A new ibuprofen derivative inhibits platelet aggregation and ros mediated platelet apoptosis

Article abstract of DOI:10.1371/journal.pone.0107182

Thrombocytopenia is a serious issue connected with the pathogenesis of several human diseases including chronic inflammation, arthritis, Alzheimer's disease, cardiovascular diseases (CVDs) and other oxidative stress-associated pathologies. The indiscriminate use of antibiotics and other biological drugs are reported to result in thrombocytopenia, which is often neglected during the treatment regime. In addition, augmented oxidative stress induced by drugs and pathological conditions has also been shown to induce thrombocytopenia, which seems to be the most obvious consequence of elevated rate of platelet apoptosis. Thus, blocking oxidative stress-induced platelet apoptosis would be of prime importance in order to negotiate thrombocytopenia and associated human pathologies. The current study presents the synthesis and platelet protective nature of novel ibuprofen derivatives. The potent anti-oxidant ibuprofen derivative 4f was selected for the study and the platelet protective efficacy and platelet aggregation inhibitory property has been demonstrated. The compound 4f dose dependently mitigates the oxidative stress-induced platelet apoptosis in both platelet rich plasma and washed platelets. The platelet protective nature of compound 4f was determined by assessing various apoptotic markers such as ROS generation, cytosolic Ca²⁺levels, PS externalization, cytochrome C translocation, Caspase activation, mitochondrial membrane depolarization, cytotoxicity, LDH leakage and tyrosine phosphorylation of cytosolic proteins. Furthermore, compound 4f dose dependently ameliorated agonist induced platelet aggregation. Therefore, compound 4f can be estimated as a potential candidate in the treatment regime of pathological disorders associated with platelet activation and apoptosis. In addition, compound 4f can be used as an auxiliary therapeutic agent in pathologies associated with thrombocytopenia.

Full text of DOI:10.1371/journal.pone.0107182

A New Ibuprofen Derivative Inhibits Platelet
Aggregation and ROS Mediated Platelet Apoptosis
Kodagahalli S. Rakesh1., Swamy Jagadish1., Ajjampura C. Vinayaka1., Mahadevappa Hemshekhar2,
Manoj Paul2, Ram M. Thushara2, Mahalingam S. Sundaram2, Toreshettahally R. Swaroop1,
Chakrabhavi D. Mohan1, Basappa3, Marilinganadoddi P. Sadashiva1, Kempaiah Kemparaju2,
Kesturu S. Girish2,4*, Kanchugarakoppal S. Rangappa1*
1 DOS in Chemistry, University of Mysore, Manasagangotri, Mysore, India, 2 DOS in Biochemistry, University of Mysore, Manasagangotri, Mysore, India, 3 Laboratory of
Chemical Biology, Department of Chemistry, Bangalore University, Bangalore, India, 4 Department of Studies and Research in Biochemistry, Tumkur University, Tumkur,
India
Abstract
Thrombocytopenia is a serious issue connected with the pathogenesis of several human diseases including chronic
inflammation, arthritis, Alzheimer’s disease, cardiovascular diseases (CVDs) and other oxidative stress-associated
pathologies. The indiscriminate use of antibiotics and other biological drugs are reported to result in thrombocytopenia,
which is often neglected during the treatment regime. In addition, augmented oxidative stress induced by drugs and
pathological conditions has also been shown to induce thrombocytopenia, which seems to be the most obvious
consequence of elevated rate of platelet apoptosis. Thus, blocking oxidative stress-induced platelet apoptosis would be of
prime importance in order to negotiate thrombocytopenia and associated human pathologies. The current study presents
the synthesis and platelet protective nature of novel ibuprofen derivatives. The potent anti-oxidant ibuprofen derivative 4f
was selected for the study and the platelet protective efficacy and platelet aggregation inhibitory property has been
demonstrated. The compound 4f dose dependently mitigates the oxidative stress-induced platelet apoptosis in both
platelet rich plasma and washed platelets. The platelet protective nature of compound 4f was determined by assessing
various apoptotic markers such as ROS generation, cytosolic Ca2+ levels, PS externalization, cytochrome C translocation,
Caspase activation, mitochondrial membrane depolarization, cytotoxicity, LDH leakage and tyrosine phosphorylation of
cytosolic proteins. Furthermore, compound 4f dose dependently ameliorated agonist induced platelet aggregation.
Therefore, compound 4f can be estimated as a potential candidate in the treatment regime of pathological disorders
associated with platelet activation and apoptosis. In addition, compound 4f can be used as an auxiliary therapeutic agent in
pathologies associated with thrombocytopenia.
Citation: Rakesh KS, Jagadish S, Vinayaka AC, Hemshekhar M, Paul M, et al. (2014) A New Ibuprofen Derivative Inhibits Platelet Aggregation and ROS Mediated
Platelet Apoptosis. PLoS ONE 9(9): e107182. doi:10.1371/journal.pone.0107182
Editor: Dermot Cox, Royal College of Surgeons, Ireland
Received May 7, 2014; Accepted August 6, 2014; Published September 19, 2014
Copyright: ß 2014 Rakesh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. Data are available from the Mysore University
Institutional Data Access/Ethics Committee for researchers who meet the criteria for access to confidential data.
Funding: KS Rangappa thanks DST-JSPS (DST/INT/JAP/P-79/09, Dated: 20/05/2009), KS Rakesh thanks BRNS (No. 2009/37/40/BRNS/2266, Dated: 23/11/2009), S
Jagadish thanks UGC-MRP (F. No. 39-106/2010 SR, Dated: 24/12/2010), AC Vinayaka thanks CSIR (SRF-Ref: 9/119 (0819) 2KR-EMR-I) for financial assistance. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: rangappaks@gmail.com (K. S. Rangappa); ksgbaboo@gmail.com (KSG)
. These authors have contributed equally to this work.
have demonstrated that platelets possess the necessary cellular
machinery to undergo apoptosis [7,8]. Besides aging, several other
factors like chemical and physical agonists, oxidative stress-
Introduction
Several human diseases including chronic inflammation,
diabetes, arthritis, cardiovascular diseases (CVDs) and other
induced pathological conditions are also shown to trigger apoptosis
oxidative stress-associated pathologies are also being linked to
mean platelet volume and reduced platelet count, in other words
in platelets [5,9,10]. They undergo apoptosis via intrinsic
apoptotic pathway; however, very few studies have reported the
thrombocytopenia [1–4]. The augmented oxidative stress as
extrinsic pathway too [5,11]. The altered platelet number and
evidenced by elevated reactive oxygen species (ROS) is shown to
functions would certainly lead to bleeding disorders and throm-
induce thrombocytopenia, which seems to be the most obvious
botic diseases. The distorted platelet functions are also responsible
consequence of elevated rate of platelet apoptosis [5]. Platelets are
for the multifactorial diseases including coronary heart disease
simple anuclear cells, yet execute a plethora of physiological
(CHD) and other cardiovascular diseases (CVDs), inflammatory
actions such as hemostasis, thrombosis and wound healing [6]. It is
and immune reactions [12–14].
no more surprising that the anuclear platelets end their life
The cytoplasmic events of intrinsic apoptotic pathway is
through apoptosis like any other nucleated cell. Several studies
instigated by elevated endogenous reactive oxygen species (ROS)
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Ibuprofen Derivative Inhibits the Platelet Activation and Apoptosis
particularly, hydrogen peroxide (H2O2). The principal target of
oxidative stress is mitochondrial damage resulting in the down-
regulation of electron transport chain, which in turn increases
ROS generation and formation of mitochondrial permeability
transition pore (MPTP). This would significantly induce depolar-
ization of mitochondrial membrane potential (DYm) causing
leakage of cytochrome c (cyt c) into the cytoplasm. Furthermore,
the leaked cyt c in association with pro-apoptotic factors like
Apoptotic Protease Activating Factor-1 (Apaf-1) mediate the
activation of caspase-9 (elicitor caspase), which in turn activates
caspase-3 (executioner caspase). Finally, phosphatidylserine (PS)
externalization occurs, which is a signal for phagocytosis [7,8,15].
