RESUMO
BACKGROUND: Proinflammatory cytokines play key roles in atherogenesis and disease progression. Because hyperhomocysteinemia is an independent risk factor for cardiovascular disease, we hypothesized that homocysteine could be atherogenic by altering the expression of specific cytokines in vascular endothelial cells. METHODS AND RESULTS: Northern blot and RNase protection assays showed that DL-homocysteine induced mRNA expression of the proinflammatory cytokines monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) in cultured human aortic endothelial cells (HAECs). Homocysteine had no effect on expression of other cytokines, namely tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor, interleukin-1beta, and transforming growth factor-beta. MCP-1 mRNA expression increased 1 hour after homocysteine treatment, reached a maximum within 2 to 4 hours, and declined to basal levels over the next 24 hours. Induction of mRNA expression for both chemokines was observed with as little as 10 micromol/L DL-homocysteine, and maximal expression was achieved with 50 micromol/L DL-homocysteine. Homocysteine also triggered the release of MCP-1 and IL-8 protein from HAECs into the culture medium. The induction was specific for homocysteine, because equimolar concentrations of L-homocystine, L-cysteine, and L-methionine had no effect on mRNA levels and protein release. Furthermore, L-homocysteine induced chemokine expression, but D-homocysteine did not, thus demonstrating enantiomeric specificity. The culture medium from homocysteine-treated HAECs promoted chemotaxis in human peripheral blood monocytes and U937 cells. Anti-human recombinant MCP-1 antibody blocked the migration. CONCLUSIONS: Pathophysiological levels of L-homocysteine alter endothelial cell function by upregulating MCP-1 and IL-8 expression and secretion. This suggests that L-homocysteine may contribute to the initiation and progression of vascular disease by promoting leukocyte recruitment.
Assuntos
Aorta Torácica/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Endotélio Vascular/efeitos dos fármacos , Homocisteína/farmacologia , Aorta Torácica/citologia , Aorta Torácica/metabolismo , Northern Blotting , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/farmacologia , Quimiotaxia/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Compostos de Enxofre/farmacologia , Fatores de Tempo , Células U937 , Doenças Vasculares/genética , Doenças Vasculares/metabolismo , Doenças Vasculares/patologiaRESUMO
Since infective endocarditis may affect individuals without pre-existing valvar heart disease, and Staphylococcus aureus is the organism most commonly involved, the binding characteristics of S aureus to several components of normal vascular endothelium and subendothelium were studied. S aureus adhered specifically to endothelial monolayers (6.08(1.10)%; p less than 0.005), fibronectin (5.43(0.81)%; p less than 0.001), fibrinogen (7.13(1.43)%; p less than 0.001), and acid soluble calf skin collagen (2.38(0.90)%; p less than 0.001). S aureus also adhered specifically to Von Willebrand factor (1.62(0.28)%, p less than 0.001). Protein A containing (Cowan I) and deficient (Wood) strains of S aureus adhered similarly to all surfaces and substrates (NS). Escherichia coli adhered poorly. Immunofluorescence microscopy of preconfluent endothelial cells identified an extensive pericellular fibronectin network at regions of cell to cell contact. Light microscopy showed S aureus binding solely within these regions. Therefore, the ability of S aureus to infect valvar endothelium may be dependent on the presence of a fibronectin receptor. The existence of specific receptor for S aureus on the endothelial cell surface itself remains undetermined.
Assuntos
Endocardite Bacteriana/microbiologia , Staphylococcus aureus/fisiologia , Animais , Aderência Bacteriana , Bovinos , Linhagem Celular , Colágeno/metabolismo , Endocardite Bacteriana/fisiopatologia , Endotélio Vascular/metabolismo , Escherichia coli/metabolismo , Escherichia coli/fisiologia , Fibrinogênio/metabolismo , Fibronectinas/metabolismo , Humanos , Staphylococcus aureus/metabolismo , Fator de von Willebrand/metabolismoRESUMO
The studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (alpha-fibrin) or both fibrinopeptides A and B (alpha beta-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX congruent 200-800 molecules/cell, KD congruent to 10(-12) M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX greater than or equal to 10(5) molecules/cell, KD greater than or equal to 10(-6) M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD congruent to 10(-10) M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (beta-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the alpha- and the beta-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with electrophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.
