Vitamin K-dependent Research Articles

Novel Gamma-Glutamyl Carboxylase Mutation Causing Hemorrhagic and Non-Hemorrhagic VKCFD1 Phenotypes

Background: Vitamin K clotting factor deficiency (VKCFD) types 1 and 2 are very rare autosomal recessive disorders associated with reduced clotting factors activities for all vitamin K dependent clotting factors. Less than 30 cases are reported with defects in GGCX (gamma-glutamyl carboxylase) leading to VKCFD Type 1 (VKCFD1) or VKORC1 (vitamin K epoxide reductase complex subunit 1) leading to VKCFD Type 2 (VKCFD2). In addition to clotting factor deficiency, GGCX mutations can be associated with skeletal abnormalities such as bone hypoplasia, conductive hearing loss, mental retardation, and skin hyperlaxity. Here, we report an Arabic family with VKCFD1 harboring an unreported, new pathogenic variant in the GGCX gene. The proband presented at 17 months of age with severe bruising following trauma and work up demonstrated deficiencies of all Vitamin K dependent (VKD) coagulation proteins. Another sibling born 8 years later was also diagnosed with the same condition with abnormal coagulation parameters at birth. Additionally, the non-hemorrhagic manifestations diagnosed in both siblings were mid-facial hypoplasia with flat nasal bridge, dental abnormalities, and shortened distal phalanges representing a keutel syndrome-like phenotype. Both patients are currently managed with oral vitamin K supplementation with resolution of bleeding symptoms. Here we present the in-vitro assessment of the mutations effects to vitamin K supplementation and potential clinical implications. Methods: The proband, affected brother and mother, all underwent genetic evaluation at the University Clinic Bonn in Germany. DNA was isolated from the patient's blood and Sanger Sequencing was performed for the genes VKORC1 and GGCX. The identified new pathogenic variant in GGCX was further characterized by the in-vitro assay published by Ghosh et al. in 2021. Results: The brothers were found to be homozygous for c.[416G>A];[416G>A] p.(Cys139Tyr), a genetic variant in exon 4 of the GGCX gene. Mutations in VKORC1 gene leading to reduced activity for the VKD clotting factors were excluded confirming the diagnosis of VKCFD Type 1 (VKCFD1). The mother was found to be heterozygous for the same GGCX variant. The affected siblings and the mother were further genotyped for the promoter single nucleotide polymorphism in VKORC1 gene NG_011564.1:g.3588G>A (VKORC1:c.-1639 G>A) since this might correlate with the severity of the phenotypes in patients with skeletal manifestations. The brothers and mother were found to harbor VKORC1:c.-1639 GA, which has been associated with a 33% reduced VKORC1 expression. Treatment with 5mg of vitamin K resulted in increased activities of the VKD clotting factors (Table 1). However, clotting factor activities did not normalized after vitamin K supplementation. The in vitro characterization of the new identified pathogenic variant GGCX:p.C139Y (Figure 1) showed reduced activities for all VKD clotting factors, which did not reach wild type levels despite increasing concentrations of vitamin K. The levels of Matrix Gla protein (MGP), Bone Gla protein (BGLAP), and Gla-rich protein (GRP) showed almost no response to increasing amount of vitamin K. Discussion: Here, we report a new pathogenic variant in exon 4 of GGCX c.[416G>A];[416G>A] p.(Cys139Tyr) leading to VKCFD1 with reduced clotting factor activities and non-hemorrhagic manifestations. The pre and post vitamin K supplementation factor levels in the patients correlate with the in-vitro data and demonstrate failure of the clotting factor levels to normalize despite vitamin K supplementation. The in vitro data for the non-hematological VKD proteins indicate that the patients might develop a pseudoxanthoma elasticum (PXE)-like phenotype resulting into hyperlaxity of skin due to the almost zero levels of carboxylated GRP. The skeletal/bone phenotypes can be explained by the almost zero values for carboxylated MGP and GRP. To assess this in-vivo, levels of human MGP and BGLAP are currently being analyzed in the patients and mothers blood samples. These patients highlight the nuances associated with the diagnosis and management of patients with VKCFD. The antenatal management and timing of Vitamin K supplementation to prevent intrauterine bleeding is also unknown, and there may be benefit to starting early to prevent the bone and skin manifestations. Further research is warranted in rare bleeding disorders such as VKCFD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

