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Platelets and Blood Cells

Protective mechanisms of adenosine 5’-monophosphate in platelet activation and thrombus formation Eduardo Fuentes1,2; Lina Badimon4; Julio Caballero3; Teresa Padró4; Gemma Vilahur4; Marcelo Alarcón1,2; Pablo Pérez1; Iván Palomo1,2 1

Summary Platelet activation is relevant to a variety of acute thrombotic events. We sought to examine adenosine 5’-monophosphate (AMP) mechanisms of action in preventing platelet activation, thrombus formation and platelet-related inflammatory response. We assessed the effect of AMP on 1) P-selectin expression and GPIIb/IIIa activation by flow cytometry; 2) Platelet aggregation and ATP secretion induced by ADP, collagen, TRAP-6, convulxin and thrombin; 3) Platelet rolling and firm adhesion, and platelet-leukocyte interactions under flow-controlled conditions; and, 4) Platelet cAMP levels, sP-selectin, sCD40L, IL-1β, TGF-β1 and CCL5 release, PDE3A activity and PKA phosphorylation. The effect of AMP on in vivo thrombus formation was also evaluated in a murine model. The AMP docking with respect to A2 adenosine receptor was determined by homology. AMP concentration-dependently (0.1 to 3 mmol/l) inhibited P-selectin expression and GPIIb/IIIa activation, platelet secretion and aggregation induced by ADP, collagen,

Correspondence to: Iván Palomo G., PhD Immunology and Haematology Laboratory Faculty of Health Sciences Universidad de Talca Casilla: 747, Talca, Chile Tel.: +56 71 200493, Fax: +56 71 20048 E-mail: [email protected]

Introduction The development and progression of cardiovascular diseases (CVD) is based in the interactive processes between atherosclerotic lesions and thrombus formation, whereby platelets play a critical role (1). Indeed, platelets are not only key mediators of thrombosis but also in inflammation by directly interacting with cells of the immune system (2-4). In fact, a large-scale, prospective human study has reported that the risk of future cardiovascular events increases as do the levels of plasma platelet-leukocyte aggregation (5). In addition circulating activated platelets and platelet-leukocyte aggregates have been shown to promote atherosclerotic lesion development in apolipoprotein E-deficient mice, further supporting their atherogenic potential (6). © Schattauer 2014

TRAP-6 and convulxin, and diminished platelet rolling and firm adhesion. Furthermore, AMP induced a marked increase in the rolling speed of leukocytes retained on the platelet surface. At these concentrations AMP significantly decreased inflammatory mediator from platelet, increased intraplatelet cAMP levels and inhibited PDE3A activity. Interestingly, SQ22536, ZM241385 and SCH58261 attenuated the antiplatelet effect of AMP. Docking experiments revealed that AMP had the same orientation that adenosine inside the A2 adenosine receptor binding pocket. These in vitro antithrombotic properties were further supported in an in vivo model of thrombosis. Considering the successful use of combined antiplatelet therapy, AMP may be further developed as a novel antiplatelet agent.

Keywords Platelets, antithrombotic, adenosine 5’-monophosphate, PDE inhibition, A2A receptor

Received: May 13, 2013 Accepted after major revision: October 28, 2013 Prepublished online: December 5, 2013 doi:10.1160/TH13-05-0386 Thromb Haemost 2014; 111: 491–507

Reports in the last decade sustain that the secretion of plateletderived pro-inflammatory molecules (sCD40L, CCL5 and sPselectin, among others) exacerbate the inflammatory response within the atherosclerotic plaque (7, 8). Given the central role of platelets in the atherosclerotic-related inflammatory response and the subsequent thrombotic event a variety of antiplatelet agents have been developed (9, 10). Although antiplatelet drugs have proven to be beneficial in patients with clinical evidence of CVD, the outcome still remains poor. As such, antiplatelet agents are frequently associated with serious adverse effects (internal bleeding and gastrointestinal adverse effects, among others) [pic](11) and their effectiveness in primary prevention is still a matter of debate (12). Epidemiological studies have provided evidence of a protective role of healthy diets in the prevention of CVD (13-15). More speThrombosis and Haemostasis 111.3/2014

