Version May 2024
INTRODUCTION — Vitamin K is a cofactor for the enzymatic conversion of select glutamic acid (Glu) residues in vitamin K-dependent proteins into gamma-carboxyglutamic acid (Gla), which has important functional implications. This process is the target of vitamin K antagonist (VKA) anticoagulants.
The functions of Gla within coagulation factors and other proteins are reviewed here, along with synthesis of Gla and the role of vitamin K and vitamin K antagonists.
Separate topics discuss:
●The clotting cascade – (See "Overview of hemostasis".)
●Vitamin K dietary requirements and deficiency – (See "Overview of vitamin K".)
●VKA mechanism of action – (See "Biology of warfarin and modulators of INR control".)
FUNCTIONS OF Gla — Gamma-carboxyglutamic acid (Gla) has primarily been studied in the vitamin K-dependent blood coagulation factors (factors II [prothrombin], VII, IX, and X), proteins that regulate blood clotting (proteins C, S, and Z), and proteins of mineralized tissue (bone Gla protein and matrix Gla protein). Several other proteins that contain Gla domains of unknown function have been identified, including Gas6 and others. (See 'Protein substrates for gamma-carboxylation' below.)
Timeline of discoveries
●Vitamin K – Vitamin K and its association with blood clotting were initially described in the 1920s and 1930s, following investigation of a hemorrhagic disease of cattle caused by ingestion of spoiled sweet clover [1]. A similar phenomenon was observed in chickens [2]. The vitamin K antagonist (VKA) activity was purified in 1941 [3]. (See 'VKA inhibition of VKOR' below.)
Subsequent studies led to the proposal of a mechanism of vitamin K-mediated enhancement of carboxylase action and regulation of enzymatic vitamin K epoxidase activity by glutamate-containing substrate [4,5].
●Gla and the carboxylase – In the 1970s, Gla was discovered in blood clotting proteins and determined to confer metal-binding properties required for membrane-protein interactions of certain clotting factors [6-9].
The enzyme activity of the vitamin K-dependent gamma-glutamyl carboxylase was reported in 1975, and the GGCX gene was published in 1991 [10,11]. Subsequent studies characterized the structure of the carboxylase enzyme and features of vitamin K-dependent proteins including the propeptide and gamma-carboxylation recognition site [10-16]. (See 'Gamma-glutamyl carboxylase enzyme' below.)
This carboxylase activity is found in essentially all mammalian tissues, and its reaction product, Gla, has been observed in both vertebrates and invertebrates [17].
●VKOR – The vitamin K recycling enzyme (vitamin K epoxide reductase [VKOR]), target of the VKA anticoagulants, and the VKORC1 gene, were published in 2004 [18,19]. Subsequent studies characterized the enzyme structure and mechanism of action. [20,21]. (See 'Vitamin K recycling by VKOR' below.)
●FSP1 – In 2022 and 2023, the FSP1 protein, encoded by the FSP1 gene, was identified as the primary warfarin-independent vitamin K reductase, enabling rescue of VKA toxicity and suppressing ferroptosis [22,23]. (See 'Ferroptosis suppressor 1 (FSP1) and other enzymes that can reduce vitamin K' below.)
Function of Gla in clotting proteins — Clotting proteins circulate in an inactive form and become activated at sites of hemostasis. The main function of Gla domains in the vitamin K-dependent clotting proteins is to facilitate their localization to phospholipid membranes, which is essential to turning on their procoagulant activity. (See 'Protein substrates for gamma-carboxylation' below and "Overview of hemostasis", section on 'Multicomponent complexes'.)
Procoagulant factors (factors II, VII, IX, and X)
●Factor II [prothrombin] is the precursor to thrombin. (See "Overview of hemostasis", section on 'Thrombin generation'.)
●Factors VII, IX, and X contribute to thrombin generation. (See "Overview of hemostasis", section on 'Clotting cascade and propagation of the clot'.)
Natural anticoagulants (proteins C, S, and Z)
●Activated protein C (aPC) inactivates coagulation factors Va and VIIIa. Protein S enhances aPC activity. (See "Protein C deficiency", section on 'Biology of protein C' and "Protein S deficiency", section on 'Biology of protein S'.)
●Protein Z and a protein Z-dependent protease inhibitor (ZPI) appear to serve as cofactors for the inhibition of activated factors X and XI [24-26]. ZPI inhibition of factor Xa bound to phospholipid membranes, but not in solution, is dependent upon an interaction between the Gla domains of protein Z and factor Xa [27]. Deficiencies of protein Z or ZPI, as with deficiencies of proteins C and S, might therefore result in a prothrombotic state or might rebalance bleeding phenotypes in hereditary hemostatic disorders [28-34].
Phospholipid membrane binding — The vitamin K-dependent clotting proteins contain 10 to 12 Gla residues in the Gla domain, which is located within the first 40 residues of the N-termini of the mature proteins. The surface of the Gla domain, in association with the adjacent aromatic amino acid stack domain, functions as a membrane-binding component of these proteins.
