Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1998 May 1;101(9):1983–1991. doi: 10.1172/JCI2006

A targeted point mutation in thrombomodulin generates viable mice with a prethrombotic state.

H Weiler-Guettler 1, P D Christie 1, D L Beeler 1, A M Healy 1, W W Hancock 1, H Rayburn 1, J M Edelberg 1, R D Rosenberg 1
PMCID: PMC508785  PMID: 9576763

Abstract

The activity of the coagulation system is regulated, in part, by the interaction of thrombin with the endothelial cell receptor thrombomodulin with subsequent generation of activated protein C and suppression of thrombin production. Our previous investigation demonstrated that ablation of the thrombomodulin gene in mice causes embryonic lethality before the assembly of a functional cardiovascular system, indicating a critical role for the receptor in early development. In the current study, we show that a single amino acid substitution in thrombomodulin dissociates the developmental function of the receptor from its role as a regulator of blood coagulation. Homozygous mutant mice with severely reduced capacity to generate activated protein C or inhibit thrombin develop to term, and possess normal reproductive performance. The above animals exhibit increased fibrin deposition in selected organs, which implies tissue specific regulation of the coagulation system that is supported by further evidence from the examination of mice with defects in fibrinolysis. The thrombomodulin-deficient animals provide a murine model to examine known or identify unknown genetic and environmental factors that lead to the development of thrombosis.

Full Text

The Full Text of this article is available as a PDF (369.1 KB).

