Platelet secretion and signaling under GPVI stimulation in Ptpn11D61G/+ mice. (A) P-selectin surface expression in resting platelets or after CRP (1 μg/mL) stimulation was analyzed by flow cytometry. Graph represents mean ± SEM of mean fluorescence intensity fold increase when comparing with resting condition (n = 4 mice of each genotype). (B) TXA2 production in resting or CRP-stimulated (1 μg/mL) platelets was analyzed by mass spectrometry by quantifying the production of its stable derivative, TXB2. Graph represents mean ± SEM (n = 4 mice of each genotype). (C) Ptpn11D61G/+ platelets were stimulated with CRP (3 μg/mL) at 37°C in nonaggregating conditions over 0.5, 1.5, and 6 minutes. Platelet lysates were analyzed by western blotting using an antiphosphotyrosine (clone 4G10) antibody (Ab). FcRγ chain and actin were used as loading controls. Representative western blots of 3 independent experiments are shown. (D) Ptpn11D61G/+ platelets were stimulated with CRP (1 μg/mL) at 37°C in nonaggregating conditions over 1, 3, and 6 minutes. Platelet lysates were analyzed by western blotting using specific Abs: phosphorylated Syk (pSyk) (Tyr525/526), pLAT (Tyr191), pAKT (Ser473), and pPLCγ2 (Tyr759). Syk, LAT, AKT, and PLCγ2 proteins were used as loading controls. Representative western blots are shown, and quantifications are represented as mean ± SEM (n = 5 mice of each genotype). *P < .05, ***P < .001 vs WT; †P < .05, ††P < .01, †††P < .001 according to 2-way ANOVA.