Peroxisome proliferator-activated receptor γ and retinoid X receptor transcription factors are released from activated human platelets and shed in microparticles

University of Rochester Medical Center, Environmental Medicine, Rochester, New York 14642, USA .
Thrombosis and Haemostasis (Impact Factor: 4.98). 02/2008; 99(1):86-95. DOI: 10.1160/TH07-05-0328
Source: PubMed


Peroxisome proliferator-activated receptor gamma (PPARgamma) and its ligands are important regulators of lipid metabolism, inflammation, and diabetes. We previously demonstrated that anucleate human platelets express the transcription factor PPARgamma and that PPARgamma ligands blunt platelet activation. To further understand the nature of PPARgamma in platelets, we determined the platelet PPARgamma isoform(s) and investigated the fate of PPARgamma following platelet activation. Our studies demonstrated that human platelets contain only the PPARgamma1 isoform and after activation with thrombin, TRAP, ADP or collagen PPARgamma is released from internal stores. PPARgamma release was blocked by a cytoskeleton inhibitor, Latrunculin A. Platelet-released PPARgamma was complexed with the retinoid X receptor (RXR) and retained its ability to bind DNA. Interestingly, the released PPARgamma and RXR were microparticle associated and the released PPARgamma/RXR complex retained DNA-binding ability. Additionally, a monocytic cell line, THP-1, is capable of internalizing PMPs. Further investigation following treatment of these cells with the PPARgamma agonist, rosiglitazone and PMPs revealed a possible transcellular mechanism to attenuate THP-1 activation. These new findings are the first to demonstrate transcription factor release from platelets, revealing the complex spectrum of proteins expressed and expelled from platelets, and suggests that platelet PPARgamma has an undiscovered role in human biology.

