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Molecular and Cellular Biochemistry
An International Journal for Chemical
Biology in Health and Disease
ISSN 0300-8177
Mol Cell Biochem
DOI 10.1007/s11010-012-1476-7
Crocin, a dietary additive protects platelets
from oxidative stress-induced apoptosis and
inhibits platelet aggregation
R.M.Thushara, M.Hemshekhar,
M.Sebastin Santhosh, S.Jnaneshwari,
S.C.Nayaka, S.Naveen, K.Kemparaju &
K.S.Girish
1 23
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Crocin, a dietary additive protects platelets from oxidative
stress-induced apoptosis and inhibits platelet aggregation
R. M. Thushara •M. Hemshekhar •M. Sebastin Santhosh •S. Jnaneshwari •
S. C. Nayaka •S. Naveen •K. Kemparaju •K. S. Girish
Received: 2 July 2012 / Accepted: 26 September 2012
ÓSpringer Science+Business Media New York 2012
Abstract Platelets are the key players in the development
of cardiovascular diseases as the microparticles generated
by apoptotic platelets and platelet aggregation contribute
actively towards the disease propagation. Thus, the aim of
this study was to demonstrate the effect of a phytochemical
which can prevent these two processes and thereby project
it as a cardio-protective compound. Crocin, a natural
carotenoid exhibits a wide spectrum of therapeutic poten-
tials through its antioxidant property. The study demon-
strated its effects on cytoplasmic apoptotic events of
mitochondrial pathway in platelets. Collagen/calcium ion-
ophore-A23187 stimulated platelets were treated with
crocin and endogenous generation of reactive oxygen
species (ROS) and hydrogen peroxide (H
2
O
2
) were mea-
sured. H
2
O
2
-induced changes in crocin-pretreated platelets
such as intracellular calcium, mitochondrial membrane
potential (DWm), caspase activity, phosphatidylserine
exposure and cytochrome c translocation were determined.
Crocin dose-dependently ameliorated collagen- and
A23187-induced endogenous generation of ROS and H
2
O
2
.
It also abolished the H
2
O
2
-induced events of intrinsic
pathway of apoptosis. Further, it hindered collagen-induced
platelet aggregation and adhesion. The current piece of
work clearly suggests its anti-apoptotic effect as well as
inhibitory effects on platelet aggregation. Thus, crocin can
be deemed as a prospective candidate in the treatment
regime of platelet-associated diseases.
Keywords Crocin Oxidative stress Platelet apoptosis
Microparticles Platelet aggregation
Cardiovascular diseases
Abbreviations
AC-DEVD-AMC Acetyl-Asp-Glu-Val-Asp-7-
amido-4-methylcoumarin
AC-LEHD-FMC-N Acetyl-Leu-Glu-His-Asp
trifluoromethylcoumarin
Apaf-1 Apoptotic protease activating
factor-1
CM-H2DCFDA or
DCF-5-(and-6)
Chloromethyl-20,70-
dichlorodihydrofluorescein
diacetate acetylester
CVD Cardiovascular diseases
cyt.c Cytochrome c
DTT Dithiothreitol
EGTA Ethylene glycol tetra acetate
HBS HEPES-buffered saline
HEPES N-(2-Hydroxyethyl)piperazine-
N0-ethanesulfonic acid
HVA Homovanillicacid
JC-1-5,50-6,60Tetrachloro-1,1
´,3,3
´-tetraethyl
benzimidazolylcarbocyanine
iodide
MP Microparticle
MPTP Mitochondrial permeability tran-
sition pore
Ox-LDL Oxidative low density lipoprotein
PMSF Phenylmethylsulfonyl fluoride
R. M. Thushara M. Hemshekhar M. S. Santhosh
S. Jnaneshwari K. Kemparaju K. S. Girish (&)
Department of Studies in Biochemistry, University of Mysore,
Mysore 570 006, Karnataka, India
e-mail: ksgbaboo@yahoo.com
S. C. Nayaka
Department of Studies in Biotechonology, University of Mysore,
Mysore 570 006, Karnataka, India
S. Naveen
Biochemistry and Nutrition Discipline, Defence Food Research
Laboratory, Siddarthanagar, Mysore 570011, India
123
Mol Cell Biochem
DOI 10.1007/s11010-012-1476-7
Author's personal copy
PRP Platelet-rich plasma
PS Phosphatidylserine
ROS Reactive oxygen species
TBST Tris-buffered saline with 0.1 %
tween 20
DWmMitochondrial membrane potential
Introduction
Platelets, the discoid-shaped anuclear blood cells derived
from megakaryocytes, are conventionally known for their
essential roles in hemostasis and thrombosis. However,
research in the field of platelet biology has gained a lot of
momentum lately, due to the critical role played by them in
human health and disease [1]. With a life span of just about
a week, they are very sensitive to various external and
internal stimuli and are a vulnerable target for the reactive
oxygen species (ROS) generated in the endothelium during
oxidative stress. It has been reported that oxidative stress is
a major contributing factor for the pathophysiology of
several diseases including cardiovascular diseases (CVDs).
Therefore, it seems plausible to inter-relate oxidative
stress, altered platelet functions and CVDs, going by the
fact that accelerated platelet activation and aggregation
eventually lead to the development of ischemia/reperfu-
sion, atherothrombosis and myocardial infarction. Conse-
quently, many of the current treatment strategies for CVDs
include antiplatelet drugs which target the various events of
platelet activation and aggregation. However, recent stud-
ies claim that high amount of circulating microparticles
(MPs) are also involved in the progression of CVDs. MPs
are phosphatidylserine (PS)-positive membrane fractions
released from apoptotic platelets. Despite their anuclear
nature, platelets undergo apoptosis, the process of pro-
grammed cell death, similar to nucleated cells, but sans the
nuclear events [2]. The apoptotic events include the
increased production and release of ROS, particularly
hydrogen peroxide (H
2
O
2
), depolarization of mitochondrial
inner transmembrane potential (DWm), release of apoptotic
factors and PS externalization [3]. Eventually they release
MPs possessing pro-inflammatory and pro-coagulatory
properties, thereby affecting vascular function and leading
to the pathogenesis of CVDs [4–6].