Besides, the process of platelet apoptosis also releases PS-positive
membrane fractions called microparticles (MPs), which signifi-
cantly contribute to the pathogenesis of atherosclerosis, central
nervous system damage and neoplasia [16].
Research laboratories Pvt Ltd., Mumbai (India). All other reagents
were of analytical grade.
Synthesis and characterization of Ibuprofen derivatives
The starting materials were commercially available and used as
received without further purification. Reactions were monitored
by TLC using precoated sheets of silica gel (G/UV-254) of
0.25 mm thickness (Merck 60F254) using UV light for visualiza-
tion. The melting points were determined on Selaco melting point
1
apparatus and are uncorrected. H and 13C NMR spectra were
recorded on an NMR spectrometer operating at 400 and
100 MHz, respectively, using the residual solvent peaks as
reference relative to SiMe4. Mass spectra were recorded using
electrospray ionization (ESI) mass spectrometry. The C, H, and N
analysis were performed using CE-400 CHN analyzer. Infrared
spectra were recorded on Shimadzu FT-IR model 8300 spectro-
photometer.
Thus, blocking oxidative stress-induced platelet apoptosis would
be of prime importance in order to combat thrombocytopenia and
associated human pathologies. Hence, there is a need for potent
molecules that can protect platelets from the premature death. Till
date there are only two reports describing the inhibition of platelet
apoptosis by phytochemicals namely, cinnamtannin-B1 and crocin
[17,18]. Both the molecules ameliorate the oxidative stress-
induced platelet apoptosis by modulating ROS mediated mito-
chondrial damage. Ibuprofen is a non-steroidal anti-inflammatory
drug (NSAID) used to treat the pathological conditions related to
inflammation. It exerts its anti-inflammatory action by blocking
cyclooxygenase-1 non-specifically and thereby thromboxane
production [19]. In the recent past, studies reported that
Ibuprofen augments the anticancer activity of cisplatin in lung
adenocarcinoma cells by blocking heat shock protein 70 [20].
However, laboratory testing of drug-induced immune thrombo-
cytopenia (DIIT) reported development of immune thrombocyto-
penia by ibuprofen [21]. Based on these facts, a series of novel
ibuprofen derivatives 4(a-f) are synthesized by modifying the
functional groups and evaluated for improved anti-oxidant
activity. Among the derivatives, the potent anti-oxidant compound
4f was investigated for the protective efficacy against platelet
apoptosis as a clinical implication to thrombocytopenia.
Ethyl 2-[4-(2-methylpropyl)phenyl]propanoate (2)
A solution of Ibuprofen 1 (2 g, 9.6 mmol) and 0.5 mL
concentrated sulfuric acid in ethanol (20 mL) was refluxed for
6 h. The solvent was removed under reduced pressure and the
residue was taken up in CHCl3 (30 mL). The solution was
extracted with saturated aqueous NaHCO3 solution (2615 mL)
and water. The organic layer was dried over anhydrous Na2SO4,
filtered and solvent was evaporated under reduced pressure,
affording 2 (2.1 g, 95%) as a clear oil.
2-[4-(2-methylpropyl)phenyl]propanehydrazide (3)
The mixture of ethyl ester of Ibuprofen 2 (2 g, 8.5 mmol) and
hydrazine hydrate (0.8 mL, 16 mmol) in absolute ethanol (30 mL)
was refluxed for 10 h. The solvent was removed under reduced
pressure and the residue was added to ice cold water. The solid
separated was filtered, washed and dried. The solid was purified by
recrystallization from ethanol to get pure product 3 (1.7 g, 96%) as
a white solid.
2-[4-(2-methylpropyl)phenyl]-N’-
(phenylsulfonyl)propanehydrazide (4a-f)
To a stirred solution of 2-[4-(2-methylpropyl)phenyl]propane-
hydrazide (0.15 g, 0.68 mmol), substituted benzene sulfonyl
chloride (0.68 mmol) in dichlorormethane (5 mL), triethylamine
(0.14 mL, 1.0 mmol) was added at 0uC. The reaction mixture was
brought to room temperature and further stirred for 2 h. After the
completion of reaction, the reaction mixture was extracted with
ethyl acetate (2610 mL). The combined organic extracts were
washed with water (365 mL), brine (1610 mL) and dried over
anhydrous Na2SO4, filtered and solvent was evaporated under
reduced pressure to get crude products 4 (a-f), which were purified
by column chromatography over silica gel using hexanes-EtOAc
as eluent.
Materials and Methods
Chemicals/Reagents
Calcium ionophore (A23187), benzamidine hydrochloride, N-
acetyl-Leu-Glu-His-Asp trifluoro methylcoumarin (AC-LEHD-
FMC), acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (AC-
DEVD-AMC), sodium orthovanadate, dimethyl sulfoxide
(DMSO), fluorescein isothiocyanate (FITC)-labeled annexin V,
5-(and-6)-chloromethyl-29,79-dichlorodihydrofluorescein diacetate
acetylester (CM-H2DCFDA), CHAPS, rhodamine 123, leupeptin
hydrochoride, N-(2-Hydroxyethyl) piperazine-N9-ethanesulfonic
acid (HEPES), fura-2/AM, monoclonal anti-phosphotyrosine
antibody, acridine orange 10-nonyl bromide (NAO) and dithio-
threitol (DTT) were from Sigma Chemicals, St. Louis (USA).
Monoclonal anti-cytochrome c antibody and anti-b-actin were
from Epitomics Burlingame, CA (USA). Anti-Caspase-3 antibody
was from Santa Cruz Biotechnology, Inc. Texas (USA). Collagen
type-I was from Chrono-log Corporation, Pennsylvania (USA). 3-
2-[4-(2-methylpropyl)phenyl]-N’-
(phenylsulfonyl)propanehydrazide (4a)
Yield 89%, white solid; MP 112–114uC; IR (KBr) n,3320,
2951, 1675, 1585, 1124; H NMR (400 MHz, CDCl3) d 7.82 (d,
1
J = 6.4 Hz, 1H, NH), 7.72–7.57 (m, 5H, Ar-H), 7.21 (d,
J = 6.4 Hz, 1H, NH), 7.07 (d, J = 7.6 Hz, 2H, Ar-H), 7.01 (d,
J = 8.4 Hz, 2H, Ar-H), 3.42 (q, J = 6.1 Hz, 1H, CH), 2.44 (d,
J = 7.6 Hz, 2H, ArCH2), 1.85 (m, 1H, CH), 1.26 (d, J = 7.2 Hz,
3H, Me), 0.88 (dd, J = 6.6 Hz, 1.0 Hz, 6H, Me2); 13C NMR
(100 MHz, CDCl3) d 177.8, 141.2, 136.4, 132.4, 130.8, 129.6,
127.2, 126.9, 125.6, 44.9, 44.4, 30.2, 22.4, 22.3, 17.9; Anal. Calcd
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) and 1, 1-diphenyl-2-picrylhdrazyl (DPPH) were from
HiMedia Laboratories, Mumbai (India). Lactate dehydrogenase
(LDH) kit was from AGAPPE diagnostics Ltd., Kerala (India). c-
glutamyl p-nitroanilide and glycylglycine were from Sisco
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Ibuprofen Derivative Inhibits the Platelet Activation and Apoptosis
for C19H24N2O3S: C 63.31, H 6.71, N 7.77. Found: C 63.32, H
6.72, N 7.78.
