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. 1990 Sep 1;270(2):419–425. doi: 10.1042/bj2700419

Functional role of the polysaccharide component of rabbit thrombomodulin proteoglycan. Effects on inactivation of thrombin by antithrombin, cleavage of fibrinogen by thrombin and thrombin-catalysed activation of factor V.

M C Bourin 1, U Lindahl 1
PMCID: PMC1131739  PMID: 2169242

Abstract

Thrombomodulin (TM), a major anticoagulant protein at the vessel wall, serves as a potent cofactor for the activation of Protein C by thrombin. Previous work has indicated that (rabbit) TM is a proteoglycan that contains a single polysaccharide chain, tentatively identified as a sulphated galactosaminoglycan, and furthermore suggested that this component may be functionally related to additional anticoagulant activities expressed by the TM molecule [Bourin, Ohlin, Lane, Stenflo & Lindahl (1988) J. Biol. Chem. 263, 8044-8052]. Results of the present study establish that (enzymic) removal of the polysaccharide chain abolishes the inhibitory effect of TM on thrombin-induced fibrinogen clotting as well as the promoting effect of TM on the inactivation of thrombin by antithrombin, but does not affect the ability of TM to serve as a cofactor in the activation of Protein C. Studies of yet another biological activity of rabbit TM, namely the ability to prevent the activation of Factor V by thrombin [Esmon, Esmon & Harris (1982) J. Biol. Chem. 257, 7944-7947], confirmed that TM markedly delays the conversion of the native 330 kDa Factor V precursor into polypeptide intermediates, and further into the 96 kDa heavy chain and 71-74 kDa light-chain components of activated Factor Va. In contrast, the activation kinetics of a similar sample of Factor V incubated with thrombin in the presence of chondroitinase ABC-digested TM did not differ from that observed in the absence of TM. It is concluded that the inhibitory effect of TM on Factor V activation also depends on the presence of the polysaccharide component on the TM molecule.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Blobel G., Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 1975 Dec;67(3):835–851. doi: 10.1083/jcb.67.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bourin M. C., Boffa M. C., Björk I., Lindahl U. Functional domains of rabbit thrombomodulin. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5924–5928. doi: 10.1073/pnas.83.16.5924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bourin M. C. Effect of rabbit thrombomodulin on thrombin inhibition by antithrombin in the presence of heparin. Thromb Res. 1989 Apr 1;54(1):27–39. doi: 10.1016/0049-3848(89)90334-4. [DOI] [PubMed] [Google Scholar]
  4. Bourin M. C., Ohlin A. K., Lane D. A., Stenflo J., Lindahl U. Relationship between anticoagulant activities and polyanionic properties of rabbit thrombomodulin. J Biol Chem. 1988 Jun 15;263(17):8044–8052. [PubMed] [Google Scholar]
  5. Dahlbäck B. Human coagluation factor V purification and thrombin-catalyzed activation. J Clin Invest. 1980 Sep;66(3):583–591. doi: 10.1172/JCI109890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Esmon C. T., Esmon N. L., Harris K. W. Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation. J Biol Chem. 1982 Jul 25;257(14):7944–7947. [PubMed] [Google Scholar]
  7. Esmon C. T. The roles of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem. 1989 Mar 25;264(9):4743–4746. [PubMed] [Google Scholar]
  8. Esmon N. L., Carroll R. C., Esmon C. T. Thrombomodulin blocks the ability of thrombin to activate platelets. J Biol Chem. 1983 Oct 25;258(20):12238–12242. [PubMed] [Google Scholar]
  9. Esmon N. L., Owen W. G., Esmon C. T. Isolation of a membrane-bound cofactor for thrombin-catalyzed activation of protein C. J Biol Chem. 1982 Jan 25;257(2):859–864. [PubMed] [Google Scholar]
  10. Hofsteenge J., Taguchi H., Stone S. R. Effect of thrombomodulin on the kinetics of the interaction of thrombin with substrates and inhibitors. Biochem J. 1986 Jul 1;237(1):243–251. doi: 10.1042/bj2370243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jakubowski H. V., Kline M. D., Owen W. G. The effect of bovine thrombomodulin on the specificity of bovine thrombin. J Biol Chem. 1986 Mar 15;261(8):3876–3882. [PubMed] [Google Scholar]
  12. Kurosawa S., Aoki N. Preparation of thrombomodulin from human placenta. Thromb Res. 1985 Feb 1;37(3):353–364. doi: 10.1016/0049-3848(85)90064-7. [DOI] [PubMed] [Google Scholar]
