INTRODUCTION — Vitamin K is a cofactor for the enzymatic conversion of glutamic acid (Glu) residues to gamma-carboxyglutamic acid (Gla) in vitamin K-dependent proteins, via the endoplasmic reticulum resident vitamin K-dependent gamma-glutamyl carboxylase. This carboxylase activity is found in essentially all mammalian tissues, and its reaction product, Gla, has been observed in both vertebrates and invertebrates; both play an important biological role in protein function [1].
The functions of Gla and the vitamin K-dependent biosynthesis of Gla will be discussed here. An overview of the blood coagulation cascade is presented separately. (See "Overview of hemostasis".)
BACKGROUND AND HISTORY — Vitamin K and its association with blood coagulation were initially described in the 1920s and 1930s, following investigation of a hemorrhagic disease of cattle caused by ingestion of spoiled sweet clover [2]. Since then, a number of observations, which will be discussed in detail below, have improved our understanding of the biological role of vitamin K [3]:
●Discovery of vitamin K antagonists and their introduction as pharmacologic agents for anticoagulation (ie, the coumarins) [4].
●Discovery of Gla in blood clotting proteins [5,6].
●Identification of Gla as an amino acid that confers metal binding properties on proteins, a requirement for protein-membrane interaction [7,8].
●Detection of an enzymatic activity (ie, the vitamin K-dependent gamma-glutamyl carboxylase) that catalyzes the incorporation of CO2 into glutamic acid to form Gla [7].
●Identification of an intervening sequence (the propeptide) between the signal peptide and the mature vitamin K-dependent protein [9].
●Discovery of the requirement for [10] and the sufficiency of [11] the gamma-carboxylation recognition site within the propeptide in directing synthesis of gamma-carboxyglutamic acid on the adjacent Gla domain of the precursor protein.
Additional advances include purification and cloning of the vitamin K-dependent carboxylase [12,13]; determination of the three-dimensional structure of the Gla domain of prothrombin and observation of an internal carboxylate-calcium network [14]; the proposal of a mechanism of vitamin K-mediated enhancement of carboxylase action [15]; and regulation of enzymatic vitamin K epoxidase activity by glutamate containing substrate [16].
BIOSYNTHESIS OF GAMMA-CARBOXYGLUTAMIC ACID — The requirement for vitamin K as an enzyme cofactor is unique to the vitamin K-dependent gamma-glutamyl carboxylase and the biosynthesis of gamma-carboxyglutamic acid (Gla). The mechanism by which vitamin K participates as a cofactor with the carboxylase is incompletely understood. The most attractive hypothesis is that an activated species of vitamin K abstracts a hydrogen from the gamma-carbon of Glu residues on specific proteins (factors VII, IX, X, and prothrombin; proteins S and C; and, in bone, osteocalcin [bone Gla protein] and matrix Gla protein), followed by transformation of the vitamin K intermediate to an epoxide. Carbon dioxide is subsequently added to the gamma-carbon of glutamic acid, utilizing the carboxylase activity of gamma-glutamyl carboxylase, to form Gla (figure 1) [17].
Based on a nonenzymatic model [15], 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) [15]. It was initially proposed that a free cysteine in the gamma-glutamyl carboxylase was responsible for deprotonation of vitamin KH2, a hypothesis that is supported by numerous publications [18-28]. However, other studies suggest that the role of a free cysteine residue in carboxylation is uncertain, and implicate two lysine residues in the gamma-glutamyl carboxylase as important for catalysis [29,30].
Gamma-glutamyl carboxylase has two actions: it promotes the formation of Gla on the Glu residues; and it promotes the formation of the highly reactive alkoxide which then collapses into the epoxide form of vitamin K [16,18,31]. Thus, under normal conditions, for each molecule of Gla generated, one molecule of vitamin K epoxide is also formed [31,32].
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 the carboxylase until a carboxylase substrate is bound to the enzyme and converts its vitamin K epoxidase function to an active state [16]. The epoxide is then recycled back to vitamin KH2 via a vitamin K reductase (see below).
