RESUMO
Fibrin formation and turnover are intimately associated with inflammation and wound healing. To explore whether fibrin(ogen)-derived peptides exert direct effects upon cells involved in inflammation and tissue repair we examined the capacity of human fibrinopeptide B (hFpB), a thrombin-derived proteolytic cleavage product of the fibrinogen B beta-chain, to stimulate neutrophils (PMN), monocytes, and fibroblasts. hFpB caused directed cell migration of PMN and fibroblasts that was optimal at approximately 10(-8) M. This chemotactic activity was blocked by preincubating hFpB with antiserum to hFpB. hFpB was not chemotactic for monocytes. The chemotactic potency of hFpB for PMN was equivalent to that of anaphylatoxin from the fifth component of human complement (C5a), leukotriene B4 (LTB4), and formyl-methionyl-leucyl-phenylalanine (fMLP), and for fibroblasts its chemotactic activity was comparable to that of platelet-derived growth factor. hFpB did not interact with PMN receptors for C5a, LTB4, or fMLP as (a) desensitization with 10(-7) M hFpB abolished chemotaxis to hFpB but had no effect upon chemotaxis to C5a, LTB4, or fMLP and (b) induction of chemotactic responses to fMLP and LTB4 in neutrophilic leukemic cells (HL-60 cells) by incubation with dimethylsulfoxide did not extend to hFpB. Like fMLP, hFpB caused a rapid, dose-dependent increase in PMN cytoskeletal associated actin, but unlike fMLP, hFpB did not cause PMN aggregation, release of lysosomal enzymes (lysozyme and beta-glucuronidase), or the production of superoxide anion. These results suggest that hFpB may have a role in recruiting PMN and fibroblasts at sites of fibrin deposition and turnover. The capacity of hFpB to cause PMN chemotaxis without causing concurrent release of lysosomal enzymes or the production of superoxide anion is further evidence for the complexity of PMN responses to chemotactic agents.
Assuntos
Quimiotaxia de Leucócito , Fibrinogênio/fisiologia , Fibrinopeptídeo A/fisiologia , Inflamação/fisiopatologia , Neutrófilos/fisiologia , Actinas/fisiologia , Agregação Celular , Membrana Celular/metabolismo , Células Cultivadas , Complemento C5/fisiologia , Complemento C5a , Dimetil Sulfóxido/farmacologia , Fibroblastos/fisiologia , Glucuronidase/metabolismo , Humanos , Leucotrieno B4/fisiologia , Lisossomos/enzimologia , N-Formilmetionina Leucil-Fenilalanina/fisiologia , Neutrófilos/ultraestrutura , Superóxidos/metabolismoRESUMO
Eight dogs were made hypoxemic by surgical construction of a right-to-left cardiac shunt; and they were given sodium ortho-iodobenzoate (OISB) before and for 3 months after operation. The P(50) at 50% saturation) rose from 27.2 +/- 0.7 to 31.2 +/- 0.6 mm Hg (p less than 0.001) during OLSB treatment before operation and increased further to 32.2 +/- 0.8 mm Hg 3 months after creation of hypoxemia. The P(50) remained elevated for an additional 3 months after OISB was stopped. Administration of OISB before operation did not alter the red blood cell 2,3-diphosphoglycreate concentration. Hypoxemia caused an increase of this metabolite from 0.91 +/- 0.21 to 1.50 +/- 0.28 moles/moles of hemoglobin (p less than 0.05); the rise was not as great as that observed in hypoxemic dogs without OISB treatment. In spite of significant hypoxemia, hematocrit rose only slightly during the period of OISB infusion. OISB increased P50 and prevented the compensatory polycythemia regularly seen when dogs are made hypoxemic. Altering oxygen transport in this fashion may increase tissue oxygen delivery in patients with conditions which result in tissue hypoxia.