Assuntos
Fibrina/metabolismo , Macrófagos/metabolismo , Receptores de Peptídeos , Marcadores de Afinidade , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatografia de Afinidade , Produtos de Degradação da Fibrina e do Fibrinogênio/isolamento & purificação , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Humanos , Fatores Imunológicos/farmacologia , Radioisótopos do Iodo , Dados de Sequência Molecular , Fragmentos de Peptídeos/farmacologia , Coelhos , Receptores de Superfície Celular/metabolismoRESUMO
Current hematologic approaches to minimize postoperative bleeding have focused principally on antifibrinolytic agents. To explore whether a need might exist to promote clot stabilization independent of steps that might be taken to prevent lysis, we followed levels of the functional A-chain of factor XIII (fibrin stabilizing factor) immunologically in 19 patients undergoing coronary artery bypass grafting. The levels of factor XIIIA together with alterations in fibrinogen were followed at five stages of operation: (1) initial catheter placement (control), (2) heparinization, (3) initiation of cardiopulmonary bypass, (4) discontinuation of cardiopulmonary bypass, and (5) heparin neutralization with protamine sulfate. Significant (p < 0.05) inverse correlations were observed between postoperative chest-tube drainage volumes and levels of XIIIA at stages 1 through 3, and borderline associations (p < 0.1) were observed for stages 4 and 5. Pronounced losses of factor XIIIA accompanied initiation of cardiopulmonary bypass, when levels fell to 43% +/- 12% (standard deviation) of the control value, significantly below the 59% +/- 9% of the control value expected from hemodilution. By comparison, fibrinogen concentrations fell only to the extent attributable to hemodilution, unaccompanied by substantial degradation as indicated by electrophoretic, functional, and immunologic assays. There was a reversible heparin-induced precipitation of fibrin complexes and fibrinogen dimers from the blood on initiation of hypothermia, but these components returned to the circulation on restoration of normothermia. This precipitation was unrelated to losses of factor XIIIA. The findings warrant inference that XIIIA supplementation in deficient states should be considered as an adjunct to other therapies for postoperative bleeding.
Assuntos
Perda Sanguínea Cirúrgica/fisiopatologia , Ponte de Artéria Coronária , Transglutaminases/análise , Adulto , Ponte de Artéria Coronária/efeitos adversos , Feminino , Fibrinogênio/análise , Hemodiluição , Humanos , MasculinoRESUMO
Our preceding study indicated that, in course of coagulation of human fibrinogen by thrombin, substantial production of the fibrin intermediate (alpha-profibrin) lacking only one fibrinopeptide A (FPA) precedes the formation of alpha-fibrin monomer lacking both FPAs. The plateau concentration of alpha-profibrin (20% of initial fibrinogen) appearing in reactions indicated, however, that the second FPA is released four times faster than the first. The study reported here confirms those findings, and provides new insight into the significance of differing rate constants for the production of alpha-profibrin and its conversion to alpha-fibrin. The intermediate could be isolated in a distinct electrophoretic band by electrophoresing partial thrombin digests at high concentrations. Its identity was verified by digesting it with CNBr and by demonstrating that its N-terminal domain, the NDSK fragment, both lacks an FPA and contains an FPA, unlike the NDSKs of the bands from fibrin which contained no FPA or the fibrinogen band that lacked no FPA. The single step isolation also enabled us to confirm the 15-20% plateau level of alpha-profibrin in course of thrombin reactions, well below the 37% maximum that would be expected if release of the first and second FPA proceeded independently with no difference in rate. The 37% maximum is observed in reactions with atroxin, and it is suggested that the abundant production of alpha-profibrin underlies the therapeutic utility of atroxin as a defibrinating agent. Gel chromatography procedures were optimized for isolation of alpha-profibrin/fibrin mixtures free of fibrinogen, the final step of which involves literal use of agarose gel as a filter to remove fibrin aggregates from the fibrinogen free fractions (aggregates are left behind in gel filtration, rather than their moving ahead in gel chromatography). Unlike human fibrinogen, rabbit fibrinogen does not yield much alpha-profibrin in course of its conversion to fibrin, less than 10% as determined by electrophoresis and comparison with abundant production with atroxin. This low production of alpha-profibrin conformed with conclusions from our early studies on the generalized Shwartzman reaction in rabbits, and we now infer that the low production of alpha-profibrin and rapid conversion to fibrin by rabbit fibrinogen underlies the unparalleled susceptibility of these animals toward fibrinoid formation in the generalized Shwartzman reaction.
