Mechanism underlying cofactor-dependent proteolysis of von Willebrand Factor

冯维勒布兰德因子辅因子依赖性蛋白水解的机制

基本信息

批准号：
10551879
负责人：
Xiaohui Zhang
金额：
$ 44.7万
依托单位：
UNIVERSITY OF KANSAS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-01 至 2026-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10551879
关键词：
ADAMTS Acquired von Willebrand disease Adhesives Affect Animal Model Aortic Valve Stenosis Atomic Force Microscopy Binding Binding Sites Biochemical Biological Assay Blood Platelets Blood Vessels Complex Deuterium Development Disease Disintegrins Endothelial Cells Enzyme-Linked Immunosorbent Assay Enzymes Event Exogenous Factors Exposure to Extracorporeal Membrane Oxygenation Factor VIII Fibrin Hemorrhage Hemostatic function Human Hydrogen Individual Inflammatory Ischemia Ischemic Stroke Laboratories Life Mammalian Cell Mass Spectrum Analysis Mechanics Megakaryocytes Metalloproteases Molecular Molecular Conformation Mutant Strains Mice Myocardial Infarction Patients Peptides Physiological Plasma Plasma Proteins Play Prevention Process Proteins Proteolysis Protomer Recombinants Regulation Risk Role Sampling Series Site Structural Models Structure Surface Plasmon Resonance System Techniques Testing Thrombosis Thrombospondins Thrombotic Thrombocytopenic Purpura Tissues Traumatic Brain Injury Variant analytical tool beta pleated sheet biophysical tools cleavage factor cofactor design in vivo laser tweezer mechanical force member molecular modeling mutant novel novel therapeutics optic tweezer recombinant antihemophilic factor VIII single molecule skills thrombogenesis thrombotic von Willebrand Disease von Willebrand Factor

项目摘要

PROJECT SUMMARY von Willebrand factor (VWF), a large multimeric plasma protein, plays a critical role in hemostasis. VWF is synthesized and secreted as ultra-large (UL) multimers that contain 25-50 protomers. If not processed by a plasma metalloprotease ADAMTS13, ULVWF can initiate the formation of life- threatening thrombosis as in thrombotic thrombocytopenic purpura (TTP). How the proteolytic cleavage of ULVWF by ADAMTS13 is regulated under physiological conditions is not fully understood. The cleavage site is buried under the central β-sheet within the A2 domain of VWF, and tensile force is required to expose the cleavage site for enzymatic cleavage to occur. Our preliminary studies have demonstrated that coagulation factor VIII (FVIII) may function as a cofactor that facilitates the cleavage of VWF by ADAMTS13 under mechanic shear. Taking advantage of our unique combination of molecular, biochemical and single-molecule biophysical tools available in both laboratories, we will test the hypothesis that the binding of FVIII to VWF-D’D3 and other adjacent domains such as the A2 domain may result in conformational changes in the central A2, thus exposing the cleavage site (Y1605-M1606) more readily to ADAMTS13 under mechanical force. In Aim 1, we will determine the mechanical unfolding profile of A2 with or without other adjacent domains in the absence and presence of FVIII; in Aim 2, we will elucidate the molecular mechanism of A2 and FVIII interactions by investigating their variants and mutants; and in Aim 3, we will determine the physiological relevance of the FVIII-dependent proteolytic cleavage of VWF under force and in animal models and human with heareditary TTP. The completion of the proposed project will help understand the molecular interactions among substrate, enzyme, and protein cofactor under physiological conditions, which provides rationales for the development of novel therapeutics for the prevention and treatment of TTP and other thrombotic and inflammatory disorders.

项目摘要大型多聚体等离子体蛋白von Willebrand因子（VWF）在止血中起关键作用。 vwf 合成并分泌为包含25-50个代理的超大（UL）多聚体。如果不在血浆金属蛋白酶ADAMTS13的处理中，ULVWF可以启动生命形成 - 威胁性血栓形成，如血栓性血小板减少紫菜（TTP）。蛋白水解裂解如何 ADAMTS13的ULVWF在身体条件下受到调节。这裂解位点被埋在VWF的A2域内的中央β-折叠下，拉伸力为需要暴露裂解位点以进行酶促切割。我们的初步研究证明凝血因子VIII（FVIII）可能是辅助裂解的辅助因子 Adamts13在机械剪切下的VWF。利用我们独特的组合分子，生化和单分子生物物理工具在两个实验室都有，我们将测试 FVIII与VWF-D'D3和其他相邻域（例如A2）结合的假设域可能会导致中央A2的会议变化，从而暴露了裂解位点（Y1605-M1606）在机械力下更容易对Adamts13。在AIM 1中，我们将确定在没有或没有其他相邻域的A2的机械展开轮廓和 FVIII的存在；在AIM 2中，我们将阐明A2和FVIII相互作用的分子机制调查它们的变体和突变体；在AIM 3中，我们将确定 VWF的FVIII依赖性蛋白水解裂解在力下，动物模型和人类，暖内TTP。拟议项目的完成将有助于了解分子相互作用在生理条件下的底物，酶和蛋白质辅因子中，为预防和治疗TTP和其他其他治疗的新疗法开发的理由血小板和炎症性疾病。