Biochimie 105 (2014) 182e191

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Research paper

Effect of 2-arachidonoylglycerol on myosin light chain phosphorylation and platelet activation: The role of phosphatidylinositol 3 kinase/AKT pathway Maria Grazia Signorello, Giuliana Leoncini* Department of Pharmacy, Biochemistry Lab, University of Genoa, Viale Benedetto XV 3, 16132 Genova, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 March 2014 Accepted 14 July 2014 Available online 25 July 2014

The endocannabinoid 2-arachidonoylglycerol (2-AG) can be considered a true agonist as it is able to activate human platelets stimulating arachidonic acid release, thromboxane B2 formation and calcium intracellular elevation. Recently we have shown that 2-AG induces a rapid myosin light chain (MLC) phosphorylation/activation, early mediated by RhoA kinase (ROCK) signalling pathway and later by myosin light chain kinase. The aim of the present study was to investigate the role of phosphatidylinositol 3 kinase (PI3K)/AKT pathway in MLC phosphorylation and some downstream events such as actin polymerization, ATP secretion and aggregation. We demonstrated that PI3K in particular the isoforms a and b and AKT have a role in MLC phosphorylation. The stimulation of PI3K/AKT pathway activates ROCK. ROCK is directly involved in the early phase of MLC activation stimulating thr18 phosphorylation. MLC activation is strengthened through the MLC phosphatase inhibition, that is accomplished through the phosphorylation of MYPT1, catalytic subunit of MLC phosphatase, overall mediated by ROCK. In addition we have found that the PI3Ka/b isoforms and AKT are involved in the downstream mechanisms leading to actin polymerization, ATP secretion and aggregation of human platelets stimulated by 2-AG. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: 2-Arachidonoylglycerol Myosin light chain RhoA/RhoA kinase Myosin light chain phosphatase Phosphatidylinositol 3 kinase/AKT pathway

1. Introduction Platelet activation is important for mediating haemostasis but also contributes to thrombosis in the arterial circulation. In fact when platelets encounter matrix proteins exposed by injury to the vessel wall, they arrest on the exposed subendothelial surface, become activated showing morphological alterations, secrete the content of their granules and aggregate. Phosphorylation of the 20 kDa myosin (MLC) is a very important step in the activation of acto-myosin events involved in shape-change and secretion [1]. In human platelets phosphorylation of MLC can be mediated by the Ca2þ/calmodulin-dependent myosin light chain kinase (MLCK) [2] and/or the serine/threonine directed RhoA/Rho kinase (ROCK) as a function of different stimuli [3]. The phosphorylation of MLC is also controlled by the myosin light chain phosphatase (MLCP), a trimeric holoenzyme composed by 37 kDa (PP1C) and 110 kDa

Abbreviation: 2-AG, arachidonoylglycerol; MLC, p20 myosin light chain; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; PI3K, phosphatydilinositol 3 kinase; ROCK, RhoA kinase. * Corresponding author. Tel.: þ39 0103538154; fax: þ39 0103538153. E-mail address: [email protected] (G. Leoncini). http://dx.doi.org/10.1016/j.biochi.2014.07.014 0300-9084/© 2014 Elsevier Masson SAS. All rights reserved.

(MYPT1) catalytic subunits, the latter specifically involved in the targeting of MLCP to myosin filaments, and a 20 kDa inhibiting subunit (CPI-17). Phosphorylation of MYPT1 by ROCK results in loss of ability of MLCP to dephosphorylate MLC [4]. In addition phosphorylation of CPI-17 by ROCK [5] and/or PKC greatly increases the ability of CPI-17 to inhibit MLCP [6]. The endocannabinoid 2-arachidonoylglycerol (2-AG) can be considered a true agonist as it is able to activate human platelets at micromolar concentrations [7,8]. Platelet treatment with 2-AG leads to the p38MAPK/cytosolic phospholipase A2 pathway stimulation [9] and to NO/cGMP pathway regulation by protein kinase C [10]. Recent studies have demonstrated that 2-AG induces a rapid MLC phosporylation/activation, early mediated by ROCK signalling pathway and later by MLCK. In addition it was shown that MLC phosphorylation is potentiated by the MLCP inhibition as the phosphorylation of both MYPT1 and CPI-17 subunits is involved in this mechanism [11]. In vitro studies using wortmannin and LY294002 put in evidence that phosphatidylinositol 3 kinase (PI3K) exerts a role in regulating a broad range of functional responses including primary platelet adhesion, cytoskeletal remodelling and aggregation. Three families of PI3K (classes I, II and III) are present in human platelets. The class I PI3K, responsible for agonist-induced