The propeptide region of the vitamin K-dependent proteins functions as a recognition sequence, binding the carboxylase to its substrate on the adjacent glutamic acid rich (Gla) domain (see below) [10,11]. It also stimulates gamma-glutamyl carboxylation [33,34].
RECYCLING OF VITAMIN K — In its naturally occurring form, vitamin K is in the quinone oxidation state and must be reduced to the hydroquinone form (vitamin KH2), the active cofactor for the vitamin K-dependent carboxylase. The enzyme responsible for this conversion is known as vitamin K epoxide reductase (VKOR) because it also reduces the vitamin K epoxide formed during the carboxylation reaction [35]. Therapeutic doses of warfarin, which shares a common ring structure with vitamin K (figure 2), inhibit this reductase by binding to the same key residues and inducing the same conformational changes required for the VKOR catalytic cycle [36], resulting in insufficient generation of vitamin K hydroquinone to support full carboxylation and therefore full function of the vitamin K-dependent proteins of blood coagulation.
The gene encoding VKOR resides on chromosome 16 and encodes a protein of 163 amino acids [37,38]. VKOR is effective at low concentrations of vitamin K epoxide and vitamin K quinone and is the physiologically important enzyme for recycling vitamin K [39,40].
Homologs of VKOR have been identified in a variety of organisms [41-43]. Like VKOR, all the homologs contain an active site CXXC motif (C132-X-X-C135 in human VKOR) that forms the redox center and can directly interact with substrates, as well as an additional pair of cysteines that are conserved [37,38]. Molecular modeling and in vitro experiments with human VKOR suggest that residues F55, N80, and F83 act in a concerted manner to localize vitamin K epoxide close to the C135 catalytic residue [44]. Bacterial VKOR homologs catalyze disulfide bond formation in secreted proteins [43].
The reducing equivalents required by the bacterial VKOR are provided by periplasmic thioredoxin-like proteins or by a thioredoxin-like domain fused to the VKOR domain. Reducing equivalents required for conversion of vitamin K quinone or epoxide to the hydroquinone by mammalian VKOR are provided by endoplasmic reticulum anchored thioredoxin-like protein, TMX [45]. Determination of the crystal structure of a naturally occurring bacterial VKOR-thioredoxin domain fusion protein has suggested an electron transfer pathway that could apply to all VKOR homologs [46]. Newly synthesized proteins in the periplasm or endoplasmic reticulum reduce the CXXC motif of a thioredoxin-like protein, which in turn reduces a conserved disulfide bridge in a loop of VKOR. The loop cysteines reduce the CXXC motif of VKOR and finally the disulfide in this CXXC motif is reestablished by reduction of a quinone [46]. However, in mammalian cells, only a minor fraction (5.6 percent) of VKOR is in a fully reduced state, with the remainder split almost equally between partially oxidized and fully oxidized [47]. VKOR catalysis and electron transfer can initiate from either the fully reduced or partially oxidized state.
A second enzyme, DT-diaphorase, an NAD(P)H dehydrogenase, reduces the quinone form of vitamin K but not vitamin K epoxide [35]. This enzyme requires high concentrations of vitamin K and probably does not play a role in recycling at physiologic tissue concentrations of vitamin K [39]. It may, however, be important when vitamin K, in the quinone form, is used to overcome warfarin intoxication or poisoning with vitamin K antagonist rodenticides [48]. These "superwarfarins" are VKOR inhibitors that are two orders of magnitude more potent than warfarin and have half-lives measured in weeks. Treatment with high doses of vitamin K quinine for several months or years is often required to reverse the coagulopathy associated with superwarfarin poisoning. (See "Overview of rodenticide poisoning", section on 'Anticoagulants (superwarfarins and warfarins)' and "Management of warfarin-associated bleeding or supratherapeutic INR".)
