Robert G. Bell

Vitamin K Activity of Phylloquinone Oxide

Abstract The biological activity of phylloquinone oxide in vitamin K-deficient rats was approximately the same as phylloquinone. In warfarin-treated animals phylloquinone was an effective antagonist of the anticoagulant whereas the oxide was not unless it was administered 15 min before warfarin. The possibility that the activity of the oxide in deficient rats is dependent on its conversion to phylloquinone and that warfarin inhibits this transformation was supported by isotope experiments in which tritiated oxide and 14 C-labeled phylloquinone were injected into normal and warfarin-treated rats. Warfarin caused a dramatic change in the metabolism of phylloquinone resulting in a large preponderance of oxide in the livers of anticoagulant-treated animals. These results prompt us to consider that phylloquinone oxide, because of structural similarity, may be an inhibitor of phylloquinone and that warfarin exerts its anticoagulant effect by causing accumulation of the oxide.

Warfarin and metabolism of vitamin K1

To further examine the hypothesis that warfarin inhibits prothrombin synthesis by interfering with the cyclic interconversion of vitamin K1 and phylloquinone epoxide, the metabolism of tracer doses of labeled vitamin K1 was studied in anticoagulant-treated rats. A tracer dose of [3H]K1 initially turned over with a half-life of 2.2 hr in the liver, but after 5 hr the degradation rate decreased considerably. Warfarin caused hepatic [3H]K1 levels to drop to 50 per cent of controls 5 min after administration of the vitamin, and over 10 hr the concentration of [3H]K1 was 26–36 per cent of control levels. This decrease in vitamin K1 was not large enough to account for the inhibition of prothrombin synthesis by warfarin. Thirty-five min after warfarin administration, there was more [3H]phylloquinone epoxide in the liver than labeled vitamin and over 10 hr the [3H]epoxide: [3H]K1 ratio varied from 1.6 to 3.0. In addition to increasing the relative amount of [3H]epoxide, warfarin and phenylindanedione also increased hepatic metabolites more polar than vitamin K1 or epoxide. However, warfarin did not increase plasma or urinary radioactivity. The urine was the principal excretory route for metabolites of vitamin K1. 2-Diethylaminoethyl-2,2, diphenylvalerate hydrochloride (SKF 525-A) inhibited the turnover of [3H]K1 but did not decrease the [3H]polar metabolites in the liver in control or warfarin-treated rats. The drug also did not inhibit the epoxidation of the vitamin to phylloquinone epoxide. The metabolism of vitamin K1 was not altered by vitamin K deficiency or chronic administration of the vitamin. After a small dose of warfarin (35 μg/100 g body wt), plasma prothrombin decreased for 14 hr and then slowly rose but did not reach 90 per cent of normal until 3 days after administration of anticoagulant. The epoxide: K1 ratio in the liver, measured by injecting a tracer dose of [3H]K1, was 0.90 at 6 hr when prothrombin synthesis was completely blocked. The ratio dropped to 0.52 just before prothrombin started to rise at 14 hr and remained around 0.5 during the slow climb of plasma prothrombin toward the normal level. These low ratios were unexpected in animals in which prothrombin synthesis was partially or completely blocked. Hepatic epoxide: K1 ratios of 11.4 and 1.3 were observed in warfarin-treated rats in which prothrombin synthesis was stimulated by injections of 0.1 mg [3H]K1 plus 0.4 mg [3H]epoxide and 0.1 mg [3H]K1 respectively. Therefore, there appears to be a lack of correlation of epoxide: K1 ratios and prothrombin synthesis.

Inhibition of vitamin K epoxidase by two non-coumarin anticoagulants.

Abstract Tetrachloro-4-pyridinol (4-TCP) and 2-chloro-3-phytyl-1,4-naphtoquinone (Cl-K), like the coumarin and the indanedione anticoagulants, caused the accumulation of prothombin precursor activity in liver microsomes. Unlike the coumarins and indanediones, 4-TCP and Cl-K interrupted the vitamin K 1 -vitamin K 1 epoxide cycle by inhibiting the epoxidation of vitamin K 1 . Epoxidase activity assayed in vitro was decreased by about 40 per cent relative to controls in rats treated 24 hr previously with 4-TCP or Cl-K. In vitro assays demonstrated that 3 × 10 −6 M Cl-K and 10 −4 M 4-TCP inhibited the epoxidation of vitamin K 1 by about 75 per cent. Inhibition of phylloquinone epoxidase activity was determined in vivo by blocking the reduction of epoxide with warfarin and measuring the conversion of [ 3 H]K 1 to [ 3 H]epoxide. Doses of Cl-K and 4-TCP which blocked prothrombin synthesis also inhibited epoxidation while doses which did not lower plasma prothrombin also had no significant effect on the K 1 -epoxide conversion. When inhibition of prothrombin synthesis by 4-TCP and Cl-K was reversed by vitamin K 1 , a minimum of 6–17 nmole of epoxide were formed in the liver for each nmole of prothrombin that appeared in plasma. The results suggest that epoxidation of vitamin K may be involved in prothrombin production and that interference with either the epoxidation or reduction step in the cycle will result in inhibition of clotting protein synthesis.

