Antiplatelet activity of nifedipine is mediated by inhibition of NF-κB activation caused by enhancement of PPAR-β/-γ activity

RESEARCH PAPER

Antiplatelet activity of

nifedipine is mediated by

inhibition of NF-κB

activation caused by

enhancement of

PPAR-β/-γ activity

Ching-Yu Shih

, I-Hsin Lin

, James-Cheng Ding

, Fu-Chi Chen

and

Tz-Chong Chou

1Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, 2School of

Post-Baccalaureate Chinese Medicine,Tzu Chi University, Hualien, Taiwan, 3Fu Wai Hospital

and Cardiovascular Institute, Chinese Academy of Medical Sciences and Peking Union Medical College, Peking, China, 4Department of Biomedical Engineering, National Defense Medical

Center, Taipei, Taiwan, 5Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan, 6Department of Biotechnology, Asia University, Taichung, Taiwan, and 7China Medical

University Hospital, China Medical University, Taichung, Taiwan

Correspondence

Tz-Chong Chou, Institute of Medical Sciences, Tzu Chi University, 6F, Xie-Li Building, No. 707, Sec. 3, Zhongyang Road, Hualien 97002, Taiwan. E-mail: [email protected]

---Keywords

nifedipine; PPARs; NF-κB; platelet aggregation; NO

---Received

4 July 2013

18 October 2013

Accepted

24 October 2013

BACKGROUND AND PURPOSE

The transcription factor NF-κB, stimulates platelet aggregation through a non-genomic mechanism. Nifedipine, a

voltage-gated L-type calcium channel blocker, is widely used to treat hypertension. Nifedipine also displays antiplatelet

activity, but the underlying mechanisms involved remain unclear. This study was designed to investigate whether the

antiplatelet effects of nifedipine are mediated by regulating NF-κB-dependent responses.

EXPERIMENTAL APPROACH

Platelet aggregation was measured turbidimetrically using an aggregometer. NF-κB and PPAR activation, intracellular Ca2+

mobilization, PKCα activity, surface glycoprotein IIb/IIIa (GPIIb/IIIa) expression and platelet activation-related signalling

pathways were determined in control and nifedipine-treated platelets in the presence or absence of PPAR antagonists or

betulinic acid, a NF-κB activator.

KEY RESULTS

Exposure of platelets to nifedipine significantly increased the PPAR-β/-γ activity in activated human platelets. Treatment with

nifedipine reduced collagen-induced NF-κB events, including the phosphorylation of IκB kinase-β, IκBα and p65NF-κB, which

were markedly attenuated by GSK0660, a PPAR-β antagonist, or GW9662, a PPAR-γ antagonist. Furthermore, the interaction

of PPAR-β/-γ with NF-κB and the PPAR-β/-γ-up-regulated NO/cGMP/PKG1 cascade may contribute to inhibition of NF-κB

activation by nifedipine. Suppressing PPAR-β/-γ activity or increasing NF-κB activation greatly reversed the inhibitory effect of

nifedipine on collagen-induced platelet aggregation, intracellular Ca2+ mobilization, PKCα

activity and surface GPIIb/IIIa expression.

CONCLUSIONS AND IMPLICATIONS

PPAR-β/-γ-dependent inhibition of NF-κB activation contributes to the antiplatelet activity of nifedipine. These findings provide

a novel mechanism underlying the beneficial effects of nifedipine on platelet hyperactivity-related vascular and inflammatory

diseases.

BJP

British Journal of

Pharmacology

DOI:10.1111/bph.12523 www.brjpharmacol.org

1490 British Journal of Pharmacology (2014) 171 1490–1500 © 2013 The British Pharmacological Society

Abbreviations

BetA, betulinic acid; GPIIb/IIIa, glycoprotein IIb/IIIa; IKKs, IκB kinases; ODQ, 1H-[1, 2, 4] oxadiazolo[4,3-a]

quinoxalin-1-one

Introduction

Traditionally, platelet hyperactivity is recognized as a crucial factor in the pathogenesis of thrombotic vascular events. Currently, it has been believed that the activated platelets also play a key role in modulating inflammatory responses (Rajagopalan et al., 2007). Therefore, inhibiting platelet hyperactivity may be a potential strategy for preventing and alleviating platelet-related thrombotic and inflammatory diseases. Nifedipine, a dihydropyridine derivative, blocks the

voltage-gated L-type α1c calcium channel (Cav1.2) and is

widely used in the treatment of hypertension and coronary heart diseases (Yamagishi et al., 2006). Interestingly, nifedipine also exhibits an antiplatelet activity (Os´miałowska et al.,

