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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
PubMed2006

Integrins in drug targeting-RGD templates in toxins.

Integrins are a family of heterodimeric receptors, which modulate many cellular processes including: growth, death (apoptosis), adhesion, migration, and invasion by activating several signaling pathways. Integrin-binding RGD (arginine-glycine-aspartic acid) is found in several important extracellular matrix proteins which serve as adhesive integrin ligands. The RGD motif has also been found in many toxins from snake venom and other sources that specifically inhibit integrin binding function and serve as potent integrin antagonists, particularly of platelet aggregation and integrin-mediated cell adhesion. Many of these proteins have potential as therapeutic agents which can target integrins directly. Structural and functional studies of several RGD-containing toxins suggest that the inhibitory potency of these proteins lies in subtle positional requirements of the tripeptide RGD at the tip of a flexible loop, a structural feature for binding to integrins. In addition, amino acid residues in this loop in close vicinity to the RGD-motif determine the integrin-binding specificity and selectivity. This review will present a review of integrin structure and function, and of disintegrin structural features responsible for their activity as antagonists of integrin function. The use of integrins in drug targeting and integrins as targets for drug delivery by using the RGD as a template structure will also be discussed.

Animals↗
PubMed2005

Snake venom metalloproteinase containing a disintegrin-like domain, its structure-activity relationships at interacting with integrins.

Snake venom disintegrins represent a family of RGD (Arg-Gly-Asp) or KGD (Lys-Gly-Asp)-containing proteins which have been reported to be unique and potentially useful tools not only for investigating integrin-ligand interactions, but also for the development of anti-thrombotic agents in terms of their anti-platelet activities. Snake venom proteins containing a disintegrin-like domain represent another super-family of proteins in which many of them have been demonstrated to have similar ability to inhibit platelet aggregation and integrin-mediated cell adhesion as the disintegrins. This super-family includes a large number of snake venom metalloproteinases and disintegrin related, RGD-containing snake venom proteins (disintegrin-like proteins) such as dendroaspin. Recently, a family of homologues of the snake venom metalloproteinases have been found in a wide variety of mammalian tissues as well as in other eukaryotic organisms termed ADAM (a disintegrin-like and metalloproteinase) proteins. ADAMs are members of the metazincins that also include the related matrix metalloprotease (MMPs). Some of ADAM proteins have now shown to interact with integrins, and the disintegrin-like domain may be crucial part in their function as proteases. A description of structure-activity relationships of snake venom proteins containing a disintegrin-like domain is outlined in this review, along with reports of the modulation of protein activity by recombinant mutation. Comparison is also made of the structural and functional features of the metalloproteinases in snakes compared with those from other species. The review is intended to provide insights in which may assist the development of new therapeutic approaches.

Animals↗
PubMed2004

Enhanced angiogenesis following allogeneic blood transfusion.

Blood transfusions are associated with recurrence of solid cancers. Angiogenesis is essential for cancer growth. Our aim was to determine for the first time in a prospective cohort study the effect of prestorage allogeneic leucodepleted SAGM (saline, adenine, glucose, mannitol) red cell transfusion on angiogenic factor levels and in vitro angiogenesis. Forty pretransfusion adult hospital inpatients were selected consecutively. Serum vascular endothelial growth factor (VEGF) and endostatin were measured in each patient before and after prestorage allogeneic leucodepleted SAGM red cell transfusion. All samples were exposed to an in vitro endothelial cell proliferation assay and 10 sample groups were also exposed to an in vitro whole angiogenesis assay. The median number of units transfused was 2 (minimum-maximum, 2-4). Twenty-nine (73%) patients had a rise in VEGF, with an overall increase of 118 pg/ml (quartiles -5, 306; P < 0.01). Twenty-eight (70%) patients had a decrease in endostatin, with an overall reduction of 1.2 ng/ml (quartiles 4.0, 0.0; P = 0.017). There was an overall 33% increase in endothelial cell proliferation (P < 0.01) and a 9.4% increase in in vitro whole assay angiogenesis (P < 0.01). Prestorage allogeneic leucodepleted SAGM red cell transfusions are associated with a favourable angiogenic factor imbalance and an elevation in in vitro angiogenesis.

Adult↗
PubMed2003

Modulation of integrin-binding selectivity by mutation within the RGD-loop of snake venom proteins: a novel drug development approach.