Vitamin K epoxide is not detectable in the plasma of normal subjects, even after a pharmacologic dose of 10 mg of vitamin K [49]. However, it is measurable following the administration of warfarin [50,51]. The finding in warfarin-treated patients of a significant positive correlation between the INR and plasma vitamin K epoxide concentrations suggests that the latter reflects the pharmacodynamic activity of warfarin in anticoagulated patients [52].
Genetic variants of the VKOR complex — Pathogenic variants affecting the VKOR complex have been described in a number of families with deficiency of the vitamin K-dependent coagulation proteins [53-56]. Consanguinity was present in one family. The defect was corrected by supplementation with vitamin K in a number of these families and partially corrected in another [55]. (See "Rare inherited coagulation disorders", section on 'Multiple vitamin K-dependent factor deficiencies'.)
Hereditary warfarin resistance — Missense mutations within the gene for VKOR (VKORC1), have been proposed to be involved in resistance to vitamin K antagonist anticoagulants [37,57-60]. In addition, common variants within the VKORC1 gene appear to modulate the mean daily dose of warfarin required to acquire target anticoagulation intensity. Despite this observation, several studies have demonstrated that genotyping to determine the initial dosing of warfarin does not improve outcomes [61]. (See "Biology of warfarin and modulators of INR control", section on 'Genetic factors'.)
Other factors that affect warfarin dosing are discussed separately. (See "Warfarin and other VKAs: Dosing and adverse effects", section on 'Warfarin resistance'.)
Variants in VKORC1 may also be associated with increased risk for arterial vascular disease [62]. 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 [63]; this is consistent with previous observations made in mice deficient in matrix Gla protein [64].
THE GAMMA-CARBOXYLATION RECOGNITION SEQUENCE — The propeptides of the vitamin K-dependent proteins contain a gamma-carboxylation recognition site [10,11]. In the vitamin K-dependent coagulation proteins (factors VII, IX, X, prothrombin) and regulatory proteins (proteins S and C), the recognition site is within the propeptide region; in contrast, the gamma-carboxylation recognition site resides within the mature protein sequence itself in matrix Gla protein [65].
The amino acids of this recognition site bind directly to the vitamin K-dependent carboxylase [66]. 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 [67].
Disruption of the carboxylation recognition site in the propeptide region of factor IX yields a final protein that either lacks or is deficient in Gla [10]. Thus, this recognition site is required for gamma-carboxylation to occur. It is most likely that this region of the blood coagulation protein precursor docks with the membrane-bound carboxylase, bringing the active site of the carboxylase in close proximity to the substrate Glu residues on the precursor form of the vitamin K-dependent proteins. A mutation in factor VII, factor VII Tokushima (Cys22 —> Gly), blocks gamma carboxylation due to a disrupted gamma-carboxylase recognition site [68]. Glu residues contribute significantly to the recognition of the protein substrate by the carboxylase [11].
To test this hypothesis concerning the function of the recognition site, a prothrombin propeptide/thrombin chimera was constructed, which had the signal peptide and gamma-carboxylation recognition site-containing propeptide of prothrombin juxtaposed to a glutamate-rich C-terminal region of thrombin. Of the eight glutamic acids within the first 40 residues of the NH2-terminus of prothrombin adjacent to the propeptide, which are not normally gamma-carboxylated, at least seven underwent complete carboxylation when this chimeric protein was expressed in CHO cells.
These results indicate that the prothrombin gamma-carboxylation recognition site on the propeptide is sufficient to direct carboxylation of adjacent Glu residues in the propeptide/thrombin chimera by the carboxylase, without regard for the sequence context of the Glu substrate or structures defined by disulfide bonds. Thus, we hypothesize that any protein will undergo gamma-carboxylation if it meets the following criteria:
●The protein includes a gamma-carboxylation recognition site that interacts with the gamma-glutamyl carboxylase
●The protein is routed through the rough endoplasmic reticulum during biosynthesis
●The cell has the carboxylase enzyme associated with the rough endoplasmic reticulum
●There are Glu sites within 40 residues of the gamma-carboxylation recognition site
●Intracellular vitamin K is present
Recognition site mutations — Additional factors may also be important for the carboxylation recognition site. As an example of the importance of alanine at position 10 as part of the carboxylation recognition site, an experimentally induced mutation at this site is associated with diminished gamma-carboxylation [10,69,70]. In addition, naturally occurring mutations of alanine 10 to threonine or valine in factor IX are associated with a marked increase in sensitivity to warfarin or phenprocoumon that is limited to the patient's factor IX activity [70-73].
