J Thromb Thrombolysis DOI 10.1007/s11239-014-1056-7

Crocin prevents sesamol-induced oxidative stress and apoptosis in human platelets Ram M. Thushara • Mahadevappa Hemshekhar • Manoj Paul • Mahalingam Shanmuga Sundaram Rohith L. Shankar • Kempaiah Kemparaju • Kesturu S. Girish

•

Ó Springer Science+Business Media New York 2014

Abstract Recent studies have reported the platelet proapoptotic propensity of plant-derived molecules such as, resveratrol, thymoquinone, andrographolide and gossypol. Meanwhile, there were also reports of phytochemicals such as cinnamtannin B1, which shows antiapoptotic effect towards platelets. Platelets are mainly involved in hemostasis, thrombosis and wound healing. However, altered platelet functions can have serious pathological outcomes that include cardiovascular diseases. Platelets are sensitive to external and internal stimuli including therapeutic and dietary components. The anuclear platelets do undergo apoptosis via mitochondrial pathway. However, exaggerated rate of platelet apoptosis could lead to thrombocytopenia and other bleeding disorders. The present study deals with ameliorative efficacy of crocin on sesamol-induced platelet apoptosis. The antiapoptotic property of crocin and the proapoptotic tendency of sesamol in platelets were previously demonstrated. Therefore, it was interesting to see how these two compounds would interact and wield their effects on human platelets. Crocin effectively inhibited sesamol-induced oxidative stress on platelets, which was evidenced by the measurement of endogenously generated reactive oxygen species, particularly hydrogen R. M. Thushara  M. Hemshekhar  M. Paul  M. Shanmuga Sundaram  K. Kemparaju  K. S. Girish (&) Department of Studies in Biochemistry, University of Mysore, Manasagangothri, Mysore 570006, Karnataka, India e-mail: [email protected]; [email protected] R. L. Shankar Department of Sericulture, Yuvaraja’s College, University of Mysore, Mysore 570006, Karnataka, India K. S. Girish Department of Studies and Research in Biochemistry, Tumkur University, Tumkur 572103, Karnataka, India

peroxide, and changes in thiol levels. Further, crocin abrogated sesamol-induced biochemical events of apoptosis in platelets, which include intracellular calcium mobilization, changes in mitochondrial membrane integrity, cytochrome c release, caspase activity and phosphatidylserine externalization. Even though sesamol has proapoptotic effects on platelets, its anti-platelet activity cannot be neglected. Thus, the study proposes that sesamol could be supplemented with crocin, an approach that could not only abolish the toxic effects of sesamol on platelets, but also enhance the quality of treatment due to their synergistic action. Keywords Oxidative stress  Platelet apoptosis  Sesamol  Crocin  Mitochondrial membrane potential  Caspases

Introduction The surfacing of the applied fields of pharmacology and drug discovery, and the large scale production of drugs was a boon to the modern man. However, the indiscriminate use of drugs resulted in frequent adverse reactions including thrombocytopenia. Thrombocytopenia is a condition wherein the platelet count drops to 100,000 platelets/lL of blood as against the normal range of 150,000–450,000 lL of blood [1, 2]. One of the reasons for drug-induced thrombocytopenia could be attributed to an enhanced rate of platelet apoptosis, which is a less investigated aspect. Recent studies have revealed the platelet proapoptotic effects of antibiotics such as, vancomycin and balhimycin, antiplatelet drug aspirin, and anticancer drug cisplatin [3–6]. Even phytochemicals that were deemed to be much safer options

