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J McDonagh

Publications and source records attributed to J McDonagh.

At least 19 recordsLinked to original sources
PubMed1992

Effects of tumor necrosis factor-alpha on peroxidation of plasma lipoprotein lipids in experimental animals and patients.

Changes in the plasma lipid composition are observed in patients and animals with malignancy and certain other diseases that are consistent with peroxidation of plasma lipoprotein lipids. These changes can be observed with water-suppressed proton (H-1) and carbon-13 (C-13) nuclear magnetic resonance spectroscopy (NMR) and gas chromatography. Gas chromatography provides evidence of a decrease in polyunsaturated fatty acids relative to monounsaturated fatty acids. This evidence is consistent with that observed by C-13 NMR spectroscopy. Mediators for these effects were sought. Cytokines, known to be released in response to malignant tumor cells and to affect lipid metabolism, were injected into normal mice and their effects on the H-1 and C-13 NMR spectra of plasma lipids were observed. Mouse recombinant tumor necrosis factor-alpha (mr-TNF-alpha) significantly decreased the H-1 methyl and methylene lipid linewidths, and the C-13 spectra indicated a decrease in the relative concentration of polyunsaturated fatty acids. The same changes were directly confirmed by gas chromatographic analysis, showing decreases in the amount of linoleic and arachidonic acids and other polyunsaturated fatty acids relative to monounsaturated fatty acids and in the ratio of polyunsaturated to monounsaturated fatty acids. Serial plasma samples from volunteers receiving an infusion of endotoxin showed similar changes in their C-13 NMR spectroscopy at times when peak TNF-alpha values were measured. In addition, in these samples the C-13 NMR spectra showed direct evidence of lipid peroxidation products. These changes were similar to those observed commonly in the plasma of cancer patients. Other cytokines (human recombinant interleukin-1 alpha [hr-IL-1 alpha], hr-IL-2, mouse recombinant interferon-gamma) did not produce these effects. We conclude that TNF-alpha is a mediator (but not necessarily the only one) of changes in plasma lipoprotein lipid composition due to peroxidation and that this is a mechanism for the changes observed in the NMR spectra of plasma from cancer patients and from normal animals injected with TNF-alpha.

Animals↗
PubMed1992

A unique factor XIII inhibitor to a fibrin-binding site on factor XIIIA.

An 81-year-old woman, who presented with sudden episodes of spontaneous bleeding, was found to have a specific inhibitor of factor XIII. Her fibrin clots had approximately 70% gamma-gamma and no alpha polymer formation, under conditions where normal fibrin was fully cross-linked; the patient's clots were soluble in urea or monochloroacetic acid. Factor XIII activity in her plasma was 24%, measured by the dansylcadaverine incorporation assay. When mixed with normal plasma, the patient's plasma inhibited fibrin cross-linking; however, in mixtures of patient and normal plasma, there was no inhibition of factor XIII activity when assayed by the incorporation of dansylcadaverine into casein. Thus, this inhibitor was active against fibrin cross-linking but not against ligation of small molecules to casein. Consequently, gel electrophoresis of reduced, sodium dodecyl sulfate-solubilized fibrin clots was a simple, quantitative method that was used to measure inhibitor activity. This inhibitor is unique and has been designated inhibitor New Haven. It was neutralized by anti-IgG and anti-kappa. It did not inhibit the activation of factor XIII but did inhibit fibrin cross-linking. There was complex formation between the inhibitor and activated factor XIII (A', A*) but not between A2 or fibrinogen. Only A', A* and the 56-Kd fragment bound to affinity columns made with this IgG. The inhibitor significantly decreased the binding of A', A* to fibrin clots. These data indicate that the epitope for this inhibitor is in a fibrin binding site. It is hidden in the zymogen and expressed on A' and A*, indicating that the conformational change occurring with the cleavage of the activation peptide is sufficient to expose the fibrin binding site.

Aged↗
PubMed1991

Fibrinogen Ledyard (A alpha Arg16----Cys): biochemical and physiologic characterization.

