RESUMO
Phytoestrogens are popular alternatives to estrogen therapy however their effects on hemostasis in post-menopausal women are unknown. The aim of this study was to determine the effect of the phytoestrogens, genistein, daidzein and equol on the expression of key genes from the hemostatic system in human hepatocyte cell models and to determine the role of estrogen receptors in mediating any response seen. HepG2 cells and Hep89 cells (expressing estrogen receptor alpha (ERα)) were incubated for 24 h with 50 nM 17ß-estradiol, genistein, daidzein or equol. Tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), Factor VII, fibrinogen γ, protein C and protein S mRNA expression were determined using TaqMan PCR. Genistein and equol increased tPA and PAI-1 expression in Hep89 cells with fold changes greater than those observed for estradiol. In HepG2 cells (which do not express ERα), PAI-1 and tPA expression were unchanged. Increased expression of Factor VII was observed in phytoestrogen treated Hep89 cells but not in similarly treated HepG2s. Prothrombin gene expression was increased in equol and daidzein treated HepG2 cells in the absence of the classical estrogen receptors. These data suggest that phytoestrogens can regulate the expression of coagulation and fibrinolytic genes in a human hepatocyte cell line; an effect which is augmented by ERα.
Assuntos
Coagulação Sanguínea/genética , Equol/farmacologia , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Hemostáticos/farmacologia , Isoflavonas/farmacologia , Fitoestrógenos/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Fator VII/genética , Fator VII/metabolismo , Fibrina/metabolismo , Expressão Gênica/efeitos dos fármacos , Genisteína/farmacologia , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Extratos Vegetais/farmacologia , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Protrombina/genética , Protrombina/metabolismo , RNA Mensageiro/metabolismo , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
Recent data has shown that hormone therapy (HT) increases the risk of cardiovascular and thromboembolic disease, particularly in users of oral HT. Phytoestrogens are popular alternatives to oestrogen therapy; however, their effects on cardiovascular risk are unknown. We investigated the effect of the phytoestrogen, genistein on the expression of genes and proteins from the haemostatic system in the liver in an ovariectomised rat model. Fifty-nine virgin female Sprague-Dawley rats were fed with soy-free chow supplemented with 17ß estradiol (E2) (daily uptake 0.19 or 0.75 mg/kg body weight), or genistein (daily uptake 6 or 60 mg/kg body weight), for three months and compared to soy-free control rats. Gene expression of prothrombin, factor VII, fibrinogen alpha and fibrinogen beta was increased with E2 and genistein compared to the soy-free control group (p<0.001). Genistein increased factor VII significantly more than E2 (p<0.005). Plasminogen mRNA was increased in both treatment groups compared to the soy-free control, with genistein expression significantly higher than E2 (p<0.001). Tissue plasminogen inhibitor (tPA), plasminogen activator inhibitor-1 (PAI-1) and C-reactive protein (CRP) expression were also increased in both groups relative the soy-free control. Results of protein analysis largely concurred with those of the mRNA. Oestrogen receptor ß (ERß) was undetected while oestrogen receptor α (ERα) was detected in each sample group. Genistein can increase the expression of coagulation and fibrinolytic genes. This effect was similar and in some cases higher than 17ß estradiol. These results suggest that genistein may not be neutral with respect to the haemostatic system.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Terapia de Reposição de Estrogênios , Genisteína/farmacologia , Fígado/efeitos dos fármacos , Ovariectomia , Fitoestrógenos/farmacologia , Animais , Coagulação Sanguínea/genética , Western Blotting , Proteína C-Reativa/genética , Proteína C-Reativa/metabolismo , Ensaio de Imunoadsorção Enzimática , Fator VII/genética , Fator VII/metabolismo , Feminino , Fibrinogênio/genética , Fibrinogênio/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Plasminogênio/genética , Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Protrombina/genética , Protrombina/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
Inherited factor VII (FVII) deficiency is considered to be a haemorrhagic disease. Nonetheless, some patients paradoxically present with venous thrombosis. We assessed whether there was a link between phenotype and genotype in seven patients with inherited FVII deficiency and thrombosis (eleven venous thrombotic events). For each patient (FVII:C < 50%), clinical data were collected, aetiological assessment of risk factors for thrombosis was investigated, and direct sequencing of the nine exons and promoter of the FVII gene (F7) was performed. We present the second series ever published on FVII patients with thrombosis. In nine of the eleven thrombotic events, there was at least one classical triggering risk factor; clinical (n = 4), familial antecedent (n = 2), or biological, defined by phospholipid-binding antibodies or elevated FVIII:C levels (n = 7). In contrast to a previous series, only two events occurred after surgery, performed both with and without replacement therapy. The thrombotic event remained unexplained in one young patient, highlighting the lack of 'protection' against venous thrombosis by low FVII:C levels. Genetic mutations were found to be heterogeneous. Among the seven F7 sequence alterations identified in the present study, only two (p.Ala354Val and p.Arg364Gln) have previously been reported in FVII-deficient patients presenting with venous thrombosis. Our genetic analyses of the F7 mutations in these patients show the complexity of FVII deficiency associated with thrombosis. These data justify a holistic, clinical and biological approach for patients with these specific symptoms. This series also strongly suggest that mild FVII deficiency should not prevent physicians from using antithrombotic prophylaxis in FVII-deficient patients.
