NOVEL ACTIVITY AND IMMUNOASSAYS FOR PAI-1

PLASMINOGEN ACTIVATION IS MODULATED BY A NUMBER OF INHIBITORS. THE FAST ACTING INHIBITOR OF T-PA AND U-PA HAS BEEN DESIGNATED PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1). THE DETECTION OF RAISED LEVELS OF THIS ACUTE PHASE PROTEIN IS INDICATIVE OF AN IMBALANCE IN THE HEMOSTATIC SYSTEM. IN ORDER TO FULLY ASSESS PAI-1 PLASMA LEVELS IT IS NECESSARY TO MEASURE THE PROTEIN BY BOTH ANTIGENIC AND ACTIVITY ASSAYS. OUR APPROACH WILL ALLOW THE MEASUREMENT OF TOTAL PLASMA ANTIGEN LEVELS, PAI-1/T-PA ANTIGEN LEVELS, PAI-1 ACTIVITY LEVELS, AND THE ACTIVITY AND ANTIGEN LEVELS OF VITRONECTIN/PAI-1 COMPLEX WHICH REPRESENTS A SPECIAL, STABLE SUBPOPULATION OF PAI-1 WHICH MAY HAVE ANTITHROMBIN ACTIVITY. THE ACITIVITY ASSAY INVOLVES THE USE OF AN ENZYME LABELLED FIBRIN MATRIX AS THE SUBSTRATE FOR T-PA; RESIDUAL ACTIVITY WILL ALLOW THE MEASUREMENT OF PAI-1 INHIBITORY ACTIVITY. WE WILL USE BOTH STANDARD ELISA ASSAY AND ULTRASENSITIVE ELISA-ELCA ASSAY TECHNIQUE. THIS IS BECAUSE LEVELS OF FREE PAI-1 ANTIGEN AND COMPLEXED PAI-1 DIFFER BY AT LEAST 10 FOLD. WITH THE PREPARATION OF REAGENTS TO CARRY OUT THESE ASSAYS WE WILL BE ABLE TO DO INITIAL TESTS ON ASSAYS FOR PLASMA COMPONENTS SUCH AS THE VIRONECTIN/PAI-1 COMPLEXES AND U-PA/PAI-1 COMPLEXES.