Biochemical Journal

Research article

Differential effect of the inhibition of Grb2–SH3 interactions in platelet activation induced by thrombin and by Fc receptor engagement

Abdelhafid SACI, Wang-Qing LIU, Michel VIDAL, Christiane GARBAY, Francine RENDU, Christilla BACHELOT-LOZA

Abstract

The adaptor protein Grb2 (growth factor receptor-bound protein 2) is involved in cell proliferation via the Ras signalling pathway. In order to study the role of Grb2 in blood platelet responses, we used a peptide containing two proline-rich sequences derived from Sos (peptidimer), which binds to Grb2—Src homology 3 domain (SH3) with a high affinity, and hence inhibits Grb2—SH3-mediated protein interactions. Platelet aggregation and 5-hydroxytryptamine (serotonin) release measured in the presence of the peptidimer were: (i) significantly decreased when induced by thrombin; and (ii) potentiated when induced by the engagement of the Fc receptor. In thrombin-activated platelets, the Grb2—SH2 domain formed an association with the β3 subunit of the αIIb—β3 integrin (GPIIb—IIIa), Shc, Syk, Src and SHP1 (SH2-containing phosphotyrosine phosphatase 1), whereas these associations did not occur after the engagement of the receptor for the Fc domain of IgG (FcγRIIa) or in resting platelets. Grb2—SH3 domains formed an association with the proline-rich sequences of Sos and Cbl in both resting and activated platelets, since the peptidimer abolished these associations. Inhibition of both fibrinogen binding and platelet aggregation by the peptide RGDS (Arg-Gly-Asp-Ser) had no effect on thrombin-induced Grb2—SH2 domain association with the aforementioned signalling molecules, indicating that these associations occurred during thrombin-induced ‘inside-out’ signalling. Platelet aggregation induced by direct activation via αIIb—β3 ('outside-in’ signalling) was potentiated by the peptidimer. The results show that inhibition of Grb2—SH3 interactions with signal-transduction proteins down-regulates thrombin-induced platelet activation, but also potentiates Fc receptor- and αIIb—β3-mediated platelet activation.

  • adaptor protein
  • extracellular-signal-regulated protein kinase (ERK)
  • integrin αIIb–β3
  • peptidimer
  • SH2/SH3

Footnotes

  • 2 Formerly known as INSERM U266.

  • Abbreviations used: ERK, extracellular-signal-regulated protein kinase; Grb2, growth factor receptor-bound protein 2; LAT, linker for activator of T cells; mAb, monoclonal antibody; MAPK, mitogen-activated protein kinase; NP-40, Nonidet P40; PI 3-kinase, phosphoinositide 3-kinase; PTP1C, protein tyrosine phosphatase 1C; RGDS, Arg-Gly-Asp-Ser; FcγRIIa, receptor for the Fc domain of IgG; SH2/3, Src homology domain 2/3; SHIP, SH2-containing inositol phosphatase; SHP1, SH2-containing phosphotyrosine phosphatase 1; WASP, Wiskott—Aldrich syndrome protein.