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PubMed1995

Differences in the interactions of lupus anticoagulant IgG with human prothrombin and bovine prothrombin.

Lupus anticoagulant (LA) IgGs have been reported to inhibit more effectively and consistently the Xa/Va/phospholipid complex-catalyzed activation of human prothrombin that the Xa/Va/phospholipid complex-catalyzed activation of bovine prothrombin. This led us to carry out studies to determine whether the ability to inhibit the activation of prothrombin of LA IgGs, separated from the plasma of 15 patients by protein A affinity chromatography, could be related to the ability of the LA IgGs to bind to prothrombin under various experimental conditions. Of 14 LA IgG preparations tested all prolonged to a variable but substantial extent the dilute Russell's viper venom time (dRVVT) of human plasma but only minimally prolonged the dRVVT of bovine plasma. In a purified prothrombin activation system with a rate limiting concentration of phospholipid, all 15 LA IgG preparations inhibited the activation of human prothrombin with the majority showing > 50% of inhibition. In contrast, only one LA IgG markedly inhibited (> 50%) the activation of bovine prothrombin and five others moderately inhibited (25-40%) the activation of bovine prothrombin. Nevertheless, the majority of LA IgG preparations bound to immobilized bovine prothrombin on a Western blot and also to immobilized bovine prothrombin on a microtiter well. In an ELISA in which phosphatidylserine (PS) was immobilized on microtiter wells, bovine prothrombin supported the binding of 10 of 15 LA IgG preparations to PS. However, the extent of binding was lower than that observed with human prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

Prothrombin Salakta: an abnormal prothrombin characterized by a defect in the active site of thrombin.

An abnormal prothrombin has been detected in a 17 yr-old female originating from Tunisia. There was no history of excessive bleeding. Prothrombin time and activated partial thromboplastin time were moderately prolonged. Prothrombin activity was 15-18% when measured using either the classical one-stage and two-stage assays, or assays with Echis carinatus venom or staphylocoagulase, whereas prothrombin antigen was 100%. In keeping with current nomenclature practices, the abnormal molecule has been designated prothrombin Salakta. The electrophoretic behaviour and calcium binding properties of the abnormal prothrombin did not differ significantly from normal, as assessed by crossed immunoelectrophoresis. Prothrombin Salakta was isolated by chromatography on DEAE-Sephadex and Dextran sulphate sepharose. Electrophoretic migration of purified prothrombin Salakta on SDS polyacrylamide gels or alkaline disc gels was normal. Upon activation by either bovine factor Xa or Echis carinatus venom, thrombin activity produced by prothrombin Salakta was only 15% of normal, even when the incubation period was prolonged for 24 hours. The pattern of factor Xa-catalyzed proteolysis of prothrombin Salakta, investigated by SDS polyacrylamide gel electrophoresis, was found to be normal. These results indicated that prothrombin Salakta was characterized by a defective thrombin enzymatic activity. Thrombin Salakta was therefore isolated by heparin-sepharose chromatography. Affinity for heparin and molecular weight of thrombin Salakta were found to be normal. Biological activity of thrombin Salakta, determined by clotting assay, was 535 u/mg versus 3 200 u/mg for normal thrombin. Amidolytic activity of thrombin Salakta parallelled its clotting activity, suggesting that the defect resides either in the catalytic site or in the residues adjacent to the catalytic site and implicated as contact residues, rather than in the fibrinogen recognition site.

Adolescent↗

PubMed2001

G20210A prothrombin gene polymorphism and prothrombin activity in subjects with or without angiographically documented coronary artery disease.

BACKGROUND: G20210A prothrombin mutation has been associated with high prothrombin levels and an increased risk of venous thrombosis. The role of this common polymorphism, as well as that of prothrombin levels, in determining the risk of arterial disease is still somewhat controversial. METHODS AND RESULTS: We determined the prevalence of the G20210A mutation and prothrombin activity in 660 individuals, of whom 436 had angiographically documented severe coronary artery disease (CAD patients) and 224 had normal coronary angiography (CAD-free control subjects). Heterozygosity for the 20210A allele was found in 5.3% of the CAD patients versus 3.1% of the CAD-free subjects (P=0.21). Similarly, no statistically significant difference was found between CAD patients with or without previous myocardial infarction (4.5% versus 5.3%, respectively; P=0.73). The genotype-phenotype correlation study showed a significant influence of the 20210A allele on prothrombin activity, with higher levels in carriers compared with noncarriers (153.2% versus 122.2%, respectively; P<0.001). Prothrombin activity was significantly higher in CAD patients than in CAD-free subjects (132.8% versus 123.3%, respectively; P<0.005). By multiple logistic regression, prothrombin activity in the upper tertile of the control distribution was significantly associated with CAD compared with prothrombin activity in the lower tertile (adjusted odds ratio 1.86, 95% CI 1.01 to 3.4). CONCLUSIONS: In a population with a clear-cut definition of the phenotype, the G20210A prothrombin mutation was not significantly associated, per se, with either angiographically documented CAD or myocardial infarction, whereas it significantly influenced prothrombin activity. In our population, high prothrombin activity itself was independently associated with CAD but not with the presence or absence of previous myocardial infarction.

