John W. Suttie

Vitamin K and maintenance of skeletal integrity in adults.

PURPOSE To determine the role of vitamin K status in the maintenance of skeletal integrity in adults. PATIENTS AND METHODS 1. Bone mineral density (BMD) was measured by quantitative digital radiography (QDR) in 50 patients taking a vitamin K antagonist (warfarin) who were recruited from a large urban cardiology practice, and 50 age-, sex- and race-matched controls recruited from the community. 2. The relationship of BMD versus indices of vitamin K status (determined by measuring levels of vitamin K and descarboxyprothrombin in the plasma) in 113 nonanticoagulated adults was assessed. RESULTS Measurements of BMD in the hip and spine were similar in anticoagulated subjects and matched controls. Multivariate analysis revealed that use of warfarin was not associated with a lower BMD. Ninety-five percent confidence intervals excluded a 0.06 g/cm2 reduction in BMD associated with the use of warfarin. Indices of vitamin K status did not correlate with BMD in normal subjects. CONCLUSIONS Patients receiving long-term maintenance therapy with a vitamin K antagonist have normal bone density. BMD is unrelated to vitamin K status in nonanticoagulated subjects. These data suggest that vitamin K does not have a major role in maintenance of skeletal integrity in adults.

The vitamin K dependent, in vitro production of prothrombin.

Postmitochondrial supernates from vitamin K deficient rats respond to the in vitro addition of vitamin K to produce prothrombin. This system is energy dependent, and is inhibited by antagonists of vitamin K, but not by cycloheximide. These observations offer further proof that the vitamin acts at a postribosomal site in promoting the synthesis of prothrombin.

Vitamin K-dependent carboxylase: Requirements for carboxylation of soluble peptide substrates and substrate specificity

Abstract Rat liver microsomes contain a triton X-100 solubilizable vitamin K-dependent carboxylase activity that converts specific glutamyl residues of precursor proteins to γ-carboxyglutamyl residues. This activity has been studied utilizing synthetic peptides as substrates for the enzyme. When compared to the carboxylation of the endogenous microsomal precursors, the peptide carboxylase activity is more sensitive to the action of various inhibitors, and requires a higher concentration of vitamin K for maximal activity. The apparent Km for the peptide Phe-Leu-Glu-Glu-Leu was found to be 4 mM. Substrate specificity depends on residues adjacent to the carboxylated Glu residues and macromolecular recognition sites.

Properties of asialo and aglycoprothrombin

Abstract Native bovine prothrombin was shown to have a circulatory half-life of 90 minutes when injected into the circulatory system of rats. Removal of sialic acid resulted in a circulatory half-life of only 9 minutes and further enzymatic degradation of the carbohydrate restored the circulatory half-life to more nearly that of intact prothrombin. These observations broaden the generality of this phenomenon displayed by some other glycoproteins and therefore the possibility that sialic acid may function in determining the circulatory life of glycoproteins.

Solubilization and characterization of vitamin K epoxide reductase from normal and warfarin-resistant rat liver microsomes☆

Two procedures have been developed for the solubilization of vitamin K epoxide reductase from rat liver microsomal membranes using the detergent Deriphat 160 at pH 10.8. The methods are applicable to both normal and Warfarin-resistant-strain rat liver microsomes and yield material suitable for further purification. The preparations retain dithiothreitol-dependent vitamin K quinone reductase activity as well as vitamin K epoxide reductase and are free of vitamin K-dependent carboxylase and epoxidase activities. Optimal epoxide reductase activity is obtained at 0.1 M KCl and pH 9 in the presence of sodium cholate. Artifactual formation of vitamin K metabolites was eliminated through the use of mercuric chloride to remove excess dithiothreitol prior to extraction and metabolite assay. Using the solubilized enzyme, valid initial velocities were measured, and reproducible kinetic data was obtained. The substrate initial velocity patterns were determined and are consistent with a ping-pong kinetic mechanism. The kinetic parameters obtained are a function of the cholate concentration, but do not vary drastically from those obtained using intact microsomal membranes. At 0.8% cholate, the enzymes solubilized from normal Warfarin-sensitive- and Warfarin-resistant-strain rat livers exhibit respective values of Vmax = 3 and 0.75 nmol/min/g liver; Km for vitamin K epoxide = 9 and 4 microM; and Km for dithiothreitol of 0.6 and 0.16 mM.