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in terms of their negligible side effects and potent therapeutic value were also shown to induce platelet apoptosis. Of late, there were reports of plant-derived molecules such as, resveratrol, thymoquinone, andrographolide and gossypol causing platelet apoptosis [7–10]. Meanwhile, there were also reports of phytochemicals such as cinnamtannin B1, which protects platelets from undergoing apoptosis [11]. Platelets, the megakaryocyte-derived anuclear blood cells are basically involved in hemostasis, thrombosis and wound healing. However, altered platelet functions can have a multitude of pathological consequences including ischemia/ reperfusion, atherothrombosis, MI, diabetes and arthritis among others [12]. With a lifespan of 7–10 days, they are susceptible to various external and internal stimuli including dietary components and drugs. In spite of lacking nucleus, platelets do undergo apoptosis. The intrinsic or mitochondrial pathway of apoptosis is well-documented in platelets [13]. Exaggerated rate of platelet apoptosis could result in a drastic drop in platelet numbers (thrombocytopenia), leading to bleeding disorders. With this background, the current study sought to demonstrate the proapoptotic effect of sesamol and its amelioration by crocin. Crocin (a diester of disaccharide gentiobiose and the dicarboxylic acid crocetin) is a natural dietary carotenoid found in the stigma of flowers of Crocus sativus L. (saffron) and fruits of Gardenia jasminoides (Fig. 1). It is the major active principle and coloring pigment of the widely used spice saffron. Studies emphasize that crocin is an excellent antioxidant with immense therapeutic potentials including tumoricidal, anticarcinogenic, antihyperlipidemic, and antidepressant properties [14–17]. Previously we had demonstrated the antiapoptotic property of crocin in platelets. It effectively impaired oxidative stress in platelets and inhibited H2O2-induced mitochondrial pathway of apoptosis [18]. Sesamol (3,4-methylenedioxyphenol or 1,3-benzodioxol-5-ol) is a phenolic antioxidant derived from sesame seed (Sesamum indicum L.) lignans and is a major constituent of sesame oil. Sesamol renders sesame oil less vulnerable to oxidative deterioration when compared to other vegetable oils. Sesamol has numerous therapeutic potentialities including antiaging, hepatoprotective, neuroprotective, anti-inflammatory, chemoprotective, chondroprotective, antiarthritic and antiplatelet properties [19–21]. We have recently reported the tendency of sesamol to induce platelet apoptosis via ROS-mediated mitochondrial pathway [22]. It would be interesting to see how these two compounds would interact and wield their effects on platelets. Even though sesamol is pro-apoptotic towards platelets, its numerous therapeutic properties cannot be ignored. Hence, it would be very advantageous if crocin could prevent the pro-apoptotic tendency of sesamol. This would pave way for a combinatorial approach wherein crocin can be supplemented with sesamol when used as a dietary supplement or therapeutic

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drug. This would not only mask the platelet toxicity of sesamol, but also further enhance its healing property.

Materials and methods Chemicals Crocin, rotenone, calcium ionophore A23187, benzamidine hydrochloride, glutaraldehyde, N-acetyl-Leu-Glu-His-Asp trifluoromethylcoumarin (AC-LEHD-FMC), acetyl-AspGlu-Val-Asp-7-amido-4-methylcoumarin (AC-DEVDAMC), sodium orthovanadate (Na3VO4), fluorescein isothiocyanate-labeled annexin V, 5-(and-6)-chloromethyl20 ,70 -dichlorodihydrofluorescein diacetate acetylester (CMH2DCFDA), dithiothreitol (DTT), 5,50 -6,60 -tetrachloro1,10 ,3,30 -tetraethyl benzimidazolylcarbocyanine iodide (JC1), leupeptin hydrochoride, N-(2-Hydroxyethyl) piperazine-N0 -ethanesulfonic acid (HEPES), enhanced chemiluminiscence detection reagents, and hyperfilm ECL were purchased from Sigma Chemicals, St. Luois, USA. Homovanillicacid (HVA) was purchased from Sisco Research laboratories Pvt Ltd., Mumbai, India. Sesamol (purity98 %) was purchased from Acros Organics, New Jersey, USA. Horseradish peroxidase-conjugated rabbit antisheep IgG antibody and anti-cytochrome c antibody were purchased from Epitomics, Inc., Burlingame, USA. Preparation of sesamol solution Sesamol was dissolved in 0.5 % dimethyl sulfoxide (DMSO) to obtain the solution of required concentration. DMSO (0.5 %) was chosen as the solvent based on literature survey, and also preliminary studies with the solvent control neither showed any adverse effects on platelets nor did it influence the results. Preparation of platelet rich plasma (PRP) and washed platelets Washed platelets were prepared according to the method of Kumar et al. [23]. Briefly, venous blood was drawn from healthy, non-smoking, drug-free human volunteers with informed consent as per the guidelines of Institutional Human Ethical Committee (IHEC–UOM No. 72/Ph.D/ 2012-13), University of Mysore, Mysore. The blood 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). It was then centrifuged at 909g for 15 min and the supernatant thus obtained was the PRP, which was centrifuged at 1,7009g for 15 min at 37 °C. The platelet pellet thus obtained was washed after suspension and incubation in Tyrode’s albumin

Oxidative stress and apoptosis in human platelets

Fig. 1 Effect of crocin on sesamol-induced ROS generation: dosedependent inhibition of sesamol (1 mM)-induced ROS generation by crocin (25–100 lM) of in PRP (A) as well as washed platelets (B) as compared to positive control calcium ionophore A23187 (10 lg/mL).

