SUBENDOTHELIAL BINDING OF VON WILLEBRAND FACTOR

血管内皮的损伤导致血管内皮细胞的快速粘附， 血小板与暴露的下层内皮细胞（SE）， 止血和动脉粥样硬化形成的起始步骤。 这 血小板粘附需要血管性血友病因子（vWf），我们 以前被证明存在于SE中。 我们确定 SE提取物中的三种vWf结合蛋白，具有表观分子量 15万、20万和8万的重量，并成功地分离出 15万蛋白质 我们发现它的特征与 一种新发现的胶原蛋白VI型胶原蛋白， 微纤维的组成部分。 我们已经开始研究， 纯化的VI型胶原蛋白，并有证据表明vWf结合型 VI胶原蛋白，并且还，初步认为：1）结合结构域 对vWf的影响不同于对纤维状胶原的报道， 和，2）结合由VI型上的球状结构域介导 胶原分子 我们现在将采取两个主要途径：1）在确定了 150 kDa蛋白，我们计划使用类似的方法，包括SDS-PAGE， 制备电洗脱，放射性配体和斑点印迹，异- 电聚焦氨基酸分析，Western blotting分离 并表征200和80 kDa的蛋白质，和2）我们将 研究vWf与VI结合的分子基础 胶原蛋白着眼于贡献信息， 可用于开发调节这种结合的方法。 的 结合机制将用单克隆抗体进行剖析 vWf上的特定结构域，以及vWf的酶和CNBr片段， vWf，沿着候选肽和其他粘合剂 糖蛋白 对VI型胶原蛋白的结合要求将 进行类似的研究。 荧光免疫定位研究 将用于确定vWf、VI型胶原蛋白和其他 相关细胞外基质蛋白和粘附糖蛋白 在血管和组织培养中共定位。 我们将开始 检查vWf与VI型结合的功能意义 静态和流动系统中的胶原蛋白，使用去内皮化血液 船舶. 阐明vWf与SE的结合将是重要的 血小板粘附机制的研究进展 动脉粥样硬化的发病机制。 这些知识可能有助于 调节血小板与损伤细胞粘附的新方法 血管

Injury to vascular endothelium results in the rapid adhesion of platelets to the exposed underlying subendothelium (SE), an early step in the initiation of hemostasis and of atherogenesis. This platelet adhesion requires von Willebrand factor (vWf), which we previously demonstrated to be present within SE. We identified three vWf binding proteins in SE extracts, with apparent molecular weights of 150,000, 200,000 and 80,000, and succeeded in isolating the 150,000 protein. We found that its characteristics match those of a newly identified form of collagen, type VI collagen, the building block of microfibrils. We have initiated studies with purified type VI collagen and have evidence that vWf binds to type VI collagen, and also, preliminarily that: 1) the binding domain on vWf is different from those reported for fibrillar collagens, and, 2) binding is mediated by a globular domain on the type VI collagen molecule. We will now pursue two major avenues: 1) Having identified the 150kDa protein, we plan to use similar methods, including SDS-PAGE, preparative electro-elution, radio-ligand and dot-blotting, iso- electric focusing amino acid analysis, Western blotting to isolate and characterize the 200 and 80kDa proteins, and 2) we will investigate the molecular basis of the binding between vWf and VI collagen with an eye toward contributing information which will be useful in developing ways of modulating this binding. The mechanism of binding will be dissected with monoclonal antibodies to specific domains on vWf, and enzymatic and CNBr fragments of vWf, along with candidate peptides and other adhesive glycoproteins. The binding requirements on type VI collagen will be similarly studied. Fluorescence immuno-localization studies will be used to determine whether vWf, type VI collagen and other relevant extracellular matrix proteins and adhesive glycoproteins co-localize in blood vessels and in tissue culture. We will begin to examine the functional significance of vWf binding to type VI collagen in static and flow systems, using de-endotheliazed blood vessels. Elucidation of the binding of vWf to SE would be an important advance in understanding the mechanism of platelet adhesion and the pathogenesis of atherosclerosis. This knowledge may contribute to new means of regulating the attachment of platelets to injured blood vessels.