GGCX mutants that impair hemostasis reveal the importance of processivity and full carboxylation to VKD protein function

Abstractγ-Glutamyl carboxylase (GGCX) generates multiple carboxylated Glus (Glas) in vitamin K–dependent (VKD) proteins that are required for their functions. GGCX is processive, remaining bound to VKD proteins throughout multiple Glu carboxylations, and this study reveals the essentiality of processivity to VKD protein function. GGCX mutants (V255M and S300F) whose combined heterozygosity in a patient causes defective clotting and calcification were studied using a novel assay that mimics in vivo carboxylation. Complexes between variant carboxylases and VKD proteins important to hemostasis (factor IX [FIX]) or calcification (matrix Gla protein [MGP]) were reacted in the presence of a challenge VKD protein that could potentially interfere with carboxylation of the VKD protein in the complex. The VKD protein in the complex with wild-type carboxylase was carboxylated before challenge protein carboxylation occurred and became fully carboxylated. In contrast, the V255M mutant carboxylated both forms at the same time and did not completely carboxylate FIX in the complex. S300F carboxylation was poor with both FIX and MGP. Additional studies analyzed FIX- and MGP-derived peptides containing the Gla domain linked to sequences that mediate carboxylase binding. The total amount of carboxylated peptide generated by the V255M mutant was higher than that of wild-type GGCX; however, the individual peptides were partially carboxylated. Analysis of the V255M mutant in FIX HEK293 cells lacking endogenous GGCX revealed poor FIX clotting activity. This study shows that disrupted processivity causes disease and explains the defect in the patient. Kinetic analyses also suggest that disrupted processivity may occur in wild-type carboxylase under some conditions (eg, warfarin therapy or vitamin K deficiency).

Vitamin K-Dependent Protein Activation: Normal Gamma-Glutamyl Carboxylation and Disruption in Disease.

Vitamin K-dependent (VKD) proteins undergo an unusual post-translational modification, which is the conversion of specific Glu residues to carboxylated Glu (Gla). Gla generation is required for the activation of VKD proteins, and occurs in the endoplasmic reticulum during their secretion to either the cell surface or from the cell. The gamma-glutamyl carboxylase produces Gla using reduced vitamin K, which becomes oxygenated to vitamin K epoxide. Reduced vitamin K is then regenerated by a vitamin K oxidoreductase (VKORC1), and this interconversion of oxygenated and reduced vitamin K is referred to as the vitamin K cycle. Many of the VKD proteins support hemostasis, which is suppressed during therapy with warfarin that inhibits VKORC1 activity. VKD proteins also impact a broad range of physiologies beyond hemostasis, which includes regulation of calcification, apoptosis, complement, growth control, signal transduction and angiogenesis. The review covers the roles of VKD proteins, how they become activated, and how disruption of carboxylation can lead to disease. VKD proteins contain clusters of Gla residues that form a calcium-binding module important for activity, and carboxylase processivity allows the generation of multiple Glas. The review discusses how impaired carboxylase processivity results in the pseudoxanthoma elasticum-like disease.

The Role of GRP and MGP in the Development of Non-Hemorrhagic VKCFD1 Phenotypes.

Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1) is a rare hereditary bleeding disorder caused by mutations in γ-Glutamyl carboxylase (GGCX) gene. The GGCX enzyme catalyzes the γ-carboxylation of 15 different vitamin K dependent (VKD) proteins, which have function in blood coagulation, calcification, and cell signaling. Therefore, in addition to bleedings, some VKCFD1 patients develop diverse non-hemorrhagic phenotypes such as skin hyper-laxity, skeletal dysmorphologies, and/or cardiac defects. Recent studies showed that GGCX mutations differentially effect γ-carboxylation of VKD proteins, where clotting factors are sufficiently γ-carboxylated, but not certain non-hemostatic VKD proteins. This could be one reason for the development of diverse phenotypes. The major manifestation of non-hemorrhagic phenotypes in VKCFD1 patients are mineralization defects. Therefore, the mechanism of regulation of calcification by specific VKD proteins as matrix Gla protein (MGP) and Gla-rich protein (GRP) in physiological and pathological conditions is of high interest. This will also help to understand the patho-mechanism of VKCFD1 phenotypes and to deduce new treatment strategies. In the present review article, we have summarized the recent findings on the function of GRP and MGP and how these proteins influence the development of non-hemorrhagic phenotypes in VKCFD1 patients.

