RESUMO
Congenital homozygous dysfibrinogenemia was diagnosed in a man with a history of 2 thrombotic strokes before age 30. His hemostatic profile was characterized by a dramatically prolonged plasma thrombin clotting time, and no clotting was observed with reptilase. Complete clotting of the abnormal fibrinogen occurred after a prolonged incubation of plasma with thrombin. The release of fibrinopeptides A and B by thrombin and of fibrinopeptide A by reptilase were both normal. Thrombin-induced fibrin polymerization was impaired, and no polymerization occurred with reptilase. The polymerization defect was characterized by a defective site "a," resulting in an absence of interaction between sites A and a, indicated by the lack of fragment D(1) (or fibrinogen) binding to normal fibrin monomers depleted in fibrinopeptide A only (Des-AA fm). By SDS-PAGE, the defect was detected on the gamma-chain and in its fragment D(1). The molecular defect determined by analysis of genomic DNA showed a single base change (A-->T) in exon VIII of the gamma-chain. The resulting change in the amino acid structure is gamma 330 aspartic acid (GAT) --> valine (GTT). It is concluded that the residue gamma-Asp(330) is essential for the normal functioning of the polymerization site a on the fibrinogen gamma-chain.
Assuntos
Fibrinogênios Anormais/genética , Adulto , Afibrinogenemia/genética , Afibrinogenemia/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Transtornos da Coagulação Sanguínea/sangue , Transtornos da Coagulação Sanguínea/complicações , Transtornos da Coagulação Sanguínea/genética , Transtornos de Proteínas de Coagulação/sangue , Transtornos de Proteínas de Coagulação/complicações , Transtornos de Proteínas de Coagulação/genética , Eletroforese em Gel de Poliacrilamida , Fibrina/genética , Fibrina/metabolismo , Fibrina/ultraestrutura , Fibrinogênios Anormais/efeitos adversos , Fibrinogênios Anormais/metabolismo , Fibrinolisina/metabolismo , Fibrinolíticos/metabolismo , Hemostáticos/metabolismo , Homozigoto , Humanos , Masculino , Mutação , Ligação Proteica , Subunidades Proteicas , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/etiologia , Trombina/efeitos dos fármacos , Trombina/metabolismo , Tempo de Trombina , Trombofilia/sangue , Trombofilia/complicações , Trombofilia/genética , Trombose/sangue , Trombose/etiologiaRESUMO
Fibrinogen Caracas V is a thrombotic dysfibrinogenemia with an Aalpha 532 Ser-->Cys mutation characterized by a tight fibrin network formed of thin fibers responsible for a less porous clot than a normal one. In the present work, fibrinogen Caracas V is further characterized in order to understand the relationship between the structural defect and thrombophilia. This thrombotic disorder has been attributed to a tight fibrin network responsible for a decreased permeation of flow through the clot, leading to defective thrombus lysis due to a diminished availability of fibrinolytic enzymes to the inner fibrin surface. Correction of clot structure anomaly, by addition of dextran 40 to fibrinogen before clotting, induces an improvement in fibrin degradation that was attributed to an increase in porosity. The pulmonary embolism observed in this family has been related to an hyper rigidity of the clot, an anomaly that is also corrected by dextran. Furthermore, this abnormal fibrinogen binds more albumin than does normal fibrinogen, a phenomenon attributed to the mutation of serine in Aalpha-532 by cysteine. Therefore, this fibrinogen shows a striking similarity to the fibrinogen Dusart, allowing us to confirm that the alphaC-terminal part of fibrinogen plays an important role in fibrin structure, and to conclude that the anomaly of fibrin network observed in fibrinogen Caracas V is responsible for a deficient thrombus lysis.
