Inhibition of FXII binding to HUVECs by antibodies to CK1, uPAR, and gC1qR.
(A) Inhibition of FITC-FXII binding to HUVEC suspensions. HUVECs (4 × 104 cells per well) in suspension were incubated with 70 nM FITC-FXII in HCB containing 10 μM ZnCl2 in the presence or absence of mouse IgG (4 μg/mL), goat IgG (300 μg/mL), affinity-purified goat anti-GPV20 (300 μg/mL), mouse anti-uPAR (4 μg/mL), mouse anti-gC1qR (4 μg/mL), mouse anti–von Willebrand factor (VWF) (4 μg/mL), or mouse anti–ICAM-1 (4 μg/mL) antibodies for 2 hours at 37°C. After the incubation, the cells were washed and the bound FITC-FXII to HUVECs was determined using a CytoFluor4000 plate reader. The results presented are the means ± SEM of 3 independent experiments. (B) Inhibition of biotin-FXII binding to HUVEC monolayers. Confluent monolayers of HUVECs were washed 3 times with HCB containing 10 μM ZnCl2. Afterward, biotin-FXII (10 nM) was added to the HUVECs in the presence or the absence of increasing concentrations of mouse IgG (▴), anti-uPAR (●), or anti-gC1qR (▪) antibodies for 90 minutes at 37°C. Biotin-FXII binding to the cells was determined using ImmunoPure streptavidin horseradish peroxidase conjugate and peroxidase-specific fast-reacting substrate, turbo-TMP (see “Materials and methods”). Bound biotin-FXII was quantified by measuring the absorbance of the reaction mixture at 450 nm using a microplate autoreader EL 311 (Bio-Tek Instruments). The results presented are the means ± SEM of 3 independent experiments.
