Human Prothrombin Research Articles

Lupus Anticoagulant IgG's (LA) Are Not Directed to Phospholipids only, but to a Complex of Lipid-Bound Human Prothrombin

Plasmas from 16 patients that were found to be positive both for anticardiolipin antibodies (ACA) and lupus anticoagulants (LA) were incubated with liposomes that contained anionic phospholipids. In 11 of these plasmas, ACA could be cosedimented with the liposomes in a dose-dependent manner, whereas LA activity of the remaining supernatant was unaffected. LA activity of purified total IgG from 6 patients was measured in three different coagulation tests, using normal plasmas from different species. Prolongation of the aPTT, KCT and dRVV clotting times was observed only with normal plasma from human origin, not with bovine, rat or sheep plasma. Highly purified coagulation factors Xa, Va and prothrombin, both of human and bovine origin, were used to establish for two patient IgG's the effect of LA on the rate of thrombin formation in the presence and absence of lipid vesicles composed of 20 mole% phosphatidylserine and 80 mole% phosphatidylcholine. A strong and dose dependent inhibition by LA was observed only when human prothrombin was used as substrate in the prothrombinase complex in the presence of lipids. No inhibition was found when bovine prothrombin was used as substrate. The inhibitory effect observed in the presence of human prothrombin was independent of the source of factors Xa and Va, and was not found in the absence of lipid. Preliminary binding studies suggest that LA only associate with a lipid surface, provided that human prothrombin and calcium ions are present. These data indicate that LA are not directed to phospholipids alone, but presumably recognize an epitope which becomes exposed upon Ca(2+)-mediated binding of human prothrombin to phospholipids.

Ncol RFLP in the human prothrombin (F2) gene

Ncol RFLP in the human prothrombin (F2) gene

Determination of Human Prothrombin Activation Fragment 1+2 in Plasma with an Antibody against a Synthetic Peptide

The present investigation describes a novel approach to prepare a specific antibody against prothrombin activation fragment 1 + 2 (F 1 + 2). The antibody discriminates between native prothrombin and F 1 + 2 in plasma. A synthetic peptide from the negatively charged region of F 1 + 2, which becomes the carboxy-terminal sequence after cleavage of prothrombin by factor Xa, was used for immunization of rabbits. Obtained antiserum was immunopurified and an enzyme-linked immunosorbent assay (ELISA) was constructed for determination of F 1 + 2. The test system follows the sandwich principle and uses two different antibodies directed against F 1 + 2 and prothrombin, respectively. The ELISA was calibrated with purified F 1 + 2 added to F 1 + 2-poor plasma. The lower limit of sensitivity of the assay was 0.02 nmol/l. Coefficients of variation of 6.9 to 10.4% (intraassay) and 6.7 to 11% (interassay) were found for F 1 + 2 concentrations between 0.08 and 4.9 nmol/l. A reference range from 0.32 to 1.2 nmol/l was calculated from 95 healthy donors (mean value +/- SD: 0.67 +/- 0.19 nmol/l). In patients with deep vein thrombosis (n = 7) confirmed by phlebography and in patients with pulmonary embolism (n = 8) confirmed by lung scan, F 1 + 2 levels were found up to 1.5 to 9.5 nmol/l. In plasma samples of patients under oral anticoagulant therapy in the stable state F 1 + 2 concentrations were found to be in the range of 0.08 to 0.5 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein E genotyping using the polymerase chain reaction and allele-specific oligonucleotide primers.

A method for apolipoprotein (apo) E genotyping was developed using the polymerase chain reaction (PCR) with allele-specific oligonucleotide primers (ASP). Synthetic oligonucleotides with base-pair mismatches at the 3' terminus were used as primers to amplify the apoE gene in subjects previously phenotyped using isoelectric focusing (IEF). Complementary primer-allele combinations were specifically amplified by PCR, together with a control pair of primers specific to the human prothrombin gene. Identification of genotype by PCR using ASP was consistent with the phenotypes that were determined by IEF for 14 healthy normolipidemic subjects. These results were achieved using DNA isolated from buccal epithelial cells obtained from a mouthwash or DNA extracted from leukocytes. Genotype identification required analysis of the PCR products on an ethidium-stained agarose gel, yielding results 3 h after DNA extraction. In comparison with other current methods, PCR using ASP is suggested as a rapid and simple noninvasive technique for determining population apoE allelic distribution.

