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DOI: 10.1160/TH04-06-0384
Accelerating ability of synthetic oligosaccharides on antithrombin inhibition of proteinases of the clotting and fibrinolytic systems Comparison with heparin and low-molecular-weight heparin
Financial support: This work was supported by National Institutes of Health Grant HL39888 (S.T.O.), Swedish Scientific Council–Medicine Grant 4212 (I.B.) and by a Research Grant from Sanofi-Synthélabo (S.T.O. and I.B.)Publication History
Received
18 June 2004
Accepted after revision
03 September 2004
Publication Date:
04 December 2017 (online)
![](https://www.thieme-connect.de/media/10.1055-s-00035024/200405/lookinside/thumbnails/10-1160-th04-06-0384-1.jpg)
Summary
The abilities of three synthetic oligosaccharides to accelerate antithrombin inhibition of ten clotting or fibrinolytic proteinases were compared with those of unfractionated, fractionated high-affinity and low-molecular-weight heparins. The results show that the anticoagulant effects of the latter three heparins under conditions approximating physiologic are exerted almost exclusively by acceleration of the inactivation of thrombin, factor Xa and factor IXa to near diffusion-controlled rate constants of ∼106 – 107 M−1·s−1. All other proteinases are inhibited with at least 20-fold lower rate constants. The anticoagulant ability of the synthetic regular (fondaparinux) and high-affinity (idraparinux) pentasaccharides is due to a common mechanism, involving acceleration of only factor Xa inhibition to rate constants of ∼106 M−1·s−1. A synthetic hexadecasaccharide, containing both the pentasaccharide sequence and a proteinase binding site, exerts its anticoagulant effect by accelerating antithrombin inactivation of both thrombin and factor Xa to rate constants of ∼106 – 107 M−1·s−1, although thrombin appears to be the more important target. In contrast, factor IXa inhibition is appreciably less stimulated. The conformational change of antithrombin induced both by the pentasaccharides and longer heparins contributes substantially, ∼150–500-fold, to accelerating the inactivation of factors Xa, IXa and VIIa and moderately, ∼50-fold, to that of factor XIIa and tissue plasminogen activator inhibition. The bridging effect due to binding of antithrombin and proteinase to the same, long heparin chain is dominating, ∼1000–3000-fold, for thrombin inhibition and is appreciably smaller, although up to ∼250-350-fold, for the inactivation of factors IXa and XIa. These results establish the proteinase targets of heparin derivatives currently used in or considered for thrombosis therapy and give new insights into the mechanism of heparin acceleration of antithrombin inhibition of proteinases.
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References
- 1 Olson ST, Björk I. Regulation of thrombin activity by antithrombin and heparin. Semin Thromb Hemost 1994; 20: 373-409.
- 2 Björk I, Olson ST. Antithrombin. A bloody important serpin. In: Church FC, Cunningham DD, Ginsburg D, Hoffman M, Stone SR, Tollefsen DM. Chemistry and Biology of Serpins. New York: Plenum Press; 1997: 17-33.
- 3 Petitou M, Casu B, Lindahl U. 1976-83, a critical period in the history of heparin: the discovery of the antithrombin binding site. Biochimie 2003; 85: 83-9.
- 4 Olson ST, Björk I, Sheffer R. et al. Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement. J Biol Chem 1992; 267: 12528-38.
- 5 Huntington JA, Olson ST, Fan BQ. et al. Mechanism of heparin activation of antithrombin. Evidence for reactive center loop preinsertion with expulsion upon heparin binding. Biochemistry 1996; 35: 8495-503.
- 6 Jin L, Abrahams JP, Skinner R. et al. The anticoagulant activation of antithrombin by heparin. Proc Natl Acad Sci U S A 1997; 94: 14683-8.
- 7 Huntington JA, McCoy A, Belzar KJ. et al. The conformational activation of antithrombin – A 2.85-Å structure of a fluorescein derivative reveals an electrostatic link between the hinge and heparin binding regions. J Biol Chem 2000; 275: 15377-83.
- 8 Chuang YJ, Swanson R, Raja SM. et al. Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence – Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin. J Biol Chem 2001; 276: 14961-71.
- 9 Chuang YJ, Swanson R, Raja SM. et al. The antithrombin P1 residue is important for target proteinase specificity but not for heparin activation of the serpin. Characterization of P1 antithrombin variants with altered proteinase specificity but normal heparin activation. Biochemistry 2001; 40: 6670-9.
- 10 Danielsson Å, Raub E, Lindahl U. et al. Role of ternary complexes, in which heparin binds both antithrombin and proteinase, in the acceleration of the reactions between antithrombin and thrombin or factor Xa. J Biol Chem 1986; 261: 15467-73.
- 11 Olson ST, Björk I. Predominant contribution of surface approximation to the mechanism of heparin acceleration of the antithrombinthrombin reaction. Elucidation from salt concentration effects. J Biol Chem 1991; 266: 6353-64.
