Download PDF
Thromb Haemost 2003; 89(01): 53-64
DOI: 10.1055/s-0037-1613543
Blood Coagulation, Fibrinolysis and Cell Haemostasis
Schattauer GmbH

Chemical crosslinking of urokinase to pulmonary surfactant protein B for targeting alveolar fibrin

Clemens Ruppert
1   Department of Internal Medicine, Justus-Liebig-University Gießen, Germany
,
Philipp Markart
1   Department of Internal Medicine, Justus-Liebig-University Gießen, Germany
,
Reinhold Schmidt
1   Department of Internal Medicine, Justus-Liebig-University Gießen, Germany
,
Friedrich Grimminger
1   Department of Internal Medicine, Justus-Liebig-University Gießen, Germany
,
Werner Seeger
1   Department of Internal Medicine, Justus-Liebig-University Gießen, Germany
,
Claus-Michael Lehr
2   Department of Biopharmaceutics and Pharmaceutical Technology, Saarland University, Saarbrücken, Germany
,
Andreas Günther
1   Department of Internal Medicine, Justus-Liebig-University Gießen, Germany
› Author Affiliations
Further Information

Publication History

Received 08 August 2002

Accepted after revision 01 November 2002

Publication Date:
09 December 2017 (online)

    Permissions and Reprints

Summary

Intraalveolar fibrin formation is a consistent finding in acute inflammatory and chronic interstitial lung disease. Polymerization of fibrin in the presence of pulmonary surfactant results in far-reaching incorporation of the hydrophobic surfactant compounds into the growing fibrin matrix, with loss of surface activity, altered fibrin structure and reduced susceptibility of the clot to fibrinolysis. For specific targeting of such alveolar fibrin, we designed a hybrid molecule consisting of the catalytic domain of urokinase (B-chain) and the hydrophobic surfactant protein B (SP-B), termed SPUC. The urokinase B-chain, obtained by limited reduction of human two-chain-urokinase (u-PA) and subsequent affinity purification, was chemically coupled to SP-B in a semi-organic solvent system using a hetero-bifunctional crosslinker. Purification of the chimeric proteins included reversed phase and cation exchange chromatography. SDS-PAGE and Western Blotting with immunostaining were employed for biochemical characterization of the conjugate. Chromogenic substrate assays, 125I-based fibrin plate assays, active site titration and surface tension measurements (pulsating bubble surfactometer) were performed to analyze the specific fibrinolytic activity of the conjugate and its surface activity. SPUC was found i) to be assembled stoichiometrically in a 1:1 fashion (SP-B:u-PA), ii) to fully retain the biophysical activity as compared to native SP-B and iii) to also retain the fibrinolytic activity. SPUC was 2-3 fold more effective in lysis of surfactant containing clots and 5-fold more resistant against plasminogen activator 1 (PAI-1) as compared to the native u-PA. We conclude that urokinase and SP-B can be chemically crosslinked, thereby yielding a fibrinolytic enzyme suitable for targeting alveolar fibrin.