1990), but the mechanisms involved remain unclear. Accumulating evidence has indicated that the antiplatelet activity

of nifedipine may be independent of the inhibition of calcium influx (Doyle and Ruegg, 1985), suggesting that other mechanisms may contribute to this property. Although platelets are non-nucleated cells, they do contain transcription factors, notably PPARs and NF-κB,

which exert negative and positive regulating effects on platelet aggregation respectively. PPARs are ligand-activated transcription factors and modulate several crucial physiological

functions such as lipid metabolism and glucose homeostasis (Rosen and Spiegelman, 2001; Yessoufou and Wahli, 2010). Three PPAR isoforms (PPAR-α, PPAR-β/-δ and PPAR-γ) have been found in human platelets (nomenclature follows

Alexander et al., 2013). Recent studies have demonstrated that PPARs inhibit platelet aggregation through a nongenomic mechanism (Ali et al., 2009; Chou et al., 2011), suggesting that compounds with PPAR-activating activity may be

a new type of antiplatelet drug. Although nifedipine reportedly enhances PPAR-γ activity in macrophages and smooth

muscle cells (Hashimoto et al., 2010; Ishii et al., 2010), no information regarding whether PPARs are involved in nifedipine-mediated antiplatelet activity is available. In unstimulated cells, NF-κB, which exists as an inactive cytoplasmic heterodimer complex composed of p50 and p65 subunits, is tightly bound to an inhibitory protein, IκB-α. Once activated by several pro-inflammatory stimuli, IκB-α is

phosphorylated by IκB kinases (IKKs), leading to rapid polyubiquitination and degradation by proteasomes, followed by

the release of NF-κB from its inhibitors. The NF-κB dimers subsequently translocate to the nucleus, where they bind to and activate the transcription of target genes (Tak and Firestein, 2001). In addition to the critical role of NF-κB in inflammatory responses (Ghosh and Hayden, 2008), NF-κB also exerts a non-genomic function to regulate platelet activation. It is known that IKKβ phosphorylation, IκBα degradation

and p65NF-κB phosphorylation in human platelets are greatly amplified in response to thrombin or collagen. Because treatment with NF-κB inhibitors impairs agonistinduced platelet aggregation and granule release (Malaver

et al., 2009; Chang et al., 2011), suppressing NF-κB activation

may be a way of inhibiting platelet aggregation. Several lines of evidence have confirmed that PPAR-γ and PPAR-β/-δ agonists exhibit anti-inflammatory activity by inhibiting NF-κB

activation through the attenuation of IKKs and the DNAbinding activity of NF-κB in activated macrophages, smooth

muscle cells and experimental periodontitis (Castrillo et al., 2000; Ikeda et al., 2000; Di Paola et al., 2011). However, the effect of PPAR-γ on NF-κB activation is controversial, and it is proposed that the described mechanisms of PPARs may be cell type and PPAR isoform specific (Ricote and Glass, 2007). To date, whether PPARs or PPAR-regulated NF-κB events

contribute to the antiplatelet activity of nifedipine remain unclear. In the present study, we demonstrated that nifedipine initially increases PPAR-β/-γ activity and subsequently

attenuates NF-κB activation in platelets, which ultimately inhibits platelet aggregation.

Methods

Determination of PPAR activity

Platelets were incubated with various drugs or solvent control for 5 min at 37°C; lysed in a buffer containing 50 μM of Tris-HCl (pH 7.4), 0.5 M of NaCl, 1 μM of EDTA, 0.05% SDS, 0.5% Triton X-100 (Sigma Chemical Company, St. Louis, MO, USA) and 1 μM of PMSF; and subsequently centrifuged at 15 000× g for 10 min at 4°C. The PPAR activity in supernatants

was determined using a PPAR transcription factor ELISA _{kit, and}

the absorbance at 450 nm was measured (Chou et al., 2011).

