Anticoagulant activity of a sulfated galactan: Serpin-independent effect and specific interaction with factor Xa

Bianca F. Glauser; Ricardo M. Rezende; Fabio R. Melo; Mariana S. Pereira; Ivo M. B. Francischetti; Robson Q. Monteiro; Alireza R. Rezaie; Paulo A. S. Mourão

doi:10.1160/TH09-04-0273

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2009; 102(06): 1183-1193
DOI: 10.1160/TH09-04-0273

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Anticoagulant activity of a sulfated galactan: Serpin-independent effect and specific interaction with factor Xa

Bianca F. Glauser

¹Laboratório de Tecido Conjuntivo, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, Brazil

²Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, Brazil

,

Ricardo M. Rezende

¹Laboratório de Tecido Conjuntivo, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, Brazil

,

Fabio R. Melo

¹Laboratório de Tecido Conjuntivo, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, Brazil

,

Mariana S. Pereira

¹Laboratório de Tecido Conjuntivo, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, Brazil

³Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, Brazil

,

Ivo M. B. Francischetti

⁴Section of Medical Entomology, Laboratory of Malaria and Vector Research, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

,

Robson Q. Monteiro

²Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, Brazil

,

Alireza R. Rezaie

⁵Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri, USA

,

Paulo A. S. Mourão

¹Laboratório de Tecido Conjuntivo, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, Brazil

²Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, Brazil

› Institutsangaben

Abstract

Summary

An algal sulfated galactan has high anticoagulant and antithrombotic activities. Its serpin-dependent anticoagulant action is due to promoting thrombin and factor (F)Xa inhibition by antithrombin and heparin cofactor II. Here, we evaluated the anticoagulant effect of the algal sulfated galactan using serpin-free plasma. In contrast to heparin, the sulfated galactan is still able to prolong coagulation time and delay thrombin and FXa generation in serpin-free plasma. We further investigated this effect using purified blood coagulation proteins, discovering that sulfated galactan inhibits the intrinsic tenase and prothrombinase complexes, which are critical for FXa and thrombin generation, respectively. We also investigated the mechanism by which sulfated galactan promotes FXa inhibition by antithrombin using specific recombinant mutants of the protease. We show that sulfated galactan interacts with the heparin-binding exosite of FXa and Arg-236 and Lys-240 of this site are critical residues for this interaction, as observed for heparin. Thus, sulfated galactan and heparin have similar high-affinity and specificity for interaction with FXa, though they have differences in their chemical structures. Similar to heparin, the ability of sulfated galactan to potentiate FXa inhibition by antithrombin is calcium-dependent. However, in contrast to heparin, this effect is not entirely dependent on the conformation of the γ-carboxyglutamic acid-rich domain of the protease. In conclusion, sulfated galactan and heparin have some similar effects on blood coagulation, but also differ significantly at the molecular level. This sulfated galactan opens new perspective for the development of antithrombotic drugs.

Keywords

Antithrombotic drug - heparin - heparin-binding exosite - prothrombinase complex - sulfated galactan - tenase complex