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Department of Clinical Biochemistry and Immunohematology, Faculty of Health Sciences, Interdisciplinary Excellence Research Program on Healthy Aging (PIEI-ES), Universidad de Talca, Talca, Chile; 2Centro de Estudios en Alimentos Procesados (CEAP), CONICYT-Regional, Gore Maule, R09I2001, Talca, Chile; 3Center for Bioinformatics and Molecular Simulations, Faculty of Engineering in Bioinformatics, Universidad de Talca, Talca, Chile; 4Centro de Investigación Cardiovascular (ICCC-CSIC), Instituto de investigación Biomédica Sant Pau (IIB-Sant Pau), Hospital de la Santa Creu i Sant Pau; Barcelona, España

Fuentes et al. AMP exerts antithrombotic and anti-inflammatory activities

cifically, a number of dietary components including some fats, antioxidants and nucleosides have shown to diminish platelet activation (16, 17). To this respect, our group has recently isolated and identified adenosine and guanosine as a bioactive compound in Solanum lycopersicum (a cherry-type tomato) with potent antiplatelet activity (17, 18). Adenosine, which is released to the circulation by the heart, endothelium, and other tissues, is an endogenous anti-aggregating substance that influences platelet function by increasing cAMP and cGMP levels (19). Yet, the mechanisms by which adenosine 5’-monophosphate (AMP) may interfere with platelet activation remain to be fully elucidated. The present study aimed to investigate the relative contribution of AMP on platelet activation, thrombus formation and platelet-related inflammatory response. Furthermore, given the high structural similitude between adenosine and AMP, we performed docking experiments on A2 adenosine (A2A) receptor in order to determine their differential antiplatelet activity at a molecular level.

Materials and methods Reagents Sodium chloride (p.a.) was obtained from Arquimed (Santiago, Chile). Adenosine 5'- diphosphate (ADP), thrombin receptor activator peptide 6 (TRAP-6), calcein-AM, collagen, acetylsalicylic acid (ASA), cilostazol, bovine serum albumin (BSA), adenosine5'-monophosphate (AMP), SQ22536 (an adenylyl cyclase inhibitor), ZM241385 and SCH58261 (A2A receptor antagonists), rose bengal, prostaglandin E1 (PGE1), thrombin and rhodamine 6G were obtained by Sigma-Aldrich (St. Louis, MO, USA). Convulxin was obtained in Santa Cruz Biotechnology (Santa Cruz, CA, USA). Luciferase-luciferin reagent was obtained in ChronoLog corp (Havertown, PA, USA) and microfluidic chambers were from Bioflux (Fluxion, San Francisco, CA, USA). Anti-phosphoPKA antibody was obtained in Santa Cruz Biotechnology. Anti γ-tubulin monoclonal antibody (4D11) was obtained in Thermo Scientific (Thermo Scientific, Pierce, Rockford, IL, USA). Antibodies (anti-CD62P-PE, anti-CD61-FITC, anti-GPIIb/IIIa-FITC PAC-1 and anti-CD61-PE) were obtained from BD Pharmingen (San Diego, CA, USA).

Preparation of human platelet suspensions Venous blood samples were taken from two young healthy volunteers-who previously signed informed consent- in 3.2% citrate tubes (9:1 v/v) by phlebotomy with vacuum tube system (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA). The protocol was authorised by the ethics committee of Universidad de Talca in accordance with the Declaration of Helsinki (approved by the 18th World Medical Assembly in Helsinki, Finland, 1964). Tubes were centrifuged (DCS-16 Centrifugal Presvac RV) at 240 g for 10 minutes (min) to obtain platelet-rich plasma (PRP). PRP was adjusted to 200 X 109 platelets/l with platelet-poor plasma (PPP) obtained by centrifugation of the original tubes at 650 g (10 min). Washed platelets were prepared in Tyrode-HEPES buffer Thrombosis and Haemostasis 111.3/2014

containing 50 ng/ml PGE1 and 1 mmol/l EDTA, pH 7.4, at a concentration of 200 X 109 platelets/l. To avoid platelet activation 50 ng/ml PGE1 was added to the PRP before centrifugation at 750 g for 10 min. Platelet counts were performed in a hematologic counter (Bayer Advia 60 Hematology System, Tarrytown, NY, USA).