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phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate production, is involved in the activation of integrin aIIbb3. The class IA (a, b and d) isoforms have p55-85 regulatory subunits and are classically regulated by tyrosine kinases, whereas the class IB (g) isoform has a p101 regulatory subunit and is activated by G protein-coupled receptors [12]. Platelets contain all classes I PI3K isoforms with lower levels of p110d [13]. It was shown that PI3Kb has an important role in ADP-induced platelet aggregation [14]. PI3Kg is also thought to be mediated by the bg complexes dissociated from Gi proteins upon receptor activation [15] and plays a significant role in ADP-induced platelet aggregation cooperating with PI3Kb [16]. In addition PI3Kd plays only a minor role in GPVI mediated platelet activation [17]. Recently it has been reported that PI3Ka plays a role in insulin-like growth factor 1-mediated AKT phosphorylation through Gi signalling [18]. Even if the G1-dependent potentiation of platelet function by 2-AG through a significant decrease of cAMP intracellular levels [7] seemed related to the adenylate cyclase inhibition, some studies have demonstrated a very important role for PI3K in this process [19,20]. One potential effector of PI3K is the serine/threonine kinase AKT, that has been detected in platelets and becomes phosphorylated after platelets stimulation by agonists [21,22]. To deeply investigate the mechanisms involved in 2-AG-mediated platelet activation we investigate the role of PI3K/AKT pathway in MLC phosphorylation, the consequent cytoskeleton reorganization, ATP secretion and aggregation. Results reported in the present study show that PI3Ka, PI3Kb and AKT have a role in MLC phosphorylation stimulated by 2-AG. The stimulation of PI3K/ AKT pathway produces ROCK activation. ROCK is directly involved in the early phase of MLC phosphorylation and/or in MLCP inhibition through the phosphorylation of MYPT1 catalytic subunit of MLCP. Moreover we have found that the PI3K/AKT pathway is involved in actin polymerization, ATP secretion and aggregation induced by 2-AG.

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collected into 130 mM aqueous trisodium citrate anticoagulant solution (9:1). The donors claimed to have not taken drugs known to interfere with platelet function during two weeks prior to blood collection, and gave their informed consent. Washed platelets were prepared centrifuging whole blood at 100  g for 25 min. The obtained platelet-rich plasma was then centrifuged at 1100  g for 15 min. Pellet was washed once with pH 5.2 ACD solution (75 mM trisodium citrate, 42 mM citric acid and 136 mM glucose), centrifuged at 1100  g for 15 min and then resuspended in calcium-free 10 mM HEPES buffer containing 145 mM NaCl, 5 mM KCl, 1 mM MgSO4, 10 mM glucose (pH 7.4). 2.3. Immunoblotting analysis of proteins Platelet suspensions (1.0  109 platelets/mL), prewarmed with saline or additions at 37  C, were stimulated with 2-AG as indicated. Incubation was stopped by adding 2  Laemmli-SDS reducing sample buffer. Samples, heated for 5 min at 100  C, were separated by 5e7.5% or 5e10% SDS-PAGE, and transferred to nitrocellulose membranes. Running was performed in the presence of Colorburst™ Electrophoresis weight markers. Blots were blocked in 6% BSA dissolved in TBST (Tris buffer saline, pH 7.6, containing 10 mM Tris, 150 mM NaCl, and 0.1% Tween 20) at 37  C for 30 min, and then incubated overnight at 4  C with anti-phospho-MLC(thr18/ser19), anti-phospho-MLC(thr18), anti-phospho-MLC(ser19), anti-phospho-MYPT1(thr696) or anti-phospho-AKT(ser473) (1:1000 dilution) antibodies. Membranes were extensively washed and incubated for 60 min at room temperature with horseradish peroxidase-conjugated secondary antibody. After further washings, blots were developed using the ECL® system, and the optical density was quantified by the Bio-Rad Chemi-Doc software package. Then nitrocellulose membranes, stripped by incubation with 62.5 mM Tris/HCl (pH 6.7), 2% SDS, 100 mM b-mercaptoethanol for 30 min at 50  C, were reprobed with anti-b-actin, anti-tubulin or anti-AKT and band intensity was quantified as detailed above.

2. Materials and methods 2.4. RhoA activation assay 2.1. Materials Apyrase, benzamidine, Colorburst™ electrophoresis markers, dithiothreithol, leupeptin, b-mercaptoethanol, PGE1 (prostaglandin E1), PMSF, protease inhibitor cocktail and all chemicals were from SigmaeAldrich, USA. ROCK activity EIA kit and TGX221 were purchased from Merck Biosciences, Germany. 2-AG, LY294002 and Y27632 were from Tocris Bioscience, UK. CH5132799, PIK-75, AS252424, IC87114 and MK2206 were from Selleck Chem. Inhibitors were diluted in saline from a stock DMSO solution immediately before each experiment. Anti-phospho-MLC (thr18/ser19), anti-phospho-AKT (ser473) and anti-AKT were from Cell Signaling Technology, USA. Anti-phospho-MLC(thr18), anti-phospho-MLC(ser19), anti-b-actin, anti-tubulin and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, USA. Anti-phospho-MYPT1(thr696), anti-MYPT1 and ATP assay kit were from Millipore, USA. G-LISA kit for RhoA activity detection was from Cytoskeleton U.S.A. ECL® system and protein G-sepharose were from GE Healthcare, USA. Nitrocellulose membranes (pore size 0.45 mm) were purchased from Bio-Rad Laboratories, USA. EnzChek® phosphate assay kit was from Molecular Probes, USA. BCA protein assay kit was from Pierce Biotechnology USA. 2.2. Blood collection and preparative procedures Freshly drawn venous blood from healthy volunteers of the “Centro Trasfusionale, Ospedale San Martino” in Genoa was

