Effects of Sulfation on Antithrombin-Thrombin/Factor Xa Interactions in Semisynthetic Low Molecular Weight Heparins

 

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ABSTRACT

Most of the biological effects of heparin and low molecular weight (LMW) heparins are related to their ability to bind to many different proteins. To gain insight into structure-activity relationships, we investigated quantitatively the interactions of a series of sulfated LMW heparins of similar molecular weights (derived from statistical desulfation of a supersulfated heparin) with the target enzymes human antithrombin (AT) and thrombin (T). In addition, we analyzed the activation of the protease inhibitor against T and factor Xa (FXa). A nonlinear correlation between the strength of the AT-heparin complex and the degree of sulfation of the LMW heparins was observed, whereas only a modest modulation of T binding to heparin occurred. The efficiency of the heparin derivatives in activating AT toward the proteases is generally high for derivatives exhibiting a low dissociation constant. Only the supersulfated LMW heparin showed serpin activation ability higher than expected from the affinity studies. These results indicate that chemical modification of the sulfation pattern of LMW heparin can be used to efficiently modulate binding affinity and activity toward biological targets.

REFERENCES

• 1 Hirsh J. Heparin.  N Engl J Med . 1991;  324 1565-1574
  CrossrefPubMedGoogle Scholar
• 2 Kjellen L, Lindahl U. Proteoglycans: structures and interactions.  Annu Rev Biochem . 1991;  60 443-475
  CrossrefPubMedGoogle Scholar
• 3 Casu B. Structure and biological activity of heparin.  Adv Carbohydr Chem Biochem . 1985;  43 51-134
  PubMedGoogle Scholar
• 4 Rosenberg R D, Lam L. Correlation between structure and function of heparin.  Proc Natl Acad Sci USA . 1979;  76 1281-1222
  PubMedGoogle Scholar
• 5 Gettings P GW, Patson P A, Olson S T. Serpins: Structure, Function and Biology.  Austin, TX: Landes; 1996
  Google Scholar
• 6 Desai R U, Petitou M, Bjork I, Olson S T. Mechanism of heparin activation of antithrombin: evidence for an induced-fit model of allosteric activation involving two interaction subsites.  Biochemistry . 1998;  37 13033-13041
  CrossrefPubMedGoogle Scholar
• 7 Carrell R W, Evans D L, Stein P E. Mobile reactive centre of serpins and the control of thrombosis.  Nature . 1991;  353 576-578
  CrossrefPubMedGoogle Scholar
• 8 Huntington J A, Olson S T, Fan B, Gettins P GW. Mechanism of heparin activation of antithrombin. Evidence for reactive center loop preinsertion with expulsion upon heparin binding.  Biochemistry . 1996;  35 8495-8503
  CrossrefPubMedGoogle Scholar
• 9 Petitou M, Herault J P, Bernat A. Synthesis of thrombin-inhibiting heparin mimetics without side effects.  Nature . 1999;  398 418-422
  PubMedGoogle Scholar
• 10 Lin L, Abrahams J P, Skinner R. The anticoagulant activation of antithrombin by heparin.  Proc Natl Acad Sci USA . 1997;  94 14683-14688
  CrossrefPubMedGoogle Scholar
• 11 Naggi A, Torri G, Casu B. ``Supersulfated'' heparin fragments, a new type of low-molecular weight heparin. Physico-chemical and pharmacological properties.  Biochem Pharmacol . 1987;  36 1895-1900
  CrossrefPubMedGoogle Scholar
• 12 Naggi A, Decristofano B, Bisio A, Torri G, Casu B. Generation of antifactor Xa active 3Osulfated glucosamine rich sequences by controlled desulfation of oversulfated heparins.  Carbohydr Res . 2001 (inpress); 
  PubMedGoogle Scholar
• 13 Casu B, Naggi A, Torri G. Modulation of sulfation patterns of glycosaminoglycans. In: Thiem J, ed. Glycoscience: Chemistry and Chemical Biology, vol. III. New York: Springer-Verlag; 2001 (in press)
  Google Scholar
• 14 Nordenman B, Nystrom C, Bjork I. The size and shape of human and bovine antithrombin III.  Eur J Biochem . 1977;  78 195-203
  CrossrefPubMedGoogle Scholar
• 15 Olson S T. Transient kinetics of heparin-catalyzed protease inactivation by antithrombin III. Linkage of protease-inhibitor-heparin interactions in the reaction with thrombin.  J Biol Chem . 1988;  263 1698-1708
  PubMedGoogle Scholar
• 16 Jesty J, Esnouf M P. The preparation of activated factor X and its action on prothrombin.  Biochem J . 1973;  131 791-799
  PubMedGoogle Scholar
• 17 Olson S T, Björk I, Shore J D. Kinetic characterization of heparin-catalyzed and uncatalyzed inhibition of blood coagulation proteinases by antithrombin.  Methods Enzymol . 1993;  22 525-559
  PubMedGoogle Scholar
• 18 Evans S A, Olson S T, Shore J D. p-Aminobenzamidine as a fluorescent probe for the active site of serine proteases.  J Biol Chem . 1982;  257 3014-3017
  PubMedGoogle Scholar
• 19 Einarsson R, Andersson L O. Binding of heparin to human antithrombin III as studied by measurements of tryptophan fluorescence.  Biochim Biophys Acta . 1976;  490 104-111
  PubMedGoogle Scholar
• 20 Rezaie A R. Calcium enhances heparin catalysis of the antithrombin-factor Xa reaction by a template mechanism. Evidence that calcium alleviates Gla domain antagonism of heparin binding to factor Xa.  J Biol Chem . 1998;  273 16824-16827
  CrossrefPubMedGoogle Scholar