TIRAP and MyD88 are involved in platelet activation by CAP-PEs. (A-B) Gel-filtered human platelets were preincubated with (A) 10 μM TIRAP blocking peptide (TIRAP BP), (B) 10 μM MyD88 blocking peptide (MyD88 BP), or control peptide (CP), followed by stimulation with CAP-PEs (CHP-PE). (Left) Histogram presentation of FACS analysis data; (right) quantified data (mean ± SD) from 3 independent experiments. (C) Murine platelets were isolated by gel filtration from WT and MyD88^−/− mice and were stimulated with CAP-PEs (CHP-PE); integrin α_IIbβ₃ activation was assessed by FACS analysis. (D) HEK-Blue-TLR2 cells were preincubated with MyD88 BP and CP for 12 hours and then stimulated with CAP-PEs (CEP-PE or CHP-PE) for 16 to 18 hours. (E) A total of 40 μg of total protein from TLR2-overexpressing cells and control cells (Null) were separated by SDS-PAGE and immunoblotted against MyD88 antibody. (F,H) Gel-filtered human platelets were stimulated with CAP-PEs (CEP-PE or CHP-PE), lysed, and (F-G) 300 μg of lysate protein was immunoprecipitated (IP) with (F) TLR2 antibody or (G) MyD88 antibody and immunoblotted against (F) TLR1 or (G) TLR2; (H) 50 μg of total protein was separated by SDS-PAGE and TLR2, and MyD88 and actin were detected by western blotting. ***P < .001, **P < .01, *P < .05, comparing either blocking vs control peptide or WT vs knockout mice or control vs treated.