Assay-Based Differentiation in the Neutralization Profile of Unfractionated Heparin, Enoxaparin, and Fondaparinux by Andexanet Alfa

Abstract

Andexanet alfa is a recombinant factor Xa decoy protein, designed to reverse bleeding associated with oral anti-Xa agents. Andexanet alfa is also reported to neutralize the effects of heparin-related drugs. This study focused on the neutralization profiles of unfractionated heparin (UFH), enoxaparin, and, a chemically synthetic pentasaccharide, fondaparinux by andexanet alfa. Whole blood clotting studies were carried out using thromboelastography (TEG) and activated clotting time (ACT). The anticoagulant profile of UFH, enoxaparin, and fondaparinux was studied using the activated partial thromboplastin time (aPTT), thrombin time (TT), and amidolytic anti-Xa, and anti-IIa methods. Thrombin generation inhibition was studied using the calibrated automated thrombogram system. Reversal of each of these agents was studied by supplementing andexanet alfa at 100 µg/mL. In the TEG, andexanet alfa produced almost a complete reversal of the anticoagulant effects of UFH and enoxaparin; however, it augmented the effects of fondaparinux. In the ACT, aPTT, and TT, UFH produced strong anticoagulant effects that were almost completely neutralized by andexanet alfa. Enoxaparin produced milder anticoagulant responses that were partially neutralized, whereas fondaparinux did not produce any sizeable effects. In the anti-Xa and anti-IIa assays, UFH exhibited partial neutralization whereas enoxaparin and fondaparinux did not show any neutralization. All agents produced varying degrees of the inhibition of thrombin generation, which were differentially neutralized by andexanet alfa. These results indicate that andexanet alfa is capable of differentially neutralizing anticoagulant and antiprotease effects of UFH and enoxaparin in an assay-dependent manner. However, andexanet alfa is incapable of neutralizing the anti-Xa effects of fondaparinux.

Andexanet Alfa–Induced Heparin Resistance: When Anticoagulation Really Remains Reversed

“…In the in-vitro study by Siddiqui et al, not only were whole blood coagulation tests assessed but also assays with pooled plasma and constant AT levels. 4 In the coagulation tests (activated partial thrombin time, thrombin time) the effect of UFH was nearly completely reversed by andexanet alfa. In contrast, in the chromogenic anti-Xa and anti-IIa assays, andexanet alfa only demonstrated a modest reversal of UFH action.…”

“…In contrast, in the chromogenic anti-Xa and anti-IIa assays, andexanet alfa only demonstrated a modest reversal of UFH action. 4 In most commercially available chromogenic assays, an excess of AT is added to the substrate to ensure adequate monitoring of heparin levels. Apparently, this was not the case in this investigation.…”

“…2,3 These reports are in line with recent in-vitro investigations showing that the effect of UFH on the activated clotting time (ACT) and R-time in thromboelastography is reversed by addition of andexanet alfa. 4 Although it is obvious that andexanet alfa should not be administered prior to cardiac surgery, 5 it is conceivable that it could be necessary in emergency situations requiring fast institution or reinstitution of cardiopulmonary bypass (CPB).…”

Andexanet Alfa, the Possible Alternative to Protamine for Reversal of Unfractionated Heparin

“…8,10,15 Andexanet is the only product available on the market with supporting data; however, there are only few published in vitro studies assessing the use of andexanet for reversal of UFH. 10,12-15…”

“…Siddiqui et al 12 reported that andexanet produced an almost complete neutralization of UFH anticoagulation effects and reversed the TEG profile to near normal. The study was done using freshly drawn blood samples from healthy individual donors and did not utilize protamine as comparator.…”