In the present medical scenario, the available antiplatelet
therapies are accompanied by several harmful side effects
including severe internal bleeding owing to reduced platelet
count or thrombocytopenia. Hence, people nowadays are
turning towards plant-based therapeutics. Besides lacking
harmful side effects, many of them are potent antioxidants,
and hence have become the choice of treatment of late for
many of the oxidative stress-induced ailments including
CVDs. But, their effect on oxidative stress-induced altera-
tions in platelet functions has not been copiously assessed.
Thereby in this study, an attempt has been made to evaluate
the effect of crocin on oxidative stress-induced platelet
apoptosis and aggregation. Crocin, a natural dietary carot-
enoid found in the stigma of flowers of Crocus sativus
L. (saffron) and fruits of Gardenia jasminoides. Chemi-
cally, it is a diester of disaccharide gentiobiose and the
dicarboxylic acid crocetin. Studies emphasize that it is an
excellent antioxidant with immense therapeutic potentials
including tumoricidal, anticarcinogenic, antihyperlipidem-
ic, and antidepressant properties [7–10]. The current piece
of work aims to project crocin as a novel antiplatelet phy-
tochemical, which protects the platelets from oxidative
stress-induced apoptosis and inhibits aggregation and
thereby assert its role as a cardioprotective compound.
Materials and methods
Chemicals/reagents
Rotenone, crocin, collagen (Type-I), benzamidine hydro-
chloride, N-acetyl-Leu-Glu-His-Asp trifluoromethylcouma-
rin (AC-LEHD-FMC), acetyl-Asp-Glu-Val-Asp-7-amido-4-
methylcoumarin (AC-DEVD-AMC), glutaraldehyde, sodium
orthovanadate (Na
3
VO
4
), fluorescein isothiocyanate (FITC)-
labeled annexin V, 5-(and-6)-chloromethyl-20,70-dichlor-
odihydrofluorescein diacetate acetylester (CM-H2DCFDA),
5,50-6,60-tetrachloro-1,10,3,30-tetraethyl benzimidazolylcarbo
cyanine iodide (JC-1), leupeptin hydrochoride, N-(2-
Hydroxyethyl)piperazine-N0-ethanesulfonic acid (HEPES),
fura-2/AM, dithiothreitol (DTT), enhanced chemilumines-
cence detection reagents and hyperfilm ECL were from Sigma
Chemicals, St. Louis (USA). Homovanillicacid (HVA) was
from Sisco Research laboratories Pvt Ltd., Mumbai (India).
Horseradish peroxidase-conjugated rabbit anti-sheep IgG
antibody and anti-cytochrome c(cyt.c) antibody were pur-
chased from Epitomics, Inc., Burlingame (USA). Collagen
was from Vitrogen 100; Cohesion, Palo Alto, CA (USA). All
other reagents were of analytical grade.
Preparation of washed platelets
Venous blood was drawn from healthy drug-free human
volunteers (non-smokers) with informed consent as per the
guidelines of Institutional Human Ethical Committee (IHEC-
UOM No. 59/Ph.D/2011-12), University of Mysore, Mysore.
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
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anti-coagulated whole blood was then centrifuged at 909gfor
15 min and the supernatant obtained was the platelet-rich
plasma (PRP). The PRP was centrifuged at 1,700 9gfor
15 min at 37 °C. The platelet pellet thus obtained was sus-
pended and incubated for 10 min in Tyrode’s albumin buffer
(145 mM NaCl, 5 mM KCl, 10 mM HEPES, 0.5 mM
Na
2
HPO
4
, 1 mM MgCl
2
, 6 mM glucose, and 0.3 % bovine
serum albumin, BSA) pH 6.5 and washed thereafter at
1,7009gfor 15 min at 37 °C. 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 sus-
pension using a Neubauer chamber and adjusted to required
number of platelets in the final suspension using Tyrode’s
albumin buffer (pH 7.4) [11].
Determination of endogenously generated ROS
Endogenous ROS production in platelets was determined
according to the method of Lopez et al. [12], with slight mod-
ifications using CMH2DCFDA, a ROS-sensitive fluorescent
probe. PRP as well as washed platelet suspension were taken
separately in polystyrene 96-well microtiter plates and treated
with collagen/A23187 (10 lg/mL), final volume was made up
to 200 lL with HEPES-buffered saline (HBS), pH 7.45, con-
taining 145 mM NaCl, 10 mM HEPES, 10 mM D-glucose,
5mMKCl,1mMMgSO
4
and supplemented with 0.1 % BSA
incubated at 37 °C for 1 h. For inhibition studies, platelets were
pre-incubated with different doses of crocin (dissolved in HBS)
for 10 min at 37 °C prior to collagen/A23187 treatment. The
control (untreated) and treated platelets were then incubated
with 10 lM CMH2DCFDA for 30 min at 37 °C, fluorescence
was recorded using a Varioskan multimode plate reader
(Thermo Scientifics, USA) by exciting the samples at 488 nm
and measuring the resulting fluorescence at 530 nm.
Determination of endogenously generated H
2
O
2
HVA, a specific H
2
O
2
-sensitive fluorescent probe was used
to detect the endogenously generated H
2
O
2
. With slight
modifications to the method of Barja [13], briefly, pre-
treated (as described in the previous section) and control
PRP and washed platelets were incubated with 100 lM
HVA for 30 min at 37 °C, centrifuged, and the pellets were
resuspended in HBS, fluorescence was recorded using a
multimode plate reader by exciting the samples at 312 nm
and measuring the resulting fluorescence at 420 nm.
