Non‐genomic effects of PPARγ ligands: inhibition of GPVI‐stimulated platelet activation

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.

Thrombin and Collagen Induce a Feedback Inhibitory Signaling Pathway in Platelets Involving Dissociation of the Catalytic Subunit of Protein Kinase A from an NFκB-IκB Complex

“…The canonical Wnt signaling was recently described in platelets and shown to be involved in inhibition of platelet adhesion, shape change, dense granule secretion, RhoA activation, and aggregation (37). Activation of peroxisome proliferator-activated receptor ␥ (PPAR␥) inhibited collagen-stimulated platelet aggregation, intracellular calcium mobilization, P-selectin exposure, and thrombus formation (38). Protease nexin-1 (PN-1), stored in ␣-granules and released during platelet activation, is involved in inhibition of tissue factor-induced thrombin generation, low-dose thrombin-induced P-selectin surface expression, and platelet aggregation (39) An adapter protein disabled-2 (DAB2), which is also associated with ␣-granules and secreted from agonist-stimulated platelets, inhibited platelet integrin ␣IIb␤3 activation and platelet aggregation by binding to the extracellular region of ␣IIb integrin (40).…”

AMP-Activated Protein Kinase Mediates the Antiplatelet Effects of the Thiazolidinediones Rosiglitazone and Pioglitazone

“…As expected, rosiglitazone and pioglitazone were capable of inhibiting platelet aggregation by activating AMPK. Besides AMPK, PPARg is also involved in the antiaggregatory effect of these drugs, as previously reported (Ali et al, 2009a;Moraes et al, 2010). However, the efficacy associated with PPARg stimulation (A, B) Activation of AMPK and inhibition of platelet aggregation by pioglitazone.…”

“…3D). As with other general antiplatelet agents, the antiaggregatory effect of the PPARg agonist 15d-PGJ 2 was inversely proportional to the strength of aggregatory stimuli and was prominent in weak aggregation (Moraes et al, 2010). Accordingly, aggregation was tested with a mild stimulus.…”

“…Notably, most of these studies regarded rosiglitazone as only a PPARg agonist and tended to ascribe these platelet effects to PPARg stimulation without proper verification (Akbiyik et al, 2004;Ali et al, 2006;Bishop-Bailey, 2010;Moraes et al, 2010;Spinelli et al, 2008). Such interpretation was also attributed to the accompanying experimental evidence-the antiaggregatory activity of the PPARg agonist 15d-PGJ 2 (Akbiyik et al, 2004;Moraes et al, 2010). There is currently sufficient evidence that stimulating PPARg causes the attenuation of aggregation.…”

“…Although the function of PPARs in platelets has not been fully elucidated, they are suggested to be related to aggregation, the inherent function of platelets (Ray et al, 2008;Ali et al, 2009b). Indeed, PPARg agonists such as 15-deoxy-D12,14-prostaglandin J 2 (15d-PGJ 2 ) and rosiglitazone were capable of inhibiting platelet aggregation and suppressing the release of thromboxane A 2 and soluble CD40-ligand (sCD40L), proinflammatory and proaggregatory mediators from platelets (Akbiyik et al, 2004;Ali et al, 2006;Khanolkar et al, 2008;Moraes et al, 2010). The PPARg-mediated antiplatelet effects were also reported in a study of the statin class of hypolipidemic drugs, such as simvastatin (Ali et al, 2009a).…”