Flow cytometry analysis for P-selectin and glycoprotein (GP)IIb/IIIa Levels of P-selectin and GPIIb/IIIa on platelet surface were analysed by double-label flow cytometry by a modification of the method previously described by Frojmovic et al. (20). Briefly, 480 µl of PRP (diluted with Tyrode-HEPES buffer) was preincubated with 20 µl of saline or AMP (0.1 to 3 mmol/l) for 3 min. Then, samples were treated for 6 min of stimulation at 37°C with ADP 8 μmol/l. To determine platelet P-selectin expression, 50 µl of sample was mixed with saturated concentrations of anti-CD62P-PE and anti-CD61-FITC and incubated for 25 min in the dark. To determine platelet GPIIb/IIIa levels, 50 µl of sample was mixed with saturated concentrations of anti-GPIIb/IIIa antibody PAC-1 and anti-CD61-PE and incubated for 25 min in the dark. Samples were then centrifuged and washed for analysis. The samples were acquired and analysed in Accuri C6 flow cytometer (BD Biosciences). Platelet populations were gated on cell size using forward scatter (FSC) vs side scatter (SSC) and CD61 positivity to distinguish it from electronic noise. The light scatter and fluorescence channels were set at logarithmic gain, and 5,000 events per sample were analysed. Fluorescence intensities of differentially stained populations were expressed as mean channel value using the BD Accuri C6 Software (BD Biosciences).

Measurement of platelet secretion Platelet secretion was determined by measuring ATP release using a luciferin/luciferase reagent (17). Luciferin/luciferase (50 µl) was added to 480 µl of PRP (adjusted to 200 X 109 platelets/l) within 2 min before stimulation. Secretion was analysed after preincubation of platelets with 20 µl of saline, ASA (0.3 mmol/l) or AMP (0.1 to 3 mmol/l) for 3 min prior to the addition of ADP 8 µmol/l, collagen 1.5 μg/ml, TRAP-6 30 µmol/l, convulxin 20 ng/ml or thrombin 0.5 U/ml. Platelet secretion was measured at real time over a 6 min period in a lumi-aggregometer (Chrono-Log, Havertown, PA, USA) at 37ºC with stirring (150 g). The concentration required to inhibit platelet ATP secretion by 50% (IC50) was calculated from the dose-response curves.

Platelet aggregation assay Platelet aggregation was monitored by light transmission aggregometry according to Born and Cross (21), using a lumi-aggregometer. Briefly, 480 µl of PRP (200 x 109 platelets/l) were pre-incubated with 20 µl of saline, ASA (0.3 mmol/l) or AMP (0.1 to 3 mmol/l) for 3 min. Thereafter, 20 µl of agonist (ADP 8 µmol/l, collagen 1.5 μg/ml, TRAP-6 30 µmol/l, convulxin 20 ng/ml or throm© Schattauer 2014

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Fuentes et al. AMP exerts antithrombotic and anti-inflammatory activities

