Biophysical Study of Collagen-von Willebrand Factor Interaction during Thrombosis

描述（由申请方提供）：血管性血友病因子（vWF）与I型和III型胶原蛋白的结合是正常止血和导致卒中和心肌梗死的致病性血栓形成的重要第一步。到目前为止，人们对这两种分子之间的分子相互作用知之甚少。虽然在血栓形成过程中有几种胶原蛋白受体参与血小板的活化，但vWF与胶原蛋白的结合是在流动条件下将血小板锚定在血管壁破裂部位所必需的。我们的长期目标是了解胶原蛋白如何与血小板上不同的胶原蛋白受体相互作用，从而调节血栓形成。本工作的目的是研究胶原与vWF的分子相互作用。胶原和成熟vWF形成多聚体分子复合物。vWF的A3结构域（vWF-A3）被鉴定为胶原结合结构域，我们已成功地在大肠杆菌中表达了vWF-A3。杆菌尽管如此，胶原蛋白三螺旋的大尺寸和独特的绳状结构使得研究两种分子之间的特定分子相互作用具有挑战性。我们假设胶原蛋白上存在多个vWF结合位点，这些位点的结合能力是异质的，并且vWF的结合需要在不同位点的联合作用。 为了验证这一假设，本研究将致力于以下具体目标：（1）利用蛋白质足迹法确定vWF-A3在I型和III型胶原中的结合位点分布;（2）通过定量表征结合相互作用的热力学和动力学性质，定量了解胶原与vWF-A3之间的异质性结合反应。拟议的工作结合了使用全链胶原蛋白和使用短合成肽的研究的优势。新方法的成功应用将导致对胶原和vWF之间的结合反应的详细和全面的理解。相关知识将为进一步研究结合反应的分子基础和血栓形成过程中多个结合位点的作用模式奠定基础。胶原-vWF相互作用代表了开发新的和新颖的干预措施的良好靶点，通过从根本上抑制该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.