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.
The goal of this research proposal is to understand how the metalloprotease ADAMTS13 prevents disease in most of us by precisely regulating platelet adhesion, and to translate this knowledge into better treatment for bleeding and thrombosis.
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|Sadler, J Evan (2017) Pathophysiology of thrombotic thrombocytopenic purpura. Blood 130:1181-1188|