Binding of factors X, Xa, VIIa, and APC to EPCR expressing cells. (A) CHO cells (black bars) or CHO cells stably transfected to express human EPCR (CHO-EPCR; gray bars) were incubated with ¹²⁵I-labeled human FVIIa (10nM and 50nM), human APC (50nM), human FXa (150nM), or human FX (150nM) in HEPES buffer (10mM HEPES, 0.15M NaCl, 4mM KCl, and 11mM glucose) containing CaCl₂ (5mM), MgCl₂ (1mM), and BSA (1 mg/mL) for 3 hours at 4°C. At the end of incubation, cells were washed 4 times with the same buffer and surface bound ligands were eluted with glycine (100mM, pH 2.3) and counted for radioactivity to determine the amount of ligand bound to the cells. (B) Same as panel A except that CHO and CHO-EPCR cells were replaced with HUVECs pretreated with EPCR blocking mAb (black bars) or control vehicle (gray bars), respectively. (C) CHO or CHO-EPCR cells were incubated with various concentrations of biotinylated active site-blocked FVIIa (□), APC (Δ) or FXa (○) in Ca²⁺/Mg²⁺ containing buffer for 3 hours at 4°C. After washing the cells to remove unbound ligands, the surface bound ligands were detected by fixing the cells and adding alkaline phosphatase coupled streptavidin followed by BluePhos phosphatase substrate system (KPL). EPCR-specific binding was calculated by subtracting the absorbance measured in CHO cells from that of CHO-EPCR cells. Biotinylated active site-inhibited FXa, FVIIa, or APC were prepared by incubating FXa, FVIIa, or APC with 10-fold molar excess of biotinylated EGR for 3 hours at room temperature and excess probe was removed by dialysis. (D) Same as panel C except that CHO and CHO-EPCR cells were substituted with HUVECs pretreated with EPCR blocking mAb or a control vehicle, respectively, and EPCR-specific binding was calculated by subtracting the absorbance obtained with HUVECs pretreated with EPCR blocking antibody from HUVECs not treated with the antibody. (E) CHO-EPCR cells were incubated with ¹²⁵I-FVIIa (10nM) in the presence of various concentrations of unlabeled competitors, FIX (◇), prothrombin (PT, ▿), FX (○), FVIIa (□), APC (Δ) for 3 hours at 4°C. The surface bound ¹²⁵I-FVIIa was eluted with low pH glycine and counted for radioactivity. (F) HUVECs, treated EPCR blocking mAb (25 μg/mL) or control vehicle (top panel) or CHO-EPCR cells (bottom panel) were exposed to FVIIa (50nM for HUVECs; 10 and 50nM for CHO-EPCR cells), APC (70nM) or FX (175nM) conjugated with fluorescent probe AF488 for 1 hour at room temperature. At the end of 1 hour, cells were washed quickly and immunostained with nonblocking EPCR mAb. Images through z-axis were acquired using LSM 510 Meta confocal system (Carl Zeiss) and reconstructed to 3D composite images. (G,H) EPCR overexpressing mice were injected intravenously with active site-inhibited human APCi (400 μg/mice) and blood samples were drawn retroorbitally pre- and postadministration of APCi. Mouse protein C level in plasma was measured using mouse protein C–specific ELISA (G) and mouse FX level was measured in a FX specific clotting assay (H).