Production Of Prothrombin Research Articles

Potential inheritance patterns of a prothrombin gene mutation in a 23-year-old female and ethical considerations of a positive diagnosis: a case report

Background: Prothrombin, also called Factor II, is a blood clotting protein found in all individuals that is necessary to form blood clots. In most individuals, a balance between bleeding and blood clot formation occurs. However, in individuals with a mutation in the prothrombin gene, the balance is disrupted due to excess production of prothrombin which leads to an increase in blood clot formation (1). Inherited predispositions to blood clot formation are termed hereditary thrombophilia. Prothrombin G20210A mutations are one of the most common hereditary gene associations. Case Description: This report examines the case of a 23-year-old female who has tested positive for the prothrombin gene mutation. The individual has an extensive history of blood clots including 8 deep vein thromboses (DVTs), 4 pulmonary embolisms with one complete infarction, 4 superficial clots, and a miscarried pregnancy attributed to her thrombophilia. The individual has a significant family history of the mutation and takes Coumadin daily for prevention of further clots. Practical and Ethical Implications: Despite a strong familial history of blood clots and related hospitalizations, the parents of the case individual do not want to get their other children tested for the mutation for fear that the children will be denied insurance coverage in their futures. The case individual will likely continue to be on blood thinners indefinitely. This will affect many aspects of her life, including dental treatment, as she will be at an increased risk for bleeding.

Thrombin Mediates Vagal Apoptosis and Dysfunction in Inflammatory Bowel Disease

BackgroundIn inflammatory bowel disease, autonomic dysfunction contributes to symptoms, morbidity, and health care resource utilization. Efferent vagal neurons, which provide the primary parasympathetic input to the gastrointestinal tract, are housed in the dorsal motor nucleus of the vagus (DMV) in the brainstem. This study seeks to characterize the effects of IBD on DMV neuronal survival and function. MethodsTNBS (picrylsulfonic acid) was administered by enema to induce colitis in rats. Brain sections through the DMV were examined for neuronal apoptosis using TUNEL labeling, and for glial cell activation by immunofluorescence. Prothrombin production was evaluated via quantitative RT-PCR from DMV tissue, as well as by double immunofluorescence in DMV sections. To investigate the effects of thrombin in the DMV, thrombin or thrombin and an antagonist to its receptor were administered into the fourth ventricle via a stereotactically placed cannula. DMV sections were then examined for apoptosis by TUNEL assay. To evaluate the effect of thrombin on DMV neuronal function, we examined calcium signaling in primary DMV neuron cultures following exposure to thrombin and other neurotransmitters. ResultsTNBS colitis is associated with significantly increased rates of DMV neuronal apoptosis, affecting 12.7 % of DMV neurons in animals with colitis, compared to 3.4 % in controls. There was a corresponding increase in DMV neuron activated caspase-3 immunoreactivity (14.8 vs. 2.6 % of DMV neurons). TNBS-treated animals also demonstrated significantly increased DMV astrocyte and microglial immunoreactivity, indicating glial cell activation. DMV prothrombin production was significantly increased in TNBS colitis, with a close anatomic relationship between prothrombin and microglia. Direct DMV exposure to thrombin replicated the apoptosis and activation of caspase-3 seen in TNBS colitis; these effects were prevented by coadministration of the PAR-1 inhibitor FR171113. Cultured DMV neurons exhibited impaired calcium signaling in response to neurotransmitters following exposure to thrombin. Glutamate-induced calcium transients decreased by 59 %, and those triggered by GABA were reduced by 61 %. PAR-1 antagonism prevented these thrombin-induced changes in calcium signaling. ConclusionsIBD is associated with DMV microglial activation and production of prothrombin. Thrombin in the DMV causes vagal neuron apoptosis and decreased sensitivity to neurotransmitters.

A Novel Mutation in VKORC1 and Its Effect on Enzymatic Activity in Japanese Warfarin-Resistant Rats