Assuntos
Antígenos/metabolismo , Deficiência do Fator VII/complicações , Deficiência do Fator VII/genética , Fator VII/genética , Trombose Venosa/complicações , Adolescente , Adulto , Idoso , Fatores de Coagulação Sanguínea/efeitos adversos , Coagulantes/efeitos adversos , Análise Mutacional de DNA , Fator VII/metabolismo , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Fenótipo , Fatores de Risco , Trombofilia/genética , Trombose Venosa/genética , Trombose Venosa/prevenção & controleRESUMO
FVII is a vitamin K dependent serine protease that plays a key role in extrinsic coagulation pathway. In this paper, we report the full-length cDNA sequences of rhesus monkey FVII. The full-length cDNA has 2424 bp, and predicts an open reading frame of 1416 bp corresponding to 472 amino acids. The deduced protein sequence of rhesus monkey FVII indicates the functional domains including signal peptide, Gla domain, two EGF domains, and catalytic domain. Rhesus monkey FVII is highly homologous to human FVII with amino acid identity of 91.0%. Comparison of three-dimensional protein structure shows high conservation between them. The important functional sites such as the N-terminal gamma-carboxyglutamic acids of the Gla domain, the Ca(2+) binding region of the EGF I domain, the TF binding region, the active site binding cleft, and the macromolecular substrate binding exosite of trypsin domain are all well conserved in FVII of rhesus monkey. Prothrombin time test shows rhesus monkey FVII has a similar clotting time with that of human. This study of rhesus monkey FVII might be helpful for understanding the function compatibility of human and rhesus monkey FVII, which is beneficial for the study of xenotransplantation.
Assuntos
Fator VII/química , Fator VII/genética , Macaca mulatta , Sequência de Aminoácidos , Animais , Sequência de Bases , Coagulação Sanguínea/genética , Clonagem Molecular , DNA Complementar , Fator VII/metabolismo , Humanos , Macaca mulatta/genética , Macaca mulatta/metabolismo , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
The aim of the present study was to investigate the effect of long-term diet and very long chain n-3 fatty acids (VLC n-3) intervention on plasma coagulation factor VII (FVII), choline-containing phospholipids (PC) and triglycerides (TG), especially related to the R353Q polymorphism of the FVII gene. The present investigation included 219 subjects from the Diet and Omega-3 Intervention Trial on atherosclerosis (DOIT), a 2x2 factorial designed study in elderly men with long-standing hypercholesterolemia. The subjects were randomly allocated to receive placebo capsules (corn oil) (control), placebo capsules and dietary advice ("Mediterranean type" diet), VLC n-3 capsules, or VLC n-3 capsules and dietary advice combined. The R353Q genotype and the levels of FVIIc, FVIIag, FVIIa, PC, and TG at baseline and after 6 months were determined. Diet intervention was followed by a significant reduction of 5.1% in the levels of FVIIag and 2.4 mU/ml in FVIIa (95% CI -7.4, -2.9, and -3.8, -1.1, respectively) (both p<0.001) compared to the no diet group, independent of genotype. No effects of diet intervention on FVIIc, PC or TG were observed. After VLC n-3 supplementation the TG levels were significantly reduced compared to placebo (p=0.01), whereas all FVII levels and PC remained unchanged. Dietary advice towards a "Mediterranean type" diet, but not VLC n-3 supplementation, was shown to reduce the levels of FVIIag and FVIIa after 6 months, independent of genotype. The results indicate the dietary advice to be more favourable in reducing this risk factor for CVD as compared to specific VLC n-3 supplementation.
