Download PDF
Thromb Haemost 2000; 84(05): 904-911
DOI: 10.1055/s-0037-1614135
Review Article
Schattauer GmbH

In situ Analysis of Tissue Factor-Dependent Thrombin Generation in Human Atherosclerotic Vessels

Andrew D. Westmuckett
1   From the Vascular Biology Laboratory, Thrombosis Research Institute, Emmanuel Kaye Building, Chelsea, London, UK
,
Cristina Lupu
1   From the Vascular Biology Laboratory, Thrombosis Research Institute, Emmanuel Kaye Building, Chelsea, London, UK
,
David A. Goulding
1   From the Vascular Biology Laboratory, Thrombosis Research Institute, Emmanuel Kaye Building, Chelsea, London, UK
,
Saroj Das
1   From the Vascular Biology Laboratory, Thrombosis Research Institute, Emmanuel Kaye Building, Chelsea, London, UK
,
Vijay V. Kakkar
1   From the Vascular Biology Laboratory, Thrombosis Research Institute, Emmanuel Kaye Building, Chelsea, London, UK
,
Florea Lupu
1   From the Vascular Biology Laboratory, Thrombosis Research Institute, Emmanuel Kaye Building, Chelsea, London, UK
› Author AffiliationsThis work was supported by stipends from the British Heart Foundation (grants PG/95927 and PG/97045, CL), Leopold Muller Estate Trust (FL), Stanley Thomas Johnson Foundation (FL), The Royal Society (FL and CL), and the Thrombosis Research Trust. We thank Dr. David O’Reagan for providing us some of the human vascular tissue segments used in this study and to Prof. Nicholas Severs for the critical reading of the manuscript.
Further Information

Publication History

Received 24 January 2000

Accepted after resubmission 30 May 2000

Publication Date:
13 December 2017 (online)

Also available at
    Permissions and Reprints

Summary

Tissue factor (TF) is expressed in human atherosclerotic plaques where it may contribute to the thrombogenicity of the lesions and their progression toward unstable syndromes and acute myocardial infarction. In this study we tested the hypothesis that thrombin generation takes place in the lesion. Localisation of TF, factor VII (FVII), factor X/Xa (FX/Xa), thrombin, thrombin receptor PAR-1 and FXa receptor EPR-1 was done by immunostaining, ligand binding, or immunogold electron microscopy. Quantitation of TF antigen was done using a modified ELISA on fixed tissue sections. The amount of antigen was correlated with the pattern and intensity of immunostaining as detected on consecutive sections using confocal microscopy. TFdependent generation of FXa on cryosections was used to assess the functional activity of TF. Active thrombin was detected using hirudin as a specific probe, followed by anti-hirudin IgG. Our light microscopy and immunogold electron microscopy results showed that the factors involved in TF-dependent coagulation are localised in atherosclerotic plaques in close proximity and colocalise with active thrombin and fibrin deposits. We have detected 3 to 7-fold increase of TF antigen and TF-dependent FXa generation in atherosclerotic vessels as compared with controls. Hirudin binding proved that active thrombin is present within the lesions. In conclusion, our data show that active coagulation factors are generated within atherosclerotic lesions and co-localise with their cellular receptors. These findings may suggest possible roles of the TF-dependent coagulation pathway in the intramural fibrin deposition and the progression of the atherosclerotic lesions.

Key words

Atherosclerosis - coagulation - immunogold - tissue factor - thrombin receptor - factor Xa receptor
 

  • References

  • 1 Fuster V, Lewis A. Conner Memorial Lecture. Mechanisms leading to myocardial infarction: insights from studies of vascular biology. Circulation 1994; 90: 2126-46.
    CrossrefPubMedGoogle Scholar
  • 2 Davies MJ, Thomas AC. Plaque fissuring – the cause of acute myocardial infarction, sudden ischaemic death, and crescendo angina. Br Heart J 1988; 53: 363-73.
    PubMedGoogle Scholar
  • 3 Constantinides P. The morphological basis for altered endothelial permeability in atherosclerosis. Adv Exp Med Biol 1977; 82: 969-74.
    PubMedGoogle Scholar
  • 4 Mora R, Lupu F, Simionescu N. Cytochemical localization of b lipoproteins and their components in succesive stages of hyperlipidemic atherogenesis in rabbit aorta. Atherosclerosis 1989; 79: 183-5.
    CrossrefPubMedGoogle Scholar
  • 5 Bell FP, Adamson IL, Schwartz CJ. Aortic endothelial permeability to albumin: focal and regional patterns of uptake and transmural distribution of 131I-albumin in the young pig. Exp Mol Pathol 1974; 20: 57-68.
    CrossrefPubMedGoogle Scholar
  • 6 Hansson GK, Bondjers G, Bylock A, Hjalmarsson L. Ultrastructural studies on the localization of IgG in the aortic endothelium and subendothelial intima of atherosclerotic and nonatherosclerotic rabbits. Exp Mol Pathol 1980; 33: 302-15.
    CrossrefPubMedGoogle Scholar
  • 7 McBane RD, Miller RS, Hassinger NL, Chesebro JH, Nemerson Y, Owen WG. Tissue prothrombin. Universal distribution in smooth muscle. Arterioscler Thromb Vasc Biol 1997; 17: 2430-6.
    CrossrefPubMedGoogle Scholar
  • 8 Smith EB, Crosbie L, Carey S. Prothrombin-related antigens in human aortic intima. Semin Thromb Hemost 1996; 22: 347-50.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 9 Bini A, Fenoglio JJ, Mesa-Tejada R, Kudryk BJ, Kaplan KL. Identification and distribution of fibrinogen, fibrin and fibrin(ogen) degradation products in atherosclerosis: use of monoclonal antibodies. Arteriosclerosis 1989; 09: 109-21.
    CrossrefPubMedGoogle Scholar
  • 10 Wilcox JN, Smith KM, Schwartz SM, Gordon D. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl Acad Sci USA 1989; 86: 2839-43.
    CrossrefPubMedGoogle Scholar
  • 11 Crawley J, Lupu F, Westmuckett AD, Severs NJ, Kakkar VV, Lupu C. Expression, localization, and activity of tissue factor pathway inhibitor in normal and atherosclerotic human vessels. Arterioscler Thromb Vasc Biol 2000; 20: 1362-73.
    CrossrefPubMedGoogle Scholar
  • 12 Fernandez-Ortiz A, Badimon JJ, Falk E, Fuster V, Meyer B, Mailhac A, Weng D, Shah PK, Badimon L. Characterization of the relative thrombogenicity of atherosclerotic plaque components: implications for consequences of plaque rupture. J Am Coll Cardiol 1994; 23: 1562-9.
    CrossrefPubMedGoogle Scholar
  • 13 Mann KG. Biochemistry and physiology of blood coagulation. Thromb Haemost 1999; 82: 165-74.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 14 Marmur JD, Thiruvikraman SV, Fyfe BS, Guha A, Sharma SK, Ambrose JA, Fallon JT, Nemerson Y, Taubman MB. Identification of active tissue factor in human coronary atheroma. Circulation 1996; 94: 1226-32.
    CrossrefPubMedGoogle Scholar
  • 15 Merlini PA, Bauer KA, Oltrona L, Arddissino D, Cattaneo M, Belli C, Mannucci PM, Rosenberg RD. Persistent activation of coaguation mechanism in unstable angina and myocardial infarction. Circulation 1994; 90: 61-8.
    CrossrefPubMedGoogle Scholar
  • 16 Stary HC, Chandler AB, Dinsmore RE, Fuster V, Glagov S, Insull W, Rosenfeld ME, Schwartz CJ, Wagner WD, Wissler RW. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Arterioscler Thromb Vasc Biol 1995; 15: 1512-31.
    CrossrefPubMedGoogle Scholar
  • 17 Lupu F, Bergonzelli GE, Heim DA, Cousin E, Genton CY, Bachmann F, Kruithof EKO. Localization and production of plasminogen activator inhibitor-1 in human healthy and atherosclerotic arteries. Arterioscler Thromb 1993; 13: 1090-100.
    CrossrefPubMedGoogle Scholar
  • 18 Lupu C, Lupu F, Dennehy U, Kakkar VV, Scully MF. Thrombin induces the redistribution and acute release of tissue factor pathway inhibitor from specific granules within human endothelial cells in culture. Arterioscler Thromb Vasc Biol 1995; 15: 2055-62.
    CrossrefPubMedGoogle Scholar
  • 19 Thiruvikraman SV, Guha A, Roboz J, Taubman MB, Nemerson Y, Fallon JT. In situ localization of tissue factor in human atherosclerotic plaques by binding of digoxigenin-labeled factors VIIa and X. Lab Invest 1996; 75: 451-61.
    PubMedGoogle Scholar
  • 20 Zacharski LR, Memoli VA, Morain WD, Schlaeppi J-M, Rousseau SM. Cellular localization of enzymatically active thrombin in intact human tissues by hirudin binding. Thromb Haemost 1995; 73: 793-7.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 21 Lupu C, Poulsen E, Roquefeuil S, Westmuckett AD, Kakkar VV, Lupu F. Cellular effects of heparin on the production and release of tissue factor pathway inhibitor in human endothelial cells in culture. Arterioscler Thromb Vasc Biol 1999; 19: 2251-62.
    CrossrefPubMedGoogle Scholar
  • 22 Lupu C, Goodwin CA, Westmuckett AD, Emeis JJ, Scully MF, Kakkar VV, Lupu F. Tissue factor pathway inhibitor in endothelial cells colocalizes with glycolipid microdomains/caveolae: regulatory mechanism(s) of the anticoagulant properties of the endothelium. Arterioscler Thromb Vasc Biol 1997; 17: 2964-74.
    CrossrefPubMedGoogle Scholar
  • 23 Valenzuela R, Shainoff JR, DiBello PM, Urbanic DA, Anderson JM, Matsueda GR, Kudryk BJ. Immunoelectrophoretic and immunohistochemical characterization of fibrinogen derivatives in atherosclerotic aortic intimas and vascular prosthesis pseudointimas. Am J Pathol 1992; 141: 861-80.
    PubMedGoogle Scholar
  • 24 Zhang Y, Cliff WJ, Schoefl GI, Higgins G. Immunohistochemical study of the intimal microvessels in coronary atherosclerosis. Am J Pathol 1993; 143: 164-72.
    PubMedGoogle Scholar
  • 25 Annex BH, Denning SM, Channon KM, Sketch MHJ, Stack RS, Morrissey JH, Peters KG. Differential expression of tissue factor protein in directional atherectomy specimens from patients with stable and unstable coronary syndromes. Circulation 1995; 91: 619-22.
    CrossrefPubMedGoogle Scholar
  • 26 Altieri DC, Edgington TS. Identification of effector cell protease receptor-1: a leukocyte-distributed receptor for serine protease factor Xa. J Immunol 1990; 145: 246-53.
    PubMedGoogle Scholar
  • 27 Bono F, Herault C, Avril P, Schaeffer P, Herbert JM. Human umbilical vein endothelial cells express high affinity receptors for factor Xa. J Cell Physiol 1997; 172: 36-43.
    CrossrefPubMedGoogle Scholar
  • 28 Herbert J-M, Bono F, Herault J-P, Avril C, Dol F, Mares A-M, Schaeffer P. Effector protease receptor 1 mediates the mitogenic activity for vascular smooth muscle cells in vitro and in vivo. J Clin Invest 1998; 101: 993-1000.
    CrossrefPubMedGoogle Scholar
  • 29 Nicholson AC, Nachman RL, Altieri DC, Summers BD, Ruf W, Edgington TS, Hajjar DP. Effector cell protease receptor-1 is a vascular receptor for coagulation factor Xa. J Biol Chem 1996; 271: 28407-13.
    CrossrefPubMedGoogle Scholar
  • 30 Ehrlich HJ, Gebbink RK, Keijer L, Linders M, Preissner KT, Pannekoek H. Alteration of serpin specificity by a protein cofactor. Vitronectin endows plasminogen activator inhibitor 1 with thrombin inhibitory properties. J Biol Chem 1990; 265: 13029-35.
    PubMedGoogle Scholar
  • 31 Stoop AA, Lupu F, Pannekoek H. Co-localization of thrombin, plasminogen activator inhibitor 1 (PAI-1) and vitronectin in the atherosclerotic vessel wall: a potential regulatory mechanism of thrombin activity by PAI-1/vitronectin complexes. Arterioscler Thromb Vasc Biol 2000; 20: 1143-9.
    CrossrefPubMedGoogle Scholar
  • 32 Ko FN, Yang YC, Huang SC, Ou JT. Coagulation factor Xa stimulates platelet-derived growth factor release and mitogenesis in cultured vascular smooth muscle cells of rats. J Clin Invest 1996; 98: 1493-501.
    CrossrefPubMedGoogle Scholar
  • 33 Noda-Heiny H, Fujii S, Sobel BE. Induction of vascular smooth muscle cell expression of plasminogen activator inhibitor-1 by thrombin. Circ Res 1993; 72: 36-43.
    CrossrefPubMedGoogle Scholar
  • 34 Emeis JJ, van den Eijnden-Schrauwen Y, van den Hoogen CM, de Priester W, Westmuckett A, Lupu F. An endothelial storage granule for tissue-type plasminogen activator. J Cell Biol 1997; 139: 245-56.
    CrossrefPubMedGoogle Scholar
  • 35 Nelken NA, Soifer SJ, O’Keefe J, Vu TK, Charo IF, Coughlin SR. Thrombin receptor expression in normal and atherosclerotic human arteries. J Clin Invest 1992; 90: 4-21.
    PubMedGoogle Scholar
  • 36 McNamara CA, Sarembock IJ, Gimple LW, Fenton JW, Coughlin SR, Owens GK. Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor. J Clin Invest 1993; 91: 94-8.
    CrossrefPubMedGoogle Scholar
  • 37 Noda-Heiny H, Sobel BE. Vascular smooth muscle cell migration mediated by thrombin and urokinase receptor. Am J Physiol 1995; 268: 1195-201.
    CrossrefPubMedGoogle Scholar
  • 38 Bini A, Fenoglio JJ, Sobel J, Owen J, Fejgl M, Kaplan KL. Immunochemical characterization of fibrinogen, fibrin I, and fibrin II in human thrombi and atherosclerotic lesions. Blood 1987; 69: 1038-45.
    PubMedGoogle Scholar
  • 39 Yutani C, Imakita M, Ishibashi-Ueda H. Histopathological study of aortocoronary bypass grafts with special reference to fibrin deposits on grafted saphenous veins. Acta Pathol Jpn 1989; 39: 425-32.
    PubMedGoogle Scholar
  • 40 Lupu F, Heim DA, Bachmann F, Hurni M, Kakkar VV, Kruithof EKO. Plasminogen activator expression in human advanced atherosclerotic lesions. Arterioscler Thromb Vasc Biol 1995; 15: 1444-55.
    CrossrefPubMedGoogle Scholar
  • 41 Smith EB. Fibrin deposition and fibrin degradation products in atherosclerotic plaques. Thromb Res 1994; 75: 329-35.
    CrossrefPubMedGoogle Scholar
  • 42 Naito M, Funaki C, Hayashi T, Yamada K, Asai K, Yoshimine N, Kuzuya F. Substrate-bound fibrinogen, fibrin and other cell attachment-promoting proteins as a scaffold for cultured vascular smooth muscle cells. Atherosclerosis 1992; 96: 227-34.
    CrossrefPubMedGoogle Scholar
  • 43 Smith EB, Crosbie L, Cochran S. The inter-relation of fibrin, lipoprotein(a) and plasminogen in human atherosclerotic lesions. Blood Coagul Fibrinolysis 1990; 01: 495-98.
    CrossrefPubMedGoogle Scholar
  • 44 Weitz JI, Hudoba M, Massel D, Maraganore J, Hirsch J. Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest 1990; 86: 385-91.
    CrossrefPubMedGoogle Scholar