A Novel Colorimetric Enzyme Assay for Fibrinolysis

Period of Performance: 09/30/1989 - 09/29/1990


Phase 2 SBIR

Recipient Firm

Amplistar, Inc.
Winston-Salem, NC 27101
Principal Investigator


Thrombolytic therapy has become an increasingly important tool in the treatment of acute myocardial infarction. The most recently approved therapeutic agent for this purpose is t-PA (tissue plasminogen activator) which has the advantages of being fibrin specific and is rapidly cleared in vivo by the liver. The detection and quantitation of this component of the fibrinolytic system as well as its inhibitors is still time consuming and is measured by lysis zone areas. We propose a new technique for the quantitation of all the components of the fibrinolytic system including t-PA, plasminogen, plasmin, alpha 2-antiplasmin, and antiactivators (eg PAI-1). The assay uses a solid phase enzyme labelled fibrin as a substrate for locally generated plasmin. The manipulation of concentrations of activators and inhibitors will directly influence the amount of plasmin produced. Quantitation of the resulting plasmin is achieved through the detection and measurement of enzyme labelled fibrin fragments released through the proteolytic action of the plasmin. This assay technique has a number of advantages over existing assays including the use of solid phase fibrin as a substrate and rate enhancer which parallels the situation in vivo. The assay is also convenient for handling multiple samples, lends itself to automation and because of its sensitivity is extremely cost effective.