Summary. Background: Vitamin K epoxide reductase subunit 1 (VKORC1) is the molecular target of coumarin anticoagulants and mutations in VKORC1 have been identified previously in individuals who required high warfarin doses. Objective: Detailed characterization of the relationship between variation in VKORC1 and the warfarin resistance phenotype. Patients and methods: Serum warfarin concentration and coagulation parameters were determined in 289 subjects who required warfarin doses >20 mg day )1. The VKORC1 sequence was studied in selected study subjects. Results: Twenty-eight out of 289 (10%) subjects had serum warfarin >2.3 mg L )1 during stable therapeutic anticoagulation indicating pharmacodynamic warfarin resistance. Detailed analysis of 15 subjects from this group showed that eight out of 15 (53%) had nucleotide substitutions in VKORC1 predictive of p.V66M, p.L128R, p.V54L or p.D36Y. VKORC1 was normal in the remaining seven out of 15 (47%) subjects and in nine out of nine (100%) subjects with high warfarin dose requirement not caused by pharmacodynamic resistance. At referral, subjects with VKORC1 mutations received a median warfarin dose of 32 mg day )1(range 22-55) and had a median serum warfarin concentration of 4.6 mg L )1 (range 2.6-9.0). VKORC1 substitutions were associated with a requirement for high warfarin doses but not with adverse clinical events. Family members with VKORC1 nucleotide substitutions and not receiving warfarin had undetectable PIVKA-II and K 1 epoxide (K 1 O). Conclusions: Nucleotide variations in VKORC1 are a common cause of pharmacodynamic warfarin resistance but are not associated with adverse outcome during anticoagulation. Mutations associated with warfarin resistance do not cause a discernible defect in VKORC1 reductase function.

1 The coumarin anticoagulant difenacoum was detected by high performance liquid chromatography (HPLC) with multi-wavelength UV detection in plasma from a 41 year old man who presented with a severe deficiency of vitamin K-dependent clotting factors of unknown aetiology. A longitudinal toxicological study of the consequent coagulopathy is described. 2 Plasma concentrations of difenacoum declined from 0.97 to 0.11 mgl-1 in 47 days with a terminal half life of 11.7 days. Rifampacin (300 mg bd) had no apparent effect on the terminal half life of the drug. Subsequently plasma concentrations of difenacoum and descarbox yprothrombin (DCP) unexpectedly increased. 3 Seven months after exposure clotting times were prolonged. The patient continued to have episodes of epistaxis, haematoma, purpurae and bruising and he required frequent treatment with Fresh Frozen Plasma in additional to oral phylloquinone (200 mg day-1). 4 Intermittent and unexpected increases in plasma concentrations of difenacoum and descarboxypro thrombin suggested that covert, repeated ingestion of the anticoagulant was the most likely cause of the poisoning. The measurement of low concentrations of plasma phylloquinone except following supervised ingestion ofthe vitamin indicated that as an outpatient, the subject was not compliant with treatment despite his protestations to the contrary. He continued to deny this even when confronted by laboratory findings and at no time did he ever admit to self-poisoning.

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...

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