Platelets in circulation adhere to sites of vascular injury, become activated by agonists such as thrombin, and aggregate primarily by binding. Inhibitors of platelet aggregation include certain murine monclonal antibodies directed against the membrane glycoprotein (GP) IIb_IIIa. Platelet aggregation is important in pathologic states, characterized by vascular occlusion, such as myocardial infarction and stroke and restenosis of arteries following angioplasty or thrombolytic therapy. Our objective is to develop an antithrombotic agent which will inhibit platelet aggregation. Our approach is to identify the primary amino acid sequence(s) of regions of GPIIb-IIIa involved in finbrinogen binding. Soluble forms of GPIIb or GPIIa that bind fibrinogen will then either be used directly as inhibitors or platelet aggregation, or as the basis for the design of novel inhibitors, such as non-hydrolyzable peptides. Recently, the amino acid sequences of GPIIb and GPIIIa have become available. Thus, it is possible to utilize synthetic peptides and expression of recombinant fragments of the proteins to analyze putative binding sites. In phase I we propose to generate novel monoclonal antibodies against GPIIb-IIIa to identify fibrinogen binding sites and evaluate these antibodies in a microtiter plate assay using purified the GPIIb-IIIa complex and radiolabeled fibrinogen.