Aged↗

PubMed1991

Modulation of human prothrombin activation on phospholipid vesicles and platelets using monoclonal antibodies to prothrombin fragment 2.

Prothrombin contains two kringle domains that are removed during activation to the blood clotting enzyme alpha-thrombin. By analogy with other kringle-containing proteins the prothrombin kringles may play a role in the protein-protein interactions necessary for prothrombin activation. Four monoclonal antibodies to prothrombin kringle 2 have been produced against human prothrombin, and a fifth monoclonal antibody was produced against a synthetic peptide consisting of amino acid residues 216-231 of kringle 2. Each antibody was tested for its ability to block prothrombin activation by factor Xa. In the presence of phosphatidylcholine/phosphatidylserine vesicles and factor Va, two of the antibodies, alpha HII-3 and alpha HII-4, inhibited prothrombin activation at a 90 and 50% level, respectively. Two other monoclonal antibodies (alpha HII-6 and alpha HII-7) and the antipeptide antibody (alpha HII-5) had no effect on prothrombin activation. When factor Xa was the catalyst alone, antibody alpha HII-3 lost the ability to inhibit prothrombin activation whereas antibody alpha HII-4 again partially inhibited the reaction. When human platelets were the reaction surface, the patterns of inhibition by the anti-fragment 2 antibodies were identical to that observed with phospholipid vesicles. These data suggest a role for prothrombin fragment 2 in activation, possibly by mediating the interaction of substrate prothrombin with factor Xa or factor Va on the phospholipid surface.

Amino Acid Sequence↗

PubMed1986

Prothrombin fragment 1 X 2 X 3, a major product of prothrombin activation in human plasma.

The conversion of the blood coagulation zymogen prothrombin to thrombin is associated with the production of several cleavage intermediates and products. In contrast to earlier studies of prothrombin cleavage in chemically defined systems, the current investigation examines the fragmentation of human prothrombin in normal plasma. Radiolabeled prothrombin was added to platelet-poor relipidated normal human plasma, and clotting was initiated with the addition of Ca(II) and kaolin. Analysis of the radiolabeled prothrombin cleavage products by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and beta-mercaptoethanol identified a heretofore unobserved product of prothrombin activation with an apparent molecular weight of 45,000. This product was identified as fragment 1 X 2 X 3, the NH2-terminal 286 amino acids of prothrombin. The product was isolated from a prothrombin digest by immunoaffinity chromatography using anti-prothrombin:Ca(II) antibodies and by preparative gel electrophoresis. Its amino-terminal sequence is identical to that of prothrombin. Digestion of this product with either Factor Xa or thrombin yields, at a minimum, fragment 1 X 2 and fragment 1. Amino-terminal sequence analysis of the products obtained by digestion with Factor Xa of the unknown activation product indicated 3 amino acid residues at each cycle consistent with the presence of fragment 1, fragment 2, and fragment 3. To unambiguously identify the COOH-terminal amino acid sequence of the product, its factor Xa digestion products were separated by reverse-phase high performance liquid chromatography. Edman degradation of one peptide revealed the complete sequence of fragment 3. On this basis, we identify the Mr 45,000 polypeptide as fragment 1 X 2 X 3 and indicate that it is a prominent product of prothrombin conversion to thrombin when activation occurs in plasma.

Amino Acid Sequence↗

PubMed1998

Comparison of plasma prothrombin and factor VII and urine prothrombin F1 concentrations in patients on long-term warfarin therapy and those in the initial phase.

Control of warfarin anticoagulation during the initial phase of therapy is difficult and empirically based. Plasma and urine samples were obtained from normal controls, patients under stable anticoagulation, and patients in the initial phase of anticoagulation. Total plasma prothrombin, des-carboxy (non-adsorbable with barium chloride) prothrombin, and native (total minus non-adsorbable) prothrombin were quantitated using Echis carinatus venom activation. Functional plasma factor VII (VII) was measured using a one-stage clotting assay. Total and des-carboxy urine prothrombin F1 (F1) were measured by ELISA. All urine F1 in normals and both anticoagulated groups was adsorbed by barium chloride. Plasma des-carboxy prothrombin concentration was similar for the two anticoagulated groups and did not correlate with 1/INR. Native prothrombin correlated with 1/INR in both the stable (r = 0.76) and initial phase (r = 0.74) groups. For any given INR, the subjects on stable anticoagulation had lower native prothrombin concentrations than the initial phase patients. Functional factor VII concentration also correlated significantly with 1/INR in both the stable (r = 0.64) and initial phase (r = 0.76) patients. Unlike native prothrombin, VII concentrations did not vary between the two cohorts for any given INR. Previous studies indicate that native prothrombin is a superior predictor of both hemorrhagic and thromboembolic complications during warfarin therapy. Our findings indicate that VII, and not prothrombin, may be the predominant factor monitored by the INR. This further supports the need to reevaluate the usefulness of the INR in the monitoring of warfarin therapy during the initial phase.

Anticoagulants↗

PubMed1993

Comparison of native prothrombin antigen with the prothrombin time for monitoring oral anticoagulant prophylaxis.

BACKGROUND: Oral anticoagulation is most frequently monitored using the prothrombin time, but an alternative approach is measurement of native, fully carboxylated, prothrombin antigen (NPA). We have correlated results of the prothrombin time and NPA with development of venous thrombosis or bleeding complications in a clinical trial of warfarin prophylaxis following total hip arthroplasty to determine the potential value of NPA measurement for monitoring oral anticoagulation. METHODS AND RESULTS: Patients in one arm of a prospective, randomized trial received warfarin prophylactically beginning 10 to 14 days before total hip arthroplasty in a dose adjusted to prolong the international normalized ratio (INR) to 1.5 on the day of surgery and 2.5 after surgery. NPA was measured by ELISA, and the prothrombin time was measured using rabbit brain thromboplastin. Samples were tested from 97 patients, and data from 81 patients who had adequate venography were analyzed to correlate test results with occurrence of thrombosis. The prothrombin time and INR were less sensitive than NPA to the lowest intensities of anticoagulation, with the prothrombin time index increasing from 1.0 to 1.3 and the INR increasing from 1.0 to 2.0, whereas the NPA concentration decreased fourfold, from 200 to 50 micrograms/mL. There was little correlation between either the prothrombin time index or the INR and the development of thrombosis, whereas NPA concentrations were significantly higher on the day of surgery and on postoperative days 1, 3, 5, and 7 in patients who developed venous thrombosis. Higher concentrations of NPA were associated with an increased risk of venous thrombosis, but there was no relation between thrombosis and the prothrombin time index or INR. There was no significant correlation between surgical blood loss and prothrombin time index, INR, or NPA concentration. However, patients who received the largest number of transfusions on the day of surgery had significantly lower NPA concentrations than patients who required no transfusion. CONCLUSIONS: These results indicate that the NPA concentration more accurately reflects the antithrombotic effect of warfarin than does prothrombin time and may be superior in monitoring prophylactic oral anticoagulation.

Administration, Oral↗

PubMed1999

CA-1 method, a novel assay for quantification of normal prothrombin using a Ca2+ -dependent prothrombin activator, carinactivase-1.

We established a novel prothrombin assay, designated CA-1 method, for quantification of normal prothrombin in application of a Ca2+ -dependent prothrombin activator, carinactivase-1 (CA-1), found in the venom of Echis carinatus leucogaster. On microplate, thrombin converted from normal prothrombin in plasma sample by CA-1 cleaves a thrombin specific chromogenic substrate, t-butoxy-Val-Pro-Arg-p-nitroanilide and liberates p-nitroaniline. Then, the normal prothrombin level is decided by measuring the velocity of p-nitroaniline liberation. Normal prothrombin levels in plasma from warfarin-treated individuals were highly correlated with coagulant activities assayed by both prothrombin time and thrombotest. CA-1 method is not only a rapid and highly sensitive chromogenic microplate assay for quantification of normal prothrombin in the range of 10-200 ng/100 microl in plasma samples but also suitable for analyses of many samples in a short time. In addition, normal prothrombin levels obtained by CA-1 method are not inhibited by EDTA and heparin, which reduce prothrombin time and thrombotest activities. CA-1 method is a novel assay for monitoring coagulant activity in warfarin-treated individuals.

