News and Publications

Regulation of the Plasminogen Activation System

J. Felez,* Y. Gong, S. Hawley, R.J. Parmer,** S. Xue, L.A. Miles

* Institut de Recerca Oncologica, Barcelona, Spain
** University of California, San Diego, CA

Cell-surface receptors for plasminogen and its activators, u-plasminogen activator (urokinase) and t-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.

Glu-plasminogen, the native circulating form of plasminogen, is cleaved to a more readily activatable proteolytic derivative, Lys-plasminogen, upon binding to cell surfaces. Using a panel of protease inhibitors, we showed that plasmin is the enzyme responsible for the formation of Lys-plasminogen on the cell surface. Northern blotting of monocytoid cells indicated that plasminogen was present, suggesting that monocytes provide a source of plasmin. In plasminogen activation assays, we found that this conversion of Glu-plasminogen to Lys-plasminogen accounts for the majority of the enhancement of plasminogen activation on cell surfaces.

Although, more than one kind of molecule can bind plasminogen to cell surfaces, only proteins that expose carboxyl-terminal lysines on the cell surface can promote plasminogen activation. Two major plasminogen-binding membrane proteins were detected on monocytoid cells. These proteins also expose carboxyl-terminal lysines on the cell surface. 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.

The plasminogen activation system is also regulated by the synthesis and secretion of components of the system. Using several chromaffin cell sources, including the rat pheochromocytoma PC-12 chromaffin cell line, primary cultures of bovine adrenal chromaffin cells, and human pheochromocytoma cells, we found that t-plasminogen activator also enters the regulated secretory pathway and is packaged in and released directly from catecholamine storage vesicles. Therefore, these vesicles may be an important reservoir, and sympathoadrenal activation may be an important physiologic mechanism for the rapid release of this plasminogen activator.

We have discovered a potential mechanism by which t-plasminogen activator may enter the regulated secretory pathway: interaction with chromogranin A, the major secretory protein in the catecholamine storage vesicle. This interaction may provide a means by which aggregated complexes of chromogranin A and t-plasminogen activator bind and are separated from constitutively secreted proteins in the trans-Golgi network, with subsequent movement into the dense core--regulated secretory vesicle.

PUBLICATIONS

Fowler, B.J., Mackman, N., Parmer, R.J., Miles, L.A. Human single chain urokinase binds to Chinese hamster ovary cells: Cloning of hamster u-PAR. Thromb. Haemost., in press.

Miles, L.A., Sebald, M.T., Fless, G.M., Scanu, A.M., Curtiss, L.K., Plow, E.F., Hoover-Plow, J.L. Interaction of lipoprotein(a) [Lp(a)] with the extracellular matrix. Fibrinolysis Proteolysis, in press.

Plow, E.F., Redlitz, A., Hawley, S.B., Xue, S., Herrin, T., Hoover-Plow, J.L., Miles, L.A. Assembly of the plasminogen system on cell surfaces. In: Handbook of Experimental Pharmacology: Fibrinolytics and Antifibrinolytics. Bachmann, F. (Ed.). Springer-Verlag, New York, in press.

Plow, E.F., Ugarova, T., Miles, L.A. Interaction of the fibrinolytic system with the vessel wall. In: Thrombosis and Hemorrhage, 2nd ed. Loscalzo, J., Schafer, A. (Eds.). Williams & Wilkins, Baltimore, 1998, p. 373.