Assuntos
Fibrina/biossíntese , Fibrinogênio/metabolismo , Animais , Western Blotting , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Fibrinogênio/isolamento & purificação , Humanos , Coelhos , Serina Endopeptidases/metabolismoRESUMO
A marked increase in the cyclic AMP content and concentration of the thoracic aorta was detected during the rise in blood pressure produced by one-kidney, one-clip and DOCA hypertension. This alteration was accompanied by the development of vascular hypertrophy and preceded the abnormal increase in DNA content observed in the arterial system during the more chronic stages of the disease. These experiments suggest the participation of cyclic AMP in the development of hypertension vascular growth.
Assuntos
AMP Cíclico/sangue , Hipertensão/metabolismo , Animais , Aorta Torácica/metabolismo , Pressão Sanguínea , DNA/metabolismo , Desoxicorticosterona , Hipertensão/etiologia , Hipertensão/fisiopatologia , Hipertensão Renovascular/etiologia , Hipertensão Renovascular/metabolismo , Hipertensão Renovascular/fisiopatologia , RatosRESUMO
We have determined the activity and protein levels of CBS in a number of cardiovascular cells and tissues by direct enzyme assay and Western blot analysis, respectively. We have also determined the activity of BHMT in these same tissues and cells and have come to the conclusion that neither enzyme is expressed. This results suggests that in the human cardiovascular system homocysteine metabolism is limited to the remethylation pathway catalyzed by MS. Thus, hyperhomocysteinemia in conjunction with a limited metabolic capacity for homocysteine in the cardiovascular system could result in cellular dysfunction.
Assuntos
Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/etiologia , Sistema Cardiovascular/metabolismo , Homocisteína/sangue , Homocisteína/metabolismo , Animais , Betaína-Homocisteína S-Metiltransferase , Vasos Sanguíneos/metabolismo , Doenças Cardiovasculares/genética , Células Cultivadas , Cistationina beta-Sintase/genética , Cistationina beta-Sintase/metabolismo , Humanos , Metiltransferases/metabolismo , Músculo Liso Vascular/metabolismo , Miocárdio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RatosRESUMO
The fibrinogen molecule consists of two sets of Aalpha, Bbeta, and gamma chains assembled into a bilateral disulfide linked (Aalpha, Bbeta, gamma)2 structure. Cleavage of the two A-fibrinopeptides (FPA, Aalpha1-16) from normal Aalpha chains with arginine at position 16 (RFPA) by thrombin or the venom enzyme atroxin transforms fibrinogen into self-aggregating fibrin monomers (alpha, Bbeta, gamma)(2). Mutant Aalpha16R-->H fibrinopeptide (HFPA) cannot be cleaved from fibrinogen by atroxin. Many studies on heterozygous dysfibrinogenemias with this mutation suggested that incorporation of the mutant chains into the molecules was ordered in a manner yielding only (1) homodimeric normal (RFPARFPA) atroxin-coagulable molecules and (2) homodimeric abnormal (H(FPA)HFPA) atroxin-incoagulable molecules in equal quantities. Although heterodimeric molecules (RFPAHFPA) could not be found in studies on the intact protein, Meh et al. demonstrated their existence by showing that CNBr digests of fibrinogens from atroxin-treated Aalpha16R-->H heterozygotic dysfibrinogenemias consistently yielded N-terminal fragments (NDSKs) with partially resolved electrophoretic bands predominantly in between the NDSKs of fibrinogen and alpha-fibrin. An opportunity to confirm and better quantify the heterodimers arose with the recent development of a method (GPRphoresis) for identifying molecules lacking only one FPA, which is applied here in study of a newly presenting case of an Aalpha16R-->H dysfibrinogenemia, "fibrinogen Amarillo." GPRphoresis uses electrophoretic shifts, staged with GPRP-NH(2) to separate the self-aggregating fibrin monomers lacking both FPAs from weakly aggregating "semifibrin" molecules lacking one FPA An antifibrin alpha17-23 antibody is used to measure and differentiate the semifibrin from fibrinogen with FPA fully intact. Applying GPRphoresis to atroxin digests of fibrinogen Amarillo clearly demonstrated RFPARFPA, RFPAHFPA, and HFPAHFPA molecules in nearly perfect Mendelian 1:2:1 proportions. In turn, the high levels of the semifibrin in the terminal atroxin digests provide genetic phenotypic evidence supporting fidelity of the GPRphoresis method.
