alleviation of viper venom induced platelet apoptosis by...

CHAPTER-V

Alleviation of Viper Venom Induced

Platelet Apoptosis by Crocin

(Crocus sativus): Implications for

Thrombocytopenia in Viper Bites

Chapter-V Oxidative stress and platelet apoptosis

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Introduction

Snakebite is regarded as a major health hazard in the tropics especially in the

South Asian countries. Despite the advancement in antivenin therapies and survival

rate of the victims, the associated secondary complications persisting for several years

leading to debilitating conditions, have puzzled the health authorities posing a huge

challenge to them. A major part in the snakebite mortality and morbidity is

contributed by Russell’s viper, particularly in India (Girish and Kemparaju, 2011).

Viper envenomations are typified by prominent local tissue damage involving

an intricate sequence of pathological modifications at the bitten site including edema,

ecchymoses, myonecrosis, blisters and extensive hemorrhage. Moreover, the victims

often suffer systemic disorders such as hemostasis, instantaneous bleeding and failure

of blood coagulation (Ushanandini et al., 2009; Sunitha et al., 2011; Sajevic et al.,

2011). These effects are induced by a variety of venom components, among which

metalloproteinases and phospholipase A2 (PLA2) play an imperative role in the

propagation of hemorrhage. The mechanism of snake venom metalloproteinase

(SVMP)-induced hemorrhage involves the degradation of vascular basement

membrane components (type-IV collagen, laminin, nidogen and perlecan). This leads

to a decline in the adhesion of the endothelial cell to the basement membrane, paving

way for the disruption of capillaries resulting in internal bleeding (Howes et al., 2007;

Fox and Serrano, 2008; Cortelazzo et al., 2010; Marinov et al., 2010). Thus, the

pathophysiology of hemorrhage can be regarded as a consequence of the altered

functions of blood coagulation factors, blood vessel endothelium and platelets; among

which platelets seem to be the major target of SVMPs in the hemostatic modifications

via their interference with platelet-collagen and/or platelet-vWF. Hemorrhage may

also result from venom-induced thrombocytopenia as reported in the case of Bothrops

asper envenomation (Kamiguti, 2005; Wang et al., 2005; Cheng et al., 2012).

Thrombocytopenia occurs due to a combination of factors such as (i) venom-induced

cytotoxicity to megakaryocytes, (ii) platelet activation and clearance due to vascular

coagulation and aggregation at the bitten site iii) reduced platelet count due to the

increased rate of platelet apoptosis, a neglected and less investigated aspect observed

during viper envenomation. Taken together all these coditions results in anticoagulant

effects observed in viper envenomations (Kamiguti, 2005; Rucavado et al., 2005).

Apoptosis being a major mode of cell death is inevitable for embryonic

development, morphogenesis and maintenance of tissue homeostasis. Despite being


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anuclear, platelets present all the classical events of the mitochondria-mediated

pathway which are triggered by elevated levels of endogenously generated reactive

oxygen species (ROS) and cytoskeletal Ca2+

mobilization. Circulating platelets have

an average life span of 7-10 days; however their life can be cut short by various

factors including toxic compounds in the circulation and they seem to be a vulnerable

target for venom components owing to their extremely sensitive nature. Therefore it is

of utmost importance to investigate the influence of venom on platelets, considering

the latter’s vital role in hemostasis and thrombosis (Leytin, 2012; Jackson and

Schoenwaelder, 2010; Kaplan and Jackson, 2011; Yamashita et al., 2011).

The anti-venom therapy is the only medically approved remedy for the

treatment of snakebites. At present about 20 different nations are involved in the

production of equine/bovine/ovine derived monovalent as well as polyvalent anti-

venoms against 55 clinically important snake venoms (http://www.toxinfo.org/anti-

venoms/Index_Product.html). The processed antibodies and their Fab and F(ab)2

fragments neutralize the venom fractions based on their specificity. Although the

available anti-venom therapy is effective in neutralizing the systemic toxicity, the

therapy has intrinsic drawbacks including unavailability; lack of information on the

bitten snake species, anaphylactic reactions and more importantly no protection

against local tissue damage (Girish and Kemparaju, 2011).

