VON WILLEBRAND FACTOR STRUCTURE-FUNCTION UNDE FLUID FLOW

描述（由申请方提供）：在血管损伤模型中，在流体流动下，血小板与基质固定的血管性血友病因子（vWF）的结合导致血小板活化和随后的血栓形成。在对血小板和血蛋白施加流体力的悬浮液测定中进行的实验也证明，机械力引起血小板膜磷脂分布的变化，并且它们增强剪切诱导的血小板活化和聚集。在我们最近对血小板活化的研究中，我们提出了一个两步细胞活化机制来解释这些观察结果的基础上血小板受体Gplb，血浆vWF和流体力的相对作用。与传统的缩放参数相反，我们开发了严格的计算方法来估计在这些条件下施加在细胞和分子上的流体力的大小和性质。此外，我们观察到，vWF经历自缔合或聚集时，在规定的条件下混合。这表明，蛋白质构象可能会改变流体剪切流动下，这可能有功能的后果。基于这些观察，本项目的具体目标是：1）确定vWF单聚体可能自缔合的生理流体剪切条件。对于这一方面，采用光散射、色谱法、蛋白质印迹分析和表面等离子体共振来检测vWF自缔合，并确定该过程的动力学/亲和力。还进行了剪切诱导血小板活化的研究，以建立vWF自结合和血小板活化之间的机制联系。2)证明vWF分子的大小和血小板Gplb受体的长度是在流体剪切下调节血小板活化率的关键参数。为了做到这一点，我们创造了不同大小的微球和纳米颗粒，它们以不同的密度携带固定化抗体和重组形式的vWF。定量这些颗粒结合和活化细胞的能力。3)表征生理流体剪切条件下vWF的构象变化。在这里，溶液结构的vWF和蛋白质的构象变化下流体剪切测量使用光，中子和X-射线散射光谱。新的数学理论被开发出来以定量地指导上述实验的解释。这项工作的成功完成将建立流体剪切可以调节生物分子的结构和功能。从长远来看，将自缔合和血小板活化联系起来的结果也可能促使对这种现象进行体内检查，并刺激针对自缔合的新药开发。

DESCRIPTION (provided by applicant): In models of vascular injury, the engagement of blood platelets by substrate-immobilized von Willebrand Factor (vWF) under fluid flow leads to platelet activation and subsequent thrombus formation. Experiments carried out in suspension assays that apply fluid forces on platelets and blood proteins also demonstrate that mechanical forces cause changes in platelet membrane phospholipid distribution, and they augment shear-induced platelet activation and aggregation. In our recent studies on platelet activation, we proposed a two-step mechanism of cell activation to explain these observations based on the relative roles of platelet receptor Gplb, plasma vWF and fluid forces. As opposed to traditional scaling arguments, we developed rigorous computational methods to estimate the magnitude and nature of fluid forces applied on cells and molecules under these conditions. Further, we observed that vWF undergoes self-association or aggregation when mixed under defined conditions. This suggests that protein conformation may change under fluid shear flow and this may have functional consequences. Based on these observations, the specific goals of this project are: 1) To determine the physiological fluid shear conditions under which vWF unimers may self-associate. For this aspect, light scattering, chromatography, western blot analysis and surface plasmon resonance are employed to detect vWF self-association, and to determine the kinetics/affinity of this process. Studies of shear- induced platelet activation are also conducted to establish a mechanistic link between vWF self- association and platelet activation. 2) To demonstrate that the size of the vWF molecule and length of platelet Gplb receptor are critical parameters regulating platelet activation rates under fluid shear. In order to do this, we create microspheres and nanoparticles of varying sizes bearing immobilized antibodies and recombinant forms of vWF at varying densities. The ability of these particles to bind and activate cells is quantified. 3) To characterize conformational changes in vWF under physiological fluid shear conditions. Here, the solution structure of vWF and protein conformational changes under fluid shear are measured using light, neutron and X-ray scattering spectroscopy. New mathematical theories are developed to quantitatively guide the interpretation of the above experiments. Successful completion of this work will establish that fluid shear may regulate bio-molecule structure and function. Results linking self-association and platelet activation, in the long run, may also prompt in vivo examination of this phenomenon and stimulate new drug development against self-association.