Evidence for increased formation of preprothrombin and the noninvolvement of vitamin K-dependent reactions in sex-linked hyperprothrombinemia in the rat.

The rate of appearance of plasma prothrombin was measured in vitamin K-deficient male and female rats after the administration of vitamin K₁, and the disappearance of prothrombin was measured in normal rats after injection of cycloheximide. The results suggest that hyperprothrombinemia in female rats is due to a faster rate of formation of the clotting protein rather than to a slower rate of its degradation. Preprothrombin activity in liver microsomes was higher in warfarin-treated female rats than in warfarintreated male rats; but the activity of preprothrombin in liver disappeared at approximately the same rate in both sexes after administration of vitamin K. The rate and extent of vitamin K-dependent formation of γ-carboxyglutamic acid and the appearance of prothrombin activity in vitro were not significantly different between the sexes. These results suggest that elevated levels of plasma prothrombin in female rats are probably due to a higher rate of synthesis of preprothrombin and not to any difference in the vitamin K-dependent step. A difference was observed in the amount of cycloheximide required to inhibit synthesis of liver microsomal protein in the two sexes.     

Vitamin K-dependent carboxylase: effect of detergent concentrations, vitamin K status, and added protein precursors on activity

Activity of the rat liver microsomal vitamin K-dependent carboxylase has been studied at various concentrations of detergent. The activity which could be solubilized by 0.25% Triton X-100 was low but could be greatly increased if vitamin K-deficient rats were given vitamin K a few minutes before they were killed. At higher concentrations of Triton, more activity was solubilized and this effect was not seen. In vitro carboxylation of endogenous microsomal proteins was decreased by 80-90% if vitamin K was administered 1 min before rats were killed, but the amount of assayable prothrombin precursor was decreased by only 20%. Decarboxylated vitamin K-dependent rat plasma proteins were not substrates for the carboxylase and did not influence peptide carboxylase activity significantly. Purified microsomal prothrombin precursors did, however, stimulate carboxylation of peptide substrate and were used as a substrate for the carboxylase in a preparation from precursor depleted vitamin K-deficient rats.     

Changes in phylloquinone epoxidase activity related to prothrombin synthesis and microsomal clotting activity in the rat

The oxidation of phylloquinone to the 2,3-epoxide (by phylloquinone epoxidase) was studied in liver from control and warfarin-resistant rats. The reaction requires microsomal fraction, soluble protein, a heat-stable soluble factor and O(2). It is not inhibited by CO or CN(-). Epoxidase activity was stimulated if plasma prothrombin was lowered either by anticoagulants or the absence of vitamin K. The activity of the enzyme rapidly returned to normal values after the administration of vitamin K to hypoprothrombinaemic rats. These differences in the activity of the enzyme occur in the microsomal fraction and not the cytosol. A thrombin-generating polypeptide that accumulates in microsomal fraction of hypothrombinaemic rats correlated directly with epoxidase activity. These data support the view that enzymic interconversion of phylloquinone and its 2,3-epoxide participates in the biological activity of vitamin K.     

A microsomal endopeptidase from liver with substrate specificity for processing proproteins such as the vitamin K-dependent proteins of plasma.

The microsomal fraction of rabbit liver contains an endopeptidase that cleaves synthetic peptides that mimic the amino acid sequences of the processing sites of many proproteins, including the vitamin K-dependent proteins. The endopeptidase (M(r) 69,000) was extracted from liver microsomes with 1% Lubrol and purified about 2,700-fold. The substrate employed for isolation and characterization of the enzyme was the decapeptide acetyl-Ala-Arg-Val-Arg-Arg-Ala-Asn-Ser-Phe-Leu (prothrombin peptide), in which hydrolysis occurred on the carboxyl side of the paired Arg-Arg residues. The purified enzyme, whose activity was enhanced 1.8-fold by 0.1 mM CoCl2, has a Km = 80 microM and Vmax = 21,000 nmol.min-1.mg-1 and a pH optimum of 8.7. Proteolytic cleavage of decapeptide substrates was dependent on an arginine residue at positions P1 and P4. The enzyme was completely inhibited by EDTA and 1,10-phenanthroline as well as by p-chloromercuriphenylsulfonic acid and Hg2+. Inhibitors of serine proteases and cysteine proteases had no effect. Based on the substrate preference, the endopeptidase appears to be a good candidate for the enzyme responsible for the precursor processing of the vitamin K-dependent proteins and a number of other proproteins that are synthesized via the secretory pathway in liver and other tissues.     

