Factor X and factor VII binding to endothelial protein C receptor differs between species

Authors

  • C. PUY,

    1. Division of Cardiovascular Sciences, Laboratory of Thrombosis and Haemostasis, Centre for Applied Medical Research, University of Navarra, Pamplona, Spain
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  • J. HERMIDA,

    1. Division of Cardiovascular Sciences, Laboratory of Thrombosis and Haemostasis, Centre for Applied Medical Research, University of Navarra, Pamplona, Spain
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  • R. MONTES

    1. Division of Cardiovascular Sciences, Laboratory of Thrombosis and Haemostasis, Centre for Applied Medical Research, University of Navarra, Pamplona, Spain
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Ramón Montes, Laboratory of Thrombosis and Haemostasis, Division of Cardiovascular Sciences, Centre for Applied Medical Research (CIMA), University of Navarra, Avenida Pío XII 55, 31008 Pamplona, Spain.
Tel.: +34 948194700; fax: +34 948194716.
E-mail: rmontes@unav.es

The endothelial protein C receptor (EPCR) has gained attention due to its involvement in the protective mechanisms exerted by activated protein C (APC). Recently, the coagulation factors VII (FVII) and X (FX) have been reported to bind to EPCR, opening the possibility of novel roles for this receptor [1–4]. Ligands of EPCR interact with the receptor through their Gla domain. Although the sequence identity between the human Gla domains of FVII and FX and their murine counterparts is high, there are subtle differences in the regions involved in EPCR binding. For this reason, we wanted to clarify the species specificity for FVII or FX reactivity with EPCR.

Recently, an article [4] was published that describes a new role for the endothelial protein C/activated protein C receptor (EPCR) as a cofactor for factor Xa (FXa) to trigger cell signaling events. The experiments used by Disse et al. to suggest that FX is a ligand for EPCR were performed using human FX (hFX) and murine, instead of human, soluble EPCR (msEPCR). They calculated a Kd of 292 nm for the interaction between them. In our opinion, these results must be considered carefully because we observed that the species origin of EPCR influences its interaction with hFX. We performed surface plasmon resonance (SPR) experiments, which confirm that msEPCR and hFX interact with a similar affinity (Kd = 397 ± 54.1 nm). However, we observed that the binding of hFX to hsEPCR was much weaker (Kd = 3.48 ± 1.55 μm) (Fig. 1A,B). This result does not support any hypothesis that the interaction between EPCR and FX has a significant role in the human setting. Additionally, we performed flow cytometry experiments to compare the ability of hFX, human activated protein C (hAPC) and hFVII to displace FITC-labeled hAPC (fl-hAPC) from the surface of human aortic endothelial cells (HAEC). Confirming a previous report [5], we found that while hFVIIa and hAPC prevented fl-hAPC binding efficiently in a dose-dependent manner, a displacing effect by FXa was hardly seen, even when this was used at 20-fold excess with respect to fl-hAPC (Fig. 1C). Consistent with this, we found that hsEPCR inhibited the hFX activation by binding to the human factor VIIa (hFVIIa)-soluble tissue factor (sTF) complex rather than by binding to hFX, as is suggested by the fact that the Kcat was reduced while the Km remained unaltered [2]. Disse et al. found that the inhibition of FX activation was due to its interaction with EPCR. However, this was probably due to the fact that they used mEPCR rather than hEPCR.

Figure 1.

 Interaction between soluble endothelial protein C receptor (sEPCR) and factor (F) X or FVII. (A). Murine soluble endothelial protein C receptor (msEPCR) was immobilized on a CM5 chip in a BIAcore X and the binding of human FX (hFX) (500, 150, 75, 25, 10 nm) was monitored as previously described [7]. One representative experiment out of three is shown (B). Human soluble endothelial protein C receptor (hsEPCR) was captured on a CM5 chip and the binding of hFX (2000, 1000, 500, 250 nm) was monitored as previously described [2]. One representative experiment out of three is shown (C). The efficiency of human activated protein C (hAPC), human FVIIa (hFVIIa) and hFX in preventing the binding of 50 nm FITC-labeled hAPC (fl-hAPC) to human aortic endothelial cells (HAEC) was studied by flow cytometry [2]. One representative experiment is shown (D). msEPCR was immobilized on the surface of a CM5 chip and the binding of 240 nm mouse protein C (mPC), 25 nm hFVIIa and 240 nm mouse FVIIa (mFVIIa) to msEPCR was monitored [7]. Molecular weights: mPC, 52 kDa; hFVIIa, 50.5 kDa; mFVIIa, 50 kDa. Thus, if the bindings were similar, the resonance units (RU) signals should also be similar due to similar molecular weights (E). The efficiency of mPC, mFVII, hFVIIa and hFX in preventing the binding of 50 nm fl-hAPC to MS-1 cells was studied by flow cytometry [2]. s, seconds.