Assuntos
Suplementos Nutricionais , Fator VII/efeitos dos fármacos , Fator VII/genética , Ácidos Graxos Ômega-3/farmacologia , Fosfolipídeos/sangue , Polimorfismo de Nucleotídeo Único , Idoso , Arteriosclerose/tratamento farmacológico , Doença das Coronárias/prevenção & controle , Fator VII/análise , Ácidos Graxos Ômega-3/administração & dosagem , Humanos , Masculino , Mutação de Sentido Incorreto , Fosfatidilcolinas/sangue , Risco , Triglicerídeos/sangueRESUMO
Factor VII is a vitamin K-dependent zymogen that plays a key role in the initiation of the extrinsic pathway. A severe factor VII deficiency was identified in a 45-year old male whose plasma factor VII antigen was less than 60 ng/ml and expressed 5.2% of normal factor VII activity. DNA sequence analysis of the patient's factor VII gene showed a thymidine to guanine transversion at nucleotide 10968 in exon VIII that results in a novel amino acid substitution of His348 to Gln. The patient was homozygous for this mutation, whereas some of his family members were heterozygous. Both wild type and mutant factor VII were transiently expressed in COS-1 cells. The level of secreted mutant factor VII antigen was only 11.0% of the level of wild type factor VII. In CHO cells stably transfected with the mutant factor VII, only 37.3% of the total labeled FVII was secreted into the conditioned media and the remainder was retained inside the cells. These data suggest this mutation leads to factor VII deficiency due to the impaired secretion of the molecule.
Assuntos
Sítios de Ligação/genética , Deficiência do Fator VII/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Testes de Coagulação Sanguínea , Células CHO , Células COS , Cricetinae , DNA Complementar , Fator VII/biossíntese , Fator VII/química , Fator VII/genética , Deficiência do Fator VII/sangue , Saúde da Família , Expressão Gênica , Hematemese/genética , Hemorragia , Homozigoto , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Radioisótopos de Enxofre , TransfecçãoRESUMO
Clotting factors synthesized by monocytes and macrophages may initiate coagulation reactions during inflammation. Functional vitamin K-dependent coagulation factors have been found to be associated with human monocytes/macrophages, but there are no reports identifying mRNA coding for vitamin K-dependent proteins in these cells. In the present studies, factor VII mRNA was found in total RNA extracted from freshly isolated human alveolar macrophages using hybridization with a complementary DNA probe. On the other hand, vitamin K-dependent carboxylase activity which is required for postribosomal modification of the protein, was not detectable in the macrophages before or after culture, and human blood mononuclear leukocytes also lacked this enzyme activity. Control human and rat hepatoma cells exhibited high levels of carboxylase activity within the same experiments. Using sensitive kinetic assays, no increase in factor VII activity was detected during culture of alveolar macrophages under conditions promoting 1.78 +/- .24 (n = 8) fold increases of tissue factor activity. These findings with freshly isolated cells demonstrate that alveolar macrophages synthesize factor VII mRNA in vivo. However, the mRNA was found in the absence of evidence for gamma-carboxylase activity or processing of the factor into a functional clotting enzyme. The results imply that functional expression of any synthesized coagulation factor VII in alveolar macrophages may be limited or prevented due to a cellular deficiency at the level of postribosomal processing.
Assuntos
Carbono-Carbono Ligases , Fator VII/genética , Regulação da Expressão Gênica , Macrófagos/análise , Alvéolos Pulmonares/análise , RNA Mensageiro/análise , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Fator X/metabolismo , Fluorescência , Humanos , Ligases/metabolismo , Neoplasias Hepáticas , Microssomos/metabolismo , Microssomos Hepáticos/metabolismo , Hibridização de Ácido Nucleico , Biossíntese de Proteínas , Alvéolos Pulmonares/citologia , Ribossomos , Células Tumorais Cultivadas/metabolismoRESUMO
Several murine monoclonal anti-human Factor VII antibodies were produced using hybridoma technology. Two noncompetitive monoclonal antibodies were used to examine by Western blotting the Factor VII cross-reactive material (CRM) in normal human plasma and three commercially available congenitally Factor VII-deficient plasmas, and to construct a facile "sandwich" immunoassay for plasma Factor VII. A second, previously undescribed, form of Factor VII CRM was detected in human plasma, which on Western blotting stained with an apparent intensity 5-8% that of Factor VII. This glycoprotein, tentatively called VII*, has a molecular weight 4,500 D less than Factor VII, lacks detectable Factor VII functional activity, does not bind to barium citrate, and is not recognized by a monoclonal antibody that recognizes Factor VII but not alpha-chymotrypsin-treated Factor VII. VII* was not proteolytically produced from Factor VII during in vitro coagulation or after infusion of human Factor VII into rabbits. As determined by Western blotting, the human hepatoma cell line, HepG2, cultured in the presence of vitamin K, secreted relatively greater levels of VII* in proportion to VII (75%) than that found in human plasma. Warfarin treatment of HepG2 cells decreased the quantity of VII secreted by 77%, whereas it only inhibited the secretion of VII* by 14%. Immunologic studies of the plasmas from a patient on chronic warfarin therapy and an individual given a short course of high dose warfarin therapy corroborated the in vitro synthetic studies obtained with HepG2 cells. The data are consistent with the production of VII* by posttranslational, proteolytic, modification of VII, that, at least in the HepG2 cells studied, occurs intracellularly. However, other mechanisms for the production of VII*, in particular, alternative RNA splicing of the transcript from a single gene, cannot be excluded.