Warfarin is a rodenticide commonly used worldwide. It inhibits coagulation of blood by inhibiting vitamin K 2,3-epoxide reductase (VKOR) activity. An inadequate supply of vitamin K blocks the production of prothrombin and causes hemorrhage. Recently, warfarin-resistant brown rats (Rattus norvegicus) were found around the Aomori area of Japan. There is no significant difference in the metabolic activity of warfarin in sensitive and resistant brown rats. To clarify the mechanism underlying warfarin resistance, we cloned the VKORC1 gene from rats and identified a novel substitution of arginine to proline at position 33 of the VKORC1 amino acid sequence. Then, we determined the differences in kinetics of VKOR activity between warfarin-resistant and sensitive rats. Hepatic microsomal VKOR-dependent activity was measured over a range of vitamin K epoxide concentrations from 6.25 to 150 µM. The Vmax values of resistant rats (0.0029 ± 0.020 nmol/min/mg) were about one tenth of those of sensitive rats (0.29 ± 0.12 nmol/min/mg). The Km values of resistant rats (47 ± 32 µM) were similar to those of sensitive rats (59 ± 18 µM). Warfarin-sensitive rats exhibited enzyme efficiencies (Vmax/Km) which were ten-fold greater than those observed in resistant rats. It may mean that VKOR activity of warfarin-resistant Aomori rats is almost lost, because their enzymatic efficiencies are very low even without warfarin. Further studies are needed to clarify how these rats can survive with a markedly reduced VKOR activity and how they simultaneously exhibit warfarin resistance.

Myocardial infarction marker levels are influenced by prothrombin and tumor necrosis factor-α gene polymorphisms in young patients

Polymorphisms of genes encoding key factors for the control and activation of inflammatory response and coagulation cascade regulation may play a role in genetic susceptibility to acute myocardial infarction (AMI). This study sought to analyze the effect of TNF −308G/A and pro-thrombin (FII) 20210G/A polymorphisms on the laboratory parameters of young patients affected by AMI. Results indicated that TNF −308A positive genotype frequencies were increased in these patients and that a genetically determined higher production of TNF-α is associated in young subjects to a more severe cardiac damage as depicted by higher levels of troponin, Creatine kinase-MB Isoenzyme (mCK-MB) and a significant increased plasma fibrinogen levels. Similar and probably additive effects on might have a genetically determined increased production of pro-thrombin even if no significant differences in genotype frequencies of pro-thrombin (FII) 20210G/A polymorphisms were observed in this study. All together these results, indicating the relationship among genetically determined TNFα and FII production and increased levels of tissue damage markers of AMI, suggest that a complex genetic background, might be involved in susceptibility to AMI in young men influencing the extension and severity of the disease.

Vitamin K3 Disrupts the Microtubule Networks by Binding to Tubulin: A Novel Mechanism of Its Antiproliferative Activity

Vitamin K3 (2-methyl-1,4-naphthoquinone), also known as menadione, is the synthetic precursor of all the naturally occurring vitamin K in the body. Vitamin K is necessary for the production of prothrombin and five other blood-clotting factors in humans. We have examined the effects of menadione on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human cervical epithelial cancer cells (HeLa) and human oral epithelial cancer cells (KB) indicated that the IC(50) values for menadione are 25.6 +/- 0.6 and 64.3 +/- 0.36 microM, respectively, in those cells. Mendione arrests HeLa cells in mitosis. Immunofluorescence studies using an anti-alpha-tubulin antibody showed a significant irreversible depolymeriztion of the interphase microtubule network and spindle microtubule in a dose-dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by menadione with an IC(50) value of 47 +/- 0.65 microM. The binding of menadione with tubulin was studied using menadione fluorescence and intrinsic tryptophan fluorescence of tubulin. Binding of menadione to tubulin is slow, taking 35 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 189.12 +/- 17 and 32.44 +/- 21 M(-1) s(-1) at 25 degrees C, respectively. The stoichiometry of menadione binding to tubulin is 1:1 (molar ratio) with a dissociation constant from 2.44 +/- 0.34 to 3.65 +/- 0.25 microM at 25 degrees C. Menadione competes for the colchicine binding site with a K(i) of 2.5 muM as determined from a modified Dixon plot. The obtained data suggested that menadione binds at the colchicine binding site to tubulin. Thus, we can conclude one novel mechanism of inhibition of cancer cell proliferation by menadione is through tubulin binding.

Pesticide resistance in wild mammals - Mechanisms of anticoagulant resistance in wild rodents -

Warfarin is commonly used worldwide as a rodenticide. It inhibits coagulation of blood by inhibiting vitamin K 2,3-epoxide reductase (VKOR) activity. An inadequate supply of vitamin K blocks the production of prothrombin and causes hemorrhage. It has been reported that repeated or long-term treatments with this drug cause resistance in wild rodents. However, the mechanism of warfarin resistance in rodents is still not known precisely. Recent studies reported and identified the function of the molecule, vitamin K epoxide reductase complex subunit 1 (VKORC1), which is the main unit of VKOR. An amino acid substitution in VKORC1 is one of the supposed mechanisms of warfarin resistance. An accelerated detoxification system involving cytochrome P450 (CYP) could also cause the rodenticide resistance. Administration of SKF-525A, a potent inhibitor for P450, increased the mortality due to reduction of warfarin metabolism in warfarin-resistant rats. Meanwhile, the appearance of warfarin-resistant rodents has led to the development of the more effective and toxic rodenticide superwarfarin, which is widely used in Europe and the USA. However, animals resistant to this second-generation rodenticide have already been reported in Europe. In this review, we focus on the mechanism and the pleiotropic effects of pesticide resistance in wild rodents.