The phenotype, factor IX levels, and factor IX carboxylation of these patients with mutations at alanine 10 were all normal; however, there was a more marked reduction in factor IX levels than in other vitamin K-dependent proteins after exposure to a vitamin K antagonist. It is possible that, with a reduced affinity for the carboxylase, the mutant profactor IX may be at a kinetic disadvantage when the concentration of the other carboxylase substrate, reduced vitamin K, is limited by coumarin-induced inhibition of the recycling enzymes. In contrast, the other unaffected vitamin K-dependent proteins can still compete effectively for enzyme and cofactor.
VITAMIN K-DEPENDENT CARBOXYLASE — The vitamin K-dependent carboxylase is an integral membrane protein, requiring carbon dioxide, molecular oxygen, and the hydroquinone form of vitamin K to convert Glu residues to Gla (figure 1) [13,74]. Molecular cloning of the human and bovine vitamin K-dependent carboxylase predicted a single chain protein of 758 amino acids with a molecular weight of about 94,000 [13,75]. The human carboxylase gene is localized on chromosome 2p12, and is encoded by 15 exons [76].
The known functional properties of this enzyme include:
●A carboxylase active site
●An epoxidase active site
●A propeptide binding site that allows substrate to attach and shares sequence similarity with the propeptide of the carboxylase substrate [77]
●A propeptide binding site that stimulates carboxylase and epoxidase activity
●A glutamate binding site [78]
●A vitamin K binding site
Mutations — Mutations of the carboxylase gene have been described in a number of families, leading to a congenital bleeding disorder with deficiency of all vitamin K-dependent coagulation factors [79-81]. A similar defect has been found in the Devon Rex cat [82], and mice carrying a null mutation of the carboxylase gene die at birth of massive intra-abdominal hemorrhage [83]. In most of the affected humans and in cats, the activity of the vitamin K-dependent coagulation factors can be normalized by vitamin K supplementation [53,82,84,85]. This normalization of activity is possible because the effect of most of the reported mutations is to weaken the binding of vitamin K to the enzyme. Thus, increasing the concentration of vitamin K raises the levels of the vitamin K-dependent coagulation factors. (See "Rare inherited coagulation disorders", section on 'Multiple vitamin K-dependent factor deficiencies'.)
INTRACELLULAR SITE OF GAMMA-CARBOXYLATION — Use of anticarboxylase antibodies has confirmed the intracellular localization of the carboxylase in both the endoplasmic reticulum and the Golgi apparatus. Studies in Chinese hamster ovary cells expressing prothrombin showed that uncarboxylated proprothrombin is completely gamma-carboxylated in the endoplasmic reticulum [86]. The carboxylated proprothrombin leaves the endoplasmic reticulum intact and is further processed in the Golgi apparatus to remove the propeptide.
Prothrombin and the other gamma-carboxylated extracellular vitamin K-dependent proteins bind to acidic membranes in the presence of calcium ions. The question therefore arises as to the mechanism which prevents the fully gamma-carboxylated intracellular precursor vitamin K-dependent proteins, such as proprothrombin, from binding to endoplasmic reticular membranes during transit through the biosynthetic pathway. The calcium concentration in the endoplasmic reticulum is high, in contrast to the cytoplasm, and is sufficient to support protein-membrane interaction.
Fully carboxylated profactor IX does not bind to membranes in the presence of calcium ions, whereas factor IX does [87]. 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 profactor IX with membranes.
