A noble function of BAY 11-7082: Inhibition of platelet aggregation mediated by an elevated cAMP-induced VASP, and decreased ERK2/JNK1 phosphorylations.

Abstract

Platelets, though anucleated, possess several transcription factors, including NF-kappaB, that exert non-genomic functions regulating platelet activation. Since platelets have not only been recognized as central players of homeostasis, but also participated in pathological conditions such as thrombosis, atherosclerosis, and inflammation, we examined rat platelet NF-kappaB expression and evaluated the effects of anti-inflammatory drug BAY 11-7082, an inhibitor of NF-kappaB activation, in platelet physiology. Western blotting revealed that rat platelets express NF-kappaB. BAY 11-7082, dose dependently, inhibited collagen- or thrombin-induced-platelet aggregation. ATP release, TXB(2) formation, P-selectin expression, and intercellular Ca(2+) concentration activated by collagen were reduced in BAY 11-7082-treated platelets. BAY 11-7082 elevated intracellular levels of cAMP, but not cGMP, and its co-incubation with cAMP-activating agent (forskolin) or its hydrolyzing enzyme inhibitor (3-isobutyl-1-methylxanthine, IBMX), synergistically inhibited collagen-induced-platelet aggregation. In addition, vasodilator-stimulated-phosphoprotein (VASP) phosphorylation was enhanced in BAY 11-7082-treated platelets, which was partially inhibited by a protein kinase A (PKA) inhibitor, H-89. Moreover, while p38 mitogen-activated protein kinase (MAPK) was not affected, BAY 11-7082 attenuated c-Jun N-terminal kinase 1 (JNK1) and extracellular-signal-regulated protein kinase 2 (ERK2) phosphorylations. In conclusion, BAY 11-7082 inhibits platelet activation, granule secretion, and aggregation, and that this effect is mediated by inhibition of JNK1 and ERK2 phosphorylations, and partially by stimulation of cAMP-dependent PKA VASP phosphorylation. The ability of BAY 11-7082 to inhibit platelet function might be relevant in cases involving aberrant platelet activation where the drug is considered as anti-atherothrombosis, and anti-inflammatory therapy.

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Molecular and Cellular Pharmacology
A noble function of BAY 11-7082: Inhibition of platelet aggregation mediated by an
elevated cAMP-induced VASP, and decreased ERK2/JNK1 phosphorylations
Hyun-Sub Leea, Sung Dae Kimb, Whi Min Leeb, Mehari Endaleb, S.M. Kamruzzamanb, Won Jun Ohb,
Jae Youl Choc, Sang Keun Kimd, Hyun-Jeong Choe, Hwa-Jin Parka,⁎, Man Hee Rheeb,⁎
aCollege of Biomedical Science and Engineering, and Regional Research Center, Inje University, Gimhae 200-701, Republic of Korea
bLaboratory of Veterinary Physiology & Signaling, College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, Republic of Korea
cSchool of Biotechnology and Bioengineering, Kangwon National University, Chuncheon 200-701, Republic of Korea
dCollege of Veterinary Medicine, Chungnam National University, Daejeon 302-305, Republic of Korea
eCollege of Medical Science, Konyang University, Daejeon 302-718, Republic of Korea
a b s t r a c ta r t i c l ei n f o
Article history:
Received 21 April 2009
Received in revised form 12 October 2009
Accepted 3 November 2009
Available online 10 November 2009
Keywords:
Platelet
BAY 11-7082
cAMP
TXA2
VASP
MAP kinase
Platelets, though anucleated, possess several transcription factors, including NF-κB, that exert non-genomic
functions regulating platelet activation. Since platelets have not only been recognized as central players of
homeostasis, but also participated in pathological conditions such as thrombosis, atherosclerosis, and
inflammation, we examined rat platelet NF-κB expression and evaluated the effects of anti-inflammatory
drug BAY 11-7082, an inhibitor of NF-κB activation, in platelet physiology. Western blotting revealed that rat
platelets express NF-κB. BAY 11-7082, dose dependently, inhibited collagen- or thrombin-induced-platelet
aggregation. ATP release, TXB2 formation, P-selectin expression, and intercellular Ca2+concentration
activated by collagen were reduced in BAY 11-7082-treated platelets. BAY 11-7082 elevated intracellular
levels of cAMP, but not cGMP, and its co-incubation with cAMP-activating agent (forskolin) or its
hydrolyzing enzyme inhibitor (3-isobutyl-1-methylxanthine, IBMX), synergistically inhibited collagen-
induced-platelet aggregation. In addition, vasodilator-stimulated-phosphoprotein (VASP) phosphorylation
was enhanced in BAY 11-7082-treated platelets, which was partially inhibited by a protein kinase A (PKA)
inhibitor, H-89. Moreover, while p38 mitogen-activated protein kinase (MAPK) was not affected, BAY 11-
7082 attenuated c-Jun N-terminal kinase 1 (JNK1) and extracellular-signal-regulated protein kinase 2
(ERK2) phosphorylations. In conclusion, BAY 11-7082 inhibits platelet activation, granule secretion, and
aggregation, and that this effect is mediated by inhibition of JNK1 and ERK2 phosphorylations, and partially
by stimulation of cAMP-dependent PKA VASP phosphorylation. The ability of BAY 11-7082 to inhibit platelet
function might be relevant in cases involving aberrant platelet activation where the drug is considered as
anti-atherothrombosis, and anti-inflammatory therapy.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
Plateletsplay notonlya key rolein hemostasis,butalsoinvolved in
pathophysiological conditions such as thrombosis, atherosclerosis,
inflammation, and tumor progression. At the site of vascular injury,
plateletsrollandadheretovariouscomponentsofthe sub-endothelial
matrix by their surface adhesive receptors (Barrett et al., 2008).
