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: 8683219
• 负责人: Matthew Auton
• 金额: $ 34.51万
• 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/8683219
• 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（由申请人提供）：血管性血液病因子（VWF）是一种由血管内皮细胞和活化的血小板分泌的多聚体聚合物糖蛋白，其功能是将血小板隔离并粘附到内皮下层并启动凝血。自最初观察到VWF的功能受血流流变剪切应力的构象调节以来，普遍的观点是，暴露于升高的血管剪切应力下，VWF会被激活成一个激活的构象，从而增加其与血小板GPIba表面受体A1结构域的结合亲和力。当我们发现在VWF的“血小板GPIba结合”A1结构域中存在一种热力学上不同的展开中间体时，它提出了许多关于该构象在健康和疾病中所起的功能作用的研究。由于VWF与男性最常见的遗传性出血性疾病血管性血友病（VWD）有关，因此值得广泛研究。2型VWD的两种亚型的特点是血小板-VWF相互作用增强（2B型）或不足（2M型），这是由于VWF A1结构域的特异性点突变。我们的研究已经确定，这些表型相反的突变对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之间的力依赖的捕获-滑动键（一种随着剪切流动增加而调节血小板在VWF上的滑动速度的特性）有不同的影响。这些突变在多大程度上影响A1结构域的这些特性揭示了1)结合亲和力与这种天然到中间产物(N？1)构象平衡；2)A1-GPIba相互作用的力/剪应力依赖性质是原生低亲和构象和中间高亲和构象之间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，该模型为本应用的中心假设奠定了基础，即A1结构域的中间构象是构成VWF活性状态的结构的一个组成部分。我们的目标是确定VWF的A1A2A3三结构域片段中的A1稳定性和结构域关联是如何热力学耦合的，以及这种调节如何与临床疾病中出血的严重程度相关联。为了实现这一目标，我们将通过调查导致血管性血友病的2B型和2M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A1A2A3三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，因为它有可能根据突变对VWF结构和功能的构象影响对突变进行分类。深入了解遗传突变引起的构象缺陷的广度对于VWF定量结构-活性关系的发展以及心血管疾病新治疗方法的开发至关重要。