35 results on '"Farrell, D H"'
Search Results
2. Fibrinogen Binds to Integrin 5 1via the Carboxyl-Terminal RGD Site of the A -Chain
- Author
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Suehiro, K., primary, Mizuguchi, J., additional, Nishiyama, K., additional, Iwanaga, S., additional, Farrell, D. H., additional, and Ohtaki, S., additional
- Published
- 2000
- Full Text
- View/download PDF
3. 12. Sedimentation equilibrium analysis of the ??A/????? fibrinogen-factor XIII complex
- Author
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Moaddel, M., primary, Farrell, D. H., additional, Mosesson, M. W., additional, Siebenlist, K. R., additional, and Fried, M. G., additional
- Published
- 1998
- Full Text
- View/download PDF
4. Role of fibrinogen alpha and gamma chain sites in platelet aggregation.
- Author
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Farrell, D H, primary, Thiagarajan, P, additional, Chung, D W, additional, and Davie, E W, additional
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- 1992
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5. A simple two-step purification of protease nexin
- Author
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Farrell, D H, Van Nostrand, W E, and Cunningham, D D
- Abstract
This paper describes a simple purification procedure for protease nexin, a serine proteinase inhibitor secreted by cultured human fibroblasts that regulates proteinase activity at and near the cell surface. The first step in the procedure takes advantage of the high-affinity binding of protease nexin to dextran sulphate-Sepharose. This step eliminates the need for prior concentration of the serum-free fibroblast-conditioned medium, since protease nexin binds to the resin in the presence of physiological saline. The use of dextran sulphate also provides an affinity resin with considerably less variability than the heparin-based resins previously used. Final purification to homogeneity involves a combination of DEAE-Sepharose in-line with dextran sulphate-Sepharose to simultaneously purify and concentrate the protein. Purified protease nexin is shown by Ouchterlony analysis and peptide mapping to be immunologically and structurally distinct from antithrombin III and heparin cofactor II, two plasma proteinase inhibitors with similar properties.
- Published
- 1986
- Full Text
- View/download PDF
6. Interaction of ~3 integrin-derived peptides 214-218 and 217-231 with a~I~I~b ~3 complex and with fibrinogen A -chain
- Author
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Wierzbicka, I., Kowalska, M. A., Lasz, E. C., Farrell, D. H., Budzynski, A. Z., and Niewiarowski, S.
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- 1997
- Full Text
- View/download PDF
7. Genetic and molecular characterization of essential components of the Vibrio anguillarum plasmid-mediated iron-transport system.
- Author
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Actis, L A, Tolmasky, M E, Farrell, D H, and Crosa, J H
- Abstract
The iron-transport genes from the pJM1 plasmid of Vibrio anguillarum have been cloned and sequenced. Five open-reading frames have been identified, one of which encodes the outer membrane receptor for ferric anguibactin, OM2. This coding region corresponds to a protein of 726 amino acids with a Mr of 78,777. The protein has a hydrophobic signal sequence of 35 amino acids and a potential membrane-associated hydrophobic region at the carboxyl terminus. A 2.3-kilobase iron-regulated mRNA was transcribed from this region in vivo. The four other open-reading frames were shown to be involved in the regulation of OM2 expression and in iron transport by the use of insertion mutagenesis and complementation analysis. One of these open-reading frames, ORF3, encodes a 40-kDa polypeptide which, as deduced from the amino acid sequence and the hydropathy plot, is likely to be membrane-associated and together with OM2 may play a role in the transport of iron into the cell cytosol.
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- 1988
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8. Glycosaminoglycans on fibroblasts accelerate thrombin inhibition by protease nexin-1
- Author
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Farrell, D H and Cunningham, D D
- Abstract
Protease nexin-1 (PN-1) is a proteinase inhibitor that is secreted by human fibroblasts in culture. PN-1 inhibits certain regulatory serine proteinases by forming a covalent complex with the catalytic-site serine residue; the complex then binds to the cell surface and is internalized and degraded. The fibroblast surface was recently shown to accelerate the rate of complex-formation between PN-1 and thrombin. The present paper demonstrates that the accelerative activity is primarily due to cell-surface heparan sulphate, with a much smaller contribution from chondroitin sulphate. This conclusion is supported by the effects of purified glycosaminoglycans on the second-order rate constant for the inhibition of thrombin by PN-1. Also, treatment of 35SO4(2-)-labelled cells with heparitin sulphate lyase or chondroitin sulphate ABC lyase demonstrated two discrete pools of 35S-labelled glycosaminoglycans; subsequent treatment of plasma membranes with these glycosidases showed that heparitin sulphate lyase treatment abolished about 80% of the accelerative activity and chondroitin sulphate ABC lyase removed the remaining 20%. These results show that two components are responsible for the acceleration of PN-1-thrombin complex-formation by human fibroblasts. Although dermatan sulphate is also present on fibroblasts, it did not accelerate the inhibition of thrombin by PN-1.
- Published
- 1987
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9. Resistance of gammaA/gamma' fibrin clots to fibrinolysis.
- Author
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Falls, L A and Farrell, D H
- Abstract
Elevated plasma fibrinogen levels are a major risk factor for thrombosis. This report shows two mechanisms by which fibrinogen can affect the fibrinolysis rate in vitro and thus may lead to thrombosis. First, the lysis rate of fibrin decreases as the initial concentration of fibrinogen increases. Second, a minor variant form of fibrinogen decreases the rate of fibrinolysis. This variant, gammaA/gamma' fibrinogen, has one altered gamma chain and is known to bind to factor XIII zymogen. In a fibrinolysis assay containing purified thrombin, fibrinogen, tissue-type plasminogen activator, and plasminogen, clots from gammaA/gammaA and gammaA/gamma' fibrinogen lysed at similar rates. However, when factor XIII was added, slower lysis was seen in gammaA/gamma' fibrin clots when compared with gammaA/gammaA fibrin clots. A D-dimer agglutination assay showed that the gammaA/gamma' clots were more highly cross-linked than the gammaA/gammaA clots. The lysis rates of gammaA/gamma' clots were similar to gammaA/gammaA clots in the presence of N-ethylmaleimide, a specific inhibitor of factor XIIIa. The gammaA/gamma' fibrin clots made in the presence of factor XIII showed increased proteolytic resistance to both plasmin and trypsin. Clots made from afibrinogenemic plasma reconstituted with gammaA/gamma' fibrinogen also showed significant resistance to lysis compared with gammaA/gammaA fibrinogen. These data demonstrate gammaA/gamma' fibrin is resistant to fibrinolysis, possibly as a result of concentrating factor XIII on the clot. The total fibrinogen concentration and the amount of gammaA/gamma' fibrinogen increase clot stability in vitro and thus may contribute independently to the risk of thrombosis in humans.
