An Antithrombin III Assay Based on Factor Xa Inhibition Provides a More Reliable Test to Identify Congenital Antithrombin III Deficiency Than an Assay Based on Thrombin Inhibition

Abstract

SummaryObjectives: To determine whether functional antithrombin III (AT-III) levels measured by a factor Xa inhibition (AT-III-Xa) assay identifies AT-III deficient individuals more reliably than functional AT-III levels measured by a thrombin inhibition (AT-III-IIa) assay.Study design: Cross-sectional study.Patient population: Sixty-seven members of a large family with type 2 AT-III deficiency.Intervention: DNA analysis was used as the reference diagnostic standard for AT-III status and subjects were classified as AT-III deficient or non deficient according to these results. Functional AT-III levels were measured in all subjects using: 1) a chromogenic substrate for thrombin and added human thrombin (AT-III-IIa), and 2) a chromogenic substrate for factor Xa and added bovine factor Xa (AT-III-Xa). Functional heparin cofactor II (HC-II) levels were measured using a commercially available kit. The proportions of 125I-α-thrombin complexed to AT-III and HC-II were measured by polyacrylamide gel electrophoresis and autoradiography.Results: Thirty-one (46%) individuals were classified as AT-III deficient and 36 (54%) as AT-III non deficient. AT-III-Xa assay measured a significantly lower mean AT-III value and a narrower range for individuals classified as AT-III deficient than the AT-III-IIa assay. Using the AT-III-IIa assay, six subjects had borderline AT-III levels compared to none with the AT-III-Xa assay. Thrombin inhibition by HC-II likely accounts for the AT-III-IIa assay giving higher values than the AT-III-Xa assay since 1) there was a significant correlation between the difference in AT-III-IIa and AT-III-Xa levels and HC-II levels, 2) the mean level of HC-II was significantly higher for individuals who had a positive difference between AT-III-IIa and AT-III-Xa levels compared to those who had a negative difference and 3) there was a significant correlation between the difference in AT-III-IIa and AT-III-Xa levels and the percentage of 125I-α-thrombin complexed to HC-II.Conclusion: The AT-III-Xa assay is a better discriminant between AT-III deficient and AT-III non deficient individuals than the AT-III-IIa assay.