Interaction of Factor VIIa with Endothelial Cell Protein C Receptor: Differences Between the Human and Mouse Systems

Abstract 1181

Recent studies from our laboratory and others have shown that both the zymogen and activated form of FVII bind to endothelial cell protein C receptor (EPCR), a cellular receptor for protein C and activated protein C (APC). Although at present the pathophysiological significance of this interaction is unclear, recent studies indicate that FVIIa binding to EPCR may facilitate FVIIa transport from blood to extravasculature, mediate FVIIa-induced cell signaling and provide endothelial barrier protection. However, at present, there is no direct evidence demonstrating FVIIa actually associates with EPCR in vivo. Further, the Gla region involved in the EPCR binding is not fully conserved in mouse FVII, which raises questions on the ability of mouse FVIIa to associate with EPCR and the validity of mouse model systems in investigating the importance of the interaction between FVIIa and EPCR. Thus, it is important to first characterize the interaction of mouse FVIIa with mouse EPCR before employing murine model systems to investigate the importance of FVIIa interaction with EPCR in hemostasis and inflammation. Measurement of plasma levels of FVII, using both clotting and antigen assays, in wild-type, EPCR-deficient and EPCR-over expressing mice showed a small but insignificant increase (∼10%) in the circulating levels of FVII in EPCR-deficient mice and a ∼15% reduction in EPCR-over expressing mice in comparison to plasma FVII levels in wild-type mice. In comparison, the plasma level of protein C was decreased by more than 60% in EPCR-over expressing mice. Infusion of high concentrations of either human APCi or human FVIIai (400 μg/mice) to EPCR-over expressing mice failed to displace and increase the plasma levels of endogenous mouse FVII, while they increased plasma levels of endogenous mouse protein C by 2–3-fold. In additional studies, either mouse FVIIa or human FVIIa (120 μg/kg, tagged with AF488 probe) was administered exogenously via tail-vein to the wild-type, EPCR-deficient and EPCR-over expressing mice and FVIIa association with EPCR was evaluated by immunohistochemistry using anti-AF488 antibodies. In the case of mice injected with human FVIIa, the staining of FVIIa was undetectable or negligible in EPCR-deficient mice, whereas visible staining for FVIIa was clearly observed on the endothelium lining of the vessel walls in the wild-type mice. Furthermore, very intense staining (more so than wild-type) of FVIIa was observed on the endothelium of EPCR-over expressing mice. Although exogenously infused mouse FVIIa also appears to associate with the endothelium, there are no discernable differences in the staining intensity among wild-type, EPCR-deficient and EPCR-over expressing mice, suggesting that mouse FVIIa association with the endothelium is unaffected by EPCR. Next, in vitro binding studies were performed using mouse and human ligands and EPCR to further evaluate differences between binding of human and mouse ligands to EPCR. Surface Plasmon Resonance binding studies showed negligible binding of mouse FVIIa to either soluble human EPCR or mouse EPCR immobilized on the sensor chip. Under identical experimental conditions, human FVIIa was shown to bind both human and mouse EPCR. Experiments conducted with mouse and human endothelial cells or CHO cells transfected to express mouse or human EPCR also showed that mouse FVIIa does not bind, in any appreciable amount, to mouse EPCR and binds very poorly to human EPCR. Conversely, human FVIIa was found to bind to both human and mouse EPCR with a similar efficiency, both of which were approximately equivalent to that of human APC binding to EPCR. Interestingly, the binding of human FVIIa and APC to mouse EPCR is more dependent on Mg²⁺ ions as compared to their binding to human EPCR, indicating that differences between human and murine EPCR may also contribute to the differential binding of human and mouse ligands to EPCR. In summary, our data indicate that significant differences exist between human and mouse FVIIa in their ability to interact with EPCR. Given that human but not mouse FVIIa binds to EPCR, it may be more appropriate to use human FVII/FVIIa in mouse model systems to properly investigate the importance of FVIIa interaction with EPCR in hemostasis and inflammation.