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PubMed1976

Pig prothrombin: purification and properties.

A procedure for the preparation of highly purified pig prothrombin is described. Compared to the initial clotting activity of the starting plasma, this protein was purified 776 times with a final yield of 8 per cent. The purified zymogen showed a specific activity of 1,460 NH units/mg of protein , a molecular weight of 65,000 as determined by SDS-polyacrylamide disc gel electroesis, E 1.0 mg/ml 1.0 cm, 280 nm = 1.45 at pH 7.0 and the following amino acid composition: Asx 51, Thr 38, Glx 62, Pro 23, Gly44, Ala 25, Half-Cys 30, Val 35, Met 3, Ile 30, Leu 32, Tyr 19, Phe 22, Lys 36, His 8, Arg 28, and Trp 13, which accounts for a minimum molecular weight of 59,370 (carbohydrates not computed). Alanine was found as the only N-terminal residue. Carboxypeptidases A and B failed to release any C-terminal residue. By hydrazinolysis however 0.4 mole of serine was released per mole of prothrombin. The activation of crude and chromatographed pig prothrombin was investigated.

Amino Acids↗
PubMed1998

DX9065a, an Xa inhibitor, inhibits prothrombin-induced A549 lung adenocarcinoma cell proliferation.

In this study we demonstrate that prothrombin activates the cell proliferation of the lung adenocarcinoma A549 cells. The A549 cell expresses factor Xa-like prothrombinase activity on its surface and prothrombin was converted to thrombin on the cell surface. Furthermore, thrombin induced the activation of PKC, increased [Ca2+]i and potentiated MAP kinase activity through thrombin receptor. The mitogenic activity of prothrombin and the conversion to thrombin were completely abolished by the synthetic coagulation factor Xa inhibitor, DX9065a. These findings suggest that DX9065a is an effective agent for a therapeutic strategy against cancer itself and prothrombotic complications associated with malignancy.

Adenocarcinoma↗
PubMed2003

Prothrombin G20210A mutation, antithrombin, heparin cofactor II, protein C, and protein S defects.

These defects are not as common as factor V Leiden, but they are more common than many other hereditary procoagulant defects. The incidence of the prothrombin gene (G20210A) mutation is not yet known with certainty, but it may approach or even exceed that of factor V Leiden. These defects also seem less common than hereditary sticky platelet syndrome; however, they are all common enough that they always should be considered in any individual with unexplained thrombosis and should be part of the work-up for patients with thrombotic disorders. Of the defects discussed herein, prothrombin G20210A mutation seems, thus far, to be more common than AT, protein C, protein S, or HC-II defects. Assessment of prothrombin gene mutation should be part of the primary evaluation of patients with unexplained thrombosis.

Antithrombins↗
PubMed1988

Prothrombin activation on phospholipid membranes with positive electrostatic potential.

The conversion of prothrombin into thrombin, which is a crucial reaction in hemostatic plug formation, is greatly stimulated by phospholipids plus calcium ions. It has been proposed that phospholipid surfaces which promote blood coagulation should have a negative surface charge [Bangham, A. D. (1961) Nature (London) 192, 1197-1198]. However, the experiments that led to this proposal were carried out with one kind of anionic phospholipid (dicetyl phosphate). Here we report that membranes, which contain phosphatidylserine (PS) as the anionic phospholipid, can be made positively charged by incorporation of stearylamine and still exhibit almost full procoagulant and prothrombin-converting activity. This suggests that electrostatic forces contribute negligibly to the binding of coagulation factors to PS-containing membranes. Introduction of stearylamine in membranes containing phosphatidyl-beta-lactate (PLac) causes considerable inhibition of their prothrombin-converting activity. Since PLac and PS only differ by the presence of an amino group in the polar head group, the much higher procoagulant activity of PS-containing vesicles is indicative of an important function of the amino group of PS in the interaction with coagulation factors. We propose that the association of coagulation factors with PS-containing membranes results from complex formation between Ca2+ ions and ligands supplied by the protein and by PS molecules. The ability to form such a complex may well explain why cell membranes with PS have such excellent procoagulant properties.

Amines↗
PubMed1988

Carbohydrate-linked asparagine-101 of prothrombin contains a metal ion protected acetylation site. Acetylation of this site causes loss of metal ion induced protein fluorescence change.

