Abstract

Unfractionated heparin (UFH), the standard anticoagulant for cardiopulmonary bypass (CPB) surgery, carries a risk of post-operative bleeding and is potentially harmful in patients with heparin-induced thrombocytopenia–associated antibodies. To improve the activity of an alternative anticoagulant, the RNA aptamer 11F7t, we solved X-ray crystal structures of the aptamer bound to factor Xa (FXa). The finding that 11F7t did not bind the catalytic site suggested that it could complement small-molecule FXa inhibitors. We demonstrate that combinations of 11F7t and catalytic-site FXa inhibitors enhance anticoagulation in purified reaction mixtures and plasma. Aptamer–drug combinations prevented clot formation as effectively as UFH in human blood circulated in an extracorporeal oxygenator circuit that mimicked CPB, while avoiding side effects of UFH. An antidote could promptly neutralize the anticoagulant effects of both FXa inhibitors. Our results suggest that drugs and aptamers with shared targets can be combined to exert more specific and potent effects than either agent alone.

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References

  1. 1.

    & The science and practice of cardiopulmonary bypass: from cross-circulation to ECMO and SIRS. Glob. Cardiol. Sci. Pract. 2013, 249–260 (2013).

  2. 2.

    & Anticoagulation management associated with extracorporeal circulation. Best Pract. Res. Clin. Anaesthesiol. 29, 189–202 (2015).

  3. 3.

    & Anticoagulant therapy during cardiopulmonary bypass. J. Thromb. Thrombolysis 26, 218–228 (2008).

  4. 4.

    & Thrombin during cardiopulmonary bypass. Ann. Thorac. Surg. 82, 2315–2322 (2006).

  5. 5.

    & Activation of the hemostatic system during cardiopulmonary bypass. Anesth. Analg. 113, 1319–1333 (2011).

  6. 6.

    Cardiopulmonary bypass in humans: bypassing unfractionated heparin. Ann. Thorac. Surg. 70, 1434–1443 (2000).

  7. 7.

    , , & Parenteral anticoagulants: antithrombotic therapy and prevention of thrombosis, 9th ed: American College of Chest Physicians evidence-based clinical practice guidelines. Chest 141, e24S–e43S (2012).

  8. 8.

    Focus on thrombin: alternative anticoagulants. Semin. Cardiothorac. Vasc. Anesth. 11, 256–260 (2007).

  9. 9.

    Inflammatory response to cardiopulmonary bypass. Ann. Thorac. Surg. 66, S12–S16 (1998).

  10. 10.

    , & Coagulation disorders of cardiopulmonary bypass: a review. Intensive Care Med. 30, 1873–1881 (2004).

  11. 11.

    Hemostatic and thrombotic issues in cardiac surgery. Semin. Thromb. Hemost. 41, 84–90 (2015).

  12. 12.

    & Alterations to hemostasis following cardiopulmonary bypass and the relationship of these changes to neurocognitive morbidity. Blood Coagul. Fibrinolysis 12, 601–618 (2001).

  13. 13.

    , , , & Clot-bound thrombin is protected from inhibition by heparin–antithrombin III but is susceptible to inactivation by antithrombin III–independent inhibitors. J. Clin. Invest. 86, 385–391 (1990).

  14. 14.

    , & Factor Xa is highly protected from antithrombin–fondaparinux and antithrombin–enoxaparin when incorporated into the prothrombinase complex. J. Thromb. Haemost. 1, 1258–1263 (2003).

  15. 15.

    & Inflammatory response to cardiopulmonary bypass. Ann. Thorac. Surg. 75, S715–S720 (2003).

  16. 16.

    et al. Measurement of thrombin generation intraoperatively and its association with bleeding tendency after cardiac surgery. Thromb. Res. 133, 488–494 (2014).

  17. 17.

    et al. Effect of combined anticoagulation using heparin and bivalirudin on the hemostatic and inflammatory responses to cardiopulmonary bypass in the rat. Anesthesiology 106, 295–301 (2007).

