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Inhibitory Effect of Allyl Isothiocyanate on Platelet Aggregation Do-Seop Lee, Tae-Ho Kim, and Yi-Sook Jung J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf4041518 • Publication Date (Web): 01 Jul 2014 Downloaded from http://pubs.acs.org on July 1, 2014

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Journal of Agricultural and Food Chemistry

Inhibitory Effect of Allyl Isothiocyanate on Platelet Aggregation

Do-Seop Lee†, Tae-Ho Kim†, Yi-Sook Jung*, †, ‡ †

College of Pharmacy, Ajou University, Suwon 443-749, Republic of Korea

‡

College of Pharmacy, Research Institute of Pharmaceutical Sciences and Technology, Ajou

University, Suwon 443-749, Republic of Korea.

AUTHOR INFORMATION * Coressponding author Yi-Sook Jung, Ph.D. College of Pharmacy, Ajou University San 5, Woncheon-dong, Youngtong-gu, Suwon 443-749, Republic of Korea Telephone: +82-31-219-3444 Fax: +82-31-219-3435 E-mail: [email protected]

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ABSTRACT

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Allyl isothiocyanate (AITC) is one of the major components of mustard. The present study

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for the first time attempted to evaluate the effect of AITC on platelet aggregation. In the in vitro

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study using platelet rich plasm (PRP) from rat and human, AITC at the concentrations of 100 and

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300 µM significantly inhibited platelet aggregation induced by collagen, thrombin, ADP, and

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arachidonic acid. AITC also attenuated thromboxane A2 production and ATP release in rat and

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human PRP. AITC elicited inhibitory effects on cellular Ca2+ increase and platelet shape change

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in rat PRP. AITC further showed inhibitory effects on the phosphorylation of PKCδ, p38, ERK,

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and Akt in rat PRP. In the rat ex vivo study, 1 and 3 mg/kg (p.o.) of AITC showed significantly

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inhibitory effect on platelet aggregation. Furthermore, AITC showed protective effect in

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thromboembolism attack model in mouse. These results suggest that AITC has remarkable anti-

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platelet effects and maybe a therapeutic potential for the prevention of aberrant platelet

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activation-related disorders.

14 15 16

Keywords

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Allyl isothiocyanate, Platelet, Aggregation, Thromboxane A2, ATP

18 19 20 21 22


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INTRODUCTION

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Platelets play an essential physiological role in hemostasis by permitting the formation of

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blood clots. However, the aberrant activation of platelets by pathological factors and subsequent

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thrombogenesis are commonly associated with atherosclerotic lesions. Indeed, thrombosis and

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embolization contribute to cardiovascular diseases such as ischemic heart disease and stroke.1

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Therefore, a suitable anti-platelet therapy is critical for preventing the progression of

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cardiovascular diseases.2 A number of anti-platelet drugs, including clopidogrel, have been

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developed and are used clinically to prevent thrombosis. However, some of the chemically

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synthesized anti-platelet agents may be related to adverse effects, such as gastrointestinal

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bleeding, and palpitation. Considering that plant constituents have a nature-friendly image and

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various safety merits, much effort recently has been devoted toward developing herbal medicines

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for the prevention of thrombosis and atherosclerotic diseases. 3, 4

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Mustard (Brassica spp.), one of the first domesticated crops, is a widely consumed vegetable

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East Asia use mustard oil as the cooking oil. Mustard and mustard oil have been reported to have

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physiological effects such as anti-inflammation6 and anti-tumor7. Recently, it has been reported

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that polyunsaturated fatty acid-rich mustard oil has anti-platelet effect in ex vivo study.8 However,

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there is little information about anti-platelet effect of mustard oil in in vitro study. In addition, its

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active constituent and inhibitory mechanism is still unknown. Allyl isothiocyanate (3-

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isothiocyanato-1-propene, AITC) is the chief constituent of mustard oil9, 10 and causes pungent

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flavor. AITC has been used as a food additive for its flavor-enhancing, antibacterial, anti-fungal

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effects.11 In addition, studies using animal models have shown that AITC inhibits bladder cancer

and currently grown on millions acres in the North America and India. Also, many people of


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growth, attenuates levels of inflammatory markers such as interleukin-1β, and reduces blood

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glucose levels.12-14 However, the anti-platelet effects of AITC have not been evaluated.

