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Biomedical subjects

M F Scully

Publications and source records attributed to M F Scully.

At least 19 recordsLinked to original sources
PubMed1995

Role of thrombin in pulmonary fibrosis.

Pulmonary fibrosis commonly develops in systemic sclerosis. We assessed the role of thrombin in promoting fibroblast proliferation in the lungs in this disorder. Bronchoalveolar lavage fluid (BALF) thrombin concentrations were higher in ten patients with systemic sclerosis than in 12 healthy controls (14.6 vs 3.6 nmol/L, p < 0.02), but values in patients with cryptogenic fibrosing alveolitis (n = 10) or sarcoidosis (n = 10) were not increased. BALF from all patients induced fibroblast proliferation. This proliferation was attenuated by thrombin inhibitors for BALF from systemic sclerosis patients only. We suggest thrombin contributes to lung fibroblast proliferation in this disorder.

Alveolitis, Extrinsic Allergic↗
PubMed1995

Bound plasminogen is rate-limiting for cell-surface-mediated activation of plasminogen by urokinase.

The ability of U937 monocyte-like cells and KATO III cells (a human gastric carcinoma line) to potentiate activation of plasminogen by single-chain urokinase-type plasminogen activator (scu-PA), as mediated by the cell receptor for urokinase (u-PAR), was compared. It was observed that, although the concentration of u-PAR on these cell lines differed considerably (U937 cells: 5000 receptors/cell, Kd 0.35 nM; KATO III cells: 400 receptors/cell, Kd 0.85 nM), the rate of activation of plasminogen by scu-PA in the presence of the same density of each cell line was equivalent. From data generated in the presence of increasing concentrations of scu-PA, the kcat, for plasminogen activation in the presence of each cell line was calculated and found to differ by 26-fold (0.36 s-1 on U937 cells; 9.25 s-1 on KATO III cells). However, the Km for plasminogen with respect to the rate of formation of plasmin was lower than the Kd for binding (0.2 microM compared with 0.5 microM on U937 cells; 0.34 microM compared with 1.6 microM on KATO III cells). A rapid transformation from Glu-plasminogen (native plasminogen with N-terminal Glu) to Lys-plasminogen (plasmin-degraded plasminogen with primarily N-terminal Lys-77) occurred on the surface of U937 cells (unlike KATO III cells), but this transition did not coincide with faster rates of plasminogen activation. From this evidence it is concluded that the accessibility of bound plasminogen acts to limit the rate of activation by cell-bound urokinase. The significance of this proposal is that the proteolytic potential of the cell-mediated activation of plasminogen would be controlled by the accessibility of plasminogen for activation rather than by the concentration of u-PAR (the latter may act to localize proteolysis to appropriate domains on the surface of the cell).

Cell Line↗
PubMed1995

Evidence for a protein S receptor(s) on human vascular smooth muscle cells. Analysis of the binding characteristics and mitogenic properties of protein S on human vascular smooth muscle cells.

The presence of specific binding sites for the coagulation factor protein S (PS) on the surface of human vascular smooth muscle cells (HVSMC) is described. The binding characteristics of 125I-PS to HVSMC were studied and found to be saturable, reversible and, as described by the Hill equation, co-operative (h 1.74; Kd 0.33 nM). Autoradiographic analysis of detergent extracts of HVSMC chemically cross-linked with 125I-PS and separated by SDS/PAGE revealed radioactivity associated with two proteins with apparent molecular masses of 220 and 230 kDa respectively. The mitogenic activity of PS on HVSMC was also investigated. Protein S was shown to stimulate DNA synthesis of growth-arrested HVSMC and to support their proliferation under low-serum conditions in a sustained and dose-dependent manner.

Adolescent↗
PubMed1995

Increased expression of procoagulant activity on the surface of human platelets exposed to heavy-metal compounds.

