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Biomedical subjects

M F Scully

Publications and source records attributed to M F Scully.

At least 37 records · Page 2Linked 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

Prothrombin cleavage by human vascular smooth muscle cells: a potential alternative pathway to the coagulation cascade.

Thrombin is a potent mitogen for human vascular smooth muscle cells (HVSMC) and its enzymatic activity is required for this function. The present study demonstrates that prothrombin is also mitogenic for HVSMC due to the generation of enzymatically active thrombin which occurs upon incubation of prothrombin with the cells. Analysis by SDS-PAGE, immunoblotting, and amino acid sequencing revealed that prothrombin incubated with HVSMC undergoes limited proteolysis. Prethrombin 1 was formed through cleavage at R155-S156. Cleavage at R271-T272 generated fragment 1.2 and prethrombin 2 whilst cleavage at R284-T285 yielded truncated prothrombin 2 (prethrombin 2'). However, cleavage at R320-I321 which, during prothrombin activation produces two-chain alpha-thrombin, was not detectable. Studies on HVSMC-conditioned medium revealed that a similar pattern of prothrombin cleavage occurred by a cell-secreted factor(s). Amidolytic activity analysis indicated that 1-3% catalytically active thrombin-like activity was generated upon incubation of prothrombin with HVSMC-conditioned medium. By treating conditioned medium with various classes of proteinase inhibitors or hirudin, it was determined that prothrombin is cleaved by a cell-derived serine proteinase-like factor(s) at R271-S272 and by alpha-thrombin at R155-S156 and R284-T285. Antibodies neutralising the activity of either urokinase, tissue plasminogen activator, or factor Xa failed to alter the prothrombin cleaving activity of conditioned medium. This activity which may catalyse an alternative pathway for the generation of thrombin, was eluted from a gel filtration column as a single peak with apparent molecular mass of 30-40 kDa.

Adolescent↗
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↗
PubMed1995

Structure/function aspects of neutral P1 residue peptide inhibitors of thrombin.

Control of thrombin by its inhibition in indications such as myocardial infarction, unstable angina or stroke has been demonstrated to be therapeutically valuable. However restoration of hemostasis by targeting thrombin while avoiding its fellow serine proteinases, (e.g. plasmin, trypsin), remains a challenge of medicinal chemistry. Tripeptide-boronates and -phosphonates with neutral P1 side chains meet these criteria. Development of novel, high yielding chemical routes furnishes a wide range of un-natural P1 functionalities, demonstrating that this indeed is a class effect with selectivity conferred by the uncharged P1 residue. For example N-benzyloxycarbonyl-D-phenylalanylprolyl-1- (3-methoxypropyl) boroglycine ester (1) has a Ki value for thrombin of 7 nM and greater than two order of magnitude higher with all other serine proteinases tested. The ester group determines the kinetics of inhibition by tripeptide phosphonates, with diphenylphosphonates being slow tight binding inhibitors, showing 50% reversibility of inhibition. Therefore this design of inhibitors offers a facile strategic approach to development as thrombin specific pharmaceutical agents.

Amino Acid Sequence↗
PubMed1994

Thrombin receptor activating peptide does not stimulate platelet procoagulant activity.

Platelets after challenge with alpha-thrombin alone, collagen alone or thrombin/collagen mixture were observed to increase the rate of activation of prothrombin by factor Xa in the presence of factor Va and calcium ion (platelet procoagulant activity) by a maximum of 25, 45 and 110 fold, respectively. The increase in platelet procoagulant activity due to these agonists has been described previously and arises from increased expression of phosphatidylserine on the platelet surface. When platelets were treated with the thrombin receptor activating peptide (TRAP) (SFLLRNPNDKYEPK), alone or in the presence of collagen or thrombin, no change in platelet procoagulant activity was observed at concentrations of TRAP sufficient to cause increased intracellular calcium levels and protein phosphorylation in a manner similar to that of thrombin. In addition, no increase in platelet procoagulant activity was seen upon treatment with TRAP in the presence of inactivated thrombin (PPACK-thrombin). These results suggest that the thrombin-mediated increase in procoagulant activity may be due to activation of a thrombin receptor distinct from the recently cloned G-protein-coupled receptor, or to other proteolytic events on the platelet surface.

Amino Acid Sequence↗
PubMed1994

Mercury compounds induce a rapid increase in procoagulant activity of monocyte-like U937 cells.