The following observations emphasize the importance of phospholipid binding to the function of these clotting proteins:
●Warfarin causes vitamin K-dependent proteins to be undercarboxylated, having Glu residues at some or all of the positions that are usually carboxylated to Gla. These proteins are either biologically inert or are misfolded in the endoplasmic reticulum and degraded by quality control pathways (protein C) or otherwise not secreted (protein S). In patients treated with warfarin, circulating coagulant activity correlates closely with the quantity of fully carboxylated proteins remaining in the circulation [35]. (See 'VKA inhibition of VKOR' below.)
●Gla differs from aspartic acid and glutamic acid (Glu) in containing two carboxyl groups in its side chain. This bivalency enables the formation of the calcium-carboxylate network that stabilizes the Gla domains and, in the vitamin K-dependent clotting proteins, allows expression of the phospholipid binding site. In most cases, neither aspartic acid nor Glu, nor a mutated form of the protein that cannot be gamma-carboxylated, will substitute for the function of Gla, emphasizing the importance of both carboxyl groups on a single amino acid [36-39].
●Structural analysis of Gla-containing vitamin K-dependent clotting proteins and regulators show that the Gla domain is highly structured and share nearly identical calcium binding sites in the internal core [40-43]. Many of the Gla side chains point inward to a linear array of internal calcium ions, several of which are completely sequestered inside the core of the Gla domain [16].
Gla domains bind specifically to membrane phosphatidylserine. The structural basis for this comes from crystallographic structure of the lysophosphatidylserine binding site in the bovine prothrombin Gla domain, which demonstrated that the serine head group binds Gla domain-bound calcium ions and Gla residues 17 and 21, which are fixed elements of the Gla domain fold [44].
An x-ray structure had shown unexpected exposure of three hydrophobic residues on the surface of these proteins, suggesting a potential role for these residues in membrane interaction [16]. Site-specific mutagenesis of homologous residues in protein C interfered with membrane-binding properties, but mutation of other hydrophobic residues did not perturb membrane binding significantly [45].
Calcium-induced conformational change — Gla domains provide a unique mechanism for protein-phospholipid membrane interaction in the presence of calcium. With the addition of calcium ions, the vitamin K-dependent clotting proteins undergo a structural transition that leads to exposure of a phospholipid binding site. Calcium-induced exposure of hydrophobic amino acids in the Gla domain is critical for membrane binding [46]. Direct comparison of the structures of the calcium-stabilized form of factor IX, which binds to membranes, and the magnesium-stabilized form of factor IX, which does not, implicated residues 1 to 11 that form a loop in the Gla domain [47].
Correlation of membrane-binding properties of the vitamin K-dependent proteins with homology considerations suggests a possible alternative membrane contact site that involves residues 11, 33, and 34 [48]. A molecular dynamics simulation study of anionic phospholipid binding to the Gla domain of bovine fragment 1 in the presence of calcium ions also suggests a phosphatidylserine binding site with the head group bound by a Gla-bound calcium ion, Gla 30 and Lys 11 [49].
Function of Gla in bone, connective tissue, and vascular proteins — The function of Gla-containing proteins in mineralized tissue, including osteocalcin and matrix Gla protein, is less well defined than for the clotting proteins. Insights from experimental animal models have suggested the following possible functions:
●Bone remodeling – Osteocalcin-deficient mice are characterized by increased bone formation, including higher bone mass and bones of improved functional quality [50]. (See "Normal skeletal development and regulation of bone formation and resorption".)
●Limiting calcification of extraosseous tissues – Matrix Gla protein-deficient mice suffer from spontaneous and ultimately fatal calcification of arteries and cartilage [51], suggesting that one of the functions of this protein is to control and limit extraosseous calcification. Decreased carboxylation of matrix Gla protein following warfarin treatment causes rapid arterial and aortic valve calcification in rats and mice [52,53].
A similar phenotype has been observed in humans with Keutel syndrome, which is caused by pathogenic variants in the BGLAP gene, which encodes matrix Gla protein and is characterized by abnormal cartilage calcification [54]. The Gla residues in matrix Gla protein prevent osteogenic differentiation and calcification via binding and inhibition of bone morphogenetic protein-4, as well as via binding to vascular smooth muscle cell-derived vesicles [55,56].
A Gla-rich protein (GRP) of unknown function is expressed in chondrocytes, chondroblasts, osteocytes and osteoblasts; in animals the highest levels are found in bone and cartilage [57]. GRP contains the highest Gla content of any known protein (16 of 74 residues) and is not homologous to any other known Gla-containing proteins. It was originally identified in the calcified cartilage of the Adriatic sturgeon and has orthologs in all vertebrates [57].
There is no evidence to support vitamin K administration in patients with osteoporosis or other interventions in patients receiving vitamin K antagonists (VKAs). The relationship of VKA use and bone health is discussed separately. (See "Drugs that affect bone metabolism", section on 'Anticoagulants' and "Overview of the management of low bone mass and osteoporosis in postmenopausal women", section on 'Therapies not recommended'.)