Washed platelets (1.0  109 platelets/mL), preincubated at 37  C with saline or varying inhibitors, were stimulated with 10 mM 2-AG at 37  C for 30 s under mild shaking without stirring. Incubation was stopped by putting suitable aliquots of platelets in ice. Samples were lysed by the addition of supplied cell lysis buffer and then centrifuged at 16,000  g for 1 min. Obtained supernatant was stored at 70  C and RhoA activity was detected in each sample by a commercial G-LISA kit following the manufacturer's instructions with appropriate modifications. The G-LISA RhoA activation assay is an ELISA-based system that measures the level of RhoA activation. In brief, the active GTP-bound form of RhoA is captured on a substrate and then detected by incubation with specific RhoA primary antibody followed by a secondary antibody conjugated to HRP. 2.5. ROCK activity assay The ROCK activity assay was performed by a commercial EIA kit with appropriate modifications. Washed platelets (1.0  109 platelets/mL), preincubated at 37  C with saline or varying inhibitors were incubated with 2-AG at 37  C for 30 s under mild shaking without stirring. Incubation was stopped by putting suitable aliquots of platelets in ice. Samples added to 1.0 mM PMSF, 10 mg/mL leupeptin and 100 mM dithiothreithol were sonicated twice for 15 s in ice and then centrifuged at 16,000  g for 5 min. In the supplied MYPT1-precoated wells, the enzyme reaction was assembled in a final volume of 100 mL mixing aliquots of the supernatant platelet cell lysate (100 mg of protein quantified in each

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samples by the BCA method [23] with 0.5 mM ATP, 75 mM MgCl2 and assay dilution buffer I). MYPT1-precoated wells and assay dilution buffer I were components of the kit. The ROCK activity was detected following the manufacturer's instructions. 2.6. MLCP activity assay The assay developed by Patil et al. [24] was adapted to platelets with suitable modifications. Briefly, washed platelets (2.0  109 platelets/mL), resuspended in pH 7.4 Hepes buffer were preincubated at 37  C with saline or additions and then incubated with 2-AG at 37  C for 30 s under mild shaking without stirring. Incubation was stopped by lysing at 4  C suitable aliquots of platelet suspension with equal volumes of lysis buffer (20 mM TriseHCl, 150 mM NaCl, 1% Triton X-100, 1 mM CaCl2, 1 mM Na3Vo4, 5 mM NaF, 1 mM PMSF, 100 mg/mL leupeptin, 10 mM benzamidine, protease inhibitor cocktail). To facilitate depolymerization of actin filaments, lysates were held at room temperature for 30 min. Remaining filaments were removed by centrifugation at 16,000 g for 1 min. Protein content of each samples was determined by BCA method [23]. Lysates, containing 500 mg protein, were pre-cleared with protein G-sepharose for 60 min at 4  C and subjected to anti-MYPT1 antibody overnight (1:250). Samples were then mixed with protein G-Sepharose beads, rocked for 2 h and washed twice with Tris-buffered saline. MLCP activity was determined in lysates immunoprecipitated with anti-MYPT1 antibody by the detection of Pi released using 2-amino-6-mercapto-7-methy purine riboside (MESG) as substrate following the manufacturer's instruction of the EnzChek® phosphate assay kit. Briefly, in the presence of Pi, the substrate MESG is converted enzymatically by purine nucleoside to ribose-1-phosphate and 2-amino-6-mercapto-7-methy purine, which produces a spectrophotometric shift from 330 to 360 nm. The values obtained in the absence of substrate, were subtracted from values of each sample performed in the presence of substrate. 2.7. F-actin content assay Washed platelets (1.0  109 platelets/mL), resuspended in pH 7.4 Hepes buffer were preincubated at 37  C with saline or agents and then incubated with 2-AG at 37  C under mild shaking without stirring. At the end of incubation suitable aliquots of samples were fixed in 2% paraformaldehyde for 30 min at 37  C. Fixed platelets, permeabilized with 0.1% Triton X-100, incubated with 10 mM fluorescein isothiocyanate (FITC)-phalloidin for 30 min at room temperature, were washed with PBS. Bound FITC-phalloidin was quantified by fluorescence with excitation at 495 nm and emission at 509 nm. 2.8. Measurements of ATP secretion Washed platelets (3.0  108 platelets/mL), resuspended in pH 7.4 Hepes buffer were preincubated at 37  C with saline or agents and then incubated with 2-AG at 37  C under mild shaking without stirring. The incubation was stopped by putting samples in ice. ATP secreted was determined by a commercial kit following manufacturer's instruction. The light produced by luciferase from ATP and luciferin was measured by luminometry. 2.9. Aggregation studies Platelet aggregation, performed in a Menarini Aggrecoder PA3210 aggregometer, was monitored according to Born's method [25], and quantified by the light transmission reached within 3 min. Washed platelets (3.0  108 platelets/mL) were preincubated with saline or varying agents at 37  C before adding 2-AG. The amplitude

