This proposal is a request for five years funding for a FIRST award, designed to identify and characterize ligand binding domains on the platelet membrane glycoprotein (GP) IIb-IIIa complex. Previous work demonstrated that GP IIb-IIIa is sufficient to account for the fibrinogen and fibronectin receptor on platelets. However, the number and location of fibrinogen and fibronectin binding sites on GP IIb-IIIa are unknown. Preliminary results suggest that GP IIb and GP IIIa each have fibrinogen binding domains.
The specific aims of this proposal are threefold. First, fibrinogen and fibronectin binding domains will be identified by testing the ability of fragments of cleaved GP IIb and GP IIIa to inhibit 125I-GP IIb-IIIa binding to immobilized ligands. This approach will be supplemented with photoaffinity labeling of GP IIb-IIIa. Fragments of GP IIb and GP IIIa containing ligand binding domains will be isolated and characterized with regard to sites recognized on fibrinogen and fibronectin and will serve as antigens in monoclonal antibody production. Second, ligands and monoclonal antibodies will be used to locate ligand binding regions on GP IIb-IIIa by rotary shadowing electron microscopy. Third, monoclonal antibodies will be used to determine if the identified ligand binding domains function in whole platelets to bind ligands and mediate aggregation. In addition, monoclonal antibodies and 125I surface labeling will be used to determine if the conversion of GP IIb-IIIa to a competent receptor on activated platelets is due to an increased exposure of the identified ligand binding domains. Completion of these studies will contribute to the long- term goal of relating GP IIb-IIIa structure to function. Such knowledge will increase our understanding of the molecular mechanisms of hemostasis and thrombosis.
| Smyth, S S; Parise, L V (1993) Regulation of ligand binding to glycoprotein IIb-IIIa (integrin alpha IIb beta 3) in isolated platelet membranes. Biochem J 292 ( Pt 3):749-58 |
| Smyth, S S; Hillery, C A; Parise, L V (1992) Fibrinogen binding to purified platelet glycoprotein IIb-IIIa (integrin alpha IIb beta 3) is modulated by lipids. J Biol Chem 267:15568-77 |
| Hillery, C A; Smyth, S S; Parise, L V (1991) Phosphorylation of human platelet glycoprotein IIIa (GPIIIa). Dissociation from fibrinogen receptor activation and phosphorylation of GPIIIa in vitro. J Biol Chem 266:14663-9 |
| Parise, L V; Criss, A B; Nannizzi, L et al. (1990) Glycoprotein IIIa is phosphorylated in intact human platelets. Blood 75:2363-8 |
