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. 1987 Jun 1;104(6):1655–1663. doi: 10.1083/jcb.104.6.1655

The platelet fibrinogen receptor: an immunogold-surface replica study of agonist-induced ligand binding and receptor clustering

PMCID: PMC2114491  PMID: 3584243

Abstract

Platelet aggregation requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma-membrane glycoproteins (GP) IIb and IIIa. Although the GPIIb-IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold-surface replica technique to study the distribution of GPIIb-IIIa and bound fibrinogen over broad areas of surface membranes in unstimulated, as well as thrombin-activated and ADP-activated human platelets. We found that the immunogold-labeled GPIIb-IIIa was monodispersed over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. On thrombin-stimulated platelets, approximately 65% of the GPIIb-IIIa molecules were in clusters within the plane of the membrane. Fibrinogen, which had been released from the alpha-granules of these cells, bound to GPIIb-IIIa on the cell surface and was similarly clustered. To determine whether the receptors clustered before ligand binding, or as a consequence thereof, we studied the surface distribution of GPIIb-IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb-IIIa without inducing the release of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding-competent receptors on ADP-stimulated platelets were monodispersed. The addition of fibrinogen caused the GPIIb-IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb-IIIa-binding domains of fibrinogen, namely the tetrapeptide Arg-Gly-Asp-Ser on the alpha-chain or the gamma-chain decapeptide gamma 402-411. These results show that receptor occupancy causes clustering of GPIIb-IIIa in activated platelets.

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Selected References

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  1. Asch A. S., Leung L. L., Polley M. J., Nachman R. L. Platelet membrane topography: colocalization of thrombospondin and fibrinogen with the glycoprotein IIb-IIIa complex. Blood. 1985 Oct;66(4):926–934. [PubMed] [Google Scholar]
  2. Banga H. S., Simons E. R., Brass L. F., Rittenhouse S. E. Activation of phospholipases A and C in human platelets exposed to epinephrine: role of glycoproteins IIb/IIIa and dual role of epinephrine. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9197–9201. doi: 10.1073/pnas.83.23.9197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bennett J. S., Vilaire G. Exposure of platelet fibrinogen receptors by ADP and epinephrine. J Clin Invest. 1979 Nov;64(5):1393–1401. doi: 10.1172/JCI109597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berridge M. J., Irvine R. F. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature. 1984 Nov 22;312(5992):315–321. doi: 10.1038/312315a0. [DOI] [PubMed] [Google Scholar]
  5. Brass L. F. Ca2+ transport across the platelet plasma membrane. A role for membrane glycoproteins IIB and IIIA. J Biol Chem. 1985 Feb 25;260(4):2231–2236. [PubMed] [Google Scholar]
  6. Bray P. F., Rosa J. P., Lingappa V. R., Kan Y. W., McEver R. P., Shuman M. A. Biogenesis of the platelet receptor for fibrinogen: evidence for separate precursors for glycoproteins IIb and IIIa. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1480–1484. doi: 10.1073/pnas.83.5.1480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Burns G. F., Cosgrove L., Triglia T., Beall J. A., López A. F., Werkmeister J. A., Begley C. G., Haddad A. P., d'Apice A. J., Vadas M. A. The IIb-IIIa glycoprotein complex that mediates platelet aggregation is directly implicated in leukocyte adhesion. Cell. 1986 Apr 25;45(2):269–280. doi: 10.1016/0092-8674(86)90391-0. [DOI] [PubMed] [Google Scholar]
