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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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Related in: MedlinePlus

Peroxisome proliferator-activated receptor-γ (PPARγ) ligands inhibit thrombus formation under arterial flow conditions. Whole blood from healthy donors was incubated for 5 min with PPARγ ligands or vehicle control and perfused through collagen-coated capillaries at a shear rate of 1000 s−1. Composite data from Z series images were obtained by confocal microscopy (Ai–iii). Analysis of thrombus volume (B) and protein concentration (C) in the presence of increasing concentrations of PPARγ ligands was performed. The PPARγ antagonist GW96622 (3 μmol L−1) was incubated for 5 min prior addition of PPARγ ligand or vehicle and thrombus volume analyzed (D). To assess the impact of exposure of pre-formed thrombi to PPARγ agonist, formed thrombi were perfused at an arterial shear rate with rosiglitazone or solvent control for 5 min, and thrombus volume measured by confocal microscopy (E). Numerical data represent percentage of inhibition compared with control, mean ± SEM (n = 4) t-test *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001.
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fig04: Peroxisome proliferator-activated receptor-γ (PPARγ) ligands inhibit thrombus formation under arterial flow conditions. Whole blood from healthy donors was incubated for 5 min with PPARγ ligands or vehicle control and perfused through collagen-coated capillaries at a shear rate of 1000 s−1. Composite data from Z series images were obtained by confocal microscopy (Ai–iii). Analysis of thrombus volume (B) and protein concentration (C) in the presence of increasing concentrations of PPARγ ligands was performed. The PPARγ antagonist GW96622 (3 μmol L−1) was incubated for 5 min prior addition of PPARγ ligand or vehicle and thrombus volume analyzed (D). To assess the impact of exposure of pre-formed thrombi to PPARγ agonist, formed thrombi were perfused at an arterial shear rate with rosiglitazone or solvent control for 5 min, and thrombus volume measured by confocal microscopy (E). Numerical data represent percentage of inhibition compared with control, mean ± SEM (n = 4) t-test *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001.

Mentions: The effect of 15d-PGJ2 and rosiglitazone on thrombus formation in whole blood was examined under arterial flow conditions in vitro. Whole blood was perfused through microcapillary tubes coated internally with collagen at a shear (laminar flow) rate of 1000 s−1 in the presence of rosiglitazone or 15d-PGJ2 (0.1, 1 and 20 μmol L−1) or vehicle [DMSO 0.1% (v/v)]. Thrombus size was calculated from the mean thrombus volume of five randomly selected fields of view. Figure 4A (i–iii) shows composite images from Z series captured and analyzed by confocal microscopy in the presence of vehicle control and rosiglitazone. Both PPARγ ligands, rosiglitazone and 15d-PGJ2 inhibited the thrombus formation significantly in a concentration-dependent manner, where 1 μmol L−1 rosiglitazone or 15d-PGJ2 were able to inhibit thrombus formation by 50.4 ± 14.7 % and 66.6 ± 2.7 % compared with the vehicle control (Fig. 4B). To measure thrombus formation along the whole capillary, lysis buffer was passed through each capillary and protein concentration measured as an indicator of thrombus size. This approach is important because, as a result of the fibrilar nature of the collagen used, coating of microslides may not be completely uniform. As this may influence data collected from selected fields, analysis of platelet recruitment along the entire capillary is quantitatively more reliable. Consistent with the thrombus volume data, PPARγ ligands resulted in reduced protein concentration compared with control (Fig. 4C), and no significant differences were noted between rosiglitazone and 15d-PGJ2 treatments. The inclusion of the PPARγ antagonist GW9662 (3 μmol L−1) was able to reverse the inhibitory effect of the PPARγ ligand 15d-PGJ2 (3 μmol L−1) on thrombus formation (Fig. 4D).


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 inhibit thrombus formation under arterial flow conditions. Whole blood from healthy donors was incubated for 5 min with PPARγ ligands or vehicle control and perfused through collagen-coated capillaries at a shear rate of 1000 s−1. Composite data from Z series images were obtained by confocal microscopy (Ai–iii). Analysis of thrombus volume (B) and protein concentration (C) in the presence of increasing concentrations of PPARγ ligands was performed. The PPARγ antagonist GW96622 (3 μmol L−1) was incubated for 5 min prior addition of PPARγ ligand or vehicle and thrombus volume analyzed (D). To assess the impact of exposure of pre-formed thrombi to PPARγ agonist, formed thrombi were perfused at an arterial shear rate with rosiglitazone or solvent control for 5 min, and thrombus volume measured by confocal microscopy (E). Numerical data represent percentage of inhibition compared with control, mean ± SEM (n = 4) t-test *P ≤ 0.05, **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

fig04: Peroxisome proliferator-activated receptor-γ (PPARγ) ligands inhibit thrombus formation under arterial flow conditions. Whole blood from healthy donors was incubated for 5 min with PPARγ ligands or vehicle control and perfused through collagen-coated capillaries at a shear rate of 1000 s−1. Composite data from Z series images were obtained by confocal microscopy (Ai–iii). Analysis of thrombus volume (B) and protein concentration (C) in the presence of increasing concentrations of PPARγ ligands was performed. The PPARγ antagonist GW96622 (3 μmol L−1) was incubated for 5 min prior addition of PPARγ ligand or vehicle and thrombus volume analyzed (D). To assess the impact of exposure of pre-formed thrombi to PPARγ agonist, formed thrombi were perfused at an arterial shear rate with rosiglitazone or solvent control for 5 min, and thrombus volume measured by confocal microscopy (E). Numerical data represent percentage of inhibition compared with control, mean ± SEM (n = 4) t-test *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001.
Mentions: The effect of 15d-PGJ2 and rosiglitazone on thrombus formation in whole blood was examined under arterial flow conditions in vitro. Whole blood was perfused through microcapillary tubes coated internally with collagen at a shear (laminar flow) rate of 1000 s−1 in the presence of rosiglitazone or 15d-PGJ2 (0.1, 1 and 20 μmol L−1) or vehicle [DMSO 0.1% (v/v)]. Thrombus size was calculated from the mean thrombus volume of five randomly selected fields of view. Figure 4A (i–iii) shows composite images from Z series captured and analyzed by confocal microscopy in the presence of vehicle control and rosiglitazone. Both PPARγ ligands, rosiglitazone and 15d-PGJ2 inhibited the thrombus formation significantly in a concentration-dependent manner, where 1 μmol L−1 rosiglitazone or 15d-PGJ2 were able to inhibit thrombus formation by 50.4 ± 14.7 % and 66.6 ± 2.7 % compared with the vehicle control (Fig. 4B). To measure thrombus formation along the whole capillary, lysis buffer was passed through each capillary and protein concentration measured as an indicator of thrombus size. This approach is important because, as a result of the fibrilar nature of the collagen used, coating of microslides may not be completely uniform. As this may influence data collected from selected fields, analysis of platelet recruitment along the entire capillary is quantitatively more reliable. Consistent with the thrombus volume data, PPARγ ligands resulted in reduced protein concentration compared with control (Fig. 4C), and no significant differences were noted between rosiglitazone and 15d-PGJ2 treatments. The inclusion of the PPARγ antagonist GW9662 (3 μmol L−1) was able to reverse the inhibitory effect of the PPARγ ligand 15d-PGJ2 (3 μmol L−1) on thrombus formation (Fig. 4D).

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