Further, the available antivenins are anti-toxins and can neutralize the venom

components in circulation, but are unable to overcome the venom-induced oxidative

stress and associated complications which arise from the accelerated rate of platelet

apoptosis. Therefore, in order to overcome the limitations of anti-venom therapy, the

venom researchers and medical practitioners are focused on the need for a

supplementary therapy involving antioxidants which can effectively scavenge the

endogenously generated ROS in response to the venom components. Phytochemicals

are a population of privileged structures selected by evolutionary pressures to interact

with wide variety of biological targets for specific purposes. These phytochemicals

could be the safe bet for the damage induced by venom components as they are not

only potential antioxidants, but also support the fact that phytochemicals have become

effective drugs in a wide variety of therapeutic indications without/less secondary

complications (Koehn and carter, 2005; Perumal samy et al., 2008). Moreover,

traditional healers world over have been exploiting medicinal plants from centuries

for snakebite treatment, some of them being effective against local manifestations


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(Santhosh et al., 2013). Recently, rapid decline in the platelet number after VR venom

injection was shown in mice models, while no such effects were observed in the

group of mice injected with VR venom pretreated with crocin (Sebastin et al., 2013).

Consequently the present study sought to investigate the effects of viper

venom on platelet apoptosis and its amelioration by crocin, a water soluble carotenoid

from the dried stigma of the flowers of Crocus sativus. Crocin is a promising

phytochemical in the therapeutic arena due to its innate antioxidant potential and

shown to be anti-carcinogenic, anti-depressant, anti-atherosclerotic, anti-

inflammatory, anti-hyperlipidemic, anti-arthritic, anti-aging and neuroprotective as

well as hepatoprotective molecule (Bakshi et al., 2009; Hemshekhar et al., 2012;

Sebastin et al., 2013).

Materials and methods

Chemicals

Vipera russelli (VR) venom was procured from Hindustan Park, Kolkata,

India. Crocin, Dichlorodihydrofluorescein diacetate (DCFDA), 5,5’-6,6’-tetrachloro-

1,1’,3,3’-tetraethyl benzimidazolyl carbocyanine iodide (JC-1), Fluorescein

isothiocyanate-labeled annexin V, N-acetyl-Asp-Glu-Val-Asp-7-amido-4-

methylcoumarin (AC-DEVD-AMC), N-(2-Hydroxyethyl) piperazine-N΄-

ethanesulfonic acid (HEPES), N-acetyl-Leu-Glu-His-Asp-7-amido-4-

methylcoumarin (AC-LEHD-AMC), CHAPS, leupeptin hydrochoride, Fura 2-AM,

Sodium orthovanadate (Na3VO4) and enhanced chemiluminiscence detection reagents

and hyperfilm ECL were from Sigma Chemical Co., St Louis, USA,

Homovanillicacid (HVA) was from Sisco Research laboratories Pvt Ltd., Horseradish

peroxidase-conjugated rabbit anti-sheep IgG antibody and anti-cytochrome c antibody

were purchased from Epitomics, Inc., Burlingame (USA). All other reagents were of

analytical grade.

Preparation of Platelets

Venous blood was drawn from healthy, non-smoking human volunteers with

informed consent as per the guidelines of Institutional Human Ethical Committee

(IHEC-UOM No. 59/Ph.D/2011-12), University of Mysore, Mysore. Platelet

suspension was prepared according to the method of Rosado et al., (2000). Briefly,

freshly drawn blood was citrated and platelet-rich plasma (PRP) was prepared by


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centrifugation at 600 rpm for 15 min. Washed platelets were prepared by centrifuging

PRP for 15 min at 8000 rpm. The pellet thus obtained was incubated for 10 min in

Tyrode’s albumin buffer (TAB) (145 mM NaCl, 5 mM KCl, 10 mM HEPES, 0.5 mM

Na2HPO4, 1 mM MgCl2, 6 mM glucose, and 0.3 % BSA pH 6.5) and washed twice by

centrifuging at 8000 rpm for 15 min at 37°C. Finally, the washed platelets were

suspended in the TAB, pH 7.4. The cell count was determined using neubauer

chamber. Final platelet concentration was adjusted to required number (4 X 107 to 5 X

107 cells/mL) using HBS buffer. All the protocols were approved and followed

according to the institutional Human Ethical Committee guidelines.

