Biotin-FXII binding to HUVEC monolayers.

(A) HUVEC monolayers (4 × 10⁴ cells per well) were incubated with 10 nM biotin-FXII for 0 to 180 minutes at 37°C in the presence (●) or absence (▪) of 10 μM Zn²⁺ or in the presence of 10 μM Zn²⁺ and 200-fold molar excess of unlabeled FXII (▴). (B) HUVEC monolayers (4 × 10⁴cells per well) were incubated with 10 nM biotin-FXII for 0 to 180 minutes at 37°C in HEPES carbonate gelatin buffer (HC-Gelatin) (●), HCB containing 40 mg/mL bovine serum albumin (HC-BSA) (▪), or FXII-deficient serum (▴) in the absence or presence of increasing concentrations of Zn²⁺ (0.1-3000 μM). (C) HUVEC monolayers (4 × 10⁴ cells per well) were incubated with increasing concentrations of biotin-FXII (1-70 nM) for 90 minutes at 37°C in HEPES carbonate gelatin buffer in the presence (●) or absence (▪) of 10 μM Zn²⁺. The specific binding curve (▴) is calculated by subtracting binding in the absence of added Zn²⁺ from that in the presence of Zn²⁺. 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.