We examined the effects of glycoprotein IIb/IIIa (GPIIb/IIIa) antagonists c7E3 Fab and DMP728 on the development of platelet prothrombinase (PT) activity. c7E3 Fab dose-dependently inhibited the rate of thrombin-stimulated thrombin generation over a 1-minute reaction time. The IC50 was 11 nM with an IC90 of 1000 nM. DMP728 inhibited PT activity maximally by 60% at 100 nM. A similar profile was observed for the inhibition of platelet tenase activity. Inhibition was platelet specific up to approximately 200 nM c7E3 Fab. Above 200 nM, inhibition was platelet independent, as shown by the inhibition of activity assembled on PS/PC vesicles. c7E3 Fab and DMP728 did not inhibit calcium ionophore-induced activity. DMP728 potency diminished with reaction time (over 6 minutes) whereas c7E3 Fab potency did not. Inhibition by 2 microM DMP728 was not further increased by 20 nM c7E3 Fab. Heparin inhibition of platelet PT activity was additive to that of c7E3 Fab. Studies with added von Willebrand factor (vWf) indicate that in the context of thrombin activation vWf activates platelets through mechanisms independent of GPIIb/IIIa to promote PT activity. Thrombin activation induced binding of FITC-Annexin V to a subpopulation of platelets which was reduced by approximately 50% by pretreatment with either c7E3 Fab or DMP728. Together, these data indicate that c7E3 Fab and DMP728 inhibit the development of GPIIb/IIIa-mediated platelet PT activity at events during platelet activation. The inhibitory activities are not additive, suggesting these agents compete for the same site or inhibit via the same mechanism. Inhibition accompanies a reduction in the number of phosphatidylserine binding sites, implying that GPIIb/IIIa receptor antagonists reduce platelet membrane scrambling induced by thrombin. The additivity of inhibition with heparin by c7E3 Fab suggests a combination of these agents might have a greater bleeding liability than the use of either agent alone.