of aggregation measured in the presence of additions was compared with that measured in a control experiment carried out under the same conditions, and the inhibition percentage was calculated. From each series of experiments an inhibition percentage curve was derived and the concentration of compound inducing 50% inhibition (IC50) was calculated. 2.10. Statistical analysis Data are mean ± SD of at least four independent experiments, each performed in duplicate. Statistical comparison between two groups was made through the unpaired Student's t-test. One-way ANOVA followed by Bonferroni's post hoc test was used to compare multiple groups. Statistical significance was defined as P < 0.05. 3. Results 3.1. The 2-AG induced MLC phosphorylation Previously we have shown that 2-AG stimulated MLC phosphorylation at thr18/ser19 in a dose- and time-dependent manner, peaking at 10 mM and 15 s after platelet incubation with 2-AG [11]. Thus to deeply investigate the mechanisms involved we wanted to verify if PI3K could participate in MLC phosphorylation. We found that MLC phosphorylation induced by 2-AG was cancelled by the generic PI3K inhibitor LY294002 and was partially reduced (by about 50%) by CH5132799 or TGX221, which can be considered specific inhibitors of the PI3Ka or PI3Kb isoform, respectively. The effect was complete in platelet pretreated with CH5132799 and TGX221 added at the same time (Fig. 1A). In like manner to CH5132799 the more widely used PI3Ka inhibitor PIK-75 produced a partial reduction of MLC phosphorylation (about 50%) and the effect was complete when PIK-75 and TGX221 were added at the same time (Fig. 1B). On the other hand MLC phosphorylation was not affected by the PI3Kg inhibitor AS252424 or the PI3Kd inhibitor IC87114 or apyrase (Fig. 1C). Moreover MLC phosphorylation was significantly reduced by MK2206, a novel allosteric inhibitor of AKT activity [26] and it was cancelled by the ROCK inhibitor Y27632 (Fig. 1A). It is known that phosphorylation of MLC occurs differentially on thr18 and ser19 with functional implications in platelets [27]. Thus we have analysed the 2-AG effect measuring MLC phosphorylation on thr18 and ser19 separately. Results reported in Fig. 2 show that the response to 2-AG occurs in a dose-dependent manner and phosphorylation on both residues peaks at 10 mM. Moreover the 2AG effect on thr18 and ser19 phosphorylation is time-dependent (Fig. 3). However thr18 phosphorylation is early and peaks at 15 s, preceding ser19 phosphorylation that occurs later reaching the maximum after 60 s of 2-AG addition. 3.2. Phosphorylation of AKT in response to 2-AG Since MLC phosphorylation was significantly reduced in platelets pretreated with MK2206 (Fig. 1A), AKT phosphorylation seemed to be involved in MLC phosphorylation. Thus to determine the kinetics of AKT phosphorylation, ser473 phosphorylation was monitored over a time range of 5e300 s. Immunoblot analysis revealed that AKT phosphorylation in response to 2-AG was rapid. The level of phosphorylation peaked at 30 s and did not change at prolonged incubation times (till to 300 s) (Fig. 4A). Moreover we exposed platelets to increasing concentrations of 2-AG and ser473 phosphorylation was measured at 30 s after the agonist addition. The AKT phosphorylation was dose-dependent and maximal phosphorylation was achieved at 10 mM 2-AG (Fig. 4B).

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Fig. 1. Effect of selected compounds on MLC phosphorylation induced by 2-AG. Washed platelets (1.0  109 platelets/mL) were preincubated at 37  C for 10 min with saline or 20 mM LY294002 (L), 10 mM CH5132799 (C), 10 mM TGX221 (T), 10 mM MK2206 (M), 20 mM Y27632 (Y), 1 mM PIK-75 (P), 1 U/mL apyrase (AP), 5 mM AS252424 (AS), 1 mM IC87114 (IC) and then stimulated for 30 s with 10 mM 2-AG. At the end of incubation suitable aliquots were immunoblotted with anti-p-MLC (thr18/ser19) as detailed in Methods. Blot is representative of four independent experiments. In the right panels the densitometric scanning ± SD of the blots is shown. Student's t-test: *P < 0.0001 vs none, #P < 0.0001 vs 2-AG.

