A specific sub-class of plasminogen receptors with carboxyl terminal lysine residues is predominantly responsible for promoting plasminogen activation. One member of this sub-class of receptors is an enolase-related molecule (ERM), t hat is present on the surface of U937 monocytoid cells. I have used 2-dimensional gel electrophoresis to separate alpha-enolase isoforms from ERM, and found that the membrane-associated ERM represents a larger pool of cell surface plasminogen binding activity that the alpha-enolase present in the cell membrane fraction. ERM contains a carboxyl terminal lysine as judged by carboxypeptidase B treatment, and is not recognized by an anti-alpha-enolase antibody. This suggests that ERM is involved in cell surface plasminogen activation and is distinct from alpha-enolase. The major hypothesis to be tested in this proposal is that ERM functions to localize the broad spectrum proteolytic activity of plasmin to monocytoid cell surfaces to enhance their fibrinolytic potential. This objective is closely associated with determining the exact identity of ERM, since the relationship between alpha-enolase and ERM is currently unknown. I an now in the process of using preparative isoelectric focusing to purify ERM for further characterization and protein sequencing. I propose to make antibodies to the ERM protein and use these to probe ERM function in in vitro assays measuring cell surface plasmin activity and fibrinogen degradation. Inhibition of these activities by an anti-ERM antibody will suggest that ERM has a major role in fibrinolysis. Ultimately, I will clone ERM, and then use the cDNA sequence in transfection studies to identify the role of ERM in enhanced plasminogen activation. Transfection of human 293 cells with the cDNA sequence for native and mutant ERM, will allow in vitro determination of ERM function as it relates to both plasmin activity and fibrinolysis.
| Miles, Lindsey A; Hawley, Stephen B; Baik, Nagyung et al. (2005) Plasminogen receptors: the sine qua non of cell surface plasminogen activation. Front Biosci 10:1754-62 |
| Lopez-Alemany, Roser; Longstaff, Colin; Hawley, Stephen et al. (2003) Inhibition of cell surface mediated plasminogen activation by a monoclonal antibody against alpha-Enolase. Am J Hematol 72:234-42 |