Platelet aggregation

Blood samples were taken from healthy human volunteers who had not taken any medicine during the preceding 2 weeks, were mixed with ACD solution (75 mM of trisodium citrate, 42 mM of citric acid and 136 mM of glucose, pH 5.2) and centrifuged at 160× g and 25°C for 10 min to produce PRP. Centrifugation was subsequently performed to produce platelet pellets and suspended in Tyrode solution (pH 7.4). To prevent the contamination of platelet samples with leukocytes, platelet suspension was filtered through a 5 μm syringe-adaptable filter to remove white blood cell contaminants as previously described (Freedman et al., 2010),

and platelet concentration was adjusted to 3.0 × 108

platelets·mL−1. Platelet aggregation was measured turbidimetrically

using an aggregometer at 37°C with constant stirring at 1000 r.p.m. (Model 560; Chrono-Log Corporation,

Havertown, PA, USA). Tyrode solution was assigned as 100% aggregation, and platelet suspension was assigned as 0% aggregation. Platelet suspensions (0.3 mL) were preincubated with various drugs or solvent control (0.1% DMSO) for 3 min,

and collagen (10 μg·mL−1) was subsequently added for 6 min.

percent-Nifedipine inhibits NF-κB activation in platelets

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British Journal of Pharmacology (2014) 171 1490–1500 1491 age of the Tyrode solution in light transmission units as described previously (Chou et al., 2011).

Determination of cGMP formation

Washed platelets were preincubated with various drugs or

solvent control for 3 min at 37°C, and collagen (10 μg·mL−1)

was subsequently added for 6 min. The reaction was stopped by adding EDTA (10 mM) followed by immediate boiling for 5 min. After centrifugation at 10 000× g for 5 min at 4°C, the

cGMP content of the supernatant was measured using an ELISA

kit.

Determination of nitrate + nitrite formation

Washed platelets were preincubated with various drugs or

solvent control for 3 min at 37°C, and collagen (10 μg·mL−1)

was subsequently added for 6 min. Centrifugation was performed at 10 000× g for 5 min at 4°C. The amount of nitrate

+ nitrite (NOx), a stable end product of NO, in the supernatants was measured using a Sievers NO analyser (Sievers 280 NOA; Sievers, Boulder, CO, USA) as described previously (Chou et al., 2008). Nitrate concentrations were calculated with reference to a standard solution of sodium nitrate.

Measurement of platelet intracellular

Ca

mobilization

One mL of PRP (3.0 × 108 platelets·mL−1) was incubated with

Fluo-4 AM (5 μM; Sigma Chemical Company) for 30 min at 37°C in the dark and subsequently centrifuged at 500× g for 10 min. The pellets were then suspended in 2 mL of Tyrode solution. The fluorescence intensity of 20 000 platelets per sample was analysed using a flow cytometer equipped with CellQuest software (FACScan; Becton Dickinson, Heidelberg, Germany) (Chou et al., 2011).

Measurement of platelet surface glycoprotein

IIb/IIIa (GPIIb/IIIa) expression

A platelet suspension (10 μL) was placed into polystyrene tubes containing 35 μL of HEPES buffer and 5 μL of CD41/ CD61-FITC that was raised against a platelet GPIIb/IIIa complex.

Various drugs were then added and incubated at 37°C

for 5 min, and collagen (10 μg·mL–1) was subsequently added

for 10 min. The reaction was stopped by adding 500 μL of 1% paraformaldehyde, and the fluorescence intensity of 20 000 platelets per sample was analysed using a flow cytometer.

Immunoprecipitation

The total cellular protein of platelets (1 × 109 cells) was

extracted and incubated with pure proteome protein A magnetic beads for 1 h at 4°C. The sample tubes were subsequently placed onto the magnetic rack, and the beads were allowed to adhere to the side to remove non-specific binding. After centrifugation and collecting the supernatant, the primary antibody

for PPAR-β or PPAR-γ was added in the presence of protein A magnetic beads and incubated overnight at 4°C. The sample tubes were subsequently placed onto the magnetic rack, and the beads were allowed to adhere to the side, which enabled the collection of the magnetic beads. Subsequently, a lysis buffer was added and boiled to separate the beads and bound protein (Chou et al., 2011). The expression of the target protein in the samples was determined usingWestern blotting.

Western blotting

Platelets (3 × 108 mL−1) were incubated with various drugs for

3 min at 37°C and were subsequently lysed in RIPA buffer (150 mM of NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM of Tris, pH 8.0) containing a mixture of protease and phosphatase inhibitors. The cell lysates were heated at 95°C for 10 min, and proteins (20 μg) were separated in 8% SDSPAGE and electrotransferred using semi-dry transfer (Bio-Rad

Laboratories, Inc., Hercules, CA, USA). Various primary antibodies were then incubated with transferred membranes for

1.5 h, and a peroxidase-conjugated secondary antibody was added in PBS Tween 20 (Sigma Chemical Company) for 1 h. The immunoreactive bands of target genes were detected using an ECL kit (Amersham International Plc., Buckinghamshire, UK) with reference to a cytoplasmic protein (β-actin).