Integrins are a family of heterodimeric class I transmembrane receptors, many of which bind to the RGD sequence in adhesive proteins and mediate the adhesive interactions of a variety of cells. The RGD motif has also been found in snake venom proteins that specifically inhibit integrin binding function and serve as potent integrin antagonists. The majority of these proteins interact with beta1 and beta3 associated integrins and their potency is at least 500-2000 times higher than short RGD peptides. Structural and functional studies suggest that the inhibitory potency of these proteins lies in subtle positional requirements of the tripeptide RGD that is harboured in a defined flexible loop. The integrin-binding specificity and selectivity of each of the proteins is controlled by amino acid residues in this loop in close vicinity to the RGD-motif. The review includes an overview of the structure and function of snake-venom integrin antagonists. The ability of these proteins to control platelet aggregation, cell adhesion and ligand binding is compared to that of short linear, cyclic RGD-peptides and RGD-containing proteins and the influence of modulation of amino acid residues flanking the RGD motif is also considered. The review is intended to provide insight into the development of novel inhibitors as drugs.

Amino Acid Sequence↗
PubMed2002

Platelets and cancer.

The presence of platelets in association with cancer deposits has been recognised for over 100 years; however, the recognition of a two-way interaction has been more recent. The link between cancer spread and platelet stimulation is pivotal to understanding of the hypercoagulable state found in most cancer patients. The assistance of platelets in cancer spread may provide opportunities to interrupt this relation, thus inhibiting metastasis.

Animals↗
PubMed2001

Inhibition of human alpha-thrombin by a phosphonate tripeptide proceeds via a metastable pentacoordinated phosphorus intermediate.

X-ray crystallographic studies of human alpha-thrombin with a novel synthetic inhibitor, an acyl (alpha-aminoalkyl)phosphonate, reveal the existence of a pentacovalent phosphorus intermediate state. Crystal structures of the complex of alpha-thrombin with the phosphonate compound were determined independently using crystals of different ages. The first structure, solved from a crystal less than seven days old, showed a pentacoordinated phosphorus moiety. The second structure, determined from a crystal that was 12 weeks old, showed a tetracoordinated phosphorus moiety. In the first structure, a water molecule, made nucleophilic by coordination to His57 of alpha-thrombin, is bonded to the pentacoordinated phosphorus atom. Its position is approximately equivalent to that occupied by the water molecule responsible for hydrolytic deacylation during normal hydrolysis. The pentacoordinated phosphorus adduct collapses to give the expected pseudo tetrahedral complex, where the phosphorus atom is covalently bonded to Ser195 O(gamma). The crystallographic data presented here therefore suggest that the covalent bond formed between the inhibitor's phosphorus atom and O(gamma) of Ser195 proceeds via an addition-elimination mechanism, which involves the formation of a pentacoordinate intermediate.

Binding Sites↗
PubMed2001

High affinity binding of heparin by necrotic tumour cells neutralises anticoagulant activity--implications for cancer related thromboembolism and heparin therapy.

We have observed a striking neutralisation of the anticoagulant activity of unfractionated heparin in the presence of a pancreatic carcinoma cell line (MIA PaCa-2) due to binding of around 9 microg of heparin per 10(7) cells (apparent Kd, 30 nM). The loss of anticoagulant activity was less marked in the presence of low molecular weight forms of heparin. Binding to the cell blocked acceleration of the thrombin:antithrombin interaction by heparin. Neutralisation of heparin activity was also shown to occur in the presence of a number of other tumour cell lines. FACS analysis demonstrated that live cells did not bind heparin and high affinity binding only occurred to dead MIA PaCa-2 cells. Heparin binding proteins accumulating in cell medium were identified as histone and ribosomal proteins that will become exposed during necrosis. The release of these proteins from cells within the necrotic core of a tumour or from cells killed during chemotherapy may abrogate the heparan sulphate/antithrombin system and possibly contribute to the idiopathic thromboembolism often associated with cancer (Trousseau's syndrome). The findings also suggest a reason for the reported advantage of LMWH over UFH in treating venous thromboembolism in cancer patients and in improving patient survival.

Adsorption↗
PubMed2000

Outcome of pediatric thromboembolic disease: a report from the Canadian Childhood Thrombophilia Registry.

