Figure 5: Platelets or platelet releasates inhibit TNF-α and IL-6 production induced by LPS in bone marrow-derived macrophages.

(a) LPS (1 ng ml⁻¹~10 μg ml⁻¹) was added with buffer or washed platelets (3 × 10⁸ per ml) to BMDMs and incubated for 6 h. TNF-α and IL-6 concentrations in the supernatant were measured. (b) Washed platelets or medium were incubated with ³H-LPS (250 ng ml⁻¹) at 37 °C for 2 h and centrifuged. Disintegrations per minute (DPM) in medium, platelet pellet or supernatant was measured. (c) LPS (100 ng ml⁻¹) was added to BMDMs in the presence or absence of platelets (3 × 10⁸ per ml) and incubated for 6 h. Washed platelets (3 × 10⁸ per ml) were also incubated with LPS (100 ng ml⁻¹) for 30 min or thrombin (Enzyme Research Laboratories, South Bend, IN, 0.1 U ml⁻¹) in aggregometer at 37 °C for 5 min and centrifuged. The resulting supernatants with LPS were added to BMDMs and incubated for 6 h. LPS plus thrombin (Throm) were added to BMDMs and incubated for 6 h as a control. TNF-α and IL-6 concentrations in the supernatant were measured. (d) Washed platelets from G_q knockout mice or wild-type littermates were incubated with thrombin in aggregometer at 37 °C for 5 min and centrifuged. Supernatants plus LPS were added to BMDMs and incubated for 6 h. (e) Platelets (3 × 10⁸ per ml) were pre-incubated with thrombin in aggregometer at 37 °C for 5 min and centrifuged. Serum amyloid A (SAA) (1 μg ml⁻¹) together with buffer or supernatant from thrombin-stimulated platelets were added to BMDMs and incubated for 6 h. Values are means±s.d. (n=3 for all). Differences between two groups were assessed using unpaired two-tailed Student’s t-test.