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PubMed1994

Prothrombin synthesis and degradation in rat hepatoma (H-35) cells: effects of warfarin.

Vitamin K is a substrate for the enzyme catalyzing the carboxylation of specific glutamyl residues to gamma-carboxyglutamyl residues in hepatic precursors of a limited number of plasma proteins, including prothrombin. The gamma-carboxylation of these proteins can be blocked by the anticoagulant warfarin; and in the bovine and human, warfarin treatment results in the secretion of under-gamma-carboxylated forms of prothrombin into plasma. In the rat, this response is not seen, but plasma prothrombin concentrations are drastically decreased. This response has now been studied in rat hepatoma (H-35) cells in which prothrombin secretion is decreased 90% by incubation in the presence of warfarin. Neither prothrombin mRNA levels nor the apparent rate of prothrombin message translation were decreased when cells were cultured in the presence of warfarin rather than of vitamin K. The pool of intracellular prothrombin precursors is increased threefold by warfarin treatment, and this pool is rapidly secreted when vitamin K is administered. In contrast, continued incubation in the presence of warfarin resulted in the degradation of 60% of this pool in 24 hours. When transport of secretory proteins to the golgi apparatus was blocked with Brefeldin A, this precursor pool was gamma-carboxylated in the presence of vitamin K and no degradation occurred. Lysosomal enzyme inhibitors did not block the degradation, and the data suggest that, in rat hepatocytes, under-gamma-carboxylated prothrombin is specifically targeted to a pathway of protein degradation located in the endoplasmic reticulum.

Animals↗

PubMed1993

Adsorption and conversion of prothrombin on a rotating disc.

In most flow systems, the rate of protein transfer from bulk solution to a macroscopic surface is site-dependent. In studies on surface-mediated protein conversion, this hampers the comparison of a proposed expression for the conversion process, such as the Michaelis-Menten equation, which actually measured overall conversion rates. However, the rotating disc is a classical example of a uniformly accessible surface and therefore was used for a quantitative analysis of prothrombin conversion by the phospholipid-bound factor Xa/factor Va complex (prothrombinase complex). A simple design of a rotating disc, adapted for ellipsometric measurement of protein adsorption, is presented. Agreement between experiment and theory was obtained for the influence of rotation velocity on the initial, transport-limited, adsorption rates of lysozyme, prothrombin, and fibrinogen. After coverage of the disc with a 20% phosphatidylserine/80% phosphatidylcholine bilayer and preadsorption of factor Va, addition of excess factor Xa and prothrombin resulted in effective conversion of prothrombin. For high (10 fmol.cm-2) surface coverage of prothrombinase, the rate of conversion equals the transport limited adsorption rate of prothrombin. For low (0.1 to 0.5 fmol.cm-2) surface concentrations of prothrombinase, the conversion rate dropped below the transport limit and the intrinsic kinetic parameters could be estimated at Km = 7.1 +/- 1.2 nM and kcat = 25 +/- 1.0 s-1 (20 degrees C). At these low surface activities of prothrombinase, the effect of the rotation rate (6 to 225 rad.s-1) on prothrombin conversion could be explained by the rotation-rate dependent prothrombin transport. This indicates that the fluid shear rate has no drastic influence on the intrinsic kinetics of prothrombin conversion.

Adsorption↗

PubMed2005

Comparative analysis of prothrombin activators from the venom of Australian elapids.