Several mechanisms support the initial adhesion of platelets and their
subsequent activation and aggregation into a thrombus. Recent
reports revealed that collagen is the most thrombogenic component
of the sub-endothelial matrix (Davi and Patrono, 2007; Nieswandt
andWatson,2003;Watsonetal.,2005;Zarbocketal.,2007);itsupports
firm platelet adhesion mediated by its two surface receptors, the
integrinα2β1andglycoprotein(GP)VI,andinducesadhesion,secretion,
aggregation (Gibbins, 2004; Nieswandt and Watson, 2003).
The secretions of platelet agonists from dense granules such as ADP
and ATP, and adhesion molecules from α-granules, which contain
fibrinogenandP-selectincausefurtherplateletactivation(Jacksonetal.,
2003; Zarbock et al., 2007). In addition, platelets generate
lipid mediators such as thromboxane A2that activate Phospholipase
C-β (PLC-β), resulting in diacylglycerol and inositol trisphosphate (IP3)
production, and subsequent protein kinase C activation and [Ca2+]i
elevation, respectively.
Thrombin activates platelets via interactions with the proteinase-
activated receptors (PAR1 and PAR4) (Kahn et al., 1999), and cleaves
fibrinogen to form fibrin. Fibrin further stabilizes adhesion at the site
of injury, resulting in stable platelet interactions with collagen, vWF,
European Journal of Pharmacology 627 (2010) 85–91
⁎ Corresponding authors. Park is to be contacted at College of Biomedical Science and
Engineering, and Regional Research Center, Inje University, Gimhae 200-701, Republic
of Korea. Tel.: +82 55 320 3538; fax: +82 55 334 3426. Rhee, Laboratory of Physiology
& Signaling, College of Veterinary Medicine, Kyungpook National University, Daegu
702-701, Republic of Korea. Tel.: +82 53 950 5967; fax: +82 53 950 5955.
E-mail addresses: mlsjpark@inje.ac.kr (H.-J. Park), rheemh@knu.ac.kr (M.H. Rhee).
0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2009.11.005
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar

Page 2

ADP, thromboxane A2, and thrombin; which in turn lead integrin
αIIbβ3activation (Brass et al., 1997).
Vasodilator-stimulated-phosphoprotein(VASP)phosphorylationcor-
relates with the inhibition of fibrinogen binding to the integrin αIIb/β3
and inhibition of platelet aggregation(Horstrup et al., 1994). Elevationof
platelet cAMP increased VASP phophorylation that negatively regulates
platelet secretion, adhesion, and aggregation (Wentworth et al., 2006).
Among mitogen-activated protein (MAP) kinases, extracellular-
signal-regulated kinase 2 (ERK2), p38, and c-Jun NH2-terminal kinase
1 (JNK1) are present and activated by platelet agonists. Reports
indicated ERK2 plays a role in ADP-induced thromboxane A2
generation (Garcia et al., 2007; Mazharian et al., 2005); and JNK1
and p38 are involved in collagen-induced-platelet aggregation
(Kauskot et al., 2007; Mazharian et al., 2005).
Recent reports demonstrate that, despite anucleated, platelets
express transcription factors including intracellular receptors such as
the retinoic X receptor (Moraes et al., 2007) and the peroxisome
proliferator-activated receptors (PPARs), PPARγ and PPARβ/δ (Ali et al.,
2006). PPARγ ligands prevent CD40L, TXA2and ATP release in platelets
(Akbiyik et al., 2004), PPARβ/δ agonists inhibit platelet aggregation (Ali
etal.,2006),andretinoicXreceptorligandsinhibitADPorTXA2induced-
platelet activation mediated through suppression of Gq signaling, and
inhibition of [Ca2+]imobilization (Moraes et al., 2007). These findings
show intracellular receptors (transcription factors) regulate non-
genomic platelet functions. NF-κB/I-κB, in platelets, have been reported
tofunctionindependentofgeneregulation,andafterplateletactivation,
I-kB is phosphorylated and degraded (Liu et al., 2002). However, the
effect of I-κB phosphorylation inhibition in platelet function is not
investigated. On the other hand, a complete understanding of associa-
tions between responses caused by platelet-derived cytokines and
endogenous inflammatory mediators may enable the development of
anti-inflammatory drug that repair aberrant platelet activation.
