Mechanism of Coumarin Action: Significance of Vitamin K Epoxide Reductase Inhibition

D. S. Whitlon, J. A. Sadowski, J. W. Suttie*

*Corresponding author for this work

Research output: Contribution to journalArticle

265 Citations (Scopus)

Abstract

Vitamin K functions in a microsomal carboxylation reaction that converts glutamyl residues in precursor proteins to γ-carboxyglutamyl residues in the products of this reaction. The same liver microsomal preparations that carry out this carboxylation also convert the vitamin to its 2,3-epoxide (epoxidase activity) and reduce the epoxide to the vitamin (epoxide reductase activity). The effect of the coumarin anticoagulant Warfarin on these reactions has been studied. The vitamin K dependent carboxylase activity in intact microsomes is dependent on either NADH or dithiothreitol as a source of reducing equivalents to form the biologically active reduced form of the vitamin. The dithiothreitol dependent reaction is inhibited by Warfarin, but the NADH dependent reaction is not. When microsomes are solubilized in detergent, dithiothreitol is no longer an effective source of reducing equivalents, and Warfarin inhibition of the carboxylase activity is lost. The vitamin K epoxide reductase will use dthiothreitol, but not NADH as a reductant, and this reaction is strongly inhibited by Warfarin. The vitamin K dependent carboxylase system will utilize vitamin K epoxide as an active form of vitamin K only if dithiothreitol is used as the reducing agent, and, under these conditions, the reaction is inhibited by Warfarin. When microsomes were prepared from a strain of rats resistant to the anticoagulant effect of Warfarin, all three of these dithiothreitol dependent activities—vitamin K dependent carboxylase, vitamin K epoxide dependent carboxylase, and vitamin K epoxide reductase—were relatively insensitive to the inhibitory effect of Warfarin. However, a second coumarin, Difenacoum, which has been shown to be an effective anticoagulant in this strain of rats was an effective inhibitor of these in vitro reactions. These data support the theory that the vitamin K-vitamin K epoxide interconversion is a physiologically important cycle of the vitamin and that the action of Warfarin as an anticoagulant might be to block this cycle. The physiologically important reducing agent which is replaced by dithiothreitol in these in vitro studies has not been identified.

Original languageEnglish (US)
Pages (from-to)1371-1377
Number of pages7
JournalBiochemistry
Volume17
Issue number8
DOIs
StatePublished - Jan 1 1978

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Vitamin K Epoxide Reductases
Warfarin
Dithiothreitol
Vitamins
Anticoagulants
Vitamin K
Epoxy Compounds
Reducing Agents
Microsomes
Carboxylation
NAD
Rats
Protein Precursors
coumarin
Detergents
Liver
Oxidoreductases

ASJC Scopus subject areas

  • Biochemistry

Cite this

Whitlon, D. S. ; Sadowski, J. A. ; Suttie, J. W. / Mechanism of Coumarin Action : Significance of Vitamin K Epoxide Reductase Inhibition. In: Biochemistry. 1978 ; Vol. 17, No. 8. pp. 1371-1377.
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abstract = "Vitamin K functions in a microsomal carboxylation reaction that converts glutamyl residues in precursor proteins to γ-carboxyglutamyl residues in the products of this reaction. The same liver microsomal preparations that carry out this carboxylation also convert the vitamin to its 2,3-epoxide (epoxidase activity) and reduce the epoxide to the vitamin (epoxide reductase activity). The effect of the coumarin anticoagulant Warfarin on these reactions has been studied. The vitamin K dependent carboxylase activity in intact microsomes is dependent on either NADH or dithiothreitol as a source of reducing equivalents to form the biologically active reduced form of the vitamin. The dithiothreitol dependent reaction is inhibited by Warfarin, but the NADH dependent reaction is not. When microsomes are solubilized in detergent, dithiothreitol is no longer an effective source of reducing equivalents, and Warfarin inhibition of the carboxylase activity is lost. The vitamin K epoxide reductase will use dthiothreitol, but not NADH as a reductant, and this reaction is strongly inhibited by Warfarin. The vitamin K dependent carboxylase system will utilize vitamin K epoxide as an active form of vitamin K only if dithiothreitol is used as the reducing agent, and, under these conditions, the reaction is inhibited by Warfarin. When microsomes were prepared from a strain of rats resistant to the anticoagulant effect of Warfarin, all three of these dithiothreitol dependent activities—vitamin K dependent carboxylase, vitamin K epoxide dependent carboxylase, and vitamin K epoxide reductase—were relatively insensitive to the inhibitory effect of Warfarin. However, a second coumarin, Difenacoum, which has been shown to be an effective anticoagulant in this strain of rats was an effective inhibitor of these in vitro reactions. These data support the theory that the vitamin K-vitamin K epoxide interconversion is a physiologically important cycle of the vitamin and that the action of Warfarin as an anticoagulant might be to block this cycle. The physiologically important reducing agent which is replaced by dithiothreitol in these in vitro studies has not been identified.",
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Mechanism of Coumarin Action : Significance of Vitamin K Epoxide Reductase Inhibition. / Whitlon, D. S.; Sadowski, J. A.; Suttie, J. W.

