Thromb Haemost 1991; 65(05): 618-623
DOI: 10.1055/s-0038-1648199
Original Article
Schattauer GmbH Stuttgart

Soluble Thrombomodulin Antigen in Conditioned Medium Is Increased by Damage of Endothelial Cells

Hidemi Ishii
The Department of Clinical Biochemistry Faculty of Pharmaceutical Sciences, Teikyo University Kanagawa, Japan
,
Hiroyuki Uchiyama
The Department of Clinical Biochemistry Faculty of Pharmaceutical Sciences, Teikyo University Kanagawa, Japan
,
Mutsuyoshi Kazama
The Department of Clinical Biochemistry Faculty of Pharmaceutical Sciences, Teikyo University Kanagawa, Japan
› Author Affiliations
Further Information

Publication History

Received 04 July 1990

Accepted after revision 09 January 1991

Publication Date:
24 July 2018 (online)

Summary

Thrombomodulin (TM) exists not only in endothelial cells but also in circulating plasma as soluble heterogeneous fragments. A release mechanism of soluble TM antigen from endothelial cells was investigated. Cultured human umbilical vein endothelial cells released about 0.6% of total cellular TM antigen into conditioned medium during 24 h. The release of TM antigen was not influenced by addition of various concentrations (0.01-5.0 μM) of monensin, which inhibits intracellular transport of secretory proteins, though the secretion of plasminogen activator inhibitor-1 from the cells was inhibited. The release of TM antigen was not increased when total cellular TM level increased 1.3- or 1.4-fold relative to control cells after stimulation with 0.1-1.0 U/ml thrombin or 3 mM dibutyryl cAMP, respectively. Exposure of endothelial cells for 6 h to mixture of 1 μM N-formyl-methionyl-leucyl-phenylalanine (FMLP) and 100 ng/ml lipopolysaccharide (LPS) decreased cellular TM level by 30% relative to control cells without increase in the TM release. The FMLP and LPS-stimulated leukocyte treatment of the cells increased the release of TM antigens into the medium in a time-dependent manner and the increased release of TM antigen paralleled the extent of cell damage as measured by 51Cr release. Hydrogen peroxide treatment of the cells increased the release of TM antigens into the medium in a time- and concentration-dependent manner. The increased release of TM antigen by hydrogen peroxide also paralleled the extent of cell damage. Soluble TM antigen in conditioned medium from the untreated control cells was composed of six heterogeneous fragments of 105, 56, 48, 33, 31 and 28 kDa observed on Western blots after sodium dodecyl sulfatepolyacrylamide gel electrophoresis under reducing conditions, and the soluble TM antigen from the hydrogen peroxide-treat-ment cells was composed of five heterogeneous fragments of 80, 56, 33, 31 and 28 kDa. These compositions were quite similar to that observed for soluble TM fragments in human plasma. It is suggested that soluble TM antigen is not secreted from endothelial cells, but results from cellular damage.

