Thermodynamics of the Conformational Activation of Von Willebrand Factor

冯维勒布兰德因子构象激活的热力学

• 批准号: 8497717
• 负责人: Matthew Auton
• 金额: $ 33.52万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-15 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8497717
• 关键词: Affect; Affinity; Behavior; Binding; Binding Proteins; Blood Platelets; Blood Vessels; Blood flow; Cardiovascular Diseases; Clinical; Coagulation Process; Complex; Coupled; Databases; Defect; Development; Diagnosis; Disease; Equilibrium; Exposure to; Glycoproteins; Goals; Health; Hemorrhage; Hemostatic function; Human; Induced Mutation; Inherited; Injury; Investigation; Learning; Link; Literature; Mechanics; Medical; Modeling; Molecular Conformation; Mutation; Physiological; Play; Point Mutation; Polymers; Population; Prevalence; Property; Publishing; Quantitative Structure-Activity Relationship; Regulation; Research Project Grants; Role; Severities; Structure; Surface; Testing; Thermodynamics; Translating; Vascular Endothelial Cell; Work; X-Ray Crystallography; base; clinical Diagnosis; clinical effect; loss of function; man; novel; predictive modeling; receptor; shear stress; von Willebrand Disease; von Willebrand Factor

DESCRIPTION (provided by applicant): Von Willebrand factor (VWF), a multimeric polymer glycoprotein secreted from vascular endothelial cells and activated platelets, functions to sequester and adhere platelets to the subendothelium and initiate coagulation. Since the initial observation that the function of VWF is conformationally regulated by the rheological shear stress of blood flow, the prevailing idea has been that exposure to elevated vascular shear stress unfolds VWF to an activated conformation that increases its binding affinity of the A1 domain for the platelet GPIba surface receptor. When we identified the presence of a thermodynamically distinct unfolding intermediate in the "platelet GPIba binding" A1 domain of VWF, it set forth numerous studies of the functional role that this conformation plays in health and disease. VWF has merited extensive study because of its association with the most common inherited bleeding disorder in man, Von Willebrand Disease (VWD). Two subtypes of type 2 VWD are characterized by either enhanced (type 2B) or deficient (type 2M) platelet-VWF interactions due to point mutations located specifically in the A1 domain of VWF. Our studies have established that these phenotypically opposite mutations differentially affect the thermodynamic stability of the A1 domain, the binding affinity of the A1 domain for platelet GPIba, and the force-dependent catch-slip bonding between A1 and GPIba, a property that regulates platelet-rolling velocities on VWF as shear flow is increased. The degree to which these mutations affect these properties of the A1 domain have revealed that 1) the binding affinity is coupled to this native to intermediate (N?I) conformational equilibrium and 2) the force/shear stress dependent properties of the A1-GPIba interaction are a direct consequence of the thermodynamic linkage between the native low affinity and intermediate high affinity conformations. These studies have resulted in the first quantitative working model for the mechanism of VWF function that forms the basis for the central hypothesis of this application that the intermediate conformation of the A1 domain forms an integral part of the structure that comprises the active state of VWF. Our objective is to identify how A1 stability and domain association within the A1A2A3 tri-domain fragment of VWF are thermodynamically coupled and how this regulation is linked to the severity of bleeding in clinical disease. To accomplish this objective we will test the Conformational Model through the investigation of a comprehensive clinical database of type 2B and 2M mutations that cause Von Willebrand disease and establish the effects of these clinical mutations on the quaternary domain interactions within the A1A2A3 tri-domain. The proposed work will be of significant value to the clinical diagnosis of VWD as it has the potential to classify mutations according to their conformational effects on VWF structure and function. A thorough understanding of the breadth of conformational defects caused by inherited mutations will be essential for the development of quantitative structure-activity relationships in VWF as well as the development of new treatments for cardiovascular disease.

描述（由申请方提供）：血管性血友病因子（VWF）是一种由血管内皮细胞和活化血小板分泌的多聚体多聚体糖蛋白，其功能是隔离血小板并使其粘附于内皮下并启动凝血。由于最初观察到VWF的功能是由血流的流变剪切应力构象调节，流行的想法是，暴露于升高的血管剪切应力展开VWF的活化构象，增加其结合亲和力的A1域的血小板GPIba表面受体。当我们确定了VWF的“血小板GPIba结合”A1结构域中存在一个独特的解折叠中间体时，它阐述了许多关于这种构象在健康和疾病中发挥的功能作用的研究。由于VWF与人类最常见的遗传性出血性疾病--血管性血友病（VWD）有关，因此值得进行广泛的研究。2型VWD的两种亚型的特征在于由于特异性位于VWF的A1结构域中的点突变而导致的血小板-VWF相互作用增强（2B型）或缺陷（2 M型）。我们的研究已经确定，这些表型相反的突变差异影响A1结构域的热力学稳定性，A1结构域对血小板GPIba的结合亲和力，以及A1和GPIba之间的力依赖性捕获滑动键合，这是一种调节血小板滚动速度的特性，随着剪切流的增加。这些突变影响A1结构域的这些性质的程度揭示了1）结合亲和力与该天然中间体（N？I）构象平衡和2）A1-GPIba相互作用的力/剪切应力依赖性性质是天然低亲和力和中间高亲和力构象之间的热力学联系的直接结果。这些研究产生了VWF功能机制的第一个定量工作模型，该模型形成了本申请的中心假设的基础，即A1结构域的中间构象形成了包含VWF活性状态的结构的组成部分。我们的目的是确定如何A1的稳定性和结构域协会内的A1 A2 A3三结构域片段的VWF的免疫耦合，以及这种调节是如何与出血的严重程度在临床疾病。为了实现这一目标，我们将通过调查导致Von Willebrand病的2B和2 M型突变的综合临床数据库来测试构象模型，并确定这些临床突变对A1 A2 A3三结构域内的四元结构域相互作用的影响。这项工作将对VWD的临床诊断具有重要价值，因为它有可能根据VWF结构和功能的构象效应对突变进行分类。深入了解遗传突变引起的构象缺陷的广度对于VWF定量构效关系的发展以及心血管疾病新治疗方法的开发至关重要。