Bimodal frequency distribution of rat hepatic vitamin K-dependent carboxylation rate

The time course of vitamin K-dependent carboxylation was studied in an in vitro rat hepatic microsomal system. This method is based on incorporation of radiolabelled CO2 into endogenous substrate proteins. Forty rats were studied in order to characterize the intrinsic formation rate (V/KM) of carboxylated vitamin K-dependent proteins and the maximum amount of endogenous substrate available for vitamin K-dependent carboxylation (P infinity; normalized for the total amount of microsomal protein harvested). The frequency distributions of V/KM and P infinity values were both well described as the sum of two Gaussian components, each representing about 40% and 60% of the populations.     

Propeptide recognition by the vitamin K-dependent carboxylase in early processing of prothrombin and factor X.

Precursors of vitamin K-dependent proteins are synthesized with a propeptide that is believed to target these proteins for gamma-carboxylation by the vitamin K-dependent carboxylase. In this study synthetic propeptides were used to investigate gamma-carboxylation of the prothrombin and factor X precursors in rat liver microsomes. The extent of prothrombin processing by the carboxylase was also investigated. Antisera raised against the human prothrombin and factor X propeptides only recognized precursors with the respective propeptide regions. The data demonstrate structural differences in the propeptide region of the prothrombin and the factor X carboxylase substrates which raises questions about the hypothesis of a common propeptide binding site on the carboxylase for all precursors of vitamin K-dependent proteins. The hypothesis of separate binding sites is supported by data which demonstrate differences in binding of the prothrombin and factor X precursors to membrane fragments from rough and smooth microsomes. gamma-Carboxylation of the prothrombin precursors in vitro was investigated with conformational specific antibodies raised against a portion of the Gla (gamma-carboxyglutamic acid) region extending from residue 15 to 24. The synthetic peptide used as antigen contains three of the ten potential Gla sites in prothrombin. It is shown that these antibodies do not recognize mature prothrombin but recognize the decarboxylated protein. It is also demonstrated that the epitope is Ca2(+)-dependent. The antibodies were used to assess gamma-carboxylation of the prothrombin precursor in membrane fragments from microsomal membranes. The results suggest that microsomal gamma-carboxylation does not involve Glu residues 16, 19 and 20 of the Gla region.     

Vitamin K-dependent carboxylase: effect of endogenous microsomal protein precursors on the rate of exogenous substrate carboxylation

Rat liver microsomes contain a Triton X-100 solubilizable vitamin K-dependent carboxylase activity that converts specific glutamyl residues of a microsomal prothrombin precursor to gamma-carboxyglutamyl residues. This activity has been studied in partially (0.25% Triton X-100) and completely (1.0% Triton X-100) solubilized rat liver microsomal preparations. The rate of vitamin K-dependent carboxylation of endogenous microsomal protein precursors was very rapid in the completely solubilized liver microsomal preparation, and carboxylation of an exogenous peptide substrate (Phe-Leu-Glu-Glu-Leu) proceeded at the same time. In the partially solubilized liver microsomal preparation, the rate of protein carboxylation was greatly reduced, and a lag in carboxylation of the exogenous substrate was observed. When microsomal preparations which were depleted of endogenous precursors were used, this lag was eliminated. These data suggest that both substrates utilize the same microsomal pool of carboxylase and that the fraction of the carboxylase bound to the endogenous precursors is not immediately available to exogenous substrates.     