Values are presented as mean ± SEM (n = 5), expressed as fold increase in DCF fluorescence relative to control (resting platelets). a* significant compared to control; b* significant compared to A23187 and sesamol (p [ 0.05)

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) pH 6.5 for 10 min at 37 °C. The platelets were again washed with Tyrode’s albumin buffer by repeating the previous step. Finally, the platelets were suspended in the Tyrode’s albumin buffer, pH 7.4. The platelet count in the washed platelet suspension was determined using Neubauer chamber. For all the following assays, the platelet count was adjusted to 5 9 107 cells/mL in the final suspension using Tyrode’s albumin buffer (pH 7.4).

platelet suspensions (as described in the previous section) were incubated with HVA (100 lM) for 30 min at 37 °C, centrifuged and the pellets were suspended in HBS. Fluorescence was recorded using the multimode plate reader by exciting the samples at 312 nm and the resulting fluorescence was measured at 420 nm.

Determination of endogenously generated ROS ROS-sensitive fluorescent probe CMH2DCFDA was used to detect endogenous ROS production as described by Lopez et al. [24]. Pretreated (with 10 lg/mL A23187 as standard agonist or with 1 mM sesamol?varying concentrations of crocin as test group for 1 h at 37 °C) and control (untreated) platelet suspensions were made up to a final volume of 200 lL with HBS, pH 7.45, containing 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), incubated with 10 lM CMH2DCFDA in a polystyrene 96-well microtiter plate for 30 min at 37 °C. Fluorescence was recorded with a Varioskan multimode plate reader (Thermo Scientifics, USA) by exciting the samples at 488 nm and the resulting fluorescence was measured at 530 nm.

Estimation of total thiol, GSH and GSSG levels The total thiol level in platelets was measured according to the method of Mokrasch and Teschke [26], with minor modifications. The assay mixture consisted of 20 lL treated (with 2 mM H2O2 as standard control/with 1 mM sesamol?varying concentrations of crocin as test group), and control PRP and washed platelet suspension were made up to 100 lL with buffered formaldehyde (1:4 V/V, 1 mL formaldehyde: 0.1 M Na2HPO4) kept at 37 °C for 5 min then 150 lL of 0.1 M phosphate buffer (pH 8.0 containing 5 mM EDTA) and 25 lL of O-phthalaldehyde (1 mg/mL) were added, incubated at 37 °C for 45 min and the fluorescence was recorded using fluorescence plate reader by exciting the sample at 340 nm and the resulting fluorescence was measured at 425 nm. For the measurement of oxidized glutathione (GSSG), equal volume of 0.04 M N-ethylmaleimide (NEM) was added to the treated platelet suspensions and incubated for 30 min (to prevent GSH from undergoing further oxidation) prior to the above procedure. The difference between total thiol and GSSG levels gives the GSH levels.

Determination of endogenously generated H2O2 Estimation of intracellular calcium According to the method of Barja [25], endogenously generated H2O2 in platelets was evaluated using HVA a specific H2O2-sensitive fluorescent probe. Pretreated and control

Intracellular Ca2? concentration was measured in platelets as described previously [27]. Treated and control platelet

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suspensions (as described in previous section) were taken in 96-well polystyrene microtiter plates and the final volume made up to 200 lL with modified Tyrode’s solution (pH 7.4) containing NaCl (150 mM), KCl (2.7 mM), KH2PO4 (1.2 mM), MgSO4 (1.2 mM), CaCl2 (1.0 mM), and HEPES (10 mM) with 1 % BSA and incubated for 1 h at 37 °C to induce the release of Ca2? from the intracellular Ca2? stores. The platelets were then incubated for 45 min at room temperature with 2 lM fura-2/AM, a fluorescence Ca2? 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 suspended in modified Tyrode’s solution. The fura-2/AM absorption was determined 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 Changes in the DWm were detected using JC-1, a cationic dye. JC-1 aggregates in mitochondria at high membrane potentials and emits red fluorescence, whereas, at low membrane potential it is converted to a green fluorescent monomeric form. Treated and control platelet suspensions were loaded with JC-1 (10 lg/mL) and incubated 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 and the emission ratios (585/516) were calculated. Changes in DWm following the treatment with the agonist were computed as the integral of the decrease in JC-1 fluorescence ratio [28].