  18. 18.

    et al. Postoperative antithrombin levels and outcome in cardiac operations. Crit. Care Med. 33, 355–360 (2005).

  19. 19.

    & Heparin-induced thrombocytopenia. Annu. Rev. Med. 61, 77–90 (2010).

  20. 20.

    et al. Effects of cardiac surgery on hemostasis. Transfus. Med. Rev. 20, 230–241 (2006).

  21. 21.

    & Bleeding complications associated with cardiopulmonary bypass. Blood 76, 1680–1697 (1990).

  22. 22.

    et al. Unfractionated heparin inhibits thrombin-catalyzed amplification reactions of coagulation more efficiently than those catalyzed by factor Xa. Biochem. J. 257, 143–150 (1989).

  23. 23.

    , & The mode of action of heparin in plasma. Thromb. Haemost. 60, 457–462 (1988).

  24. 24.

    & The limited importance of factor Xa inhibition to the anticoagulant property of heparin in thromboplastin-activated plasma. Blood 72, 2048–2052 (1988).

  25. 25.

    et al. An anticoagulant RNA aptamer that inhibits proteinase–cofactor interactions within prothrombinase. J. Biol. Chem. 285, 5212–5223 (2010).

  26. 26.

    et al. Probing the coagulation pathway with aptamers identifies combinations that synergistically inhibit blood clot formation. Chem. Biol. 21, 935–944 (2014).

  27. 27.

    , & Oral direct factor Xa inhibitors. Circ. Res. 111, 1069–1078 (2012).

  28. 28.

    et al. A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa. Nat. Med. 19, 446–451 (2013).

  29. 29.

    et al. Andexanet alfa for the reversal of factor Xa inhibitor activity. N. Engl. J. Med. 373, 2413–2424 (2015).

  30. 30.

    et al. A rapid pro-hemostatic approach to overcome direct oral anticoagulants. Nat. Med. 22, 924–932 (2016).

  31. 31.

    Identification of basic residues in the heparin-binding exosite of factor Xa critical for heparin and factor Va binding. J. Biol. Chem. 275, 3320–3327 (2000).

  32. 32.

    et al. Discovery of the novel antithrombotic agent 5-chloro-N-((5S)-2-oxo-3- [4-(3-oxomorpholin-4-yl)phenyl]-1,3-oxazolidin-5-ylmethyl)thiophene- 2-carboxamide (BAY 59-7939): an oral, direct factor Xa inhibitor. J. Med. Chem. 48, 5900–5908 (2005).

  33. 33.

    , & Novel factor Xa inhibitors: a patent review. Expert Opin. Ther. Pat. 19, 1535–1580 (2009).

  34. 34.

    et al. Crystal structure of the prothrombinase complex from the venom of Pseudonaja textilis. Blood 122, 2777–2783 (2013).

  35. 35.

    , , & Aptamers as therapeutics. Annu. Rev. Pharmacol. Toxicol. 57, 61–79 (2017).

  36. 36.

    et al. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol. Haemost. Thromb. 33, 4–15 (2003).

  37. 37.

    et al. Thrombelastography. Transfus. Apheresis Sci. 40, 119–123 (2009).

  38. 38.

    et al. Monitoring thrombin generation with prothrombin fragment 1.2 assay during cardiopulmonary bypass surgery. Thromb. Res. 84, 45–54 (1996).

  39. 39.

    et al. Elevated levels of prothrombin fragment 1 + 2 indicate high risk of thrombosis. Clin. Appl. Thromb. Hemost. 14, 279–285 (2008).

  40. 40.

    , , , & Activation of the kallikrein–kinin system by cardiopulmonary bypass in humans. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281, R1059–R1070 (2001).

  41. 41.

    et al. A novel antidote-controlled anticoagulant reduces thrombin generation and inflammation and improves cardiac function in cardiopulmonary bypass surgery. Mol. Ther. 14, 408–415 (2006).