Propeptide recognition by the vitamin K-dependent carboxylase in early processing of prothrombin and factor X

Precursors of vitamin K-dependent proteins are synthesized with a propeptide that is believed to target these proteins for gamma-carboxylation by the vitamin K-dependent carboxylase. In this study synthetic propeptides were used to investigate gamma-carboxylation of the prothrombin and factor X precursors in rat liver microsomes. The extent of prothrombin processing by the carboxylase was also investigated. Antisera raised against the human prothrombin and factor X propeptides only recognized precursors with the respective propeptide regions. The data demonstrate structural differences in the propeptide region of the prothrombin and the factor X carboxylase substrates which raises questions about the hypothesis of a common propeptide binding site on the carboxylase for all precursors of vitamin K-dependent proteins. The hypothesis of separate binding sites is supported by data which demonstrate differences in binding of the prothrombin and factor X precursors to membrane fragments from rough and smooth microsomes. gamma-Carboxylation of the prothrombin precursors in vitro was investigated with conformational specific antibodies raised against a portion of the Gla (gamma-carboxyglutamic acid) region extending from residue 15 to 24. The synthetic peptide used as antigen contains three of the ten potential Gla sites in prothrombin. It is shown that these antibodies do not recognize mature prothrombin but recognize the decarboxylated protein. It is also demonstrated that the epitope is Ca2(+)-dependent. The antibodies were used to assess gamma-carboxylation of the prothrombin precursor in membrane fragments from microsomal membranes. The results suggest that microsomal gamma-carboxylation does not involve Glu residues 16, 19 and 20 of the Gla region.

Coagulation impact on chemotactic activity generation for polymorphonuclear leukocytes

Chemotactic technique in agarose gel has exposed the attractive properties of human αthrombin with respect to human polymorphonuclear leukocytes. The observed chemotaxis is maximal between 1.4.10-5 M and 1.6.10-5 M but extends from 2.8.10-6 M to 2.2.10-5 M. Human prothrombin, in an identical concentration zone as that studied for thrombin shows no chemotactic activity on the polymorphonuclear leukocytes. During coagulation the formed α thrombin attracts the polymorphonuclear leukocytes to it's formation site.

Chemical Modification ofγ-Carboxyglutamic Acid Residues in Prothrombin Elicits a Conformation Similar to That of Abnormal (Des-γ-Carboxy)Prothrombin1

Chemical modification of gamma-carboxyglutamic acid (Gla) residues in human prothrombin to gamma-methyleneglutamic acid (gamma-MGlu) residues elicited a conformation similar, if not identical, to that of des-gamma-carboxy prothrombin or PIVKA-II, i.e., prothrombin molecules induced by vitamin K antagonists or vitamin K deficiency states. The reaction seems to proceed sequentially by preferentially modifying a Gla at residue 32 that is located innermost among 10 Gla residues of human prothrombin. The initial modification resulted in nearly 50% losses of barium salt adsorption, the procoagulant activity and thrombin generation by the prothrombinase complex. The subsequent modification of two Gla residues at positions 6 and 16 gave rise to the immunoreactivity to an established monoclonal antibody that specifically recognizes the des-gamma-carboxy prothrombin. Further modification of Gla residues increased the reactivity to the antibody, indicating that the conformation recognized by the antibody was stabilized so as to more readily fit the recognition site of the antibody. The appearance of the immunoreactivity was obviously related to the modification of Gla residues in prothrombin, since all other similarly treated derivatives of prothrombin lacking the Gla-domain failed to react with the antibody. Such chemically modified prothrombins may serve as models for studying abnormal des-gamma-carboxy prothrombin produced in vitamin K deficiency states.