Analysis of platelet rolling and firm adhesion under controlled flow conditions Experiments under flow were performed in a BioFlux-200 flow system (Fluxion, San Francisco, CA, USA) with high shear plates (48 wells, 0-20 dyne/cm2) (22). The microfluidic chambers were coated with 20 µl of collagen 200 µg/ml at a wall shear rate of 200 s-1 for 1 hour (h). Then the collagen-coated plate surface was allowed to dry at room temperature (RT) for 1 h. Thereafter, channels were perfused with phosphate-buffered saline (PBS) for 10 min to remove the interface and blocked with bovine serum albumin (BSA) 5% for 10 min (RT, wall shear rate 200 s-1). Whole blood, anticoagulated with sodium citrate, was labelled with calcein-AM (4 μmol/l) and incubated with saline, ASA (0.3 mmol/l) or AMP (0.1 to 3 mmol/l) for 15 min (RT) before being added into the inlet of the well. Chambers were perfused for 10 min at RT at a wall shear rate of 1,000 s-1. The plates were mounted on the stage of an inverted fluorescence microscope (TE200, NIKON, Japan). Platelet deposition was observed and recorded at real-time with a CCD camera (QICAM, QIMaging, Surrey, BC, Canada). Realtime visualisation of platelet-collagen interactions was performed using a bright field and fluorescence microscopy. Platelets and collagen interaction over a 30 seconds (s) interval was defined as rolling whereas platelet immobilisation for more than 30 s was defined as firm adhesion. For each flow experiment, fluorescent images were analysed off-stage by quantifying the area covered by platelets with the ImageJ software (version 1.26t, NIH, Bethesda, MD, USA).

Leukocyte rolling over collagen-bound platelet monolayer under controlled flow The collagen-coated microfluidic chambers were rinsed in PBS buffer and perfused with whole blood, (anticoagulated with sodium citrate) labelled with calcein-AM 4 μmol/l at a shear rate of 200 s-1 for 10 min at RT. Under these conditions, a homogeneous carpet of spread platelets was formed. The remaining blood was washed out with saline, ASA (0.3 mmol/l) or AMP (0.1 to 3 mmol/l) for 3 min. Thereafter, leukocytes rolling and firm adhesion were visualised on the platelet layer with an inverted fluorescence microscope. To this end, leukocytes were previously labelled with rhodamine 6G (50 μl of 0.05%) Digital movies were captured, and translocation velocity was calculated by image analysis (Image J) (23).

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Measurement of cAMP levels in platelets The effect of AMP (0.1 to 3 mmol/l) on cAMP platelet levels was evaluated in PRP samples (500 µl) after a 5-min incubation period without stirring. Platelet reaction was stopped in cold-ice 10% trichloroacetic acid and precipitated proteins removed by centrifugation. Thereafter, HCl 1 mol/l (150 µl) was added and supernatants subjected to 6 ether extractions (v/v) and lyophilised. Samples were stored at -70°C until analysis. Before determination, samples were dissolved in 200 µl PBS at a pH 6.2. cAMP Direct Immunoassay Kit (BioVision Research Products, Mountain View, CA, USA) was performed.

Measurement of sP-selectin, sCD40L, interleukin (IL)-1β, transforming growth factor (TGF)-β1 and CCL5 levels Soluble P-selectin (sP-selectin) was determined by ELISA according to the manufacturer´s instructions (Invitrogen, Carlsbad, CA, USA) and soluble CD40 ligand (sCD40L), CCL5, IL-1β and TGF-β1 were determined using human quantikine ELISA kits (R&D systems, Minneapolis, MN, USA). Briefly, washed platelets were pretreated with saline, ASA (0.3 mmol/l) or AMP (0.1 to 3 mmol/l) for 15 min at 37°C and then stimulated with thrombin (2 U/mL) for 45 at 37 °C. Finally, the supernatants were collected after centrifugation (2,000 g, 10 min, 4°C) and stored at −70°C until use.

Effects of SQ22536 on ADP-induced platelet aggregation, cAMP intraplatelet levels, sP-selectin, sCD40L, CCL5, IL-1β and TGF-β1 release To elucidate whether AMP antiaggregant activity was mediated by the adenylyl cyclase pathway, PRP was pretreated with SQ22536 (200 and 400 µmol/l) for 3 min before the addition of AMP (3 mmol/l). Then, platelet aggregation was challenged by ADP 8 µmol/l. In addition, to study a potential effect on cAMP intraplatelet levels, platelets were pretreated with SQ22536 (200 and 400 µmol/l) before the addition of AMP (3 mmol/l). Moreover, washed platelets were pretreated with SQ22536 (200 and 400 µmol/l) and then with saline or AMP (3 mmol/l) for 15 min at 37°C before thrombin (2 U/ml) stimulation (45 min at 37°C). sP-selectin, sCD40L, CCL5, IL-1β and TGF-β1 release were assessed as described above. Platelets firstly exposed to SQ22536 and then ADP was used for control purposes.

