IRAK4, TRAF6, and SFKs are involved in signaling pathway induced by CAP-PEs. (A) Gel-filtered human platelets were stimulated with CAP-PEs (CHP-PE) for the indicated times. Phosphorylation of IRAK4 was detected by western blotting. Gel-filtered human platelets were preincubated with (B) 10 μM TRAF6 blocking peptide (TRAF6 BP) or CP, (C) pan-Src kinase family inhibitor PP2 (10 μM), followed by CAP-PEs (CEP-PE) stimulation. P-selectin expression was assessed by FACS analysis. (D-E) Murine platelets were isolated by gel filtration from (D) Lyn^−/− and (E) Src^−/− mice and stimulated with CAP-PEs (CHP-PE) and platelets’ integrin α_IIbβ₃ activation was assessed by FACS. (F) Gel-filtered human platelets were preincubated with PP2, followed by CAP-PEs (CEP-PE) stimulation for 5 minutes. (G-H) Gel-filtered murine platelets isolated from Src^−/− and Lyn^−/− mice were stimulated with CAP-PEs (CHP-PE) for 5 minutes. (I-K) Gel-filtered human platelets are pretreated with either TLR2 (I) or TLR1 blocking antibody (Ab) (J) or its isotype-matched control IgG; (K) either MyD88 BP or the CP and then stimulated with CAP-PEs (CEP-PE) for 5 minutes. Src phosphorylation was determined by western blot analysis using phospho-specific antibody. ***P < .001, *P < .05, comparing either blocking antibody vs IgG or blocking vs control peptide.