Data analysis

Experimental results are expressed as means ± SEM. A oneway

analysis. Results were considered significant difference at a value of P < 0.05.

Materials

NG-nitro L-arginine methyl ester (L-NAME), 1H-[1, 2, 4]

oxadiazolo[4,3-a] quinoxalin-1-one (ODQ) and other chemical agents were obtained from Sigma Chemical Company (St. Louis, MO, USA). Collagen (type I, equine tendon) was obtained from Chrono-Log Corporation (Broomall, PA, USA). RIPA buffer was obtained from Pierce Biotechnology Inc. (Rockford, IL, USA). A PPAR transfactor kit and PPAR-γ (NR1C3) antibody were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). GW6471, GSK0660, GW9662 and betulinic acid (BetA) were purchased from

Tocris (Avonmouth, Bristol, UK). An enhanced chemiluminescence (ECL) reagent was purchased from Upstate Biotechnology (Lake Placid, NY, USA). PPAR-β (NR1C2) and β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-p65NF-κB, total-p65NF-κB, phospho-IKK and total-IKK antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Nifedipine and rosiglitazone were purchased from Sigma Chemical Company, dissolved in DMSO and diluted with Tyrode solution; the final concentration of DMSO was fixed at 0.1%.

Other chemical agents were obtained from Sigma Chemical Company.

Results

Nifedipine increases PPAR-β/-γ activity in

human platelets

Nifedipine (1 and 5 μM) concentration-dependently increased PPAR-β and PPAR-γ activity but did not affect PPAR-α (NRIC1) activity in collagen-stimulated platelets. Adding GW7647 (20 μM), a PPAR-α agonist; GW0742

(20 μM), a PPAR-β agonist; or rosiglitazone (20 μM), a PPAR-γ agonist, as positive controls, markedly enhanced the activity of PPAR-α, PPAR-β and PPAR-γ respectively (Figure 1A).

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C-Y Shih et al.

Moreover, nifedipine significantly attenuated collageninduced PPAR-γ phosphorylation (Figure 1B).

PPAR-β/-γ involve nifedipine-mediated

inhibition of NF-κB activation

Collagen-induced NF-κB events, including the phosphorylation of IKK-β, IκBα and p65NF-κB in human platelets, were

significantly inhibited by nifedipine, GW0742 and rosiglitazone. However, the decreased NF-κB events caused by nifedipine were reversed by GSK0660 (5 μM), a PPAR-β antagonist, or GW9662 (5 μM), a PPAR-γ antagonist (Figure 2). In addition, nifedipine concentration-dependently attenuated p65NF-κB phosphorylation in the PPAR-β/-γ-NF-κB complexes of collagen-stimulated platelets (Figure 3A).

Figure 1

Effect of nifedipine on PPAR activity in activated platelets. (A) Platelets were incubated with GW7647 (20 μM), GW0742 (20 μM), rosiglitazone

(20 μM) or nifedipine (1 or 5 μM) for 5 min followed by addition of collagen (10 μg·mL−1) for 6 min and

lysed. PPAR activity was measured as

described. (B) Platelets were preincubated with nifedipine (5 μM) at 37°C for 3 min followed by

addition of collagen (10 μg·mL−1) for 6 min; the

PPAR-γ phosphorylation was detected by Western blotting. Platelets treated with vehicle alone served as controls (resting). Data were expressed

as mean ± SEM (n = 4). ***P < 0.001, significantly different from collagen-stimulated platelets.

Nifedipine inhibits NF-κB activation in platelets

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British Journal of Pharmacology (2014) 171 1490–1500 1493

Role of PPAR-β/-γ and NF-κB in

nifedipine-mediated antiplatelet activity

Nifedipine (1, 5 μM) concentration dependently inhibited collagen-induced platelet aggregation, which was markedly attenuated by GSK0660, GW9662 and BetA (10 μM), a NF-κB activator (Figure 3B). These findings were also observed in thrombin-stimulated platelets (Supporting Information; Figure S1). Moreover, the PPAR-β/-γ antagonists and BetA did

Figure 2

Effects of PPAR-β/-γ on nifedipine-mediated suppression of NF-κB activation in activated platelets. Platelets were pretreated with GW0742,

37°C for 3 min followed by addition of

collagen (10 μg·mL−1) for 6 min. Then, the expression of (A) phospho-IKK, (B) phospho-IκBα and (C)

phospho-p65NF-κB were determined by

Western blot. Data were expressed as means ± SEM (n = 4). ***P < 0.001, significantly different from

collagen-stimulated platelets; +++P < 0.001,

significantly different from collagen + nifedipine-treated platelets.

