title: Chemical crosslinking of urokinase to pulmonary surfactant protein B for targeting alveolar fibrin.
abstract: 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, (125)I-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.
authors.affiliation: Department of Internal Medicine, Justus-Liebeg University Giessen, Germany.
authors.forename: Clemens
authors.initials: C
authors.lastname: Ruppert
authors.forename: Philipp
authors.initials: P
authors.lastname: Markart
authors.forename: Reinhold
authors.initials: R
authors.lastname: Schmidt
authors.forename: Friedrich
authors.initials: F
authors.lastname: Grimminger
authors.forename: Werner
authors.initials: W
authors.lastname: Seeger
authors.forename: Claus-Michael
authors.initials: CM
authors.lastname: Lehr
authors.forename: Andreas
authors.initials: A
authors.lastname: Günther
mesh_terms: D002863:Chromogenic Compounds; D003432:Cross-Linking Reagents / Q000737:chemistry; D015195:Drug Design; D004355:Drug Stability; D005337:Fibrin / Q000187:drug effects*; D005342:Fibrinolysis / Q000187:drug effects; D005343:Fibrinolytic Agents / Q000138:chemical synthesis / Q000494:pharmacology; D006801:Humans; D008171:Lung Diseases / Q000188:drug therapy* / Q000517:prevention & control; D011650:Pulmonary Alveoli; D037701:Pulmonary Surfactant-Associated Protein B / Q000737:chemistry* / Q000627:therapeutic use; D011663:Pulmonary Surfactants / Q000737:chemistry; D019851:Thrombophilia / Q000188:drug therapy; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry* / Q000627:therapeutic use
chemical_list: D002863:Chromogenic Compounds; D003432:Cross-Linking Reagents; D005343:Fibrinolytic Agents; D037701:Pulmonary Surfactant-Associated Protein B; D011663:Pulmonary Surfactants; D005337:Fibrin; D014568:Urokinase-Type Plasminogen Activator