RESUMO
Routine coagulation assays are performed with platelet-poor plasma obtained after centrifugation of whole citrated blood. Usually clinical laboratories centrifuge blood from 2,000 to 2,500 g for 15-30 minutes. Thirty two blood samples routinely submitted to coagulation tests, were assayed for the prothrombin time, activated partial thromboplastin time, and fibrinogen level, in order to compare results obtained using 2 types of centrifugation : centrifugation at 2,500 g for 15 minutes and rapid centrifugation on StatSpin Express 2 at 4,440 g for 2 minutes. A good correlation was observed for the prothrombin time, activated partial thromboplastin time, and fibrinogen levels being respectively 1,009, 0,908 and 1. We concluded that rapid centrifugation at 4,440 g for 2 minutes does not modify results and contributes, by decreasing duration of the pre-analytical variable to reduce the completion time of these tests.
Assuntos
Testes de Coagulação Sanguínea/métodos , Centrifugação/métodos , Coleta de Amostras Sanguíneas/métodos , Fibrinogênio/metabolismo , Hemostasia , Humanos , Tempo de Tromboplastina Parcial , Tempo de Protrombina , Valores de ReferênciaRESUMO
BACKGROUND: Budd-Chiari syndrome (BCS) is associated with parenchymal changes leading to major architecture remodelling. In order to gain further insight into the pathogenesis of BCS, we investigated expression of a set of genes involved in the course of chronic liver diseases. METHODS: Quantitative expression of 35 selected genes involved in extracellular matrix regulation, growth factors, and angiogenesis was investigated in 13 cases of BCS and compared with 10 normal livers and 13 cirrhosis cases by real time reverse transcription-polymerase chain reaction. Differential gene expression was considered significant for genes showing at least a twofold variation, with p < 0.05. RESULTS: Expression of 14 genes was significantly increased in BCS versus normal liver, with the highest increase in superior cervical ganglion 10 (SCG10) gene. BCS cases were classified according to their evolution and morphological pattern as either acute or chronic in six and seven cases, respectively. Unsupervised hierarchical clustering of acute and chronic BCS cases on the basis of similarity in gene expression pattern led to distinction between the two groups. Expression of three genes was significantly different in acute versus chronic BCS (increase in matrix metalloproteinase 7 and SCG10, decrease in thrombospondin-1 for chronic BCS). Seventeen and 10 genes, mainly involved in extracellular matrix and vascular remodelling, were significantly deregulated in acute BCS versus normal liver and cirrhosis, respectively. CONCLUSION: These results show that BCS cases display a specific gene expression profile that is different from that of normal liver and cirrhosis; the molecular configuration of BCS can be readily distinguished by its evolution and morphological pattern.
Assuntos
Síndrome de Budd-Chiari/genética , Doença Aguda , Adulto , Indutores da Angiogênese/metabolismo , Síndrome de Budd-Chiari/metabolismo , Síndrome de Budd-Chiari/patologia , Doença Crônica , Progressão da Doença , Matriz Extracelular/metabolismo , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Substâncias de Crescimento/genética , Substâncias de Crescimento/metabolismo , Humanos , Fígado/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
We have recently identified (Akhavan S et al., Thromb Haemost 2000; 84: 989-97) a patient with a mild bleeding diathesis associated to an homozygous mutation in the thrombin B chain (Gly25Ser, chymotrypsinogen numbering, i.e. position 330 in human prothrombin numbering). Transient transfection of wild-type prothrombin (FII-WT) and mutant prothrombin (designated FII-G25(330)S) cDNA in COS-7 cells showed a mild reduction (50%) in FII-G25(330)S production. Recombinant proteins, stably expressed in Chinese hamster ovary cells, were isolated and activated by Taipan snake or Echis carinatus venoms. We show that the G25(330)S mutation results in a decrease in the rate of prothrombin proteolytic activation. The mutation also significantly decreases (i) the catalytic activity of thrombin with a 9-fold reduction in catalytic efficiency of the mutant toward S-2238; (ii) the interaction with benzamidine; (iii) the rate of inhibition by TLCK and antithrombin; and (iv) the rate of hydrolysis of macromolecular substrates (fibrinogen, protein C). In contrast, exosite I does not appear to be affected by the molecular defect. These results, together with molecular modeling and dynamics, indicate that Gly25(330) is important for proper expression and probably proper folding of prothrombin, and also plays a critical role in both the alignment of the catalytic triad and the flexibility of one of the activation segments of prothrombin.
