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PubMed2002

Commercial variants of the prothrombin time test as a screening test of acquired coagulation factor II, VII, and X deficiencies in dogs.

The aim of this study was to acquire the single factor sensitivity of three commercial variants of the prothrombin time test (PT(Va1)-Thrombotest, PT(Va2)-Normotest, PT(Va3)-Hepato Quick) for canine plasma. For this purpose, 38 samples from animals with coumarin intoxication followed by vitamin K(1) treatment who had a reduced activity of the coagulation factors II, VII, and/or X (experiment 1) as well as 25 plasma samples from animals with moderately reduced activity of the coagulation factors II, V, VII, and/or X caused by a hepatogenic synthesis disorder or disseminated intravascular coagulation (experiment 2), were examined. Measurements for all tests were performed according to the instructions of the manufacturer and also with higher plasma dilution. Furthermore, control measurements were performed with the conventional prothrombin time test (standard test; PT(ST)). The prothrombin time measured with the PT(Va3), which was prolonged in all samples from experiments 1 and 2, especially reflected with high sensitivity the reduced activity of the coagulation factors. The measurements with PT(Va2) and PT(Va1) showed only an insignificantly lower sensitivity (> or =0.88) with a maximum of 1 (experiment 1) and 3 (experiment 2) false negative test results, respectively. Compared to the already high sensitivity, a higher dilution of the sample material did not lead to a significantly higher sensitivity (P>0.05) for any of the tested commercial variants of the prothrombin time assay. While there was no significant difference (P>0.05) between the number of false negative test results in the variants of the prothrombin time assay, in both experiments a significantly higher number of false negative test results was found for the PT(ST) (P<0.001). The results show that, in contrast to the PT(ST), all three tested commercial prothrombin time variants are suitable for the detection of deficiencies of coagulation factors II, VII, and X in canine plasma.

Animals↗
PubMed2007

Corneal activation of prothrombin to form thrombin, independent of vascular injury.

PURPOSE: Two major functions of thrombin observed in the cornea are activation of thrombin-sensitive, proteinase-activated receptors and cleavage of fibrinogen to fibrin. The purpose of this study was to determine whether the normal human cornea itself is competent to convert prothrombin to thrombin and synthesizes the mRNA for the proteins required. METHODS: Human corneas were processed for immunolocalization studies or separated into epithelial, stromal, and endothelial layers for proteins and RNA isolation. The protein extracts were used for Western blots, prothrombin time, and activated partial thromboplastin time assays and fibrinopeptide A generation tests. RNA was used for RT-PCR. Apoptosis of cultured human corneal cells was induced with sodium nitroprusside or camptothecin and activation of prothrombin tested. RESULTS: Prothrombin and its mRNA were present in all three layers of human donor cornea. It was found to be associated with the cells and the extracellular matrix at similar levels across the cornea. With corneal stromal extracts, activation of either the intrinsic or extrinsic coagulation pathways resulted in thrombin activation and fibrin formation with fibrinopeptide A release. Detection of key components of the coagulation cascades confirmed noninjured human corneas contain factors required for prothrombin activation. In addition, mRNAs for representative factors and inhibitors were detected by RT-PCR and confirmed by sequencing. Apoptotic corneal stromal cells provide a surface for prothrombin activation. CONCLUSIONS: These studies suggest that the normal avascular human cornea contains and synthesizes the components required for thrombin generation and that this process does not depend on a breech in the limbal vascular endothelium.

Blood Coagulation Factors↗
PubMed1994

The influence of N-acetylcysteine on the measurement of prothrombin time and activated partial thromboplastin time in healthy subjects.

