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Thromb Haemost 1990; 64(02): 297-301
DOI: 10.1055/s-0038-1647305
Original Article
Schattauer GmbH Stuttgart

Localization and Biosynthesis of Functional Thrombomodulin in Human Megakaryocytes and a Human Megakaryoblastic Cell Line (MEG-01)

Michinori Ogura
1   The First Dept. of Internal Medicine, Nagoya University School of Medicine, Japan
*   The Present address: the Department of Hematology and Cancer Chemotherapeutics, Aichi Cancer Center Hospital, Nagoya, Japan
,
Takahiko Ito
1   The First Dept. of Internal Medicine, Nagoya University School of Medicine, Japan
,
Ikuro Maruyama
2   The Third Dept. of Internal Medicine, Kogoshima University School of Medicine, Japan
,
Junki Takamatsu
1   The First Dept. of Internal Medicine, Nagoya University School of Medicine, Japan
,
Shuji Yamamoto
3   The Bio-Science Laboratory, Life Science Research Laboratories, Asahi Chemical Industry, Japan
,
Kohei Ogawa
3   The Bio-Science Laboratory, Life Science Research Laboratories, Asahi Chemical Industry, Japan
,
Hiroshi Nagura
4   The Second Dept. of Pathology, Tohoku University School of Medicine, Japan
,
Hidehiko Saito
1   The First Dept. of Internal Medicine, Nagoya University School of Medicine, Japan
› Author Affiliations
Further Information

Publication History

Received 27 February 1990

Accepted after revision28 May 1990

Publication Date:
25 July 2018 (online)

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Summary

The localization and biosynthesis of functiosnal thrombomodulin (TM) on the cell surfaces of human platelets, megakaryocytes and a human megakaryoblastic cell line (MEG-01) were investigated. TM was demonstrated on the cell surfaces and in cytoplasms of human platelets, megakaryocytes and MEG-01 by an indirect immunofluorescent technique using monospecific rabbit anti-human TM serum. Immunoelectronmicroscopic analysis revealed that TM was localized in plasma membranes of MEG-01 cells as well as human megakaryocytes. 125I-monoclonal antithrombomodulin IgG binding assay showed that one MEG-01 cell possessed approximately 78,000 TM molecules on its cell surface. Thrombin-dependent protein C activating-cofactor activity was demonstrated on MEG-01 cells. Northern hybridization technique using cDNA probe of TM revealed that poly(A)+-RNA from MEG-01 cells showed a single band of 3.8 kb similar to that from human endothelial cells. These data suggest that human megakaryocytes synthesize functional TM, and thereby platelets possess TM on their surfaces. TM on platelets may participate in the activation of protein C at the site of a hemostatic plug.

 

