1. Functional study of the vitamin K cycle in mammalian cells
by Jian-Ke Tie, Da-Yun Jin, David L. Straight, and Darrel W. Stafford
Blood
Volume 117(10):
March 10, 2011
©2011 by American Society of Hematology

2. Vitamin K cycle.
Vitamin K cycle. During vitamin K–dependent carboxylation of glutamic acid to γ-carboxyglutamic acid, the reduced form of vitamin K (KH2) is oxidized to KO by GGCX. KO is reduced to vitamin K by VKOR using the enzyme's 2 active-site cysteine residues. This reaction is sensitive to warfarin inhibition. The reduction of vitamin K to KH2 is carried out in 2 pathways. One pathway is sensitive to warfarin inhibition and also involves 2 free cysteine residues in the enzyme active site (VKOR). The second pathway is resistant to warfarin and uses NAD(P)H as a cofactor.
Jian-Ke Tie et al. Blood 2011;117:
©2011 by American Society of Hematology

3. Effect of vitamin K, KO, and warfarin on FIXgla-PC carboxylation and secretion.
Effect of vitamin K, KO, and warfarin on FIXgla-PC carboxylation and secretion. Carboxylated (■) and total () FIXgla-PC secreted from HEK293 cells under different culture conditions was measured by ELISA as described in “FIXgla-PC measurement in cell-culture medium using ELISA.” Control, complete medium (no added vitamin K); K, complete medium with 11μM vitamin K; KO, complete medium with 5μM KO; KO + W, complete medium with 5μM KO + 2μM warfarin
Jian-Ke Tie et al. Blood 2011;117:
©2011 by American Society of Hematology

4. Effect of warfarin on FIXgla-PC carboxylation in HEK293 cells.
Effect of warfarin on FIXgla-PC carboxylation in HEK293 cells. (A) Cells grown in culture medium with either 11μM vitamin K (○) or 5μM KO (●) were incubated for 48 hours with increasing concentrations of warfarin. The concentration of carboxylated FIXgla-PC in the medium was measured by ELISA. The data are presented as percentages to make the first concentration points of vitamin K and KO coincide. (B) Cells were grown with (○) or without (●) 2μM warfarin in increasing concentrations of vitamin K for 48 hours. The concentration of carboxylated FIXgla-PC in the culture medium was measured by ELISA.
Jian-Ke Tie et al. Blood 2011;117:
©2011 by American Society of Hematology

5. Effect of warfarin on FIXgla-PC carboxylation in AV12 cells.
Effect of warfarin on FIXgla-PC carboxylation in AV12 cells. (A) Cells grown in culture medium with either 11μM vitamin K (○) or 5μM KO (●) were incubated for 48 hours with increasing concentrations of warfarin. The concentration of carboxylated FIXgla-PC in the medium was detected by ELISA. The data are presented as percentages to make the first concentration points of vitamin K and KO coincide. (B) Cells were grown with (○) or without (●) 2μM warfarin in increasing concentrations of vitamin K for 48 hours. The concentration of carboxylated reporter protein in the culture medium was measured by ELISA.
Jian-Ke Tie et al. Blood 2011;117:
©2011 by American Society of Hematology

6. Endogenous VKOR and GGCX activity in HEK293 and AV12 cells as measured by in vitro enzymatic activity assay.
Endogenous VKOR and GGCX activity in HEK293 and AV12 cells as measured by in vitro enzymatic activity assay. (A) Endogenous GGCX activity of 1 × 106 HEK293 or AV12 cells was determined as described in “GGCX activity assay” using FLEEL as a substrate in the presence of propeptide. (B) Endogenous VKOR activity of 1 × 107 HEK293 or AV12 cells was determined as described in “VKOR activity assay” using K1(25) as the HPLC internal standard.
Jian-Ke Tie et al. Blood 2011;117:
©2011 by American Society of Hematology

7. Effect of dicoumarol on the carboxylation of the FIXgla-PC in HEK293 and AV12 cells.
Effect of dicoumarol on the carboxylation of the FIXgla-PC in HEK293 and AV12 cells. Increasing concentrations of dicoumarol were added to the cell-culture medium with either 11μM K (○) or 5μM KO (●) and incubated with HEK293 (A) or AV12 (B) cells for 48 hours. The concentration of carboxylated FIXgla-PC in the medium was measured by ELISA. The data are presented as percentages to make the first concentration points of vitamin K and KO coincide.
Jian-Ke Tie et al. Blood 2011;117:
©2011 by American Society of Hematology

8. Effect of the warfarin-resistant VKOR mutant on FIXgla-PC carboxylation.
Effect of the warfarin-resistant VKOR mutant on FIXgla-PC carboxylation. (A) The warfarin-resistant VKOR-Y139F mutant was transiently expressed in HEK293 (■) and AV12 cells (). Thirty hours after transfection, cells were cultured in medium containing 5μM KO and 2μM warfarin for 48 hours. The concentration of carboxylated FIXgla-PC in the medium was measured by ELISA. The control was the cell line transfected with empty vector, representing endogenous VKOR. (B) AV12 cells (■) or AV12 cells that were stably expressing the warfarin-resistant VKOR-Y139F mutant () were cultured in medium containing 5μM KO (KO) or 5μM KO + 2μM warfarin (KO + W) for 48 hours. The concentration of carboxylated FIXgla-PC in the medium was measured by ELISA.
Jian-Ke Tie et al. Blood 2011;117:
©2011 by American Society of Hematology