Fibrinogen Ledyard was discovered in a 10-year-old boy with a mild bleeding history. His father had the same defect and a bleeding history after surgery. Both patients were heterozygous. The plasma fibrinogen concentration was normal immunologically (335 mg/dL) and very low functionally (52 mg/dL). Purified fibrinogen Ledyard had a prolonged polymerization, which was somewhat corrected by addition of Ca2+ ions. High performance liquid chromatography (HPLC) analyses of the fibrinopeptides released by thrombin showed 1 mol of fibrinopeptide A (FPA) and 2 mol of fibrinopeptide B (FPB) released per mole of fibrinogen Ledyard. Steady-state kinetic parameters were evaluated for release of FPA by thrombin. When the concentration of fibrinogen Ledyard was corrected to 50% of total protein, because only 50% of fibrinogen Ledyard can release FPA, the kinetic constants were similar to those of control fibrinogen (Km = 7.5 mumol/L for A alpha chain, kcat = 54 s-1). This finding indicates that the cleavage site of the A alpha chain in these abnormal molecules may not interact with the catalytic site of thrombin. The three chains of fibrinogen Ledyard were isolated on reverse-phase C4-HPLC. The sequence of the amino terminus of A alpha chain showed that Arg in position 16 was replaced by Cys in the abnormal molecules. Approximately half of fibrinogen Ledyard (52%) was clotted by reptilase, suggesting that fibrinogen Ledyard may consist of 50% normal homodimers (A alpha Arg16 . A alpha Arg16) and 50% abnormal homodimers (A alpha Cys16 . A alpha Cys16). Abnormal molecules could form disulfide bond between the A alpha Cys16 residues. Thus, the abnormal molecules have a different structure that does not bind to thrombin. Probably the abnormality of polymerization of fibrinogen Ledyard results from the interaction of the abnormal molecules with normal fibrin monomers, so that the growth of fibrin protofibrils is inhibited. This abnormal fibrinogen supports adenosine diphosphate-induced platelet aggregation in a normal manner.

Amino Acid Sequence↗
PubMed1991

Selective cytotoxicity of low-density lipoprotein to helper T cells of cutaneous T-cell lymphoma after photoperoxidation with 8-methoxypsoralen.

Lymphocyte-containing plasma subjected to photolysis in the presence of 8-methoxypsoralen (methoxsalen, 8-MOP) has previously been shown to be effective against cutaneous T-cell lymphoma and the AIDS-related complex. The mechanism of this effect was thought to involve photoreaction of 8-MOP with DNA, based on certain in vitro experiments. The results of this study suggest a different mechanism. Low-density lipoprotein (LDL) from fresh human plasma was photosensitized by addition of 8-MOP and exposure to UV light (mp-LDL), and the reactions of the LDL lipids and the chemical actions induced by these reactions were monitored. In a separate procedure, LDL was peroxidized with hydrogen peroxide and peroxidase (p-LDL). mp-LDL and p-LDL were then tested in cytotoxicity assays on HuT-78 helper T cells of cutaneous T-cell lymphoma. These results indicate that (a) LDL in plasma in the presence of very low concentrations of 8-MOP (200 ng/mL) can be peroxidized by UV light; (b) this photoperoxidized LDL is cytotoxic to helper T cells of cutaneous T-cell lymphoma in a dose-dependent manner; but (c) it does not kill normal lymphocytes under similar conditions. The findings also suggest alternative therapeutic strategies for treatment of cutaneous T-cell lymphoma, such as direct utilization of peroxidized LDL.

Cell Survival↗
PubMed1991

C-13 NMR spectroscopy of plasma reduces interference of hypertriglyceridemia in the H-1 NMR detection of malignancy. Application in patients with breast lesions.

We have previously described the application of water-suppressed proton nuclear magnetic resonance (H-1 NMR) spectroscopy of plasma for detection of malignancy. Subsequently, hypertriglyceridemia has been identified as a source of false positive results. We now describe a confirmatory, adjunctive technique--analysis of the carbon-13 (C-13) NMR spectrum of plasma--which also identifies the presence of malignancy but is not sensitive to the plasma triglyceride level. Blinded plasma samples from 480 normal donors and 208 patients scheduled for breast biopsy were analyzed by water-suppressed H-1 and C-13 NMR spectroscopy. Triglyceride levels were also measured. Among the normal donors, there were 38 individuals with hypertriglyceridemia of whom 18 had results consistent with malignancy by H-1 NMR spectroscopy. However, the C-13 technique reduced the apparent H-1 false positive rate from 7.0% to 0.6%. Similarly, in the breast biopsy cohort, C-13 reduced the false positive rate from 2.8% to 0.9%. Furthermore, the accuracy of the combined H-1/C-13 test in this blinded study was greater than 96% in 208 patients studied.

