Novel Solution and Membrane-Based Assays Reveal Lack of Interaction Between Murine FVIIa and Murine EPCR

Abstract 531

Generation of anticoagulant activated protein C (aPC) is enhanced by the presentation of protein C (PC) on the endothelial protein C receptor (EPCR) to the thrombin/thrombomodulin complex. Apart from its role in anticoagulation, EPCR also binds human coagulation Factor VIIa (FVIIa). However, the physiological consequences of this interaction either in vitro or in vivo are still unclear. In lieu of the widespread use of high-dose human FVIIa in hemophilic patients with anti Factor VIII or FIX antibodies as well as off-label applications, in vivo experimentation in appropriate mouse models is necessary to further define of the role of the FVIIa-EPCR interaction. Towards this, the aim of this study was to characterize the interaction of murine FVIIa with murine EPCR (mEPCR) in vitro. We used two novel assays that we have developed to study this interaction either in solution (by isothermal titration calorimetry [ITC]) or on the cell surface of mEPCR-expressing cells. The choice of ITC was based on its ability to provide a complete thermodynamic profile of protein-protein interaction in a single experiment: binding constant (K), stoichiometry (n), enthalpy (δH) and entropy (δS), parameters that cannot be determined with other methodologies in a single experiment. We first generated stable cells lines expressing recombinant murine soluble EPCR (msEPCR) or recombinant mFVIIa. The latter was generated as we have previously shown by insertion of a furin intracellular cleavage site between the light and heavy chains of murine FVII (mFVII). This resulted in a molecule secreted and purified in the activated form (mFVIIa) with ∼100% extrinsic activity vs. human FVIIa. Recombinant purified msEPCR had an apparent molecular weight of ∼46 kDa (vs. a predicted ∼26kDa size) which was consistent with extensive carbohydrate modifications. This was confirmed by expressing msEPCR in CHO cells modified to limit the size of the attached N-linked glycan chains: expressed msEPCR from such cells showed a reduction in its apparent molecular size (∼ 38kDa). Next, to validate ITC for the study the FVIIa-EPCR interaction, we used msEPCR and plasma-derived human PC (pd-hPC), a known msEPCR binder, that interacted with msEPCR with a Kd of ∼200nM, a stoichiometry of ∼1:1 and a ΔH and ΔS of −2733 ± 62 cal/mol and 21.3 cal/mol/deg, respectively. Initial ITC experiments with purified recombinant murine PC (mPC) also confirmed its binding to msEPCR. In contrast, we did not observe an interaction between recombinant mFVIIa and msEPCR. This finding was not due to protein degradation, as confirmed by Coomassie protein staining prior to and after ITC. Subsequently, to investigate the mFVIIa-msEPCR interaction in the context of a membrane-anchored murine EPCR, we generated a CHO-K1 cell line that expressed surface-exposed, full-length mEPCR, as verified by flow cytometry. These cells were incubated with pd-hPC or recombinant mFVIIa (50nM) in the presence of physiological Ca²⁺ (1.6mM) and Mg²⁺ (0.6mM) ion concentration. The bound protein was eluted with 10mM EDTA, electrophoresed and detected by western blotting. Despite the low femtomolar sensitivity of this assay (corresponding to 0.4% and 0.16% of total mFVIIa and pd-hPC used, respectively), we did not observe mFVIIa binding on mEPCR expressing CHO-K1 cells. Corroborating the ITC data, pd-hPC exhibited detectable binding to mEPCR expressing CHO-K1 cells. As a control, neither pd-hPC nor mFVIIa bound to naïve CHO-K1 cells (no mEPCR expression). In conclusion, we have established two novel assays to study the putative mFVIIa-mEPCR binding and clearly documented the lack of such interaction either in solution or on the cell-membrane. This is in good agreement with recently-published data obtained with surface plasmon resonance. In contrast to human FVIIa-EPCR, our results suggest that extrapolation of certain EPCR-dependent pathologic/physiologic processes using the mouse may be biased by species-specific effects. However, if the mEPCR binding capacity can be engineered to mFVIIa (as seen for human FVIIa-EPCR binding), such mFVIIa molecule(s) may facilitate the dissection of processes involving FVIIa and EPCR using the mouse as a human surrogate in vivo system. The assays developed here can easily assess such possibilities.