A key component of the venom of many Australian snakes belonging to the elapid family is a toxin that is structurally and functionally similar to that of the mammalian prothrombinase complex. In mammals, this complex is responsible for the cleavage of prothrombin to thrombin and is composed of factor Xa in association with its cofactors calcium, phospholipids, and factor Va. The snake prothrombin activators have been classified on the basis of their requirement for cofactors for activity. The two major subgroups described in Australian elapid snakes, groups C and D, are differentiated by their requirement for mammalian coagulation factor Va. In this study, we describe the cloning, characterization, and comparative analysis of the factor X- and factor V-like components of the prothrombin activators from the venom glands of snakes possessing either group C or D prothrombin activators. The overall domain arrangement in these proteins was highly conserved between all elapids and with the corresponding mammalian clotting factors. The deduced protein sequence for the factor X-like protease precursor, identified in elapids containing either group C or D prothrombin activators, demonstrated a remarkable degree of relatedness to each other (80%-97%). The factor V-like component of the prothrombin activator, present only in snakes containing group C complexes, also showed a very high degree of homology (96%-98%). Expression of both the factor X- and factor V-like proteins determined by immunoblotting provided an additional means of separating these two groups at the molecular level. The molecular phylogenetic analysis described here represents a new approach for distinguishing group C and D snake prothrombin activators and correlates well with previous classifications.

Amino Acid Sequence↗

PubMed1995

Normotest and abnormal prothrombin in liver transplantation.

Postoperative changes in coagulation parameters, including the abnormal plasma prothrombin level, were studied in 95 patients who underwent liver transplantation, and the results were compared with the clinical outcome. The patients were classified into four groups: Group I had a satisfactory postoperative course, (n = 76), Group II suffered graft failure or death at 31 days or more after transplantation (n = 9); Group III suffered graft failure or death from 8 to 30 days after transplantation (n = 4); and Group IV suffered graft failure or death within 7 days of transplantation (n = 6). The Normotest, which closely reflected liver graft function, showed an increase immediately after transplantation in Group I, II, and III, but showed a marked decrease in Group IV. In patients with severe acute cellular rejection, the plasma level of abnormal prothrombin (des-gamma-carboxy prothrombin) was compared with the histology of the liver biopsy specimen. When liver graft function was good after orthotopic transplantation, the Normotest value recovered to the normal range of 70% or more. Subsequently, graft function remained good when the des-gamma-carboxy prothrombin level stayed low, whereas acute cellular rejection was indicated by an elevation of des-gamma-carboxy prothrombin was not produced by graft with early failure, the des-gamma-carboxy prothrombin level also remained low. Thus, the Normotest value and the des-gamma-carboxy prothrombin level were both useful parameters for assessing hepatic function and rejection after transplantation.

Adult↗

PubMed1988

Inhibition of the anticoagulant activity of protein S by prothrombin.

Protein S is a vitamin K-dependent protein cofactor to the anticoagulant, activated protein C (APC). This study examines the inhibition of human protein S anticoagulant activity by prothrombin. In the absence of protein S, the anticoagulant activity of APC measured in a Factor Xa recalcification time, was comparable using normal or plasma adsorbed with Al(OH)3. Protein S was an effective cofactor to APC in Al(OH)3-adsorbed plasma, but was significantly less active in normal plasma. Analysis of the difference in the two plasmas revealed that normal plasma contained an inhibitor to the anticoagulant activity of protein S that was removed by Al(OH)3 adsorption. Purification of this inhibitory activity demonstrated that it was mediated by the vitamin K-dependent protein, prothrombin. Prothrombin purified by conventional techniques caused immediate, dose-dependent inhibition of the cofactor activity of protein S in the presence of phospholipids or platelets, but had no effect on the anticoagulant activity of APC. The inhibition was demonstrable using a Factor Xa recalcification time, and studies of the rates of inactivation of purified Factor Va. Increasing concentrations of protein S overcame the inhibition by prothrombin and kinetic analysis of the interaction demonstrated that prothrombin acted as a competitive inhibitor to protein S. Immunoabsorption of prothrombin from plasma using immobilized antiprothrombin antibodies was associated with the complete removal of the protein S inhibitory activity. We conclude that the anticoagulant activity of protein S is modulated by prothrombin and that this may represent another regulatory mechanism of the natural anticoagulant system.

Blood Coagulation↗

PubMed1979

Spectrophotometric assays of prothrombin in plasma of patients using oral anticoagulants.

