Figure 5.

Extracellular LPA generated by platelet activation stimulates PPRE reporter expression in monocytic cells. (a) Activated platelets generate an agonist that stimulates a CD36 reporter through its PPRE. Human platelet-rich plasma was treated, or not, with activated thrombin, and platelet-free serum was recovered and tested, as a 10% addition to the medium, for the ability to stimulate luciferase expression in RAW264.7 cells previously transfected with CD36⁻²⁷³ or CD36⁻²⁶¹ reporters that do or do not, respectively, contain the PPRE. Rosiglitazone and oleoyl LPA were the positive controls for PPARγ activation. (b) Accumulation of a PPARγ agonist(s) in platelet-rich plasma is time-dependent. Human platelet-rich plasma was treated with activated thrombin for the stated times before addition, at a 100-fold dilution, to RAW264.7 cells transfected with acyl-CoA oxidase PPRE luciferase and SV40-β-galactosidase reporters. (c) LPA acyltransferase expression suppresses the response of a PPRE reporter to the supernatants of thrombin-activated platelets. RAW264.7 cells were transfected with acyl-CoA oxidase PPRE and SV40-β-galactosidase reporters and, for some cells, with an LPA acyltransferase (LPAAT) expression construct. These cells were treated with the supernatant from unactivated or thrombin-activated platelets as in a.