Warfarin Resistance Is Associated with a Protein Component of the Vitamin K 2,3-Epoxide Reductase Enzyme Complex in Rat Liver

Summary

Warfarin, the most used drug in the world in long-term anticoagulation prophylaxis, targets the vitamin K 2,3-epoxide reductase (VKOR) of the vitamin K cycle in liver. Recently, the enzyme has been identified as a multicomponent lipid-protein enzyme system in the endoplasmic reticulum (ER) membrane (17). As the first step towards understanding genetic resistance to warfarin, we present in this paper data on VKOR from normal and a strain of warfarin resistant laboratory rats maintained in the United States. Metal induced in vitro assembly of the enzyme complex demonstrates that the glutathione-S-transferase (GST) enzyme component of the complex loses its GST activity upon formation of VKOR. Less VKOR activity is measured upon assembly of the complex from warfarin resistant rats. The GST activity measured in warfarin resistant rats, before assembly of the complex, is 10-fold less sensitive to warfarin inhibition than the GST activity measured in normal rats. Microsomal epoxide hydrolase (mEH) is the second component of VKOR. When incubated with the components of VKOR before assembly of the complex, antibodies raised against mEH prevented formation of the enzyme complex. Sequencing of mEH cDNAs from normal and warfarin resistant rats revealed identical sequences. The data suggest that the mutation responsible for genetic warfarin resistance is associated with the GST component of VKOR.

