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Effects of peroxisome proliferator-activated receptor-γ agonists on the generation of microparticles by
Tommaso Neri1, Cinzia Cordazzo, Yuri Carmazzi
1Laboratory of Respiratory Cell Biology, Cardiac, Thoracic and Vascular Department, University of Pisa, Via Paradisa, 2, 56124 Pisa, Italy.
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.
Insights
Certain peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonists increase procoagulant microparticle generation in monocytes/macrophages. This effect involves ERK phosphorylation and calcium signaling, highlighting a potential link to thrombotic risk.
Area of Science:
- Cell biology
- Hematology
- Pharmacology
Background:
- Microparticles are cell-derived vesicles implicated in thrombosis.
- Peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonists regulate lipid metabolism and have diverse cellular effects.
- Some PPAR-γ agonists are associated with an increased risk of thrombotic diseases.
Purpose of the Study:
- To investigate the effect of PPAR-γ agonists on the generation of procoagulant microparticles by human monocytes/macrophages.
- To elucidate the signaling pathways involved in this process.
Main Methods:
- Human monocytes/macrophages were treated with PPAR-γ agonists (rosiglitazone, pioglitazone, 15-deoxy-Δ(12,14)-prostaglandin J(2)).
- Microparticle generation was quantified by measuring phosphatidylserine exposure and tissue factor content.
- Intracellular calcium levels and extracellular signal-regulated kinase (ERK) phosphorylation were assessed.
Main Results:
- Rosiglitazone and 15-deoxy-Δ(12,14)-prostaglandin J(2), but not pioglitazone, significantly increased tissue factor-bearing microparticle generation in a dose-dependent manner.
- These agonists also elevated intracellular calcium concentrations and induced ERK phosphorylation.
- Inhibition of calcium signaling (EGTA) or ERK phosphorylation (PD98059), and blockade of PPAR-γ (GW9662), reduced microparticle generation.
Conclusions:
- PPAR-γ agonists rosiglitazone and 15-deoxy-Δ(12,14)-prostaglandin J(2) promote the generation of procoagulant microparticles from human monocytes/macrophages.
- This procoagulant effect is mediated by ERK phosphorylation and intracellular calcium mobilization, with direct PPAR-γ activation also playing a role.
- These findings suggest a mechanism by which certain PPAR-γ agonists may contribute to thrombotic risk.
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