Gla proteins of unknown function
●Gas6 – Gas6 (growth-arrest-specific gene 6) has marked sequence homology in the Gla domain to the vitamin K-dependent blood coagulation and regulatory proteins, in particular protein S [58]. Gas6 is released from and potentiates the growth of vascular smooth muscle cells. When synthesized in the presence of warfarin and therefore lacking Gla, Gas6 demonstrates no thrombin-inducible growth potentiating activity or receptor binding ability [59]. In contrast, Gas6 lacking the entire Gla domain is a functional growth factor. This observation suggests that the Gla domain may be a negative regulator of the structure of a growth factor domain located elsewhere on the molecule.
●Vitamin K-dependent single-pass integral membrane proteins – This family of four proteins was identified by searching a database with a consensus sequence derived from analysis of known Gla domains. They all contain the PPXY motif involved in diverse cellular functions. There are two potential subclasses based on their gene organization and protein sequence.
•Proline rich Gla proteins PRGP1 and PRGP2 [60].
•Transmembrane Gla proteins TMG3 and TMG4 [61]. The gene for TMG4 is one of those deleted in the 11p14-p12 chromosome region deletion associated with WAGR syndrome (Wilms tumor, Aniridia, Genitourinary anomalies, and Range of developmental delays) and falls into the linkage region of 11p13-p12 as an autism candidate gene [62]. (See "Microdeletion syndromes (chromosomes 1 to 11)", section on '11p13 deletion syndrome (WAGR syndrome)'.)
●Endoplasmic reticulum Gla protein – This gamma-carboxylated calcium-binding protein was identified in the endoplasmic reticulum of pancreatic beta-cells [63]. Gamma-carboxylation of regulates calcium homeostasis in beta-cells and is critical for their capacity to adapt insulin secretion to metabolic needs. Glucose upregulates carboxylase activity.
Gla SYNTHESIS — Gamma carboxyglutamic acid (Gla) is an alternative amino acid created by modification of the amino acid glutamic acid (Glu, also called glutamate).
●Gamma-glutamyl carboxylase (GGCX) – GGCX is the enzyme that catalyzes the conversion of Glu to Gla. (See 'Gamma-glutamyl carboxylase enzyme' below.)
●Vitamin K – Vitamin K is a cofactor in the carboxylation reaction. (See 'Vitamin K requirement' below.)
Protein substrates for gamma-carboxylation — The following proteins require gamma carboxylation of glutamic acid residues to produce gamma-carboxyglutamic acid (Gla):
Gla residues in the vitamin K-dependent clotting factors and natural anticoagulants facilitate membrane binding. (See 'Function of Gla in clotting proteins' above.)
●Coagulation factors – These are the vitamin K-dependent clotting factors.
•Factor II (prothrombin)
•Factor VII
•Factor IX
•Factor X
●Proteins that regulate coagulation – These are natural anticoagulants.
•Protein C
•Protein S
•Protein Z
●Bone and matrix proteins
•Osteocalcin (also called bone Gla protein)
•Matrix Gla protein
•Gla-rich protein
These proteins may function in bone remodeling or preventing calcification of extraosseous tissues and vasculature. (See 'Function of Gla in bone, connective tissue, and vascular proteins' above.)
●Others – Functions of additional Gla proteins are unclear. (See 'Gla proteins of unknown function' above.)
•Gas6
•Vitamin K-dependent single-pass integral membrane proteins
•Endoplasmic reticulum Gla protein
●Carboxylase – The GGCX enzyme itself has Glu residues in a region of sequence homology with the region of matrix Gla protein containing the gamma-carboxylation recognition site [64]. Under some conditions, the carboxylase can become carboxylated, although the role of Gla in this enzyme remains to be determined [65]. (See 'Gamma-glutamyl carboxylase enzyme' below.)
●Cone snail conotoxin – Only one invertebrate, the cone snail, has been found to contain Gla in toxins used to sting prey (conotoxins); many of the conotoxins are channel blockers [66-69]. (See "Marine envenomations from corals, sea urchins, fish, or stingrays", section on 'Cone snail'.)
Gamma-carboxylation recognition site
●Clotting factors and regulatory proteins – The propeptide region of the vitamin K-dependent clotting proteins (factors II [prothrombin], VII, IX, and X) and the regulatory proteins (proteins S, C, and Z) functions as a recognition sequence, binding the carboxylase to its substrate on the adjacent glutamic acid rich domain (referred to as a "Gla domain") [12,13]. It also stimulates gamma-glutamyl carboxylation [70,71].
●Bone and matrix Gla proteins – For the matrix Gla protein, the gamma-carboxylation recognition site resides within the mature protein sequence itself in matrix Gla protein [72]
The propeptide of osteocalcin (bone Gla protein) is unable to support carboxylation of blood coagulation factors [73]; this suggests that the previously established determinants of clotting factor carboxylation may not apply to all Gla proteins.
