Conformational activation of von Willebrand factor

项目总结 大的多聚体血浆蛋白von Willebrand因子(VWF)关键地介导止血和 通过感知和响应血液剪切流而形成血栓。在低剪切条件下，VWF倍增器 由于VWF之间弱相互作用，在循环中采用松散盘绕、浓缩形状 单体。在临界剪切速率以上，VWF多聚体沿拉伸流方向延伸， 体验张力，它有两个相反的效果。紧张会导致周围的结构变化 VWF A1结构域促进与血小板膜糖蛋白(GP)Ibα结合，支持血小板黏附和 激活。张力还会展开VWF A2结构域，暴露出Tyr-Met多肽键，该键被 金属蛋白酶ADAMTS13，从而释放黏附的血小板，降低VWF的反应性。 因此，VWF的大小和反应性处于微妙调节的平衡中。这种平衡的破坏是一种 出血或血栓形成的常见原因。以前的研究已经证实，在低切变情况下 VWF和ADAMTS13的条件都是自动抑制的。然而，潜在的分子机制 对于自身抑制的研究尚不清楚，这严重限制了人们对剪切诱导的影响的理解。 以及它与GPIBα和ADAMTS13的相互作用。尽管许多原子的结构 VWF和ADAMTS13的结构域已经确定，全长VWF和ADAMTS13是大的和 灵活多变。结果，每个蛋白质中关键的结构域间相互作用，以及VWF-ADAMTS13相互作用， 是X射线结晶学所不能达到的。我们通过一个组合绕过了这一限制 氢-氚交换质谱仪(HDX-MS)，电子显微镜(EM)，小角 X射线散射(SAXS)、分析超速离心法(AUC)和分子模拟。在这个项目中，我们 将使用这些方法来描述VWF、ADAMTS13、 和GPIBα，建议如下2个具体目标。目的1阐明VWF的发病机制。 自我抑制和激活。我们将描述A1结构域是如何被自身抑制所掩盖的 VWF多聚体和各种重组片段中的模块(AIM)，并确定 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 ADAMTS13的结构，并表征ADAMTS13的构象变化 模拟未折叠A2结构域的VWF片段的变构激活。我们期待着发展 详细的分子模型，将解释这台非凡的分子机器的关键功能 VWF/ADAMTS13/GPIBα以前所未有的细节。研究结果将为下一步操作提供基础 用于治疗目的的血小板黏附和血小板黏附的反馈抑制。

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.