- Published
- 1997
10. Human fibroblasts accelerate the inhibition of thrombin by protease nexin.
- Author
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Farrell, D H and Cunningham, D D
- Abstract
Protease nexin (PN) is a protein protease inhibitor secreted by human fibroblasts in culture that complexes and inhibits certain regulatory serine proteases. The PN-protease complexes then bind to these cells and are rapidly internalized and degraded. This report shows that the fibroblast surface accelerates the formation of PN-thrombin complexes. In contrast, it did not accelerate the formation of complexes between thrombin and antithrombin III, a closely related protease inhibitor found in plasma. These results support a role for PN in the regulation of certain proteases in the extravascular compartment at and near the surface of tissue cells. The activity that accelerated PN-thrombin complex formation was membrane-associated, since fixed cells, purified membranes, and extracellular matrix preparations all contained this activity. The ability of cells to accelerate the reaction between PN and thrombin was inhibited by protamine, suggesting that the activity was similar to that of heparin. Heparitinase digestion of plasma membranes prior to assay reduced the activity by about 80%, suggesting that heparan sulfate may account for most of the accelerative activity.
- Published
- 1986
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11. Elevated plasma fibrinogen gamma' concentration is associated with myocardial infarction: effects of variation in fibrinogen genes and environmental factors.
- Author
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Mannila MN, Lovely RS, Kazmierczak SC, Eriksson P, Samnegård A, Farrell DH, Hamsten A, and Silveira A
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- Female, Fibrin chemistry, Fibrinogen chemistry, Genetic Predisposition to Disease, Humans, Male, Models, Genetic, Polymorphism, Genetic, Protein Isoforms, RNA, Messenger metabolism, Risk, Risk Factors, Fibrinogen genetics, Myocardial Infarction blood, Peptide Fragments blood, Peptide Fragments genetics
- Abstract
Background: Fibrinogen gamma', a fibrinogen gamma-chain variant generated via alternative mRNA processing, has been associated with susceptibility to thrombotic disease., Objective: The present case-control study searched for potential determinants of the plasma fibrinogen gamma' concentration and examined the relationship between this variant and risk of myocardial infarction (MI)., Patients and Methods: The Stockholm Coronary Artery Risk Factor study, comprising 387 postinfarction patients and 387 healthy individuals, was employed. The fibrinogen gamma (FGG) 9340T > C [rs1049636], fibrinogen alpha (FGA) 2224G > A [rs2070011] and fibrinogen beta (FGB) 1038G > A [rs1800791] polymorphisms were determined. The plasma fibrinogen gamma' concentration was measured by enzyme-linked immunosorbent assay. The multifactor dimensionality reduction method was used for interaction analyses on risk of MI., Results: The FGG 9340T > C and FGA 2224G > A polymorphisms, total plasma concentrations of fibrinogen, insulin and high-density lipoprotein, and gender appeared to be independent determinants of plasma fibrinogen gamma' concentration in patients, and the corresponding determinants in controls included FGG 9340T > C and FGA 2224G > A polymorphisms and plasma fibrinogen concentration. An elevated plasma fibrinogen gamma' concentration proved to be an independent predictor of MI [adjusted odds ratio (OR) (95% CI): 1.24 (1.01, 1.52)]. The plasma fibrinogen gamma' concentration was involved in a high-order interaction with total plasma fibrinogen and the FGG 9340T > C and FGA 2224G > A polymorphisms, associated with a further increased risk of MI [OR (95% CI): 3.22 (2.35, 4.39)]., Conclusions: Plasma fibrinogen gamma' concentration influences the risk of MI, and this relationship seems to be strengthened by the presence of an elevated total plasma fibrinogen concentration and the FGG 9340T and FGA 2224G alleles.
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- 2007
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12. Influence of gamma' fibrinogen splice variant on fibrin physical properties and fibrinolysis rate.
- Author
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Collet JP, Nagaswami C, Farrell DH, Montalescot G, and Weisel JW
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- Alternative Splicing genetics, Fibrin metabolism, Fibrin ultrastructure, Fibrinogen metabolism, Fibrinogen ultrastructure, Fibrinolysis genetics, Humans, Microscopy, Electron, Scanning, Protein Isoforms chemistry, Protein Isoforms genetics, Structure-Activity Relationship, Alternative Splicing physiology, Fibrin chemistry, Fibrinogen chemistry, Fibrinolysis physiology
- Abstract
Objective: A splice variant of fibrinogen, gamma', has an altered C-terminal sequence in its gamma chain. This gammaA/gamma' fibrin is more resistant to lysis than gammaA/gammaA fibrin. Whether the physical properties of gamma' and gammaA fibrin may account for the difference in their fibrinolysis rate remains to be established., Methods and Results: Mechanical and morphological properties of cross-linked purified fibrin, including permeability (Ks, in cm2) and clot stiffness (G', in dyne/cm2), were measured after clotting gammaA and gamma' fibrinogens (1 mg/mL). gamma'/gamma' fibrin displayed a non-significant decrease in the density of fibrin fibers and slightly thicker fibers than gammaA/gammaA fibrin (12+/-2 fiber/10(-3) nm3 versus 16+/-2 fiber/10(-3) nm3 and 274+/-38 nm versus 257+/-41 nm for gamma'/gamma' and gammaA/gammaA fibrin, respectively; P=NS). This resulted in a 20% increase of the permeability constant (6.9+/-1.7 10(-9) cm2 versus 5.5+/-1.9 10(-9) cm2, respectively; P=NS). Unexpectedly, gamma' fibrin was found to be 3-times stiffer than gammaA fibrin (72.6+/-2.6 dyne/cm2 versus 25.1+/-2.3 dyne/cm2; P<0.001). Finally, there was a 10-fold decrease of the fibrin fiber lysis rate., Conclusions: Fibrinolysis resistance that arises from the presence of gammaA/gamma' fibrinogen in the clot is related primarily to an increase of fibrin cross-linking with only slight modifications of the clot architecture.
- Published
- 2004
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13. Fibrinogen gamma' chain binds thrombin exosite II.
- Author
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Lovely RS, Moaddel M, and Farrell DH
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- Amino Acid Sequence, Animals, Binding Sites, Binding, Competitive, Cell Line, DNA chemistry, DNA metabolism, Fibrinogen chemistry, Heparin chemistry, Heparin metabolism, Hirudins chemistry, Hirudins metabolism, Humans, Kinetics, Mice, Molecular Sequence Data, Protein Binding, Protein Isoforms genetics, Protein Isoforms metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Substrate Specificity, Thrombin chemistry, Transfection, Tyrosine chemistry, Tyrosine metabolism, Fibrinogen genetics, Fibrinogen metabolism, Thrombin metabolism
- Abstract
A high-affinity thrombin-binding site in an alternately processed fibrinogen variant, the gammaA/gamma' isoform, is characterized in this report. The binding site has been shown to be situated between gamma' 414 and 427, and Tyr418 and 422 in this part of the gamma' chain are known to be sulfated. A synthetic peptide corresponding to the gamma' chain carboxyl terminus is shown to bind thrombin with a Kd = 0.63 +/- 0.16 micro mol L-1. Maximum binding of this peptide requires negative charges on Tyr418 and 422. Competitive binding studies with hirudin peptides, heparin and DNA aptamers specific for thrombin exosites I or II indicate thrombin binds to the gamma' peptide via exosite II. Thus, thrombin binding to the gamma' chain leaves exosite I and the active site accessible to substrates. This may explain why fibrin-bound thrombin can retain enzymatic activity, and why fibrin-bound thrombin is heparin-resistant.