  42. 42.

    et al. Inhibition of factor IXa by the pegnivacogin system during cardiopulmonary bypass: a potential substitute for heparin. A study in baboons. Eur. J. Cardiothorac. Surg. 49, 682–689 (2016).

  43. 43.

    et al. Effect of factor Xa inhibitors on thrombin formation and complement and neutrophil activation during in vitro extracorporeal circulation. Circulation 94 Suppl, II341–II346 (1996).

  44. 44.

    et al. Recombinant desulfatohirudin as a substitute for heparin during cardiopulmonary bypass. J. Thorac. Cardiovasc. Surg. 108, 381–382 (1994).

  45. 45.

    , & Thrombin generation during cardiac surgery: is heparin the ideal anticoagulant? Thromb. Haemost. 70, 259–262 (1993).

  46. 46.

    , & Thrombin in myocardial ischemia–reperfusion during cardiac surgery. Ann. Thorac. Surg. 88, 318–325 (2009).

  47. 47.

    et al. Complex formation with nucleic acids and aptamers alters the antigenic properties of platelet factor 4. Blood 122, 272–281 (2013).

  48. 48.

    , , & Crystal structure of an RNA aptamer bound to thrombin. RNA 14, 2504–2512 (2008).

  49. 49.

    et al. Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor. Nat. Chem. Biol. 12, 709–716 (2016).

  50. 50.

    , , & Targeting two coagulation cascade proteases with a bivalent aptamer yields a potent and antidote-controllable anticoagulant. Nucleic Acid Ther. 26, 1–9 (2016).

  51. 51.

    & Role of the activation peptide domain in human factor X activation by the extrinsic Xase complex. J. Biol. Chem. 271, 16126–16134 (1996).

  52. 52.

    , & Extended interactions with prothrombinase enforce affinity and specificity for its macromolecular substrate. J. Biol. Chem. 277, 46191–46196 (2002).

  53. 53.

    , & Formation of factors IXa and Xa by the extrinsic pathway: differential regulation by tissue factor pathway inhibitor and antithrombin III. J. Biol. Chem. 279, 17241–17249 (2004).

  54. 54.

    , , , & Nematode anticoagulant protein c2 reveals a site on factor Xa that is important for macromolecular substrate binding to human prothrombinase. J. Biol. Chem. 277, 26689–26698 (2002).

  55. 55.

    , & Human factor VIIIa subunit structure. Reconstruction of factor VIIIa from the isolated A1/A3-C1-C2 dimer and A2 subunit. J. Biol. Chem. 266, 8957–8962 (1991).

  56. 56.

    , , , & Prothrombin. Methods Enzymol. 80, 286–302 (1981).

  57. 57.

    , & Activation of human factor X (Stuart factor) by a protease from Russell's viper venom. Biochemistry 16, 5253–5260 (1977).

  58. 58.

    , , , & Interaction of clotting factor V heavy chain with prothrombin and pre-thrombin 1 and role of activated protein C in regulating this interaction: analysis by analytical ultracentrifugation. Biochemistry 28, 2348–2354 (1989).

  59. 59.

    , & Activation of human factor IX (Christmas factor). J. Clin. Invest. 61, 1528–1538 (1978).

  60. 60.

    , , , & Factor VIII accelerates proteolytic cleavage of von Willebrand factor by ADAMTS13. Proc. Natl. Acad. Sci. USA 105, 7416–7421 (2008).

  61. 61.

    Xds. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

  62. 62.

    & Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

  63. 63.

    et al. Molecular recognition at the active site of factor Xa: cation–π interactions, stacking on planar peptide surfaces and replacement of structural water. Chemistry 18, 213–222 (2012).

  64. 64.

    et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

  65. 65.

    , , , & Correcting pervasive errors in RNA crystallography through enumerative structure prediction. Nat. Methods 10, 74–76 (2013).

  66. 66.

    , , & Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

  67. 67.