Prothrombin Synthesis in Rat Hepatoma H4IIEC3 Cells: Response to the Proposed “Coagulopoietin Factor”

Based on studies in intact animals, the presence of humoral factors, "coagulopoietins", which regulate the synthesis of vitamin K-dependent plasma proteins has been proposed. These proposed factors are produced in response to vitamin K deficiency, coumarin treatment, or specific antibody depletion of vitamin K-dependent clotting factors. The production of prothrombin by rat hepatoma H4IIEC3 cells has now been shown to be dependent on the source of bovine serum in the media. Cells grown in serum from cows treated with dicoumarol produce about 20% more prothrombin in 24 h than those cells grown in control serum. The humoral factor causing this response is present early in the course of dicoumarol treatment, and the increase in prothrombin production is dependent on the amount of serum from a dicoumarol-treated cow in the media. Based on membrane filtration studies, the factor appears to be associated with the protein fraction of serum.

Vitamin K1 metabolism and the production of des-carboxy prothrombin and protein C in the term and premature neonate

This study investigated the incidences of undercarboxylated (protein induced by vitamin K absence: PIVKA) prothrombin and protein C in 496 neonates across a wide range of gestational ages. These findings are related to vitamin K1 levels (an indicator of cofactor availability) and vitamin K1 epoxide levels (a measure of the efficiency of the hepatic vitamin K cycle). PIVKA protein C was present in at least trace amounts in 27% of infants; whereas, PIVKA prothrombin was present in 7% of infants. PIVKA prothrombin and protein C were present at high plasma concentrations in 2% to 3% of term and preterm neonates and both PIVKA protein C and prothrombin increased with gestational age. Despite elevated plasma concentrations of PIVKA protein C and diminished levels of normally carboxylated protein C, clinical thrombosis was not observed. The mean (+/- SD) vitamin K1 level in the study population was 0.009 +/- 0.02 nmol/L (adult reference interval: 0.3 to 2.6 nmol/L) with no clear relationship between vitamin K1 levels and production of PIVKA protein C or prothrombin. By comparison with adults, the epoxide form of the vitamin comprised an abnormally high proportion of total vitamin K1; this suggests possible inefficiencies in hepatic reductase cycling.

Vitamin K1 Metabolism and the Production of Des-Carboxy Prothrombin and Protein C in the Term and Premature Neonate

This study investigated the incidences of undercarboxylated (protein induced by vitamin K absence: PIVKA) prothrombin and protein C in 496 neonates across a wide range of gestational ages. These findings are related to vitamin K1 levels (an indicator of cofactor availability) and vitamin K1 epoxide levels (a measure of the efficiency of the hepatic vitamin K cycle). PIVKA protein C was present in at least trace amounts in 27% of infants; whereas, PIVKA prothrombin was present in 7% of infants. PIVKA prothrombin and protein C were present at high plasma concentrations in 2% to 3% of term and preterm neonates and both PIVKA protein C and prothrombin increased with gestational age. Despite elevated plasma concentrations of PIVKA protein C and diminished levels of normally carboxylated protein C, clinical thrombosis was not observed. The mean (+/- SD) vitamin K1 level in the study population was 0.009 +/- 0.02 nmol/L (adult reference interval: 0.3 to 2.6 nmol/L) with no clear relationship between vitamin K1 levels and production of PIVKA protein C or prothrombin. By comparison with adults, the epoxide form of the vitamin comprised an abnormally high proportion of total vitamin K1; this suggests possible inefficiencies in hepatic reductase cycling.