- 12 Bedsted T, Swanson R, Chuang Y-J. et al. Heparin and calcium ions dramatically enhance antithrombin reactivity with factor IXa by generating new interaction exosites. Biochemistry 2003; 42: 8143-52.
- 13 Wiebe EM, Stafford AR, Fredenburgh JC. et al. Mechanism of catalysis of inhibition of factor IXa by antithrombin in the presence of heparin or pentasaccharide. J Biol Chem 2003; 278: 35767-74.
- 14 Conrad HE. Heparin-binding proteins. San Diego, CA: Academic Press; 1998
- 15 Warkentin TE, Chong BH, Greinacher A. Heparin-induced thrombocytopenia: towards consensus. Thromb Haemost 1998; 79: 1-7.
- 16 Devlin VJ, Einhorn TA, Gordon SL. et al. Total hip arthroplasty after renal transplantation. Long-term follow-up study and assessment of metabolic bone status. J Arthroplasty 1988; 03: 205-13.
- 17 Barrowcliffe TW. Low molecular weight heparin(s). Br J Haematol 1995; 90: 1-7.
- 18 van Boeckel CAA, Petitou M. The unique antithrombin III binding domain of heparin: A lead to new synthetic antithrombotics. Angew Chem 1993; 32: 1671-90.
- 19 Petitou M, Hérault JP, Bernat A. et al. Synthesis of thrombin-inhibiting heparin mimetics without side effects. Nature 1999; 398: 417-22.
- 20 Choay J, Petitou M, Lormeau JC. et al. Structure-activity relationship in heparin: a synthetic pentasaccharide with high affinity for antithrombin III and eliciting high antifactor Xa activity. Biochem Biophys Res Commun 1983; 116: 492-9.
- 21 Turpie AGG, Gallus AS, Hoek JA. A synthetic pentasaccharide for the prevention of deepvein thrombosis after total hip replacement. N Engl J Med 2001; 344: 619-25.
- 22 Eriksson BI, Bauer KA, Lassen MR. et al. Fondaparinux compared with enoxaparin for the prevention of venous thromboembolism after hip-fracture surgery. N Engl J Med 2001; 345: 1298-304.
- 23 Bauer KA, Eriksson BI, Lassen MR. et al. Fondaparinux compared with enoxaparin for the prevention of venous thromboembolism after elective major knee surgery. N Engl J Med 2001; 345: 1305-10.
- 24 Turpie AGG, Bauer KA, Eriksson BI. et al. Fondaparinux versus enoxaparin for the prevention of venous thromboembolism in major orthopedic surgery. A meta-analysis of 4 randomized double-blind studies. Arch Intern Med 2002; 162: 1833-40.
- 25 Eriksson BI, Bauer KA, Lassen MR. et al. Influence of the duration of fondaparinux (Arixtra®) prophylaxis in preventing venous thromboembolism following major orthopedic surgery. J Thromb Haemost 2003; 01: 383-4.
- 26 Herbert JM, Hérault JP, Bernat A. et al. Biochemical and pharmacological properties of SANORG 34006, a potent and long-acting synthetic pentasaccharide. Blood 1998; 91: 4197-205.
- 27 Herbert JM, Hérault JP, Bernat A. et al. SR123781A, a synthetic heparin mimetic. Thromb Haemost 2001; 85: 852-60.
- 28 Danielsson A, Björk I. Binding to antithrombin of heparin fractions with different molecular weights. Biochem J 1981; 193: 427-33.
- 29 Olson ST, Björk I, Shore JD. Kinetic characterization of heparin-catalyzed and uncatalyzed inhibition of blood coagulation proteinases by antithrombin. Methods Enzymol 1993; 222: 525-60.
- 30 Nordenman B, Nyström C, Björk I. The size and shape of human and bovine antithrombin III. Eur J Biochem 1977; 78: 195-203.
- 31 Bock PE, Day DE, Verhamme IM. et al. Analogs of human plasminogen that are labeled with fluorescence probes at the catalytic site of the zymogen. Preparation, characterization, and interaction with streptokinase. J Biol Chem 1996; 271: 1072-80.
- 32 Melhado LL, Peltz SW, Leytus SP. et al. p-Guanidinobenzoic acid esters of fluorescein as active-site titrants of serine proteases. J Am Chem Soc 1982; 104: 7299-306.
- 33 Schägger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 1987; 166: 368-79.
- 34 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
- 35 Arocas V, Bock SC, Olson ST. et al. The role of Arg46 and Arg47 of antithrombin in heparin binding. Biochemistry 1999; 38: 10196-204.
- 36 Björk I, Jackson CM, Jörnvall H. et al. The active site of antithrombin. Release of the same proteolytically cleaved form of the inhibitor from complexes with factor IXa, factor Xa, and thrombin. J Biol Chem 1982; 257: 2406-11.
