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Non-genomic effects of PPARgamma ligands: inhibition of GPVI-stimulated platelet activation.

Moraes LA, Spyridon M, Kaiser WJ, Jones CI, Sage T, Atherton RE, Gibbins JM - J. Thromb. Haemost. (2009)

Bottom Line: PPAR(gamma) ligands inhibited collagen-stimulated platelet aggregation that was accompanied by a reduction in intracellular calcium mobilization and P-selectin exposure.The incorporation of GW9662 reversed the inhibitory actions of PPAR(gamma) agonists, implicating PPAR(gamma) in the effects observed.Furthermore, PPAR(gamma) ligands were found to inhibit tyrosine phosphorylation levels of multiple components of the GPVI signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cardiovascular & Metabolic Research, School of Biological Sciences, University of Reading, Reading, UK. l.a.moraes@reading.ac.uk

ABSTRACT

Background: Peroxisome proliferator-activated receptor-(gamma) (PPAR(gamma)) is expressed in human platelets although in the absence of genomic regulation in these cells, its functions are unclear.

Objective: In the present study, we aimed to demonstrate the ability of PPAR(gamma) ligands to modulate collagen-stimulated platelet function and suppress activation of the glycoprotein VI (GPVI) signaling pathway.

Methods: Washed platelets were stimulated with PPAR(gamma) ligands in the presence and absence of PPAR(gamma) antagonist GW9662 and collagen-induced aggregation was measured using optical aggregometry. Calcium levels were measured by spectrofluorimetry in Fura-2AM-loaded platelets and tyrosine phosphorylation levels of receptor-proximal components of the GPVI signaling pathway were measured using immunoblot analysis. The role of PPAR(gamma) agonists in thrombus formation was assessed using an in vitro model of thrombus formation under arterial flow conditions.

Results: PPAR(gamma) ligands inhibited collagen-stimulated platelet aggregation that was accompanied by a reduction in intracellular calcium mobilization and P-selectin exposure. PPAR(gamma) ligands inhibited thrombus formation under arterial flow conditions. The incorporation of GW9662 reversed the inhibitory actions of PPAR(gamma) agonists, implicating PPAR(gamma) in the effects observed. Furthermore, PPAR(gamma) ligands were found to inhibit tyrosine phosphorylation levels of multiple components of the GPVI signaling pathway. PPAR(gamma) was found to associate with Syk and LAT after platelet activation. This association was prevented by PPAR(gamma) agonists, indicating a potential mechanism for PPAR(gamma) function in collagen-stimulated platelet activation.

Conclusions: PPAR(gamma) agonists inhibit the activation of collagen-stimulation of platelet function through modulation of early GPVI signalling.

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Inhibitory effect of collagen-stimulated platelet aggregation by peroxisome proliferator-activated receptor-γ (PPARγ) ligands is not platelet endothelial cell adhesion molecule-1 (PECAM-1) dependent. Washed platelets obtained from wild-type (WT) mice (A) and PECAM-1-deficient mice (B) were treated with PPARγ ligand rosiglitazone (1, 20 μmol L−1) or vehicle [DMSO 0.1% (v/v)] and stimulated with collagen (1.0 μg mL−1). Aggregation was measured under constant stirring conditions at 37 °C. Representative aggregation traces (A–B) and cumulative data (C) represent the percentage of inhibition compared with control. Numerical data represent, mean ± SEM (n = 3) t-test **P ≤ 0.01.
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fig08: Inhibitory effect of collagen-stimulated platelet aggregation by peroxisome proliferator-activated receptor-γ (PPARγ) ligands is not platelet endothelial cell adhesion molecule-1 (PECAM-1) dependent. Washed platelets obtained from wild-type (WT) mice (A) and PECAM-1-deficient mice (B) were treated with PPARγ ligand rosiglitazone (1, 20 μmol L−1) or vehicle [DMSO 0.1% (v/v)] and stimulated with collagen (1.0 μg mL−1). Aggregation was measured under constant stirring conditions at 37 °C. Representative aggregation traces (A–B) and cumulative data (C) represent the percentage of inhibition compared with control. Numerical data represent, mean ± SEM (n = 3) t-test **P ≤ 0.01.

Mentions: Consistent with previous reports [30,36], platelets derived from PECAM-1-deficient mice exhibit a mildly exaggerated GPVI-mediated aggregation response to collagen when compared with wild-type mouse platelets (controls Fig. 8A,B: reduced lag phase and faster initial kinetics). Collagen-stimulated platelet aggregation in wild-type and PECAM-1-deficient platelets was inhibited in the presence of PPARγ ligand rosiglitazone, when compared with the vehicle control (Fig. 8A–C). This indicates that the acute (i.e. non-genomic) inhibitory effects of rosiglitazone on platelet function are not dependent on the presence or function of PECAM-1.


