The influence of HK and PK on pro-urokinase and plasminogen activation. (A) Pro-urokinase activation. Empty microtiter plate wells or wells coated with a monolayer of endothelial cells (HUVECs) were incubated with HK (20 nmol/L) or buffer for 1 hour. Unbound HK was removed and the cells were incubated with PK (20 nmol/L) for another 1 hour and washed. Pro-UK (20 nmol/L) and 0.6 mmol/L S2444 were added to empty wells or wells coated with HUVECs and hydrolysis was monitored continuously over 75 minutes at 37°C. In one set of experiments, 0.4 mg/mL of a neutralizing antibody to FXII was added along with the PK. Formation of tcuPA was determined by comparing substrate hydrolysis on cells with known concentrations of soluble tcuPA. The data presented are the mean ± SEM of three experiments. (B) Plasminogen activation. Empty microtiter plate wells or wells coated with a monolayer of HUVECs were incubated for 1 hour with 1 μmol/L plasminogen (PLG) before 0.3 mmol/L S2251 was added either alone or in the presence of 2 nmol/L Pro-UK. In other experiments, HUVEC-coated wells were incubated for 1 hour with 20 nmol/L HK. After removal of the HK, the wells were incubated with 20 nmol/L PK for another 1 hour. After removal of the excess PK, the cells were incubated with 1 μmol/L plasminogen (PLG) for a third hour. As indicated, in one case, 0.4 mg/mL of a neutralizing antibody to FXII was added along with the PK. Hydrolysis of the substrate was measured over 210 minutes at 37°C. Plasmin formation was determined using a standard curve made by adding known amounts of purified plasmin to S2251 (see the Materials and Methods). The data shown are mean ± SEM of four independent experiments. The absence of standard error bars in some columns indicates that the variation was too little to portray visually.