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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) interacts with Syk and LAT upon platelet stimulation with collagen. Washed platelets were incubated with rosiglitazone or vehicle control for 15 min and then stimulated with collagen (25 μg mL−1) for 90 s. Syk (A) and LAT (B) were immunoprecipitated from cell lysates and immunoblotted to detect PPARγ levels. Equivalent protein loading was verified by reprobing for Syk and LAT. Densitometry analyses were performed on replicate experiments using blood from four different donors, and data normalized for protein loading levels expressed as a percentage of change in Syk-PPARγ (A) and LAT-PPARγ association (B). GW9662 (3 μmol L−1) was incubated with platelets for 5 min prior rosiglitazone or vehicle control for 15 min and then stimulated with collagen (25 μg mL−1) for 90 s (C). Blots are representative of three different experiments (n = 3) [mean ± SEM (n = 4), t-test **P ≤ 0.01 and ***P ≤ 0.001].
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fig07: Peroxisome proliferator-activated receptor-γ (PPARγ) interacts with Syk and LAT upon platelet stimulation with collagen. Washed platelets were incubated with rosiglitazone or vehicle control for 15 min and then stimulated with collagen (25 μg mL−1) for 90 s. Syk (A) and LAT (B) were immunoprecipitated from cell lysates and immunoblotted to detect PPARγ levels. Equivalent protein loading was verified by reprobing for Syk and LAT. Densitometry analyses were performed on replicate experiments using blood from four different donors, and data normalized for protein loading levels expressed as a percentage of change in Syk-PPARγ (A) and LAT-PPARγ association (B). GW9662 (3 μmol L−1) was incubated with platelets for 5 min prior rosiglitazone or vehicle control for 15 min and then stimulated with collagen (25 μg mL−1) for 90 s (C). Blots are representative of three different experiments (n = 3) [mean ± SEM (n = 4), t-test **P ≤ 0.01 and ***P ≤ 0.001].

Mentions: As in the presence of PPARγ agonists tyrosine phosphorylation of Syk remained unaffected, while downstream LAT phosphorylation was inhibited significantly, it was hypothesized that PPARγ may interact with Syk and/or LAT. In order to test this, Syk and LAT were immuno-precipitated from platelets treated with rosiglitazone (10–100 μmol L−1) for 15 min prior to their stimulation with collagen (25 μg mL−1) and immunoblot analyses were conducted to detect PPARγ. PPARγ was found to interact with Syk and LAT when platelets were stimulated with collagen in the absence of PPARγ ligands (Fig. 7A–B). In the presence of the PPARγ ligand rosiglitazone, this interaction with both Syk and LAT was inhibited. The inhibitory effect on the PPARγ–Syk interaction was prevented by the addition of the PPARγ antagonist GW9662 (3 μmol L−1) (Fig. 7C), indicating that this effect is PPARγ activation dependent. GW9662 also prevented rosiglitazone-dependent inhibition of PPARγ–LAT interactions (data not shown).


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) interacts with Syk and LAT upon platelet stimulation with collagen. Washed platelets were incubated with rosiglitazone or vehicle control for 15 min and then stimulated with collagen (25 μg mL−1) for 90 s. Syk (A) and LAT (B) were immunoprecipitated from cell lysates and immunoblotted to detect PPARγ levels. Equivalent protein loading was verified by reprobing for Syk and LAT. Densitometry analyses were performed on replicate experiments using blood from four different donors, and data normalized for protein loading levels expressed as a percentage of change in Syk-PPARγ (A) and LAT-PPARγ association (B). GW9662 (3 μmol L−1) was incubated with platelets for 5 min prior rosiglitazone or vehicle control for 15 min and then stimulated with collagen (25 μg mL−1) for 90 s (C). Blots are representative of three different experiments (n = 3) [mean ± SEM (n = 4), t-test **P ≤ 0.01 and ***P ≤ 0.001].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig07: Peroxisome proliferator-activated receptor-γ (PPARγ) interacts with Syk and LAT upon platelet stimulation with collagen. Washed platelets were incubated with rosiglitazone or vehicle control for 15 min and then stimulated with collagen (25 μg mL−1) for 90 s. Syk (A) and LAT (B) were immunoprecipitated from cell lysates and immunoblotted to detect PPARγ levels. Equivalent protein loading was verified by reprobing for Syk and LAT. Densitometry analyses were performed on replicate experiments using blood from four different donors, and data normalized for protein loading levels expressed as a percentage of change in Syk-PPARγ (A) and LAT-PPARγ association (B). GW9662 (3 μmol L−1) was incubated with platelets for 5 min prior rosiglitazone or vehicle control for 15 min and then stimulated with collagen (25 μg mL−1) for 90 s (C). Blots are representative of three different experiments (n = 3) [mean ± SEM (n = 4), t-test **P ≤ 0.01 and ***P ≤ 0.001].
Mentions: As in the presence of PPARγ agonists tyrosine phosphorylation of Syk remained unaffected, while downstream LAT phosphorylation was inhibited significantly, it was hypothesized that PPARγ may interact with Syk and/or LAT. In order to test this, Syk and LAT were immuno-precipitated from platelets treated with rosiglitazone (10–100 μmol L−1) for 15 min prior to their stimulation with collagen (25 μg mL−1) and immunoblot analyses were conducted to detect PPARγ. PPARγ was found to interact with Syk and LAT when platelets were stimulated with collagen in the absence of PPARγ ligands (Fig. 7A–B). In the presence of the PPARγ ligand rosiglitazone, this interaction with both Syk and LAT was inhibited. The inhibitory effect on the PPARγ–Syk interaction was prevented by the addition of the PPARγ antagonist GW9662 (3 μmol L−1) (Fig. 7C), indicating that this effect is PPARγ activation dependent. GW9662 also prevented rosiglitazone-dependent inhibition of PPARγ–LAT interactions (data not shown).

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