Abstract 18218: In Vitro Characterization of Andexanet Alfa (PRT064445), a Specific fXa Inhibitor Antidote versus Aripazine (PER977), a Non-specific Reversal Agent

Abstract

New oral fXa inhibitors have been increasingly adopted for VTE or AF treatment in the outpatient setting instead of warfarin. Andexanet alfa (AnXa) is a modified, recombinant human fXa molecule developed as a specific antidote to reverse anticoagulant activity of fXa inhibitors during episodes of serious bleeding or before urgent surgery. PER977, a small molecule under development by Perosphere, Inc., is reported to reverse the effect of a broad range of anticoagulants (fXa and thrombin inhibitors, LMWH). In order to compare AnXa and PER977 mechanisms of action, we studied the in vitro activity of both agents in the presence or absence of fXa inhibitors rivaroxaban, apixaban, edoxaban or enoxaparin. In a buffer system containing purified human fXa with physiologic Ca 2+ (5 mM), AnXa dose-dependently reversed oral fXa inhibitor activity. Reversal activity was not observed for any fXa inhibitor with PER977 over a wide concentration range (≤2 mM). In human plasma, AnXa reversed both direct and ATIII-dependent fXa inhibitors, whereas no reversal effect by PER977 was detected. In the absence of a fXa inhibitor, PER977 potentiated fX activation by fIXa in a buffer system (purified human fX, fIXa and 5mM Ca 2+ ). Similar cofactor activity was observed with polylysine, indicating that PER977 may have properties similar to poly-cationic molecules. Procoagulant activity was observed in human plasma with PER977 at low concentrations (≤100 μM) as measured by clotting (aPTT) or thrombin generation, but inhibition in these assays was seen at higher concentrations ( ~1 mM). PER977 also potentiated human platelet activation as determined by P-selectin expression induced by 10 μM ADP. Platelet aggregation, whole blood hemolysis, or complement activation testing showed no effect with either AnXa or PER977. These data indicate that PER977 does not reverse the anticoagulant activity of a direct or indirect fXa inhibitor in vitro. Its observed in vivo effect on blood loss in animals may not be mediated by direct interaction of PER977 with the inhibitor, but may be in part attributed to an off-target effect, suggested by its potential procoagulant activity. In contrast, AnXa acts as a specific antidote to fXa inhibitors by sequestering them with a defined stoichiometry (1:1 molar ratio).

Differential Neutralization of Unfractionated Heparin and Enoxaparin by Andexanet Alfa

“…Our results differed from the previous study conducted on LMWH, 15 in that we did not find a significant reversal of LMWH anticoagulation. One difference is in the methodology and administration of andexanet alfa, where our study was run in vitro , and the other study was performed in vivo in rats.…”

“…A case report from 2019 noted that a patient was unresponsive to UFH anticoagulation during surgery following an andexanet alfa reversal. 13,14 Previous research has also shown that recombinant factor Xa may be effective at reversing LMWH 15 and UFH. 16 Furthermore, it appears that andexanet alfa binds directly with UFH and LMWH and interestingly, binds to heparin-activated antithrombin.…”

The methods for removal of direct oral anticoagulants and heparins to improve the monitoring of hemostasis: a narrative literature review

“…No binding to FIIa and FXa, plasma proteins, or other drugs was found [75]. Lu et al suggested a possible increase in human platelet activation by ciraparantag using P-selectin expression induced by 10 µM adenosine diphosphate [76]. In this study, ciraparantag did not bind DOACs in vitro, which may depend on the assay.…”