Effects of peroxisome proliferator-activated receptor-γ agonists on the generation of microparticles by monocytes/macrophages.

AIMS

Microparticles are membrane vesicles shed by cells upon activation and/or apoptosis. Microparticles are involved in several processes, including blood coagulation and thrombosis. In addition to their role in the regulation of lipid metabolism, peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists exert other effects, both dependent on and independent of PPAR-γ activation. Some PPAR-γ agonists have been linked to an increased risk of thrombotic diseases. We aimed to investigate the potential role of PPAR-γ agonists on the generation of procoagulant microparticles by human monocytes/macrophages.

METHODS AND RESULTS

Monocytes/macrophages were isolated from the buffy coats of normal donors. Cells were incubated with three structurally unrelated PPAR-γ agonists, namely, rosiglitazone, pioglitazone, and 15-deoxy-Δ(12,14)-prostaglandin J(2). Microparticle generation was assessed as phosphatidylserine concentration by a prothrombinase assay, after capturing the microparticles onto annexin V-coated wells. Intracellular calcium concentration was assessed by a fluorescent probe. Extracellular signal-regulated kinase (ERK) phosphorylation was assessed by western blot. Tissue factor expression on microparticles was measured with a one-stage clotting assay. Rosiglitazone and 15-deoxy-Δ(12,14)-prostaglandin J(2), but not pioglitazone, caused a dose-dependent, significant increase in intracellular calcium mobilization and tissue factor-bearing microparticle generation. EGTA inhibited microparticle generation. The specific PPAR-γ inhibitor, GW9662, also inhibited microparticle generation.  Finally, rosiglitazone and 15-deoxy-Δ(12,14)-prostaglandin J(2) caused phosphorylation of ERK; inhibition of ERK by PD98059 inhibited microparticle generation.

CONCLUSION

The PPAR-γ agonists rosiglitazone and 15-deoxy-Δ(12,14)-prostaglandin J(2), but not pioglitazone, caused an increase in procoagulant, tissue factor-bearing microparticle generation by human monocytes/macrophages. The effect was dependent on ERK phosphorylation and partly mediated through intracellular calcium mobilization; however, direct activation of the PPAR-γ ligand was also involved.