Synthesis of a fibrinolytic activator and inhibitor by endothelial cells.
Vascular endothelial cells derived from rabbit vena cava and maintained in continuous culture exhibited properties characteristic of the intact endothelium. These cells were used as a model for characterizing the fibrinolytic components specified by the endothelium. Endothelial cells in culture digested radiolabeled fibrinogen. Digestion resulted from the synthesis and secretion of a plasminogen activator. Fibrinolysis was not detected when cells were grown in medium lacking plasminogen, indicating the absence of plasminogen-independent fibrinolytic enzymes. Phorbol-myristate-acetate increased extracellular plasminogen activator activity dramatically. This increase was prevented when actinomycin D or cycloheximide was included in the growth medium, indicating that new gene expression was required for it. Intracellular plasminogen activator could not be detected unless the cell extracts were exposed briefly to mildly acidic conditions. Mixing experiments between acid-treated and untreated extracts suggested that the cells contained a potent, acid-labile inhibitor of fibrinolysis. As little as 10 mug of protein from whole cell extracts inhibited both cell and urokinase-mediated fibrinolysis by more than 70%. Cell fractionation studies localized the inhibitor to the cytosol whereas plasminogen activator activity was restricted to the membrane-rich fraction. This membrane fraction did not require acidification for activity, suggesting that the inhibitor had been removed and that acidification did not activate a plasminogen proactivator. These observations demonstrate that regulation of endothelial fibrinolytic activity is far more complex than had been anticipated and raise several uncertainties in regard to detecting the presence of plasminogen activators in cells and tissues.
D000818:Animals; D002478:Cells, Cultured; D004727:Endothelium / Q000378:metabolism; D004789:Enzyme Activation; D005341:Fibrinolysin / Q000096:biosynthesis*; D005342:Fibrinolysis; D007700:Kinetics; D010958:Plasminogen / Q000378:metabolism; D011817:Rabbits; D013347:Subcellular Fractions / Q000378:metabolism; D014684:Venae Cavae / Q000378:metabolism
D010958:Plasminogen; D005341:Fibrinolysin