Figure 2:

Miropin is an efficient inhibitor of human plasmin as indicated by its stoichiometry of inhibition (SI) (A), association rate constant (k_ass) (B), and ability to inhibit the fibrinolytic activity of plasmin (C). A) Plasmin was preincubated with increasing miropin concentrations for 15 min, and the residual enzymaticactivity was measured and plotted against the miropin:plasmin molar ratio. B) Plasmin was added to mixtures 6 containing a constant amount of substrate and increasing miropin concentrations. Changes in fluorescence (RFU) were then recorded. Based on progress curve analysis, the k_obs values were plotted as a function of the miropin concentration, and the k_ass was determined from the slope of the linear curve fit to the data points and corrected for the stoichiometry factor and K_M. C) α₂AP and miropin were added to thrombin-induced fibrin mesh, and the plasmin-mediated clot degradation was monitored by measuring the absorbance at 350 nm. The miropin K368A mutant (miropin^K368A), which is inactive against plasmin, was used as a control. The results presented (A, B) are the mean ± SD.