GGCX variants leading to biallelic deficiency to γ-carboxylate GRP cause skin laxity in VKCFD1 patients.

γ-Glutamyl carboxylase (GGCX) catalyzes the γ-carboxylation of 15 different vitamin K dependent (VKD) proteins. Pathogenic variants in GGCX cause a rare hereditary bleeding disorder called Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1). In addition to bleedings, some VKCFD1 patients develop skin laxity and skeletal dysmorphologies. However, the pathophysiological mechanisms underlying these non-hemorrhagic phenotypes remain elusive. Therefore, we have analyzed 20 pathogenic GGCX variants on their ability to γ-carboxylate six non-hemostatic VKD proteins in an in vitro assay, where GGCX variants were expressed in GGCX-/- cells and levels of γ-carboxylated co-expressed VKD proteins were detected by a functional ELISA. We observed that GGCX variants causing markedly reduced γ-carboxylation of Gla rich protein (GRP) in vitro were reported in patients with skin laxity. Reduced levels of γ-carboxylated Matrix gla protein (MGP) are not exclusive for causing skeletal dysmorphologies in VKCFD1 patients. In silico docking of vitamin K hydroquinone on a GGCX model revealed a binding site, which was validated by in vitro assays. GGCX variants affecting this site result in disability to γ-carboxylate VKD proteins and hence are involved in the most severe phenotypes. This genotype-phenotype analysis will help to understand the development of non-hemorrhagic phenotypes and hence improve treatment in VKCFD1 patients.

Naturally occurring UBIAD1 mutations differentially affect menaquinone biosynthesis and vitamin K-dependent carboxylation.

UbiA prenyltransferase domain-containing protein-1 (UBIAD1) is responsible for the biosynthesis of menaquinone-4 (MK-4), a cofactor for extrahepatic carboxylation of vitamin K-dependent (VKD) proteins. Genetic variations of UBIAD1 are mainly associated with Schnyder corneal dystrophy (SCD), a disease characterized by abnormal accumulation of cholesterol in the cornea. Results from in vitro studies demonstrate that SCD-associated UBIAD1 mutations are defective in MK-4 biosynthesis. However, SCD patients do not exhibit typical phenotypes associated with defects of MK-4 or VKD carboxylation. Here, we coupled UBIAD1's biosynthetic activity of MK-4 with VKD carboxylation in HEK293 cells that stably express a chimeric VKD reporter protein. The endogenous Ubiad1 gene in these cells was knocked out by CRISPR-Cas9-mediated genome editing. The effect of UBIAD1 mutations on MK-4 biosynthesis and VKD carboxylation was evaluated in Ubiad1-deficient reporter cells by determining the production of MK-4 or by measuring the efficiency of reporter-protein carboxylation. Our results show that the hot-spot mutation N102S has a moderate impact on MK-4 biosynthesis (retained ˜ 82% activity) but does not affect VKD carboxylation. However, the G186R mutation significantly affected both MK-4 biosynthesis and VKD carboxylation. Other mutations exhibit varying degrees of effects on MK-4 biosynthesis and VKD carboxylation. These results are consistent with in vivo results obtained from gene knock-in mice and SCD patients. Our findings suggest that UBIAD1's MK-4 biosynthetic activity does not directly correlate with the phenotypes of SCD patients. The established cell-based assays in this study provide a powerful tool for the functional studies of UBIAD1 in a cellular milieu.

