FIG 8.

CspA-bound plasminogen is converted to plasmin by uPA. Microtiter plate wells were coated with 10 μg/ml purified plasminogen (PLG) (A), BSA (B), CspA LW2 (C), CspA PKo (D), or CspA A14S (E). BSA and CspA orthologs were subsequently incubated with 20 μg/ml plasminogen. Upon washing, the plasminogen activator uPA was added (2.5 μg/ml) together with the chromogenic substrate S-2251 (D-Val-Leu-Lys-p-nitroanilide dihydrochloride) (■). As negative controls, either uPA (▲) or plasminogen (●) was omitted or the plasminogen inhibitor tranexamic acid (T) (50 mM) was added to plasminogen (◆). Microtiter plates were incubated for approximately 15 h, and absorbance at 405 nm was measured at 30-min intervals. Three separate experiments were performed; data shown are from a representative experiment.