The purpose of the study was to evaluate whether the infusion of N-acetylcysteine decreased the measurement of prothrombin time and activated partial thromboplastin time (APTT) in healthy persons. N-acetylcysteine was administered intraveneously 10 mg kg-1 as a loading dose and then at a rate of 10 mg kg-1 h-1 for 32 h in six male subjects. The intrinsic, extrinsic and common pathway of coagulation were monitored with activated partial thromboplastin time (APTT), and prothrombin time, respectively. In addition, the extrinsic coagulation pathway was monitored with the clotting activity of single factors II, VII, and X. No effect on the intrinsic coagulation pathway was observed. There was a significant and rapid decrease in prothrombin time. Coagulation factors II, VII and X, the three components of prothrombin time, decreased significantly to different degrees. We conclude that infusion of N-acetylcysteine intraveneously decreases the prothrombin time in healthy subjects. Thus, one should not make conclusions which are too far-reaching based on prothrombin time alone in patients who have been treated recently with N-acetylcysteine intraveneously.

Acetylcysteine↗
PubMed1998

Prothrombin Greenville, Arg517-->Gln, identified in an individual heterozygous for dysprothrombinemia.

A 64-year-old white male was referred for evaluation of prolonged prothrombin time (PT) and activated partial thromboplastin time (aPTT) obtained before elective surgery with initial PT and PTT results of 14.9 and 38.4 seconds, respectively, which corrected to normal in 1:1 mixes with normal plasma. Functional prothrombin assay indicated a level of 51% with thromboplastin as an activator. The prothrombin antigen was 102%. This discordance in the functional and immunologic prothrombin levels was evidence for dysprothrombinemia. Western blotting showed that thrombin was formed at a normal rate in diluted plasma consistent with a mutation within the thrombin portion of prothrombin. DNA was isolated from leukocytes and the thrombin exons were amplified by polymerase chain reaction, cloned, and sequenced. For exon 13, eight clones were sequenced with four clones showing a point mutation in the codon for Arg517, which would result in substitution by Gln. Arg517 is part of the Arg-Gly-Asp(RGD) sequence in thrombin and contributes to an ion cluster with aspartic acid residues 552 and 554. Mutation at this residue most probably distorts the structure of the Na+ binding site in thrombin. This is the first report indicating the critical role of Arg517 in the normal physiological interaction of thrombin with fibrinogen. This dysprothrombin is designated Prothrombin Greenville.

Arginine↗
PubMed2000

Prevalence of the G1691A mutation in the factor V gene (factor V Leiden) and the G20210A prothrombin gene mutation in the Thai population.

We investigated the prevalence of a genetic variation in the factor V gene (G1691A Leiden mutation) and the prothrombin gene (G20210A) using polymerase chain reaction techniques in samples from 500 normal Thai population and among 50 unselected Thai patients with an objectively confirmed history of deep venous thrombosis. The prevalence of factor V Leiden and the prothrombin G20210A gene mutation in a group of 500 healthy controls was 0.2% in both groups (allele frequency of 0.1%). Of the 50 adult patients studied, none was a carrier of factor V Leiden or the prothrombin G20210A gene mutation. Our findings confirm that the prevalence of factor V Leiden and prothrombin G20210A gene mutation is lower among Asians than Caucasians and that the distribution of factor V Leiden is similar to that of the prothrombin G20210A variant. The low prevalence of these two mutations can, at least in part, account for the lower frequency of deep venous thrombosis reported in the Thai population. Screening for factor V Leiden and prothrombin gene mutation is of limited benefit and may not be cost-effective in Thai patients with the first episode of deep venous thrombosis.

Adolescent↗
PubMed2001

Primary thrombophilia in Mexico. II. Factor V G1691A (Leiden), prothrombin G20210A, and methylenetetrahydrofolate reductase C677T polymorphism in thrombophilic Mexican mestizos.

