Reteplase (r-PA), a truncated variant of tissue-type plasminogen activator (t-PA) consisting of the second kringle and protease domains, is now used for treatment of acute myocardial infarction (AMI). At equally effective doses, r-PA causes more fibrinogen (Fg) consumption than t-PA. Recently, we demonstrated that Fg stimulates t-PA-mediated plasminogen (Pg) activation, albeit weakly, because t-PA binds Fg via its second kringle domain. In this study, we set out to determine whether kringle-dependent interactions contribute to Fg consumption induced by r-PA. The fibrinolytic potentials of r-PA and tPA were compared by incubating I25l-labeled plasma clots with clinically relevant concentrations of r-PA or t-PA. After 1 h, residual clot radioactivity was used as a measure of thrombolysis. Three-fold higher concentrations of r-PA are required to produce the same clot lysis as t-PA. Binding of r-PA or t-PA to fibrin was measured to determine if the reduced fibrinolytic potential of r-PA reflects lower affinity for fibrin. Whereas t-PA binds fibrin via one high affinity, finger-dependent site (Kd = 0.050 jiM) and one low affinity, kringledependent site (Kd = 2.5 nM), r-PA binds fibrin through a single class of low affinity, kringle-dependent sites (Ka = 0.92 (iM). The fibrinogenolytic activities of r-PA and t-PA were compared by incubating various concentrations of r-PA or t-PA in plasma for l h and measuring levels of Bβl-42, a sensitive marker of plasmin-mediated Fg degradation. At concentrations that produce equivalent clot lysis, Bβl-42 generation with r-PA is up to 14-fold greater than that with t-PA. A detailed kinetic analysis on the effect of Fg on GluPg activation by r-PA or t-PA was performed. In the presence of Fg, the catalytic efficiency (k 4/KM) of r-PA-mediated Pg activation is 35-fold greater than in its absence (1.8 x 102 and 5.1 x 104 nM 's ', respectively). Likewise, Fg increases the catalytic efficiency of t-PAmediated Pg activation 30-fold (from 9.0 x 10A to 2.9 x 10J JiM-'s'1). Binding of r-PA or tPA to Fg was measured using light scattering spectroscopy. r-PA and t-PA bind Fg with similar affinities (Ka = 0.87 and 0.80 (iM, respectively) and their interactions are kringledependent. With similar affinities for Fg, both activators are stimulated to the same extent by Fg. However, 3-fold higher concentrations of r-PA are needed to effect the same thrombolysis as t-PA because r-PA has lower affinity for fibrin. Together, these data explain why r-PA produces more Fg consumption than t-PA when used at doses that are equally effective for AMI treatment.