Coumarins and the vitamin K-K epoxide cycle. Lack of resistance to coumatetralyl in warfarin-resistant rats

Abstract In female warfarin-resistant rats, coumatetralyl at 0.5 mg/100 g body wt blocked prothrombin synthesis and interrupted the vitamin K1-K1 epoxide cycle by almost completely blocking the conversion of epoxide to vitamin K1. In contrast, prothrombin synthesis and the epoxide-K1 conversion were unaffected by warfarin at the same dose, although at 2 mg/100 g body wt warfarin also blocked prothrombin synthesis and the conversion. In Sprague-Dawley rats, the anticoagulants inhibited prothrombin synthesis about equally well over a range of doses. At 0.05 mg/100 g body wt or greater, warfarin and coumatetralyl severely inhibited both prothromhin synthesis and the reduction of the epoxide to K1, while at 0.01 mg/100 g body wt the anticoagulants had little effect. Metabolic studies with tracer doses of [3H]K1 and [3H]epoxide indicated that resistant rats have hepatic epoxide: K1 ratios 5–6-fold greater than in Sprague-Dawley animals. The hepatic concentrations of [3H]K1 in male and female resistant rats were 41 and 26 per cent, and plasma prothrombin concentrations 17 and 39 per cent respectively, of those values in Sprague-Dawley rats. When resistant rats were injected with vitamin K1 plasma, prothrombin increased while the hepatic epoxide: K1 ration decreased. Two days later prothrombin and the ration had returned to their original values. These results are consistent with the idea that the K1-epoxide cycle is involved in clotting protein synthesis and that the site of action of coumarins is the epoxide-K1 conversion. The impaired epoxide-K1 conversion may explain why warfarin-resistant rats have a lowered rate of prothrombin synthesis.

Inhibition of prothrombin synthesis and epoxidation of vitamin K1 by anticoagulants invitro

Summary A number of 4-hydroxycoumarin and indanedione anticoagulatns completly inhibited prothrombin synthesis in vitro at concentrations of less than 10 mM. Since the high concentration of vitamin K1 used to stumulate prothrombin synthesis made it unlikely that the anticoagulants were inhiting prothrombin synthesis by prevention the regeneration of vitamin from vitamin K1 epoxide, this suggested a second site of action of anticoagulants. This second site may be the epoxication of vitamin K since all the anticoagulants inhibited the epoxidation at concentrations less than 7 mM. 3-Phenyl-4-hydroxycoumarin, Dicumarol, Marcoumar and phenylindanedione were the most potent in inhibiting prothrombin synthesis and epoxidation while Warfarin, 4-hydrocoumarin and indanedione were the least effective.

Vitamin K-dependent carboxylation of glutamic acid residues to γ-carboxyglutamic acid in lung microsomes☆

Abstract Vitamin K-dependent carboxylation of glutamic acid residues to γ-carboxyglutamic acid was demonstrated in proteins of lung microsomes. The carboxylation was 12% of that in liver microsomes per milligram of mierosomal protein. Carboxylation was very low with microsomes of untreated rats but increased with time up to 42 h after warfarin administration. Carboxylation was highest with microsomes from rats fed a vitamin K-deficient diet. This suggests that a protein(s) accumulates which can be carboxylated in vitro/J. Lung microsomes also catalyzed the vitamin K-dependent carboxylation of the peptide Phe-Leu-Glu-Glu-Leu. The peptide carboxylase activity was 9% of that obtained with liver microsomes. Vitamin K-dependent protein carboxylation required NADH or dithioerythritol, suggesting that vitamin K had to be reduced to the hydroquinone. Accordingly, vitamin K 1 hydroquinone had carboxylating activity without added reducing agents. Menaquinone-3 was considerably more active than phylloquinone. The temperature optimum for carboxylation was around 27 °C.

