Serpin-independent anticoagulant activity of a fucosylated chondroitin sulfate

Summary

Fucosylated chondroitin sulfate is a glycosaminoglycan from sea cucumber composed of a chondroitin sulfate-like core with branches of sulfated fucose. This glycosaminoglycan has high anticoagulant and antithrombotic activities. Its serpin-dependent anticoagulant activity is mostly due to activating thrombin inhibition by heparin cofactor II. Here, we evaluated the anticoagulant activity of fucosylated chondroitin sulfate using antithrom-bin- and heparin cofactor II-free plasmas. In contrast to mammalian heparin, the invertebrate glycosaminoglycan is still able to prolong coagulation time and delay thrombin and factor Xa generation in serpin-free plasmas. These observations suggest that fucosylated chondroitin sulfate has a serpin-independent anticoagulant effect. We further investigated this effect using purified blood coagulation proteins. Clearly, fucosylated chondroitin sulfate inhibits the intrinsic tenase and prothrombinase complexes, which are critical for thrombin generation. It is possible

that the invertebrate chondroitin sulfate inhibits interactions between cofactor Va and factor Xa. We also employed chemically modified polysaccharides in order to trace a structure versus activity relationship. Removal of the sulfated fucose branches, but not reduction of the glucuronic acid residues to glucose, abolished its activity. In conclusion, fucosylated chondroitin sulfate has broader effects on the coagulation system than mammalian glycosaminoglycans. In addition to its serpin-dependent inhibition of coagulation protease, it also inhibits the generation of factor Xa and thrombin by the tenase and prothrombinase complexes, respectively. In plasma systems, the serpin-independent anticoagulant effect of fucosylated chondroitin sulfate predominates over its serpin-dependent action. This glycosaminoglycan opens new avenues for the development of antithrombotic agents.

Keywords

Glycosaminoglycan - tenase complex - prothrombinase complex - sea cucumber - heparin

• References

• 1 American Heart Association 2002. Heart and Stroke. Statistical update.
• 2 Friedman RJ. Optimal duration of prophylaxis for venous thromboembolism following total hip arthroplasty and total knee arthroplasty. J Am Acad Orthop Surg 2007; 15: 148-155.
• 3 Mourão PAS, Pereira MS. Searching for alternatives to heparin. Trends Cardiovasc Med 1999; 09: 225-232.
• 4 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.
• 5 Mulloy B, Mourão PA, Gray E. Structure/function studies of anticoagulant sulphated polysaccharides using NMR. J Biotechnol 2000; 77: 123-135.
• 6 Mourão PA, Pereira MS, Pavão MS. et al. Structure and anticoagulant activity of a fucosylated chondroitin sulfate from echinoderm. J Biol Chem 1996; 271: 23973-23984.
• 7 Pacheco RG, Vicente CP, Zancan P. et al. Different antithrombotic mechanisms among glycosaminoglycans revealed with a new fucosylated chondroitin sulfate from an echinoderm. Blood Coagul Fibrinolysis 2000; 11: 563-573.
• 8 Fonseca RJC, Mourão PA S. Fucosylated chondroitin sulfate as a new antithrombotic agent. Thromb Haemost 2006; 96: 822-829.
• 9 Zancan P, Mourão PA. Venous and arterial thrombosis in rat models: dissociation of the antithrombotic effects of glycosaminoglycans. Blood Coagul Fibrinolysis 2004; 15: 45-54.
• 10 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.
• 11 Ngai PK, Chang JY. A novel one-step purification of human alpha-thrombin after direct activation of crude prothrombin enriched from plasma. Biochemistry J 1991; 280: 805-806.
• 12 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.
• 13 Pereira MS, Melo FR, Mourão PA S. Is there a correlation between structure and anticoagulant action of sulfated galactans and sulfated fucans?. Glycobiology 2002; 12: 573-580.
• 14 Butenas S, Mann KG. Blood coagulation. Biochemistry 2002; 67: 3-12.
• 15 Furie B, Furie BC. The molecular basis of blood coagulation. Cell 1988; 53: 505-518.
• 16 Stenflo J. Structure-function relationships of epidermal growth factor modules in vitamin K-dependent clotting factors. Blood 1991; 78: 1637-1651.
• 17 Rezaie AR. Heparin-binding exosite of factor Xa. Trends Cardiovasc Med 2000; 10: 333-338.
• 18 Sheehan JP, Sadler JE. Molecular mapping of the heparin-binding exosite of thrombin. Proc Natl Acad Sci USA 1994; 91: 5518-5522.
• 19 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.
• 20 Sheehan JP, Walke EN. Depolymerized holothurian glycosaminoglycan and heparin inhibit the intrinsic tenase complex by a common antithrombin-independent mechanism. Blood 2006; 107: 3876-3882.
• 21 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.
• 22 Björk I, Lindahl U. Mechanism of the anticoagulant action of heparin. Mol Cell Biochem 1982; 48: 161-182.
• 23 Bauer KA. New anticoagulants. Hematology Am Soc Hematol Educ Program 2006; 450-456.
• 24 Bauer KA. New anticoagulants: anti IIa vs anti Xa--is one better?. J Thromb Thrombolysis 2006; 21: 67-72.
• 25 Wienbergen H, Zeymer U. Management of acute coronary syndromes with fondaparinux. Vasc Health Risk Manag 2007; 03: 321-329.
• 26 Eriksson BI, Dahl OE, Büller HR. et al. BISTRO II Study Group. A new oral direct thrombin inhibitor, dabigatran etexilate, compared with enoxaparin for prevention of thromboembolic events following total hip or knee replacement: the BISTRO II randomized trial. J Thromb Haemost 2005; 03: 103-111.
• 27 Holrner E. Heparin. Chemical and biological properties, clinical applications 1989; 575-595.
• 28 Leizorovicz A, Haugh MC, Chapuis FR. et al. Low molecular weight heparin in prevention of perioperative thrombosis. Br Med J 1992; 305: 913-920.
• 29 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.
• 30 Thomas DP, Merton RE, Barrowcliffe TW. et al. Effects of heparin oligosaccharides with high affinity for antithrombin III in experimental venous thrombosis. Thromb Haemost 1982; 47: 244-248.
• 31 Rand MD, Lock JB, van’tVeer C. et al. Blood clotting in minimally altered whole blood. Blood 1996; 88: 3432-3445.
• 32 Hockin MF, Jones KC, Everse SJ. et al. A model for the stoichiometric regulation of blood coagulation. J Biol Chem 2002; 277: 18322-18333.
• 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 Sie P, Ofosu F, Fernandez F. et al. Activerole of anti-thrombin III and heparin cofactor II in the in vitro anticoagulant effect of heparin and of various sulfated polysaccharides. Br J Haematol 1986; 64: 707.
• 35 Nagase H, Enjyoji K, Minamiguchi K. et al. De-polymerized 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.
• 36 Buchanan MR, Ofosu FA, Fernandez F. et al. Lack of relationship between enhanced bleeding induced by heparin and other sulfated polysaccharides and enhanced catalysis of thrombin inhibition. Semin Thromb Hemost 1986; 12: 324-327.
• 37 He L, Vicente CP, Westrick RJ. et al. Heparin cofactor II inhibits arterial thrombosis after endothelial injury. J Clin Invest 2002; 109: 213-219.