BIOMECHANICS OF UNFOLDING THE A2 DOMAIN OF VON WILLEBRAND FACTOR

冯维勒布兰德因子 A2 结构域的生物力学

• 批准号: 8264964
• 负责人: JIN-YU SHAO
• 金额: $ 22.8万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8264964
• 关键词: Adhesions; Affect; Binding; Biomechanics; Blood; Blood Clot; Blood Platelets; Blood Vessels; Blood coagulation; Chemicals; Cleaved cell; Collaborations; Cystine; Disease; Drug Design; Endothelial Cells; Excision; Glycoproteins; Goals; Hematology; Hemorrhage; Hemostatic function; Injury; Link; Mechanics; Mediating; Megakaryocytes; Metalloproteases; Methodology; Molecular Biology; Plasma; Polysaccharides; Process; Recombinant Proteins; Recombinants; Research Project Grants; Screening procedure; Site; Stretching; Structure; Techniques; Technology; Testing; Thrombosis; Thrombotic Thrombocytopenic Purpura; Universities; Urea; Washington; Wound Healing; effective intervention; glycosylation; improved; medical schools; multidisciplinary; novel; optical traps; public health relevance; shear stress; von Willebrand Disease; von Willebrand Factor

DESCRIPTION (provided by applicant): Von Willebrand Factor (VWF) is a multimeric plasma glycoprotein that is critical for both hemostasis and thrombosis. Normal VWF function depends on its size distribution in plasma, regulated via proteolytic cleavage by ADAMTS13. On the one hand, insufficient VWF cleavage leads to thrombotic thrombocytopenic purpura, a disease characterized by microvascular thrombosis; on the other hand, excessive cleavage of VWF leads to Von Willebrand disease, a potentially-fatal bleeding disorder manifested by lack of large VWF multimers in plasma. VWF cleavage by ADAMTS13 is regulated by many factors including denaturing agents like urea, shear stress in blood, VWF glycosylation state, ST2 (a two-domain fragment of ADAMTS13), and VWF domains like A1. It has been found that a single recombinant A2 domain can be unfolded by an increasing pulling force and the unfolding force depends on the force loading rate. A2 unfolding facilitates its cleavage by ADAMTS13. However, mechanics of A2 unfolding was rarely studied in the context of a VWF multimer. How ST2, A1, and glycosylation state regulate A2 unfolding and cleavage has never been examined from a mechanical perspective. Therefore, our overall goal of this project is to characterize force-induced A2 unfolding and its relationship with VWF cleavage by ADAMTS13, as well as how this relationship is influenced by ST2, A1, and VWF glycosylation state. Our specific aims are: 1) determine whether A2 is unfolded first among 14 VWF domains when a VWF multimer is stretched by force, 2) determine whether A1 stabilizes A2 mechanically, manifested by larger unfolding force and less A2 cleavage in the presence of A1, and 3) determine whether ST2 and deglycosylation affect A2 unfolding and cleavage in an additive fashion, manifested by additive A2 unfolding forces. These studies will be carried out mainly with the optical trap and the recombinant protein technology. The successful completion of these studies will improve our understanding of how VWF mechanics is related to VWF cleavage. It will also provide a platform for our multidisciplinary effort of exploring novel methodologies for screening drugs and designing effective interventions for VWF-related diseases. PUBLIC HEALTH RELEVANCE: This research project investigates how the A2 domain of von Willebrand factor is unfolded under a pulling force and how A2 unfolding is related to VWF cleavage by ADAMTS13. Excessive VWF cleavage results in von Willebrand disease (VWD), whereas insufficient VWF cleavage results in thrombotic thrombocytopenic purpura (TTP), a disease characterized by microvascular thrombosis.

描述（由申请人提供）：von Willebrand因子（VWF）是一种多聚体等离子体糖蛋白，对止血和血栓形成至关重要。正常的VWF函数取决于其在血浆中的大小分布，这是通过ADAMTS13通过蛋白水解裂解调节的。一方面，VWF裂解不足会导致血小板性血小板减少性紫癜，这种疾病以微血管血栓形成为特征。另一方面，VWF的过度切割导致冯·威勒氏疾病，冯·威勒布兰疾病是一种潜在的致命性出血疾病，表现为血浆中缺乏大量的VWF多聚体。 ADAMTS13的VWF裂解受许多因素的调节，包括尿素，血液中的剪切应力，VWF糖基化状态，ST2（ADAMTSS13的两域片段）和VWF域等许多因素。已经发现，单个重组A2结构域可以通过增加的拉力来展开，而展开的力取决于力载荷速率。 A2展开促进了Adamts13的乳沟。但是，在VWF多聚机的背景下，很少研究A2展开的力学。从机械的角度，从未对ST2，A1和糖基化状态调节A2的展开和分裂。因此，该项目的总体目标是表征力引起的A2展开及其与ADAMTS13的VWF裂解的关系，以及这种关系如何受ST2，A1和VWF糖基化状态的影响。 Our specific aims are: 1) determine whether A2 is unfolded first among 14 VWF domains when a VWF multimer is stretched by force, 2) determine whether A1 stabilizes A2 mechanically, manifested by larger unfolding force and less A2 cleavage in the presence of A1, and 3) determine whether ST2 and deglycosylation affect A2 unfolding and cleavage in an additive fashion, manifested by additive A2 unfolding力量。这些研究将主要使用光学陷阱和重组蛋白技术进行。这些研究的成功完成将提高我们对VWF力学与VWF裂解如何相关的理解。它还将为我们的多学科努力提供一个平台，以探索筛查药物并设计有效干预措施的新方法。 公共卫生相关性：该研究项目研究了von Willebrand因素的A2域是如何在拉力部队下展开的，以及A2的展开与Adamts13的VWF裂解有关。过度的VWF裂解导致von Willebrand疾病（VWD），而VWF裂解不足会导致血栓性血栓细胞减少紫癜（TTP），这种疾病是微血管血栓形成的特征。