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References
1 Fareed J, Hoppensteadt DA. Heparins in the new millennium: Will unfractionated heparin survive?. Semin Thromb Haemost 2000; 26: 87-88.
2 Béguin S, Lindhout T, Hemker HC. The mode of action of heparin in plasma. Thromb Haemost 1988; 60: 457-462.
3 Blossom DB. et al. Outbreak of adverse reactions associated with contaminated heparin. N Engl J Med 2008; 359: 2674-2684.
4 Mourão PAS, Pereira MS. Searching for alternatives to heparin. Trends Cardiovasc Med 1999; 09: 225-232.
5 Farias WRL, Valente AP, Pereira MS. et al. Structure and anticoagulant activity of sulfated galactans Isolation of a unique sulfated galactan from the red algae Botryocladia occidentalis and comparison of its anticoagulant action with that of sulfated galactans from invertebrates. J Biol Chem 2000; 275: 29299-29307.
6 Pereira MS, Mulloy B, Mourão PAS. Structure and anticoagulant activity of sulfated fucans – Comparison between the regular, repetitive, and linear fucans from echinoderms with the more heterogeneous and branched polymers from brown algae. J Biol Chem 1999; 274: 7656-7667.
7 Pereira MS, Melo FR, Mourão PAS. Is there a correlation between structure and anticoagulant action of sulfated galactans and sulfated fucans?. Glycobiology 2002; 12: 573-580.
8 Pereira MS, Vilela-Silva ACES, Valente AP. et al. A 2-sulfated, 3-linked alpha-L-galactan is an anticoagulant polysaccharide. Carbohydr Res 2002; 337: 2231-2238.
9 Melo FR, Pereira MS, Foguel D. et al. Antithrombin-mediated anticoagulant activity of sulfated polysaccharides — different mechanisms for heparin and sulfated galactans. J Biol Chem 2004; 279: 20824-20835.
10 Shipp EL, Hsieh-Wilson LC. Profiling the sulfation specificities of glycosaminoglycans interactions with growth factor and chemotactic proteins using microarrays. Chem Biol 2007; 14: 195-208.
11 Gama CI, Tully SE, Sotogaku N. et al. Sulfation patterns of glycosaminoglycans encode molecular recognition and activity. Nat Chem Biol 2006; 02: 467-473.
12 Farias WRL, Nazareth RA, Mourão PAS. Dual effects of sulfated D-galactans from the red algae Botryocladia occidentalis preventing thrombosis and inducing platelet aggregation. Thromb Haemostasis 2001; 86: 1540-1546.
13 Melo FR, Pereira MS, Monteiro RQ. et al. Sulfated galactan is a catalyst of antithrombin-mediated inactivation of a-thrombin. Biochim Biophys Acta 2008; 1780: 1047-1053.
14 Astermark J, Hogg PJ, Bjork I. et al. Effects of gamma-carboxyglutamic acid and epidermal growth factor-likemodules of factor IX on factor X activation. Studies using proteolytic fragments of bovine factor IX. J Biol Chem 1992; 267: 3249-3256.
15 Ngai PK, Chang JY. A novel one-step purification of human alpha-thrombin after direct activation of crude prothrombin enriched from plasma. Biochem J 1991; 280: 805-806.
16 Rezaie AR. Identification of basic residues in the heparin-binding exosite of factor Xa critical for heparin and factor Va binding. J Biol Chem 2000; 275: 3320-3327.
17 Nazareth RA, Tomaz LS, Ortiz-Costa S. et al. Antithrombotic properties of Ixolaris, a factor VIIa/tissue factor complex inhibitor. Thromb Haemost 2006; 96: 7-13.
18 Monteiro RQ, Rezaie AR, Bae JS. et al. Ixolaris binding to factor X reveals a precursor state of factor Xa heparin-binding exosite. Protein Sci 2008; 17: 146-153.
19 Monteiro RQ, Zingali RB. Bothrojaracin, a proexosite I ligand, inhibits factor Va-accelerated prothrombin activation. Thromb Haemost 2002; 87: 288-293.
20 Barrow RT, Parker ET, Krishnaswamy S. et al. Inhibition by heparin of the human blood coagulation intrinsic pathway factor X activator. J Biol Chem 1994; 269: 26796-26800.
21 Stenflo J. Structure-function relationships of epidermal growth factor modules in vitamin K-dependent clotting factors. Blood 1991; 78: 1637-1651.
22 Rezaie AR. Heparin-binding exosite of factor Xa. Trends Cardiovasc Med 2000; 10: 333-338.
23 Sheehan JP, Sadler JE. Molecular mapping of the heparin-binding exosite of thrombin. Proc Natl Acad Sci (USA) 1994; 91: 5518-5522.
24 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: 16824-16827.
25 Monteiro RQ, Rezaie AR, Ribeiro JMC. et al. Ixolaris: a Factor Xa heparin-binding exosite inhibitor. Biochem J 2005; 387: 871-877.
26 Sie P, Ofosu F, Fernandez F. et al. Respective role of antithrombin III and heparin cofactor II in the in vitro anticoagulant effect of heparin and of various sulfated polysaccharides. Br J Haematol 1986; 64: 707.
27 Glauser BF, Pereira MS, Monteiro RQ. et al. Serpin-independent anticoagulant activity of a fucosylated chondroitin sulfate. Thromb Haemost 2008; 100: 420-428.
28 Nagase H, Enjyoji K, Minamiguchi K. et al. Depolymerized holothurian glycosaminoglycan with novel anticoagulant actions: antithrombin III- and heparin cofactor II-independent inhibition of factor X activation by factor IXa-factor VIIIa complex and heparin cofactor II-dependent inhibition of thrombin. Blood 1995; 85: 1527-1534.
29 Kishimoto TK, Viswanathan K, Ganguly T. et al. Contaminated heparin associated with adverse clinical events and activation of the contact system. N Engl J Med 2008; 358: 2457-2467.
30 Holmer E. In: Heparin. Chemical and Biological Properties, Clinical Applications. 1989: 575-595.
31 Leizorovicz A, Haugh MC, Chapuis FR. et al. Low molecular weight heparin in prevention of perioperative thrombosis. Br Med J 1992; 305: 913-920.
32 Nurmohamed MT, Rosendaal FR, Büller HR. et al. Low-molecular-weight heparin versus standard heparin in general and orthopaedic surgery: a meta-analysis. Lancet 1992; 340: 152-156.
33 Anderson JA, Fredenburgh JC, Stafford AR. et al. Hypersulfated low molecular weight heparin with reduced affinity for antithrombin acts as an anticoagulant by inhibiting intrinsic tenase and prothrombinase. J Biol Chem 2001; 276: 9755-9761.
34 Melo FR, Mourão PAS. An algal sulfated galactan has an unusual dual effect on venous thrombosis due to activation of factor XII and inhibition of the coagulation proteases. Thromb Haemost 2008; 99: 531-538.