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Conformational activation of von Willebrand factor

血管性血友病因子的构象激活

基本信息

批准号：
10183306
负责人：
Renhao Li
金额：
$ 61.89万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-01 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10183306
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

项目摘要

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.

项目概要大的多聚体血浆蛋白血管性血友病因子 (VWF) 关键介导止血和通过感知和响应血液剪切流来形成血栓。在低剪切条件下，VWF多聚体由于 VWF 之间的弱相互作用，循环中的颗粒呈松散盘绕、浓缩的形状单体。高于临界剪切速率时，VWF 多聚体沿拉伸流动方向延伸，受到拉力，拉力有两种相反的作用。张力引起周围的结构变化 VWF A1 结构域促进与血小板糖蛋白 (GP)Ibα 的结合并支持血小板粘附和激活。张力还会展开 VWF A2 结构域，暴露出 Tyr-Met 肽键，该键被金属蛋白酶 ADAMTS13，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确调节的平衡中。破坏这种平衡是出血或血栓形成的常见原因。先前的研究已经证实，在低剪切力下 VWF 和 ADAMTS13 均处于自抑制状态。然而，潜在的分子机制对于自抑制的作用尚不清楚，这严重限制了对剪切诱导效应的理解 VWF 及其与 GPIbα 和 ADAMTS13 的相互作用。尽管许多原子结构 VWF和ADAMTS13的结构域已经确定，全长VWF和ADAMTS13很大并且灵活的。因此，每个蛋白质中关键的域间相互作用以及 VWF-ADAMTS13 相互作用， X射线晶体学无法检测到。我们通过组合规避了这个限制氢-氘交换质谱 (HDX-MS)、电子显微镜 (EM)、小角度 X 射线散射 (SAXS)、分析超速离心 (AUC) 和分子建模。在这个项目中我们将采用这些方法来表征 VWF、ADAMTS13 内部和之间的动态相互作用，和 GPIbα，如以下 2 个具体目标中所提议。目标1是阐明VWF的机制自抑制和激活。我们将描述 A1 结构域如何被自动抑制掩盖 VWF多聚体和各种重组片段中的模块（AIM），并确定影响因素破坏或稳定 AIM-A1 相互作用及其对 A1 与 GPIbα 结合的影响。目标 2 是确定力如何调节 VWF 和 ADAMTS13 之间的相互作用。我们将确定自抑制 ADAMTS13 的结构，并表征 ADAMTS13 的构象变化通过模拟未折叠的 A2 结构域的 VWF 片段进行变构激活。我们期望发展详细的分子模型将解释这种非凡的分子机器的关键功能 VWF/ADAMTS13/GPIbα 的细节前所未有。结果将为操纵提供基础血小板粘附，以及血小板粘附的反馈抑制，用于治疗目的。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Autoinhibitory module underlies species difference in shear activation of von Willebrand factor.

DOI：
10.1111/jth.15837
发表时间：
2022-11
期刊：
JOURNAL OF THROMBOSIS AND HAEMOSTASIS
影响因子：
10.4
作者：
Arce, Nicholas A.;Liu, Yi;Chen, Wenchun;Zhang, X. Frank;Li, Renhao
通讯作者：
Li, Renhao

Activation of von Willebrand factor via mechanical unfolding of its discontinuous autoinhibitory module.

DOI：
10.1038/s41467-021-22634-x
发表时间：
2021-04-21
期刊：
Nature communications
影响因子：
16.6
作者：
Arce NA;Cao W;Brown AK;Legan ER;Wilson MS;Xu ER;Berndt MC;Emsley J;Zhang XF;Li R
通讯作者：
Li R

Removal of single-site N-linked glycans on factor VIII alters binding of domain-specific monoclonal antibodies.