NF-κB induction can be abrogated using its inhibitor, BAY 11-7082
((E) 3-[(4-methylphenyl)-sulfonil]-2-propenenitrile), which is an
irreversible inhibitor of I-κBα phosphorylation resulting in down-
regulation of the cytokine-induced NF-κB activation (Pierce et al.,
1997). Given the significance of NF-κB and platelets in atherosclerosis,
inflammation, and tumor progression, we investigated the role of BAY
11-7082 induced-NF-κB inhibition in platelet function.
2. Materials and methods
2.1. Materials
Thrombin, Fura-2/AM, BAY 11-7082, and 3-isobutyl-1-methyl xan-
thine (IBMX) were obtained from Sigma (St. Louis, MO). Collagen was
procuredfromChronolog(Havertown,PA,USA).CyclicAMP,cyclicGMP,
and thromboxane B2EIA Kits were from Cayman Chemical (MI, USA).
AntibodiestototalERK1/2,phospho-ERK1/2,totalp38mitogen-activated
protein kinase (MAPK), phospho-p38 MAPK, JNK1, and phospho-JNK1,
monoclonal antibodies against the phosphorylated Ser239site of VASP,
16C2, and against the phosphorylated ser157site of VASP, 5C6 and anti-
NF-κB/P65 were from Cell Signaling (Beverly, MA). Mouse monoclonal
antibody to CD62P and goat polyclonal antibody to mouse IgG (FITC)
were obtained from abcam (Cambridge, UK). ATP assay kit was from
SUNY at Buffalo (NY, USA). All other chemicals were of reagent grade.
2.2. Platelet preparation
The preparation of platelets has been described previously (Kim
et al., 2006). Male Sprague Dawely rats of 60 days age and weighing
from 240 to 250 g were obtained from Orient Co. (Seoul, Korea) and
maintained in a standard laboratory animal facility with free access to
feed, water and acclimated for at least two weeks before use. Rat
blood (8 ml) was obtained using the venipuncture method (insertion
of a 23-g needle into the abdominal aorta) and transferred to a 15 ml
test tube containing 1 ml of a citrate phosphate dextrose solution
(CPD: 90 mM of Na3C6H5O7·2H2O, 14 mM of C6H8O7·H2O, 128.7 mM
of NaH2PO4·H2O, and 2.55 g/100 ml dextrose). Blood was centrifuged
at 170×g for 7 min to obtain platelet-rich plasma (PRP). In order to
remove residual erythrocytes, the platelet-rich plasma samples were
again centrifuged at 120×g for 7 min. In order to isolate the platelets
and remove the CPD solution, platelet-rich plasma was centrifuged
twice at 350×g with a washing buffer for 10 min. The supernatant was
allocated to a platelet-poor plasma fraction, which was used as a
referencesolution in aggregation assays. The platelets of the precipitate
wereadjustedtothepropernumber[108/mlfortheaggregationassayin
Tyrode buffer (137 mM of NaCl, 12 mM of NaHCO3, 5.5 mM of glucose,
2 mM of KCl, 1 mM of MgCl2, 0.3 mM of NaHPO4, pH 7.4)]. All platelet
preparations were conducted at room temperature, and all experimen-
tal procedures and protocols used in this investigation were reviewed
and approved by the Ethics Committee of our University.
2.3. Platelet aggregation assays
Platelet aggregation was performed as previously described (Kim
et al., 2006). Aggregation was monitored by measuring light
transmission in an aggregometer (Chronolog, Havertown, PA, USA).
The washed platelets were pre-incubated at 37 °C for 3 min with
either BAY 11-7082 or vehicle and stimulated with agonists for 5 min.
The reaction mixture was further incubated for 8 min with stirring at
170 g. The concentration of the vehicle was held at less than 0.5%.