A novel vitamin K derived anticoagulant tolerant to genetic variations of vitamin K epoxide reductase

BackgroundVitamin K antagonists (VKAs), such as warfarin, have remained the cornerstone of oral anticoagulation therapy in the prevention and treatment of thromboembolism for more than half a century. They function by impairing the biosynthesis of vitamin K‐dependent (VKD) clotting factors through the inhibition of vitamin K epoxide reductase (VKOR). The challenge of VKAs therapy is their narrow therapeutic index and highly variable dosing requirements, which are partially the result of genetic variations of VKOR. ObjectivesThe goal of this study was to search for an improved VKA that is tolerant to the genetic variations of its target enzyme. MethodsA series of vitamin K derivatives with benzyl and related side‐chain substitutions at the 3‐position of 1,4‐naphthoquinone were synthesized. The role of these compounds in VKD carboxylation was evaluated by mammalian cell‐based assays and conventional in vitro activity assays. ResultsOur results showed that replacing the phytyl side‐chain with a methylene cyclooctatetraene (COT) moiety at the 3‐position of vitamin K1 converted it from a substrate to an inhibitor for VKD carboxylation. Strikingly, this COT‐vitamin K derivative displayed a similar inhibition potency in warfarin‐resistant VKOR mutations whose warfarin resistance varied more than 400‐fold. Further characterization of COT‐vitamin K for the inhibition of VKD carboxylation suggested that this compound targets multiple enzymes in the vitamin K redox cycle. Importantly, the anticoagulation effect of COT‐vitamin K can be rescued with high doses of vitamin K1. ConclusionWe discovered a vitamin K analogue that functions as a VKA and is tolerant to genetic variations in the target enzyme.

γ-Glutamyl carboxylase mutations differentially affect the biological function of vitamin K–dependent proteins

Abstractγ-Glutamyl carboxylase (GGCX) is an integral membrane protein that catalyzes posttranslational carboxylation of a number of vitamin K–dependent (VKD) proteins involved in a wide variety of physiologic processes, including blood coagulation, vascular calcification, and bone metabolism. Naturally occurring GGCX mutations are associated with multiple distinct clinical phenotypes. However, the genotype–phenotype correlation of GGCX remains elusive. Here, we systematically examined the effect of all naturally occurring GGCX mutations on the carboxylation of 3 structure–function distinct VKD proteins in a cellular environment. GGCX mutations were transiently introduced into GGCX-deficient human embryonic kidney 293 cells stably expressing chimeric coagulation factor, matrix Gla protein (MGP), or osteocalcin as VKD reporter proteins, and then the carboxylation efficiency of these reporter proteins was evaluated. Our results show that GGCX mutations differentially affect the carboxylation of these reporter proteins and the efficiency of using vitamin K as a cofactor. Carboxylation of these reporter proteins by a C-terminal truncation mutation (R704X) implies that GGCX's C terminus plays a critical role in the binding of osteocalcin but not in the binding of coagulation factors and MGP. This has been confirmed by probing the protein–protein interaction between GGCX and its protein substrates in live cells using bimolecular fluorescence complementation and chemical cross-linking assays. Additionally, using a minigene splicing assay, we demonstrated that several GGCX missense mutations affect GGCX's pre–messenger RNA splicing rather than altering the corresponding amino acid residues. Results from this study interpreted the correlation of GGCX's genotype and its clinical phenotypes and clarified why vitamin K administration rectified bleeding disorders but not nonbleeding disorders.

A cell-based high-throughput screen identifies drugs that cause bleeding disorders by off-targeting the vitamin K cycle

AbstractDrug-induced bleeding disorders contribute to substantial morbidity and mortality. Antithrombotic agents that cause unintended bleeding of obvious cause are relatively easy to control. However, the mechanisms of most drug-induced bleeding disorders are poorly understood, which makes intervention more difficult. As most bleeding disorders are associated with the dysfunction of coagulation factors, we adapted our recently established cell-based assay to identify drugs that affect the biosynthesis of active vitamin K–dependent (VKD) coagulation factors with possible adverse off-target results. The National Institutes of Health (NIH) Clinical Collection (NCC) library containing 727 drugs was screened, and 9 drugs were identified, including the most commonly prescribed anticoagulant warfarin. Bleeding complications associated with most of these drugs have been clinically reported, but the pathogenic mechanisms remain unclear. Further characterization of the 9 top-hit drugs on the inhibition of VKD carboxylation suggests that warfarin, lansoprazole, and nitazoxanide mainly target vitamin K epoxide reductase (VKOR), whereas idebenone, clofazimine, and AM404 mainly target vitamin K reductase (VKR) in vitamin K redox cycling. The other 3 drugs mainly affect vitamin K availability within the cells. The molecular mechanisms underlying the inactivation of VKOR and VKR by these drugs are clarified. Results from both cell-based and animal model studies suggest that the anticoagulation effect of drugs that target VKOR, but not VKR, can be rescued by the administration of vitamin K. These findings provide insights into the prevention and management of drug-induced bleeding disorders. The established cell-based, high-throughput screening approach provides a powerful tool for identifying new vitamin K antagonists that function as anticoagulants.

Improvement of the recombinant human coagulation factor IX expression by co-expression of a novel transcript of Drosophila γ carboxylase in a human cell line.

Mammalian cells as the main host for production of human proteins are incapable of complete γ-carboxylation of over-expressed Vitamin K Dependent (VKD) proteins. The Drosophila γ-glutamyl carboxylase (DγC) has been shown to be more efficient than its human counterpart in γ-carboxylation of human substrates, in vitro. Considering the Drosophila γ-carboxylase (DγC) efficiency, in comparison with its human counterpart, for recognition and γ-carboxylation of a human substrate in vitro, we were determined to study the effect of the DγC on the hFIX expression in a mammalian cell line. With this aim, we examined co-expression of the DγC with the hFIX, in a human cell line. While the co-expression of a complete DγC cDNA reduced the hFIX expression, a truncated form of DγC could improve both the expression level (up to 1211ng/106 cells/ml on the 4th day of post-transfection) and carboxylation of the expressed hFIX, significantly (p < 0.009). Our findings provided evidences for potential of a partial fragment of the DγC for improvement of the γ-carboxylation of a human substrate in a mammalian cell. Our experimental data, in accordance with in silico analysis suggested that the DγC C-terminal fragment, with the advantage of a Kozak-like element has the potential of being expressed as a separate internal translation unit, to generate a peptide with appropriate γ-carboxylase activity.

Enhanced functional recombinant factor IX production by human embryonic kidney cells engineered to overexpress VKORC1.

Replacement therapy with recombinant drugs is the main therapeutic strategy for hemophilia B patients. To reduce the production costs of recombinant coagulation factors, improvement of their expression and activity by enhancement of γ-carboxylation might be of interest. The expression and functional activity of vitamin K-dependent (VKD) coagulation proteins rely, in part, on the VKD process of γ-carboxylation that is mediated by the enzymes γ-carboxylase and vitamin K epoxide reductase (VKOR). Since the recombinant production of VKD proteins is hampered by the inefficiency of this enzymatic process, we specifically have examined the stable expression of functional blood coagulation factor IX (FIX) in HEK293 cells following transient overexpression of VKORC1 as an important part of VKOR component. Recombinant hFIX-producing human embryonic kidney (HEK) cells were transfected to overexpress VKORC1. Following reverse transcription polymerase chain reaction (RT-PCR) analysis, expression efficiency of the active hFIX was analyzed by performing enzyme-linked immunosorbent assay and coagulation test. In addition, to quantify γ-carboxylated recombinant FIX, the barium citrate method was used. Overexpression of VKORC1 in FIX-producing HEK cells, resulting in a 3.2-fold higher expression of functional FIX, which displayed a 1.4-fold enhanced specific activity. Moreover, a 3.9-fold enhanced recovery of fully γ-carboxylated FIX following barium citrate adsorption was achieved. Collectively, these findings indicate that the overexpression of VKORC1 results in the production of higher levels of functional hFIX in HEK293 cells. The increase of the VKORC1 as a supplier of γ-carboxylase seems to play a significant role in increasing the amount and efficiency of recombinant FIX production, thereby reducing the production costs.

Exon 2 skipping eliminates γ‐glutamyl carboxylase activity, indicating a partial splicing defect in a patient with vitamin K clotting factor deficiency

