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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 20, 2005 as doi:10.1096/fj.05-4395fje. |
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Unit of Critical Care Medicine, National Heart and Lung Institute, Imperial College, London, UK;
* William Harvey Research Institute, Barts and the London, Queen Mary’s School of Medicine and Dentistry, Charterhouse Square, London, UK
1 Correspondence: Unit of Critical Care Medicine, National Heart and Lung Institute, Imperial College School of Medicine, Dovehouse St., London SW3 6LY, UK. E-mail: j.a.mitchell{at}ic.ac.uk
SPECIFIC AIMS
Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that heterodimerize with the retinoid X receptor and then modulate the function of many target genes. Three PPARs are known: 
, ß (sometimes called 
), and 
. The better studied are PPAR and PPAR, which are activated by fibrates and thiazolidinediones/glitazones, respectively. It is now believed that activation of the PPARs could be associated with the prevention of heart attack and stroke in humans. PPARß is less well studied, but is known to be ubiquitously expressed and important in plaque formation. PPARß is a putative receptor for prostacyclin. Prostacyclin is an important antithrombotic hormone that synergizes with nitric oxide (NO) to inhibit platelet aggregation. However, because platelets have no nuclei, the notion that PPARß may be involved in antithrombotic effects has not previously been addressed. Indeed, the antithrombotic properties of prostacyclin are currently thought to be mediated exclusively via activation of cell surface prostacyclin (IP) receptors, followed by stimulation of adenylyl cyclase. However, it has recently been shown in a single isolated publication that PPAR is present in the cytoplasm of platelets and that a PPAR ligand can inhibit platelet aggregation. In the current study, we have investigated the presence and function of PPARß in human platelets. Since the antithrombotic effects of prostacyclin act in synergy with those of NO, we have also investigated if a similar relationship exists between PPARß and NO donors.
PRINCIPAL FINDINGS
1. Human platelets and meg-01 megakaryocytes express PPARß
PPAR- and PPARß-like immunoreactivity was detected in platelet precursor megakaryocytes (meg-01 cells), purified platelets, and in whole blood extracts.
2. Effects of PPAR ligands on platelet aggregation
Ligands for PPARß (GW0742 and L-165041), PPAR (rosiglitazone) and PPAR (fenofibrate) inhibited ADP-induced platelet aggregation in whole blood. GW0742 and L-165041 were more potent inhibitors than rosiglitazone or fenofibrate (all drugs at 10–5 M) on platelet aggregation in whole blood (29.5%±9.4, 27.6%±3.5, 13.3%±6.7, and 16.8%±2.2 inhibition of aggregation compared with control when stimulated with 10–5 M ADP, respectively). GW0742 also inhibited platelet aggregation in PRP stimulated with 10–6 M ADP (10–5 M 14.7±5.3% inhibition; 4x10–5 M 42.0±10.5%). To demonstrate that the effects of GW0742 were not due to PPAR activation, the effects of GW0742 on platelet aggregation in the presence of the PPAR antagonist GW9662 were investigated. GW9662 had no effect on the inhibition caused by GW0742 on ADP induced platelet aggregation in whole blood.
In addition to ADP, GW0742 similarly inhibited aggregation induced by collagen (2x10–5g/mL), thrombin (1unit/mL) and 14:0 lyso phosphatidic acid (LPA; 10–6 M).
3. Effects of GW0742 on calcium release in platelets
GW0742 caused a concentration dependent decrease in the amount of calcium released from platelets when stimulated with 10–5 M ADP. 10–5 M GW0742 caused a total inhibition in calcium release (Fig. 1
).
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4. PPAR activation in platelets
The activation of PPARß, , and DNA binding consensus sequences were examined in platelets incubated for 5 min with either GW0742, L-165041, or rosiglitazone (all at 10–5 M) or treprostinil sodium (10–11 M). Both GW0742 and L-165041 were found to activate the PPARß receptor without affecting PPAR and activity. The prostacyclin mimetic, treprostinil sodium, was able to activate the PPARß receptor to similar levels as the PPARß ligands.
5. Interactions between PPARß, NO, and prostacyclin on platelet aggregation
The NO donor, sodium nitroprusside (10–7–10–4 M), induced a concentration-dependent inhibition of platelet aggregation induced by ADP. Similarly, the prostacyclin-mimetic, treprostinil sodium (10–12–10–8 M), inhibited in a concentration-dependent manner aggregation induced by ADP, collagen, thrombin, and LPA. In support of previous reports for other prostacyclin-mimetics, treprostinil sodium at threshold concentrations (10–12 M) acted in synergy with sodium nitroprusside to inhibit platelet aggregation.
Similarly to observations made with treprostinil sodium, GW0742 acted in synergy with sodium nitroprusside to inhibit platelet aggregation (Fig. 2
). GW0742 also synergized with treprostinil sodium but not with rosiglitazone to inhibit platelet aggregation.
