Von Willebrand Factor structure and function under fluid flow

描述（由申请人提供）：人血液蛋白血管性血友病因子（VWF）在血栓形成和止血过程中发挥关键作用，通过在裸露的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。这种在受伤血管壁上的血小板聚集导致血管堵塞和栓塞形成。VWF功能的几个方面受到流体或水动力剪切的调节：i)组成活性的血液金属蛋白酶ADAMTS-13切割VWF，蛋白质水解速率受到流体剪切的严格调节。ii)血小板表面VWF与GpIba的结合通过流体剪切增强，血管损伤部位的血小板募集也以剪切依赖的方式发生。iii)除了ADAMTS-13外，流体剪切促进了VWF的自关联，这是调节循环中VWF大小的另一个机制。由于VWF的多种功能是由相似大小的水动力作用调节的，我们认为这些功能是由共同/重叠的结构变化调节的。这些变化可能发生在VWF的球状头部部分，该部分含有蛋白质的D'D3, A1， A2和A3结构域。特别是，我们的具体目标确定：1)如果VWF-A1结构域被VWF-D'D3掩盖有助于减少天然蛋白的细胞粘附，流体剪切揭开这种分子相互作用。2) ADAMTS-13与VWF的结合是否改变了a2结构域的构象，是否与流体剪切协同作用以调节蛋白水解动力学。3)如果VWF自结合先于并提高剪切驱动VWF- a2蛋白水解的速率，并且这种蛋白质聚集过程是否也提高了剪切作用下VWF- gpib1结合的快慢。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域、双域和多聚vwf结构。还制作了新的单域和多聚vwf FRET蛋白面板。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。表面等离子体共振（SPR）提供了分子结合亲和/动力学的测量。串联质谱法用于分析剪切引起的结构变化。就这些不同实验模式之间的桥梁而言，流体动力学建模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，特别强调在全人血液环境中验证所提出的假设，以及在存在生理/病理剪切应力的情况下。一些假设也在动脉血栓形成的小鼠模型中得到了验证。总之，这些研究旨在提供流体剪切在调节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，它是由血流调节的。

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.