STRUCTURAL/FUNCTIONAL CHARACTERIZATION OF VWF-AL DOMAIN

描述：（改编自研究者摘要。）血小板粘附部位 内皮细胞损伤对于维持血管的完整性是至关重要的。 脉管系统这个过程的一个重要部分是冯维勒布兰德 vWF通过在血小板和血管内皮细胞之间形成“桥梁”来促进血小板沉积。 暴露的内皮下基质和血小板糖蛋白Ib/IX/V（GpIb/IX/V） 受体复合物在这个大的A1结构域中自然发生的突变 已经描述了一种复杂的糖蛋白，其干扰血小板结合，但不 其形成高分子量多聚体（2 M型）的能力。大量的研究 一直致力于这一领域的功能，但进展受到阻碍 通过1）不能表达和纯化足够量的功能性多肽， 重组vWF-A1蛋白，和2）缺乏体内小鼠模型，以更好地 在生理学相关研究中表征vWF-A1：GpIb/IX/V相互作用 设置.申请人已成功地采用定点诱变， 定义了GpIb/IX/V与重组人抗体结合所必需的残基 vWF-A1（rvWF-A1）蛋白。但他发现， rvWF-A1不支持小鼠血小板的粘附，这妨碍了其在小鼠体内的表达。 在体内使用。本申请的基本目的是表征 小鼠vWF和GpIb/IX/V之间的分子相互作用，以改善我们的研究。 了解参与促进体内血栓形成的粘附机制。 将使用申请方克隆的鼠vWF-A1 cDNA进行拟定研究， 针对两个具体目标。第一个目标是确定关键结构 具有促进与GpIb/IX/V相互作用的鼠vWF-A1结构域的元件。 为了实现这一目标，候选残基的定点诱变，基于 人vWF-A1的晶体结构，将被执行和功能 使用体外流动室确定细菌表达的蛋白质。 还将进行人和小鼠vWF A1结构域的同源诱变 进一步确定人vWF-A1中的GpIb/IX/V结合位点。第二个目标是 在急性血管性心脏病中， 损伤使用活体显微镜，重组vWF-A1蛋白的能力 而改变小动脉损伤部位血小板行为的抗体将被 测定小鼠将被基因工程改造，以表达绿色荧光 蛋白（GFP）的血小板，以促进这些在体内研究。

DESCRIPTION: (Adapted from investigator's abstract.) Platelet adhesion to sites of endothelial cell injury is critical for maintaining the integrity of the vasculature. An essential part of this process is the ability of von Willebrand factor (vWF) to promote platelet deposition by forming a "bridge" between the exposed subendothelial matrix and platelet glycoprotein Ib/IX/V (GpIb/IX/V) receptor complex. Naturally occurring mutations in the A1 domain of this large complex glycoprotein have been described that perturb platelet binding but not its ability to form high molecular weight multimers (Type 2M). Much research has concentrated on the function of this domain, but progress has been hampered by 1) the inability to express and purify sufficient quantities of a functional recombinant vWF-A1 protein, and 2) lack of an in vivo mouse model to better characterize vWF-A1:GpIb/IX/V interactions in a physiologically relevant setting. The applicant has successfully employed site directed mutagenesis to define residues that are essential for GpIb/IX/V binding to recombinant human vWF-A1 (rvWF-A1) protein under flow conditions. Yet, he has found that human rvWF-A1 does not support the adhesion of mouse platelets, which precludes its use in vivo. The fundamental purpose of this application is to characterize the molecular interactions between murine vWF and GpIb/IX/V in order to improve our understanding of adhesive mechanisms involved in promoting thrombosis in vivo. The proposed studies, using murine vWF-A1 cDNA cloned by the applicant, will be directed at two specific aims. The first aim is to identify key structural elements with murine vWF-A1 domain that promote interactions with GpIb/IX/V. To achieve this goal, site directed mutagenesis of candidate residues, based on the crystal structure of human vWF-A1, will be performed and the function of bacterial expressed protein ascertained using an in vitro flow chamber. Homologue mutagenesis of human and mouse vWF‑A1 domains will also be done to further define the GpIb/IX/V binding site in human vWF-A1. The second aim is to characterize interactions mediated by vWF-A1 in a setting of acute vascular injury. Using intravital microscopy, the ability of recombinant vWF-A1 protein and antibodies to alter platelet behavior at sites of arteriolar injury will be determined. Mice will be genetically engineered to express green fluorescent protein (GFP) in platelets in order to facilitate these in vivo studies.