Association of a Plasminogen Activator Inhibitor (PAI-1) with the Growth Substratum and Membrane of Human Endothelial Cells

• Published in: The Journal of cell biology 1987-12, Vol.105 (6), p.2543-2549
• Main Authors: Levin, Eugene G., Santell, Lydia
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Cell lines; Cell Membrane - physiology; Cell metabolism, cell oxidation; Cell physiology; Cells; Cells, Cultured; Cultured cells; Cytosol; Cytosol - analysis; Endothelial cells; endothelium; Endothelium, Vascular - cytology; Female; Fibrinolysis; Fundamental and applied biological sciences. Psychology; Gels; Glycoproteins - isolation & purification; Glycoproteins - physiology; Humans; Immunoprecipitation; man; Molecular and cellular biology; Molecular Weight; Particulate matter; plasminogen activator; Plasminogen activators; Plasminogen Activators - antagonists & inhibitors; Plasminogen Inactivators; Umbilical Veins; veins
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.105.6.2543

We have studied the distribution of the plasminogen activator inhibitor type 1 (PAI-1) in cultures of confluent human umbilical vein endothelial cells. Plasminogen activator inhibitor activity measured by the 125 I-fibrin plate assay was detected in the cytosol (2.85 ± 0.16 U), 100,000 g particulate fraction (1.26 ± 0.30 U), and in the growth substratum (9.82 ± 1.80 U). Characterization of the protein responsible for this activity by reverse fibrin autography, immunoprecipitation, and immunoblotting demonstrated that it had an M r of 46,000 and was antigenically related to PAI-1. Only the active form of the inhibitor was found in all three fractions. Inhibitor in the cytosol and particulate fraction converted to the latent form during 37°C incubation while the substratum inhibitor remained fully active. Extracellular PAI-1 was detected in the growth substratum before its appearance in conditioned medium and represented the major protein deposited beneath the cells. The inhibitor was only transiently localized in the substratum, disappearing within 6 h and concomitantly appearing in the culture medium. Incubation of isolated metabolically labeled substratum with tissue plasminogen activator (tPA) resulted in the appearance and release of an immunologically related inactive 44,000 M r form as well as the tPA-PAI-1 complex (110,000 M r). PAI-1 was also converted into its 44,000-M r form and released by treatment of the substratum with human leukocyte elastase. The rapid deposition and predominance of PAI-1 in the underlying compartment of endothelial cells may explain how the basement membrane is protected from proteolytic degradation by plasmin-generating enzymes.