N’-[(4-methoxyphenyl)sulfonyl]-2-[4-(2-
methylpropyl)phenyl]propanehydrazide (4f)
Yield 94%, White solid; MP 96–98uC; IR (KBr) n,3315, 2953,
1673, 1597, 1154; 1H NMR (400 MHz, CDCl3) d 7.82 (d,
J = 6.4 Hz, 1H, NH), 7.68 (dt, J = 8.4 Hz, 3.2 Hz, 2.0 Hz, 2H, Ar-
H), 7.24 (d, J = 6.4 Hz, 1H, NH), 7.07 (d, J = 7.6 Hz, 2H, Ar-H),
7.01 (d, J = 8.4 Hz, 2H, Ar-H), 6.84 (dt, J = 8.4 Hz, 3.2 Hz,
2.0 Hz, 2H, Ar-H), 3.84 (s, 3H, OMe), 3.40 (q, J = 6.4 Hz, 1H,
CH), 2.44 (d, J = 7.6 Hz, 2H, ArCH2), 1.85 (m, 1H, CH), 1.28 (d,
J = 7.2 Hz, 3H, Me), 0.88 (dd, J = 6.6 Hz, 1.0 Hz, 6H, Me2); 13C
NMR (100 MHz, CDCl3) d 177.8, 163.8, 141.2, 136.4, 130.8,
129.6, 127.2, 127.1, 114.1, 55.6, 44.9, 44.4, 30.2, 22.4, 22.3, 17.9;
MS m/z 391 [M+H]; Anal. Calcd for C20H26 N2O4S: C 61.52, H
6.71, N 7.17. Found: C 61.54, H 6.73, N 7.18.
N’-[(4-fluorophenyl)sulfonyl]-2-[4-(2-methylpropyl)
phenyl]propanehydrazide (4b)
Yield 91%, white solid, MP 121–123uC; IR (KBr) n,3312,
2960, 1679, 1592, 1139; H NMR (400 MHz, CDCl3) d 7.85 (d,
1
J = 6.2 Hz, 1H, NH), 7.79 (d, J = 7.8 Hz, 2H, Ar-H), 7.62 (d,
J = 7.8 Hz, 2H, Ar-H), 7.26 (d, J = 6.2 Hz, 1H, NH), 7.07 (d,
J = 7.6 Hz, 2H, Ar-H), 7.01 (d, J = 8.4 Hz, 2H, Ar-H), 3.42 (q,
J = 6.1 Hz, 1H, CH), 2.44 (d, J = 7.6 Hz, 2H, ArCH2), 1.85 (m,
1H, CH), 1.26 (d, J = 7.2 Hz, 3H, Me), 0.88 (dd, J = 6.6 Hz,
1.0 Hz, 6H, Me2); 13C NMR (100 MHz, CDCl3) d 177.8, 168.8,
141.2, 136.4, 131.8, 129.6, 128.4, 127.2, 117.3, 44.9, 44.4, 30.2,
22.4, 22.3, 17.9; Anal. Calcd for C19H23FN2O3S: C 60.30, H
6.13, N 7.40. Found: C 60.31, H 6.15, N 7.43.
Determination of reducing ability
Reducing ability of Ibuprofen and its derivatives (4a, 4b, 4c, 4d,
4e and 4f) along with quercetin as positive control were
determined according to the method of Hsieh and Yan [22] with
slight modifications. Briefly, samples (0–100 mM) were mixed with
2.5 mL of 200 mM sodium phosphate buffer, pH 6.6 containing
1% potassium ferricyanide and the mixture was incubated at 50uC
for 20 min. At the end of incubation, 2.5 mL of 5% TCA was
added and centrifuged at 4506g for 10 min. Further, 2.5 mL of
supernatant was taken and mixed with 0.5 mL of aqueous 0.1%
ferric chloride. The absorbance was measured at 700 nm against
blank using UV/Vis spectrophotometer (BioMate 3S, Thermo
scientifics).
N’-[(4-bromophenyl)sulfonyl]-2-[4-(2-
methylpropyl)phenyl]propanehydrazide (4c)
Yield 81%, red solid, MP 116–118uC; IR (KBr) n,3340, 2949,
1668, 1598, 1162; 1H NMR (400 MHz, CDCl3) d 7.85 (d,
J = 6.2 Hz, 1H, NH), 7.72 (d, 2H, J = 7.2 Hz, Ar-H), 7.68 (d, 2H,
J = 7.2 Hz, Ar-H), 7.25 (d, J = 6.2 Hz, 1H, NH), 7.07 (d,
J = 7.6 Hz, 2H, Ar-H), 7.01 (d, J = 8.4 Hz, 2H, Ar-H), 3.42 (q,
J = 6.1 Hz, 1H, CH), 2.44 (d, J = 7.6 Hz, 2H, ArCH2), 1.85 (m,
1H, CH), 1.26 (d, J = 7.2 Hz, 3H, Me), 0.88 (dd, J = 6.6 Hz,
1.0 Hz, 6H, Me2); 13C NMR (100 MHz, CDCl3) d 177.6, 141.1,
135.8, 131.1, 130.4, 129.6, 128.3, 127.1, 126.9, 44.9, 44.4, 30.2,
22.4, 22.3, 17.9; Anal. Calcd for C19H23BrN2O3S: C 51.94, H
5.28, N 6.38. Found: C 51.95, H 5.31, N 6.39.
Antioxidant activity
The free radical scavenging activity of Ibuprofen and its
derivatives (4a, 4b, 4c, 4d, 4e and 4f) along with quercetin as
positive control were determined using 1, 1-diphenyl-2-picrylh-
drazyl (DPPH) radical according to the method of Yamaguchi et
al. [23] with slight modifications. Briefly, Samples (0–100 mM)
were taken in test tubes with 1 mL of freshly prepared 0.1 mM
DPPH solution and the final volume was made up to 2 mL using
methanol. Samples were incubated for 20 min at room temper-
ature in dark and the resulting absorbance was recorded at
517 nm against blank using UV/Vis spectrophotometer.
N’-[(4-nitrophenyl)sulfonyl]-2-[4-(2-
methylpropyl)phenyl]propanehydrazide (4d)
Yield 78%, yellow solid, MP 142-144uC; IR (KBr) n,3319,
2971, 1678, 1582, 1145; H NMR (400 MHz, CDCl3) d 7.88 (d,
1
J = 6.2 Hz, 1H, NH), 7.71–7.63 (m, 4H, Ar-H), 7.29 (d,
J = 6.2 Hz, 1H, NH), 7.07 (d, J = 7.6 Hz, 2H, Ar-H), 7.01 (d,
J = 8.4 Hz, 2H, Ar-H), 3.40 (q, J = 6.4 Hz, 1H, CH), 2.44 (d,
J = 7.6 Hz, 2H, ArCH2), 1.85 (m, 1H, CH), 1.28 (d, J = 7.2 Hz,
3H, Me), 0.88 (dd, J = 6.6 Hz, 1.0 Hz,6H, Me2); 13C NMR
(100 MHz, CDCl3) d 177.8, 152.8, 144.1, 141.2, 136.4, 129.6,
128.6, 127.2, 125.1, 44.9, 44.4, 30.2, 22.4, 22.3, 17.9; Anal. Calcd
for C19H23N3O5S: C 56.28, H 5.72, N 10.36. Found: C 56.29, H
5.74, N 10.38.
