Andexanet alfa (PRT4445, marketed as Andexxa®) is a recombinant modified human factor Xa (FXa) designed to mimic the activity of FXa (Lu G et al., 2013). Andexanet alfa competitively binds to FXa inhibitors, preventing their interaction with native FXa (reviewed by Kaatz S et al., 2017). Compared to native FXa, this recombinant FXa variant lacks: (a) the activation peptide of native FX, which is replaced by a hexapeptide RKRRKR linker to enable expression of the mature FXa; (b) a 34-residue fragment in the membrane-binding Gla domain containing 11 Gla residues; and (c) catalytic activity due to a serine-to-alanine (S419A) substitution in the active site of FXa (Lu G et al., 2013). Modifications (b) and (c) result in a catalytically inactive FXa variant that cannot convert prothrombin to thrombin. The absence of the Gla domain prevents andexanet alfa from competing with native FXa for plasma membrane binding and assembly of a membrane-bound prothrombinase complex (Lu G et al., 2013; reviewed by Kaatz S et al., 2017).
Clotting and chromogenic FXa activity assays have demonstrated that andexanet alfa effectively reverses the anticoagulant effects of FXa inhibitors, including direct inhibitors such as rivaroxaban and apixaban, as well as indirect inhibitors like unfractionated heparin (UFH), enoxaparin, and fondaparinux (Lu G et al., 2013; Kalathottukaren MT et al., 2018; Siddiqui F et al., 2020). Direct FXa inhibitors bind andexanet alfa with nanomolar affinities similar to their affinities for native FXa (Lu G et al., 2013; Kalathottukaren MT et al., 2018). Indirect FXa inhibitors, such as unfractionated heparin (UFH), low molecular weight heparins (LMWH), and heparin derivatives, act via binding to antithrombin III (AT), an endogenous anticoagulant encoded by the SERPINC1 gene. Heparin binding induces conformational changes in antithrombin III (SERPINC1), enhancing its ability to inhibit FXa catalytic activity by 1,000-fold (Izaguirre G et al., 2007). Isothermal titration calorimetry (ITC) assays have shown that the relatively weak direct binding of andexanet alfa to enoxaparin is enhanced in the presence of antithrombin III (Kalathottukaren MT et al., 2018). High binding affinity has also been observed for the antithrombin III:fondaparinux complex (Lu G et al., 2013). These findings suggest that andexanet alfa neutralizes the anticoagulant effects of heparin-based drugs by binding to the antithrombin III:heparin complex, supporting its potential therapeutic use (Maneno JN & Ness GL, 2021).
This Reactome event illustrates the binding of andexanet alfa to heparin‑based anticoagulants such as UFH, enoxaparin, and fondaparinux. However, further experimental studies are necessary to evaluate the reversal effects of andexanet alfa on the anticoagulant activity of indirect FXa inhibitors (Siddiqui F et al., 2020; Lewis J et al., 2022). Additional research is also required to assess its effectiveness in treating uncontrolled bleeding caused by heparin therapy in patients (Müther M et al., 2022; Apostel HJCL et al., 2021; Erdoes G et al., 2021).