Assuntos
Glicina/química , Trombina/química , Animais , Sítios de Ligação , Células CHO , Células COS , Catálise , Cricetinae , Análise Mutacional de DNA , DNA Complementar/metabolismo , Fibrina/química , Fibrinogênio/química , Homozigoto , Humanos , Hidrólise , Cinética , Modelos Moleculares , Mutação , Conformação Proteica , Estrutura Terciária de Proteína , Protrombina/química , Protrombina/genética , Proteínas Recombinantes/química , Venenos de Serpentes , Serpentes , Trombina/antagonistas & inibidores , Fatores de Tempo , TransfecçãoRESUMO
According to a recent hypothesis, venous thrombosis results from the concurrence of several factors. This hypothesis was assessed in patients with portal or hepatic venous thrombosis by simultaneously investigating most of the currently identified prothrombotic disorders, local precipitating factors, and other risk factors such as oral contraceptive use. Patients with a tumorous obstruction and patients with cirrhosis with portal vein thrombosis were excluded. The prothrombotic disorders that were investigated included classical and occult myeloproliferative disorders; antiphospholipid syndrome; protein C; protein S and antithrombin deficiency; factor V Leiden; factor II; and methylene-tetrahydrofolate-reductase gene mutations. We found 1 or several prothrombotic disorders and a local precipitating factor in 26 and 10 of the 36 patients with portal vein thrombosis, respectively; and in 28 and none of the 32 patients with hepatic vein thrombosis, respectively. We found a combination of prothrombotic disorders in 5 and 9 patients with portal and hepatic vein thrombosis, respectively, whereas such a combination is expected in less than 1% of asymptomatic subjects. Of the 10 patients with a local precipitating factor, 8 had a prothrombotic disorder. Of the 13 patients who use oral contraceptives, 10 had a prothrombotic disorder. We conclude that portal or hepatic venous thrombosis should be regarded as an index for 1 or several prothrombotic disorders, whether or not local precipitating factors or oral contraceptive use are found. Concurrence of prothrombotic disorders is more common than expected. Extensive investigation of prothrombotic disorders and anticoagulation should be considered in patients with portal or hepatic venous thrombosis.
Assuntos
Síndrome de Budd-Chiari/etiologia , Veia Porta , Trombose Venosa/etiologia , Síndrome de Budd-Chiari/complicações , Síndrome de Budd-Chiari/genética , Fator V/metabolismo , Feminino , Humanos , Masculino , Gravidez , Prognóstico , Proteína C/metabolismo , Fatores de Risco , Trombose Venosa/complicações , Trombose Venosa/genéticaRESUMO
We have previously identified and characterized a potent and specific thrombin inhibitor, isolated from Bothrops jararaca, named bothrojaracin. Bothrojaracin interacts with the two positively charged recognition sites of thrombin referred to as exosite 1 and exosite 2, whereas it does not interact with the thrombin active site. Consequently, bothrojaracin inhibits thrombin-induced fibrinogen to fibrin conversion and platelet activation, without inhibition of thrombin-catalyzed cleavage of small synthetic substrates. In the present study, we show that bothrojaracin exerts an anticoagulant effect in plasma, illustrated by the prolongation of the aPTT. Using purified proteins, we observed that the anticoagulant effect of bothrojaracin was not only due to the inhibition of fibrinogen to fibrin conversion, but in addition to the inhibition of factor V activation by thrombin. Bothrojaracin decreased the rate of thrombin-catalyzed proteolysis of factor V and concurrently the generation of factor Va cofactor activity measured in a prothrombinase assay. We compared the effect of bothrojaracin with that of ligands binding specifically exosite 1 (hirudin C-terminal peptide SH54-65) or exosite 2 (heparin, prothrombin fragment 2). SH54-65 delayed thrombin catalyzed factor V activation whereas heparin or prothrombin fragment 2 did not. The thrombin derivatives beta- and gamma-thrombin, which are defective in their exosite 1, but present with a normally exposed exosite 2, had a reduced capacity to activate factor V, which was not further impaired by the exosite 2 ligands, bothrojaracin, heparin or prothrombin fragment 2. Altogether, our results provide further insight into the anticoagulant effect of bothrojaracin showing that it is a potent inhibitor of the feedback activation of factor V by thrombin, and thus of the up-regulation of its own production by thrombin. Inhibition of thrombin-catalyzed factor V activation by bothrojaracin is mainly mediated through the interaction of the inhibitor with thrombin exosite 1, whereas contribution of the interaction with exosite 2 does not appear to play a direct role in factor V recognition by thrombin.