Two spectrophotometric assays for prothrombin have been developed and compared with a one stage coagulant and an immunological assay. One of these assays (called the XAPC assay) uses a combination of factor Xa, phospholipid, Ca2+ and factor V as activator of prothrombin, and measures only normal prothrombin. The second (the ECAR assay) uses Echis carinatus venom as activator. This assay measures both normal prothrombin and PIVKA II (protein induced by vitamin K antagonists/absence). Combination of the results obtained by the XAPC and ECAR assays provides rapid and reliable information on the degree of "subcarboxylation" of prothrombin (oral anticoagulation, vitamin K deficiency). For patients on long term anticoagulant treatment the prothrombin time (Thrombotest) shows better correlation with the ratio prothrombin/prothrombin plus PIVKA II (XAPC/ECAR) than with the factor II concentration. For patients starting the anticoagulant treatment there is no correlation between the Thrombotest time and the XAPC/ECAR ratio. It seems doubtful that (a) spectrophotometric factor II assay(s) will be as useful as the prothrombin time in the control of oral anticoagulation.

Administration, Oral↗

PubMed1987

Warfarin therapy. The effect of heparin on prothrombin times.

Sharp decreases in the prothrombin time after discontinuing heparin have been reported in patients undergoing oral anticoagulant therapy. Twenty-five patients receiving continuous intravenously administered heparin and orally or intravenously administered warfarin were studied. All patients had prothrombin times greater than 1.40 times control, and activated partial thromboplastin times 1.5 to three times control before discontinuing heparin therapy. Prothrombin times on the heparin infusion and four to six hours after it was discontinued were compared. The mean change in the prothrombin time was -1.60 s with a range of +0.8 to -5.5 s. Eight (32%) of 25 patients had a decrease of greater than 2 s. The decrease in prothrombin time correlated poorly with heparin dose or activated partial thromboplastin time in patients taking heparin. Since the change in prothrombin time is unpredictable, a repeated prothrombin time is recommended after stopping heparin therapy prior to discharging a patient.

Adult↗

PubMed1998

Regulation of prothrombin, thrombin receptor, and protease nexin-1 expression during development and after denervation in muscle.

Prothrombin, thrombin receptor (ThR), and protease nexin-1 (PN-1) mRNA levels in mouse muscle were quantified using competitive reverse transcriptase-polymerase chain reaction during development and after denervation to examine the possible role of thrombin in activity-dependent synapse elimination at the neuromuscular junction. The results showed that the levels of prothrombin and ThR were maximal at birth and decreased by two orders of magnitude by postnatal day 20 (P20). The level of PN-1 mRNA was fairly constant during development except for a 4-fold to 5-fold downregulation at P10 and P15, the periods of maximal synapse elimination at the rodent neuromuscular junction. The expression of prothrombin mRNA in muscle at birth was 41-fold and 22-fold lower than those of ThR and PN-1, respectively, and the level of difference between prothrombin and PN-1 reached almost three orders of magnitude at adulthood. Denervation of adult muscle resulted in a reversal of the relative expression levels of the three genes. There were rapid 8-fold and 10-fold increases in prothrombin and ThR mRNA, respectively, and a 2-fold decrease in PN-1 mRNA. The changes in mRNA levels of the three genes after denervation indicated that these genes were regulated in a innervation-dependent manner and that nerve activity may play an important regulatory role in the expression of prothrombin, ThR, and PN-1. The concurrent regulation of prothrombin and ThR suggests that thrombin-mediated cellular activities in muscle may be affected via the activation of ThR. An elevated level of local thrombin or thrombin-like activity might result from the decreased inhibitory activity of PN-1 during the period of peak synapse elimination in muscle development.

Amyloid beta-Protein Precursor↗

PubMed1995

Role of platelet factor Xa in chylomicron-prothrombin complexes induced platelet activation.