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Available from: Neil Blumberg, Feb 10, 2014
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    • "Platelets were also activated with thrombin (1 U/ml), collagen (10 µg/ml), cross-linked CRP-peptide (CRP-XL) (1 µg/ml, a kind gift from Professor Richard Farndale, University of Cambridge, UK), ADP (60 µM, Sigma-Aldrich) and TRAP-6 (10 µM, Bachem AG, Bubendorf, Switzerland) at +37°C for 30 min. The concentrations and time points for the activation were chosen by previous literature to allow maximal platelet vesiculation (7,19,21,29,30). Platelets and cell debris were removed with a centrifugation for 5 min at 5,000×g, followed by 1 min at 11,000×g to obtain a tight pellet followed by transfer into new tubes and a centrifugation for 15 min at 2,500×g at RT (Eppendorf 5415D with F45-24-11 rotor, Eppendorf AG, Hamburg, Germany). "
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    ABSTRACT: Background Platelet-derived extracellular vesicles (EVs) participate, for example, in haemostasis, immunity and development. Most studies of platelet EVs have targeted microparticles, whereas exosomes and EV characterization under various conditions have been less analyzed. Studies have been hampered by the difficulty in obtaining EVs free from contaminating cells and platelet remnants. Therefore, we optimized an EV isolation protocol and compared the quantity and protein content of EVs induced by different agonists. Methods Platelets isolated with iodixanol gradient were activated by thrombin and collagen, lipopolysaccharide (LPS) or Ca2+ ionophore. Microparticles and exosomes were isolated by differential centrifugations. EVs were quantitated by nanoparticle tracking analysis (NTA) and total protein. Size distributions were determined by NTA and electron microscopy. Proteomics was used to characterize the differentially induced EVs. Results The main EV populations were 100–250 nm and over 90% were <500 nm irrespective of the activation. However, activation pathways differentially regulated the quantity and the quality of EVs, which also formed constitutively. Thrombogenic activation was the most potent physiological EV-generator. LPS was a weak inducer of EVs, which had a selective protein content from the thrombogenic EVs. Ca2+ ionophore generated a large population of protein-poor and unselectively packed EVs. By proteomic analysis, EVs were highly heterogeneous after the different activations and between the vesicle subpopulations. Conclusions Although platelets constitutively release EVs, vesiculation can be increased, and the activation pathway determines the number and the cargo of the formed EVs. These activation-dependent variations render the use of protein content in sample normalization invalid. Since most platelet EVs are 100–250 nm, only a fraction has been analyzed by previously used methods, for example, flow cytometry. As the EV subpopulations could not be distinguished and large vesicle populations may be lost by differential centrifugation, novel methods are required for the isolation and the differentiation of all EVs.
    08/2014; 3. DOI:10.3402/jev.v3.24692
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    • "In activated macrophages and vascular smooth muscle cells, agonists of PPARγ inhibit the expression of many pro-inflammatory genes [17], [18]. PPARγ is highly expressed in platelets, and PPARγ activation can inhibit platelet activation and decrease CD40L release [19], [20]. Nevertheless, the role of PPARγ in countering the adverse effects of hypertension on platelet function has not been studied. "
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    ABSTRACT: Hypertension is known to be associated with platelet overactivity, but the direct effects of hydrostatic pressure on platelet function remain unclear. The present study sought to investigate whether elevated hydrostatic pressure is responsible for platelet activation and to address the potential role of peroxisome proliferator-activated receptor-γ (PPARγ). We observed that hypertensive patients had significantly higher platelet volume and rate of ADP-induced platelets aggregation compared to the controls. In vitro, Primary human platelets were cultured under standard (0 mmHg) or increased (120, 180, 240 mmHg) hydrostatic pressure for 18 h. Exposure to elevated pressure was associated with morphological changes in platelets. Platelet aggregation and PAC-1 (the active confirmation of GPIIb/IIIa) binding were increased, CD40L was translocated from cytoplasm to the surface of platelet and soluble CD40L (sCD40L) was released into the medium in response to elevated hydrostatic pressure (180 and 240 mmHg). The PPARγ activity was up-regulated as the pressure was increased from 120 mmHg to 180 mmHg. Pressure-induced platelet aggregation, PAC-1 binding, and translocation and release of CD40L were all attenuated by the PPARγ agonist Thiazolidinediones (TZDs). These results demonstrate that platelet activation and aggregation are increased by exposure to elevated pressure and that PPARγ may modulate platelet activation induced by high hydrostatic pressure.
    PLoS ONE 02/2014; 9(2):e89654. DOI:10.1371/journal.pone.0089654 · 3.23 Impact Factor
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    • "Megakaryocytes and platelets express PPARγ (Akbiyik et al., 2004), and recently we reported that PPARγ1 is released from activated platelets and in platelet microparticles as an active transcription factor complex with RXR (Ray et al., 2008). Internalization of PPARγcontaining platelet microparticles elicits a transcellular attenuation of THP-1 monocytic cell activation in the presence of the PPARγ agonist, rosiglitazone (Ray et al., 2008). PPARγ activation exerts anti-inflammatory effects in nucleated cells via nongenomic mechanisms (Ray et al., 2006). "
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    ABSTRACT: Widely known for its role in adipogenesis and energy metabolism, PPARgamma also plays a role in platelet function. To further understand functions of platelet-derived PPARgamma, we produced rabbit polyclonal (PoAbs) and mouse monoclonal (MoAbs) antibodies against PPARgamma 14mer/19mer peptide-immunogens. Unexpectedly, our work produced two key findings. First, MoAbs but not PoAbs produced against PPARgamma peptide-immunogens displayed antigenic crossreactivity with highly conserved PPARalpha and PPARbeta/delta. Similarly, Santa Cruz PoAb sc-7196 was monospecific for PPARgamma while MoAb sc-7273 crossreacted with PPARalpha and PPARbeta/delta. Second, immunized rabbits and mice exhibited unusual pathology including cachexia, excessive bleeding, and low platelet counts leading to thrombocytopenia. Spleens from immunized mice were fatty, hemorrhagic and friable. Although passive administration of anti-PPARgamma PoAbs failed to induce experimental thrombocytopenia, megakaryocytopoiesis was induced 4-8-fold in mouse spleens. Similarly, marrow megakaryocytopoiesis was enhanced 1.8-4-fold in immunized rabbits. These peptide-immunogens are 100% conserved in human, rabbit and mouse; thus, immune-mediated platelet destruction via crossreactivity with platelet-derived PPARgamma likely caused bleeding, thrombocytopenia, and compensatory megakaryocytopoiesis. Such overt pathology would cause significant problems for large-scale production of anti-PPARgamma PoAbs. Furthermore, a major pitfall associated with MoAb production against closely related molecules is that monoclonicity does not guarantee monospecificity, an issue worth further scientific scrutiny.
    Journal of Biotechnology 11/2010; 150(3-3):417-27. DOI:10.1016/j.jbiotec.2010.09.955 · 2.87 Impact Factor
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