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Combination of aptamer and drug for reversible anticoagulation in cardiopulmonary bypass

Nature Biotechnology volume 36, pages 606–613 (2018)Cite this article

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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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Figure 1: X-ray crystal structures of aptamer 11F7t bound to GD-FXaSer195Ala.
Figure 2: Inhibitory effects of 11F7t and apixaban, and anticoagulant reversal.
Figure 3: Inhibitory effects of 11F7t and a FXa catalytic site inhibitor.
Figure 4: Anticoagulant effects during extracorporeal circulation of human blood.
Figure 5: Thrombin generation during extracorporeal circulation of human blood.

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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.

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Author notes

  1. Ruwan Gunaratne and Shekhar Kumar: These authors contributed equally to this work.

Authors and 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.

Corresponding authors

Correspondence to Sriram Krishnaswamy or Bruce A Sullenger.

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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.

Integrated supplementary information

Supplementary Figure 1 Structural definition of ligands bound to GD-FXaS195A.

Composite omit maps following simulated annealing and contoured to 1.5 σ illustrate the unbiased data defining 11F7t (Panel A, structure 5VOE) and rivaroxaban (Panel B, structure 5VOF) bound to GD-XaS195A. Panel A: Sidechains of the proteinase domain making hydrogen bonds with nucleotide bases in 11F7t are shown in blue and labelled. Panel B: Sidechains of the catalytic triad are labelled in red, D189 at the bottom of the primary specificity pocket is in gold, Ca2+ is in yellow and Na+ is in cyan. Elements of bound 11F7t are visible in the upper portion of the panel.

Supplementary Figure 2 Superposition of the 11F7t/GD-FXaS195A X-ray structure with the structure of the P. textilis FX-FV complex.

11F7t and GD-FXaS195A are illustrated in orange and red respectively. In the complex of FV and FX from P. textilis (4BXW), the FV component is in blue and FX is in yellow. The right panel is enlarged to focus on FX/FXa in the standard orientation. Overlay of the two structures illustrates that 11F7t inhibits prothrombinase assembly by competing with the a2 polypeptide for binding to FXa, rather than interfering with interaction between the cofactor and the 165 helix of the protease or by interfering with other aspects of positioning the proteinase adjacent to the cofactor.

Supplementary Figure 3 Inhibitory effects of UFH, 11F7t, or various FXa catalytic site inhibitors on thrombin generation in plasma following extrinsic pathway activation.

Calibrated Automated Thrombogram (CAT) assays were performed to measure thrombin generation in platelet rich plasma (150,000 platelets/μL) following addition of 50 pM TF in the presence of no anticoagulant or one of the indicated anticoagulation strategies, and the computed area under the curve or endogenous thrombin potential (ETP) is depicted: (a) UFH alone (n = 4 independent experiments) or (b) 11F7t alone (n = 3), rixaroxaban (riva) alone (n = 3), edoxaban (edox) alone (n = 3), or apixaban (apix) alone (n = 3) at varying concentrations. n denotes the number of independent experiments performed for each condition with plasma from a different donor. Within an individual experiment, each condition was evaluated with 3 replicates. Error bars denote standard error of the mean.

Supplementary Figure 4 Inhibitory effects of 11F7t and a FXa catalytic site inhibitor, alone or in combination, on thrombin generation in plasma following extrinsic pathway activation.

Calibrated Automated Thrombogram (CAT) assays were performed to measure thrombin generation in platelet rich plasma (150,000 platelets/μL) following addition of 50 pM TF in the presence of no anticoagulant (n = 6 independent experiments) or varying concentrations of 11F7t (n = 3) and/or rivaroxaban (n = 3) (a), 11F7t (n = 3) and/or apixaban (n = 3) (b), and 11F7t (n = 3) and/or edoxaban (n = 3) (c), and the calculated area under the curve or endogenous thrombin potential (ETP) is depicted. n denotes the number of independent experiments performed for each condition with plasma from a different donor. Within an individual experiment, each condition was evaluated with 3 replicates. Error bars denote standard error of the mean. Statistical analysis of anticoagulant synergy was performed by two-way ANOVA.

Supplementary Figure 5 Inhibitory effects of 11F7t and a FXa catalytic site inhibitor, alone or in combination, on whole blood clot formation following intrinsic pathway activation.