- Published
- 2003
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14. The role of gamma A/gamma ' fibrinogen in plasma factor XIII activation.
- Author
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Moaddel M, Falls LA, and Farrell DH
- Subjects
- Amino Acid Sequence, Dimerization, Enzyme Activation, Fibrin metabolism, Humans, Intercellular Signaling Peptides and Proteins, Kinetics, Models, Biological, Molecular Sequence Data, Peptides metabolism, Thrombin metabolism, Factor XIII metabolism, Fibrinogen metabolism, Protein Isoforms metabolism
- Abstract
Factor XIII zymogen activation is a complex series of events that involve fibrinogen acting in several different roles. This report focuses on the role of fibrinogen as a cofactor in factor XIII activation by thrombin. We demonstrate that fibrinogen has two distinct activities that lead to an increased rate of factor XIII activation. First, the thrombin proteolytic activity is increased by fibrin. The cleavage rates of both a small chromogenic substrate and the factor XIII activation peptide are increased in the presence of either the major fibrin isoform, gammaA/gammaA fibrin, or a minor variant form, gammaA/gamma' fibrin. This enhancement of thrombin activity by fibrin is independent of fibrin polymerization and requires only cleavage of the fibrinopeptides. Subsequently, gammaA/gamma' fibrinogen accelerates plasma factor XIII activation by a non-proteolytic mechanism. This increased rate of activation results in a slightly more rapid cross-linking of fibrin gammaA and gamma' chains and a significantly more rapid cross-linking of fibrin alpha chain multimers. Together, these results show that although both forms of fibrin increase the rate of activation peptide cleavage by thrombin, gammaA/gamma' fibrinogen also increases the rate of factor XIII activation in a non-proteolytic manner. A revised model of factor XIII activation is presented below.
- Published
- 2000
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15. Expression of antisense to integrin subunit beta 3 inhibits microvascular endothelial cell capillary tube formation in fibrin.
- Author
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Dallabrida SM, De Sousa MA, and Farrell DH
- Subjects
- Angiogenesis Inhibitors genetics, Angiogenesis Inhibitors physiology, Antibodies immunology, Antibodies pharmacology, Blotting, Western, Capillaries cytology, Capillaries drug effects, Capillaries ultrastructure, Cell Line, Collagen metabolism, Dermis blood supply, Down-Regulation, Drug Combinations, Endothelium, Vascular cytology, Endothelium, Vascular drug effects, Endothelium, Vascular ultrastructure, Fibrin ultrastructure, Gene Expression, Genetic Therapy, Humans, Laminin metabolism, Microscopy, Electron, Neovascularization, Pathologic genetics, Neovascularization, Pathologic therapy, Polymerase Chain Reaction, Proteoglycans metabolism, RNA, Antisense genetics, RNA, Antisense therapeutic use, Receptors, Vitronectin genetics, Receptors, Vitronectin immunology, Transfection, Capillaries growth & development, Endothelium, Vascular growth & development, Fibrin metabolism, Neovascularization, Physiologic drug effects, RNA, Antisense physiology, Receptors, Vitronectin metabolism
- Abstract
alpha(v)beta(3) antagonists are potent angiogenesis inhibitors, and several different classes of inhibitors have been developed, including monoclonal antibodies, synthetic peptides, and small organic molecules. However, each class of inhibitor works by the same principal, by blocking the binding of ligands to alpha(v)beta(3). In an effort to develop an alpha(v)beta(3) inhibitor that down-regulates the actual level of alpha(v)beta(3), we developed an antisense strategy to inhibit alpha(v)beta(3) expression in vitro. beta(3) antisense expressed in endothelial cells specifically down-regulated alpha(v)beta(3) and inhibited capillary tube formation, with the extent of down-regulation correlating with the extent of tube formation inhibition. This inhibition was matrix-specific, since tube formation was not inhibited in Matrigel. These findings support the notion that alpha(v)beta(3) is required for an essential step of angiogenesis in fibrin, namely capillary tube formation. These results suggest that pseudogenetic inhibition of beta(3) integrins using antisense techniques may ultimately provide a therapeutic means to inhibit angiogenesis in vivo.
- Published
- 2000
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16. Fibrinogen binds to integrin alpha(5)beta(1) via the carboxyl-terminal RGD site of the Aalpha-chain.
- Author
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Suehiro K, Mizuguchi J, Nishiyama K, Iwanaga S, Farrell DH, and Ohtaki S
- Subjects
- Amino Acid Sequence, Antibodies, Monoclonal immunology, Antibodies, Monoclonal pharmacology, Binding, Competitive, Cations, Divalent metabolism, Cations, Divalent pharmacology, Fibrinogen genetics, Fibrinogen immunology, Humans, Manganese metabolism, Manganese pharmacology, Molecular Sequence Data, Mutation, Oligopeptides genetics, Oligopeptides immunology, Peptide Fragments chemistry, Peptide Fragments genetics, Peptide Fragments immunology, Peptide Fragments metabolism, Protein Binding drug effects, Recombinant Proteins metabolism, Substrate Specificity, Temperature, Fibrinogen chemistry, Fibrinogen metabolism, Oligopeptides metabolism, Receptors, Fibronectin metabolism
- Abstract
Fibrinogen interactions with vascular endothelial cells are implicated in various physiological and pathophysiological events, including angiogenesis and wound healing. We have shown previously that integrin alpha(5)beta(1) is a fibrinogen receptor on endothelial cells [Suehiro, K., Gailit, J., and Plow, E.F. (1997) J. Biol. Chem. 272, 5360-5366]. In the present study, we have characterized fibrinogen interactions with purified alpha(5)beta(1) and have identified the recognition sequence in fibrinogen for alpha(5)beta(1). The binding of fibrinogen to immobilized alpha(5)beta(1) was selectively supported by Mn(2+). Fibrinogen bound to purified alpha(5)beta(1) in a time-dependent, specific, and saturable manner in the presence of Mn(2+), and the binding was blocked completely by Arg-Gly-Asp (RGD)-containing peptides and by anti-alpha(5) and anti-alpha(5)beta(1) monoclonal antibodies. A monoclonal antibody directed to the C-terminal RGD sequence at Aalpha572-574 significantly inhibited the binding of fibrinogen to alpha(5)beta(1), whereas monoclonal antibodies directed to either the N-terminal RGD sequence at Aalpha95-97 or the C-terminus of the gamma-chain did not. Furthermore, substituting RGE for RGD at position Aalpha95-97 in recombinant fibrinogen had a minimal effect on binding, whereas substituting RGE for RGD at position Aalpha572-574 decreased binding by 90%. These results demonstrate that the C-terminal RGD sequence at Aalpha572-574 is required for the interaction of fibrinogen with alpha(5)beta(1).
