Supplementary Materials

Supplementary Material for:

Affinity-based design of a synthetic universal reversal agent for heparin anticoagulants

Rajesh A. Shenoi, Manu Thomas Kalathottukaren, Richard J. Travers, Benjamin F. L. Lai, A. Louise Creagh, Dirk Lange, Kai Yu, Marie Weinhart, Ben H. Chew, Caigan Du, Donald E. Brooks, Cedric J. Carter, James H. Morrissey, Charles A. Haynes, Jayachandran N. Kizhakkedathu*

*Corresponding author. E-mail: jay{at}pathology.ubc.ca

Published 29 October 2014, Sci. Transl. Med. 6, 260ra150 (2014)
DOI: 10.1126/scitranslmed.3009427

This PDF file includes:

  • Study design
  • Materials and Methods
  • Fig. S1. Synthetic scheme for UHRA (A) and MPEG-R (B).
  • Fig. S2. 1H NMR spectrum (CDCl3, 300 MHz) of polymer scaffold (23 kD).
  • Fig. S3. GPC-MALLS profile of polymer scaffold (23 kD) in 0.1 M NaNO3.
  • Fig. S4. 1H NMR spectrum (CDCl3, 300 MHz) of UHRA-7.
  • Fig. S5. 13C NMR spectrum (CDCl3, 150 MHz) of UHRA-7.
  • Fig. S6. GPC-MALS profile of UHRA-7 in 1 M NaNO3.
  • Fig. S7. Reverse-phase HPLC traces for UHRA and starting materials.
  • Fig. S8. 1H NMR spectrum (CDCl3, 300 MHz) of MPEG-R.
  • Fig. S9. Raw data and the integral heats for the titration of MPEG-R (A), UHRA-1 (B), and UHRA-7 (C) into UFH, and MPEG-R (D), UHRA-1 (E), and UHRA-7 (F) into enoxaparin.
  • Fig. S10. Blood clot characteristics for the neutralization of fondaparinux (1.2 IU/ml) with UHRA-7 studied by TEG (mean ± SD, n = 5).
  • Fig. S11. Neutralization of fondaparinux (1.2 IU/ml) by UHRA-6 and UHRA-7 in human plasma.
  • Fig. S12. Thrombin generation assay in human plasma for the reversal of (A) UFH (2 IU/ml), (B) tinzaparin (2 IU/ml), and (C) fondaparinux (1.2 IU/ml) with UHRA-7.
  • Fig. S13. Blood clot characteristics for the neutralization of semuloparin (0.75 IU/ml) with UHRA-7 studied by TEG (mean ± SD, n = 5).
  • Fig. S14. Fractionation of UHRAs and the heparin neutralization activity of each fraction.
  • Fig. S15. Structure of the polymer control sample (23 kD) used for the biocompatibility study.
  • Fig. S16. Clot strength of recalcified human whole blood in the presence of UHRAs (0.5 mg/ml) measured by thromboelastography.
  • Fig. S17. Effect of MW of UHRAs on biocompatibility.
  • Fig. S18. Optical microscopic images of human whole blood incubated with UHRA-7 or protamine.
  • Fig. S19. RBC hemolysis in the presence of UHRAs (5 mg/ml) measured by Drabkin’s method.
  • Fig. S20. Serum LDH activity in mice administered with UHRA-7.
  • Fig. S21. Biodistribution in female Balb/C mice after intravenous administration of tritiated UHRA-4 (20 mg/kg) or UHRA-4/UFH complex.
  • Fig. S22. Impact of UHRA/UFH and protamine/UFH complexes on complement activation in human serum in vitro.
  • Fig. S23. Average size of UHRA/UFH and protamine/UFH complexes (1:1) in PBS measured by AFM.
  • Fig. S24. Intrinsic fluorescence measurements on AT-UFH complex with and without added UHRA.
  • Fig. S25. Proposed mechanism of action of UHRA.
  • Table S1. In vitro neutralization of heparins with UHRAs measured by APTT clotting time.
  • Table S2. Neutralization of fondaparinux and semuloparin: TEG and chromogenic FXa assay.
  • Table S3. Thrombin generation assay parameters after incubation of UHRA-7 and heparins with human PRP.
  • Table S4. Thrombin generation assay parameters for the neutralization of UFH and tinzaparin with UHRA-7 in human PRP.
  • Table S5. Thrombin generation assay parameters for the neutralization of fondaparinux with UHRA-7 in human PRP.
  • Table S6. In vivo neutralization of UFH and enoxaparin by UHRA-7.
  • Table S7. Bleeding time and hemoglobin loss for the mouse tail bleeding studies with UHRA.
  • Table S8. Blood compatibility of UHRAs.
  • Table S9. Body weight of individual mouse after intravenous administration of UHRA-7 or protamine.
  • Table S10. LDH activity in mice serum after intravenous administration of UHRA-7.
  • Table S11. Biodistribution of UHRA or UHRA/UFH complex in mice after intravenous administration.
  • Table S12. Clearance data after intravenous administration of tritiated UHRA-4 and UHRA-4/UFH complex in female Balb/C mice.
  • References (58, 59)

[Download PDF]