Platelet aggregation is known to play a crucial role in hemostasis and thrombosis. Considerable literature suggests that platelet aggregation is a fibrinogen-dependent process, and that platelet cell-surface glycoproteins IIb and IIIa are intimately involved in the formation of the platelet cell-surface fibrinogen receptor. It has been shown that when these glycoproteins are extracted from the platelet membrane with nonionic detergents, they exist as a calcium-dependent complex, dissociable by the action of EDTA. There is also evidence to suggest that GPIIb/GPIIIa complex formation can occur on the platelet surface, and that formation of this complex may be a regulated event. We have produced 4 hybridoma antibodies reactive with GPIIb and GPIIIa. Preliminary experiments strongly suggest that 3 of these hybridoma are specific for new antigenic determinants created by the association of GPIIb with GPIIIa in a calcium-dependent complex, being unreactive with either dissociated glycoprotein. Additionally, 2 of these hybridomas totally inhibit ADP-induced platelet aggregation. In view of these potentially exciting properties, we will directly determine if these 3 hybridomas are GPIIb/GPIIIa complex-specific by studying their ability to bind to the dissociated or complexed forms of these glycoproteins prepared by crossed immunoelectrophoresis. If they are complex specific, we will use them as probes to study the appearance of the GPIIb/IIIa complex on the platelet surface by examining the ability of the hybridomas to bind to resting and activate platelets. We will also determine if GPIIb/GPIIIa purified by use of our hybridomas are able to bind fibrinogen. We will also determine which subregions of the GPIIb/GPIIIa complex might mediate complex formation or fibrinogen binding by proteolytically generating fragments of the GPIIb/GPIIIa complex, and characterize the fragments able to bind to complex-specific hybridomas or fibrinogen. Finally, with these hybridomas we have developed an assay which has demonstrated the presence of antibodies to GPIIb/GPIIIa in the plasma of some patients with ITP. We will use this assay to characterize the frequency and specificity of such antibodies.
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