2.4. Determining the [Ca2+]i
The [Ca2+]iwas determined with fura-2/AM as described previ-
ously(Choetal.,2006).Briefly,theplatelet-richplasmawasincubated
with5 µM offura-2/AMfor60 min at 37 °C. Thefura-2-loadedwashed
platelets (108/ml) were then pre-incubated with BAY 11-7082 for
3 min at 37 °C in the presence of 1 mM CaCl2. Next, the platelets were
stimulated withcollagenfor 5 min. Fura-2 fluorescence wasmeasured
in a spectrofluorometer (F-2500, Hitachi, Japan) with an excitation
wavelengththat ranges from 340 nm to 380 nm, changing every 0.5 s;
the emission wavelength was 510 nm. The [Ca2+]iwas calculated by
the method of Schaeffer (Schaeffer and Blaustein, 1989): [Ca2+]iin
cytosol=224 nM×(F−Fmin)/(Fmax−F), where 224 nM is the disso-
ciation constant of the fura-2-Ca2+complex, and Fmin and Fmax
represent the fluorescence intensity levels at very low and very high
Ca2+concentrations, respectively. In our experiment, Fmax is the
fluorescence intensity of the fura-2-Ca2+complex at 510 nm after
the platelet suspensioncontaining1 mM of CaCl2had been solubilized
by Triton X-100 (0.1%). Fminis the fluorescence intensity of the fura-2-
Ca2+complex at 510 nm, after the platelet suspension containing
20 mM Tris/3 mM of EGTA had been solubilized by Triton X-100
(0.1%). F represents the fluorescence intensity of the fura-2-complex
at 510 nm after the platelet suspension was stimulated by collagen,
with and without BAY 11-7082, in the presence of 1 mM CaCl2.
2.5. Measurement of TXB2
Washed platelets were pre-incubated with or without BAY 11-
7082 for 3 min in the presence of 1 mM CaCl2, stirring at 170×g and
activated for 5 min with collagen. The reactions were terminated by
the addition of ice-cold EDTA (5 mM) and indomethacin (0.2 mM).
Samples were centrifuged and the TXB2levels in the supernatants
weremeasuredusing TXB2EIA Kit (AnnArbor,MI,USA)in SynergyHT
Multi-Model Microplate Reader (BioTek Instrument, Winooski, USA).
2.6. Measurement of cAMP and cGMP
Washedplateletswerepre-incubatedfor3 minat37 °Cwithvarious
concentrationsofBAY11-7082orvehicleinthepresenceof1 mMCaCl2,
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and then stimulated with collagen for 5 min in a platelet aggregometer.
The reaction was terminated by addition of equal volumes of 80% ice-
cold ethanol. Samples were then centrifuged at 2000×g for 10 min at
4 °C, andthesupernatantcAMPand cGMPlevels weredeterminedwith
a cyclic AMP and cyclic GMP EIA Kit (Ann Arbor, MI, USA).
2.7. ATP release assay
Washed platelets were pre-incubated for 3 min at 37 °C with
various concentrations of BAY 11-7082 and then stimulated with
collagen. The reaction was terminated, samples centrifuged and
supernatants were used for the assay. ATP release was measured in a
luminometer (GloMax 20/20, Promega, Madison, USA) using ATP
assay kit (Biomedical Research Service Center, Buffalo, USA).
2.8. Flow cytometry
Washed platelets, treated either with BAY 11-7082 or vehicle, were
stimulated with collagen and incubated for 5 min at 37 °С. The reaction
wasterminatedandcellscentrifugedfollowedbyresuspensioninice-cold
PBS, containing 10% FCS and 1% sodium azide. Cells were then incubated
withCD62Pprimaryantibodyin3%BSA/PBSfor30 minat4 °Сinthedark.
Next,plateletswerefixedandwashed3-timesbycentrifugationat400×g
for 5 min and resuspended in ice-cold PBS followed by FITC-conjugated
secondary antibody incubation in 3% BSA/PBS for 30 min at 4 °С in the
dark.Plateletswereagainwashed3-timesbycentrifugationat400×gfor
5 minandresuspendedinice-coldPBS,3%BSAand1%sodiumazide.Flow
cytometry analysis was performed on a FACSCalibur flow cytometer®
using CellQuest software (BD Biosciences, San Jose, UAS).
2.9. Immunoblotting
Platelets treated with BAY 11-7082 or vehicle were stimulated with
collagen and incubated for 5 min in an aggregometer before the reaction
wasterminatedandcellswerecentrifuged.Lysateswerethenpreparedby
solubilizingandcentrifugingplateletsinsamplebuffer(0.125MTris–HCl
at pH 6.8, 2% SDS, 2% β-mercaptoethanol, 20% glycerol, 0.02% bromophe-
nolblueinthepresenceof proteaseinhibitors, 1mMphenylmethylsulfo-
nylfluoride (PMSF), 2 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml and
pepstatin A). Protein concentration was determined using BSA Assay
(PRO-MEASURE, iNtRON Biotechnology, Korea). Equal volume of platelet
proteins were resolved in a 10% SDS-PAGE and transferred to nitrocel-
lulosemembraneinatransferbuffer(25 mMTris(pH8.5),0.2 Mglycerin,
and20%methanol).ImmunoblotswereblockedwithTBS-Tcontaining5%
nonfatdrymilkandincubatedwithprimaryantibodydilutedinablocking
solution. The immunoblots were again incubated with HRP secondary
antibody and the membranes were visualized using enhanced chemilu-
minescence, ECL (iNtRON Biotechnology, Korea).