Adult↗
PubMed1990

Alteration of aliphatic lipid proton NMR linewidths by malignant tumors in guinea pigs.

Water-suppressed proton nuclear magnetic resonance spectroscopy was used to observe plasma lipoprotein lipid methyl and methylene resonances from guinea pigs which had been injected with viable or heat-killed line 1 or line 10 tumor cells or sterile oil. It was shown that the widths of these resonances became significantly sharper as the number of tumor cells grew. Plasma from tumor-free control animals showed no change in the NMR linewidths. It is concluded that the changes observed reflect a specific host response to viable tumor cells, and in these models there is a reciprocal relationship between the number of viable tumor cells and the linewidths of plasma lipoprotein methyl and methylene resonances.

Animals↗
PubMed1989

Electron microscopy and hydrodynamic properties of factor XIII subunits.

Factor XIII is a transglutaminase important in blood coagulation and fibrinolysis. Its function is to catalyze peptide bond formation between the gamma-carboxamide group of glutamines in one protein and the epsilon-amino group of lysine in another. There are two zymogenic forms of factor XIII: one is a noncovalent, intracellular dimer (A2); the other is a noncovalent, extracellular tetramer (A2B2). The catalytic function resides in the activated A chain (A2.). Purified forms of factor XIII (A2B2, A2, A2.B2, B) were prepared and analyzed by electron microscopy, gel filtration, and gradient centrifugation. Hydrodynamic constants were derived. Electron microscopy of rotary-shadowed molecules showed A2 to consist of two globular particles each about 6 x 9 nm in size. The A2 dimer is significantly elongated, 18 nm long and 6 nm in diameter. Sedimentation and gel filtration of the A2 dimer are consistent with this asymmetric structure. B protein is a filamentous, flexible strand with kinks, with a contour length of 30 nm and a diameter of approximately 2-3 nm. The sedimentation and gel filtration behavior of the B subunit are characteristic of a highly asymmetric molecule. The observed structure of the B subunit, combined with data for its amino acid sequence, suggests a modular structure. The B subunit is a member of a family of proteins composed of tandem, repeating structures (referred to as GP-I domains); the structure seen by electron microscopy for B subunit is probably applicable to all proteins in this family. Plasma and platelet factor XIII zymogens are tetrameric and dimeric, but B protein, in the absence of A protein, appears to be monomeric. Our model for the A2B2 zymogen has the elongated A2 dimer forming the core and the two B strands wrapping around the outside.

Centrifugation, Density Gradient↗
PubMed1989

Diagnosis of disseminated intravascular coagulation. Role of D-dimer.

Detection of the cross-linked fibrin degradation fragment, D-dimer, in patients at risk for disseminated intravascular coagulation (DIC) is strong evidence for the diagnosis. D-dimer confirms that both thrombin generation and plasmin generation have occurred. Patients at risk for DIC (58) and normal controls (7) were studied. Thirty-three patients had DIC--with fragment D-dimer identified in their serum by immunoblotting. Latex agglutination measurements of fibrin(ogen) degradation products (FDPs) and D-dimer were compared with immunoblotting in the detection of D-dimer. FDP measurement was extremely sensitive but not specific. D-dimer measurement was less sensitive but highly specific. Used in tandem, screening with FDP and confirming with D-dimer, sensitivity and specificity were maximized, rendering a predictive value of a confirmed FDP of 100% in this cohort. D-dimer is a valuable adjunct for the laboratory diagnosis of DIC but is most appropriately used as a confirmatory test for the very sensitive FDP test.

Adult↗
PubMed1989

A simple, automated functional assay for protein C.