Estimation of intracellular calcium
Intracellular Ca
2?
concentration was measured in PRP and
washed platelets as described previously [14]. Both PRP
and washed platelets, taken in 96-well polystyrene micro-
titer plates were treated with H
2
O
2
(2 mM) and the final
volume made up to 200 lL with modified Tyrode’s solu-
tion (pH 7.4) containing NaCl (150 mM), KCl (2.7 mM),
KH
2
PO
4
(1.2 mM), MgSO
4
(1.2 mM), CaCl
2
(1.0 mM),
and HEPES (10 mM) with 1 % BSA and incubated for 1 h
at 37 °C to induce the release of Ca
2?
from the intracel-
lular Ca
2?
stores. Inhibition studies with crocin were done
as described in the previous section. The platelets were
then incubated for 45 min at room temperature with 2 lM
fura-2/AM, a fluorescence Ca
2?
indicator. The cells were
subsequently washed twice with the modified Tyrode’s
solution to remove the dye from the extracellular fluid and
finally the platelet pellet was suspended in modified
Tyrode’s solution. The fura-2/AM absorption was deter-
mined by exciting the cells 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 changes in mitochondrial membrane
potential (DWm)
The cationic dye JC-1 was used to detect changes in the
DWmaccording to the method of Salvioli et al. [15]. JC-1
accumulates in mitochondria as red fluorescent aggregates
at high membrane potentials, whereas it exists as a green
fluorescent monomeric form at low membrane potential.
Treated (as described in the previous section) and control
PRP/washed platelets were loaded with 10 lg/mL JC-1 at
37 °C for 10 min. The cells were then excited at 488 nm
and emission was detected at 585 nm for JC-1 aggregates
and 516 nm for JC-1 monomers using multimode plate
reader. Data were presented as emission ratios (585/516).
Agonist-induced changes in DWmwere quantified as the
integral of the decrease in JC-1 fluorescence ratio.
Preparation of platelet lysate
Platelet lysate was prepared by immediately adding an
equal volume of 29Triton buffer (2 % TritonX-100, 2 mM
ethylene glycol tetra acetate, EGTA, 100 mM Tris/HCl—
pH 7.2, 100 lg/mL leupeptin, 2 mM PMSF, 10 mM ben-
zamidine, 2 mM Na
3
VO
4
) to the treated and control plate-
lets from PRP and washed platelet suspension and allowed
to undergo lysis for 30 min at 4 °C. The lysate was cen-
trifuged at 16,0009gfor 5 min. The pellet thus obtained is
the cytoskeleton-rich (triton-insoluble) fraction, which was
subjected to caspase activity and western blotting [16].
Detection of cyt.c release
Cytochrome crelease was detected by western blotting
samples from cytosolic fractions of washed platelets [17].
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Platelets were pretreated with different concentrations of
crocin (0–100 lg/mL) for 10 min and then stimulated with
H
2
O
2
(2 mM) for 1 h at 37 °C. Cytosolic proteins were
separated by 10 % SDS-PAGE and electrophoretically
transferred on to a nitrocellulose membrane for 1 h at 50 V
using a wet blotter. Blots were then incubated overnight
with 10 % BSA in tris-buffered saline with 0.1 % tween 20
(TBST) to block residual protein-binding sites. Membranes
were incubated with anticytochrome cantibody (1:1,000)
in TBST for 2 h. Blots were incubated with horseradish-
peroxidase (HRP)-conjugated anti IgG antibody (1:10,000)
in TBST and exposed to enhanced chemiluminescence for
3 min. Finally, the blots were exposed to photographic
films. b-actin was used as loading control.
Assay of caspases 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 lM caspase substrate (AC-DEVD-AMC for
caspase-3 and AC-LEHD-AFC for caspase-9) for 2 h at
37 °C[18]. Substrate cleavage was measured with a mul-
timode plate reader (excitation wavelength 360 nm and
emission at 460 nm).
Determination of PS externalization
Samples of treated and control PRP/washed platelets were
transferred to equal volume of ice-cold 1 % (w/v) glutar-
aldehyde in HBS for 10 min, and then incubated for 10 min
with annexin V FITC (0.6 lg/mL) in HBS. The cells were
collected by centrifugation for 60 s at 3,0009gand resus-
pended in HBS. Cell staining was measured in a multimode
plate reader by exciting the samples at 496 nm and emission
was recorded at 560 nm [19].
Platelet aggregation
Platelet aggregation was determined by turbidimetric
method [11] with a dual channel Chrono-log model 700-2
aggregometer (Havertown, USA). Briefly, 240 lL of PRP
was incubated at 37 °C in a siliconized glass cuvette and
pre-incubated with different concentrations of crocin
(0–200 lg/mL) in 5 lL HBS for 3 min, and the aggrega-
tion was initiated by the addition of collagen (10 lg/mL)/
ADP (10 lM)/epinephrine (5 lM). The aggregation was
then followed with constant stirring at 900 rpm for 6 min.
Aggregation induced by collagen/ADP/epinephrine alone
was considered as 100 % aggregation.
Platelet adhesion assay
Platelet adhesion assay was carried out according to the
method of Bellavite et al. [20]. Briefly, collagen was
immobilized on to 96-well polystyrene microtiter plates by
adding 20 lg of collagen type I in 200 lL phosphate buf-
fered saline (PBS) to each of the wells and left overnight at
4°C. Following which, 200 lL of 1 % (w/v) BSA in PBS
was added to block the wells and incubated at 37 °C for 1 h.
The wells were then washed three times with PBS. In the
first set of experiments, crocin (10–200 lg/mL) was
directly added to the collagen-coated wells, pre-incubated
for 10 min, and then PRP was added. In the second set of
experiments, PRP pre-treated with crocin (10–200 lg/mL)
for 10 min was added to the collagen-coated wells. Total
reaction volume was made up to 200 lL. The reaction
mixture was incubated at 37 °C for 90 min and then washed
three times with PBS. The adherent platelets were then
lysed with 150 lL lysis buffer (0.1 M citrate buffer pH 5.4
containing 5 mM p-nitrophenyl phosphate and 0.1 % Triton
X-100) by incubating at 37 °C for 90 min. The reaction was
terminated by inactivating the platelet membrane acid
phosphatase activity with the addition of 100 lL stopping
reagent (2 N NaOH). The color developed was measured at
405 nm. Platelet adhesion was expressed as percent adhe-
sion, considering PBS-treated platelet suspension as 100 %.
