Determination of a region on FXII that binds to HUVECs.
(A) Inhibition of FITC-FXII binding to HUVEC suspensions by FXII peptides. HUVECs in suspension were incubated with 70 nM FITC-FXII in HCB containing 10 μM ZnCl2 in the absence or presence of 100 μM of peptides YHK9, GYK9, FPF9, IPP19, or HKH20 for 2 hours at 37°C. After the incubation, the cells were washed and the bound FITC-FXII to HUVECs was monitored 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 by FXII peptides. Confluent monolayers of HUVECs were washed 3 times with HCB containing 10 μM ZnCl2. Biotin-FXII (10 nM) was added to HUVECs in the absence or presence of increasing concentrations of peptides YHK9 (●), FPF9 (▪), or IPP19 (▴) for 90 minutes at 37°C. After the incubation, the cells were washed and the relative binding of biotin-FXII binding to cells was determined using ImmunoPure streptavidin horseradish peroxidase conjugate followed by peroxidase-specific fast-reacting substrate, turbo-TMP. Bound biotin-FXII was quantified by measuring the absorbance of the reaction mixture at 450 nm using a microplate autoreader EL 311. The results presented are the means ± SEM of 3 independent experiments.
