Summary
CD63 is a 53 kDa lysosomal membrane glycoprotein that has been identified as a platelet activation molecule. We investigated the localization of CD63 antigen in platelets and in three megakaryocytic cell lines (K562, HEL and CMK11-5) using flow cytometry and immunoelectron microscopy. Flow cytometry showed that a monoclonal antibody directed against CD63 bound to 8.1% of unstimulated platelets and 59.2% of thrombin-stimulated platelets. Immunoelectron microscopy demonstrated that CD63 antigen was distributed randomly inside unstimulated platelets, while it was localized in the open canalicular system of washed platelets and on the cell membranes of thrombin-stimulated platelets. Flow cytometry detected CD63 on 16.4% of HEL cells, 31.2% of K562 cells, and 43.2% of CMK11-5 cells. Immunoelectron microscopy demonstrated that CD63 was localized in the granules and on the surface membranes of HEL cells, in the vesicles and on the membranes of K562 cells, and in the granules and vesicles as well as on the membranes of CMK11-5 cells. Thus, the distribution of CD63 differed markedly among these three megakaryocytic cell lines.
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Azorsa, D. O., Hymen, J. A. & Hildreth, J. E. K. (1991) CD63/Pltgp40: a platelet activation antigen identical to the stage-specific, melanoma-associated antigen ME491. Blood 78, 280–4.
Coller, B. S. (1989) Activation-specific platelet antigens. In Platelet Immunology: Molecular and Clinical Aspects (edited by Kuniki, T. J. & George, J. N.), pp. 166–89 Philadelpha: Lippincott.
Knol, E. F., Mul, F. P. J., Jansen, H., Calafat, J. & Roos, D. (1991) Monitoring human basophil activation via CD63 monoclonal antibody 435. J. Allergy Clin. Immunol. 88, 328–38.
Kuijipers, T. W., Tool, A. T. J., Van DerSchoot, C. E., Ginsel, L. A., Onderwater, J. J. M., Roos, D. & Verhoeven, A. J. (1991) Membrane surface antigen expression on neutrophils: a reappraisal of the use of surface marker for neutrophil activation. Blood 78, 1105–11.
Lozzio, C. B. & Lozzio, B. B. (1975) Human chronic myelogenous leukemia cell line with positive Philadelphia chromosome. Blood 45, 3211–34.
Martin, P. & Papayannopoulou, T. (1982) HEL cells: a new human erythroleukemia cell line with spontaneous and induced globlin expression. Science 216, 1233–35.
McEver, R. P. & Martin, N. M. (1984) A monoclonal antibody to a membrane glycoprotein binds only to activation platelets. J. Biol. Chem. 259, 9799–804.
Metezelaar, M. J., Sixma, J. J. & Nieuwenhuis, H. K. (1989) A new cluster of activation-dependent mAb recognizing a 53- kDa lysosome-like granule protein, expressed on the plasma membrane after activation. In Leukocyte Typing IV (edited by Knapp, W., et al.) pp. 1043–44. New York: Oxford.
Metezelaar, M. J., Sixma, J. J. & Nieuwenhuis, H. K. (1990) Detection of platelet activation using activation specific monoclonal antibodies. Blood Cells 16, 85–96.
Metezelaar, M. J., Wijngaard, P. L. J., Peters, P. J., Sixma, J. J., Nieuwenhuis, H. K. & Cleversh, C. (1991) CD63 antigen: a novel lysosomal membrane glycoprotein, cloned by a screening procedure for intracellular antigens in eukaryotic cells. J. Biol. Chem. 266, 3239–45.
Nieuwenhis, H. K., VanOosterhout, J. J. G., Rozemuller, E., VanIwaarden, F. & Sixma, J. J. (1987) Studies with a monoclonal antibody against activated platelets: evidence that a secreted 53 000-molecular weight lysosome-like granule protein is exposed on the surface of activated platelets in the circulation. Blood 70, 838–45.
Nomura, S. & Kokawa, T. (1990) Recent advance on analysis of antiplatelet antibody - special reference to application of flow cytometry. In Automation and New Technology in the Clinical Laboratory (edited by Okuda, K.), pp 287–94. Oxford: Blackwell Scientific Publications.
Nomura, S., Suzuki, M., Kido, H., Yamaguchi, K., Fukuroi, T., Soga, T., Nagata, H., Kokawa, T. & Yasunaga, K. (1992) Differences between platelet and microparticle glycoprotein IIb/IIIa. Cytometry 13, 621–9.
Ohga, S., Hamamoto, K., Kitajima, H., Nagata, H., Nomura, S., Kimura, T., Fujitake, H., Kokawa, T. & Yasunaga, K. (1992) The ultrastructure and distribution of fibrinogen, von Willebrand factor and glycoprotein IIb/IIIa complex in platelet alpha granules by cryo-ultramicrotomy. Inter. J. Hematology 54, 331–9.
Peschel, C., Broxmeyer, H. E., Campos, L., Konwalinka, G., Strauss, L. C. & Tabilio, A. (1989) Reactivity of selected new myeloid workshop antibodies with haemopoietic progenitor cells. In Leukocyte Typing IV (edited by Knapp, W., et al.) pp. 906–9. New York: Oxford University Press.
Peters, P. J., Borst, J., Oorschot, V., Fukuda, M., Krahenbuhl, O., Tschopp, J., Slot, J. W. & Geuze, H. J. (1991) Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perform and granzymes. J. Exp. Med. 173, 1099–109.
Rakowicz-Szulczynska, E. M. & Koprowski, H. (1989) Nuclear uptake of monoclonal antibody to a surface glycoprotein and its effect on transcription. Arch. Biochem. Biophys. 271, 366–79.
Sato, T., Fuse, A., Eguchi, M., Hayashi, Y., Ryo, R., Adachi, M., Kishimoto, Y., Teramura, M., Mizoguchi, H., Shima, Y., Komori, I., Sunami, S., Okimoto, Y. & Nakajima, H. (1989) Establishment of a human leukaemic cell line (CMK) with megakaryocytic characteristics from a Down's syndrome patient with acute megakaryoblastic leukaemia. Br. J. Haematology 72, 184–90.
Sixma, J. J., Van DenBerg, A., Hasilik, A., VonFigura, K. & Geuze, H. J. (1985) Immunoelectron microscopical demonstration of lysosomes in human blood platelets and megakaryocytes using anti-cathepsin D. Blood 65, 1287–91.
Steen, V. M. & Holmsen, H. (1987) Current aspects on human platelet activation and responses. Eur. J. Haematol. 38, 383–99.
Tokuyasu, K. T. (1989) Use of poly(vinyl pyrrolidone) and poly(vinylalcohol) for cryoultramicrotomy. Histochem. J. 21, 163–71.
Wencel-Drake, J. D. (1990) Plasma membrane GPIIb/IIIa: evidence for a cycling receptor pool. Am. J. Pathol. 136, 61–70.
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Hamamoto, K., Ohga, S., Nomura, S. et al. Cellular distribution of CD63 antigen in platelets and in three megakaryocytic cell lines. Histochem J 26, 367–375 (1994). https://doi.org/10.1007/BF00157770
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DOI: https://doi.org/10.1007/BF00157770