The outcome for children with deep vein thrombosis (DVT) and pulmonary embolism (PE) is unknown. An understanding of morbidity and mortality of DVT/PE is crucial to the development of rational treatment protocols. The Canadian Childhood Thrombophilia Registry has followed 405 children aged 1 mo to 18 y with DVT/PE for a mean of 2.86 y (range, 2 wk to 6 y) to assess outcome. The all-cause mortality was 65 of 405 children (16%). Mortality directly attributable to DVT/PE occurred in nine children (2.2%), all of whom had central venous line-associated thrombosis. Morbidity was substantial, with 33 children (8.1%) having recurrent thrombosis, and 50 children (12.4%) having postphlebitic syndrome. Recurrent thrombosis and postphlebitic syndrome were more common in older children, although deaths occurred equally in all age groups. The incidence of recurrent thrombosis and postphlebitic syndrome are likely underestimated because of difficulties in diagnosis, especially in younger children. The significant mortality and morbidity found in our study supports the need for international multicenter randomized clinical trials to determine optimal prophylactic and therapeutic treatment for children with DVT/PE.

Adolescent↗
PubMed1998

Bifunctional peptide boronate inhibitors of thrombin: crystallographic analysis of inhibition enhanced by linkage to an exosite 1 binding peptide.

The affinity of the hirudin49-64 segment for exosite 1 of thrombin has been used previously to enhance the potency of simple competitive inhibitors [DiMaio, J., Gibbs, B., Munn, D., Lefebvre, J. , Ni, F., Konishi, Y. (1990) J. Biol. Chem. 265, 21698-21703., and Maraganore, J. M., Bourdon, P., Jablonski, J., Ramachandran, K. L., and Fenton, J. W., II (1990) Biochemistry 29, 7095-7087.]. Using a similar approach, we have enhanced the activity of two active site directed thrombin inhibitors by attaching this segment via a novel reverse oriented linker to each of two tripeptide boronate inhibitors. At P1, compound 1 contains an arginine-like, isothiouronium, side chain, while compound 2 contains an uncharged, bromopropyl residue. Inhibition of human alpha-thrombin by compound 1 shows slow, tight-binding competitive kinetics (final Ki of 2.2 pM, k1 of 3.51 x 10(7) M-1 s-1, and k-1 of 1.81 x 10(-)4 s-1). The addition of hirugen peptide (20 microM) competes for exosite 1 binding and restores the k1 and k-1 to that of the analogous tripeptide, 0.29 x 10(7) M-1 s-1 and 0.13 x 10(-)4 s-1, respectively. Compound 1 has enhanced specificity for thrombin over trypsin with KiTry/KiThr of approximately 900 compared to the analogous tripeptide, with KiTry/KiThr of approximately 4. Compound 2 acts as a competitive inhibitor (KiThr of 0.6 nM) and is highly selective with no effect on trypsin. Crystallographic analysis of complexes of human alpha-thrombin with compound 1 (1.8 A) and compound 2 (1.85 A) shows a covalent bond between the boron of the inhibitor and Ser195 (bond lengths B-O of 1.55 and 1.61 A, respectively). The isothiouronium group of compound 1 forms bidentate interactions with Asp189. The P2 and P3 residues of the inhibitors form interactions with the S2 and S3 sites of thrombin similar to other D-Phe-Pro based inhibitors [Bode, W., Turk, D., and Karshikov, A. (1992) Protein Sci. 1, 426-471.]. The linker exits the active site cleft of thrombin forming no interactions, while the binding of Hir49-64 segment to exosite 1 is similar to that previously described for hirudin [Rydel, T. J., Tulinsky, A., and Bode, W. (1991) J. Mol. Biol. 221, 583-601.]. Because of the similarity of binding at each of these sites to that of the analogous peptides added alone, this approach may be used to improve the inhibitory activity of all types of active site directed thrombin inhibitors and may also be applicable to the design of inhibitors of other proteases.

Amino Acid Sequence↗
PubMed1997

A cyclic peptide analogue of loop III of PDGF-BB causes apoptosis in human fibroblasts.

A cyclic peptide analogue of platelet-derived growth factor-BB (PDGF-BB), P1 [77IVRKK81-C-73RKIE76], has recently been shown to inhibit specifically [125I]PDGF-BB/receptor binding, and PDGF-BB-induced DNA synthesis in cells expressing PDGF receptors. Here we demonstrate that P1 induces apoptosis in exponentially growing human fibroblasts as confirmed by characteristic changes in cell and nuclear morphology, by TUNEL staining and by flow cytometry. Following incubation with P1 (100 microM), the percentage of cells exhibiting DNA fragmentation increased from 24% after 8 h to 76% after 28 h as exponentially growing cells progressed through the cell cycle. We conclude from these findings taken together that apoptosis accounts for the major proportion of P1-induced cell death. Omission of the Cys residue from P1 or replacement by Ser did not alter the potency of the peptide confirming that peptide dimerisation is not important for its activity. PDGF-BB, EGF, FGF, thrombin and foetal bovine serum were not able to rescue cells from the effects of P1. P1 is a useful tool for investigation of the balance of cellular proliferation/apoptosis in wound healing, atherosclerosis and restenosis, and constitutes a basis from which to design compounds with greater potency.