Assuntos
Anticoagulantes/farmacologia , Venenos de Crotalídeos/farmacologia , Fator V/efeitos dos fármacos , Trombina/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Catálise/efeitos dos fármacos , Retroalimentação/efeitos dos fármacos , Hirudinas/farmacologia , Humanos , Tempo de Tromboplastina Parcial , Fragmentos de Peptídeos/farmacologia , Trombina/farmacologiaRESUMO
In the present study we examined if, among other mechanisms, the abnormal exposure of phosphatidylserine at the surface of sickle red blood cells (RBCs) contributes to the hypercoagulability which characterizes homozygous sickle cell disease (SCD). The question was addressed by comparison of the procoagulant properties of RBCs from subjects with various phenotypes (SS, SC and AS) that differ in clinical presentation. As previously reported, SS-RBCs accelerated the prothrombin activation by factor Xa, by decreasing the Km of the reaction compared to normal RBCs. SC-RBCs and AS-RBCs also promoted prothrombin activation although their procoagulant properties were milder compared to SS-RBCs. A significant increase of the thrombin-antithrombin complexes was observed in SS subjects. Prothrombin fragment 1+2 (F1+2) was elevated in half of the SS subjects, but the difference with controls did not reach significance. Elevated levels of thrombin-antithrombin complexes were observed in a number of SC (4/11) and AS (3/12) subjects, but the difference with controls was not significant. A significant correlation was observed between the plasma levels of thrombin-antithrombin complexes in the subjects with SS, AS and AA phenotypes, and the procoagulant properties of RBCs. Our results strongly suggest that the procoagulant properties which characterize SS-RBCs also affect SC-RBCs and AS-RBCs, and that exposure of phosphatidylserine by RBCs contributes to the hypercoagulable state observed in SCD.
Assuntos
Anemia Falciforme/sangue , Coagulação Sanguínea/fisiologia , Eritrócitos/metabolismo , Protrombina/fisiologia , Adulto , Antitrombina III/metabolismo , Fator Xa/metabolismo , Feminino , Hemoglobina Fetal/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeo Hidrolases/metabolismoRESUMO
In Caucasians, the R506Q mutation in exon 10 of the factor V gene (FV Leiden) confers an increased risk of thromboembolism. We have scanned this region of the gene for possible mutations in 450 subjects from populations at risk for sickle cell disease (SCD). The R506Q mutation was absent in subjects from sub-Saharan Africa, whereas its allelic frequency was 2.5% in the West Indies. Only one other substitution with no functional consequences in vitro (R485K) was found (32.4% allelic frequency) in sub-Saharan Africa. Thus, we found no mutations in exon 10 of the FV gene constituting an additional risk factor for thrombosis in SCD in sub-Saharan Africa. This suggests that the putative selective advantage conferred by R506Q does not exist in these populations, unless R485K has functional consequences in vivo. If further suggests that R506Q in American Africans is of Caucasian origin. Our data are the first to document ethnic variations in the frequency of the R485K polymorphism.
