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PubMed2000

Association of autoantibodies against the phosphatidylserine-prothrombin complex with manifestations of the antiphospholipid syndrome and with the presence of lupus anticoagulant.

OBJECTIVE: To clarify the association of autoantibodies against prothrombin with the clinical manifestations of the antiphospholipid syndrome (APS) and with the presence of lupus anticoagulant (LAC). METHODS: We examined 265 patients who visited our autoimmune disease clinic. IgG and IgM antiprothrombin antibodies were tested by enzyme-linked immunosorbent assay (ELISA) as either antiphosphatidylserine-prothrombin complex (aPS/PT) antibodies or as antibodies against prothrombin coated on irradiated ELISA plates (as antigen) (aPT). IgG, IgM, and IgA anticardiolipin (aCL) antibodies and their beta2-glycoprotein I (beta2GPI) dependency were also evaluated by ELISA. LAC was tested by 3 different methods. RESULTS: The presence of aPS/PT, but not of aPT, significantly correlated with the clinical manifestations of APS (odds ratio [OR] 4.39, 95% confidence interval [95% CI] 2.06-9.38), and aPS/PT antibodies were as specific as beta2GPI-dependent aCL for APS (93.1% for both). IgG aPS/PT strongly correlated with the presence of LAC as detected using the dilute Russell viper venom time test (OR 38.2, 95% CI 13.4-109.1). CONCLUSION: Antiprothrombin antibodies are heterogeneous and their clinical relevance depends on the method of detection applied. Positive results on the aPS/PT test can serve as a marker of thrombotic events in patients with autoimmune diseases.

Adolescent↗
PubMed1997

A rapid and highly sensitive chromogenic microplate assay for quantification of rat and human prothrombin.

A rapid and highly sensitive chromogenic microplate assay for quantification of rat and human prothrombin in subcellular fractions and large series of plasma samples has been developed. The assay is based on the conversion of prothrombin to thrombin, using Echis carinatus venom as an activator, and the subsequent cleavage of a chromogenic thrombin specific substrate, D-cyclohexylglycyl-L-alanyl-L-arginine-p-nitroanilide dihydroacetate. para-Nitroaniline being released by the cleavage is then measured at 410 nm with a microplate reader. The method is suitable for analyses of a large number of samples in a short time, measuring prothrombin in the nanogram range (0.3-2.4 ng/40 microliters of sample).

Animals↗
PubMed1993

Crystal matrix protein is related to human prothrombin.

Crystal matrix protein (CMP) is the principal protein found in calcium oxalate (CaOx) crystals precipitated from whole human urine. It is a potent inhibitor of crystal aggregation and may therefore be important in the aetiology of kidney stone disease. CMP was isolated from CaOx crystals by EDTA dissolution and purified by Sephacryl S-200 column chromatography and reversed-phase high pressure liquid chromatography. Edman degradation revealed 81.8% sequence identity of the 11 N-terminal amino acids of CMP with the N-terminus of human prothrombin, which contains 10 gamma-carboxyglutamic acid residues in the first 32 amino acids. The apparent relationship between CMP and prothrombin was confirmed when an antibody to human prothrombin reacted with CMP upon Western blotting of sodium dodecyl sulphate polyacrylamide gels of the protein.

Amino Acid Sequence↗
PubMed1995

The pH-dependence of the equilibrium between the fast- and slow-folding forms of bovine prothrombin fragment 1: a role for pH-induced self-association?

Examination of the pH- and ionic strength (mu)-dependence of the equilibrium between fast- and slow-folding forms of bovine prothrombin fragment 1 reveals a sharp dependence of Keq ([% fast-folding form]/[% slow-folding form]) and % fluorescence quenching (%Q) on pH at low mu, and the absence of a pH-dependence of Keq at high mu (0.1 M NaCl) and much reduced pH-dependence of %Q, suggesting that the ionization process is coupled to other processes, such as self-association. The observed low mu pH effect on Keq amounts to a 10% increase in the % of the fast-folding form of prothrombin at low pH. We hypothesize the existence of a pH-dependent self-association of bovine prothrombin fragment 1. This process is associated with a conformation change involving an ionizing group with pKa in the neighborhood of 6.5 and a change in Keq from 0.27 to 0.45.