Preparation of platelet-rich plasma and washed platelets
Venous blood was drawn from healthy drug-free human
volunteers (non-smokers) approved by the Institutional Human
Ethical Committee (IHEC-UOM No. 95/Ph.D/2013-14) Univer-
sity of Mysore, Mysore. Written consent were obtained from the
healthy volunteers as per the guidelines of Institutional Human
Ethical Committee (IHEC-UOM No. 95/Ph.D/2013-14). It was
immediately mixed with acid citrate dextrose (ACD) anticoagulant
(85 mM sodium citrate, 78 mM citric acid and 111 mM D-
glucose) in the ratio 6:1 (blood: ACD v/v). The anti-coagulated
whole blood was then centrifuged at 906g for 15 min and the
supernatant thus obtained was the platelet-rich plasma (PRP). The
PRP was centrifuged at 1,7006g for 15 min at 37uC. The platelet
pellet thus obtained was suspended and incubated for 10 min in
Tyrode’s albumin buffer [145 mM NaCl, 5 mM KCl, 10 mM
HEPES, 0.5 mM Na2HPO4, 1 mM MgCl2, 6 mM glucose, and
0.3% bovine serum albumin (BSA), pH 6.5] and washed
thereafter at 1,7006g for 15 min at 37uC. The previous washing
step was repeated one more time. Finally, the washed platelets
were suspended in the Tyrode’s albumin buffer (pH 7.4). The cell
count was determined in both PRP and washed platelet suspension
using a Neubauer chamber and adjusted to 56108 cells/ml in the
N’-[(2,5-dichlorophenyl)sulfonyl]-2-[4-(2-
methylpropyl)phenyl]propanehydrazide (4e)
Yield 85%, white solid, MP 102–104uC; IR (KBr) n,3364,
2983, 1658, 1518, 1084; H NMR (400 MHz, CDCl3) d 7.82 (d,
1
J = 6.4 Hz, 1H, NH), 7.90 (s, 1H, Ar-H), 7.71 (d, J = 8.0 Hz, 1H,
Ar-H), 7.65(d, J = 8.0 Hz, 1H, Ar-H) 7.24 (d, J = 6.4 Hz, 1H, NH),
7.07 (d, J = 7.6 Hz, 2H, Ar-H), 7.01 (d, J = 8.4 Hz, 2H, Ar-H),
3.40 (q, J = 6.4 Hz, 1H, CH), 2.44 (d, J = 7.6 Hz, 2H, ArCH2),
1.85 (m, 1H, CH), 1.28 (d, J = 7.2 Hz, 3H, Me), 0.88 (dd,
J = 6.6 Hz, 1.0 Hz, 6H, Me2); 13C NMR (100 MHz, CDCl3) d
177.8, 143.4, 141.2, 136.4, 134.2, 132.8, 131.3, 130.8, 129.6,
128.5, 127.2, 44.9, 44.4, 30.2, 22.4, 22.3, 17.9; Anal. Calcd for
C19H22 Cl2N2O3S: C 53.15, H 5.16, N 6.52. Found: C 53.16, H
5.18, N 6.54.
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Ibuprofen Derivative Inhibits the Platelet Activation and Apoptosis
final suspension using platelet poor plasma/Tyrode’s albumin
buffer (pH 7.4) [24].
inhibition studies. After incubation, samples were then loaded with
0.2 mM rhodamine 123 followed by 15 min incubation at 37uC
and the fluorescence was recorded by exciting the samples at
502 nm and the resulting emission was recorded at 527 nm.
Determination of endogenously generated reactive
oxygen species (ROS)
Assay of Caspases activity
Endogenous ROS production in platelets was determined
according to the method of Lopez et al. [25] with slight
modifications using CMH2DCFDA, a ROS-sensitive fluorescent
probe. PRP as well as washed platelet suspensions were
independently treated with calcium ionophore (A23187, 10 mM)
as agonist. For inhibition studies, pre-loaded platelets with agonist
were incubated with 4f in increasing doses (0–100 mM) and the
final volume was made up to 200 mL with HEPES-buffered saline
[HBS, 145 mM NaCl, 10 mM HEPES, 10 mM D-glucose, 5 mM
KCl, 1 mM MgSO4 and supplemented with 0.1% bovine serum
albumin (BSA), pH 7.45] and incubated at 37uC for 1 h. The
control (untreated) and treated platelets were then incubated with
10 mM CMH2DCFDA for 30 min at 37uC, fluorescence was
recorded using Varioskan multimode plate reader (Thermo
Scientifics, USA) by exciting the samples at 488 nm and
measuring the resulting fluorescence at 530 nm.
PRP and washed platelets were processed as discussed
previously with A23187 (10 mM) as agonist and 4f in increasing
doses for inhibition studies along with A23187 as agonist. Platelet
lysate was prepared by adding an equal volume of 2X Triton
buffer (2% Triton X-100, 2 mM EGTA, 100 mM Tris-HCl -
pH 7.2, 10 mg/mL leupeptin, 2 mM PMSF, 10 mM benzami-
dine, 2 mM Na3VO4) to the treated and control platelets and
allowed to undergo lysis for 30 min at 4uC. The lysate was
centrifuged at 160006g for 5 min. The pellet thus obtained is the
cytoskeleton-rich (Triton-insoluble) fraction, which was subjected
to caspase activity.
Caspase activity was determined by incubating cell lysate in a
microtitre plate with substrate solution (20 mM HEPES, pH 7.4,
2 mM EDTA, 0.1% CHAPS, 5 mM DTT and 8.25 mM caspase
substrate (AC-DEVD-AMC for caspase-3 and AC-LEHD-AFC
for caspase-9) for 2 h at 37uC. Substrate cleavage was measured
with a multimode plate reader (excitation wavelength 360 nm and
emission at 460 nm) [29].
Estimation of intracellular calcium
Intracellular Ca2+ concentration was measured in PRP and
washed platelets according to the method of Asai et al. [26] with
slight modifications. Briefly, PRP and washed platelet suspensions
were independently treated with A23187 (10 mM) as agonist. For
inhibition studies, pre-loaded platelets with agonist were incubated
with 4f in increasing doses (0–100 mM) and the final volume was
made up to 200 mL with modified Tyrode’s solution (150 mM
NaCl, 2.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.0 mM
CaCl2, 10 mM HEPES with 0.1% bovine serum albumin, pH 7.4)
and incubated for 1 h at 37uC to induce the release of Ca2+ from
the intracellular Ca2+ stores. Samples were then incubated for
45 min at room temperature with 2 mM fura-2/AM, a fluores-
cence Ca2+ indicator. The cells were subsequently washed twice
with modified Tyrode’s solution to remove the unbound dye and
finally the platelet pellet was suspended in modified Tyrode’s
solution. The fura-2/AM absorption was determined by exciting
the samples at 340 and 380 nm and the resulting fluorescence was
measured at 500 nm. Data were presented as absorption ratios
(340/380 nm).
Determination of PS externalization
Both PRP and washed platelets were processed as discussed
above with A23187 (10 mM) as agonist. For inhibition studies, pre-
loaded platelets with agonist were incubated with 4f in increasing
concentrations (0–100 mM) and incubated at 37uC for 1 h.
Further, samples of treated and control PRP/washed platelets
were transferred to equal volume of ice-cold 1% (v/v) glutaral-
dehyde in HBS for 10 min, and then incubated for 10 min with
FITC labeled-annexin V (0.6 mg/mL) in HBS. The cells were
collected by centrifugation for 60 s at 30006g and resuspended in
HBS. Cell staining was measured in a multimode plate reader by
exciting the samples at 496 nm and emission was recorded at
516 nm [30].
Detection of cytochrome c release
Cytochrome
c release was detected by western blotting
technique. Washed platelets (56108/ml) were treated with
A23187 (10 mM) as agonist and for inhibition studies, pre-loaded
platelets with agonist were incubated with 4f in increasing
concentrations (0–100 mM) and incubated at 37uC for 1 h. Then
the samples were lysed using freeze-thaw method whereby, cells
were freezed for 10 min at 280uC and kept for 10 min at room
temperature for thawing. The cycle was repeated for 4 times to get
platelet lysate. Cytosolic and cytoskeleton fractions were separated
by centrifugation at 10,5006g for 15 min. Cytosolic proteins were
separated on SDS-PAGE (10%) and electroblotted on to PVDF
membrane for 1 h at 50 V using wet blotter. Blots were then
incubated overnight with 5% skimmed milk powder in tris-
buffered saline with 0.1% tween 20 (TBST) to block residual
protein binding sites. Later membrane was incubated with anti-
cytochrome c antibody (1:1,000) in TBST for 3 h followed by
incubation with horseradish-peroxidase (HRP)-conjugated anti-
IgG antibody (1:10,000) in TBST. Blots were then developed by
enhanced chemiluminescence method (ECL) [18] b-actin was used
as loading control.