Unlabelled Box BackgroundMutations in the γ‐glutamyl carboxylase (GGCX), which is required for vitamin K‐dependent (VKD) protein activation, can result in vitamin K clotting factor deficiency (VKCFD1). A recent report described a VKCFD1 patient with a homozygous carboxylase mutation that altered splicing and deleted exon 2 (Δ2GGCX). Only Δ2GGCX RNA was observed in the patient. ObjectivesLoss of exon 2 results in the deletion of carboxylase sequences thought to be important for membrane topology and consequent function. Carboxylase activity is required for life, and we therefore tested whether the Δ2GGCX mutant is active. MethodsHEK 293 cells were edited by the use of CRISPR‐Cas9 to eliminate endogenous carboxylase. Recombinant wild‐type GGCX and recombinant Δ2GGCX were then expressed and tested for carboxylation of the VKD protein factor IX. A second approach was used to monitor carboxylation biochemically, using recombinant carboxylases expressed in insect cells that lack endogenous carboxylase. Results and conclusionsΔ2GGCX activity was undetectable in both assays, which is strikingly different from the low levels of carboxylase activity observed with other VKCFD1 mutants. The similarity in clotting function between patients with Δ2GGCX and these mutations must therefore arise from a novel mechanism. Low levels of properly spliced carboxylase RNA that produce full‐length protein would not have been observed in the previous study. The results suggest that the splicing defect is incomplete. Δ2GGCX RNA has been detected in normal human liver, and has been designated carboxylase isoform 2; however, Δ2GGCX protein was not observed in normal human liver. The lack of activity and protein expression suggest that isoform 2 is not physiologically relevant to normal VKD protein carboxylation.

Pathway to malonic acid-containing metabolites

A crucial malonic acid moiety in the antibiotic malonomycin has been shown to be installed by a bacterial vitamin K-dependent (VKD) carboxylase orthologue.

Warfarin alters vitamin K metabolism: a surprising mechanism of VKORC1 uncoupling necessitates an additional reductase

AbstractThe anticoagulant warfarin inhibits the vitamin K oxidoreductase (VKORC1), which generates vitamin K hydroquinone (KH2) required for the carboxylation and consequent activation of vitamin K–dependent (VKD) proteins. VKORC1 produces KH2 in 2 reactions: reduction of vitamin K epoxide (KO) to quinone (K), and then KH2. Our dissection of full reduction vs the individual reactions revealed a surprising mechanism of warfarin inhibition. Warfarin inhibition of KO to K reduction and carboxylation that requires full reduction were compared in wild-type VKORC1 or mutants (Y139H, Y139F) that cause warfarin resistance. Carboxylation was much more strongly inhibited (∼400-fold) than KO reduction (two- to threefold). The K to KH2 reaction was analyzed using low K concentrations that result from inhibition of KO to K. Carboxylation that required only K to KH2 reduction was inhibited much less than observed with the KO substrate that requires full VKORC1 reduction (eg, 2.5-fold vs 70-fold, respectively, in cells expressing wild-type VKORC1 and factor IX). The results indicate that warfarin uncouples the 2 reactions that fully reduce KO. Uncoupling was revealed because a second activity, a warfarin-resistant quinone reductase, was not present. In contrast, 293 cells expressing factor IX and this reductase activity showed much less inhibition of carboxylation. This activity therefore appears to cooperate with VKORC1 to accomplish full KO reduction. Cooperation during warfarin therapy would have significant consequences, as VKD proteins function in numerous physiologies in many tissues, but may be poorly carboxylated and dysfunctional if the second activity is not ubiquitously expressed similar to VKORC1.

VKOR paralog VKORC1L1 supports vitamin K-dependent protein carboxylation in vivo.

Vertebrates possess 2 proteins with vitamin K oxidoreductase (VKOR) activity: VKORC1, whose vitamin K reduction supports vitamin K-dependent (VKD) protein carboxylation, and VKORC1-like 1 (VKORC1L1), whose function is unknown. VKD proteins include liver-derived coagulation factors, and hemorrhaging and lethality were previously observed in mice lacking either VKORC1 or the γ-glutamyl carboxylase (GGCX) that modifies VKD proteins. Vkorc1-/- mice survived longer (1 week) than Ggcx-/- mice (midembryogenesis or birth), and we assessed whether VKORC1L1 could account for this difference. We found that Vkorc1-/-;Vkorc1l1-/- mice died at birth with severe hemorrhaging, indicating that VKORC1L1 supports carboxylation during the pre- and perinatal periods. Additional studies showed that only VKORC1 sustains hemostasis beyond P7. VKORC1 expression and VKOR activity increased during late embryogenesis and following birth, while VKORC1L1 expression was unchanged. At P0, most (>99%) VKOR activity was due to VKORC1. Prothrombin mRNA, protein, and carboxylation also increased during this period, as did mRNA levels of coagulation factors encoding genes F7, F9, and F10. VKORC1L1 levels in Vkorc1-/- mouse liver may therefore be insufficient for supporting carboxylation beyond day 7. In support of this conclusion, VKORC1L1 overexpression in liver rescued carboxylation and hemostasis in adult Vkorc1-/- mice. These findings establish that VKORC1L1 supports VKD protein carboxylation in vivo.

