SURFACE AND LIGAND RECEPTOR INTERACTIONS OF VWF

血管性血友病因子（vWF）在止血调节中起着重要作用 和血栓形成，通过参与血液凝固和促进 血小板的粘附和聚集。双重角色是不可或缺的， 血管损伤后或存在以下情况下维持止血 一种植入的心血管装置虽然一般序列 涉及vWF的功能性事件是已知的，相应的结构性 vWF的变化尚未在水环境下在分子尺度上确定。 条件拟议的研究重点是阐明这三个（3D） 在生理和生理条件下vWF的三维（三级）结构 病理生理剪切条件下使用的新技术的原子 力显微镜（AFM）。要检验的中心假设是， vWF的构象状态与 蛋白质的功能结合结构域的表达。具体目标 本课题的主要研究内容包括：（1）vWF的三维构象测定 在不同的粘附基质（胶原、肝素、人工 表面）在血流动力学条件下增加剪切应力;（2） 在所述细胞中鉴定因子VIII功能性结合结构域， 表面吸附vWF的三级结构;（3）鉴定和作图 在亚纳米尺度上的vWF的暴露表面电荷域;和（4） 分子间粘附力的定量参与的具体 vWF配体-受体相互作用，包括Al的肽段 结构域和RGDX配体与血小板整合膜受体的结合 GPIb和GPIIb-IIIa以及胶原或肝素化基质。独特的 AFM测量3D结构和分子间力的能力 将是这些研究中使用的核心技术。一系列体外 从停滞到病理生理剪切的动态流动条件 将使用旋转盘系统来量化和控制应力 和层流室。构象研究将直接 与vWF的功能状态相关， 包括通过高分辨率荧光测定的血小板粘附 显微镜下，并通过FITC标记和金珠标记的单克隆抗体， 已知vWF表位的抗体。这些研究将有助于新的 vWF结构-功能关系的见解和理解， 剪切条件的范围，并在发展一个全面的了解 血栓形成

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.