Figure 6: Platelets inhibit TNF-α production by macrophages through the COX1 signalling pathway.

(a) Washed platelets were pre-incubated with aspirin (1 mM) or buffer for 5 min and then incubated with thrombin in an aggregometer at 37 °C for 5 min and centrifuged. LPS plus the supernatants of platelets were added to BMDMs and incubated for 6 h. Thrombin or thrombin plus aspirin were added to the wells with LPS in the absence of platelet supernatant as controls. (b) Platelets from COX1 knockout mice (COX1^−/−) or wild-type littermates (COX1^+/+) were incubated with buffer (resting) or thrombin in an aggregometer at 37 °C for 5 min. LPS plus the supernatants of platelets were added to BMDMs and incubated for 6 h. (c) Washed platelets from C57BL/6J mice were incubated with LPS for 30 min or thrombin in an aggregometer at 37 °C for 5 min. PGE₂ in the supernatant were measured by an enzyme-linked immunosorbent assay (Cayman Chemical, Ann Arbor, MI). (d) BMDMs were pre-incubated with buffer, SC-51089 10 μM, AH 6809 10 μM, pertusis toxin (PTX) 0.5 μg ml⁻¹, or GW627368X (GW62) at 37 °C for 15 min. BMDMs were then added with LPS or LPS plus supernatant from thrombin-activated platelets and incubated at 37 °C 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.