Fig. 2. Time-dependence of 2-AG effect on thr18 or ser19 MLC phosphorylation. Washed platelets (1.0  109 platelets/mL), prewarmed at 37  C, were incubated for the indicated time with 10 mM 2-AG. At the end of incubation suitable aliquots were immunoblotted with anti-p-MLC(thr18), panel A, or anti-p-MLC(ser19), panel B, as detailed in Methods. Blot is representative of four independent experiments. In the lower panels the densitometric scanning ± SD of the blots is shown. One way ANOVA-Bonferroni's post hoc test: *P < 0.001, # P < 0.01.

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Fig. 3. Dose-dependence of 2-AG effect on thr18 or ser19 MLC phosphorylation. Washed platelets (1.0  109 platelets/mL), prewarmed at 37  C, were incubated for 30 s with varying concentrations of 2-AG, as indicated. At the end of incubation suitable aliquots were immunoblotted with anti-p-MLC(thr18), panel A, or anti-p-MLC(ser19), panel B, as detailed in Methods. Blot is representative of four independent experiments. In the lower panels the densitometric scannings ± SD of the blots are shown. One way ANOVA-Bonferroni's post hoc test: *P < 0.001.

Fig. 4. Effect of 2-AG on AKT phosphorylation. Washed platelets (1.0  109 platelets/mL), prewarmed at 37  C, were stimulated with 10 mM 2-AG. The time-course of AKT phosphorylation is reported in panel A. In panel B washed platelets (1.0  109 platelets/mL), prewarmed at 37  C, were incubated for 30 s with varying concentrations of 2-AG. In panel C the effect of several compounds on AKT phosphorylation was tested. Washed platelets (1.0  109 platelets/mL) were preincubated at 37  C for 10 min with saline or 20 mM LY294002 (L), 10 mM CH5132799 (C), 10 mM TGX221 (T), 10 mM MK2206 (M), 20 mM Y27632 (Y) and then stimulated for 30 s with 10 mM 2-AG. At the end of incubation suitable aliquots were immunoblotted with anti-p-AKT as detailed in Methods. Blots are representative of four independent experiments. In the right panels the densitometric scanning ± SD of the blots is shown. One way ANOVA-Bonferroni's post hoc test: *P < 0.001, #P < 0.01. Student's t-test: *P < 0.0001 vs none, #P < 0.0001 vs 2-AG.

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3.3. Effect of PI3K inhibitors on AKT phosphorylation induced by 2-AG Data shown in Fig. 1A suggest that PI3K stimulated by 2-AG is involved in AKT phosphorylation. To better understand the role of individual isoforms PI3K-selective inhibitors were evaluated on 2AG-mediated AKT phosphorylation. The 2-AG induced AKT phosphorylation was significantly inhibited in the presence of PI3Ka or PI3Kb inhibitors CH5132799 or TGX221, respectively and was cancelled when both were added at the same time to platelets (Fig. 4C), but it was not affected by the PI3Kg inhibitor AS252424, and the PI3Kd inhibitor IC87114 (data not shown). Obviously no effect was shown in the presence of Y27632 or MK2206 (Fig. 4C) which is an allosteric inhibitor of AKT activity [26], but it does not affect PI3K induced AKT phosphorylation. These data indicate that MLC phosphorylation is dependent on PI3K/AKT pathway activation. 3.4. The RhoA/ROCK signalling pathway activation by 2-AG Previously it was reported that in vascular smooth muscle PI3K regulates RhoA and myosin phosphatase activities [28]. Moreover we have shown that ROCK is involved in MLC phosphorylation induced by 2-AG [11]. Since results reported in the present study put in evidence that PI3K participates to MLC phosphorylation (Fig. 1A), we decided to investigate the probable connection between PI3K/AKT and RhoA/ROCK pathway in human platelets stimulated by 2-AG. Thus to explore the upstream mechanisms involved in RhoA activation we measured RhoA activity stimulated by 2-AG in the presence of the PI3K inhibitor LY294002. We found that in these conditions RhoA activity was reduced by 81%. In addition to clarify which isoform of PI3K was involved in the effect we performed experiments in platelets pretreated with CH5132799 or TGX221. Results reported in Fig. 5A put in evidence that CH5132799 and TGX221 reduced the 2-AG stimulated RhoA activity by 40 or 48%, respectively. Moreover RhoA activity was cancelled (about 95% of the total) by platelet treatment with CH5132799 and TGX221 added at the same time. In addition a significant (p < 0.001) reduction in RhoA activity stimulated by 2-AG was observed in the presence of the AKT allosteric inhibitor MK2206. A main target of activated RhoA is ROCK, which regulates the level of active MLC either by its direct phophorylation and/or by inactivation of MLCP [29]. Thus assays of ROCK activity demonstrated that 2-AG stimulated ROCK activity, as previously described [11]. This effect was cancelled by LY294002 and by Y27632 and it was reduced by 44 or 54% in platelets treated with CH5132799 or TGX221, respectively, while the contemporary addition of CH5132799 and TGX221 cancelled the 2-AG-stimulated ROCK activity. Moreover ROCK activity was significantly reduced (p < 0.0005) by MK2206 (Fig. 5B).