- 37 Björk I, Fish WW. Production in vitro and properties of a modified form of bovine antithrombin, cleaved at the active site by thrombin. J Biol Chem 1982; 257: 9487-93.
- 38 Olson ST. Heparin and ionic strength-dependent conversion of antithrombin III from an inhibitor to a substrate of alpha-thrombin. J Biol Chem 1985; 260: 10153-60.
- 39 Stürzebecher J, Kopetzki E, Bode W. et al. Dramatic enhancement of the catalytic activity of coagulation factor IXa by alcohols. FEBS Lett 1997; 412: 295-300.
- 40 Olson ST, Swanson R, Day D. et al. Resolution of Michaelis complex, acylation, and conformational change steps in the reactions of the serpin, plasminogen activator inhibitor-1, with tissue plasminogen activator and trypsin. Biochemistry 2001; 40: 11742-56.
- 41 Björk I, Ylinenjärvi K, Olson ST. et al. Decreased affinity of recombinant antithrombin for heparin due to increased glycosylation. Biochem J 1992; 286: 793-800.
- 42 Turk B, Brieditis I, Bock SC. et al. The oligosaccharide side chain on Asn-135 of α-antithrombin, absent in β-antithrombin, decreases the heparin affinity of the inhibitor by affecting the heparin-induced conformational change. Biochemistry 1997; 36: 6682-91.
- 43 Desai U, Swanson R, Bock SC. et al. Role of arginine 129 in heparin binding and activation of antithrombin. J Biol Chem 2000; 275: 18976-84.
- 44 Olson ST, Sheffer R, Francis AM. High molecular weight kininogen potentiates the heparin-accelerated inhibition of plasma kallikrein by antithrombin: Role for antithrombin in the regulation of kallikrein. Biochemistry 1993; 32: 12136-47.
- 45 Pixley RA, Schapira M, Colman RW. Effect of heparin on the inactivation rate of human activated factor XII by antithrombin III. Blood 1985; 66: 198-203.
- 46 Rezaie AR. 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: 16824-7.
- 47 Rezaie AR, Olson ST. Calcium enhances heparin catalysis of the antithrombin-factor Xa reaction by promoting the assembly of an intermediate heparin-antithrombin-factor Xa bridging complex. Demonstration by rapid kinetics studies. Biochemistry 2000; 39: 12083-90.
- 48 Ruf W, Kalnik MW, Lund-Hansen T. et al. Characterization of factor VII association with tissue factor in solution. High and low affinity calcium binding sites in factor VII contribute to functionally distinct interactions. J Biol Chem 1991; 266: 15719-25.
- 49 Lormeau JC, Herault JP, Herbert JM. Antithrombin-mediated inhibition of factor VIIa-tissue factor complex by the synthetic pentasaccharide representing the heparin binding site to antithrombin. Thromb Haemost 1996; 76: 5-8.
- 50 Desai UR, Petitou M, Björk I. et al. Mechanism of heparin activation of antithrombin - Role of individual residues of the pentasaccharide activating sequence in the recognition of native and activated states of antithrombin. J Biol Chem 1998; 273: 7478-87.
- 51 Holmer E, Kurachi K, Söderstrom G. The molecular-weight dependence of the rateenhancing effect of heparin on the inhibition of thrombin, factor Xa, factor IXa, factor XIa, factor XIIa and kallikrein by antithrombin. Biochem J 1981; 193: 395-400.
- 52 Zhao MM, Abdel-Razek T, Sun MF. et al. Characterization of a heparin binding site on the heavy chain of factor XI. J Biol Chem 1998; 273: 31153-9.
- 53 Badellino KO, Walsh PN. Localization of a heparin binding site in the catalytic domain of factor XIa. Biochemistry 2001; 40: 7569-80.
- 54 Scott CF, Colman RW. Factors influencing the acceleration of human factor XIa inactivation by antithrombin III. Blood 1989; 73: 1873-9.
- 55 Wuillemin WA, Eldering E, Citarella F. et al. Modulation of contact system proteases by glycosaminoglycans - Selective enhancement of the inhibition of factor XIa. J Biol Chem 1996; 271: 12913-8.
- 56 Izaguirre G, Zhang W, Swanson R. et al. Localization of an antithrombin exosite that promotes rapid inhibition of factors Xa and IXa dependent on heparin activation of the serpin. J Biol Chem 2003; 278: 51433-40.
- 57 Schoen P, Lindhout T, Hemker HC. Ratios of anti-factor Xa to antithrombin activities of heparins as determined in recalcified human plasma. Br J Haematol 1992; 81: 255-62.
- 58 Streusand VJ, Björk I, Gettins PGW. et al. Mechanism of acceleration of antithrombinproteinase reactions by low affinity heparin. Role of the antithrombin binding pentasaccharide in heparin rate enhancement. J Biol Chem 1995; 270: 9043-51.