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. 1985 Apr;75(4):1169–1173. doi: 10.1172/JCI111812

Antithrombotic properties in rabbits of heparin and heparin fragments covalently coupled to human antithrombin III.

C Mattsson, M Hoylaerts, E Holmer, T Uthne, D Collen
PMCID: PMC425441  PMID: 3988937

Abstract

Clinical grade heparin is a very heterogeneous mucopolysaccharide, containing molecules with Mr ranging from 6,000 to 30,000 that have either a high affinity or a low affinity for antithrombin III (AT). In this study, the antithrombotic properties of intact high-affinity heparin (Mr = 15,000) and of two heparin fragments (h16, a 16-monosaccharide fragment, with Mr = 4,300, and h12, a 12-monosaccharide fragment, with Mr = 3,200) and of their functional covalent stoichiometric complexes with human AT were compared in a venous thrombosis stasis model in rabbits. Thrombosis was induced by injection of glass-activated human plasma and measured in a segment of the jugular vein that was isolated between two vascular clamps for 10 min. Injections of 55 micrograms/kg resulted in a clear antithrombotic effect for intact heparin, but not for the two fragments. Equivalent amounts (carbohydrate moiety) of covalent complexes of heparin or of both heparin fragments with human AT resulted in an antithrombotic effect lasting for 45-60 min. Injection of 110 micrograms/kg of heparin and of the heparin fragments yielded an antithrombotic effect, lasting 45-60 min; the corresponding amounts of covalent complexes caused an anti-thrombotic effect for 60-120 min. The free and conjugated fragments produced equal antithrombotic effects at equal plasma levels of anti-Factor Xa activity, but the specific antithrombotic activities of free and complexed intact heparin, on a molar basis, were 10-20-fold greater than those of the free and complexed heparin fragments. The plasma half-life of the covalent complexes of the heparin fragments with AT is, however, 10 times longer than that of the complex between intact heparin and AT and 30 times longer than that of free intact heparin. Covalent complexes between AT and heparin fragments could, therefore, be useful to maintain more stable levels of antithrombotic activity in plasma.