Assuntos
Fibrinogênios Anormais/genética , Dimerização , Eletroforese em Gel de Poliacrilamida , Saúde da Família , Fibrina/metabolismo , Fibrinogênios Anormais/metabolismo , Fibrinopeptídeo A/metabolismo , Heterozigoto , Humanos , Cinética , Mutação Puntual , Serina Endopeptidases/metabolismoRESUMO
High plasma homocysteine (tHcy) is a risk factor for cardiovascular disease and stroke and Alzheimer's disease (AD). An inverse relationship has been reported between tHcy and plasma B12 and folate levels. Seventy-nine AD patients and 156 controls from three Arab villages in northern Israel participated. Plasma tHcy, B12 and folate levels were determined. Data were analyzed using univariate statistical tests and logistical regression with confounders. tHcy was significantly higher in AD patients (20.6+/-8.7 micromol/l) than in controls (16.4+/-6.5 micromol/l) (p=0.03) after correction for year of birth, gender and smoking status. Plasma B12 (322.9+/-136.0/350.5+/-175.3 pmol/l) and plasma folate (4.5+/-3.8/4.9+/-2.6 nmol/l) levels did not differ significantly between AD patients and controls. Subjects in the highest tHcy tertile or in the lowest B12 and folate tertiles did not have greater risk to develop AD. In this population residing in Arab villages in northern Israel, tHcy levels were significantly higher among AD patients than in controls. Plasma B12 and folate levels were lower among cases but were not significant. There was not a significant association between plasma tHcy, B12 and folate levels in controls or AD patients. High levels of tHcy may suggest the need for folate and vitamin B12 supplementation in this population.
Assuntos
Doença de Alzheimer/sangue , Ácido Fólico/sangue , Homocisteína/sangue , Vitamina B 12/sangue , Idoso , Idoso de 80 Anos ou mais , Árabes , Estudos de Casos e Controles , Intervalos de Confiança , Feminino , Humanos , Israel/epidemiologia , Israel/etnologia , Masculino , Razão de ChancesRESUMO
An electrophoretic method for determining (i) cross-linked fibrin-complexes, (ii) fibrin-monomer, (iii) fibrinogen-dimers, (iv) normal fibrinogen and (v) degradation products in plasma, has been devised. The technique is based on differences in their migration characteristics in the presence and absence of Gly-Pro-Arg (GPR), a specific inhibitor of fibrin aggregation. In buffer containing 2.5 mM GPR, fibrin monomer and fibrinogen co-migrate anodally, but, unlike fibrinogen which does not depend on GPR for solubility, the fibrin monomers precipitate when they traverse a boundary between buffer containing and buffer lacking GPR. By limiting the GPR to a 2 cm zone of buffer under the conditions employed, the precipitation of fibrin monomer occurs in a sharp band 4 mm anodally to the sample application point. Cross-linked fibrin complexes have slower mobility than fibrin monomer and precipitate in a broad band behind the monomer. Dimeric fibrinogen, like fibrinogen itself but unlike the fibrin complexes, is not constrained to migration within the GPR boundary and passes through it, but behind the band for normal fibrinogen due to sieving by the gel. Fibrinogen and all but low molecular weight degradation products can be specifically precipitated within electrophoregrams by heat denaturation at 47 degrees C. After washing unrelated protein away, the fibrin(ogen) derivatives can be measured by staining with Coomassie blue. Since the method does not depend on immunoprobing for specific staining, it provides an inexpensive and rapid means for differential assessment of the prevalence of the fibrinogen derivatives in disease states and in models of disease, regardless of animal species.
