Determination of the warfarin inhibition constant Ki for vitamin K 2,3-epoxide reductase complex subunit-1 (VKORC1) using an in vitro DTT-driven assay

Background

Warfarin directly inhibits vitamin K 2,3-epoxide reductase (VKOR) enzymes. Since the early 1970s, warfarin inhibition of vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1), an essential enzyme for proper function of blood coagulation in higher vertebrates, has been studied using an in vitro dithiothreitol (DTT) driven enzymatic assay. However, various studies based on this assay have reported warfarin dose–response data, usually summarized as half-maximal inhibitory concentration (IC₅₀), that vary over orders of magnitude and reflect the broad range of conditions used to obtain VKOR assay data.

Methods

We standardized the implementation of the DTT-driven VKOR activity assay to measure enzymatic Michaelis constants (K_m) and warfarin IC₅₀ for human VKORC1. A data transformation is defined, based on the previously confirmed bi bi ping-pong mechanism for VKORC1, that relates assay condition-dependent IC₅₀ to condition-independent K_i.

Results

Determination of the warfarin K_i specifically depends on measuring both substrate concentrations, both Michaelis constants for the VKORC1 enzyme, and pH in the assay.

Conclusion

The K_i is not equal to the IC₅₀ value directly measured using the DTT-driven VKOR assay.

General significance

In contrast to warfarin IC₅₀ values determined in previous studies, warfarin inhibition expressed as K_i can now be compared between studies, even when the specific DTT-driven VKOR assay conditions differ. This implies that warfarin inhibition reported for wild-type and variant VKORC1 enzymes from previous reports should be reassessed and new determinations of K_i are required to accurately report and compare in vitro warfarin inhibition results.     

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1) mediates vitamin K-dependent intracellular antioxidant function

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1), expressed in HEK 293T cells and localized exclusively to membranes of the endoplasmic reticulum, was found to support both vitamin K 2,3-epoxide reductase (VKOR) and vitamin K reductase enzymatic activities. Michaelis-Menten kinetic parameters for dithiothreitol-driven VKOR activity were: K(m) (μM) = 4.15 (vitamin K(1) epoxide) and 11.24 (vitamin K(2) epoxide); V(max) (nmol·mg(-1)·hr(-1)) = 2.57 (vitamin K(1) epoxide) and 13.46 (vitamin K(2) epoxide). Oxidative stress induced by H(2)O(2) applied to cultured cells up-regulated VKORC1L1 expression and VKOR activity. Cell viability under conditions of no induced oxidative stress was increased by the presence of vitamins K(1) and K(2) but not ubinquinone-10 and was specifically dependent on VKORC1L1 expression. Intracellular reactive oxygen species levels in cells treated with 2,3-dimethoxy-1,4-naphthoquinone were mitigated in a VKORC1L1 expression-dependent manner. Intracellular oxidative damage to membrane intrinsic proteins was inversely dependent on VKORC1L1 expression and the presence of vitamin K(1). Taken together, our results suggest that VKORC1L1 is responsible for driving vitamin K-mediated intracellular antioxidation pathways critical to cell survival.     

Probing for missing links in the binary and ternary V(V)-citrate-(H2O2) systems: Synthetic efforts and in vitro insulin mimetic activity studies

In a pH-specific fashion, V₂O₅ and citric acid in the absence and presence of H₂O₂ reacted and afforded, in the presence of NaOH and (CH₆N₃)₂CO₃, two new dinuclear V(V) binary non-peroxo (CH₆N₃)₆[V₂O₄(C₆H₄O₇)₂] · 2H₂O (1) and ternary peroxo (CH₆N₃)₄[V₂O₂(Ο₂)₂(C₆H₅O₇)₂] · 6Η₂Ο (2) species, respectively. Complexes 1 and 2 were further characterized by elemental analysis, UV/Vis, FT-IR, NMR (solution and solid state Cross Polarization-Magic Angle Spinning (CP-MAS)) and Raman spectroscopies, cyclic voltammetry, and X-ray crystallography. Both 1 and 2 are members of the family of dinuclear V(V)-citrate species bearing citrate with a distinct coordination mode and degree of deprotonation, with 2 being the missing link in the family of pH-structural variants of the ternary V(V)-peroxo-citrate system. Given that 1 and 2 possess distinct structural features, relevant binary V(III), V(IV) and V(V), and ternary V(V) species bearing O- and N-containing ligands were tested in in vitro cell cultures to assess their cellular toxicity and insulin mimetic capacity. The results project a clear profile for all species tested, earmarking the importance of vanadium oxidation state and its ligand environment in influencing further binary and ternary interactions of vanadium arising with variable mass cellular targets, ultimately leading to a specific (non)toxic phenotype and glucose uptake ability.