Purpose: Graft procoagulant activity is determined by thrombin (IIa) and activated factor X (Xa) that binds to thrombus. Thrombus-associated factor Xa and thrombin are resistant to antithrombin III-dependent therapy (heparin). To avoid complications and costs associated with systemic administration, we evaluated whether locally applied antithrombotic agents inhibit prosthetic graft procoagulant activity under no-flow and low-flow conditions. Methods: Four-millimeter-diameter collagen-coated grafts were preclotted in recalcified human plasma, washed, immersed in antithrombotic agents (either 100 nm hirudin, 20 μm D-Phe-L-Pro-L-Arg chloromethylketone, 5 μm tick anticoagulant peptide or 5 or 10 μg/ml tissue factor pathway inhibitor) or saline solution, and extensively rewashed. Grafts were exposed to recalcified plasma either in multiwell plates or underwent perfusion at 1 ml/min flow rate. Fibrinopeptide A, which reflects fibrin elaboration, was measured as a marker of thrombin activity. Results: Inhibitors reduced fibrinopeptide generation at 8 minutes by 55% (tissue factor pathway inhibitor), 57% (hirudin), or 63% (tick anticoagulant peptide and D-Phe-L-Pro-L- Argchloromethylketone) compared with the control agents (p < 0.05). Under low-flow conditions tissue factor pathway inhibitor and hirudin reduced fibrinopeptide generation at 13 minutes by 61% and 49%, respectively, when compared with control agents (p < 0.05). Conclusions: Graft-associated inhibitors targeted at factors IIa, Xa, or tissue factor/VIIa/Xa complex effectively reduce procoagulant activity on prosthetic grafts. The success of local application of antithrombotic agents in attenuating early fibrin formation suggests that this strategy could favorably influence acute graft patency, and we speculate these agents may improve long-term graft patency as well.