Animals↗
PubMed1993

Prothrombin fragment F 1 + 2 plasma concentrations in patients with gynecologic malignancies.

Malignant diseases have been shown to cause hypercoagulable states. Elevated prothrombin fragment F 1 + 2 plasma levels occur, when prothrombin is converted into thrombin by factor Xa. In order to evaluate the role of prothrombin fragment F 1 + 2 as an indicator for hemostatic disorders in patients with gynecologic malignancies, plasma levels of 76 patients with various malignancies of the female genital tract and those of 25 healthy subjects were investigated. Fifty-two of 76 (68.4%) patients with malignant diseases showed elevated F 1 + 2 plasma concentrations, whereas no elevated plasma levels were observed in the control group. Mean values of F 1 + 2 in the study group and the controls were 1.94 +/- 1.63 and 0.89 +/- 0.07 nmol/liter, respectively (P < 0.01). These results provide further evidence that gynecologic malignancies can induce hypercoagulable states.

Adult↗
PubMed2000

Modes of evolution in the protease and kringle domains of the plasminogen-prothrombin family.

Phylogenetic analysis of protease domains of the vertebrate plasminogen-prothrombin family revealed two major subfamilies: (1) a subfamily containing macrophage-stimulating protein (MSP), hepatocyte growth factor (HGF), plasminogen, and apolipoprotein(a) (APOA); and (2) a subfamily containing prothrombin, HGF activator, and plasminogen activators. There was evidence that these two subfamilies diverged prior to the divergence of amphibians and amniotes. The phylogeny indicated a close relationship of APOA from the European hedgehog, rhesus monkey, and human with plasminogen. Phylogenetic analysis of repeated kringle domains supported the hypothesis that APOA evolved independently in hedgehog and primates through numerous duplications of different kringle domains of the ancestral plasminogen. Phylogenies of kringle domains revealed two modes of evolution: (1) a conservative mode, whereby duplication of kringle domains occurred prior to cladogenesis and the same kringle structure has been maintained in different lineages (exemplified by plasminogen and prothrombin); and (2) a concerted mode, whereby kringle domains have duplicated since cladogenesis and thus orthologous relationships do not exist between kringles of different lineages (exemplified by APOA).

Animals↗
PubMed1996

Overexpression of human prothrombin in permanent cell lines using a dominant selection/amplification fusion marker.

Human prothrombin was overexpressed in transformed eukaryotic cells using a dominant bifunctional selection and amplification marker. The marker consists of the murine wild-type dihydrofolate reductase (dhfr) cDNA and the Escherichia coli hygromycin phosphotransferase gene fused in frame. The gene of interest is connected by the encephalomyocarditis virus 5' untranslated region to the fusion marker gene, forming a dicistronic transcription unit. The human prothrombin gene (FII) was used to monitor expression after initial selection for hygromycin B resistance and DHFR activity. In Chinese hamster ovary (CHO) cells, 5-15 mU prothrombin/10(6) cells per 24 h was obtained; in human 293 kidney cells levels of 20-50 mU/ 10(6) cells per 24 h were obtained. Methotrexate-mediated amplification of the foreign gene in CHO cells resulted in a more than 10-fold increase in FII expression, while in the presence of methotrexate, 293 cells expressed 200-250 mU/10(6) cells per 24 h. The use of this fusion marker within a dicistronic transcription unit allowed efficient dominant selection of cell clones and amplification of the gene of interest. Stably transfected cell lines that were able to secrete high levels of processed gamma-carboxylated human prothrombin were thus obtained.

Animals↗
PubMed1992

A comparison of the relative sensitivities of factor VII and prothrombin time measurements in detecting drug interactions with warfarin.

We have studied the comparative abilities of the prothrombin time and factor VII clotting activity, measured using a chromogenic assay, to detect drug interactions with warfarin. Pharmacokinetic and pharmacodynamic data were collected from studies involving the single administration of 25 mg of warfarin in the absence and presence of fengabin, cimetidine, ranitidine, and enoxacin. Fengabin caused changes in both the pharmacokinetics and pharmacodynamics of warfarin, whereas cimetidine and enoxacin only caused changes in its pharmacokinetics. Ranitidine had no effect on either the pharmacokinetics or pharmacodynamics of warfarin. In general, factor VII clotting activity showed greater sensitivity but also greater variability than the prothrombin time to changes in clotting activity. Consequently, factor VII clotting activity did not have greater discriminatory power than the prothrombin time in detecting drug interactions involving warfarin.

Adult↗
PubMed1996

Increased prothrombin fragment 1 + 2 and D-dimer in first-degree relatives of type 2 diabetic patients. Prethrombotic state in relatives of type 2 diabetic patients.

To evaluate whether or not activated coagulation is present in the preclinical phases of type 2 diabetes mellitus, we studied 46 non-diabetic first-degree relatives of type 2 diabetic patients and 21 matched controls with no family history of diabetes. We determined the plasma levels of prothrombin fragment 1 + 2, D-dimer, fibrinogen, plasminogen activator inhibitor type 1, tissue plasminogen activator, von Willebrand factor and coagulation factors VII and VIII. Glucose tolerance, beta-cell function and insulin sensitivity were assessed in all subjects by a continuous glucose infusion of 5 mg.kg ideal body weight-1.min-1 for 60 min with model assessment of glucose, insulin and C-peptide values. Plasma levels of prothrombin fragment 1 + 2 (median 1.24 vs 0.68 nmol.l-1; P = 0.0001) and D-dimer (331 vs 254 micrograms.l-1 UEF; P = 0.018) were higher in relatives, without significant differences in the other haemostatic variables. Relatives showed higher fasting (5.5 vs 4.9 mmol.l-1, P = 0.004) and post-infusion (9.3 vs 8.3 mmol.l-1, P = 0.02) serum glucose, no differences in insulin or C-peptide levels, lower beta-cell function (122% vs 147%; P = 0.02) and no significant differences in insulin sensitivity. Fifteen relatives were glucose-intolerant and had lower beta-cell function and insulin sensitivity than glucose-tolerant relatives. Both subsets of relatives exhibited higher levels of prothrombin fragment 1 + 2 and D-dimer than control subjects. Thus, first-degree relatives of type 2 diabetic patients present an activated coagulation, even in the absence of minor degrees of glucose intolerance. These abnormalities can play a role in the pathogenesis of cardiovascular diseases frequently seen at diagnosis of type 2 diabetes.

Adult↗
PubMed1975

The effect of several viper venoms on prothrombin Padua.

The effect of four viper venoms (Oxyuranus scutellatus, Notechis scutatus scutatus, Echis carinatus, Naja nigricollis) on prothrombin Padua has been studied. Using Oxyuranus scutellatus venom and Notechis scutatus scutatus venom, prothrombin activity resulted to be moderately decreased similarly to what observed with other one-stage and two-stage methods. On the contrary, using Echis carinatus venom a normal level was obtained. No clotting was observed using the Naja nigricollis venom, regardless of the concentration used. The normal level of factor II obtained with Echis carinatus venom as compared with the low levels obtained with the other venoms, suggests that it acts on a different site of the prothrombin molecule.

Animals↗
PubMed1987

The genetic relationships between the kringle domains of human plasminogen, prothrombin, tissue plasminogen activator, urokinase, and coagulation factor XII.

