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

• 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
• 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

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 血管性血友病因子（vonWillebrand factor，VWF）是一种大的多聚体血浆蛋白，在止血中起关键作用。VWF 作为含有25-50个原聚体的超大（UL）多聚体合成和分泌。如果不是 通过血浆金属蛋白酶ADAMTS 13处理，ULVWF可以启动生命的形成， 如血栓性血小板减少性紫癜（TTP）中的威胁性血栓形成。蛋白水解裂解 在生理条件下，ADAMTS 13对ULVWF的调节作用尚不完全清楚。的 切割位点被埋在VWF的A2结构域内的中心β-折叠下，并且张力是 需要暴露切割位点以发生酶促切割。我们的初步研究表明 证明凝血因子VIII（FVIII）可能作为辅助因子，促进切割 在机械剪切作用下，ADAMTS 13对VWF的影响。利用我们独特的 分子，生物化学和单分子生物物理工具，在这两个实验室，我们将测试 假设FVIII与VWF-D ′ D3和其它邻近结构域如A2 结构域可能导致中心A2的构象变化，从而暴露切割位点 （Y1605-M1606）在机械力作用下更容易与ADAMTS 13结合。在目标1中，我们将确定 在不存在和不存在其它相邻结构域的情况下， 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： 研究它们的变体和突变体;在目标3中，我们将确定 在强制下以及在动物模型和人中， 心脏性TTP该项目的完成将有助于了解分子间的相互作用 在底物、酶和蛋白质辅因子之间， 开发预防和治疗TTP和其他疾病的新疗法的基本原理 血栓形成和炎性疾病。