Animals↗
PubMed1997

Identification of a cyclic peptide inhibitor of platelet-derived growth factor-BB receptor-binding and mitogen-induced DNA synthesis in human fibroblasts.

Peptides corresponding to residues from Loops I and III of platelet-derived growth factor-BB (PDGF-BB) were examined for their potential to act as PDGF antagonists. We have identified two peptides which directly stimulated DNA synthesis in human dermal fibroblasts and a cyclic peptide which inhibited PDGF-induced DNA synthesis. The inhibitory action of cyclic PDGF-BB(73-81), on DNA synthesis was shown to be restricted to cells which express PDGF receptors. Also cyclic PDGF-BB(73-81) specifically competed for 125I-labelled PDGF-BB but not for 125I-labelled EGF binding to their respective cellular receptors. The cyclic peptide therefore provides a minimum structure to investigate PDGF/receptor interactions and our findings confirm the importance of the loop configuration of PDGF-BB(73-81) in the native molecule. The cyclic peptide may constitute a basis for developing more potent inhibitors of PDGF action.

Amino Acid Sequence↗
PubMed1997

Tissue factor pathway inhibitor in endothelial cells colocalizes with glycolipid microdomains/caveolae. Regulatory mechanism(s) of the anticoagulant properties of the endothelium.

Tissue factor pathway inhibitor (TFPI), the main downregulator of the procoagulant activity of tissue factor.factor VIIa complex, locates in human endothelial cells (EC) in culture as well-defined clusters uniformly distributed both on the cell surface and intracellularly. We here demonstrate by immunofluorescence that TFPI colocalizes in EC with caveolin, urokinase-type plasminogen activator receptor, and glycosphingolipids. The localization of TFPI in caveolae in resting endothelium is proved by double immunogold electron microscopy for TFPI and caveolin. After ultracentrifugation of rat lung or EC homogenates through density gradients of Nycodenz, TFPI was highly enriched at densities of 1.05 to 1.08 g/mL, together with caveolin and alkaline phosphatase. By ELISA, more than half of the cellular TFPI was detected in Triton X-100-insoluble extracts of EC. TFPI incorporates [1-3H]ethanolamine and is cleaved from the cell surface by phosphatidylinositol-phospholipase C, indicating a specific glycosylphosphatidylinositol-anchorage mechanism for TFPI in the plasma membrane. Clustering of TFPI and its localization in caveolae are dependent on the presence of cholesterol in the membrane. Agonist-induced stimulation of EC caused marked changes of distribution for both TFPI and caveolin at subcellular level, with subsequent increase of the cell surface-associated inhibitory activity toward tissue factor.factor VIIa. Our findings suggest that, beside their function in transcytosis, potocytosis, cell surface proteolysis, and regulation of signal transduction, caveolae also play a direct role in the regulation of EC anticoagulant properties.

Animals↗
PubMed1997

Decrease in factor VII coagulant activity during percutaneous transluminal coronary angioplasty by heparin-mediated lipolytic action.

Levels of factor VII coagulant activity (FVII:C) and two-chain factor VIIa antigen (FVIIa:Ag) were measured in ten patients before and up to 6 h after receiving a bolus of heparin during percutaneous transluminal coronary angioplasty (PTCA). A significant and sustained post-heparin fall in the level of FVII:C was observed (approximately 30%) without any change in the level of FVIIa:Ag. The level of tissue factor antigen within the circulation remained unchanged. The observed decrease in FVII:C coincided with a significant decrease in triglyceride levels presumably due to lipoprotein and hepatic lipase released by the heparin. These findings appear to demonstrate a lipid (triglyceride) dependence of FVII:C. Thus, heparin may act indirectly as antithrombotic agent by limiting a lipid-dependent activation of the extrinsic pathway of coagulation.

Aged↗
PubMed1996

Heparin enhances the catalytic activity of des-ETW-thrombin.

