Endothelial protein C receptor is required for tissue factor ternary complex signaling in the mouse.

The endothelial protein C receptor (EPCR) is widely recognized for its role in the activation of the anticoagulant protein C (PC) pathway [1] and as a co-receptor for activated PC-mediated protease activated receptor (PAR) 1 cytoprotective signaling [2]. EPCR also binds coagulation factor VIIa (VIIa) [3;4], regulates transendothelial delivery of therapeutic doses of VIIa [5], and supports tissue factor (TF) independent endothelial signaling by VIIa [6]. EPCR binds coagulation factor X/Xa in a strictly Mg2+-dependent manner and our recent study identified EPCR as a functional component of ternary TF-VIIa-Xa complex signaling through PAR1 and PAR2 [7]. In experimental systems consisting of human proteins, we showed with function blocking antibodies or by reconstitution into EPCR-deficient cells that EPCR is required for ternary complex signaling independent of interaction with the 4-carboxyglutamic acid-rich (Gla) domain of VIIa. Furthermore, genetic approaches in murine cells provided evidence that EPCR is necessary for ternary complex signaling initiated by murine VIIa and human X. Puy and colleagues [8] recently demonstrated species differences in the binding affinities between X and EPCR using surface plasmon resonance (SPR) experiments. Because human X bound somewhat tighter to murine EPCR than to human EPCR, this study questioned the significance of X binding to human EPCR and the biological significance of our reported signaling experiments that used human X in combination with murine cells [7]. Consistent with a previous study [9], we had confirmed that human soluble EPCR (sEPCR) attenuated human X activation by human sTF-VIIa in a lipid-free assay system [7]. As shown in Fig. 1A, this inhibitory effect of human sEPCR was reversed by monoclonal anti-human EPCR antibody 1535, known to block activated PC binding to EPCR [10], but not by the non-blocking antibody 1500. Importantly, mutagenesis of critical VIIa Gla-domain residues shown by biochemical or cellular studies to be required for binding to EPCR [3;7;9] had no effect on the inhibition of the human sTF-VIIa extrinsic Xase complex by human EPCR (Fig. 1A). These data are consistent with our prior results using murine sEPCR [7], exclude a major effect of species mismatch, and provide clear evidence for a functionally relevant interaction of human EPCR with the human ternary coagulation initiation complex in a lipid-free system. However, the effect of heterologous cellular expression of EPCR on TF procoagulant reactions is minimal [7;11], suggesting that EPCR primarily regulates other functions of the cell surface TF ternary complex. Figure 1 A, Inhibition of Xa generation by 1 μM human sEPCR [15] in the presence of human sTF/VIIa (1 μM/10 nM) was reversed by 2 μM inhibitory monoclonal anti-human EPCR antibody 1535 but not the non-inhibitory anti-human EPCR antibody ... Based on poor affinity of murine VIIa for murine sEPCR determined by SPR analysis and lack of an appreciable effect of murine sEPCR to prolong the prothrombin time in mouse plasma, Puy et al. further concluded that murine VIIa and X are not crucial ligands for murine EPCR. For the interpretation of this finding, it is important to point out that complex formation of murine VIIa with murine sTF in our hands increased the affinity for immobilized sEPCR in similar SPR experiments [7]. In addition, measurements of binary complex formation are not necessarily predictive for functionally relevant interactions in multi-subunit complexes. In this respect, substrate interactions of X with TF-VIIa can significantly stabilize the binding of the X Gla-domain with EPCR. We experimentally approached the open question whether the association of murine X with a completely species-matched murine sTF-VIIa complex was regulated by murine EPCR. Under experimental conditions previously applied to the study of human X [7], the rate of murine X activation by murine sTF-VIIa was significantly decreased in the presence of murine sEPCR (Fig. 1B). Thus, in a completely species-matched murine system sEPCR showed effects consistent with a functional interaction with the ternary complex, although the anticoagulant effects were less pronounced as seen with human proteins. We next asked whether murine EPCR supports PAR2 cleavage by murine X activated by cell surface expressed TF-VIIa. As described for human EPCR [7], CHO/TF cells expressing human TF were transfected with murine EPCR. The resulting clonal lines expressed high levels of both, TF and murine EPCR (Fig. 1C). These cells were transiently transfected with amino-terminal FLAG-tagged murine PAR2 (mPAR2) and PAR2 receptor cleavage was monitored as described [12]. In accordance with previous results in human EPCR-transfected CHO/TF cells [7], expression of murine EPCR significantly enhanced cleavage of murine PAR2 by the ternary complex assembled by adding murine VIIa and murine X (Fig. 1D). The nematode inhibitor NAPc2 stabilizes the TF-VIIa-Xa complex without blocking the catalytic activity of Xa [13]. As previously shown for human VIIa and X, NAPc2 traps newly generated Xa in a signaling active complex with TF-VIIa and abolishes the requirement for EPCR expression. This was also the case with murine proteins. Taken together, these data support the conclusion that murine EPCR is functionally equivalent to human EPCR to stabilize the signaling conformation of the ternary TF complex during initiation of coagulation in the mouse. In order to exclude the potential caveat that murine EPCR-dependent ternary complex signaling was a peculiarity of human TF expressed in CHO cells or of heterologous overexpression, we investigated ternary complex signaling in primary murine smooth muscle cells (SMC) that were isolated from lungs and express physiological levels of TF and EPCR, as described [7]. Cells from mice that carried conditional alleles of murine EPCR (Procrflox [14]) were transduced with adenovirus for Cre-mediated specific gene deletion of EPCR without reducing TF expression levels [7]. We had previously shown that protease signaling in SMC is primarily mediated by PAR1 [7]. TF ternary complex signaling induced by murine VIIa and murine X was therefore compared with the PAR1 activator thrombin in a mitogen activated protein kinase (MAPK) phosphorylation assay. Deletion of EPCR from primary SMC abolished ternary complex signaling, whereas the thrombin response was unchanged (Fig. 1E). Ternary complex signaling was abolished by the Xa-specific inhibitor NAP5, demonstrating that similar to human cells ternary complex signaling in murine cells occurs in a Xa- and EPCR-dependent manner. Collectively, the presented data suggest that binding affinities between individual components of multi-subunit complexes are poor predictors for functionally relevant interactions of cell surface-expressed EPCR. Our findings confirm in entirely species matched systems that EPCR is a required component of ternary complex signaling in humans and the mouse. These results emphasize that mouse models should be further considered as valid tools to study EPCR involvement in TF signaling pathways in vivo.