Assuntos
Transtornos de Proteínas de Coagulação/fisiopatologia , Fibrinogênios Anormais/metabolismo , Albuminas/metabolismo , Substituição de Aminoácidos , Coagulação Sanguínea/efeitos dos fármacos , Coagulação Sanguínea/genética , Transtornos de Proteínas de Coagulação/sangue , Transtornos de Proteínas de Coagulação/genética , Dextranos/farmacologia , Fibrina/genética , Fibrina/metabolismo , Fibrina/ultraestrutura , Fibrinogênios Anormais/genética , Fibrinólise/efeitos dos fármacos , Fibrinólise/genética , Humanos , Microscopia Confocal , Mutação , Trombofilia/sangue , Trombofilia/genéticaRESUMO
Thrombosis represents a major issue during arterial local delivery. We evaluated the occurrence of thrombosis after adenovirus (Ad)-mediated gene transfer into normal and atherosclerotic arteries. A replication-deficient Ad vector expressing the beta-galactosidase reporter gene (Ad.RSV betagal; 4 x 10(9) PFU) was injected into normal and atherosclerotic arteries (n = 11 in both groups). The contralateral artery received either an Ad vector carrying no transgene (Ad.MLPnull) (n = 7 in both groups, 4 x 10(9) PFU) or vehicle buffer (n = 4 in normal group, n = 8 in atherosclerotic group). Animals were sacrificed 3 days following gene transfer for thrombus detection and assessment of beta-galactosidase activity. Thrombus was absent in normal arteries and in atherosclerotic arteries injected with vehicle buffer only. In contrast, nonocclusive thrombus was present in atherosclerotic arteries injected with either Ad.RSV betagal (5 of 11) or Ad.MLPnull (3 of 7). Beta-galactosidase activity was predominantly found in the endothelial layer of the transfected arteries. Gene transfer and expression occurred despite the presence of the thrombus (4 of 5), and its efficiency did not significantly differ regardless of the thrombus. We conclude that thrombus frequently occurred in atherosclerotic arteries after Ad-mediated gene transfer. Further studies are warranted to identify the mechanisms of thrombus generation after Ad-mediated gene transfer into atherosclerotic arteries.
Assuntos
Adenoviridae/genética , Arteriosclerose/complicações , Técnicas de Transferência de Genes/efeitos adversos , Vetores Genéticos/genética , Trombose/etiologia , Animais , Artérias , Arteriosclerose/patologia , Vírus Defeituosos/genética , Orelha/irrigação sanguínea , Vetores Genéticos/administração & dosagem , Coelhos , Replicação Viral , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The urokinase receptor (u-PAR), a protein anchored to cell membrane by a glycosyl phosphatidylinositol, plays a central role in cancer cell invasion and metastasis by binding urokinase plasminogen activator (u-PA), thereby facilitating plasminogen activation. Plasmin can promote cell migration either directly or by activating metalloproteinases that degrade some of the components of the extra cellular matrix. However, the IGR-OV1-Adria cell line contains the u-PAR but does not migrate even in the presence of exogenous u-PA, although the parental IGR-OV1 cell line migrates normally in the presence of u-PA. We therefore investigated the role of cell signalling for u-PA induced cell locomotion. We show that cell migration induced by u-PA-u-PAR complex is always associated with tyrosine kinase activation for the following reasons: (1) the blockade of the u-PAR by a chimeric molecule (albumin-ATF) inhibits not only the u-PA-induced cell migration, but also the signalling in IGR-OV1 line; (2) the binding of u-PA to u-PAR on non-migrating IGR-OV1-Adria cells was not associated with tyrosine kinase activation; (3) the inhibition of tyrosine kinase also blocked cell migration of IGR-OV1. Therefore tyrosine kinase activation seems to be essential for the u-PA-induced cell locomotion possibly by the formation of a complex u-PAR-u-PA with a protein whose transmembrane domain can ensure cell signalling. Thus, IGR-OV1 and IGR-OV1-Adria cell lines represent a good model for the analysis of the mechanism of u-PA-u-PAR-induced cell locomotion.
Assuntos
Movimento Celular/fisiologia , Fosfotirosina/metabolismo , Receptores de Superfície Celular/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Western Blotting , Movimento Celular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Feminino , Fibrinolisina/metabolismo , Glicosilfosfatidilinositóis/fisiologia , Humanos , Imuno-Histoquímica , Microscopia Confocal , Neoplasias Ovarianas , Fosforilação , Inibidor 1 de Ativador de Plasminogênio/análise , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Transdução de Sinais , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
High levels of fibrinogen are recognized as an important vascular risk factor; however, it is not known if the increase of plasma fibrinogen is directly responsible for this risk, or is only a marker of vascular inflammation. To support this second hypothesis, Oncostatin M (OSM) is a potent stimulator of fibrinogen biosynthesis and induces smooth muscle cell proliferation. In the same way, we analysed whether interleukin-4 (IL-4), interleukin-10 (IL-10) or interleukin-13 (IL-13), which protect vessel walls from monocytes injuries leading to atherosclerosis, could influence fibrinogen biosynthesis. The two levels of regulation of fibrinogen biosynthesis were tested: firstly, the direct effect of these cytokines on fibrinogen production by the hepatoma cell line Hep G2, and secondly their effect on the secretion of hepatocyte stimulating factor (HSF) activity in the supernatant of lipopolysaccharide (LPS)-activated monocytes. IL-4 and IL-13 added to Hep G2 cells down-regulated both the increase of fibrinogen secretion induced by IL-6 and fibrinogen mRNA levels, this effect being more pronounced when Hep G2 were preincubated with the two cytokines before IL-6 addition. The effect of IL-10 was evidenced only on mRNA expression. IL-10 and IL-13 dose-dependently decrease HSF activity secreted by LPS-activated monocytes, whereas IL-4 had no effect. However, the three cytokines decreased HSF activity when monocytes were incubated with the cytokines before LPS activation. The effects of these cytokines on HSF activity are related to variations of IL-6 and OSM secretion. Our data strengthen the hypothesis that the fibrinogen level is a marker of vascular disease, since cytokines which have a protective vascular effect down-regulate fibrinogen production.
