TLR9 signaling results in type C CpG sequestration, increased CD62P surface expression, and PLT clumping. (A and B) Flow cytometric analysis showing representative forward versus side scatter profiles of human washed PLTs under resting conditions and after incubation with synthetic unmethylated type C CpG ODNs (characteristic of bacterial/viral DNA) before (A) and after (B) type IV collagen preincubation. Outlines represent PLT gate used for sample thresholding by forward and side scatter. (C) Quantification of the percentage of PLTs expressing TLR9, CD62P, and type C CpG was normalized to resting levels to resolve the difference on agonist exposure over time. Incubation of resting washed human PLTs with type C CpG ODNs resulted in a 40% increase in TLR9 surface expression followed by a 30% increased type C CpG sequestration and CD62P surface expression over 20 min. (D) Type IV collagen preincubation resulted in considerably increased type C CpG sequestration, CD62P surface expression above resting PLT controls, and significant PLT clumping within 30 s of type C CpG addition. (E) Mouse PLTs show a more pronounced ODN sequestration and comparable CD62P expression after type C CpG incubation. Unlike in human PLTs, TLR9 surface expression is not changed in mice. Although TLR9 KO mice show reduced levels of ODN sequestration and CD62P surface expression 20 min after type C CpG addition, preincubation with type IV collagen still resulted in immediate PLT clumping when type C CpG was added (not depicted). Statistical significance for marked pairings was established using a one-tailed Student’s t test for paired samples (*, P < 0.05; **, P < 0.01). Error bars represent one standard deviation about the mean.