Keywords

Surfactant protein B - fibrinolysis - urokinase - fibrosis - ARDS
 

  • References

  • 1 Ashbaugh DG, Bigelow DB, Petty TL, Levine BE. Acute respiratory distress in adults. Lancet 1967; 2: 319-23.
    CrossrefPubMedGoogle Scholar
  • 2 Bachofen M, Weibel ER. Structural alterations of lung parenchyma in the adult respiratory distress syndrome. Clin Chest Med 1982; 3: 35-56.
    PubMedGoogle Scholar
  • 3 Burkhardt A. Alveolitis and collapse in the pathogenesis of pulmonary fibrosis. Am Rev Respir Dis 1989; 140: 513-24.
    CrossrefPubMedGoogle Scholar
  • 4 Chapman HA, Allen CL, Stone OL. Abnormalities in pathways of alveolar fibrin turnover among patients with interstitial lung disease. Am Rev Respir Dis 1986; 133: 437-43.
    PubMedGoogle Scholar
  • 5 Hallman M, Maasilta P, Kivisaari L, Mattson K. Changes in surfactant in bronchoalveolar lavage fluid after hemithorax irradiation in patients with mesothelioma. Am Rev Respir Dis 1990; 141: 998-1005.
    CrossrefPubMedGoogle Scholar
  • 6 Jackson LK. Idiopathic pulmonary fibrosis. Clin Chest Med 1982; 3: 579-92.
    PubMedGoogle Scholar
  • 7 Gross TJ, Simon RH, Sitrin RG. Tissue factor procoagulant expression by rat alveolar epithelial cells. Am J Respir Cell Mol Biol 1992; 6: 397-403.
    CrossrefPubMedGoogle Scholar
  • 8 Hasday JD, Bachwich PR, Lynch JP, Sitrin RG. Procoagulant and plasminogen activator activities of bronchoalveolar fluid in patients with pulmonary sarcoidosis. Exp Lung Res 1988; 14: 261-78.
    CrossrefPubMedGoogle Scholar
  • 9 Idell S, Koenig KB, Fair DS, Martin TR, McLarty J, Maunder RJ. Serial abnormalities of fibrin turnover in evolving adult respiratory distress syndrome. Am J Physiol 1991; 261: L240-8.
    PubMedGoogle Scholar
  • 10 Idell S, Gonzalez K, Bradford H, Mac Arthur CK, Fein AM, Maunder RJ, Garcia JG, Griffith DE, Weiland J, Martin TR. et al. Pro-coagulant activity in bronchoalveolar lavage in the adult respiratory distress syndrome. Contribution of tissue factor associated with factor VII. Am Rev Respir Dis 1987; 136: 1466-74.
    CrossrefPubMedGoogle Scholar
  • 11 Idell S, James KK, Levin EG, Schwartz BS, Manchanda N, Maunder RJ, Martin TR, McLarty J, Fair DS. Local abnormalities in coagulation and fibrinolytic pathways predispose to alveolar fibrin deposition in the adult respiratory distress syndrome. J Clin Invest 1989; 84: 695-705.
    CrossrefPubMedGoogle Scholar
  • 12 Nakstad B, Lyberg T, Skjonsberg OH, Boye NP. Local activation of the coagulation and fibrinolysis systems in lung disease. Thromb Res 1990; 57: 827-38.
    CrossrefPubMedGoogle Scholar
  • 13 Nakstad B, Boye NP, Lyberg T. Procoagulant activities in human alveolar macrophages. Eur J Respir Dis 1987; 71: 459-71.
    PubMedGoogle Scholar
  • 14 Günther A, Mosavi P, Ruppert C, Heinemann S, Temmesfeld-Wollbrück B, Velcovsky HG, Morr H, Grimminger F, Walmrath D, Seeger W. Enhanced tissue factor pathway activity and fibrin turnover in the alveolar compartment of patients with interstitial lung disease. Thromb Haemost 2000; 83: 853-60.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 15 Günther A, Mosavi P, Heinemann S, Ruppert C, Muth H, Markart P, Grimminger F, Walmrath D, Temmesfeld-Wollbrück B, Seeger W. Alveolar fibrin formation caused by enhanced procoagulant and depressed fibrinolytic capacities in severe pneumonia – comparison with the acute respiratory distress syndrome. Am J Respir Crit Care Med 2000; 161: 454-62.