- Published
- 2000
- Full Text
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17. Interactions of human fibrinogens with factor XIII: roles of calcium and the gamma' peptide.
- Author
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Moaddel M, Farrell DH, Daugherty MA, and Fried MG
- Subjects
- Binding, Competitive, Edetic Acid, Electrophoresis, Polyacrylamide Gel, Enzyme Activation, Fibrinogen genetics, Humans, Peptide Fragments metabolism, Protein Isoforms chemistry, Protein Isoforms genetics, Ultracentrifugation, Calcium metabolism, Factor XIII chemistry, Fibrinogen chemistry
- Abstract
Plasma factor XIII is the zymogen of the transglutaminase factor XIIIa. This enzyme catalyzes the formation of isopeptide cross-links between fibrin molecules in nascent blood clots that greatly increase the mechanical stability of clots and their resistance to thrombolytic enzymes. We have characterized the solution interactions of factor XIII with two variants of fibrinogen, the soluble precursor of fibrin. Both the predominant fibrinogen gamma(A)/gamma(A) and the major variant gamma(A)/gamma' form complexes with a 2 fibrinogen:1 factor XIII ratio. The absence of detectable concentrations of 1:1 complexes in equilibrium mixtures containing free factor XIII and 2:1 complexes suggests that this interaction is cooperative. Factor XIII binds fibrinogen gamma(A)/gamma' approximately 20-fold more tightly than fibrinogen gamma(A)/gamma(A), and the interaction with fibrinogen gamma(A)/gamma' (but not fibrinogen gamma(A)/gamma(A)) is accompanied by a significant release of Ca(2+). Taken together, these results suggest that the strikingly anionic gamma' C-terminal sequence contains features that are important for factor XIII binding. Consistent with this notion, a synthetic 20-residue polypeptide containing the gamma' sequence was found to associate with factor XIII in a 2:1 molar ratio and act as an efficient competitor for fibrinogen gamma(A)/gamma' binding.
- Published
- 2000
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18. Factor XIIIa supports microvascular endothelial cell adhesion and inhibits capillary tube formation in fibrin.
- Author
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Dallabrida SM, Falls LA, and Farrell DH
- Subjects
- Cell Adhesion drug effects, Cell Adhesion physiology, Cell Movement drug effects, Cells, Cultured, Fibrin, Humans, Neovascularization, Physiologic drug effects, Transglutaminases pharmacology, Cell Movement physiology, Endothelium, Vascular cytology, Endothelium, Vascular physiology, Transglutaminases physiology
- Abstract
Coagulation factor XIIIa is a transglutaminase that catalyzes covalent cross-link formation in fibrin clots. In this report, we demonstrate that factor XIIIa also mediates adhesion of endothelial cells and inhibits capillary tube formation in fibrin. The adhesive activity of factor XIIIa was not dependent on the transglutaminase activity, and did not involve the factor XIIIb-subunits. The adhesion was inhibited by 99% using a combination of monoclonal antibodies directed against integrin alpha(v)beta(3) and beta(1)-containing integrins, and was dependent on Mg(2+) or Mn(2+). Soluble factor XIIIa also bound to endothelial cells in solution, as detected by flow cytometry. In addition, factor XIIIa inhibited endothelial cell capillary tube formation in fibrin in a dose-dependent manner. Furthermore, the extent of inhibition differed in 2 types of fibrin. The addition of 10 to 100 microg/mL factor XIIIa produced a dose-dependent reduction in capillary tube formation of 60% to 100% in gammaA/gammaA fibrin, but only a 10% to 37% decrease in gammaA/gamma' fibrin. These results show that factor XIIIa supports endothelial cell adhesion in an integrin-dependent manner and inhibits capillary tube formation. (Blood. 2000;95:2586-2592)
- Published
- 2000
19. The contribution of the three hypothesized integrin-binding sites in fibrinogen to platelet-mediated clot retraction.
- Author
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Rooney MM, Farrell DH, van Hemel BM, de Groot PG, and Lord ST
- Subjects
- Adenosine Diphosphate pharmacology, Afibrinogenemia blood, Animals, Binding Sites, CHO Cells, Cricetinae, Fibrinogen chemistry, Humans, Mutagenesis, Site-Directed, Oligopeptides metabolism, Platelet Aggregation drug effects, Recombinant Fusion Proteins metabolism, Blood Platelets physiology, Clot Retraction, Fibrinogen metabolism, Platelet Glycoprotein GPIIb-IIIa Complex metabolism
- Abstract
Fibrinogen is a plasma protein that interacts with integrin alphaIIb beta3 to mediate a variety of platelet responses including adhesion, aggregation, and clot retraction. Three sites on fibrinogen have been hypothesized to be critical for these interactions: the Ala-Gly-Asp-Val (AGDV) sequence at the C-terminus of the gamma chain and two Arg-Gly-Asp (RGD) sequences in the Aalpha chain. Recent data showed that AGDV is critical for platelet adhesion and aggregation, but not retraction, suggesting that either one or both of the RGD sequences are involved in clot retraction. Here we provide evidence, using engineered recombinant fibrinogen, that no one of these sites is critical for clot retraction; fibrinogen lacking all three sites still sustains a relatively normal, albeit delayed, retraction response. Three fibrinogen variants with the following mutations were examined: a substitution of RGE for RGD at position Aalpha 95-97, a substitution of RGE for RGD at position Aalpha 572-574, and a triple substitution of RGE for RGD at both Aalpha positions and deletion of AGDV from the gamma chain. Retraction rates and final clot sizes after a 20-minute incubation were indistinguishable when comparing the Aalpha D97E fibrinogen or Aalpha D574E fibrinogen with normal recombinant fibrinogen. However, with the triple mutant fibrinogen, clot retraction was delayed compared with normal recombinant fibrinogen. Nevertheless, the final clot size measured after 20 minutes was the same size as a clot formed with normal recombinant fibrinogen. Similar results were observed using platelets isolated from an afibrinogenemic patient, eliminating the possibility that the retraction was dependent on secretion of plasma fibrinogen from platelet alpha-granules. These findings indicate that clot retraction is a two-step process, such that one or more of the three putative platelet binding sites are important for an initial step in clot retraction, but not for a subsequent step. With the triple mutant fibrinogen, the second step of clot retraction, possibly the development of clot tension, proceeds with a rate similar to that observed with normal recombinant fibrinogen. These results are consistent with a mechanism where a novel site on fibrin is involved in the second step of clot retraction.
