VON WILLEBRAND FACTOR STRUCTURE AND FUNCTION UNDER FLUID FLOW

描述（由申请人提供）：在血管损伤模型中，液体流动下基质固定化血管性血友病因子（vWF）与血小板的接触导致血小板活化和随后的血栓形成。悬浮液试验中对血小板和血液蛋白施加流体力的实验也表明，机械力会导致血小板膜磷脂分布的变化，并增强剪切诱导的血小板活化和聚集。在我们最近关于血小板活化的研究中，我们基于血小板受体Gplb、血浆vWF和流体力的相对作用，提出了细胞活化的两步机制来解释这些观察结果。与传统的缩放论点相反，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在特定条件下混合时会发生自关联或聚集。这表明，在流体剪切流动下，蛋白质构象可能发生改变，这可能具有功能后果。基于这些观察结果，本项目的具体目标是：1)确定vWF分子可能自我关联的生理流体剪切条件。为此，采用光散射、层析、western blot分析和表面等离子体共振等方法检测vWF自结合，并确定该过程的动力学/亲和性。剪切诱导血小板活化的研究也被进行，以建立vWF自关联与血小板活化之间的机制联系。2)证明vWF分子大小和血小板Gplb受体长度是调节血小板在流体剪切作用下活化率的关键参数。为了做到这一点，我们制造了不同大小的微球和纳米颗粒，以不同的密度承载固定抗体和重组形式的vWF。这些颗粒结合和激活细胞的能力是量化的。3)表征生理性流体剪切条件下vWF的构象变化。本文利用光、中子和x射线散射光谱测量了流体剪切作用下vWF溶液结构和蛋白质构象的变化。新的数学理论的发展，定量指导上述实验的解释。这项工作的成功完成将证明流体剪切可以调节生物分子的结构和功能。从长远来看，将自我关联和血小板激活联系起来的结果也可能促使对这一现象的体内研究，并刺激针对自我关联的新药开发。

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.