HK (70 nmol/L) was preincubated with HUVEC for 60 minutes at 4°C, and then excess HK was removed. The complete system for the generation of plasmin consists of HEPES-BSA buffer, pH 7.4, Zn²⁺ (50 μmol/L), HUVEC-bound HK, PK (50 nmol/L), plasminogen (60 nmol/L), and pro-UK (4.5 nmol/L) and the plasmin substrate (H-D-Val-Leu-Lys-amino methyl coumarin, 50 μmol/L). The resulting solution was incubated for 46 minutes at 23°C. The fluorescence emission intensity was recorded at 450 nm (λ_excit = 360 nm) and used to calculate the amount of substrate hydrolyzed. HEPES-BSA buffer containing ZnCl₂ (50 μmol/L) was used for all experiments.

PK contains 1% to 2% kallikrein.