  13. Kurosawa S., Stearns D. J., Jackson K. W., Esmon C. T. A 10-kDa cyanogen bromide fragment from the epidermal growth factor homology domain of rabbit thrombomodulin contains the primary thrombin binding site. J Biol Chem. 1988 May 5;263(13):5993–5996. [PubMed] [Google Scholar]
  14. Lindahl U., Thunberg L., Bäckström G., Riesenfeld J., Nordling K., Björk I. Extension and structural variability of the antithrombin-binding sequence in heparin. J Biol Chem. 1984 Oct 25;259(20):12368–12376. [PubMed] [Google Scholar]
  15. Lu R. L., Esmon N. L., Esmon C. T., Johnson A. E. The active site of the thrombin-thrombomodulin complex. A fluorescence energy transfer measurement of its distance above the membrane surface. J Biol Chem. 1989 Aug 5;264(22):12956–12962. [PubMed] [Google Scholar]
  16. Lundblad R. L., Uhteg L. C., Vogel C. N., Kingdon H. S., Mann K. G. Preparation and partial characterization of two forms of bovine thrombin. Biochem Biophys Res Commun. 1975 Sep 16;66(2):482–489. doi: 10.1016/0006-291x(75)90536-7. [DOI] [PubMed] [Google Scholar]
  17. Marcum J. A., Atha D. H., Fritze L. M., Nawroth P., Stern D., Rosenberg R. D. Cloned bovine aortic endothelial cells synthesize anticoagulantly active heparan sulfate proteoglycan. J Biol Chem. 1986 Jun 5;261(16):7507–7517. [PubMed] [Google Scholar]
  18. Maruyama I., Salem H. H., Ishii H., Majerus P. W. Human thrombomodulin is not an efficient inhibitor of the procoagulant activity of thrombin. J Clin Invest. 1985 Mar;75(3):987–991. doi: 10.1172/JCI111800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nesheim M. E., Mann K. G. Thrombin-catalyzed activation of single chain bovine factor V. J Biol Chem. 1979 Feb 25;254(4):1326–1334. [PubMed] [Google Scholar]
  20. Owen W. G., Esmon C. T. Functional properties of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. J Biol Chem. 1981 Jun 10;256(11):5532–5535. [PubMed] [Google Scholar]
  21. Preissner K. T., Delvos U., Müller-Berghaus G. Binding of thrombin to thrombomodulin accelerates inhibition of the enzyme by antithrombin III. Evidence for a heparin-independent mechanism. Biochemistry. 1987 May 5;26(9):2521–2528. doi: 10.1021/bi00383a018. [DOI] [PubMed] [Google Scholar]
  22. Rodgers G. M., Shuman M. A. Prothrombin is activated on vascular endothelial cells by factor Xa and calcium. Proc Natl Acad Sci U S A. 1983 Nov;80(22):7001–7005. doi: 10.1073/pnas.80.22.7001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Salem H. H., Maruyama I., Ishii H., Majerus P. W. Isolation and characterization of thrombomodulin from human placenta. J Biol Chem. 1984 Oct 10;259(19):12246–12251. [PubMed] [Google Scholar]
  24. Stearns D. J., Kurosawa S., Esmon C. T. Microthrombomodulin. Residues 310-486 from the epidermal growth factor precursor homology domain of thrombomodulin will accelerate protein C activation. J Biol Chem. 1989 Feb 25;264(6):3352–3356. [PubMed] [Google Scholar]
  25. Stenflo J. A new vitamin K-dependent protein. Purification from bovine plasma and preliminary characterization. J Biol Chem. 1976 Jan 25;251(2):355–363. [PubMed] [Google Scholar]
  26. Suzuki K., Kusumoto H., Deyashiki Y., Nishioka J., Maruyama I., Zushi M., Kawahara S., Honda G., Yamamoto S., Horiguchi S. Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation. EMBO J. 1987 Jul;6(7):1891–1897. doi: 10.1002/j.1460-2075.1987.tb02448.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Suzuki K., Kusumoto H., Hashimoto S. Isolation and characterization of thrombomodulin from bovine lung. Biochim Biophys Acta. 1986 Jul 16;882(3):343–352. doi: 10.1016/0304-4165(86)90257-6. [DOI] [PubMed] [Google Scholar]
  28. Suzuki K., Nishioka J., Hayashi T., Kosaka Y. Functionally active thrombomodulin is present in human platelets. J Biochem. 1988 Oct;104(4):628–632. doi: 10.1093/oxfordjournals.jbchem.a122523. [DOI] [PubMed] [Google Scholar]
  29. Tracy P. B., Nesheim M. E., Mann K. G. Coordinate binding of factor Va and factor Xa to the unstimulated platelet. J Biol Chem. 1981 Jan 25;256(2):743–751. [PubMed] [Google Scholar]
  30. Walker F. J., Sexton P. W., Esmon C. T. The inhibition of blood coagulation by activated Protein C through the selective inactivation of activated Factor V. Biochim Biophys Acta. 1979 Dec 7;571(2):333–342. doi: 10.1016/0005-2744(79)90103-7. [DOI] [PubMed] [Google Scholar]
  31. Wen D. Z., Dittman W. A., Ye R. D., Deaven L. L., Majerus P. W., Sadler J. E. Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene. Biochemistry. 1987 Jul 14;26(14):4350–4357. doi: 10.1021/bi00388a025. [DOI] [PubMed] [Google Scholar]

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