Circadian rhythms of blood clotting time and coagulation factors II, VII, IX and X in rats

The 24-hr variations in clotting times and vitamin K-dependent blood coagulation factors were studied in rats kept on a 12-hr light-dark cycle (light on: 0600-1800 hours). Clotting times were determined under a binocular microscope by measuring the time required for the formation of the first fibrin thread. Factors II, VII and X were analyzed by the prothrombin test while the factor IX was quantified using the activated partial thromboplastin time assay. Results indicated that the clotting times were significantly longer during the dark (activity) period with a peak at 1:00 and a trough at 17:00. Similarly, a variation was found in factor activity levels: prothrombin (II), factor VII and factor X had higher activities during the light span (rest period). The highest activities found at 13:00 and 09:00 were statistically different from the minimum activity levels obtained at 21:00. Factor IX did not show a significant circadian variation.     

Prolongation by warfarin of "template" bleeding time in rats: a vitamin K-dependent effect

Administration of warfarin to rats induced not only the well-known anticoagulant effect, but also an impairment of primary hemostasis as reflected by a significant prolongation of the "template" bleeding time. This effect was very closely associated with lowering of the prothrombin complex level and was reversed by administration of vitamin K. It is suggested that some of the clotting factors known to be vitamin K-dependent also play a role in primary hemostasis; alternatively, a putative vascular "bleeding factor" could be modulated by vitamin K availability.     

In vitro prothrombin synthesis from a purified precursor protein. II. Partial purification of bovine carboxylase

In this paper, we describe the isolation and partial purification of an enzyme system that converts bovine decarboxyfactor II (PIVKA-II) into prothrombin (factor II). It is shown that the increase in factor II activity occurs in parallel with 14CO2 incorporation into BaSO4 adsorbable proteins. The system is not strictly vitamin K-dependent because it is obtained from the livers of normal healthy cows. By preincubating the enzyme(s) with an excess of warfarin, an absolute vitamin K1-dependence can be obtained. The reaction is inhibited by its own product, factor II.     

The vitamin K dependent, in vitro production of prothrombin

Postmitochondrial supernates from vitamin K deficient rats respond to the $\text{in vitro}$ 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.     

Separation of human vitamin K-dependent coagulation proteins using hydrophobic interaction chromatography

A rapid and simple method was developed to separate human vitamin K-dependent plasma proteins from each other, yielding virtually homogeneous pools. The purification technique is based on the single use of hydrophobic interaction chromatography, starting from prothrombin concentrate (PC or DEFIX, also termed factor IX concentrate) as initial material. Phenyl-sepharose HP demonstrated optimal separation by comparing several hydrophobic resins as well as resins used in standard procedures like immobilised heparin and Cibacron blue. Under ideal conditions, factor X could be separated in a single step as well as prothrombin. Factor IX co-eluted with other minor proteins. Focus was given only on these three proteins due to their relative abundance. Complete separation of all proteins present in the starting material was achieved by MonoQ anion-exchange chromatography following the phenyl-sepharose run. The resulting purified material could be demonstrated to be of equal or higher purity than using described methods. This strategy employing hydrophobic interaction chromatography for blood macromolecules could be of immense value for purifying the human vitamin K-dependent proteins and represents a considerable simplification over other purification schemes. It not only involves minimal sample handling but also can be readily up-scaled and is a cost-efficient alternative.     

Vitamin K-dependent carboxylase. Requirements of the rat liver microsomal enzyme system.

