performed some of the binding studies; RG conducted animal experiments; C.T.E. experimental methods. Radioligand binding studies did not show any significant difference in the binding of FX or FXa between CHO and CHO-EPCR cells (Physique 1A). Furthermore, pretreatment of CHO or CHO-EPCR cells with an EPCR blocking mAb did not diminish the basal binding of FX or FXa to these cells (data not shown). Similarly, blockade of EPCR on HUVECs with this EPCR blocking mAb did not have a significant effect on FX or FXa binding (Figure 1B). FVIIa and APC binding studies performed in parallel with FX or FXa experiments clearly demonstrated that both FVIIa and APC bound to cells to a similar degree and in an EPCR-specific manner LRIG2 antibody (Figure 1A-B). Analysis of the binding of biotinylated, active-site blocked FXa, FVIIa and APC to EPCR on CHO-EPCR and HUVECs revealed that little FXa was bound to EPCR on cell surfaces compared with FVIIa or APC (Figure 1C-D). In this assay, FVIIa and APC both bound to EPCR with an apparent Kd of 15 to 25nM, whereas the Kd for FXa was 1M. Consistent with these data that FX does not bind appreciably to EPCR, even a 100-fold molar NRA-0160 excess of unlabeled FX (1M) failed to compete effectively with the binding of 125I-FVIIa (10nM) to CHO-EPCR cells (Figure 1E). Analysis of FX binding to EPCR expressing cells by confocal fluorescence microscopy did not show any detectable fluorescence, either at the cell surface or intracellularly, in CHO-EPCR cells or HUVECs exposed to FX tagged with a fluorescence dye (AF488; Figure 1F). In additional studies, we measured plasma levels of mouse factor X and protein C in EPCR overexpressing mice that received a high dose of active-site inhibited human APC. EPCR overexpression has been found to decrease circulating levels of protein C, while administration of human protein C has been shown to increase mouse protein C levels in circulation by displacing endogenous protein C from EPCR on the endothelium.6 As expected from this study,6 administration of human APCi increased plasma levels of mouse protein C (Figure 1G). In contrast to protein C, there was not a detectable increase in plasma levels of mouse FX after APCi administration (Figure NRA-0160 1H). These data indicate that FX does not effectively interact with EPCR in vivo, at least in regards to the mouse system. Overall our data indicate that FX binding to EPCR, if any, is minimal and likely physiologically insignificant. Our findings do not exclude the possibility that FX/FXa could indirectly interact with EPCR as suggested by others.7 Open in a separate window Figure 1 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 4C. At the end of incubation, cells were washed NRA-0160 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 () or FXa () in Ca2+/Mg2+ containing buffer for 3 hours at 4C. 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 NRA-0160 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.