Measurement of antithrombin III in normal and pathologic states using chromogenic substrate S-2238. Comparison with immunoelectrophoretic and factor Xa inhibition assays

Synthetic substrates have recently been developed for the assay of several of the proteolytic Enzymes required for blood coagulation, One of these substrates, S-2238, is specific for Thrombin and hence can also be used to measure antithrombin-heparin co-factor (AT-III). The Thrombin remaining after neutralization by AT-III cleaves a chromophore, p-nitroaniline, from the substrate, which can then te quantified in a spectrophotometer. This new assay for AT-III inhibitory activity was evaluated for normal subjects and for patients who had certain pathologic conditions associated with reduced levels of AT-III. Results from this assay were compared with those from AT-III assays by Laurell immunoelectrophoresis (an immunologic method) and antithrombin inhibition of Factor Xa (a biologic clotting method). The chromogenic assay using S-2238 was found to be sensitive, accurate, and easy to perform; its results for both normal subjects and patients correlated extremely well with those of the immunologic and Factor Xa inhibition assays (r = 0.94-0.98). Measurement of AT-III using this synthetic substrate should be a valuable addition to routine assays in a clinical coagulation laboratory. These and future substrates may prove extremely useful for assays of factors involved in blood coagulation and fibrinolysis.