We have shown that in Mexican mestizo patients with clinical features of primary thrombophilia, 39% have activated protein C resistance phenotype, 5% protein C deficiency, and 2% protein S deficiency. In the present study, in a group of 37 thrombophilic Mexicans and 50 normal controls, we assessed the factor V G1691A (Leiden), the prothrombin G20210A, and the methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphisms. Four patients were found to be heterozygous for factor V Leiden, 5 heterozygous for the prothrombin 20210, 16 heterozygous, and 6 homozygous for the MTHFR 677. There were four individuals with co-segregation of alleles: two heterozygotes for the factor V Leiden/prothrombin 20210, one heterozygote for prothrombin 20210/MTHFR 677, and one heterozygote for prothrombin 20210/homozygote for MTHFR 677. For factor V Leiden, prothrombin 20210, and MTHFR 677 mutations, the allele frequencies were respectively 1% (+/-0.2%, alpha = 0.05), <1% and 51% (+/-5%, alpha = 0.05), with calculated relative risks for thrombosis of 5.94 (P = 0.08), >7.66 (P < 0.05), and 0.44 (P NS), respectively. In Mexican mestizo thrombophilic patients, the low prevalence of the factor V Leiden mutation (10.8%) and the high prevalence of the prothrombin 20210 mutation (13.5%) contrast with those identified in Caucasian thrombophilic patients (21% and 6%, respectively; P < 0.01). On the other hand, the high prevalence of the MTHFR 677 mutation gene both in normal controls (78%) and thrombophilic patients (61%) does not support a role of this mutation in the thrombogenesis of Mexican mestizo patients.

3' Untranslated Regions↗
PubMed1981

Prothrombin Gainesville. A dysprothrombinemia in a pair of identical twins.

A pair of identical twins was evaluated for menometrorrhagia and mild post-operative bleeding. Each had a slightly prolonged partial thromboplastin time and prothrombin time. Full hemostatic evaluation was normal, except that plasma prothrombin activity was 23% and 25% of normal with a Russell's viper venom assay. Each twin had 70% of normal plasma prothrombin antigen. Prothrombin kinetic data were obtained before and during hysterectomy. In these twins, infused prothrombin (Konyne) was found to have a volume of distribution of approximately one and one-half times the plasma volume and a half-life of 34 hours. The previously reported dysprothrombins are briefly reviewed. Prothrombin Gainesville is the twelfth such disorder reported.

Adult↗
PubMed2004

Factor V Leiden and prothrombin gene G20210A mutations in Italian patients with Behçet's disease and deep vein thrombosis.

OBJECTIVE: To evaluate the frequency and type of vascular lesions and to study the association of factor V gene G1691A (Leiden) and prothrombin gene G20210A polymorphisms with venous thrombosis in Italian patients with Behçet's disease (BD). METHODS: Included were 118 consecutive Italian BD patients followed over a 3-year period (1997-1999) who satisfied the International Study Group criteria for BD. The control group consisted of 132 healthy Italian blood donors. All BD patients and controls were genotyped by polymerase chain reaction and allele-specific restriction enzyme techniques for factor V Leiden and prothrombin gene G20210A polymorphisms. RESULTS: Vascular lesions were observed in 37 (31.4%) patients. The 2 most common lesions were subcutaneous thrombophlebitis (10.2%) and deep vein thrombosis (DVT) of the legs (22.8%). No significant demographic and clinical differences between patients with and without DVT were present. The distribution of allele and genotype frequencies of prothrombin gene G20210A and factor V Leiden polymorphisms did not differ significantly between BD patients and healthy controls. The frequencies of carriage rates of prothrombin gene G20210A and factor V Leiden polymorphisms in BD patients with and without DVT were similar. However, the frequency of 20210A allele was significantly higher in BD patients with ocular disease than in those without, particularly in the patients with posterior uveitis/retinal vasculitis. CONCLUSIONS: The frequency and types of vascular lesions in Italian BD patients were similar to those reported in studies from other countries. No association between factor V Leiden mutation and G20210A mutation in the 3'-untranslated region of the prothrombin gene with DVT was found. However, a prothrombin gene G20210A mutation may influence the development and severity of ocular involvement in BD.

Adolescent↗
PubMed1985

Immunochemical detection by specific antibody to thrombin of prothrombin conformational changes upon adsorption to artificial surfaces.

