Inhibition of biotin-HK binding and PK activation on HUVECs by antibodies to kininogen HUVEC membrane-binding proteins.

(A) HUVECs were incubated with biotin-HK (7 nM) with increasing concentrations of goat anti-GPV20 (●) and goat IgG (s) for 1 hour at 37°C. HUVECs were also incubated with biotin-HK (7 nM) and various concentrations of mouse anti-uPAR (■), mouse anti-gC1qR (⧫), or mouse IgG (t) for 1 hour at 37°C. After incubation, the level of bound biotin-HK was determined. (B) HUVECs were incubated with HK (20 nM) and increasing concentrations of goat anti-GPV20 (●) or goat IgG (s) for 1 hour at 37°C. HUVECs were also incubated with HK (20 nM) and various concentrations of mouse anti-uPAR (■), mouse anti-gC1qR (⧫), or mouse IgG (t) for 1 hour at 37°C. At the conclusion of incubation, the cells were washed and incubated with PK (20 nM) for an additional hour at 37°C. The level of membrane-associated kallikrein was determined by the addition of the chromogenic substrate H-D-Pro-Phe-Arg-pNA (0.8 mM) as described in “Materials and methods.” The data are mean ± SEM of triplicate determinations from 3 different experiments.