2.10. Statistical analysis
Data wereanalyzed bya one-wayanalysis of variance, followedbya
post-hoc Dunnett's testin ordertodeterminethe statisticalsignificance
of the differences. All data are presented as means±S.E.M. p values of
0.05 or less were considered to be statistically significant.
3. Results
3.1. Rat platelets express NF-κB
Western blotting studies show that rat platelets express NF-κB/
p65. In Fig. 1C (lanes 1–4), anti-p65 antibody directed against NF-κB
revealed a clear band in platelet preparations from four different rats.
3.2. BAY 11-7082 inhibited collagen- and thrombin-induced-platelet
aggregation
In our model, we previously determined that collagen (2.5 μg/ml)
and thrombin (0.1 U/ml) induced complete platelet aggregation and
the present study employed those agonist concentrations for platelet
activation. As shown in Fig. 1A, BAY 11-7082 potently inhibited
collagen-induced-platelet aggregation in a concentration-dependent
manner. The IC50of BAY 11-7082 against collagen-activated platelets
was 5.6±0.2 μM. We also tested the title compound against
thrombin-induced-platelet aggregation. BAY 11-7082 dose depen-
dently inhibited platelet aggregation with an IC50 of 5.7±1.7 μM
(Fig. 1B).
3.3. Effect of BAY 11-7082 on cAMP and cGMP generations
Since platelet activation has been known to inhibit cAMP/cGMP
generation and cyclic nucleotide dependent protein kinase activity
(Weber et al., 1999; Li et al., 2003; Sudo et al., 2003), we next
investigated whether BAY 11-7082 influences platelet cAMP/cGMP
concentration. BAY 11-7082 significantly elevated cAMP levels in
collagen-activated platelets (Fig. 2A). However, BAY 11-7082 had no
discernable effect on the cGMP levels (Fig. 2B). In addition, co-
incubation of BAY 11-7082 with forskolin (2.5 µM), adenylyl cyclase
Fig. 1. Platelets express NF-κB and BAY 11-7082 inhibits collagen or thrombin-induced-
platelet aggregation. Washed platelets were analyzed after agonist stimulation in a
Lumi aggregometer. Platelets were stirred in an aggregometer with vehicle or BAY 11-
7082 (at the concentrations indicated) for 3 min prior to activation by collagen
(2.5 µg/ml)(A),andpriortoactivationbythrombin(0.1 U/ml)(B).BAY11-7082,dose
dependently, inhibited collagen or thrombin-induced-platelet aggregation. (C) Represen-
tative immunoblot of three independent NF-κB/p65 expressions from four different
rats in (panel 1–4). Results in (A) and (B) are summary of at least nine independent
experiments performed and bar graphs presented as mean±S.E.M. ⁎: pb0.05 vs. agonist-
induced control, ⁎⁎: pb0.01 vs. control.
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activator, and IBMX (50 µM), broad spectrum cyclic phosphodiester-
ase inhibitor, highly potentiated BAY 11-7082 evoked anti-platelet
activity, and therefore, augmented each of the individual anti-platelet
effects (Fig. 3A and B).
3.4. BAY 11-7082 inhibited collagen-induced TXA2 generation and
intracellular Ca2+concentration
We next examined the effect of BAY 11-7082 on collagen-induced
TXB2, a stable metabolite of TXA2, and interacellular Ca2+concentra-
tions [Ca2+]i, which are known to be positive and critical regulators of
platelet aggregation. We found that pre-incubation with BAY 11-7082
dose dependently suppressed the production of TXB2generation in
collagen-activated platelets (Fig. 4A). In addition, collagen-induced
[Ca2+]i spike (258.3±33.1 nM), relative to basal level (135.8
±3.0 nM), was dose dependently inhibited in BAY-treated platelets
(Fig. 4B). At a maximal dose of 10 μM, BAY 11-7082 reduced the
intracellular Ca2+concentration to sub-basal levels.
3.5. BAY 11-7082 inhibited ATP release and surface P-selectin expression
in collagen-activated platelets
Since granule secretions are critical markers of platelet activation
prior to aggregation (Zarbock et al., 2007; Barrett et al., 2008;
Mackman, 2008), we examined whether BAY 11-7082 pre-incubation
affects collagen-induced-platelet granule secretion. As such, agonist-
induced ATP release was significantly inhibited in BAY 11-7082-
treated-platelets (Fig. 5A). This suggests that the drug attenuates
platelet dense-granule secretion. Moreover, BAY 11-7082 pretreat-
ment markedly inhibited agonist-induced P-selectin expression
(Fig. 5B). At a dose of 10 µM, the compound completely inhibited
the expression of this surface marker protein, suggesting that platelet
α-granule secretary activity was impaired by the drug.
Fig. 2. Effects BAY 11-7082 on collagen-induced cyclic AMP and cyclic GMP elevation.