Assesment of cardiolipin peroxidation
NAO, a fluorescent probe was used to detect peroxidation of
cardiolipin. NAO loses its affinity for peroxidised cardiolipin
resulting in decreased fluorescence [27]. Both PRP and washed
platelets were processed as discussed above with either A23187
(10 mM) as agonist or pre-loaded platelets with agonist were
incubated with 4f in increasing concentrations (0–100 mM) for
inhibition studies. After incubation, samples were loaded with
NAO (5 mM) for 30 min at 37uC. After incubation fluorescence
was recorded by exciting the samples at 499 nm and emission was
recorded at 530 nm.
Determination of changes in mitochondrial membrane
potential (DYm)
Changes in DYm were determined using cationic dye,
rhodamine 123 in which the intensity of the fluorescence decreases
proportionally with decrease in DYm [28]. Both PRP and washed
platelets were processed as discussed above with either A23187
(10 mM) as agonist or pre-loaded platelets with agonist were
incubated with 4f in increasing concentrations (0–100 mM) for
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Ibuprofen Derivative Inhibits the Platelet Activation and Apoptosis
with different concentrations of compound 4f (0–100 mM), and the
aggregation was initiated by the addition of collagen (2 mg/mL)/
ADP (10 mM)/epinephrine (10 mM). The aggregation was then
followed with constant stirring at 1200 rpm for 6 min [24].
Detection of protein tyrosine phosphorylation and
caspase-3 by immunoblots
Washed platelets (56108/ml) were stimulated by adding
collagen (1 mg/ml) as agonist for protein tyrosine phosphorylation,
A23187 (10 mM) as agonist for caspase-3 activation along with
different doses of 4f (0–100 mM) for inhibition studies. After 3 min
of incubation for protein tyrosine phosphorylation and 1 h for
caspase-3 activation, platelet suspensions were lysed by adding
10 mL of HEPES buffer containing 10% SDS, 20 mM N-
ethylmaleimide, 20 mM sodium o-vanadate, 50 mM EDTA and
10 mM PMSF. Following centrifugation, the supernatants were
separated on SDS-PAGE (10%) and electroblotted on to a PVDF
membrane. After blocking with 5% skimmed milk powder in
TBST, the blots were probed with monoclonal anti-phosphotyr-
osine antibody (1:1,000) for protein tyrosine phosphorylation and
anti-caspase-3 antibody for caspase-3 in TBST for 3 h. Blots were
then incubated with horseradish-peroxidase (HRP)-conjugated
anti-IgG antibody (1:10,000) in TBST and blots were finally
developed by ECL method [24] b-actin was used as loading
control.
Platelet adhesion assay
Platelet adhesion assay was carried out according to the method
of Bellavite et al. [33]. Briefly, collagen was immobilized on to 96-
well polystyrene microtiter plates by adding 20 mg of collagen type
I in 200 mL phosphate buffered saline (PBS, 10 mM, pH 7.4) to
each well and left overnight at 4uC. Following which, 200 mL of
1% (w/v) BSA in PBS was added to block the wells and incubated
at 37uC for 1 h. The wells were then washed three times with PBS.
In the first set of experiment, 4f (0–100 mM) was directly added to
the collagen-coated wells, pre-incubated for 10 min, washed three
times with PBS and then PRP was added. In the second set of
experiment, PRP pre-treated with 4f (0–100 mM) for 10 min at
37uC was added to the collagen-coated wells. Total reaction
volume was made up to 200 mL with PBS. The reaction mixture
was incubated at 37uC for 90 min and then washed three times
with PBS. The adherent platelets were then lysed with 150 mL lysis
buffer (100 mM citrate buffer pH 5.4 containing 5 mM p-
nitrophenyl phosphate and 0.1% Triton X-100) at 37uC for
90 min. The reaction was terminated by inactivating the platelet
membrane acid phosphatase activity with the addition of 100 mL
stopping reagent (2 N NaOH). The color developed was measured
at 405 nm. Platelet adhesion was expressed as percent adhesion,
considering PBS-treated platelet suspension as 100%.
Determination of cytotoxicity by MTT assay
MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] colorimetric assay was performed to assess cell viability.
PRP was taken separately in polystyrene 96-well microtiter plates
and treated with A23187 (10 mM) as agonist and 4f (0–100 mM)
for inhibition studies along with agonist and the final volume was
made up to 200 mL with HBS. After 1 h of incubation 250 mM of
MTT was added and incubated for additional 3 h. Thereafter,
MTT was removed and remaining formazan crystals were
completely dissolved in DMSO. Afterwards, the absorbance was
recorded at 570 nm, using Varioskan multimode plate reader
[31].
Protein estimation
Protein estimation was carried out according to the method of
Lowry et al. [34] using BSA as standard.
Statistical analysis
Measurement of LDH leakage
Results were expressed as mean 6 SEM of five independent
experiments. Statistical significance among groups was determined
by one way analysis of variance (ANOVA) followed by Tukey’s test
for comparison of mean [n = 5, p,0.05 (#), p,0.01 (**/##), p,
0.001 (***/###); *: significant compared to control platelets and
#: significant compared to A23187 treated platelets].
PRP was treated with different doses of 4f (0–100 mM) along
with A23187 (10 mM) as agonist for 1 hr. Following, platelets were
pelleted by centrifugation at 17006g for 10 min. Supernatants
were used to detect LDH release by kit method, according to the
manufacturer’s protocol. The assay was performed in a time
course of decrease in NADH absorbance at 340 nm for 3 min.
Results
Measurement of c-glutamyltransferase (GGT) activity
To determine the GGT activity, washed platelets were
suspended in tubes with 100 mL HBS, each containing
26106 platelets/mL. Further, platelets were treated with A23187
(10 mM) as agonist and for inhibition studies 4f in increasing
concentrations (0–100 mM) along with agonist and incubated for
1 h. After incubation, platelets were pelleted, suspended in distilled
water and lysed by sonication. The resulting lysate was used to
determine GGT activity. Platelet-GGT activity was done accord-
ing to the method of Sener et al. [32], which included assay
mixture containing 4 mM c-glutamyl p-nitroanilide and 40 mM
glycylglycine in 185 mM Tris-HCl buffer, pH 8.2. The results
were calculated using molar extinction coefficient of p-nitroanilide
(9,900 M21cm21) at 405 nm and expressed as mM p-nitroanilide
formed/min/mg protein.
Synthesis of new Ibuprofen derivatives
The novel derivatives of Ibuprofen 1 described in this study,
were prepared by the reaction sequence as shown in Fig. 1.
Esterification of parent compound (Ibuprofen) using ethanol in
presence of sulfuric acid furnished ethyl 2-[4-(2-methylpropyl)-
phenyl]propanoate. Upon refluxing the resulting ester 2 with
alcoholic hydrazine hydrate gave 2-(4-isobutylphenyl)propanehy-
drazide (3). Further, sulfonation of 3 with various sulfonyl
chlorides generated N’-(2-(4-isobutylphenyl)propanoyl)-4-methox-
ybenzenesulfonohydrazide 4(a-f). The synthesized compounds
were chemically characterized (Table 1).
Free radical scavenging potential of Ibuprofen and its
derivatives
Determination of anti-oxidant properties of Ibuprofen and its
derivatives prior to the evaluation of their anti-apoptotic properties
is essential, as molecules with strong anti-oxidant properties are
ideally preferred in combating against oxidative stress induced
platelet apoptosis. Thus, Ibuprofen and its derivatives (4a-4f) were
evaluated for their anti-oxidant properties. To begin with,
Platelet aggregation
Platelet aggregation was determined by turbidimetric method
with a dual channel Chrono-log model 700-2 aggregometer
(Havertown, USA). Briefly, 250 mL of PRP were taken in
siliconized glass cuvette and pre-incubated for 3 min at 37uC
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Ibuprofen Derivative Inhibits the Platelet Activation and Apoptosis
Figure 1. Reaction sequence for the synthesis of new Ibuprofen derivatives.
doi:10.1371/journal.pone.0107182.g001
reducing ability of Ibuprofen and its derivatives on Fe3+ to Fe2+
was evaluated by ferric reducing power assay. It was observed that
the compound 4f had significant and dose dependently increasing
reducing ability (Fig. 2A). Further, DPPH radical scavenging
potential of Ibuprofen and its derivatives were also evaluated.