Assuntos
Anemia Falciforme/genética , Fator V/genética , Polimorfismo Genético , Trombose/genética , África Subsaariana , África do Norte , Anemia Falciforme/complicações , Europa (Continente) , Éxons , Humanos , Risco , Trombose/complicações , Índias OcidentaisRESUMO
It has recently been proved that, in vitro, red blood cells (RBCs) from patients with homozygous beta-thalassemia behave as procoagulant cells. The procoagulant activity of beta-thalassemia RBCs might be the result of an increased exposure of procoagulant phospholipids (i.e. phosphatidylserine) in the outer leaflet of the membrane. In order to test this hypothesis, we compared the catalytic properties of RBCs of patients with beta-thalassemia and homozygous sickle cell disease (SS-RBCs) with that of controls. The catalytic parameters (Km, kcat) of prothrombin activation by factor Xa were determined both in the absence and in the presence of RBCs. The turn-over number (kcat) of the reaction was not modified by normal, SS- or beta-thalassemia RBCs. The Km was lower in the presence of normal RBCs (mean value: 9.1 microM) than in the absence of cells (26 microM). The Km measured in the presence of either SS-RBCs (mean value: 1.6 microM) or beta-thalassemia RBCs (mean value: 1.5 microM) was significantly lower compared to normal RBCs (p < 0.001). No significant difference was observed between SS-RBCs and beta-thalassemia RBCs. Annexin V, a protein with high affinity and specificity for anionic phospholipids, inhibited the procoagulant activity of both SS-RBCs and beta-thalassemia RBCs, in a dose-dependent manner. More than 95% inhibition was achieved at nanomolar concentrations of annexin V. These results indicate that the procoagulant activity of both beta-thalassemia RBCs and SS-RBCs may be fully ascribed to an abnormal exposure of phosphatidylserine at the outer surface of the red cells.
Assuntos
Anemia Falciforme/sangue , Eritrócitos/patologia , Talassemia beta/sangue , Adolescente , Adulto , Anemia Falciforme/genética , Coagulação Sanguínea , Criança , Pré-Escolar , Feminino , Homozigoto , Humanos , Lactente , Masculino , Talassemia beta/genéticaRESUMO
An enzyme-linked immunosorbent assay (ELISA) was developed for measuring human hypocarboxyprothrombin, a protein induced by vitamin K antagonists (PIV KA-II). A specific monoclonal antibody (P1-2-B9) was prepared and used for coating a microELISA plate, and revelation proceeded with a rabbit polyclonal anti-human prothrombin antibody-peroxidase conjugate. This assay allowed the measurement of PIVKA-II at concentrations ranging from 2 to 200 ng/ml, with an intraassay coefficient of variation of less than 5.2% and an interassay coefficient of variation of less than 7.4%. This assay permits the direct evaluation of PIVKA-II in citrated plasma as well as in serum. The concentration of PIVKA-II is expressed in nanograms per milliliter. It offers specific measurement of PIVKA-II without any cross-reactivity from native prothrombin: the specificity for PIVKA-II respective to prothrombin is > 10(5). No reactivity was observed with other vitamin K-dependent proteins, whether fully active or decarboxylated. Complementary studies have demonstrated that the monoclonal antibody used was preferentially directed to 3 to 6 Gla hypocarboxylated prothrombin. PIVKA-II concentration was below 2.4 ng/ml in normal individuals (n = 59). In 61 patients given dicoumarol for more than 90 days (receiving a stable therapy), the measured concentrations of PIVKA-II ranged from 750 to 13,400 ng/ml. Combined with the assay of alpha-fetoprotein (AFP), measurement of PIVKA-II in patients with liver diseases introduces a complementary exploration for hepatocellular carcinoma (HCC). In a study in 59 patients with HCC, the assay sensitivity was 49.1% for PIVKA-II, 47.5% for AFP, and 71% for both markers combined.