  • References

  • 1 Esmon CT. The regulation of natural anticoagulant pathways. Science 1987; 235: 1348-1352
    CrossrefPubMedGoogle Scholar
  • 2 Clouse LH, Comp PC. The regulation of hemostasis. N Engl J Med 1986; 314: 1298-1304
    CrossrefPubMedGoogle Scholar
  • 3 Comp PC. Hereditary disorders predisposing to thrombosis. In: Progress in Hemostasis and Thrombosis Coller B. (ed) Grune & Stratton; Philadelphia: 1986. vol 8 p 71
  • 4 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
    CrossrefPubMedGoogle Scholar
  • 5 Walker FJ, Sexton PW, 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
    CrossrefPubMedGoogle Scholar
  • 6 Suzuki K, Stenflo J, Dahlbäck B, Teodorsson G. Inactivation of human coagulation factor V by activated protein C. J Biol Chem 1983; 258: 1914-1920
    PubMedGoogle Scholar
  • 7 Fulcher CA, Gardiner JE, Griffin JH, Zimmerman TS. Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V. Blood 1984; 63: 486-489
    PubMedGoogle Scholar
  • 8 Maruyama I, Bell CE, Majerus PW. Thrombomodulin is found on endothelium of arteries, veins, capillaries, and lymphatics, and on syncytiotrophoblast of human placenta. J Cell Biol 1985; 101: 363-371
    CrossrefPubMedGoogle Scholar
  • 9 Schwarz HP, Heep MJ, Wencel-Drake JD, Griffin JH. Identification and quantitation of protein S in human platelets. Blood 1985; 66: 1452-1455
    PubMedGoogle Scholar
  • 10 Ogura M, Tanabe N, Nishioka J, Suzuki K, Saito H. Biosynthesis and secretion of functional protein S by a human megakaryoblastic cell line (MEG-01). Blood 1987; 70: 301-306
    PubMedGoogle Scholar
  • 11 Ogura M, Takamatsu J, Tanabe N, Maruyama I, Saito H. Localization of thrombomodulin in human platelets, megakaryocytes and megakaryoblastic cell line (MEG-01). Blood 1987 70. 357 (abstr)
    PubMedGoogle Scholar
  • 12 Suzuki K, Nishioka J, Hayashi T, Kosaka Y. Functionally active thrombomodulin is present in human platelets. J Biochem 1988; 104: 628-632
    CrossrefPubMedGoogle Scholar
  • 13 Handagama PJ, George JN, Shuman MA, McEver RP, Bainton DF. Incorporation of a circulating protein into megakaryocyte and platelet granules. Proc Natl Acad Sci USA 1987; 84: 861-865
    CrossrefPubMedGoogle Scholar
  • 14 Ogura M, Morishima Y, Ohno R, Kato Y, Hirabayashi N, Nagura H, Saito H. Establishment of a novel human megakaryoblastic leukemia cell line, MEG-01, with positive Phildelphia chromosome. Blood 1985; 66: 1384-1392
    PubMedGoogle Scholar
  • 15 Ogura M, Morishima Y, Okumura M, Hotta T, Takamoto S, Ohno R, Hirabayashi N, Nagura H, Saito H. Functional and morphological differentiation induction of a human megakaryoblastic leukemia cell line (MEG-01) by phorbol diesters. Blood 1988; 72: 49-60
    PubMedGoogle Scholar
  • 16 Ogura M, Tanabe N, Hamaguchi M, Hotta T, Saito H. Biosynthesis and secretion of p-thromboglobulin by a human megakaryoblastic cell line (MEG-01). Thromb Haemostas 1987 58. 492 (Abstr)
    PubMedGoogle Scholar
  • 17 Maruyama I, Majerus PW. The turnover of thrombin-thrombomodulin complex in cultured human umbilical vein endothelial cells and A549 lung cancer cells. J Biol Chem 1985; 260: 15432-15438
    PubMedGoogle Scholar
  • 18 Tangen O, Berman HJ, Marfey P. A new technique for separation of blood platelets from plasma. Thromb Diath Haemorrh 1971; 25: 268-278
    PubMedGoogle Scholar
  • 19 Kisiel W. Human plasma protein C. J Clin Invest 1979; 64: 761-769
    CrossrefPubMedGoogle Scholar
  • 20 Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonu-clease. Biochemistry 1979; 18: 5294-5299
    CrossrefPubMedGoogle Scholar
  • 21 Davis LG, Dibner MD, Battey JF. Selection of poly(A+)-RNA on oligo(dT) cellulose. In: Basic Methods in Molecular Biology Elsevier; New York, NY: 1986. pp 139-142
  • 22 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 endothelial acting as a cofactor for protein C activation. EMBO J 1987; 6: 1891-1897
    PubMedGoogle Scholar
  • 23 Debault LE, Esmon NL, Esmon CT. The relationship between thrombomodulin expression and cell cycle stages in cultured rabbit endothelial cells. Cytometry 1984; 5: 386-391
    CrossrefPubMedGoogle Scholar
  • 24 Tollefsen DM, Majerus PW. Evidence for single class of thrombin-binding sites on human platelets. Biochemistry 1976; 15: 2144-2149
    CrossrefPubMedGoogle Scholar
  • 25 Ganguly P, Sonnichen WJ. Binding of thrombin to human platelets and its possible significance. Br J Haematol 1976; 34: 291-301
    CrossrefPubMedGoogle Scholar
  • 26 Workman Jr EF, White GC, Lundblad RL. Structure-function relationship in the interaction of a-thrombin with blood platelets. J Biol Chem 1977; 252: 7119-7123
    PubMedGoogle Scholar
  • 27 Takamatsu J, Horne MK, Gralnick HR. Identification of the thrombin receptor on human platelets by chemical cross-linking. J Clin Invest 1986; 77: 362-369
    CrossrefPubMedGoogle Scholar
  • 28 Granguly P, Fossett NG. Inhibition of thrombin-induced platelet aggregation by a derivative of wheat germ agglutinin. Evidence for a physiological receptor of thrombin in human platelets. Blood 1981; 57: 343-352
    PubMedGoogle Scholar
  • 29 Chelladurai M, Fossett NG, Granguly P. A novel thrombin-reactive protein complex in human platelets. J Biol Chem 1983; 258: 1407-1410
    PubMedGoogle Scholar
  • 30 Moore KL, Andreoli SP, Esmon NL, Esmon CT, Bang NU. Endotoxin endhances tissue factor and suppresses thrombomodulin expression of human vascular endothelium in vitro. J Clin Invest 1987; 79: 124-130
    CrossrefPubMedGoogle Scholar
  • 31 Nawroth PP, Handley DA, Esmon CT, Stern DM. Interleukin 1 induced endothelial cell procoagulant while suppressing cell-surface anticoagulant activity. Proc Natl Acad Sci USA 1986; 83: 3460-3464
    CrossrefPubMedGoogle Scholar
  • 32 Nawroth PP, Stem DM. Modulation of endothelial cell hemostatic properties by tumor necrosis factor. J Exp 1986; 163: 740-745
    CrossrefPubMedGoogle Scholar