Platelet adhesion to subendothelial constituents and platelet aggregation are important in hemostasis and thrombosis, and are mediated in part by the binding of 3 adhesive proteins which contain Arg-Gly-Asp (RGD) sequences: fibrinogen, fibronectin and von Willebrand factor. GPIIb-IIIa is a receptor for these adhesive proteins and binds to peptides containing the RGD sequence. Moreover, GPIIb-IIIa is a member of widely distributed adhesion receptor superfamily termed Integrins whose members are comprised of two non-identical alpha and beta subunits. Besides GPIIb-IIIa, certain other integrins also recognize the RGD sequence. We recently identified a platelet membrane protein of Mr approximately 160 kDa (P160) which binds to peptides containing the RGD sequence. This proposal will test the hypothesis that P160 forms a heterodimer complex with another platelet membrane protein to constitute a novel platelet integrin. To determine if P160 forms a heterodimer with a beta subunit, we will analyze the polypeptide content of immunoprecipitates formed with antibodies reactive with purified P160. In addition, we will examine the hydrodynamic properties of purified P160 to establish its Mr under non- denaturing conditions. Furthermore, we will investigate whether the sequence of P160 is similar to known integrins by direct sequencing of the amino-terminus and fragments of P160. Similar studies will be performed with the putative beta subunit of P160 should one be identified. The RGD-binding domain of P160 will be localized and characterized by affinity chromatography of proteolytic digests of purified P160, and by chemical crosslinking of RGD peptides to P160 followed by proteolytic cleavage. Since several membrane proteins which bind RGD serve adhesive functions, we will test the hypothesis that P160 plays a role in platelet adhesive reactions. Firstly, this will be accomplished by reconstituting purified P160 into radioactive liposomes and examining its binding to various RGD-containing adhesive proteins in a solid phase assay. Secondly, we will identify antibodies and RGD peptides which preferentially block the binding of P160 to immobilized RGD peptides. The capacity of these antibodies and peptides to inhibit platelet adhesive functions will be assessed. These studies will provide fundamental information about a membrane protein likely to be involved in mechanisms underlying platelet adhesion or aggregation.