Vitamin K dependent formation of γ-carboxyglutamate residues in tumor microsomes

Abstract Vitamin K stimulated the incorporation of 14 C into proteins when microsomes from melanoma, mammary gland, mast cell and lymphoma tumors were incubated with Na 2 14 CO 3 . The 14 C label in the [ 14 C] proteins was identified as [ 14 C] γ-carboxyglutamate (Gla), which is formed by carboxylation of glutamic acid residues. Carboxylation in tumor microsomes ranged from 2 to 19% of the carboxylation in normal liver microsomes per mg of microsomal protein. Carboxylation in microsomes was completely blocked by 10 μM Warfarin. SDS-polyacrylamide gel analysis of the melanoma [ 14 C] Gla protein(s) revealed one major peak of 14 C with an apparent MW of less than 6,000.

Inhibition by warfarin enantiomers of prothrombin synthesis, protein carboxylation, and the regeneration of vitamin K from vitamin K epoxide

Abstract S (−)-Warfarin was about twice as effective as R (+)-warfarin at inhibiting the vitamin K 1 stimulated synthesis of prothrombin in vitamin K deficient rats. It has been proposed [R.G. Bell, Fedn Proc. 37 , 2599 (1978)] that warfarin blocks prothrombin synthesis by inhibiting the regeneration of vitamin K from its chief metabolite, vitamin K epoxide. Consistent with this idea, S -warfarin was more effective than R -warfarin at inhibiting the epoxide to vitamin K conversion in vivo with an anticoagulant dose of 50 μg/100g body wt. S -Warfarin was 1.9 to 3.5 times more effective than R -warfarin at inhibiting the epoxide to K 1 conversion in hepatic microsomal and post-mitochondrial supernatant fractions. Similarly, S -warfarin was 1.9 to 3.3 times more effective at inhibiting epoxide-dependent carboxylation in these preparations. S -Warfarin was 1.4 to 3.0 times more effective than the R -enantiomer at inhibiting vitamin K dependent carboxylation in post-mitochondrial supernatant fractions. We have concluded that the greater anticoagulant potency of S -warfarin was due to two factors of almost equal importance: the slower turnover and the higher intrinsic activity of the S -enantiomer in inhibiting the regeneration of vitamin K from the epoxide.

Vitamin K-Dependent Processes in Tumor Cells

Tumor cells are known to interfere with blood coagulation pathways of the host by producing procoagulants and other substances, thereby deriving certain advantages relating to tumor growth, metastasis, and angiogenesis. Anticoagulants may diminish these advantages under certain conditions. The interaction between coumarin anticoagulants and tumor cells has been reviewed with respect to procoagulants and their vitamin K-dependent properties. Evidence is also presented which suggests that vitamin K-dependent protein carboxylation is a general property of tumor cells.

Mechanism for potentiation of warfarin by phenylbutazone: Inhibition of vitamin K-dependent carboxylation and prothrombin synthesis by phenylbutazone in preparations from rat liver☆

Phenylbutazone potentiated the anticoagulant effects of racemic warfarin and of the individual enantiomers to similar extents in the rat. This indicates that the phenylbutazone did not act stereospecifically on the enantiomers, as it does in humans. Phenylbutazone doubled the turnover rate of warfarin in plasma, but it did not increase the amount of the anticoagulant in liver or the amount excreted in urine. The drug had no effect on plasma disappearance of [3H] or on hepatic levels of [3H] vitamin K1 or of its chief metabolite, [3H] vitamin K1 epoxide, after injection of [3H] vitamin K1. Phenylbutazone, however, at concentrations of 0.5 to 2.8 mM inhibited vitamin K-dependent carboxylation of a synthetic pentapeptide substrate in liver microsomes by 40-88 per cent. Vitamin K-dependent protein carboxylation was also inhibited by about 40 per cent in microsomes and post-mitochondrial supernatant fluid at drug concentrations of 2.8 to 4.8 mM. Most importantly, prothrombin synthesis was inhibited in post-mitochondrial supernatant fractions by 19 and 39 per cent at drug concentrations of 2.8 and 4.8 mM respectively. The inhibition of both carboxylation and prothrombin synthesis appears to have been of sufficient magnitude to account for the potentiation by phenylbutazone observed in vivo. The calculated hepatic level of phenylbutazone during potentiation was around 3 mM, a concentration that produced inhibition in vitro.