Experimental design

The PRP was categorized in to seven different groups. Group I- control, Group

II- H2O2 alone, Group III- VR venom alone, Group IV & V - VR venom pre-

incubated with crocin for 10 min at 37°C (1:0.25 and 1:0.5 ratio respectively (venom:

crocin; w/w), Group VI - H2O2 pre-incubated with crocin for 10 min at 37°C (1:0.25;

venom: crocin; w/w) ratio and Group VII- crocin alone (100 µg/ml). The experiments

were done according to the following methods.

Determination of endogenously generated reactive oxygen species (ROS):

Endogenous ROS production in platelets was determined according to the

method of Lopez et al., (2007) with slight modifications. PRP (20 µl) taken in

polystyrene 96-well microtiter plate was treated with VR venom (50 µg), and the final

volume was made up to 200 µL with HEPES-buffered saline (HBS), pH 7.4,

containing 145 mM NaCl, 10 mM HEPES, 10 mM D-glucose, 5 mM KCl, 1 mM

MgSO4 supplemented with 0.1% BSA and incubated for 1h at 37°C followed by the

addition of 10 μM of DCF-DA. 100mM H2O2 (20 μl) was used as a positive control.

The reaction mixture was incubated at 37°C for 30 min and the resulting fluorescence

was measured using Varioskan multimode plate reader with excitation and emission

wavelengths at 480 and 530 nm expressed as ρmol DCF formed/min/mg protein. For

inhibition studies, the venom samples were pre-incubated with different

concentrations of crocin for 10 min at 37˚C.


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Measurement of intracellular Ca2+

concentration

Intracellular Ca2+

concentration in PRP was measured according to the method

of Asai et al., (2008) with slight modifications. Briefly, the treated cells were

incubated at 37˚C for 1h with modified Tyrode’s solution (pH 7.4) containing 150mM

Nacl, 2.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.0 mM CaCl2, and 10 mM

HEPES with 1% BSA and 2 μM fura-2-AM as a fluorescence Ca2+

indicator. The

cells were washed twice with the HBSS to remove the dye and the absorption shift

that occurs upon calcium binding was measured spectrofluorimetrically with

excitation at 340/380 nm and emission at 510 nm and data are presented as (340/380)

ratio of fluorescence intensity. 100 mM H2O2 (20 μl) was used as a positive control.

For inhibition studies, the venom samples were pre-incubated with different

concentrations of crocin for 10 min at 37˚C.

Determination of changes in mitochondrial membrane potential (ΔΨm)

Changes in the mitochondrial membrane potential were registered using the

cationic dye JC-1. In the presence of physiological ΔΨm, JC-1 forms aggregates that

fluoresce with an emission peak at 588 nm, loss of membrane potential favors the

monomeric form of JC-1, which has an emission peak at 530 nm. In brief, cells were

incubated with venom for 1h at 37˚C followed by the addition of freshly diluted JC-1

(5 µg/mL in DMSO). After incubation at 37˚C for 15 min, the cells were washed with

HBS. JC-1-loaded cells were excited at 488 nm, and emission was detected at 585 nm

(JC-1 aggregates) and 516 nm (JC-1 monomers) using spectrofluorimeter. Data are

presented as emission ratios (585/516 nm) (Salvioli et al., 1997). 100 mM H2O2 (20

μl) was used as a positive control. For inhibition studies, the venom samples were pre-

incubated with different concentrations of crocin at 37˚C for 10 min.

Preparation of platelet lysate

Platelet lysate was prepared by freeze-thaw method for 4-5 cycles. The

resulting lysates were centrifuged at 14000 rpm for 15 min at 4°C. The pellet thus

obtained is the cytoskeleton-rich (Triton-insoluble) fraction, which was subjected to

caspase activity and western blotting as described below.


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Detection of Cytochrome c release

Release of cytochrome c (Cyt-c) from mitochondria was measured by

immunoblotting in samples from cytosolic fractions (Lopez et al., 2007). Cytosolic

proteins were separated by 10% SDS-PAGE and electrophoretically transferred on to

a nitrocellulose membrane for 1h at 50V 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 anti-

cyt-c antibody (1:1000) in TBST for 2h. Blots were incubated with horseradish-

peroxidase (HRP)-conjugated anti IgG antibody (1:10,000) in TBST and exposed to

enhanced chemiluminescence for 3min. Finally the blots were exposed to

photographic films.