Assuntos
Biomarcadores , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/diagnóstico , Precursores de Proteínas/análise , Protrombina/análise , Anticorpos Monoclonais , Carcinoma Hepatocelular/sangue , Dicumarol/uso terapêutico , Ensaio de Imunoadsorção Enzimática/estatística & dados numéricos , Humanos , Neoplasias Hepáticas/sangue , Valores de Referência , Sensibilidade e Especificidade , alfa-Fetoproteínas/análiseRESUMO
A factor X molecular variant was identified in a 55-year-old woman at a routine preoperative coagulation screening. Plasma factor X antigen was normal, whereas factor X activity was decreased when factor X was activated by either the extrinsic pathway (21%), the intrinsic pathway (21%) or the factor X activator from Russell viper venom, RVV-X (26%). Factor XMarseille was isolated from plasma by immunoaffinity chromatography and compared with normal factor X purified by the same method. Activation of factor XMarseille by factor IXa or by RVV-X in a purified system showed that the rate of cleavage was decreased, whereas once produced, factor XaMarseille had a normal catalytic efficiency for either the peptide substrate S-2765 (D-Arg-Gly-Arg-NH-Np) or prothrombin. The rate of inhibition of factor XaMarseille by antithrombin III was also normal. Defective proteolysis of factor XMarseille by factor IXa or by RVV-X was the consequence of a threefold decrease in the kcat for the activation of factor XMarseille while the Km of RVV-X or factor IXa for factor X was normal. We have determined the molecular basis of the defect in the factor XMarseille gene by amplification of all eight exons, single-strand conformational polymorphism analysis of the amplified exons and subsequent sequence analysis. The patient was homozygous for a T-->C mutation in exon VIII, resulting in the substitution of Ser334 by proline. From comparison of three-dimensional models of various serine proteases, it appears that Ser334 is located within a surface-exposed variable region of factor X. This observation suggests that the Ser334-->Pro mutation either is responsible for a misalignment of the active sites of specific factor X activators in close proximity to the cleavage site, or that the Ser-->Pro mutation alters the spatial orientation of the cleavage site by nonlocal modifications of factor X structure.
Assuntos
Fator X/metabolismo , Mutação Puntual , Prolina/genética , Serina/genética , Animais , Sequência de Bases , Cálcio/metabolismo , Bovinos , DNA , Primers do DNA , Fator Va/metabolismo , Fator X/genética , Deficiência do Fator X/sangue , Fator Xa/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fosfolipídeos/metabolismo , Protrombina/metabolismo , CoelhosRESUMO
A model of thrombin interaction with distinct substrates or ligands has been derived from the crystallographic studies of thrombin-inhibitors complexes, and buttressed by functional studies with mutant thrombins, thrombin proteolytic derivatives or antibodies against thrombin. The unique specificity of thrombin for its substrates and ligands may be ascribed to multiple interactions with both the active site cleft and exosite(s) distinct from the active site. Two prominent insertion loops around Trp 50 and Trp 148 project over the active site cleft and play an important role in the substrates selection. Several substrates (fibrinogen, thrombin receptor, heparin cofactor II) or ligands (thrombomodulin, glycoprotein Ib) interact with a large exosite located on the surface of the loop segment 65-76, mainly constituted of basic amino acids, designated anion binding exosite 1. Interaction with these various macromolecules appears to involve a limited number of residues within the large exosite 1. It is conceivable that exosite 1 contains distinct subsites, although most of them may overlap. A second basic exosite (anion binding exosite 2) is located close to the carboxy-terminal B chain helix. Exosite 2 interacts with heparin, the chondroitin sulfate moiety of thrombomodulin and prothrombin activation fragment 2. Interaction of ligands with either exosite 1 or exosite 2 leads to conformational changes of the thrombin molecule, that may be important determinants of thrombin specificity. Whether exosite 2 cooperates with exosite 1 for thrombin interaction with fibrin(ogen) or the thrombin receptor remains to be determined.
Assuntos
Trombina/metabolismo , Sequência de Aminoácidos , Animais , Arginina , Autoanticorpos/imunologia , Sítios de Ligação , Fatores de Coagulação Sanguínea/química , Fatores de Coagulação Sanguínea/metabolismo , Fibrinogênio/química , Fibrinogênio/metabolismo , Humanos , Isoanticorpos/imunologia , Lisina , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutação Puntual , Inibidores de Proteases/metabolismo , Ligação Proteica , Receptores de Trombina/metabolismo , Especificidade por Substrato , Trombina/química , Trombina/genética , Trombina/imunologiaRESUMO
The objective of this study was to look for HBV precore mutations in three patients with chronic active hepatitis B who developed HBV-DNA-positive/HBeAg-negative reactivation after HBe seroconversion induced by interferon therapy. Direct sequencing of polymerase chain reaction products was performed on serum collected before and after HBe seroconversion. In two patients precore sequence showed only wild-type HBV before and after interferon therapy. In one patient, precore sequence showed only wild-type HBV before interferon therapy and a mixed infection by wild-type HBV and precore mutant viruses (1858 and 1896 nucleotide mutations) after treatment. The presence of HBeAg/anti-HBe immune complexes was found after HBe seroconversion in all cases. Our results suggest that: 1) precore mutations are not always found in patients with chronic hepatitis B who develop HBV DNA-positive/HBeAg-negative reactivation; and 2) HBeAg negativity, despite the presence of wild-type HBV, might be due to HBeAg/anti-HBe immune complexes. We speculate that the production of these immune complexes may be favored by interferon therapy.