Prothrombin fragment 1 (prothrombin residues 1-156) contains two acetylation sites that are protected from derivatization by calcium. The first site was protected by only calcium [Welsch, D. J., & Nelsestuen, G. L. (1988) Biochemistry (second of three papers in this issue)] while the second site was protected by magnesium as well. To identify this second acetylation site, fragment 1 was first acetylated with unlabeled reagent in the presence of magnesium. Metal ions were removed, and the protein was acetylated with radiolabeled reagent. The incorporated radiolabel was stable over long periods of time and at acidic or basic pH as long as elevated temperatures were avoided. The radiolabel was removed by treatment of the protein at pH 10 and 50 degrees C or with 0.2 M hydroxylamine at 50 degrees C for at least 30 min. Proteolytic degradation of the protein showed that the radioactivity appeared in a tryptic peptide corresponding to residues 94-111 of prothrombin. The Lys-97 in this peptide was acetylated but did not contain radiolabel. Amino acid sequence analysis revealed that the radiolabel was associated with an unextracted sequence product. Aglycofragment 1, produced by treatment of fragment 1 with HF, was radiolabeled by this procedure; peptide 94-111 was isolated and was further digested with protease. The major radiolabeled product contained Asn101-Ser102 along with the expected chitobiose attached to Asn-101. NMR analysis revealed the presence of three acetate groups which would correspond to two from the chitobiose plus the incorporated acetate residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMed2005

Functional mapping of charged residues of the 82-116 sequence in factor Xa: evidence that lysine 96 is a factor Va independent recognition site for prothrombin in the prothrombinase complex.

It has been hypothesized that two antiparallel structures comprised of residues 82-91 and 102-116 in factor Xa (fXa) may harbor a factor Va- (fVa-) dependent prothrombin recognition site in the prothrombinase complex. There are 11 charged residues in the 82-116 loop of human fXa (Glu-84, Glu-86, Lys-90, Arg-93, Lys-96, Glu-97, Asp-100, Asp-102, Arg-107, Lys-109, and Arg-115). With the exception of Glu-84, which did not express, and Asp-102, which is a catalytic residue, we expressed the Ala substitution mutants of all other residues and evaluated their proteolytic and amidolytic activities in both the absence and presence of fVa. K96A and K109A activated prothrombin with 5-10-fold impaired catalytic efficiency in the absence of fVa. All mutants, however, exhibited normal activity toward the substrate in the presence of fVa. K109A also exhibited impaired amidolytic activity and affinity for Na(+); however, both fVa and higher Na(+) restored the catalytic defect caused by the mutation. Analysis of the X-ray crystal structure of fXa indicated that Glu-84 may interact by a salt bridge with Lys-109, explaining the lack of expression of E84A and the lower activity of K109A in the absence of fVa. These results suggest that none of the residues under study is a fVa-dependent recognition site for prothrombin in the prothrombinase complex; however, Lys-96 is a recognition site for the substrate independent of the cofactor. Moreover, the 82-116 loop is energetically linked to fVa and Na(+) binding sites of the protease.

Alanine↗
PubMed2003

Biochemical markers of fibrosis in patients with chronic hepatitis C: a comparison with prothrombin time, platelet count, and age-platelet index.

As an alternative to liver biopsy, an index of five biochemical markers (alpha2-macroglobulin, apolipoprotein A1, haptoglobin, total bilirubin, gamma-glutamyl-transpeptidase) has been shown to predict the severity of hepatitis C-related fibrosis. The objective of this study was to compare this index with other markers frequently used for this purpose (prothrombin time, platelets, age-platelet index). In 323 hepatitis C-infected patients, the discriminative values of these markers for F2-F4 fibrosis (by the METAVIR classification) were compared. By multiple logistic regression analysis, only the five-marker index (P < 0.0001) and prothrombin time (P = 0.02) were independently predictive of F2-F4 fibrosis. For this outcome, the area under the receiver operating characteristic curve was significantly higher for the five-marker index (0.836 +/- 0.024) than the age-platelet index (P = 0.002), and the platelet count and prothrombin time (P < 0.001), indicating greater diagnostic value. The addition of the latter markers to the five-marker index proved unhelpful for increasing its accuracy. In conclusion, an index of five biochemical markers accurately predicts significant hepatitis C-related fibrosis and is superior to traditional markers.