Caspase activity

The caspase activity was determined according to the method described by

Amor et al., (2006) with slight modification. In brief, platelet lysates were incubated

with the substrate solution (20 mM HEPES, pH 7.4, 2 mM EDTA, 0.1% CHAPS, 5

mM DTT and 8.25 µM of caspase substrate) for 2h at 37˚C. Substrate cleavage was

measured fluorimetrically with an excitation and emission wavelength of 360 nm and

460 nm for caspase 3 and 400 nm and 505 nm for caspase 9. The activities of

caspases-3 and -9 were calculated from the cleavage of the respective specific

fluorogenic substrate (AC-DEVD-AMC for caspase-3 and AC-LEHD-AFC for

caspase-9). 100 mM H2O2 (20 μl) was used as a positive control. For inhibition

studies, the venom samples were pre-incubated with different concentrations of crocin

for 10 min at 37˚C.

Determination of Phosphatidylserine (PS) externalization

The PS externalization was determined according to the method described by

Rosado et al., (2006) with minor modifications. Briefly, the final concentration of

PRP (5x104) in 500 µl were incubated with venom for 1h at 37˚C followed by the

addition of 500 µl ice-cold 1% (w/v) glutaraldehyde in phosphate-buffered saline

(PBS). The cells were then incubated for 10 min with annexin V-fluorescein

isothiocyanate (FITC) (0.6 µg/ml) in PBS supplemented with 0.5% (w/v) BSA and 2

mM CaCl2 protected from light. Cells were collected by centrifugation for 60s at 8000

rpm and resuspended in HBS. 100 mM H2O2 (20 μl) was used as a positive control.


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Cell staining was measured spectrofluorimetrically with excitation at 496 nm and

emission at 516 nm.

Statistical analysis

Results are expressed as mean ± SEM of three independent experiments.

Statistical significance among groups was determined by one way analysis of variance

(ANOVA) followed by Tukey’s test for comparison of means among the groups.

Here, data were shown as mean ± SEM. p < 0.05 (*) and p < 0.01 (**) were

considered to be significant.

Results

Effect of crocin on VR venom-induced ROS generation

The efficacy of crocin was evaluated for its antioxidant effect on VR venom

induced endogenous generation of ROS levels in platelets. A marked enhancement in

the ROS was apparent in platelets when treated with VR venom (50 μg). The VR

venom induced elevation in ROS levels by 81±5% whereas H2O2 by 128.8± 7%

compared to control cells. Pre-treatment with crocin offered 52±4% inhibition at

1:0.25 ratio (venom: crocin; w/w) while complete inhibition was observed at both

venom and H2O2 induced elevation at 1:0.5 ratio (venom: crocin; w/w), whereas no

such alterations were observed in crocin treated cells (Fig. 5.1).

Effect of crocin on VR venom induced increased intracellular Ca2+

levels

The VR venom induced release of intracellular Ca2+

levels were measured

using fluorescent Ca2+

indicator, Fura 2-AM. Treatment of Platelets with VR venom

(100 µg) resulted in the elevated Ca2+

levels by 80±6% and 105±8% with H2O2 while

crocin alone did not cause any significant changes in the Ca2+

levels. Inhibition

studies with crocin resulted in the dose-dependent amelioration in Ca2+

generation.

Pre-treatment with crocin resulted in reduction in the intracellular levels of Ca2+

by

55±3% and 88±5.3% respectively at 1:0.25 and 1:0.5 ratio (venom: crocin; w/w)

compared to venom treated platelets while complete inhibition with H2O2 induced

elevated Ca2+

levels (Fig 5.2).


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Effect of crocin on VR venom-induced activation of caspases-3 and -9

Caspase activation was determined using specific fluorescent substrates AC-

DEVD-AMC and AC-LEHD-AFC for caspase-3 and -9 respectively. Treatment of

platelets with VR venom resulted in the significant increase in the activities of

caspase 3 and 9 by 95±4.5% and 77±3.4% respectively with respect to an increase of

112± 8% and 90±6% in H2O2 alone group. Pretreatment of venom with crocin

significantly reduced venom induced activation of caspases. Crocin dose dependently

neutralized the caspase-3 activating property of the venom by 52±1.8 % and 90

±3.4% while for caspase -9 the inhibition range was 65±6% and 86±5.4% at 1:0.25

and 1:0.5 ratio (venom: crocin; w/w) respectively while complete neutralization in

H2O2 induced group (Fig. 5.3).

