Regulation of von Willebrand factor processing

描述（由申请人提供）：血管性血友病因子是血浆中的一种多聚体蛋白，通过介导血管损伤部位的血小板粘附，在原发性止血中起重要作用。血管性血友病因子的止血功能取决于其亚基的多聚程度。内皮细胞和血小板分泌的部分血管性血友病因子含有高度聚合形式的血管性血友病因子，可自发凝集血小板，在循环中形成病理性闭塞血栓。这些高度聚合形式的血管性血友病因子在血浆中被金属蛋白酶ADAMTS13以剪切依赖的方式蛋白水解加工成低聚合形式。因此，血浆中血管性血友病因子的分泌后加工是调节血管性血友病因子功能的关键步骤。虽然剪切应力和ADAMTS13的存在对加工至关重要，但这一过程是如何调节的尚不清楚。本申请拟开展的研究将重点关注von Willebrand因子亚基内各结构域之间的分子内相互作用，以及von Willebrand因子与其他大分子（包括辅助因子）之间的分子间相互作用如何调节ADAMTS13对von Willebrand因子的蛋白水解加工。在目的1中，我们将利用von Willebrand因子的重组和突变片段，确定risstocetin结合位点在调节ADAMTS13切割A2结构域的可及性中的作用。在目标2中，我们将研究von Willebrand因子亚基内三个“A”结构域之间的变构相互作用及其与其他大分子的相互作用如何调节裂解位点的暴露和可及性。在目标3中，我们将描述肝素和肝素样辅助因子如何通过ADAMTS13调节血管性血友病因子的分裂。对这一过程的进一步了解将为预防和治疗由血管性血友病因子异常、不充分或过度处理引起的血栓性和出血性疾病提供新的策略。

DESCRIPTION (provided by applicant): Von Willebrand factor is a multimeric protein in plasma that plays an essential role in primary hemostasis by mediating platelet adhesion at sites of vascular injury. The hemostatic function of von Willebrand factor depends on the degree of multimerization of its subunits. A fraction of the von Willebrand factor secreted from endothelial cells and platelets contains a highly polymerized form of von Willebrand factor that would spontaneously agglutinate platelets to form pathological occlusive thrombi in the circulation. These highly polymerized forms of von Willebrand factor are proteolytically processed to a less polymerized form in a shear-dependent manner by the metalloprotease ADAMTS13 in plasma. Thus, post-secretion processing of von Willebrand factor in plasma is a critical step in regulating the function of von Willebrand factor. Although shear stress and the presence of ADAMTS13 are essential for processing, how this process is regulated is not known. Studies proposed in this application will focus on how intramolecular interactions among the various domains within the von Willebrand factor subunit, and intermolecular interactions between von Willebrand factor and other macromolecules, including that of a cofactor, regulate the proteolytic processing of von Willebrand factor by ADAMTS13. In aim 1, by use of recombinant and mutant fragments of von Willebrand factor, we will determine the role of the ristocetin binding site in regulating the accessibility of the A2 domain for cleavage by ADAMTS13. In aim 2, we will study how allosteric interactions among the three 'A' domains within the von Willebrand factor subunit and their interactions with other macromolecules modulate the exposure and accessibility of the cleavage site. In aim 3, we will characterize how heparin and a heparin-like cofactor regulate the cleavage of von Willebrand factor by ADAMTS13. An improved understanding of this process would lead to new strategies for the prevention and treatment of thrombotic and bleeding diseases caused by aberrant, inadequate or excessive processing of von Willebrand factor. PUBLIC HEALTH RELEVANCE: The proposed research is focused on understanding how a plasma metalloprotease regulates the function of the adhesive protein von Willebrand factor, which initiates platelet adhesion and aggregation at the site of vascular injury. These studies will further our understanding of how the metalloprotease and a newly identified cofactor regulate the function of von Willebrand factor and thrombi formation in health and in disease.