Negative regulation of Gq-mediated pathways in platelets by G(12/13) pathways through Fyn kinase

Abstract

Platelets contain high levels of Src family kinases (SFKs), but their functional role downstream of G protein pathways has not been completely understood. We found that platelet shape change induced by selective G(12/13) stimulation was potentiated by SFK inhibitors, which was abolished by intracellular calcium chelation. Platelet aggregation, secretion, and intracellular Ca(2+) mobilization mediated by low concentrations of SFLLRN or YFLLRNP were potentiated by SFK inhibitors. However, 2-methylthio-ADP-induced intracellular Ca(2+) mobilization and platelet aggregation were not affected by PP2, suggesting the contribution of SFKs downstream of G(12/13), but not G(q)/G(i), as a negative regulator to platelet activation. Moreover, PP2 potentiated YFLLRNP- and AYPGKF-induced PKC activation, indicating that SFKs downstream of G(12/13) regulate platelet responses through the negative regulation of PKC activation as well as calcium response. SFK inhibitors failed to potentiate platelet responses in the presence of G(q)-selective inhibitor YM254890 or in G(q)-deficient platelets, indicating that SFKs negatively regulate platelet responses through modulation of G(q) pathways. Importantly, AYPGKF-induced platelet aggregation and PKC activation were potentiated in Fyn-deficient but not in Lyn-deficient mice compared with wild-type littermates. We conclude that SFKs, especially Fyn, activated downstream of G(12/13) negatively regulate platelet responses by inhibiting intracellular calcium mobilization and PKC activation through G(q) pathways.

FIGURE 1.

Effect of Src kinase inhibition on platelet shape change induced by YFLLRNP. A, aspirin-treated, washed human platelets were preincubated at 37 °C with DMSO (vehicle), 10 μm Y27632, 10 μm PP1, 10 μm PP2, or 10 μm PP3 for 5 min prior to platelet stimulation with 60 μm YFLLRNP. B and C, the time to half-complete shape change (point at which half-maximal light absorbance was reached) (B) and the initial rate of light absorbance during platelet shape change (change in absorbance/sec) (C) are presented. D, aspirinated human platelets were pretreated at 37 °C with either 10 μm PP3 or 10 μm PP2. Platelet samples that were preincubated with 10 μm Y27632 or 10 μm 5,5′-dimethyl-BAPTA were stimulated with 60 μm YFLLRNP as noted. All tracings are representative of at least three experiments from different donors.

FIGURE 2.

Effect of Src kinase inhibition on platelet aggregation and secretion induced by SFLLRN and 2-MeSADP. Aspirinated, washed human platelets were preincubated at 37 °C with DMSO (vehicle), 10 μm PP1, 10 μm PP2, or 10 μm PP3 for 5 min prior to platelet stimulation with different concentrations of either (A) SFLLRN or (B) 2-MeSADP as noted, and platelet aggregation and ATP secretion were measured as described under “Experimental Procedures.” Results shown are representative of at least three experiments from different donors.

FIGURE 3.

Effect of Src kinase inhibition on platelet aggregation and secretion mediated by the simultaneous stimulation of G_12/13 and G_i/G_z pathways. Washed human platelets were preincubated at 37 °C with DMSO (vehicle), 10 μm PP1, 10 μm PP2, or 10 μm PP3 for 5 min prior to platelet stimulation with either (A) 60 μm YFLLRNP + 100 nm 2-MeSADP + MRS2179 (100 μm) or (B) YFLLRNP (60 μm) + epinephrine (10 μm), and platelet aggregation and ATP secretion were measured. The data shown are representative of at least three experiments.

FIGURE 4.

Effect of PP2 on agonist-induced intracellular calcium mobilization and PKC activation. A, Fura-2 (2 μm)-loaded, aspirinated, washed human platelets were stimulated with different concentrations of YFLLRNP or 2-MeSADP in the presence of either 10 μm PP3 or 10 μm PP2 as noted. Ca²⁺ was measured by recording fluorescence at 510 nm following excitation at 340 and 380 nm. The results show composite data from three experiments, and values are mean ± S.E. (error bars) (n = 3). B, washed platelets were stimulated at 37 °C for 30 s with different concentrations of YFLLRNP in the absence and presence of PP2 (10 μm). Platelet proteins were separated by SDS-PAGE followed by Western blot analysis using Ser(P) PKC substrate or anti-β-actin (lane loading control) antibody. The blot shown is a representative of three independent experiments.

FIGURE 5.

Effect of 5,5′-dimethyl-BAPTA and GF109203X on the potentiation of platelet aggregation and secretion in the presence of PP2. Washed platelets were pretreated with either DMSO or 10 μm PP2. Platelets that were preincubated with 10 μm GF109203X or 10 μm 5,5′-dimethyl-BAPTA were stimulated with 1 μm SFLLRN. All aggregation tracings are representative of at least three experiments.

FIGURE 6.

Role of G_q signaling in G_12/13-induced platelet shape change in the presence of Src kinase inhibitors. A, G_q-deficient mouse platelets were preincubated at 37 °C with DMSO, 10 μm PP1, 10 μm PP2, or 10 μm PP3 for 5 min prior to platelet stimulation with AYPGKF (500 μm). B, washed human platelets were preincubated with either 10 μm PP3 or 10 μm PP2. Platelet samples that were preincubated with 100 nm YM254890 or 10 μm Y27632 were stimulated with 60 μm YFLLRNP as noted. All tracings are representative of at least three experiments from different donors.

FIGURE 7.

Platelet aggregation and PKC activation in Fyn- and Lyn-deficient platelets. Platelets were isolated from Fyn^−/−, Lyn^−/−, or their wild-type littermates. A, platelets isolated from Fyn^−/− and Lyn^−/− mice were stimulated with 80 μm AYPGKF and different concentration of convulxin as indicated, and platelet aggregation was measured. B, platelets isolated from Fyn^−/− mice were preincubated with either 10 μm PP3 or 10 μm PP2 and stimulated with 60 μm AYPGKF and platelet aggregation were measured. C and D, platelets isolated from Fyn^−/− (C) and Lyn^−/− (D) mice were stimulated at 37 °C for 30 s with AYPGKF (100 μm). Equal amounts of proteins were analyzed by Western blot analysis with Ser(P) PKC substrate or anti-β-actin (lane loading control) antibody. Densitometric measurement of Ser(P) PKC is shown, expressed as percentage of agonist control in wild-type platelets. E and F, washed human platelets preincubated with 100 nm YM254890 were stimulated for the time point indicated with 500 μm AYPGKF, and platelet proteins were analyzed by Western blot analysis with phospho-Fyn, phospho-Lyn, or anti-β-actin (lane loading control) antibodies. The Western blot analysis shown is a representative of three independent experiments. Error bars, S.E.