Assuntos
Antígenos E da Hepatite B/sangue , Vírus da Hepatite B/genética , Hepatite B/virologia , Interferons/uso terapêutico , Mutação/genética , Adulto , Sequência de Bases , Doença Crônica , Hepatite B/imunologia , Hepatite B/terapia , Vírus da Hepatite B/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência MolecularRESUMO
The objective of this study was to evaluate the role of hepatitis B virus (HBV) precore mutations in patients with anti-HBe-positive chronic hepatitis B with or without previous known HBe antigen (HBeAg) viremic phase, and to assess the potential implication of precore mutants in HBeAg-negative reactivation after loss of HBeAg. Nineteen patients were studied: 7 had a previous HBeAg-positive phase and had spontaneous or therapeutically induced loss of HBeAg (group A); 12 had no previous HBeAg-positive phase (group B). Direct sequencing of PCR products was performed on serum collected during the anti-HBe-positive phase in the two groups. In group A, precore sequencing showed that 5 patients were infected by wild-type virus, 1 patient was infected with a precore mutant, and 1 patient was found to be infected by a mixture of wild-type and precore mutant viruses. In group B, precore sequencing showed that only 1 patient was infected with wild-type virus and that 11 were infected with precore mutants. In a few patients, the presence of HBeAg within immune complexes may explain HBeAg negativity. In conclusion, our results show that, in patients with anti-HBe-positive chronic hepatitis B: (1) precore mutations creating a stop codon are more frequently found in those without known previous HBeAg positivity; (2) after loss of HBeAg, the patients who have anti-HBe-positive reactivation are infected by wild-type virus, which suggests that reactivation is not related to precore mutations; (3) HBeAg negativity may be caused by immune complexes formation.
Assuntos
Anticorpos Anti-Hepatite B/análise , Antígenos E da Hepatite B/imunologia , Vírus da Hepatite B/genética , Hepatite B/imunologia , Hepatite B/virologia , Mutação , Adulto , Idoso , Sequência de Bases , Doença Crônica , Vírus da Hepatite B/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/genética , Ativação ViralRESUMO
Limited proteolysis of human alpha-thrombin by various proteases has been efficiently used to demonstrate the importance of two insertion loops located on the surface of this molecule. In the present study, we demonstrate that two-chain urokinase (tcu-PA) specifically cleaves the B chain of alpha-thrombin giving rise to a transient derivative, consisting of two non-covalently linked subunits. Although the thrombin derivative conserves its activity towards the synthetic substrate S-2238 (Km = 8.4 microM and kcat = 145 s-1 versus respectively 4.5 microM and 149 s-1 for alpha-thrombin), most of its coagulant activity is lost (140 NIH u/mg versus 3000 NIH u/mg) and its ability to activate platelets is considerably reduced (threshold for full platelet aggregation 2.5 nM versus 0.25 nM). The thrombin fragments were separated by HPLC and after reduction and S-carboxyamidemethylation were digested with a lysylendopeptidase; the resulting peptides were separated by HPLC and sequenced. One fragment corresponded to B chain fragment 1-73 and the second to B chain fragment 74-259 covalently linked to the A chain, indicating that tcu-PA cleaves selectively the peptide bond Arg 73-Asn 74 in the B chain. The proteolytic derivative obtained, designated beta u-thrombin, is therefore identical to the transient proteolytic derivative, beta 1-thrombin, produced by trypsin. Prolonged incubation with tcu-PA resulted in further conversion in a derivative analogous to gamma t-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Trombina/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Arginina , Asparagina , Humanos , Hidrólise , Dados de Sequência Molecular , Trombina/química , Trombina/efeitos dos fármacosRESUMO
