Andexanet Alfa Corrects the Interference of Direct Factor Xa Inhibitors on Lupus Anticoagulant Testing

Background

Direct Factor Xa (FXa) inhibitors (e.g. rivaroxaban or apixaban) are widely used oral anticoagulants and are known to interfere with dilute Russell's viper venom time (dRVVT) and aPTT-based lupus anticoagulant (LA) testing, often yielding false-positive LA results. A false positive LA result might incorrectly diagnose a patient with the anti-phospholipid antibody syndrome and lead to a wrong decision for long-term anticoagulation to prevent recurrent thrombosis.

Andexanet alfa is a recombinant modified human FXa decoy protein that has been specifically designed as an antidote to neutralise the effect of the direct and indirect FXa inhibitors.

Aim

To determine whether the addition of andexanet alfa can overcome the interference of rivaroxaban and apixaban on LA testing in plasma samples from both LA positive and LA negative patients.

Methods

After written informed consent, citrated blood (20mL) was collected from 12 patients on therapeutic doses of rivaroxaban (n=6) or apixaban (n=6) with previously known LA status (3/6 LA positive in each group). Samples were double centrifuged at 3,000 x g for 10 minutes and aliquots were frozen at -40^oC until analysed. Normal pooled plasma and rivaroxaban and apixaban calibrated plasma was obtained from a commercial source (Diagnostica Stago) or control plasma from healthy volunteers, prepared as above. IgG was purified by a Protein G column from a known LA positive patient and a control healthy volunteer.

Rivaroxaban (Bayer) and apixaban (Bristol-Myers Squibb) tablets were crushed and dissolved in DMSO. Andexanet alfa (Portola Pharmaceuticals, Inc.) was dissolved in distilled water and aliquots were frozen at -40^oC until used. Plasma samples were spiked with varying concentrations of FXa inhibitors and andexanet alfa.

LA-sensitive aPTT, PT, TCT, dRVVT (Siemens Healthcare Diagnostics) and anti-FXa level (Diagnostica Stago) were performed with and without andexanet alfa on the Sysmex CS5100 analyser.

Andexanet alfa (at a final concentration of 250µg/mL) was added to LA positive and LA negative plasma samples to assess the impact on the dRVVT normalised ratio.

Results

Andexanet alfa demonstrated a concentration-dependent increase in LA sensitive aPTT and dRVVT clotting times, with negligible impact on PT or TCT. Andexanet alfa was capable of reversing FXa inhibition in a concentration-dependent manner in plasma samples containing rivaroxaban or apixaban.

When rivaroxaban is added to normal plasma it significantly prolongs the dRVVT without phospholipid more than with excess phospholipid leading to a false-positive LA ratio at final concentrations >100ng/mL. This level of rivaroxaban is in the therapeutic range. The addition of andexanet alfa reduced the measurable anti-FXa level and returned the normalised dRVVT ratio to LA negative result (<1.25). In contrast, apixaban and andexanet alfa proportionately prolongs the dRVVT with or without excess phospholipid and even at high concentrations, the normalised LA ratio remains negative (<1.25).

Andexanet alfa converted the false positive dRVVT rivaroxaban patient plasmas LA ratio to negative. True LA positive patients remained positive after the addition of andexanet alfa. These results were confirmed by adding LA positive and control purified IgG to known concentrations of rivaroxaban and apixaban (200ng/ml) in normal plasma.

Conclusion

Andexanet alfa corrects the false positive LA ratio in patients on rivaroxaban without affecting the true diagnosis in LA positive patients. Apixaban and andexanet alfa proportionately prolong the dRVVT with and without phospholipid, so the LA ratio remains negative. For this reason true LA positive patients can be diagnosed whilst on apixaban at therapeutic concentrations without the addition of andexanet alfa.