One of the essential roles for platelets in haemostasis is in the potentiation of blood clotting due to the contribution of anionic phospholipid from the surface of the cells, as an essential cofactor to the proteolytic reactions of coagulation (platelet procoagulant activity). Only a limited number of agonists are known to initiate platelet procoagulant activity. In this study the rate of thrombin formation on the platelet surface was observed to increase in a dose-dependent manner upon treatment of washed platelets with heavy-metal compounds. Unlike the immediate increase observed upon treatment of platelets with calcium ionophore, A23187, the change due to these agents was progressive, approaching a maximum after 10 min. The maximum-fold acceleration of the rate of thrombin formation compared with control platelets was calculated for HgCl2 (56-fold), AgNO3 (42-fold) phenylmercuriacetate (24-fold) and thimerosal (14-fold), compared with 70-fold observed for calcium ionophore. The increase in procoagulant activity due to HgCl2 coincided with a large increase in intracellular calcium and phosphorylation of 22 and 45 kDa proteins. It is considered that the mechanism responsible for the increase in procoagulant activity is exposure of anionic phospholipids. This was detected by a 2-fold increase in the binding of 125I-annexin V upon addition of HgCl2, compared with resting platelets (3-fold on treatment of platelets with calcium ionophore). In contrast to the generation of activity by A23187 and other known agonists of this reaction, heavy-metal compounds appeared to cause little or no release of microparticles from the platelet surface. Since HgCl2 did not cause aggregation of platelets or significant release of serotinin, these findings may give further support to the need for exposure and ligation of glycoprotein IIb:IIIa for vesiculization to occur. Treatment of platelets with heavy metals may constitute a new approach to investigating the early changes in the cell membrane which lead to increased expression of anionic phospholipid.

Annexin A5↗
PubMed1995

Tissue factor expression correlates with histological grade in human pancreatic cancer.

The transmembrane cellular receptor tissue factor (TF) is the primary initiator of the coagulation cascade in man. Expression of TF was determined in 55 specimens of ductal adenocarcinoma of the pancreas and was found to correlate strongly with the degree of histological differentiation. A significant linear trend was observed with stronger immunoreactivity observed in poorly differentiated tumours (chi 2 = 6.69, P = 0.0098). No TF staining was seen in pancreatic samples from normal controls (n = 18). As expression of TF may be associated with tumour progression, its analysis could provide useful prognostic information in patients with pancreatic malignancy.

Adult↗
PubMed1995

Thrombin induces the redistribution and acute release of tissue factor pathway inhibitor from specific granules within human endothelial cells in culture.

Tissue factor pathway inhibitor (TFPI) is a vascular anticoagulant that regulates the tissue (TF)-dependent pathway of coagulation. The majority of intravascular TFPI is thought to be noncovalently bound to the vessel wall. Our immunolocalization studies in cultures of human umbilical vein endothelial cells (HUVEC) and immortalized EA.hy926 cells that TFPI is located in well-defined granules evenly spread over the cell surface and with apical polarization within the cytoplasm. These granules are smaller than and distinct from Weibel-Palade bodies. Upon treatment of cultured cells with low concentrations of thrombin (0.01 to 1 NIH U/mL), a marked redistribution of TFPI, occurred with patching in focal points and increased exposure of both TFPI antigen and anticoagulant activity on the surface of the stimulated endothelial cells. This redistribution was paralleled by an acute release of TFPI in the cell medium. EA.hy926 cells responded more readily to thrombin stimulation than HUVECs. The process was inhibited by both hirudin and anti-thrombin receptor antibody. Our findings demonstrate a novel mechanism by which thrombin may exert a negative feedback control on blood coagulation. Therefore, this pathway can be physiological importance in controlling TF-mediated thrombin generation.

Biological Transport↗
PubMed1994

Beta-thalassemia intermedia in a Lebanese child due to homozygosity for the -88 (C-->T) mutation.

We report a case of beta-thalassemia intermedia involving a 3-year-old male child of Lebanese descent. Molecular studies of the family showed that he is homozygous for the -88 (C-->T) beta (+)-thalassemia mutation. This mutation is the second most common cause of beta-thalassemia in Black populations, and has also been reported in Asian Indians. A review of Lebanese beta-thalassemia cases revealed considerable mutation heterogeneity and excess homozygosity due to consanguinity.