Compound 4f showed marked radical scavenging potential owing
it to be a potent anti-oxidant molecule compared with other
derivatives of Ibuprofen (Fig. 2B). Based on these results,
compound 4f was proved to possess strong anti-oxidant property
and appeared to be promising for the evaluation of its anti-
apoptotic nature and platelet aggregation inhibitory properties.
Quercetin is being used as a standard anti-oxidant compound to
compare the efficacy of test molecules.
of 4f. Platelets treated with standard agonist A23187, evoked
significant ROS levels with 138% in PRP and 119% in washed
platelets respectively (Fig. 3A). Compound 4f was able to
significantly reduce A23187 evoked ROS in a concentration
dependent manner with marked reduction of ROS levels was
observed at 50 and 100 mM concentrations. Platelets undergoing
oxidative stress are associated with elevated levels of intracellular
calcium which drives platelets towards intrinsic apoptotic pathway.
Consequently, 4f was scrutinized for its effects on dwindling
intracellular calcium levels in platelets where, A23187 was used as
an agonist in increasing intracellular calcium concentration in
platelets. Compound 4f was able to reduce A23187 induced
increase in intracellular calcium in a dose dependent manner and
significant reduction was observed at 100 mM in both PRP and
washed platelets (Fig. 3B).
Compound 4f alleviates A23187 induced ROS and
intracellular calcium levels
Effect of compound 4f on DYm depolarization and
peroxidation of cardiolipin
As mitochondria are the primary sources of ROS production,
making them highly susceptible to ROS attacks which includes
dissipation of DYm and peroxidation of cardiolipin which is a
Platelets being anuclear generally undergo apoptosis via
intrinsic pathway mediated by mitochondria where mitochondrial
generated ROS play imperative role in triggering apoptosis.
Therefore, in order to investigate the anti-apoptotic properties of
compound 4f, it is ideal to analyze the ROS scavenging properties
Table 1. Physical and chemical characteristics of new Ibuprofen derivatives.
Melting point
Entry
Ibuprofen derivatives
Yield (%) ( C)
6
Molecular formula
19H24N2O3S
Molecular weight
4a
4b
2-[4-(2-methylpropyl)phenyl]-N’-(phenylsulfonyl)propanehydrazide
89
91
112–114
121–123
C
360
378
N’-[(4-fluorophenyl)sulfonyl]-2-[4-(2-methylpropyl)
phenyl]propanehydrazide
C19H23FN2O3S
C19H23BrN2O3S
C19H23N3O5S
C19H22Cl2N2O3S
C20H26N2O4S
4c
4d
4e
4f
N’-[(4-bromophenyl)sulfonyl]-2-[4-(2-
methylpropyl)phenyl]propanehydrazide
81
78
85
94
116–118
142–144
102–104
96–98
438
405
428
390
N’-[(4-nitrophenyl)sulfonyl]-2-[4-(2-
methylpropyl)phenyl]propanehydrazide
N’-[(2,5-dichlorophenyl)sulfonyl]-2-[4-(2-
methylpropyl)phenyl]propanehydrazide
N’-[(4-methoxyphenyl)sulfonyl]-2-[4-(2-
methylpropyl)phenyl]propanehydrazide
doi:10.1371/journal.pone.0107182.t001
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Ibuprofen Derivative Inhibits the Platelet Activation and Apoptosis
Figure 2. Efficacy of Ibuprofen and its derivatives measured in terms of (A) Reduction potential (B) DPPH radical scavenging
activity in comparison with Quercetin. Values are presented as mean 6 SEM (n = 5).
doi:10.1371/journal.pone.0107182.g002
major component of mitochondrial membrane. Therefore, 4f was
evaluated for its efficacy in preventing DYm depolarization and
cardiolipin peroxidation. A23187 was used as agonist in inducing
DYm depolarization which dissipated DYm significantly. Besides,
4f markedly restored dissipation of DYm in a concentration
dependent manner and at 100 mM concentration there was
complete restoration of DYm to basal levels (Fig. 3C). Further, 4f
was assessed for its effects on cardiolipin peroxidation with A23187
as agonist which showed significant increase in cardiolipin
peroxidation. 4f which was proved to be potent in reducing
oxidative stress in the previous set of experiments also showed
marked abrogation of A23187 induced peroxidation of cardiolipin
in a concentration dependent manner. At 100 mM concentration
of 4f there was complete inhibition of cardiolipin peroxidation
(Fig. 3D).
Figure 3. Effect of compound 4f on A23187 induced (A) Endogenous generation of ROS (B) Intracellular calcium levels (C)
Mitochondrial membrane depolarization and (D) Peroxidation of cardiolipin in PRP and washed platelets. Values are presented as
mean 6 SEM (n = 5), expressed as percentage increase in DCF fluorescence (for ROS), percentage increase in Fura-2/AM fluorescence (intracellular
calcium), Rhodamine 123 fluorescence (DYm) and NAO fluorescence (cardiolipin), relative to control. ***p,0.001; significant compared to control.
#p,0.05, ##p,0.01, ###p,0.001; significant compared to A23187.
doi:10.1371/journal.pone.0107182.g003
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Ibuprofen Derivative Inhibits the Platelet Activation and Apoptosis
there was significant reduction in activated form of caspase-3 in
platelets treated with 4f (Fig. 4A).
Arresting of cytosolic cytochrome c expression by
compound 4f
Release of cytochrome c from mitochondria to cytosol is
mediated by the formation of MPTP. Altered redox conditions
and peroxidation of cardiolipin play critical role in the formation
of MPTP and the release of cyt c into the cytosol. A23187 was
used as agonist in inducing the release of cyt c to the cytosol. On
the other hand, 4f was also able to diminish the expression of
cytosolic cyt c induced by A23187 in a dose dependent manner
and at 100 mM concentration basal levels of cyt c was restored
(Fig. 4A).
Mitigation of PS externalization by compound 4f
Cells undergoing apoptosis are finally marked by the scrambling
of PS from inner plasma membrane to outer plasma membrane.
A23187 was used as agonist in inducing PS externalization in
platelets and its inhibition by 4f was evaluated. As expected, there
was dose dependent inhibition of PS externalization by 4f in a
concentration dependent fashion and significant inhibition was
observed at 100 mM concentration in both PRP and washed
platelets (Fig. 4D).
Inhibition of caspase activity by compound 4f
Suppression of protein phosphorylation by compound 4f
Recently, it has been revealed that sites for protein phosphor-
ylation are exposed by caspase cleavage activity. Therefore, 4f was
evaluated for its effects on protein phosphorylation with collagen
as agonist. There was significant increase in protein phosphory-
lation in platelets treated with collagen whereas, 4f markedly
reduced collagen induced protein phosphorylation in a concen-
tration dependent manner and at 100 mM concentration protein
phosphorylation was restored up to basal levels (Fig. 5A).