FUNCTION OF GAMMA-CARBOXYGLUTAMIC ACID — Gamma-carboxyglutamic acid has been primarily studied in two protein families: the vitamin K-dependent blood coagulation (factors VII, IX, X, and prothrombin) and regulatory (proteins C and S) proteins; and proteins of mineralized tissue (bone Gla protein and matrix Gla protein). In addition, several proteins that contain Gla domains homologous to those of blood coagulation but of unknown function have been identified. These include Gas6 and four proteins that contain transmembrane regions as well as a Gla domain.
Gamma-carboxyglutamic acid distinguishes itself from aspartic acid and glutamic acid by containing two carboxyl groups in its side chain. The bivalent nature of Gla is similar to the immunoglobulins or fibrinogen. In these cases, a structural framework is formed via the linking of one ligand to another through a common bivalent, symmetrical molecule. As is described below, this also applies to the formation of the calcium-carboxylate network that stabilizes the Gla domains and, in the vitamin K-dependent proteins of blood coagulation, allows expression of the phospholipid binding site.
Plasma proteins of blood coagulation — The vitamin K-dependent blood coagulation and regulatory 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 Gla domain, in association with the adjacent aromatic amino acid stack domain, functions as a membrane binding component of these proteins.
Gla is an amino acid that confers metal binding properties on the vitamin K-dependent proteins. With the addition of calcium ions, these proteins undergo a structural transition that leads to exposure of a phospholipid binding site. In most cases, neither aspartic acid nor Glu will substitute for the function of Gla, emphasizing the importance of the presence of both carboxyl groups on a single amino acid.
The inability of Glu to substitute for Gla and the importance of vitamin K are best illustrated clinically by the usefulness of warfarin. Warfarin prevents the recycling of vitamin K from the oxidized (epoxide) to the reduced form (hydroquinone), leading to decreased gamma-glutamyl carboxylation because of insufficient active cofactor (the hydroquinone) (figure 1). The abnormal forms of the vitamin K-dependent proteins that result from the use of warfarin are undercarboxylated, having Glu residues at some or all of the positions that are usually carboxylated (ie, Gla). These proteins are for the most part biologically inert. Thus, in patients treated with warfarin, circulating coagulant activity correlates closely with the quantity of fully carboxylated proteins remaining in the circulation [88].
Elimination of even one Gla residue can significantly reduce the biological function of these proteins [89]. In addition, systematic mutation of individual carboxylatable Glu residues in recombinant prothrombin [89] or protein C [90-92] has yielded molecules with modest to severe functional defects, again indicating the importance of the bifunctional Gla side chain.
Prothrombin — The x-ray crystal structure of prothrombin fragment 1 indicates that the Gla domain is highly structured; many of the Gla side chains point inward to a linear array of internal calcium ions [14]. Several of these calcium ions are completely sequestered inside the core of the Gla domain.
Factor IX — The factor IX Gla domain is characterized by a fold of the polypeptide backbone, similar to that seen in prothrombin [93]. The location of the calcium binding sites in the internal core structure is nearly identical in prothrombin and factor IX, although there are some important distinguishing features [94,95].
Factor VII — The crystal structure of the factor VII-tissue factor complex indicates that the structure of the Gla domain of factor VII in the presence of calcium ions is nearly identical to its homolog in prothrombin [96].
Phospholipid membrane binding site — The phospholipid membrane binding site of the vitamin K-dependent proteins is expressed on the surface of the Gla domain. 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 [14]. Site-specific mutagenesis of homologous residues in protein C interfered with the membrane binding properties of this protein, but mutation of other hydrophobic residues did not perturb membrane binding significantly [97].
It has been concluded that calcium-induced exposure of hydrophobic amino acids in the Gla domain is critical for membrane binding [98]. 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 [99]. 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 [100].
