Coagulant versus amidolytic properties of human and bovine thrombins: implications in standardization and diagnostic...

Since the introduction of synthetic peptide substrates for thrombin, many amidolytic methods for the determination of AT III, heparin cofactor II, prothrombin, thrombin, platelet factor 4, and absolute levels of heparin have been proposed. All of these methods utilize thrombin that has been standardized in coagulant assays using either fibrinogen (human or bovine) or citrated plasma substrates. These thrombin preparations may contain noncoagulant forms of thrombin, prothrombin fragments, and other serine protease enzymes. Impurities other than variant forms of thrombin in commercial preparations may interact with antithrombin and other reagents altering the results of an assay. Similarly, the noncoagulant forms of thrombin contribute to amidolytic but not coagulant activity. If these parameters are not properly controlled, the assays based on amidolysis are seriously affected. Our studies on the amidolytic and coagulant properties of commercial thrombins suggest that, although these preparations are assigned their potency in NIH units, they vary greatly and do not truly exhibit the same potency as designated in the coagulant assays. In addition, these thrombin preparations show wide variations in their amidolytic actions toward synthetic chromogenic and fluorogenic peptide substrates. We propose that thrombin preparations for chromogenic and fluorogenic peptide assays should be standardized in terms of their amidolytic activity under defined conditions. In addition, further studies should be conducted to prove their efficacy in providing reliable diagnostic information in clinical laboratory assays.