A Simple Amidolytic Method for the Determination of Functionally Active Antithrombin III

A simple amidolytic method for the determination of the concentration of functionally active antithrombin III is described. Plasma is diluted with buffer containing EDTA and Polybrene®. In stage I, diluted plasma is incubated with thrombin. EDTA retards fibrin polymerization, and plasma fibrinogen does not influence the assay. Polybrene makes the assay result independent of heparin. In stage II, remaining thrombin is determined with the chromogenic substrate benzoyl-Phe-Arg-p-NA. The method is simpler and has a higher accuracy than clotting methods. There is a close correlation between the results obtained with this assay and with immunoassay of antithrombin III.