Protein C is an important anticoagulant in circulating blood. The balance between the procoagulant and anticoagulant pathways determines whether a patient is at risk for thrombosis or hemorrhage, and low levels of protein C may result in thrombotic disease. It is important for clinical coagulation laboratories to measure protein C. For this purpose, a simple functional assay is needed. This article describes an automated assay for functional protein C with activation by copperhead venom. The authors have automated this assay for a centrifugal analyzer (COBAS Bio) and have proved the specificity of the assay by comparing values obtained amidolytically with those obtained with a radioimmunoassay for protein C.

Blood Coagulation Disorders↗
PubMed1988

Thrombin binding to the A alpha-, B beta-, and gamma-chains of fibrinogen and to their remnants contained in fragment E.

In order to study thrombin interaction with fibrinogen, thrombin binding to fragments D and E (prepared by plasmin digestion of fibrinogen) and to intact S-carboxymethylated chains of fibrinogen (A alpha, B beta, and gamma) was analyzed by autoradiography, immunoblotting, and affinity chromatography. Complex formation was observed between late fragment E and thrombin but not with fragment D. The three reduced chain remnants of fragment E all formed complexes with thrombin. Also, thrombin bound to the intact, separated A alpha, B beta, and gamma chains of fibrinogen as well as to the alpha and beta chains of fibrin. In these experiments the extended substrate-binding site, but not the catalytic-binding site, was being examined because fragment E had as its amino-terminal amino acids Val20 in the alpha chain, Lys54 in the beta chain, and Tyr1 in the gamma chain. Also, thrombin inhibited in its active center by D-phenyl-alanyl-L-prolyl-L-arginine-chloromethyl ketone bound to fragment E and to the separated chains in the same manner as unmodified thrombin. A lysine residue to thrombin was essential for its binding to fibrinogen. Thrombin attached to CNBr-activated Sepharose through its amino groups did not bind to fragment E, but when thrombin was attached through its carboxyl groups, it bound fragment E.

Amino Acid Sequence↗
PubMed1988

Macrophage migration in fibrin gel matrices. II. Effects of clotting factor XIII, fibronectin, and glycosaminoglycan content on cell migration.

We investigated the migration of oil-induced, guinea pig peritoneal macrophages in three-dimensional fibrin matrices, with particular attention to variables which modified fibrin gel structure and/or its adhesive properties for cells. The variables studied were fibrin concentration, gel cross-linking, and fibronectin and glycosaminoglycan content. Macrophage migration was an inverse linear function of fibrinogen concentration. Little or no fibrinolysis accompanied macrophage migration; rather, macrophages migrated through fibrin gels by an active process associated with marked distortions of cell shape and specialized plasma membrane contacts with fibrin strands. Fibrin matrices prepared from fibrinogen that had been depleted of clotting factor XIII and/or fibronectin provided a superior matrix for macrophage migration. Both the number of migrating cells and distance of migration were reduced when the gel matrix included fibronectin and was cross-linked by factor XIII. A hexapeptide containing the fibronectin cell-binding RGDS sequence reversed this migration inhibition, suggesting that fibronectin immobilized by cross-linking to fibrin may have bound macrophages and restricted cell migration. Hyaluronic acid, heparin, and heparan sulfate inhibited macrophage migration in cross-linked fibrin-fibronectin gels over a range of concentrations. These data are relevant to an understanding of macrophage migration in vivo where cross-linked fibrin-fibronectin gels containing variable amounts of glycosaminoglycans are deposited in tissues in immunologic reactions and in many other types of pathology.

Animals↗
PubMed1988

Plasminogen San Antonio: an abnormal plasminogen with a more cathodic migration, decreased activation and associated thrombosis.

An abnormal plasminogen (San Antonio) has been isolated from a patient with axillary vein thrombosis. A decreased level of fibrinolytic activity was detected in both plasma and a purified system. The molecular abnormalities were investigated with both functional and immunological tests. Slightly decreased antigen concentration was noted in plasma. By crossed immunoelectrophoresis, the patient and his two children had a second small arc and the primary arc migrated more cathodically. A distinct isozyme was detected in the abnormal plasminogen. Functionally, this abnormal plasminogen is characterized by failure to enhance maximal conversion to plasmin, especially by plasminogen activators, which are enhanced by fibrin or fibrin degradation products. The proband and his children are heterozygous for this abnormal plasminogen.

