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：
10376469
负责人：
Xiaohui Zhang
金额：
$ 45.98万
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/10376469
关键词：
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 Exposure to Extracorporeal Membrane Oxygenation F8 gene 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 base beta pleated sheet biophysical tools cleavage factor cofactor design in vivo laser tweezer mechanical force member molecular modeling mutant novel novel therapeutics single molecule 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.

项目概要血管性血友病因子 (VWF) 是一种大型多聚体血浆蛋白，在止血中发挥着关键作用。被合成并分泌为含有 25-50 个原聚体的超大 (UL) 多聚体。通过血浆金属蛋白酶 ADAMTS13 处理，ULVWF 可以启动生命的形成血栓性血小板减少性紫癜（TTP）中的威胁性血栓形成如何蛋白水解。 ADAMTS13 对 ULVWF 在生理条件下的调节尚不完全清楚。切割位点埋藏在 VWF A2 结构域内的中央 β 片层下方，张力为我们的初步研究表明，需要暴露酶促裂解的裂解位点。证明凝血因子 VIII (FVIII) 可能作为促进裂解的辅助因子利用我们独特的组合，在机械剪切下通过 ADAMTS13 进行 VWF。两个实验室均提供分子、生化和单分子生物物理工具，我们将测试假设 FVIII 与 VWF-D’D3 和其他相邻结构域（例如 A2）结合结构域可能会导致中央 A2 的构象变化，从而暴露切割位点 (Y1605-M1606) 在机械力下更容易适应 ADAMTS13 在目标 1 中，我们将确定。在不存在或不存在其他相邻结构域的情况下，A2 的机械展开轮廓 FVIII 的存在；在目标 2 中，我们将通过以下方式阐明 A2 和 FVIII 相互作用的分子机制：在目标 3 中，我们将确定以下因素的生理相关性：在动物模型和人类中，VWF 在受力下发生 FVIII 依赖性蛋白水解裂解拟议项目的完成将有助于理解分子相互作用。在生理条件下，底物、酶和蛋白质辅因子之间的相互作用，提供了开发预防和治疗 TTP 及其他疾病的新疗法的基本原理血栓和炎症性疾病。