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.
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