    CrossrefPubMedGoogle Scholar
  • 16 Bertozzi P, Astedt B, Zenzius L, Lynch K, LeMaire F, Zapol W, Chapman Jr HA. Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome. N Engl J Med 1990; 322: 890-7.
    CrossrefPubMedGoogle Scholar
  • 17 Akiyama SK, Hasegawa E, Hasegawa T, Yamada KM. The interaction of fibronectin fragments with fibroblastic cells. J Biol Chem 1985; 260: 13256-60.
    PubMedGoogle Scholar
  • 18 Aota S, Nagai T, Olden K, Akiyama SK, Yamada KM. Fibronectin and integrins in cell adhesion and migration. Biochem Soc Trans 1991; 19: 830-5.
    CrossrefPubMedGoogle Scholar
  • 19 Bitterman PB, Rennard SI, Adelberg S, Crystal RG. Role of fibronectin as a growth factor for fibroblasts. J Cell Biol 1983; 97: 1925-32.
    CrossrefPubMedGoogle Scholar
  • 20 Campbell EJ, Senior RM, Welgus HG. Extracellular matrix injury during lung inflammation. Chest 1987; 92: 161-7.
    CrossrefPubMedGoogle Scholar
  • 21 Couchman JR, Austria MR, Woods A. Fibronectin-cell interactions. J Invest Dermatol 1990; 94: 7S-14S.
    CrossrefPubMedGoogle Scholar
  • 22 Yamada KM. Adhesive recognition sequences. J Biol Chem 1991; 266: 12809-12.
    PubMedGoogle Scholar
  • 23 Hernandez Rodriguez NA, Cambrey AD, Harrison NK, Chambers RC, Gray AJ, Southcott AM, duBois RM, Black CM, Scully MF, McAnulty RJ. et al. Role of thrombin in pulmonary fibrosis. Lancet 1995; 346: 1071-3.
    CrossrefPubMedGoogle Scholar
  • 24 Ohba T, McDonald JK, Silver RM, Strange C, LeRoy EC, Ludwicka A. Scleroderma bronchoalveolar lavage fluid contains thrombin, a mediator of human lung fibroblast proliferation via induction of platelet-derived growth factor alpha-receptor. Am J Respir Cell Mol Biol 1994; 10: 405-12.
    CrossrefPubMedGoogle Scholar
  • 25 Tani K, Yasuoka S, Ogushi F, Asada K, Fujisawa K, Ozaki T, Sano N, Ogura T. Thrombin enhances lung fibroblast proliferation in bleomycin-induced pulmonary fibrosis. Am J Respir Cell Mol Biol 1991; 5: 34-40.
    CrossrefPubMedGoogle Scholar
  • 26 Gray AJ, Reeves JT, Harrison NK, Winlove P, Laurent GJ. Growth factors for human fibroblasts in the solute remaining after clot formation. J Cell Sci 1990; 96: 271-4.
    PubMedGoogle Scholar
  • 27 Gray AJ, Bishop JE, Reeves JT, Mecham RP, Laurent GJ. Partially degraded fibrin(ogen) stimulates fibroblast proliferation in vitro. Am J Respir Cell Mol Biol 1995; 12: 684-90.
    CrossrefPubMedGoogle Scholar
  • 28 Lewis JF, Jobe AH. Surfactant and the adult respiratory distress syndrome. Am Rev Respir Dis 1993; 147: 218-33.
    CrossrefPubMedGoogle Scholar
  • 29 Günther A, Ruppert C, Schmidt R, Markart P, Grimminger F, Walmrath D, Seeger W. Surfactant alteration and replacement in acute respiratory distress syndrome. Respir Res 2001; 2: 353-64.
    CrossrefPubMedGoogle Scholar
  • 30 Gregory TJ, Longmore WJ, Moxley MA, Whitsett JA, Reed CR, Fowler AA, Hudson LD, Maunder RJ, Crim C, Hyers TM.. Surfactant chemical composition and biophysical activity in acute respiratory distress syndrome. J Clin Invest 1991; 88: 1976-81.
    CrossrefPubMedGoogle Scholar
  • 31 Hallman M, Spragg R, Harrell JH, Moser KM, Gluck L. Evidence of lung surfactant abnormality in respiratory failure. Study of bronchoalveolar lavage phospholipids, surface activity, phospholipase activity, and plasma myoinositol. J Clin Invest 1982; 70: 673-83.