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3.5. Effect of 2-AG on MLCP activity and MYPT1 phosphorylation It was found that in human platelets stimulated with 2-AG MLCP activity was inhibited [11]. The very rapid effect of endocannabinoid was dose-dependent. A significant decrease (p < 0.0005) of MLCP activity was measured 30 s after the addition of 10 mM 2-AG (Fig. 6A). MLCP activity was partially recovered (about 50%) in platelets pretreated with CH5132799 and TGX221, while a complete effect was observed in the presence of both or in the presence of LY294002. Moreover MLCP activity was recovered by 76% or by 80% in platelets treated with Y27632 or with MK2206 respectively (Fig. 6A). MLCP activity is regulated by two subunits: the 110 kDa (MYPT1) and the 20 kDa (CPI-17) subunits. Both are able to inhibit MLCP. In particular phosphorylation of thr696 residue in the human MYPT1 isoform is able to inhibit MLCP activity. Data reported in Fig. 6B show that 2-AG stimulates MYPT1 phosphorylation [11]. Moreover the 2-AG induced MYPT1 phosphorylation is significantly reduced by LY294002, Y27632 or MK2206. The PI3Ka specific inhibitor CH5132799 and the PI3Kb specific inhibitor TGX221 had a minor effect, but cancelled the 2-AG stimulated MYPT1 phosphorylation when were added to platelets at the same time. 3.6. 2-AG induced actin polymerization and ATP secretion MLC phosphorylation initiates myosin Mg2þ-ATPase activity, causes the binding of myosin to F-actin [30,31] and leads to the initiation of platelet shape change response [32]. Thus the 2-AG effect on actin filament polymerization was investigated. It was found that 2-AG in a dose- and time-dependent manner (Fig. 7A) produced a significant increase in F-actin, in agreement with Malorni et al. [33]. Moreover CH5132799 and TGX221, the a and b PI3K isoforms specific inhibitors, produced a quite similar inhibition (more than 50%) on 2-AG induced actin polymerization while added together cancelled the effect. In addition a complete inhibition was measured in the presence of LY294002, while MK2206 and Y27632 produced only a partial inhibiting effect (60 or 50% respectively) (Fig. 7B). When platelets are activated with agonists in the absence of stirring they change shape and secret ATP from dense bodies [34]. Thus experiments carried out to measure ATP secretion have shown that 2-AG effect on ATP secretion was timeand dose-dependent. The ATP secreted peaked at 60 s, decreasing rapidly at prolonged incubation times. The maximal effect was observed at 10 mM 2-AG (Fig. 8A). ATP secretion was almost abolished by Y27632 and by LY294002, while CH5132799 and TGX221 had a partial effect (Fig. 8B), in accordance with data obtained testing other parameters such as MLC phosphorylation or actin polymerization.

Fig. 5. The 2-AG effect on RhoA and ROCK activities. Washed platelets (1.0  109 platelets/mL) were preincubated with saline or 20 mM LY294002 (L), 10 mM CH5132799 (C), 10 mM TGX221 (T), 10 mM MK2206 (M), 20 mM Y27632 (Y) for 10 min, then stimulated with 2-AG for 30 s. At the end of incubation samples were lysed and RhoA (panel A) or ROCK (panel B) activities were detected in supernatants. As detailed in Methods RhoA activity was quantified by a G-LISA kit and ROCK activity by an EIA kit, according to manufacturer's instructions. Each bar represents the mean ± SD of four independent experiments. Student's t-test: xp < 0.0005 vs none; *p < 0.0005, **p < 0.001, ##p < 0.0025, #p < 0.025 vs 2-AG.

