PAI-749 is a small molecule inhibitor of PAI-1 with proven antithrombotic efficacy in several preclinical models. PAI-1 inactivation by PAI-749 using purified components can result from a dual mechanism of action. First, PAI-749 binds directly to PAI-1, blocks PAI-1 from accessing the active site of tPA, and abrogates formation of the SDS-stable tPA/PAI-1 complex. Second, binding of PAI-749 to PAI-1 renders PAI-1 vulnerable to plasmin-mediated proteolytic degradation. Despite its efficacy in a purified human system and in preclinical models of thrombosis, the ex vivo clinical study suggests that PAI-749 does not affect thrombus formation or fibrinolysis in a range of established human plasma and whole blood-based systems.