The effects of chylomicron-prothrombin complexes on platelet activation, including platelet aggregation, serotonin release, arachidonic acid release and increases of platelet cytosolic [Ca2+]i were examined. Furthermore the role of platelet factor Xa on the conversion of chylomicron bound prothrombin to thrombin was studied by using a synthetic inhibitor of factor Xa, TenStop. The chylomicron-prothrombin complexes could induce platelet aggregation and enhance the platelet serotonin release and arachidonic acid release in contrast to native chyle chylomicrons. An increase of platelet [Ca2+]i was observed during incubation with chylomicron-prothrombin complexes. TenStop inhibited platelet aggregation and serotonin release that were induced by chylomicron-prothrombin complexes in a dose-dependent manner, whereas the TenStop itself did not inhibit the platelet aggregation induced by thrombin and collagen. It is concluded that platelet activation induced by chylomicron-prothrombin complexes is related to the platelet factor Xa that could be the key factor in the conversion of chylomicron bound prothrombin to thrombin.

Animals↗

PubMed1998

Human prothrombin fragment 1 and 2 inhibit bFGF-induced BCE cell growth.

Previously, we reported that the rabbit prothrombin fragment 2 (kringle 2 domain) has an anti-endothelial cell proliferative effect (Lee et al., J. Biol. Chem., in press). In this report, we show that not only rabbit prothrombin fragment 2 but also human prothrombin fragment 1 and 2 have an inhibitory effect on bFGF-stimulated BCE cell growth. Human prothrombin fragment 1 and 2 obtained as proteolytic fragments of human prothrombin display potent inhibitory effects on bovine capillary endothelial cells with a half-maximal concentration (ED50) of approximately 100 nM and 120 nM, respectively. As rabbit prothrombin fragment 2, the human prothrombin fragment 1 and 2 also inhibit angiogenesis in the chorioallantoic membrane (CAM) of chick embryos.

Allantois↗

PubMed1975

Activation of a pro-enzyme by a stoichiometric reaction with another protein. The reaction between prothrombin and staphylocoagulase.

The reaction between prothrombin and staphylocoagulase was investigated and the following conclusions were drawn: (a) Optimal amounts of the active reaction product (coagulase-thrombin) are found when equimolar amounts of prothrombin and staphylocoagulase are added together. (b) The molecular weight of coagulase-thrombin equals the sum of the molecular weights of staphylocoagulase and prothrombin when estimated both by gelfiltration and by sodijm dodecylsulphate-polyacrylamide gel electrophoresis. (c) The amino acid composition of coagulase-thrombin cannot be distinguished from the sum of the amino acid compositions of prothrombin and staphylocoagulasd. (d)in a preparation of coagulase-thrombin the N-terminal amino acids are those of prothrombin (alanin) and staphylocoagulase (aspartic acid). (e) An antibody against coagulase-thrombin precipitates prothrombin and staphylocoagulase but not thrombin. (f) We put forward the hypothesis that the thrombin activity in coagulasethrombin is the result of a stoichiometric reaction between one molecule of prothrombin and one molecule of staphylocoagulase, and limited proteolysis does not play a role in this mechanism.

Amino Acids↗

PubMed1978

The mechanism of activation of human prothrombin by an activator isolated from Dispholidus typus venom.

Purified human prothrombin was activated, both in the absence and in the presence of thrombin inhibitors (diisopropylfluorophosphate or hirudin), by a coagulant principle isolated from Dispholidus typus venom. The process of activation was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the absence of thrombin inhibitor, prolonged incubation of prothrombin with the purified venom yielded thrombin, fragment 1 (F 1) and fragment 2 (F 2). In the presence of diisopropylfluorophosphate, which in the experimental conditions used inhibited only partially the thrombin generated activity, products obtained upon activation of prothrombin by venom were F 1 and a two-chain, disulfide-bridged protein of 58 000 daltons called meizothrombin (des F 1). In the presence of hirudin, which fully inhibited thrombin generated activity, prothrombin activation by the venom did not liberate any fragment, but prothrombin was converted to a derivative composed of two disulfide-bridged polypeptide chains of 48 000 and 37 000 daltons, called meizothrombin. These results are similar to those reported by others when studying the process of prothrombin activation by Echis carinatus venom and allow to conclude that Dispholidus typus venom cleaves a bond linking the A and B chains of thrombin, converting prothrombin into meizothrombin. This enzyme is then responsible for the cleavage of the bond linking F 1 and F 2 and the bond linking F2 the A chain of thrombin.