Non-genomic effects of PPARgamma ligands: inhibition of GPVI-stimulated platelet activation.

Moraes LA, Spyridon M, Kaiser WJ, Jones CI, Sage T, Atherton RE, Gibbins JM - J. Thromb. Haemost. (2009)

Inhibitory effect of collagen-stimulated platelet aggregation by peroxisome proliferator-activated receptor-γ (PPARγ) ligands is not platelet endothelial cell adhesion molecule-1 (PECAM-1) dependent. Washed platelets obtained from wild-type (WT) mice (A) and PECAM-1-deficient mice (B) were treated with PPARγ ligand rosiglitazone (1, 20 μmol L−1) or vehicle [DMSO 0.1% (v/v)] and stimulated with collagen (1.0 μg mL−1). Aggregation was measured under constant stirring conditions at 37 °C. Representative aggregation traces (A–B) and cumulative data (C) represent the percentage of inhibition compared with control. Numerical data represent, mean ± SEM (n = 3) t-test **P ≤ 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298645&req=5

fig08: Inhibitory effect of collagen-stimulated platelet aggregation by peroxisome proliferator-activated receptor-γ (PPARγ) ligands is not platelet endothelial cell adhesion molecule-1 (PECAM-1) dependent. Washed platelets obtained from wild-type (WT) mice (A) and PECAM-1-deficient mice (B) were treated with PPARγ ligand rosiglitazone (1, 20 μmol L−1) or vehicle [DMSO 0.1% (v/v)] and stimulated with collagen (1.0 μg mL−1). Aggregation was measured under constant stirring conditions at 37 °C. Representative aggregation traces (A–B) and cumulative data (C) represent the percentage of inhibition compared with control. Numerical data represent, mean ± SEM (n = 3) t-test **P ≤ 0.01.
Mentions: Consistent with previous reports [30,36], platelets derived from PECAM-1-deficient mice exhibit a mildly exaggerated GPVI-mediated aggregation response to collagen when compared with wild-type mouse platelets (controls Fig. 8A,B: reduced lag phase and faster initial kinetics). Collagen-stimulated platelet aggregation in wild-type and PECAM-1-deficient platelets was inhibited in the presence of PPARγ ligand rosiglitazone, when compared with the vehicle control (Fig. 8A–C). This indicates that the acute (i.e. non-genomic) inhibitory effects of rosiglitazone on platelet function are not dependent on the presence or function of PECAM-1.

Bottom Line: PPAR(gamma) ligands inhibited collagen-stimulated platelet aggregation that was accompanied by a reduction in intracellular calcium mobilization and P-selectin exposure.The incorporation of GW9662 reversed the inhibitory actions of PPAR(gamma) agonists, implicating PPAR(gamma) in the effects observed.Furthermore, PPAR(gamma) ligands were found to inhibit tyrosine phosphorylation levels of multiple components of the GPVI signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cardiovascular & Metabolic Research, School of Biological Sciences, University of Reading, Reading, UK. l.a.moraes@reading.ac.uk

ABSTRACT

Background: Peroxisome proliferator-activated receptor-(gamma) (PPAR(gamma)) is expressed in human platelets although in the absence of genomic regulation in these cells, its functions are unclear.

Objective: In the present study, we aimed to demonstrate the ability of PPAR(gamma) ligands to modulate collagen-stimulated platelet function and suppress activation of the glycoprotein VI (GPVI) signaling pathway.

Methods: Washed platelets were stimulated with PPAR(gamma) ligands in the presence and absence of PPAR(gamma) antagonist GW9662 and collagen-induced aggregation was measured using optical aggregometry. Calcium levels were measured by spectrofluorimetry in Fura-2AM-loaded platelets and tyrosine phosphorylation levels of receptor-proximal components of the GPVI signaling pathway were measured using immunoblot analysis. The role of PPAR(gamma) agonists in thrombus formation was assessed using an in vitro model of thrombus formation under arterial flow conditions.

Results: PPAR(gamma) ligands inhibited collagen-stimulated platelet aggregation that was accompanied by a reduction in intracellular calcium mobilization and P-selectin exposure. PPAR(gamma) ligands inhibited thrombus formation under arterial flow conditions. The incorporation of GW9662 reversed the inhibitory actions of PPAR(gamma) agonists, implicating PPAR(gamma) in the effects observed. Furthermore, PPAR(gamma) ligands were found to inhibit tyrosine phosphorylation levels of multiple components of the GPVI signaling pathway. PPAR(gamma) was found to associate with Syk and LAT after platelet activation. This association was prevented by PPAR(gamma) agonists, indicating a potential mechanism for PPAR(gamma) function in collagen-stimulated platelet activation.

Conclusions: PPAR(gamma) agonists inhibit the activation of collagen-stimulation of platelet function through modulation of early GPVI signalling.

Show MeSH
Related in: MedlinePlus