Effect of crocin on VR venom induced Cyt-c levels

VR venom induced activation of caspase pathway was further confirmed by

measuring the released Cyt-c levels in platelets. VR venom resulted in the rapid

release of Cyt-c from the mitochondria when incubated for 1 h at 37˚C, while the

levels remained unaltered in the control cells as well as the crocin treated cells. In

contrast, a considerable inhibition of Cyt-c release was evident in the pre-incubation

group suggesting the role of VR venom on caspase activation via direct or indirect

effects on mitochondria (Fig. 5.4).

Efficacy of Crocin in diminishing the VR venom induced mitochondrial

membrane depolarization and PS scrambling

The altered mitochondrial membrane depolarization in platelets was

determined by monitoring the decrease in JC-1 fluorescence ratio (585/516 nm). H2O2

resulted in the drastic decline in the membrane potential (108.8±8%) compared VR

Venom (78±4%) alone, while crocin alone did not altered the membrane potential.

Pretreatment with crocin resulted in restoring the changes by 62±4% and 87± 5.5% at

1:0.25 and 1:0.5 ratio (venom: crocin; w/w) respectively compared to venom treated

cells while it offered 90±5.5% protection against H2O2 induced decline in membrane

potential (Fig. 5.5).

The PS externalization was quantified by surface annexin V staining. From the

fig. 5.6, it is clear that a significant increase in the surface PS exposure was observed

in the H2O2 (120.6 ±8.5%) and VR venom (78 ±4.5%) treated group compared to


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control. On the other hand crocin alone did not alter the PS levels. In the pre-

treatment group crocin was able to prevent dose dependently the externalization of PS

by 58.3±4.5 % and 90±6.5 % at 1:0.25 and 1:0.5 ratios (venom: crocin;w/w)

respectively compared to venom treated cells while crocin completely inhibited the

H2O2 induced PS externalization at 1:0.5 ratio (Fig 5.6).

Discussion

Envenomation due to viper bites are often linked to altered hemostasis and

associated disorders that might be due to altered functions of clotting factors, platelets

or damage to blood vessels. Venom (SVMPs and PLA2)-induced alterations in the

endothelial environment, which drastically affect platelets, lead to serious

consequences resulting due to the drop in platelet count. From our preliminary studies

VR venom was found to be pro-coagulant in vitro; in contrast to its anti-coagulant

activity in vivo. Similar effects were also observed in many studies which reports the

observed anti-coagulant property may be due to the depletion of fibrinogen

concentration below the threshold level for clotting activity (Cheng et al., 2012). In

addition, some of the studies have demonstrated that the systemic bleeding during

viper envenomation is due to the action of SVMPs on coagulation and platelets

resulting in hypovolemia which leads to cardiovascular shock, the leading cause of

mortality. Further, C-type lectin-like protein aspercetin can induce thrombocytopenia

by interacting with platelet receptor GPIb (Rucavado et al., 2005; Gutierrez et al.,

2009; Luna et al., 2011). Many of the case studies have reported the incidence of

moderate to severe thrombocytopenia in individuals following viper envenomation

which led to death in many cases (Hung et al., 2002; Marinov et al., 2010; Herath et

al., 2012; Monteiro et al., 2012). Recently, rapid decline in the platelet number after

VR venom injection was shown in mice models, which was normalized when

pretreated with crocin (Sebastin et al., 2013). In view of this, the current study

hypothesizes that venom-induced thrombocytopenia might be due to an exaggerated

rate of platelet apoptosis.

Venom components have been reported to induce apoptosis in various cell

lines via either death receptor activation or through the intrinsic mitochondria-

dependent pathway (Park et al., 2009; Samel et al., 2012). Platelets being anuclear

have been found to undergo apoptosis via intrinsic pathway in response to oxidative

stress in the endothelium. As such, it seems plausible to inter-relate venom-induced


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oxidative stress and exacerbated platelet apoptosis. Moreover, the available antivenins

do not seem to offer protection against venom-induced secondary complications

including the endogenous/endothelial oxidative stress. As a result there is a necessity

for secondary treatment regime which could protect the victim from oxidative stress-

induced disorders including CVDs, diabetes, hypertension and platelet loss. Off late,

phytochemicals have acquired the stake in the treatment of oxidative stress-induced

ailment because of their inherent antioxidant potentiality. Therefore, the present study

explored the effects of viper venom on platelet apoptosis and its mitigation by crocin,

a potent antioxidant carotenoid derived from the dietary colorant saffron.

