Analysis of tPA-catalyzed plasmin digestion of fibrin and tPA-mediated plasmin generation on fibrin surfaces. (A) Normal or patient fibrinogen was incubated with plasminogen, tPA, FXIII, and thrombin in the presence of calcium. After the indicated time periods, samples were heated at 98°C in the presence of 2% SDS and 10 mM DTT for 5 minutes to terminate the reaction and then analyzed by SDS-PAGE. (B) Normal fibrinogen (•) or patient fibrinogen (○) was incubated with plasminogen, tPA, FXIII, thrombin, and S-2251 in the presence of 2 mM calcium. As a control, normal fibrinogen (▴) was incubated in buffer containing plasminogen, tPA, and S-2251 or in buffer containing plasminogen, tPA, and thrombin. The changes in absorbance at 405 nm were monitored. Background absorbance determined with samples incubated in the absence of S-2251 at 405 nm were subtracted from all values.