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This study for the first time evaluated the effects of AITC on platelet aggregation and its

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mechanism of action, focusing on Thromboxane A2 (TXA2) production and ATP release in

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platelet from rat and human blood. In addition, its ex vivo anti-platelet effect in rat and its in vivo

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protective effect from thromboembolism attack in mouse were also investigated.

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MATERIALS AND METHODS

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Reagents. Allyl isothiocyanate (AITC) at 95% purity, apyrase, acetylsalicylic acid (ASA),

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bovine serum albumin (BSA), fura 2-AM, polyethylene glycol (PEG), β-nicotinamide adenine

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dinucleotide (reduced form, β-NADH), U46619 and pyruvic acid were obtained from Sigma

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Chemical Co. (St. Louis, MO, USA). Collagen, thrombin, adenosine diphosphate (ADP), and

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arachidonic acid (AA), and luciferin-luciferase reagent were purchased from Chrono-Log Co.

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(Harvertown, PA, USA). Thromboxane B2 EIA kits were purchased from Cayman Chemical Co.

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(Ann Arbor, MI, USA). Antibodies against PLCγ, phosphor-PLCγ, PKCδ, phospho-PKCδ, ERK,

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phospho-ERK, p38, phospho-p38, Akt, and phospho-Akt were purchased from Cell Signaling

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(Danvers, MA, USA).

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Animals. Animals were purchased from Samtako Laboratory Animal Center (Suwon,

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Korea). Spargue-Dawley (SD) rats (8 weeks, 220-240 g) and ICR mouse (8 weeks, 34-40 g)

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were used for experiments. They were housed in a conventional animal facility with free access

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to food and water in a temperature- and relative humidity-controlled environment under a 12-

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/12-h lighting schedule. The animals were acclimatized at least 7 days before experiments.

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Animal study protocols conformed to the guidelines in the Guide for the Care and Use of


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Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-

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23, revised 1996), and were approved by the Committee on Animal Research at Ajou Medical

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Center, Ajou University

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Experimental treatment. For in vitro experiments, AITC and ASA were dissolved in 70%

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PEG-saline. The final concentration of 70% PEG-saline never exceeded 0.1%. For ex vivo and in

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vivo experiments, AITC and ASA were dissolved in 70% PEG-saline. 70% PEG-saline solution

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did not influence platelet aggregation. All agents were prepared just before use.

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Preparation of platelet rich plasma (PRP) from rat and human blood. PRP from rat

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blood was prepared as described previously.15, 16 Briefly, Sprague-Dawley (SD) rats weighing

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200–240 g were lightly anesthetized with diethyl ether. A volume of 8–10 ml of blood was

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collected from the abdominal aorta into a syringe containing 3.8% sodium citrate. The ratio of

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blood to 3.8% sodium citrate was adjusted to 1:9 v/v. After centrifugation at 150 g for 10 min at

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room temperature, supernatants (PRP) were used for the aggregation study. To prepare washed

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platelets (WP), blood was collected into a syringe containing acid citrate dextrose (ACD: 2.5%

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trisodium citrate, 2% dextrose, and 1.5% citric acid) and adjusted to a ratio of 1:9 v/v. After

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centrifugation at 150 g for 10 min at room temperature, the platelets were isolated and then

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centrifuged again at 150 g for 10 min in washing buffer (Tyrode’s solution containing 10% ACD

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buffer and 0.3% BSA). Platelets were diluted with Tyrode’s solution (NaCl 11.9 mM, KCl 2.7

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mM, MgCl2 2.1 mM, NaH2PO4 0.4 mM, NaHCO3 11.9 mM, glucose 11.1 mM, and bovine

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serum albumin 3.5 mg/ml, pH 7.2). To make platelet-poor plasma (PPP) from rat blood, PRP

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were centrifuged at 1200 g for 10 min at room temperature and supernatants were obtained as

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PPP. For PRP from human blood, the human platelet suspensions were supplied from Red Cross


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(Suwon, Korea). The human blood was collected from healthy human volunteers who had not

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taken medicine indicated by Red Cross guide.