- Published
- 1998
20. Human fibroblast adhesion to fibrinogen.
- Author
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Farrell DH and al-Mondhiry HA
- Subjects
- Amino Acid Sequence, Cells, Cultured, Fibroblasts cytology, Fibroblasts physiology, Humans, Infant, Newborn, Kinetics, Male, Recombinant Proteins, Skin cytology, Cell Adhesion drug effects, Fibrinogen, Oligopeptides pharmacology, Skin Physiological Phenomena
- Abstract
Fibrinogen and fibrin mediate the adhesion of many cell types. In this report, the adhesion sites for human dermal fibroblasts on fibrinogen are identified and characterized. Fibroblasts showed a time- and dose-dependent adhesion to fibrinogen. Using a combination of synthetic peptide mimetics, monoclonal antibodies, and recombinant fibrinogens, two major classes of adhesive sites were identified. One class was RGD-dependent and involved the RGD sites in the alpha chain of fibrinogen. alpha V integrins present on fibroblasts appeared to mediate this adhesion. Inhibition studies showed that the RGD-independent site was blocked by an ICAM-1 antagonist peptide. Furthermore, the inhibition was additive with RGD peptide inhibition and accounted for essentially all of the fibroblast adhesion. Together, these results suggest that fibroblast adhesion to fibrinogen is mediated by both alpha V integrins and ICAM-1.
- Published
- 1997
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21. Interaction of beta 3 integrin-derived peptides 214-218 and 217-231 with alpha IIb beta 3 complex and with fibrinogen A alpha-chain.
- Author
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Wierzbicka I, Kowalska MA, Lasz EC, Farrell DH, Budzynski AZ, and Niewiarowski S
- Subjects
- Adenosine Diphosphate metabolism, Blood Platelets metabolism, Enzyme-Linked Immunosorbent Assay, Humans, Integrin beta3, Antigens, CD metabolism, Fibrinogen metabolism, Integrins metabolism, Peptide Fragments metabolism, Platelet Glycoprotein GPIIb-IIIa Complex metabolism, Platelet Membrane Glycoproteins metabolism
- Abstract
beta 3 integrin-derived peptides 214-218 and 217-231 have been shown previously to inhibit platelet aggregation and fibrinogen binding to platelets and to purified receptor. In this paper we study the activity of both peptides in inhibition of binding of biotinylated fibrinogen to activated platelets and to immobilized alpha IIb beta 3 receptor. We found that the mechanism of this inhibition by both peptides is different 125I-labeled 214-218 peptide binds to alpha IIb beta 3 but in contrast, 125I-labeled 217-231 peptide binds to the A alpha-chain of native and gamma' fibrinogen, as judged by the cross-linking study. In solid phase assay both purified alpha IIb beta 3 and 217-231 peptide bound extensively to native and recombinant fibrinogen, and to fibrinogen with either D574E or D97E mutations in the A alpha-chain. Binding of purified alpha IIb beta 3 to gamma' fibrinogen was markedly impaired whereas binding of 217-231 was only slightly impaired in comparison with native fibrinogen. Binding of 217-231 to fibrinogen fragment X was also reduced suggesting that sequences other than RGDS and RGDF may represent binding sites for this peptide. We hypothesize that the close vicinity of fibrinogen binding site (217-231) and of the site participating in conformational changes of the alpha IIb beta 3 receptor (214-218) may facilitate fibrinogen interaction with its receptor.
- Published
- 1997
- Full Text
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22. Adhesion of platelets to surface-bound fibrinogen under flow.
- Author
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Zaidi TN, McIntire LV, Farrell DH, and Thiagarajan P
- Subjects
- Alprostadil pharmacology, Amino Acid Sequence, Animals, Cattle, Collagen metabolism, Fibrinogen genetics, Hemorheology, Humans, Molecular Sequence Data, Mutagenesis, Site-Directed, Recombinant Proteins metabolism, Theophylline pharmacology, Fibrinogen metabolism, Platelet Adhesiveness drug effects
- Abstract
After platelet activation, fibrinogen mediates platelet-platelet interactions leading to platelet aggregation. In addition, fibrinogen can also function as a cell adhesion molecule, providing a substratum for adhesion of platelets and endothelial cells. In this report, we studied the adhesion of platelets to surface-immobilized fibrinogen under flow in different shear rates. Heparinized whole blood containing mepacrine-labeled platelets was perfused for two minutes at various wall shear rates from 250 to 2,000 s-1 in a parallel plate flow chamber. The number of adherent fluorescent platelets was quantitated every 15 seconds with an epifluorescent videomicroscope and digital image processing system. When compared with platelet adhesion and aggregation seen on glass surfaces coated with type I bovine collagen, a significant increase in platelet adhesion was observed on immobilized fibrinogen up to wall shear rates of 800 s-1. The adherent platelets formed a single layer on fibrinogen-coated surfaces. Under identical conditions, no significant adhesion was observed on fibronectin- or vitronectin-coated surfaces. Although platelet adhesion to collagen was substantially inhibited by the platelet inhibitors prostaglandin E1 and theophylline, these inhibitors had no effect on platelet adhesion to fibrinogen. Platelets adhered to recombinant homodimeric wild-type (gamma 400-411) fibrinogen, but not to the recombinant homodimeric gamma' variant of fibrinogen. Platelet adhesion to recombinant fibrinogen with RGD to RGE mutations at positions alpha 95-97 and alpha 572-574 was similar to that with plasma-derived fibrinogen. These results show that platelets adhere to fibrinogen-coated surfaces under moderate wall shear rates, that the interaction is mediated by the fibrinogen 400-411 sequence at the carboxy-terminus of the gamma chain, and that the interaction is independent of platelet activation and the RGD sequences in the alpha chain.
