The Miles Laboratory

  Cell surface receptors for plasminogen and its activators, urokinase and tissue plasminogen activator, positively regulate the plasminogen activation system by enhancing activation of plasminogen and protecting plasmin from inactivation by protease inhibitors. These receptors also localize the proteolytic activity of plasmin on cell surfaces to promote degradation of the extracellular matrix, which is crucial for processes that require cell migration. We are investigating the structure, function and regulation of these receptors. Although more than one molecular species interacts with plasminogen on the cell surface, only proteins that expose carboxyl-terminal lysines on the cell surface can promote plasminogen activation. On some cell types alpha-enolase functions as a plasminogen receptor. Purified alpha-enolase binds plasminogen, promotes plasminogen activation and protects plasmin from its inhibitor, alpha-2-antiplasmin. We are currently studying the mechanism by which alpha-enolase (which lacks a classical signal sequence) is associated with the cell membrane. Two other major plasminogen-binding membrane proteins also expose carboxyl terminal lysines on monocytoid cell surfaces. We have purified and sequenced these proteins and are now cloning them.

  The plasminogen activation system is negatively regulated by molecules that interfere with binding of plasminogen to its substrates and regulatory molecules. Lipoprotein(a), which is associated with atherosclerosis and with disease processes involving thrombosis, contains an apoprotein with a sequence highly homologous to the amino acid sequence of plasminogen. Hence, lipoprotein(a) binds directly to cells, fibrin and the extracellular matrix and competes for the binding of plasminogen to these regulatory surfaces. These interactions may contribute to the proatherothrombogenic consequences of high levels of lipoprotein(a). We found that multiple domains within apoprotein(a) may modulate these interactions. We expressed these isolated domains in both bacterial and mammalian systems and found that the domains interact directly with fibrin and that the intact lipoprotein(a) particle can inhibit the interaction. We are using mutagenesis to determine the key residues within these constructs that mediate this interaction.