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.
Department of Biological Sciences, University of Wollongong, NSW, Australia.
M J
MJ
O'Mullane
M S
MS
Baker
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology*; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000494:pharmacology; D016923:Cell Death; D002470:Cell Survival; D003513:Cycloheximide / Q000494:pharmacology; D004789:Enzyme Activation; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007700:Kinetics; D008239:Lysine; D009000:Monocytes / Q000166:cytology / Q000378:metabolism*; D010958:Plasminogen / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017396:Plasminogen Activator Inhibitor 2 / Q000494:pharmacology; D015849:Plasminogen Inactivators / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D014148:Tranexamic Acid / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D017396:Plasminogen Activator Inhibitor 2; D015849:Plasminogen Inactivators; D011500:Protein Synthesis Inhibitors; C086298:glutamyl-glycyl-arginyl-chloromethyl ketone; D014148:Tranexamic Acid; D010958:Plasminogen; D003513:Cycloheximide; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; D014568:Urokinase-Type Plasminogen Activator; D008239:Lysine