Aniline Compounds↗

PubMed1998

Prothrombin antigen levels in symptomatic and asymptomatic carriers of the 20210A prothrombin variant.

A recently discovered variant in the prothrombin gene (20210A) has been found in approximately 5-10% of patients with venous thromboembolism. It has been shown that patients with this variant present with high levels of prothrombin in plasma and this is maintained to be the most likely mechanism by which the risk of thrombosis is increased. We have evaluated prothrombin antigen levels in 50 carriers of the 20210A allele and compared with non-carriers. 327 subjects were subdivided according to deficiency status and previous thrombosis. 30 symptomatic and 20 asymptomatic carriers had increased mean prothrombin levels as compared to symptomatic (n=178) or asymptomatic (n=99) non-carriers. The percentage of subjects with prothrombin levels above cut-off values of 1.15u/ml or 1.30u/ml was significantly higher in carriers of the prothrombin variant as compared to non-carriers, regardless of a previous thrombosis. However, among non-carriers the percentage of those with prothrombin levels above cut-off values was significantly higher in the group of symptomatic as compared to asymptomatic individuals. In conclusion, increased prothrombin antigen levels, as detected by a specific ELISA, were found among 50 symptomatic and asymptomatic carriers of the 20210A prothrombin variant as well as among a large group of symptomatic non-carriers. The data are in agreement with those found by using functional tests for the determination of prothrombin levels in these patients.

Adult↗

PubMed2001

Complexes of anti-prothrombin antibodies and prothrombin cause lupus anticoagulant activity by competing with the binding of clotting factors for catalytic phospholipid surfaces.

We investigated the mechanism by which anti-prothrombin antibodies cause lupus anticoagulant (LAC) activity. Addition of affinity-purified anti-prothrombin antibodies from LAC-positive plasma samples (alpha-FII-LAC+) to normal plasma induced LAC activity. Upon increasing the phospholipid concentration, LAC activity was neutralized. Addition of purified alpha-FII-LAC+ to normal plasma strongly inhibited factor Xa formation. No inhibition was measured when alpha-FII-LAC+ were added to prothrombin-deficient plasma or when purified anti-prothrombin antibodies from LAC-negative plasma samples (alpha-FII-LAC-) were added. When a combination of prothrombin and alpha-FII-LAC+ was added to the purified clotting complex, a strong inhibition of factor Xa and IIa formation was seen. The alpha-FII-LAC+ alone or a combination of prothrombin and alpha-FII-LAC- did not show inhibition. Ellipsometry studies showed that, in the presence of alpha-FII-LAC+, the affinity of prothrombin for a phospholipid surface increased dramatically, whereas a much lower increase was observed with alpha-FII-LAC-. Our results show that complexes of prothrombin and anti-prothrombin antibodies with LAC activity inhibit both prothrombinase and tenase. The antibodies increase the affinity of prothrombin for the phospholipid surface, thereby competing with clotting factors for the available catalytic phospholipid surface, a mechanism similar to that of anti-beta2-glycoprotein I antibodies.

Antiphospholipid Syndrome↗

PubMed2001

Determination of plasma prothrombin level by Ca2+-dependent prothrombin activator (CA-1) during warfarin anticoagulation.

BACKGROUND AND AIM OF THE STUDY: The carinactivase-1 (CA-1) test is a new method for monitoring plasma prothrombin levels during warfarin anticoagulation therapy. METHODS: A total of 192 patients were allocated to two groups. Group A patients (n = 42) were controls (no warfarin); group B patients (n = 150) received warfarin. A Ca2+-ion and Boc-Val-Pro-Arg-pNA (a chromogenic substrate for thrombin) were added to 10-fold diluted plasma, after which prothrombin was activated with CA-1. Prothrombin levels were determined by measuring the extent of p-nitoroaniline liberation. RESULTS: The mean prothrombin level was 112.8 +/- 20.0 microg/ml in group A (Gaussian distribution), and 53.3 +/- 19.6 microg/ml in group B. In group B, correlations were found between the CA-1 test and prothrombin levels measured by prothrombin time (PT; r = 0.61, p <0. 01), PT-INR (r = 0.61, p <0.01), Thrombotest (TT; r = 0.57, p <0.01) and Hepaplastin test (HPT; r = 0.69, p <0.01). CONCLUSION: The CA-1 test represents a viable method of monitoring the coagulation system. CA-1 recognized the Gla-domain of prothrombin, and activated prothrombin. The CA-1 test required only 10 microl of diluted blood plasma, and took approximately 30 min to complete. The CA-1 test also measures prothrombin levels, correlates excellently with other tests for coagulation, and compares well with currently available methods for determining the efficacy of warfarin.

