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. 1997 Jan 15;16(2):252–259. doi: 10.1093/emboj/16.2.252

Rapid Ca2+-mediated activation of Rap1 in human platelets.

B Franke 1, J W Akkerman 1, J L Bos 1
PMCID: PMC1169632  PMID: 9029146

Abstract

Rap1 is a small, Ras-like GTPase whose function and regulation are still largely unknown. We have developed a novel assay to monitor the active, GTP-bound form of Rap1 based on the differential affinity of Rap1GTP and Rap1GDP for the Rap binding domain of RalGDS (RBD). Stimulation of blood platelets with alpha-thrombin or other platelet activators caused a rapid and strong induction of Rap1 that associated with RBD in vitro. Binding to RBD increased from undetectable levels in resting platelets to >50% of total Rap1 within 30 s after stimulation. An increase in the intracellular Ca2+ concentration is both necessary and sufficient for Rap1 activation since it was induced by agents that increase intracellular Ca2+ and inhibited by a Ca2+-chelating agent. Neither inhibition of translocation of Rap1 to the cytoskeleton nor inhibition of platelet aggregation affected thrombin-induced activation of Rap1. In contrast, prostaglandin I2 (PGI2), a strong negative regulator of platelet function, inhibited agonist-induced as well as Ca2+-induced activation of Rap1. From our results, we conclude that Rap1 activation in platelets is an important common event in early agonist-induced signalling, and that this activation is mediated by an increased intracellular Ca2+ concentration.

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Selected References

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

  1. Altschuler D. L., Peterson S. N., Ostrowski M. C., Lapetina E. G. Cyclic AMP-dependent activation of Rap1b. J Biol Chem. 1995 May 5;270(18):10373–10376. doi: 10.1074/jbc.270.18.10373. [DOI] [PubMed] [Google Scholar]
  2. Ammit A. J., O'Neill C. Rapid and selective measurement of platelet-activating factor using a quantitative bioassay of platelet aggregation. J Pharmacol Methods. 1991 Aug;26(1):7–21. doi: 10.1016/0160-5402(91)90050-f. [DOI] [PubMed] [Google Scholar]
  3. Authi K. S., Bokkala S., Patel Y., Kakkar V. V., Munkonge F. Ca2+ release from platelet intracellular stores by thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone: relationship to Ca2+ pools and relevance in platelet activation. Biochem J. 1993 Aug 15;294(Pt 1):119–126. doi: 10.1042/bj2940119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benton A. M., Gerrard J. M., Michiel T., Kindom S. E. Are lysophosphatidic acids or phosphatidic acids involved in stimulus activation coupling in platelets? Blood. 1982 Sep;60(3):642–649. [PubMed] [Google Scholar]
  5. Burgering B. M., Pronk G. J., van Weeren P. C., Chardin P., Bos J. L. cAMP antagonizes p21ras-directed activation of extracellular signal-regulated kinase 2 and phosphorylation of mSos nucleotide exchange factor. EMBO J. 1993 Nov;12(11):4211–4220. doi: 10.1002/j.1460-2075.1993.tb06105.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Cavallini L., Alexandre A. Ca2+ efflux from platelets. Control by protein kinase C and the filling state of the intracellular Ca2+ stores. Eur J Biochem. 1994 Jun 1;222(2):693–702. doi: 10.1111/j.1432-1033.1994.tb18914.x. [DOI] [PubMed] [Google Scholar]
  8. Cavallini L., Coassin M., Alexandre A. Two classes of agonist-sensitive Ca2+ stores in platelets, as identified by their differential sensitivity to 2,5-di-(tert-butyl)-1,4-benzohydroquinone and thapsigargin. Biochem J. 1995 Sep 1;310(Pt 2):449–452. doi: 10.1042/bj3100449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cook S. J., McCormick F. Inhibition by cAMP of Ras-dependent activation of Raf. Science. 1993 Nov 12;262(5136):1069–1072. doi: 10.1126/science.7694367. [DOI] [PubMed] [Google Scholar]
  10. Cook S. J., Rubinfeld B., Albert I., McCormick F. RapV12 antagonizes Ras-dependent activation of ERK1 and ERK2 by LPA and EGF in Rat-1 fibroblasts. EMBO J. 1993 Sep;12(9):3475–3485. doi: 10.1002/j.1460-2075.1993.tb06022.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dash D., Aepfelbacher M., Siess W. Integrin alpha IIb beta 3-mediated translocation of CDC42Hs to the cytoskeleton in stimulated human platelets. J Biol Chem. 1995 Jul 21;270(29):17321–17326. doi: 10.1074/jbc.270.29.17321. [DOI] [PubMed] [Google Scholar]
  12. Doni M. G., Cavallini L., Alexandre A. Ca2+ influx in platelets: activation by thrombin and by the depletion of the stores. Effect of cyclic nucleotides. Biochem J. 1994 Oct 15;303(Pt 2):599–605. doi: 10.1042/bj3030599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fischer T. H., Gatling M. N., Lacal J. C., White G. C., 2nd rap1B, a cAMP-dependent protein kinase substrate, associates with the platelet cytoskeleton. J Biol Chem. 1990 Nov 15;265(32):19405–19408. [PubMed] [Google Scholar]
  14. Fischer T. H., Gatling M. N., McCormick F., Duffy C. M., White G. C., 2nd Incorporation of Rap 1b into the platelet cytoskeleton is dependent on thrombin activation and extracellular calcium. J Biol Chem. 1994 Jun 24;269(25):17257–17261. [PubMed] [Google Scholar]
  15. Fox J. E. The platelet cytoskeleton. Thromb Haemost. 1993 Dec 20;70(6):884–893. [PubMed] [Google Scholar]
  16. Frech M., John J., Pizon V., Chardin P., Tavitian A., Clark R., McCormick F., Wittinghofer A. Inhibition of GTPase activating protein stimulation of Ras-p21 GTPase by the Krev-1 gene product. Science. 1990 Jul 13;249(4965):169–171. doi: 10.1126/science.2164710. [DOI] [PubMed] [Google Scholar]
  17. Gabig T. G., Crean C. D., Mantel P. L., Rosli R. Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation. Blood. 1995 Feb 1;85(3):804–811. [PubMed] [Google Scholar]
  18. Grünberg B., Kruse H. J., Negrescu E. V., Siess W. Platelet rap1B phosphorylation is a sensitive marker for the action of cyclic AMP- and cyclic GMP-increasing platelet inhibitors and vasodilators. J Cardiovasc Pharmacol. 1995 Apr;25(4):545–551. doi: 10.1097/00005344-199504000-00006. [DOI] [PubMed] [Google Scholar]
  19. Hartwig J. H. Mechanisms of actin rearrangements mediating platelet activation. J Cell Biol. 1992 Sep;118(6):1421–1442. doi: 10.1083/jcb.118.6.1421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hata Y., Kikuchi A., Sasaki T., Schaber M. D., Gibbs J. B., Takai Y. Inhibition of the ras p21 GTPase-activating protein-stimulated GTPase activity of c-Ha-ras p21 by smg p21 having the same putative effector domain as ras p21s. J Biol Chem. 1990 May 5;265(13):7104–7107. [PubMed] [Google Scholar]
  21. Herrmann C., Horn G., Spaargaren M., Wittinghofer A. Differential interaction of the ras family GTP-binding proteins H-Ras, Rap1A, and R-Ras with the putative effector molecules Raf kinase and Ral-guanine nucleotide exchange factor. J Biol Chem. 1996 Mar 22;271(12):6794–6800. doi: 10.1074/jbc.271.12.6794. [DOI] [PubMed] [Google Scholar]
  22. Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. doi: 10.1016/0092-8674(92)90115-s. [DOI] [PubMed] [Google Scholar]
  23. Inazu T., Taniguchi T., Ohta S., Miyabo S., Yamamura H. The lectin wheat germ agglutinin induces rapid protein-tyrosine phosphorylation in human platelets. Biochem Biophys Res Commun. 1991 Feb 14;174(3):1154–1158. doi: 10.1016/0006-291x(91)91541-j. [DOI] [PubMed] [Google Scholar]
  24. Kroll M. H., Schafer A. I. Biochemical mechanisms of platelet activation. Blood. 1989 Sep;74(4):1181–1195. [PubMed] [Google Scholar]
  25. Lapetina E. G., Lacal J. C., Reep B. R., Molina y Vedia L. A ras-related protein is phosphorylated and translocated by agonists that increase cAMP levels in human platelets. Proc Natl Acad Sci U S A. 1989 May;86(9):3131–3134. doi: 10.1073/pnas.86.9.3131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nakamura K., Kimura M., Aviv A. Role of cyclic nucleotides in store-mediated external Ca2+ entry in human platelets. Biochem J. 1995 Aug 15;310(Pt 1):263–269. doi: 10.1042/bj3100263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nassar N., Horn G., Herrmann C., Scherer A., McCormick F., Wittinghofer A. The 2.2 A crystal structure of the Ras-binding domain of the serine/threonine kinase c-Raf1 in complex with Rap1A and a GTP analogue. Nature. 1995 Jun 15;375(6532):554–560. doi: 10.1038/375554a0. [DOI] [PubMed] [Google Scholar]
  28. Noda M. Structures and functions of the K rev-1 transformation suppressor gene and its relatives. Biochim Biophys Acta. 1993 May 25;1155(1):97–109. doi: 10.1016/0304-419x(93)90024-7. [DOI] [PubMed] [Google Scholar]
  29. Okamoto Y., Ninomiya H., Miwa S., Masaki T. Capacitative Ca2+ entry in human platelets is resistant to nitric oxide. Biochem Biophys Res Commun. 1995 Jul 6;212(1):90–96. doi: 10.1006/bbrc.1995.1940. [DOI] [PubMed] [Google Scholar]
  30. Polakis P., Rubinfeld B., McCormick F. Phosphorylation of rap1GAP in vivo and by cAMP-dependent kinase and the cell cycle p34cdc2 kinase in vitro. J Biol Chem. 1992 May 25;267(15):10780–10785. [PubMed] [Google Scholar]
  31. Quinn M. T., Parkos C. A., Walker L., Orkin S. H., Dinauer M. C., Jesaitis A. J. Association of a Ras-related protein with cytochrome b of human neutrophils. Nature. 1989 Nov 9;342(6246):198–200. doi: 10.1038/342198a0. [DOI] [PubMed] [Google Scholar]
  32. Rubinfeld B., Crosier W. J., Albert I., Conroy L., Clark R., McCormick F., Polakis P. Localization of the rap1GAP catalytic domain and sites of phosphorylation by mutational analysis. Mol Cell Biol. 1992 Oct;12(10):4634–4642. doi: 10.1128/mcb.12.10.4634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shen T. Y., Winter C. A. Chemical and biological studies on indomethacin, sulindac and their analogs. Adv Drug Res. 1977;12:90–245. [PubMed] [Google Scholar]
  34. Siess W., Grünberg B. Phosphorylation of rap1B by protein kinase A is not involved in platelet inhibition by cyclic AMP. Cell Signal. 1993 Mar;5(2):209–214. doi: 10.1016/0898-6568(93)90071-s. [DOI] [PubMed] [Google Scholar]
  35. Siess W., Lapetina E. G. Prostacyclin inhibits platelet aggregation induced by phorbol ester or Ca2+ ionophore at steps distal to activation of protein kinase C and Ca2+-dependent protein kinases. Biochem J. 1989 Feb 15;258(1):57–65. doi: 10.1042/bj2580057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Siess W. Molecular mechanisms of platelet activation. Physiol Rev. 1989 Jan;69(1):58–178. doi: 10.1152/physrev.1989.69.1.58. [DOI] [PubMed] [Google Scholar]
  37. Siess W., Winegar D. A., Lapetina E. G. Rap1-B is phosphorylated by protein kinase A in intact human platelets. Biochem Biophys Res Commun. 1990 Jul 31;170(2):944–950. doi: 10.1016/0006-291x(90)92182-y. [DOI] [PubMed] [Google Scholar]
  38. Spaargaren M., Bischoff J. R. Identification of the guanine nucleotide dissociation stimulator for Ral as a putative effector molecule of R-ras, H-ras, K-ras, and Rap. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12609–12613. doi: 10.1073/pnas.91.26.12609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tamaoki T. Use and specificity of staurosporine, UCN-01, and calphostin C as protein kinase inhibitors. Methods Enzymol. 1991;201:340–347. doi: 10.1016/0076-6879(91)01030-6. [DOI] [PubMed] [Google Scholar]
  40. Torti M., Lapetina E. G. Role of rap1B and p21ras GTPase-activating protein in the regulation of phospholipase C-gamma 1 in human platelets. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7796–7800. doi: 10.1073/pnas.89.16.7796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Torti M., Lapetina E. G. Structure and function of rap proteins in human platelets. Thromb Haemost. 1994 May;71(5):533–543. [PubMed] [Google Scholar]
  42. 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]
  43. Watson S. P., Poole A., Asselin J. Ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and the tyrphostin ST271 inhibit phospholipase C in human platelets by preventing Ca2+ entry. Mol Pharmacol. 1995 Apr;47(4):823–830. [PubMed] [Google Scholar]
  44. Wittinghofer A., Herrmann C. Ras-effector interactions, the problem of specificity. FEBS Lett. 1995 Aug 1;369(1):52–56. doi: 10.1016/0014-5793(95)00667-x. [DOI] [PubMed] [Google Scholar]
  45. Wu J., Dent P., Jelinek T., Wolfman A., Weber M. J., Sturgill T. W. Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate. Science. 1993 Nov 12;262(5136):1065–1069. doi: 10.1126/science.7694366. [DOI] [PubMed] [Google Scholar]
  46. Yoshida Y., Kawata M., Miura Y., Musha T., Sasaki T., Kikuchi A., Takai Y. Microinjection of smg/rap1/Krev-1 p21 into Swiss 3T3 cells induces DNA synthesis and morphological changes. Mol Cell Biol. 1992 Aug;12(8):3407–3414. doi: 10.1128/mcb.12.8.3407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zhang Z., Vuori K., Wang H., Reed J. C., Ruoslahti E. Integrin activation by R-ras. Cell. 1996 Apr 5;85(1):61–69. doi: 10.1016/s0092-8674(00)81082-x. [DOI] [PubMed] [Google Scholar]
  48. van Willigen G., Hers I., Gorter G., Akkerman J. W. Exposure of ligand-binding sites on platelet integrin alpha IIB/beta 3 by phosphorylation of the beta 3 subunit. Biochem J. 1996 Mar 15;314(Pt 3):769–779. [PMC free article] [PubMed] [Google Scholar]

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