MOLECULAR BIOLOGY OF THE THROMBOCYTE FIBRINOGEN RECEPTOR

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The long-term objectives of this research proposal are to acquire further insight into the identity and nature of the recognition site(s) on IIb-IIIa for RGD peptides and H12, to characterize interactions between these recognition sites that might support fibrinogen binding and platelet aggregation, and to further understand the nature of the conformational change(s) of IIb-IIIa that is initiated by platelet stimulation. To address these questions, we have formulated three specific aims: 1) To develop and characterize prototypes of antibody-based ligands and to identify key structural requirements for specific, affinity and dependence upon IIb-IIIa activation. Op-G2 and CP3, like PAC-1, contain the sequence RYD in H3 and thus mimic RGD-containing protein ligands. Unlike PAC-1, OP-G2 and CP3 bind to IIb-IIIa in nonactivated platelets. A comparison of the sequence and structure of these three antibodies as well as a number of other antibodies which contain similar sequences in H3 but fail to bind to IIb-IIIa is a basis for structural models that can be used to study the mechanics of RGD or H12 recognition by IIb- IIIa. Other antibodies, e.g., AP2, uses alternative binding motifs to inhibit fibrinogen binding and platelet aggregation. The molecular characterization of the specificity of such antibodies may elucidate alternative recognition sites on IIb-IIIa. 2) To characterize the epitopes recognized by AP2, OP-G2 and other murine monoclonal antibodies that bind at or near IIb-IIIa recognition sites. We feel that the identification of such conformationally- constrained, "complex-dependent" epitopes will provide important insights about the topography of the IIb-IIIa complex and the architecture of its RGD and H12 recognition "pockets." This specific aim is now feasible because of our recombinant antibody expression system. 3) To utilize recombinant IIb-IIIa expression systems and site-directed mutagenesis to confirm the identification of reactive sites. We propose to express beta3 integrin constructs in an appropriate expression system. Recombinant integrins will be used as a source of antigens for confirmation of epitope identity. The successful completion of these studies will contribute to our understanding of the molecular basis for the specificity, affinity and activation-dependence of ligands which bind to RGD and dodacepeptide recognition sites of the integrin IIb-IIIa.