Intrinsic apoptotic pathway in principally is mediated by
caspase-9 which is activated by itself bound to apoptosome
complex along with cytosolic cyt c. The activated caspase-9 in turn
activates caspase-3 which orchestrates platelet apoptosis. There-
fore, in order to investigate the anti-apoptotic properties of 4f,
inhibition of caspase-9 and caspase-3 activity is critically impor-
tant. Compound 4f which was proved to be effective in
ameliorating oxidative stress, was able to significantly inhibit both
caspase-9 and caspase-3 activities induced by A23187 in a
concentration dependent manner (Fig. 4B & Fig. 4C). Further,
inhibition of caspase-3 was confirmed by western blot wherein,
Figure 4. Effect of compound 4f on A23187 induced (A) Translocation of cytosolic cytochrome C and activation of caspase-3 (B)
Caspase-9 and (C) Caspase-3 activities and (D) PS externalization in PRP and washed platelets. Values are presented as mean 6 SEM
(n = 5), expressed as percentage increase in (B & C) caspase activity and (D) Annexin V-FITC fluorescence and expressed as percentage increase in
apoptotic platelets expressing PS relative to control. ***p,0.001; significant compared to control. #p,0.05, ##p,0.01, ###p,0.001; significant
compared to A23187.
doi:10.1371/journal.pone.0107182.g004
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Ibuprofen Derivative Inhibits the Platelet Activation and Apoptosis
Figure 5. Effect of compound 4f on (A) Collagen induced protein phosphorylation (B) A23187 induced c-glutamyltransferase
activity (C) MTT cell viability assay (D) LDH release in platelets. (A) Lane I- resting platelets (untreated). Lane II- platelets treated with
Collagen (1 mg/mL). Lanes III, IV and V- pre-loaded platelets with collagen and incubated with 4f in increasing concentration of 25, 50 and 100 mM
respectively. Values are presented as mean 6 SEM (n = 5). ***p,0.001; significant compared to control. #p,0.05, ##p,0.01, ###p,0.001; significant
compared to agonist.
doi:10.1371/journal.pone.0107182.g005
Inhibition of c-glutamyl transferase (GGT) activity by
compound 4f
Effect of compound 4f on agonist induced platelet
aggregation and platelet adhesion
GGT is involved in GSH homeostasis and its expression is
increased during oxidative stress. In the previous set of experi-
ments 4f was proved to be anti-apoptotic by reducing oxidative
stress in the platelets. Accordingly, 4f was analyzed for its effects on
GGT activity. There was significant increase in GGT activity in
platelets treated with A23187 as agonist and as expected there was
concentration dependent reduction of GGT activity in agonist
activated platelet suggesting that 4f serves as anti-apoptotic
molecule basically by reducing oxidative stress in platelets
(Fig. 5B).
Further, platelets being the key mediators in maintaining the
integrity of endothelium, effect of compound 4f on platelet
aggregation and platelet adhesion with collagen was evaluated. In
order to stimulate platelet aggregation collagen/ADP/epinephrine
were used as agonist. Compound 4f was able to significantly
inhibit agonist induced platelet aggregation to varied extent. Of all
the agonists tested, the epinephrine-induced aggregation was
abolished at 100 mM concentration of compound 4f. On the other
hand, compound 4f alone did not exhibit any effect on platelet
aggregation up to the tested concentrations (Fig. 6A & Fig. S1–
S3).
In order to determine the effect of compound 4f on the platelet
receptors for agonists, collagen adhesion was performed using pre-
coated collagen microtiter wells. The compound 4f untreated/PBS
treated platelet suspension served as control and accounted for
100% adhesion. In contrast, 4f pre-treated platelets did not adhere
efficiently to collagen immobilized on the microtiter wells.
Compound 4f was able to prevent the binding of platelet with
collagen in a concentration dependant fashion in both collagen
pre-treated with compound 4f and PRP pre-treated with
compound 4f. At 250 mM concentration, only 54% adhesion
Cyto-protective effects of compound 4f
In order to investigate whether 4f had cyto-protective effect,
MTT assay was performed. A23187 was used as standard agonist,
which reduced cell viability from 93% to 21%. However, treating
platelets with 4f restored its survivability up to 75% in agonist
activated platelets (Fig. 5C). Further, cyto-protective nature of 4f
was confirmed by measure the leakage of LDH into the medium.
Treatment of platelets with A23187 increased LDH release in to
the medium whereas, 4f was able to significantly reduce LDH
release indicating its cyto-protective properties (Fig. 5D).
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Ibuprofen Derivative Inhibits the Platelet Activation and Apoptosis
Figure 6. Effect of compound 4f on (A) Platelet aggregation induced by Collagen/ADP/Epinephrine and (B) Platelet adhesion on
immobilized collagen type I with compound 4f pre-treated collagen and PRP pre-treated with compound 4f. Values are presented as
mean 6 SEM (n = 5), expressed as percentage decrease in aggregation and increase in platelet adhesion. **p,0.01, ***p,0.001; significant compared
to control.
doi:10.1371/journal.pone.0107182.g006
was observed, and thus affecting the collagen binding property of
platelets (Fig. 6B).
potent antioxidant and effectively inhibited oxidative stress-
induced apoptosis in platelets.
In the first set of experiments, the effect of compound 4f on
oxidative stress was assessed. A23187 was used to stimulate the
endogenous generation of ROS in platelets. When the platelets
were pretreated with 4f prior to A23187 treatment, the ROS
generation was found to decrease in a dose-dependent manner. It
is reported that it is specifically H2O2 that initiates the apoptotic
events in platelets through the intrinsic or mitochondrial pathway.
Thus, the results highlight the ability of compound 4f to diminish
oxidative stress in platelets and thereby protect them from
undergoing an early death. This property may be attributed to
the anti-inflammatory and anti-platelet efficacy of the parent
compound ibuprofen. Previously it has been reported that
ibuprofen treatment decreases the levels of lipid peroxidation,
tyrosine nitration, protein oxidation and ROS production in
murine model of Alzheimer’s disease [39].
Discussion
Reduced platelet count or thrombocytopenia is the medical
condition where the platelet count drops drastically to 50,000/mL
of blood as against the normal count of 150,000 to 450,000 plate-
lets/mL of blood. It can be caused by a variety of clinical
conditions including 1) decreased production of platelets or 2)
augmented platelet destruction or 3) increased splenic sequestra-
tion of platelets. Among these reasons, augmented platelet
destruction or apoptosis can be seen in a number of medical
and pathological conditions [1,4,35]. The platelet apoptosis is
mediated either by immune related or non-immune related causes.
The immune thrombocytopenia is a common bleeding disorder
characterized by autoantibody-mediated platelet destruction. The
auto-antibodies primarily target the membrane receptors and
integrin complex of platelets including glycoproteins GPIIb/IIIa
and GPIb/IX [36]. In contrast, platelets undergo oxidative stress-
mediated apoptosis in non-immune related thrombocytopenia.
The adverse effects of thrombocytopenia may lead to morbidity
and mortality from severe surgical hemorrhage and delay in the
normal process of clotting. Several studies reported oxidative stress
or immunologic reaction-induced platelet apoptosis by a wide
range of biologicals including antibiotics, anticancer drugs,
phytochemicals and hormones [5,8,9]. Therefore, the clinical
implication of thrombocytopenia in various human pathologies
has strongly demanded the need for small molecular weight
natural/synthetic platelet protective molecules. Till date only two
phytochemicals (cinnamtannin B1 and crocin) are shown to inhibit
platelet apoptosis [17,18]. Hence, there continues the search for
potent small molecular weight natural/synthetic molecules to
manage thrombocytopenia associated with various human pathol-
ogies.
For the next set of experiments, A23187 was used as the agonist
to induce the different events of apoptosis. In order to assess the
effect of compound 4f on mitochondria, the central players in the
intrinsic pathway of apoptosis, changes in DYm were analyzed.
A23187 was used as the positive control to induce changes in
DYm. Compound 4f markedly diminished A23187-induced
changes in DYm and reinstates the membrane potential. From
the result it can be stated that 4f has the capacity to inhibit platelet
apoptosis by protecting mitochondria from oxidative stress. In the
recent past, a study by Sanz-Blasco et al. [40] demonstrated that
ibuprofen prevents mitochondrial Ca2+ overload, cyt c release and
thus prevent neurotoxicity [40]. Thus, to verify whether the same
was true in the case of the effect of 4f on platelets, the levels of
intracellular Ca2+ and cytosolic cyt c were measured. It was found
that compound 4f could inhibit the increase in intracellular Ca2+
and cytosolic cyt c levels in a dose-dependent manner. Release of
cyt c from mitochondrial intermembrane space to the cytosol due
to formation of a channel MPTP is a key event that initiates
morphological changes during apoptosis. The morphological
changes are arbitrated by the activated caspases, finally resulting
in PS externalization [7,8,15].