Assuntos
Fibrinogênio/biossíntese , Interleucina-10/farmacologia , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Animais , Arteriosclerose/etiologia , Linhagem Celular , Regulação para Baixo , Humanos , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Monócitos/metabolismo , Oncostatina M , Peptídeos/farmacologia , RNA Mensageiro/metabolismoAssuntos
Fibroblastos/imunologia , Ceratocone/imunologia , Estudos de Casos e Controles , Células Cultivadas/imunologia , Dinoprostona/imunologia , Fibroblastos/enzimologia , Humanos , Inflamação , Ceratocone/enzimologia , Linfotoxina-alfa/imunologia , Prostaglandina-Endoperóxido Sintases/imunologia , Receptores de Interleucina-1/imunologia , Ativador de Plasminogênio Tecidual/imunologiaAssuntos
Doenças Cardiovasculares/epidemiologia , Fibrinogênio/análise , Fígado/metabolismo , Adolescente , Adulto , Biomarcadores , Carcinoma Hepatocelular , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/etiologia , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Fibrinogênio/biossíntese , Fibrinogênio/fisiologia , Inibidores do Crescimento/farmacologia , Humanos , Interleucina-6/farmacologia , Fator Inibidor de Leucemia , Fígado/efeitos dos fármacos , Neoplasias Hepáticas , Linfocinas/farmacologia , Oncostatina M , Peptídeos/farmacologia , Proteínas Recombinantes/farmacologia , Fatores de Risco , Células Tumorais CultivadasRESUMO
Previous study showed that the secretion of urokinase (UK) by monoblastic cell line U 937 and the number of binding sites for urokinase and for plasminogen (Plg) on the cell surfaces were augmented by interferon gamma (INF tau). This induction led to an increase in fibrinolytic activity on cell surfaces. A similar increase was also observed when treating the U 937 cells with 1,25-dihydroxyvitamin D3 (1,25 (OH)2D3. Here we report that the combination of these two agents induced a 2.7 fold increase in the plasminogen activator activity on U 937 cell surfaces in comparison with 1 fold increase induced by INF tau and 1.3 fold increase by 1,25(OH)2D3. As evaluated by a flow cytometer, the increased fibrinolytic activity induced by the combination of INF tau and 1,25(OH)2D3 could be attributed to the increase of the number of binding sites both for UK (3.7 x 10(4) vs 1.2 x 10(4) per cell) and for Plg (16.2 x 10(4) vs 3.6 x 10(4) per cell), accompanied by an increased expression of CD 14, which is an antigen of differentiation on cell surfaces. These results suggest that the expression of urokinase receptors and plasminogen receptors may be coupled together by unknown intracellular mechanisms during cell differentiation, and support the idea that the concomitant regulation of these two receptors for UK and Plg is an important aspect in cell associated-fibrinolytic activity.
Assuntos
Calcitriol/farmacologia , Fibrinólise/efeitos dos fármacos , Interferon gama/farmacologia , Calcitriol/administração & dosagem , Diferenciação Celular , Linhagem Celular , Membrana Celular/metabolismo , Sinergismo Farmacológico , Humanos , Interferon gama/administração & dosagem , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Plasminogênio/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
As fibrinogen is an independent risk factor for arterial thrombosis we were interested in analysing the mechanism controlling fibrinogen biosynthesis. In this work, we showed that incubation of monocytes with lymphocytes increased hepatocyte stimulating factor (HSF) production. Different mechanisms are involved and our results demonstrated that this effect is in part mediated by an increase in interleukin 6 (IL-6) production. However, IL-6 cannot account for the whole effect and other cytokines could be implicated. In addition, we observed a stimulation of urokinase-type plasminogen activator (u-PA) associated with monocytes when these cells were incubated with lymphocytes for 18 h at 37 degrees C. By producing fragment D (fibrinogen degradation product) and D-dimer (fibrin degradation product) this fibrinolytic activity might also contribute to fibrinogen biosynthesis by hepatocytes.
