Thromb Haemost 1995; 73(02): 291-296
DOI: 10.1055/s-0038-1653767
Original Article
Platelets
Schattauer GmbH Stuttgart

Internalization of an Anti-Glycoprotein IIb/IIIa Antibody by HEL Cells

Kenjiro Hamamoto
The First Department of Internal Medicine, Kansai Medical University, Osaka, Japan
,
Shosaku Nomura
The First Department of Internal Medicine, Kansai Medical University, Osaka, Japan
,
Masahiko Suzuki
The First Department of Internal Medicine, Kansai Medical University, Osaka, Japan
,
Shigetoshi Ohga
The First Department of Internal Medicine, Kansai Medical University, Osaka, Japan
,
Shirou Fukuhara
The First Department of Internal Medicine, Kansai Medical University, Osaka, Japan
› Author Affiliations
Further Information

Publication History

Received 04 February 1994

Accepted after resubmission 28 October 1994

Publication Date:
09 July 2018 (online)

Summary

Platelets are known to internalize monoclonal antibodies directed against the glycoprotein (GP) IIb/IIIa complex. We investigated whether an antibody directed against this complex (NNKY 2-11) was transported from the surface membrane to the intracellular pool in HEL cells. Flow cytometry showed that the percent binding of NNKY 2-11 to the surface membrane of HEL cells was decreased after incubation for 24 h compared with 1 h, while the binding of an anti-GPIb antibody (NNKY 5-5) did not change. It did not seem likely that the GP Ilb/IIIa complex antibody was shed from the surface membrane of the HEL cells during incubation, because the medium conditioned by incubation with these cells for 24 h showed almost no binding to washed platelets. In addition, immunoelectron microscopy demonstrated that GP IIb/IIIa complex antibodies were incorporated into the intracellular pool of HEL cells and were associated with alpha granules. These findings indicated that an anti-GP IIb/IIIa antibody could be internalized by megakaryocytes, as has been previously shown with platelets, suggesting that megakaryocyte GP IIb/IIIa may act as a carrier for various adhesion proteins.

