Von Willebrand Factor structure and function under fluid flow

描述（由申请方提供）：人血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与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。在受损血管壁上的这种血小板募集有助于血管系统中的栓塞和栓子形成。VWF功能的几个方面由流体或流体动力学剪切调节：i）组成型活性血液金属蛋白酶ADAMTS-13切割VWF，其中蛋白水解速率由流体剪切严格调节。ii）VWF与血小板表面GpIba的结合通过流体剪切而增强，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，流体剪切促进VWF的自缔合，并且这是调节循环中VWF大小的另外的机制。由于VWF的多种功能是由类似大小的应用流体动力学力调节，我们认为这些功能是由共同/重叠的结构变化调节。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。特别地，我们的具体目标确定：1）VWF-D 'D3对VWF-A1结构域的掩蔽是否有助于天然蛋白中细胞粘附的降低，其中流体剪切揭示了这种分子相互作用。2)如果ADAMTS-13与VWF的结合改变了A2结构域的构象，并且如果这与流体剪切协同作用以调节蛋白水解动力学。3)如果VWF自缔合先于剪切驱动的VWF-A2蛋白水解并增强剪切驱动的VWF-A2蛋白水解的速率，并且如果该蛋白聚集过程也增强剪切下VWF-GpIb 1结合的亲合力。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。还制备了新的单结构域和多聚体-VWF FRET蛋白的组。进行功能/结构研究以使用流式细胞术和荧光/共聚焦显微镜测量VWF结合、蛋白质构象变化、血小板粘附和活化。表面等离子体共振（SPR）提供了分子结合亲和力/动力学的测量。串联质谱法用于阐明剪切促进的结构变化。在这些不同的实验模式之间的桥梁，流体动力学建模应用于估计各种流体剪切条件下施加的力的大小和性质。为了确认的生理相关性的工作，特别强调的是放在验证所提出的假设，在环境中的全血，并在存在的生理/病理剪切应力。一些假设也在小鼠动脉血栓形成模型中得到验证。总之，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。这项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，这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.