Download PDF
Thromb Haemost 2009; 102(05): 865-873
DOI: 10.1160/TH09-02-0081
Theme Issue Article
Schattauer GmbH

Structural features of low-molecular-weight heparins affecting their affinity to antithrombin

Antonella Bisio
1   Institute for Chemical and Biochemical Research “G. Ronzoni”, Milan, Italy
,
Davide Vecchietti
1   Institute for Chemical and Biochemical Research “G. Ronzoni”, Milan, Italy
,
Laura Citterio
1   Institute for Chemical and Biochemical Research “G. Ronzoni”, Milan, Italy
,
Marco Guerrini
1   Institute for Chemical and Biochemical Research “G. Ronzoni”, Milan, Italy
,
Rahul Raman
2   Harvard-MIT Division of Health Sciences & Technology, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
,
Sabrina Bertini
1   Institute for Chemical and Biochemical Research “G. Ronzoni”, Milan, Italy
,
Giorgio Eisele
1   Institute for Chemical and Biochemical Research “G. Ronzoni”, Milan, Italy
,
Annamaria Naggi
1   Institute for Chemical and Biochemical Research “G. Ronzoni”, Milan, Italy
,
Ram Sasisekharan
2   Harvard-MIT Division of Health Sciences & Technology, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
,
Giangiacomo Torri
1   Institute for Chemical and Biochemical Research “G. Ronzoni”, Milan, Italy
› Author Affiliations
Financial support: This work was supported in part by the National Institutes of Health Grant HL080278.
Further Information

Publication History

Received: 05 February 2009

Accepted after major revision: 09 July 2009

Publication Date:
27 November 2017 (online)

   Permissions and Reprints

Summary

As part of a more extensive investigation on structural features of different low-molecular-weight heparins (LMWHs) that can affect their biological activities, Enoxaparin,Tinzaparin and Dalteparin were characterised with regards to the distribution of different chain length oligosaccharides as determined by size-exclusion (SE) chromatography, as well as their structure as defined by 2D-NMR spectra (HSQC). The three LMWHs were also fractionated into high affinity (HA) and no affinity (NA) pools with regards to their ability to bind antithrombin (AT).The HA fractions were further subfractionated and characterised. For the parent LMWHs and selected fractions,molecular weight parameters were measured using a SE chromatographic system with a triple detector (TDA) to obtain absolute molecular weights. The SE chromatograms clearly indicate that Enoxaparin is consistently richer in shorter oligosaccharides than Tinzaparin and Dalteparin. Besides providing the content of terminal groups and individual glucosamine and uronic acid residues with different sulfate substituents, the HSQC-NMR spectra permitted us to evaluate and correlate the content of the pentasaccharide, AT-binding sequence A-G-A*-I-A (AT-bs) through quantification of signals of the disaccharide sequence G-A*.Whereas the percent content of HA species is approximately the same for the three LMWHs, substantial differences were observed for the chain distribution of AT-bs as a function of length, with the AT-bs being preferentially contained in the longest chains of each LMWH. The above information will be useful in establishing structure-activity relationships currently under way. This study is therefore critical for establishing correlations between structural features of LMWHs and their AT-mediated anticoagulant activity.

Keywords

Low-molecular-weight heparin - structure - affinity fractionation - size fractionation - 2D-NMR
 