A computer-based statistical evaluation of the optimal alignments of the kringle domains of human plasminogen, human prothrombin, human tissue plasminogen activator, human urokinase, and human coagulation Factor XIIa, as well as the putative kringle of human haptoglobin, has been performed. A variety of different alignments has been examined and scores calculated in terms of the number of standard deviations (SD) of a given match from randomness. With the exception of human haptoglobin, it was found that very high alignment scores (8.9-23.0 SD from randomness) were obtained between each of the kringles, with the kringle 1 and kringle 5 regions of human plasminogen displaying the highest similarity, and the S kringle of human prothrombin and the human Factor XII kringle showing the least similarity. The relationships obtained were employed to construct an evolutionary tree for the kringles. The predicted alignments have also allowed nucleotide mutations in these regions to be evaluated more accurately. For those regions for which nucleotide sequences are known, we have employed the maximal alignments from the protein sequences to assess nucleotide sequence similarities. It was found that a range of approximately 40-55% of the nucleotide bases were placed at identical positions in the kringles, with the highest number found in the alignment of the two kringles of human tissue plasminogen activator and the lowest number in the alignment of the S kringle of prothrombin with the second kringle of tissue plasminogen activator. From both protein and nucleotide alignments, we conclude that haptoglobin is not statistically homologous to any other kringle. Secondary structural comparisons of the kringle regions have been predicted by a combination of the Burgess and Chou-Fasman methods. In general, the kringles display a very high number of beta-turns, and very low alpha-helical contents. From analysis of the predicted structures in relationship to the functional properties of these domains, it appears as though many of their functional differences can be related to possible conformational alterations resulting from amino acid substitutions in the kringles.

Amino Acid Sequence↗
PubMed2002

Factor V Leiden and prothrombin 20210 G-A mutations in controls and in patients with thromboembolic events during pregnancy or the puerperium.

Factor V Leiden and prothrombin 20210 G-A mutations are independent risk factors for venous thrombosis. We studied the frequency of these mutations in 35 patients who had thromboembolic events during pregnancy and puerperium, and in 32 women who had a history of uncomplicated pregnancy, delivered either vaginally or by cesarean section, and did not have a past history of thromboembolism. Factor V Leiden mutation was present in 7 patients (20%) in the study group. Of these 7 patients, 1 was homozygote, whereas the remaining 6 were heterozygote for the mutation. Prothrombin 20210 G-A mutation was present in 2 patients (5.7%) in the study group. In the control group none of the 32 patients was positive for the factor V Leiden and prothrombin 20210 G-A mutations. Our findings indicate that the factor V Leiden mutation is an important risk factor for thromboembolic disease during pregnancy or puerperium.

Adult↗
PubMed2006

Recombinant human prothrombin kringle-2 induces bovine capillary endothelial cell cycle arrest at G0-G1 phase through inhibition of cyclin D1/CDK4 complex: modulation of reactive oxygen species generation and up-regulation of cyclin-dependent kinase inhibitors.

Prothrombin is a plasma glycoprotein involved in blood coagulation and, as we have previously reported, prothrombin kringles inhibit BCE (bovine capillary endothelial) cell proliferation. To reveal the mechanism, we investigated the influence of rk-2 (recombinant human prothrombin kringle-2) on the BCE cell cycle progression and ROS (reactive oxygen species) generation using FACS (fluorescence-activated cell sorter) analysis. Cell cycle analysis showed a decrease of G(1) phase cells in cells treated with bFGF (basic fibroblast growth factor) and an increase in cells treated with rk-2, as compared with the control cells. But, the portion of the S phase was reversed. In Western blot analysis, bFGF induced cytoplasmic translocation of p21(Waf1/Cip1) and p27(Kip1) and phosphorylation of p27(Kip1) but rk-2 treatment inhibited translocation of p21(Waf1/Cip1) and p27(Kip1) from nucleus to cytoplasm and phosphorylation of p27(Kip1). Also, rk-2 induced up-regulation of p53 and nuclear p21(Waf1/Cip1) and inhibited the cyclin D1/CDK4 (cyclin-dependent kinase 4) complex. The ROS level of rk-2-treated BCE cells was increased 2-fold when compared with the control, but treatment with NAC (N-Acetyl-L: -cysteine), an anti-oxidant, decreased ROS generation about 55% as compared with the rk-2 treatment. NAC treatment also restored cell cycle progression inhibited by rk-2 and down-regulated p53 and nuclear p21(Waf1/Cip1) expression induced by rk-2.These data suggest that rk-2 induces the BCE cell cycle arrest at G(0)-G(1) phase through inhibition of the cyclin D1/CDK4 complex caused by increase of ROS generation and nuclear cyclin-dependent kinase inhibitors.

Animals↗
PubMed1985

Calcium-dependent changes in properties of human prothrombin: a study using high-performance size-exclusion chromatography and gel-permeation chromatography.

