Antifactor Xa activity measured with amidolytic methods

Methods for the assay of antifactor Xa activity in the presence and absence of heparin are described. Diluted plasma is incubated with bovine, activated factor X (Xa) in stage I, and remaining Xa is measured with the chromogene substrate Bz-Ile-Glu-Gly-Arg-pNA in stage II. In the presence of heparin, the inactivation is completed in 30 sec, and this method measures total Xa-inactivating capacity in diluted plasma (Method I). In a clincal material, this capacity showed a strong positive correlation (r=0.85) to the thrombin-inactivating capacity of diluted heparinized plasma (heparin cofactor activity) and apparently reflects antithrombin III (At-III) concentration. In the absence of heparin, the inactivation of factor xa occurs slowly. With an incubation of 5 min, about 25% of Xa is inactivated, and this assay reflects initial inactivation of Xa (Method II). With this method, a positive, but less strong correlation to the thrombin-inactivating capacity was found (r=0.58), indicating that inhibitors different from At-III accounts for a minor part of the initial inactivation. Determinations in plasma, in which At-III was removed by immunoadsorption, indicated that At-III accounts for about 80% of the initial inactivation. The results of the assays are not significantly influenced by varying concentrations of fibrinogen, fibrinogen degradation products or heparin in the test plasma.