Progression of arterial thrombosis partly depends on thrombus-associated thrombin and activated factor X (Xa) activity. However, whether Xa or thrombin is the most appropriate target for inhibition of recurrent thrombosis is unknown. This study was designed to determine whether inhibition of Xa results in more sustained attenuation of thrombus-associated precoagulant activity than does inhibition of thrombin. Clots prepared ex vivo from human whole blood and pathological arterial thrombi from patients were preincubated in citrated plasma containing no inhibitor, 0.5 to 1 U/mL heparin, 0.5 to 1 mumol/L hirudin, 5 to 10 mumol/L tick anticoagulant peptide (TAP), 0.15 to 3 mumol/L tissue factor pathway inhibitor (TFPI), or a combination of 1 mumol/L hirudin and 10 mumol/L TAP for 2 hours. After preincubation the clots were removed from first-stage plasma, extensively washed in phosphate-buffered saline, and added to nonanticoagulated whole blood. Clots preincubated in plasma without inhibitors induced marked activation of the coagulation system in whole blood, as characterized by greater increases in the concentration of fibrinopeptide A (FPA) over 7 minutes than in blood without added clots (1522 +/- 568 compared with 117 +/- 170 ng/mL, P < .01). Preincubation of clots with heparin or hirudin did not attenuate the increases in FPA in wholeblood. In contrast, compared with incubation without an inhibitor, preincubation of clots with TAP or TFPI markedly attenuated the increases in FPA when clots were added to whole blood (551 +/- 316 and 508 +/- 208 ng/mL, respectively, P < .01). Similar results were obtained with arterial thrombi from patients. Inhibition of Xa but not of thrombin results in sustained attenuation of thrombus-associated procoagulant activity. Uninhibited thrombus-associated Xa activity may account for the increases in thrombin activity that are commonly observed in clinical trials after discontinuation of thrombin inhibitors.