Measurement of antithrombin activity by thrombin-based and by factor Xa-based chromogenic substrate assays

Functionally active antithrombin can be quantified by chromogenic substrate assays utilizing the heparin cofactor activity of antithrombin and the inh ibition rates of thrombin or of activated factor X (FXa). Thrombin-based as says but not FXa-based assays may overestimate the antithrombin activity du e to their sensitivity toward heparin cofactor II. We focused on the questi on whether an overestimation of antithrombin activity by thrombin-based ass ays involves the risk of misdiagnosing antithrombin-deficient individuals a s being non-deficient. We determined antithrombin using two thrombin-based assays and one FXa-based assay in 27 plasma samples from patients with acqu ired antithrombin deficiency spiked with lepirudin, in antithrombin-deficie nt plasma and in mixtures of antithrombin-deficient plasma and normal plasm a. We also measured antithrombin in healthy subjects, in patients with inhe rited and acquired antithrombin deficiency and in patients under high-dose heparin treatment. At therapeutic final concentrations of lepirudin, antith rombin activities were considerably overestimated by the thrombin-based ass ays but not by the FXa-based assay. The residual antithrombin activities in antithrombin-deficient plasma determined by the thrombin-based assays were markedly higher than the corresponding values obtained with the FXa-based assay. The thrombin-based assays also overestimated antithrombin activity i n patients under high-dose heparin. However, the degree of overestimation i n the range between 50 and 100 IU/dl was too low to misidentify individuals with inherited or acquired antithrombin deficiency as normal. We conclude that functionally active antithrombin can be reliably determined using FXa- based chromogenic substrate assays in all settings examined. Thrombin-based assays must not be used in patients under treatment with hirudin or other direct thrombin inhibitors. Blood Coag Fibrinol 11:127-135 (C) 2000 Lippinc ott Williams & Wilkins.