Figure 4

Plasminogen-binding and activation and fibrin degradation assays. (A) Paracoccidioides yeast cells and the rFBA were incubated with hPlg in the presence or absence of tPA and εACA. We used a plasmin substrate (D-valyl-L-lysyl-p-nitroaniline hydrochloride) (Sigma-Aldrich) to dose the amidolytic activity of the reaction of converting plasminogen into plasmin. (B) In competition experiments we added to the wells increasing concentrations of εACA (50 mM to 1 M), followed by the addition of hPlg. Experiments were performed in triplicate as described in Materials and Methods. The error bars indicate the standard deviations between the results. *: results significantly different from control, at a p value < 0.05. (C) The fibrinolytic activity of Paracoccidioides was analysed by the observation of clear hydrolysis haloes within the opaque jellified-fibrin-containing matrix. Panel 1: Paracoccidioides yeast cells in the absence of hPlg; 2: the fungus after binding to hPlg; 3: Similar to 1 and 2, but reflecting the presence of tPA. The fungus was incubated in the presence of hPlg and tPA, with the addition of anti-rFBA (panel 4) and proteases inhibitors, aprotinin and PMSF (panels 5 and 6). Controls consisting of plasminogen and tPA (panel 7).