Latent tissue plasminogen activator produced by human endothelial cells in culture: evidence for an enzyme-inhibitor complex.
Conditioned medium from cultures of human umbilical vein endothelial cells was analyzed for the presence of tissue plasminogen activator (tPA) and urokinase. Immunoprecipitation studies using metabolically labeled conditioned medium and anti-tPA IgG revealed a single band on autoradiographs corresponding to a Mr of 100,000. No bands were observed after immunoprecipitation with anti-urokinase IgG. The Mr 100,000 tPA was found to be inactive and did not bind to fibrin clots. However, exposure of this tPA to 1% NaDodSO4 resulted in the appearance of plasminogen activator activity with no apparent change in its Mr. Treatment with 10 mM diisopropylfluorophosphate prior to NaDodSO4 activation did not inhibit the NaDodSO4-induced appearance of plasminogen activator activity, indicating that the active site was not available for diisopropylfluorophosphate binding. The possibility that the properties of this Mr 100,000 tPA reflected a tPA-inhibitor complex was examined. Attempts to dissociate such a complex by denaturation, reduction, or extremes of temperature were not successful. However, after treatment of conditioned medium with 1 M hydroxylamine in the presence of 0.1% NaDodSO4, the Mr of the anti-tPA immunoprecipitable material declined by 40,000 to Mr 60,000, a Mr consistent with that of other human tPAs. Hydroxylamine has been shown previously to dissociate covalently coupled serine protease-inhibitor complexes. Furthermore, incubation of purified human melanoma cell tPA with conditioned medium resulted in an increase in its Mr by 40,000 with a concomitant decline in tPA activity. The data suggest that the latent tPA present in the conditioned medium of endothelial cells is composed of a Mr 60,000 tPA associated with an inhibitor.
D002478:Cells, Cultured; D004727:Endothelium / Q000378:metabolism; D005260:Female; D005337:Fibrin / Q000378:metabolism; D006801:Humans; D007531:Isoflurophate / Q000494:pharmacology; D008970:Molecular Weight; D010960:Plasminogen Activators / Q000096:biosynthesis* / Q000302:isolation & purification; D011247:Pregnancy; D011485:Protein Binding; D014471:Umbilical Veins / Q000378:metabolism*
D007531:Isoflurophate; D005337:Fibrin; D010960:Plasminogen Activators