Assuntos
Eletroforese/métodos , Fibrina/análise , Oligopeptídeos , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Fibrinogênio/análise , Desnaturação Proteica , SolubilidadeRESUMO
Cross-linked fibrin(ogen) dimers are known to be elevated in the plasma of subjects with occlusive vascular disease, and are thought to be fibrin dimers. Immunoelectrophoretic analyses of the dimers, however, indicate that (1) they are predominantly fibrinogen rather than fibrin dimers, and (2) they contain cross-linked A alpha-chains (A alpha-dyads) instead of the gamma-chain dyads that are rapidly formed by factor XIII during blood coagulation. Furthermore, the mobilities of the A alpha-dyads differ from the cross-linked alpha-chain products that accompany the gamma-chain cross-linking by factor XIII. Instead, the mobilities coincide with the distinct A alpha-dyads that are produced by tissue transglutaminase, an intracellular enzyme not normally present in plasma. The intimal fibrinogen deposits in atherosclerotic aortas also possess fibrinopeptide A and cross-linked A alpha-chains. Thus, both the plasma fibrinogen dimers and the intimal fibrinogen deposits appear to derive from the action of released tissue transglutaminase more so than factor XIII. It is proposed that, in the absence of other indications of cytolytic processes, the levels of A alpha-dyads in plasma reflect ongoing cellular injury accompanying atherogenesis. The extent to which gamma-dyads accompany the A alpha-dyads may signal progression of the disease to advanced stages in which ulcerations and occlusive lesions trigger thrombotic complications.
Assuntos
Arteriosclerose/metabolismo , Fibrinogênio/análise , Trombose/metabolismo , Biomarcadores , Eletroforese em Gel de Ágar/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Imunoeletroforese/métodos , Tromboflebite/sangue , Transglutaminases/metabolismoRESUMO
A fully automated immunoassay for total plasma homocysteine assay was evaluated at four centers. To measure total homocysteine, oxidized forms of homocysteine in serum and plasma were reduced by dithiothreitol and assayed by a competitive fluorescence polarization technique. The assay had within-run precision from 0.9 to 3.0% and total precision from 2.8 to 4.1% for control materials with homocysteine concentrations of approximately 7, 12.5, and 25 micromol/L, a sensitivity of 0.35 micromol/L, good parallelism upon dilution, and analytical recovery ranging from 97.4 to 103.8%. The immunoassay correlated with four different HPLC assays for homocysteine, yielding a slope of 0.98, an intercept of -0.19 micromol/L, and a correlation coefficient of 0.966 for 440 paired samples. The reference range, determined with plasma samples from 609 males and 600 females, yielded a mean of 9.17+/-2.86 micromol/L, with a central 95% range of 4.78-15.43 micromol/L. The immunoassay is a suitable alternative to HPLC and may be useful in screening persons with high risk of coronary artery disease.
Assuntos
Doença das Coronárias/diagnóstico , Homocisteína/análise , Homocisteína/sangue , Imunoensaio/métodos , Química Clínica/métodos , Química Clínica/normas , Cromatografia Líquida de Alta Pressão , Doença das Coronárias/sangue , Estudos de Avaliação como Assunto , Humanos , Imunoensaio/normas , Valores de Referência , Análise de Regressão , Sensibilidade e EspecificidadeRESUMO
Direct immunoprobing of electrophoregrams of plasma and intimal protein on glyoxyl agarose and composite-gels with polyacrylamide have uncovered novel modes of cross-linking of fibrinogen that differ from those previously characterized. These modes of cross-linking involve the A alpha-chains of fibrinogen and hybrid cross-linking of alpha- and gamma-chains.
