Vanilloid-Like Agents Inhibit Aggregation of Human Platelets Safa Y. Almaghrabi, Dominic P. Geraghty, Kiran D.K. Ahuja, Murray J. Adams PII: DOI: Reference:

S0049-3848(14)00319-3 doi: 10.1016/j.thromres.2014.05.038 TR 5549

To appear in:

Thrombosis Research

Received date: Revised date: Accepted date:

16 February 2014 2 May 2014 30 May 2014

Please cite this article as: Almaghrabi Safa Y., Geraghty Dominic P., Ahuja Kiran D.K., Adams Murray J., Vanilloid-Like Agents Inhibit Aggregation of Human Platelets, Thrombosis Research (2014), doi: 10.1016/j.thromres.2014.05.038

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ACCEPTED MANUSCRIPT Category Original Article

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Vanilloid-Like Agents Inhibit Aggregation of Human Platelets

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Title

Authors

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Safa Y Almaghrabi, Dominic P Geraghty, Kiran DK Ahuja, and Murray J Adams

Affiliation

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School of Health Sciences, University of Tasmania, Bag 1320 Launceston, Tasmania,

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Australia, 7250

Word Count

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2220 (excluding abstract and references)

Address for correspondence:

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Murray Adams PhD MAIMS FFSc(RCPA) School of Human Life Sciences University of Tasmania Bag 1320 Launceston Tasmania, 7250, Australia Phone +61 3 6324 5483 Fax

+61 3 6324 3658

Email: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT Introduction Plant-derived and endogenous vanilloid-like agents exert their effects on cells through

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transient receptor potential vanilloid-1 (TRPV1). Little is known about the effects of these agents on platelet aggregation. We investigated the effect of various vanilloid-like agents on

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in-vitro platelet aggregation and tested whether this action is mediated through TRPV1. Understanding the mechanism of action of these compounds in platelets is important in that

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these compounds may be developed as novel anti-platelet agents.

Materials and Methods

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The effects of plant-derived (capsaicin; dihydrocapsaicin, DHC) and endogenous vanilloidlike agents (N-oleoyldopamine, OLDA; N-arachidonoyl-dopamine, NADA) on platelet

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aggregation were investigated using ADP (5, 10μM), collagen (4, 8g/mL) and arachidonic acid (AA, 300, 400µg/mL) as agonists. The direct effects of these agents on platelet viability

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were also determined using an LDH release assay.

Results

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Capsaicin, OLDA and NADA inhibited ADP-induced platelet aggregation in a concentrationdependent manner. OLDA and NADA, but not capsaicin and DHC, inhibited collageninduced aggregation, whereas AA-induced aggregation was inhibited by capsaicin, DHC and NADA, but not OLDA. Inhibition of aggregation was not due to direct toxicity of these agents towards platelets. The TRPV1 antagonist, SB-452533, did not affect inhibition of ADP-induced platelet aggregation by capsaicin and OLDA.

Conclusions These results demonstrate that the endovanilloids, OLDA and NADA, and plant-derived vanilloid, capsaicin, inhibit ADP-induced platelet aggregation. Collagen-induced aggregation was inhibited only by endovanilloids, whereas AA-induced aggregation was inhibited by 2

ACCEPTED MANUSCRIPT capsaicin, DHC and NADA. This inhibition was not due to direct toxic effects of these agents, nor was inhibition of ADP-induced aggregation TRPV1 mediated.

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KEYWORDS

ABBREVIATIONS AA,

arachidonic

acid;

ADP,

adenosine

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Capsaicin, dihydrocapsaicin, N-oleoyldopamine, N-arachidonoyl-dopamine, TRPV1

diphosphate;

CB,

cannabinoid;

DHC,

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transient receptor potential vanilloid-1,

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dihydrocapsaicin; OLDA, N-oleoyldopamine; NADA, N-arachidonoyl-dopamine; TRPV1,

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ACCEPTED MANUSCRIPT Platelets play an essential role in the pathogenesis of atherosclerosis and in the development of acute thrombotic events [1, 2]. Their importance in coronary disease and acute coronary syndromes is indirectly confirmed by the benefit of antiplatelet agents in treating these

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disorders. Natural compounds have been studied as potential antiplatelet agents with evidence to suggest that the active ingredient of the hot chili pepper, capsaicin, inhibits in vitro platelet

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aggregation [3-5].