Axillary Vein↗
PubMed1988

B protein of factor XIII: differentiation between free B and complexed B.

Plasma factor XIII is a complex of A and B proteins noncovalently linked in a tetramer, A2B2, Enzyme-linked immunosorbent assays (ELISA) were developed to measure the separate factor XIII proteins and the complex. All of the A protein in plasma is in the zymogen complex. The B assay measures the total amount of B protein in plasma (both free B and complexed B). This was confirmed by nondenaturing gel electrophoresis and immunoblotting, which showed two bands for B in plasma with this antibody. Two assays were developed to measure A2B2 complex specifically. One assay used a monoclonal antibody to B to bind antigen and measured B protein in the zymogen complex only and hence the concentration of the complex. The specificity of this antibody was also shown by immunoblotting. In the second assay, the capture antibody was to B and the tag antibody was to A. These two assays gave identical results for the concentration of A2B2 (0.07 mumol/L, 21.6 micrograms/mL in normal plasma). Thus, for the first time, differentiation and quantitation of free B and complexed B in plasma was possible. The assays were used to measure factor XIII proteins in plasma from normal controls, homozygous-deficient factor XIII patients, and their heterozygous relatives. The normal concentration of A in plasma is 0.13 to 0.16 mumol/L (approximately 11 micrograms/mL), all of which is in A2B2. The total B concentration is 0.26 to 0.28 mumol/L (approximately 21 micrograms/mL), half of which is complexed. The free B concentration is 0.13 mumol/L (approximately 10 micrograms/mL). Homozygous-deficient patients have essentially no A protein, but their free B concentration is 0.11 mumol/L. Heterozygotes have decreased A2B2, but their free B is 0.11 mumol/L. These results indicate that the concentration of free B is remarkably constant and does not depend on the concentration of A2 or A2B2.

Antibodies, Monoclonal↗
PubMed1987

Fibrinogen-sepharose interaction with prothrombin, prethrombin 1, prethrombin 2 and thrombin.

Binding of prothrombin, prethrombin 1, prethrombin 2 and thrombin to fibrinogen-Sepharose was studied. Thrombin and prethrombin 2 bound to fibrinogen-Sepharose, while prethrombin 1 and prothrombin did not. Bound thrombin and prethrombin 2 were recovered from the column by eluting with 0.1 M NaCl/0.05 M Tris-HCl buffer (pH 7.4). The affinity of thrombin and prethrombin 2 to fibrinogen-Sepharose depended on ionic strength and reached a maximum at 50 mm concentration. Prethrombin 2 interacts with fibrinogen as well as thrombin; and prothrombin fragment 1.2 is not important in the formation of this complex. Thus, prethrombin 2, which is a precursor of thrombin without measurable enzymatic activity and which lacks the single cleavage at Arg-322-Ile-323 present in thrombin, has the same or very similar structural conformation as thrombin and has the same macromolecular substrate recognition site. These results confirm the earlier results that active center is not necessary in fibrinogen-thrombin interaction.

Amino Acid Sequence↗
PubMed1987

Inhibited thrombins. Interactions with fibrinogen and fibrin.