Statistical analysis
Results were expressed as mean ±SEM of five indepen-
dent experiments. Statistical significance among groups was
determined by one-way analysis of variance (ANOVA)
followed by Tukey’s test for comparison of means. Here,
data were shown as mean ±SEM (n=5), p\0.05 (*).
Results
Effect of crocin on ROS and H
2
O
2
Crocin was firstly evaluated for its efficacy on collagen/
A23187-induced endogenous generation of ROS, H
2
O
2
in
platelets. Treatment of PRP with collagen induced 2.25- and
1.61-fold increase in the generation of ROS and H
2
O
2
,
respectively, as compared to the control. Treatment of washed
platelets with A23187 induced 1.6- and 2.36-fold increase in
the generation of ROS and H
2
O
2
, respectively, as compared to
the control. Inhibition studies with crocin at different con-
centration ratios of 1:0.5, 1:1, 1:1.5, 1:2 (collagen:crocin;
w/w) significantly reduced both collagen-evoked ROS as well
H
2
O
2
generation in a concentration-dependent manner in
PRP. Similarly, crocin at different concentration ratios of
1:0.25, 1:0.5, 1:0.75, 1:1, 1:2 (A23187:crocin; w/w),
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significantly reduced both A23187-evoked ROS as well as
H
2
O
2
generation in a concentration-dependent manner in
washed platelets. At the ratio 1:1, there was complete inhibi-
tion of ROS and at the ratio 1:2, there was 98 % inhibition of
H
2
O
2
generation in both PRP as well as washed platelets
(Fig. 1).
Effect of crocin on H
2
O
2
-induced generation
of intracellular calcium
Treatment with 10 lg/mL A23187 and 2 mM H
2
O
2
,
respectively, resulted in 1.5- and 1.4-fold increase in
intracellular Ca
2?
in PRP, whereas in washed platelets it
resulted in 1.5- and 2.26-fold, respectively. Inhibition
studies with crocin in the concentration range 0–50 lg/mL
resulted in the dose-dependent amelioration in Ca
2?
gen-
eration. The Ca
2?
concentration was restored to basal level
in A23187- and H
2
O
2
-treated PRP at crocin concentration
25 and 50 lg/mL, respectively. In the case of washed
platelets, inhibition studies were carried out at concentra-
tion range 0–150 lg/mL of crocin. The Ca
2?
concentration
was restored to 80 and 87 % of the basal level in A23187-
and H
2
O
2
-treated PRP at crocin concentration 100 and
150 lg/mL, respectively (Fig. 2).
Effect of crocin on H
2
O
2
-induced activation
of caspases-9 and -3 in platelets
Caspase activation was analyzed using two specific fluo-
rescent substrates AC-DEVD-AMC and AC-LEHD-AFC
for caspase-3 and -9, respectively. Treatment of platelets
with 2 mM H
2
O
2
induced the activation of caspase-3 by
1.3- and 1.6-fold in PRP and washed platelets, respectively.
Activation of caspase-9 by H
2
O
2
was to a greater extent
than that of caspase-3, reaching a maximal activity after 2 h
of stimulation with 1.8- and 2.0-fold increase, respectively,
in PRP and washed platelets. Pretreatment of platelets for
with crocin in the concentration range 25–100 lg/mL sig-
nificantly reduced H
2
O
2
-evoked activation of caspase-3 and
-9 in a concentration-dependent manner. At a concentration
of 100 lg/mL, there was almost complete neutralization of
both caspase-9 and -3 (Fig. 3).
Crocin diminishes the effect of H
2
O
2
-induced
mitochondrial membrane potential (DWm)
depolarization and PS scrambling
Mitochondrial membrane depolarization was detected by
the decrease in JC-1 fluorescence ratio (585/516 nm).
Treatment with 10 lM rotenone induced maximal decrease
in JC-1 fluorescence ratio and was considered as 100 %
DWmdissipation. Treatment with 2 mM H
2
O
2
resulted in
87 and 62 % DWmdissipation in PRP and washed platelets
as compared to that of rotenone. Pretreatment with crocin
at 0–100 lg/mL induced a dose-dependent restoration of
membrane potential. At 100 lg/mL concentration, crocin
was able to restore H
2
O
2
-induced changes in DWmto 96 %
in PRP as compared to control. In the case of washed
platelets, 50 lg/mL crocin was able to restore the DWmup
to 99 % (Fig. 4).
As shown in Fig. 5, treatment of platelets with 2 mM
H
2
O
2
induced a 1.6- and 2.15-fold increase in PS exposure
in PRP and washed platelets, respectively, when compared
to control. Pre-treatment of platelets with crocin at con-
centrations between 0 and 200 lg/mL significantly reduced
H
2
O
2
-evoked PS externalization in a concentration-depen-
dent manner and at the concentration 100 and 200 lg/mL,
crocin completely abolished the PS externalization in PRP
and washed platelets, respectively.
Fig. 1 a Effect of crocin on endogenous generation of ROS and
H
2
O
2
induced by collagen (10 lg/mL) in PRP. bEffect of crocin on
endogenous generation of ROS and H
2
O
2
induced by calcium
ionophore-A23187 (10 lg/mL) in washed platelets. Values are
presented as mean ±SEM (n=5), expressed as fold increase in
DCF fluorescence (for ROS) and HVA fluorescence (for H
2
O
2
)
relative to control. a* Significant compared to control. b* Significant
compared to collagen/A23187-treated (p\0.05)
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Effect of crocin on H
2
O
2
-induced cytochrome c release
Treatment with 2 mM H
2
O
2
for 1 h at 37 °C induces cyt.c
release into the cytosol that was detected by western
blotting technique. However, pretreatment with crocin at
concentrations of 50 and 100 lg/mL inhibited the cyt.c
release by the platelets in a concentration-dependent
fashion. Crocin, at the dose of 100 lg/mL, showed com-
plete inhibition of cyt.c expression (Fig. 6).