 
  • References

  • 1 George JN, Nurden AT, Phillips DR. Molecular defects that cause abnormalities of platelet-vessel wall interaction. N Engl J Med 1984; 311: 1084-1098
  • 2 Plow EF, McEver RP, Coller BS, Woods VL, Marguerie GA, Ginsberg MH. Related binding mechanisms for fibrinogen, fibronectin, von Wille-brand factor and thrombospondin on thrombin-stimulated platelets. Blood 1985; 66: 724-727
  • 3 Nachman RL, Leung LL K. Complex formation of platelet membrane glycoprotein IIb and/or IIIa fibrinogen. J Clin Invest 1982; 69: 263-269
  • 4 Gogstad GO, Brosstad F, Krutanes M-B, Hagen I, Solum NO. Fibrinogen binding proteins of the human platelet glycoprotein IIb-IIIa complex: A study using crossed radio immunoelectrophoresis. Blood 1982; 60: 663-671
  • 5 Wencel-Drake JD, Plow EF, Kuniki TJ, Woods VL, Keller DM, Ginsberg HH. Localization of internal, pools of membrane glycoproteins involved in platelet adhesive responses. Am J Pathol 1982; 124: 324-334
  • 6 Santoso S, Zimmermann U, Neppert J, Muller-Eckhardt C. Receptor patching and capping of platelet membranes induced by monoclonal antibodies. Blood 1986; 67: 343-349
  • 7 Saunders PG W, Durack BE, Narang HK. Observations on the distribution of the ZWa(PIAI)antigen on fixed and unfixed platelets. Br J Haematol 1986; 62: 631-634
  • 8 Morgenstem E, Ruf A, Patscheke H. Transport of anti-glycoprotein IIb/IIIa-antibodies into the alpha-granules of unstimulated human blood platelets. Thromb Haemost 1992; 67: 121-125
  • 9 Martin P, Papayannopoulou T. HEL cells: A new human erythroleukemia cell line with spontaneous and induced globulin expression. Science 1982; 216: 1233-1235
  • 10 Bonner WN, Lasky RA. A film detection method for tritium-labelled protein and nucleic acid in polyacrylamide gels. Eur J Biochem 1974; 46: 83-88
  • 11 Shattil SJ, Cunningham M, Hoxie JA. Detection of activated platelets in whole blood using activation dependent monoclonal antibodies and flow cytometry. Blood 1987; 70: 307-315
  • 12 Nomura S, Nagata H, Sone N, Oda K, Kokawa T, Yasunaga K. Analysis of platelet antigen for anti-platelet antibodies in idiopathic thrombocytopenic purpura using flow cytometry. Acta Haematol Jpn 1988; 51: 118-129
  • 13 Nomura S, Kokawa T. Recent advance on analysis of antiplatelet antibody - special reference to application of flow cytometry. In: Automation and new technology in the clinical laboratory. Okuda K. ed Blackwell Scientific Publications, Oxford; 1990. PP 287 294
  • 14 Nomura S, Suzuki M, Kido H, Yamaguchi K, Fukuroi T, Yanabu M, Soga T, Nagata H, Kokawa T, Yasunaga K. Differences between platelet and microparticle glycoprotein IIb/IIIa. Cytometry 1992; 13: 621-629
  • 15 Takada Y, Huang C, Hemler ME. Fibronectin receptor structures within the VLA family of heterodimers. Nature 1987; 326: 607-609
  • 16 Ohga S, Hamamoto K, Kitajima H, Nagata H, Nomura S, Kimura T, Fujitake H, Kokawa T, Yasunaga K. The ultrastructure and distribution of fibrinogen, von Willebrand factor and glycoprotein IIb/IIIa complex inplatelet alpha granules by cryo-ultramicrotomy. Int J Hematol 1992; 54: 331-339
  • 17 Hamamoto K, Ohga S, Nomura S, Yasunaga K. Cellular distribution of CD63 in platelets and in three megakaryocytic cell lines. Histochem J 1994; 26: 367-375
  • 18 Stenberg PE, McEver RP, Shuman MA, Jacques YV, Bainton DF. A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J Cell Biol 1985; 101: 880-886
  • 19 Berman CL, Yeo EL, Wencel-Drake JD, Furie BC, Ginsberg MH, Furie BA. A platelet alpha granule membrane protein that is associated with the plasma membrane after activation. Characterization and subcellular localization of platelet activation-dependent granule-external membrane protein J Clin Invest 1986; 78: 130-137
  • 20 Cramer EM, Debili N, Martin JF, Gladwin AM, Breton-Gorius J, Harrison P, Savidge GF, Vainchenker W. Uncoordinated expression of fibrinogen compared with thrombospondin and von Willebrand factor in maturing megakaryocytes. Blood 1989; 73: 1123-1129
  • 21 Handagama PJ, Shuman MA, Bainton DF. Incorporation of intravenously injected albumin, immunoglobulin G, and fibrinogen in guinea pig megakaryocyte granules. J Clin Invest 1989; 84: 73-82
  • 22 Harrison P, Wilboum BR, Debili N, Vainchenker W, Lawrie AS, Breton- Gorius J, Masse JM, Savidge GF, Cramer EM. Uptake of plasma fibrinogen into the alpha granules of human megakaryocytes and platelets. J Clin Invest 1989; 84: 1320-1324
  • 23 Wencel-Drake JD. Plasma membrane GPIIb/IIIa. Evidence for a cycling receptor pool Am J Pathol 1990; 137: 1393-1399
  • 24 Suzuki M, Kawakatsu T, Nagata H, Hamamoto K, Iwata K, Ohga S, Nomura S, Kokawa T, Yasunaga K. Effects of injected antibody against the platelet glycoprotein IIb/IIIa complex on monkey platelet fibrinogen. Thromb Haemost 1992; 67: 578-581
  • 25 Harrison P, Wilboum B, Cramer E, Faint R, Mackie IJ, Bhattacharya S, Lahiri A, Tenza D, Machin SJ, Savidge GF. The influence of therapeutic blocking of GP IIb/IIIa on platelet alpha granular fibrinogen. Br J Haematol 1992; 82: 721-728
  • 26 Tabilio A, Rosa J-P, Testa U, Kieffer N, Nurden AT, Del Canizo MC, Breton-Gorius J, Vainchenker W. Expression of platelet membrane glycoproteins and alpha-granule proteins by a human erythroleukemia cell line (HEL). EMBOJ 1984; 3: 453-459
  • 27 Handagama P, Bainton DF, Jacques Y, Conn MT, Lazarus RA, Shuman MA. Kistrin, an integrin antagonist, blocks endocytosis of fibrinogen into guinea pig megakaryocyte and platelet alpha-granules. J Clin Invest 1993; 91: 193-200