Characterization of the cDNA Coding for Mouse Prothrombin and Localization of the Gene on Mouse Chromosome 2

A series of overlapping cDNAs coding for mouse prothrombin (coagulation factor II) have been isolated and the composite DNA sequence has been determined. The complete prothrombin cDNA is 1,987 bp in length [excluding the poly(A) tail] and codes for 18 bp of 5' untranslated sequence, an open reading frame coding for 618 amino acids, a stop codon, and a 3' untranslated region of 112 bp followed by a poly(A) tail. The translated amino acid sequence predicts a molecular weight of 66,087, which includes 10 residues of gamma-carboxyglutamic acid. There are five potential N-linked glycosylation sites. Mouse prothrombin is 81.4% and 77.3% identical to the human and bovine proteins, respectively. Comparison of the cDNA coding for mouse prothrombin to the human and bovine cDNAs indicates 79.9% and 76.5% identity, respectively. Amino acid residues important for the structure and function of human prothrombin are conserved in the mouse and bovine proteins. In the adult mouse and rat, prothrombin is primarily synthesized in the liver, where is constitutes 0.07% of total mRNA as determined by solution hybridization analysis. The genetic locus for mouse prothrombin, Cf-2, has been mapped using an interspecies backcross and DNA fragment differences between the two species. The prothrombin locus lies on mouse chromosome 2, 1.8 +/- 1.3 map units proximal to the catalase locus. The gene order in this region is Cen-Acra-Cf-2-Cas-1-A-Tel. This localization extends the proximal boundary of the known region of homology between mouse chromosome 2 and human chromosome 11p from Cas-1 about 2 map units toward the centromere.

Prothrombin Levels Are Increased in the Estrogen-Treated Immature Rat Uterus*

An estrogen-responsive uterine proenzyme of a proteinase in the immature rat uterus has been known for some time. Its mol wt is 77,000, its N-terminal amino acid sequence is the same as prothrombin's for 15 residues, it contains gamma-carboxyl glutamate residues, its biosynthesis is prevented by warfarin, it cross-reacts with antibodies to human and rat prothrombin, and it can be activated by human factor Xa or a uterine procoagulant. The products of activation, when separated on sodium dodecyl sulfate-gels, react with antibodies to human or rat prothrombin to give bands that have mol wt corresponding to those of the products of activation of prothrombin. These activation intermediates hydrolyze synthetic substrates specific for thrombin and have the same mol wt as the activation products of prothrombin. The proteinase generated in the activation has the following properties of thrombin: it is inhibited by hirudin and PheProArg-chloromethyl ketone, it has kinetic constants similar to those of thrombin with tripeptide p-nitroanilides as substrates, and it digests actin to give the same peptides as thrombin. We conclude that the uterine proenzyme is prothrombin. The time course of the prothrombin response to estrogen suggests that prothrombin enters the uterus as part of the transudation of plasma proteins that occurs after estrogen stimulation. A membrane-bound uterine procoagulant that activates uterine prothrombin also increases in response to estrogen stimulation. We propose that the simultaneous increase in these two activities results in a localized generation of thrombin, a well characterized mitogen in fibroblasts and epithelial cells. Our results suggest that thrombin may have a vital function as a mitogen in the early steps of the estrogen-stimulated hypertrophy and hyperplasia of the immature uterus.