then incubated overnight with 10 % BSA in tris-buffered saline with 0.1 % tween 20 (TBST) to block residual protein binding sites followed by incubation with anticytochrome c antibody (1:1,000) in TBST for 2 h. Finally, they were incubated with HRP-conjugated anti IgG antibody (1:10,000) in TBST, exposed to enhanced chemiluminescence for 3 min and then exposed to photographic films. b-actin was used as loading control. Assay of caspase activity Caspase activity was determined by the method of Amor et al. [30]. The cell lysate (from treated and control platelets) is incubated in a 96-well polystyrene micro-titer 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 or ACLEHD-AFC for caspase 9) for 2 h at 37 °C. Substrate cleavage was measured with a multimode plate reader (excitation wavelength 360 nm and emission at 460 nm). Determination of phosphatidylserine externalization PS scrambling was determined by the method of Rosado et al. [31]. Treated and control platelet suspension samples were transferred to equal volumes of ice-cold 1 % (w/v) glutaraldehyde in HBS for 10 min, and then incubated in 0.6 lg/mL buffered aqueous solution of annexin V fluorescein isothiocyanate for 10 min. This was followed by centrifugation for 60 s at 3,0009g and the cells were collected by suspending the pellet in HBS. Cell staining was analyzed in a multimode plate reader by exciting the samples at 496 nm and emission was recorded at 560 nm.

Preparation of platelet lysate Statistical analysis Platelet lysate was prepared by immediately lysing the treated and control platelets from PRP and washed platelet suspension with an equal volume of 29 Triton buffer (2 % TritonX-100, 2 mM EGTA, 100 mM Tris/HCl—pH 7.2, 100 lg/mL leupeptin, 2 mM PMSF, 10 mM benzamidine, 2 mM Na3VO4) at 4 °C for 30 min. The lysate was centrifuged at 16,0009g for 5 min. The pellet thus obtained is the cytoskeleton-rich (Triton-insoluble) fraction, which was subjected to caspase activity and western blotting [29].

Results are expressed as mean ± 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 means. Here, data were shown as mean ± SEM (n = 5), p \ 0.05 (a*) was considered to be significant compared to control and (b*) was considered to be significant compared to A23187 and sesamol.

Detection of cytochrome c release Results Cytochrome c release was measured by Western blot technique. The cytosolic fractions from the treated and control platelets were subjected to immunoblotting. 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

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Crocin scavenges sesamol-induced endogenous generation of ROS and H2O2 Treatment of PRP and washed platelets with positive control A23187 (10 lg/mL) and sesamol (1 mM) induced

Oxidative stress and apoptosis in human platelets Table 1 Crocin inhibits sesamol-induced ROS and H2O2 generation, PS externalization, and caspase activity—the results for sesamol (1 mM)-induced ROS and H2O2 generation, caspase activity, and PS externalization (expressed as increase in folds) and their inhibition by 100 lM crocin (expressed as % inhibition), in both platelet-rich plasma (PRP) and washed platelets (WP) Platelet apoptosis parameter

Positive control (fold increase)

Sesamol (1 mM) treatment (fold increase)

% Inhibition by crocin (100 lg/mL)

ROS in PRP

2.84

4.33

100.0

ROS in WP

3.04

6.50

99.0

H2O2 in PRP

1.9

2.33

97.5

H2O2 in WP

2.07

2.56

99.0

Caspase 9 in PRP

1.82

3.00

92.5

Caspase 9 in WP

2.7

4.4

89.0

Caspase 3 in PRP

1.45

2.3

95.5

Caspase 3 in WP

1.58

3.8

90.5

PS externalization in PRP

1.55

1.98

88.5

PS externalization in WP

1.84

2.16

93.5

a remarkable increase in the generation of ROS, as well as H2O2. Crocin (25–100 lM) dose-dependently scavenged sesamol-induced ROS and H2O2 generation. Crocin at concentration 100 lM was able to inhibit ROS and H2O2 generation significantly (Table 1; Figs. 1, 2).