  8. Charo I. F., Fitzgerald L. A., Steiner B., Rall S. C., Jr, Bekeart L. S., Phillips D. R. Platelet glycoproteins IIb and IIIa: evidence for a family of immunologically and structurally related glycoproteins in mammalian cells. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8351–8355. doi: 10.1073/pnas.83.21.8351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Coller B. S. A new murine monoclonal antibody reports an activation-dependent change in the conformation and/or microenvironment of the platelet glycoprotein IIb/IIIa complex. J Clin Invest. 1985 Jul;76(1):101–108. doi: 10.1172/JCI111931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fitzgerald L. A., Charo I. F., Phillips D. R. Human and bovine endothelial cells synthesize membrane proteins similar to human platelet glycoproteins IIb and IIIa. J Biol Chem. 1985 Sep 15;260(20):10893–10896. [PubMed] [Google Scholar]
  11. Fitzgerald L. A., Phillips D. R. Calcium regulation of the platelet membrane glycoprotein IIb-IIIa complex. J Biol Chem. 1985 Sep 15;260(20):11366–11374. [PubMed] [Google Scholar]
  12. Fujimoto T., Ohara S., Hawiger J. Thrombin-induced exposure and prostacyclin inhibition of the receptor for factor VIII/von Willebrand factor on human platelets. J Clin Invest. 1982 Jun;69(6):1212–1222. doi: 10.1172/JCI110560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gardner J. M., Hynes R. O. Interaction of fibronectin with its receptor on platelets. Cell. 1985 Sep;42(2):439–448. doi: 10.1016/0092-8674(85)90101-1. [DOI] [PubMed] [Google Scholar]
  14. Gartner T. K., Bennett J. S. The tetrapeptide analogue of the cell attachment site of fibronectin inhibits platelet aggregation and fibrinogen binding to activated platelets. J Biol Chem. 1985 Oct 5;260(22):11891–11894. [PubMed] [Google Scholar]
  15. George J. N., Nurden A. T., Phillips D. R. Molecular defects in interactions of platelets with the vessel wall. N Engl J Med. 1984 Oct 25;311(17):1084–1098. doi: 10.1056/NEJM198410253111705. [DOI] [PubMed] [Google Scholar]
  16. George J. N., Pickett E. B., Saucerman S., McEver R. P., Kunicki T. J., Kieffer N., Newman P. J. Platelet surface glycoproteins. Studies on resting and activated platelets and platelet membrane microparticles in normal subjects, and observations in patients during adult respiratory distress syndrome and cardiac surgery. J Clin Invest. 1986 Aug;78(2):340–348. doi: 10.1172/JCI112582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Griffiths G., Hoppeler H. Quantitation in immunocytochemistry: correlation of immunogold labeling to absolute number of membrane antigens. J Histochem Cytochem. 1986 Nov;34(11):1389–1398. doi: 10.1177/34.11.3534077. [DOI] [PubMed] [Google Scholar]
  18. Hopkins C. R., Miller K., Beardmore J. M. Receptor-mediated endocytosis of transferrin and epidermal growth factor receptors: a comparison of constitutive and ligand-induced uptake. J Cell Sci Suppl. 1985;3:173–186. doi: 10.1242/jcs.1985.supplement_3.17. [DOI] [PubMed] [Google Scholar]
  19. Houdijk W. P., Sakariassen K. S., Nievelstein P. F., Sixma J. J. Role of factor VIII-von Willebrand factor and fibronectin in the interaction of platelets in flowing blood with monomeric and fibrillar human collagen types I and III. J Clin Invest. 1985 Feb;75(2):531–540. doi: 10.1172/JCI111729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hourdillé P., Hasitz M., Belloc F., Nurden A. T. Immunocytochemical study of the binding of fibrinogen and thrombospondin to ADP- and thrombin-stimulated human platelets. Blood. 1985 Apr;65(4):912–920. [PubMed] [Google Scholar]
  21. Isenberg W. M., McEver R. P., Shuman M. A., Bainton D. F. Topographic distribution of a granule membrane protein (GMP-140) that is expressed on the platelet surface after activation: an immunogold-surface replica study. Blood Cells. 1986;12(1):191–204. [PubMed] [Google Scholar]
  22. Jennings L. K., Phillips D. R. Purification of glycoproteins IIb and III from human platelet plasma membranes and characterization of a calcium-dependent glycoprotein IIb-III complex. J Biol Chem. 1982 Sep 10;257(17):10458–10466. [PubMed] [Google Scholar]
