Differential Neutralization of Unfractionated Heparin, Enoxaparin and Fondaparinux by Andexanet Alpha
Authors: Fareed Jawed , Walenga Jeanine , Jeske Walter , Bontekoe Emily , Siddiqui Fakiha , Kouta Ahmed , Tafur Alfonso , Hoppensteadt Debra 
Published: June 2021

Introduction

Andexanet Alpha (Coagulation factor Xa recombinant, inactivated Zh‐zo; AA, Portola Pharmaceuticals) is a recombinant factor Xa decoy protein which is designed to reverse the inhibition of factor Xa in humans to control bleeding associated with newer oral anti‐Xa agents. The molecular modification in this recombinant protein involves the substitution of serine active site by alanine and the removal of the gamma‐carboxyglutamic acid (GLA) domain to restrict its assemblage into prothrombinase complex. Beside the reversal of the effects of anti‐Xa agents AA is also reported to neutralize the effects of heparin and related drugs. The purpose of this study is to compare the relative neutralization profile of heparin (UFH), a low molecular weight heparin enoxaparin (E) and a chemically synthetic pentasaccharide, Fondaparinux (F) by AA.

Materials and Methods

API versions of UFH, E and F were commercially obtained in powdered forms and dissolved in saline at a working dilution of 1mg/ml. AA was dissolved in saline to obtain a 10mg/ml working solution. The anticoagulant profile of UFH, E and F was studied using the activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombinase activated clotting time (PICT) in a concentration range of 0 – 10 ug/ml in pooled human plasma. The anti‐Xa and anti‐IIa studies were carried out in amidolytic assays in the same concentration range. The thrombin generation inhibition was studied using calibrated automated thrombin generation systems (CAT, Diagnostica Stago). The effect of AA on the reversal of the anticoagulant and anti‐protease effects of each of these agents was studied by supplementing this agent at 100 ug/ml.

Results

All agents produce a concentration dependent effect in the anticoagulant and anti‐protease assays with the exception of F which showed mild anticoagulant effects, and very weak anti‐IIa actions and strong anti‐Xa activity. In the anti‐Xa assay the IC‐50 for UFH was 2.1ug/ml (0.13 um), E 4.3 ug/ml (0.95 um) and F 0.7 ug/ml (0.41 um) upon supplementation of AA the IC50s for UFH was increased to 5 ug/ml (0.31 um) and for E 5 ug/ml (1.11 um). There was no change in the IC50 for F. The anticoagulant effects of UFH and E were neutralized at variable levels and were assay dependent. However there was no neutralization of the anti‐Xa effects of the F by AA. At high concentrations of greater than 2.5 ug/ml (1.47 um) the mild anticoagulant effects of F were neutralized in an assay dependent fashion.

Conclusion

These results indicate that AA is capable of differentially neutralizing anticoagulant and antiprotease effects of UFH and E in an assay dependent manner. However AA is incapable of neutralizing the anti‐Xa effects of F. This may be due to the relatively strong affinity of F AT complex to factor Xa rendering it inhibited in the presence of AA.

Support or Funding Information

None.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.