Regulation of FXII binding to HUVECs.
(A) Inhibition of FITC-FXII binding to HUVEC suspensions by HK or vitronectin. HUVECs (4 × 104 cells per well) in suspension were incubated in HCB, pH 7.4, containing 10 μM Zn2+ with 70 nM FITC-FXII in the presence or absence of 1- to 100-fold molar excess of HK (●), FXII (▪) (10-7000 nM), urokinase (uPA) (▴) (10-3500 nM), or vitronectin (▾) (10-3500 nM) for 2 hours at 37°C. At the conclusion of 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) The influence of albumin on biotin-FXII binding to HUVEC monolayers. Washed human platelets (0.1 × 109 to 3.5 × 109/mL) in HCB containing 3.5 mg/mL bovine serum albumin and 50 μM Zn2+, pH 7.4, were incubated over washed HUVEC monolayers in microtiter plate cuvette wells in the same buffer in the presence of 10 nM biotin-FXII and the presence or absence of 5 μg/mL collagen for 90 minutes at 37°C on a rotating shaker. At the completion of the incubation, the microtiter plate cuvette wells were washed and the amount of biotin-FXII specifically bound to the HUVECs was determined as described in the legend to Figure 1. The data are presented as a bar graph of specific biotin-FXII binding determined by subtracting the level of binding seen with unactivated platelets from that occurring after collagen-activated platelets. The column “Biotin-FXII + Zn2+” represents the level of specific biotin-FXII binding alone to HUVEC monolayers in HCB, pH 7.4, and 10 μM Zn2+. These data are the mean ± SEM of 5 individual experiments.
