To evaluate the effect of thrombin on the dynamics of thrombolysis, we infused rabbits with heparin or hirudin alone or in conjunction with tissue-type plasminogen activator (t-PA) and monitored the kinetics of fibrinolysis and changes in ex vivo platelet aggregation responses over time. Both heparin and hirudin enhanced total fibrinolysis in an ex vivo arteriovenous shunt preparation: 82±2% and 79±2%, respectively, compared with 51±8% for t-PA alone (p<0.05) and 50±4% for t-PA plus aspirin (p<0.05). Heparin coadministered with t-PA significantly reduced the half-time for clot lysis compared with t-PA alone (p<0.05), whereas hirudin coadministered with t-PA significantly reduced the half-time for clot lysis compared with that for t-PA alone, t-PA plus aspirin, and t-PA plus heparin (5.5±0.6 versus 12.1±2.0 versus 12.6±2.2 versus 10.0±0.8 minutes, respectively; p<0.05). Both heparin and hirudin prevented the increase in ADP-induced platelet aggregation normally seen with t-PA alone (p<0.01 byttest; p<0.05 by two-way analysis of variance). These data demonstrate that selective, antithrombin III-independent thrombin inhibitors can enhance the efficacy of thrombolysis by modulating the dynamics of the process and preventing platelet activation associated with plasminogen activator therapy.