Adult↗
PubMed2004

Effect of lupus anticoagulant on the interaction of prothrombin with platelets.

We studied binding of (125)I-labeled prothrombin to platelets in the presence of circulating endogenous lupus anticoagulant. Lupus anticoagulant modulated the interaction and increased affinity of prothrombin for calcium-independent binding sites on platelets. The number of these sites decreased, while the total number of calcium-dependent binding sites increased. Our results indicate that lupus anticoagulant plays a role in the binding of prothrombin to nonactivated platelets.

Binding Sites↗
PubMed2003

Echis time, under-carboxylated prothrombin and vitamin K status in intensive care patients.

Vitamin K deficiency is a known cause of coagulopathy in hospitalized patients, but the extent of the problem has not been well assessed. This noninterventional, prospective observational study of 35 adults was undertaken in the intensive care unit (ICU) and examined the incidence of and the methods for diagnosing vitamin K deficiency. Measurements of prothrombin time, Echis time and plasma concentrations of under-carboxylated prothrombin (proteins induced in vitamin K absence or antagonism, PIVKA-II), vitamin K1 and ferritin were made during the 48 h after admission to the unit and repeated if coagulopathy developed later. Plasma vitamin K1 was low in 15 admissions (43%), in 11 cases of patients with coagulopathy and in four cases without coagulopathy. PIVKA-II was present in 12 cases (34%), of whom four had low vitamin K1 levels. All of the eight patients with raised PIVKA-II but normal vitamin K concentration were hyperferritinaemic. We conclude that low plasma vitamin K levels, suggestive of low tissue stores, are common in intensive care patients with or without coagulopathy. As 34% of patients had a raised PIVKA-II, this suggests that vitamin K stores may be insufficient to maintain full gamma-carboxylation of prothrombin and emphasize the need to anticipate vitamin K deficiency in the ICU setting by appropriate supplementation.

Adolescent↗
PubMed1998

Prevalence and significance of anticardiolipin, anti-beta2 glycoprotein I and anti-prothrombin antibodies in chronic hepatitis C.

Antiphospholipid antibodies have been demonstrated in chronic hepatitis C, but their clinical and pathogenetic significance remains elusive. We prospectively studied 115 patients (85 men, mean age 36.9 years) with chronic hepatitis C without cirrhosis and treated by alpha-interferon (alpha-IFN). Antiphospholipid determinations comprised anticardiolipin (ACA), anti-beta2-glycoprotein I and anti-prothrombin antibodies of the IgG and IgM classes. At entry, 24 patients (21%) were found to possess low to moderate ACA levels (18 IgG, two IgM and four both isotypes) compared with only 4/115 age- and sex-matched control subjects (3.5% P=0.001). ACA positivity rate increased to 31% (P=0.01) after a 6-month course of alpha-IFN treatment. In contrast, the prevalence of anti-beta2-glycoprotein I and anti-prothrombin antibodies was not significantly different from controls at either time point. The presence of ACA correlated with that of antinuclear antibodies (P=0.0002), but was not associated with parameters such as histological activity, viral burden and response to alpha-IFN, nor with a history of thrombosis or pregnancy loss. However, a non-significant trend of higher incidence of mild thrombocytopenia among ACA-positive patients was observed. We conclude that low-titre ACA positivity is a common finding in patients with chronic hepatitis C, especially following alpha-IFN treatment, but does not select a category with different clinical features. These data are in keeping with the absence of associated anti-beta2GPI and anti-prothrombin antibodies, and do not support a role for HCV infection in the pathogenesis of the antiphospholipid syndrome.

Adult↗
PubMed1999

Prothrombin carora: hypoprothrombinaemia caused by substitution of Tyr-44 by Cys.

Two members of a family from Carora, Venezuela, were found to have prothrombin activity levels at 4% of normal and undetectable antigen levels. All exons of the prothrombin gene from the proband were sequenced and a mutation at nucleotide 1305 was identified that would result in the substitution of Cys for Tyr at residue 44. Residue 44 is present in the aromatic stack region of the protein. Substitution of a Cys in this region would result in an abnormal folding of the protein which could be the cause for the observed lack of secretion of the abnormal prothrombin.