The vitamin K-dependent carboxylase binds directly to the amino acids of the propeptide recognition site [74]. Although no consensus sequence prevails in the carboxylation recognition sites, these sites are best defined by a Z-F-Z-X-X-X-X-A motif, where Z is an aliphatic hydrophobic residue (isoleucine, valine, or leucine), F is phenylalanine, A is alanine, and X is any amino acid. Phenylalanine at residue 16 is preferred in carboxylase substrates, but leucine, valine, and lysine at this position also support carboxylation [75].
Evidence from point mutations illustrates how the carboxylation recognition site in the propeptide region of vitamin K-dependent clotting factors is required for gamma carboxylation [12,13,76-81].
This carboxylation site region in clotting factor precursors likely docks with the membrane-bound carboxylase, bringing the active site of the carboxylase in proximity to the substrate Glu residues on the clotting factor precursor.
A protein will likely undergo gamma-carboxylation if it meets the following criteria:
●It has a gamma-carboxylation recognition site that interacts with the carboxylase
●Glu residues are present within 40 residues of the recognition site
●The cell contains reduced vitamin K
●The cell has the carboxylase enzyme associated with the rough endoplasmic reticulum (RER), and the protein is routed through the RER during biosynthesis
Gamma-glutamyl carboxylase enzyme
GGCX enzyme function — The gamma-glutamyl carboxylase (GGCX, also called the vitamin K-dependent carboxylase) is an integral membrane protein that converts glutamic acid (Glu) amino acid residues to Gla (figure 1) [10,82].
Cofactors required for the carboxylation reaction include:
●Carbon dioxide.
●Molecular oxygen.
●Vitamin K in the hydroquinone form. (See 'Vitamin K requirement' below.)
The enzyme catalyzes two coupled reactions [4,83,84]:
●It promotes the formation of Gla on the Glu residues.
●It promotes the formation of the highly reactive alkoxide, which then collapses into the epoxide form of vitamin K.
The enzyme has the following features:
●A carboxylase active site.
●An epoxidase active site.
●A propeptide binding site (shares sequence similarity with the propeptide of the carboxylase substrate) [85].
●A propeptide binding site that stimulates carboxylase and epoxidase activity.
●A glutamate binding site [86].
●A vitamin K binding site.
The human carboxylase gene, GGCX, is a 15-exon gene located on chromosome 2p12 [87]. Variants in GGCX can cause a bleeding disorder due to lack of vitamin K-dependent clotting factors. (See 'Genetic deficiency of GGCX (vitamin K-dependent clotting factor deficiency type I)' below.)
Intracellular localization — GGCX is an integral membrane protein localized to the endoplasmic reticulum and Golgi apparatus. Immunofluorescence studies using anti-GGCX antibodies confirm the carboxylase resides in these organelles.
Studies in Chinese hamster ovary cells expressing prothrombin showed that uncarboxylated pro-prothrombin is completely gamma-carboxylated in the endoplasmic reticulum [88]. The carboxylated pro-prothrombin leaves the endoplasmic reticulum intact and is further processed in the Golgi apparatus to remove the propeptide to create prothrombin. (See 'Protein substrates for gamma-carboxylation' above.)
Many substrates of GGCX are extracellular proteins (clotting factors and natural anticoagulants).
●Prothrombin and the other gamma-carboxylated extracellular vitamin K-dependent proteins bind to negatively charged phospholipids in the presence of calcium ions.
●Fully carboxylated pro-factor IX does not bind to membranes in the presence of calcium ions, whereas mature factor IX does [89]. Thus, the propeptide attached to factor IX appears to prevent proper folding of the Gla domain, expression of the phospholipid binding site, and the interaction of pro-factor IX with membranes.
VITAMIN K REQUIREMENT
Reduced vitamin K (KH2) is the cofactor for GGCX — Vitamin K in the reduced/hydroquinone form (vitamin KH2) is a cofactor for the gamma glutamyl carboxylase (GGCX) enzyme. GGCX is the only enzyme that requires vitamin K.
The mechanism by which vitamin K participates as a cofactor for the GGCX enzyme is incompletely understood. An attractive hypothesis is that an activated form of vitamin K extracts a hydrogen atom from the gamma carbon of Glu residues on specific proteins (see 'Protein substrates for gamma-carboxylation' above), in a reaction that converts the vitamin K to vitamin K epoxide.
Carbon dioxide is subsequently added to the gamma-carbon of Glu by the gamma-glutamyl carboxylase to form Gla (figure 1) [90]. For each molecule of Gla generated, one molecule of vitamin K epoxide is also formed [84,91].
A "base strength amplification mechanism" has been proposed to explain the conversion of the weak base form of vitamin K (vitamin KH2) into an oxygenated intermediate (the alkoxide) of sufficient basicity to abstract a hydrogen from the gamma-carbon of Glu (figure 1) [5].
It was initially proposed that a free cysteine in GGCX was responsible for deprotonation of vitamin KH2, a hypothesis that is supported by numerous publications [83,92-101]. However, other studies question the role of a free cysteine residue in carboxylation and implicate two lysine residues in the gamma-glutamyl carboxylase as important for catalysis [102,103].
