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The Fibrinolytic System of Cultured Endothelial Cells

  • David J. Loskutoff
  • Raymond R. Schleef
  • Michael Sawdey
Part of the GWUMC Department of Biochemistry Annual Spring Symposia book series (GWUN)

Abstract

Abnormal thrombus formation and dissolution are associated with several cardiovascular diseases including atherosclerosis and both thromboembolic and hemorrhagic conditions. The walls of blood vessels undoubtedly contribute to the pathogenesis of these disorders. More specifically, vascular endothelial cells have been shown to influence both the coagulation and fibrinolytic systems. We have been studying cultured bovine aortic endothelial cells (BAEs) as a model of endothelium. The fibrinolytic activity of these cells results from the production of multiple, functionally distinct forms of plasminogen activator (PA), including both urokinaselike (uPA) and tissue-type (tPA) molecules. The overall fibrinolytic activity of these cells changes with their growth state and in response to the presence of a variety of agents including activated protein C. Although it has been suggested that such changes reflect changes in PA, our recent and unexpected finding that these cells also synthesize an unusually stable fibrinolytic inhibitor makes accurate interpretation of such results difficult. For example, do the altered fibrinolytic states in these cells following various treatments or, for that matter, in blood in certain human diseases reflect changes in PA, inhibitor, or both? The inhibitor is a major product of BAEs, accounting for 2-12% of the total protein secreted by the cells in a 24-h period. It is a single-chain glycoprotein of M r 50,000, isoelectric point 4.5–5.0, and migrates in the ßI region when analyzed by agarose zone electrophoresis. It is an antiactivator and can neutralize the activity of both uPAs and tPAs. Inhibition is associated with the formation of an enzyme-inhibitor complex that survives SDS-PAGE. Preliminary kinetic analysis of the interaction between the purified PAI and tPA established an apparent K a of 5 × 107 M-1 sec-1 and k d of 10-13 M. The PAI is active after incubation in the presence of 0.1% SDS or at pH 2.7 but is rapidly inactivated by H2O2 and other oxidants. Activity can be restored by treating the inactive PAI with methionine sulfoxide reductase, suggesting that the loss of activity is caused by oxidation of a critical methionine residue. Interestingly, both a latent form of PAI (representing 95% of the total) and an active form (5%) can be detected in cell samples. The latent form can be converted into the active one by treatment with SDS and other denaturants. Antiserum to the BAE PAI removes the rapidly acting PAI activity from human platelets, serum, and plasma. These results indicate that the BAE PAI and the PAIs present in these blood samples are immunologically related and suggest that the ßi-PAI may be the physiologically relevant inhibitor of PA in the vascular system. The PAI gene has been cloned and sequenced. Its relation to other protease inhibitors is discussed

Keywords

Plasminogen Activator Plasminogen Activator Inhibitor Fibrinolytic Activity Fibrinolytic System Bovine Aortic Endothelial Cell 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Plenum Press, New York 1987

Authors and Affiliations

  • David J. Loskutoff
    • 1
  • Raymond R. Schleef
    • 1
  • Michael Sawdey
    • 2
    • 3
  1. 1.Scripps Clinic and Research FoundationLa JollaUSA
  2. 2.Department of BiologyUniversity of CaliforniaSan DiegoUSA
  3. 3.Scripps Clinic and Research FoundationLa JollaUSA

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