Animals↗

PubMed1986

On the quantification of prothrombin from different species using Echis carinatus as activator.

The generation of thrombin-like activity from rat, human, bovine and mouse prothrombin by Echis carinatus venom (ECV) treatment was compared using a partially purified system (i.e. whole ECV and isolated prothrombin). A rapid increase in coagulant activity was obtained within 0.5 to 2 min., being constant upon further incubation for 60 min. A large variation in coagulant activity of the ECV generated thrombin from the four species was found, whereas no differences were found for the amidolytic activities. The coagulant activities of the ECV generated thrombin was also low compared with the corresponding thrombin activities obtained by physiological activation. Coagulant activity of the ECV generated thrombin levelled off at increasing concentration of prothrombin in the sample as measured by the one-stage coagulation assay. By measuring amidolytic activity a linear relationship to the concentration of prothrombin was found, however. These findings indicate that ECV converts prothrombin from the four different species to a thrombin-like protein with properties distinct from alpha-thrombin. The lack of linearity in the ECV generated clot activity with increasing concentration of prothrombin could be explained by assuming a dimerization of the thrombin-like protein molecules making them less accessible to fibrinogen. The significance of these observations for the quantification of prothrombin from different species is discussed.

Animals↗

PubMed1992

Non-specific effects of aquaMEPHYTON (vitamin K1) on prothrombin expression in human hepatoblastoma (HepG2) cells.

In order to determine the effects of vitamin K1 on prothrombin production, we have treated cultures of human hepatoblastoma cells with an aqueous colloidal suspension of vitamin K1. Dose-response analysis demonstrated increases in secreted prothrombin antigen levels ranging from 3 to 3.7-fold over controls. Time-course analysis demonstrated increases in secreted prothrombin antigen levels over controls up to 6 hours of treatment. Between 6 and 24 hours, secreted prothrombin antigen levels increased at a rate parallel to controls. Vitamin K1 treatment also resulted in a parallel increase in total secreted protein levels. Prothrombin mRNA size (approximately 2.1 kb) and levels (ranging from 390-480 prothrombin mRNA molecules per cell) were determined by Northern and quantitative solution hybridization analysis, respectively, and were unaffected by vitamin K1 treatment. The increases in secreted prothrombin antigen levels most likely result from non-specific effects of vitamin K1 or agents used to emulsify vitamin K1 on protein release from HepG2 cells.

Carcinoma, Hepatocellular↗

PubMed1995

Binding of prothrombin to chyle chylomicrons: effects of temperature and calcium ions, and role of surface phospholipids.

The ability of chyle chylomicrons to bind prothrombin has been studied. Rat chyle chylomicrons were incubated with human 125I-prothrombin and binding was examined by separating the chylomicrons from free 125-I-prothrombin by density-gradient ultracentrifugation, and by gel filtration on Sepharose CL-2B. A significant binding of prothrombin to chyle chylomicrons occurred. The complex formation was calcium dependent, and decreased markedly when the temperature was lowered from 37 degrees C to 20 degrees C and when PH was raised above 8. The time course for the binding at 37 degrees C in presence of 2 mmol/L CaCl2 exhibited an initial lag phase at about 10 minutes. Thereafter most of the binding occurred within 30 minutes. Bound prothrombin could not be removed from chyle chylomicrons by treatment with EDTA, suggesting that this binding is not a simple Ca2+ dependent association between prothrombin and chyle chylomicrons. Inclusion of 1% purified human serum albumin caused a 50% decrease in binding, half of which was reversed by increasing the Ca2+ ion concentration. Addition of pancreatic phospholipase A2 (PLA2) in doses sufficient to hydrolyze more than 95% of the phosphatidylethanolamine (PE) and 37% of the phosphatidylcholine (PC) decreased the binding by 50%. Doses of PLA2 that hydrolyze more than 95% of the phosphatidylethanolamine (PE) and 37% of the phosphatidylcholine (PC) decreased the binding by 50%. Doses of PLA2 that hydrolyzed 60-80% of (PE and 4-10% of the PC decreased the binding by only 7-15%. It is suggested that the binding of prothrombin to chyle chylomicrons is in part mediated by negatively charged phospholipids of the chylomicron surface, although a specific role of the PE could not be demonstrated.

