IDENTIFICATION OF PLASMINOGEN IN MATRIGEL(TM) AND ITS ACTIVATION BY RECONSTITUTION OF THIS BASEMENT-MEMBRANE EXTRACT

Matrigel(TM) a basement membrane (BM) extract of the Engelbreth-Holm-S warm (EHS) sarcoma, used in tumor invasion assays, was found to contai n plasminogen. Plasminogen was identified, using Western blot analysis and casein zymograms, by comparison with human plasminogen. Matrigel contained approximately 20-100 ng of plasminogen per 100 mu g of prote in as determined by these assays. Matrigel reconstitution and incubati on at 37 degrees C caused activation of plasminogen, which was serine protease dependent and involved tissue plasminogen activator (tPA) as an anti-tPA antibody which inhibited activation. This reconstitution a nd incubation also caused leupeptin-inhibitable degradation of Matrige l components as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Degradation of the BM extract copolymerized in zymogr ams was caused by human plasminogen and plasminogen in the Matrigel. M aximal plasmin activity following incubation of Matrigel at 37 degrees C for 16 h, was equivalent to approximately 10 ng of purified plasmin using the plasmin substrate D-Val-Leu-Lys p-nitroanilide. Matrigel, t herefore, contained all the components of the plasmin-generating syste m, including plasminogen. The plasmin generated degraded Matrigel comp onents and exogenous substrates. Our data suggest that, since this tum or BM acts as a reservoir for enzymes of the plasmin-generating system , caution should be taken by investigators interpreting data concernin g the effects of Matrigel on cell behavior and in particular cellular invasion.