Effect of propeptide amino acid substitution in γ-carboxylation, activity and expression of recombinant human coagulation factor IX.

The production of recombinant vitamin K dependent (VKD) proteins for therapeutic purposes is an important challenge in the pharmaceutical industry. These proteins are primarily synthesized as precursor molecules and contain pre-propeptide sequences. The propeptide is connected to γ-carboxylase enzyme through the γ-carboxylase recognition site for the direct γ-carboxylation of VKD proteins that has a significant impact on their biological activity. Propeptides have different attitudes toward γ-carboxylase and certain amino acids in propeptide sequences are responsible for the differences in γ-carboxylase affinity. By aiming to replace amino acids in hFIX propeptide domain based on the prothrombin propeptide, pMT-hFIX-M14 expression cassette, containing cDNA of hFIX with substituted -14 residues (Asp to Ala) was made. After transfection of Drosophila S2 cells, expression of the active hFIX was analyzed by performing ELISA and coagulation test. A 1.4-fold increase in the mutant recombinant hFIX expression level was observed in comparison with that of a native recombinant hFIX. The enhanced hFIX activity and specific activity of the hFIXD-14A (2.2 and 1.6 times, respectively) were further confirmed by comparing coagulation activity levels of substituted and native hFIX. Enrichment for functional, fully γ-carboxylated hFIX species via barium citrate adsorption demonstrated 2-fold enhanced recovery in the S2-expressing hFIXD-14A relative to that expressed native hFIX. These results show that changing -14 residues leads to a decrease in the binding affinity to substrate, increase in γ-carboxylation and activity of recombinant hFIX. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:515-520, 2018.

Osteocalcin: The extra-skeletal role of a vitamin K-dependent protein in glucose metabolism

The role of vitamin K in the body has long been associated with blood clotting and coagulation. In more recent times, its role in a range of physiological processes has been described including the regulation of bone and soft tissue calcification, cell growth and proliferation, cognition, inflammation, various oxidative processes and fertility, where osteocalcin is thought to up-regulate the synthesis of the enzymes needed for the biosynthesis of testosterone thereby increasing male fertility. Vitamin K dependent proteins (VKDP) contain γ-carboxyglutamic acid residues which require post-translational, gamma-glutamyl carboxylation by the vitamin K-dependent (VKD) gamma-glutamyl carboxylase enzyme for full functionality. These proteins are present both hepatically and extrahepatically. The role of bone-derived osteocalcin has many physiological roles including, maintenance of bone mass with more recent links to energy metabolism due to the role of the skeleton as an endocrine organ. It has been proposed that insulin binds to bone forming cells (osteoblasts) promoting osteocalcin production which in turn promotes β-cell proliferation, insulin secretion and glucose control. However much of this research has been conducted in animal models with equivocal findings in human studies. This review will discuss the role of osteocalcin in relation to its role in human health, focusing specifically on glucose metabolism.

During warfarin induction, the Fiix‐prothrombin time reflects the anticoagulation level better than the standard prothrombin time