Adult↗
PubMed1993

Activation of protein C and its distribution between its inhibitors, protein C inhibitor, alpha 1-antitrypsin and alpha 2-macroglobulin, in patients with disseminated intravascular coagulation.

Activation and inactivation of protein C during the clinical course of disseminated intravascular coagulation (DIC) was studied in three patients by qualitative (Western blotting) and quantitative (ELISA) analysis and the intensity of procoagulant activity monitored by the measurement of thrombin and factor Xa antithrombin III complexes. In one patient, inhibitor complexes of APC with protein C inhibitor (PCI) and alpha 1-antitrypsin (alpha 1-AT) were observed and the latter predominated at presentation. Both disappeared during the development of remission but the loss of alpha 1-AT complexes preceded PCI complexes which on Western blotting appeared to increase in intensity prior to disappearance. The two other patients bled to death from uncontrollable haemorrhage. In both cases, APC/inhibitor complexes with alpha 2-macroglobulin (alpha 2-M) in addition to PCI and alpha 1-AT were detected and persisted until death. Although PCI appeared to be the primary inhibitor in all three cases, alpha 1-antitrypsin and particularly alpha 2-macroglobulin appeared to assume greater roles in the two fatal cases. These data are similar to previous findings in an experimental animal model of DIC that suggested that alpha 2-macroglobulin and alpha 1-antitrypsin become more important inhibitors of APC as the primary inhibitor PCI is consumed in the face of a sustained procoagulant challenge.

Adult↗
PubMed1992

Non-specific influence of chemical modification upon the properties of antithrombin III:modification of carboxyl groups.

The ability of antithrombin III to inhibit thrombin was observed to be rapidly inactivated upon specific modification of carboxyl groups. The loss of activity, upon treatment with nitrotyrosyl ester in the presence of 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate, was concomitant to the incorporation of 2 moles of nitrotyrosine per mole of inhibitor indicating the modification of only two carboxyl groups. Moreover, the modification occurred with loss, also, of the ability of the native protein to bind tightly to heparin. The modified antithrombin III retained a reduced affinity for heparin (eluting at 0.3M NaCl from heparin Agarose) and was observed to be a competitive inhibitor of the heparin-dependent rate of inhibition of thrombin by native antithrombin III. However, FAB-MS (fast atom bombardment mass spectroscopy) analysis of digests of modified material gave no indication that modification was localized to specific Asp or Glu residues. It is concluded that the loss of activity is due to deleterious change in conformation during modification. These findings, together with our previous report upon tryptophan modification of antithrombin III [1] suggest that the nature of the molecule is such that considerable care must be taken in interpretation of results when investigating the structure/function relationships of this protein by chemical modification.

Antithrombin III↗
PubMed1992

Generation of forms of fragment E with differing thrombin-binding properties during digestion of fibrinogen by plasmin.

Two principal forms of fragment E are generated upon digestion of fibrinogen by plasmin, according to the concentration of enzyme used. At a high concentration of plasmin (above 10 micrograms/ml), a form lacking fibrinopeptide A (FpA) at the N-terminus of the A alpha-chain was generated. This form of fragment E caused a dose-dependent increase in thrombin clotting times but had no measurable inhibitory activity towards thrombin cleavage of D-phenylalanyl-L-pipecolyl-L-arginine p-nitroanilide. At a low concentration of plasmin (less than 1 microgram/ml), fragment E containing 35-40% of the original amount of FpA was present in the terminal digest. The FpA-containing form of fragment E inhibited thrombin cleavage of fibrinogen, inhibited amidolytic activity and bound to the enzyme with an affinity 3-fold tighter than fibrinogen itself (Kd 4.1 +/- 0.3 microM as opposed to 12.7 +/- 1.8 microM). During digestion of fibrinogen at low plasmin concentration, up to 65% of the FpA was cleaved just subsequent to the progressive release of B beta-(1-42)-peptide, and the Arg-16-Gly-17 bond of the A alpha-chain became relatively stable towards plasmin action when present in fragment E (and possibly fragment Y). It is proposed that both forms of fragment E can inhibit clotting by binding to the fibrin(ogen)-recognition site (anion-binding exosite) of thrombin. The FpA-containing form of fragment E can also inhibit binding that occurs distal to the P1 site and thereby interfere with amidolysis of the peptide substrate. Our finding of a lability of the Arg-16-Gly-17 bond in the early phase of digestion may provide an alternative explanation of the increased FpA concentrations observed during thrombolytic therapy.