High-performance size-exclusion chromatography using a TSK 3000 SW column and aqueous gel filtration with Sephacryl S-200 SF have been used to characterize the effects of calcium ions on the hydrodynamic properties of human prothrombin and prethrombin 1. The results suggest that the effective hydrodynamic radius of prothrombin is less in the presence than in the absence of calcium ions. In addition, when using the TSK-3000 SW column, Ca2+-dependent formation of a hydrophobic site in the fragment 1 region of prothrombin results in an apparent further decrease in hydrodynamic radius.

Calcium↗
PubMed1976

A variant of prothrombin induced in cattle by prolonged administration of warfarin.

Cows on long term Warfarin therapy produce a form of prothrombin which, although it binds to barium citrate, has a low biological activity. Activation experiments on this form of prothrombin show that it is only slowly converted to thrombin in the presence of Ca2+, although the thrombin produced has normal activity. Further experiments show that the Fragment 1 region of the molecule has a reduced calcium binding capacity. The results indicate the existance of a partially carboxylated form of prothrombin.

Animals↗
PubMed1977

The N-terminal activation fragment of bovine prothrombin. Immunological studies leading to a one step purification.

Some immunological studies on prothrombin fragment 1 from bovine prothrombin and its warfarin-induced precursor acarboxyprothrombin are reported. Based on the results, a rapid and simple immunoadsorption method for the isolation of prothrombin fragment 1 in good yield has been established. The method exploits the conformational change induced in the fragment by removal of Ca2+. The principle may be applicable to other gamma-carboxyglutamyl-containing proteins or fragments therof.

Apoproteins↗
PubMed1994

Structure of the non-covalent complex of prothrombin kringle 2 with PPACK-thrombin.

Prothrombin fragment 2 (the second kringle) has been co-crystallized with PPACK (D-Phe-Pro-Arg)-thrombin and the structure of the non-covalent complex has been determined and refined (R = 0.16) at 3.2 A resolution using X-ray crystallographic methods. The kringles interact with thrombin at a site that has previously been proposed to be the heparin binding region. The latter is a highly electropositive surface near the C-terminal helix of thrombin abundant in arginine and lysine residues. These form salt bridges with acidic side chains of kringle 2. Somewhat unexpectedly, the negative groups of the kringle correspond to an enlarged anionic center of the lysine binding site of lysine binding kringles such as plasminogen K1 and K4 and TPA K2. The anionic motif is DGDEE in prothrombin kringle 2. The corresponding cationic center of the lysine binding site region has an unfavorable Arg71Phe substitution but Lys35 is conserved. However, the folding of fragment 2 is different from that of prothrombin kringle 1 and other kringles: the second outer loop possesses a distorted two-turn helix and the hairpin beta-turn of the second inner loop pivots at V64 and D70 by 60 degrees. The Lys35 is located on a turn of the helix, which causes it to project into solvent space in the fragment 2-thrombin complex, thereby devastating the cationic center of the lysine binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Chloromethyl Ketones↗
PubMed1982

A sensitive colorimetric assay for thrombin, prothrombin and antithrombin III in human plasma using a new synthetic substrate.

Benzoyl-L-leucyl-L-alanyl-L-arginine-alpha-naphthylester (Bz-Leu-Ala-Arg-NE) was synthesized as a new substrate for use in the assay of thrombin. In the assay alpha-naphthol released by the enzyme reaction was measured colorimetrically. With Bz-Leu-Ala-Arg-NE as substrate, the minimum detectable concentration of human thrombin was 0.0025 U. This assay using Bz-Leu-Ala-Arg-NE is a highly sensitive method for detecting prothrombin, thrombin and antithrombin III in human plasma. Prothrombin could be determined with 0.2 microliter of human plasma using Echis carinatus venom (ECV) as activator. Antithrombin III activity could be determined with 2 microliter of human plasma using human thrombin and heparin as cofactor. A zymogram of human prothrombin was prepared with Bz-Leu-Ala-Arg-NE as substrate. The preparation gave one band (pI 4.9) on polyacrylamide disc gel isoelectrophoresis.

Antithrombin III↗