Capsaicin is one of several capsaicinoids, the ‘hot’ compounds present in placental tissues of

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Capsicum fruits, that are responsible for capsicum’s pungency and the sensation of burning pain experienced due to stimulation of sensory neurons that convey the noxious stimuli to the

resiniferatoxin,

and

endogenous

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central nervous system [6, 7]. Capsaicinoids and other vanilloid-like agents, e.g., vanilloid-like

lipids

(‘endovanilloids’),

e.g.,

N-

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oleoyldopamine (OLDA) and N-arachidonoyl-dopamine (NADA), exert their actions primarily by activating transient receptor potential vanilloid-1 (TRPV1), a non-selective

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cation channel [8, 9]. Several endovanilloids derived from arachidonic acid have different

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affinities for TRPV1 and are recognized as an important group of signalling molecules

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affecting tissue injury, pain and inflammation [9].

Although TRPV1 has been reported to be present in platelets [10], there are few reports that have investigated the effects of vanilloid-like agents on platelet function, with conflicting data generated from these studies [3-5, 10, 11]. The aims of the present study were to therefore investigate; 1) the effects of plant-derived vanilloids (capsaicin, DHC) and endovanilloids (NADA, OLDA) on in vitro platelet activation and aggregation, 2) the effect of these agents on the release of lactate dehydrogenase (LDH) from platelets, as a measure of cytotoxicity, and, 3) whether the effects of these agents on platelets are mediated through TRPV1.

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ACCEPTED MANUSCRIPT MATERIALS AND METHODS Ethics This study was approved by the Tasmanian Health and Medical Human Research Ethics

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Committee (approval number: H00011414). Informed written consent was obtained from all

Materials

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participants.

Adenosine diphosphate (ADP), arachidonic acid (AA) and collagen were from Helena Beaumont,

USA.

Capsaicin,

N-arachidonoyl-dopamine

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Laboratories,

(NADA),

N-

oleoyldopamine (OLDA) and the TRPV1 antagonist, SB452533 (N-(2-Bromophenyl)-N'-[2-

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[ethyl(3-methylphenyl)amino]ethyl]-urea), were from Tocris, Bristol, UK. Dihydrocapsaicin

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(DHC) was from Sigma-Aldrich, St Louis, USA.

Sample collection and processing

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Venous whole blood was collected by venepuncture using minimal stasis from forty-eight

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healthy subjects (aged 18-65 years) with no previously diagnosed haemostatic abnormality. Blood was collected into 3.2% sodium citrate anticoagulant (1:9 ratio of anticoagulant to

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whole blood). Subjects avoided aspirin and antiplatelet medications for at least 10 days and dietary chili for at least two days, prior to blood collection. Samples were centrifuged at 150 g for 10 minutes at room temperature (RT) to obtain platelet rich plasma (PRP). The remaining blood was then centrifuged at 2000 g for 20 minutes at RT to obtain platelet poor plasma (PPP).

Platelet aggregometry All experiments were conducted using a four-channel AggRAM platelet aggregometer (Helena Laboratories, Beaumont, USA) at 600 rpm and 37°C as described previously [3, 12, 13]. ADP and AA were dissolved in deionized water and stored in 1M aliquots at -20C. Capsaicin, DHC and OLDA were dissolved in 100% ethanol and stored as 0.1 M aliquots at 5

ACCEPTED MANUSCRIPT 20C. NADA was stored as 1.14mM aliquots at -20C. Aliquots of all vanilloids were diluted as required in buffered normal saline (BNS; pH 7.1). PPP from the same sample was used as the blank for each experiment and to adjust the platelet count of PRP to 250 x 109/L. PRP

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(225 μL) was activated with 25 μL of ADP (5 or 10 M), collagen (4 or 8 g/mL) or AA (300

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or 400 g/mL), in the presence and absence of capsaicin, DHC, NADA or OLDA (final concentrations, 0-100 M). At the highest concentration of ethanol used (0.1%) in these

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studies, platelet aggregation was not affected.

