Plasminogen activation by longistatin.

(A) Longistatin (40, 80, 160, 320 and 640 nM) was incubated without or with plasminogen (0.24 units) adding fibrin CNBr fragments (4 µg) in a total volume of 200 µl of buffer (50 mM Tris–HCl, pH 7.5; 100 mM NaCl and 5 mM CaCl₂) at 25°C for 2 h. Then, plasmin-specific fluorogenic substrate (100 µM, final concentration) was added and substrate hydrolysis was monitored by measuring excitation and emission wavelengths of 360 nm and 460 nm, respectively, at 15 min intervals. Inset, initial rate of plasminogen activation at different concentrations of longistatin. (B) Effects of fibrin CNBr fragments on the activation of plasminogen by longistatin. Plasminogen (0.24 units) was incubated with longistatin (640 nM) in the absence or presence of fibrin CNBr fragments (0.25, 1 and 4 µg) as described in Materials and Methods. All assays were performed in triplicate.