The structure of the lysophosphatidylserine binding site in the bovine prothrombin Gla domain was determined by x-ray crystallography [101]. The serine head group binds Gla domain-bound calcium ions and Gla residues 17 and 21, fixed elements of the Gla domain fold, predicting the structural basis for phosphatidylserine specificity among Gla domains. A molecular dynamics simulation study of anionic phospholipid binding to the Gla domain of bovine fragment 1 in the presence of calcium ions has identified a second phosphatidylserine binding site with the head group bound by a Gla-bound calcium ion, Gla 30 and Lys 11 [102]. It can be concluded that Gla domains provide a unique mechanism for protein-phospholipid membrane interaction.
Gla-containing proteins of mineralized tissue — Whereas the role of Gla is well defined for the plasma coagulation proteins, its function remains less clear in proteins/peptides outside of this family. There have been no successful studies to date on the structure of Gla-containing bone proteins, including osteocalcin (bone Gla protein), the most abundant osteoblast-specific noncollagenous protein, and matrix Gla protein. (See "Normal skeletal development and regulation of bone formation and resorption".)
There is also no evidence to support the use of vitamin K administration in patients with osteoporosis or, in a patient receiving a vitamin K antagonist, to support the use of additional interventions over and above those routinely used for osteoporosis prevention. These subjects are discussed in more detail separately. (See "Overview of the management of osteoporosis in postmenopausal women", section on 'Therapies not recommended' and "Prevention of osteoporosis" and "Drugs that affect bone metabolism", section on 'Anticoagulants'.)
Some insights into the function of osteocalcin and matrix Gla protein have come from experimental models lacking these proteins.
●Osteocalcin deficient mice are characterized by increased bone formation, including higher bone mass and bones of improved functional quality; these observations suggest an important role for osteocalcin in the regulation of bone remodeling [103].
●Matrix Gla protein deficient mice suffer from spontaneous and ultimately fatal calcification of arteries and cartilage [64], suggesting that one of the functions of this protein is to control and limit extraosseous calcification. The Gla residues in matrix Gla protein prevent osteogenic differentiation and calcification via binding and inhibition of bone morphogenetic protein-4 [104], as well as via binding to vascular smooth muscle cell-derived vesicles [105].
Other mammalian vitamin K-dependent proteins — Several other families of vitamin K-dependent proteins described in mammalian systems are Gas6 and the proline-rich Gla proteins.
Gas6 protein — 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 [106]. 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 [107]. 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, identified by searching a database with a consensus sequence derived from analysis of known Gla domains, can be divided into two subclasses based on their gene organization and protein sequence.
●Proline rich Gla proteins, PRGP1 and PRGP2, are named for their proline-rich Gla protein characteristic [108].
●Transmembrane Gla proteins, TMG3 and TMG4, were subsequently identified [109].
All of these proteins are expressed variably in fetal and adult tissue. While their functions are unknown, all of these proteins contain the PPXY motif involved in diverse cellular functions. The gene for TMG4 is one of those deleted in the 11p14-p12 chromosome region deletion associated with WAGR syndrome and falls into the linkage region of 11p13-p12 as an autism candidate gene [110].
Gamma-glutamyl carboxylase — The vitamin K-dependent gamma-glutamyl carboxylase, which converts Glu residues to Gla, has sequence homology with the region of matrix Gla protein containing the gamma-carboxylation recognition site [111]. Under some conditions, the carboxylase can become carboxylated, although the role of Gla in this enzyme remains to be determined [112].
Protein Z — Protein Z is a 62,000 molecular weight vitamin K-dependent plasma glycoprotein whose structure is similar to coagulation factors VII, IX, X, and proteins C and S [113]. Protein Z and a protein Z-dependent protease inhibitor (ZPI) appear to serve as cofactors for the inhibition of activated factor X and activated factor XI by a number of different mechanisms [114-116]. 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 [117]. Deficiencies of protein Z or ZPI, as with deficiencies of proteins C and S, might therefore result in a prothrombotic state (eg, arterial thrombosis, pregnancy complications, venous thromboembolism) [118-123].
In support of this supposition are the following highly preliminary observations:
●Protein Z deficiency dramatically increases the severity of the prothrombotic phenotype of factor V Leiden in a transgenic mouse model [113]. Similarly, protein Z deficiency or specific polymorphisms within the protein Z gene appear to influence clinical symptoms of thromboembolism in human subjects with factor V Leiden [124,125].