Thromboelastography (TEG) assays were performed on human whole blood containing no anticoagulant or one of the indicated anticoagulation strategies. The time until detectable clot formation following kaolin-initiated coagulation is depicted for varying concentrations of (a) 11F7t, (b) rivaroxaban, (c) edoxaban, (d) 11F7t or rivaroxaban, alone or in combination, and (f) 11F7t or edoxaban, alone or in combination, with the maximum time limit of a TEG assay being 180 minutes. n denotes the number of independent experiments performed for each condition. Error bars denote standard error of the mean. Statistical analysis of anticoagulant synergy was performed by two-way ANOVA.

Supplementary Figure 6 Inhibitory effects of bivalirudin on whole blood clot formation following intrinsic pathway activation.

Thromboelastography (TEG) assays were performed on human whole blood containing no anticoagulant or varying concentrations of bivalirudin. The time until detectable clot formation following kaolin-initiated coagulation is depicted. n denotes the number of independent experiments performed for each condition. Error bars denote standard error of the mean.

Supplementary Figure 7 Evidence of gross clotting during extracorporeal circulation of human blood anticoagulated with 11F7t or a FXa catalytic site inhibitor alone.

(a) Photographic depiction of a large clot that developed in the circuit reservoir during circulation of blood anticoagulated with 11F7t (2 μM) alone. (b) Photographic depiction of clot formation on the bottom surface of the oxygenator membrane after circulation of blood anticoagulated with rivaroxaban (2 μM) alone.

Supplementary Figure 8 Inhibitory effects of 11F7t and fondaparinux, alone or in combination, on thrombin generation in plasma following extrinsic pathway activation and on whole blood clot formation following intrinsic pathway activation.

(a-b) Calibrated Automated Thrombogram (CAT) assays were performed to measure thrombin generation in platelet rich plasma (150,000 platelets/μL) following addition of 50 pM TF in the absence of an anticoagulant (n = 6) or in the presence of one of the indicated anticoagulation strategies and the area under the curve or endogenous thrombin potential (ETP) is depicted: varying concentrations of fondaparinux alone (n = 3) (a) or combinations of 11F7t plus fondaparinux (n = 3) (b). n denotes the number of independent experiments performed for each condition with plasma from a different donor. Within an individual experiment, each condition was evaluated with 3 replicates. (c-d) Thromboelastography (TEG) assays were performed on human whole blood containing no anticoagulant or one of the indicated anticoagulation strategies, and the time until detectable clot formation following kaolin-initiated coagulation varying concentrations of fondaparinux (c) or combinations of 11F7t plus fondaparinux (d), with the maximum time limit of a TEG assay being 180 minutes. n denotes the number of independent experiments performed for each condition. In all panels, error bars denote standard error of the mean. Statistical analysis of anticoagulant synergy was performed by two-way ANOVA.

Supplementary Figure 9 Dual anti-FXa anticoagulation strategy limits bradykinin generation during extracorporeal circulation as well as UFH.

Pre- and post-circulation plasma bradykinin levels measured in blood anticoagulated with UFH or one of the indicated combinations of 11F7t plus either rivaroxaban (riva), apixaban (apix), or edoxaban (edox). For each anticoagulant strategy, n = 3 independent experiments were performed. Error bars indicate standard error of the mean. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparisons test. NS denotes “not significant.” Exact p values are provided as follows; A vs B: p = 0.96, A vs C: p = 0.82; A vs D: p = 0.63; B vs C: p = 0.56; B vs D: p = 0.88; C vs D: p = 0.2.

Supplementary Figure 10 Assessment of platelet activation induced by IgG samples from HIT patients in the presence of UFH, 11F7t, or combinations of 11F7t plus a FXa catalytic site inhibitor.

Maximum light transmission observed upon incubation of purified IgG (25-100 μg/mL) from 3 HIT patients with platelet-rich plasma (PRP) in the presence of UFH (0.05 and 2 U/mL), 11F7t (0.1, 0.5, 1, and 5 μM), 11F7t plus edoxaban (0.1, 1, and 5 μM), 11F7t plus fondaparinux (0.1, 1, and 5 μM), or no anticoagulant. Each bar represents the mean ± standard error of three measurements performed using purified IgG from each of the three HIT patients tested and using PRP from 3 different healthy donors. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparisons test. Exact p values are provided as follows; (2) vs (1), (2) vs (3), (2) vs (4), (2) vs (5), (2) vs (6), (2) vs (7), (2) vs (8), (2) vs (9), (2) vs (10), (2) vs (11), (2) vs (12), (2) vs (13): p < 0.0001.

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Gunaratne, R., Kumar, S., Frederiksen, J. et al. Combination of aptamer and drug for reversible anticoagulation in cardiopulmonary bypass. Nat Biotechnol 36, 606–613 (2018). https://doi.org/10.1038/nbt.4153

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