Cell-based carboxylation activity assay with 2 reporter proteins. (A) Domain structure of the reporter proteins. Catalytic domain, region containing the serine protease catalytic triad; Gla domain, residues 1 through 46 of human factor IX containing 12 Gla residues; Kringles, regions of internal sequence homology; propeptide, residue −18 to −1 of human prothrombin. Arrow indicates the site that is proteolytically cleaved for protein maturation. MGP retains its propeptide within the mature protein and has Gla residues scattered around the propeptide. (B) Schematic diagram of reporter-protein carboxylation, secretion from HEK293 cells, and detected by sandwich-based ELISA. (C) FIXgla-ProT carboxylation in HEK293 cells in response to increasing concentrations of vitamin K1. (D) MGP carboxylation in HEK293 cells in response to increasing concentrations of vitamin K1.