●In a comparison of 200 women with one unexplained primary episode of early fetal loss (10th to 15th week of gestation) and 200 controls who had normal pregnancies, protein Z deficiency was significantly more common in the women with fetal loss (22 versus 4 percent) [126].
●In two studies, nonsense mutations of the ZPI gene were found in a significantly higher percent of patients with thrombosis compared with controls, with odds ratios of 5.7 and 3.3, respectively [116,127].
The relationships between protein Z levels and haplotypes and ischemic stroke, Sneddon's syndrome, and atherosclerotic disease are unclear [128-134]. The significance of low levels of protein Z in patients with antiphospholipid antibodies is similarly unknown [135,136].
Gla-rich proteins — The Gla-rich protein (GRP), so-named because it contains the highest Gla content of any known protein (16 of 74 residues), was identified in the calcified cartilage of the Adriatic sturgeon [137]. The Gla containing region of GRP is not homologous to any other known Gla containing protein. GRP has orthologs in all taxonomic groups of vertebrates. GRP mRNA was found in virtually all rat and sturgeon tissue with the highest levels in cartilage and bone [137]. The protein is expressed in chondrocytes, chondroblasts, osteocytes and osteoblasts. The function of this protein is unknown.
Invertebrate Gla-containing proteins — Only one invertebrate, the cone snail, has been found to contain Gla [138]. This tropical snail stings its prey, whether fish, molluscs, or worms, via a harpoon that injects conotoxins. Many of these conotoxins are Gla-containing peptides that are channel blockers [139,140]. The presence of Gla in cysteine-free conotoxins known as conomarphins appear to preferentially stabilize certain conomarphin conformations, which may be associated with their target specificity and selectivity [141].
SUMMARY
●Vitamin K dependent carboxylase and synthesis of Gla – The requirement for vitamin K as an enzyme cofactor is unique to the vitamin K-dependent gamma-glutamyl carboxylase and the biosynthesis of gamma-carboxyglutamic acid (Gla). The vitamin K-dependent carboxylase is an integral membrane protein, requiring carbon dioxide, molecular oxygen, and the hydroquinone form of vitamin K to convert glutamic acid (Glu) residues to Gla (figure 1). Mutations of the carboxylase gene have been described, leading to a congenital bleeding disorder with deficiency of all vitamin K-dependent coagulation factors. (See 'Biosynthesis of gamma-carboxyglutamic acid' above and 'Vitamin K-dependent carboxylase' above.)
●Vitamin K recycling – For each molecule of Gla generated, one molecule of vitamin K epoxide is also formed. Vitamin K epoxide reductase (VKOR) is responsible for the conversion of vitamin K to the active cofactor for the vitamin K-dependent carboxylase; it also reduces the vitamin K epoxide formed during the carboxylation reaction. Therapeutic doses of warfarin inhibit this reductase, resulting in insufficient generation of vitamin K hydroquinone to support full carboxylation and therefore full function of the vitamin K-dependent proteins of blood coagulation. (See 'Recycling of vitamin K' above.)
●Functions of Gla in clotting – Gla is a post-translationally modified glutamic acid that confers metal binding properties on the vitamin K-dependent proteins. With the addition of calcium ions, these proteins undergo a structural transition that leads to exposure of a phospholipid binding site. Factors that require Gla for their function include prothrombin (factor II) and factors VII, IX, and X, as well as proteins C and S. (See 'Function of gamma-carboxyglutamic acid' above.)
●Other functions of Gla – Other Gla-requiring proteins (eg, the bone and matrix Gla proteins, protein Z) are discussed above. (See 'Gla-containing proteins of mineralized tissue' above and 'Other mammalian vitamin K-dependent proteins' above.)
ACKNOWLEDGMENT — The UpToDate editorial staff acknowledges Barbara C Furie, PhD, who contributed to an earlier version of this topic review.