Effect of crocin on collagen-induced platelet
aggregation and adhesion
Pre-treatment of PRP with crocin at different concentrations
prior to collagen treatment resulted in a dose-dependent
inhibition of platelet aggregation. Crocin at a concentration
25 lg/mL was able to inhibit aggregation by 97 %, and at
higher concentrations there was 100 % inhibition (Fig. 7).
However, it was not able to inhibit the aggregation induced
by ADP and epinephrine (data not shown). On the other
hand crocin was able to prevent the binding of platelets to
collagen in a dose-dependent manner. In the case of crocin
pre-treated collagen coated well, the platelet adhesion was
brought down to 45 ±5 % and in the case of crocin pre-
treated PRP, the adhesion was brought down to 65 % as
against 100 % adhesion with PBS-treated PRP.
Fig. 2 Dose-dependent inhibition of H
2
O
2
(2 mM)- and A23187
(10 lg/mL)-induced increase in intracellular Ca
2?
in PRP (a) and in
washed platelets (b). Values are presented as mean ±SEM (n=5),
expressed as percentage increase fura-2/AM fluorescence relative to
control. a* Significant compared to control. b* Significant compared
to H
2
O
2
/A23187-treated (p\0.05)
Fig. 3 Concentration-
dependent inhibition of H
2
O
2
(2 mM)-induced caspases-9 and
-3 activation by crocin in PRP
(a) and in washed platelets (b).
Values are presented as
mean ±SEM (n=5),
expressed as fold increase
caspase activity relative to
control. a* Significant
compared to control.
b* Significant compared to
H
2
O
2
-treated (p\0.05)
Fig. 4 Dose-dependent inhibition of H
2
O
2
(2 mM)-induced
DWmdepolarization by crocin in PRP (a) and in washed platelets (b).
Values are presented as mean ±SEM (n=5), expressed as percentage
increase (JC-1) fluorescence relative to DWmdepolarization induced by
10 lM rotenone (considered as 100 %). a* Significant compared to
control. b* Significant compared to H
2
O
2
-treated (p\0.05)
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Discussion
In view of the current trend of people choosing plant-based
therapeutics for treatment of various ailments including
CVDs, this study investigated the influence of crocin on
human platelets. Crocin, being a natural antioxidant, has
been claimed to have remarkable curative properties. It is the
pigment responsible for the coloration of saffron, which by
itself is used in many traditional systems of medicine
including Chinese and Iranian. In the traditional Ayurveda
system of medicine, it is used as a medication to improve
blood circulation and libido and also used as a supplement in
the diet of pregnant women. Therefore, an attempt has been
made to probe the effect of crocin on oxidative stress-
induced platelet apoptosis as well as platelet aggregation.
The study was based on the fact that one of the causes for
platelet apoptosis is oxidative stress induced by free radicals
and ROS such as H
2
O
2
as evidenced by recent studies and
that crocin being a potential antioxidant, a fact validated by
several independent studies, may possibly attenuate the free
radical-induced platelet apoptosis. Since platelets are anu-
clear, the effects of crocin on the cytoplasmic events of
programmed cell death, which are the parameters for platelet
apoptosis, were evaluated.
In the first set of experiments, collagen/A23187 was
used to induce the endogenous generation of ROS in
platelets. When the platelets were pretreated with crocin
prior to collagen/A23187 treatment; there was impairment
in the ROS generation in a dose-dependent manner.
According to the study by Lopez et al. [12], H
2
O
2
in par-
ticular triggers the events of apoptosis in platelets through
the intrinsic or mitochondrial pathway by altering
DWm. Therefore, the endogenous generation of H
2
O
2
was
also measured, where in crocin-pretreated platelets upon
collagen/A23187 treatment exhibited a dose-dependent
decrease in H
2
O
2
generation. Carotenoids including crocin
have long been investigated for their antioxidant functions,
which have been shown to involve radical scavenging,
quenching, and enzyme-inhibiting actions. Thereby the
results of this work are in tune with the fact that crocin is
an excellent antioxidant and further validate this fact. In
addition, several existing studies suggest that the endo-
thelial cell dysfunction is associated with the pathogenesis
of atherosclerosis. Vascular endothelial cells are highly
sensitive to ROS, because they are in close contact with the
constituents of blood (e.g., leucocytes) and activated
Fig. 6 The expression of cytosolic cyt.c was determined by immuno-
blot (I). Platelets were pretreated with different concentrations of
crocin (0–100 lg/mL) for 10 min and then stimulated with H
2
O
2
(2 mM) for 1 h at 37 °C. The cytosolic proteins were separated by
10 % SDS-PAGE and transferred on to a nitrocellulose membrane.
Membranes were incubated with anticytochrome cantibody (1:1,000)
in TBST for 2 h followed by HRP-conjugated anti IgG antibody
(1:10,000) in TBST and exposed to enhanced chemiluminescence.
Lane 1 represents control platelets (untreated). Lane 2 represents 2 mM
H
2
O
2
-treated platelets. Lanes 3, 4, and 5 represent platelets pretreated
with 50, 75, and 100 lg/mL of crocin, respectively, and then treated
with H
2
O
2
. Histograms represent the expression levels of cyt.c in
respective groups. a* Significant compared to control. b* Significant
compared to collagen-treated (p\0.05 (II) Expression of b-actin)
Fig. 5 Concentration-dependent inhibition of H
2
O
2
(2 mM)-induced
PS externalization in PRP (dashed line) and in washed platelets (solid
line). Values are presented as mean ±SEM (n=5), expressed as
percentage increase in annexin V-FITC fluorescence. a* Significant
compared to control. b* Significant compared to H
2
O
2
-treated
(p\0.05)
Mol Cell Biochem
123
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leucocytes release ROS including H
2
O
2
for biophylaxis,
which damage endothelial cells. This in turn facilitates
adhesion of platelets and other blood cells to the vascular
wall, which is considered as an initial cause of athero-
genesis. In a recent study, it was found that crocin could
prevent endothelial cells from apoptosis induced by H
2
O
2
,
which suggested that treatment of atherosclerosis by crocin
might correlate with its anti-apoptotic effect [21]. For
further experiments H
2
O
2
was used as the agonist. Rote-
none was used as standard agonist to induce changes in
DWm. Crocin could effectively prevent H
2
O
2
-induced
changes in DWmand restore the membrane potential up to
96 % of the original membrane potential in the case of
PRP. While in washed platelets it could almost completely
restore the DWm. Mitochondrial membrane depolarization
is the initial step of the intrinsic/mitochondrial pathway of
apoptosis and hence is the most crucial one [22]. From the
result, it can be stated that crocin has the capacity to inhibit
H
2
O
2
-induced intrinsic pathway of platelet apoptosis. To
further ascertain the efficacy of crocin in protecting DWm,
its effect on intracellular Ca
2?