The thrombin mutant, des-ETW-thrombin, lacking Glu(146), Thr(147), and Trp(148) within a unique insertion loop located at the extreme end of the primary specificity pocket, has been shown previously to exhibit reduced catalytic activity with respect to macromolecular and synthetic thrombin substrates and reduced or enhanced susceptibility to inhibition. Investigation of the hydrolysis of peptidyl p-nitroanilide substrates by des-ETW-thrombin showed increased activity in the presence of heparin and other sulphated glycosaminoglycans. No effect was observed upon the activity of wild-type thrombin. Heparin was found to decrease the K(m) for cleavage of four thrombin-specific substrates by des-ETW-thrombin by 3-4-fold. Similarly, pentosan polysulphate (PPS) decreased the K(m) with these substrates by 8-10-fold. Heparin also increased the rate of inhibition of des-ETW-thrombin by antithrombin III and D-phenylalanyl-prolyl-arginylchloromethane (PPACK). The inhibition of des-ETW-thrombin by a number of thrombin-specific peptide boronic acids also showed significant reduction in the final K(i) in the presence of heparin, due to reduction in the off-rate. A peptide analogue of a sequence of hirudin which binds thrombin tightly to exosite I (fibrinogen recognition site) potentiated the activity of des-ETW-thrombin against peptide p-nitroanilide substrates in a manner similar to heparin. The K(i) for the inhibition of des-ETW-thrombin by p-aminobenzamidine was decreased by these ligands from 9.7 mM to 7.5 mM, 5.1 mM, and 2.5 mM in the presence of heparin, hirudin peptide and PPS respectively, suggesting the increased catalytic activity is due to enhanced access to the primary specificity pocket. The positive influence of these ligands on des-ETW-thrombin was reversed in the presence of ATP or ADP; the latter has previously been shown to inhibit thrombin activity by blocking initial interaction with fibrinogen at exosite 1. Because the effect of heparin and PPS is similar to that of hirudin peptide, it is proposed that the most likely mechanism is that binding at the heparin-binding site (thrombin exosite 2) facilitates interaction at exosite 1 causing a conformational change which partially corrects the defective ground-state binding of the mutant thrombin. Although no effect was observed upon the activity of wild-type thrombin, our findings do provide further evidence of an allosteric property of thrombin which may control the geometry of, and access to, the primary specificity pocket.

Amino Acid Sequence↗
PubMed1996

Evidence for cultured human vascular smooth muscle cell heterogeneity: isolation of clonal cells and study of their growth characteristics.

The monoclonal theory of atherosclerosis postulates that the initial vascular smooth muscle cell (VSMC) proliferative event involves the expansion of a single cell or a sub-population of cells thus implying differences in the replicative potential of VSMC. Using the technique of limited dilution, VSMC clones derived from animal tissues have been previously isolated and shown to be morphologically heterogeneous. However, the same technique applied to human VSMC (HVSMC) has been unsuccessful, possibly because HVSMC do not grow when plated at very low densities. In this report, the anchorage-independent growth factor-BB (PDGF-BB) and to lesser extent PDGF-AB and basic fibroblast growth factor (bFGF) induced colony formation. This assay provided a tool for the isolation of HVSMC clones. In terms of their growth characteristics and responsiveness to several growth factors, isolated HVSMC clones and the original parental cell population exhibited marked heterogeneity.

Cell Division↗
PubMed1996

The potential thrombogenic action of a nonionic radiographic contrast medium used during coronary angiography is offset by heparin during coronary angioplasty.

Iohexol sodium, a nonionic radiographic contrast medium, used in invasive imaging techniques has been shown to be potentially thrombogenic. In the present study, the effect of iohexol sodium on haemostatic factors was evaluated in 20 patients, 16 male and 4 female, 10 undergoing coronary angiography and another 10 undergoing coronary angioplasty. All the patients had angiographically-assessed coronary artery disease. The patients undergoing coronary angioplasty received a significantly larger quantity of the dye as compared with the patients undergoing coronary angiography. The former group of patients also received a bolus of 20,000 units of standard heparin in addition. The levels of thrombin-antithrombin-III complex (TAT), prothrombin fragments 1 and 2 (F1F2), D-dimer and the functional activity of tissue factor pathway inhibitor (TFPI) were assayed. While the baseline and 30-min post-dye levels of TAT and F1F2 were comparable in patients undergoing coronary angioplasty, the 30 min levels were significantly elevated in patients undergoing coronary angiography. The post-dye levels of TFPI activity were significantly increased in the former group due to the heparin-induced release of TFPI. It is concluded that the thrombogenic potential of iohexol sodium was overcome by heparin used routinely during coronary angioplasty.

Angioplasty, Balloon, Coronary↗