Characteristics of the vitamin K-dependent carboxylating system in human placenta.

Gamma-carboxyglutamic acid, formed during the post-translational vitamin K-dependent carboxylation of glutamic acid residues in polypeptides has been identified not only in coagulation factors II (prothrombin),, VII, IX and X [1--4], but also in several other plasma proteins [3,5,6] and in protein of bone [7,8] and kidney [9]. In rat liver, carboxylation is mediated through an enzyme system located in the microsomal membrane [10]. The enzyme system requires CO2, O2 and the reduced (hydroquinone) form of the vitamin, as well as a suitable substrate [10,11]. Rat liver microsomes also convert vitamin K1 (phylloquinone) to its stable 2,3-epoxide [12]. Several studies suggest a link between carboxylation and the formation of the epoxide [12--14]. In one of these [14], a survey of rat tissues for vitamin K1 epoxidation revealed that, in addition to liver, this activity was also possessed by kidney, bone, spleen and placenta. In preliminary experiments, vitamin K-dependent carboxylating systems have been found in rat and chick kidney [9], in chick bone [15] and in rat spleen and placenta (unpublished observations). In this communication, we describe some of the basic characteristics of the vitamin K-dependent carboxylating system as found in human placental microsomes.     

The intracellular distribution of [1-3H]-cholecalciferol in the intestine of vitamin D-deficient and supplemented rats

1. [1-(3)H]Cholecalciferol was administered orally at two dosages to vitamin D-deficient and -supplemented rats, and the intracellular distribution of the vitamin in the intestinal mucosa studied. 2. The concentration of cholecalciferol was highest in a fraction consisting of brush borders and nuclei. The microsomal fraction contained a higher concentration of the vitamin than the mitochondrial fraction in deficient rats, irrespective of the dose, whereas in the vitamin D-supplemented rats the concentration was the same in the two fractions. 3. Appreciable metabolism of the cholecalciferol occurred only in the supplemented rats and the metabolites were found predominantly in the mitochondrial fraction. 4. The cholecalciferol is more tightly bound to the microsomal fraction than to the mitochondrial fraction. 5. Experiments conducted in vitro have shown that all the intracellular particles combine with the vitamin either when dissolved in ethanol or bound to albumin. However, such an uptake does not account for the high concentration of radioactivity found in vivo in the fraction containing nuclei and brush border, nor for the tightly bound vitamin in the microsomal fraction.     

Vitamin K and the biosynthesis of prothrombin. V. Gamma-carboxyglutamic acids, the vitamin K-dependent structures in prothrombin.

Tryptic peptides obtained from normal prothrombin have been compared with those obtained from prothrombin synthesized by cattle given the vitamin K antagonist dicumarol. Two peptides were found which contain vitamin K-dependent structures. These peptides contain residues 4 through 10 and residues 12 through 44, respectively. One of these (residues 4 through 10) has previously been shown to contain gamma-carboxyglutamic acid residues. Digestion of this peptide with aminopeptidase M and carboxypeptidase B yielded a tetrapeptide (residues 6 through 9). Mass spectra of this peptide showed that it has the structure Leu-Glu(CO2)-Glu(CO2)-Val. The structure of the peptide containing residues 12 through 44 was determined by automated degradation in a peptide sequenator. The modified glutamic acid residues were identified by mass spectrometric comparison with the thiohydantoin derivatives of synthetic gamma-carboxyglutamic acid. This approach unequivocally demonstrated that all of the first 10 glutamic acid residues in prothrombin are carboxylated to form gamma-carboxyglutamic acid residues. Evidence is also presented that indicates that these gamma-carboxyglutamic acid residues constitute the entire vitamin K-dependent modification of prothrombin.     

