SURFACE AND LIGAND RECEPTOR INTERACTIONS OF VWF

血管性血友病因子(VWF)在止血调节中的核心作用 通过参与凝血和促进血栓形成 血小板的黏附和聚集。这两个角色是 血管损伤后或在有血管损伤的情况下维持止血 一个植入的心血管装置。而大体顺序是 涉及vWF的功能事件是已知的，对应的结构 在水溶液中，vWF的变化还没有在分子水平上确定 条件。拟议的研究重点是阐明这三个(3D) 生理和心理状态下vWF的空间(三级)结构 应用原子切变新技术的病理生理切变条件 力显微镜(AFM)。需要检验的中心假设是 VWF的构象状态与VWF的构象状态密切相关 表达该蛋白的功能结合域。具体目标 本项目的主要工作包括：(1)确定vWF的三维构象 不同粘合剂底物上的多聚体(胶原蛋白、肝素、人造 表面)在增加切应力的血流动力学条件下；(2) 重组人凝血因子VIII功能结合域的鉴定 表面吸附的vWF的三级结构；(3)鉴定和作图 关于vWF裸露表面电荷域的亚纳米尺度；和(4) 分子间粘附力的定量研究 包括A1多肽片段的VWF配体-受体相互作用 结构域和RGDX配体与血小板整膜受体的结合 GPIB和GPIIb-IIIa以及胶原蛋白或肝素化底物。独一无二的 原子力显微镜同时测量三维结构和分子间作用力的能力 将是这些研究中使用的核心技术。一系列体外培养 从淤积到病理生理切变的动态流动条件 将使用旋转圆盘系统来量化和控制应力 和一个层流流室。构象研究将直接 与vWF功能状态相关的补充分析 包括高分辨率荧光法测定的血小板粘附性 显微镜下，并用FITC标记和金珠标记的单抗 针对已知vWF表位的抗体。这些研究将做出新的贡献 对vWF结构-功能关系的洞察和理解 剪切条件的范围和发展一个全面的理解 血栓形成的可能性。

von Willebrand Factor (vWF) plays central role in regulation of hemostasis and thrombosis by participating in blood coagulation and facilitating adhesion and aggregation of platelets. The dual roles are integral to the maintenance of hemostasis after vasculature injury or in the presence of an implanted cardiovascular device. While the general sequence of functional events involving vWF is known, the corresponding structural changes in vWF have not been determined on a molecular scale under aqueous conditions. The proposed studies focus on elucidation of the three (3D) dimensional (tertiary) structure of vWF under physiologic and pathophysiologic shear conditions using the novel technique of atomic force microscopY (AFM). The central hypothesis to be examined is that there is a close relationship between the conformational state of vWF and expression of the protein's functional binding domains. The specific aims of this project include: (1) determination of the 3D conformation of vWF multimers on different adhesive substrata (collagens, heparin, artificial surfaces) under hemodynamic conditions of increasing shear stress; (2) identification of the Factor VIII functional binding domain in the tertiary structure of surface adsorbed vWF; (3) identification and mapping on a sub-nanometer scale of exposed surface charge domains of vWF; and (4) quantification of the intermolecular adhesive forces involved in specific vWF ligand-receptor interactions including peptide segments of the Al domain and RGDX ligand binding to platelet integral membrane receptors GPIb and GPIIb-IIIa and to collagen or heparinized substrata. The unique capabilities of AFM to measure both 3D structure and intermolecular forces will be the central technique used in these studies. A range of in vitro dynamic flow conditions extending from stasis to pathophysiologic shear stresses will be quantified and controlled using a rotating disk system and a laminar flow chamber. Conformational studies will be directly correlated to the functional state of vWF using complimentary assays including platelet adhesion determined by high resolution fluorescence microscopy, and by FITC labeled and gold bead labeled monoclonal antibodies to known vWF epitopes. These studies will contribute new insights and understanding of vWF structure-function relations under a range of shear conditions and in developing a comprehensive understanding of thrombogenesis.