Animals↗

PubMed1989

Interaction of human gamma-carboxyglutamic acid domainless prothrombin with phospholipids.

The conversion of prothrombin into thrombin occurs at the surface of stimulated platelets. In order to see the influence of gamma-carboxyglutamic acid in the interaction of prothrombin with phospholipid, we investigated the direct interaction of the peptide 1-41 and prothrombin (des 1-44) with phospholipid monolayers of various compositions. Adsorption of the labeled proteins was determined by surface radioactivity measurements. Penetration of the proteins into the lipid layers was inferred from capacitance variation of the monolayer, measured by a.c. polarography. Prothrombin (des 1-44) was found both to adsorb and to penetrate the lipid monolayers, in the presence and in the absence of Ca++. The effects are higher on 100% PS than on 25% PS. This protein was also found to increase the permeability of vesicles containing 25% PS to T1+ ions, in the presence and in the absence of Ca++. Comparison with prothrombin shows that Gla residues are clearly involved in the interaction at 25% PS; nevertheless, the peptide 1-41 does not penetrate. A model of interaction of prothrombin with phospholipid, including both adsorption of prothrombin by Gla residues and its penetration by another domain, is proposed.

1-Carboxyglutamic Acid↗

PubMed1992

Activation of prothrombin accompanying thrombolysis with recombinant tissue-type plasminogen activator.

Increases in thrombin activity in patients given fibrinolytic agents for acute myocardial infarction have been shown to be important in limiting the ultimate success of coronary thrombolysis. The present study was designed to determine whether increases in thrombin activity reflect, in part, activation of prothrombin accompanying thrombolysis. Plasma concentrations of prothrombin fragment 1.2, a polypeptide released when prothrombin is activated by factor Xa, were measured in 22 patients with acute myocardial infarction before and after treatment with 100 mg of recombinant tissue-type plasminogen activator (rt-PA). Concentrations of prothrombin fragment 1.2 increased from 0.83 +/- 1.1 nM (mean +/- SD) before rt-PA infusion to 1.5 +/- 1.5 nM 2 h after initiation of the infusion (p less than 0.05). After a 5,000-U intravenous dose of heparin given at the end of the infusion of rt-PA, concentrations of prothrombin fragment 1.2 decreased from 1.8 +/- 1.5 to 1.1 +/- 0.9 nM (n = 20, p less than 0.05), although values were still increased compared with concentrations before rt-PA. These results indicate that thrombin activity increases in patients given rt-PA at least in part because of activation of the coagulation system leading to activation of prothrombin. Thus, inhibition of the reactions involving coagulant proteins that lead to activation of prothrombin may be of value as conjunctive treatment to potentiate the efficacy of pharmacologic thrombolysis.

Blood Coagulation↗

PubMedDate not supplied

Knockdown of prothrombin in zebrafish.

Thrombin is a serine protease generated from its zymogen, prothrombin, and plays a central role in the coagulation cascade. It is also important for mammalian development. The zebrafish has now been established as an excellent genetic model for studies on mammalian hemostasis and development. In this report, we used prothrombin-specific antisense morpholinos to knock down the levels of prothrombin to characterize the effects of prothrombin deficiency in the zebrafish embryo. Prothrombin morpholino-injected zebrafish embryos yielded an early phenotype exhibiting severe abnormalities that later showed occasional bleeding. In a second late phenotype, the embryos had no observable morphological abnormalities in early stages, but showed occasional bleeding at later stages. These phenotypes resembled characteristics shown by prothrombin knockout mice. Laser-induced vascular injury on some of the normal appearing phenotypic larvae showed a prolonged time to occlusion, and recombinant zebrafish prothrombin injected into these larvae restored a normal time to occlusion thus showing the specificity of the morpholino effect. The system developed here should be useful for investigation of the role of thrombin in vertebrate development.

Animals↗