Table 2 Crocin restores sesamol-induced depletion of GSH:GSSG ratio—the results for sesamol (1 mM)-induced depletion of GSH:GSSG ratio and its restoration by 100 lM crocin (expressed as % ratio of GSH:GSSG) in both platelet-rich plasma (PRP) and washed platelets (WP) Platelet treatment

Control (resting/ untreated platelets %)

Positive control (2 lM A23187)

Sesamol (1 mM)

Sesamol (1 mM) ? crocin (100 lM)

PRP

97.25

70.40

67.00

96.90

WP

94.40

86.20

81.90

95.88

platelets and A23187-treated platelets (positive control). Crocin (25–100 lM) was able to restore GSH:GSSG ratio to normal (Table 2; Fig. 3A). Sesamol (1 mM) was also found to deplete in the total thiol levels in both PRP and washed platelets as compared to control platelets and A23187-treated platelets (positive control). Crocin (25–100 lM) was found to refurbish the thiol levels in a dose-dependent manner (Fig. 3B, C). Crocin impairs sesamol-induced increase in intracellular calcium

Sesamol (1 mM) brought down the GSH:GSSG ratio in PRP as well as washed platelets as compared to control

Considering the increase in intracellular Ca2? induced by A23187 (positive control) to be 100 %, sesamol at the concentration of 1 mM induced the mobilization of intracellular Ca2? by 77 and 93 % respectively in PRP and washed platelets. Crocin (25–100 lM) was able to inhibit sesamol-induced Ca2? release in a dose-dependent manner. Crocin at the concentration 100 lM was found to impair Ca2? release to a maximum extent (Table 3; Fig. 4A, B).

Fig. 2 Effect of crocin on sesamol-induced H2O2 generation: dosedependent amelioration of sesamol-induced generation of H2O2 by crocin (25–100 lM) in PRP (A) as well as washed platelets (B) as compared to positive control calcium ionophore A23187 (10 lg/mL).

Values are presented as mean ± SEM (n = 5), expressed as fold increase in HVA fluorescence relative to control (resting platelets). a* significant compared to control; b* significant compared to A23187 and sesamol (p [ 0.05)

Crocin restores sesamol-induced depletion of total thiols and GSH:GSSG ratio

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Fig. 3 Effect of crocin on sesamol-induced depletion of GSH:GSSG ratio and total thiol: crocin (25–100 lM) restores sesamol (1 mM)induced depletion of GSH:GSSG ratio in PRP as well as washed platelets (A) in a dose-dependent manner as compared to positive control A23187 (2 lM). Values are presented as mean ± SEM (n = 5), expressed as % ratio of GSH:GSSG concentration. Crocin

(25–100 lM) restores sesamol (1 mM)-induced decrease in total thiol level in PRP (B) as well as washed platelets (C) as compared to positive control A23187 (2 lM). Values are presented as mean ± SEM (n = 5), expressed as concentration of thiols in lg/mL. a* significant compared to control; b* significant compared to A23187 and sesamol (p [ 0.05)

Table 3 Crocin ameliorates sesamol-induced intracellular Ca2? release and changes in mitochondrial membrane potential—the results for inhibition of sesamol (1 mM)-induced intracellular Ca2? release, and changes in DWm by crocin

with 1 mM sesamol induced a significant elevation in caspase activities, which was effectively impaired in a dose-dependent manner by crocin (25–100 lM) (Table 1; Fig. 5A, B).

Platelet apoptosis parameter

Positive control (% increase)

Sesamol triggers mitochondrial membrane depolarization and PS scrambling

Ca2? in PRP

100

Ca2? in WP

100

93

97

DWm Depolarization in PRP

100

105

98

DWm Depolarization in WP

100

126

91.5

Sesamol (1 mM) treatment (% increase) 77

% Inhibition by crocin (100 lM) 97.5

Effect of sesamol on caspase activity Treatment of PRP and washed platelets with 2 mM H2O2 (positive control), at 37 °C for 2 h, induced the activation of caspases 9 and 3. Stimulation of PRP and washed platelets

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Rotenone (10 lM) treatment (positive control) for 1 h at 37 °C evoked maximal decrease in JC-1 fluorescence and was considered as 100 % DWm dissipation. Treatment of PRP and washed platelets with 1 mM sesamol evoked DWm dissipation and was found to be more potent than rotenone. Crocin (25–100 lM) treatment was found to significantly restore the normal membrane potential (Table 3; Fig. 6A, B). Treatment of PRP and washed platelets with 2 mM H2O2 (positive control) for 1 h at 37 °C induced an increase in the rate of externalization of PS. Treatment of platelets with 1 mM sesamol evoked a significant increase in PS exposure and was found to be more effective than the