  • References

  • 1 Warkentin TE, Levine MN, Hirsh J. et al. Heparin induced thrombocytopenia in patients treated with low molecular weight heparin or unfractionated heparin. N Engl J Med 1995; 332: 1330-1336.
    CrossrefPubMedSearch in Google Scholar
  • 2 Bara L, Samama MM. Pharmacokinetics of low molecular weight heparins. Acta Chir Scand 1988; 543: 65-72.
    PubMedSearch in Google Scholar
  • 3 Handeland GF, Abildgaard U, Holm HA. et al. Dose adjusted heparin treatment of deep venous thrombosis: a comparison of unfractionated and low molecular weight heparin. Eur J Clin Pharmacol 1990; 39: 107-112.
    CrossrefPubMedSearch in Google Scholar
  • 4 Harenberg J. Pharmacology of low molecular weight heparins. Semin Thromb Hemost 1990; 16: 12-18.
    PubMedSearch in Google Scholar
  • 5 Hirsh J. Low molecular weight heparin: a review of the results of recent studies of the treatment of venous thromboembolism and unstable angina. Circulation 1998; 98: 1575-1582.
    CrossrefPubMedSearch in Google Scholar
  • 6 Gray E, Mulloy B, Barrowcliffe TW. Heparin and low-molecular-weight-heparin. Thromb Haemost 2008; 99: 807-818.
    Thieme ConnectPubMedSearch in Google Scholar
  • 7 Jeske WP, Walenga JM, Hoppensteadt DA. et al. Differentiating low-molecular-weight heparins based on chemical, biological, and pharmacologic properties: implications for the development of generic versions of low-molecular-weight heparins. Semin Thromb Hemost 2008; 34: 74-85.
    Thieme ConnectPubMedSearch in Google Scholar
  • 8 Casu B. Structure and active domains of heparin. In: Chemistry and Biology of Heparin and Heparan Sulfate. Oxford: Elsevier; 2005: 1-28.
    Search in Google Scholar
  • 9 Riesenfeld J, Thunberg L, Höök M. et al. Antithrombin-binding sequence of heparin. Location of essential N-sulfate groups. J Biol Chem 1981; 256: 2389-2394.
    PubMedSearch in Google Scholar
  • 10 Casu B, Lindahl U. Structure and biological interactions of heparin and heparan sulfate. Adv Carbohydr Chem Biochem 2001; 57: 159-206.
    PubMedSearch in Google Scholar
  • 11 Petitou M, Casu B, Lindahl U. 1976-1983, a critical period in the history of heparin. The discovery of the antithrombin binding site. Biochimie 2003; 85: 83-89.
    CrossrefPubMedSearch in Google Scholar
  • 12 Linhardt RJ, Gunay NS. Production and chemical processing of low molecular weight heparins. Semin Thromb Hemost 1999; 25 (Suppl. 03) 5-16.
    Thieme ConnectPubMedSearch in Google Scholar
  • 13 Guerrini M, Guglieri S, Naggi A. et al. Low molecular weight heparins: structural differentiation by bidimensional nuclear magnetic resonance spectroscopy Semin Thromb Hemost 2007; 33: 478-487.
    Thieme ConnectPubMedSearch in Google Scholar
  • 14 Mascellani G, Guerrini M, Torri G. et al. Characterization of di-and monosulfated, unsaturated heparin disaccharides with terminal N-sulfated 1,6-anhydro-β-D-glucosamine or N-sulfated 1,6-anhydro-β-D-mannosamine residues. Carbohydr Res 2007; 342: 835-842.
    CrossrefPubMedSearch in Google Scholar
  • 15 Guerrini M, Guglieri S, Casu B. et al. Antithrombin-binding octasaccharides and role of extensions of the active pentasaccharide sequence in the specificity and strength of interaction. J Biol Chem 2008; 283: 26662-26675.
    CrossrefPubMedSearch in Google Scholar
  • 16 Wagenvoord R, Al Dieri R, van Dedem G. et al. Linear diffusion of thrombin and factor Xa along the heparin molecule explains the effects of extended heparin chain lengths. Thromb Res 2008; 122: 237-245.
    CrossrefPubMedSearch in Google Scholar
  • 17 Petitou M, Imberty A, Duchaussoy P. et al. Experimental proof for the structure of a thrombin-inhibiting heparin molecule. Chemistry 2001; 07: 858-873.
    CrossrefPubMedSearch in Google Scholar
  • 18 de Kort M, Buijsman RC, van Boeckel CAA. Synthetic heparin derivatives as new anticoagulant drugs. Drug Discov Today 2005; 10: 769-779.
    CrossrefPubMedSearch in Google Scholar
  • 19 Jeske W, Fareed J. In vitro studies on the biochemistry and pharmacology of low molecular weight heparins. Semin Thromb Hemost 1999; 25: 27-33.
    Thieme ConnectPubMedSearch in Google Scholar
  • 20 Bertini S, Bisio A, Torri G. et al. Molecular weight determination of heparin and dermatan sulfate by size exclusion chromatography with a triple detector array. Biomacromol 2005; 06: 168-173.
    CrossrefPubMedSearch in Google Scholar
  • 21 Guerrini M, Naggi A, Guglieri S. et al. Complex glycosaminoglycans: profiling substitution patterns by two-dimensional nuclear magnetic resonance spectroscopy. Anal Biochem 2005; 337: 35-47.
    CrossrefPubMedSearch in Google Scholar
  • 22 Goodger SJ, Robinson CJ, Murphy KJ. et al. Evidence that heparin saccharides promote FGF2 mitogenesis through two distinct mechanisms. J Biol Chem 2008; 283: 13001-13008.
    CrossrefPubMedSearch in Google Scholar
  • 23 Höök M, Björk I, Hopwood J. et al. Anticoagulant activity of heparin: separation of high-activity and lowactivity heparin species by affinity chromatography on immobilized antithrombin. FEBS Lett 1976; 66: 90-93.
    CrossrefPubMedSearch in Google Scholar
  • 24 Bitter T, Muir HM. Quantitative determination of uronic acids with m-hydroxydiphenyl. Anal Biochem 1962; 04: 330-334.
    CrossrefPubMedSearch in Google Scholar
  • 25 Iacomini M, Casu B, Guerrini M. et al. Linkage region sequences of heparins and heparan sulfates. Detection and quantification by NMR spectroscopy. Anal Biochem 1999; 274: 50-58.
    CrossrefPubMedSearch in Google Scholar
  • 26 Rodèn L, Ananth S, Campbell P. et al. Heparin - an introduction. In: Heparin and Related Polysaccharides. New York: Plenum Press; 1992: 1-20.
    Search in Google Scholar
  • 27 Kusche M, Torri G, Casu B. et al. Biosynthesis of heparin. Availability of 3-O-sulfation sites. J Biol Chem 1990; 265: 7292-7300.
    PubMedSearch in Google Scholar
  • 28 Casu B, Torri G. Structural characterization of low molecular weight heparins. Semin Thromb Hemost 1999; 25 (Suppl. 03) 17-25.
    Thieme ConnectPubMedSearch in Google Scholar
  • 29 Fareed J, Ma Q, Florian M. et al. Differentiation of low molecular-weight heparins: impact on the future of the management of thrombosis. Semin Thromb Hemost 2004; 30 (Suppl. 01) 89-104.
    Thieme ConnectPubMedSearch in Google Scholar