Vascular Pharmacology 62 (2014) 162–166

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Vascular Pharmacology

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Review

Mechanism of antiplatelet action of hypolipidemic, antidiabeticand antihypertensive drugs by PPAR activation

PPAR agonists: New antiplatelet agents

Eduardo Fuentes ⁎, Iván Palomo ⁎Department of Clinical Biochemistry and Immunohaematology, Faculty ofHealth Sciences, Interdisciplinary Excellence Research ProgramonHealthy Aging (PIEI-ES), Universidad de Talca, Talca, ChileCentro de Estudios en Alimentos Procesados (CEAP), CONICYT-Regional, Gore Maule, R09I2001, Chile

⁎ Corresponding authors at: Immunology and HaemHealth Sciences, Universidad de Talca, Casilla, 747, Talcfax: +56 71 20048.

E-mail addresses: edfuentes@utalca.cl (E. Fuentes), ipa

http://dx.doi.org/10.1016/j.vph.2014.05.0081537-1891/© 2014 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 20 February 2014Received in revised form 8 May 2014Accepted 15 May 2014Available online 27 May 2014

Keywords:AntiplateletHypolipidemicAntidiabeticAntihypertensive peroxisomeproliferator-activated receptors

Given the prevalence of cardiovascular disease in patients with cardiovascular risk factors (i.e., hypertension, dia-betes, smoking and obesity) and that platelet activation plays an important pathogenic role in cardiovascular dis-eases, it is very important to identify the drugs that havemultiple targets. In this sense, the present article describesthe mechanism of antiplatelet action of hypolipidemic (statins and fibrates), antidiabetic (thiazolidinediones) andantihypertensive (nifedipine) drugs via peroxisome proliferator-activated receptor (PPAR) activation. Themecha-nism of antiplatelet action of the drugs is by direct activation of PPARs with the inhibition of cyclooxygenase-1,protein kinase C-alpha, calcium mobilization, thromboxane A2, sCD40L, platelet microparticles and cAMP-phosphodiesterase, and the stimulation of proteins kinase G and A. Thus, these observations highlight PPARs as anovel therapeutic target for the treatment and prevention of cardiovascular diseases.

© 2014 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1622. PPARs and atherothrombosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1633. Mechanism of antiplatelet action by PPAR activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1634. Drugs and platelet inhibition: role of PPAR activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

4.1. Statins and fibrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1634.2. Thiazolidinediones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1644.3. Nifedipine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165Declaration of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165

1. Introduction

Cardiovascular diseases (CVD) (i.e., acute myocardial infarction,cerebrovascular disease and peripheral arterial thrombosis) havesignificantly increased in recent years [1,2]. The most frequent andindependent risk factors for CVD are cigarette smoking, elevated

atology Laboratory, Faculty ofa, Chile. Tel.: +56 71 200493;

lomo@utalca.cl (I. Palomo).

blood pressure, elevated serum total cholesterol and diabetes,among others [3,4].

In most cases, CVD is the consequence of an atherosclerotic plaquerupture and thrombus formation. Accelerated atherosclerosis and an in-creased risk of thrombotic vascular events in diabetes may result fromdyslipidemia, endothelial dysfunction and platelet hyperreactivity [5,6]. Platelets from patients with type 1 and type 2 diabetes exhibit en-hanced platelet aggregation activity early in the disease course thatmay precede CVD development [7,8]. Thus, platelets adhere; secretetheir granule contents, aggregate and initiate thrombus formation fol-lowing an atheromatous plaque rupture, [9,10].

163E. Fuentes, I. Palomo / Vascular Pharmacology 62 (2014) 162–166

Given the central role of platelets in the atherosclerotic-related in-flammatory response and the subsequent thrombotic event, a varietyof antiplatelet agents have been developed [11,12]. Although antiplate-let drugs have been proven to be beneficial in patients with clinical ev-idence of CVD, outcomes still remain poor [13]. In this way, plateletsfrom diabetic patients release greater amounts of thromboxane A2(TXA2) than platelets from non-diabetic patients receiving treatmentwith low-dose aspirin [14]. This is because all the current available an-tiplatelet agents only target one signal pathway and moderately inhibitplatelet activation (e.g. aspirin and clopidogrel) [15]. Therefore, there ismuch room for further improvement of antiplatelet treatment and theneed to identify the mechanisms by which hypolipidemic (statins andfibrates), antidiabetic (thiazolidinediones) and antihypertensive (nifed-ipine) drugs inhibit platelet activation. It has also been observed thatthese drugs are ligands for peroxisome proliferator-activated receptors(PPARs) in cells which have a nucleus [16–18].

