We described an amidolytic method for determining the anticoagulant activity of commercially available low molecular-weight heparin (LMWH) with the use of factor Xa (FXa) and thrombin (FIIa), and a chromogenic peptidyl substrate, S-2222 or S-2238. The procedures were based on the photometric determination of the inactivation of FXa and FIIa after incubation with LMWH in the presence of antithrombin III (AT III). At first, the optimal condition of FXa and FIIa activities with respect to pH, temperature, and the amounts of AT III and LMWH-International Standards (LMWH-IS) were determined. And then, the anticoagulant activities of various LMWHs were determined under the established conditions. In the comparative study with LMWHs, significant differences were found in anti FXa and anti FIIa activities, and their ratios. The anti FIIa activity was reconfirmed to decrease with decreasing molecular weight of LMWHs. On the other hand, the anti Xa activity was, however, less dependent on the molecular weight of LMWHs.