BACKGROUND: An important marker for hepatocellular carcinoma is the presence of des-gamma-carboxy (abnormal) prothrombin. However, the molecular basis for the reduced carboxylation of prothrombin is unknown. METHODS: Two groups of patients were defined according to the absence (Group I, n = 7) or presence (Group II, n = 8) of des-gamma-carboxy prothrombin. The enzymatic activity of gamma-carboxylase and the total microsomal prothrombin concentration were determined in all tumors. The kinetic parameters for the synthetic peptide Phe-Leu-Glu-Glu-Leu (FLEEL) were measured in eight tumors. The gamma-carboxylase mRNA expression was evaluated by Northern blot analysis in 12 of 15 tumors. In addition, the total vitamin K content (K1, K1 epoxide, and menaquinones 4-10) in 10 tumors was investigated by high performance liquid chromatography. RESULTS: Concentrations of menaquinones 4-10 were normal in the nontumorous part of the liver but significantly decreased (P = 0.02) in all the tumors (Groups I and II). This decrease was more severe in Group II (P = 0.02). The tumors in Group I had normal or increased gamma-carboxylase activity and increased mRNA expression (P < 0.02) as compared with their nontumorous counterparts. The tumors in Group II were heterogeneous. Five tumors displayed low gamma-carboxylase activity, associated with low mRNA expression in two, whereas two others had high gamma-carboxylase activity and mRNA expression. The concentration of FLEEL at half-maximal velocity was normal in all the tumors examined (Groups I and II), and a relation was found between the level of expression of gamma-carboxylase and the maximal velocity for FLEEL carboxylation in the tumors in Group II (r = 0.98; P < 0.01). The microsomal content of normal prothrombin was within normal limits in all tumors (Groups I and II). CONCLUSIONS: Tumor vitamin K content has a critical role in the synthesis of des-gamma-carboxy prothrombin. Furthermore, the gamma-carboxylase defect, which is observed in some secreting tumors, is the result of the defective gene expression of a normal enzyme and not the consequence of the presence of a competitive inhibitor. It is possible that a 75% reduction in gamma-carboxylase gene expression could take a part in the secretion of des-gamma-carboxy prothrombin, but this mechanism is not predominant.
Assuntos
Biomarcadores , Carbono-Carbono Ligases , Carcinoma Hepatocelular/metabolismo , Ligases/metabolismo , Neoplasias Hepáticas/metabolismo , Precursores de Proteínas/metabolismo , Protrombina/metabolismo , Vitamina K/metabolismo , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Fator V/análise , Regulação Neoplásica da Expressão Gênica , Humanos , Ligases/análise , Ligases/genética , Fígado/enzimologia , Fígado/metabolismo , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Microssomos Hepáticos/enzimologia , Precursores de Proteínas/análise , Precursores de Proteínas/genética , Protrombina/análise , Protrombina/genética , RNA/análise , RNA/genética , RNA Neoplásico/análise , RNA Neoplásico/genética , Vitamina K/análogos & derivados , Vitamina K/análise , Vitamina K 1/análogos & derivados , Vitamina K 1/análise , Vitamina K 2/análogos & derivados , alfa-Fetoproteínas/análiseRESUMO
The prevalence of hepatitis C virus (HCV) infection was studied prospectively in pregnant women in France and their children by detection of anti-HCV with second-generation ELISA (ELISA2). In ELISA2-positive women, anti-HCV was detected with second- and third-generation RIBA (RIBA2 and RIBA3) and serum HCV RNA was detected with PCR. Among 670 women, anti-HIV1-negative, 26 (3.9%) were positive with ELISA2. RIBA2 was positive in 13 and HCV RNA was found in 10. Ten ELISA2-positive women had a further evaluation with assessment of HCV infection in their children. Among the 10 children born to the index pregnancy, only one was positive with ELISA2 and RIBA2 but negative with RIBA3 and PCR; the nine other children were ELISA2, RIBA2, RIBA3, and PCR negative. All 26 siblings (2-16 years old), of whom 14 were born to PCR-positive mothers, were ELISA2 and RIBA2 negative. We conclude that among anti-HIV1-negative pregnant women with normal serum ALT levels, the prevalence of HCV infection is relatively high but the risk for mother-to-infant transmission of HCV seems to be low.