Assuntos
Eritropoese , Hipóxia/fisiopatologia , Iodobenzoatos/farmacologia , Oxigênio/sangue , Oxiemoglobinas , Animais , Artérias/transplante , Dióxido de Carbono/sangue , Ácidos Difosfoglicéricos/sangue , Cães , Átrios do Coração/cirurgia , Lactatos/sangue , Métodos , Oxiemoglobinas/análise , Piruvatos/sangue , Transplante Homólogo , Veia Cava Inferior/cirurgiaRESUMO
Simplified procedures were developed to produce and isolate the plasmin-derived B beta 1-42 fragment from human fibrinogen. Peptides generated from fibrinogen by plasmin were separated by reverse phase HPLC chromatography. Two distinct peaks were identified as having amino acid compositions identical to B beta 1-42. The average final yield of HPLC-purified B beta 1-42 was 1.2 mg per 100 mg of fibrinogen. Recovery of the peptide (40-45% when compared to the theoretical yield) was significantly higher than yields obtained by open column techniques demonstrating the advantages of this simple one-step HPLC procedure.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Produtos de Degradação da Fibrina e do Fibrinogênio/isolamento & purificação , Fibrinogênio/análise , Fragmentos de Peptídeos , Aminoácidos/análise , HumanosRESUMO
Elimination of endogenous digoxin-like immunoreactive factors (DLIF) that interfere with accurate measurement of digoxin requires use of a highly specific anti-digoxin antibody, or that DLIF be separated from digoxin before immunoassay. Several commercial digoxin-assay kits include a step for separating serum proteins and other substances from digoxin before immunoassay. We tested six different immunoassay methods (some having pretreatment steps) for their ability to detect DLIF in serum from patients in renal failure, pregnant women, and neonates, all of whom were not taking digoxin. Extracting digoxin on a column of derivatized silicagel eliminated detectable DLIF from serum as measured by enzyme immunoassay (EMIT; Syva Co.), but recovery of added digoxin was quantitative. In contrast, protein precipitation with 5-sulfosalicylic acid left significant amounts of DLIF in samples, most probably because the procedure (TDx assay; Abbott Labs.) disrupted protein-DLIF binding. A glass-bead radioimmunoassay (Immophase; Corning Medical) had the most digoxin-specific antisera. By preparative silica-gel-chromatography of serum we could eliminate or significantly minimize inaccurate digoxin measurements attributable to endogenous DLIF.
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Saponinas , Cardenolídeos , Cromatografia em Gel/métodos , Digoxina/análise , Feminino , Humanos , Técnicas Imunoenzimáticas , Recém-Nascido , Falência Renal Crônica/sangue , Gravidez , Terceiro Trimestre da Gravidez , Radioimunoensaio , Valores de ReferênciaRESUMO
Use of immunoassays that do not detect endogenous digoxin-like immunoreactive factors (DLIF) in serum significantly improves the between-assay correlation of digoxin results for patients. We investigated five different immunoassay methods (Abbott, Clinical Assays, Corning, Du Pont, and Syva), measuring digoxin by all five assays in sera from 38 patients in renal failure and in 40 patients with normal renal function, all taking digoxin. The mean standard error of the estimate (Sy X x) of digoxin results (compared for all five assays) were significantly lower for patients with normal renal function than for patients in renal failure (0.148 vs 0.293 microgram/L, P less than 0.001). Assays previously shown (Clin Chem 1987;33:401) to be the least sensitive to DLIF (Syva and Corning) gave the lowest mean scatter about the regression (Sy X x = 0.192 microgram/L, renal failure; 0.114 microgram/L, normal renal function) for all 10 assay correlations. Evidently, discrepancies between digoxin values as measured by different immunoassay kits for patients with renal disease can be attributed to DLIF. Moreover, because inaccurate digoxin results attributed to DLIF may not be limited exclusively to groups of patients with known increased concentrations of DLIF, the possibility of "latent" DLIF interference may be a problem in many other human subjects.
