A Chromogenic Anti-FIIa Assay for the Assessment of Melagatran Activity in Plasma

Abstract number: P0143

Reber G, de Moerloose P

Although no coagulation monitoring is needed during treatment with ximelagatran, the assessment of its plasma activity (as melagatran) may be necessary in emergency situations e.g. overdosage. Prothrombin time and aPTT can be used for qualitative but are not well suited for quantitative assessment of anticoagulant activity of oral direct thrombin inhibitors (DTI) in plasma. Methods relying on discrete steps of the coagulation cascade have been developed (ecarin clotting time, prothrombin induced clotting time, etc.). These methods are based on the activation and subsequent inhibition of coagulation factors, cofactors and fibrinogen present in the sample. Their level as well as the presence of other anticoagulants may interfere with the assessment of DTIs. For DTIs assessment, chromogenic anti-FIIa assays are independent from plasma components.

Methods:  We have adapted a hirudin chromogenic anti-FIIa assay (Hirudin-Test/Assay, Dade Behring, Marburg, Germany) for the assessment of melagatran, the active form of ximelagatran (AstraZeneca, Mölndal, Sweden) in plasma samples. The test design is a one-stage competitive format and was performed with a BCS centrifugal analyser (Dade Behring). The reaction mixture contained 6 U bovine thrombin dissolved in Tris buffer and 0.5 mmol/l thrombin substrate (tos-gly-pro-arg-ANBA-IPA) in a final volume of 0.23 ml. The increase in absorbance at 405 nm was recorded between 15 and 40 sec after mixing. Sample volume was 30 µl.

Results:  The calibration curve spanned from 0 to 1 µmol/l and the range of measurement 2.2 to 1.1 OD units/min. Within-run coefficients of variation were 8.8% and 4% at 0.2 and 0.8 µmol/l melagatran, respectively. The corresponding between-run figures were 9.1% and 3.4%. The chromogenic anti-FIIa assay is independent on coagulation status of the patient, except for the presence of other thrombin inhibitors such as unfractionated heparin or hirudin.

Conclusion:  The chromogenic anti-FIIa assay tested appears well suited for assessment of melagatran effect in plasma.