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. 1996 Mar 15;314(Pt 3):769–779.

Exposure of ligand-binding sites on platelet integrin alpha IIB/beta 3 by phosphorylation of the beta 3 subunit.

G van Willigen 1, I Hers 1, G Gorter 1, J W Akkerman 1
PMCID: PMC1217123  PMID: 8615768

Abstract

The exposure of ligand-binding sites for adhesive proteins on platelet integrin alpha IIB/beta 3 (glycoprotein IIB/IIIA) by platelet-activating (PAF) is transient, whereas sites exposed by alpha-thrombin remain accessible. The same difference is seen in the phosphorylation of the beta 3 subunit. Inhibition of protein kinases (1 microM staurosporine) and protein kinase C (10 microM bisindolylmaleimide) closes binding sites exposed by both agonists and induces dephosphorylation of beta 3. Inhibition of Tyr-kinases (20 microM Herbimycin A) has only a slight effect. Inhibition of Ser/Thr-phosphatases (1 microM okadaic acid, 30 s preincubation) changes the transient exposure and beta phosphorylation by PAF into the 'permanent' patterns induced by alpha-thrombin. Inhibition of Tyr-phosphatases (100 microM vanadate) has little effect. Preincubation with okadaic acid makes exposed binding sites and phosphorylated beta 3 insensitive to staurosporine, resulting in exposed alpha IIB/beta 3 independent of concurrent phosphorylation/dephosphorylation. The stoichiometry of beta 3 phosphorylation by alpha-thrombin is 0.80+/-0.10. Thus, one of the mechanisms that regulates exposure and closure of ligand-binding sites on the alpha IIb/beta 3 is phosphorylation/dephosphorylation of a Ser/Thr-residue in the beta 3 subunit.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akkerman J. W., Ebberink R. H., Lips J. P., Christiaens G. C. Rapid separation of cytosol and particle fraction of human platelets by digitonin-induced cell damage. Br J Haematol. 1980 Feb;44(2):291–300. doi: 10.1111/j.1365-2141.1980.tb01211.x. [DOI] [PubMed] [Google Scholar]
  2. Bryckaert M. C., Rendu F., Tobelem G., Wasteson A. Collagen-induced binding to human platelets of platelet-derived growth factor leading to inhibition of P43 and P20 phosphorylation. J Biol Chem. 1989 Mar 15;264(8):4336–4341. [PubMed] [Google Scholar]
  3. Calvete J. J. Clues for understanding the structure and function of a prototypic human integrin: the platelet glycoprotein IIb/IIIa complex. Thromb Haemost. 1994 Jul;72(1):1–15. [PubMed] [Google Scholar]
  4. Chatila T. A., Geha R. S., Arnaout M. A. Constitutive and stimulus-induced phosphorylation of CD11/CD18 leukocyte adhesion molecules. J Cell Biol. 1989 Dec;109(6 Pt 2):3435–3444. doi: 10.1083/jcb.109.6.3435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen Y. P., Djaffar I., Pidard D., Steiner B., Cieutat A. M., Caen J. P., Rosa J. P. Ser-752-->Pro mutation in the cytoplasmic domain of integrin beta 3 subunit and defective activation of platelet integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) in a variant of Glanzmann thrombasthenia. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10169–10173. doi: 10.1073/pnas.89.21.10169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen Y. P., O'Toole T. E., Ylänne J., Rosa J. P., Ginsberg M. H. A point mutation in the integrin beta 3 cytoplasmic domain (S752-->P) impairs bidirectional signaling through alpha IIb beta 3 (platelet glycoprotein IIb-IIIa). Blood. 1994 Sep 15;84(6):1857–1865. [PubMed] [Google Scholar]
  7. Clark E. A., Shattil S. J., Brugge J. S. Regulation of protein tyrosine kinases in platelets. Trends Biochem Sci. 1994 Nov;19(11):464–469. doi: 10.1016/0968-0004(94)90131-7. [DOI] [PubMed] [Google Scholar]