- Published
- 1996
23. Alternative adhesion sites in human fibrinogen for vascular endothelial cells.
- Author
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Thiagarajan P, Rippon AJ, and Farrell DH
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Cattle, Cells, Cultured, Cricetinae, Fibrinogen isolation & purification, Haplorhini, Humans, Integrins metabolism, Kinetics, Mice, Molecular Sequence Data, Mutagenesis, Site-Directed, Oligopeptides, Rats, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Species Specificity, Swine, Umbilical Veins, Cell Adhesion, Endothelium, Vascular physiology, Fibrinogen chemistry, Fibrinogen metabolism
- Abstract
Fibrinogen mediates endothelial cell adhesion, spreading, and angiogenesis through integrin alphavbeta3. Previous studies by several investigators have suggested that the Arg-Gly-Asp (RGD) site at position 572-574 on the alpha chain of human fibrinogen can bind to alphavbeta3. However, this RGD sequence is absent in fibrinogen from most other species, including bovine, hamster, monkey, mouse, pig, and rat fibrinogen. In these species, an RGD site exists at the equivalent of position alpha252-254, which has the sequence RGG in humans. In addition, the role of an integrin binding site on the gamma chain at position 400-411 has been an issue of controversy. In the present studies, recombinant fibrinogen molecules with mutations in the potential endothelial cell binding sites have been used to test the role of these sites directly. The results show that the RGD at alpha572-574 is the primary adhesion site, and that the gamma chain site plays no significant role. Human and bovine plasma fibrinogens were also assayed for their ability to support adhesion of human and bovine vascular endothelial cells. The results show that although the two types of fibrinogen have RGD sequences at widely divergent sites, there is no significant difference in their ability to support endothelial cell adhesion. Furthermore, a chimeric human fibrinogen molecule with an RGD sequence at the bovine site, position alpha252-254, also supported adhesion. These results indicate that an RGD site in human fibrinogen at either position alpha252-254 or position alpha572-574 can mediate endothelial cell adhesion.
- Published
- 1996
- Full Text
- View/download PDF
24. Further studies on the interaction of migrating keratinocytes with fibrinogen.
- Author
-
Donaldson DJ, Mahan JT, Amrani DL, Farrell DH, and Sobel JH
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Monoclonal, Binding Sites, Cell Movement drug effects, Fibrinogen genetics, Keratinocytes metabolism, Male, Mutation physiology, Notophthalmus viridescens, Oligopeptides pharmacology, Recombinant Proteins biosynthesis, Recombinant Proteins metabolism, Fibrinogen metabolism, Keratinocytes cytology
- Abstract
If glass implants placed under one edge of a skin wound in the adult newt are coated with fibrinogen (FGN), keratinocytes from the wound periphery migrate onto the implant. To learn more about the site(s) in FGN that permits this migration, we exposed keratinocytes to implants coated with forms of FGN containing modifications or deletions in the 3 most commonly studied cell binding sites; the RGDF sequence at A alpha 95-98, RGDS at A alpha 572-575 and the carboxy terminal 12 amino acids in the gamma A chain. Recombinant FGN with either RGD sequence altered to RGE supported migration as well as unmodified FGN did. Replacement of the carboxy terminal 4 amino acids in the gamma A chain by a 20 amino acid sequence that disrupts the ability of the gamma terminus to mediate platelet aggregation (the gamma' variant) likewise had no effect. Nor did simultaneous antibody blockade of the RGDS, RGDF, and gamma A sites have any effect. At its best, Dhem1, a fragment containing the RGDS and gamma A sites, produced only about half as much migration as the maximum obtained on intact FGN. Dhem2, a fragment differing from Dhem1 only by having a gamma' variant in place of gamma A, was even less active. Two other D fragments, both of which were missing a large part of the A alpha chain, and one of which contained none of the three major binding sites, supported considerable migration, suggesting that loss of the A alpha chain COOH terminus reveals a site that was not exposed in Dhem1 and 2. A alpha chain fragments containing the RGDF or RGDS sequence were active, but a much larger fragment without RGD was inactive. A soluble peptide consisting of the sequence, RGDS, was a potent inhibitor of migration on FGN but RGDF and the gamma A pentapeptide, KQAGD, were minimally effective. Longer versions of these peptides decreased the effectiveness in all cases. These results suggest that under certain circumstances, newt keratinocytes may interact with each of the 3 major binding sites in FGN as well as a site outside these sequences.
- Published
- 1994
- Full Text
- View/download PDF
25. Binding of recombinant fibrinogen mutants to platelets.
- Author
-
Farrell DH and Thiagarajan P
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Blood Platelets cytology, Cell Adhesion, Cell Line, Cricetinae, Fibrinogen genetics, Humans, Molecular Sequence Data, Mutagenesis, Site-Directed, Platelet Aggregation, Protein Binding, Recombinant Proteins genetics, Recombinant Proteins metabolism, Blood Platelets metabolism, Fibrinogen metabolism
- Abstract
Platelet aggregation is mediated by the interaction of fibrinogen with platelet membrane glycoprotein IIb-IIIa, a member of the integrin family (integrin alpha IIb beta 3). Three different binding sites on fibrinogen for IIb-IIIa have been proposed, two RGD-containing sequences in the alpha chain and one dodecapeptide sequence at the carboxyl terminus of the gamma chain. However, recent evidence shows that mutations in either of the alpha chain sequences have no effect on platelet aggregation, whereas the substitution of a variant gamma chain (gamma') for the gamma chain results in a major reduction in platelet aggregation activity. The present investigation demonstrates that the gamma' chain shows decreased binding to IIb-IIIa as measured by direct binding experiments. In addition, adhesion studies indicate that the binding of both stimulated and unstimulated platelets to immobilized fibrinogens is mediated primarily through the gamma chain carboxyl terminus. Furthermore, a peptide corresponding to the carboxyl terminus of the gamma chain inhibits fibrinogen binding and platelet adhesion, whereas a peptide corresponding to the carboxyl terminus of the gamma' chain is significantly less inhibitory. These data show that the defective platelet aggregation activity of the fibrinogen gamma' chain is due to decreased binding to platelet glycoprotein IIb-IIIa.
- Published
- 1994
26. Processing of the carboxyl 15-amino acid extension in the alpha-chain of fibrinogen.
- Author
-
Farrell DH, Huang S, and Davie EW
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Blood Coagulation, Cell Line, Cricetinae, DNA metabolism, Fibrinogen biosynthesis, Genetic Vectors, Humans, Kidney, Kinetics, Macromolecular Substances, Mesocricetus, Molecular Sequence Data, Mutagenesis, Site-Directed, Oligodeoxyribonucleotides, Recombinant Proteins biosynthesis, Transfection, Tumor Cells, Cultured, DNA genetics, Fibrinogen genetics
- Abstract
The cDNA for the alpha-chain of human fibrinogen codes for 15 amino acids at the carboxyl terminus that are not found in the native protein circulating in plasma. In the present studies, experiments were designed to test whether the carboxyl extension was required for fibrinogen assembly or was simply the result of limited proteolysis and maturation of the protein during or following secretion. Baby hamster kidney cells were transfected with cDNAs coding for the alpha-, beta-, and gamma-chains, and the recombinant fibrinogen secreted into the cell culture medium was identified either with an antibody against the mature molecule or with an antibody directed toward the carboxyl extension of the alpha-chain. The secreted protein contained primarily two species of alpha-chains, including one with the carboxyl extension and one that was the same as that in plasma fibrinogen. A mutant fibrinogen, in which Arg-611 at the putative cleavage site in the alpha-chain was converted to Gly, was also readily assembled, and, in this case, the protein was secreted with the alpha-chain carboxyl extension. Similarly, a mutant fibrinogen that completely lacked the carboxyl extension of the alpha-chain was assembled and secreted from the transfected baby hamster kidney cells. These two mutants with modified alpha-chains were readily clotted by thrombin and cross-linked by factor XIIIa. These results indicate that the alpha-chain carboxyl extension in fibrinogen is not required for assembly or secretion of the molecule. Accordingly, it was concluded that the cleavage of the alpha-chain removing the carboxyl-terminal 15 amino acids is a normal and specific processing event occurring during the maturation of the protein.
