Biophysical Study of Collagen-von Willebrand Factor Interaction during Thrombosis

描述(由申请人提供)：血管性血友病因子(VWF)与I型和III型胶原的结合是正常止血和导致中风和心肌梗死的病理性血栓形成的关键第一步。到目前为止，人们对这两个分子之间的分子相互作用知之甚少。在血栓形成过程中，多种胶原蛋白受体参与了血小板的活化，但在血流条件下，vWF与胶原蛋白的结合是在血管壁断裂处锚定血小板所必需的。我们的长期目标是了解胶原如何与血小板上不同的胶原受体相互作用，从而调节血栓形成。本工作的目的是研究胶原蛋白与vWF的分子相互作用。胶原蛋白与成熟的vWF形成多聚体分子复合体。VWF的A3结构域(vWF-A3)已被鉴定为胶原结合域，并在大肠杆菌中成功表达。尽管如此，胶原蛋白三螺旋的大小和独特的绳状结构使得研究这两个分子之间的特定分子相互作用具有挑战性。我们假设胶原上有多个vWF结合位点，它们的结合能力是不同的，并且vWF的结合需要不同位点的联合作用。 为了验证这一假说，本工作将追求以下具体目标：(1)使用蛋白质足迹方法确定vWF-A3结合位点在I型和III型胶原中的分布；(2)通过定量表征结合作用的热力学和动力学性质，定量了解胶原与vWF-A3之间的非均相结合反应。这项拟议的工作结合了使用全链胶原的研究和使用短合成多肽的研究的优点。新方法的成功应用将使人们对胶原和vWF之间的结合反应有详细和一般的了解。相关知识将为进一步研究结合反应的分子基础和多个结合部位在血栓形成过程中的作用方式奠定基础。胶原蛋白-vWF的相互作用是开发新的和新的干预措施的良好靶点，通过从根本上抑制血栓形成的过程来有效和安全地控制血栓形成。

DESCRIPTION (provided by applicant): The binding of von Willebrand factor (vWF) to collagen type I and type III is the essential first step in normal hemostasis and in pathogenic thrombosis leading to stroke and myocardial infarction. So far the molecular interactions between the two molecules are poorly understood. While several collagen receptors are involved in the activation of platelets during thrombosis, the binding of vWF to collagen is required for anchoring platelets at the site of rupture of blood vessel wall under flow conditions. Our long-term goal is to understand how collagen interacts with the different collagen receptors on platelet and thereby modulates thrombosis. The objective of this work is to investigate molecular interactions of collagen with vWF. Both collagen and the mature vWF form multimeric molecular complexes. The A3 domain of vWF (vWF-A3) has been identified as the collagen binding domain, and we have successfully expressed the vWF-A3 in E. coli. Still, the large size and the unique rope-like structure of collagen triple helix make the investigation of specific molecular interactions between the two molecules challenging. We hypothesized that there are multiple vWF binding sites on collagen that are heterogeneous in binding capacity, and that the binding of vWF requires the joint action at the different sites. In order to test this hypothesis the proposed work will pursue the following specific aims: (1) to define the distribution of vWF-A3 binding sites in both type I and type III collagen using protein footprinting approach, and (2) to gain a quantitative understanding of the heterogeneous binding reactions between collagen and the vWF-A3 by quantitatively characterizing the thermodynamic and the kinetic properties of the binding interactions. The proposed work combines the strengths of the studies using the full-chain collagen and those using short synthetic peptides. The successful applications of the new approaches will lead to both detailed and general understanding of the binding reactions between collagen and vWF. The related knowledge will set up the stage for the further investigation of the molecular basis of the binding reaction and of the mode of action of the multiple binding sites during thrombosis. The collagen-vWF interaction represents a good target for the development of new and novel interventions for effective and safe control of thrombosis by inhibiting the process at its root.