Effect of ZM241385 and SCH58261 on ADP-induced platelet aggregation To elucidate whether AMP anti-aggregant activity was mediated by the interaction with A2A receptor, PRP was pretreated with antagonists selective for the A2A receptor (ZM241385 30 µmol/l and SCH58261 30 µmol/l) for 3 min before the addition of AMP (3 mmol/l). Then, platelet aggregation was challenged by ADP 8 Thrombosis and Haemostasis 111.3/2014

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bin 0.5 U/ml) was added and platelet aggregation registered during 6 min. All measurements were performed in triplicate. The result of platelet aggregation (maximal amplitude [%]) was determined by the software AGGRO/LINK (Chrono-Log). AMP inhibition of the maximal platelet aggregation was expressed as a percentage of the control (saline). The concentration required to inhibit platelet aggregation by 50% (IC50) was calculated from the dose-response curves.

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µmol/l. Platelets firstly exposed to ZM241385 and SCH58261, and then to ADP were used for control purposes.

Enzyme activity assay for cAMP- phosphodiesterase 3A (PDE3A) AMP (0.1 to 3 mmol/l) induced PDE3A activity was assessed by measuring the amount of cAMP hydrolysed as previously described (24, 25). Briefly, the enzyme was added to a buffer (137 mmol/l NaCl, 2.7 mmol/l KCl, 8.8 mmol/l Na2HPO4, 1.5 mmol/l KH2PO4, 1 mmol/l CaCl2, 1 mmol/l MgCl2 and 200 µM cAMP, pH 7.4). Reaction mixture, with and without enzyme, was incubated

A

C

B

D

Figure 1: Effects of AMP on P-selectin expression and GPIIb/IIIa activation. PRP (diluted with Tyrode-HEPES buffer) was preincubated with saline (control) or AMP (0.1 to 3 mmol/l) for 3 min. Then, samples were treated for 6 min with ADP 8 μmol/l. P-selectin expression (A) and GPIIb/IIIa activation (B) were determined by flow cytometry. C) Dot plots demonstrating the simplified gating strategy for P-selectin expression and GPIIb/IIIa activation in human platelet. Platelets were confirmed in terms of CD61 ex-

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for 15 min (37°C) and catalysis was terminated by addition of ZnSO4 (0.2 mol/l) and Ba(OH)2 (0.2 mol/l), which precipitates AMP but not cAMP. After centrifugation, supernatants containing cAMP were measured at 256 nm. Cilostazol (a phosphodiesterase inhibitor; 0.1 mmol/l) was employed as positive control. Inhibition of PDE activity was calculated according to the equation % inhibition of PDE3A activity = (1 - cAMP levels ) x 100%.

Western blotting study Washed platelets (200 X 109 platelets/l) were pre-incubated with saline, AMP (0.1 to 3 mmol/l) or SQ22536 (400 µmol/l) and AMP

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pression (R1) and these CD61(+) were finally analysed in terms of the PAC-1(+) and CD62(+). D) Representative dot plots of the effects of AMP on platelet P-selectin expression and GPIIb/IIIa activation. Results were expressed as mean ± SEM of n=3. Asterisk denotes statistically significant difference when compared with control + ADP analysed by Student’s t-test (* p

Protective mechanisms of adenosine 5'-monophosphate in platelet activation and thrombus formation.

Platelet activation is relevant to a variety of acute thrombotic events. We sought to examine adenosine 5'-monophosphate (AMP) mechanisms of action in...
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