For ADP-induced aggregation, the effects of the TRPV1 antagonist, SB452533, were also

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investigated. SB452533 was dissolved in 100% ethanol and stored in 10 M aliquots at -20C, then diluted for experiments to final concentrations of 1 and 10 M. SB-452533 (1, 10 μM),

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ADP (5 μM) and capsaicin or OLDA (50 μM) were added to PRP, and platelet aggregation

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recorded.

Aggregometry data were generated by the HemoRAM 1.1.0. software package (Helena

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Laboratories, Beaumont, USA). Parameters included percentage of the maximum aggregation (%MAX), percentage of the area under the curve (%AUC) and slope (rate) of ADP-,

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collagen- and AA-induced aggregation. Data for %MAX and %AUC were similar for all experiments therefore only %AUC data is presented. The lag time was also recorded for collagen- and AA-induced platelet aggregation. Each data set is presented as the mean of four experiments, using platelets collected from four separate donors.

Release of lactate dehydrogenase (LDH) from platelets The measurement of LDH is a well-established indicator of cell viability. In the present studies, the measurement of LDH release from platelets in the presence of vanilloid-like agents was performed using a LDH cytotoxicity kit according to manufacturer’s instructions (Clontech Laboratories, Mountain View, USA).

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ACCEPTED MANUSCRIPT Statistical analysis All data was collated and figures were generated using GraphPad Prism (version 5; San Diego, USA). Comparison of different concentrations of the specific vanilloids was made

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with repeated measures ANOVA using general linear modelling (STATA version 12SE, StataCorp LP, College Station, USA). Post-estimation Holm test analysis was then used to

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adjust the p values for multiple comparisons. Statistical significance was set at p < 0.05.

RESULTS

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Plant derived vanilloids and endovanilloids inhibit ADP-induced platelet aggregation Capsaicin inhibited ADP (5 M and 10 M) induced platelet aggregation in a concentration

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dependent manner (Fig. 1A and 1B). DHC also inhibited aggregation induced by 10 M (Fig. 1B) but not 5 M ADP (Fig. 1A) in a concentration-dependent manner. NADA and OLDA

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slightly enhanced aggregation induced by 5 M and 10 M ADP at a concentration of 3.125

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M but significantly inhibited it in a concentration-dependent manner at higher concentrations (Fig. 1A and 1B). The slope (rate) for both 5 and 10 M ADP-induced

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platelet aggregation curve was significantly reduced by 100 M OLDA (Fig. 1C and 1D).

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Endovanilloids inhibit collagen-induced platelet aggregation NADA and OLDA significantly inhibited 4 g/mL (Fig. 2A) but not 8 g/mL (Fig. 2B) collagen-induced platelet aggregation. In contrast, capsaicin and DHC had no effect on any of the measured parameters of platelet aggregation induced by 4 or 8 g/mL collagen (Fig. 2). Furthermore, NADA and OLDA both increased the lag time of 4 g/mL collagen-induced platelet aggregation (BNS vs endovanilloid; OLDA, 2313.2s vs 74.521.8s; NADA, 2612s vs 72.831.2s).

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ACCEPTED MANUSCRIPT Plant derived vanilloids and NADA inhibit AA-induced platelet aggregation Capsaicin and DHC significantly inhibited platelet aggregation induced by 300 and 400 g/mL AA (Fig. 3). NADA inhibited AA-induced (300 and 400 g/mL) platelet aggregation,

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but to a lesser extent than the plant-derived vanilloids (Fig. 3A and 3B). The lag time of 300

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g/mL AA-induced platelet aggregation was decreased by capsaicin and DHC (BNS vs vanilloid-like agent; capsaicin, 33.59.7s vs 10.87.2s; DHC, 23.413.7s vs 10.94.3s). Furthermore, the lag time of AA-induced (400 g/mL) platelet aggregation was prolonged by

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NADA (BNS vs NADA, 8.33.9s vs 39.215.2s). OLDA had no significant effect on AA-

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induced platelet aggregation, even at the highest concentration tested (100 μM).

Plant-derived vanilloids and endovanilloids are not directly cytotoxic to platelets

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When platelets were exposed to concentrations of up to 100 M for each of capsaicin, DHC, OLDA and NADA, there was no change to the release of lactate dehydrogenase (LDH) (data

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not shown).

mediated

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Inhibition of ADP-induced platelet aggregation by vanilloid-like agents is not TRPV1-

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To determine whether the inhibitory effects of these agents toward ADP-induced platelet aggregation was mediated through TRPV1, the effects of the potent and selective antagonist, SB452533, on aggregation was investigated. The presence of SB452533 (10 μM) did not affect the inhibition of ADP-induced aggregation by either capsaicin or OLDA (Fig. 4).

