Fibronectin (Fn) has been implicated as an important co-factor for platelet adhesion in flowing blood and may participate in platelet aggregation. Studies supported by this grant have led to the first quantitative assessment of Fn binding to a cell (platelets), the initial identification of polypeptide components of an Fn receptor (GPIIb-IIIa), the suggestion that platelets possess a binding site which recognizes common features of Fn, fibrinogen, and VWF, and the demonstration that Arg-Gly-Asp (RGD) containing synthetic peptides inhibit Fn binding to a cell. These peptides inhibit platelet adhesive functions and may therefore be prototypes of short acting inhibitors of platelet function. Continued support is requested to test 3 hypotheses arising from previous work. To test the hypothesis that RGD peptides competitively inhibit the binding of RGD sequence in adhesive proteins to a common receptor we will directly assess the interaction of such peptides with platelets by quantitative ligand binding and affinity chromatography. We will analyze the apparent affinity and concentration of peptide binding sites, their specificity, activation dependence and divalent cation dependence. These measurements will be related to the capacity of these peptides to inhibit platelet binding of Fn. Secondly, we will test the hypothesis that a definable domain of GPIIb-IIIa recognizes the RGD sequence by efforts to characterize and isolate such domains. This will be done by affinity chromatography of proteolytic digests of GPIIb-IIIa and by chemical crosslinking of bound synthetic peptides to GPIIb-IIIa followed by proteolytic cleavage. We will produce antibodies reactive with idiotopes of anti-RGD peptide antibodies which have the properties expected of an internal image of the RGD recepter as a third approach to identification of RGD binding domains in GPIIb-IIIa. Finally, to test the hypothesis that a second platelet binding site is present in Fn, we will undertake to isolate and characterize a chymotryptic fragment of Fn which appears to lack RGD, but inhibits Fn binding to platelets. The mechanism of inhibition by this fragment will be established and its receptor will be identified by employing the same approaches utilized for the RGD binding site.
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