• References

• 1 Suttie JW. Vitamin K-dependent carboxylase. Ann Rev Biochem 1985; 54: 459-77.
• 2 Furie B, Furie C. The molecular basis of blood coagulation. Cell 1988; 53: 505-18.
• 3 Suttie JW, Vitamin K. In: Handbook of lipid research. Deluca HF. ed. New York: Plenum Press; 1978. pp 211-77.
• 4 Wallin R, Martin LF. Warfarin poisoning and vitamin K antagonism in rat and human liver; design of a system in vitro that mimics the system in vivo. Biochem J 1987; 241: 389-96.
• 5 Wallin R, Gebhardt O, Prydz H. NAD(P)H dehydrogenase and its role in the vitamin K-dependent carboxylation reaction. Biochem J 1978; 169: 95-101.
• 6 Wallin R, Martin LF. Vitamin K-dependent carboxylation and vitamin K metabolism in liver: effects of warfarin. J Clin Invest 1985; 76: 1879-84.
• 7 Suttie JW. The biochemical basis of warfarin therapy. In: Advances in Experimental Medicine and Biology. Wessler S, Becker CG, Nemerson Y. eds. New York: Plenum Press; 1987. pp 3-16.
• 8 Hirsh J, Dalen JE, Deykin D, Poller L. Oral Anticoagulants; Mechanism of Action, Clinical Effectiveness, and Optimal Therapeutic Range. Chest 1992; 102: 312S-26S.
• 9 O’Reilly RA, Aggeler PM, Hoag MS. et al. Hereditary transmission of exceptional resistance to coumarin anticoagulant drugs. N Engl J Med 1983; 308: 1229-30.
• 10 Alving BM, Strickler MP, Knight RD. et al. Hereditary warfarin resistance. Arch Intern Med 1985; 145: 499-501.
• 11 Meehan AP. In: Rats and Mice, their Biology and Control. Meehan AP. ed. Mentokil Limited; Felcourt, East Grinstead, W. Sussex RH19 2JY: 1984
• 12 Hadler MR, Stadbolt RS. Novel 4-hydroxycoumarin anticoagulants active against resistant rats. Nature 1988; 253: 275-7.
• 13 Fasco MJ, Principe LM, Walsh WA, Friedman PA. Warfarin inhibition of Vitamin K 2,3-epoxide reductase in rat liver. Biochemistry 1983; 22: 5655-60.
• 14 Lee JJ, Fasco M. Metabolism of vitamin K and vitamin K 2,3-epoxide via interaction with a common disulfide. Biochemistry 1984; 23: 2246-52.
• 15 Hildebrandt EF, Suttie JW. Mechanism of Coumarin Action: Sensitivity of Vitamin K Metabolizing Enzymes of Normal and Warfarin-Resistant Rats. Biochemistry 1982; 21: 2406-11.
• 16 Wallin R, Guenthner TM. Purification of Warfarin-Sensitive Vitamin K Epoxide Reductase. Met Enzymol 1997; 282: 395-403.
• 17 Cain D, Hutson SM, Wallin R. Assembly of the Warfarin Sensitive Vitamin K 2,3-Epoxide Reductase Enzyme Complex in the Endoplasmic Reticulum Membrane. J Biol Chem 1997; 272: 29068-75.
• 18 Tishler M, Fieser LF, Wender NL. Hydro oxide and other derivatives of vitamin K1 and related compounds. J Amer Chem Soc 1940; 62: 2866-71.
• 19 Andersson C, Piemonte F, Mosialou E, Weinander R, Sun T-H, Lundquist G, Adang AEP, Morgenstern R. Kinetic studies on rat liver microsomal glutathione transferase: consequences of activation. Biochem Biophys Acta 1995; 1247: 277.
• 20 Wallin R, Culp E, Coleman DB, Goodman SR. A structural model of human erythrocyte band 2.1: Alignmemt of chemical and functional domains. Proc Natl Acad Sci USA 1984; 81: 4095-9.
• 21 Porter TD, Beck TW, Kasper CB. Complementary DNA and Amino Acid Sequence of Rat Liver Microsomal, Xenobiotic Epoxide Hydrolase. Arch Biochem Biophys 1986; 248: 121-9.
• 22 Laemmli UK. Cleavage of structured proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
• 23 Wallin R, Patrick SD, Martin L. Rat and Human Liver Vitamin K Epoxide Reductase: Inhibition by Thiol Blockers and Vitamin K1. Int J Biochem 1987; 19: 1063-8.
• 24 Chen CJ, Traugh JA. Expression of recombinant elongation factor 1 rabbit in Escherichia coli. Phosphorylation by casein kinase II. Biochim Biophys Acta 1995; 1264: 303-11.
• 25 Hayes JD, Pulford DJ. The glutathione S-transferase supergene family: Regulation of GST and the Contribution of the Isoenzymes to Cancer Chemoprotection and Drug Resistance. Crit Rev Biochem Mol Biol 1995; 30: 446-800.
• 26 Jackson WB, Ashton AD, Delventhal K. Overview of anticoagulant rodenticide usage and resistance. In: Current Advances in Vitamin K Research. Suttie JW. ed. New York. Elsevier; 1988. pp 381-8.
• 27 Ansell JE. Oral anticoagulant therapy – 50 years later. Arch Intern Med 1993; 153: 586-96.
• 28 Seidegard J, Depierre JW. Microsomal epoxide hydrolase: properties, regulation and function. Biochim Biophys Acta 1983; 695: 251-70.
• 29 Alves C, Von Dippe P, Amoui M, Levy D. Bile acid transport into hepatocytes smooth endoplasmic reticulum vesicles is mediated by microsomal epoxide hydrolase, a membrane protein exhibiting two distinct topological orientations. J Biol Chem 1990; 268: 20148-55.
• 30 Thijssen HHW, Baars LGM. Microsomal warfarin binding and vitamin K 2,3-epoxide reductase. Biochem Pharmacol 1989; 38: 1115-20.
• 31 Abramovitz M, Wong E, Cox ME, Richardson CD, Li C, Vickers PJ. 5-Lipoxygenase-activating protein stimulates the utilization of arachidonic acid by 5-lipoxygenase. Eur J Biochem 1993; 215: 105-11.
• 32 Morgenstern R, Guthenberg C, Mannervik B, DePierre JW. The amount and nature of glutathione transferases in rat liver microsomes determined by immunochemical methods. FEBS Lett 1983; 160: 264-8.
• 33 Wu S-M, Morris DP, Stafford DW. Identification and purification to near homogeneity of the vitamin K-dependent carboxylase. Proc Natl Acad Sci USA 1991; 88: 2236-40.