  8. Clark E. A., Trikha M., Markland F. S., Brugge J. S. Structurally distinct disintegrins contortrostatin and multisquamatin differentially regulate platelet tyrosine phosphorylation. J Biol Chem. 1994 Sep 2;269(35):21940–21943. [PubMed] [Google Scholar]
  9. Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem. 1989;58:453–508. doi: 10.1146/annurev.bi.58.070189.002321. [DOI] [PubMed] [Google Scholar]
  10. Dahl S. C., Grabel L. B. Integrin phosphorylation is modulated during the differentiation of F-9 teratocarcinoma stem cells. J Cell Biol. 1989 Jan;108(1):183–190. doi: 10.1083/jcb.108.1.183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Daniel J. L., Molish I. R., Holmsen H. Radioactive labeling of the adenine nucleotide pool of cells as a method to distinguish among intracellular compartments. Studies on human platelets. Biochim Biophys Acta. 1980 Oct 15;632(3):444–453. doi: 10.1016/0304-4165(80)90240-8. [DOI] [PubMed] [Google Scholar]
  12. Daniel J. L., Robkin L., Molish I. R., Holmsen H. Determination of the ADP concentration available to participate in energy metabolism in an actin-rich cell, the platelet. J Biol Chem. 1979 Aug 25;254(16):7870–7873. [PubMed] [Google Scholar]
  13. Du X. P., Plow E. F., Frelinger A. L., 3rd, O'Toole T. E., Loftus J. C., Ginsberg M. H. Ligands "activate" integrin alpha IIb beta 3 (platelet GPIIb-IIIa). Cell. 1991 May 3;65(3):409–416. doi: 10.1016/0092-8674(91)90458-b. [DOI] [PubMed] [Google Scholar]
  14. Fox J. E. Regulation of platelet function by the cytoskeleton. Adv Exp Med Biol. 1993;344:175–185. doi: 10.1007/978-1-4615-2994-1_13. [DOI] [PubMed] [Google Scholar]
  15. Fox J. E. The platelet cytoskeleton. Thromb Haemost. 1993 Dec 20;70(6):884–893. [PubMed] [Google Scholar]
  16. Freed E., Gailit J., van der Geer P., Ruoslahti E., Hunter T. A novel integrin beta subunit is associated with the vitronectin receptor alpha subunit (alpha v) in a human osteosarcoma cell line and is a substrate for protein kinase C. EMBO J. 1989 Oct;8(10):2955–2965. doi: 10.1002/j.1460-2075.1989.tb08445.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Haimovich B., Lipfert L., Brugge J. S., Shattil S. J. Tyrosine phosphorylation and cytoskeletal reorganization in platelets are triggered by interaction of integrin receptors with their immobilized ligands. J Biol Chem. 1993 Jul 25;268(21):15868–15877. [PubMed] [Google Scholar]
  18. Hannun Y. A., Greenberg C. S., Bell R. M. Sphingosine inhibition of agonist-dependent secretion and activation of human platelets implies that protein kinase C is a necessary and common event of the signal transduction pathways. J Biol Chem. 1987 Oct 5;262(28):13620–13626. [PubMed] [Google Scholar]
  19. Hawiger J., Parkinson S., Timmons S. Prostacyclin inhibits mobilisation of fibrinogen-binding sites on human ADP- and thrombin-treated platelets. Nature. 1980 Jan 10;283(5743):195–197. doi: 10.1038/283195a0. [DOI] [PubMed] [Google Scholar]
  20. Haystead T. A., Sim A. T., Carling D., Honnor R. C., Tsukitani Y., Cohen P., Hardie D. G. Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature. 1989 Jan 5;337(6202):78–81. doi: 10.1038/337078a0. [DOI] [PubMed] [Google Scholar]
  21. Hillery C. A., Smyth S. S., Parise L. V. Phosphorylation of human platelet glycoprotein IIIa (GPIIIa). Dissociation from fibrinogen receptor activation and phosphorylation of GPIIIa in vitro. J Biol Chem. 1991 Aug 5;266(22):14663–14669. [PubMed] [Google Scholar]
  22. Holmsen H., Setkowsky C. A., Day H. J. Effects of antimycin and 2-deoxyglucose on adenine nucleotides in human platelets. Role of metabolic adenosine triphosphate in primary aggregation, secondary aggregation and shape change of platetets. Biochem J. 1974 Nov;144(2):385–396. doi: 10.1042/bj1440385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Holmsen H., Storm E., Day H. J. Determination of ATP and ADP in blood platelets: a modification of the firefly luciferase assay for plasma. Anal Biochem. 1972 Apr;46(2):489–501. doi: 10.1016/0003-2697(72)90323-5. [DOI] [PubMed] [Google Scholar]
  24. Kloprogge E., Akkerman J. W. Platelet-activating factor (PAF-acether) induces high- and low-affinity binding of fibrinogen to human platelets via independent mechanisms. Biochem J. 1986 Dec 1;240(2):403–412. doi: 10.1042/bj2400403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kloprogge E., Hasselaar P., Akkerman J. W. PAF-acether (1-O-hexadecyl/octadecyl-2-acetyl-sn-glycero-3-phosphocholine)-induced fibrinogen binding to platelets depends on metabolic energy. Biochem J. 1986 Sep 15;238(3):885–891. doi: 10.1042/bj2380885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kloprogge E., Hasselaar P., Gorter G., Akkerman J. W. Stimulus-aggregation coupling in platelets activated with PAF-acether. Biochim Biophys Acta. 1986 Aug 6;883(1):127–137. doi: 10.1016/0304-4165(86)90143-1. [DOI] [PubMed] [Google Scholar]
  27. Kloprogge E., Mommersteeg M., Akkerman J. W. Kinetics of platelet-activating factor 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine-induced fibrinogen binding to human platelets. J Biol Chem. 1986 Aug 25;261(24):11071–11076. [PubMed] [Google Scholar]
  28. Kornecki E., Cooper B. A., Ehrlich Y. H. Identification of a unique type of thrombopathy of human platelets: defect in the exposure of active fibrinogen receptors in a patient with Friedreich's ataxia. J Lab Clin Med. 1988 Jun;111(6):618–626. [PubMed] [Google Scholar]
  29. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  30. Lerea K. M., Tonks N. K., Krebs E. G., Fischer E. H., Glomset J. A. Vanadate and molybdate increase tyrosine phosphorylation in a 50-kilodalton protein and stimulate secretion in electropermeabilized platelets. Biochemistry. 1989 Nov 28;28(24):9286–9292. doi: 10.1021/bi00450a008. [DOI] [PubMed] [Google Scholar]
  31. Linder B. L., Chernoff A., Kaplan K. L., Goodman D. S. Release of platelet-derived growth factor from human platelets by arachidonic acid. Proc Natl Acad Sci U S A. 1979 Aug;76(8):4107–4111. doi: 10.1073/pnas.76.8.4107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Loh E., Beaverson K., Vilaire G., Qi W., Poncz M., Bennett J. S. Agonist-stimulated ligand binding by the platelet integrin alpha IIb beta 3 in a lymphocyte expression system. J Biol Chem. 1995 Aug 4;270(31):18631–18636. doi: 10.1074/jbc.270.31.18631. [DOI] [PubMed] [Google Scholar]
  33. Morii N., Teru-uchi T., Tominaga T., Kumagai N., Kozaki S., Ushikubi F., Narumiya S. A rho gene product in human blood platelets. II. Effects of the ADP-ribosylation by botulinum C3 ADP-ribosyltransferase on platelet aggregation. J Biol Chem. 1992 Oct 15;267(29):20921–20926. [PubMed] [Google Scholar]
  34. Nievelstein P. F., D'Alessio P. A., Sixma J. J. Fibronectin in platelet adhesion to human collagen types I and III. Use of nonfibrillar and fibrillar collagen in flowing blood studies. Arteriosclerosis. 1988 Mar-Apr;8(2):200–206. doi: 10.1161/01.atv.8.2.200. [DOI] [PubMed] [Google Scholar]
  35. O'Toole T. E., Katagiri Y., Faull R. J., Peter K., Tamura R., Quaranta V., Loftus J. C., Shattil S. J., Ginsberg M. H. Integrin cytoplasmic domains mediate inside-out signal transduction. J Cell Biol. 1994 Mar;124(6):1047–1059. doi: 10.1083/jcb.124.6.1047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. O'Toole T. E., Mandelman D., Forsyth J., Shattil S. J., Plow E. F., Ginsberg M. H. Modulation of the affinity of integrin alpha IIb beta 3 (GPIIb-IIIa) by the cytoplasmic domain of alpha IIb. Science. 1991 Nov 8;254(5033):845–847. doi: 10.1126/science.1948065. [DOI] [PubMed] [Google Scholar]
  37. Paris S., Pouysségur J. Further evidence for a phospholipase C-coupled G protein in hamster fibroblasts. Induction of inositol phosphate formation by fluoroaluminate and vanadate and inhibition by pertussis toxin. J Biol Chem. 1987 Feb 15;262(5):1970–1976. [PubMed] [Google Scholar]
  38. Parise L. V., Criss A. B., Nannizzi L., Wardell M. R. Glycoprotein IIIa is phosphorylated in intact human platelets. Blood. 1990 Jun 15;75(12):2363–2368. [PubMed] [Google Scholar]
  39. Schoenwaelder S. M., Jackson S. P., Yuan Y., Teasdale M. S., Salem H. H., Mitchell C. A. Tyrosine kinases regulate the cytoskeletal attachment of integrin alpha IIb beta 3 (platelet glycoprotein IIb/IIIa) and the cellular retraction of fibrin polymers. J Biol Chem. 1994 Dec 23;269(51):32479–32487. [PubMed] [Google Scholar]
  40. Shattil S. J., Brass L. F. Induction of the fibrinogen receptor on human platelets by intracellular mediators. J Biol Chem. 1987 Jan 25;262(3):992–1000. [PubMed] [Google Scholar]
  41. Shaw L. M., Messier J. M., Mercurio A. M. The activation dependent adhesion of macrophages to laminin involves cytoskeletal anchoring and phosphorylation of the alpha 6 beta 1 integrin. J Cell Biol. 1990 Jun;110(6):2167–2174. doi: 10.1083/jcb.110.6.2167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Smyth S. S., Hillery C. A., Parise L. V. Fibrinogen binding to purified platelet glycoprotein IIb-IIIa (integrin alpha IIb beta 3) is modulated by lipids. J Biol Chem. 1992 Aug 5;267(22):15568–15577. [PubMed] [Google Scholar]
  43. Speiser-Ellerton S., Weiss H. J. Studies on platelet protein phosphorylation in patients with impaired responses to platelet agonists. J Lab Clin Med. 1990 Jan;115(1):104–111. [PubMed] [Google Scholar]
  44. Torti M., Sinigaglia F., Ramaschi G., Balduini C. Platelet glycoprotein IIb-IIIa is associated with 21-kDa GTP-binding protein. Biochim Biophys Acta. 1991 Nov 18;1070(1):20–26. doi: 10.1016/0005-2736(91)90141-t. [DOI] [PubMed] [Google Scholar]
  45. Toullec D., Pianetti P., Coste H., Bellevergue P., Grand-Perret T., Ajakane M., Baudet V., Boissin P., Boursier E., Loriolle F. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem. 1991 Aug 25;266(24):15771–15781. [PubMed] [Google Scholar]
  46. Verhoeven A. J., Cook C. A., Holmsen H. Use of actin-bound adenosine 5'-diphosphate as a method to determine the specific 32P-radioactivity of the gamma-phosphoryl group of adenosine 5'-triphosphate in a highly compartmentalized cell, the platelet. Anal Biochem. 1988 Nov 1;174(2):672–678. doi: 10.1016/0003-2697(88)90072-3. [DOI] [PubMed] [Google Scholar]
  47. Verhoeven A. J., Mommersteeg M. E., Akkerman J. W. Metabolic energy is required in human platelets at any stage during optical aggregation and secretion. Biochim Biophys Acta. 1984 Aug 21;800(3):242–250. doi: 10.1016/0304-4165(84)90402-1. [DOI] [PubMed] [Google Scholar]
  48. van Willigen G., Akkerman J. W. Protein kinase C and cyclic AMP regulate reversible exposure of binding sites for fibrinogen on the glycoprotein IIB-IIIA complex of human platelets. Biochem J. 1991 Jan 1;273(Pt 1):115–120. doi: 10.1042/bj2730115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. van Willigen G., Akkerman J. W. Regulation of glycoprotein IIB/IIIA exposure on platelets stimulated with alpha-thrombin. Blood. 1992 Jan 1;79(1):82–90. [PubMed] [Google Scholar]

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