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In vitro platelet aggregation and shape change in rat PRP. The platelet aggregation study

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was performed as previously described.17, 18 Briefly, PRP was diluted with Tyrode’s solution

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containing 0.3% BSA and adjusted to about 2 × 108 platelets/ml. PRP was stimulated to

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aggregate by using different aggregating agents at the following final concentrations: collagen, 2

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µg/ml; thrombin, 0.2 U/ml; ADP, 2.5 µM; AA, 100 µM and U46619 1 µM. We decided the

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concentrations of agonists based on the results from preliminary experiments (data not shown).

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Platelet aggregation was recorded for 5 min after stimulation by using whole blood/optical Lumi-

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Aggregometer (chrono-log, Havertown, PA). Aggregations were measured and expressed as percent

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changes in light transmission with respect to PPP. Data of light transmission assay was calculated to

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percent change by using chronolog-software. In the in vitro study, PRP was pre-incubated with

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AITC or ASA at 37°C for 3 min before being stimulated with the aggregating agents. To monitor

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shape change, platelet shape change study was performed as previously described.19 Platelets (2

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× 108 platelets/ml) were pre-incubated with apyrase (2 U/ml), indomethacin (10 µM) and EGTA

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(1 mM), before addition of collagen.

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Determination of cytotoxicity in rat PRP. To determine the cytotoxicity of AITC, lactate

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dehydrogenase (LDH) release from platelets was measured as previously described.20 After

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incubating PRP (2 × 108 platelets/ml) with 70% PEG-saline or different concentrations of AITC

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for 3 min at 37°C, PRP was centrifuged at room temperature at 10,000 × g for 1 min. A 25-µl

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aliquot of the supernatant was mixed with 100 µl of NADH solution (0.03% β-NAD, reduced

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form of the disodium salt in phosphate buffer) and 25 µl of pyruvate solution (22.7 mM pyruvic

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acid in phosphate buffer) at room temperature. Reductions in absorbance at 340 nm because of

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conversion of NADH to NAD+ were measured to determine LDH activity in supernatants. LDH


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release is expressed as the percentage of the total enzyme activity in platelets completely lysed

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with 0.2% triton X-100.

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Measurement of TXA2 production and ATP release in rat and human PRP.

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Measurement of TXA2 production was conducted using a TXB2 EIA kit (Cayman Chemical Co,

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Ann Arbor, MI, USA) as previously described.21 PRP (2 × 108 platelets/ml) was incubated for 3

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min in the presence or absence of samples, and then collagen (2 µg/ml) was added and PRP was

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incubated at 37°C for 5 min with stirring. EDTA (10 mM) was added to stop TXA2 production.

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After centrifugation at 12,000 g for 3 min, the amount of TXB2 was measured according to the

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manufacturer’s instructions.

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Detection of ATP release was determined as previously described.22 PRP (2 × 108

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platelets/ml) was incubated at 37°C with stirring at 1,200 rpm. After addition of the luciferin-

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luciferase reagent, collagen (2 µg/ml) was added to initiate the reaction. PRP was incubated with

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AITC (30, 100, 300 µM) for 3 min before the addition of luciferin-luciferase reagent.

