A major objective of this research is to elucidate molecular mechanisms by which fibrinogen interacts with its receptor on the platelet surface resulting in platelet aggregation. Several lines of investigation strongly implicate the roles of the surface glycoprotein (GPIIIa) in the function of the fibrinogen receptor. Our studies have revealed a 66,000 Mr protein that is present on the surface of chymotrypsin- and pronase-treated platelets. This protein, precipitated by polyclonal antibodies which block fibrinogen-induced platelet aggregation and 125I-fibrinogen binding, is formed during limited proteolysis of GPIIIa by chymotrypsin or pronase. The formation of this 66,000 Mr protein derived from GPIIIa on the surface of proteolytically-treated platelets correlated with fibrinogen-induced platelet aggregation and 125I-fibrinogen binding to these platelets. We propose a hypothesis that the Mr = 66,000 is a part of glycoprotein IIIa present on the surface of proteolytically-treated platelets and it may function in fibrinogen binding and fibrinogen-induced platelet aggregation, and that the 66,000-dalton region of glycoprotein IIIa in intact platelets may represent the fibrinogen-binding domain of glycoprotein IIIa. The purpose of the research proposed here is to identify and characterize the fibrinogen-binding domains(s) of GPIIIa with emphasis on its 66,000 Mr protein fragment.
Our specific aims are: 1) further development and characterization of polyclonal antibodies directed against purified human platelet membrane proteins associated with the fibrinogen receptor; 2) development of monoclonal antibodies to GPIIIa, the 66,000 Mr derivative of GPIIIa, and proteolytic fragments present on the surface of chymotrypsin- and pronase-treated platelets; 3) use of polyclonal and monoclonal antibodies in the purification of fibrinogen receptors from platelet membranes with special emphasis on GPIIIa and the 66,000 Mr derivative of GPIIIa; 4) study the interaction of these membrane glycoproteins with fibrinogen at the cellular level and in the purified system; and 5) use of a library of antibodies developed against GPIIIa and proteolytic digest products of GPIIIa in studies on the mechanism of exposure of the fibrinogen receptor during platelet aggregation. My long term goals are to determine the chemicl structure of the fibrinogen receptor and to study the mechanism of fibrinogen receptor exposure and activation by ADP, epinephrine, and thrombin during platelet aggregation. These studies will serve to elucidate basic mechanisms involved in thrombosis and hemostasis.
