SELECTIVE CAPTURE AND QUANTITATION OF SERINE PROTEASES

THROMBOSIS AND THROMBOLYSIS ARE MAINTAINED IN A DELICATE BALANCE BY A SERIES OF ENZYMATIC REACTIONS. THE COMPONENT ENZYMES ARE PRODUCED AS NEEDED FROM ZYMOGENS, INACTIVE FORMS THAT CIRCULATE IN THE BLOOD AT RELATIVELY HIGH CONCENTRATIONS. IDENTIFYING AND EVALUATING PATIENTS AT RISKFOR CLINICAL THROMBOTIC DISORDERS WILL REQUIRE ACCURATE MEASUREMENT OF PLASMA LEVELS OF THESE ACTIVE PRO- AND ANTICOAGULANT FACTORS. SPECIFIC PROBES HAVE BEEN USED TO BIND TO AND IRREVERSIBLY INHIBIT SERINE PROTEASES, SUCH AS THOSE ASSOCIATED WITH THROMBOSIS AND THROMBOLYSIS. HAEMATOLOGIC TECHNOLOGIES, INC., HAS SYNTHESIZED A FORM OF ONE SUCH PROBE CONTAINING A SECONDARY RECOGNITION SITE THAT CAN BE USED TO CAPTURE THE ENZYME ONTO A SOLID PHASE. SUCH A PROBE HAS BEEN USED, ALONG WITH AN ENZYME-CONJUGATED MONOCLONAL ANTIBODY SPECIFICFOR TISSUE-TYPE PLASMINOGEN ACTIVATOR (TPA), TO PREPARE A PROTOTYPE ASSAY FOR THIS SERINE PROTEASE. THIS PHASE I PROJECT HAS THE FOLLOWING SPECIFIC AIMS: (1) TO OPTIMIZE THE CONDITIONS FOR PROBE PRODUCTION, (2) TO DETERMINE OPTIMAL CONDITIONS FOR USE OF THE PROBE IN WHOLE BLOOD AND PLASMA, AND (3) TO FURTHER DEVELOP THE TPA ASSAY FOR USE WITH CLINICAL SPECIMENS.