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型胶原蛋白的结合是正常止血和致病性血栓形成导致中风和心肌梗死必不可少的第一步。到目前为止，人们对这两种分子之间的相互作用知之甚少。虽然血栓形成过程中血小板的活化涉及多种胶原受体，但在血流条件下，vWF与胶原蛋白的结合是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受体相互作用，从而调节血栓形成。本研究的目的是研究胶原蛋白与vWF的分子相互作用。胶原蛋白和成熟的vWF形成多聚分子复合物。vWF的A3结构域（vWF-A3）已被确定为胶原结合结构域，并在大肠杆菌中成功表达。尽管如此，胶原蛋白三螺旋的大尺寸和独特的绳状结构使得研究两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，且这些位点的结合能力各不相同，vWF的结合需要不同位点的联合作用。

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.