Fibrin-monomer-Sepharose was used to study thrombin binding to fibrin and the role of the enzyme active centre in this interaction. Binding properties of preformed enzyme-inhibitor complexes, as well as inhibition of thrombin already adsorbed to fibrin monomer, were investigated. No apparent difference was found in binding properties of phenylmethanesulphonyl fluoride-, D-Phe-Pro-Arg-CH2Cl- and dansylarginine NN-(3-ethylpentane-1,5-diyl)amide-inhibited thrombins. Also, the elution profile of phenylmethane-sulphonyl fluoride-inhibited thrombin from fibrinogen-Sepharose was identical with that of active thrombin from fibrin-monomer-Sepharose. Thus far the only low-Mr inhibitor that prevents thrombin from binding to fibrin monomer is pyridoxal 5'-phosphate. Preformed hirudin-thrombin complexes do not interact with fibrin. The extent to which the active centre of thrombin associated with fibrin is still accessible to substrates and inhibitors was also studied. Thrombin bound to fibrin hydrolyses a synthetic substrate at the same rate as the free enzyme. Water-soluble low-Mr inhibitors such as D-Phe-Pro-Arg-CH2Cl and dansylarginine NN-(3-ethylpentane-1,5-diyl)amide can readily modify the active centre of the fibrin-associated enzyme, and the active centre is exposed to the degree that displacement of dansylarginine NN-(3-ethylpentane-1,5-diyl)amide by D-Phe-Pro-Arg-CH2Cl is possible without disturbing the binding. Hirudin disrupts the affinity between thrombin and fibrin. These data indicate that the active centre of thrombin associated with fibrin through extended binding is fully exposed and freely accessible. It is possible that extended binding may play a regulatory role in the activation of Factor XIII by thrombin, as well as inactivation of this enzyme by antithrombin III.

Amino Acid Chloromethyl Ketones↗
PubMed1987

Characterization of thrombospondin as a substrate for factor XIII transglutaminase.

Thrombin activation of platelets induces the release of a high molecular weight glycoprotein, thrombospondin. On treatment with factor XIII transglutaminase and [3H]putrescine, thrombospondin undergoes specific incorporation of this labeled amine, with 2-3 mol of putrescine being incorporated per mol of thrombospondin. Analysis of plasmin digests of [3H]putrescine-thrombospondin showed that the Mr 53,000-core peptide contains the glutamine site for amine incorporation. In the absence of amine substrate, thrombospondin was found to provide both donor (glutamine) and acceptor (lysine) sites for intermolecular cross-links by factors XIIIa, and high molecular weight protein complexes were formed. Homopolymers of thrombospondin were also observed by electron microscopy. Thrombin-cleaved thrombospondin has more cross-linking sites accessible for [3H]putrescine incorporation or for cross-linkage to itself than does the uncleaved native protein. Examination of thrombospondin cross-linkage in the presence of other protein substrates (fibronectin, collagen, fibrinogen, and von Willebrand factor) for factor XIIIa, resulted in reduced thrombospondin polymer formation. Electron microscopy and autoradiography of fibrin clots formed in the presence of 125I-thrombospondin showed an association of thrombospondin with fibrin fibrils. However, confirmation that this association involves covalent epsilon-(gamma-glutamyl)lysyl cross-links between thrombospondin and fibrin was not obtained.

Autoradiography↗
PubMed1987

Fibrin containing gels induce angiogenesis. Implications for tumor stroma generation and wound healing.

Fibrin deposition is a consistent early event in solid tumors and healing wounds and precedes new blood vessel ingrowth in both. We now demonstrate that fibrin gels of themselves induce an angiogenic response in the absence of tumor cells or platelets. Angiogenesis was enhanced when certain chemoattractants or mitogens were included in the fibrin gel. Newly devised, inert plastic chambers with one porous surface were filled with varying contents and were implanted in the subcutaneous space of guinea pigs. Chambers filled with cross-linked homologous fibrin or plasma induced an angiogenic response within 4 days. Vessels entered chambers through the surface pores and flared out radially; angiogenesis was quantitated by point counting. Vessels were functional and matured along a gradient that proceeded from distal (least mature) to proximal. The intensity of the angiogenic response was enhanced when zymosan activated serum, an N-formylmethionine tripeptide, or platelet-derived growth factor was included in the fibrin matrix. Prior aldehyde fixation or boiling of fibrin-filled chambers inhibited angiogenesis, as did high concentrations of hyaluronic acid. Chambers filled with type I collagen or agarose did not induce new blood vessel formation, but addition of collagen did not reduce fibrin's capacity to initiate angiogenesis. The novel assay introduced here offers several advantages that should facilitate the study of angiogenesis. These include reproducibility, low background, objective and quantitative scoring, and the capacity to evaluate native molecules in animals of several species. Taken together, our findings strongly implicate fibrin or related proteins in the pathogenesis of angiogenesis and offer a new approach for elucidating the underlying molecular mechanisms.

Animals↗