Aged↗

PubMed1987

Homo- and heterodimer formation with prothrombin and prothrombin fragment 1 in the presence of calcium ions.

The purpose of the current study is to present further evidence for prothrombin self-association as assessed by chemical crosslinking. When the self-association (evaluated by covalent crosslinking with dithiobis(succinimidylpropionate) of prothrombin or fragment 1 was evaluated at the same molar concentration of protein, similar rates of dimer formation were observed for either protein. When prothrombin and fragment 1 were incubated together with the crosslinking reagent and calcium ions, a heterodimer consisting of prothrombin and fragment 1 was observed in addition to prothrombin dimer and fragment 1 dimer. Similar experiments with prethrombin 1 showed neither significant self-association nor effect on prothrombin self-association. Comparison of the formation of prothrombin fragment 1 heterodimer formation with the effect of fragment 1 on prothrombin activation by factor Xa suggests that the anticoagulant activity of fragment 1 is not solely a result of the formation of a heterodimer between prothrombin and fragment 1.

Animals↗

PubMed1991

Fourier transform infrared spectroscopic study of Ca2+ and membrane-induced secondary structural changes in bovine prothrombin and prothrombin fragment 1.

Fourier transform infrared (FTIR) spectroscopy was used to monitor secondary structural changes associated with binding of bovine prothrombin and prothrombin fragment 1 to acidic lipid membranes. Prothrombin and prothrombin fragment 1 were examined under four different conditions: in the presence of (a) Na2EDTA, (b) 5 mM CaCl2, and in the presence of CaCl2 plus membranes containing 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC) in combination with either (c) bovine brain phosphatidyl-serine (bovPS) or (d) 1,2-dioleoyl-phosphatidylglycerol (DOPG). The widely reported Ca(2+)-induced conformational change in bovine prothrombin fragment 1 was properly detected by our procedures, although Ca(2+)-induced changes in whole prothrombin spectra were too small to be reliably interpreted. Binding of prothrombin in the presence of Ca2+ to procoagulant POPC/bovPS small unilamellar vesicles produced an increase in ordered secondary structures (2% and 3% increases in alpha-helix and beta-sheet, respectively) and a decrease of random structure (5%) as revealed by spectral analysis on both the original and Fourier-self-deconvolved data and by difference spectroscopy with the undeconvolved spectra. Binding to POPC/DOPG membranes, which are less active as procoagulant membranes, produced no detectable changes in secondary structure. In addition, no change in prothrombin fragment 1 secondary structure was detectable upon binding to either POPC/bovPS or POPC/DOPG membranes. This indicates that a membrane-induced conformational change occurs in prothrombin in the nonmembrane-binding portion of the molecule, part of which is activated to form thrombin, rather than in the membrane-binding fragment 1 region. The possible significance of this conformational change is discussed in terms of differences between the procoagulant activities of different acidic lipid membranes.

Animals↗

PubMed1995

The second kringle domain of prothrombin promotes factor Va-mediated prothrombin activation by prothrombinase.