RFLP for the human prothrombin (F2) gene

RFLP for the human prothrombin (F2) gene

Measurement of Immunoreactive Prothrombin Precursor and Vitamin-K-Dependent Gamma-Carboxylation in Human Hepatocellular Carcinoma Tissues: Decreased Carboxylation of Prothrombin Precursor as a Cause of Des-Gamma-Carboxyprothrombin Synthesis

Des-gamma-carboxyprothrombin is an abnormal prothrombin which is drastically increased in the plasma of patients with hepatocellular carcinoma. To investigate the process of the abnormal prothrombin synthesis, the amount of prothrombin precursor was measured with an enzyme-linked immunosorbent assay using a specific antibody directed to human prothrombin; the vitamin-K-dependent gamma-carboxylation of prothrombin precursor was determined in human liver tissues. The tissue content of prothrombin precursor was increased in hepatoma tissues compared with noncancerous liver tissues, while the vitamin-K-dependent carboxylation of prothrombin precursor was markedly decreased in hepatoma tissues of the patients with increased plasma des-gamma-carboxyprothrombin. The present study indicates that in hepatocellular carcinoma an increase in prothrombin precursor concentration does not induce vitamin-K-dependent carboxylase activity, which is ordinarily observed in normal liver; probably an overproduction of prothrombin precursor with reduced gamma-carboxylation causes an increase in plasma des-gamma-carboxyprothrombin in patients with hepatocellular carcinoma.

Protein Structural Requirements and Properties of Membrane Binding by γ-Carboxyglutamic Acid-containing Plasma Proteins and Peptides

Abstract The membrane-binding characteristics of a number of modified vitamin K-dependent proteins and peptides showed a general pattern of structural requirements. The amino-terminal peptides from human prothrombin (residues 1-41 and 1-44, 60:40) bovine factor X (residues 1-44), and bovine factor IX (residues 1-42), showed a general requirement for a free amino-terminal group, an intact disulfide, and the tyrosine homologous to Tyr44 of factor X for membrane binding. Consequently, the peptide from factor IX did not bind to membranes. Any of several modifications of the amino terminus, except reaction with trinitrobenzenesulfonic acid, abolished membrane binding by the factor X and prothrombin peptides. Calcium, but not magnesium, protected the amino terminus from chemical modification. The requirement for a free amino terminus was also shown to be true for intact prothrombin fragment 1, factor X, and factor IX. Although aggregation of the peptide-vesicle complexes greatly complicated accurate estimation of equilibrium binding constants, results with the factor X peptide indicated an affinity that was not greatly different from that of the parent protein. The most striking difference shown by the peptides was a requirement for about 10 times as much calcium as the parent proteins. In a manner similar to the parent proteins, the prothrombin and factor X peptides showed a large calcium-dependent quenching of tryptophan fluorescence. This fluorescence quenching in the peptides also required about 10 times the calcium needed by the parent proteins. Thus, the 1-45 region of the vitamin K-dependent proteins contained most of the membrane-binding structure but lacked component(s) needed for high affinity calcium binding. Protein S that was modified by thrombin cleavage at Arg52 and Arg70 showed approximately the same behavior as the amino-terminal 45-residue peptides. That is, it bound to membranes with overall affinity that was similar to native protein S but required high calcium concentrations. These results suggested that the second disulfide loop of protein S (Cys47-Cys72) and prothrombin (Cys48-Cys61) were involved in high affinity calcium binding. Since factor X lacks a homologous disulfide loop, an alternative structure must serve a similar function. A striking property of protein S was dissociation from membranes by high calcium. While this property was shared by all the vitamin K-dependent proteins, protein S showed this most dramatically and supported protein-membrane binding by calcium bridging.

Evidence for a common metal ion-dependent transition in the 4-carboxyglutamic acid domains of several vitamin K-dependent proteins