Oxidative stress and apoptosis in human platelets

Fig. 4 Effect of crocin on sesamol-induced release of intracellular calcium: inhibition of Sesamol (1 mM)-induced mobilization of calcium ions by crocin (25–100 lM) in a dose-dependent manner in PRP (A) as well as washed platelets (B) as compared to positive

control A23187 (10 lg/mL). Values are presented as mean ± SEM (n = 5), expressed as percentage increase in Fura-2AM fluorescence. a* significant compared to control; b* significant compared to A23187 and sesamol (p [ 0.05)

Fig. 5 Effect of crocin on sesamol-induced caspase activation: crocin (25–100 lM) dose-dependently impairs sesamol (1 mM)-induced increase in caspase activity in PRP (A) as well as washed platelets (B) as compared to positive control H2O2 (2 mM). Values are

presented as mean ± SEM (n = 5), expressed as fold increase in caspase activity. a* significant compared to control; b* significant compared to A23187 and sesamol (p [ 0.05)

positive control. Sesamol-induced PS externalization was hindered by crocin (25–100 lM) in a concentrationdependent manner (Table 1; Fig. 7).

Discussion

Effect of sesamol on cytochrome c release Treatment with 2 mM H2O2 (positive control) for 1 h at 37 °C induced cytochrome c release into the cytosol and was detected by Western blotting technique. Treatment with 1 mM sesamol triggered the cytochrome c release more effectively than positive control, and it was inhibited by crocin (25–100 lM) in a dose-dependent manner (Fig. 8A). Figure 8B represents the immunoblot of b-actin, which was used as the loading control.

Platelets play an essential role in human health and disease. Aberrations in platelets are involved in the pathophysiology of many diseases including CVDs, diabetes mellitus and thrombocytopenia [12]. They are particularly susceptible to apoptotic stimuli such as, therapeutic drugs as well as, nutritional components [8]. Since they lack nucleus, mitochondria play a major role in platelet apoptosis. Elevated rate of platelet apoptosis can have two key consequences: generation of a huge number of microparticles (MPs) into the circulation, and thrombocytopenia. Plateletderived MPs participate in thrombus formation as well as, atherosclerotic plaque formation [32] Moreover, MPs

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Fig. 6 Effect of crocin on sesamol-induced changes in mitochondrial membrane potential: crocin (25–100 lM) dose-dependently restores sesamol (1 mM)-induced alteration in mitochondrial membrane potential in PRP (A) as well as washed platelets (B) and as compared

Fig. 7 Effect of crocin on sesamol-induced phosphatidylserine externalization: crocin (25–100 lM) dose-dependently inhibits sesamol (1 mM)-induced increase in PS externalization in PRP as well as washed platelets (WP) and as compared to positive control H2O2 (2 mM). Values are presented as mean ± SEM (n = 5), expressed as fold increase in Annexin-V FITC fluorescence ratio. a* significant compared to control; b* significant compared to A23187 and sesamol (p [ 0.05)

further worsen certain disease conditions including, CVD—cancer and diabetes [33]. Alternatively, thrombocytopenia leads to bleeding disorders and failure of blood clot. Of late, purified phytochemicals are being exploited as therapeutic drugs to treat a number of diseases including CVDs, mainly due to their exceptional antioxidant properties [34]. Though they are believed to be quite safe with negligible side effects, there were few reports on their

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to positive control rotenone (10 lM). Values are presented as mean ± SEM (n = 5), expressed as percentage increase in JC-1 fluorescence ratio. a* significant compared to control; b* significant compared to A23187 and sesamol (p [ 0.05)