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1494 British Journal of Pharmacology (2014) 171 1490–1500 not affect the platelet aggregation in both resting and collagen-stimulated platelets (Supporting Information; Figure S2).

The PPAR-β/-γ-regulated NO/cGMP/PKG1

cascade involves NF-κB inactivation

by nifedipine

Treatment with nifedipine further enhanced NOx and cGMP formation in collagen-stimulated platelets, compared with platelets stimulated with collagen alone (Figure 4A,B). Adding GSK0660 or GW9662 significantly diminished the enhancement of NOx and cGMP formation by nifedipine. In the presence of L-NAME (100 μM), an inhibitor of NOS; ODQ (10 μM), an inhibitor of soluble GC; or KT-5823 (5 μM), a PKG1 inhibitor, the inhibitory effect of nifedipine on collagen-induced p65NF-κB phosphorylation was markedly decreased (Figure 4C).

The PPAR-β/-γ/NF-κB cascade involves the

nifedipine-mediated reduction of intracellular

calcium mobilization and surface

GPIIb/IIIa expression

Nifepidine concentration-dependently reduced collageninduced intracellular calcium mobilization (Figure 5A) and

surface GPIIb/IIIa expression (Figure 5B). These effects of nifedipine were decreased after adding GSK0660, GW9662 or BetA. Similarly, the PPARs antagonists and BetA did not affect the parameters measured in this study.

Discussion and conclusions

Several studies have reported that activation of NF-κB induces platelet activation, and blocking NF-κB activation by BAY

11-7082 or Ro106-9920 inhibits platelet aggregation via suppressing

the PLA2-TxA2 cascade and surface GPIIb/IIIa expression

(Malaver et al., 2009; Chang et al., 2011). These results suggest that agents able to inhibit NF-κB activation may exhibit antiplatelet activity. The present study is the first to demonstrate that nifedipine attenuates NF-κB activation in activated human platelets, possibly providing a novel mechanism underlying the antiplatelet activity of nifedipine.

The molecular mechanisms underlying the inhibitory

effect of nifedipine on NF-κB activation were then investigated. A critical finding of this study is that nifedipine

increases both PPAR-β and PPAR-γ activity in human platelets. Our supplemental data show that other calcium channel blockers (CCBs) such as amlodipine, but not lacidipine, are PPAR-β activators in platelets (Supporting Information; Figure S3). The effects of different CCBs on PPAR activity in platelets is diverse and chemical structure specific. Ishii et al. (2010) reported that activation of ERK1/2 suppressed PPAR-γ activity through the phosphorylation of PPAR-γ.We observed that nifedipine inhibited collagen-induced PPAR-γ phosphorylation accompanied by an attenuation of ERK1/2 activation

(data not shown), which may be a possible mechanism of increasing the PPAR-γ activity. In the work described here, to determine the role of PPAR-β/-γ in regulating NF-κB activation, PPAR-β/-γ antagonists were added. In the presence of

GSK0660 or GW9662, nifedipine’s inhibition of NF-κB events, including the phosphorylation of IKK, IκBα and p65NF-κB in activated platelets, were markedly reduced, effects comparable to those observed in nucleated cells (Castrillo et al., 2000; Ikeda et al., 2000). Accordingly, PPAR-β/-γ are upstream negative regulators of NF-κB activation. We then investigated whether PPAR-β/-γ and NF-κB were involved in the antiplatelet activity of nifedipine. Blocking PPAR-β/-γ activity using GSK0660 or GW9662 or activating

Figure 2

Continued

British Journal of Pharmacology (2014) 171 1490–1500 1495

Figure 3

The interaction of PPAR-β/-γ with NF-κB and role of PPAR-β/-γ and NF-κB on nifedipine-mediated antiplatelet activity. Platelets were treated with

various drugs for 3 min at 37°C followed by addition of collagen (10 μg·mL−1) for 6 min. (A) The

extracted protein was immunoprecipitated (IP)

with PPAR-β or PPAR-γ. Then, the expression of target genes in the IP complexes was determined by Western blotting (WB). (B) The changes of

platelet aggregation in various groups were measured, and the representative image was shown. Data were expressed as means ± SEM (n = 4).