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Determining the [Ca2+]i in rat PRP. The intracellular Ca2+ was determined with fura-2/AM

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as described previously.23 Briefly, the PRP was incubated with 6µM of fura-2/AM for 60 min at

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37℃. The fura-2-loaded washed platelets (108 ml-1) were then pre-incubated with AITC for 3

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min with 1 mM CaCl2. Next, WPs were stimulated with collagen 2 µg/ml for 5 min. Fura-2

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fluorescence was measured in a spectrofluorometer with an excitation wavelength that ranges

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from 340 to 380 nm, changing every 0.5s, and emission wavelength of 510 nm. The [Ca2+]i was

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calculated.24

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[Ca2+]i in cytosol = 224 nM x (F – Fmin) / (Fmax – F)

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224 nM: dissociation constant of the fura-2-Ca2+ complex and Fmin and Fmax represent the

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fluorescence intensity levels at very low and very high Ca2+ concentrations respectively. Fmin is


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the fluorescence intensity of the Fura-2-Ca2+ complex at 510 nm, after the platelet suspension

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containing 20 mM Tris/10 mM of EGTA had been solubilized by 0.1% Triton X-100. Fmax is the

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fluorescence intensity of the Fura-2-Ca2+ complex at 510 nm, after the platelet suspension

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containing 1 mM CaCl2 had been solubilized by 0.1% Triton X-100. F indicates the fluorescence

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intensity of the fura-2-complex at 510 nm after WPs were stimulated by collagen, with 1mM

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CaCl2 and AITC or vehicle.

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Western blotting. The immunoassay was performed as previously described.4 The WPs (2

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× 108 platelets/ml) were stimulated by collagen (2 µg/ml) in the presence or absence of AITC

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(300 µM). Reactions were terminated by adding 10 mM EDTA and centrifuged at 12,000 g for 3

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min. After adding ice-cold platelet lysis buffer (1% NP40, 15 mM HEPES, 75 mM NaCl, 1 mM

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EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium fluoride (NAF), 1 mM

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Na3VO4, 1 µg/ml leupeptin, and 1 µg/ml aprotinin; pH 7.4), lysates were centrifuged at 13,000 g

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for 10 min at 4°C. The supernatants were collected and protein concentration was determined

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using a BCA protein assay (Thermo Scientific, IL, USA). Equal volumes of protein were

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resolved using SDS-PAGE (10%) and transferred to PVDF membranes. The PVDF membranes

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were blocked with 5% nonfat dry milk in TBS and incubated with the primary antibody diluted

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in TBS buffer (1:1000 ratio, 4°C overnight). Immunoblots were then incubated again with a

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horse radish peroxidase (HRP)-conjugated secondary antibody and then the immune-reactive

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bands were visualized with a LAS 4000 (Fuji, Honshu, Japan) using a western blot detection

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system (WestZol, Intron, Seoul, Korea).

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Ex vivo platelet aggregation study in rat. Two hours before experiments, SD rats were

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orally administered 70% PEG-saline, AITC (0.3, 1, 3 mg/kg), or ASA (50 mg/kg). Rat blood

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samples were collected and the platelet aggregation study was performed as described above.


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In vivo acute pulmonary thromboembolism study in mouse. Acute pulmonary

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thromboembolism model was performed as previously described.25 Male ICR mouse weighing

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35-40 g were fasted for 6 hour before experiments. AITC and ASA were dissolved in 70% PEG-

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saline. Mice were orally administered AITC or ASA before 2 hours performed experiments.

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Collagen 500 µg/kg plus epinephrine 25 µg/ml was injected to the tail vein to induce thrombosis.

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Each mouse watched for 15 min whether the mouse was paralyzed, dead or recovered from acute

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thrombotic challenge.

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In vivo mouse tail bleeding time assay. Mouse tail bleeding times were recorded as

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previously described.26,

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experiments. Two hours after the oral administration of AITC (0.3, 1, 3 mg/kg) or ASA (50

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mg/kg), the mice were anesthetized with ketamine 100 mg/kg and xylazine 10 mg/kg. Thereafter,

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the mice were individually placed on a hotplate to control body temperature and tails were

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transected 3 mm from the tips by using a razor blade. The tail was then immersed in a 15-ml

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clear conical tube containing warmed saline (37°C). The time until the cessation of blood flow

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was then measured. Bleeding time was defined as the time taken for bleeding stopped for 15 s.

Male ICR mice weighing 35–40 g were fasted for 16 h before

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Statistical analysis. In thromboembolism experiment, the chi-square test was used to

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determine significant differences between the vehicle and treated groups. Statistical significance

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was considered at P

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