Non-genomic effects of PPARgamma ligands: inhibition of GPVI-stimulated platelet activation.
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.
Moraes
L A
LA
Institute for Cardiovascular & Metabolic Research, School of Biological Sciences, University of Reading, Reading, UK. l.a.moraes@reading.ac.uk
Spyridon
M
M
Kaiser
W J
WJ
Jones
C I
CI
Sage
T
T
Atherton
R E L
RE
Gibbins
J M
JM
D048868:Adaptor Proteins, Signal Transducing / Q000097:blood; D000813:Anilides / Q000494:pharmacology; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000097:blood; D003094:Collagen / Q000097:blood; D004305:Dose-Response Relationship, Drug; D005343:Fibrinolytic Agents / Q000494:pharmacology*; D006801:Humans; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins / Q000097:blood; D008024:Ligands; D008565:Membrane Proteins / Q000097:blood; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D019007:P-Selectin / Q000097:blood; D047495:PPAR gamma / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000097:blood; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000187:drug effects*; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000097:blood / Q000235:genetics; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D015230:Prostaglandin D2 / Q000031:analogs & derivatives* / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000097:blood; D000077154:Rosiglitazone; D015398:Signal Transduction / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D000072377:Syk Kinase; D045162:Thiazolidinediones / Q000494:pharmacology*; D013927:Thrombosis / Q000097:blood / Q000517:prevention & control*; D013997:Time Factors; D014443:Tyrosine / Q000097:blood
C477819:15-deoxyprostaglandin J2; C457499:2-chloro-5-nitrobenzanilide; D048868:Adaptor Proteins, Signal Transducing; D000813:Anilides; D005343:Fibrinolytic Agents; D047908:Intracellular Signaling Peptides and Proteins; C110810:LAT protein, human; D008024:Ligands; D008565:Membrane Proteins; D019007:P-Selectin; D047495:PPAR gamma; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D010980:Platelet Membrane Glycoproteins; D045162:Thiazolidinediones; C098341:platelet membrane glycoprotein VI; D000077154:Rosiglitazone; D014443:Tyrosine; D003094:Collagen; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606897:Syk protein, mouse; D015230:Prostaglandin D2; D002118:Calcium