Discriminating between cell surface and intracellular plasminogen-binding proteins: Heterogeneity in profibrinolytic plasminogen-binding proteins on monocytoid cells

When plasminogen binds to cell surfaces, its activation is markedly enhance d compared to soluble plasminogen. Although several distinct molecules may contribute to plasminogen binding to a given cell type, the subset of plasm inogen receptors responsible for enhancing plasminogen activation expose a carboxyl-terminal lysine on the cell surface and are sensitive to proteolys is by carboxypeptidase B (CpB). To distinguish this subset of plasminogen r eceptors from plasminogen-binding proteins that are not profibrinolytic, we treated intact U937 monocytoid cells and peripheral blood monocytes with C pB to remove exposed carboxyl-terminal lysines, and subjected the membrane proteins to two-dimensional gel electrophoresis followed by ligand blotting with I-125-plasminogen, Western blotting was performed with antibodies aga inst previously characterized candidate plasminogen receptors to identify p lasminogen-binding proteins on the two-dimensional ligand blots. Densitomet ry of autoradiograms of the I-125-plasminogen ligand blots of U937 cell mem branes revealed that membrane-associated alpha -enolase, actin and annexin II showed minimal changes in I-125-plasminogen binding following CpB treatm ent of intact cells, suggesting that these proteins an not accessible to Cp B on the U937 cell surface and most likely do nor serve as profibrinolytic plasminogen receptors on U937 cells. In contrast, densitometry of autoradio grams of I-125-plasminogen ligand blots of monocyte membranes revealed that I-125-plasminogen binding to alpha -enolase was reduced 71% by treatment o f intact cells with CpB, while binding to annexin II was reduced 14%.