Assuntos
Proteínas Sanguíneas , Digoxina/sangue , Nefropatias/sangue , Saponinas , Cardenolídeos , Humanos , Imunoensaio , Análise de RegressãoRESUMO
The gamma-carboxyglutamic acid (Gla)-domain region of factor X (residues 1-44 of the light chain) was selectively removed by limited proteolysis with alpha-chymotrypsin. The Gla-domainless factor X was then activated by the factor X coagulant protein of Russell's viper venom. Apparent dissociation constants Kd' values for the interaction of factor Va with either factor Xa or Gla-domainless factor Xa were determined kinetically using prothrombin as the substrate. In the absence of phospholipid, factor Va interacted with Gla-domainless factor Xa with lower affinity (Kd' 4 X 10(-6) M) than with factor Xa (Kd' = 5 X 10(-8) M). At saturating concentrations of factor Va, maximal rates of thrombin formation were similar for either enzyme. The addition of phospholipid increased the affinity of factor Va for factor Xa approximately 75-fold (Kd' = 3.3 X 10(-10) M). In contrast, phospholipid had no effect on the affinity of Gla-domainless factor Xa for factor Va (Kd' = 4 X 10(-6) M). The maximal rate of thrombin formation increased approximately 300-fold with the addition of phospholipid to the factor Xa-factor Va system. Under the same conditions, phospholipid had no effect on the rate of thrombin formation when Gla-domainless factor Xa was the enzymatic moiety. These results demonstrate phospholipid has little or no effect on factor Va function when factor Xa has lost its Gla-mediated Ca2+-binding sites.
Assuntos
Fator V/metabolismo , Fator X/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Bovinos , Fator Xa , Cinética , Ligação Proteica , Protrombina/metabolismoRESUMO
O-Iodobenzoate interacts non-covalently with hemoglobin and lowers the oxygen affinity of the protein. In contrast to 2,3-diphosphoglycerate or inositol hexaphosphate, its interaction does not depend upon the presence of free amino groups at the beta-chain amino terminals. Lysine beta82 is one of its oxygenation linked binding sites. As with the organic phosphates, the halogenated benzoate reacts preferentially with deoxy-hemoglobin to shift the allosteric equilibrium from R to T.
Assuntos
Hemoglobinas , Iodobenzoatos/farmacologia , Regulação Alostérica , Oxigênio , OxiemoglobinasRESUMO
The formation and degradation of fibrin play a central role in hemostasis, but other activities have been associated with fibrin(ogen)-derived peptides, which suggests that products of fibrin(ogen) turnover may be involved in inflammation and wound healing. The present study was undertaken to determine whether the plasmic fibrinogen-derived peptide B beta 1-42 has effects on inflammatory cells and fibroblasts (FB). B beta 1-42 was found to be a potent chemotaxin for neutrophils (PMN) and FB, maximally stimulating PMN migration at 10(-9) mol/L peptide. Unlike the chemotactic factors f-Met-Leu-Phe and C5a, B beta 1-42 did not induce the release of lysosomal hydrolases and superoxide anion from PMN, nor did it stimulate directed movement of monocytes (MN). These features of B beta 1-42 resemble the properties of human fibrinopeptide B (hFpB), the 14-reside, thrombin-cleaveable fragment that constitutes the amino terminus of B beta 1-42, and suggested that the chemotactic effects of B beta 1-42 are mediated through its hFpB domain. Against this conclusion, however, were observations that (a) desensitization of PMN with 10(-7) mol/L hFpB ablated chemotaxis to hFpB without affecting chemotaxis to B beta 1-42; (b) antiserum to hFpB, which recognizes the B beta 1-14 sequence both free and bound to larger fragments of the B beta chain, blocked hFpB chemotactic activity but did not affect B beta 1-42-mediated chemotaxis; (c) desensitization of PMN with equimolar amounts of hFpB and beta 15-42 (10(-7) mol/L), the isolated carboxyterminal sequence of B beta 1-42 remaining after the removal of hFpB, completely inhibited B beta 1-42-mediated chemotaxis; and (d) beta 15-42 itself was chemotactic for PMN. These data indicate that PMN recognize several independent domains within the amino terminal region of the human fibrinogen B beta chain and that these biologic effects extend to mesenchymal cells.