Rat washed platelets were stirred with either vehicle or BAY 11-7082 (at the
concentrations indicated) in an aggregometer and stimulated with collagen for 5 min
prior to the termination of the reaction and then cAMP and cGMP enzyme
immunoassays were performed as described in the Materials and methods section.
(A) BAY 11-7082 significantly increased cAMP accumulation in a dose dependent
manner. (B) BAY had no significant effect on cGMP levels. Results are summary of at
least 3 independent experiments performed and bar graphs presented as mean±S.E.M.
⁎: pb0.05 vs. control, ⁎⁎: pb0.01 vs. collagen-activated.
Fig. 3. BAY potentiates forskolin or IMBX induced cAMP elevation and VASP
phosphorylation. Co-stimulation of BAY 11-7082 with forskolin (A) or IBMX (B) at the
concentrations indicated displayed significant synergism in collagen-induced-platelet
aggregation. Results are summary of at least 3 independent experiments performed and
presented as mean±S.E.M. ⁎⁎: pb0.01 vs. agonist activated control.
Fig.4.BAYinhibitedcollagen-inducedthromboxaneA2generationandcytosolic[Ca2+]i.
(A) Platelets were pre-incubated with BAY 11-7082 (at the concentrations indicated
under the bar graph), and stirred in an aggregometer for 2 min prior to collagen
stimulation for 5 min, and then the reactions were terminated. (A) Thromboxane B2
generation was assayed as described in the Materials and methods section, and
BAY 11-7082 significantly inhibited collagen (2.5 μg/ml) induced TXB2generation.
(B). Washedplatelets were incubatedwithcalcium fluorophore (fura-2AM), stimulated
with collagen and intracellular Ca2+concentration was determined as described in
the Materials and methods section for which BAY 11-7082 significantly inhibited
collagen-activated [Ca2+]i. Results are summary of at least 3 independent experiments
performed and bar graphs presented as mean±S.E.M. ⁎: pb0.05 vs. agonist activated
control, ⁎⁎: pb0.01 vs. agonist activated control.
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3.6. BAY 11-7082 enhanced vasodilator-stimulated-phosphoprotein
(VASP) phosphorylation
It is reported that VASP is a substrate of cyclic nucleotide (cAMP/
cGMP)-dependent protein kinases (PKA/PKG) (Reinhard et al., 1992;
Aszodi et al., 1999; Pula et al., 2006). Although basal phosphor-VASP
(Ser239) expression was not detectable (Fig. 6), BAY 11-7082
treatment increased its phosphorylation in a dose dependent manner.
A similar expression pattern was observed in Phospho-VASP (Ser157)
phosphorylation. Forskolin induced VASP phosphorylation was as a
positive control and H-89, a potent PKA inhibitor (de Rooij et al.,
1998), partially attenuated both the BAY 11-9082 and forskolin
induced VASP phosphorylation. This suggests that the drug may be
involved in a cyclic nucleotide dependent protein kinase induced
VASP phosphorylation.
3.7. BAY 11-7082 attenuated collagen-activated platelet JNK1 and ERK2
phosphorylations
Since it has been a well established fact that ERK2, p38, and JNK1
are present in platelets and activated by various agonists, we
determined whether agonist induced-platelet JNK1, ERK2 and p38
phosphorylation are modulated as a signaling pathway in the drug's
anti-platelet activity. BAY 11-7082 significantly and dose dependently
suppressedagonist-induced JNK1and ERK2 phosphorylations withno
significant effect on p38 expression (Fig. 7). This result suggests that
the compound effect might involve MAPK signaling pathway.