Adolescent↗
PubMed1999

Contribution of the glycoprotein Ia 807TT, methylene tetrahydrofolate reductase 677TT and prothrombin 20210GA genotypes to prothrombotic risk among factor V 1691GA (Leiden) carriers.

The cooperative effects of the GPIa 807TT, MTHFR 677TT and prothrombin 20210GA genotypes with the FV Leiden 1691GA (FVL) genotype were evaluated by comparing these genotype frequencies in 77 asymptomatic and 156 symptomatic heterozygous FVL carriers. The GPIa 807TT and MTHFR 677TT genotypes did not segregate within the symptomatic FVL carrier group and did not contribute to venous thrombotic risk in this patient cohort. There was no difference in the prothrombin 20210GA genotype frequency between asymptomatic FVL carriers and a random Caucasian control group; however, the prothrombin 20210GA genotype was nearly 5 times as prevalent (19/156 v 2/77; P < 0.02) in the symptomatic FVL carriers (odds ratio 5.21; 95% confidence interval 1.20-47.62), demonstrating that this important prothrombotic risk factor acts synergistically with FVL.

Factor V↗
PubMed2003

A simple method to discriminate between beta2-glycoprotein I- and prothrombin-dependent lupus anticoagulants.

Lupus anticoagulants (LAC) are a heterogeneous group of autoantibodies that prolong phospholipid-dependent clotting assays. The autoantibodies that cause LAC activity are predominantly directed against beta2-glycoprotein I (beta2GPI) or prothrombin. In the present study, we describe a method to differentiate between LAC caused by antibodies directed against beta2GPI or prothrombin. Monoclonal antibodies, affinity purified patient antibodies, and selected patient samples were used to show that in an aPTT-based clotting assay (PTT-LA; Diagnostica Stago), the use of cardiolipin vesicles in the neutralization procedure discriminates between beta2GPI- or prothrombin-dependent LAC activities. Addition of cardiolipin vesicles shortened the prolonged clotting time caused by anti-beta2GPI antibodies with LAC activity, whereas this procedure further prolonged clotting times caused by antiprothrombin antibodies with LAC activity. In contrast, addition of phosphatidylcholine/phosphatidylserine vesicles corrected prolonged clotting times caused by either anti-beta2GPI or antiprothrombin antibodies with LAC activity. The effects of cardiolipin (CL) on beta2GPI-induced LAC activity were specific for contact activation mediated clotting assays. Possible explanations for these findings are the relatively high affinity of beta2GPI for cardiolipin, as determined by surface plasmon resonance analysis, and inhibition by anti-beta2GPI antibodies of the CL-induced prolongation of the PTT-LA.

Autoantibodies↗
PubMedDate not supplied

[Decreased prothrombin coagulation activities in breast-fed children?].

Intracranial bleeding due to vitamin K deficiency in infants without prior symptoms of disease has been reported. All of these children were fully breastfed. To evaluate a potential deficit of vitamin K dependent coagulation factors in fully breastfed infants we determined the prothrombin clotting times in healthy babies on the 5th day (n = 204) and in the 5th week of life and correlated the results to the feeding. On the fifth day of life prothrombin coagulation times in fully breastfed infants were significantly longer than in babies receiving formula exclusively or additionally. Vitamin K supply in fully breastfed neonates should therefore be reinvestigated. In the 5th week of life, however, there was no difference in prothrombin coagulation times between fully breastfed infants and infants with exclusive or supplementary formula feeding.

Breast Feeding↗
PubMed1989

[Hereditary dysprothrombinemia with a mild bleeding tendency (prothrombin Magdeburg)].

Seven members of one family over three generations were found to have a characteristic reduction to about 0.5 of the ratio between factor II clotting activity and factor II concentration, which was not seen when measuring prothrombin after activation with staphylocoagulase. In addition to the "normal" prothrombin, two abnormal prothrombins, with higher molecular weights and lower isoelectric points, were found by SDS-polyacrylamide gel electrophoresis. This is an autosomal hereditary dysprothrombinemia, the affected persons being heterozygotes. Five of the seven persons had a slightly increased bleeding tendency which manifested itself especially in more marked or prolonged posttraumatic and postoperative bleedings.

