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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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Peroxisome proliferator-activated receptor-γ (PPARγ) ligands 15d-PGJ2 and rosiglitazone signal through PPARγ on platelets. Washed human platelets were treated for 5 min with (A) PPARγ antagonist GW9662 (1, 3 μmol L−1) or (B) GW9662 (1 μmol L−1) followed by incubation for 15 min with PPARγ ligands 15d-PGJ2 or rosiglitazone (3 μmol L−1) prior to stimulation for 90 s with collagen (1.0 μg mL−1) and aggregation measured at 37 °C with constant stirring. Data represents percentage of (A) aggregation and (B) recovery of aggregation compared with control. Numerical data represent, mean ± SEM (n = 3), t-test *P ≤ 0.05 and ***≤ 0.001.
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fig03: Peroxisome proliferator-activated receptor-γ (PPARγ) ligands 15d-PGJ2 and rosiglitazone signal through PPARγ on platelets. Washed human platelets were treated for 5 min with (A) PPARγ antagonist GW9662 (1, 3 μmol L−1) or (B) GW9662 (1 μmol L−1) followed by incubation for 15 min with PPARγ ligands 15d-PGJ2 or rosiglitazone (3 μmol L−1) prior to stimulation for 90 s with collagen (1.0 μg mL−1) and aggregation measured at 37 °C with constant stirring. Data represents percentage of (A) aggregation and (B) recovery of aggregation compared with control. Numerical data represent, mean ± SEM (n = 3), t-test *P ≤ 0.05 and ***≤ 0.001.

Mentions: To establish whether the effects of PPARγ ligands on platelets are mediated by the receptor (PPARγ), similar aggregation assays were carried out in the presence of the PPARγ antagonist GW9662. Washed human platelets were treated for 5 min with PPARγ antagonist GW9662 alone or followed incubation for 15 min with PPARγ ligands 15d-PGJ2 or rosiglitazone prior to stimulation for 90 s with collagen (1 μg mL−1). The PPARγ antagonist GW9662 alone (1, 3 μmol L−1) did not modulate the levels of collagen-stimulated aggregation (Fig. 3A). GW9662 (1 μmol L−1) did, however, cause a significant suppression of the inhibition of collagen-stimulated platelet aggregation by 15d-PGJ2 and rosiglitazone (3 μmol L−1) (Fig 3B). These data indicate that the effects of 15d-PGJ2 and rosiglitazone are mediated, at least in part, through binding to PPARγ in platelets.


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)

Peroxisome proliferator-activated receptor-γ (PPARγ) ligands 15d-PGJ2 and rosiglitazone signal through PPARγ on platelets. Washed human platelets were treated for 5 min with (A) PPARγ antagonist GW9662 (1, 3 μmol L−1) or (B) GW9662 (1 μmol L−1) followed by incubation for 15 min with PPARγ ligands 15d-PGJ2 or rosiglitazone (3 μmol L−1) prior to stimulation for 90 s with collagen (1.0 μg mL−1) and aggregation measured at 37 °C with constant stirring. Data represents percentage of (A) aggregation and (B) recovery of aggregation compared with control. Numerical data represent, mean ± SEM (n = 3), t-test *P ≤ 0.05 and ***≤ 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: Peroxisome proliferator-activated receptor-γ (PPARγ) ligands 15d-PGJ2 and rosiglitazone signal through PPARγ on platelets. Washed human platelets were treated for 5 min with (A) PPARγ antagonist GW9662 (1, 3 μmol L−1) or (B) GW9662 (1 μmol L−1) followed by incubation for 15 min with PPARγ ligands 15d-PGJ2 or rosiglitazone (3 μmol L−1) prior to stimulation for 90 s with collagen (1.0 μg mL−1) and aggregation measured at 37 °C with constant stirring. Data represents percentage of (A) aggregation and (B) recovery of aggregation compared with control. Numerical data represent, mean ± SEM (n = 3), t-test *P ≤ 0.05 and ***≤ 0.001.
Mentions: To establish whether the effects of PPARγ ligands on platelets are mediated by the receptor (PPARγ), similar aggregation assays were carried out in the presence of the PPARγ antagonist GW9662. Washed human platelets were treated for 5 min with PPARγ antagonist GW9662 alone or followed incubation for 15 min with PPARγ ligands 15d-PGJ2 or rosiglitazone prior to stimulation for 90 s with collagen (1 μg mL−1). The PPARγ antagonist GW9662 alone (1, 3 μmol L−1) did not modulate the levels of collagen-stimulated aggregation (Fig. 3A). GW9662 (1 μmol L−1) did, however, cause a significant suppression of the inhibition of collagen-stimulated platelet aggregation by 15d-PGJ2 and rosiglitazone (3 μmol L−1) (Fig 3B). These data indicate that the effects of 15d-PGJ2 and rosiglitazone are mediated, at least in part, through binding to PPARγ in platelets.

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