Recombinant mFVIIa chimeras bind to mEPCR on CHO-K1 cells, in contrast to mFVIIa. Increasing concentration of ligand (10-1000 nM) (A) mFVIIa, (B) mFVIIa-FMR, or (C) mFVIIa-(1-43 mPC) was incubated on CHO-K1 or CHO-K1-mEPCR cells in the presence of 1.6 mM Ca²⁺ and 0.6 mM Mg²⁺ for 1 hour at 4°C. Bound fraction was eluted and visualized by fluorescence-based western blotting (top, images shown in greyscale) and band intensities were quantified and corrected for nonspecific binding (on CHO-K1 cells). Integrated densities (bottom) were plotted, and equilibrium dissociation constants (K_d) were calculated using a 1-site–specific binding curve fitting with GraphPad Prism v5.0b (GraphPad Software). Conversion to nanomolar was done using the integrated densities of known amounts of ligand in buffer C, electrophoresed, and blotted as described in the Methods, and values were normalized for cell numbers. Depicted curves for each protein are fitted from 3 independent experiments. Data points are shown as mean ± standard deviation (SD). The y-axis of all graphs depicts FVIIa bound material.