On the other hand, Disse et al. claim that mFVIIa-sTF binds to msEPCR with reasonable affinity. They studied this interaction by SPR and obtained a Kd of 160 nm. We have found that mFVII/VIIa is a rather weak ligand for msEPCR (Fig. 1D). After the immobilization of msEPCR on the chip, the injection of mFVIIa resulted in a very weak signal, notably lower than that observed for mPC or hFVIIa. The increment in the signal was so small that it was not possible to calculate a Kd accurately. The same result was obtained when mFVII instead of mFVIIa was used. We could not observe any mFVIIa binding to hsEPCR either (not shown). Additionally, we performed flow cytometry experiments to compare the ability of mFVII, mPC, hFX and hFVIIa to displace fl-hAPC from the surface of a murine endothelial cell line (MS-1). We observed that mPC and hFVIIa prevented fl-hAPC binding efficiently in a dose-dependent manner, while mFVII was not able to displace fl-hAPC binding (Fig. 1E). An explanation for the inability of mFVIIa to bind to EPCR may rely in the fact that Phe at position 4 of hFVII, hPC, hFX and mPC, which is critical for the interaction with EPCR [2,6], is replaced by Leu in mFVII.

In order to validate the findings described above by an alternative method, we performed clotting time experiments. While we had previously shown that the prothrombin time in human plasma was prolonged by human soluble endothelial protein C receptor (hsEPCR) [2], we have now demonstrated that msEPCR is not able to prolong the prothrombin time in murine plasma (data not shown). This result argues against the view that there is a relevant interaction between mEPCR and mFVII/VIIa or mFX. Furthermore, we compared the efficiency of hsEPCR and msEPCR in delaying the prothrombin time in human plasma. In confirmation of our previous observations [2], a dose-dependent effect of hsEPCR could be seen. However, msEPCR exerted a notably higher anticoagulant effect than hsEPCR: the lag time to clotting start, which was 29.5 ± 0.7 min, was increased to 37.9 ± 1.9 and 59.2 ± 4.2 min with 200 nm hsEPCR and msEPCR, respectively. Moreover, the time to reach 50% maximum clotting, which was 32.6 ± 0.7 min, was increased to 42.7 ± 2.8 and 72.2 ± 5.6 min with human and murine sEPCR, respectively. Because our SPR results indicated that msEPCR and hsEPCR bind to hFVIIa with a similar affinity [7], the better effect of msEPCR should be attributed to its superior affinity for hFX in comparison to hsEPCR.

Finally, Disse et al. performed appealing experiments exploring the role of EPCR in the cell signaling functions of FX in different cellular settings. Their results make us think of a scenario that fits both their findings and ours. At least in the human setting, the affinity between FXa and EPCR is relevant, provided that the former is incorporated into the FVIIa-TF complex. In the absence of TF, EPCR would preferably bind to PC or FVII rather than FX.

In summary, EPCR may play a hitherto unexpected role by allowing new ligands to exert cell signaling actions. Due to the differences in species specificity of the reactivity of EPCR with its ligands, care must be taken in the design of experimental models for its investigation.

Acknowledgements

We thank J. López-Sagaseta for his support with surface plasmon resonance and E. Molina for the production and purification of reagents. This work was supported through the Unión Temporal de Empresas project CIMA and by grants from Instituto de Salud Carlos III (PI05/1178, Red Temática de Investigación RECAVA RD/0014/0008), Fundación Mutua Madrileña and from the Health Department, Gobierno de Navarra (12/2006).

Disclosure of Conflict of Interests

The authors state that they have no conflict of interest.

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