4. Discussion
A large body of studies indicates that platelets secrete immune cell
activators, induce inflammatory cytokines production, and thus are
important amplifiers of acute inflammation. However, studies on NF-
κB expression and the possible non-genomic anti-platelet activity of
the transcription factor in platelet physiology are limited. In this study
using western blotting, we determined that rat platelets express NF-
κB protein. Our results for the first time show that BAY 11-7082, an
inhibitorofNF-κB, potentlyinhibitedcollagenandthrombin-induced-
platelet aggregation. The ability of the compound to suppress platelet
responses stimulated by other agonists such as ADP, was also similar
and consistent (data not shown). This conclusion was further
confirmed in that BAY 11-7082 suppressed different collagen evoked
platelet responses such as ATP release, TXA2formation, P-selectin
expression, and intercellular Ca2+concentration. These observations
suggest that NF-κB may be involved in the regulation of the various
steps of platelet activation. The regulatory effects of the transcription
factor can be evidenced from the inhibitory effects of BAY 11-7082, a
prototype NF-κB inhibitor, on agonist induced-platelet activity. In
addition, the BAY 11-7082 elevated intracellular cyclic AMP, but not
cyclic GMP, levels. Besides, BAY 11-7082 co-incubation with forskolin,
adenylyl cyclase activating and cAMP-elevating agent; or 3-isobutyl-
1-methylxanthine, cAMP hydrolyzing cyclic nucleotide phosphodies-
terase inhibitor, synergistically suppressed collagen-induced-platelet
aggregation. Further, cyclic AMP elevation was accompanied by cyclic
nucleotide protein kinase-dependent vasodilator-stimulated phos-
phoprotein (VASP) phosphorylation in BAY 11-7082-treated-plate-
lets. The compound induced VASP phosphorylation was partially
inhibited by H-89, PKA inhibitor, suggesting that BAY 11-7082
Fig. 5. Effects of BAY 11-7082 on collagen-activated platelet granule secretions. Washed
platelets were pre-incubated with BAY 11-7082 (at the concentrations indicated), and
stirred in an aggregometer for 2 min prior to collagen stimulation for 5 min, and then
the reactions were terminated followed by granule secretion assay. (A) ATP release in
response to agonist stimulation was performed as described in the Materials and
methods section and BAY 11-7082, dose dependently, suppressed collagen (2.5 μg/ml)
stimulated platelet ATP release. (B) Collagen-induced surface P-selected expression
was analyzed as described in the Materials and methods section. BAY 11-7082
significantly attenuated (at higher dose, 10 µM) abolished collagen-activated platelet
surface P-selectin expression. Bar graphs show mean±S.E.M of at least 3 independent
experiments performed. ⁎: pb0.05 vs. agonist-activated control, ⁎⁎: pb0.01 agonist
activated control.
Fig. 6. Effect of BAY 11-7082 on VASP phosphorylation of resting platelet. Washed rat
platelets were stirred in an aggregometer with vehicle or BAY 11-7082, alone, with
H-89 or forskolin and H-19 (at the concentrations indicated) for 5 min prior to
termination. Proteins were extracted, separated, transferred to nitrocellulose and
immunoblotted using either an anti phospho-VASP Ser239or Ser157. Blots were
visualized by ECL and the immunoblot panels are representative of 3–4 similar
experiments.
Fig. 7. Effects of BAY 11-7082 on agonist-induced MAP kinase phosphorylation. Washed
platelets were stirred in an aggregometer with vehicle or BAY 11-7082 at the
concentration indicated for 3 min prior to the addition of collagen (2.5 µM) for 5 min
before termination of the reactions. Bay 11-7082, dose dependently, attenuated JNK1
and ERK2 phosphorylations but not p38 expression. Proteins were extracted, separated
by SDS-PAGE transferred to nitrocellulose and immunoblotted with the indicated
corresponding antibodies. Blots were visualized by ECL and all immunoblots are
representative of 3 to 4 similar experiments.
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Page 6

phosphorylates VASP through cAMP elevation and PKA activation.
Sudo et al. (2003), using cilostazol, phosphodiesterase-3 (PDE3)
inhibitor, reported a similar result to our findings and suggested that
thedrug's anti-plateleteffect is mediatedthroughan increasein cAMP
level, PKA activation, and enhanced VASP phosphorylation. Previous
studies reported that VASP is an actin- and profilin-binding protein
that is expressed in platelets at high levels, and plays a major role in
negatively regulating secretary and adhesive events in these cells
(Wentworth et al., 2006). In addition, Aszodi et al. (1999) indicated
that, in the absence of VASP, cAMP- and cGMP-mediated inhibition of
platelet aggregation were significantly reduced. The authors sug-
gested two mechanisms: 1) VASP-independent mechanism for
inhibition of calcium mobilization and granule release. 2) VASP-
dependent mechanism for inhibition of platelet aggregation that may
be involved in regulation of integrin function. Another report revealed
that platelet integrin αIIbβ3is inhibited by cyclic nucleotide-elevating
agents that stimulate VASP phosphorylation, and concluded that
cAMP- and cGMP-dependent protein kinase mediated phosphoryla-
tion of VASP correlates well with αIIbβ3inhibition (Horstrup et al.,
1994).
MAPK are a family of serine/threonine protein kinases comprised
of three subgroups that include ERKs, p38, and JNKs. Among the MAP
kinases, ERK2, p38, and JNK1 are reported to be present in platelets
and activated by various agonists (Toth-Zsamboki et al., 2003). Our
findings in this study show that BAY 11-7082 attenuated JNK1 and
ERK2 phosphorylation, but p38 MAPK was not affected in collagen-
activated platelets. Similarly, Kauskot et al. (2007) reported JNK1
involvement in ADP dependent collagen-induced-platelet aggrega-
tion. They further provided evidence that during rolling and adhesion
of platelets to vWF, GPIb-vWF interaction triggers αIIbβ3activation in
a JNK1-dependent manner. Extensive studies, using various experi-
mental setups, have indicated the role of ERK2 in platelet aggregation.