    CrossrefPubMedGoogle Scholar
  • 32 Pison U, Seeger W, Buchhorn R, Joka T, Brand M, Obertacke U, Neuhof H, Schmidt Neuerburg KP. Surfactant abnormalities in patients with respiratory failure after multiple trauma. Am Rev Respir Dis 1989; 140: 1033-9.
    CrossrefPubMedGoogle Scholar
  • 33 Günther A, Siebert C, Schmidt R, Ziegler S, Grimminger F, Yabut M, Temmesfeld B, Walmrath D, Morr H, Seeger W. Surfactant alterations in severe pneumonia, acute respiratory distress syndrome, and cardiogenic lung edema. Am J Respir Crit Care Med 1996; 153: 176-84.
    CrossrefPubMedGoogle Scholar
  • 34 Günther A, Schmidt R, Nix F, Yabut Perez M, Guth C, Rosseau S, Siebert C, Grimminger F, Morr H, Velcovsky HG. Seeger. Surfactant abnormalities in idiopathic pulmonary fibrosis, hypersensitivity pneumonitis and sarcoidosis. Eur Respir J 1999; 14: 565-73.
    CrossrefPubMedGoogle Scholar
  • 35 Cockshutt AM, Weitz J, Possmayer F. Pulmonary surfactant-associated protein A enhances the surface activity of lipid extract surfactant and reverses inhibition by blood proteins in vitro. Biochemistry 1990; 29: 8424-9.
    CrossrefPubMedGoogle Scholar
  • 36 Fuchimukai T, Fujiwara T, Takahashi A, Enhorning G. Artificial pulmonary surfactant inhibited by proteins. J Appl Physiol 1987; 62: 429-37.
    PubMedGoogle Scholar
  • 37 Holm BA, Enhorning G, Notter RH. A biophysical mechanism by which plasma proteins inhibit lung surfactant activity. Chem Phys Lipids 1988; 49: 49-55.
    CrossrefPubMedGoogle Scholar
  • 38 Holm BA, Notter RH. Effects of hemoglobin and cell membrane lipids on pulmonary surfactant activity. J Appl Physiol 1987; 63: 1434-42.
    CrossrefPubMedGoogle Scholar
  • 39 Seeger W, Günther A, Thede C. Differential sensitivity to fibrinogen inhibition of SP-C-vs. SP-B-based surfactants. Am J Physiol 1992; 262: L286-91.
    PubMedGoogle Scholar
  • 40 Seeger W, Thede C, Günther A, Grube C. Surface properties and sensitivity to protein-inhibition of a recombinant apoprotein C-based phospholipid mixture in vitro – comparison to natural surfactant. Biochim Biophys Acta 1991; 1081: 45-52.
    CrossrefPubMedGoogle Scholar
  • 41 Seeger W, Stöhr G, Wolf HR, Neuhof H. Alteration of surfactant function due to protein leakage: special interaction with fibrin monomer. J Appl Physiol 1985; 58: 326-38.
    CrossrefPubMedGoogle Scholar
  • 42 Seeger W, Elssner A, Günther A, Krämer HJ, Kalinowski HO. Lung surfactant phospho-lipids associate with polymerizing fibrin: loss of surface activity. Am J Respir Cell Mol Biol 1993; 9: 213-20.
    CrossrefPubMedGoogle Scholar
  • 43 Günther A, Kalinowski M, Rosseau S, Seeger W. Surfactant incorporation markedly alters mechanical properties of a fibrin clot. Am J Respir Cell Mol Biol 1995; 13: 712-8.
    CrossrefPubMedGoogle Scholar
  • 44 Günther A, Kalinowski M, Elssner A, Seeger W. Clot-embedded natural surfactant: kinetics of fibrinolysis and surface activity. Am J Physiol 1994; 267: L618-24.
    PubMedGoogle Scholar
  • 45 Günther A, Markart P, Kalinowski M, Ruppert C, Grimminger F, Seeger W. Cleavage of surfactant-incorporating fibrin by different fibrinolytic agents – kinetics of lysis and rescue of surface activity. Am J Respir Cell Mol Biol 1999; 21: 738-45.
    CrossrefPubMedGoogle Scholar
