Fibronectin and Platelet Function

描述(由申请人提供)：重新提交的竞争性更新的目的是根据结果表明，黏附的血小板组装血浆纤维连接蛋白的能力是由介导血小板黏附的配体决定的。因此，黏附于纤维蛋白原、玻璃体粘连蛋白或von Willebrand因子的血小板组装纤维连接蛋白的能力受到抑制，而黏附于纤维蛋白、层粘连蛋白、胶原蛋白或纤维连接蛋白本身的血小板则牢固地组装纤维连接蛋白。此外，在体外血流系统中，血小板对纤维连接蛋白的组装是决定血小板在纤维蛋白或胶原基质上形成血栓程度的重要因素。这些假设是：(I)血小板整合素和其他细胞表面蛋白识别黏附配体的关键特征，启动细微不同的信号通路，支持或抑制黏附的血小板随后组装的纤维连接蛋白；(Ii)迄今尚未意识到的血小板黏附的微观和生化后果的差异与黏附的血小板组装纤维连接蛋白的能力相关；以及(Iii)纤维连接蛋白的N-末端部分与某些蛋白质的非结构化延伸特异地相互作用，其中包括黏附的血小板上的细胞表面分子和驱动纤维连接蛋白组装的成纤维细胞。具体目标是：1.确定纤维连接蛋白、纤维蛋白原/纤维蛋白、玻璃体连接蛋白和von Willebrand因子的特性，这些特性可解释它们的支持或抑制活性。2.除组装纤维连接蛋白的能力外，区分血小板与支持性和抑制性配体的差异的特征。3.发现纤维连接蛋白组装位点的分子特征存在于有粘附性组装能力的血小板上，而不存在于无粘附性组装能力的血小板上。实现这些目标的方法包括体外诱变以剖析支持性和抑制性粘附性配体的结构/功能，组装能力强和组装能力不强的粘附性血小板的显微和蛋白质组学表征，利用可逆交联法鉴定与支持性和抑制性粘附性配体相互作用的血小板表面蛋白，以及开发激活的凝血因子XIII作为鉴定启动纤维连接蛋白组装的血小板表面分子的工具。

DESCRIPTION (provided by applicant): The aims of this resubmitted competitive renewal follow on results demonstrating that the ability of adherent platelets to assemble plasma fibronectin is determined by the ligands that mediate platelet adhesion. Thus, platelets adherent of fibrinogen, vitronectin, or von Willebrand factor are suppressed in their ability to assemble fibronectin whereas platelets adherent to fibrin, laminin, collagen, or fibronectin itself assemble fibronectin robustly. Further, assembly of fibronectin by platelets in an ex vivo flow system is a strong determinant of the extent of platelet thrombus formation on matrices of fibrin or collagen. The hypotheses are (i) platelet integrins and other cell surface proteins recognize critical features of adhesive ligands, initiating subtly different signaling pathways that support or suppress subsequent assembly of fibronectin by adherent platelets; (ii) as yet unappreciated differences in the microscopic and biochemical consequences of platelet adhesion correlate with the ability of adherent platelets to assemble fibronectin; and (iii) the N- terminal portion of fibronectin interacts specifically with unstructured stretches of certain proteins, among which are the cell surface molecules on adherent platelets and fibroblasts that drive fibronectin assembly. Specific aims are to: 1. Identify features of fibronectin, fibrinogen/fibrin, vitronectin, and von Willebrand factor that account for their supportive or suppressive activity. 2. Characterize differences other than ability to assemble fibronectin that distinguish platelets adherent to supportive and suppressive ligands. 3. Discover the molecular features of fibronectin assembly sites present on adherent assembly- competent platelets and absent on adherent assembly-incompetent platelets. Methods to accomplish these aims include in vitro mutagenesis to dissect the structure/function of the supportive and suppressive adhesive ligands, microscopic and proteomic characterization of assembly- competent and assembly-incompetent adherent platelets, utilization of a reversible cross-linking strategy to identify the platelet surface proteins that interact with supportive and suppressive adhesive ligands, and development of activated blood coagulation Factor XIII as a tool to identify the platelet surface molecules that initiate fibronectin assembly.