**P < 0.01, ***P < 0.001, significantly different from collagen-stimulated platelets; ++P < 0.01, +++P <

0.001, significantly different from

corresponding collagen + nifedipine-treated platelets.

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1496 British Journal of Pharmacology (2014) 171 1490–1500 NF-κB using BetA significantly decreased the inhibitory effect of nifedipine on platelet aggregation. Therefore, the PPAR-β/-γ-dependent attenuation of NF-κB activation contributed to the antiplatelet activity of nifedipine in our system.

The activation of PKCα is crucial for stimulating platelet secretion and aggregation (Konopatskaya et al., 2009). Our recent study confirmed that PPAR-α/-γ-mediated inhibition of PKCα activity in activated platelets is associated with an

interaction of PPAR-α/-γ with PKCα (Chou et al., 2011), suggesting that protein–protein interaction is a novel mechanism

in which they exert their functions. In this study, we

demonstrated for the first time that nifedipine increases the interaction of PPAR-β/-γ with NF-κB in collagen-stimulated platelets accompanied by decreased p65NF-κB phosphorylation in the complex. Thus, the association of PPAR-β/-γ with

NF-κB may play a role in the attenuation of NF-κB activation caused by nifedipine.

Up-regulation of the NO/cGMP/PKG1 signalling pathway reportedly inhibits platelet activation by modulating the dynamics of actin filaments, integrin activation and

intracel-Figure 4

in collagen-stimulated platelets were

determined. (C) The effects of L-NAME (100 μM), ODQ (10 μM) or KT-5823 (5 μM) on nifedipine-mediated inhibition of p65NF-κB phosphorylation

were examined. Data were expressed as means ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from

collagen-stimulated platelets; +P < 0.05, ++P < 0.01, +++P < 0.001, significantly different from

corresponding collagen + nifedipine-treated platelets.

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British Journal of Pharmacology (2014) 171 1490–1500 1497

lular Ca2+ mobilization, in turn suppressing PLC and PKC

activity (Antl et al., 2007; Chou et al., 2008). Our results showed that nifedipine treatment significantly increased NO and cGMP formation in activated platelets, which was markedly reduced by PPAR-β/-γ antagonists, indicating that

nifedipine-induced NO/cGMP formation is regulated by PPAR-β/-γ. Furthermore, Liang et al. (2009) and Jimenez et al. (2010) have proposed that activation of the PI3K/Akt signalling pathway is involved in PPAR-β/-γ-induced NO/cGMP

production. Suppressing the NO/cGMP/PKG1 cascade with appropriate inhibitors reversed the inhibition of p65NF-κB phosphorylation induced by nifedipine. Overall, the molecular mechanisms underlying decreased NF-κB activation by nifedipine may involve a direct association of PPAR-β/-γ with NF-κB and consequent up-regulation of the NO/cGMP/PKG1 pathway.

Figure 5

Role of PPAR-β/-γ and NF-κB in nifedipine-mediated attenuation of intracellular Ca2+ mobilization and

GPIIb/IIIa expression in activated platelets.

Platelets were pretreated with nifedipine or nifedipine combination with GSK0660 (5 μM), GW9662 (5 μM) or BetA (10 μM) for 3 min followed

by addition of collagen (10 μg·mL−1) for 6 min. The intracellular Ca2+ mobilization (A) and surface

GPIIb/IIIa expression (B) were determined. Data

were expressed as means ± SEM (n = 4). ***P < 0.001 as compared with collagen-stimulated alone

platelets. ++P < 0.01, +++P < 0.001 as compared

with respective collagen + nifedipine-treated alone platelets.