Assuntos
Interleucina-6/metabolismo , Interleucina-6/farmacologia , Linfócitos/fisiologia , Monócitos/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Células Cultivadas/metabolismo , Produtos de Degradação da Fibrina e do Fibrinogênio , Fibrinogênio/biossíntese , Humanos , Monócitos/efeitos dos fármacos , Ativadores de Plasminogênio/metabolismoRESUMO
This work provides evidence that the production by monocytes of hepatocyte stimulating factor(s) for fibrinogen biosynthesis was dramatically increased when monocytes were exposed to Adriamycin. This effect was related to an increased production of leukaemia inhibiting factor (LIF), a cytokine known to stimulate fibrinogen biosynthesis by hepatic cells. Adriamycin also induces an increase in membrane-associated urokinase on monocytes. These results are consistent with the clinical observation in patients with ovarian cancer that when the CA-125 tumour marker decreases during chemotherapy, an increased level of D-dimer is a marker of good prognosis.
Assuntos
Doxorrubicina/farmacologia , Interleucina-6/metabolismo , Monócitos/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Meios de Cultura/análise , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio , Fibrinogênio/biossíntese , Inibidores do Crescimento/metabolismo , Humanos , Fator Inibidor de Leucemia , Linfocinas/metabolismo , Monócitos/metabolismo , Neoplasias Ovarianas/fisiopatologiaRESUMO
In patients with ovarian cancer before they receive chemotherapy, the level of fibrin degradation products (D-dimer), is correlated with the tumor load. In this study, the evolution of D-dimer was compared in patients receiving antineoplastic therapy with the evolution of the disease. The patients could be classified into three groups. In Group 1 (nine patients), both plasma CA 125 (a tumor-associated antigen) and D-dimer remained elevated; the prognosis was always poor. In Group 2 (eight patients), CA 125 and D-dimer decreased simultaneously, complete remission was observed in two patients, and significant residual tumor was observed in the others. In Group 3 (nine patients), despite an important decrease in CA 125, D-dimer remained elevated during therapy. In this group, complete remission was observed in six patients, and three others showed a large decrease in their tumor load. The combination of a decrease in CA 125 levels with a continuous enhanced level of D-dimer during chemotherapy identified a subgroup of patients with a favorable prognosis.
Assuntos
Antígenos Glicosídicos Associados a Tumores/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Neoplasias Ovarianas/sangue , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Valor Preditivo dos Testes , PrognósticoRESUMO
S-antigen (also named arrestin or 48K protein) is a protein abundant in photoreceptor cells of vertebrates and invertebrates. The presently known function of this protein in retina is to arrest the enzymatic cascade of phototransduction in retinal rods, through its binding to photoactivated and phosphorylated rhodopsin. Proteins closely related to S-antigen were recently demonstrated in several non photosensitive cells. In this work, we demonstrated the presence of a protein similar to retinal S-antigen with regards to its immunoreactivity with a panel of monoclonal antibodies and its molecular weight in soluble extracts of human platelets. This protein was purified by immunoaffinity chromatography using a rabbit antibody to retinal S-antigen. This S-antigen-like protein could have a regulatory function in G-protein-mediated transduction of chemical signals in platelets, similar to arrestin function in phototransduction.
Assuntos
Antígenos/isolamento & purificação , Plaquetas/química , Proteínas do Olho/isolamento & purificação , Anticorpos Monoclonais , Antígenos/química , Arrestina , Western Blotting , Proteínas do Olho/química , Humanos , Técnicas de Imunoadsorção , Peso Molecular , Transdução de SinaisRESUMO
In this work, fibrinogen evolution was analysed by testing the reactivity of fibrinogen from different species with monoclonal antibodies against human fibrinogen fragment D. One epitope concerning the fibrin polymerization site 'a' and two epitopes responsible for tPA binding to fibrin were conserved in all mammalian fibrogens tested but not in crab coagulogen or pleurodella fibrinogen. In these two species, some epitopes which are not implicated in fibrinogen function were conserved. Therefore, we can conclude that polymerizing site 'a' and tPA binding sites have not been modified for at least 80 million years.
Assuntos
Anticorpos Monoclonais/imunologia , Fibrina/metabolismo , Fibrinogênio/imunologia , Fibrinólise , Animais , Evolução Biológica , Proteínas Sanguíneas/imunologia , Reações Cruzadas , Produtos de Degradação da Fibrina e do Fibrinogênio/imunologia , Fibrinogênio/genética , Caranguejos Ferradura/genética , Caranguejos Ferradura/imunologia , Humanos , Mamíferos/genética , Mamíferos/imunologia , Salamandridae/genética , Especificidade da Espécie , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
In this study, we have shown that the secretion of plasminogen activators (PA) by corneal fibroblasts from Schnyder dystrophy is much lower than that secreted by normal corneal fibroblasts. This defective secretion of PA was noted both in the supernatant of cell culture and on the cell surface. This anomaly is found in cultured fibroblasts and therefore is not related to environment modification. Since PA was implicated in remodeling of extracellular matrix, we hypothesized that this anomaly should be responsible for the corneal dystrophy.