Binding of heparin to plasma proteins and endothelial surfaces is inhibited by covalent linkage to antithrombin

Summary

Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are used for prophylaxis and treatment of thrombosis. However, UFH has a short plasma half-life and variable anticoagulant response in vivo due to plasma or vessel wall protein binding and LMWH has a decreased ability to inactivate thrombin, the pivotal enzyme in the coagulation cascade. Covalent linkage of antithrombin to heparin gave a complex (ATH) with superior anticoagulant activity compared to UFH and LMWH, and longer intravenous half-life compared to UFH. We found that plasma proteins bound more to UFH than ATH, and least to LMWH. Also, UFH bound significantly more to endothelial cells than ATH, with 100% of UFH and 94% of ATH binding being on the cell surface and the remainder was endocytosed. Competition studies with UFH confirmed that ATH binding was likely through its heparin moiety. These findings suggest that differences in plasma protein and endothelial cell binding may be due to available heparin chain length. Although ATH is polydisperse, the covalently-linked antithrombin may shield a portion of the heparin chain from association with plasma or endothelial cell surface proteins. This model is consistent with ATH’s better bioavailability and more predictable dose response.

Keywords

Anticoagulant - vessel wall - protein binding - heparinoid - endocytosis

• References

• 1 Choay J, Petitou M. The chemistry of heparin: A way to understand its mode of action. Med J Austr 1986; 144: 7-10.
• 2 Johnson EA, Mulloy B. The molecular-weight range of mucosal-heparin preparations. Carbohydr Res 1976; 51: 119-27.
• 3 Fareed J, Fu K, Yang LH, Hoppensteadt DA. Pharmacokinetics of low molecular weight heparins in animal models. Semin Thromb Hemost 1999; 25: 51-5.
• 4 Fareed J, Haas S, Sasahar A. Past, present and future considerations on low molecular weight heparin differentiation: an epilogue. Semin Thromb Hemost 1999; 25: 145-7.
• 5 Hirsh J, Warkentin TE, Shaughnessy SG, Anand SS. et al. Heparin and low-molecularweight heparin: mechanisms of action, pharmacokinetics, dosing, monitoring, efficacy, and safety. Chest 2001; 119: 64S-94S.
• 6 Rosenberg RD. Actions and interactions of antithrombin and heparin. N Engl J Med 1975; 292: 146.
• 7 Rosenberg RD, Bauer KA. The heparin-antithrombin system: A natural anticoagulant mechanism. In: Coleman RW, Hirsh J, Marder VJ, Salzman EW. editors. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Philadelphia: J.B. Lippincott Company; 1994: 837-60.
• 8 Mille B, Watton J, Barrowcliffe TW. et al. N- and C-terminal amino acids in antithrombin binding to pentasaccharide. J Biol Chem 1994; 269: 29435-43.
• 9 Byun Y, Jacobs HA, Kim SW. Mechanism of thrombin inactivation by immobilized heparin. J Biomed Mater Res 1996; 30: 423-7.
• 10 Lindahl U, Thunberg L, Backstrom G. et al. Extension and structural variability of the antithrombin-binding sequence in heparin. J Biol Chem 1994; 259: 12368-76.
• 11 Thunberg L, Backstrom G, Lindahl U. Further characterization of antithrombin-binding sequence in heparin. Carbohydr Res 1982; 100: 393-410.
• 12 Hirsh J. Heparin. N Engl J Med 1991; 324: 1565-74.
• 13 Weitz JI. Low molecular weight heparins. N Engl J Med 1997; 337: 688-98.
• 14 Lane DA, Pijler G, Flynn AM. Neutralization of heparin related saccharides by histidinerich glycoprotein and platelet factor 4. J Biol Chem 1986; 261: 3980-6.
• 15 Preissner KT, Muller-Berghaus G. Neutralization and binding of heparin by S-protein/vitronectin in the inhibition of factor Xa by antithrombin III. J Biol Chem 1987; 262: 12247-53.
• 16 Mosesson MW. The roles of fibrinogen and fibrin in hemostasis and thrombosis. Semin Hematol 1992; 29: 177-88.
• 17 Zammit A, Pepper DS, Dawes J. Interaction of immobilised unfractionated and LMW heparins with proteins in whole human plasma. Thromb Haemost 1993; 70: 951-8.
• 18 Young E, Wells P, Holloway S. et al. Ex-vivo and in-vitro evidence that low molecular weight heparins exhibit less binding to plasma proteins than unfractionated heparin. Thromb Haemost 1994; 71: 300-4.
• 19 Cosmi B, Fredenburgh JC, Rischke J. et al. Effect of nonspecific binding to plasma proteins on the antithrombin activities of unfractionated heparin, low-molecular-weight heparin, and dermatan sulfate. Circulation 1997; 95: 118-24.
• 20 Manson L, Weitz JI, Podor TJ. et al. The variable anticoagulant response to unfractionated heparin in vivo reflects binding to plasma proteins rather than clearance. J Lab Clin Med 1997; 130: 649-55.
• 21 Andrew M, Mitchell L, Vegh P. et al. Thrombin regulation in children differs from adults in the absence and presence of heparin. Thromb Haemost 1994; 72: 836-42.
• 22 Chan AKC, Berry LR, Monagle PT. et al. Decreased concentrations of heparinoids are required to inhibit thrombin generation in plasma from newborns and children compared to plasma from adults due to reduced thrombin potential. Thromb Haemost 2002; 87: 606-13.
