In recent years, therapeutic intervention into myocardial infarction has focused on the use of plasminogen activators. The penultimate step in these modalities of thrombolytic therapy is the proteolytic conversion of plasminogen to plasmin. The accurate assessment of the extent of this conversion, as well as the quantitation of circulating levels of therapeutic plasminogen activators is crucial to development of safe and effective treatment protocols. There exists a need for improved assays to assess plasma levels of plasminogen, plasmin and urokinase that can distinguish between inactive and active forms of these enzymes. Our proposed assay format would combine the advantages of conventional antigen specific assays with active-site specific assays. Phase I of the proposed research will draw on our experience in producing bifunctional probes for the active sites of serine proteases. These probes consist of peptide chloromethyl ketones of limited specificity linked to a biotin moiety. These inhibitors covalently label the active site of serine proteases with biotin. We plan to extend this technology to assay systems for plasminogen, plasmin and urokinase.
