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.

Liver X receptors (LXRs) are transcription factors involved in the regulation of cholesterol homeostasis. LXR ligands have athero-protective properties independent of their effects on cholesterol metabolism. Platelets are involved in the initiation of atherosclerosis and despite being anucleate express nuclear receptors. We hypothesized that the athero-protective effects of LXR ligands could be in part mediated through platelets and therefore explored the potential role of LXR in platelets. Our results show that LXR-␤ is present in human platelets and the LXR ligands, GW3965 and T0901317, modulated nongenomically platelet aggregation stimulated by a range of agonists. GW3965 caused LXR to associate with signaling components proximal to the collagen receptor, GPVI, suggesting a potential mechanism of LXR action in platelets that leads to diminished platelet responses. Activation of platelets at sites of atherosclerotic lesions results in thrombosis preceding myocardial infarction and stroke. Using an in vivo model of thrombosis in mice, we show that GW3965 has antithrombotic effects, reducing the size and the stability of thrombi. The atheroprotective effects of GW3965, together with its novel antiplatelet/thrombotic effects, indicate LXR as a potential target for prevention of athero-thrombotic disease. (Blood. 2011;117(21):5751-5761) IntroductionLiver X receptors (LXRs) belong to the nuclear receptor superfamily, and their natural ligands, oxysterols, are cholesterol derivatives. 1 LXRs play key roles in cholesterol homeostasis by regulating the transcription of genes, such as cytochrome P450 7␣-hydroxylase 1 (Cyp7a1) and apoliprotein E (ApoE), involved in cholesterol catabolism to bile acids 2,3 and cholesterol efflux back to the liver, 4-6 respectively. LXRs have also been shown to have anti-inflammatory 7,8 and athero-protective effects after ligand stimulation. 9,10 Platelets are anucleate blood cells with a central role in hemostasis but are also involved in inflammation and atherosclerosis. [11][12][13][14] Their primary function is to prevent hemorrhage at sites of vascular injury, where exposed extracellular matrix proteins, such as collagen, interact with platelet surface receptors, resulting in platelet activation, aggregation, and thrombus formation. Although platelets are essential for the preservation of vascular integrity, inappropriate platelet activation, for example, at sites of atherosclerotic lesions, causes arterial thrombosis, leading to ischemic stroke and myocardial infarction, 2 of the main causes of morbidity and mortality in the industrialized world. 15 Low-density lipoprotein is the main therapeutic target for the prevention of atherothrombosis, 16 and low-density lipoprotein-lowering medications, such as statins, appear additionally to display antiplatelet actions. 17,18 Platelet function is also affected by cholesterol derivatives, although there is no consensus on whether these molecules have inhibitory or activatory roles. 19 The initial entrapment of platelets on subendothelial c...

Background: Platelet activation by collagen depends on signals transduced by the glycoprotein (GP)VI–Fc receptor (FcR)γ-chain collagen receptor complex, which involves recruitment of phosphatidylinositol 3-kinase (PI3K) to phosphorylated tyrosines in the linker for activation of T cells (LAT). An interaction between the p85 regulatory subunit of PI3K and the scaffolding molecule Grb-2-associated binding protein-1 (Gab1), which is regulated by binding of the Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) to Gab1, has been shown in other cell types to sustain PI3K activity to elicit cellular responses. Platelet endothelial cell adhesion molecule-1 (PECAM-1) functions as a negative regulator of platelet reactivity and thrombosis, at least in part by inhibiting GPVI–FcRγ-chain signaling via recruitment of SHP-2 to phosphorylated immunoreceptor tyrosine-based inhibitory motifs in PECAM-1. Objective: To investigate the possibility that PECAM-1 regulates the formation of the Gab1–p85 signaling complexes, and the potential effect of such interactions on GPVI-mediated platelet activation in platelets. Methods: The ability of PECAM-1 signaling to modulate the LAT signalosome was investigated with immunoblotting assays on human platelets and knockout mouse platelets. Results: PECAM-1-associated SHP-2 in collagen-stimulated platelets binds to p85, which results in diminished levels of association with both Gab1 and LAT and reduced collagen-stimulated PI3K signaling. We therefore propose that PECAM-1-mediated inhibition of GPVI-dependent platelet responses result, at least in part, from recruitment of SHP-2–p85 complexes to tyrosine-phosphorylated PECAM-1, which diminishes the association of PI3K with activatory signaling molecules, such as Gab1 and LAT.

Background Intravenous iron is often used to treat iron deficiency anaemia in non-dialysis chronic kidney disease (ND-CKD), but the optimal dosing regimen remains unclear. We evaluated the impact of high- versus low-dose intravenous iron isomaltoside on the probability of retreatment with intravenous iron in iron-deficient ND-CKD patients. Methods This real-world, prospective, observational study collected data from 256 ND-CKD patients treated for anaemia in the UK. Following an initial course of iron isomaltoside, patients were followed for ≥12 months. Iron dose and the need for retreatment were determined at the investigators’ discretion. The primary study outcome was the need for retreatment at 52 weeks compared between patients who received >1000 mg of iron during Course 1 and those who received ≤1000 mg. Safety was evaluated through adverse drug reactions. Results The probability of retreatment at Week 52 was significantly lower in the >1000 mg iron group (n = 58) versus the ≤1000 mg group (n = 198); hazard ratio (95% confidence interval [CI]): 0.46 (0.20, 0.91); p = 0.012. Mean (95% CI) haemoglobin increased by 6.58 (4.94, 8.21) g/L in the ≤1000 mg group and by 10.59 (7.52, 13.66) g/L in the >1000 mg group (p = 0.024). Changes in other blood and iron parameters were not significantly different between the two groups. Administering >1000 mg of iron isomaltoside saved 8.6 appointments per 100 patients compared to ≤1000 mg. No serious adverse drug reactions were reported. Of the patients who received ≤1000 mg of iron in this study, 82.3% were eligible for a dose >1000 mg. Conclusions The >1000 mg iron isomaltoside regimen reduced the probability of retreatment, achieved a greater haemoglobin response irrespective of erythropoiesis-stimulating agent treatment, and reduced the total number of appointments required, compared to the ≤1000 mg regimen. Many of the patients who received ≤1000 mg of iron were eligible for >1000 mg, indicating that there was considerable underdosing in this study. Trial registration ClinicalTrials.gov NCT02546154, 10 September 2015.

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