Figure 8.

ODN-activated PLTs do not form thrombi on collagen type IV and endocytose type C CpG to distinct granules that do not colocalize with TLR9. (A–C) Washed human whole-blood PLTs (A) and PRP (B) were pretreated with 5 µM control ODN, type C CpG ODN, or a vehicle control and perfused at a shear rate of 200 s⁻¹ (flow rate of 18.7 µl/min) over a surface coated with type IV collagen or type I collagen (C) for 10 min. Although addition of ODN to PLTs resulted in PLT clumping in the presence of type IV collagen (white arrows), these did not form thrombi as compared with type I collagen (positive control, black arrows). Addition of type C CpG did not result in increased thrombus formation relative to the ODN control in PRP on the type I collagen-coated surface. (D) PLTs were incubated with FITC-conjugated type C CpG at 37°C and 5% CO₂ for a period of ≤4 h. PLTs were subsequently spun down onto poly-l-lysine–coated glass cover slides and probed for TLR9. After 1 h of incubation, the majority of FITC-conjugated type C CpG was associated with the PLT surface and did not colocalize with TLR9. (E) Samples were examined by wide-field fluorescence microscopy. After 4 h of incubation, FITC-conjugated type C CpG became endocytosed by resting human PLTs into distinct granules that showed minimal colocalization with TLR9. Insets represent (from left to right) type C CpG labeling, TLR9 labeling, and colabeling of magnified region outlined by the yellow boxes in the images. (insets) Bars, 2 µm.