PPARs are involved inmany biological processes, including lipid andenergymetabolism inflammation responses, and atherosclerotic plaqueformation [19–21]. Selective agonists through their action on nuclearreceptors (e.g. PPARs) regulate platelet function despite the absence ofa nucleus in platelets. [22]. In this sense, the present article describesthe antiplatelet action mechanism of hypolipidemic, antidiabetic andantihypertensive drugs via PPAR activation.

2. PPARs and atherothrombosis

PPARs consist of a family of three nuclear receptor isoforms (γ, β/δ,andα) [23]. PPARs are the key regulators of metabolism and inflamma-tion, and play an important role in chronic inflammatory disease pro-cesses [24,25]. Accumulating evidence suggests that PPAR activation isa key mechanism to reduce atherosclerosis and improve cardiovascularfunction [26–28].

Recent studies suggest that PPAR-γ activation decreases atherogen-esis, not only by correcting metabolic disorders, but also through its di-rect effects on the vascular wall [29]. In addition, PPAR-α and PPAR-β/δhave also been suggested in the regulation of inflammation and slowatherosclerosis progression [20,30].

Ligands of PPAR-α are important regulators of lipid and lipoproteinmetabolism, thereby positively affecting plasma lipid risk factors relatedto atherosclerosis [31]. In this context, the lipid-lowering effect offenofibrate is achieved by activating PPAR-α andAMP-activated proteinkinase (AMPK) signaling pathway that results in increasing lipolysis andfatty acid β-oxidation [32].

PPARs appear to play a major role in the regulation of atherogenesisby countering the inflammation-provoking action of platelet adhesionand activation [33].

3. Mechanism of antiplatelet action by PPAR activation

Nuclear receptors are transcription factors that are activated by li-gands and subsequently bind to regulatory regions in target genes;allowing the organism to integrate signals coming from the environ-ment [34]. While steroid/nuclear receptors are recognized via for theirrole in gene regulation, increasing evidence supports nongenomic ac-tions of these receptors [35,36]. Thus, although platelets lack a nucleus,they express a number of transcription factors including steroid/nuclearreceptors such as PPARs [37,38], glucocorticoid receptor (GR) [39],retinoic X receptor (RXR) [41], estrogen receptor (ER) [40] and nuclearfactor kappa B (NF-κB) [42]. Thus, selective ligands for these receptorsregulate platelet aggregation and activation [22,43].

Whilemany reports focus on PPARexpression in the nucleus [44,45],in this article we focus on the role of PPAR-cytoplasm in platelet func-tion. Recent studies have provided the first evidence that human bonemarrow megakaryocytes and human platelets express PPAR-γ [37].PPAR-γ activation decreases platelet aggregation and delays intra-arterial thrombus formation in rats, at least partially, by an increase in

the expression of nitric oxide synthase (NOS) and thrombomodulin[46]. PPAR-γ activation also inhibits platelet function induced by colla-gen through the modulation of early glycoprotein (GP) VI signaling, atthe level of Syk and LAT [47]. In addition, platelet incubation with a nat-ural PPAR-γ agonist (15d-PGJ(2)) or with a potent synthetic PPAR-γ li-gand (rosiglitazone) not only attenuates platelet activation, but alsodecreases the release of platelet proinflammatory and procoagulantme-diators (sCD40L and TXA2) [33,37].

Agonists of PPAR-β, including GW0742 and L-165041, inhibit plate-let activation after 5min of incubation. Clearlywith such acute exposureand as platelets have no nucleus, PPAR-β is an active antithromboticpathway in platelets, whose effects are independent of the nucleus [38].

Moreover, PPAR-α activators may exert vasculo-protective actionthrough suppression of PDGF-BB production in amegakaryocyte/plateletpathway [48]. Prostacyclin formed by the consecutive actions ofcyclooxygenase-1 (COX-1) and prostacyclin synthase is arguably themost important endogenous antiplatelet hormone identified to date[49]. Prostacyclin inhibits platelet function via stimulation of a surfaceprostanoid receptor linked to the activation of adenylyl cyclase and in-creased intracellular levels of cyclic adenosine monophosphate (cAMP)[50]. Moreover, prostacyclin activates PPAR-β/δ in human platelets inthe low 10−9 M range. Therefore, an additional mechanism of antiplate-let action by prostacyclin occurs by activation of PPAR-β/δ and its effectson platelet aggregation is synergistic with nitric oxide (NO) [38,51].

4. Drugs and platelet inhibition: role of PPAR activation

Given the prevalence of CVD in patients with cardiovascular riskfactors (i.e., hypertension, diabetes, smoking and obesity) and thatplatelet activation plays an important pathogenic role in athero-thrombosis [3,52], it is very important to identify the drugs thathave multiple targets. In this sense, the present article describesthe mechanism of antiplatelet action of hypolipidemic (statins andfibrates), antidiabetic (thiazolidinediones) and antihypertensive(nifedipine) drugs by PPAR activation.

4.1. Statins and fibrates

The 3-hydroxy-3-methylglutaryl-coenzyme (HMG-CoA) reductaseinhibitors, also known as statins, are the most effective class of drugsfor lowering serum low-density lipoprotein cholesterol (LDL-C) concen-trations [53]. In this way, another class of drug called fibrates reducestriglycerides and increases high-density lipoprotein cholesterol (HDL-C) [54]. In this article, we discuss how statins and fibrates appear tohave more biological effects than those originally targeted. Statins andfibrates can exert beneficial anti-inflammatory and antithrombotic ef-fects in after patients with a high risk of coronary artery disease asearly as 3 days after therapy [55]. Thus, statins (simvastatin, atorvastat-in, or cerivastatin) decrease morbidity and mortality in patients withCVD by their effects on proinflammatory cytokines: interleukin 6 (IL-6), IL-8, and monocyte chemoattractant protein-1 (MCP-1) [56]. Mean-while, fibrates by PPAR-activation may inhibit atherosclerosis develop-ment in vivo [57].

Interestingly both statins and fibrates inhibit platelet function [54].Inhibition of platelet aggregation by simvastatin involves the activationof the cAMP-eNOS/NO-cGMP pathway, resulting in the inhibition of thePLC-γ2/PKC/p38 MAPK/TXA2 cascade [58,59]. Therefore, the direct in-hibitory effects of statins and fibrates on platelet activation are mediat-ed by PPAR activation and this new finding reveals some of thepleiotropic effects of these drugs [60]. In platelets, PPAR signaling path-way activation involves binding and repression of PKC, and increasing ofcAMP and cGMP levels [60]. The increase of intraplatelet levels of cAMPis due to the fact that the repression of PKC allows greater activity ofadenylyl cyclase, which converts ATP to cAMP [61,62]. In addition,cAMP induced inhibition of platelet P-selectin expression, platelet

Fig. 1. Mechanism of antiplatelet action of PPAR ligands. AA = arachidonic acid; AKT = known as protein kinase B; AMP = adenosine monophosphate; cAMP = cyclic adenosinemonophosphate; cGMP= cyclic guanosinemonophosphate; COX-1=cyclooxygenase-1;NO= nitric oxide;NOS= nitric oxide synthase; PDE3=phosphodiesterase-3; PGH2= prostaglan-dinH2; PGG2= prostaglandinG2; PKA= protein kinaseA; PKC= protein kinase C; PKG= protein kinaseG; PLA2= phospholipaseA2; PMP= plateletmicroparticles; PPARs= peroxisomeproliferator-activated receptors; TXA2 = thromboxane A2; VASP = vasodilator-stimulated phosphoprotein; VASP-P = vasodilator-stimulated phosphoprotein phosphorylation.

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aggregation and release of sCD40L, for the large part, is mediatedthrough the activation of protein kinase A (PKA) [63–66].

Platelet microparticles (PMP) have an average diameter of 0.1 μm[67], and are the most abundant type of microparticles (MP) in humanblood (representing 70–90%) and contribute to many biologically pro-cesses [68]. PMP levels are greatly elevated in patients (range from~3000–11,000/μL) with diseases including venous thromboembolism[69], hypertension [70], diabetes [71], cancer [72], immunologic plateletdisorders and arterial thrombosis [73,74]. In humans, plasma levels ofcirculating PMP are known to increase with the presence of cardiovas-cular risk factors and progression of atherosclerosis [75]. Even after ini-tiation of antithrombotic treatment, PMP are still elevated after sixmonths [76]. Possibly because a low dose of aspirinmight not be strongenough to suppress the shedding of PMP in the microcirculation [77].With respect to the role of statins, MP from hypercholesterolemia pa-tients with statin treatment had reduced markers of activated platelets(GPIIb-IIIa), activated inflammatory cells (Mac-1) and tissue factor [78].Meanwhile atorvastatin reduces thrombin generation and expression oftissue factor, GPIIIa and P-selectin on PMP in patients with peripheralvascular disease [79]. In this way statins may inhibit thrombotic eventgeneration partly through a PMP dependent mechanism.

4.2. Thiazolidinediones

The thiazolidinediones (TZDs) are a class of oral antidiabetic drugsthat exert effects through a mechanism that involves an activation ofPPAR-γ [80]. TZDs (rosiglitazone, pioglitazone and troglitazone) havebeen shown to protect LDL-C from oxidative modification in patientswith type 2 diabetes that were treated for 8 weeks, one of the key initialevents in atherogenesis [81]. In addition, TZD activation of PPAR-γ re-duced the progression of subclinical atherosclerosis by normalizing

proatherogenic metabolic abnormalities of the insulin resistance, andthrough an inhibition of vascular cell growth and movement [82].

Recent studies suggest that TZDs may also be clinically beneficial toinflammatory mediators [83]. Matrix metalloproteinase-9 (MMP-9) iscritically involved in the development of unstable plaques [84]. AlsoMMP-9 levels are increased in type 2 diabetes patients with coronaryartery disease, and the treatment of these patients with the antidiabeticPPARγ-activator rosiglitazone significantly reduces the serum levels ofMMP-9, tumor necrosis factor alpha (TNF-α) and serumamyloid A [85].

Troglitazonehas a potent inhibitory effect on platelet aggregation viathe suppression of the thrombin-induced activation of phosphoinositidesignaling in human platelets [86]. Based on the function of other cells(astrocyte cells), the TZDmode of action is likely related to the elevationof the intracellular cAMP level by PPAR-γ activation [87]. This could ex-plain why the treatment with TZDs reduces sCD40L serum levels in pa-tients with type 1 and 2 diabetes and coronary artery disease [88,89].

PPAR-γ containing PMP are quickly released fromα-granule into ex-tracellular parts when platelets are activated by collagen [90]. Then,PMP-PPAR-γ can be internalized by the monocytic cell line (THP-1). Inthis way, THP-1 cell activation can be attenuated in the presence of aPPAR-γ agonist (rosiglitazone) [91,92].

In addition, one possible mechanism explaining the anti-inflammatory activity of TZDs is its ability to activate GR [93]. Inthis way, classic anti-inflammatory glucocorticoids are due to thefact that human platelets contain GR [39].

4.3. Nifedipine

Nifedipine, a L-type calcium channel blocker, is widely used in thetreatment of hypertension and coronary heart disease [94,95]. Nifedi-pine increases the activity and intracellular expression of PPAR-β/γfromactivated platelets. Therefore, themechanism of antiplatelet action

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of nifedipine is PAR-β/γ-dependent with upregulation of the PI3K/Akt/NO/cGMP/PKG pathway, and the inhibition of PKC activity and intracel-lular Ca2+ mobilization in platelets [96]. Taking into consideration allthese findings in vivo evidence of such in vitro-related nifedipine-antithrombotic effects is provided. Indeed, using a murine model ofthrombus formation, the administration of nifedipine substantiallyinhibitedfluorescein sodium-induced vessel thrombus formation. In ad-dition, its antithrombotic effect is considerably reduced in the presenceof PPAR-β/γ antagonists [96].

Finally, themechanismof antiplatelet action of statins, fibrates, TZDsand nifedipine by direct activation of PPARs is mediated by inhibition ofCOX-1 [37], PKC [62] and Ca2+mobilization [47], and indirect inhibitionof TXA2, sCD40L, PMP, P-selectin and cAMP-phosphodiesterase, andstimulation of PKG (increased levels of cGMP) and PKA (increased levelsof cAMP) [63,97] (Fig. 1).

5. Conclusions

The data presented in this article has demonstrated that the ability ofstatins, fibrates, TZDs and nifedipine to inhibit platelet activation is like-ly attributable to their influence on PPAR activation. Thus, these obser-vations highlight PPARs as a novel therapeutic target for the treatmentand prevention of cardiovascular risk factors and CVD.

Declaration of interest

The authors report no declarations of interest.

Acknowledgments

This work was funded by CONICYT REGIONAL/GORE MAULE/CEAP/R09I2001, Programa de Investigación de Excelencia Interdisciplinariaen Envejecimiento Saludable (PIEI-ES), and supported by grant no.1130216 (I.P., M.G., R.M., M.A., J.C.) from Fondecyt, Chile.

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