Endothelial Cell Protein C Receptor Acts as a Cellular Receptor for Factor VIIa on Endothelium

Abstract

Although factor VII/factor VIIa (FVII/FVIIa) is known to interact with many non-vascular cells, activated monocytes, and endothelial cells via its binding to tissue factor (TF), the interaction of FVII/FVIIa with unperturbed endothelium and the role of this interaction in clearing FVII/FVIIa from the circulation are unknown. To investigate this, in the present study we examined the binding of radiolabeled FVIIa to endothelial cells and its subsequent internalization. 125 I-FVIIa bound to non-stimulated human umbilical vein endothelial cells (HUVEC) in time-and dose-dependent manner. The binding is specific and independent of TF and negatively charged phospholipids. Protein C and monoclonal antibodies to endothelial cell protein C receptor (EPCR) blocked effectively 125 I-FVIIa binding to HUVEC. FVIIa binding to EPCR is confirmed by demonstrating a marked increase in 125 I-FVIIa binding to CHO cells that had been stably transfected with EPCR compared with the wild-type. Binding analysis revealed that FVII, FVIIa, protein C, and activated protein C (APC) bound to EPCR with similar affinity. FVIIa binding to EPCR failed to accelerate FVIIa activation of factor X or protease-activated receptors. FVIIa binding to EPCR was shown to facilitate FVIIa endocytosis. Pharmacological concentrations of FVIIa were found to impair partly the EPCR-dependent protein C activation and APC-mediated cell signaling. Overall, the present data provide convincing evidence that EPCR serves as a cellular binding site for FVII/FVIIa. Further studies are needed to evaluate the pathophysiological consequences and relevance of FVIIa binding to EPCR.Despite the progress that has been made in understanding the biochemistry and pathophysiology of the coagulation cascade events, the clearance mechanism of the various coagulation proteins from the circulation is still unclear. The marked differences in circulating half-lives of factor VII (FVII) 4 and FVIIa compared with those of zymogen and the enzyme forms of other vitamin K-dependent coagulation proteins (1-7) suggest that there may be a specific and distinct clearance mechanism for FVII/FVIIa. Although tissue factor (TF), the * This work was supported by Grants HL 58869 and HL 65500 from the National Institutes of Health. 4 The abbreviations used are: FVII, factor VII; FVIIa, activated factor VII; TF, tissue factor; EPCR, endothelial cell protein C receptor; APC, activated protein C; PAR, protease-activated receptor; AP, alkaline phosphatase; TFPI, tissue factor pathway inhibitor; AT, antithrombin; FACS, fluorescence-activated cell sorter; TNF, tumor necrosis factor; IL, interleukin. NIH Public Access Cell CulturePrimary human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex Bio Science (Walkersville, MD). The cells were cultured to confluency at 37 °C and 5% CO 2 in a humidified incubator in EBM-2 basal media supplemented with growth supplements (Cambrex Bio Science) and 5% fetal bovine serum. Endothelial cell passages between 3 and 10 were used in the present stud...

Factor VIIa Interaction With Tissue Factor and Endothelial Cell Protein C Receptor on Cell Surfaces

“…They also showed that blocking the EPCR binding site on the endothelial cell surface increased the generation of FXa. Further investigation is needed on this subject, as these results conflict with our own published (31) and unpublished data. At present, it is unknown whether FVIIa binding to EPCR either on non-stimulated or stimulated endothelium facilitates TFPI inhibition of FVIIa.…”

“…More FVIIa is endocytosed in cells expressing EPCR than in cells that lack EPCR. Furthermore, blocking EPCR on endothelial cells significantly reduces FVIIa internalization in HUVEC (31), suggesting that FVIIa association with EPCR facilitates FVIIa endocytosis. If this is confirmed in in vivo experiments using animal model systems, then novel therapeutic options to prolong circulatory levels of FVIIa might arise.…”

“…EPCR-FVIIa complexes neither activate FX (31) nor induce cell signaling through activation of PAR1 or PAR2 (31,36). FVIIa binding to EPCR has no influence on the formation of TF-FVIIa complexes on activated endothelial cells (31).…”

“…Our recent studies revealed that FVIIa binds specifically to non-stimulated HUVEC, and that EPCR-a known receptor for protein C/ APC-provides the binding site for FVII/FVIIa (31). FVIIa interacts with EPCR on endothelial cell surfaces with a similar affinity to that of protein C/APC (32).…”

Factor VIIa binding to endothelial cell protein C receptor

“…Studies with unstimulated HUVEC showed that FVIIa associates with endothelial cells in a time-, temperature-and dose-dependent manner. The binding is calcium dependent but independent of TF and negatively charged phospholipids [3]. The N-terminus domain that contains γ-carboxyglutamic acid (Gla) residues is essential for FVIIa binding to endothelial cells.…”