Vitamin K dependent in vitro production of prothrombin

During prothrombin biosynthesis, glutamyl residues in prothrombin precursor proteins are carboxylated to gamma-carboxyglutamyl residues by a vitamin K dependent carboxylase. Calcium-dependent and calcium-independent rat prothrombin antibody subpopulations have been produced and utilized to study the liver microsomal precursors of prothrombin that accumulate when vitamin K action is blocked. A substantial portion of the precursor pool accumulating in the vitamin K deficient or warfarin-treated rat will react with a Ca2+-dependent antibody at high calcium concentration and appears to be partially carboxylated. During in vitro incubation in the presence of vitamin K, the fraction of the precursor pool which is tightly bound to the microsomal membrane appears to be the preferred substrate for the vitamin K dependent carboxylation. A small amount of completely carboxylated rather than a large amount of partially carboxylated products are produced during these incubations. Treatment with a Sepharose-bound prothrombin antibody demonstrated that about 20-25% of the total carboxylated microsomal protein precursor pool consists of prothrombin precursors. This treatment removes an equal amount of total carboxylase activity, and the enzyme is active in this carboxylase precursor-antibody complex.     

The in vivo effects of acenocoumarol, phenprocoumon and warfarin on vitamin K epoxide reductase and vitamin K-dependent carboxylase in various tissues of the rat

In rats the in vivo effects of a chronic low-dose treatment (+/- 60 micrograms/rat per day) with different coumarins (acenocoumarol, phenprocoumon and warfarin) on hepatic and non-hepatic vitamin K-dependent enzyme systems were compared. The plasma concentrations of the three coumarins differed largely but these differences were not reflected in the microsomal coumarin contents. The non-hepatic microsomes contained less than 20% of the coumarins found in liver microsomes. No substantial differences were observed between the following effects of the three anticoagulant treatments. The blood coagulation factor activities were about 10% of normal. The hepatic microsomal vitamin K epoxide reductase activity was diminished to about 35% of control values. The vitamin K epoxide reductase activities present in kidney, lung, spleen, testis and brain microsomes were less influenced by the coumarin treatments; activities ranged between 45 and 65% of normal. In the liver microsomes a 15-fold accumulation of non-carboxylated precursor proteins was found; in the non-hepatic microsomes this effect was less pronounced but still present. The hepatic vitamin K-dependent carboxylase activity was enhanced but the corresponding non-hepatic enzyme activities were slightly or not affected. In addition, the effects of a chronic low-dose warfarin treatment were compared with those after an acute high dose of the drug.     

Synthesis of clotting factors by a cell-free system from rat liver in response to the addition of vitamin K1 in vitro.

A cell free system from the liver of vitamin K-deficient rats will form clotting factors after addition of vitamin K₁ in vitro. The response requires both microsomal pellet and supernatant. It is not energy dependent and no co-factor requirement could be demonstrated. Immunological tests and the response to vitamin K₁ analogues demonstrate the physiological nature of the response. It has been recently claimed that vitamin K is required for the formation of calcium binding sites by carboxylation of glutamyl residues. Failure to demonstrate an energy requirement in this system suggests that either vitamin K-dependent carboxylation proceeds by a mechanism hitherto unknown in biology or that the vitamin K-dependent reaction is not directly coupled to carboxylation.     

Association of folic acid with rat liver microsomes

Summary Shin et al. (Biochim Biophys Acta 444: 794–801, 1976) described the subcellular location of [³H]folic acid after injection into rats. The microsomal fraction of the liver contained relatively large amounts of tracer initially but lower amounts at later times. Because of the heterogeneous nature of the microsomal fraction of the liver we re-examined the nature of the folate binding fraction. The location of injected [³H]folic acid resembled that of the microsomes derived from the plasma membrane, where ultracentrifugal analysis was conducted in the presence and absence of cesium ions. The location of the folate did not resemble that of microsomes derived from the endoplasmic reticulum (ER). One of the marker enzymes of the ER was the vitamin K-dependent carboxylase. A simple method for reducing vitamin K is described.     