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Fig. 6. The 2-AG effect on MLCP activity and MYPT1 phosphorylation. MLCP activity results are reported in panel A. Briefly washed platelets (1.0  109 platelets/mL), preincubated at 37  C for 10 min with 20 mM LY294002 (L), 10 mM CH5132799 (C), 10 mM TGX221 (T), 10 mM MK2206 (M), 20 mM Y27632 (Y), were stimulated with 10 mM 2-AG for further 30 s. At the end of incubation samples were lysed and immunoprecipitated with anti-MYPT1 and assayed for Pi released from 2-amino-6-mercapto-7-methypurine riboside (MESG) substrate as detailed in Methods. Each bar represents the mean ± SD of four independent experiments. Student's t-test: *p < 0.0005 vs none, **p < 0.0005 vs 2-AG. In panel B the effect of several compounds on MYPT1 phosphorylation was tested. Washed platelets (1.0  109 platelets/mL), preincubated at 37  C for 10 min with 20 mM LY294002 (L), 10 mM CH5132799 (C), 10 mM TGX221 (T), 10 mM MK2206 (M), 20 mM Y27632 (Y), were treated for 30 s with 10 mM 2-AG. At the end of incubation suitable aliquots were immunoblotted with anti-pMYPT1 as detailed in Methods. Blot is representative of four independent experiments. In the lower panel of B the densitometric scanning ± SD of the blots is shown. Student's ttest: *P < 0.0001 vs none, #P < 0.0001 vs 2-AG.

3.7. Effect of PI3K inhibitors on 2-AG induced platelet aggregation

4. Discussion

Data above reported put in evidence that PI3K, AKT and ROCK play an important role in MLC phosphorylation and actin polymerization induced by 2-AG. Thus to evaluate the role of this pathway on platelet activation, we tested the effect of PI3K, AKT and ROCK inhibitors on platelet aggregation stimulated by 2-AG. Data reported in Table 1 show that all tested inhibitors are effective. In particular TGX221 or CH5132799, specific inhibitors of PI3Kb or PI3Ka, are powerful, being the IC50 values 2.8 ± 0.8 or 4.2 ± 1.4 mM respectively. On the other hand in platelets pretreated with the AKT inhibitor MK2206 or the ROCK inhibitor Y27632 the IC50 value was 6.1 ± 1.9 or 15.1 ± 4.6 mM, respectively.

Recently we have shown that 2-AG, which is considered a true platelet agonist [33], was able to induce the rapid MLC phosphorylation stimulating early ROCK and later MLCK [11]. MLC phosphorylation was strengthened through the MLCP inhibition that was overall accomplished through the RhoA/ROCK mediated phosphorylation of the MLCP catalytic subunit MYPT1 [11]. Since RhoA/ROCK pathway plays a very important role in MLC phosphorylation we investigated the mechanisms leading to RhoA/ ROCK activation induced by 2-AG, with particular reference to the PI3K/AKT pathway. Previously it was shown that PI3K inhibitors suppress agonist-induced Rho activation, MYPT1 phosphorylation,

Fig. 7. Actin polymerization induced by 2-AG. Washed platelets (1.0  109 platelets/mL), prewarmed at 37  C, were incubated with varying concentrations of 2-AG, as indicated (panel A). In panel B the effect of several compounds is shown. Washed platelets (1.0  109 platelets/mL) were preincubated at 37  C for 10 min with saline or 20 mM LY294002 (L), 10 mM CH5132799 (C), 10 mM TGX221 (T), 10 mM MK2206 (M), 20 mM Y27632 (Y) and then stimulated for 60 s with 10 mM 2-AG. Samples were then fixed, permeabilized and incubated with FITC-phalloidin. Actin polymerization was quantified measuring bound FITC-phalloidin by fluorescence. Curves reported in panel A are representative of four independent experiments. In panel B each bar represents the mean ± SD of four independent experiments. Student's t-test: *p < 0.0005 vs 2-AG.

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Fig. 8. The 2-AG effect on ATP secretion. Washed platelets (1.0  109 platelets/mL), prewarmed at 37  C, were incubated with varying concentrations of 2-AG (panel A). In panel B the effect of several compounds was tested. Washed platelets (1.0  109 platelets/mL) were preincubated at 37  C for 10 min with saline or 20 mM LY294002 (L), 10 mM CH5132799 (C), 10 mM TGX221 (T), 10 mM MK2206 (M), 20 mM Y27632 (Y) and then stimulated for 60 s with 10 mM 2-AG. ATP secretion was determined by a commercial kit following manufacturer's instruction by luminometry. Curves reported in panel A are representative of four independent experiments. Each bar reported in panel B represents the mean ± SD of four independent experiments. Student's t-test: xp < 0.0001 vs none, *p < 0.0005, #p < 0.025, **p < 0.05 vs 2-AG.

