MOLECULAR ANALYSIS OF INTEGRIN RECEPTORS OF PLATELET AND ENDOTHELIAL CELLS

拟议研究的长远目标是阐明某些 血小板GP IIb-IIIa受体的结构和功能域。 这种血小板聚集受体与细胞外基质有关 内皮细胞（EC）上的粘附受体。 血小板GP IIb-IIIa EC整合素的复杂和改变的功能与 在ARDS中观察到的血管损伤的病理进展。 微血栓在ARDS中常见，血小板和 它们的分泌和代谢产物影响着 白细胞与EC和EC本身的关系。 整合素是 该方案具有同源的结构元件，但配体不同， 粘合和其他功能特性。 血小板GP IIb-IIIa 与相关的玻连蛋白和纤连蛋白相比， 受体（VnR，FnR）在EC上的亚基组成，配体结合 机制和对血小板激动剂活化的依赖性。 具体目标 #1和#2通过以下方式解决这些独特的GP IIb-IIIa功能： 开发基于以下的异源细胞表达系统： GP IIb的嵌合形式，以确定 配体结合和亚基缔合。 表达GP的稳定细胞系 将测定IIb-IIIa及其变体在以下方面的差异：（i）配体 在血小板聚集的可溶性肽拮抗剂中 和细胞粘附，以及（iii）它们结合两种配体和 未受刺激的血小板以诱导型或组成型的方式。 这些研究与血小板生成等过程直接相关， 聚集和血栓形成的新机制 未受刺激的血小板募集。 本提案中的具体目标#3是 以确定是否通过促进剂在体外扰动EC 炎症和形态学变化引起的改变， 整合素的mRNA表达水平和表面受体分布 受体（例如，VnR、FnR）是细胞外基质粘附所必需的。

The long term objectives of the proposed study are to elucidate certain structural and functional domains of the platelet GP IIb-IIIa receptor. This platelet aggregation receptor is related to extracellular matrix adhesion receptors on endothelial cells (EC). The platelet GP IIb-IIIa complex and altered functions of EC integrins are relevant to the pathological progression of vascular injury observed in ARDS. Microthrombi are commonly seen in ARDS and the presence of platelets and their secretory and metabolic products influence both the interactions of leukocytes with the EC and the EC itself. The integrins central to this proposal have homologous structural elements, but differ in ligand- binding and other functional properties. The platelet GP IIb-IIIa complex is unique compared to the related vitronectin and fibronectin receptor (VnR, FnR) on EC in terms of subunit composition, ligand-binding mechanism, and dependence on platelet agonist activation. Specific Aims #1 and 2 address these unique GP IIb-IIIa functions through the development of a heterologous cell expression system that is based on chimeric forms of GP IIb to determine specific sequences required for ligand-binding and subunit association. Stable cell lines expressing GP IIb-IIIa and its variants will be assayed for differences in; (i) ligand recognition both in soluble peptide antagonists of platelet aggregation and cell adhesion, and (iii) their ability to bind to both ligands and unstimulated platelets in either an inducible or constitutive manner. Such studies are directly relevant to such processes as platelet aggregation and novel mechanisms of thrombus formation involving unstimulated platelet recruitment. Specific Aim #3 in this proposal is to determine whether EC perturbation in vitro by agents promoting inflammation and morphological changes cause alterations in the expression levels of mRNA and surface receptor distribution for integrin receptors (eg., VnR, FnR) necessary for extracellular matrix adhesion.