Assuntos
Soronegatividade para HIV , Hepatite C/epidemiologia , Hepatite C/transmissão , Troca Materno-Fetal , Complicações Infecciosas na Gravidez/epidemiologia , Adulto , Ensaio de Imunoadsorção Enzimática , Feminino , França/epidemiologia , Hepacivirus/isolamento & purificação , Anticorpos Anti-Hepatite/sangue , Humanos , Immunoblotting , Reação em Cadeia da Polimerase , Gravidez , Prevalência , Estudos Prospectivos , RNA Viral/sangue , Viremia/epidemiologia , Viremia/transmissãoRESUMO
Fibrinogen contains at least two independent sites having demonstrable affinity for alpha-thrombin. One of these two sites, located in the fibrin E domain, binds to structures within the anion-binding exosite of alpha-thrombin. Taking advantage of its solubility, we have used late-fibrin(ogen) fragment E in competition experiments to examine its effect on alpha-thrombin specificity. We show that fragment E modulates alpha-thrombin enzymic activity towards small synthetic substrates, suggesting that fibrin-thrombin interaction might induce subtle changes in the conformation near the catalytic center of the enzyme. In addition, fragment E behaved as a competitive inhibitor of alpha-thrombin-catalyzed fibrinopeptide-A cleavage (Ki = 5.2 +/- 1.3 microM), indicating that alpha-thrombin interaction with the fibrin moiety of fibrinogen makes a major contribution to the efficacy of fibrinogen hydrolysis. Fragment E inhibited alpha-thrombin-induced serotonin release by platelets (concentration required to obtain 50% inhibition, IC50 = 10 microM) and alpha-thrombin binding to GPIb. Fragment E competitively inhibited alpha-thrombin binding to thrombomodulin (Ki = 18.3 +/- 0.8 microM) but did not inhibit protein-C activation in the absence of thrombomodulin. The data are consistent with the proposal that fibrin, platelet GPIb and thrombomodulin bind to overlapping, but probably non-identical sites, while protein C binds to an independent site on alpha-thrombin.
Assuntos
Produtos de Degradação da Fibrina e do Fibrinogênio/farmacologia , Trombina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Dados de Sequência Molecular , Agregação Plaquetária/efeitos dos fármacos , Proteína C/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Trombina , Trombina/farmacologiaRESUMO
A percentage ranging from 60 to 80% of hepatocarcinomas are associated with increased levels of alphafetoprotein (AFP). In the three years following surgical resection there was a 80% possibility of recidivation. The aims of the present study were: a) to evaluate the significance of preoperative AFP assay as a prognostic index of recidivation; b) to evaluate the importance of repeated assays during the postoperative period in order to ensure an early diagnosis of recidivation. Between 1982 and 1989, 62 patients underwent surgery for hepatocarcinoma. Thirty-one patients who had undergone so-called curative surgery were periodically controlled for a period varying between 6 and 55 months, and were included in the present study. The remaining 32 patients were excluded for the following reasons: palliative surgery, postoperative death, postoperative complications unrelated to tumoral recidivation. In all cases AFP assay was carried out preoperatively, one month after surgery, and then every six months. Recidivation was always confirmed on the basis of tomodensitometric and arteriographic data. Before surgery out of a group of 30 patients, 11 showed normal AFP levels (below 20 mg/ml), while 19 had levels between 49 and 7350 mg/ml. Twenty-three patients (74%) reported one case of recidivation during the period between 6 and 40 months. Among the 11 patients who had showed normal preoperative AFP levels, 5 had a recidivation between 12 and 36 months, and 3 of these showed high AFP levels. In 18 out of the 19 patients (90%) with high preoperative AFP levels recidivation was diagnosed between 4 and 40 months following surgery; 4 of these were not associated with a rise in AFP.(ABSTRACT TRUNCATED AT 250 WORDS)