Plasminogen (Pg) is the zymogen of the fibrinolytic enzyme plasmin (Pn). Fibrin (Fn) promotes the activation of Pg by tissue-type plasminogen activator (t-PA) by serving as a template that brings t-PA and Pg into close proximity. In addition, proteolysis of Fn by Pn generates carboxyl-terminal lysine residues that provide nascent high affinity binding sites for Glu-Pg, thereby promoting Pg activation. Pg activation is also enhanced when Glu-Pg is converted to Lys-Pg, a derivative with higher Fn affinity. Therefore, kinetic and functional data suggest that Pg binding to Fn is a key aspect of efficient Pg activation. To explore this concept, we performed binding and kinetic analyses with Mini-Pg, an elastase-derived fragment of Glu-Pg with reduced Fn affinity. Binding of Glu-Pg, Lys-Pg and Mini-Pg to immobilized fibrinogen (Fg) and fibrin monomer (Fm) was monitored by surface plasmon resonance. The affinity of Glu-Pg for Fm is 4-fold higher than that for Fg with Kd values of 3.1 and 12.5 μM, respectively, whereas Lys-Pg binds with high affinity to both Fg and Fm (Kd values of 0.25 and 0.21 μM, respectively). In contrast, Mini-Pg demonstrates weak binding to Fg and Fm with Kd values of 10.5 and 24.5 μM, respectively. To complement the binding experiments, kinetic studies of Pg activation by t-PA were performed in the absence or presence of native Fn clots by monitoring Pn formation using a Pn-directed chromogenic substrate. As expected, the catalytic efficiency of Lys-Pg or Mini-Pg activation by t-PA in the absence of cofactor was higher than that of Glu-Pg (kcat/KM values of 1.3, 0.325, and 0.026 μM−1 min−1, respectively). The catalytic efficiency of Glu-Pg activation by t-PA is 500-fold higher in the presence of Fn than it is in its absence. The stimulatory effect of fibrin was maintained with Lys and Mini-Pg with over 100-fold enhancement in catalytic efficiency of activation. The fibrin-dependent increase in catalytic efficiency was expressed predominantly through a decrease in KM, with values of >20 and 0.2 μM for Glu-Pg activation in the absence and presence of fibrin, respectively. Lys and Mini-Pg also expressed similar enhancements in catalytic efficiency through a decrease in KM. Thus, despite a 100-fold range in their affinities for Fn, the activation of Mini-Pg, Glu-Pg and Lys-Pg by t-PA are all enhanced by at least 2 orders of magnitude in the presence of Fn. These results demonstrate that substrate binding to Fn is not essential for Fn-mediated stimulation of Pg activation by t-PA. To investigate the importance of the Fn-plasminogen activator interaction, activation studies were carried out using urokinase-type plasminogen activator (u-PA), an activator without Fn affinity. In contrast to the results with t-PA, Fn did not enhance u-PA-mediated activation of Glu, Lys or Mini-Pg. The lack of fibrin stimulation of Pg activation by u-PA suggests that the Pg-Fn interaction is not essential to Pg activation. Therefore, the cofactor role of Fn is expressed predominantly through interaction and stimulation of t-PA. These findings support the hypothesis that Fn binding to t-PA may expose cryptic binding sites in the activator that stabilize the formation of the enzyme-substrate complex.