Binding Sites↗
PubMed1991

Influence of intrinsic and extrinsic plasminogen upon the lysis of thrombi in vitro.

When the rate of lysis of artificial thrombi (prepared from plasma or whole blood) was expressed according to the concentration of tissue type plasminogen activator (t-PA) or single chain urokinase type plasminogen activator (sc-uPA) then bell-shaped dose response curves were obtained, low rates being observed at concentrations of activator greater than 500 units/ml. Bell-shaped dose response curves were not observed for rate of lysis of artificial thrombi over the concentrations of streptokinase tested (SK) or for the lysis of plasma gel clots by any of the activators tested. Further investigation indicated that the preponderant mechanism for dissolution of thrombi at 500 units/ml of t-PA was by activation of the plasminogen within the thrombus (intrinsic) since the plasminogen present in the plasma perfusing the thrombus (extrinsic) rapidly became depleted. On the other hand, at 50 units/ml t-PA the lysis was observed to be due preponderantly to the action of plasmin arising from extrinsic rather than intrinsic plasminogen. If "plasminogen enriched" thrombi were prepared in the presence of Lys plasminogen (Lys-Plg) faster rates of lysis occurred and bell-shaped biometric curves were not observed.

Fibrin↗
PubMed1991

Influence of tryptophan modification upon digestion of antithrombin III by elastase.

Chemical modification of tryptophan residues in antithrombin III by dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide (HNBSB) generates products with similar levels of modification (equivalent to 0.9 mole 2-hydroxy-5-nitrobenzyl [HNB] incorporated/mole of antithrombin III) but with high or low affinity for heparin-Sepharose. Upon digestion with pancreatic or neutrophil elastase the low affinity forms generate a product of molecular weight form (55 kDa) not seen in digests of native antithrombin III or modified forms with high affinity for heparin. When measured as loss of activity the observed rate of digestion of the latter in the absence of heparin was more rapid than that of native antithrombin III. The differences in digestion are considered to be related to conformation at differences between the various forms.

Antithrombin III↗
PubMed1990

Properties of thrombolytic agents in vitro using a perfusion circuit attaining shear stress at physiological levels.

A perfusion circuit was designed to investigate in vitro some of the factors which may influence the success of thrombolytic treatment in vivo. The rate of lysis of clotted plasma and different types of artificial thrombi (fibrin thrombi or whole blood thrombi) was measured in citrated plasma or whole blood under static conditions or under shear stress equivalent to the arterial or venous circulation. With both streptokinase (SK) and tissue-type plasminogen activator (t-PA) the rate of lysis of fibrin thrombi and whole blood thrombi was reduced significantly, when compared to the conventional plasma gel clot model (25-fold and 8-fold, respectively). This occurred particularly with SK which showed a reduction (4-fold) in potency relative to t-PA under these conditions. Lysis of thrombi by both activators was observed to be faster in plasma than whole blood, and also faster with whole blood thrombi than fibrin thrombi. High shear stress, generally, caused a reduction in the rate of lysis of fibrin thrombi and an increase in the rate of lysis of whole blood thrombi compared to lysis rates under static conditions. Under all conditions of flow the lysis rate observed at 50 units t-PA per ml was much faster than that at 500 units per ml unlike the conventional plasma gel clot model.

Fibrin↗