MOLECULAR ANALYSIS OF INTEGRIN RECEPTORS OF PLATELET AND ENDOTHELIAL CELLS

拟议研究的长远目标是澄清某些 血小板膜糖蛋白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)上的黏附受体。血小板糖蛋白IIb-IIIa EC整合素的复杂和改变的功能与 急性呼吸窘迫综合征血管损伤的病理进展。 微血栓常见于急性呼吸窘迫综合征和血小板和 它们的分泌和代谢产物影响着两者的相互作用。 白细胞与EC和EC本身的关系。以整合素为中心的 这一提议具有相同的结构元素，但在配体- 绑定和其他功能属性。血小板糖蛋白IIb-IIIa 与相关的玻璃体连接蛋白和纤维连接蛋白相比，复合体是独一无二的 内皮细胞上受体(VNR、FNR)的亚基组成、配体结合 机制，以及依赖于血小板激动剂的激活。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 一种异源细胞表达系统的建立 GP IIb的嵌合形式以确定所需的特定序列 配基结合和亚基结合。稳定表达糖蛋白的细胞系 将分析IIB-IIIa及其变异体在以下方面的差异：(I)配体 可溶性多肽拮抗剂对血小板聚集的识别作用 和细胞黏附，以及(Iii)它们与配体和 未刺激的血小板以可诱导的或构成的方式。 这类研究与血小板等过程直接相关 凝集与血栓形成的新机制 未受刺激的血小板募集。本提案的第三个具体目标是 确定促进剂在体外是否对EC产生干扰 炎症和形态变化导致血管内皮细胞的改变 整合素的mRNA表达水平和表面受体分布 细胞外基质黏附所必需的受体(如VNR、FNR)。

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.