Vitamin K is required in an enzymatic reaction which carboxylates glutamyl residues in a microsomal protein precursor of plasma prothrombin to form gamma-carboxyglutamic acid residues. The partial requirements of this microsomal, vitamin K-dependent carboxylase system have been determined. A requirement of the system for cytosolic factors appears to be due primarily to the presence of reduced pyridine nucleotides or a reduced pyridine nucleotide-generating system in the cytosol. The hydroquinone of vitamin K has been demonstrated to be the enzymatically active form of the vitamin. When vitamin K1 hydroquinone is added to the carboxylase system, no NAD(P)H is needed for maximum activity. The carboxylase activity is half-maximally stimulated by 0.25 mug of vitamin K1/ml in the presence of cytosolic components but requires at least 10 times as much vitamin when microsomes are incubated in a cytosol-free buffer. Menadione is inactive as a vitamin source in this system, and the carboxylase activity is inhibited by the 2-chloro analog of vitamin K1 and by Warfarin. The ATP analog, AMP-P(NH)P, inhibited the carboxylase activity, but a dependence on exogenous ATP or an ATP-generating system could not be demonstrated. Carboxylase activity was found to be dependent on an O2-containing gas phase, and upon the HCO3- concentration.     

Ineffectiveness of phylloquinone epoxide as an inhibitor of prothrombin synthesis in the rat

Phylloquinone epoxide (vitamin K₁-oxide), a metabolite of phylloquinone, does not inhibit prothrombin synthesis when administered in high doses to Sprague-Dawley and warfarin-resistant rats. Further, it does not accumulate to presumed inhibitory levels in the livers of rats given physiological doses of ³H-phylloquinone when they are anticoagulated with warfarin. These data do not support the Bell-Matschiner hypothesis that warfarin exerts its action by inhibiting the vitamin K oxide reductase which results in the accumulation of vitamin K oxide and the inhibition of vitamin K at its active site. Rather, our data support the view that vitamin K and warfarin combine at different sites with a single regulatory protein which serves as a conformational switch for prothrombin synthesis.     

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.     

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.     

Atypical Prothrombins induced by Dicoumarol

VITAMIN K maintains normal plasma prothrombin concentration. The effects of this vitamin as well as of its antagonist, dicoumarol, have been studied, often with the aid of inhibitors of protein synthesis, such as puromycin and cycloheximide^1–8. It is generally accepted that the site of action of vitamin K is not at the DNA level but at the late ribosomal stage; whether the vitamin acts in the de novo synthesis of prothrombin or in the completion of “unfinished” molecules is uncertain. Dicoumarol may cause release of unfinished material, or of “modified” molecules which are converted to the normal form by vitamin K. Conflicting evidence may be due to differences in dosage of protein inhibitors or of dicoumarol, differences in sampling time, differences in degree of purification, species variation, or differences in physical characteristics and chemical behaviour of unfinished or modified molecules which would demand that methods be specially adapted for their isolation.     

Status of liver mitochondria and rat tissue creatine kinase during administration of antivitamin K]

It has been shown that 16-18 days administration of antivitamin K (pelentan) leads to two-fold increase of prothrombin time in adult rats but does not influence the soluble brain, renal, heart, muscle and serum creatine kinase activity. No effect on metabolic function of isolated liver mitochondria has been found in contrast to the vitamin K deficient rats. Mitochondria were characterized by high value of respiration control; substance oxidation rates and internal mitochondrial Ca2+ content do not differ in the level from those of control animals. From the obtained results and data published in literature a conclusion can be drawn about only particular similarity (prothrombin time) between the antivitamin K administration and alimentary vitamin K deficit.     

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.     

Synthesis of 2-methyl-1,4-naphthoquinones with higher gamma-glutamyl carboxylase activity than MK-4 both in vitro and in vivo

Vitamin K is the collective term for compounds that share a 2-methyl-1,4-naphthoquinone ring, but differ in the side-chain at the 3-position. We synthesized novel 2-methyl-1,4-naphthoquinone derivatives with different side chain length at the 3-position. Derivatives with C-14 and C-16 tails showed the highest in vitro bioactivity resulting in 2.5 and 2-fold higher carboxylated osteocalcin synthesis in MG63 cells than menaquinone-4 (MK-4, form of vitamin K2). Longer side chain lengths resulted in lower bioactivity. The in vivo vitamin K activity of the C-14 tail derivative was further tested in WKY rats receiving a vitamin K-deficient diet that resulted in a 40% decrease of prothrombin activity. The C-14 tail derivative was able to counteract the effects on vitamin K deficiency induced by the diet and resulted in the complete restoration of prothrombin activity. Compared to naturally occurring forms of vitamin K, synthetic vitamin K derivatives may have higher bioactivity and different pharmacological characteristics that are more favorable for use as supplements or in clinical settings.     

