VON WILLEBRAND FACTOR STRUCTURE AND FUNCTION UNDER FLUID FLOW

描述(申请人提供)：在血管损伤模型中，在流体流动下，底物固定化的von Willebrand因子(VWF)与血小板的结合导致血小板激活和随后的血栓形成。在悬浮分析中对血小板和血液蛋白质施加流体力的实验也表明，机械力会引起血小板膜磷脂分布的变化，并增强剪切诱导的血小板激活和聚集。在我们最近关于血小板激活的研究中，我们提出了细胞激活的两步机制，基于血小板受体Gplb、血浆vWF和流体力的相对作用来解释这些观察结果。与传统的比例论证相反，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经历自缔合或聚集。这表明，在流体剪切流的作用下，蛋白质的构象可能会发生变化，这可能会产生功能上的影响。基于这些观察，本项目的具体目标是：1)确定vWF单体可能自结合的生理流体剪切条件。为此，采用光散射、层析、Western印迹分析和表面等离子体共振等方法检测vWF的自缔合作用，并确定这一过程的动力学/亲和力。我们还对剪切诱导的血小板活化进行了研究，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受体的长度是调节流体剪切下的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，它们携带着不同密度的固定化抗体和重组形式的vWF。对这些颗粒结合和激活细胞的能力进行了量化。3)表征生理性流体剪切条件下vWF的构象变化。在这里，我们用光、中子和X射线散射光谱测量了vWF的溶液结构和蛋白质在流体剪切作用下的构象变化。发展了新的数学理论来定量地指导上述实验的解释。这项工作的成功完成将确立流体剪切可以调节生物分子结构和功能。结果从长远来看，将自结合与血小板激活联系起来，也可能促使体内对这一现象的研究，并刺激针对自结合的新药开发。

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.