肝癌細胞における異常プロトロンビンＰＩＶＫＡ‐ＩＩの産生に関する検討

諸種の肝癌細胞株及びその他の細胞株での異常プロトロンビンPIVKA-IIの産生に関する検討を行なったところ,huH-1, huH-2, PLC/PRF/5, Hep G2, Hep 3Bの5種の肝癌細胞株の培養上清中にPIVKA-IIが検出され,しかも経日的にPIVKA-II値が増加した.またvitamin Kをあらかじめ添加した場合にはいずれもPIVKA-II値が著減した.胃癌由来細胞株のAZ-521及びMKN 7,膵癌由来細胞株のMIAPaCa-2ではその培養上清中にはPIVKA-IIは検出されなかった.以上よりPIVKA-IIは肝癌細胞で産生され,しかもその産生はvitamin Kの存在に左右される事が判明した.huH-2を使用して肝癌細胞におけるPIVKA-IIの産生機序に関する検討を行なったところ,プロトロンビン産生に関与する3つの酵素vitamin K-epoxide-reductase, vitamin K-reductase (vitamin K cycle)及びγ-glutamyl-carboxylaseのすべてが働いている事が示唆され,その機序としてはプロトロンビン前駆体の産生亢進にともなうvitamin K cycle及びγ-carboxylation systemの不均衡によるものと推測された.

Production of abnormal prothrombin (des- γ-carboxy prothrombin) by hepatocellular carcinoma : A clinical and experimental study

We measured plasma abnormal prothrombin (des-gamma-carboxy prothrombin; DCP) levels in normal subjects and in patients with hepatocellular carcinoma and other various diseases using the enzyme-linked immunosorbent assay developed by Motohara et al. (Pediatr Res 1985; 19: 354-357). Fifty-eight percent of 52 patients with hepatocellular carcinoma had elevated DCP levels; 24 of 28 patients with advanced or moderately advanced hepatocellular carcinoma were positive. By contrast, 50 normal controls, 13 pregnant women and 10 patients with acute hepatitis had normal levels. Three of 55 patients with chronic liver disease, and 6 of 32 patients with other malignancies, showed a slight increase. Thus, increased plasma DCP appears useful for the diagnosis of hepatocellular carcinoma. To elucidate the mechanism for the increase of DCP in hepatocellular carcinoma, we cultured a human hepatoma cell line, huH-2, and measured the levels of this abnormal prothrombin in the medium. The huH-2 cells produced large amounts of DCP in the medium without added vitamin K. It increased in a cell concentration- and time-dependent fashion. These cells produced no detectable amount of DCP in the medium with added vitamin K. Thus, human hepatoma cell line huH-2 produces DCP, and its production is dependent on the amount of vitamin K available in the medium. Des-gamma-carboxy prothrombin may be a useful tumor marker for the diagnosis of hepatocellular carcinoma.

Vitamin K deficiency in chronic alcoholic males.

Twenty male alcoholic subjects were studied initially within 1 day after stopping alcohol and again after about 1 week. Vitamin and mineral measurements were made on blood and abnormal prothrombin molecules quantitated for vitamin K status. Nine of the 20 patients received menadiol after the initial blood sample. Twelve of the alcoholics had significant elevations of abnormal prothrombin. Of these 12, all five who received vitamin K reduced the abnormal prothrombin levels toward normal but no change was observed in the seven who did not receive vitamin K. All nine patients receiving vitamin K lowered the abnormal prothrombin level significantly whereas there was no change in those 11 who did not receive vitamin K. The prothrombin time by the one-stage technique was normal in all patients. These data suggest that the production of abnormal prothrombin is frequently present in alcoholics and this may represent a subclinical vitamin K deficiency.

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.

PROTHROMBIN SYNTHESIS IN THE DOG.

Liver parenchymal cells were found to be the sites for production of prothrombin. Dogs were employed and their rate of prothrombin synthesis was modified as desired by giving either Coumadin or vitamin K1. The fluorescent antibody technique was found a valuable aid in evaluating liver cell function. Under normal conditions there is periodic synthesis of prothrombin with only 10% of the parenchymal cells actively participating at any one time. Prothrombin-deficient or normal dogs can be stimulated to more vigorous activity by giving vitamin K1. In such circumstances all the liver parenchymal cells were able to synthesize prothrombin. A regulatory mechanism promotes either synthesis, storage, or release of prothrombin by liver parenchymal cells. No other cell types of the liver, spleen, or bone marrow appeared capable of synthesizing prothrombin.

Cellular site for prothrombin synthesis.

The cell type responsible for synthesis of any of the trace plasma proteins concerned in blood coagulation has not been identified before. In this study the fluorescent antibody technique was used to find out the cellular site for prothrombin synthesis. Fluorescent antiprothrombin precipitated out on bovine liver parenchymal cells containing adequate concentrations of prothrombin. The specificity of the reaction was established using various absorptive and blocking techniques. The antiprothrombins produced were species specific. Not all parenchymal cells reacted uniformly with fluorescent antiprothrombin so that groups of brilliantly fluorescing cells were seen among cells displaying dim natural fluorescence. This may mean that only a certain type of parenchymal cell produces prothrombin or that there is cyclic production of prothrombin.

