Factor X binding to endothelial cell protein C receptor: comparison with factor VIIa and activated protein C

To the editor:

Endothelial cell protein C receptor (EPCR) is the cellular receptor for protein C (PC) and activated protein C (APC). Studies from our laboratory¹  and others^2,3  have shown that FVII/FVIIa also binds to EPCR in a true-ligand fashion. Recently, it has been suggested that FX⁴  and FXa⁵  can bind to EPCR and these interactions may play a role in regulating TF-FVIIa-FXa– or FXa-mediated cell signaling via activation of protease activated receptors (PARs). This reported observation that FX/FXa binds to EPCR is somewhat surprising given that the specific Gla domain residues of protein C necessary for molecular recognition of EPCR are not completely conserved in human FX.²  As reported recently,⁵  if FXa binds to EPCR with an affinity similar to that of APC binding to EPCR, then this interaction may not only influence the activation of FX and FXa-mediated cell signaling, but could also modulate EPCR-dependent functions of APC and FVIIa by competing with APC and FVIIa for EPCR binding.

To evaluate the physiologic relevance of FX binding to EPCR, we have reexamined FX binding to EPCR on endothelial as well as CHO-EPCR cells using plasma concentrations of FX and multiple experimental approaches. Radioligand binding studies did not indicate any significant difference in the binding of FX or FXa between CHO and CHO-EPCR cells (Figure 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 (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 > 1μM. Consistent with these data that FX does not bind appreciably to EPCR, even a 100-fold molar excess of unlabeled FX (1μM) 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 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