Beta-hydroxyaspartic acid in vitamin K-dependent plasma proteins from scorbutic and warfarin-treated guinea pigs

beta- Hydroxyaspartic acid is a rare amino acid, present in all vitamin K-dependent plasma proteins except prothrombin, and is formed by a post-translational hydroxylation of aspartic acid. We have now investigated whether this hydroxylation, like that of proline in collagen, is vitamin C-dependent. The vitamin K-dependent plasma proteins were isolated from normal and scorbutic guinea pig plasma by barium citrate adsorption and the beta- hydroxyaspartic acid content was determined. Compared with normal animals, scorbutic animals showed no significant reduction of beta- hydroxyaspartic acid content. In warfarin-treated animals there was a decreased content of both beta- hydroxyaspartic acid and gamma-carboxyglutamic acid in the barium citrate adsorbed fraction. It was concluded that the post-translational hydroxylation of aspartic acid is unlikely to be vitamin C-dependent.     

The role of vitamin K in the interaction of 1,25-dihydroxyvitamin D3 receptors with DNA

Vitamin K deficiency in rats caused a rise of in vivo occupied 1,25(OH)2D3 receptor level in chromatin of the intestinal mucosa and a marked (2-2.5-fold) increase of intestinal cytosolic 1,25(OH)2D3-receptor complex binding with heterologous DNA, whereas maximum binding capacity and equilibrium dissociation constant of cytosolic 1,25 (OH)2D3 receptors did not change. Preincubation of renal and intestinal cytosol of vitamin K-deficient rats with microsomal vitamin K-dependent gamma-carboxylating system reduced sharply 1,25(OH)2D3-receptor complex binding with DNA. In rats treated by vitamin K antagonist along with a low calcium diet, no dramatic decrease of occupied 1,25(OH)2D3 receptors occurred after the animals were maintained with a high calcium diet. No such effect was observed in vitamin K-replete rats. The data demonstrate vitamin K-dependent Ca-sensitive qualitative modification of 1,25(OH)2D3 receptor dropping its binding performance to DNA.     

A method for systematic purification from bovine plasma of six vitamin K-dependent coagulation factors: prothrombin, factor X, factor IX, protein S, protein C, and protein Z

A systematic purification scheme is presented for the isolation of six vitamin K-dependent coagulation factors from bovine plasma in a functionally and biochemically pure state. The vitamin K-dependent proteins concentrated by the ordinary barium citrate adsorption were first separated into four fractions, fractions A, B, C, and D, by DEAE-Sephadex A-50 chromatography. From the pooled fraction A, protein S, factor IX, and prothrombin were purified by column chromatography on Blue-Sepharose CL-6B. Heparin-Sepharose chromatography of the pooled fraction B provided mainly pure factor IX, in addition to homogeneous prothrombin. A high degree of resolution of protein C and prothrombin from the pooled fraction C was obtained with a Blue-Sepharose column. This dye-ligand chromatographic procedure was also very effective for the separation of protein Z and factor X contained in the pooled fraction D. Thus, these preparative procedures allowed high recovery of milligram and gram quantities of six vitamin K-dependent proteins from 15 liters of plasma in only two chromatographic steps, except for protein S, which required three (the third step was rechromatography on Blue-Sepharose CL-6B).     

