The long-term objective of this project is to understand the enzymatic properties of the vitamin K epoxide reductase (VKOR) and its role in vitamin K-dependent carboxylation. Carboxylation is an essential post-translational modification for proteins important in several physiologic functions, including blood coagulation and bone metabolism. Recently, researchers in this laboratory identified the warfarin-sensitive VKOR gene. VKOR is the target of warfarin, the most widely prescribed anti-coagulant for thromboembolic disorders. Although estimated to prevent twenty strokes per induced bleeding episode, warfarin is under-utilized because of fear of bleeding. Research funded by this grant hopefully will lead to therapies that will alleviate some of the problems involved in warfarin treatment. Specifically, we propose to accomplish the following during the tenure of this grant: (1) express the isoforms of the VKOR gene to determine which have activity;(2) purify the VKOR using the amino acid tag we have inserted and the antibody to this tag;(3) investigate different cell expression systems to determine which is best for production of recombinant VKOR;(4) investigate the status of cysteines in VKOR, the glycosylation sites and other potential post-translational modifications, and the warfarin binding site by mass spectrometry;and (5) investigate the effect of vitamin K epoxide reductase (VKOR) gene polymorphisms on response to warfarin. This study of VKOR should lead to a better understanding of the mechanism of warfarin inhibition. It may also result in more accurate dosing of warfarin and in the design of new anti-coagulants.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hemostasis and Thrombosis Study Section (HT)
Program Officer
Link, Rebecca P
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University of North Carolina Chapel Hill
Schools of Arts and Sciences
Chapel Hill
United States
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Tie, J-K; Stafford, D W (2016) Structural and functional insights into enzymes of the vitamin K cycle. J Thromb Haemost 14:236-47
Jin, Da-Yun; Vermeer, Cees; Stafford, Darrel W et al. (2016) Splice-Site Mutation of Exon 3 Deletion in the Gamma-Glutamyl Carboxylase Gene Causes Inactivation of the Enzyme. J Invest Dermatol 136:2314-2317
Tie, Jian-Ke; Carneiro, Jorge D A; Jin, Da-Yun et al. (2016) Characterization of vitamin K-dependent carboxylase mutations that cause bleeding and nonbleeding disorders. Blood 127:1847-55
Wu, S; Tie, J-K; Stafford, D W et al. (2014) Membrane topology for human vitamin K epoxide reductase. J Thromb Haemost 12:112-4
Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W (2014) Conserved loop cysteines of vitamin K epoxide reductase complex subunit 1-like 1 (VKORC1L1) are involved in its active site regeneration. J Biol Chem 289:9396-407
Ingram, Brian O; Turbyfill, Jared L; Bledsoe, Peggy J et al. (2013) Assessment of the contribution of NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) to the reduction of vitamin K in wild-type and NQO1-deficient mice. Biochem J 456:47-54
Tie, J-K; Jin, D-Y; Tie, K et al. (2013) Evaluation of warfarin resistance using transcription activator-like effector nucleases-mediated vitamin K epoxide reductase knockout HEK293 cells. J Thromb Haemost 11:1556-64
Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W (2012) Human vitamin K epoxide reductase and its bacterial homologue have different membrane topologies and reaction mechanisms. J Biol Chem 287:33945-55
Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W (2012) Mycobacterium tuberculosis vitamin K epoxide reductase homologue supports vitamin K-dependent carboxylation in mammalian cells. Antioxid Redox Signal 16:329-38
Wu, Sangwook; Liu, Shubin; Davis, Charles H et al. (2011) A hetero-dimer model for concerted action of vitamin K carboxylase and vitamin K reductase in vitamin K cycle. J Theor Biol 279:143-9

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