  23. Kahn C. R., Baird K. L., Jarrett D. B., Flier J. S. Direct demonstration that receptor crosslinking or aggregation is important in insulin action. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4209–4213. doi: 10.1073/pnas.75.9.4209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kloczewiak M., Timmons S., Lukas T. J., Hawiger J. Platelet receptor recognition site on human fibrinogen. Synthesis and structure-function relationship of peptides corresponding to the carboxy-terminal segment of the gamma chain. Biochemistry. 1984 Apr 10;23(8):1767–1774. doi: 10.1021/bi00303a028. [DOI] [PubMed] [Google Scholar]
  25. Leeksma O. C., Zandbergen-Spaargaren J., Giltay J. C., van Mourik J. A. Cultured human endothelial cells synthesize a plasma membrane protein complex immunologically related to the platelet glycoprotein IIb/IIIa complex. Blood. 1986 Apr;67(4):1176–1180. [PubMed] [Google Scholar]
  26. Marguerie G. A., Plow E. F., Edgington T. S. Human platelets possess an inducible and saturable receptor specific for fibrinogen. J Biol Chem. 1979 Jun 25;254(12):5357–5363. [PubMed] [Google Scholar]
  27. McEver R. P., Baenziger N. L., Majerus P. W. Isolation and quantitation of the platelet membrane glycoprotein deficient in thrombasthenia using a monoclonal hybridoma antibody. J Clin Invest. 1980 Dec;66(6):1311–1318. doi: 10.1172/JCI109983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McEver R. P., Bennett E. M., Martin M. N. Identification of two structurally and functionally distinct sites on human platelet membrane glycoprotein IIb-IIIa using monoclonal antibodies. J Biol Chem. 1983 Apr 25;258(8):5269–5275. [PubMed] [Google Scholar]
  29. McLean I. W., Nakane P. K. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem. 1974 Dec;22(12):1077–1083. doi: 10.1177/22.12.1077. [DOI] [PubMed] [Google Scholar]
  30. Menon A. K., Holowka D., Webb W. W., Baird B. Cross-linking of receptor-bound IgE to aggregates larger than dimers leads to rapid immobilization. J Cell Biol. 1986 Feb;102(2):541–550. doi: 10.1083/jcb.102.2.541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Painter R. G., Ginsberg M. Concanavalin A induces interactions between surface glycoproteins and the platelet cytoskeleton. J Cell Biol. 1982 Feb;92(2):565–573. doi: 10.1083/jcb.92.2.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Painter R. G., Prodouz K. N., Gaarde W. Isolation of a subpopulation of glycoprotein IIb-III from platelet membranes that is bound to membrane actin. J Cell Biol. 1985 Feb;100(2):652–657. doi: 10.1083/jcb.100.2.652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Parise L. V., Phillips D. R. Reconstitution of the purified platelet fibrinogen receptor. Fibrinogen binding properties of the glycoprotein IIb-IIIa complex. J Biol Chem. 1985 Sep 5;260(19):10698–10707. [PubMed] [Google Scholar]
  34. Peerschke E. I., Zucker M. B. Fibrinogen receptor exposure and aggregation of human blood platelets produced by ADP and chilling. Blood. 1981 Apr;57(4):663–670. [PubMed] [Google Scholar]
  35. Phillips D. R., Jennings L. K., Edwards H. H. Identification of membrane proteins mediating the interaction of human platelets. J Cell Biol. 1980 Jul;86(1):77–86. doi: 10.1083/jcb.86.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pidard D., Montgomery R. R., Bennett J. S., Kunicki T. J. Interaction of AP-2, a monoclonal antibody specific for the human platelet glycoprotein IIb-IIIa complex, with intact platelets. J Biol Chem. 1983 Oct 25;258(20):12582–12586. [PubMed] [Google Scholar]
  37. Plow E. F., Ginsberg M. H. Specific and saturable binding of plasma fibronectin to thrombin-stimulated human platelets. J Biol Chem. 1981 Sep 25;256(18):9477–9482. [PubMed] [Google Scholar]
  38. Plow E. F., Srouji A. H., Meyer D., Marguerie G., Ginsberg M. H. Evidence that three adhesive proteins interact with a common recognition site on activated platelets. J Biol Chem. 1984 May 10;259(9):5388–5391. [PubMed] [Google Scholar]
  39. Polley M. J., Leung L. L., Clark F. Y., Nachman R. L. Thrombin-induced platelet membrane glycoprotein IIb and IIIa complex formation. An electron microscope study. J Exp Med. 1981 Oct 1;154(4):1058–1068. doi: 10.1084/jem.154.4.1058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Powling M. J., Hardisty R. M. Glycoprotein IIb-IIIa complex and Ca2+ influx into stimulated platelets. Blood. 1985 Sep;66(3):731–734. [PubMed] [Google Scholar]
  41. Pytela R., Pierschbacher M. D., Ginsberg M. H., Plow E. F., Ruoslahti E. Platelet membrane glycoprotein IIb/IIIa: member of a family of Arg-Gly-Asp--specific adhesion receptors. Science. 1986 Mar 28;231(4745):1559–1562. doi: 10.1126/science.2420006. [DOI] [PubMed] [Google Scholar]
  42. Robenek H., Schmitz G., Assmann G. Topography and dynamics of receptors for acetylated and malondialdehyde-modified low-density lipoprotein in the plasma membrane of mouse peritoneal macrophages as visualized by colloidal gold in conjunction with surface replicas. J Histochem Cytochem. 1984 Oct;32(10):1017–1027. doi: 10.1177/32.10.6481148. [DOI] [PubMed] [Google Scholar]
  43. Ruoslahti E., Pierschbacher M. D. Arg-Gly-Asp: a versatile cell recognition signal. Cell. 1986 Feb 28;44(4):517–518. doi: 10.1016/0092-8674(86)90259-x. [DOI] [PubMed] [Google Scholar]
  44. Schechter Y., Hernaez L., Schlessinger J., Cuatrecasas P. Local aggregation of hormone-receptor complexes is required for activation by epidermal growth factor. Nature. 1979 Apr 26;278(5707):835–838. doi: 10.1038/278835a0. [DOI] [PubMed] [Google Scholar]
  45. Schreiber A. B., Libermann T. A., Lax I., Yarden Y., Schlessinger J. Biological role of epidermal growth factor-receptor clustering. Investigation with monoclonal anti-receptor antibodies. J Biol Chem. 1983 Jan 25;258(2):846–853. [PubMed] [Google Scholar]
  46. Shattil S. J., Hoxie J. A., Cunningham M., Brass L. F. Changes in the platelet membrane glycoprotein IIb.IIIa complex during platelet activation. J Biol Chem. 1985 Sep 15;260(20):11107–11114. [PubMed] [Google Scholar]
  47. Stenberg P. E., McEver R. P., Shuman M. A., Jacques Y. V., Bainton D. F. A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J Cell Biol. 1985 Sep;101(3):880–886. doi: 10.1083/jcb.101.3.880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Stenberg P. E., Shuman M. A., Levine S. P., Bainton D. F. Redistribution of alpha-granules and their contents in thrombin-stimulated platelets. J Cell Biol. 1984 Feb;98(2):748–760. doi: 10.1083/jcb.98.2.748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wakshull E. M., Wharton W. Stabilized complexes of epidermal growth factor and its receptor on the cell surface stimulate RNA synthesis but not mitogenesis. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8513–8517. doi: 10.1073/pnas.82.24.8513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Weiss H. J., Turitto V. T., Baumgartner H. R. Platelet adhesion and thrombus formation on subendothelium in platelets deficient in glycoproteins IIb-IIIa, Ib, and storage granules. Blood. 1986 Feb;67(2):322–330. [PubMed] [Google Scholar]
  51. Wencel-Drake J. D., Plow E. F., Kunicki T. J., Woods V. L., Keller D. M., Ginsberg M. H. Localization of internal pools of membrane glycoproteins involved in platelet adhesive responses. Am J Pathol. 1986 Aug;124(2):324–334. [PMC free article] [PubMed] [Google Scholar]
  52. Wheeler M. E., Gerrard J. M., Carroll R. C. Reciprocal transmembranous receptor-cytoskeleton interactions in concanavalin A-activated platelets. J Cell Biol. 1985 Sep;101(3):993–1000. doi: 10.1083/jcb.101.3.993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zidovetzki R., Yarden Y., Schlessinger J., Jovin T. M. Microaggregation of hormone-occupied epidermal growth factor receptors on plasma membrane preparations. EMBO J. 1986 Feb;5(2):247–250. doi: 10.1002/j.1460-2075.1986.tb04205.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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