title: Measurement of antithrombin activity by thrombin-based and by factor Xa-based chromogenic substrate assays.
abstract: Functionally active antithrombin can be quantified by chromogenic substrate assays utilizing the heparin cofactor activity of antithrombin and the inhibition rates of thrombin or of activated factor X (FXa). Thrombin-based assays but not FXa-based assays may overestimate the antithrombin activity due to their sensitivity toward heparin cofactor II. We focused on the question whether an overestimation of antithrombin activity by thrombin-based assays involves the risk of misdiagnosing antithrombin-deficient individuals as being non-deficient. We determined antithrombin using two thrombin-based assays and one FXa-based assay in 27 plasma samples from patients with acquired antithrombin deficiency spiked with lepirudin, in antithrombin-deficient plasma and in mixtures of antithrombin-deficient plasma and normal plasma. We also measured antithrombin in healthy subjects, in patients with inherited and acquired antithrombin deficiency and in patients under high-dose heparin treatment. At therapeutic final concentrations of lepirudin, antithrombin activities were considerably overestimated by the thrombin-based assays 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 in patients under high-dose heparin. However, the degree of overestimation in 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.
authors.affiliation: Institute of Hemostaseology and Transfusion Medicine, City Hospital, Ludwigshafen, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Beeck
authors.forename: D
authors.initials: D
authors.lastname: Nagel
authors.forename: G
authors.initials: G
authors.lastname: Pindur
authors.forename: I
authors.initials: I
authors.lastname: Scharrer
authors.forename: A
authors.initials: A
authors.lastname: Preiss
authors.forename: D
authors.initials: D
authors.lastname: Seiler
authors.forename: P
authors.initials: P
authors.lastname: Hellstern
mesh_terms: D000328:Adult; D000368:Aged; D000925:Anticoagulants / Q000097:blood; D000991:Antithrombins / Q000172:deficiency / Q000187:drug effects / Q000378:metabolism*; D002863:Chromogenic Compounds / Q000592:standards*; D003276:Contraceptives, Oral / Q000097:blood / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015951:Factor Xa; D005260:Female; D006493:Heparin / Q000097:blood / Q000494:pharmacology; D006629:Hirudins / Q000031:analogs & derivatives / Q000097:blood; D006801:Humans; D008297:Male; D008722:Methods; D008875:Middle Aged; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins / Q000097:blood; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013917:Thrombin
chemical_list: D000925:Anticoagulants; D000991:Antithrombins; D002863:Chromogenic Compounds; D003276:Contraceptives, Oral; D006629:Hirudins; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins; D006493:Heparin; D013917:Thrombin; D015951:Factor Xa; C083544:lepirudin