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.