was investigated. The
presence of high concentration of Ca
2?
in the cytosol is one
of the factors responsible for the changes in DWmand
formation of mitochondrial permeability transition pore
(MPTP). Yet again crocin could inhibit the increase in
intracellular calcium. In support, further experiments were
carried out, which included the protein expression levels of
cytosolic cyt.c. Leakage of mitochondrial membrane cyt.c
following the membrane depolarization is the next event of
intrinsic apoptotic pathway. Crocin was able to dose-
dependently hinder cyt.c expression. Release of apopto-
genic cyt.c from mitochondrial intermembrane space to the
cytosol due to formation of a channel MPTP, in the outer
mitochondrial membrane, serves a regulatory function as it
precedes morphological changes associated with apoptosis.
Once cyt.c is released it binds with apoptotic protease
activating factor-1 (Apaf-1) and ATP, which then bind to
pro-caspase-9 to create a protein complex known as
apoptosome. The apoptosome cleaves the pro-caspase to its
active form caspase-9, which in turn activates the effector
caspase-3. Hence, cyt.c release can be regarded as a pivotal
event in the intrinsic pathway of apoptosis.
Caspases are a group of cysteine-dependent aspartate-
directed proteases that cleave proteins at aspartic acid res-
idues and play a vital role in apoptosis, inflammation and
cell necrosis. Generally, caspases are expressed in an
Fig. 7 a Concentration-dependent inhibition of collagen (2 lg/mL)-
induced platelet aggregation by crocin: (i) control (collagen alone),
(ii) crocin 2 lg/mL, (iii)10lg/mL, (iv)25lg/mL. bGraphical
representation of the above data showing the percentage platelet
aggregation. cEffect of platelet adhesion on the immobilized collagen
type 1 with crocin pre-treated collagen and crocin pre-treated PRP.
Values are presented as mean ±SEM (n=5), expressed as
percentage increase in platelet adhesion. a* Significant compared to
control
Mol Cell Biochem
123
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inactive proenzyme form known as procaspsases and once
activated, can activate other procaspases, allowing initiation
of a protease cascade. The proteolytic cascade amplifies the
apoptotic signaling pathway and thus leads to rapid cell
death. There are ten major caspases, which are classified
into initiators (caspase-2, -8, -9, -10), effectors or execu-
tioners (caspase-3, -6, -7) and inflammatory caspases (cas-
pase-1, -4, -5). Crocin was able to impair the H
2
O
2
-induced
activation of both the caspases-9 and -3 in a concentration-
dependent manner. These findings further confirm the anti-
apoptotic effects of crocin in platelets. Consistent with this,
it was shown that H
2
O
2
was able to induce PS exposure,
another biochemical feature of apoptosis [23]. The
expression of cell surface markers results in the early
phagocytic recognition of apoptotic cells, permitting quick
phagocytosis. This event is achieved by the movement of
the normal inward-facing PS of the cell’s lipid bilayer to
expression on the outer layers of the plasma membrane.
H
2
O
2
-induced PS externalization was clearly abrogated by
crocin treatment. All these results firmly highlight the anti-
apoptotic effects of crocin on human platelets.
Platelets undergoing apoptosis expose negatively charged
PS on their surface and also release PS-positive membrane
fractions called MPs, both of which cause fibrin deposition
by providing competent surfaces for the assembly of coag-
ulation factors and thrombus formation. They have a potent
pro-inflammatory effect, promote coagulation and affect
vascular function. These processes are all involved in the
development of CVDs. Circulating MP numbers are also
altered in many CVDs. Hence, MPs play a major role in the
pathogenesis of CVDs including transient ischemia and
myocardial infarction, and post surgery complications in
cardiopulmonary bypass patients. There are numerous
studies, which signify the protective role of crocin against
CVDs such as the anti-atherosclerotic effects of crocin
through decreasing the oxidative low density lipoprotein
(Ox-LDL) levels, the cardioprotective role of crocin due to
its ability to maintain redox potential and thus prevent ath-
erosclerosis, inflammation, cardiotoxicity, and its hypoten-
sive, anti-diabetic effects [24–28]. Also, a recent study
claims it to be a potential therapeutic candidate in the treat-
ment of cerebral ischemia [29]. This study suggests another
possible mechanism through which this compound can be
considered as a therapeutic constituent in the treatment of
CVDs, i.e., crocin through its anti-apoptotic effect on
platelets can possibly reduce the extent of generation of MPs
and thus prevent CVDs. Moreover, recent study by Hem-
shekhar et al. [30] demonstrated that crocin by reducing
oxidative stress and inflammation in arthritic rats, acts as a
potential anti-arthritic molecule. Arthritis is accompanied
with reduction in platelet count and a concurrent increase in
the platelet-derived MPs in the synovial joints resulting in
the aggravation of inflammation. Therefore, it can be
articulated that crocin by blocking platelet apoptosis controls
the production of MPs and consequently reduces inflam-
mation at the joints.