  • 46 Hawgood S, Benson BJ, Schilling J, Damm D, Clements JA, White RT. Nucleotide and amino acid sequences of pulmonary surfactant protein SP 18 and evidence for cooperation between SP 18 and SP 28-36 in surfactant lipid adsorption. Proc Natl Acad Sci USA 1987; 84: 66-70.
    CrossrefPubMedGoogle Scholar
  • 47 Schägger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 1987; 166: 368-79.
    CrossrefPubMedGoogle Scholar
  • 48 Unkeless JC, Gordon S, Reich E. Secretion of plasminogen activator by stimulated macrophages. J Exp Med 1974; 139: 834-50.
    CrossrefPubMedGoogle Scholar
  • 49 Geiger M, Binder BR. Tissue-type plasminogen activator and urokinase: differences in the reaction pattern with the active-site titrant 4-methylumbelliferyl-p-guanidinobenzoate hydrochloride. Biochim Biophys Acta 1987; 912: 34-40.
    CrossrefPubMedGoogle Scholar
  • 50 Enhorning G. Pulsating bubble technique for evaluating pulmonary surfactant. J Appl Physiol 1977; 43: 198-203.
    CrossrefPubMedGoogle Scholar
  • 51 Ruppert C, Schmidt R, Grimminger F, Suzuki Y, Seeger W, Lehr CM, Günther A. Chemical coupling of a monoclonal antisurfactant protein-B antibody to human urokinase for targeting surfactant-incorporationg alveolar fibrin. Bioconjugate Chem 2002; 13: 804-11.
    CrossrefPubMedGoogle Scholar
  • 52 Tanaka K, Einaga K, Tsuchiyama H, Tait JF, Fujikawa K. Preparation and characterization of a disulfide-linked bioconjugate of annexin V with the B-chain of urokinase: an improved fibrinolytic agent targeted to phospholipid-containing thrombi. Biochemistry 1996; 35: 922-9.
    CrossrefPubMedGoogle Scholar
  • 53 Robbins KC, Tanaka Y. Covalent molecular weight approximately 92,000 hybrid plasminogen activator derived from human plas-min amino-terminal and urokinase carboxyl-terminal domains. Biochemistry 1986; 25: 3603-11.
    CrossrefPubMedGoogle Scholar
  • 54 Possmayer F. A proposed nomenclature for pulmonary surfactant-associated proteins. Am Rev Respir Dis 1988; 138: 990-8.
    CrossrefPubMedGoogle Scholar
  • 55 Diemel RV, Bader D, Walch M, Hotter B, van Golde LMG, Amann A, Haagsmann HP, Putz G. Functional tests for the characterization of surfactant Protein B (SP-B) and a fluorescent SP-B analog. Arch Biochem Biophys 2001; 385: 338-47.
    CrossrefPubMedGoogle Scholar
  • 56 Johansson J, Curstedt T, Jornvall H. Surfactant protein B: disulfide bridges, structural properties, and kringle similarities. Biochemistry 1991; 30: 6917-21.
    CrossrefPubMedGoogle Scholar
  • 57 Schermuly RT, Günther A, Ermert M, Ermert L, Ghofrani HA, Weissmann N, Grimminger F, Seeger W, Walmrath D. Conebulization of surfactant and urokinase restores gas exchange in perfused lungs with alveolar fibrin formation. Am J Physiol 2001; 280: L792-800.
    PubMedGoogle Scholar
  • 58 Günther A, Lübke N, Weissmann N, Quantz K, Weimar B, Schermuly R, Ruppert C, Muth H, Grimminger F, Seeger W. Restoration of lung compliance and prevention of lung fibrosis in a rabbit model of bleomycin induced lung fibrosis upon aerosol application of heparin and urokinase. Am J Respir Crit Care Med 2000; 161: A 751 (Abstr.)
    PubMedGoogle Scholar
  • 59 Hattori N, Degen JL, Sisson TH, Liu H, Moore BB, Pandrangi RG, Simon RH, Drew AF. Bleomycin-induced pulmonary fibrosis in fibrinogen-null mice. J Clin Invest 2000; 106: 1341-50.
    CrossrefPubMedGoogle Scholar