Chemical Science
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(1200 IU kgꢂ1, PBS) or fondaparinux (0.5 mg kgꢂ1, PBS). Next, 13 G. L. Montalvo, Y. Zhang, T. M. Young, M. J. Costanzo,
mice received an intravenous injection (via tail vein) of DMSO–
H2O solution (control), protamine (16 mg kgꢂ1, PBS), or oxalyl
surfen (6 or 16 mg kgꢂ1, DMSO–water) 60 min aer UFH and 10
K. B. Freeman, J. Wang, D. J. Clements, E. Magavern,
R. W. Kavash, R. W. Scott, D. Liu and W. F. DeGrado, ACS
Chem. Biol., 2014, 9, 967–975.
min aer fondaparinux administration. Five min aer admin- 14 S. Choi, D. J. Clements, V. Pophristic, I. Ivanov,
istration of the reversal compound, blood was collected via
S. Vemparala, J. S. Bennett, M. L. Klein, J. D. Winkler and
submandibular bleeds into 3.2% sodium citrate tubes. Plasma
W. E. DeGrado, Angew. Chem., Int. Ed., 2005, 44, 6685–6689.
was collected immediately (2200 g for 15 min) and stored at 15 L. D'Ilario, I. Francolini, A. Martinelli and A. Piozzi, Dyes
ꢂ80 ꢀC. A heparin anti-FXa colorimetric assay was used to
Pigm., 2009, 80, 343–348.
analyze the neutralization effect of protamine and oxalyl surfen 16 M. Schmidtke, A. Karger, A. Meerbach, R. Egerer, A. Stelzner
(7). Their activity was compared to a heparin control without
and V. Makarov, Virology, 2003, 311, 134–143.
reversal agent as described above. The change in the absorption 17 H.-C. Selinka, L. Florin, H. D. Patel, K. Freitag,
at 405 nm for each sample was compared with a standard
calibration curve to determine the amount of heparin present.
M. Schmidtke, V. A. Makarov and M. Sapp, J. Virol., 2007,
81, 10970–10980.
The purpose of this assay is to analyze the amount of UFH or 18 N. Harris, F. Y. Kogan, G. Il'kova, S. Juhas, O. Lahmy,
fondaparinux remaining in plasma by indirectly measuring the
residual activity of FXa. Heparinized plasma (3 mL) was mixed
Y. I. Gregor, J. Koppel, R. Zhuk and P. Gregor, Biochim.
Biophys. Acta, Gen. Subj., 2014, 1840, 245–254.
with antithrombin, forming an AT–heparin complex. The 19 M. Schuksz, M. M. Fuster, J. R. Brown, B. E. Crawford,
amount of UFH or fondaparinux remaining in plasma was
plotted against the reversal agent dosage.
D. P. Ditto, R. Lawrence, C. A. Glass, L. Wang, Y. Tor and
J. D. Esko, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 13075–13080.
20 N. R. Roan, S. Sowinski, J. Muench, F. Kirchhoff and
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Acknowledgements
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This work was supported by grants from the NIH (GM077471 to
Y. T. and J. D. E. and HL107150 to J. D. E.). We are grateful to Drs
Beth Wilson and Manuela Schuksz for their advice with the
binding assays, Patrick Secrest for help with the mice injections,
and Qiongyu Chen (UCSD Murine Hematology, Chemistry and
Coagulation Core) for help with the factor Xa chromogenic
assays. We also thank Drs Curtis Moore and Arnold Rheingold
(UCSD Chemistry and Biochemistry X-ray Facility) for their help.
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