More on: endoplasmic reticulum loop VKORC1 substitutions cause warfarin resistance but do not diminish gamma‐carboxylation of the vitamin K‐dependent coagulation factors

Abstract

To cite this article: Harrington DJ, Siddiq S, Allford SL, Shearer MJ, Mumford AD. More on: endoplasmic reticulum loop VKORC1 substitutions cause warfarin resistance but do not diminish gamma-carboxylation of the vitamin K-dependent coagulation factors. J Thromb Haemost 2011; 9: 1093-5.See also Watzka M, Geisen C, Bevans CG, Sittinger K, Spohn G, Rost S, Seifried E, Mnller CR, Oldenburg J. Thirteen novel VKORC1 mutations associated with oral anticoagulant resistance: insights into improved patient diagnosis and treatment. J Thromb Haemost 2011; 9: 109-18.Watzka et al. [1] have recently reported the phenotype of patients with therapeutic resistance to 3-substituted-4-hydroxycoumarin oral anticoagulants (OACs) and with point mutations in VKORC1, which encodes vitamin K epoxide reductase subunit 1 (VKORC1). This study extends the repertoire of naturally occurring VKORC1 variants and provides valuable confirmation that VKORC1 is a molecular target of OACs. Watzka et al. also present a new topology model of VKORC1 and demonstrate that most mutations associated with OAC resistance predict substitutions within, or adjacent to, the VKORC1 endoplasmic reticulum (ER) loop (residues 30-79). This model also identifies the VKORC1 active site (CXXY; residues 132-135) that mediates de-epoxidation of vitamin K 2,3-epoxide (KO) to vitamin K (K) and the reduction of K to K hydroquinone (KH 2 ) and a putative 4-hydroxycoumarin binding motif (TYA; residues 138-140).Several groups have proposed that OACs inhibit VKORC1 reductase function by binding irreversibly at the VKORC1 active site to prevent the formation of transition state complexes that are formed normally during reduction of KO to K [2]. It is a plausible extension of this model to suggest that in OAC resistance, structural disruption of VKORC1 diminishes the affinity of OAC binding and thereby reduces this inhibition. Watzka et al. argue further that structural disruptions of VKORC1 sufficient to reduce OAC binding are also likely to reduce the binding of the KO transition state complexes and thereby, impair VKORC1 function. This is supported by previous data from ex vivo expression studies that indicate that most OAC-resistant VKORC1 variants showed a markedly impaired KO de-epoxidase activity compared with wild-type controls [1].However, some OAC-resistant ER-loop variants showed similar, or even increased, KO de-epoxidase function compared with wild-type controls [1]. Mutagenesis of the conserved Cys residues at 51 and 43 in VKORC1 did not diminish dithiothreitol-driven KO de-epoxidase and K reductase activities [3], although there was marked loss of both activities compared with wild-type VKORC1 when reduction was supported by thioredoxin oxidoreductase [4]. These data highlight continued uncertainty about the mechanism of OAC resistance and how structural variation in VKORC1 contributes to substrate and OAC interactions.We contribute to this debate by presenting phenotypic data from a subject with a previously unreported Ala34Pro substitution and two subjects with th...

Functional Study of the Vitamin K Cycle Enzymes in Live Cells

“…Results from the DGKO cell-based assay show that all these VKOR mutants are fully active (Tie et al, 2013). This is consistent with a recent clinical study showing that patients with warfarin-resistant VKOR mutations have normal VKD clotting factors in the absence of warfarin (Harrington, Siddiq, Allford, Shearer, & Mumford, 2011). Compared with the wild-type VKOR, five VKOR mutants showed increased warfarin resistance.…”

VKORC1-1639G/A and 1173 C/T Genetic Polymorphisms Influence Individual Differences in Warfarin Maintenance Dose

“…Thus, warfarin dose requirement is also related to genes encoding other proteins participating in the vitamin K cycle (Cabral et al, 2013;Solvik et al, 2014). VKORC1 is a key enzyme in vitamin K recycling and its inhibition prevents generation of reduced vitamin K, suppressing the activation of vitamin K-dependent coagulation factors (de Visser et al, 2011;Harrington et al, 2011).…”

Adaptative evolution of theVkorc1gene inMus musculus domesticusis influenced by the selective pressure of anticoagulant rodenticides

“…This variability being partly due to genetic polymorphisms in the Vkorc1 gene, numerous studies have been performed to detect new Vkorc1 mutations in patients resistant to AVK. These studies allowed to detect in humans 27 mutations in the coding sequence of the Vkorc1 gene (Bodin, Horellou, Flaujac, Loriot, & Samama, 2005; Bodin, Perdu, Diry, Horellou, & Loriot, 2008; D'Andrea et al., 2005; Harrington, Siddiq, Allford, Shearer, & Mumford, 2011; Harrington et al., 2008; Loebstein et al., 2007; Osman, Enström, Arbring, Söderkvist, & Lindahl, 2006; Peoc'h, Pruvot, Gourmel, Dit Sollier, & Drouet, 2009; Rieder et al., 2005; Rishavy, Usubalieva, Hallgren, & Berkner, 2011; Rost et al., 2004; Schmeits et al., 2010; Watzka et al., 2011; Wilms, Touw, Conemans, Veldkamp, & Hermans, 2008). In rats, 15 missense mutations in the Vkorc1 gene of Rattus norvegicus have been described in Europe (Grandemange, Lasseur, Longin‐Sauvageon, Benoit, & Berny, 2010; Haniza et al., 2015; Pelz et al., 2005, 2012; Rost et al., 2004).…”