The study evaluated the various parameters of the mitochondria-mediated

apoptotic events in platelets induced by venom and their concurrent inhibition by

crocin. The exposure of human platelets to VR venom induced a dramatic elevation in

the levels of endogenous ROS as well as Ca2+

, which were effectively impaired when

the venom was pre-incubated with crocin. Studies have shown that the platelet

apoptosis could be triggered by ROS and elevated Ca2+

either through intrinsic or

mitochondrial pathway by altering ΔΨm (Rosado et al., 2000; Lopez et al., 2007).

Recent studies have reported the apoptotic effect of various venom toxins on different

cancer cell lines and are shown to undergo apoptosis via up-regulation of ROS- and

JNK-mediated death receptor expression on treatment with venom components (Liu

and Chang, 2009; Park et al., 2012). Mitochondrial membrane depolarization is the

initial and crucial step of the intrinsic/ mitochondrial pathway of apoptosis (Leytin et

al., 2009). The current results clearly affirm the drastic reduction in the membrane

depolarization after treating of platelets with VR venom. Increased ROS along with

the Ca2+

overload facilitate the formation of the mitochondrial membrane transition

pore (MPTP) responsible for depolarization of mitochondrial inner transmembrane

potential ΔΨm, permeabilization of inner and outer mitochondrial membrane, and

subsequent release of proapoptotic proteins (Cyt-c and apoptosis inducing factor)

from mitochondria to cytosol (Danial and Korsmeyer, 2004). To further ascertain the

pro-apoptotic effect of VR venom, the other parameters such as Cyt-c translocation to

cytosol, caspases 9 and 3 activation and PS externalization were evaluated. The

current results firmly indicate the activation of these events in venom-treated platelets

and their abrogation by crocin. The release of Cyt-c from the inner membrane of

mitochondria into the cytosol seems to be pivotal event in the intrinsic pathway of

apoptosis. The cytosolic Cyt-c along with apoptotis activating factor Apaf1, activates


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the elicitor caspase-9. Caspase-9 which is the key initiator caspase in the intrinsic

pathway activates the downstream effecter caspase-3, resulting in the proteolytic

degradation of the cytoskeleton, culminating in the PS externalization.

The study firmly highlights one of the poorly investigated aspects of venom-

induced secondary complications i.e., endogenous ROS generation and subsequent

platelet apoptosis. Thereby, it brings to light the underlying reason for venom-induced

thrombocytopenia, systemic hemorrhage and in vivo anticoagulant effect. In addition,

the present study proposes the use of phytochemicals as a supplementary therapy in

addition to available antivenins during treatment regime (Santhosh et al., 2013). The

results validate the claims that plant-based therapeutics is more efficient in tackling

the secondary complications of snakebites compared to the antivenin therapies.

Further, antivenins merely neutralize the toxic components of venom and thereby

increase the chances of victim survival, but are incapable of curing the oxidative

stress and associated complications including platelet apoptosis. Phytochemicals by

virtue of their radical-scavenging properties might be able to effectively overcome the

venom-induced oxidative stress. Crocin, a well known antioxidant was used in the

study to demonstrate its potent inhibitory effects on venom-induced oxidative stress

and platelet apoptosis. Additional clinical studies are necessary to evaluate the

involvement of VR venom induced oxidative stress in vivo as a determinant of

platelet apoptosis and the administration of phytochemicals as a therapeutic strategy

for platelet-derived diseases involving oxidative stress and apoptosis. The study has

further scope as the influence of venom on the death receptor-mediated pathway of

apoptosis in platelets is not known and moreover the underlying molecular

mechanisms of venom-induced platelet apoptosis and its precise role in the

promulgation of hemorrhage is yet to be elucidated.


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Figures and legends

Fig 5.1

Fig 5.1 Effect of crocin on the VR venom induced generation of ROS in platelets.