Salt-tolerant cation exchanger-containing sulfate groups as a viable alternative for mixed-mode type and heparin-based affinity resins

Ion-exchange chromatography is still one of the most popular protein separation techniques. Before chromatographic separation, the high salt concentration in various samples necessitates additional steps. Therefore, low salt tolerance of ion-exchange resins is a drawback that needs to be addressed. Herein, the differences in salt tolerance and hydrophobicity of strong cation-exchange TOYOPEARL resins of sulfonium and sulfate-types were investigated. Despite only a minor structural difference, differences in selectivity and salt tolerance between the sulfate and sulfonic groups were detected. In silico calculations were also carried out for model substances representing the sulfonium and sulfate groups, wherein significant differences in hydrophobicity was observed. These experiments confirmed the hypothesis that the salt tolerance, higher affinity, and selectivity for certain vitamin K dependent clotting factors are interrelated and dependent on the presence of the sulfate group. Separation of clotting factor IX from the prothrombin complex concentrate further to confirmed the affinity for these proteins. The results show that the use of only a resin with the sulfate ligand and not with the sulfonic acid ligand allows for a facile and rapid separation of clotting factor IX and other vitamin K dependent clotting factors.     

Congenital deficiency of factor VII in a canine family

Prolonged prothrombin time in the blood coagulation test was seen in some beagle dogs whose activated partial prothrombin times were distributed within the normal range. This phenomenon suggested possible abnormalities in coagulation factors II, V, VII, and/or X. Therefore, a revised cross-matching test was given and a determination of coagulation factors related to the extrinsic system was performed. We also determined whether or not factor VII inhibitor was present. The results were as follows: 1) In the revised cross-matching test, the prolonged prothrombin times were revised when normal canine serum was added to the plasma that showed prolongation of prothrombin time, but not when pooled normal canine plasma absorbed with BaSO4 was added to it. 2) The level of factor VII in the plasma with prolonged prothrombin time was 5 approximately 10% of the level in normal canine plasma. 3) Factor VII inhibitor was not detected in the plasma with prolonged prothrombin time or in normal plasma. Consequently, the prolongation of prothrombin time was attributed to a deficiency in factor VII. This abnormality was confirmed to be congenital.     

Estrogen and prothrombin synthesis. The prothrombinogenic action of estrogen

Vitamin K deficient castrate male rats exhibit more rapid prothrombin depletion after injection of 100 μg of methyltestosterone, decreasing from 25% to 8% of normal plasma prothrombin levels. During the same 96 hour period, control castrate male rats showed a decline from 25% to 16%. Injection of 100 μg ethynylestradiol to similar vitamin K deficient, castrate male rats increased plasma prothrombin levels from 23% to approximately 45% within 96 hours. The effect of estradiol on biosynthesis of prothrombin was investigated by measuring incorporation of [³H]L-amino acids into the electrophoretically separable prothrombin. We conclude that the observed estrogen effect is due to a prothrombinogenic, rather than prothrombin-sparing mechanism.     

Vitamin K status in peritoneally dialyzed patients with chronic kidney disease

Abnormal vitamin K status was documented in patients with chronic kidney diseases (CKD), especially those undergoing hemodialysis. The data related to patients undergoing peritoneal dialysis (PD) are contradictory. Therefore, in the present study we aimed to evaluate vitamin K status in patients with CKD who are treated with continuous ambulatory PD. Twenty-eight patients entered into the study. Dialysis vintage ranged from 3 to 89 months. Vitamin K status was assessed in all subjects using undercarboxylated prothrombin measurement (PIVKA-II). In addition, total protein and albumin levels, total cholesterol, LDL cholesterol, triglyceride, calcium, urea and creatinine concentrations were determined. PIVKA-II concentrations were abnormal in 13 (46.4 %) subjects. BMI values, both total and LDL cholesterol concentrations were significantly higher in patients with than those without vitamin K deficiency. Moreover, PIVKA II levels correlated with BMI values (r = 0.441, p < 0.019), LDL cholesterol (r = 0.434, p < 0.021) and creatinine (r = 0.406, p< 0.032) concentrations. However, through the use of logistic regression analysis and multiple regression analysis, no clinical factor was documented to be the independent risk factor of vitamin K deficiency. In conclusion, vitamin K deficiency is a frequent condition in peritoneally dialyzed patients. Assessment of vitamin K status should become a standard procedure in this group of patients.     