The murine monoclonal antibody H-11 binds a conserved epitope found at the amino terminal of the vitamin K-dependent blood proteins prothrombin, factors VII and X, and protein C. The sequence of polypeptide recognized by antibody H-11 contains 2 residues of gamma-carboxyglutamic acid, and binding of the antibody is inhibited by divalent metal ions. By using a solid-phase immunoassay with 125I-labeled antibody and immobilized vitamin K-dependent protein, binding of the antibody to the vitamin K-dependent proteins was inhibited by increasing concentrations of calcium, manganese, and magnesium ion. The transition midpoints for antibody binding were in the millimolar concentration range and were different for each metal ion. In general, the transition midpoints were lowest for manganese ion, intermediate for calcium ion, and highest for magnesium ion. Antibody H-11 bound specifically to a synthetic peptide corresponding to residues 1-12 of human prothrombin that was synthesized as the gamma-carboxyglutamic acid-containing derivative. Binding of the antibody to the peptide was not inhibited by calcium ion. These data suggest that inhibition of antibody H-11 binding by divalent metal ions is not due simply to neutralization of negative charge by Ca2+. This transition which is conserved in vitamin K-dependent proteins containing the H-11 antigenic site is likely due to a structural transition of the amino-terminal polypeptide possibly from a random (accessible) to ordered (inaccessible) structure.

A new model to describe extrinsic protein binding to phospholipid membranes of varying composition: application to human coagulation proteins.

We introduce here a new model to describe the binding of extrinsic membrane proteins to acidic lipid membranes. In this view, macroscopic binding affinity is determined by two processes: nonspecific adsorption of protein to the membrane surface and association of acidic lipids with specific sites on the bound protein. We apply this model here to compare the binding of human prothrombin and factor X/Xa to phosphatidylglycerol (PG)- and phosphatidylserine (PS)-containing small unilamellar vesicles measured via relative light scattering. This comparison was undertaken because model membranes containing PS are much more effective in supporting thrombin formation than are membranes containing PG. Analysis of binding isotherms in terms of a traditional membrane binding model gave apparent dissociation constants systematically varying from 0.1 to 10 microM over a range of 8-65 mol% negatively charged phospholipid. With our new description of membrane binding, the dependence of binding data on the acidic lipid surface concentration revealed that only two or three acidic lipid molecules were associated with each surface-bound factor X/Xa or prothrombin molecule. Assuming four independent and equivalent acidic lipid binding sites per protein, it was possible to adjust the values of only the nonspecific adsorption equilibrium constant and the equilibrium constant describing binding of each species of acidic lipid to individual sites on the protein and thereby obtain a good simulation of log-linear binding isotherms for the full range of acidic lipid surface concentrations. The protein-associated binding sites had a greater affinity for PS than for PG; i.e., a lower surface concentration of PS was required to fill the binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformational changes in human prothrombin as detected by antibody populations.

The amino-terminal peptides of human prothrombin corresponding to residues 1-51 and 52-156 have been isolated from a thrombin digest of prothrombin fragment 1. The products of digestion were purified by means of barium citrate and ammonium sulfate precipitations, followed by gel filtration and hydroxyapatite chromatographies. They were identified by their molecular sizes as well as their amino acid compositions. Peptides 1-51 (F1A) and 52-156 (F1B) were used as affinity ligands for the isolation of antibody populations from antisera that were elicited against human prothrombin or prothrombin fragment 1. These antibody populations displayed restricted specificity for the respective ligands as shown by competitive radioimmunoassays. They were used to study the conformational changes in prothrombin and fragment 1. The F1A-specific antibody populations detected a conformational change which is stabilized by calcium ions and which has a transition midpoint at approximately 0.2 mM calcium ion concentration. The F1B-specific antibody populations identified a different conformational change which is destabilized by calcium ions and which has a transition midpoint at approximately 0.5 mM calcium.

Antithrombin III-dependent anti-prothrombinase activity of heparin and heparin fragments