The incorporation of factor Xa into the prothrombinase complex, factor Xa-factor Va-phospholipid-Ca(II), results in an approximately 10(5)-fold higher rate of substrate activation than that of the enzyme alone. To examine the role of the prothrombin kringle domains in the interaction with prothrombinase we have employed site-directed mutagenesis to produce prothrombin species that lack either the first kringle domain, PT/delta K1, or the second kringle domain, PT/delta K2. Previously, we have shown that these proteins are fully carboxylated and that they bind to phospholipid vesicles. In this investigation we demonstrate that cleavage at Arg271-Thr272 and Arg320-Ile321 peptide bonds occurs upon activation with prothrombinase to yield normal thrombin from both PT/delta K1 and PT/delta K2. In the absence of factor Va, the Km(app) for the activation of PT/delta K1, PT/delta K2, or plasma-derived prothrombin by factor Xa-phospholipid-Ca(II) are equivalent. The Km(app) for the activation of PT/delta K2 by prothrombinase is approximately 4-5-fold higher than that obtained for plasma-derived prothrombin or PT/delta K1. These data demonstrate that the prothrombin kringle domains do not contribute significantly to the binding affinity of the substrate-enzyme interaction. In the absence of factor Va, equivalent kcat values were obtained for all of the prothrombin species when they were activated by factor Xa-Ca(II)-phospholipid. In contrast, a 7-fold lower kcat value was obtained for the activation of PT/delta K2 by prothrombinase as compared with that obtained for plasma prothrombin or PT/delta K1. Collectively, these data suggest that determinants within the second prothrombin kringle domain interact with factor Va to elicit a significant acceleration in the catalytic rate of substrate turnover. Indeed, in contrast to plasma-derived prothrombin, no direct binding of PT/delta K2 to factor Va to form the PT/delta K2-factor Va complex could be demonstrated by 90 degrees light scattering.

Animals↗

PubMed1990

Chemical modification of gamma-carboxyglutamic acid residues in prothrombin elicits a conformation similar to that of abnormal (des-gamma-carboxy)prothrombin.

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.

1-Carboxyglutamic Acid↗

PubMed1990

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.

Adult↗

PubMed2000

Linkage analysis demonstrates that the prothrombin G20210A mutation jointly influences plasma prothrombin levels and risk of thrombosis.

Association studies suggest that the G20210A mutation (G to A substitution at nucleotide position 20210) in the prothrombin gene (PT) is associated with increased plasma prothrombin activity and with increased risk for venous thromboembolism. To test directly for linkage between this PT variant and plasma prothrombin activity we performed a family-based study. The G20210A genotypes and plasma prothrombin activity levels were determined in 435 individuals belonging to 22 extended Spanish families. The sample was composed of 388 homozygous (G/G) normal individuals and 43 heterozygote (G/A) and 4 homozygote (A/A) carriers for the G20210A mutation. The results of variance-component linkage analysis yielded a highly significant lod score of 3.6 (P = 2.4 x 10(-5)) between this mutation and a quantitative trait locus (QTL) that influences prothrombin activity. Importantly, a conditional linkage analysis that simultaneously accounted for association with the G20210A variant completely eliminated the linkage signal, which indicates that this mutation affects the function of the prothrombin gene. Additionally, a bivariate linkage analysis of plasma prothrombin activity and thrombosis significantly improved the linkage signal for prothrombin activity (lod score = 4.7; P = 1.5 x 10(-6)) and provided strong evidence that this QTL has a pleiotropic effect on the risk of thrombosis (lod score = 2.43; P =.0004). These results represent the first direct genetic evidence that a QTL in the PT gene influences prothrombin activity levels and susceptibility to thrombosis and strongly support the conclusion that G20210A is a functional polymorphism. (Blood. 2000;95:2780-2785)

Adult↗

PubMed2001

Polymorphisms in the prothrombin gene and their association with plasma prothrombin levels.

To find genetic causes of high plasma prothrombin levels, an established prothrombotic risk factor. we searched for sequence variations in the prothrombin gene. We selected subjects with the 20210-GG genotype (since the 20210-A allele is already known to be associated with high levels) and elevated prothrombin levels (> or = 130 U/dl) from the Leiden Thrombophilia Study (LETS). No mutations were found in the 1 kb promoter region of the prothrombin gene in seven individuals with an isolated high prothrombin level. Comparison of the allelic frequencies of four different polymorphisms in the prothrombin gene in healthy volunteers and in the control subjects among the selected LETS individuals indicated a higher frequency of the 19911-G allele in the latter group (allele frequency 52 vs. 78%, respectively). Homozygous carriers of the 19911-G allele had 8 U/dl higher plasma prothrombin levels than 19911-AA carriers. This difference in prothrombin levels did not affect the thrombotic risk in 20210-GG carriers. In heterozygous 20210-A carriers the odds ratio increased from 1.6 (95% CI: 0.6-4.3) in subjects with 19911-A to 4.7 (1.6-14.0) in subjects with 19911-G on the other prothrombin allele.

Adult↗