Polyclonal antihuman alpha-thrombin antibodies produced in rabbits reacted minimally (less than 0.05%) in solution with human prothrombin. However, when prothrombin was adsorbed to artificial surfaces such as polyvinyl chloride (PVC), the cross-reactivity of surface-bound prothrombin with antibody IgG to thrombin (greater than 95% purity) was shown to be significantly enhanced. On PVC, the molar ratios of antibody IgG to thrombin/prothrombin approached the same level as that of antibody IgG to thrombin/thrombin when thrombin was adsorbed to the same material. The analyses of antigen-antibodies interaction, in solution with a direct binding assay by immune precipitation at high-speed centrifugation (160,000 g, 30 min), and on solid-phase PVC, were accomplished by use of double-labeling technique, i.e., 131I-thrombin (or 131I-prothrombin) and 125I-antibody IgG to thrombin. The results appear to suggest that prothrombin adsorption to PVC has resulted in some molecular conformational changes so that immunologically the adsorbed prothrombin resembles that of adsorbed thrombin on the same PVC surface.

Adsorption↗
PubMed1999

Acetylated prothrombin as a substrate in the measurement of the procoagulant activity of platelets: elimination of the feedback activation of platelets by thrombin.

Human prothrombin was acetylated to produce a modified prothrombin that upon activation by platelet-bound prothrombinase generates a form of thrombin that does not activate platelets but retains its amidolytic activity on a chromogenic peptide substrate. If normal prothrombin is used in such an assay, the thrombin that is generated activates the platelets in a feedback manner, accelerating the rate of thrombin generation and thereby preventing accurate measurement of the initial platelet procoagulant activity. Acetylation of prothrombin was carried out over a range of concentrations of sulfo-N-succinimidyl acetate (SNSA). Acetylation by 3 mM SNSA at room temperature for 30 min at pH 8.2 in the absence of metal ions produced a modified prothrombin that has <0.1% clotting activity (by specific prothrombin clotting assay), but it is activated by factor Xa (in the presence of either activated platelets or factor Va + anionic phospholipid) to produce thrombin activity that is measurable with a chromogenic substrate. Because the feedback action on the platelets is blocked, thrombin generation is linear, allowing quantitative measurement of the initial platelet activation state.

Acetylation↗
PubMed1982

Prothrombin determination in plasma and in liver biopsies in chronic liver diseases.

Following ribosomal synthesis of the protein moiety of prothrombin, postribosomal carboxylation in the liver is necessary to obtain prothrombin ready for physiologic activation. Both decarboxyprothrombin and carboxyprothrombin can be determined by means of specific activators. Using synthetic substrates for thrombin, a micro-prothrombin assay suitable for functional determinations of decarboxy- and carboxy-prothrombin in liver biopsies was evolved. Decarboxyprothrombin was not found in plasma, independent of the kind of liver disease. Prothrombin determination in 31 liver samples showed no relation between the absolute concentration of carboxyprothrombin and decarboxyprothrombin and the kind of disease. The carboxyprothrombin fraction of total prothrombin was correlated with the activity of histologically estimated fibrosis in the liver.

Biopsy, Needle↗
PubMed1995

Intramolecular domain-domain interactions and intermolecular self-association in bovine prothrombin. A potentiometric and laser light-scattering study.

The interaction of bovine prothrombin with Ca2+ and Mg2+ ions was investigated by following H+ release as a function of metal ion concentration at pH 6 and pH 7.4 at high and low ionic strength. Prothrombin Ca2+ and Mg2+ binding is characterized by high- and low-affinity sites. M2+ binding at these sites is associated with intramolecular conformational changes and also with intermolecular self-association. The pH dependence of H+ release by M2+ is bell shaped and consistent with controlling pKa values of 4.8 and 6.5. At pH 6 and low ionic strength, both Ca2+ and Mg2+ titrations following H+ release clearly show independent low- and high-affinity binding sites. Laser light scattering reveals that at pH 7.4 and low ionic strength, and at pH 6.0 and high ionic strength, the prothrombin molecular weight is between 73 and 98 kD. At pH 7.4 and high ionic strength, prothrombin is monomeric in the absence of metal ions, but appears to dimerize in the presence of M2+. At pH 6.0 and low ionic strength prothrombin exists as a dimer in the absence of metal ions and is tetrameric in the presence of Ca2+ and remains dimeric in the presence of Mg2+. These results and those for metal ion-dependent H+ release indicate that H+ release occurs concomitantly with association processes involving prothrombin.