Coagulation mechanism and effect of heparin in hemorrhagic shock.

Hemorrhagic shock was produced in anesthetized and unanesthetized dogs by bleeding to fixed arterial pressure levels for specified periods using Wiggers' method. Blood was drawn through an ion exchange resin and at the end of the hypotension was reinfused simultaneously with the removed ions. Early in the hypotension there was usually some shortening of blood coagulation time. In the late hypotension and reinfusion period 75% of the dogs had a distinct prolongation of clotting time. Prothrombin activity and blood platelets showed a marked progressive decline during the shock period. Special tests indicated that the coagulation defect was mainly due to a decrease of labile factor in conjunction with a second deficiency, most likely prothrombin. There was no discernible deficiency of stable factor nor any release of heparinoids during the shock procedure. It is suggested that the hepatic anoxia and the resultant diminished production of labile factor and prothrombin contributes importantly to the clotting defect. Prior administration of heparin (10 mg/ kg) tended to lower mortality and increase survival time but did not afford critical protection against the development of irreversibility.

On the Influence of Dicumarol on Serum Protein Fraction

An electrophoretic study by means of the Tiselius apparatus, was made on the influence of Dicumarol on normal human serum protein fraction.100-150mg of Dicumarl of was administered, orally, for three consecutive days, and the serum protein fractions were compared before and after administration of the drug.Two significant changes were noted.1) A decrease in serum total protein. Although clinically, liver function is said to be unaffected by a therapeutic dose of Dicumarol, it may be that together with the inhibition of the production of prothrombin in the liver, some other factors may be influenced by Dicumarol.2) A decrease in β-globulin. The amount of prothrombin in the blood is only 0.02g/dl. Hence an average decrease of 6.16g/dl as obtained in this experiment cannot be explained solely by decrease in the prothrombin which is associated with the β-globulin. Mann and many other investigators, as opposed to Quick, report that Dicumarol influences other factors than prothrombin. Although we cannot as yet specify the factors involved, this experiment appears to support the former view. It is also reported by Muto et al., that a minute quantity (0.002g/dl) of Heparin causes an increase in the albumin fraction when measured electrophoretically. The explanation given by them is an interaction between the heparin and albumin. However, further experiments are necessary to give a definite conclusion on the cause of the β-globulin decrease, noted in this experiment.

Severe hemorrhage due to tromexan; report of three cases.

TROMEXAN, a new anticoagulant, has recently been accepted for general use. Chemically, it is described as ethyl biscoumacetate (3,3′ carboxymethylenebis- [4-hydroxycoumarin] ethyl ester). It is most widely recognized as Tromexan but is also known as Pelentan, B.O.E.A. and D.E.A. Dicumarol1 and Tromexan2 are related members of the coumarin series of drugs. Each is presumed to act in similar fashion, by interfering with the normal production of prothrombin by the liver. The exact mode of action is as yet unknown.3 , 4 The possibility of hemorrhage after Dicumarol administration is well known, and precautions for its use have been repeatedly stressed.5 6 7 Utilizing an . . .

PROTHROMBIN LEVEL OF PERIPHERAL BLOOD AND STERNAL MARROW

Despite the tremendous strides made in recent years in the understanding of the properties of prothrombin, its metabolic cycle remains a matter of conjecture. As far as is known, the liver is the site of production of prothrombin, and the precursor of thrombin is liberated into the circulating blood continuously and in excess. Of the fate and possible additional functions of prothrombin practically nothing is known. The manner in which vitamin K and dicoumarin (3,3′-methylene-bis-[4-hydroxycoumarin] ) exert their respective influences on the prothrombin-elaborating system likewise remains obscure. The evidence to prove that the liver is the major, if not the sole, locus for the synthesis of prothrombin is as yet neither complete nor conclusive. The conception is based on the demonstration that hepatocellular damage, artificially induced or occurring clinically, is accompanied with prothrombinopenia. Which of the components of the liver, the hepatic parenchymatous cells or the elements of the reticuloendothelial system

Plasma Prothrombin Concentration in Dogs Given 3,3′-Methylenebis(4-Hydroxycoumarin) and Purified Beef Prothrombin

Purified beef prothrombin will antidote the hypoprothrombinemia induced in dogs by oral administration of 3,3'-methylenebis (4-hydroxyeoumarin). If the hypothesis is proven that this chemical compound acts only by preventing the production of prothrombin in the liver then the life of injected beef prothrombin is probably of the same duration as that of the dog's own plasma prothrombin; namely, 2 to 3 days.