Mechanism of the abnormal vitamin k‐dependent γ‐carboxylation process in human hepatocellular carcinomas

Background. An important marker for hepatocellular carcinoma is the presence of des‐γ‐carboxy (abnormal) prothrombin. However, the molecular basis for the reduced carboxylation of prothrombin is unknown.

Control of prothrombin and factor VII biosynthesis by vitamin K

Abstract The production of the plasma proteins, prothrombin and factor VII, which are essential in the blood clotting process, is dependent on the action of vitamin K. In intact vitamin K-deficient rats, administration of actinomycin D did not block the vitamin K-induced increase in plasma prothrombin. An isolated perfused rat liver preparation that released factor VII activity to the perfusate in response to vitamin K administration was developed. The factor VII production in this system was inhibited by puromycin but not by actinomycin D. These data indicate that the site of action of the vitamin is at some in step protein biosynthesis subsequent to the formation of a specific messenger RNA and prior to release of the complete protein from the liver.

A rat liver protein with potential thrombin activity: Properties and partial purification☆

Abstract A protein which generates thrombin activity when treated with the venom from Echis carinatus has been shown to build up in the liver microsomes of vitamin K-deficient or anticoagulant-treated rats. The amount of this protein decreases rapidly when vitamin K is given to these animals. The protein has been partially purified. It differs from prothrombin in its ability to adsorb to BaSO4, its electrophoretic mobility, and chromatography on DEAE-cellulose. It does, however, react with antibody prepared against rat prothrombin, and it apparently contains the thrombin portion of prothrombin. These observations are consistent with the hypothesis that this protein represents a liver precursor protein which is converted to plasma prothrombin in a vitamin K-dependent step.

Effect of n-methyl-thiotetrazole on vitamin k epoxide reductase

Clinical use of antibiotics containing a N-methyl-thiotetrazole (NMTT) side chain has been reported to be associated with an increased incidence of a vitamin K-responsive hypoprothrombinemia. Administration of NMTT to rats decreased the activity of the liver microsomal vitamin K epoxide reductase, increased the liver ratio of vitamin K epoxide to vitamin K, and decreased the rate of metabolism of injected vitamin K epoxide. These responses are the same as those observed following the administration of coumarin anticoagulants. In contrast to the effect of coumarin anticoagulants, NMTT did not inhibit the vitamin K epoxide reductase in vitro. These data suggest that the hypoprothrombinemia which has been observed following use of these antibiotics results from the inactivation of the liver vitamin K epoxide reductase by NMTT or a NMTT metabolite.

The effect of cycloheximide administration on vitamin K-stimulated prothrombin formation.

Abstract The administration of vitamin K to severely hypoprothrombinemic, vitamin K-deficient rats results in the appearance of about 50% of the normal steady state levels of plasma prothrombin within 1 hr, followed by a slower rate of repletion. This rapid initial burst of prothrombin is inhibited only about 25% by a dose of cycloheximide which almost completely blocks total plasma protein synthesis. The same level of cycloheximide treatment will block the synthesis of prothrombin which is seen between 1 and 2 hr after vitamin K administration, and will also block the hydrocortisone-induced synthesis of tyrosine amino transferase in the same animals. It has been reported that in a rat liver perfusion system there is a specific antagonism between the action of vitamin K and cycloheximide. No evidence for such a relationship could be seen in intact animals. The data strongly suggest that the action of vitamin K may involve the conversion of some precursor protein to prothrombin in a step that does not involve ribosomal protein synthesis.