- Published
- 1993
27. Biosynthesis of human fibrinogen. Subunit interactions and potential intermediates in the assembly.
- Author
-
Huang S, Mulvihill ER, Farrell DH, Chung DW, and Davie EW
- Subjects
- Animals, Cell Line, Cricetinae, Electrophoresis, Polyacrylamide Gel, Fibrinogen genetics, Humans, Recombinant Proteins biosynthesis, Recombinant Proteins genetics, Transfection, Fibrinogen biosynthesis
- Abstract
Stable transfected baby hamster kidney (BHK) cells expressing human alpha, beta, and gamma fibrinogen chains together, in various combinations of any two, or individually, were established. Several types of subunit interactions were observed in the intracellular extracts of the transfected BHK cell lines as well as in Hep G2 cells. These included: 1) formation of alpha gamma dimers linked by a disulfide bond(s), 2) formation of beta gamma dimers linked by a disulfide bond(s), 3) formation of alpha beta gamma half-molecules linked by disulfide bonds, and 4) formation of mature fibrinogen, which was also secreted into the cell culture medium. Analysis of the chain composition confirmed the stoichiometry of the alpha gamma, beta gamma, and alpha beta gamma complexes. These data are consistent with the proposal that the alpha gamma and beta gamma dimers as well as the alpha beta gamma half-molecules are intermediates in the assembly and biosynthesis of fibrinogen. In contrast, disulfide-linked alpha beta complexes were not found in transfected BHK cells or in Hep G2 cells, suggesting that the formation of disulfide bonds between these two chains most likely occurs when alpha beta gamma half-molecules are formed from alpha gamma and/or beta gamma complexes and when alpha beta gamma half-molecules dimerize to generate the mature molecule. Dimers, trimers, and larger oligomers of each individual chain linked by disulfide bonds were also identified when each chain was expressed in the absence of the other two chains. Preferential formation of alpha gamma and beta gamma complexes, rather than oligomers of individual chains, suggested that the oligomers were less likely to be intermediates in the assembly of fibrinogen. A model for fibrinogen assembly is presented based on these results.
- Published
- 1993
28. Regulation of protease nexin-1 activity by heparin and heparan sulfate.
- Author
-
Cunningham DD, Wagner SL, and Farrell DH
- Subjects
- Amyloid beta-Protein Precursor, Animals, Cell Membrane metabolism, Extracellular Matrix metabolism, Humans, Protease Inhibitors metabolism, Protease Nexins, Receptors, Cell Surface, Serpin E2, Thrombin antagonists & inhibitors, Carrier Proteins metabolism, Heparin pharmacology, Heparitin Sulfate pharmacology
- Published
- 1992
- Full Text
- View/download PDF
29. Recombinant human fibrinogen and sulfation of the gamma' chain.
- Author
-
Farrell DH, Mulvihill ER, Huang SM, Chung DW, and Davie EW
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cell Line, Cloning, Molecular, Cricetinae, Fibrinogen metabolism, Genetic Vectors, Humans, Molecular Sequence Data, Molecular Weight, Oligonucleotides chemistry, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Sulfates chemistry, Tyrosine chemistry, Fibrinogen chemistry
- Abstract
Human fibrinogen and the homodimeric gamma'-chain-containing variant have been expressed in BHK cells using cDNAs coding for the alpha, beta, and gamma (or gamma') chains. The fibrinogens were secreted at levels greater than 4 micrograms (mg of total cell protein)-1 day-1 and were biologically active in clotting assays. Recombinant fibrinogen containing the gamma' chain incorporated 35SO4 into its chains during biosynthesis, while no incorporation occurred in the protein containing the gamma chain. The identity of the sulfated gamma' chain was verified by its ability to form dimers during clotting. In addition, carboxypeptidase Y digestion of the recombinant fibrinogen containing the gamma' chain released 96% of the 35S label from the sulfated chain, and the radioactive material was identified as tyrosine O-sulfate. These results clarify previous findings of the sulfation of tyrosine in human fibrinogen.
- Published
- 1991
- Full Text
- View/download PDF
30. Purification and characterization of a secreted protease from the pathogenic marine bacterium Vibrio anguillarum.
- Author
-
Farrell DH and Crosa JH
- Subjects
- Ammonium Sulfate, Calcium Chloride pharmacology, Chemical Precipitation, Chlorides pharmacology, Chromatography, High Pressure Liquid, Hydrogen-Ion Concentration, Metalloendopeptidases isolation & purification, Molecular Weight, Protease Inhibitors pharmacology, Vibrio drug effects, Zinc pharmacology, Endopeptidases isolation & purification, Vibrio enzymology, Zinc Compounds
- Abstract
Vibrio anguillarum is a pathogenic marine bacterium which causes the disease vibriosis in salmonid fish, which is characterized by a fatal hemorrhagic septicemia accompanied by massive tissue destruction. In this paper, the purification of the major caseinolytic extracellular protease from V. anguillarum is presented. The purification steps include ammonium sulfate precipitation, DEAE-Sepharose chromatography, Sephacryl S-200 chromatography, and DEAE high-pressure liquid chromatography. The purified protease migrates with Mr = 38,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A slightly larger protease of Mr 40,000 is also separated by this procedure, but accounts for only a minor fraction of the caseinolytic activity. The Mr 38,000 protease displays a broad pH activity profile in the neutral to basic range. It is not inhibited by serine, cysteine, or acid protease inhibitors, but is inhibited by EDTA and 1,10-phenanthroline, suggesting that it is a metalloprotease. The activity of the EDTA-inactivated protease could be partially restored by the addition of Ca2+ and Zn2+ together. The molecular weight and inhibition data show some similarities with proteases isolated from other Vibrio species such as Vibrio cholerae and Vibrio vulnificus.
- Published
- 1991
- Full Text
- View/download PDF
31. A regulatory gene, angR, of the iron uptake system of Vibrio anguillarum: similarity with phage P22 cro and regulation by iron.
- Author
-
Farrell DH, Mikesell P, Actis LA, and Crosa JH
- Subjects
- Amino Acid Sequence, Base Sequence, Gene Expression Regulation, Bacterial, Molecular Sequence Data, Protein Conformation, Regulatory Sequences, Nucleic Acid, Salmonella Phages genetics, Bacterial Proteins genetics, DNA-Binding Proteins genetics, Genes, Bacterial, Genes, Regulator, Iron metabolism, Transcription Factors genetics, Vibrio genetics
- Abstract
The angR locus in Vibrio anguillarum encodes a trans-acting transcriptional activator which modulates several Fe2(+)-regulated loci in the anguibactin biosynthesis gene cluster. In this paper, the complete nucleotide (nt) sequence of the angR gene and deduced amino acid (aa) sequence of the AngR protein are presented. A region upstream from the angR gene is shown to have similarity with Fe2(+)-regulated operators in Escherichia coli which bind the Fur protein. The involvement of a Fur-like regulator is supported by transcription analysis which show that angR itself is Fe2(+)-regulated. The aa sequence of the AngR protein predicts a helix-turn-helix motif which shows striking homology with prokaryotic DNA-binding proteins, particularly the lambda and P22 Cro proteins. In addition, there are two 18-nt regions, upstream from the angR gene, which show similarity with the OR1 and OR2 operators of P22 cro. These regions overlap with, respectively, the -35, -10 region and the putative Fur-binding region upstream from angR. These results suggest that AngR may be a DNA-binding protein which modulates Fe2(+)-regulated transcription and is itself Fe2(+)-regulated at the transcriptional level.
- Published
- 1990
- Full Text
- View/download PDF
32. Localization of protease nexin-1 on the fibroblast extracellular matrix.
- Author
-
Farrell DH, Wagner SL, Yuan RH, and Cunningham DD
- Subjects
- Amyloid beta-Protein Precursor, Enzyme-Linked Immunosorbent Assay, Fibroblasts ultrastructure, Humans, Immunologic Techniques, Protease Nexins, Receptors, Cell Surface, Serpin E2, Carrier Proteins metabolism, Extracellular Matrix metabolism, Fibroblasts metabolism
- Abstract
Protease nexin-1 (PN-1) is a protease inhibitor that is secreted by fibroblasts and several other cultured cells. PN-1 forms complexes with certain serine proteases in the extracellular environment including thrombin, urokinase, and plasmin. The complexes then bind to the cells and are rapidly internalized and degraded. This report demonstrates that PN-1 is present on the surface of fibroblasts, bound to the extracellular matrix. Immunofluorescent studies showed that PN-1 colocalized with fibronectin on both intact cells and in preparations of extracellular matrix made from these cells. In contrast, PN-1 did not colocalize with the epidermal growth factor receptor, a plasma membrane marker. An enzyme-lined immunosorbent assay was developed which showed that the extracellular matrix contained at least 60-80% of the cellular immunoreactive PN-1. Extraction of the matrix with 2 M NaCl removed PN-1 in a form which reacted with 125I-thrombin to form complexes which were immunoprecipitated by anti-PN-1 IgG and were of identical size as complexes made from soluble PN-1 and 125I-thrombin. These data indicate that in addition to its role as a soluble protease inhibitor, PN-1 is also a component of the extracellular matrix and might control its proteolysis.
- Published
- 1988
- Full Text
- View/download PDF
33. Development of lymphocytotoxic and platelet reactive antibodies: a prospective study in patients with acute leukaemia.
- Author
-
Pamphilon DH, Farrell DH, Donaldson C, Raymond PA, Brady CA, and Bradley BA
- Subjects
- Adolescent, Adult, Aged, Child, Child, Preschool, Female, Humans, Infant, Leukemia, Myeloid, Acute blood, Male, Middle Aged, Precursor Cell Lymphoblastic Leukemia-Lymphoma blood, Prospective Studies, Antilymphocyte Serum analysis, Blood Platelets immunology, Isoantibodies biosynthesis, Leukemia, Myeloid, Acute immunology, Precursor Cell Lymphoblastic Leukemia-Lymphoma immunology
- Abstract
Lymphocytotoxic (LCT) and platelet reactive (PR) antibody (Ab) responses were serially determined in 49 patients with acute leukaemia. LCTAb were found in 20 patients and occurred in 13 patients with acute myeloid leukaemia and 7 patients with acute lymphoblastic leukaemia. Four differing patterns of LCTAb responses could be defined. Thirteen of 22 subjects showed marked reduction or loss of LCTAb. Indirect platelet immunofluorescence, measured by flow cytometry, provided the most convenient means of detecting PRAb which were found in 11 subjects and generally showed moderate or weak reactivity.
- Published
- 1989
- Full Text
- View/download PDF
34. Interactions of serine proteases with cultured fibroblasts.
- Author
-
Cunningham DD, Van Nostrand WE, Farrell DH, and Campbell CH
- Subjects
- Amyloid beta-Protein Precursor, Binding Sites, Carrier Proteins metabolism, Cell Membrane metabolism, Cells, Cultured, Extracellular Matrix metabolism, Humans, Pancreatic Elastase metabolism, Protease Inhibitors, Protease Nexins, Receptors, Cell Surface, Serine Endopeptidases, Thrombin metabolism, Urokinase-Type Plasminogen Activator metabolism, Endopeptidases metabolism, Fibroblasts metabolism
- Abstract
This review summarizes the mechanisms by which several serine proteases, particularly urokinase, thrombin, and elastase, interact with cultured fibroblasts. Many of these studies were prompted by findings that interactions of these proteases with cells and the extracellular matrix are important in a number of physiologic and pathologic processes. Two main pathways have been identified for specific interactions of these proteases with fibroblasts. One involves surface binding sites for the free protease that appear to bind only one particular protease. An unusual feature collectively shared by the binding sites for urokinase, thrombin, and elastase is that the bound protease is not detectably internalized by the fibroblasts. The other pathway by which serine proteases interact with fibroblasts involves proteins named protease nexins (PNs). Three PNs have been identified. They are secreted by fibroblasts and inhibit certain serine proteases by forming a covalent complex with the protease catalytic site serine. The complexes then bind back to the fibroblasts via the PN portion of the complex and are internalized and degraded. Recent studies showing that the fibroblast surface and extracellular matrix accelerate the inactivation of thrombin by PN-1 support the hypothesis that the PNs control protease activity at and near the cell surface. The PNs differ from plasma protease inhibitors in their molecular properties, absence in plasma, site of synthesis, and site of clearance of the inhibitor:protease complexes.
- Published
- 1986
- Full Text
- View/download PDF
35. Thrombin interactions with cultured fibroblasts: relationship to mitogenic stimulation.
- Author
-
Cunningham DD and Farrell DH
- Subjects
- Amyloid beta-Protein Precursor, Animals, Cell Division, Cells, Cultured, Humans, In Vitro Techniques, Protease Nexins, Receptors, Cell Surface physiology, Carrier Proteins physiology, Fibroblasts cytology, Mitogens, Thrombin physiology
- Published
- 1986
- Full Text
- View/download PDF
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