Conformational activation of von Willebrand factor

血管性血友病因子的构象激活

• 批准号: 9754253
• 负责人: Renhao Li
• 金额: $ 72.79万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9754253
• 关键词: Adhesions; Adopted; Allosteric Regulation; Behavior; Binding; Blood; Blood Circulation; Blood Platelets; C-terminal; Cleaved cell; Complex; Cryoelectron Microscopy; Deuterium; Disease; Dissociation; Electron Microscopy; Elements; Enzymes; Equilibrium; Exposure to; Feedback; Foundations; Hemorrhage; Hemostatic function; Hydrogen; Length; Link; Maps; Masks; Mass Spectrum Analysis; Measures; Mediating; Metalloproteases; Methods; Modeling; Molecular; Molecular Conformation; Molecular Machines; Mutagenesis; N-terminal; Pathogenesis; Pathway interactions; Peptides; Plasma Proteins; Platelet Glycoproteins; Process; Proteins; Recombinants; Regulation; Ristocetin; Roentgen Rays; Shapes; Site; Structure; Therapeutic; Thrombosis; Variant; X-Ray Crystallography; analytical ultracentrifugation; base; design; experience; flexibility; molecular modeling; monomer; reconstruction; von Willebrand Disease; von Willebrand Factor

PROJECT SUMMARY Large, multimeric plasma protein von Willebrand factor (VWF) critically mediates hemostasis and thrombosis by sensing and responding to blood shear flow. Under low shear conditions, VWF multimers in circulation adopt a loosely coiled, condensed shape as a result of weak interactions between VWF monomers. Above a critical shear rate, VWF multimers extend in the direction of elongational flow and experience tensile force, which has two opposing effects. Tension induces structural changes around the VWF A1 domain that promote binding to platelet glycoprotein (GP)Ibα and support platelet adhesion and activation. Tension also unfolds the VWF A2 domain to expose a Tyr-Met peptide bond that is cleaved by the metalloprotease ADAMTS13, thereby releasing adherent platelets and reducing the VWF reactivity. Thus, VWF size and reactivity are in an exquisitely regulated balance. Disruption of this balance is a common cause of bleeding or thrombosis. Previous studies have established that under low shear conditions both VWF and ADAMTS13 are autoinhibited. However, the underlying molecular mechanisms for autoinhibition are not clear, which has severely limited understanding of shear-induced effects on VWF as well as its interactions with GPIbα and ADAMTS13. Although atomic structures of many domains of VWF and ADAMTS13 have been determined, full-length VWF and ADAMTS13 are large and flexible. As a result, critical interdomain interactions in each protein, and VWF-ADAMTS13 interactions, are not accessible to X-ray crystallography. We have circumvented this limitation through a combination of hydrogen-deuterium exchange mass spectrometry (HDX-MS), electron microscopy (EM), small angle X-ray scattering (SAXS), analytical ultracentrifugation (AUC), and molecular modeling. In this project we will employ these methods to characterize the dynamic interactions in and between VWF, ADAMTS13, and GPIbα, as proposed in the following 2 Specific Aims. Aim 1 is to elucidate the mechanism of VWF autoinhibition and activation. We will characterize how the A1 domain is masked by the autoinhibitory module (AIM) in VWF multimers and various recombinant fragments, and determine the factors that disrupt or stabilize the AIM-A1 interaction and their impacts on A1 binding to GPIbα. Aim 2 is to determine how force regulates interactions between VWF and ADAMTS13. We will determine the structure of autoinhibited ADAMTS13, and characterize the conformational changes in ADAMTS13 upon allosteric activation by VWF fragments that simulate the unfolded A2 domain. We expect to develop detailed, molecular models that will explain key functions of this remarkable molecular machine of VWF/ADAMTS13/GPIbα in unprecedented detail. The results will provide a foundation to manipulate platelet adhesion, and the feedback inhibition of platelet adhesion, for therapeutic purposes.

项目总结