Selected References

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

  1. Bertina R. M., Koeleman B. P., Koster T., Rosendaal F. R., Dirven R. J., de Ronde H., van der Velden P. A., Reitsma P. H. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature. 1994 May 5;369(6475):64–67. doi: 10.1038/369064a0. [DOI] [PubMed] [Google Scholar]
  2. Bugge T. H., Kombrinck K. W., Flick M. J., Daugherty C. C., Danton M. J., Degen J. L. Loss of fibrinogen rescues mice from the pleiotropic effects of plasminogen deficiency. Cell. 1996 Nov 15;87(4):709–719. doi: 10.1016/s0092-8674(00)81390-2. [DOI] [PubMed] [Google Scholar]
  3. Bugge T. H., Xiao Q., Kombrinck K. W., Flick M. J., Holmbäck K., Danton M. J., Colbert M. C., Witte D. P., Fujikawa K., Davie E. W. Fatal embryonic bleeding events in mice lacking tissue factor, the cell-associated initiator of blood coagulation. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6258–6263. doi: 10.1073/pnas.93.13.6258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carmeliet P., Mackman N., Moons L., Luther T., Gressens P., Van Vlaenderen I., Demunck H., Kasper M., Breier G., Evrard P. Role of tissue factor in embryonic blood vessel development. Nature. 1996 Sep 5;383(6595):73–75. doi: 10.1038/383073a0. [DOI] [PubMed] [Google Scholar]
  5. Carmeliet P., Schoonjans L., Kieckens L., Ream B., Degen J., Bronson R., De Vos R., van den Oord J. J., Collen D., Mulligan R. C. Physiological consequences of loss of plasminogen activator gene function in mice. Nature. 1994 Mar 31;368(6470):419–424. doi: 10.1038/368419a0. [DOI] [PubMed] [Google Scholar]
  6. Clarke J. H., Light D. R., Blasko E., Parkinson J. F., Nagashima M., McLean K., Vilander L., Andrews W. H., Morser J., Glaser C. B. The short loop between epidermal growth factor-like domains 4 and 5 is critical for human thrombomodulin function. J Biol Chem. 1993 Mar 25;268(9):6309–6315. [PubMed] [Google Scholar]
  7. Conard J., Bauer K. A., Gruber A., Griffin J. H., Schwarz H. P., Horellou M. H., Samama M. M., Rosenberg R. D. Normalization of markers of coagulation activation with a purified protein C concentrate in adults with homozygous protein C deficiency. Blood. 1993 Aug 15;82(4):1159–1164. [PubMed] [Google Scholar]
  8. Cross J. C., Werb Z., Fisher S. J. Implantation and the placenta: key pieces of the development puzzle. Science. 1994 Dec 2;266(5190):1508–1518. doi: 10.1126/science.7985020. [DOI] [PubMed] [Google Scholar]
  9. Cui J., O'Shea K. S., Purkayastha A., Saunders T. L., Ginsburg D. Fatal haemorrhage and incomplete block to embryogenesis in mice lacking coagulation factor V. Nature. 1996 Nov 7;384(6604):66–68. doi: 10.1038/384066a0. [DOI] [PubMed] [Google Scholar]
  10. Dahlbäck B. The protein C anticoagulant system: inherited defects as basis for venous thrombosis. Thromb Res. 1995 Jan 1;77(1):1–43. doi: 10.1016/0049-3848(94)00138-4. [DOI] [PubMed] [Google Scholar]
  11. Dittman W. A., Majerus P. W. Structure and function of thrombomodulin: a natural anticoagulant. Blood. 1990 Jan 15;75(2):329–336. [PubMed] [Google Scholar]
  12. Dvorak H. F., Form D. M., Manseau E. J., Smith B. D. Pathogenesis of desmoplasia. I. Immunofluorescence identification and localization of some structural proteins of line 1 and line 10 guinea pig tumors and of healing wounds. J Natl Cancer Inst. 1984 Nov;73(5):1195–1205. [PubMed] [Google Scholar]
  13. 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]
  14. Esmon C. T., Owen W. G. Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2249–2252. doi: 10.1073/pnas.78.4.2249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Faioni E. M., Merati G., Peyvandi F., Bettini P. M., Mannucci P. M. The G1456 to T mutation in the thrombomodulin gene is not frequent in patients with venous thrombosis. Blood. 1997 Feb 15;89(4):1467–1467. [PubMed] [Google Scholar]
  18. Ford V. A., Stringer C., Kennel S. J. Thrombomodulin is preferentially expressed in Balb/c lung microvessels. J Biol Chem. 1992 Mar 15;267(8):5446–5450. [PubMed] [Google Scholar]
  19. Healy A. M., Rayburn H. B., Rosenberg R. D., Weiler H. Absence of the blood-clotting regulator thrombomodulin causes embryonic lethality in mice before development of a functional cardiovascular system. Proc Natl Acad Sci U S A. 1995 Jan 31;92(3):850–854. doi: 10.1073/pnas.92.3.850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hui K. Y., Haber E., Matsueda G. R. Monoclonal antibodies to a synthetic fibrin-like peptide bind to human fibrin but not fibrinogen. Science. 1983 Dec 9;222(4628):1129–1132. doi: 10.1126/science.6648524. [DOI] [PubMed] [Google Scholar]
  21. Kennel S. J., Lankford T., Hughes B., Hotchkiss J. A. Quantitation of a murine lung endothelial cell protein, P112, with a double monoclonal antibody assay. Lab Invest. 1988 Nov;59(5):692–701. [PubMed] [Google Scholar]
