Elevated plasminogen receptor expression occurs as a degradative phase event in cellular apoptosis.

Plasminogen activation (PA) is involved in a variety of extracellular proteolytic events, such as fibrinolysis, cell migration (e.g. angiogenesis, tumour cell invasion, inflammation, wound healing, bacterial invasion), ovulation, tissue remodelling and the activation of other protease classes and growth factors. These diverse roles are due to the specific localization of components of the PA system to extracellular matrices, basement membranes, fibrin and cell surfaces. We have previously reported that PA is dramatically elevated during cycloheximide (CHX)-induced apoptosis in U937 cells due to a concomitant increase in both plasminogen receptors (PLG-R; i.e. specific PLG binding) and cell-surface urokinase plasminogen activator. We now extend this study by showing that the increase in PLG-R (resulting in an increase in specific PLG binding) is a late apoptotic event coincident with propidium iodide uptake and internucleosomal DNA fragmentation but occurring after elevations in phosphatidylserine exposure. Plasminogen was also observed to dramatically increase the rate of CHX-induced apoptosis. We conclude that PA may play a role in the degradative (i.e. late-stage) events of cellular apoptosis.

Increased plasminogen binding is associated with metastatic breast cancer cells: differential expression of plasminogen binding proteins.

Overexpression of urokinase-type plasminogen activator and its receptor correlates with metastatic capacity in breast cancer. In this study we show that the urokinase/urokinase receptor-overexpressing, metastatic human breast cancer cell line MDA-MB-231 (1) bound significantly more cell-surface plasminogen in a lysine-dependent manner and (2) was capable of generating large amounts of plasmin compared with the non-metastatic cell lines MCF-7 and T-47D. In addition, distinct plasminogen binding proteins were detected in the plasma membranes of the cell lines, suggesting heterogeneity of binding proteins. Plasminogen binding was analysed using a combination of dual-colour fluorescence flow cytometry and ligand histochemistry (for comparative and cellular localization of ligand binding), and fluorimetry (for Scatchard analysis). Apart from revealing the greater plasminogen binding capacity of MDA-MB-231 cells, flow cytometry and histochemistry also revealed that, in all three cell lines, non-viable or permeabilized cells bound significantly more plasminogen in a lysine-dependent manner than viable or non-permeabilized cells. Viable MDA-MB-231 cells bound plasminogen with moderate affinity and high capacity (Kd = 1.8 microM, receptor sites per cell 5.0 x 10(7). Our results indicate that differences in cell surface-specific plasminogen binding capacity between cell lines may not be detectable with binding techniques that cannot distinguish between viable and non-viable cells.

Loss of cell viability dramatically elevates cell surface plasminogen binding and activation.

The plasminogen activation cascade is focused at the cell surface by virtue of the presence of plasminogen and plasminogen activator receptors. We have utilized flow cytometric plasminogen (plg) binding and activation assays to examine both plasminogen binding and activation on the surface of specific subpopulations of U937 cells (viable, apoptotic, and dead cells). A direct relationship was found to exist between cell viability (propidium iodide uptake) and the magnitude of lysine-dependent plasminogen binding, with apoptotic and dead subpopulations of cells binding up to 100-fold more plasminogen than viable cells. Despite the high level of lysine-dependent plasminogen binding on dead cells, plasminogen activation was minimal due to low levels of cell-surface urokinase plasminogen activator. Plasminogen activation readily occurred on the surface of apoptotic cells because of a dramatic increase in both lysine-dependent plasminogen binding and endogenous urokinase plasminogen activator. These results indicate that colocalization of plasminogen and urokinase plasminogen activator are paramount for plasminogen activation to proceed on the cell surface. Our data also strongly implicate the involvement of the plasminogen activation cascade in apoptosis, especially on urokinase plasminogen activator-expressing cell types. The current study clearly supports the important role of flow cytometry in cellular plasminogen binding and activation studies.