Background The prothrombin time (PT) is equally prolonged by reduction of each of the vitamin K-dependent (VKD) factors (F) II, VII and X. The Fiix-PT is only affected by FII and FX, the main contributors to thrombin generation (TG). Objective To test the hypothesis that variability in warfarin anticoagulation is reduced early during monitoring with the normalized PT-ratio calculated from Fiix-PT (Fiix-International Normalized Ratio [INR]) compared with traditional PT-INR monitoring. Also, that because of its insensitivity to FVII, Fiix-PT more accurately reflects TG when Fiix-INR and PT-INR are discrepant. Methods Samples from Fiix-trial participants monitored with either Fiix-PT or PT were used. VKD coagulation factors and TG were measured in samples from 40 patients during stable anticoagulation and in serial samples obtained from 26 patients during warfarin induction. TG was assessed in relation to selective reduction in single VKD factors. Results During Fiix-warfarin induction full anticoagulation measured as FII or FX activity was achieved at a similar rate to that with PT-warfarin but subsequently stabilized better. Fiix-INR but not PT-INR mirrored total TG during initiation. During induction, FII (R2 = 0.66) and FX (R2 = 0.52) correlated better with TG and with a steeper slope than did FIX (R2 = 0.37) and in particular FVII (R2 = 0.21). In vitro, FII and FX were the main determinants of TG at concentrations observed during VKA anticoagulation, whereas FVII and FIX had little influence. Conclusions Fiix-PT monitoring reduces anticoagulation variability, suggesting that monitoring FVII has a limited role during VKA management. TG is better reflected by Fiix-PT.

Computational simulation and comparison of prothrombin complex concentrate dosing schemes for warfarin reversal in cardiac surgery

Prothrombin complex concentrate (PCC) is increasingly used for acute warfarin reversal. We hypothesized that computational modeling of thrombin generation (TG) could be used to optimize the timing and dose of PCC during hemodilution induced by cardiopulmonary bypass (CPB). Thrombin generation patterns were modeled in anticoagulated patients (n=59) using a published computational model. Four dosing schemes were evaluated including single full dose (median, 41.2IU/kg) of PCC before or after CPB, ½-dose before and after CPB, or 1/3-dose before CPB plus 2/3-dose after CPB. Hemodilution was modeled as 40 or 60% dilution of factors from baseline. The lag time (s) of TG, and peak thrombin level (nM) were evaluated. Prolonged lag time, and reduced peak TG were due to low vitamin K-dependent (VKD) factors, and pre-CPB PCC dose-dependently restored TG to near-normal or normal range. After 40% dilution, TG parameters were similar among 4 regimens at the end of therapy. The recovery of VKD factors was less when PCC was given before CPBafter 60 % dilution, but TG parameters were considered hemostatically effective (>200nM) with any regimen. Withholding the full dose of PCC until post-CPB resulted in severely depressed TG peak (median, 47nM) after 60 % dilution, and some supra-normal TG peaks after treatment. Pre-CPB administration of full or divided doses of PCC prevents extremely low TG peak during surgery, and maintains hemostatic TG peaks in both 40 and 60% hemodilution models. Although PCC's hemostatic activity appears to be highest using the full dose after CPB, hypercoagulability may develop in some cases.

Vitamin K antagonists favourably modulate fibrin clot properties in patients with atrial fibrillation as early as after 3 days of treatment: Relation to coagulation factors and thrombin generation

IntroductionAtrial fibrillation (AF) increases the risk of thromboembolism that is reduced by vitamin K antagonists (VKAs). We sought to investigate changes in plasma fibrin clot phenotype at the onset of oral anticoagulation. Materials and methodsForty consecutive AF patients (aged 45–83 years, CHA2DS2-VASc score 3.0±1.5) who started therapy with warfarin or acenocoumarol were studied. Plasma fibrin clot permeability (Ks), clot lysis time (CLT), along with clotting factors (F), thrombin generation (TG) profiles and protein C (PC) levels were determined on days 3, 5, 7, 28 and 56±1 since the first dose. ResultsAF patients had 16% higher median of Ks and 15% lower median of CLT as early as on day 3 of VKA therapy compared with the baseline (both p<0.001), reaching the plateau values on day 7 and 5, respectively. Higher Ks values on days 1 and 3 were found in AF patients with further stable anticoagulation (both p<0.05). Moreover, FIX explained 32% of the total variability in Ks. Multivariate analysis adjusted for potential confounders including time as a predictor showed that vitamin K-dependent (VKD) factors, PC and TG parameters were the predictors of Ks (all p<0.0001), while only the lag phase of TG and thrombin peak predicted CLT (both p<0.05) in AF patients. Regression analysis of time-series showed however, that CLT was also predicted by VKD factors and PC (all p<0.05). ConclusionsPlasma fibrin clot properties in AF patients are favourably modified as early as after 3days of VKA administration, which might contribute to antithrombotic effectiveness.