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（由申请人提供）：ADAMTS13 识别并切割血管性血友病因子 (VWF) 中的一个神秘位点，当 VWF 多聚体因流体剪切应力而拉伸时，该位点会暴露出来，严重的 ADAMTS13 缺陷会损害这种调节机制，并导致血栓性血小板减少性紫癜。 ADAMTS13 裂解 VWF 会导致冯维勒布兰德病的某些变异体出血(VWD) 和获得性血管性血友病综合征 (AVWS) 例如，几乎所有使用左心室辅助装置 (LVAD) 的患者都会出现 AVWS，其中大约三分之一有大出血，因此我们最近发现 ADAMTS13 被折叠成两半。其远端 T8-CUB 结构域抑制近端金属蛋白酶结构域，此外，VWF 与远端 ADAMTS13 结构域的结合可缓解这种自抑制作用，因此，VWF 会促进其自身抑制。我们的目标是了解这种以前未曾怀疑的调节机制的结构基础，并将这些知识转化为更好的出血和血栓形成治疗方法。在具体目标 1 中，我们将描述 VWF 和 ADAMTS13 之间必要的相互作用。我们将利用新型荧光底物的诱变、结合和酶动力学来测试特定 ADAMTS13 结构域与 ADAMTS13 结构域之间相互作用的假设。 VWF 上的同源位点会导致 ADAMTS13 结构发生变化，从而缓解 ADAMTS13 蛋白酶活性的自身抑制。在特定目标 2 中，我们将使用 SAXS 和 EM 来表征 ADAMTS13 变构调节的结构基础。复合体，我们将测试远端 ADAMTS13 结构域直接接触近端结构域的假设，并且ADAMTS13 依赖于这些接触，我们将表征伴随变构激活的构象变化，结果将提供一个结构框架来了解 ADAMTS13 如何在富含血小板的血栓中被激活，在特定目标 3 中，我们将定义并在药理学上抑制变构贡献。我们发现 ADAMTS13 变构激活抑制剂会将 ADAMTS13 活性降低至不会诱导的安全水平。我们将使用磁镊的高度并行数据收集来表征一类独特的抑制剂，这些抑制剂会干扰 ADAMT13 变构激活的关键步骤，并分析这些单分子结果如何发挥作用。 ADAMTS13 抑制剂在体外和离体 LVAD 模型中改变流体剪切应力下 VWF 多聚体裂解的动力学。结果将展示如何改变。 ADAMTS13 在不断增长的血栓中精确地在需要的地方达到最大效率，并展示了如何在治疗上调节这些相互作用。