STRUCTURAL/FUNCTIONAL CHARACTERIZATION OF VWF-AL DOMAIN

描述：(改编自调查人员摘要。)血小板与部位的黏附 内皮细胞损伤对维持血管内皮细胞损伤的完整性至关重要 脉管系统。这一过程的一个重要部分是冯·威尔布兰德的能力 促进血小板沉积的因子(VWF)通过在 暴露的内皮下基质和血小板膜糖蛋白Ib/IX/V(GPIB/IX/V) 受体复合体。这种大型病毒A1结构域自然发生的突变 复杂的糖蛋白已被描述为干扰血小板结合，但不是 其形成高分子量多聚体(2M型)的能力。大量研究 专注于这一领域的功能，但进展受阻 由于1)不能表达和纯化足够数量的功能性 重组vWF-A1蛋白，以及2)缺乏体内小鼠模型以更好 表征vWF-A1：生理相关的GPIB/IX/V相互作用 布景。申请人已成功地利用定点突变技术 确定GPIB/IX/V与重组人结合所必需的残基 流动条件下的vWF-A1(rvWF-A1)蛋白。然而，他发现人类 RvWF-A1不支持小鼠血小板的黏附，这排除了其 在体内使用。此应用程序的基本目的是将 小鼠vWF与GPIB/IX/V的分子相互作用 了解体内促进血栓形成的黏附机制。 建议的研究，使用申请人克隆的小鼠vWF-A1基因，将是 针对两个特定的目标。第一个目标是确定关键的结构 含有小鼠vWF-A1结构域的元件，可促进与GPIB/IX/V的相互作用 为了实现这一目标，对候选残基进行定点突变，基于 人vWF-A1的晶体结构将被执行，其功能是 利用体外流动室确定细菌表达的蛋白。 还将对人和小鼠的vWF&A1结构域进行同源突变。 进一步确定人vWF-A1中的GPIB/IX/V结合位点。第二个目标是 急性血管病变中vWF-A1介导的相互作用特征 受伤。利用活体显微镜观察重组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是 下定决心。将通过基因工程使小鼠表达绿色荧光 血小板中的蛋白质(GFP)，以促进这些体内研究。

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.