By using low-dose collagen, Roger et al. (2004), and thrombin, Falker
et al. (2004), independently reported involvement of ERK2 activation
in platelet aggregation and demonstrated that collagen or thrombi-
induced ERK2 activation depends on TXA2formation and ADP release.
Roger et al. (2004) and Toth-Zsamboki et al. (2003) further indicated
that ERK2 acts on platelet secretion and activation of myosin light
chain kinase.
Our findings show that BAY 11-7082 induced adenylyl cyclase
activation and inhibits collagen-induced-ERK2 and JNK1 phosphory-
lation.The mechanism of actionmayincludean increaseof cAMPlevel
with enhanced VASP phosphorylation and subsequent inhibition of
intracellular Ca2+mobilization, thromboxane A2formation, and ATP
release during the platelet aggregation. This is in agreement with a
reported study of adenylyl cyclase activation inhibits low-dose
collagen-induced MAP kinase phosphorylation both prior, and distal,
to thromboxane release (Jackson and McNicol, in press).
As such, large body of evidence revealed that platelet activation
and inhibitory signaling pathways are dependent on the actions of a
number of tyrosine and serine/threonine kinases, lipid kinases,
phosphatases, adapter proteins,calcium mobilization andcytoskeletal
reorganization, which in a highly coordinated manner organize
platelet spreading, secretion, integrin regulation, aggregation and
thrombus formation (Gibbins, 2004; Jackson et al., 2003; Moroi and
Jung, 2004; Nieswandt and Watson, 2003; Watson et al., 2005;
Zarbock et al., 2007).
We anticipated that a complete understanding of associations
between responses caused by platelet-derived cytokines and endog-
enous inflammatorymediators might enable the development of anti-
inflammatory drug that repair aberrant platelet activation. On the
other hand, Liu et al. (2002) reported that NF-κB/I-κB is expressed in
platelets with a function independent of gene regulation, and they
further stated that up on platelet activation, I-κB is phosphorylated
and degraded. Other previous studies with nucleated cells agree with
their finding of separate pathways of NF-κB activation, through either
the thrombin receptor or direct activation of protein kinase C
(Nakajima et al., 1994; Maruyama et al., 1997). Interestingly, in our
study BAY 11-7082 decreased P-selectin expression suggesting that
the non-genomic role of NF-κB in platelets could be associated with
the regulation of both hemostatic- and inflammatory-mediated
responses. Since platelets are recognized as inflammatory response
mediators, the expression of P-selectin on the membrane of activated
platelets is the main link between platelets and inflammatory cells
(Zarbock et al., 2007). Salanova et al. (2007) using acquired GPIIb/IIIa
(αIIβ3) receptors showed that platelet receptors colocalized with β2-
integrins and cooperated in NF-κB activation, and they implicated
GPIIb/IIIa receptors as new therapeutic targets in neutrophil-induced
inflammation. They also have shown that Src and Syk non-receptor
tyrosine kinases, as well as the actin cytoskeleton, control NF-κB
activation. Platelets are also reported to be mediators of CD40L-CD40
signaling, at the interface between inflammatory responses, athero-
sclerosis,hemostasisand thrombosis, highlighting anotherpotentially
beneficial therapeutic route (Zarbock et al., 2007).
Together with this, our results suggest that NF-κB could be a
mediator of platelet function, and its inhibitor, BAY 11-7082 can be
considered as therapeutic agent to inhibit aberrant platelet activation,
although a concern about the use of pharmacological inhibitors with
potential side effects exists. However, BAY 11-7082 is not a general
inhibitor of cytokine mediated phosphorylation, rather it selectively
inhibits the phosphorylation of I-kBα (Pierce et al., 1997). Even
though it is likely that the inhibitory effect of this drug on platelet
function could be due to a selective inhibition of the NF-κB pathway,
we do not rule out the possibility that this drug may also have an
alternative mode of action.
In conclusion, the main findings of this study suggest that the
inhibitory effects of BAY 11-7082 in platelet aggregation possibly
involve (i) cyclic AMP elevation and VASP phosphorylation and
(ii) inhibition of the ERK2 and JNK1 MAP kinase phosphorylations in
activated platelets. And the ability of BAY 11-7082 to inhibit [Ca2+]i-
mobilization, thromboxane A2formation, ATP release and P-selectin
expression during platelet aggregation, in combination with its anti-
inflammatoryeffects as an NF-κB inhibitor, suggests that BAY 11-7082
could be considered as an anti-atherothrombosis and anti-inflamma-
tory agent.
Acknowledgments
This research was supported by a grant from the Ministry of
Commerce, Industry and Energy(MOCIE) and the Korean Institute of
Industrial Technology Evaluation & Planning(ITEP), through the
Biohealth Products Research Center (BPRC) of Inje University, and
Ministry of Food, Agriculture, Forestry and Fisheries (No 20080439).
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