Conformational activation of von Willebrand factor

血管性血友病因子的构象激活

• 批准号: 9982098
• 负责人: Renhao Li
• 金额: $ 62.13万
• 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/9982098
• 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.

项目摘要 大的多聚体血浆蛋白血管性血友病因子（VWF）关键介导止血和 血栓形成通过感知和响应血液剪切流。在低剪切条件下，VWF多聚体 在循环中，由于VWF之间弱相互作用， 单体。在临界剪切速率以上，VWF多聚体在伸长流动的方向上延伸， 会受到张力的作用，这会产生两种相反的效果。紧张局势引起了周围的结构变化， 促进与血小板糖蛋白（GP）Ibα结合并支持血小板粘附的VWF A1结构域， activation.张力还使VWF A2结构域展开以暴露Tyr-Met肽键，该Tyr-Met肽键被VWF A2结构域切割。 金属蛋白酶ADAMTS 13，从而释放粘附的血小板并降低VWF反应性。 因此，VWF大小和反应性处于精细调节的平衡中。打破这种平衡是一种 出血或血栓形成的常见原因。先前的研究已经确定，在低剪切下， 条件下VWF和ADAMTS 13均被自动抑制。然而，潜在的分子机制 对于自身抑制的作用尚不清楚，这严重限制了对剪切诱导的 VWF及其与GPIbα和ADAMTS 13的相互作用。虽然许多原子结构 已经确定了VWF和ADAMTS 13的结构域，全长VWF和ADAMTS 13很大， 灵活.因此，每种蛋白质中的关键结构域间相互作用，以及VWF-ADAMTS 13相互作用， 无法用X射线晶体学检测我们通过一种结合 氢-氘交换质谱（HDX-MS）、电子显微镜（EM）、小角度 X射线散射（SAXS）、分析超离心（AUC）和分子建模。在这个项目中， 将采用这些方法来表征VWF，ADAMTS 13， 和GPIbα，如以下2个具体目标所建议。目的1为阐明VWF的作用机制 自抑制和激活。我们将描述A1结构域是如何被自抑制蛋白所掩盖的。 模块（AIM）的VWF多聚体和各种重组片段，并确定的因素， 破坏或稳定AIM-A1相互作用及其对A1与GPIbα结合的影响。目标二是 确定力如何调节VWF和ADAMTS 13之间的相互作用。康贝特人将以 自抑制ADAMTS 13的结构，并表征ADAMTS 13的构象变化， 通过模拟未折叠A2结构域的VWF片段的变构激活。我们希望发展 详细的，分子模型，将解释这一显着的分子机器的关键功能， VWF/ADAMTS 13/GPIbα的研究。研究结果将为进一步的研究提供基础 血小板粘附和血小板粘附的反馈抑制。