1498 British Journal of Pharmacology (2014) 171 1490–1500 When platelets are activated by agonists, an increase in

intracellular Ca2+ concentration as a result of either Ca2+

influx and/or Ca2+ release from intracellular stores is essential

for platelet activation. Nifedipine exhibited a similar inhibitory

potency on the collagen-evoked rise of intracellular Ca2+

mobilization either in the presence of extracellular calcium

(1 mmol·L−1) or in a Ca2+-free solution, implying that the

inhibition of intracellular Ca2+ mobilization may predominantly

be caused by the attenuation of Ca2+ release from

intracellular Ca2+ stores. Previous studies have indicated that

blocking NF-κB activation reduces collagen-induced platelet

intracellular Ca2+ mobilization by inhibiting PLCγ2-derived

inositol 1,4,5-trisphosphate receptor formation (Singer et al., 1997; Ragab et al., 2007; Chang et al., 2011). Activation of

PPARs decreased intracellular Ca2+ mobilization in activated

platelets through elevation of the NO/cGMP/PKG1 signalling cascade (Masuda et al., 2010; Shih and Chou, 2012). As expected, adding PPAR-β/-γ inhibitors or BetA significantly

reduced inhibition of intracellular Ca2+ mobilization by

nifedipine, suggesting that PPAR-β/-γ-mediated NF-κB

inactivation contributed to the decrease of intracellular Ca2+

mobilization.

The binding of fibrinogen to the surface GPIIb/IIIa

complex is a critical step for platelet aggregation (Shattil et al., 1998; Salanova et al., 2007). Inhibiting NF-κB activation reduces the outside-in/inside-out signalling of GPIIb/IIIa and fibrinogen binding (Malaver et al., 2009). Interestingly, the GPIIb/IIIa complex is required for NF-κB activation (Joo, 2012), suggesting that there is a mutual regulation between NF-κB and GPIIb/IIIa. Therefore, PPAR-β/-γ-dependent attenuation of NF-κB activation may in part account for the decreased surface GPIIb/IIIa expression by nifedipine. This is supported by the fact that inhibiting PPAR-β/-γ activity or enhancing NF-κB activation displays higher surface GPIIb/ IIIa expression than that of collagen + nifedipine-treated

platelets. Arachidonic acid and COX-derived TXA2 formation

data showed that nifedipine inhibits platelet activity

in the presence of arachidonic acid (Supporting Information; Table 1). Moreover, production of arachidonic acid or

collagen-induced TXB2, a stable metabolite of TXA2, was

concentration-dependently decreased by nifedipine (Supporting Information; Figure S4). However, adding GSK0660

or GW9662 did not affect the inhibitory effects of nifedipine on platelet COX-1 activity and arachidonic acid-induced

TXB2 formation, but partly attenuated the inhibition of

collagen-induced TXB2 formation by nifedipine. Accordingly,

PPAR-β/-γ may inhibit the release of arachidonic acid rather than directly affect COX-1 activity, subsequently attenuating

TXB2 formation, which may be another mechanism involved

in the antiplatelet activity of nifedipine. Although several

studies have confirmed that nifedipine inhibits platelet aggregation

in vitro and in vivo (Takahara et al., 1985; Os´miałowska et al., 1990), there is one contradictory finding conflicting

with previous studies (Murphy et al., 1985). Reasons for this discrepancy remain unclear, but the concentration of nifedipine used, treatment duration, the different condition of

human subjects chosen and preparation of platelet samples may all contribute to the differences reported. In conclusion, we have demonstrated that the antiplatelet activity of nifedipine was, at least in part, mediated by the inhibition of NF-κB

activation in a PPAR-β/-γ-dependent manner. These findings not only provide a novel molecular mechanism accounting for the antiplatelet activity of nifedipine but also suggest that nifedipine may have potential in the treatment of inflammation-related vascular disease therapy, because it modulates NF-κB activation.

Acknowledgements

This study was partly supported by a research grant from the National Science Council of Taiwan, Republic of China (NSC 97–2320-B-016-008-MY3).

Conflict of interest

The authors declare no conflict of interest.

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Nifedipine inhibits NF-κB activation in platelets

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Supporting information

Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: http://dx.doi.org/10.1111/bph.12523

Figure S1 Effects of PPAR-β/-γ antagonists on nifedipinemediated inhibition of thrombin-induced platelet aggregation

(A) and phosphorylation of p65NF-κB (B) in platelets.

Figure S2 Effect of PPAR-β/-γ antagonists and BetA on collagen-induced platelet aggregation.

Figure S3 Effects of various CCBs on platelet PPARs activity (A) and PPARs antagonists on their antiplatelet activity (B).

Figure S4 Effects of PPAR-β/-γ antagonists on nifedipinemediated

attenuation of TXB2 formation in AA or collagenstimulated

platelets.

Table S1 Effects of PPAR-β/-γ antagonists on nifedipinemediated attenuation of COX-1 activity in platelets.

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