Glycoproteins VI and Ib-IX-V stimulate tyrosine phosphorylation of tyrosine kinase Syk and phospholipase Cγ2 at distinct sites

Glycoproteins GPVI and GPlb-IX-V stimulate robust tyrosine phosphorylation of Syk and PLCgamma2 (phospholipase Cgamma2) in washed platelets, but only the former stimulates pronounced activation of phospholipase. Using phospho-specific antibodies, we demonstrate that GPVI, but not GPlb-IX-V, stimulates significant tyrosine phosphorylation of Syk at the autophosphorylation site pY525/526, a marker of Syk activity. In addition, GPVI stimulates tyrosine phosphorylation of PLCgamma2 at Tyr(753) and Tyr(759), whereas GPIb-IX-V only induces significant phosphorylation at Tyr(753). Both receptors stimulate tyrosine phosphorylation of Btk at the regulatory Tyr(223) and Tyr(551). Syk and Btk phosphorylate peptides from PLCgamma2 containing Tyr(753) and Tyr(759) respectively, suggesting that they may stimulate phosphorylation at these sites in phospholipase. Studies using PLCgamma2-deficient platelets demonstrated that phospholipase is not required for the activation of integrin aIIbbeta3 by GPIb-IX-V. Our results demonstrate fundamental differences between GPVI and GPIb-IX-V in the regulation of tyrosine phosphorylation of Syk and PLCgamma2 consistent with the functional impairment of phospholipase in signalling by GPIb-IX-V.