Assuntos
Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Doenças Vasculares/sangue , Eletroforese em Gel de Ágar/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Fibrinogênio/isolamento & purificação , Glioxilatos , Humanos , Indicadores e Reagentes , Substâncias Macromoleculares , SefaroseRESUMO
Electrophoretic profiles of the molecular weight distributions of fibrinogen derivatives in blood provide a tool for combined assessment of coagulation and fibrinolysis in the course of vascular disease. Profiles obtained in studies on an experimental model of hypertension and in humans with occlusive vascular disease are discussed. In the experimental studies elevations in the level of cross-linked dimers provided a more reliable means for predicting development of malignant hypertension than did many other criteria, especially near the outset when blood pressure changed to similar degrees in rats with malignant and benign hypertension. Similarly, we find that levels of dimeric and occasionally trimeric forms of fibrinogen are more prominently elevated than degraded forms of fibrinogen in patients with occlusive vascular disease.
Assuntos
Fibrinogênio/análise , Hipertensão Maligna/sangue , Animais , Fibrina/análise , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Humanos , Imunoeletroforese , Peso Molecular , RatosRESUMO
Cross-linking of human fibrin by fibrin stabilizing factor (factor XIIIa) and tissue transglutaminase (ti-TG) was examined by immunoprobing electrophoregrams for positive identification of the cross-linked chains. The immunoprobing was carried out by a new, direct staining technique employing composite gels of a porous protein immobilizing matrix (glyoxyl agarose) blended with a removable polyacrylamide filler that eliminates need for Western blotting. We find that the known rapid cross-linking of gamma-chains into gamma 2-dyads by XIIIa is accompanied by co-cross-linking of the gamma 2-dyads with alpha-chains to form hybrid alpha gamma 2-triads. Little or no cross-linking of relatively abundant alpha- and gamma-chain monads into hybrid alpha gamma-dydads accompanies formation of the alpha gamma 2-triads. Thus, formation of the gamma 2-dyads accelerates the hybrid cross-linking. This acceleration is viewed as demonstrating a previously unknown mode of cooperative interaction between alpha- and gamma-chains arising from cross-linking of the D-domains of the molecules. This strengthened interaction is not critically dependent on fibrinopeptide-release, because alpha gamma 2-triads are similarly formed when fibrinogen is cross-linked by XIIIa. Also observed in the study with XIIIa was the formation of small amounts of homologous gamma 3 and gamma 4 oligomers which had been predicted by others to contribute to branching of fibrin strands. Unlike XIIIa, ti-TG acts preferentially on alpha-chains rather than gamma-chains as known. As alpha gamma-dyad, not seen in reactions with XIIIa, is produced concurrent with the homologous alpha-chain cross-linking. Also, three different species of alpha 2-dyads were produced by ti-TG, two of which were not seen in reactions with XIIIa. The differences in product formation revealed by the specific staining are viewed as providing criteria for distinguishing products of XIIIa and ti-TG in biologic specimens.
Assuntos
Fibrina/metabolismo , Fibrinogênio/metabolismo , Transglutaminases/metabolismo , Reagentes de Ligações Cruzadas , Eletroforese em Gel de Poliacrilamida , Fibrina/imunologia , Fibrinogênio/imunologia , Humanos , Imuno-Histoquímica , Testes de PrecipitinaRESUMO
A preparative method for isolating centigram quantities of high molecular weight polypeptide chains with high resolution and recovery uses linear polyacrylamide/agarose composite (LPAC) gels as electrophoretic media from which the polypeptides can be easily extracted. The composites are prepared in a manner yielding linear copolymers of acrylamide and 1-allyloxy-2,3-propanediol within 2% agarose gels. After electrophoresis in sodium dodecyl sulfate (SDS), protein bands were rapidly visualized for excision by briefly immersing the gel in cold 0.1 M KCl which precipitates the protein-associated SDS. The gel slices are then freeze-thawed to disrupt the agarose matrix and promote syneresis of fluid upon centrifugation. The polypeptides are then separated from the polyacrylamide in the supernatant solution by precipitating with either acidic isopropanol, trichloroacetic acid, ammonium sulfate or other general protein precipitants. As determined with polypeptide chains of fibrinogen and its cross-linked derivatives, recoveries were virtually complete (95.4% +/- 2.2%), and were independent of molecular weights over the range tested (10(4) --10(6)).
Assuntos
Eletroforese em Gel de Ágar/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Peptídeos/isolamento & purificação , Modelos Logísticos , Microquímica , Peso Molecular , Reprodutibilidade dos TestesRESUMO
Disulfide forms of homocysteine account for >98% of total homocysteine in plasma from healthy individuals. We recently reported that homocysteine reacts with albumin-Cys(34)-S-S-cysteine to form homocysteine-cysteine mixed disulfide and albumin-Cys(34) thiolate anion. The latter then reacts with homocystine or homocysteine-cysteine mixed disulfide to form albumin-bound homocysteine (Sengupta, S., Chen, H., Togawa, T., DiBello, P. M., Majors, A. K., Büdy, B., Ketterer, M. E., and Jacobsen, D. W. (2001) J. Biol. Chem. 276, 30111-30117). We now extend these studies to show that human albumin, but not ceruloplasmin, mediates the conversion of homocysteine to its low molecular weight disulfide forms (homocystine and homocysteine-cysteine mixed disulfide) by thiol/disulfide exchange reactions. Only a small fraction of homocystine is formed by an oxidative process in which copper bound to albumin, but not ceruloplasmin, mediates the reaction. When copper is removed from albumin by chelation, the overall conversion of homocysteine to its disulfide forms is reduced by only 20%. Ceruloplasmin was an ineffective catalyst of homocysteine oxidation, and immunoprecipitation of ceruloplasmin from human plasma did not inhibit the capacity of plasma to mediate the conversion of homocysteine to its disulfide forms. In contrast, ceruloplasmin was a highly efficient catalyst for the oxidation of cysteine and cysteinylglycine to cystine and bis(-S-cysteinylglycine), respectively. However, when thiols (cysteine and homocysteine) that are disulfide-bonded to albumin-Cys(34) are removed by treatment with dithiothreitol to form albumin-Cys(34)-SH (mercaptalbumin), the conversion of homocysteine to its disulfide forms is completely blocked. In conclusion, albumin mediates the formation of disulfide forms of homocysteine by thiol/disulfide exchange, whereas ceruloplasmin converts cysteine to cystine by copper-dependent autooxidation.
Assuntos
Ceruloplasmina/fisiologia , Cisteína/biossíntese , Cisteína/química , Dissulfetos/química , Homocisteína/análogos & derivados , Homocisteína/química , Homocistina/biossíntese , Albumina Sérica/fisiologia , Ceruloplasmina/química , Ceruloplasmina/farmacologia , Cromatografia Líquida de Alta Pressão , Cobre/química , Cobre/farmacologia , Relação Dose-Resposta a Droga , Humanos , Modelos Biológicos , Oxigênio/metabolismo , Ligação Proteica , Albumina Sérica/química , Albumina Sérica/metabolismo , Compostos de Sulfidrila/química , Fatores de TempoRESUMO
An elevated concentration of plasma total homocysteine is an independent risk factor for cardiovascular disease. Greater than 80% of circulating homocysteine is covalently bound to plasma protein by disulfide bonds. It is known that albumin combines with cysteine in circulation to form albumin-Cys(34)-S-S-Cys. Studies are now presented to show that the formation of albumin-bound homocysteine proceeds through the generation of an albumin thiolate anion. Incubation of human plasma with l-(35)S-homocysteine results in the association of >90% of the protein-bound (35)S-homocysteine with albumin as shown by nonreduced SDS-polyacrylamide gel electrophoresis. Treatment of the complex with beta-mercaptoethanol results in near quantitative release of the bound l-(35)S-homocysteine, demonstrating that the binding of homocysteine to albumin is through a disulfide bond. Furthermore, using an in vitro model system to study the mechanisms of this disulfide bond formation, we show that homocysteine binds to albumin in two steps. In the first step homocysteine rapidly displaces cysteine from albumin-Cys(34)-S-S-Cys, forming albumin-Cys(34) thiolate anion and homocysteine-cysteine mixed disulfide. In the second step, albumin thiolate anion attacks homocysteine-cysteine mixed disulfide to yield primarily albumin-Cys(34)-S-S-Hcy and to a much lesser extent albumin-Cys(34)-S-S-Cys. The results clearly suggest that when reduced homocysteine enters circulation, it attacks albumin-Cys(34)-S-S-Cys to form albumin-Cys(34) thiolate anion, which in turn, reacts with homocysteine-cysteine mixed disulfide or homocystine to form albumin-bound homocysteine.
