Regulation of ADAMTS13 Activity

描述(由申请人提供)：这项建议的目标是了解ADAMTS13特异性的结构基础，并将这一知识转化为血栓性血小板减少性紫癜的更好治疗，这是一种主要发生在30至50岁之间的女性的医疗紧急情况。ADAMTS13是一种在血液中发现的金属蛋白酶，它能裂解von Willebrand因子(VWF)，VWF是一种多聚体蛋白，介导血小板在血管损伤部位的黏附。血栓性血小板减少性紫癜通常是由抗ADAMTS13的自身抗体引起的，这种抗体阻止了VWF的切割。由此导致的ADAMTS13缺陷导致了不受控制的血小板黏附和广泛的微血管血栓形成，表明正常的止血依赖于ADAMTS13对VWF功能的抑制。这种非常精确的反馈抑制机制涉及VWF的识别和切割，特别是当它受到流动的血液力的张应力时，就像在不断增长的血栓中发生的那样。提出了几种实验策略来研究ADAMTS13，并利用结果来推进患者护理。具体目标1是表征流体剪切力、辅因子和VWF结构对ADAMTS13的调节。血小板膜GPIB或肝素可协同流体切应力促进VWF的切割。相应的生化机制将在血小板悬浮液和内皮细胞表面的流体剪应力条件下表征。VWF和ADAMTS13的参与结构域将通过突变和重组蛋白的分析来鉴定。具体目标2是表征ADAMTS13识别VWF的结构基础。当受到拉伸应力时，VWF显示出与ADAMTS13上的多个Exosite相互作用的独特功能组合。这些广泛的接触加强了ADAMTS13在体内对VWF的严格特异性。一种全面的生化方法将确定VWF和ADAMTS13上互补位点之间的结合位置、结构和能量。具体目标3是开发改进的ADAMTS13活性的临床检测方法。血栓性血小板减少性紫癜的及时诊断是至关重要的，因为如果不治疗，几乎所有患者都会死亡，而血浆置换治疗可以将存活率提高到80%。了解ADAMTS 13如何识别和切割VWF将有助于开发快速的临床ADAMTS 13检测方法，这将有助于ADAMTS13缺乏引起的血栓性血小板减少性紫癜患者的诊断和治疗。

DESCRIPTION (provided by applicant): The goal of this proposal is to understand the structural basis of ADAMTS13 specificity and to translate this knowledge into better treatment for thrombotic thrombocytopenic purpura, a medical emergency that strikes mainly women between 30 and 50 years of age. ADAMTS13 is a metalloprotease found in the blood that cleaves von Willebrand factor (VWF), a multimeric protein that mediates the adhesion of platelets at sites of vascular injury. Thrombotic thrombocytopenic purpura usually is caused by autoantibodies against ADAMTS13 that prevent it from cleaving VWF. The resulting ADAMTS13 deficiency leads to unchecked platelet adhesion and widespread microvascular thrombosis, demonstrating that normal hemostasis depends on the inhibition of VWF function by ADAMTS13. This wonderfully precise feedback inhibitory mechanism involves the recognition and cleavage of VWF specifically when it is subjected to tensile stress by the force of flowing blood, as occurs in a growing thrombus. Several experimental strategies are proposed to investigate ADAMTS13 and make use of the results to advance patient care. Specific Aim 1 is to characterize the regulation of ADAMTS13 by fluid shear stress, cofactors, and VWF structure. Platelet membrane GPIb or heparin can cooperate with fluid shear stress to promote VWF cleavage. The corresponding biochemical mechanisms will be characterized under conditions of fluid shear stress in platelet suspensions and on endothelial cell surfaces. Participating structural domains of VWF and ADAMTS13 will be identified by mutagenesis and analysis of recombinant proteins. Specific Aim 2 is to characterize the structural basis for the recognition of VWF by ADAMTS13. When subjected to tensile stress, VWF displays a unique combination of features that interact with multiple exosites on ADAMTS13. These extensive contacts enforce the strict specificity of ADAMTS13 for VWF in vivo. A comprehensive biochemical approach will define the location, structure and energetics of binding between complementary sites on VWF and ADAMTS13. Specific Aim 3 is to develop improved clinical assays of ADAMTS13 activity. Prompt diagnosis of thrombotic thrombocytopenic purpura is essential because almost all patients die if untreated and plasma exchange therapy increases survival to >80%. Understanding how ADAMTS 13 recognizes and cleaves VWF will enable the development of rapid clinical ADAMTS 13 assays, which will facilitate the diagnosis and treatment of patients with thrombotic thrombocytopenic purpura caused by ADAMTS13 deficiency.