News and Publications

Regulation of the Plasminogen Activation System

N. Andronicos, J. Felez,* F. Garcia, N. Gingles, A. Gutierrez-Fernandez, N. Mackman,** R.J. Parmer,*** L. Teyton,** L. Zhang, L.A. Miles

* Institute de Recerca Oncologica, Barcelona, Spain
** Department of Immunology, TSRI
*** University of California, San Diego, CA

Interactions of proteins of the plasminogen activation system with cell surfaces modulate cellular functions, including cell migration and prohormone processing. In a reciprocal fashion, cell-surface receptors for plasminogen promote plasminogen activation, for further modulation of cellular functions. We are investigating the regulation of plasmin activity at several levels: gene expression, cellular receptors, and competition for plasmin activity by molecular mimicry.

Recently, we found that localization of Glu-plasminogen on cell surfaces enhances its cleavage to Lys-plasminogen by exogenous plasmin, which removes the amino terminus of the Glu-plasminogen molecule. This cleavage stimulates plasminogen activation because Lys-plasminogen is more readily activated by plasminogen activators than is Glu-plasminogen. Glu-plasminogen was converted to Lys-plasminogen on monocytoid cells in the absence of exogenous plasmin, although the conversion was inhibited by plasmin inhibitors, aprotinin, a₂-antiplasmin, and an anticatalytic antiplasminogen monoclonal antibody. Our results suggest that plasmin converts cell-bound Glu-plasminogen to Lys-plasminogen and that plasmin is produced by activation of monocytoid plasminogen by endogenous monocytoid plasminogen activators to enhance plasminogen activation on the monocytoid cell surface.

Identification and characterization of cell-surface plasminogen receptors are confounded because dead cells bind approximately 10- to 50-fold more plasminogen than viable cells do. Using a proteomics approach, we are developing a method to specifically isolate cell-surface plasminogen receptors from viable cells. We found that the plasminogen-binding and activation capacity of viable peripheral blood monocytes increases 28-fold as the cells differentiate into tissue macrophages in response to colony-stimulating factor 1, thereby arming the cells with plasmin, an important protease involved in cell migration.

High levels of lipoprotein(a) are a major risk factor for development of atherosclerosis. Lipoprotein(a) resembles low-density lipoprotein except that lipoprotein(a) has an additional apoprotein, apoprotein(a). Apoprotein(a) is highly homologous to plasminogen; it contains 10 disulfide-bonded kringle domains homologous to plasminogen kringle 4, 1 domain homologous to plasminogen kringle 5, and a protease-like domain (that cannot develop proteolytic activity). The mechanism by which apoprotein(a) promotes atherothrombosis is thought to be competitive interference with the functions of plasminogen. We and others found previously that apoprotein(a) kringle 4-type 10 competes for the interactions of the plasminogen molecule with fibrinogen, cells, and other regulatory molecules. However, the affinity of these interactions does not account for the entire binding affinity of the native lipoprotein(a) molecule. Using surface plasmon resonance, we found that the apoprotein(a) kringle 5 domain interacts directly with fibrinogen and with plasmin-treated fibrinogen. Our results suggest that apoprotein(a) kringle 5 may be involved in the interaction of the native lipoprotein(a) particle with fibrinogen.

Our recent findings suggest that plasmin activity can be regulated at the level of plasminogen gene expression. We characterized the murine plasminogen promoter and 5´ flanking region. The major transcription start site was identified at -83 bp relative to the ATG translational initiation codon. A 106-bp 5´ flanking fragment was sufficient to confer liver-specific transcription. Treatment with IL-6 stimulated luciferase activity driven by the 5´ flanking region and a region spanning -1063 to -700 bp was required for maximal stimulation by this cytokine. These results indicate the presence of regulatory elements in the 5´ flanking region of the murine plasminogen promoter that may regulate murine plasminogen gene expression and hence plasmin activity.

PUBLICATIONS

Gutierrez, A., Garcia-Bannach, F., Zhang, L., Jenkins, G.R., Parmer, R.J., Miles, L.A. Regulation of plasminogen gene expression. In: Plasminogen Regulation. Waisman, D. (Ed.). Kluwer Academic/Plenum, New York, in press.

Jiang, Q., Taupenot, L., Mahata, S.K., Mahata, M., O'Connor, D.T., Miles, L.A., Parmer, R.J. Proteolytic cleavage of chromogranin A (CgA) by plasmin: selective liberation of a specific bioactive CgA fragment that regulates catecholamine release. J. Biol. Chem. 276:25022, 2001.

Jiang, Q., Yasothornsrikul, S., Taupenot, L., Miles, L.A., Parmer, R.J. The local chromaffin cell plasminogen/plasmin system and the regulation of catecholamine secretion. Ann. N. Y. Acad. Sci., in press.

Miles, L.A., Castellino, F.J., Gong, Y. Conversion of Glu-plasminogen to Lys-plasminogen is necessary for optimal stimulation of plasminogen activation on the endothelial cell surface. Trends Cardiovasc. Sci., in press.

Miles, L.A., Castellino F.J., Gong, Y. The role of lys-pg in cell-mediated plasmin production. In: Plasminogen Regulation. Waisman, D. (Ed.). Kluwer Academic/Plenum, New York, in press.

Miles, L.A., Hawley S.B., Parmer, R.J. Chromaffin cell plasminogen receptors. Ann. N. Y. Acad. Sci., in press.

Tarui, T., Miles, L.A., Takada, Y. Specific interaction of angiostatin with integrin a_vb₃ in endothelial cells. J. Biol. Chem. 276:39562, 2001.

Xue, S., Madison, E.L., Miles, L.A. The kringle V-protease domain is a fibrinogen binding region within Apo(a). Thromb. Haemost. 36:1229, 2001.

Yoshinaga, H., Nakahara, M., Koyama, T., Shibamiya, A., Nakazawa, F., Miles, L.A., Hirosawa, S., Aoki, N. A single thymine nucleotide deletion responsible for congenital deficiency of plasmin inhibitor. Thromb. Haemost. 88:144, 2002.

Zhang, L., Seiffert, D., Fowler, B.J., Jenkins, G.R., Thinnes, T.C., Loskutoff, D.J., Parmer, R.J., Miles, L.A. Plasminogen has a broad extrahepatic distribution. Thromb. Haemost. 87:493, 2002.