Abstract
BackgroundPeroxisome proliferator-activated receptor-γ (PPARγ) is expressed in human platelets although in the absence of genomic regulation in these cells, its functions are unclear.ObjectiveIn the present study, we aimed to demonstrate the ability of PPARγ ligands to modulate collagen-stimulated platelet function and suppress activation of the glycoprotein VI (GPVI) signaling pathway.MethodsWashed platelets were stimulated with PPARγ ligands in the presence and absence of PPARγ 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 PPARγ agonists in thrombus formation was assessed using an in vitro model of thrombus formation under arterial flow conditions.ResultsPPARγ ligands inhibited collagen-stimulated platelet aggregation that was accompanied by a reduction in intracellular calcium mobilization and P-selectin exposure. PPARγ ligands inhibited thrombus formation under arterial flow conditions. The incorporation of GW9662 reversed the inhibitory actions of PPARγ agonists, implicating PPARγ in the effects observed. Furthermore, PPARγ ligands were found to inhibit tyrosine phosphorylation levels of multiple components of the GPVI signaling pathway. PPARγ was found to associate with Syk and LAT after platelet activation. This association was prevented by PPARγ agonists, indicating a potential mechanism for PPARγ function in collagen-stimulated platelet activation. Conclusions: PPARγ agonists inhibit the activation of collagen-stimulation of platelet function through modulation of early GPVI signalling.
Highlights
Diabetes mellitus is a major risk factor for vascular diseases and is associated with atherosclerosis and thrombotic complications [1]
We investigated the effects of Peroxisome proliferator-activated receptor-c (PPARc) agonists, 15d-PGJ2 and rosiglitazone on collagen-stimulated platelet activation, signaling and on thrombus formation
We demonstrate that PPARc ligands modulate the activity of the glycoprotein VI (GPVI) collagen receptor-stimulated signaling pathway resulting in reduced levels of platelet activation, aggregation and thrombus formation under arterial flow conditions
Summary
Diabetes mellitus is a major risk factor for vascular diseases and is associated with atherosclerosis and thrombotic complications [1]. Collagen binding to the platelet receptor glycoprotein VI (GPVI) results in clustering thereby triggering the tyrosine phosphorylation of the associated transmembrane protein, the Fc receptor c-chain by the Src-family kinases Lyn and Fyn [6,7]. Phosphoinositide 3-kinase (PI3-K) is recruited and through the generation of phosphatidylinositol (3, 4, 5)-trisphosphate, influences the recruitment and activation of phospholipase Cc2 (PLCc2), which liberates the second messengers 1,2-diacylglycerol and inositol 1,4,5-trisphosphate. The formation of these molecules is responsible for the mobilization of calcium from intracellular stores and activation of isoforms of protein kinase C (PKC) leading to secretion and aggregation. PI3-K activity results in the regulation of protein kinase B (PKB), which is important for platelet function and thrombus formation [2,8,9]
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