Diets enriched in (n-3) fatty acids affect rat coagulation factors dependent on vitamin K

The effects of dietary lipids on haemostasis were investigated in rats fed high fat diets enriched in saturated fatty acids (SAT), oleic acid (OLEIC), MaxEPA^® oil (MaxEPA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) and results were compared to those for rats fed standard chow (ST). Coagulant activities of factor IIc and factor VII-Xc were reduced by about 70 % in the MaxEPA group and 50 % in the EPA and DHA groups relative to the OLEIC, SAT and ST groups. Liver vitamin K levels were five times lower in the experimental groups than in the ST group, which would indicate an effect of high fat diets on vitamin K metabolism. However, only (n-3) fatty acids prolonged the prothrombin time. These components could act at the post-transiational modification level of vitamin K-dependent plasma clotting factors. The changes in haemostatic factors found in the MaxEPA group were counteracted by vitamin K supplementation.     

Metabolism of vitamin K and prothrombin synthesis: anticoagulants and the vitamin K--epoxide cycle.

Vitamin K is primarily located in hepatic microsomes, where the vitamin K-dependent carboxylation in prothrombin synthesis occurs. Recent evidence supports the idea that the carboxylation is linked to the metabolism of the vitamin--specifically the cyclic interconversion of vitamin K and vitamin K epoxide. The primary site of action of coumarin and indandione anticoagulants appears to be an inhibition of the epoxide-to-vitamin K conversion in this cycle. There is a correlation between the inhibition of prothrombin synthesis and the regeneration of vitamin K from the epoxide by anticoagulants. In hamsters and warfarin-resistant rats prothrombin synthesis and the epoxide-K conversion are less sensitive to warfarin than in the normal rat. The epoxide-K conversion is impaired in resistant rats, which may explain their high vitamin K requirement. There is also a correlation between vitamin K epoxidation and vitamin K-dependent carboxylation, but the apparent link may be because vitamin K hydroquinone is an intermediate in the formation of the epoxide and also the active form in carboxylation. The vitamin K-epoxide cycle is found in extrahepatic tissues such as kidney, spleen, and lung and is inhibited by warfarin.     

Chemical modification of gamma-carboxyglutamic acid residues in prothrombin elicits a conformation similar to that of abnormal (des-gamma-carboxy)prothrombin

Chemical modification of gamma-carboxyglutamic acid (Gla) residues in human prothrombin to gamma-methyleneglutamic acid (gamma-MGlu) residues elicited a conformation similar, if not identical, to that of des-gamma-carboxy prothrombin or PIVKA-II, i.e., prothrombin molecules induced by vitamin K antagonists or vitamin K deficiency states. The reaction seems to proceed sequentially by preferentially modifying a Gla at residue 32 that is located innermost among 10 Gla residues of human prothrombin. The initial modification resulted in nearly 50% losses of barium salt adsorption, the procoagulant activity and thrombin generation by the prothrombinase complex. The subsequent modification of two Gla residues at positions 6 and 16 gave rise to the immunoreactivity to an established monoclonal antibody that specifically recognizes the des-gamma-carboxy prothrombin. Further modification of Gla residues increased the reactivity to the antibody, indicating that the conformation recognized by the antibody was stabilized so as to more readily fit the recognition site of the antibody. The appearance of the immunoreactivity was obviously related to the modification of Gla residues in prothrombin, since all other similarly treated derivatives of prothrombin lacking the Gla-domain failed to react with the antibody. Such chemically modified prothrombins may serve as models for studying abnormal des-gamma-carboxy prothrombin produced in vitamin K deficiency states.