The Use of ITC and the Software AFFINImeter for the Quantification of the Anticoagulant Pentasaccharide in Low Molecular Weight Heparin

  • Eva MuñozEmail author
  • Juan Sabín
Part of the Methods in Molecular Biology book series (MIMB, volume 1964)


In this chapter, we describe an original protocol based on ITC experiments and data analysis with the software AFFINImeter to get information of heparin-AT interactions relevant for the elucidation of the anticoagulant activity of heparins. This protocol is used to confirm the presence of the bioactive pentasaccharide with anticoagulant activity in heparins and to determine the amount of this pentasaccharide in the sample. Here we have applied this protocol to the characterization of low molecular weight heparins.

Key words

ITC AFFINImeter Low molecular weight heparin Pentasaccharide Antithrombin Data analysis Heterogeneous mixtures Anticoagulant activity Biosimilars 



The development of this protocol has been part of a work carried out with the pharmaceutical company Laboratorios Farmacéuticos Rovi S.A. (Madrid, Spain). We thank them for providing the LMWH, UFH, and AT samples. We also thank Prof. Robert Linhardt and Prof. Fuming Zhang (Rensselaer Polytechnic Institute, Troy, NY) for providing UFH.


  1. 1.
    Holdgate GA, Ward WHJ (2005) Measurements of binding thermodynamics in drug discovery. Drug Discov Today 10:1543–1550CrossRefGoogle Scholar
  2. 2.
    Falconer RJ (2015) Applications of isothermal titration calorimetry – the research and technical developments from 2011 to 2015. J Mol Recognit 29:1099–1352Google Scholar
  3. 3.
    Dumas P, Ennifar E, Da Veiga C, Bec G, Palau W, Di Primo C, Piñeiro A, Sabin J, Muñoz E, Rial J (2016) Extending ITC to kinetics with kinITC. Methods Enzymol 567:157–180CrossRefGoogle Scholar
  4. 4.
    Brown A (2009) Analysis of cooperativity by isothermal titration calorimetry. Int J Mol Sci 10:3457–3477CrossRefGoogle Scholar
  5. 5.
    For more information about the software AFFINImeter.
  6. 6.
    Lever R, Page CP (2012) Non-anticoagulant effects of heparin: an overview. In: Lever R, Mulloy B, Page CP (eds) Heparin—a century of progress, Handbook of experimental pharmacology, vol 207. Springer, Berlin, Germany, pp 281–305CrossRefGoogle Scholar
  7. 7.
    Muñoz EM, Linhardt RJ (2004) Heparin-binding domains in vascular biology. Arterioscler Thromb Vasc Biol 24:1549–1557CrossRefGoogle Scholar
  8. 8.
    Ye H, Toby TK, Sommers CD, Ghasriani H, Trehy ML, Ye W, Kolinski RE, Buhse LF, Al-Hakim A, Keire DA (2013) Characterization of currently marketed heparin products: key tests for LMWH quality assurance. J Pharm Biomed Anal 85:99–107CrossRefGoogle Scholar
  9. 9.
    Muñoz E, Xu D, Avci F, Kemp M, Liu J, Linhardt RJ (2006) Enzymatic synthesis of heparin-related polysaccharides on sensor chips: Rapid screening of heparin–protein interactions. Biochem Biophys Res Comm 339:597–602CrossRefGoogle Scholar
  10. 10.
    Dutta AK, Rösgen J, Rajarathnam K (2015) Using isothermal titration calorimetry to determine thermodynamic parameters of protein–glycosaminoglycan interactions. Methods Mol Biol 1229:315–324CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.AFFINImeter Scientific & Development Team, Software 4 Science DevelopmentsS. L. Ed. EmprendiaSantiago de CompostelaSpain

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