  22. Lane D. A., Mannucci P. M., Bauer K. A., Bertina R. M., Bochkov N. P., Boulyjenkov V., Chandy M., Dahlbäck B., Ginter E. K., Miletich J. P. Inherited thrombophilia: Part 1. Thromb Haemost. 1996 Nov;76(5):651–662. [PubMed] [Google Scholar]
  23. Lawson C. A., Yan S. D., Yan S. F., Liao H., Zhou Y. S., Sobel J., Kisiel W., Stern D. M., Pinsky D. J. Monocytes and tissue factor promote thrombosis in a murine model of oxygen deprivation. J Clin Invest. 1997 Apr 1;99(7):1729–1738. doi: 10.1172/JCI119337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lodge P. A., Haisch C. E., Thomas F. T. A simple method of vascular endothelial cell isolation. Transplant Proc. 1992 Dec;24(6):2816–2817. [PubMed] [Google Scholar]
  25. Miletich J. P., Prescott S. M., White R., Majerus P. W., Bovill E. G. Inherited predisposition to thrombosis. Cell. 1993 Feb 26;72(4):477–480. doi: 10.1016/0092-8674(93)90063-v. [DOI] [PubMed] [Google Scholar]
  26. Norlund L., Holm J., Zöller B., Ohlin A. K. A common thrombomodulin amino acid dimorphism is associated with myocardial infarction. Thromb Haemost. 1997 Feb;77(2):248–251. [PubMed] [Google Scholar]
  27. Ohlin A. K., Marlar R. A. The first mutation identified in the thrombomodulin gene in a 45-year-old man presenting with thromboembolic disease. Blood. 1995 Jan 15;85(2):330–336. [PubMed] [Google Scholar]
  28. Orthner C. L., Kolen B., Drohan W. N. A sensitive and facile assay for the measurement of activated protein C activity levels in vivo. Thromb Haemost. 1993 May 3;69(5):441–447. [PubMed] [Google Scholar]
  29. Presky D. H., Wilkinson V. L., Flannery M. D., Korkmaz E., Walsky R., Mondini-Minetti L. J., Fotouhi N., Levin W. Production of monoclonal antibodies to prostromelysin (ProMMP-3) and establishment of a quantitative prostromelysin ELISA assay. Biochem Biophys Res Commun. 1993 May 28;193(1):364–370. doi: 10.1006/bbrc.1993.1632. [DOI] [PubMed] [Google Scholar]
  30. Sappino A. P., Huarte J., Belin D., Vassalli J. D. Plasminogen activators in tissue remodeling and invasion: mRNA localization in mouse ovaries and implanting embryos. J Cell Biol. 1989 Nov;109(5):2471–2479. doi: 10.1083/jcb.109.5.2471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stearns-Kurosawa D. J., Kurosawa S., Mollica J. S., Ferrell G. L., Esmon C. T. The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex. Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10212–10216. doi: 10.1073/pnas.93.19.10212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Strickland S., Richards W. G. Invasion of the trophoblasts. Cell. 1992 Oct 30;71(3):355–357. doi: 10.1016/0092-8674(92)90503-5. [DOI] [PubMed] [Google Scholar]
  33. Suh T. T., Holmbäck K., Jensen N. J., Daugherty C. C., Small K., Simon D. I., Potter S., Degen J. L. Resolution of spontaneous bleeding events but failure of pregnancy in fibrinogen-deficient mice. Genes Dev. 1995 Aug 15;9(16):2020–2033. doi: 10.1101/gad.9.16.2020. [DOI] [PubMed] [Google Scholar]
  34. Swank R. T., Munkres K. D. Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Anal Biochem. 1971 Feb;39(2):462–477. doi: 10.1016/0003-2697(71)90436-2. [DOI] [PubMed] [Google Scholar]
  35. Toomey J. R., Kratzer K. E., Lasky N. M., Stanton J. J., Broze G. J., Jr Targeted disruption of the murine tissue factor gene results in embryonic lethality. Blood. 1996 Sep 1;88(5):1583–1587. [PubMed] [Google Scholar]
  36. Weiler-Guettler H., Aird W. C., Husain M., Rayburn H., Rosenberg R. D. Targeting of transgene expression to the vascular endothelium of mice by homologous recombination at the thrombomodulin locus. Circ Res. 1996 Feb;78(2):180–187. doi: 10.1161/01.res.78.2.180. [DOI] [PubMed] [Google Scholar]
  37. Weiler-Guettler H., Aird W. C., Rayburn H., Husain M., Rosenberg R. D. Developmentally regulated gene expression of thrombomodulin in postimplantation mouse embryos. Development. 1996 Jul;122(7):2271–2281. doi: 10.1242/dev.122.7.2271. [DOI] [PubMed] [Google Scholar]
  38. Zhang Y., Weiler-Guettler H., Chen J., Wilhelm O., Deng Y., Qiu F., Nakagawa K., Klevesath M., Wilhelm S., Böhrer H. Thrombomodulin modulates growth of tumor cells independent of its anticoagulant activity. J Clin Invest. 1998 Apr 1;101(7):1301–1309. doi: 10.1172/JCI925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zöller B., Hillarp A., Berntorp E., Dahlbäck B. Activated protein C resistance due to a common factor V gene mutation is a major risk factor for venous thrombosis. Annu Rev Med. 1997;48:45–58. doi: 10.1146/annurev.med.48.1.45. [DOI] [PubMed] [Google Scholar]
  40. van der Velden P. A., Krommenhoek-Van Es T., Allaart C. F., Bertina R. M., Reitsma P. H. A frequent thrombomodulin amino acid dimorphism is not associated with thrombophilia. Thromb Haemost. 1991 May 6;65(5):511–513. [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES