Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1999 Aug 15;342(Pt 1):171–177.

A collagen-related peptide regulates phospholipase Cgamma2 via phosphatidylinositol 3-kinase in human platelets.

J M Pasquet 1, R Bobe 1, B Gross 1, M P Gratacap 1, M G Tomlinson 1, B Payrastre 1, S P Watson 1
PMCID: PMC1220450  PMID: 10432314

Abstract

The collagen receptor glycoprotein VI (GPVI) induces platelet activation through a similar pathway to that used by immune receptors. In the present study we have investigated the role of phosphatidylinositol 3-kinase (PI 3-kinase) in GPVI signalling. Our results show that collagen-related peptide ¿CRP: [GCP*(GPP*)(10)GCP*G](n); P*=hydroxyproline¿, which is selective to GPVI, induces formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 3,4-bisphosphate [PI(3, 4)P(2)] in platelets. The increase in the two 3-phosphorylated lipids is inhibited completely by wortmannin and by LY294002, two structurally unrelated inhibitors of PI 3-kinase. The formation of inositol phosphates and phosphatidic acid (PA), two markers of phospholipase C (PLC) activation, by CRP are inhibited by between 50 and 85% in the presence of wortmannin and LY294002. This is associated with inhibition of elevation of intracellular Ca(2+) ([Ca(2+)](i)) and aggregation. Wortmannin and LY294002 also partially inhibit elevation of Ca(2+) by CRP in murine megakaryocytes. Microinjection of the pleckstrin-homology PH domain of Bruton's tyrosine kinase, which binds selectively to PI(3,4, 5)P(3), but not the R28C (Arg(28)-->Cys) mutant which binds to PI(3, 4,5)P(3) with low affinity, also inhibits elevation of [Ca(2+)](i) in megakaryocytes, suggesting that it is this lipid species which mediates the action of the PI 3-kinase pathway. Studies in platelets show that the action of wortmannin and LY294002 is not mediated through an alteration in tyrosine phosphorylation of PLCgamma2. These results demonstrate that PI 3-kinase is required for full activation of PLCgamma2 by GPVI in platelets and megakaryocytes.

Full Text

The Full Text of this article is available as a PDF (197.0 KB).

Selected References

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

  1. Asselin J., Gibbins J. M., Achison M., Lee Y. H., Morton L. F., Farndale R. W., Barnes M. J., Watson S. P. A collagen-like peptide stimulates tyrosine phosphorylation of syk and phospholipase C gamma2 in platelets independent of the integrin alpha2beta1. Blood. 1997 Feb 15;89(4):1235–1242. [PubMed] [Google Scholar]
  2. Bae Y. S., Cantley L. G., Chen C. S., Kim S. R., Kwon K. S., Rhee S. G. Activation of phospholipase C-gamma by phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem. 1998 Feb 20;273(8):4465–4469. doi: 10.1074/jbc.273.8.4465. [DOI] [PubMed] [Google Scholar]
  3. Barker S. A., Caldwell K. K., Pfeiffer J. R., Wilson B. S. Wortmannin-sensitive phosphorylation, translocation, and activation of PLCgamma1, but not PLCgamma2, in antigen-stimulated RBL-2H3 mast cells. Mol Biol Cell. 1998 Feb;9(2):483–496. doi: 10.1091/mbc.9.2.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blake R. A., Asselin J., Walker T., Watson S. P. Fc gamma receptor II stimulated formation of inositol phosphates in human platelets is blocked by tyrosine kinase inhibitors and associated with tyrosine phosphorylation of the receptor. FEBS Lett. 1994 Mar 28;342(1):15–18. doi: 10.1016/0014-5793(94)80575-x. [DOI] [PubMed] [Google Scholar]
  5. Bolland S., Pearse R. N., Kurosaki T., Ravetch J. V. SHIP modulates immune receptor responses by regulating membrane association of Btk. Immunity. 1998 Apr;8(4):509–516. doi: 10.1016/s1074-7613(00)80555-5. [DOI] [PubMed] [Google Scholar]
  6. Derman M. P., Toker A., Hartwig J. H., Spokes K., Falck J. R., Chen C. S., Cantley L. C., Cantley L. G. The lipid products of phosphoinositide 3-kinase increase cell motility through protein kinase C. J Biol Chem. 1997 Mar 7;272(10):6465–6470. doi: 10.1074/jbc.272.10.6465. [DOI] [PubMed] [Google Scholar]
  7. Domin J., Waterfield M. D. Using structure to define the function of phosphoinositide 3-kinase family members. FEBS Lett. 1997 Jun 23;410(1):91–95. doi: 10.1016/s0014-5793(97)00617-0. [DOI] [PubMed] [Google Scholar]
  8. Ezumi Y., Shindoh K., Tsuji M., Takayama H. Physical and functional association of the Src family kinases Fyn and Lyn with the collagen receptor glycoprotein VI-Fc receptor gamma chain complex on human platelets. J Exp Med. 1998 Jul 20;188(2):267–276. doi: 10.1084/jem.188.2.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Falasca M., Logan S. K., Lehto V. P., Baccante G., Lemmon M. A., Schlessinger J. Activation of phospholipase C gamma by PI 3-kinase-induced PH domain-mediated membrane targeting. EMBO J. 1998 Jan 15;17(2):414–422. doi: 10.1093/emboj/17.2.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fukuda M., Kojima T., Kabayama H., Mikoshiba K. Mutation of the pleckstrin homology domain of Bruton's tyrosine kinase in immunodeficiency impaired inositol 1,3,4,5-tetrakisphosphate binding capacity. J Biol Chem. 1996 Nov 29;271(48):30303–30306. doi: 10.1074/jbc.271.48.30303. [DOI] [PubMed] [Google Scholar]
  11. Giuriato S., Payrastre B., Drayer A. L., Plantavid M., Woscholski R., Parker P., Erneux C., Chap H. Tyrosine phosphorylation and relocation of SHIP are integrin-mediated in thrombin-stimulated human blood platelets. J Biol Chem. 1997 Oct 24;272(43):26857–26863. doi: 10.1074/jbc.272.43.26857. [DOI] [PubMed] [Google Scholar]
  12. Gratacap M. P., Payrastre B., Viala C., Mauco G., Plantavid M., Chap H. Phosphatidylinositol 3,4,5-trisphosphate-dependent stimulation of phospholipase C-gamma2 is an early key event in FcgammaRIIA-mediated activation of human platelets. J Biol Chem. 1998 Sep 18;273(38):24314–24321. doi: 10.1074/jbc.273.38.24314. [DOI] [PubMed] [Google Scholar]
  13. Gross B. S., Lee J. R., Clements J. L., Turner M., Tybulewicz V. L., Findell P. R., Koretzky G. A., Watson S. P. Tyrosine phosphorylation of SLP-76 is downstream of Syk following stimulation of the collagen receptor in platelets. J Biol Chem. 1999 Feb 26;274(9):5963–5971. doi: 10.1074/jbc.274.9.5963. [DOI] [PubMed] [Google Scholar]
  14. Hendricks-Taylor L. R., Motto D. G., Zhang J., Siraganian R. P., Koretzky G. A. SLP-76 is a substrate of the high affinity IgE receptor-stimulated protein tyrosine kinases in rat basophilic leukemia cells. J Biol Chem. 1997 Jan 10;272(2):1363–1367. doi: 10.1074/jbc.272.2.1363. [DOI] [PubMed] [Google Scholar]
  15. Heraud J. M., Racaud-Sultan C., Gironcel D., Albigès-Rizo C., Giacomini T., Roques S., Martel V., Breton-Douillon M., Perret B., Chap H. Lipid products of phosphoinositide 3-kinase and phosphatidylinositol 4',5'-bisphosphate are both required for ADP-dependent platelet spreading. J Biol Chem. 1998 Jul 10;273(28):17817–17823. doi: 10.1074/jbc.273.28.17817. [DOI] [PubMed] [Google Scholar]
  16. Kehrel B., Wierwille S., Clemetson K. J., Anders O., Steiner M., Knight C. G., Farndale R. W., Okuma M., Barnes M. J. Glycoprotein VI is a major collagen receptor for platelet activation: it recognizes the platelet-activating quaternary structure of collagen, whereas CD36, glycoprotein IIb/IIIa, and von Willebrand factor do not. Blood. 1998 Jan 15;91(2):491–499. [PubMed] [Google Scholar]
  17. Lee S. B., Rao A. K., Lee K. H., Yang X., Bae Y. S., Rhee S. G. Decreased expression of phospholipase C-beta 2 isozyme in human platelets with impaired function. Blood. 1996 Sep 1;88(5):1684–1691. [PubMed] [Google Scholar]
  18. Mahajan S., Fargnoli J., Burkhardt A. L., Kut S. A., Saouaf S. J., Bolen J. B. Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase. Mol Cell Biol. 1995 Oct;15(10):5304–5311. doi: 10.1128/mcb.15.10.5304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Melford S. K., Turner M., Briddon S. J., Tybulewicz V. L., Watson S. P. Syk and Fyn are required by mouse megakaryocytes for the rise in intracellular calcium induced by a collagen-related peptide. J Biol Chem. 1997 Oct 31;272(44):27539–27542. doi: 10.1074/jbc.272.44.27539. [DOI] [PubMed] [Google Scholar]
  20. Morton L. F., Hargreaves P. G., Farndale R. W., Young R. D., Barnes M. J. Integrin alpha 2 beta 1-independent activation of platelets by simple collagen-like peptides: collagen tertiary (triple-helical) and quaternary (polymeric) structures are sufficient alone for alpha 2 beta 1-independent platelet reactivity. Biochem J. 1995 Mar 1;306(Pt 2):337–344. doi: 10.1042/bj3060337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Poole A., Gibbins J. M., Turner M., van Vugt M. J., van de Winkel J. G., Saito T., Tybulewicz V. L., Watson S. P. The Fc receptor gamma-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen. EMBO J. 1997 May 1;16(9):2333–2341. doi: 10.1093/emboj/16.9.2333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Quek L. S., Bolen J., Watson S. P. A role for Bruton's tyrosine kinase (Btk) in platelet activation by collagen. Curr Biol. 1998 Oct 8;8(20):1137–1140. doi: 10.1016/s0960-9822(98)70471-3. [DOI] [PubMed] [Google Scholar]
  23. Rameh L. E., Arvidsson A. k., Carraway K. L., 3rd, Couvillon A. D., Rathbun G., Crompton A., VanRenterghem B., Czech M. P., Ravichandran K. S., Burakoff S. J. A comparative analysis of the phosphoinositide binding specificity of pleckstrin homology domains. J Biol Chem. 1997 Aug 29;272(35):22059–22066. doi: 10.1074/jbc.272.35.22059. [DOI] [PubMed] [Google Scholar]
  24. Rameh L. E., Rhee S. G., Spokes K., Kazlauskas A., Cantley L. C., Cantley L. G. Phosphoinositide 3-kinase regulates phospholipase Cgamma-mediated calcium signaling. J Biol Chem. 1998 Sep 11;273(37):23750–23757. doi: 10.1074/jbc.273.37.23750. [DOI] [PubMed] [Google Scholar]
  25. Rittenhouse S. E. Phosphoinositide 3-kinase activation and platelet function. Blood. 1996 Dec 15;88(12):4401–4414. [PubMed] [Google Scholar]
  26. Salim K., Bottomley M. J., Querfurth E., Zvelebil M. J., Gout I., Scaife R., Margolis R. L., Gigg R., Smith C. I., Driscoll P. C. Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase. EMBO J. 1996 Nov 15;15(22):6241–6250. [PMC free article] [PubMed] [Google Scholar]
  27. Scharenberg A. M., El-Hillal O., Fruman D. A., Beitz L. O., Li Z., Lin S., Gout I., Cantley L. C., Rawlings D. J., Kinet J. P. Phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3)/Tec kinase-dependent calcium signaling pathway: a target for SHIP-mediated inhibitory signals. EMBO J. 1998 Apr 1;17(7):1961–1972. doi: 10.1093/emboj/17.7.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Takata M., Kurosaki T. A role for Bruton's tyrosine kinase in B cell antigen receptor-mediated activation of phospholipase C-gamma 2. J Exp Med. 1996 Jul 1;184(1):31–40. doi: 10.1084/jem.184.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Toker A., Cantley L. C. Signalling through the lipid products of phosphoinositide-3-OH kinase. Nature. 1997 Jun 12;387(6634):673–676. doi: 10.1038/42648. [DOI] [PubMed] [Google Scholar]
  30. Watson S. P., Gibbins J. Collagen receptor signalling in platelets: extending the role of the ITAM. Immunol Today. 1998 Jun;19(6):260–264. doi: 10.1016/s0167-5699(98)01267-5. [DOI] [PubMed] [Google Scholar]
  31. Zhang W., Sloan-Lancaster J., Kitchen J., Trible R. P., Samelson L. E. LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell. 1998 Jan 9;92(1):83–92. doi: 10.1016/s0092-8674(00)80901-0. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES