PAI-749, [1-Benzyl-3-pentyl-2[6-(1H-tetrazol-5-ylmethoxy)naphthalen-2-yl]-1H-indole], is a potent, selective, and reversible PAI-1 antagonist that preserved tPA and uPA activities in the presence of PAI-1 (EC50 values are 156 and 86 nM, respectively). PAI-749 quenched the fluorescence (Fl) signal of fluorophore-tagged PAI-1 (PAI-NBD119) (apparent Kd ~ 254 nM). Analogs of PAI-749 displayed the same potency rank order for neutralization of PAI-1 inhibitory activity and perturbation of the PAI-NBD119 Fl signal. These data establish that (a) PAI-749 bound directly to PAI-1 and (b) the binding event altering the Fl signal of PAI-NBD119 was linked to PAI-1 inactivation. PAI-749 abolished formation of the SDS-stable tPA/PAI-1 complex and induced PAI-1 polymerization. PAI-749 does not appreciably turn PAI-1 into a substrate for tPA; however, PAI-749 promotes plasmin-mediated degradation of PAI-1. In the FeCl3 rat model of arterial injury, oral dosing of PAI-749 significantly delayed the time to carotid artery thrombosis. PAI-749 when dosed orally was also efficacious in the rat venous thrombosis model, significantly reducing FeCl3-induced thrombus weight in the vena cava. PAI-749 had no effect on tail bleeding time or thrombin clotting time in the rat. Orally-dosed PAI-749 significantly prolonged time to coronary occlusion and elicited spontaneous reperfusion in a canine model of electrolytic injury-induced thrombosis. The observed antithrombotic efficacy of PAI-749 may arise from a dual mechanism of action: (a) PAI-749 binds directly to PAI-1, triggers PAI-1 polymerization, and blocks the ability of PAI-1 to inactivate tPA and (b) binding of PAI-749 to PAI-1 makes PAI-1 vulnerable to plasmin-mediated proteolytic degradation. Both of these effects should work in concert to exert an antithrombotic effect and maintain vessel patency at sites of vascular injury.