CAP-PEs are recognized by TLR2 and activate platelets via TLR2/TLR1. (A-B) Human platelets were isolated by gel filtration and preincubated with either blocking antibody (for TLR2 and TLR1) or nonimmune isotype-matched control IgG and then stimulated with CAP-PEs (CEP-PE). P-selectin expression was assessed by FACS analysis. (Right) Quantification of the data. (C) Murine platelets were isolated from WT and TLR2^−/− mice by gel filtration, stimulated by 20 μM CAP-PEs (CHP-PE or CEP-PE) and integrin α_IIbβ₃ activation was assessed by FACS analysis. (D) CEP-PE, CHP-PE, or PE binding to TLR2 in the presence of increasing concentrations of CAP-PEs was determined by competitive enzyme-linked immunosorbent assay as described in the “Methods” section. (E) HEK-Blue-TLR2 and HEK-Blue-Null cells were incubated with 20 μM CAP-PEs (CEP-PE or CHP-PE) and TLR2 agonists Pam3CSK4 (25 ng/mL) or LTA (5 μg/mL) or control buffer (NT) for 16 to 18 hours. (F) HEK-Blue-TLR9 and HEK-Blue-Null cells were incubated with CAP-PEs (CEP-PE) and positive control CpG ODN2006 (100 nM). (G) HEK-Blue-TLR2 cells were preincubated with TLR2- or TLR1-blocking antibody or both the blocking antibodies, or IgG control for 12 hours, then incubated with the agonists for 16 to 18 hours. ***P < .001, **P < .01, comparing IgG vs blocking antibody or WT vs TLR2^−/− or treated with NT.