 
  • References

  • 1 Esmon CT, Owen WG. Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. Proc Natl Acad Sci USA 1981; 78: 2249-2252
  • 2 Walker FJ, Sexton PE, Esmon CT. The inhibition of blood coagulation by activated protein C through the selective inactivation of activated factor V. Biochim Biophys Acta 1979; 571: 333-342
  • 3 Fulcher CA, Gardiner JE, Griffin JH, Zimmerman TS. Proteolytic inactivation of human factor VIII procoagulant protein by activated protein C and its analogy to factor V. Blood 1984; 63: 486-489
  • 4 Maruyama I, Bell CE, Majerus PW. Thrombomodulin is found on endothelium of arteries, veins, capillaries, lymphatics, and syncytio-trophoblast of human placenta. J Cell Biol 1985; 101: 363-371
  • 5 Ishii H, Salem HH, Bell CE, Laposata EA, Majerus PW. Thrombomodulin, an endothelial anticoagulant protein, is absent from the human brain. Blood 1986; 67: 362-365
  • 6 Ishii H, Majerus PW. Thrombomodulin is present in human plasma and urine. J Clin Invest 1985; 76: 2178-2181
  • 7 Ishii H, Nakano M, Tsubouchi J, Ishikawa T, Uchiyama H, Hiraishi S, Tahara C, Miyajima Y, Kazama M. Establishment of enzyme immunoassay of human thrombomodulin in plasma and urine using monoclonal antibodies. Thromb Haemostas 1990; 63: 157-162
  • 8 Oida K, Takai H, Maeda H, Tamai T, Nakai T, Miyabo S, Ishii H. Plasma thrombomodulin concentration in diabetes mellitus. Thromb Haemostas 1989 62. 276 (Abstr)
  • 9 Giddings JC, Coles P, Williams BD. Comparison of thrombomodulin and von Willebrand factor antigen in human plasma in various diseases. Thromb Haemostas 1989 62. 333 (Abstr)
  • 10 Salem HH, Maruyama I, Ishii H, Majerus PW. Isolation and characterization of thrombomodulin from human placenta. J Biol Chem 1984; 259: 12246-12251
  • 11 Lindmark R, Thren-Tolling K, Sjoquist J. Protein A - an immunoglobulin-binding cell wall protein in Staphylococcus. J Immunol Methods 1983; 62: 1-13
  • 12 Suzuki K, Stenflo J, Dahlback B, Teodorsson B. Inactivation of human coagulation factor V by activated protein C. J Biol Chem 1983; 258: 1914-1920
  • 13 Lundblad RL, Kingdon HS, Mann KG. Thrombin. In: Methods in Enzymology, Vol 45 Lorand L. (ed) Academic Press; New York: 1976. p 156
  • 14 Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 1973; 52: 2745-2756
  • 15 Boyum A. Separation of white blood cells. Nature 1964; 204: 793-794
  • 16 Varani J, Bendelow MJ, Sealey DE, Kunkel SL, Gannon DE, Ryan US, Ward PA. Tumor necrosis factor enhances susceptibility of vascular endothelial cells to neutrophil-mediated killing. Lab Invest 1988; 59: 292-295
  • 17 Laemmli VK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
  • 18 Kooistra T, Sprengers ED, Van Hinsbergh VWM. Rapid inactivation of the plasminogen-activator inhibitor upon secretion from cultured endothelial cells. Biochem J 1986; 239: 497-503
  • 19 Tartakoff AM, Vassalli P. Comparative studies of intracellular transport of secretory proteins. J Cell Biol 1978; 79: 694-707
  • 20 Levin EG, Marzec U, Anderson J, Harker L. Thrombin stimulates tissue plasminogen activator release from cultured human endothelial cells. J Clin Invest 1984; 74: 1988-1995
  • 21 DeGroot PG, Gonsalves MD, Loesberg C, Van Buul-Wortelboer MF, Van Aken WG, Van Mourik JA. Thrombin-induced release of von Willebrand factor from endothelial cells is mediated by phospholipid methylation. J Biol Chem 1984; 259: 13329-13333
  • 22 Imada S, Yamaguchi H, Nagumo M, Katayanagi S, Iwasaki H, Imada M. Identification of fetomodulin, a surface marker protein of fetal development, as thrombomodulin by gene cloning and functional Assays. Dev Biol 1990; 140: 113-122
  • 23 Imada M, Imada S, Iwasaki H, Kume A, Yamaguchi H, Moore EE. Fetomodulin: Marker surface protein of fetal development which is modulatable by cyclic AMP. Dev Biol 1987; 122: 483-491
  • 24 Smedly LA, Tommesen MG, Sandhaus RA, Haslett C, Gurthrie LA, Johston Jr RB, Henson PM, Worthen GS. Neutrophil-mediated injury to endothelial cells. Enhancement by endotoxin and essential role of neutrophil elastase. J Clin Invest 1986; 77: 1233-1243
  • 25 Moore KL, Andreoli SP, Esmon NL, Esmon CT, Bang NU. Endotoxin enhances tissue factor and suppressed thrombomodulin expression of human vascular endothelium in vitro. J Clin Invest 1987; 79: 124-130
  • 26 Moore KL, Esmon CT, Esmon NL. Tumor necrosis factor leads to the internalization and degradation of thrombomodulin from the surface of bovine aortic endothelial cells in culture. Blood 1989; 73: 159-165
  • 27 Nawroth PP, Handley DA, Esmon CT, Stem DM. Interleukin-1 induces endothelial cell procoagulant while suppressing cell-surface anticoagulant activity. Proc Natl Acad Sci USA 1986; 83: 3460-3464
  • 28 Kurosawa S, Galvin JB, Esmon NL, Esmon CT. Proteolytic formation and properties of functional domains of thrombomodulin. J Biol Chem 1987; 262: 2206-2212