The short-lived highly reactive alkoxide is potentially toxic, and it would be undesirable for it to be generated in the absence of Glu residues. Evidence indicates that no highly reactive vitamin K intermediate is generated by GGCX until a carboxylase substrate is bound to the enzyme and converts its vitamin K epoxidase function to an active state [4]. The epoxide is then recycled back to vitamin KH2 via a vitamin K epoxide reductase (VKOR). (See 'Vitamin K recycling by VKOR' below.)
Vitamin K recycling by VKOR — Vitamin K can occupy three distinct redox states:
●Fully oxidized (two disulfides): vitamin K epoxide
●Partially oxidized (one disulfide): vitamin K quinone
●Reduced (no disulfides): vitamin K hydroquinone (KH2)
Only a small fraction (5.6 percent) of vitamin K is in a fully reduced (active cofactor) state, with the remainder split almost equally between partially oxidized and fully oxidized [104].
Vitamin K hydroquinone (vitamin KH2), the most reduced form of vitamin K, is the active cofactor for the gamma-glutamyl carboxylase. During the conversion of Glu to Gla, vitamin KH2 is oxidized to vitamin K epoxide, the most oxidized form of vitamin K [105]. (See 'Gla synthesis' above.)
Gamma carboxylation causes oxidation of vitamin K. The vitamin must be recycled from the inactive (oxidized) to the active (reduced) form of the cofactor after every gamma carboxylation reaction. (See 'GGCX enzyme function' above.)
●VKOR – The enzyme vitamin K epoxide reductase (VKOR) catalyzes the sequential reduction of vitamin K epoxide to vitamin K quinone (the naturally occurring form of the vitamin) and then further to vitamin KH2, completing the reductive arm of the vitamin K cycle. These reductions are both inhibited by warfarin and other VKAs. (See 'VKA inhibition of VKOR' below.)
VKOR is effective at low concentrations of vitamin K epoxide and vitamin K quinone and is the physiologically important enzyme for recycling vitamin K [106,107].
VKOR is a 163 amino acid protein encoded by the VKORC1 gene, located on chromosome 16 [18,19]. It is most highly expressed in the liver (the site of vitamin K-dependent clotting factor production) and is also detected in other tissues.
●Sequence features – Alignment of functional VKORs reveals five absolutely conserved residues across all species, including two pairs of cysteine residues (C43 and C51, C132 and C135) and a serine (S57). The C132 and C135 cysteine pair contributes a canonical CXXC motif integral to many cellular redox reactions [108].
●Three-dimensional structure – Crystal structures of VKOR and its homologues in other species demonstrate a shared structure in which four transmembrane segments surround a central vitamin K molecule with its redox active quinone ring oriented to the C132-X-X-C135 motif [20,109].
An extracellular loop between transmembrane segments one and two forms a cap over the quinone binding pocket and contains the second pair of conserved cysteines (C43 and C51) [20].
Vitamin K epoxide and quinone bind preferentially to the fully reduced and partly oxidized states, with covalent or near covalent interaction between the C135 thiolate that enables electron transfer to vitamin K [20].
●Source of reducing equivalents – The source of reducing equivalents for VKOR is of great interest. Early in vitro studies used reducing agents such as dithiothreitol (DTT), which can directly reduce the active site cysteines. Lipoic acid and most dithiols show some activity in vitro [110]. Glutathione is an important redox buffer in the endoplasmic reticulum [111].
Studies of bacterial and plant VKOR homologs that catalyze disulfide bond formation in secreted proteins may shed light on the mechanism of VKOR activity [109,112]
•Bacterial VKOR homologs that are naturally fused to an extracellular (periplasmic) thioredoxin-like domain containing a CXXC motif as the redox partner have been crystallized, and the crystal structure of the complex in an arrested state of electron transfer between the thioredoxin-like domain and the VKOR homolog has suggested an electron transfer pathway that could apply to other VKOR homologs [109,112].
•Newly synthesized proteins in the periplasm reduce the CXXC motif of a thioredoxin-like protein, which in turn reduces the conserved disulfide bridge in the extracellular loop of VKOR; the loop cysteines then reduce the conserved CXXC motif of VKOR and, finally, the disulfide in this CXXC motif is reestablished by reduction of the quinone [109]. Mammalian VKOR likely functions similarly as a parallel or redundant system to catalyze oxidative protein folding in the endoplasmic reticulum [113].
•Thioredoxin domain containing proteins from bacteria can also support mammalian VKOR-catalyzed reduction of vitamin K epoxide or quinone, and endoplasmic reticulum anchored thioredoxin-like protein TMX forms an identical mixed-disulfide intermediate with human VKOR [21,114]. The extent to which these mechanisms contribute to redox reactions relative to glutathione in vivo remains to be determined.
●VKOR-like protein – VKOR-like protein is a distinct protein encoded by the VKORC1L1 gene on chromosome 7. This enzyme also catalyzes reduction of vitamin K epoxide and vitamin K quinone to vitamin KH2. It shares all of the essential catalytic residues with VKOR and based on structural studies, it appears to adopt the same overall three-dimensional configuration. It is expressed at low levels across tissues but not enriched in liver, which may explain its inability to compensate for VKOR deficiency. (See 'Genetic deficiency of VKOR (vitamin K-dependent clotting factor deficiency type II)' below.)
VKOR-like protein may significantly contribute to the gamma-carboxylation of extrahepatic vitamin K-dependent proteins with roles beyond coagulation.
Ferroptosis suppressor 1 (FSP1) and other enzymes that can reduce vitamin K — VKOR and VKOR-like protein are the only enzymes known to reduce vitamin K epoxide, but other enzymes are able to reduce vitamin K quinone to the hydroquinone form. These include NQO1 (DT-diaphorase), an NAD(P)H dehydrogenase, and the warfarin-independent, NAD(P)H-dependent vitamin K-reductase encoded by FSP1 [22,23].
These enzymes, particularly FSP1, enable excess vitamin K to restore gamma-carboxylation in the setting of VKA excess. (See 'Rescue of VKA toxicity by vitamin K' below.)
Ferroptosis is a form of cell death due to iron-induced lipid peroxidation that has been implicated in several neurodegenerative disorders, cancer, and ischemia-reperfusion injury. (See "Regulation of iron balance", section on 'Iron and the brain'.)
The ferroptosis suppressor protein FSP1 also acts as a vitamin K reductase, although it cannot reverse all steps of vitamin K antagonist (VKA) activity. (See 'Rescue of VKA toxicity by vitamin K' below.)
One or more additional warfarin-independent vitamin K reductases have yet to be identified [22].
Vitamin K antagonist (VKA) mechanisms of action
VKA inhibition of VKOR — Warfarin and other vitamin K antagonist (VKA) anticoagulants block the vitamin K epoxide reductase (VKOR), preventing vitamin K recycling. This in turn produces nonfunctional vitamin K-dependent clotting factors. (See 'Vitamin K recycling by VKOR' above and "Biology of warfarin and modulators of INR control", section on 'Mechanism of action'.)
Warfarin shares a common ring structure with vitamin K.
●Warfarin and other VKAs bind to many of the same key residues of VKOR as vitamin K and induce the same conformational changes required for the VKOR catalytic cycle [20]. This results in insufficient generation of vitamin K hydroquinone to support full carboxylation and therefore full function of the vitamin K-dependent proteins of blood coagulation.
●Warfarin preferentially binds to the fully oxidized (two disulfides) and partially oxidized (one disulfide) states of VKOR [115]. Warfarin binding triggers closure of the cap, sealing VKOR in the closed state [20].
●Vitamin K epoxide is not detectable in normal plasma, even after a pharmacologic dose of 10 mg of vitamin K [116]. However, it is measurable following the administration of warfarin [117,118]. The finding in warfarin-treated patients of a significant positive correlation between the degree of anticoagulation and plasma vitamin K epoxide concentrations suggests that this mechanism reflects the pharmacodynamic activity of warfarin in anticoagulated patients [119].
Rescue of VKA toxicity by vitamin K — Vitamin K is a logical, widely used, and effective antidote to VKA overdose [120]. (See "Management of warfarin-associated bleeding or supratherapeutic INR", section on 'Vitamin K dose, route, formulation'.)
Because the effect is not immediate, individuals with bleeding or requiring emergency surgery require a source of pre-formed vitamin K-dependent clotting factors. (See "Management of warfarin-associated bleeding or supratherapeutic INR", section on 'Treatment of bleeding'.)
The bypass mechanism by which vitamin K reverses warfarin anticoagulation may be partly attributable to the NAD(P)H-dependent reductase NQO1 (DT-diaphorase) [121]; however, this enzyme requires high concentrations of vitamin K and probably does not play a major role at physiologic tissue concentrations of vitamin K [106]. NQO1-deficient mice treated with warfarin can be rescued with vitamin K, suggesting this is not the essential warfarin-independent vitamin K reductase [122].
Major insights emerge from the discovery that the NAD(P)H quinone oxidoreductase ferroptosis suppressor protein 1 (FSP1) is an efficient vitamin K reductase [22,23]. (See 'Ferroptosis suppressor 1 (FSP1) and other enzymes that can reduce vitamin K' above.)
●FSP1 is not sensitive to inhibition by warfarin, and the prolonged prothrombin time (PT) in warfarin-treated FSP1-deficient mice is not rescued by administration of high dose vitamin K [23].
●FSP1, therefore, acts parallel to VKOR and catalyzes the major warfarin-resistant pathway enabling reduction of vitamin K quinone to the hydroquinone form required for gamma-carboxylation. FSP1 is unable to reduce vitamin K epoxide to enable vitamin K recycling, but it provides an essential mechanism to bypass VKA toxicity and preserve blood coagulation [22,23]. This is an important observation that explains why high dose vitamin K can rescue or reverse warfarin overdose. (See 'Poisoning by rodenticide superwarfarins' below.)
CLINICAL RELEVANCE — Deficiency of vitamin K-dependent clotting factors due to pathogenic variants affecting the gamma-glutamyl carboxylase enzyme (GGCX gene) or the vitamin K recycling enzyme (VKOR gene) cause a heritable bleeding disorder with reduced factor activity levels and prolongation of the prothrombin time (PT) and activated partial thromboplastin time (aPTT). (See "Rare inherited coagulation disorders", section on 'Multiple vitamin K-dependent factor deficiencies'.)
Pathogenic variants in GGCX
Genetic deficiency of GGCX (vitamin K-dependent clotting factor deficiency type I) — Pathogenic variants affecting the gamma-glutamyl carboxylase gene (GGCX) have been described in a number of kindreds with vitamin K-dependent clotting factor deficiency [VKDCFD] type I, with deficiency of all vitamin K-dependent coagulation factors and a heritable bleeding disorder [123-125]. (See "Rare inherited coagulation disorders", section on 'Multiple vitamin K-dependent factor deficiencies'.)
A similar disorder has been found in the Devon Rex cat [126]. Mice carrying a null mutation of the carboxylase gene die at birth of massive intraabdominal hemorrhage [127].
In most of the affected humans and in cats, activity of the vitamin K-dependent coagulation factors can be normalized by vitamin K supplementation [126,128-130]. This normalization of activity is possible because the effect of most of the reported pathogenic variants is to weaken the binding of vitamin K to the enzyme. As basal vitamin K concentrations are below the Km of the carboxylase for vitamin K hydroquinone, increasing the concentration of vitamin K raises the levels of the vitamin K-dependent coagulation factors.
Syndromes with aberrant calcification — Some pathogenic variants in GGCX cause aberrant calcification in addition to a mild bleeding disorder, consistent with a role for the carboxylase in preventing calcification of the vasculature [131-134]. (See 'Function of Gla in bone, connective tissue, and vascular proteins' above.)
Patients with a pseudoxanthoma elasticum-like syndrome may have excessive skin folding. Skin biopsies indicate disruption of elastin fibers, abundant calcification, and decreased matrix Gla protein carboxylation. (See "Pseudoxanthoma elasticum".)
Most of the variants causing these diseases are in uncharacterized regions of GGCX. (See 'GGCX enzyme function' above.)
Pathogenic variants in VKORC1
Genetic deficiency of VKOR (vitamin K-dependent clotting factor deficiency type II) — Vitamin K-dependent clotting factor deficiency (VKDCFD) type II is a rare autosomal recessive disorder caused by loss-of-function variants in the VKORC1 gene, which encodes the vitamin K reducing enzyme vitamin K epoxide reductase (VKOR) [128,135-137]. (See 'Vitamin K recycling by VKOR' above and "Rare inherited coagulation disorders", section on 'Multiple vitamin K-dependent factor deficiencies'.)
All known cases VKDCFD type II are caused by an Arg98Trp substitution that disrupts endoplasmic reticulum localization of VKOR and leads to its premature degradation [138]. Many patients present with perinatal intracranial hemorrhage or mucosal or soft tissue bleeding in childhood, but rare individuals do not come to clinical attention until adulthood [135,139].
Levels of factors II, VII, IX, and X are reduced to approximately 20 to 60 percent of normal, as reflected by prolongation of the PT and aPTT [139].
The disorder can be fully corrected by supplementation with daily oral vitamin K in most patients [136].
VKDCFD types I and II can be distinguished by genetic testing and/or by accumulation of vitamin K epoxide after vitamin K supplementation in patients with VKDCFD type II [136].
Vascular disease associated with VKORC1 variants — Variants in VKORC1 may also be associated with increased risk for arterial vascular disease [140]. Missense mutation of VKORC1 coupled with limited vitamin K ingestion in rats leads to areas of massive vascular calcification associated with increased expression of uncarboxylated matrix Gla protein [141]. Similar observations were made in mice deficient in matrix Gla protein [51].
Hereditary warfarin resistance — Missense mutations in VKORC1 have been proposed to be involved in resistance to warfarin and other vitamin K antagonist (VKA) anticoagulants [18,142-145].
Warfarin resistance maps to variants in two regions of the VKOR enzyme and to the VKORC1 promotor [18,20]:
●Quinone binding pocket – This is the region where Asn80 and Tyr139 (the commonly mutated residue that confers rodenticide resistance in rats) form hydrogen bonds with warfarin.
●Cap – The cap that seals the quinone/VKA binding site (or residues interacting with the cap) are sites of warfarin binding. The cap contains a pair of conserved cysteines (C43 and C51). (See 'Vitamin K recycling by VKOR' above.)
●Promotor – Common variants within the VKORC1 gene such as the promotor polymorphism -1639G>A influence VKOR expression and the mean daily dose of warfarin required to acquire target anticoagulation intensity [146].
Despite the role of genotype in warfarin sensitivity, randomized trials that evaluated genotyping to determine the initial dosing of warfarin did not demonstrate improved outcomes with genotype-guided dosing [147]. (See "Warfarin and other VKAs: Dosing and adverse effects", section on 'Baseline testing' and "Overview of pharmacogenomics", section on 'Warfarin and VKORC1 polymorphisms'.)
Factors that affect warfarin dosing are discussed separately. (See "Warfarin and other VKAs: Dosing and adverse effects", section on 'Warfarin resistance'.)
Poisoning by rodenticide superwarfarins — An extreme case of VKA toxicity is poisoning by "superwarfarins"; these rodenticides are VKOR inhibitors that are two orders of magnitude more potent than warfarin and have half-lives measured in weeks. (See "Overview of rodenticide poisoning", section on 'Anticoagulants (superwarfarins and warfarins)'.)
Tight binding of superwarfarins to VKOR is enhanced by insertion of bulky substituents into the hydrophobic groove otherwise occupied by the long isoprenoid tail of vitamin K [20].
Treatment with high doses of vitamin K quinone for several months or years is often required to reverse the coagulopathy associated with superwarfarin poisoning [148]. The exact dose of vitamin K required must be determined empirically and titrated to ensure a normal prothrombin time (PT); in most cases of poisoning by the superwarfarin brodifacoum, typical total daily doses of vitamin K can range from 25 mg to 100 mg [149]. This likely bypasses VKOR via FSP1 to ensure availability of the hydroquinone KH2. (See "Management of warfarin-associated bleeding or supratherapeutic INR", section on 'Superwarfarin poisoning'.)
SUMMARY
●Functions of Gla – In the presence of calcium ions, the amino acid gamma-carboxyglutamic acid (Gla) in Gla domains undergo a conformational change that exposes a phospholipid binding site and allows binding to membrane phosphatidylserine. The resulting membrane localization is essential to the function of vitamin K-dependent clotting proteins and natural anticoagulants. Gla-containing proteins in mineralized tissue may contribute to bone remodeling and may prevent calcification of extraosseous tissues by a less-well understood mechanism. Functions of Gla in other proteins is unclear. (See 'Functions of Gla' above and "Overview of hemostasis", section on 'Clotting cascade and propagation of the clot' and "Overview of hemostasis", section on 'Control mechanisms and termination of clotting'.)
●Conversion of Glu to Gla – The amino acid glutamate (Glu, also called glutamic acid) is converted to gamma-carboxyglutamic acid by a vitamin K-dependent carboxylase enzyme. Substrates include vitamin K-dependent clotting factors (II [prothrombin], VII, IX, and X), natural anticoagulants (proteins C, S, and Z), bone and matrix proteins, and other proteins of unknown function. (See 'Gla synthesis' above and 'Protein substrates for gamma-carboxylation' above.)
•Carboxylase – The carboxylase is an integral membrane protein. Cofactors include carbon dioxide, molecular oxygen, and the hydroquinone form of vitamin K (figure 1). (See 'Gamma-carboxylation recognition site' above and 'Gamma-glutamyl carboxylase enzyme' above.)
•Vitamin K – For each molecule of Gla generated, one molecule of vitamin K is oxidized to vitamin K epoxide. Vitamin K epoxide reductase (VKOR) reduces vitamin K epoxide to vitamin K quinone (intermediate form) and vitamin K hydroquinone (KH2, the most reduced form and active cofactor for GGCX). (See 'Vitamin K requirement' above and 'Vitamin K recycling by VKOR' above.)
Dietary vitamin K requirement and food sources are discussed separately. (See "Overview of vitamin K".)
•VKAs – Vitamin K antagonists (VKAs) such as warfarin inhibit VKOR, resulting in dysfunctional vitamin K-dependent clotting proteins. FSP1 is a warfarin-independent vitamin K reductase that enables generation of sufficient amounts of reduced vitamin K with administration of excess vitamin K to act as an antidote for overdose of warfarin and other VKAs. FSP1 also protects against ferroptosis. (See "Biology of warfarin and modulators of INR control", section on 'Mechanism of action' and 'Ferroptosis suppressor 1 (FSP1) and other enzymes that can reduce vitamin K' above.)
●Clinical implications
•Vitamin K-dependent clotting factor deficiency – Genetic deficiency of vitamin K-dependent clotting factors can be caused by pathogenic variants in genes that encode the gamma glutamyl carboxylase (GGCX gene) or the vitamin K recycling enzyme (VKORC1 gene). (See 'Genetic deficiency of GGCX (vitamin K-dependent clotting factor deficiency type I)' above and 'Genetic deficiency of VKOR (vitamin K-dependent clotting factor deficiency type II)' above.)
•Vascular calcification – Some GGCX and VKORC1 variants can also cause aberrant calcification and other vascular abnormalities. (See 'Syndromes with aberrant calcification' above.)
•VKA resistance or overdose – Some VKORC1 variants cause resistance to vitamin K antagonists. Poisoning by superwarfarins used in rat poison may require prolonged vitamin K administration. (See 'Hereditary warfarin resistance' above and 'Poisoning by rodenticide superwarfarins' above and "Overview of rodenticide poisoning", section on 'Anticoagulants (superwarfarins and warfarins)'.)
ACKNOWLEDGMENT — The UpToDate editorial staff acknowledges Bruce Furie, MD, Beth A Bouchard, MD, and Barbara C Furie, PhD, who contributed to an earlier version of this topic review.
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