Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa

Abstract

Inhibitors of coagulation factor Xa (fXa) have emerged as a new class of antithrombotics but lack effective antidotes for patients experiencing serious bleeding. We designed and expressed a modified form of fXa as an antidote for fXa inhibitors. This recombinant protein (r-Antidote, PRT064445) is catalytically inactive and lacks the membrane-binding γ-carboxyglutamic acid domain of native fXa but retains the ability of native fXa to bind direct fXa inhibitors as well as low molecular weight heparin–activated antithrombin III (ATIII). r-Antidote dose-dependently reversed the inhibition of fXa by direct fXa inhibitors and corrected the prolongation of ex vivo clotting times by such inhibitors. In rabbits treated with the direct fXa inhibitor rivaroxaban, r-Antidote restored hemostasis in a liver laceration model. The effect of r-Antidote was mediated by reducing plasma anti-fXa activity and the non–protein bound fraction of the fXa inhibitor in plasma. In rats, r-Antidote administration dose-dependently and completely corrected increases in blood loss resulting from ATIII-dependent anticoagulation by enoxaparin or fondaparinux. r-Antidote has the potential to be used as a universal antidote for a broad range of fXa inhibitors.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Design of r-Antidote and protein expression in CHO cells.
Figure 2: r-Antidote reverses the inhibitory activity of three direct fXa inhibitors.
Figure 3: Sustained reversal of whole-blood INR by r-Antidote in rats treated with direct fXa inhibitors.
Figure 4: Mitigation of blood loss caused by rivaroxaban-induced anticoagulation with r-Antidote in a rabbit liver laceration model.
Figure 5: Reversal of ATIII-dependent fXa inhibitors by r-Antidote in the rat tail transection model.

References

  1. Lassen, M.R. et al. Rivaroxaban versus enoxaparin for thromboprophylaxis after total knee arthroplasty. N. Engl. J. Med. 358, 2776–2786 (2008).

    Article  CAS  Google Scholar 

  2. Granger, C.B. et al. Apixaban versus warfarin in patients with atrial fibrillation. N. Engl. J. Med. 365, 981–992 (2011).

    Article  CAS  Google Scholar 

  3. Weitz, J.I. et al. Randomised, parallel-group, multicentre, multinational phase 2 study comparing edoxaban, an oral factor Xa inhibitor, with warfarin for stroke prevention in patients with atrial fibrillation. Thromb. Haemost. 104, 633–641 (2010).

    Article  CAS  Google Scholar 

  4. Connolly, S.J. et al. Apixaban in patients with atrial fibrillation. N. Engl. J. Med. 364, 806–817 (2011).

    Article  CAS  Google Scholar 

  5. Chawla, L.S., Moore, G. & Seneff, M.G. Incomplete reversal of enoxaparin toxicity by protamine: implications of renal insufficiency, obesity, and low molecular weight heparin sulfate content. Obes. Surg. 14, 695–698 (2004).

    Article  Google Scholar 

  6. Crowther, M.A. & Warkentin, T.E. Bleeding risk and the management of bleeding complications in patients undergoing anticoagulant therapy: focus on new anticoagulant agents. Blood 111, 4871–4879 (2008).

    Article  CAS  Google Scholar 

  7. Crowther, M.A. & Warkentin, T.E. Managing bleeding in anticoagulated patients with a focus on novel therapeutic agents. J. Thromb. Haemost. 7 (suppl. 1), 107–110 (2009).

    Article  CAS  Google Scholar 

  8. Bershad, E.M. & Suarez, J.I. Prothrombin complex concentrates for oral anticoagulant therapy-related intracranial hemorrhage: a review of the literature. Neurocrit. Care 12, 403–413 (2010).

    Article  CAS  Google Scholar 

  9. Levi, M., Bijsterveld, N.R. & Keller, T.T. Recombinant factor VIIa as an antidote for anticoagulant treatment. Semin. Hematol. 41 (suppl. 1), 65–69 (2004).

    Article  CAS  Google Scholar 

  10. Lauritzen, B. et al. Recombinant human factor VIIa and a factor VIIa-analogue reduces heparin and low molecular weight heparin (LMWH)-induced bleeding in rats. J. Thromb. Haemost. 6, 804–811 (2008).

    Article  CAS  Google Scholar 

  11. Pryzdial, E.L. & Kessler, G.E. Kinetics of blood coagulation factor Xaα autoproteolytic conversion to factor Xaβ. Effect on inhibition by antithrombin, prothrombinase assembly, and enzyme activity. J. Biol. Chem. 271, 16621–16626 (1996).

    Article  CAS  Google Scholar 

  12. Pryzdial, E.L. & Kessler, G.E. Autoproteolysis or plasmin-mediated cleavage of factor Xaα exposes a plasminogen binding site and inhibits coagulation. J. Biol. Chem. 271, 16614–16620 (1996).

    Article  CAS  Google Scholar 

  13. Sinha, U. et al. Antithrombotic activity of PRT54021, a potent oral direct factor Xa inhibitor, can be monitored sing a novel prothrombinase inhibition bioassay. Blood 108 Abstract 907 (2006).

  14. Perzborn, E. et al. In vitro and in vivo studies of the novel antithrombotic agent BAY 59–7939–an oral, direct Factor Xa inhibitor. J. Thromb. Haemost. 3, 514–521 (2005).

    Article  CAS  Google Scholar 

  15. Pinto, D.J. et al. Discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H -pyrazolo[3,4-c]pyridine-3-carboxamide (apixaban, BMS-562247), a highly potent, selective, efficacious, and orally bioavailable inhibitor of blood coagulation factor Xa. J. Med. Chem. 50, 5339–5356 (2007).