If platelet apoptosis exceeds beyond the threshold level
the platelet count may drop rapidly leading to a condition
called thrombocytopenia. This condition is presumed to be
serious if the platelet count is 50,000/lL as against the
normal count of 150,000 to 450,000 platelets/lL of blood.
The adverse effects of thrombocytopenia include sponta-
neous bleeding from organs and delay in the normal pro-
cess of clotting. In this context, it is worth mentioning the
beneficial impact of crocin in reversing the apoptosis-
induced drop in platelet count.
Though there are several studies on platelet apoptosis
and its causes, there is only one report of phytochemical
exhibiting anti-apoptotic effects in human platelets, i.e.,
cinnamtannin B-1 from bay wood extract [31]. To further
highlight crocin’s role as a cardio protective compound, its
influence on platelet aggregation was scrutinized. Platelets
are deemed to play a critical role in the development of
atherothrombotic disease. During vascular injury, they ini-
tiate the formation of hemostasis plug and mediate patho-
physiological thrombosis, which in turn promulgates a
multiplicity of CVDs. Platelet adherence and aggregation
are involved in the instigation of intraluminal thrombosis
and thus hasten myocardial infarction, peripheral vascular
occlusions, and stroke. Therefore, platelet aggregation plays
a critical role in the propagation of CVDs [32]. The results
obtained firmly suggest that it has the ability to mitigate
collagen-induced platelet aggregation. The inhibitory effect
of crocin on aggregation was probably due to its interaction
with collagen, which was verified with the platelet adhesion
assay. In the case of crocin pre-treated collagen-coated
wells, the inhibition of platelet adhesion was better com-
pared to crocin pre-treated PRP directly added to collagen-
coated wells. This was probably due to the existence of an
interaction between crocin and collagen. According to a
study by Hadley et al. [33], glycation of collagen alters the
charge distribution and influences the quaternary structure
as well as the interaction of the glycated collagen with other
proteins. Thus, it has been hypothesized that the disaccha-
ride gentiobiose present in crocin may cause glycation of
collagen and thereby prevents its interaction with collagen
receptors present on the platelets.
Conclusion
In conclusion it can be articulated that crocin is a promising
molecule, which can be implemented in the treatment strategy
for CVDs as it has been clearly demonstrated in this study. It
exerts excellent anti-apoptotic effects on oxidative stress-
induced platelets, besides inhibiting platelet aggregation. It
Mol Cell Biochem
123
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has the ability to retard the various events of the intrinsic
pathway probably mediated through its antioxidant activity.
Although crocin has been shown to have proapoptotic effects
on tumor cells and manyreports claim crocin to be a promising
cancer therapeutic agent, this study signifies its anti-apoptotic
effects in normal cells [34]. This study has further scope
because crocin’s effects on the extrinsic pathway of apoptosis
in platelets. Thus crocin, a component of saffron used in
various traditional system of medicine, appears to be a
potential molecule and nature’s answer to combat a host of
modern life style- and stress-associated ailments such as
CVDs.
Acknowledgments Dr. K. S. Girish thanks the University Grant
Commission, New Delhi, India, for financial assistance (F-36/276/
2008 (SR) dated 26/3/2009).
References
1. Ferroni P, Vazzana N, Riondino S, Cuccurullo C, Guadagni F,
Davı‘G (2012) Platelet function in health and disease: from
molecular mechanisms, redox considerations to novel therapeutic
opportunities. Antioxid Redox Signal 17(10):1447–1485
2. Leytin V (2011) Apoptosis in the anucleate platelet. Blood Rev
26(2):51–63
3. Gyulkhandanyan AV, Mutlu A, Freedman J, Leytin V (2012)
Markers of platelet apoptosis: methodology and applications.
J Thromb Thrombolysis 33(4):397–411
4. Morel O, Jesel L, Freyssinet JM, Toti F (2011) Cellular mecha-
nisms underlying the formation of circulating microparticles.
Arterioscler Thromb Vasc Biol 31:15–26
5. Mallat Z, Benamer H, Hugel B, Benessiano J, Steg PG, Freys-
sinet JM, Tedgui A (2000) Elevated levels of shed membrane
microparticles with procoagulant potential in the peripheral cir-
culating blood of patients with acute coronary syndromes. Cir-
culation 101(8):841–843
6. VanWijk MJ, VanBavel E, Sturk A, Nieuwland R (2003) Micropar-
ticles in cardiovascular diseases. Cardiovasc Res 59(2):277–287
7. Chen Y, Zhang H, Tian X, Zhao C, Cai L, Liu Y, Jia L, Yin H-X,
Chen C (2008) Antioxidant potential of crocins and ethanol
extracts of Gardenia jasminoides ELLIS and Crocus sativus L: a
relationship investigation between antioxidant activity and crocin
contents. Food Chem 109:484–492
8. Abdullaev FI (2002) Chemopreventive and tumoricidal properties
of saffron (Crocus sativus L.). Exp Biol Med 227:20–25
9. Lee IA, Lee JH, Baek NI, Kim DH (2005) Antihyperlipidemic
effect of crocin isolated from the fructus of Gardenia jasminoides
and its metabolite crocetin. Biol Pharm Bull 28(11):2106–2110
10. Wang Y, Han T, Zhu Y, Zheng CJ, Ming QL, Rahman K, Qin LP
(2010) Antidepressant properties of bioactive fractions from the
extract of Crocus sativus L. J Nat Med 64(1):24–30
11. Kumar MS, Girish KS, Vishwanath BS, Kemparaju K (2011) The
metalloprotease, NN-PF3 from Naja naja venom inhibits platelet
aggregation primarily by affecting a2b1 integrin. Ann Hematol
90(5):569–577
12. Lopez JJ, Salido GM, Go
´mez-Arteta E, Rosado JA, Pariente JA
(2007) Thrombin induces apoptotic events through the generation
of reactive oxygen species in human platelets. J Thromb Haemost
5(6):1283–1291
13. Barja G (2002) The quantitative measurement of H
2
O
2
generation
in isolated mitochondria. J Bioenerg Biomembr 34(3):227–233
14. Asai M, Takeuchi K, Uchida S, Urushida T, Katoh H, Satoh H,
Yamada S, Hayashi H, Watanabe H (2008) Misinterpretation of
the effect of amlodipine on cytosolic calcium concentration with
fura-2 fluorospectrometry. Naunyn Schmiedebergs Arch Phar-
macol 377(4–6):423–427
15. Salvioli S, Ardizzoni A, Franceschi C, Cossarizza A (1997) JC-1,
but not DiOC6(3) or rhodamine 123, is a reliable fluorescent
probe to assess delta psi changes in intact cells: implications for
studies on mitochondrial functionality during apoptosis. FEBS
Lett 411:77–82
16. Rosado JA, Graves D, Sage SO (2000) Tyrosine kinases activate
store-mediated Ca
2?