When monocytic leukaemia line U937 cells were incubated in the presence of HgCl2 there was a rapid increase in tissue factor (TF)-dependent procoagulant activity, reaching a maximum (equivalent to the total TF activity observed when cells had been subjected to a freeze/thaw cycle) after 15 min at 50 microM HgCl2 and after 30 min at 10 microM HgCl2. Two other heavy metal compounds, AgNO3 and phenylmercuric acetate, caused a similar increase in TF activity. The increase was independent of protein synthesis. Other reagents tested, CdCl2, ZnCl2, NiCl2, ADP, FMLP and monocyte chemotactic factor (MCF-1), did not cause a rapid increase in functional activity, when tested under the same experimental conditions. The addition of HgCl2 to the cells causes, in a concentration-dependent manner, a 10-12-fold increase in intracellular calcium (Cai) which coincides with increase in TF activity. Calcium ionophore also caused an increase in TF activity of the U937 cells. Upon treatment with HgCl2 the cell surface of U937 cells showed a large increase in the level of phosphatidylserine (PS) on the cell surface (as measured by potentiation of the rate of activation of prothrombin by factor Xa-factor Va) but with no change in the level of TF antigen on the cell surface. We consider that the TF is present on the cell surface of the monocyte but relatively inactive towards the physiological substrate, factor X (FX), until HgCl2 causes a change in the polarity of the cell membrane exposing PS on the outer leaflet by a mechanism likely to be enhanced by the increase in intracellular calcium.

Blood Coagulation↗
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↗
PubMed1994

Annexin V as a probe of the contribution of anionic phospholipids to the procoagulant activity of tumour cell surfaces.

The ability of anionic phospholipids (especially phosphatidylserine, PS) on the outer membrane leaflet of four tumour cell lines to support different stages of the extrinsic pathway of coagulation was probed using annexin V as an inhibitor. The procoagulant activity of two tumorigenic (MKN-28, human gastric carcinoma, Hep3B, human hepatoblastoma) and two non-tumorigenic (HepG2, human hepatocellular, HOC-1, human ovarian carcinoma) cell lines were observed to be inhibited by annexin V, although significant differences (observed as IC50 with respect to annexin V) were noted for each stage of coagulation and between different cell types. This was considered to suggest a restricted accessibility of PS in the vicinity of coagulation factors on the surface of the cell. PS levels, as estimated by binding of 125I-annexin V, were high on two of the cell lines tested, equivalent to 24 x 10(6) sites per cell for HepG2 (Kd 128 nM) and 6.5 x 10(6) sites per cell for MKN-28 (Kd 50 nM). During 9 days' culturing of HepG2 and MKN-28, the number of sites per cell remained constant. However, perhaps supporting a proposal of reduced availability, there was an observed fall in PS-dependent procoagulant activity of HepG2 and MKN-28 cells, subsequent to a peak on reaching confluency at 3 days. Both prothrombinase activity and total procoagulant activity fell, even though the number of 125I-annexin V binding sites remained constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Anions↗
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↗
PubMed1993

In vitro and in vivo characterization of a neutral boron-containing thrombin inhibitor.

Peptide boronic acid derivatives have proven to be very potent inhibitors of serine proteases with boroarginine derivatives being particularly potent thrombin inhibitors. The importance of the charged side chain of arginine has been investigated by synthesizing a derivative in which this side chain has been replaced by a neutral one. This boronic acid derivative, D-benzyloxycarbonyl (Z)-Phe-Pro-methoxypropylglycine-pinanediol (MpgC10H16), inhibited thrombin by a competitive mechanism with an inhibition constant (Ki) of 8.9 nM. In comparison to boroarginine derivatives, Z-D-Phe-Pro-boroMpgC10H16 displayed higher selectivity for thrombin over trypsin (Ki = 1.1 microM) and plasmin (Ki = 15.7 microM). Prolongation of thrombin time and activated partial thromboplastin time were observed with micromolar concentrations of Z-D-Phe-Pro-boroMpgC10H16. In a thrombin-dependent in vitro aggregation assay with human platelets, Z-D-Phe-Pro-boroMpgC10H16 inhibited aggregation with an IC50 of 85 nM. When tested in a thrombin-dependent platelet accumulation model in the rat, a bolus injection of (Z)-D-Phe-Pro-boroMpgC10H16 (0.3-3 mg/kg) inhibited platelet accumulation. Thus, the substitution of the charged guanidino group in the P1 side chain by the neutral methoxy group resulted in a potent and highly selective thrombin inhibitor with an interesting pharmacological profile with in vitro as well as in vivo models.

Animals↗
PubMed1993

Benzyloxycarbonyl-D-Phe-Pro-methoxypropylboroglycine: a novel inhibitor of thrombin with high selectivity containing a neutral side chain at the P1 position.

Thrombin, the blood-clotting enzyme, is a serine proteinase with trypsin-like specificity and is able to cleave Arg-Xaa peptide bonds but only in a very limited number of substrates (and sites therein). For the prevention and treatment of thrombosis the control of thrombin activity is a key target, and a variety of synthetic inhibitors have been introduced recently, all of which have a positive charge at the P1 site. We report the synthesis of the first example of a new class of inhibitor containing a neutral side chain at the P1 site, the peptide benzyloxycarbonyl-D-Phe-Pro- methoxypropylboroglycine. The peptide is a potent inhibitor of thrombin [Ki (limiting) = 7 nM] and is highly selective for its target enzyme in respect of other serine proteinases. This may be expected to confer considerable advantage in terms of specificity of action and reduced toxicity over conventional, positively charged, inhibitors.

Amino Acid Sequence↗