This is a revised application to investigate the mechanism by which the interaction of plasminogen with specific cellular receptors functions to enhance fibrinolysis. The proposal is based on data establishing that plasminogen interacts with cells in a structurally specific manner, and that this interaction promotes plasminogen activation, and establishes a local nidus of protected plasmin activity. The high capacity of cells for plasminogen suggests that several cell surface molecules may interact with plasminogen. The Applicant has identified a specific subclass of plasminogen receptors with carboxyl terminal lysyl residues that are predominantly responsible for promoting plasminogen activation. She has identified and characterized one member of this subclass, ERM, an alpha-enolase related molecule. ERM exhibits profibrinolytic properties in that it: enhances plasminogen activation, competes for the interaction with the major plasmin inhibitor, a2-antiplasmin and is present on cell surfaces at high capacity. However, despite its localization on the cell surface, it does not have a clearable signal peptide. Hence the first Specific Aim will be to determine whether the alpha-enolase related molecule on the cell surface is structurally distinct from cytoplasmic alpha-enolase, and to test potential mechanisms by which ERM becomes associated with the surface. The Applicant has also noted three additional cell surface proteins which have exposed carboxyl terminal lysyl residues and bind plasminogen. The profibrinolytic function of these proteins will be evaluated in Specific Aim 2, and they will be further characterized, if warranted.
In Specific Aim 3, mechanisms of regulation of plasminogen binding site expression will be investigated. The roles of adhesion and specific intracellular signalling pathways in up-regulation of plasminogen receptor expression will be tested, and up-regulation of specific plasminogen receptors will be addressed. Functional consequences of receptor modulation will be also be investigated. Finally, in Specific Aim 4, the Applicant proposes to test the hypothesis that the transition from glu-plasminogen to lys plasminogen is required for the enhancement of plasminogen activation on cell surfaces, and to isolate the cellular enzymes repsonsible for this pro-fibrinolytic activity.
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