Apixaban inhibition of factor Xa: Microscopic rate constants and inhibition mechanism in purified protein systems and in human plasma

Abstract

Apixaban is a potent, direct, selective, and orally active inhibitor of coagulation factor Xa. Rate constants for apixaban binding to free and prothrombinase-bound factor Xa were measured using multiple techniques. The inhibition mechanism was determined in purified systems and in a plasma prothrombin clotting time assay. Apixaban inhibits factor Xa with a K_i of 0.25 nM at 37°C, an association rate constant of approximately 20 μM⁻¹ s⁻¹, and a dissociation half-life of 1–2 min. Under physiological conditions apixaban exhibits mixed-type inhibition and maintains high factor Xa affinity with a K_i of 0.62 nM and association rate constant of 12 μM⁻¹ s⁻¹ for prothrombinase, and a K_i of 1.7 nM and association rate constant of 4 μM⁻¹ s⁻¹ for the prothrombinase:prothrombin complex. Experiments in prothrombin depleted human plasma showed that the mechanism and kinetics of inhibition are maintained in plasma. The mechanistic detail derived from these experiments can be used to understand and interpret the pharmacodynamic action of apixaban.

Keywords::

• Factor Xa
• anticoagulant thrombosis

Acknowledgements

Charles Kettner, Ross Stein, and Lawrence Mersinger conducted initial enzyme kinetic studies with apixaban, phospholipid membrane preparations, and interpretation of the mechanistic observations. Mark Hixon provided a detailed derivation of Equation 1 validating its application to the prothrombin activation experiments. Jeff Bozarth provided technical assistance.

Declaration of interest

The authors are current or former employees of Bristol-Myers Squibb. This study was sponsored by Bristol-Myers Squibb and Pfizer Inc.