In: Biochemistry, Vol. 17, No. 8, 01.01.1978, p. 1371-1377.

Research output: Contribution to journalArticle

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T1 - Mechanism of Coumarin Action

T2 - Significance of Vitamin K Epoxide Reductase Inhibition

AU - Whitlon, D. S.

AU - Sadowski, J. A.

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PY - 1978/1/1

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N2 - Vitamin K functions in a microsomal carboxylation reaction that converts glutamyl residues in precursor proteins to γ-carboxyglutamyl residues in the products of this reaction. The same liver microsomal preparations that carry out this carboxylation also convert the vitamin to its 2,3-epoxide (epoxidase activity) and reduce the epoxide to the vitamin (epoxide reductase activity). The effect of the coumarin anticoagulant Warfarin on these reactions has been studied. The vitamin K dependent carboxylase activity in intact microsomes is dependent on either NADH or dithiothreitol as a source of reducing equivalents to form the biologically active reduced form of the vitamin. The dithiothreitol dependent reaction is inhibited by Warfarin, but the NADH dependent reaction is not. When microsomes are solubilized in detergent, dithiothreitol is no longer an effective source of reducing equivalents, and Warfarin inhibition of the carboxylase activity is lost. The vitamin K epoxide reductase will use dthiothreitol, but not NADH as a reductant, and this reaction is strongly inhibited by Warfarin. The vitamin K dependent carboxylase system will utilize vitamin K epoxide as an active form of vitamin K only if dithiothreitol is used as the reducing agent, and, under these conditions, the reaction is inhibited by Warfarin. When microsomes were prepared from a strain of rats resistant to the anticoagulant effect of Warfarin, all three of these dithiothreitol dependent activities—vitamin K dependent carboxylase, vitamin K epoxide dependent carboxylase, and vitamin K epoxide reductase—were relatively insensitive to the inhibitory effect of Warfarin. However, a second coumarin, Difenacoum, which has been shown to be an effective anticoagulant in this strain of rats was an effective inhibitor of these in vitro reactions. These data support the theory that the vitamin K-vitamin K epoxide interconversion is a physiologically important cycle of the vitamin and that the action of Warfarin as an anticoagulant might be to block this cycle. The physiologically important reducing agent which is replaced by dithiothreitol in these in vitro studies has not been identified.

AB - Vitamin K functions in a microsomal carboxylation reaction that converts glutamyl residues in precursor proteins to γ-carboxyglutamyl residues in the products of this reaction. The same liver microsomal preparations that carry out this carboxylation also convert the vitamin to its 2,3-epoxide (epoxidase activity) and reduce the epoxide to the vitamin (epoxide reductase activity). The effect of the coumarin anticoagulant Warfarin on these reactions has been studied. The vitamin K dependent carboxylase activity in intact microsomes is dependent on either NADH or dithiothreitol as a source of reducing equivalents to form the biologically active reduced form of the vitamin. The dithiothreitol dependent reaction is inhibited by Warfarin, but the NADH dependent reaction is not. When microsomes are solubilized in detergent, dithiothreitol is no longer an effective source of reducing equivalents, and Warfarin inhibition of the carboxylase activity is lost. The vitamin K epoxide reductase will use dthiothreitol, but not NADH as a reductant, and this reaction is strongly inhibited by Warfarin. The vitamin K dependent carboxylase system will utilize vitamin K epoxide as an active form of vitamin K only if dithiothreitol is used as the reducing agent, and, under these conditions, the reaction is inhibited by Warfarin. When microsomes were prepared from a strain of rats resistant to the anticoagulant effect of Warfarin, all three of these dithiothreitol dependent activities—vitamin K dependent carboxylase, vitamin K epoxide dependent carboxylase, and vitamin K epoxide reductase—were relatively insensitive to the inhibitory effect of Warfarin. However, a second coumarin, Difenacoum, which has been shown to be an effective anticoagulant in this strain of rats was an effective inhibitor of these in vitro reactions. These data support the theory that the vitamin K-vitamin K epoxide interconversion is a physiologically important cycle of the vitamin and that the action of Warfarin as an anticoagulant might be to block this cycle. The physiologically important reducing agent which is replaced by dithiothreitol in these in vitro studies has not been identified.

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