ALLOSTERIC REGULATION OF ADAMTS13

 描述(由申请人提供)：ADAMTS13识别并切割von Willebrand因子(VWF)中的一个隐蔽位置，当VWF多聚体被流体剪应力拉伸时，该位置会暴露出来。严重的ADAMTS13缺乏会损害这一调节机制，并导致血栓性血小板减少性紫癜。相反，ADAMTS13对VWF的过度切割会导致von Willebrand病(VWD)和获得性von Willebrand综合征(AVWS)的某些变体出血。例如，几乎所有使用左心室辅助装置(LVAD)的患者都会发展为AVWS，其中约三分之一有大出血。我们最近发现ADAMTS13被折叠成两半，因此它的远端T8-CUB结构域抑制了近端的金属蛋白酶结构域。此外，VWF与远端ADAMTS13结构域的结合解除了这种自我抑制。因此，VWF通过变构激活ADAMTS13来促进自身的破坏。我们的目标是了解这一先前未知的调控机制的结构基础，并将这一知识转化为更好地治疗出血和血栓形成。在特定的目标1中，我们将表征VWF和ADAMTS13之间的相互作用，这是变构调控ADAMTS13活性所必需的。我们将利用突变、结合和与新的荧光底物的酶动力学来检验这一假设，即特定的ADAMTS13结构域与VWF上的同源位点之间的相互作用会导致ADAMTS13结构的变化，从而解除ADAMTS13蛋白酶活性的自动抑制。在特定目标2中，我们将表征VWF对ADAMTS13变构调控的结构基础。用SAXS和EM显示ADAMTS13和ADAMTS13-VWF复合体的结构，我们将检验ADAMTS13远端结构域直接接触近端结构域的假设，以及ADAMTS13的结构依赖于这些接触。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理解ADAMTS13是如何在富含血小板的血栓中被激活的。在具体目标3中，我们将定义并从药理上抑制变构对剪应力下VWF多聚体切割的影响。我们假设ADAMTS13变构激活的抑制剂将ADAMTS13的活性降低到安全水平，不会导致血栓性微血管病变，但可以改善AVWS的止血。我们将使用高度平行的磁镊子数据收集来表征一类独特的抑制剂，这些抑制剂干扰ADAMT13变构激活的关键步骤。我们将利用这些单分子结果来分析这些ADAMTS13抑制剂如何在体外和体外的LVAD模型中改变流体切应力下VWF多聚体的切割动力学。这些结果将展示ADAMTS13如何在不断增长的血栓中精确地在需要的地方达到最大效率，并展示如何通过治疗来调节这些相互作用。

 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.