The Application of Thrombin Generation Test Is Compromised By Antithrombin III Deficiency

Background: Antithrombin III (ATIII) is a stoichiometric inhibitor of factor Xa and thrombin and a major physiological regulator of coagulation. Reduction of ATIII levels, for example in patients with congenital ATIII deficiency, causes a rise in active thrombin which is associated with risk of venous and arterial thrombosis (1, 2). The calibrated automated thrombogram (CAT) assay is widely used to measure thrombin generation in human plasma. Therewith, the fluorogenic substrate Z-G-G-R-AMC is cleaved by the thrombin emerging over time in the plasma sample. Thus, an increase in fluorescence signal reflects the amount of thrombin provided that fluorogenic substrate is in excess over thrombin. To account for substrate consumption, plasma color, and 'inner filter' effect, the fluorescence signal is compared to alpha-2-macroglobulin thrombin complex, the thrombin calibrator. In this complex, thrombin is protected from its physiological inhibitors, but retains its ability to cleave small substrates (3).

Aim: We assessed the technical feasibility of measuring the effect of ATIII reduction on thrombin generation in normal and FVIII-inhibited human plasma in the tissue factor (TF) triggered CAT assay.

Methods: CAT was performed in ATIII deficient plasma with 1 pM of TF to trigger thrombin generation. The plasma was supplemented with 0.125 - 2.5 µM ATIII to achieve ATIII plasma levels of 5 - 100% of normal. The ATIII titration was also done in ATIII deficient plasma inhibited with a goat polyclonal anti-human FVIII antibody to simulate hemophilia A conditions. Raw data analysis focused on the velocity of fluorescence signal increase, which is usually automatically converted to nM of thrombin by the Thrombinoscope software.

Results: Reduced ATIII levels resulted in an apparent concentration dependent increase in thrombin generation in both normal and FVIII inhibited human plasma. For example, a reduction from 2.5 to 1.25 µM ATIII resulted in an apparent increase of 45 - 55% in thrombin peak and ETP values. Most importantly, analysis of the raw data showed that the fluorescence signal reached a plateau after 30 min (normal plasma) and 50 min (FVIII-inhibited plasma) in samples with <1.25 µM ATIII. The plateau (at ~1700 FU) is due to complete depletion of fluorescent substrate. In comparison, samples with ³1.25 µM ATIII did not reach a plateau and show substrate conversion until the end of the assay. In summary, a plasma level of at least 1.25 µM ATIII (50% of normal) was required to obtain thrombin generation profiles which were not compromised by fluorogenic substrate depletion. Below 1.25 µM ATIII, it is not possible to distinguish between inhibition of thrombin and excessive consumption of fluorescence substrate, and therefore, data may be misinterpreted.

Conclusion: Thrombin generation at low ATIII plasma levels is difficult to interpret as the physiological inhibition of the generated thrombin is reduced. Consequently, the thrombin substrate is consumed, resulting in substrate depletion. Therefore, we conclude that the CAT assay is technically not suitable to assess the effect of ATIII levels < 1.25 µM ATIII corresponding to 50% of normal plasma.