toxicity to platelets. Despite being good antioxidants with various beneficial properties, resveratrol, thymoquinone, andrographolide and gossypol were shown to exert proapoptotic effects on platelets [7–10]. Based on these facts, the effect of sesamol on platelets was investigated and it was shown to induce platelet apoptosis at concentrations above 0.25 mM, which was previously reported from this lab [22]. Meanwhile, the antiapoptotic effect of crocin on oxidative stress-induced platelets was also demonstrated [18]. It became clear that sesamol and crocin have contrasting effects on platelets. Thus, the present study investigated whether crocin could prevent apoptosis in sesamol-treated platelets. In the first set of experiments, the effect of crocin on sesamol-induced oxidative stress was assessed. Crocin effectively abrogated the sesamol-induced endogenously generated ROS and H2O2 in the platelet suspension. Crocin being a carotenoid, it is obvious that it has free radicalquenching properties, a fact that has been validated by several studies [14]. Sesamol, by virtue of its wide range of therapeutic properties, is being used as an ingredient in dietary supplements. The current results suggest that the chronic use and high dosage of sesamol might induce oxidative stress to vascular cells, especially platelets, leading to functional alterations. Dysfunctional platelets contribute to the development of CVDs, and generation of MPs which could further aggravate the disease condition. Mitochondria play an indispensible role in the intrinsic pathway of platelets. Various stimuli, including oxidative stress could affect the mitochondrial redox balance, resulting in the stimulation of apoptosis [35]. Therefore,

Oxidative stress and apoptosis in human platelets

Fig. 8 Immunoblot of the effect of crocin on sesamol-induced expression of cytosolic cytochrome c: (A) dose-dependent amelioration of sesamol (1 mM)-induced expression of cytosolic cytochrome c by crocin (25–100 lM) (B) loading control b-actin. Lane I control platelets (untreated); lane II H2O2 (2 mM)-treated platelets; lane III sesamol (1 mM)-treated platelets; lanes IV and V sesamol (1 mM)-

pre-treated platelets treated crocin at concentrations 25, 50 lM respectively. Histograms represent the expression levels of cytochrome c in respective groups measured in terms of integrated density. a* significant compared to control; b* significant compared to A23187 and sesamol (p [ 0.05)

measurement of DWm is an essential step in assessing the apoptotic state of a cell. In the present study, the effect of crocin on sesamol-induced DWm depolarization was investigated. Crocin was able to significantly restore the DWm to normal, suggesting its protective effect on the mitochondria. To further establish the protective efficiency of crocin, its effect on sesamol-induced release of intracellular Ca2? was probed. High level of Ca2? in the cytosol also contributes to changes in and formation of mitochondrial permeability transition pore (MPTP) [36]. Crocin very effectively mitigated the release of intracellular Ca2?. Additional experiments were conducted, which included the protein expression levels of cytosolic cytochrome c. Release of cytochrome c from the mitochondrial membrane subsequent to DWm depolarization is a crucial event of intrinsic apoptotic pathway. Crocin was able to dosedependently hinder sesamol-induced cytochrome c expression in the cytosol. Cytochrome c release due to formation of a channel MPTP in the outer mitochondrial membrane, acts as regulatory step as it paves the way for further morphological alterations associated with apoptosis. The cytosolic cytochrome c aids in the activation of caspase 9, which in turn activates the effector caspase 3. Therefore, the next step was to investigate the effect of crocin on sesamol-induced caspase activation. Caspases are a group of cysteine-dependent aspartate-directed proteases that cleave proteins at aspartic acid residues and play a vital role in apoptosis, inflammation and cell necrosis [37]. Crocin was able to impair the activation of both the caspases 9 and 3 in a concentration-dependent manner. These findings further confirm the antiapoptotic effects of crocin in platelets. Consequently, it was also shown that crocin was able to mitigate sesamol-induced PS exposure, another

biochemical feature of apoptosis [38]. The expression of cell surface markers including PS aids the phagocytic cells to recognize apoptotic cells, paving way to rapid phagocytosis. Thus, the findings of the current study firmly highlight the protective efficacy of crocin against sesamolinduced platelet apoptosis. Thus, the study proposes that sesamol could be supplemented with crocin, an approach that could not only abolish the toxic effects of sesamol on platelets, but also enhance the quality of treatment due to their synergistic action, on the lines of combinatorial drug therapy. The present study also puts forward the necessity for a thorough screening of phytochemicals for their adverse effects on platelets. There is also a threat from the accelerated production of procoagulant MPs from apoptotic platelets, which can reverse the positive effects of therapeutic drugs and thus worsen the disease condition. Therefore, we need to look for such bioactive compounds from plants, which can be exploited in the treatment regimen without the fear of toxicity to platelets. They could also be combined with other therapeutic drugs to further boost the effectiveness of the treatment. Acknowledgments Authors thank Institute of Excellence (IOE) facilities, University of Mysore. Conflict of interest of interest.

The authors declare that there are no conflicts

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