ALLOSTERIC REGULATION OF ADAMTS13

ADAMTS13 的变构调节

• 批准号: 9011725
• 负责人: J Evan Sadler
• 金额: $ 42.17万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9011725
• 关键词: Adhesions; Allosteric Regulation; Architecture; Binding; Biochemical; Blood Coagulation Factor VII; Blood Platelets; Blood Vessels; Blood flow; Cleaved cell; Coenzymes; Complex; Data; Data Collection; Defect; Diagnostic; Disease; Disintegrins; Distal; Enzyme Kinetics; Feedback; Fluorogenic Substrate; Foundations; Goals; Hemorrhage; Hemostatic Agents; Hemostatic function; In Vitro; Injury; Kinetics; Knowledge; Life; Liquid substance; Magnetism; Metalloproteases; Modeling; Mutagenesis; Patients; Peptide Hydrolases; Research Proposals; Roentgen Rays; Site; Stretching; Structure; Syndrome; System; Testing; Therapeutic; Thrombin; Thrombomodulin; Thromboplastin; Thrombosis; Thrombotic Thrombocytopenic Purpura; Thrombus; Translating; Variant; base; cofactor; factor IXa-factor VIIIa; improved; inhibitor/antagonist; innovation; left ventricular assist device; novel; prevent; public health relevance; shear stress; single molecule; von Willebrand Disease; von Willebrand Factor

 DESCRIPTION (provided by applicant): ADAMTS13 recognizes and cleaves a cryptic site in von Willebrand factor (VWF) that is exposed when VWF multimers are stretched by fluid shear stress. Severe ADAMTS13 deficiency impairs this regulatory mechanism and causes thrombotic thrombocytopenic purpura. Conversely, exaggerated cleavage of VWF by ADAMTS13 causes bleeding in some variants of von Willebrand disease (VWD) and acquired von Willebrand syndrome (AVWS). For example, almost all patients with left ventricular assist devices (LVADs) develop AVWS and approximately one-third of them have major bleeding. We recently discovered that ADAMTS13 is folded in half, so that its distal T8-CUB domains inhibit the proximal metalloprotease domain. Furthermore, binding of VWF to distal ADAMTS13 domains relieves this autoinhibition. Thus, VWF promotes its own destruction by allosterically activating ADAMTS13. Our goal is to understand the structural basis of this previously unsuspected regulatory mechanism and to translate this knowledge into better treatment for bleeding and thrombosis. In Specific Aim 1 we will characterize the interactions between VWF and ADAMTS13 that are necessary for allosteric regulation of ADAMTS13 activity. We will use mutagenesis, binding and enzyme kinetics with novel fluorogenic substrates to test the hypothesis that interactions between specific ADAMTS13 domains and cognate sites on VWF cause changes in ADAMTS13 structure that relieve the autoinhibition of ADAMTS13 protease activity. In Specific Aim 2 we will characterize the structural basis for allosteric regulation of ADAMTS13 by VWF. Using SAXS and EM to visualize the structure of ADAMTS13 and ADAMTS13-VWF complexes, we will test the hypothesis that distal ADAMTS13 domains directly contact proximal domains, and that the architecture of ADAMTS13 depends on these contacts. We will characterize the conformational changes that accompany allosteric activation, and the results will provide a structural framework to understand how ADAMTS13 is activated within platelet-rich thrombi. In Specific Aim 3 we will define and pharmacologically inhibit the allosteric contribution to the cleavage of VWF multimers under shear stress. We hypothesize that inhibitors of ADAMTS13 allosteric activation will reduce ADAMTS13 activity to a safe level that does not induce thrombotic microangiopathy but can improve hemostasis in AVWS. We will use the highly parallel data collection of magnetic tweezers to characterize a unique class of inhibitors that interfere with critical steps in the allosteric activation of ADAMT13. We will exted these single molecule results to analyze how these ADAMTS13 inhibitors alter the kinetics of VWF multimer cleavage under fluid shear stress in vitro and in an LVAD model ex vivo. The results will demonstrate how ADAMTS13 reaches maximal efficiency precisely where needed, within a growing thrombus, and show how to modulate these interactions therapeutically.

 描述（由适用提供）：ADAMTS13识别并切割了Von Willebrand因子（VWF）中的加密位点，当VWF多聚体被流体剪切应力拉伸时，该位置会暴露出来。严重的ADAMTS13缺乏症会损害这种调节机制，并导致血栓性血小板减少性紫癜。相反，ADAMTS13对VWF的夸张裂解导致冯·威勒疾病（VWD）的某些变体出血，并获得了冯·威勒布兰德综合征（AVWS）。例如，几乎所有患有左心室辅助装置（LVAD）的患者会出现AVW，其中大约三分之一的患者出血。我们最近发现ADAMTS13折叠了一半，因此其远端T8-CUB结构域抑制了近端金属蛋白酶结构域。此外，VWF与远端ADAMTS13结构域的结合可以缓解这种自身抑制作用。这，VWF通过变构激活ADAMTS13促进了自己的破坏。我们的目标是了解这种先前未经刺激的调节机制的结构基础，并将这些知识转化为出血和血栓形成的更好治疗。在特定目标1中，我们将表征VWF和ADAMTS13之间的相互作用，这对于ADAMTS13活性的变构调节所必需。我们将使用新型的荧光底物使用诱变，结合和酶动力学来检验以下假设：在VWF上，特定的ADAMTS13结构域与同源位点之间的相互作用会导致ADAMTS13结构的变化，从而挽救了ADAMTS13蛋白酶活性的自身抑制。在特定目标2中，我们将表征VWF对ADAMTS13的变构调节的结构基础。使用SAXS和EM可视化ADAMTS13和ADAMTS13-VWF复合物的结构，我们将测试以下假设：远端ADAMTS13域直接接触代理域，并且ADAMTS13的体系结构取决于这些接触。我们将表征发生变构激活的会议变化，结果将提供一个结构框架，以了解如何在富含血小板的血栓中激活ADAMTS13。在特定的目标3中，我们将定义和药物抑制对剪切应力下VWF多中部裂解的变构贡献。我们假设ADAMTS13的抑制剂变构激活将使ADAMTS13活性降低到不会诱导血栓性微血管病，但可以改善AVWS止血的安全水平。我们将使用磁性镊子的高度平行数据收集来表征一类独特的抑制剂，这些抑制剂干扰ADAMT13的变构激活中的关键步骤。扩展了这些单个分子的结果，以分析这些ADAMTS13抑制剂如何在体外和LVAD模型中在流体剪切应力下改变VWF多聚体裂解的动力学。结果将证明ADAMTS13如何精确地达到最大效率，在需要的情况下，在成长的血栓中，并显示如何热调节这些相互作用。