    , , & PDB_REDO: constructive validation, more than just looking for errors. Acta Crystallogr. D Biol. Crystallogr. 68, 484–496 (2012).

  68. 68.

    & Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

  69. 69.

    et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

  70. 70.

    The PyMOL Molecular Graphics System Version 1.8 (2015).

  71. 71.

    et al. Role of platelet surface PF4 antigenic complexes in heparin-induced thrombocytopenia pathogenesis: diagnostic and therapeutic implications. Blood 107, 2346–2353 (2006).

  72. 72.

    et al. Atomic description of the immune complex involved in heparin-induced thrombocytopenia. Nat. Commun. 6, 8277 (2015).

  73. 73.

    et al. Decision analysis for use of platelet aggregation test, carbon 14–serotonin release assay and heparin–platelet factor 4 enzyme-linked immunosorbent assay for diagnosis of heparin-induced thrombocytopenia. Am. J. Clin. Pathol. 111, 700–706 (1999).

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Acknowledgements

We would like to thank D. Monroe, M. Hoffman and G. Pitoc for useful discussions and T. Slotkin for statistical advice. This work was supported by US National Institutes of Health (NIH) grants HL-74124 (S. Krishnaswamy), HL-125422 (S. Krishnaswamy), HL-065222 (B.A.S.), F30 HL-127977 (R.G.) and T32 GM-007171 (R.G.). The NE-CAT 24-ID-C beamline is funded by NIH grant GM103403, and the Pilatus 6M detector is funded by a NIH–ORIP HEI grant (RR029205). This research used resources of the Advanced Photon Source, which is operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357.

Author information

Author notes

    • Ruwan Gunaratne
    •  & Shekhar Kumar

    These authors contributed equally to this work.

Affiliations

  1. Department of Pharmacology and Cancer Biology, Duke University, Durham, North Carolina, USA.

    • Ruwan Gunaratne
    • , Charlene V Chabata
    •  & Bruce A Sullenger
  2. Medical Scientist Training Program, Duke University, Durham, North Carolina, USA.

    • Ruwan Gunaratne
  3. Department of Surgery, Duke University, Durham, North Carolina, USA.

    • Ruwan Gunaratne
    • , James W Frederiksen
    • , Jens L Lohrmann
    • , Kristin M Bompiani
    •  & Bruce A Sullenger
  4. Research Institute, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

    • Shekhar Kumar
    • , Nabil K Thalji
    • , Michelle D Ho
    • , Rodney M Camire
    •  & Sriram Krishnaswamy
  5. Department of Pediatrics, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Steven Stayrook
    • , Nabil K Thalji
    • , Rodney M Camire
    •  & Sriram Krishnaswamy
  6. Northeastern Collaborative Access Team (NE-CAT) and Departments of Chemistry and Chemical Biology, Cornell University, Argonne National Laboratory, Argonne, Illinois, USA.

    • Kay Perry
  7. Department of Medicine, Duke University, Durham, North Carolina, USA.

    • Gowthami Arepally

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Contributions

R.G., S. Kumar, J.W.F., J.L.L., S. Krishnaswamy and B.A.S. designed the experiments; R.G., S. Kumar, J.W.F., J.L.L., K.M.B. and C.V.C. performed the experiments; S.S. and K.P. participated in the collection of the X-ray diffraction data; N.K.T., M.D.H. and R.M.C. generated critical reagents; R.G., S. Kumar, J.W.F., J.L.L., G.A., R.M.C., S. Krishnaswamy and B.A.S. interpreted the data; and R.G., S. Kumar, J.W.F., S. Krishnaswamy and B.A.S. wrote the manuscript.

Competing interests

Duke University (J.W.F., J.L.L., K.M.B. and B.A.S.) has applied for a U.S. patent (US20140275226A1) on this dual-anticoagulant strategy.

Corresponding authors

Correspondence to Sriram Krishnaswamy or Bruce A Sullenger.

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https://doi.org/10.1038/nbt.4153