DISCUSSION We have demonstrated for the first time that the endovanilloids, OLDA and NADA, as well as the plant-derived vanilloid-like agent capsaicin, inhibit in vitro ADP-induced platelet aggregation in a concentration-dependent manner. The neuronal effects of plant-derived and endovanilloids are mediated through the non-selective cation channel, TRPV1 [7]. However, in the present study the effects of both classes of agents on ADP-induced aggregation were 8

ACCEPTED MANUSCRIPT not due to a direct interaction with TRPV1, nor were they directly toxic toward platelets. Endovanilloids were also shown to significantly inhibit collagen-induced platelet aggregation,

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whereas the plant-derived vanilloids inhibited AA-induced platelet aggregation.

Platelets play an important role in thrombogenesis, with antiplatelet medications such as the

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cyclooxygenase inhibitors, e.g., aspirin, some of the most effective drugs for the treatment and prevention of atherothromobotic diseases [14]. These drugs, however, have a number of side effects such as gastrointestinal bleeding and urticaria [15, 16]. Naturally-occurring

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antiplatelet compounds have the potential to cause fewer adverse effects than existing medications and may be useful as an adjunct or alternative to conventional antiplatelet

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medications. Capsaicin has previously been shown to possess antiplatelet activity [8], but the

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precise mechanism(s) of action on platelets are poorly understood.

Two previous studies have reported an inhibitory effect of capsaicin on in vitro ADP-induced

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platelet aggregation [3, 11]. Using the same in vitro aggregometry technique as the present

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study, we showed that DHC inhibited the aggregation of normal human platelets at higher concentrations, but enhanced aggregation at lower concentrations [3]. More recently, it was

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reported that collagen-induced aggregation of canine platelets was completely inhibited by capsaicin [11]. A similar effect on human platelets has also been reported [5]. These findings are in contrast to those in the present study, where neither capsaicin nor DHC significantly affected collagen-induced platelet aggregation. Moreover, the current study has demonstrated that OLDA and NADA significantly inhibit platelet aggregation induced by 4g/mL but not 8g/mL collagen, an effect that may be due to the inability of these agents to overcome a higher concentration of collagen (a ‘strong’ platelet agonist) in this in vitro model.

Although the neuronal actions of endovanilloids usually occur in the nmol/L range, the effects of these agents on other cell types occur at concentrations up to 500 µmol/L [17, 18]. Indeed, vanilloid-like agents have been shown to be cytotoxic at high concentrations [19]. Therefore, 9

ACCEPTED MANUSCRIPT to determine whether the inhibition of platelet aggregation by vanilloid-like agents may instead be artefactual due to a direct cytotoxic effect, we measured the release of LDH from platelets following exposure to ADP, in the absence and presence of vanilloid-like agents.

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None of the agents tested (capsaicin, DHC, NADA and OLDA, at concentrations up to 100

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M) had an effect on LDH release, strongly suggesting that their inhibitory effects on platelets are not due to direct cytotoxicity.

The effects of both plant-derived and endogenous vanilloid-like agents on neurons are

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primarily mediated through TRPV1, a non-selective cation channel with a modest preference

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for calcium [7]. TRPV1 plays an important role in a range of physiological processes including calcium homeostasis and calcium dependent cell signalling [7]. To determine whether the inhibitory effects of these agents toward ADP-induced platelet aggregation were

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mediated through TRPV1, the effects of the potent and selective antagonist, SB452533 [20], on aggregation were investigated. The inhibitory effects of a plant-derived (capsaicin) and an

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endogenous (OLDA) vanilloid-like agent on ADP-induced aggregation were not affected by

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the potent and selective TRPV1 antagonist, SB452533, suggesting that the effects of these agents on platelets are independent of TRPV1 activation. The results presented here are

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similar to those of another recent study that investigated the effect of a different TRPV1 antagonist, A-993610, on capsaicin-inhibition of collagen-induced aggregation of canine platelets [11]. However, our results are in contrast to another study that demonstrated that capsaicin enhances platelet aggregation in the absence of ADP or other platelet agonists, by increasing intracellular Ca2+ [10].

The data from the current study suggests that although plant-derived and endogenous vanilloid-like agents inhibit platelet aggregation, this is not due to cytotoxic effects, nor mediated through TRPV1. It is therefore interesting to speculate on other potential mechanism(s) of action. Our results suggest that capsaicin, NADA and OLDA, may act through blocking of the ADP receptors, P2Y1 and/or P2Y12, or by interfering with ADP10

ACCEPTED MANUSCRIPT mediated intracellular signalling. Furthermore, NADA and OLDA dampened 4 µg/mL collagen-induced platelet aggregation, suggesting firstly that this in vitro inhibitory effect is concentration dependent, and secondly that collagen receptors such as glycoprotein VI and

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integrin α2β1, may be implicated. Finally, capsaicin and DHC completely inhibited AAinduced platelet aggregation, a mechanism that may be mediated through the inhibition of

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cyclooxygenase-1 [5].

Other potential mechanisms of inhibition of platelet aggregation by vanilloid-like agents may

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include direct insertion into platelet plasma membranes, altering permeability and fluidity [4], or through the involvement of other receptors for endovanilloids, such as cannabinoid (CB1 CB2)

receptors.

Interestingly,

it

has

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and

been

shown

that

anandamide

(arachidonoylethanlamide), an endogenous cannabinoid receptor ligand, activates rabbit

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platelets through the AA pathway [21]. In contrast, anandamide appears to activate human platelets through a mechanism that is independent of the AA pathway [22]. Later, the same

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group reported that 2-arachidonoylglycerol, an endocannabinoid that is an endogenous

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agonist of the CB1 receptor, mediated platelet aggregation via an uncharacterized CB receptor [23]. The explanation for these conflicting reports is unclear, but it may be due to

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differences in the mechanisms of action of individual cannabinoids, and/or methodological and species differences between the various studies.

In conclusion, the present study showed that the endovanilloids, OLDA and NADA, as well as the plant-derived vanilloid, capsaicin, significantly inhibit in vitro ADP-induced platelet aggregation in a concentration-dependent manner. Furthermore, NADA and OLDA inhibited collagen-induced aggregation, whereas capsaicin and DHC inhibited AA-induced aggregation. Moreover, the inhibitory effects of the vanilloids were not due to platelet destruction, nor mediated by TRPV1 activation. To the best of our knowledge, this is the first study to systematically investigate the effects of plant-derived (capsaicin, DHC) and endogenous vanilloids (OLDA, NADA) on platelet aggregation using an array of platelet 11

ACCEPTED MANUSCRIPT agonists, including ADP, collagen and AA. Further research is warranted to determine precise mechanism(s) of action so that these vanilloid-like agents, and/or related molecules or

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compounds, may be investigated for their suitability for therapeutic applications.

ACKNOWLEDGEMENTS

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We would like to thank Merrilyn Johnson for her excellent technical assistance, participants who provided blood samples for platelet aggregation experiments, and King Abdul Aziz University, Jeddah, Saudi Arabia, who supported SYA with a postgraduate scholarship. This

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work was supported in part by a grant from the Clifford Craig Medical Research Trust,

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Launceston, Tasmania, Australia.

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ACCEPTED MANUSCRIPT FIGURE LEGENDS

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Fig. 1. Plant-derived vanilloids and endovanilloids inhibit ADP-induced platelet aggregation. (Top) Representative aggregation curves showing the effect of N-oleoyldopamine (OLDA) on 10 µM ADP-induced aggregation, green 0 µM (saline control); brown 25 µM; blue 50 µM; red 100 µM OLDA. Data are presented as percent area under curve (%AUC, normalized to aggregation in the absence of vanilloid) (A,B), and slope of the propagation phase of curve (slope) (C,D), for capsaicin (CAP), dihydrocapsaicin (DHC), Narachidonoyl-dopamine (NADA) and N-oleoyldopamine (OLDA). Points are the mean ± SEM of 4 experiments. *p

Vanilloid-like agents inhibit aggregation of human platelets.

Plant-derived and endogenous vanilloid-like agents exert their effects on cells through transient receptor potential vanilloid-1 (TRPV1). Little is kn...
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