Of late, Ibuprofen, a well known NSAID was reported to
interfere with platelet functions and viability thereby, causing
thrombocytopenia, nevertheless it is also demonstrated to have
anti-cancer, analgesic, anti-pyretic and anti-platelet properties
[19,37,38]. Thus, as an attempt to reduce ibuprofen side effects
including platelet damage property, in the present study we have
synthesized a series of ibuprofen derivatives (4a-f) with improved
antioxidant activity. Among the derivatives, compound 4f was a
Therefore, the next set of experiments targeted the influence of
compound 4f over caspase activity and PS exposure. The results of
the study firmly highlight the inhibitory effect of compound 4f on
the activities of caspase -9 and -3. Further, it was also shown that
compound 4f was to impair PS exposure, the hallmark of an
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Ibuprofen Derivative Inhibits the Platelet Activation and Apoptosis
apoptotic cell. Thus, the current study underscores anti-apoptotic
effects of compound 4f on human platelets. It was recently
reported that the parent compound possesses chemo-preventive
property via the stimulation of apoptosis [21,41]. Besides, it was
also reported that it causes thrombocytopenia, which is a common
effect of most of the anti-cancer drugs [21]. Consequently, it would
be amazing if compound 4f could wield anti-cancer effect. It has
the potential to be developed as an anti-cancer molecule, which
has platelet-protective properties.
stroke [3]. In this context, platelet protective and anti-aggregant
molecules like compound 4f, crocin and cinnamtannin B1 [17,18]
could be used in the treatment regime of neurological disorders as
auxiliary therapeutic molecules in order to mitigate the platelet
apoptosis and altered hemostasis.
Taken together, the therapeutic drug-induced dysregulated
platelet apoptosis is one of the major causes for thrombocytopenia.
Furthermore, the formation of MPs during platelet apoptosis
might worsen the clinical condition [5]. Platelet-derived MPs
(PMPs) are shown to regulate several pathophysiological functions
including cell proliferation, differentiation, vascular remodeling,
angiogenesis, inflammation and apoptosis. The augmented levels
of circulating PMPs have shown to be associated with many
pathological conditions including CVDs, thromboembolism,
diabetes, rheumatoid arthritis, and cancer [5,16]. Therefore,
biologicals with anti-platelet property as well as platelet protective
efficacy stand better and thus could receive a lot of attention by the
medical practitioners in order to treat thrombolytic disorders.
From the results of the present study, it can be noted that
compound 4f (100 mM) is as effective as cinnamtannin B1 and
crocin, the previously reported compounds with platelet protective
properties at the same concentration [17,18]. Thus, compound 4f
can be taken into account as a potential candidate in the treatment
regime of altered platelet function-associated pathologies. In
addition, compound 4f can be used as an auxiliary therapeutic
agent in order to mitigate the side effects of therapeutic drugs
especially platelet apoptosis and microparticle generation. Future
studies related to the molecular mechanism of platelet protection
and aggregation inhibition of compound 4f is highly exciting.
Furthermore, effect of compound 4f on platelet aggregation was
evaluated to delineate its cardio-protective action. Platelet
aggregation plays a central role in the perpetuation of CVDs
and atherothrombotic disorders. It triggers intraluminal thrombo-
sis and thereby promulgates myocardial infarction, stroke and
peripheral vascular occlusions [42,43]. Thus, inhibition of platelet
aggregation is crucial in prevention of CVDs and associated
complications. The results demonstrated that, compound 4f
significantly inhibited various agonists (Collagen/ADP/Epineph-
rine)-induced platelet aggregation to varied extent. Among the
agonists, compound 4f showed better inhibition towards the
epinephrine-induced platelet aggregation. The role of compound
4f in blocking epinephrine receptor on platelets is not clear. The
receptors for the above-mentioned agonists are high molecular
weight glycoproteins and compound 4f might interfere with the
binding of agonists to receptors by interacting directly with the
membrane proteins or membrane lipids. In continuance, platelet
adhesion assay was carried out to verify whether compound 4f
prevents the interaction of platelets with collagen. This interaction
is crucial for platelet aggregation. The results indicate that
compound 4f markedly decreases the collagen-binding efficacy
of platelets, suggesting the mechanism through which the
compound exerts its antiplatelet effect.
Supporting Information
Figure S1 Effect of compound 4f on Collagen induced
platelet aggregation. Concentration dependent inhibition of
collagen induced platelet aggregation by compound 4f: (i) Control
(Collagen-2 mg/mL), (ii) 50 mM and (iii) 100 mM compound 4f
respectively and aggregation was performed as described in
materials and methods section.
It has been reported that ibuprofen has mild anti-platelet effect,
which is very less when compared to the other standard anti-
platelet drugs such as aspirin and ketoprofen. However, from the
current results the derivative of ibuprofen, compound 4f has been
demonstrated to be a very effective anti-platelet agent. Aspirin is
the most common anti-platelet agent used in the primary
prevention of cardiovascular events due to atherothrombosis.
Aspirin inhibits the platelet aggregation by reducing the produc-
tion of thromboxane (Tx) A2 due to inhibition of cyclooxygenase-1
in platelets [44]. This could be the possible reason for aspirin-
associated bleeding during chronic intake of aspirin. In addition,
aspirin could also aggravate platelet GP Ib and GPV ectodomain
shedding, which is yet another possible mechanism for hemor-
rhagic conditions [45]. Recent reports suggested the apoptotic
tendency of aspirin toward many cell types including platelets [46].
Zhao et al. [47] demonstrated pro-apoptotic nature of aspirin on
platelets and found that aspirin (2.5–20 mM) induced apoptosis in
platelets via COX-independent pathway [47]. This could be the
possible mechanism for aspirin induced hemorrhage in susceptible
patients. In contrast, the compound 4f inhibited the agonist-
induced platelet aggregation as well as protected the platelets from
oxidative stress-induced apoptosis.
(TIF)
Figure S2 Effect of compound 4f on ADP induced
platelet aggregation. Concentration dependent inhibition of
ADP induced platelet aggregation by compound 4f: (i) Control
(ADP-10 mM), (ii) 50 mM and (iii) 100 mM compound 4f
respectively and aggregation was performed as described in
materials and methods section.
(TIF)
Figure S3 Effect of compound 4f on epinephrine
induced platelet aggregation. Concentration dependent
inhibition of epinephrine induced platelet aggregation by com-
pound 4f: (i) Control (Epinephrine-10 mM), (ii) 50 mM and (iii)
100 mM compound 4f respectively and aggregation was performed
as described in materials and methods section.
(TIF)
Platelets are known to express the amyloid precursor protein
(APP) and exhibit the complete enzymatic machinery to process
APP proteins into amyloid-b (Ab) peptides through the same
pathway described in the brain. A recent study demonstrated the
amyloid-b (Ab) peptide-induced platelet apoptosis by assessing
ROS generation, increase of cytosolic Ca2+ levels, mitochondrial
depolarization, caspase-3 activation, cell shrinkage and cell
membrane scrambling. In neurological pathologies, uncontrolled
activation of platelets by oxidative stress and other inducers can
lead to acute vessel occlusion leading to myocardial infarction and
Acknowledgments
Authors thank Mr. SK Naveen Kumar for his kind help during the study
and also thank Central Instrumentation Facility, Institute of Excellence
(IOE), University of Mysore, Mysore.
Author Contributions
Conceived and designed the experiments: K. S. Rangappa KSG MPS KK.
Performed the experiments: K. S. Rakesh SJ ACV MSS CDM B TRS.
Analyzed the data: K. S. Rakesh SJ MP RMT MH. Contributed reagents/
PLOS ONE | www.plosone.org
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September 2014 | Volume 9 | Issue 9 | e107182

Ibuprofen Derivative Inhibits the Platelet Activation and Apoptosis
materials/analysis tools: K. S. Rangappa KSG KK MH RMT.
Contributed to the writing of the manuscript: K. S. Rangappa KSG
MH RMT.
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