Blood Coagulation Disorders↗
PubMed1980

Efficacy of prothrombin-complex concentrates in hemophiliacs with antibodies to factor VIII: a multicenter therapeutic trial.

The therapeutic efficacy of prothrombin-complex concentrates in patients with hemophilia and inhibitors (antibodies) to factor VIII has been increasingly debated. We therefore entered 51 hemophiliacs with factor VIII inhibitors into a double-blind randomized crossover study to compare two commercial prothrombin-complex concentrates (Konyne and Proplex) and an albumin placebo. Acute hemarthrosis of the elbow, knee, or ankle was treated with a single dose of a test preparation and assessed six hours later with objective and subjective criteria. In all measurements the concentrates were significantly more effective than the placebo. The data indicate that although prothrombin-complex concentrates, when used in a single dose, are only partially effective in the treatment of joint hemorrhage in hemophiliacs with inhibitors, their continued use for acute hemarthrosis is justified in the absence of any other effective and readily available therapy for this disorder.

Acute Disease↗
PubMed1996

Rational design, recombinant preparation, and in vitro and in vivo characterization of human prothrombin-derived hirudin antagonists.

A mutant derivative of human prothrombin in which active site aspartate at position 419 is replaced by an asparagine (D419N-prothrombin) has been designed, expressed in recombinant Chinese hamster ovary cells, and purified to homogeneity. D419N-prothrombin was converted to the related molecules D419N-meizothrombin and D419N-thrombin by limited proteolysis by Echis carinatus and Oxyuranus scutellatus venom protease, respectively, and affinity-purified using an immobilized modified C-terminal hirudin-derived peptide. Neither D419N-thrombin nor D419N-meizothrombin exhibited thrombin activity. Titration resulted in no detection of the active site, but binding to the most specific thrombin inhibitor, hirudin, was conserved in both proteins. In vitro examinations showed that D419N-thrombin and D419N-meizothrombin bind to immobilized hirudin, neutralize hirudin in human blood plasma as well as in the purified system, and reactivate the thrombin-hirudin complex. Animal model studies confirmed that D419N-thrombin and D419N-meizothrombin act as hirudin antagonist in blood circulation without detectable effects on the coagulation system. Thus, both D419N-thrombin and D419N-meizothrombin combine for the first time hirudin-neutralizing properties with the advantages of recombinant production of human coagulation factors.

Animals↗
PubMed2000

Directed glycosylation of human coagulation factor X at residue 333. Insight into factor Va-dependent prothrombin catalysis.

Based on homology, amino acids 326-336 (143-154 in chymotrypsin numbering) of factor X (fX) comprise a flexible surface loop, which is susceptible to self-proteolysis and influences substrate catalysis. To investigate the role of this autolysis loop in fX function, a recombinant variant with a new site for asparagine-linked glycosylation has been produced by changing glutamine 333 to asparagine. Q333N fX is activated normally by factor VIIa and tissue factor, factors IXa and VIIIa, and Russell's viper venom. Proteolysis of the loop is prevented by the mutation. Reactivity of the free enzyme toward substrates and inhibitors is attenuated 4-20-fold; relative to wild type fXa, Spectrozyme Xa(TM) hydrolysis is 25%, inhibition by antithrombin III and the tissue factor pathway inhibitor is approximately 20%, and prothrombin activation in the absence of the cofactor Va is only 5%. Surprisingly, activities of the variant and wild type enzymes are equivalent when part of the prothrombinase complex. N-Glycanase cleaves the new oligosaccharide from Q333N fXa leaving aspartic acid. Q333D fXa is approximately 1.6-fold more reactive with Spectrozyme Xa(TM), antithrombin III and tissue factor pathway inhibitor, and prothrombin than its glycosylated counterpart, Q333N fXa, but still quite abnormal relative to wild type fXa. Like Q333N fXa, Q333D fXa is fully functional as part of the prothrombinase complex. We conclude that Gln-333 is geographically close to a site of proteolytic degradation but not to activator, cofactor, or membrane binding sites. Mutation of Gln-333 impairs catalytic function, but given normal prothrombin activation by the complexed enzyme, the importance of Gln-333 for catalysis is not manifest in the prothrombinase assembly, suggesting a conformational change in complexed fXa.

Amino Acid Substitution↗