    Article  CAS  Google Scholar 

  16. Sinha, U. et al. Expression, purification, and characterization of inactive human coagulation factor Xa (Asn322Ala419). Protein Expr. Purif. 3, 518–524 (1992).

    Article  CAS  Google Scholar 

  17. Izaguirre, G. et al. Mechanism by which exosites promote the inhibition of blood coagulation proteases by heparin-activated antithrombin. J. Biol. Chem. 282, 33609–33622 (2007).

    Article  CAS  Google Scholar 

  18. Mann, K.G. et al. Surface-dependent reactions of the vitamin K–dependent enzyme complexes. Blood 76, 1–16 (1990).

    Article  CAS  Google Scholar 

  19. Huntington, J.A. Mechanisms of glycosaminoglycan activation of the serpins in hemostasis. J. Thromb. Haemost. 1, 1535–1549 (2003).

    Article  CAS  Google Scholar 

  20. Olson, S.T., Bjork, I. & Bock, S.C. Identification of critical molecular interactions mediating heparin activation of antithrombin: implications for the design of improved heparin anticoagulants. Trends Cardiovasc. Med. 12, 198–205 (2002).

    Article  CAS  Google Scholar 

  21. Andrassy, K., Eschenfelder, V. & Weber, E. Neutralization of the anticoagulant activity of low molecular weight heparin LU 47311 (Clivarin) in man by protamine chloride. Thromb. Res. 73, 85–93 (1994).

    Article  CAS  Google Scholar 

  22. Bang, C.J., Berstad, A. & Talstad, I. Incomplete reversal of enoxaparin-induced bleeding by protamine sulfate. Haemostasis 21, 155–160 (1991).

    CAS  PubMed  Google Scholar 

  23. Crowther, M.A. et al. Mechanisms responsible for the failure of protamine to inactivate low-molecular-weight heparin. Br. J. Haematol. 116, 178–186 (2002).

    Article  CAS  Google Scholar 

  24. Dementiev, A. et al. The ternary complex of antithrombin-anhydrothrombin-heparin reveals the basis of inhibitor specificity. Nat. Struct. Mol. Biol. 11, 863–867 (2004).

    Article  CAS  Google Scholar 

  25. Kuziej, J. et al. In vivo neutralization of unfractionated heparin and low-molecular-weight heparin by a novel salicylamide derivative. Clin. Appl. Thromb. Hemost. 16, 377–386 (2010).

    Article  CAS  Google Scholar 

  26. Bianchini, E.P. et al. Development of a recombinant antithrombin variant as a potent antidote to fondaparinux and other heparin derivatives. Blood 117, 2054–2060 (2011).

    Article  CAS  Google Scholar 

  27. Mega, J.L. et al. Rivaroxaban in patients with a recent acute coronary syndrome. N. Engl. J. Med. 366, 9–19 (2012).

    Article  CAS  Google Scholar 

  28. Eerenberg, E.S. et al. Reversal of rivaroxaban and dabigatran by prothrombin complex concentrate: a randomized, placebo-controlled, crossover study in healthy subjects. Circulation 124, 1573–1579 (2011).

    Article  CAS  Google Scholar 

  29. Smith, T.W. et al. Reversal of advanced digoxin intoxication with Fab fragments of digoxin-specific antibodies. N. Engl. J. Med. 294, 797–800 (1976).

    Article  CAS  Google Scholar 

  30. Carothers, A.M. et al. Structure of the dihydrofolate reductase gene in Chinese hamster ovary cells. Nucleic Acids Res. 11, 1997–2012 (1983).

    Article  CAS  Google Scholar 

  31. Carothers, A.M. et al. Splicing mutations in the CHO DHFR gene preferentially induced by (+/−)-3 α,4 β-dihydroxy-1 α,2 α-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene. Proc. Natl. Acad. Sci. USA 87, 5464–5468 (1990).

    Article  CAS  Google Scholar 

  32. Di Scipio, R.G., Hermodson, M.A. & Davie, E.W. Activation of human factor X (Stuart factor) by a protease from Russell's viper venom. Biochemistry 16, 5253–5260 (1977).

    Article  CAS  Google Scholar 

  33. Paolucci, F. et al. Fondaparinux sodium mechanism of action: identification of specific binding to purified and human plasma-derived proteins. Clin. Pharmacokinet. 41 (suppl. 2), 11–18 (2002).

    Article  CAS  Google Scholar 

  34. Godier, A. et al. Evaluation of prothrombin complex concentrate and recombinant activated factor VII to reverse rivaroxaban in a rabbit model. Anesthesiology 116, 94–102 (2012).

    Article  CAS  Google Scholar 

  35. Dejana, E., Villa, S. & de Gaetano, G. Bleeding time in rats: a comparison of different experimental conditions. Thromb. Haemost. 48, 108–111 (1982).

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Contributions

G. Lu, S.J.H. and U.S. conceived of the study, designed experiments and wrote the manuscript. G. Lu, F.R.D., M.J.K., K.A., G. Lee, P.L., A.H. and M.I. designed and conducted experiments. P.B.C. discussed and interpreted data with G. Lu, M.J.K. and P.L. D.R.P. provided expertise, designed experiments and interpreted data.

Corresponding author

Correspondence to Uma Sinha.

Ethics declarations

Competing interests

G. Lu, F.R.D., S.J.H., M.J.K., K.A., G. Lee, P.L., A.H., M.I., P.B.C., D.R.P. and U.S. are current or former employees of Portola Pharmaceuticals Inc.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 (PDF 316 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lu, G., DeGuzman, F., Hollenbach, S. et al. A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa. Nat Med 19, 446–451 (2013). https://doi.org/10.1038/nm.3102

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.3102

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing