Von Willebrand Factor structure and function under fluid flow

描述（由申请人提供）：人类血液蛋白血管性血友病因子（VWF）通过在裸露血管壁上暴露的细胞外基质蛋白和血流中的血小板之间形成分子桥，在血栓形成和止血过程中发挥关键作用。受损血管壁上的这种血小板募集有助于脉管系统中的堵塞和栓塞形成。 VWF 功能的几个方面受到流体或流体动力剪切的调节： i) 组成型活性血液金属蛋白酶 ADAMTS-13 裂解 VWF，蛋白水解速率受到流体剪切的严格调节。 ii) 流体剪切增强了 VWF 与血小板表面 GpIba 的结合，并且血管损伤部位的血小板募集也以剪切依赖性方式发生。 iii) 除了 ADAMTS-13 之外，流体剪切还促进 VWF 的自缔合，这是调节循环中 VWF 大小的附加机制。由于 VWF 的多种功能受到相似大小的施加水动力的调节，因此我们建议这些功能受到共同/重叠的结构变化的调节。这些变化可能发生在 VWF 的球状头部部分，其中包含蛋白质的 D'D3、A1、A2 和 A3 结构域。特别是，我们的具体目标确定：1) VWF-D'D3 对 VWF-A1 结构域的掩蔽是否会导致天然蛋白质中细胞粘附的减少，而流体剪切力会揭示这种分子相互作用。 2) ADAMTS-13 与 VWF 的结合是否改变了 A2 结构域的构象，并且这是否与流体剪切协同作用以调节蛋白水解动力学。 3) VWF自缔合是否先于并增强剪切驱动的VWF-A2蛋白水解的速率，并且该蛋白质聚集过程是否也增强剪切下VWF-GpIb1结合的亲和力。为了实现这些目标，在哺乳动物表达系统中产生了一系列单结构域、双结构域和多聚体 VWF 构建体。还制备了一系列新型单结构域和多聚体 VWF FRET 蛋白。使用流式细胞术和荧光/共聚焦显微镜进行功能/结构研究，以测量 VWF 结合、蛋白质构象变化、血小板粘附和激活。表面等离子共振 (SPR) 提供分子结合亲和力/动力学的测量。串联质谱用于阐明剪切促进的结构变化。作为这些不同实验模式之间的桥梁，流体动力学模型用于估计在各种流体剪切条件下施加的力的大小和性质。为了确认这项工作的生理相关性，特别强调在全人类血液环境中以及存在生理/病理剪切应力的情况下验证所提出的假设。一些假设也在动脉血栓形成的小鼠模型中得到了验证。这些研究旨在提供关于流体剪切在调节 VWF 结构、大小和功能中的作用的基本见解。这一应用的成功可能会刺激对循环中分子相互作用的进一步研究，除了 VWF 之外，这些分子相互作用是由流动的血液调节的。

DESCRIPTION (provided by applicant): The human blood protein Von Willebrand Factor (VWF) plays a critical role during thrombotic and hemostatic processes by forming a molecular bridge between extra-cellular matrix proteins exposed on the denuded blood vessel wall and platelets in the flow stream. Such platelet recruitment on the injured vessel wall contributes to plug and emboli formation in the vasculature. Several aspects of VWF function are regulated by fluid or hydrodynamic shear: i) The constitutively active blood metalloprotease ADAMTS-13 cleaves VWF, with proteolysis rate being tightly regulated by fluid shear. ii) VWF binding to GpIba on platelet surface is augmented by fluid shear, and platelet recruitment at sites of vascular injury also occurs in a shear-dependent manner. iii) In addition to ADAMTS-13, fluid shear promotes the self-association of VWF and this is an additional mechanism regulating VWF size in circulation. Since multiple functions of VWF are regulated by similar magnitudes of applied hydrodynamic forces, we suggest that these functions are regulated by common/overlapping structural changes. These changes likely occur in the globular head section of VWF that contains the D'D3, A1, A2 and A3 domains of the protein. In particular, our specific aims determine: 1) if the masking of the VWF-A1 domain by VWF-D'D3 contributes to reduced cell adhesion in the native protein, with fluid shear unmasking this molecular interaction. 2) if the binding of ADAMTS-13 to VWF changes the conformation of the A2-domain and if this acts in synergy with fluid shear to regulate proteolysis kinetics. 3) if VWF self-association precedes and enhances the rate of shear driven VWF-A2 proteolysis, and if this protein aggregation process also enhances the avidity of VWF-GpIb1 binding under shear. To address these aims, a series of single-domain, dual-domain and multimeric-VWF constructs are produced in mammalian expression systems. Panels of novel single-domain and multimeric-VWF FRET proteins are also made. Functional/structural studies are carried out to measure VWF binding, protein conformation change, platelet adhesion and activation using both flow cytometry and fluorescence/confocal microscopy. Surface plasmon resonance (SPR) provides measures of molecular binding affinity/kinetics. Tandem mass spectrometry is applied to elucidate structural changes promoted by shear. In terms of a bridge between these different experimental modalities, hydrodynamic modeling is applied to estimate the magnitude and nature of force applied under the variety of fluid shear conditions. In order to confirm the physiological relevance of the work, particular emphasis is placed on validating the proposed hypotheses in the milieu of whole human blood, and in the presence of physiological/pathological shear stress. Some hypotheses are also validated in a mouse model of arterial thrombosis. Together, the studies are designed to provide fundamental insight on the role of fluid shear in regulating VWF structure, size and function. Success in this application may spur additional investigations on molecular interactions in circulation, besides VWF, that are conditioned by flowing blood.