In vitro platelet activation under GPVI stimulation in Ptpn11^T468M/+ mouse platelets. (A) Ptpn11^T468M/+ platelets were stimulated with CRP (0.5 and 1 μg/mL), collagen (0.5 and 1 μg/mL), CLEC-2 antibody (Ab) (5 and 10 μg/mL), TXA2 analog (U46619) (0.1 μM), or thrombin (0.1 U/mL). Representative aggregation curves of 5 mice of each genotype are shown. Graphs represent the percentage of maximal aggregation at different times and area under curve (AUC) (mean ± SEM). (B) P-selectin surface expression in resting platelets or after CRP (1 μg/mL) stimulation was analyzed by flow cytometry. Graph represents mean ± SEM of the mean fluorescence intensity (MFI) fold increase when comparing with resting condition (n = 3 mice of each genotype). (C) 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). (D) JON/A-phycoerythrin Ab binding to resting or stimulated platelets with CRP (1 μg/mL) was analyzed by cytometry. Graph represents mean ± SEM of MFI. (E) Ptpn11^T468M/+ 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 = 8 mice of each genotype). *P < .05, **P < .01, ***P < .001 vs WT or WT resting according to 2-way ANOVA; †P < .05 according to 2-way ANOVA; ‡P < .05, ‡‡P < .01, ‡‡‡P < .001 vs Ptpn11^T468M/+ resting according to 2-way ANOVA.