A chemically‐modified inactive antithrombin as a potent antagonist of fondaparinux and heparin anticoagulant activity

Summary Background Heparin and its analogs, mediating their anticoagulant activity through antithrombin ( AT ) activation, remain largely used for the preventive and curative treatment of thrombosis. The major adverse reaction of these drugs is the bleeding risk associated with overdose. Unfractionnated heparin ( UFH ) can be efficiently and rapidly neutralized by protamine sulfate, but this reversal partially neutralizes low‐molecular‐weight heparin ( LMWH ) and is inefficient in reversing fondaparinux. To secure administration of AT ‐mediated anticoagulants and counteract bleeding disorders, we previously designed a recombinant inactive AT as an antidote to heparin derivatives. Objectives To get around the limited production level of recombinant AT , we propose in this study an alternative strategy to produce a chemically modified inactive AT , exhibiting increased heparin affinity, as an antagonist of heparin analogs. Methods Plasma‐derived AT was chemically modified with 2,3 butanedione, a diketone known to specifically react with the arginine side chain. The chemical reaction was conducted in the presence of heparin to preserve basic residues within the heparin binding site from modifications. Results AT treated by butanedione and selected for its high heparin affinity ( AT ‐ BD ) was indeed modified on reactive Arg393 and thus exhibited decreased anticoagulant activity and increased heparin affinity. AT ‐ BD was able to neutralize anticoagulant activity of heparin derivatives in vitro and in vivo and was devoid of intrinsic anticoagulant activity, as assessed by activated partial thromboplastin time assay. Conclusions AT ‐ BD appears to be as efficient as protamine to neutralize UFH in vivo but could be more largely used because it also reverses fondaparinux and LMWH .