Assuntos
Albuminas/química , Ânions , Homocisteína/química , Albumina Sérica/química , Albuminas/metabolismo , Cromatografia Líquida de Alta Pressão , Cisteína/química , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Homocisteína/análogos & derivados , Homocisteína/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Mercaptoetanol/farmacologia , Modelos Químicos , Ácido Pentético/farmacologia , Ligação Proteica , Conformação Proteica , Albumina Sérica/metabolismo , Fatores de TempoRESUMO
The thrombin-catalyzed cleavage of N-terminal fibrinopeptide A (FPA) from the two Aalpha-chains of fibrinogen exposes aggregation sites with the critical sequence GPR located just behind FPA. It is well known that exposure of both GPR sites transforms fibrinogen into self-aggregating, fully coagulable alpha-fibrin monomers, but the fibrin precursor with one site exposed and one FPA intact has eluded description. The formation of this "alpha-profibrin" in the course of thrombin reactions and its distribution among both the aggregating and non-aggregating components of the reactions are characterized here by immunoprobing electrophoretic and gel chromatographic separations using monoclonal antibodies specific for FPA and for exposed GPR sites. These analyses show alpha-profibrin to be a non-aggregating derivative indistinguishable from fibrinogen in solutions that are rich in fibrinogen relative to dissolved fibrin. But alpha-profibrin forms soluble complexes with alpha-fibrin monomer under conditions in which it and fibrin predominate over fibrinogen. It was isolated as a complex with fibrin by gel chromatography of cryoprecipitates and then separated from the fibrin either by electrophoretic gel shifts induced with a peptide analog of the GPR aggregation site or by chromatographic gel shifts induced with monoclonal anti-FPA antibody. The weak aggregation of alpha-profibrin with itself and with fibrinogen conforms with prior indications that coupled interactions through the paired GPR sites on fibrin monomers are pivotal to their aggregation. It is suggested that alpha-profibrin may be a hypercoagulable fibrin precursor because it is converted to alpha-fibrin monomer faster than fibrinogen converts to monomer.
Assuntos
Fibrina/química , Fibrinogênio/química , Fibrinopeptídeo A/química , Sequência de Aminoácidos , Eletroforese em Gel de Ágar/métodos , Fibrina/metabolismo , Fibrinopeptídeo A/metabolismo , Humanos , Cinética , Substâncias Macromoleculares , Oligopeptídeos/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Trombina/metabolismoRESUMO
Cadaveric aortic intimas with uncomplicated atherosclerosis were examined to determine the distribution and polypeptide chain composition of fibrinogen-related protein. Immunohistochemical staining showed deposits rich in fibrinopeptides A and B. The deposits were usually disseminated throughout intimas of moderate thickness < 0.7 mm, but were distributed focally in elongate patches bounded both lumenally and medially by deposit-free tissue in thick atheromas. Saline extracts generally showed undegraded monomers and dimers by electrophoresis. The residual protein contained A alpha and gamma-chains that were cross-linked predominantly (>80%) into unresolved high M(r) (>200 kd) derivatives, whereas B beta-chains were left non-cross-linked, as occurs in late stages of cross-linking by transglutaminases. The resolved components had electrophoretic mobilities corresponding to characteristic products of both factor XIIIa and tissue-transglutaminase. A greater incorporation of alpha- rather than gamma-chains into cross-linked products implicated tissue-transglutaminase as contributing heavily. By contrast, vascular graft pseudo-intimas and a cadaveric clot were rich in degraded fibrin devoid of fibrinopeptide A, and cross-linked in patterns typical of XIIIa with gamma 2 dimers constituting the principal product. The findings indicate that the fibrinogen in the aortic intima is comparatively well protected from thrombin and plasmin, and that much of it is deposited through direct cross-linking by tissue-transglutaminase without being converted to fibrin.