Assuntos
Fatores Quimiotáticos/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Produtos de Degradação da Fibrina e do Fibrinogênio/farmacologia , Neutrófilos/efeitos dos fármacos , Fragmentos de Peptídeos , Complemento C5/farmacologia , Complemento C5a , Citoesqueleto/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/ultraestruturaRESUMO
Sodium o-iodobenzoate (OISB) was given intravenously to 15 dogs to test the in vivo effect of this drug on the oxyhemoglobin dissociation curve. Administration of a single dose of 500 mg/kg was followed by an average increase in P50 (PO2 at 50% oxyhemoglobin saturation) of 3.6 mmHg from 26.8 +/- 0.5 to 30.4 +/- 1.8 mmHg (corrected to pH 7.4). This elevation was sustained for 7 days. During intravenous infusions of 200 mg/kg every other day for 3 wk, there was a sustained increase in P50 of 2.6 mmHg from 27.8 +/- 1.1 to 30.4 +/- 0.9 mmHg. All dogs survived the experiment and no ill effects of the drug were noted. An increase in serum lactate and pyruvate occurred in all animals following acute or chronic exposure to the drug. There was no significant change in whole blood pH, 2,3-diphosphoglycerate concentrations, intracellular pH, or serum total phosphate. Multiple infusions of sodium cyanate (50 mg/kg per day) reduced P50 by an average of 12.2 +/- 0.3 mmHg. A subsequent single infusion of OISB (500 mg/kg) failed to increase P50. Our preliminary data indicate that pharmacological manipulation of hemoglobin O2 affinity is possible with organic compounds unrelated to erythrocyte metabolism.
Assuntos
Hemoglobinas/metabolismo , Iodobenzoatos/farmacologia , Oxigênio/sangue , Animais , Cianatos/farmacologia , Cães , Lactatos/sangue , Pressão Parcial , Piruvatos/sangueRESUMO
The activation by XCP of coagulation factor X and a factor X species lacking the Gla-domain was studied in the presence and absence of Ca2+. Both proteins could be activated at low rates in the absence of Ca2+. The activation of the unmodified factor X was stimulated by the addition of Ca2+, whereas GD factor X activation was insensitive to Ca2+. The stimulatory effect of Ca2+ seen with the unmodified factor X correlated strongly with a calcium-dependent change in intrinsic protein fluorescence. This conformational change required the Gla-domain as the fluorescence emission of GD factor X was the same with or without Ca2+. Fluorescence changes which accompanied activation were the same for both factor X and GD factor X. This suggests that the Gla-domain does not participate in the structural changes which accompany activation.
Assuntos
Ácido 1-Carboxiglutâmico , Cálcio/farmacologia , Fator X/metabolismo , Glutamatos , Venenos de Víboras/farmacologia , Animais , Bovinos , Fator Xa , Cinética , Espectrometria de FluorescênciaRESUMO
A fully automated, random access method for the determination of cannabinoids (UTHC) was developed for the Dimension AR and XL clinical chemistry systems. The method utilizes Abuscreen ONLINE reagents and a multianalyte liquid calibrator containing 11-nor-Delta(9)-THC-9-carboxylic acid. Within-run and total reproducibility, determined using NCCLS protocol EP5- T2, was less than 0.6% and 1.6% CV, respectively, at all concentrations. Calibration stability was retained for at least 30 days. An extensive evaluation of non-structurally related drugs and various physiological substances indicated lack of interference in the method. No sample carry-over was observed following a specimen containing 1886 ng/ml 11-nor-Delta(9)-THC-9-carboxylic acid. A 99.1% agreement (N = 445 samples) was found between an EMIT based method on the aca discrete clinical analyser and the Dimension UTHC method.Dimension clinical chemistry system and aca discrete clinical analyser are registered trademarks of Dade International.