Animals↗
PubMed1992

Relationship between pathologic prognostic factors and abnormal levels of des-gamma-carboxy prothrombin and alpha-fetoprotein in hepatocellular carcinoma.

The relationship between pathologic prognostic factors and abnormal levels of des-gamma-carboxy prothrombin and alpha-fetoprotein was investigated in 42 patients with resectable hepatocellular carcinoma. The frequencies of macroscopic massive type, intrahepatic metastasis, and portal vein tumor thrombus were significantly higher in patients with positive des-gamma-carboxy prothrombin (p less than 0.05) but not with alpha-fetoprotein. Other histologic factors in tumorous and nontumorous tissues were not significantly different irrespective of the positivity of these markers. In patients with tumors not more than 6 cm in diameter, the frequency of intrahepatic metastasis was positively correlated with the positivity of des-gamma-carboxy prothrombin (p less than 0.05) and inversely with that of alpha-fetoprotein (p less than 0.05). Furthermore, intrahepatic metastasis was most frequently observed in patients with positive des-gamma-carboxy prothrombin and negative alpha-fetoprotein (eight of nine) and least frequently in cases with negative des-gamma-carboxy prothrombin and positive alpha-fetoprotein (one of eight). These findings indicated that both des-gamma-carboxy prothrombin and alpha-fetoprotein might be useful markers for the prediction of intrahepatic spread of hepatocellular carcinoma.

Biomarkers↗
PubMed1975

On the presence of a heat-stable, macromolecular inhibitor of the thrombin-fibrinogen reaction in rat liver microsomes and its separation from prothrombin.

By concentrating sonicates from rat liver microsomes containing prothrombin, the activity as measured by the one-stage prothrombin assay (Hjort, P., Rapaport, S. J. and Owren, P. A.(1965), J. Lab. Clin. Med. 46, 89-97) gradually decreased. Nearly a complete loss of prothrombin activity was found in sonicates being concentrated 10-fold. By adsorption of prothrombin on barium citrate and dissolving the precipitate in a solution of sodium citrate. NaCl and EDTA followed by gel filtration on a Sephadex G-50 column, the inhibitory effect on the bioassay of prothrombin disappeared. An inhibitor of the thrombin-fibrinogen reaction could be isolated from the supernatant after adsorption of prothrombin on barium citrate. The inhibitor was excluded from a Sephadex G-50 column equilibrated with 4 M NaCl in veronal buffer (pH 7.4) and was heat stable (70 degrees C for 10 min). No proteolytic or antitrypsin activity could be detected in the inhibitor preparation. The importance of removing the inhibitor of the microsomal sonicates prior to any bioassay of coagulation factors based on the thrombin-fibrinogen reaction is emphasized.

Animals↗
PubMed1993

Induction of prothrombin biosynthesis and inhibition of gamma carboxylase activity in experimental models of liver regeneration and fibrosis.

1. When acute liver damage is induced in rats treated with CCl4, both prothrombin biosynthesis and gamma carboxylase activity decreases, originating a prolongation of prothrombin time in plasma. 2. Then, during the regenerative process, prothrombin biosynthesis increases higher than normal, but gamma carboxylase activity remain decreased. In this case, prolongation of prothrombin time occurs in spite of high levels of descarboxylated prothrombin in plasma. 3. An increase of descarboxylated prothrombin in plasma is also detected in the CCl4-liver fibrosis model.

