Development of a recombinant antithrombin variant as a potent antidote to fondaparinux and other heparin derivatives.
Heparin derivative-based therapy has evolved from unfractionated heparin (UFH) to low-molecular-weight heparins (LMWHs) and now fondaparinux, a synthetic pentasaccharide. Contrary to UFH or LMWHs, fondaparinux is not neutralized by protamine sulfate, and no antidote is available to counteract bleeding disorders associated with overdosing. To make the use of fondaparinux safer, we developed an antithrombin (AT) variant as a potent antidote to heparin derivatives. This variant (AT-N135Q-Pro394) combines 2 mutations: substitution of Asn135 by a Gln to remove a glycosylation site and increase affinity for heparins, and the insertion of a Pro between Arg393 and Ser394 to abolish its anticoagulant activity. As expected, AT-N135Q-Pro394 anticoagulant activity was almost abolished, and it exhibited a 3-fold increase in fondaparinux affinity. AT-N135Q-Pro394 was shown to reverse fondaparinux overdosing in vitro in a dose-dependent manner through a competitive process with plasma AT for fondaparinux binding. This antidote effect was also observed in vivo: administration of AT-N135Q-Pro394 in 2.5-fold molar excess versus plasma AT neutralized 86% of the anti-Xa activity within 5 minutes in mice treated with fondaparinux. These results clearly demonstrate that AT-N135Q-Pro394 can reverse the anticoagulant activity of fondaparinux and thus could be used as an antidote for this drug.
Université Paris-Sud, Laboratoire d'hématologie, Châtenay-Malabry, France. elsa.bianchini@u-psud.fr
Elsa P
EP
Bianchini
Judicael
J
Fazavana
Veronique
V
Picard
Delphine
D
Borgel
D019943:Amino Acid Substitution; D000818:Animals; D000925:Anticoagulants / Q000037:antagonists & inhibitors* / Q000633:toxicity; D000931:Antidotes / Q000494:pharmacology*; D058833:Antithrombin Proteins / Q000235:genetics* / Q000494:pharmacology*; D000991:Antithrombins / Q000494:pharmacology*; D015195:Drug Design; D005260:Female; D000077425:Fondaparinux; D057809:HEK293 Cells; D006470:Hemorrhage / Q000139:chemically induced / Q000188:drug therapy; D006494:Heparin Antagonists / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D011134:Polysaccharides / Q000037:antagonists & inhibitors* / Q000633:toxicity; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology
D000925:Anticoagulants; D000931:Antidotes; D058833:Antithrombin Proteins; D000991:Antithrombins; D006494:Heparin Antagonists; D011134:Polysaccharides; D011994:Recombinant Proteins; D000077425:Fondaparinux