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Selected References

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  1. Andersson L. O., Barrowcliffe T. W., Holmer E., Johnson E. A., Sims G. E. Anticoagulant properties of heparin fractionated by affinity chromatography on matrix-bound antithrombin iii and by gel filtration. Thromb Res. 1976 Dec;9(6):575–583. doi: 10.1016/0049-3848(76)90105-5. [DOI] [PubMed] [Google Scholar]
  2. Andersson L. O., Barrowcliffe T. W., Holmer E., Johnson E. A., Söderström G. Molecular weight dependency of the heparin potentiated inhibition of thrombin and activated factor X. Effect of heparin neutralization in plasma. Thromb Res. 1979;15(3-4):531–541. doi: 10.1016/0049-3848(79)90159-2. [DOI] [PubMed] [Google Scholar]
  3. BITTER T., MUIR H. M. A modified uronic acid carbazole reaction. Anal Biochem. 1962 Oct;4:330–334. doi: 10.1016/0003-2697(62)90095-7. [DOI] [PubMed] [Google Scholar]
  4. Barrowcliffe T. W., Merton R. E., Havercroft S. J., Thunberg L., Lindahl U., Thomas D. P. Low-affinity heparin potentiates the action of high-affinity heparin oligosaccharides. Thromb Res. 1984 Apr 15;34(2):125–133. doi: 10.1016/0049-3848(84)90069-0. [DOI] [PubMed] [Google Scholar]
  5. Beeler D., Rosenberg R., Jordan R. Fractionation of low molecular weight heparin species and their interaction with antithrombin. J Biol Chem. 1979 Apr 25;254(8):2902–2913. [PubMed] [Google Scholar]
  6. Björk I., Nordenman B. Acceleration of the reaction between thrombin and antithrombin III by non-stoichiometric amounts of heparin. Eur J Biochem. 1976 Sep 15;68(2):507–511. doi: 10.1111/j.1432-1033.1976.tb10838.x. [DOI] [PubMed] [Google Scholar]
  7. Carter C. J., Kelton J. G., Hirsh J., Cerskus A., Santos A. V., Gent M. The relationship between the hemorrhagic and antithrombotic properties of low molecular weight heparin in rabbits. Blood. 1982 Jun;59(6):1239–1245. [PubMed] [Google Scholar]
  8. Ceustermans R., Hoylaerts M., De Mol M., Collen D. Preparation, characterization, and turnover properties of heparin-antithrombin III complexes stabilized by covalent bonds. J Biol Chem. 1982 Apr 10;257(7):3401–3408. [PubMed] [Google Scholar]
  9. Choay J., Lormeau J. C., Petitou M., Sinay P., Casu B., Oreste P., Torri G., Gatti G. Anti-Xa active heparin oligosaccharides. Thromb Res. 1980 May 1;18(3-4):573–578. doi: 10.1016/0049-3848(80)90356-4. [DOI] [PubMed] [Google Scholar]
  10. Ehrlich J., Stivala S. S. Chemistry and pharmacology of heparin. J Pharm Sci. 1973 Apr;62(4):517–544. doi: 10.1002/jps.2600620402. [DOI] [PubMed] [Google Scholar]
  11. Holmer E. Anticoagulant properties of heparin and heparin fractions. Scand J Haematol Suppl. 1980;36:25–39. doi: 10.1111/j.1600-0609.1980.tb02511.x. [DOI] [PubMed] [Google Scholar]
  12. Holmer E., Kurachi K., Söderström G. The molecular-weight dependence of the rate-enhancing effect of heparin on the inhibition of thrombin, factor Xa, factor IXa, factor XIa, factor XIIa and kallikrein by antithrombin. Biochem J. 1981 Feb 1;193(2):395–400. doi: 10.1042/bj1930395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Holmer E., Mattsson C., Nilsson S. Anticoagulant and antithrombotic effects of heparin and low molecular weight heparin fragments in rabbits. Thromb Res. 1982 Mar 15;25(6):475–485. doi: 10.1016/0049-3848(82)90089-5. [DOI] [PubMed] [Google Scholar]
  14. Hoylaerts M., Holmer E., de Mol M., Collen D. Covalent complexes between low molecular weight heparin fragments and antithrombin III - inhibition kinetics and turnover parameters. Thromb Haemost. 1983 Apr 28;49(2):109–115. [PubMed] [Google Scholar]
  15. Hoylaerts M., Owen W. G., Collen D. Involvement of heparin chain length in the heparin-catalyzed inhibition of thrombin by antithrombin III. J Biol Chem. 1984 May 10;259(9):5670–5677. [PubMed] [Google Scholar]
  16. Jordan R. E., Oosta G. M., Gardner W. T., Rosenberg R. D. The kinetics of hemostatic enzyme-antithrombin interactions in the presence of low molecular weight heparin. J Biol Chem. 1980 Nov 10;255(21):10081–10090. [PubMed] [Google Scholar]
  17. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  18. Lijnen H. R., 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 Mar 25;258(6):3803–3808. [PubMed] [Google Scholar]
  19. Lindahl U., Bäckström G., Hök M., Thunberg L., Fransson L. A., Linker A. Structure of the antithrombin-binding site in heparin. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3198–3202. doi: 10.1073/pnas.76.7.3198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lindahl U., Bäckström G., Thunberg L. The antithrombin-binding sequence in heparin. Identification of an essential 6-O-sulfate group. J Biol Chem. 1983 Aug 25;258(16):9826–9830. [PubMed] [Google Scholar]
  21. Ockelford P. A., Carter C. J., Mitchell L., Hirsh J. Discordance between the anti-Xa activity and the antithrombotic activity in an ultra-low molecular weight heparin fraction. Thromb Res. 1982 Nov 1;28(3):401–409. doi: 10.1016/0049-3848(82)90121-9. [DOI] [PubMed] [Google Scholar]
  22. Teien A. N., Lie M., Abildgaard U. Assay of heparin in plasma using a chromogenic substrate for activated factor X. Thromb Res. 1976 Mar;8(3):413–416. doi: 10.1016/0049-3848(76)90034-7. [DOI] [PubMed] [Google Scholar]
  23. Teien A. N., Lie M. Evaluation of an amidolytic heparin assay method: increased sensitivity by adding purified antithrombin III. Thromb Res. 1977 Mar;10(3):399–410. doi: 10.1016/0049-3848(77)90150-5. [DOI] [PubMed] [Google Scholar]
  24. Thomas D. P., Barrowcliffe T. W., Johnson E. A. The influence of tissue source, salt and molecular weight and heparin activity. Scand J Haematol Suppl. 1980;36:40–49. doi: 10.1111/j.1600-0609.1980.tb02512.x. [DOI] [PubMed] [Google Scholar]
  25. Thomas D. P., Merton R. E., Barrowcliffe T. W., Thunberg L., Lindahl U. Effects of heparin oligosaccharides with high affinity for antithrombin III in experimental venous thrombosis. Thromb Haemost. 1982 Jun 28;47(3):244–248. [PubMed] [Google Scholar]
  26. Thunberg L., Lindahl U., Tengblad A., Laurent T. C., Jackson C. M. On the molecular-weight-dependence of the anticoagulant activity of heparin. Biochem J. 1979 Jul 1;181(1):241–243. doi: 10.1042/bj1810241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. WESSLER S., REIMER S. M., SHEPS M. C. Biologic assay of a thrombosis-inducing activity in human serum. J Appl Physiol. 1959 Nov;14:943–946. doi: 10.1152/jappl.1959.14.6.943. [DOI] [PubMed] [Google Scholar]

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