Animals↗
PubMed1982

Cleavage and activation of human prothrombin by Echis carinatus venom.

The cleavage of human prothrombin by partially purified Echis carinatus venom (ECV) was investigated in the present report. Incubation of prothrombin with ECV resulted in the rapid cleavage of prothrombin to alpha-thrombin, with the release of fragment-1 and fragment-2. When dansyl arginine-N-(3-ethyl-1,5-pentanediyl) amide (DAPA), a very effective inhibitor of thrombin, was included in the ECV-prothrombin solution, meizothrombin was rapidly formed. Only small amounts of meizothrombin-1 could be detected. Prolonged incubation (23 h) in the presence of DAPA, however, resulted in nearly quantitative conversion of meizothrombin to meizothrombin-1 and fragment-1. Kinetic studies strongly suggested that the conversion of meizothrombin to meizothrombin-1 was due to ECV and not meizothrombin autolysis. In addition, EDTA, which inhibits ECV, blocked the cleavage of meizothrombin. Amino terminal sequence analysis indicated that ECV cleaves human prothrombin at two sites; Gly158-Ser159 and Arg322-Ile323. The former site differs from the site of autolytic cleavage of meizothrombin which occurs at Arg155-Ser156. In contrast to reports in the literature, the results of the present study indicate that the release of fragment-1 does not precede activation of human prothrombin by ECV.

Amino Acid Sequence↗
PubMed1983

The action of prothrombin activated by Ecarin on fibrinogen.

Highly purified human prothrombin was activated by Ecarin, a prothrombin activating principle of Echis carinatus venom and the generated thrombin-like activity was investigated. Kinetics of the release of fibrino-peptides A and B (FPA, FPB) from human fibrinogen was estimated using radioimmunoassay technique. The direct proteolytic action of Ecarin on fibrinogen was studied by means of polyacrylamide gel electrophoresis and by radioimmunoassay for fragment B beta 15-42 of fibrinogen. In a system containing Ecarin, fibrinogen and prothrombin both fibrinopeptides were cleaved off at a rate that was essentially similar to that observed with thrombin, the cleavage off of FPB being in the initial stage always slower than the release of FPA. At physiological concentration of prothrombin and at low concentration of Ecarin all FPB were liberated while less than 1% of B beta 15-42 immunoreactivity was released. It was possible to accelerate the release of FPB when prothrombin was activated by Ecarin in the presence of Fibrin I gel (formed from fibrinogen by Batroxobin which liberates FPA only) instead of fibrinogen. Fibrin I thus constituted a more favourable substrate than fibrinogen with regard to release of FPB. The results indicate that the coagulant activity generated from prothrombin by Ecarin is thrombin-like. No intermediate product capable of marked cleavage off of FPA only was detected.

Batroxobin↗
PubMed1987

Neutralization of heparin by prothrombin activation products.

The neutralization of heparin by active site blocked meizothrombin and thrombin, prothrombin fragment 1.2, fragment 1 and fragment 2 was probed by the heparin-dependent factor Xa inactivation by antithrombin III (AT III). Meizothrombin had no effect on the inactivation of factor Xa, whereas thrombin had an inhibitory effect (IC50 = 700 nM). After factor Xa catalyzed cleavage of meizothrombin, the resulting products, prothrombin fragment 1.2 plus thrombin, did not show any heparin neutralizing properties. However, after isolation of the reaction products, both thrombin and prothrombin fragment 1.2 exhibited heparin neutralizing properties in the factor Xa inactivation reaction. The IC50-values were 700 nM and 100 nM, respectively. Prothrombin fragment 1, when present at 125 nM, caused a 50% reduction of the heparin-dependent rate of inactivation of factor Xa and prothrombin fragment 2 had no effect at all. From this we conclude that, in addition to the thrombin part of the prothrombin molecule, the fragment 1 region also exhibits a rather high affinity for heparin.

Animals↗