Secretion of PP/Gla domain of PZ and/or FX fused to MetLuc. (A) PZ_PPmut was fused with MetLuc, and its expression vector (PZ_PPmut^MetLuc) was transfected to HEK 293 cells with a pGL3-control. Twenty-four hours after incubation with warfarin (W, □) or Vit.K (K, ■), MetLuc activity in the medium (top panel) and barium citrate-adsorbate (bottom panel) were assayed. Values of means (± SD) for 3 transfections were calculated relative to the activity of PZ^MetLuc treated with Vit.K (arbitrarily set at 1-fold; *P = .01; **P < .001). (B) Combinations of PP and Gla domains of PZ (gray boxes) and FX (white boxes) were fused to amino-terminal–truncated MetLuc (black boxes), as illustrated at the bottom. Fifteen micrograms of expression vector for each construct were transfected to HEK 293 cells together with the pGL3-control, and the cells were incubated with warfarin (W, □) or Vit.K (K, ■) for 24 hours. MetLuc activity in the medium (top panel) and barium citrate adsorbate (bottom panel) was measured and normalized to the activity of firefly luciferase in the cell lysates. Values of means (± SD) for 3 transfections were calculated relative to the activity of Z_PPZ_Gla^MetLuc treated with Vit.K (arbitrarily set at 1-fold; **P < .005; ***P < .001).