MLCP activity, MLC phosphorylation and contraction in vascular smooth muscle [28]. In particular PI3Ka plays a critical role in the activation of the RhoA/ROCK dependent regulation of MLCP in isolated vascular smooth muscle cells [35]. The PI3K isoform b appears to be critically involved in platelet activation as induces exposure of platelet to fibrinogen through GPIIb/IIIa activation and triggers the outside-in signalling pathway which stabilizes the cytoskeleton and contributes to shape change [36,37]. Moreover PI3Kb plays an important role in ADP-induced platelet aggregation, AKT, ERK phosphorylation and TXA2 generation [38]. In addition PI3Ka and b isoforms both contribute to GPVI induced platelet signalling and thrombus formation [39]. As the generic PI3K inhibitor LY294002 abolishes RhoA/ROCK activation, MLC phosphorylation and MLCP inhibition induced by 2-AG, data presented in this study indicate that PI3K could be involved in the upstream mechanisms regulating RhoA/ROCK activation. In particular results obtained in the presence of selective inhibitors suggest that PI3Ka and PI3Kb are both involved with identical power in the RhoA/ ROCK pathway activation and PI3K phosphorylation. Stimuli which activate ROCK have been shown to increase MLC phosphorylation through inactivation of MLCP. In agreement with data previously reported [11], 2-AG was shown to be able to activate ROCK (Fig. 5B) and inhibit MLCP (Fig. 6A). The RhoA/ROCK pathway activated by 2AG induces thr696 phosphorylation of the catalytic subunit MYPT1 of MLCP (Fig. 6B) decreasing MLCP activity (Fig. 6A). Moreover data obtained in the presence of PI3K, AKT or ROCK inhibitors (Fig. 6) suggest that the phosphorylation of MLCP catalytic subunit MYPT1 occurs through the PI3K/AKT pathway stimulation and that PI3Ka and b isoforms are involved in ROCK activation and its downstream signalling events. In addition the specific inhibitors of PI3Ka and PI3Kb isoforms are powerful on blocking actin polymerization, ATP

Table 1 Effect of selected compounds on aggregation induced by 2-AG. Addition

IC50 (mM)

LY294002 CH5132799 TGX221 MK2206 Y27632

13.7 4.2 2.8 6.1 15.1

± ± ± ± ±

2.8 1.4 0.8 1.9 4.6

Washed platelets (3.0  108 platelets/mL) were preincubated with the agent for 2 min at 37  C and then stimulated with 10 mM 2-AG for 3 min. Values are the mean ± SD of five separate determinations.

secretion and aggregation induced by 2-AG (Figs. 7B and 8B and Table 1). PI3K is un upstream regulator of AKT [40]. PI3K products phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate trigger the phosphorylation of AKT by phosphatidylinositol-dependent kinase [41]. AKT is activated by various agonists including thrombin, U46619, collagen and ADP in platelets [22,42e47]. Hirsch et al. [16] found that AKT phosphorylation by ADP is at least partially dependent on PI3Kg, while thrombin-mediated AKT phosphorylation is largely normal in the absence of PI3Kg. Likely the activation of AKT could help to promote secretion of ADP and of other mediators from platelet dense granules and a-granules as previously reported by Woulfe et al. [43]. Results reported in Fig. 5 indicate that RhoA/ROCK activation is partially dependent on the PI3K/AKT pathway although the G12/13dependent mechanism could be also suggested to contribute to 2AG mediated RhoA/ROCK stimulation [11,47]. The precise mechanism by which stimulation of PI3K/AKT leads to RhoA/ROCK activation remains to be elucidated. However it was shown that PI3K/ AKT contributes to leptin-induced hypertrophy via the RhoA/ROCK pathways and actin polymerization [48]. Platelet shape change that is one of the earliest effect detectable in response to stimuli, results from a rapid reorganization of the cytoskeleton in which MLC phosphorylation plays a pivotal role as phosphorylated myosin interacts with actin filaments to form the myosin-actin complex involved in granule centralization process [30,32,34]. Our data along with those published previously in platelets stimulated with agonists show that phosphorylation of AKT, predominantly PI3Kdependent, is involved in 2-AG mediated processes of platelet activation/aggregation. Data of this study indicate that 2-AGinduced MLC phosphorylation occurs largely on thr18 residue via RhoA/ROCK pathway activation. This finding is strengthened by data of the present report showing that MLC phosphorylation is abolished by Y27632 (Fig. 1A). Thus the RhoA/ROCK signalling pathway is essential for MLC phosphorylation on thr18 residue. We found that in human platelets stimulated by 2-AG MLC phosphorylation on thr18 peaks at 15 s (Fig. 2A) and precedes MLC phosphorylation on ser19 that reaches the maximum level 60 s after agonist addition (Fig. 2B). The Ca2þ-independent MLC phosphorylation of thr18 residue is downstream of G12/13 regulated RhoA/ ROCK pathway and ser19 phosphorylation is a downstream Gq/ Ca2þ-dependent mechanism [27]. The MLC phosphorylation at thr18 seems to be crucial for ATP secretion [48]. According to data of this study 2-AG stimulates ATP secretion in a dose and timedependent fashion (Fig. 8A). ATP secretion is significantly (p < 0.0005) inhibited by LY294002, MK2206 and Y27632 (Fig. 8B)

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