Effects of diffusible products of peroxidation of rat liver microsomal lipids

The effects on cellular structures of products of peroxidation of rat liver microsomal lipids were investigated. A system containing actively peroxidizing liver microsomal fraction was separated from a revealing or target system by a dialysis membrane. The target system, contained in the dialysis tube, consisted of either intact cells (erythrocytes) or subcellular fractions (liver microsomal fraction). When liver microsomal fractions were incubated with NADPH (or an NADPH-generating system), lipid peroxidation, as measured by the amount of malonaldehyde formed, occurred very rapidly. The malon-aldehyde concentration tended to equilibrate across the dialysis membrane. When the target system consisted of erythrocytes, haemolysis occurred abruptly after a lag phase. The lysis was greatly accelerated when erythrocytes from vitamin E-deficient rats were used, but no haemolysis was observed when erythrocytes from vitamin E-treated rats were used. When, in the same system, freshly prepared liver microsomal fractions were exposed to diffusible factors produced by lipid peroxidation, the glucose 6-phosphatase activity markedly decreased. A similar decrease in glucose 6-phosphatase activity, as well as a smaller but significant decrease in cytochrome P-450, was observed when the target microsomal fractions were exposed to diffusible factors derived from the peroxidation of liver microsomal lipids in a separate preincubation step. These and additional experiments indicated that the toxicological activity is relatively stable. Experiments in which the hepatic microsomal fractions destined for lipid peroxidation contained radioactively labelled arachidonic acid, previously incorporated into the membranes, showed that part of the radioactivity released from the microsomal fraction into the incubation medium entered the dialysis tube and was recovered bound to the constituents of the microsomal fractions of the target system. These results indicate that during the course of the peroxidation of liver microsomal lipids toxic products are formed that are able to induce pathological effects at distant loci.     

A new carboxylation reaction. The vitamin K-dependent incorporation of H-14-CO3- into prothrombin.

The bovine plasma zymogen prothrombin contains a number of gamma-carboxyglutamic acid residues which are not found in an abnormal prothrombin produced when cattle are given the vitamin K antagonist dicoumarol. These modified glutamic acid residues appear to be formed post-translationally by a reaction which requires vitamin K. It has been shown that postmitochondrial supernates from vitamin K-deficient rats incorporate added H-14-CO3- minus into microsomal proteins upon the addition of vitamin K. This incorporation is dependent upon the presence of the prothrombin precursor in the microsomal preparations, and upon factors which are present in the postmicrosomal supernatant. Most of the radioactive protein which can be obtained from the microsomal pellet by extraction with 0.25% Triton X-100 has been identified as prothrombin and it can be shown that all of the radioactivity is in the amino-terminal activation fragment of prothrombin. This portion of the protein has previously been shown to contain the gamma-carboxyglutamic acid residues. Hydrolysis of the purified radioactive prothrombin resulted in a loss of 50% of the radioactivity and subsequent chromatography of the amino acid hydrolyzate demonstrated that the remaining radioactivity was entirely in glutamic acid. These results are consistent with the hypothesis that all of the H-14-CO3- minus was incorporated into the carboxyl groups of gamma-carboxyglutamic acid residues.     

Identification of vitamin K-dependent carboxylase activity in lung type II cells but not in lung macrophages.

Fluorography of 14C-labelled glutamic acid residues in vitamin K-dependent protein precursors in lung microsomes (microsomal fractions) shows that the lung has several substrates that are not found in the liver. These precursor proteins unique to the lung have apparent molecular masses of 65, 53, 50, 36, 31 and 13 kDa. Type II epithelial cells appear to synthesize most of the vitamin K-dependent proteins in the lung. The 36 and the 31 kDa precursors also found in Type-II-cell microsomes have a similar molecular mass to those of surfactant-associated proteins, and we have previously shown [Rannels, Gallaher, Wallin & Rannels (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5952-5956] that the 36 kDa protein is one of the precursors for these proteins. Immunoblotting of membrane fragments of Type-II-cell microsomes with plasma prothrombin antibodies identified two prothrombin-like antigens of apparent molecular masses 68 and 65 kDa. This raises the question as to whether Type II cells are also a potential site for synthesis of prothrombin and possibly other vitamin K-dependent clotting factors. Pulmonary macrophages appear to be devoid of vitamin K-dependent carboxylase activity. However, Type II epithelial cells have significant activity, and this activity was unaltered when these cells were maintained in primary culture for 3 days, suggesting that carboxylase activity is expressed in lung alveolar epithelium independently of culture-induced changes in cellular differentiation. Carboxylase activity in Type II cells was enhanced 2-fold when cells were cultured for 24 h in the presence of 50 microM-warfarin. Type II cells, therefore, resemble hepatocytes with regard to their response to coumarin anticoagulant drugs.