entry in human platelets through the reorga-
nization of the actin cytoskeleton. Biochem J 351(Pt 2):429–437
17. Lo
´pez JJ, Salido GM, Pariente JA, Rosado JA (2006) Interaction
of STIM1 with endogenously expressed human canonical TRP1
upon depletion of intracellular Ca
2?
stores. J Biol Chem 281(38):
28254–28264
18. Amor NB, Pariente JA, Salido GM, Rosado JA, Bartegi A (2006)
Thrombin-induced caspases 3 and 9 translocation to the cyto-
skeleton is independent of changes in cytosolic calcium in human
platelets. Blood Cells Mol Dis 36(3):392–401
19. Rosado JA, Lopez JJ, Gomez-Arteta E, Redondo PC, Salido GM,
Pariente JA (2006) Early caspase-3 activation independent of
apoptosis is required for cellular function. J Cell Physiol 209(1):
142–152
20. Bellavite P, Andrioli G, Guzzo P, Arigliano P, Chirumbolo S,
Manzato F, Santonastaso C (1994) A colorimetric method for the
measurement of platelet adhesion in microtiter plates. Anal Bio-
chem 216:444–450
21. Xu GL, Qian ZY, Yu SQ, Gong ZN, Shen XC (2006) Evidence of
crocin against endothelial injury induced by hydrogen peroxide
in vitro. J Asian Nat Prod Res 8(1–2):79–85
22. Leytin V, Allen DJ, Mutlu A, Gyulkhandanyan AV, Mykhaylov
S, Freedman J (2009) Mitochondrial control of platelet apoptosis:
effect of cyclosporin A, an inhibitor of the mitochondrial per-
meability transition pore. J Lab Investig 89(4):374–384
23. Schoenwaelder SM, Yuan Y, Josefsson EC, White MJ, Yao Y,
Mason KD, O’Reilly LA, Henley KJ, Ono A, Hsiao S, Willcox A,
Roberts AW, Huang DC, Salem HH, Kile BT, Jackson SP (2009)
Two distinct pathways regulate platelet phosphatidylserine
exposure and procoagulant function. Blood 114(3):663–666
24. He SY, Qian ZY, Tang FT, Wen N, Xu GL, Sheng L (2005)
Effect of crocin on experimental atherosclerosis in quails and its
mechanisms. Life Sci 77(8):907–921
25. Nam KN, Park YM, Jung HJ, Lee JY, Min BD, Park SU,
Jung WS, Cho KH, Park JH, Kang I, Hong JW, Lee EH (2010)
Anti-inflammatory effects of crocin and crocetin in rat brain
microglial cells. Eur J Pharmacol 648(1–3):110–116
26. Goyal SN, Arora S, Sharma AK, Joshi S, Ray R, Bhatia J, Kumari S,
Arya DS (2010) Preventive effect of crocin of Crocus sativus on
hemodynamic, biochemical, histopathological and ultrastuctural
alterations in isoproterenol-induced cardiotoxicity in rats. Phy-
tomedicine 17(3–4):227–232
27. Imenshahidi M, Hosseinzadeh H, Javadpour Y (2010) Hypoten-
sive effect of aqueous saffron extract (Crocus sativus L.) and its
constituents, safranal and crocin, in normotensive and hyperten-
sive rats. Phytother Res 24(7):990–994
28. Mousavi SH, Tayarani NZ, Parsaee H (2010) Protective effect of
saffron extract and crocin on reactive oxygen species-mediated
high glucose-induced toxicity in PC12 cells. Cell Mol Neurobiol
30(2):185–191
29. Zheng YQ, Liu JX, Wang JN, Xu L (2007) Effects of crocin on
reperfusion-induced oxidative/nitrative injury to cerebral micro-
vessels after global cerebral ischemia. Brain Res 1138:86–94
30. Hemshekhar M, Sebastin santhosh, Sunitha K, Thushara RM,
Kemparaju K, Rangappa KS, Girish KS (2012) A dietary colorant
Mol Cell Biochem
123
Author's personal copy
crocin mitigates arthritis and associated secondary complications
by modulating cartilage deteriorating enzymes, inflammatory
mediators and antioxidant status. Biochimie. doi:10.1016/j.biochi.
2012.08.013)
31. Bouaziz A, Romera-Castillo C, Salido S, Linares-Palomino PJ,
Altarejos J, Bartegi A, Rosado JA, Salido GM (2007) Cinnam-
tannin B-1 from bay wood exhibits antiapoptotic effects in human
platelets. Apoptosis 12(3):489–498
32. Ueno M, Kodali M, Tello-Montoliu A, Angiolillo DJ (2011) Role
of platelets and antiplatelet therapy in cardiovascular disease.
J Atheroscler Thromb 18(6):431–442
33. Hadley JC, Meek KM, Malik NS (1998) Glycation changes the charge
distribution of type I collagen fibrils. Glycoconjug J 15:835–840
34. Mousavi SH, Moallem SA, Mehri S, Shahsavand S, Nassirli H,
Malaekeh-Nikouei B (2011) Improvement of cytotoxic and
apoptogenic properties of crocin in cancer cell lines by its
nanoliposomal form. Pharm Biol 49(10):1039–1045
Mol Cell Biochem
123
Author's personal copy