Dots assigned with letter a and b are statistically significant compared to control and

VR venom alone respectively. ** p<0.001 and * p<0.05, each value represents the

mean ± SEM of three independent experiments. Group I- Control; Group II- Platelets

treated with H2O2 alone; Group III- Platelets treated with venom; Group IV & V -

Platelets treated with venom pre-incubated with crocin at 1:0.25 and 1:0.5 ratio

(venom: crocin; w/w) respectively; Group VI - H2O2 pre-incubated with crocin for 10

min at 37°C (1:0.25; venom: crocin; w/w) ratio and Group VII- crocin alone (100

µg/ml).


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Fig 5.2

Fig 5.2: Inhibition of VR venom induced increase in intracellular Ca2+

in

platelets by crocin. Dots assigned with letter a and b are statistically significant

compared to control and VR venom alone respectively. ** p<0.001 and * p<0.05,

each value represents the mean ± SEM of three independent experiments. Group I-

Control; Group II- Platelets treated with H2O2 alone; Group III- Platelets treated with

venom; Group IV & V -Platelets treated with venom pre-incubated with crocin at

1:0.25 and 1:0.5 ratio (venom: crocin; w/w) respectively; Group VI - H2O2 pre-

incubated with crocin for 10 min at 37°C (1:0.25; venom: crocin; w/w) ratio and

Group VII- crocin alone (100 µg/ml).


133

Fig 5.3

Fig 5.3 Concentration-dependent inhibition of VR venom induced caspase -9 and

caspase -3 activation by crocin in platelets. Bars assigned with letter a and b are

statistically significant compared to control and VR venom alone respectively. **

p<0.001 and * p<0.05, each value represents the mean ± SEM of three independent

experiments. Group I- Control; Group II- Platelets treated with H2O2 alone; Group III-

Platelets treated with venom; Group IV & V -Platelets treated with venom pre-

incubated with crocin at 1:0.25 and 1:0.5 ratio (venom: crocin; w/w) respectively;

Group VI - H2O2 pre-incubated with crocin for 10 min at 37°C (1:0.25; venom:

crocin; w/w) ratio and Group VII- crocin alone (100 µg/ml).


134

Fig 5.4

Fig 5.4 Effect of crocin on the VR venom induced translocation of Cyt-c in

platelets. Platelets were treated with venom pre-treated with/without different

concentrations of crocin and then stimulated 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 primary and secondary antibodies in TBST and

exposed to enhanced chemiluminescence. (A) Cyt-c levels (B) β-actin. Lane I-

Control; Lane II- Platelets treated with H2O2 alone; Lane III- Platelets treated with

venom; Lane IV & V - Platelets treated with venom pre-incubated with crocin at

1:0.25 and 1:0.5 ratio (venom: crocin; w/w) respectively; Lane VI - H2O2 pre-

incubated with crocin for 10 min at 37°C (1:0.25; venom: crocin; w/w) ratio and Lane

VII- crocin alone (100 µg/ml). Histograms represent the expression levels of Cyt-c in

respective groups. a*: significant compared to control; b*: significant compared to

VR venom treated (P < 0.05).


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Fig 5.5

Fig 5.5 Dose-dependent inhibition of VR venom induced ΔΨm depolarization by

crocin in platelets. Dots assigned with letter a and b are statistically significant

compared to control and VR venom alone respectively. ** p<0.001 and * p<0.05,

each value represents the mean ± SEM of three independent experiments. Group I-

Control; Group II- Platelets treated with H2O2 alone; Group III- Platelets treated with

venom; Group IV & V -Platelets treated with venom pre-incubated with crocin at

1:0.25 and 1:0.5 ratio (venom: crocin; w/w) respectively; Group VI - H2O2 pre-

incubated with crocin for 10 min at 37°C (1:0.25; venom: crocin; w/w) ratio and



136

Fig 5.6

Fig 5.6 Concentration-dependent inhibition of VR venom induced PS

externalization in platelets. Dots assigned with letter a and b are statistically

significant compared to control and VR venom alone respectively. ** p<0.001 and

* p<0.05, each value represents the mean ± SEM of three independent experiments.

Group I- Control; Group II- Platelets treated with H2O2 alone; Group III- Platelets

treated with venom; Group IV & V -Platelets treated with venom pre-incubated with

crocin at 1:0.25 and 1:0.5 ratio (venom: crocin; w/w) respectively; Group VI - H2O2

pre-incubated with crocin for 10 min at 37°C (1:0.25; venom: crocin; w/w) ratio and


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