• 23 Schmidt B, Ofosu FA, Mitchell L. et al. Anticoagulant effects of heparin in neonatal plasma. Pediatr Res 1989; 25: 405-8.
• 24 Andrew M, Paes B, Johnston M. Development of the hemostatic system in the neonate and young infant. Am J Pediatr Hematol Oncol 1990; 12: 95-104.
• 25 Andrew M, Paes B, Milner R. et al. Development of the human coagulation system in the full-term infant. Blood 1987; 70: 165-72.
• 26 Andrew M, Vegh P, Johnston M. et al. Maturation of the hemostatic system during childhood. Blood 1992; 80: 1998-2005.
• 27 de Swart CAM, Nijmeijer B, Roelefs JMM. et al. Kinetics of intravenously administered heparin in humans. Blood 1982; 60: 1251-8.
• 28 van Rijn JLML, Trillou M, Mardiguian J. et al. Selective binding of heparins to human endothelial cells. Implications for pharmacokinetics. Thromb Res 1987; 45: 211-22.
• 29 Glimelius B, Busch C, Hook M. Binding of heparin on the surface of cultured human endothelial cells. Thromb Res 1978; 12: 773-82.
• 30 Barzu T, van Rijn JLML, Petitou M. et al. Endothelial binding sites for heparin specificity and role in heparin neutralization. Biochem J 1986; 238: 847-54.
• 31 Chan AK, Berry L, O’Brodovich H. et al. Covalent antithrombin-heparin complexes with high anticoagulant activity: Intravenous, subcutaneous and intratracheal administration. J Biol Chem 1997; 272: 22111-7.
• 32 Berry L, Stafford A, Fredenburgh J. et al. Investigation of the anticoagulant mechanisms of a covalent antithrombin-heparin complex. J Biol Chem 1998; 273: 34730-6.
• 33 Paredes N, Wang A, Berry LR. et al. Mechanisms responsible for catalysis of the inhibition of factor Xa or thrombin by antithrombin using a covalent antithrombinheparin complex. J Biol Chem 2003; 278: 23398-409.
• 34 Chan AKC, Berry L, Klement P. et al. A novel antithrombin-heparin covalent complex: Antithrombotic and bleeding studies in rabbits. Blood Coagul Fibrinolysis 1998; 09: 587-95.
• 35 Vlassara H, Brownlee M, Cerami A. Nonenzymatic glycosylation: Role in the pathogenesis of diabetic complications. Clin Chem 1986; 32: B37-B41.
• 36 Berry LR, Becker DL, Chan AK. Inhibition of fibrin-bound thrombin by a covalent antithrombin-heparin complex. J Biochem (Tokyo) 2002; 132: 167-76.
• 37 Lijnen HR, Hoylaerts M, Collen D. Heparin binding properties of human histidine-rich glycoprotein: Mechanism and role in the neutralization of heparin in plasma. J Biol Chem 1983; 258: 3803-8.
• 38 Cruickshank MK, Levine MN, Hirsh J. et al. A standard heparin nomogram for the management of heparin therapy. Arch Intern Med 1991; 151: 333-7.
• 39 Young E, Prins M, Levine MN. et al. Heparin binding to plasma proteins, an important mechanism for heparin resistance. Thromb Haemost 1992; 67: 639-43.
• 40 Emanuele RM, Fareed J. The effect of molecular weight on the bioavailability of heparin. Thromb Res 1987; 48: 591-6.
• 41 Dahlback K, Lofberg H, Alumets J. et al. Immunohistochemical demonstration of agerelated deposition of vitronectin (S-protein of complement) and terminal complement complex on dermal elastic fibers. J Invest Dermatol 1989; 92: 727-33.
• 42 Maulen-Radovan I, Gutierrez-Castrellon P. Plasma fibronectin in pediatric patients. Rev Invest Clin 1996; 48: 105-9.
• 43 Vannucchi S, Pasquali F, Porciatti F. et al. Binding, internalization and degradation of heparin and heparin fragments by cultured endothelial cells. Thromb Res 1988; 49: 373-83.
• 44 Young E, Venner T, Ribau J. et al. The binding of unfractionated heparin and low molecular weight heparin to thrombin-activated human endothelial cells. Thromb Res 1999; 96: 373-81.
• 45 Barzu T, Molho P, Tobelem G. et al. Binding and endocytosis of heparin by human endothelial cells in culture. Biochim Biophys Acta 1985; 845: 196-203.
• 46 Raub TJ, Newton CR. Recycling kinetics and transcytosis of transferrin in primary cultures of bovine brain microvessel endothelial cells. J Cell Physiol 1991; 149: 141-51.
• 47 Barzu T, van Rijn JLML, Petitou M. et al. Heparin degradation in the endothelial cells. Thromb Res 1987; 47: 601-9.
• 48 Laurent TC, Tengblad A, Thunberg L. et al. The molecular-weight-dependence of the anticoagulant activity of heparin. Biochem J 1978; 175: 691-701.
• 49 McAvoy TJ. The biologic half-life of heparin. Clin Pharmacol Ther 1979; 25: 372-9.
• 50 Collen D, Schetz J, De Cock F. et al. Metabolism of antithrombin III (heparin cofactor) in man: Effects of venous thrombosis and of heparin administration. Eur J Clin Invest 1977; 07: 27-35.
• 51 Chan AKC, Rak J, Berry LR. et al. Antithrombin-heparin covalent complex: a possible alternative to heparin for arterial thrombosis prevention. Circulation 2002; 106: 261-5.
• 52 Pletcher CH, Nelsestuen GL. The rate-determining step of the heparin-catalyzed antithrombin/thrombin reaction is independent of thrombin. J Biol Chem 1982; 257: 5342-5.