Heparin and heparin fragments in the molecular mass range 1,700-20,000 Da were examined for their ability to accelerate the antithrombin III (AT III)-dependent inhibition of human factor Xa and the prothrombin converting complex (prothrombinase) during human prothrombin activation. The prothrombinase reaction was modeled by a 3-parameter 2-exponential equation to determine the initial rate of prothrombin activation and the pseudo-first order rate constants of inhibition of prothrombinase and in situ generated thrombin activity. The catalytic specific activities of the heparins increased with increasing molecular size for both the inhibition of prothrombinase and factor Xa. A 10-fold increase over the entire Mr range was found. In contrast to results obtained by others (Ellis, V., Scully, M. F., and Kakkar, V. V. (1986) Biochem. J. 233, 161-165; Barrowcliffe, T. W., Havercroft, S. J., Kemball-Cook, G., and Lindahl, U. (1987) Biochem. J. 243, 31-37), all the heparins showed a 5-fold higher rate of inhibition of factor Xa when compared with the inhibition of prothrombinase, indicating that the factor Va-mediated protection of factor Xa from inhibition by AT III/heparin is independent of the molecular size of the heparin. Our original approach has also revealed a hitherto unrecognized phenomenon, namely, in addition to the accelerating effect of the heparins on the rate of formation of the inactive AT III-factor Xa complex, heparins with Mr greater than 4,500 reduce the initial rate of thrombin generation in the presence of AT III in a concentration-dependent way. We hypothesize that the formation of the dissociable ternary AT III-heparin-factor Xa complex results in a (partial) loss of factor Xa activity towards its natural substrate prothrombin.

Substitution of valine for glycine-558 in the congenital dysthrombin thrombin Quick II alters primary substrate specificity

Thrombin Quick II is one of two dysfunctional forms of thrombin derived from the previously described congenital dysprothrombin prothrombin Quick. Thrombin Quick II does not clot fibrinogen, hydrolyze p-nitroanilide substrates of thrombin, or bind N2-[5-(dimethylamino)naphthalene-1-sulfonyl]arginine N,N-(3-ethyl-1,5-pentanediyl)amide, a high-affinity competitive inhibitor of thrombin. To determine the structural alteration in thrombin Quick II, the reduced, carboxymethylated protein was hydrolyzed by a lysyl endopeptidase. A peptide not present in a parallel thrombin hydrolysate was identified by reverse-phase chromatography. The peptide was purified by rechromatography and subjected to Edman degradation which showed that Gly-558 of human prothrombin had been replaced by Val. This corresponds to a point mutation of the Gly codon GGC to GUC. This Gly residue, which is highly conserved in the chymotrypsin family of serine proteases, forms part of the substrate binding pocket for bulky aromatic and basic side chains in chymotrypsin and trypsin, respectively. However, in porcine elastase 1, the corresponding residue is threonine. Consistent with the identified structural alteration, thrombin Quick II incorporates [3H]diisopropyl fluorophosphate stoichiometrically and hydrolyzes the elastase substrate succinyl-Ala-Ala-Pro-Leu-p-nitroanilide with a relative kcat/KM of 0.14 when compared to thrombin. This results from a 3-fold increase in KM and a 2.5-fold decrease in kcat for thrombin Quick II when compared to thrombin acting on the same substrate. These results and those of other investigators studying mutant trypsins support the conclusion that the catalytic activity of serine proteases is very sensitive to structural alterations in the primary substrate binding pocket.

4769320 Immunoassay means and methods useful in human native prothrombin and human abnormal prothorombin determinations: Bruce Furie, Barbara C Furie, Rita Blanchard assigned to New England Medical Center Hospitals Inc

4769320 Immunoassay means and methods useful in human native prothrombin and human abnormal prothorombin determinations: Bruce Furie, Barbara C Furie, Rita Blanchard assigned to New England Medical Center Hospitals Inc

Interaction of human γ-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 γ-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 absorb 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.

A hydrophobic site in human prothrombin present in a calcium-stabilized conformer

Human prothrombin and prothrombin fragment 1 were demonstrated to bind to Phenyl-TSK columns in the presence of 5.0 mM calcium ions but not in the presence of either magnesium ions or manganese ions. The calcium-dependent interaction of prothrombin fragment 1 is markedly reduced upon oxidation of approximately one mole of tryptophan per mole of protein. The ability of prothrombin fragment 1 to inhibit prothrombin activation by factor Xa in the presence of calcium ions and phospholipid is also markedly reduced by reaction with N-bromosuccinimide. These results provide the first demonstration of a calcium-specific site in prothrombin outside of the "GLA domain".