起的灾难性并发症突出了该蛋白酶在调节止血和预防血栓性血小板减少性紫癜（TTP）综合征中发生的微血管循环血栓形成方面的生物学重要性。ADAMTS 13金属蛋白酶编码基因的鉴定和编码蛋白中结构元件的确定为理解锌金属蛋白酶在止血和血栓形成调节中的作用开辟了一个新时代，并为解决ADAMTS 13的基本（病理）生物学问题提供了必要的工具，包括其生物合成，活化，底物识别，细胞内分选和自身抗体相互作用。本研究的目的是：具体目标1。通过1）分析野生型ADAMTS 13和ADAMTS 13的各种突变体对血管性血友病因子及其类似物VWF 73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法测定VWF或VWF 73与野生型或突变型ADAMTS 13之间的结合动力学，确定ADAMTS 13底物识别。具体目标2。通过1）确定MDCK细胞中全长和突变型ADAMTS 13分泌的极性; 2）确定ADAMTS 13分选的分选信号和机制; 3）确定先天性TTP患者中发现的ADAMTS 13基因点突变或截短对ADAMTS 13分泌极性的影响，确定ADAMTS 13的细胞内分选。具体目标3。确定抗ADAMTS 13自身抗体结合的ADATMTS 13结构域（或位点）以及ADAMTS 13金属蛋白酶与抗ADAMTS 13自身抗体之间相互作用的动力学参数（亲和力和特异性），并将临床病程和结局与TTP患者中鉴别的不同类型的抗ADAMTS 13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.