At present, it is debatable whether FVIIa interaction with EPCR alters FVIIa’s coagulant activity. Ghosh et al. [3
] showed that FVIIa binding to EPCR on the endothelial cell surface had no influence on FVIIa’s coagulant activity, suggesting that EPCR, unlike TF, fails to induce the necessary allosteric conformational changes in FVIIa. However, Lopez-Sagaseta et al. [18
] reported a detectable influence of sEPCR on the amidolytic activity of FVIIa, suggesting that FVIIa may undergo a structural rearrangement involving its active site. It had also been reported that sEPCR reduced the ability of FVIIa-sTF to activate factor X, and the EPCR expressed on the endothelial cell surface down-regulated TF-FVIIa activation of factor X at the cell surface [18
]. These observations were somewhat surprising as FVII/FVIIa binds to TF with a much higher affinity than it binds to EPCR, and the levels of TF and EPCR on endothelial cells would be very limited. Under these conditions, FVIIa binding to EPCR should not impede FVIIa binding to TF.
APC binding to EPCR has been shown to provide cytoprotective activity through PAR1-mediated cell signaling. There is no evidence at present that EPCR-FVIIa activates PAR1 or PAR2. FVIIa binding to EPCR failed to cleave PAR1 or PAR2 reporter constructs in which alkaline phosphatase was fused to the N-terminus of PARs to monitor the cleavage [3
]. Unlike APC, FVIIa failed to activate p44/42 MAPK in CHO cells expressing EPCR [3
]. Recent studies of Bae et al. [26
] also suggested that FVIIa binding to EPCR does not induce PAR1-mediated cell signaling as it failed to prevent thrombin-induced cell permeability. However, these studies alone do not completely rule out a role for EPCR-FVIIa in cell signaling. Emerging evidence from various groups suggests that the effects of various coagulation proteases on PAR-mediated cell signaling are complex and multifaceted; even undetectable amounts of PAR activation may lead to robust cellular responses [27
]. Moreover, the mode of FVIIa-EPCR-induced PAR-mediated cell signaling may differ from that of other proteases. Therefore, further studies are needed to draw a firm conclusion on whether FVIIa binding to EPCR has any relevance for cell signaling.
FVII/FVIIa interaction with EPCR could play an important role in the clearance of FVII/FVIIa from the circulation and/or transport of FVIIa to extravasculature. FVIIa bound to EPCR on endothelial cells was shown to be endocytosed [3
]. However, since a majority of the internalized FVIIa appeared to recycle back to the cell surface [3
], it is unlikely that EPCR-mediated FVIIa internalization could account fully for the FVIIa clearance. Consistent with this notion, blocking the interaction of FVIIa with EPCR by EPCR blocking antibodies only delayed the clearance of FVIIa, particularly in the early phase, but did not prevent the clearance [22
]. Recent in vitro
and in vivo
studies suggest that EPCR-mediated endocytosis/recycling could facilitate the transport of FVII/FVIIa from the lumen of blood vessels to the sub-endothelial space [22
]. FVIIa administered to mice, immediately following the administration, was found to be associated with the endothelial lining of large blood vessels. Within one hour, FVIIa bound to the endothelium was transferred to the perivascular tissue surrounding the blood vessels and thereafter diffused throughout the tissue. This could facilitate FVII/FVIIa coming into contact with TF in the absence of vascular injury. Trace formation of TF-FVIIa complexes in the sub-endothelial space may prime TF-dependent blood coagulation or propagate basal TF-FVIIa signaling. Matrix associated proteins that are induced by TF-FVIIa signaling [28
] may play a role in maintaining vascular integrity [27
]. Interestingly, neither FVIIa binding to EPCR nor transport of FVIIa across the endothelium was evident in lung and brain [23
], the tissues that are very rich with TF and where thrombin generation could have devastating thrombotic effects. However, recent studies demonstrated EPCR-assisted transport of APC across the mouse blood-brain barrier [29
]. It is unclear at present whether real differences exist between EPCR-mediated transport of FVIIa and APC, or if variations in the experimental designs/detection techniques contributed to these differences.
Recombinant FVIIa is widely used for treatment of bleeding episodes in hemophilia patients with inhibitors. The recommended dose of rFVIIa for the treatment varies from 90 to 270 μg/kg [30
]. Under these therapeutic conditions, it is conceivable that FVIIa levels in plasma could reach close to protein C levels, resulting in effective competition between protein C and FVIIa for limited EPCR sites on the endothelium. Reduced protein C binding to EPCR in the presence of pharmacological doses of FVIIa would lead to reduced APC generation, as protein C association with EPCR is crucial for its activation by thrombin-thrombomodulin [11
]. Experiments performed with cell model systems in which therapeutic concentrations of FVIIa were shown to inhibit thrombin/thrombomodulin-mediated APC generation support this hypothesis [3
]. Down-regulation of APC generation by FVIIa allows FVa to continue to support factor Xa activation of prothrombin in hemophilia leading to desired thrombin generation. This mechanism may contribute to the lasting hemostatic effect afforded by rFVIIa therapy in hemophilia (). It will be interesting to test the validity of this hypothesis in the future.
Fig. 1 A potential role for EPCR and FVIIa interaction in augmenting the hemostatic effect of rFVIIa in hemophilia. Pharmacological doses of FVIIa can bind weakly to activated platelets and directly activate factor X to factor Xa. Factor Xa upon forming complex (more ...)