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CONCLUSIONS AND SIGNIFICANCE
This study shows for the first time that platelets, cell fragments that contain no nucleus, express the nuclear receptor PPARß, as does the platelet precursor megakaryocyte cell line (meg-01). We show that at concentrations expected in the plasma at therapeutic doses (i.e., 10–6 M range) PPARß agonists activate PPARß, but not PPAR or PPAR in human washed platelets and inhibit platelet activation (calcium increase) and aggregation. In nucleated cells, PPARß has been shown to be a receptor for prostacyclin. In the current study, we show a prostacyclin mimetic (in the 10–9 M range) activates PPARß selectively. Finally, we show that, like prostacyclin mimetics, PPARß ligands synergize with NO donors to inhibit platelet aggregation.
Recently it has been shown that PPAR is expressed in the cytoplasm of cells and is not restricted to the nucleus. Along with this, PPAR has been found to be expressed in platelets and meg-01 cells. Together, these findings suggest that the fragments of nucleus present in platelets may have some function. Indeed, platelets are able to synthesize IL-1ß from mRNA stores during fibrin clot formation, and this IL-1ß can stimulate further inflammatory responses from the fibrin mesh. Since PPARß is expressed in platelets, it could well be that this receptor plays some major role in the function of the platelet.
We found that two PPARß ligands, GW0742 and L-165041, significantly inhibited platelet aggregation after just 5 min incubation. These observations were made in isolated platelet suspensions (PRP) as well as in whole blood and indicate that PPARß is an active antithrombotic pathway in platelets whose effects are independent of the nucleus and new gene induction. While our studies in PRP firmly establish that PPARß is active in platelets (as opposed to other blood elements) our observations using whole blood demonstrate that this phenomenon is robust and reproducible in a physiological assay system. In addition to PPARß ligands, we also found that the PPAR ligand, rosiglitazone, or the PPAR ligand, fenofibrate, inhibited platelet aggregation, although GW0742 and L-165041 were the most potent. Since ligands for each PPAR class inhibited aggregation, it could be argued that the effects we see with PPARß ligands are nonspecific. However, at anti-aggregatory concentrations, both GW0742 and L-165041 activated PPARß, but not PPAR or PPAR in platelets. Furthermore, a PPAR antagonist did not affect the abilities of PPARß ligands to inhibit aggregation. These observations may implicate PPARß as the dominant PPAR in platelet function.
The precise identification of endogenous ligands for PPAR or PPAR is currently debated; however, it is known that prostacyclin is a ligand for PPARß. Moreover, prostacyclin formed by the consecutive actions of cyclo-oxygenase-1 and prostacyclin synthase is arguably the most important endogenous antiplatelet hormone identified. Currently it is thought that prostacyclin inhibits platelet function via stimulation of surface prostacyclin IP receptors, linked to activation of adenylyl cyclase, and increased intracellular levels of cAMP. However, our data challenge this view. We show that the prostacyclin mimetic treprostinil sodium activates PPARß in human platelets in the low 10–9M range. We show that GW0742 shares a key property with prostacyclin mimetics in its effects on platelet aggregation. Specifically, like prostacyclin mimetics, the anti-aggregatory effects of PPARß ligands are synergistic with NO (provided via the NO donor sodium nitroprusside). In contrast, GW0742 did not synergize with rosiglitazone to inhibit platelet aggregation, suggesting the effects of PPARß and NO are specific.
We found that PPARß activation inhibited aggregation in response to ADP, collagen, thrombin, and the platelet-derived LPA. These stimuli act through phosopholipase C (PLC), which results in calcium influx and platelet aggregation. These observations suggest that the anti-aggregatory effects of PPARß ligands are ubiquitous and not dependent on mode of platelet stimulation.
On the face of it, our observations are controversial. How can a nuclear receptor have effects in non-nucleated platelets? However, our data clearly show that activation of PPARß (and for comparison, PPAR and ) inhibits platelet aggregation acutely and via a nongenomic mechanism. These observations are made in the low 10–6 M range for ligands, which is consistent with the predicted plasma concentrations of PPAR ligands. This concept has implications beyond the PPARs and is therefore of interest to the general scientific community. Our data clearly show that PPARß is expressed in human platelets and that its activation inhibits aggregation. Our data suggest that some of the antithrombotic properties of prostacyclin may be mediated via PPARß. These observations highlight PPARß as a novel therapeutic target for the treatment and prevention of cardiovascular disease.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4395fje;
This article has been cited by other articles:
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  J. A. Mitchell, F. Ali, L. Bailey, L. Moreno, and L. S. Harrington Role of nitric oxide and prostacyclin as vasoactive hormones released by the endothelium Exp Physiol, January 1, 2008; 93(1): 141 - 147. [Abstract] [Full Text] [PDF]
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 L. A. Moraes, K. E. Swales, J. A. Wray, A. Damazo, J. M. Gibbins, T. D. Warner, and D. Bishop-Bailey Nongenomic signaling of the retinoid X receptor through binding and inhibiting Gq in human platelets Blood, May 1, 2007; 109(9): 3741 - 3744. [Abstract] [Full Text] [PDF]
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