theg -coupledp2y12receptorregulates diacylglycerol ... · pdf...

The Gi-coupled P2Y12 Receptor RegulatesDiacylglycerol-mediated Signaling in Human Platelets*

Received for publication, February 27, 2008, and in revised form, August 27, 2008 Published, JBC Papers in Press, August 28, 2008, DOI 10.1074/jbc.M801588200

Gianni F. Guidetti‡1, Paolo Lova‡1, Bruno Bernardi‡, Francesca Campus‡, Gianluca Baldanzi§, Andrea Graziani§,Cesare Balduini‡, and Mauro Torti‡2

From the ‡Center of Excellence for Applied Biology, Department of Biochemistry, University of Pavia, via Bassi 21, 27100 Pavia, Italyand the §Department of Clinical and Experimental Medicine, University Amedeo Avogadro, via Solaroli 17, 28100 Novara, Novara, Italy

Stimulation of Gq-coupled receptors activates phospholipaseC and is supposed to promote both intracellular Ca2� mobiliza-tion andprotein kinaseC (PKC) activation.We found thatADP-induced phosphorylation of pleckstrin, the main platelet sub-strate for PKC, was completely inhibited not only by anantagonist of the Gq-coupled P2Y1 receptor but also uponblockade of the Gi-coupled P2Y12 receptor. The role of Gi onPKC regulation required stimulation of phosphatidylinositol3-kinase rather than inhibition of adenylyl cyclase. P2Y12antagonists also inhibited pleckstrin phosphorylation, Rap1bactivation, and platelet aggregation induced upon Gq stimula-tion by the thromboxane A2 analogue U46619. Importantly,activation of phospholipase C and intracellular Ca2� mobiliza-tion occurred normally. Phorbol 12-myristate 13-acetate over-came the inhibitory effect of P2Y12 receptor blockade on PKCactivation but not on Rap1b activation and platelet aggregation.By contrast, inhibition of diacylglycerol kinase restored bothPKC and Rap1b activity and caused platelet aggregation. Stim-ulation of P2Y12 receptor or direct inhibition of diacylglycerolkinase potentiated the effect of membrane-permeable sn-1,2-dioctanoylglycerol on platelet aggregation and pleckstrin phos-phorylation, in association with inhibition of its phosphoryla-tion to phosphatidic acid. These results reveal a novel andunexpected role of the Gi-coupled P2Y12 receptor in the regu-lation of diacylglycerol-mediated events in activated platelets.

It is generally accepted that, with a very few exceptions, plate-let response to soluble agonists originates from the conver-gence of at least two different signal transduction pathwaystypically initiated by the heterotrimeric G-proteins Gq and Gi.Several receptors for platelet agonists are coupled toGq, includ-ing the P2Y1 receptor forADP, theTP� receptor for the throm-boxane A2 (TxA2),3 and the PAR1 and PAR4 receptors for

thrombin (1, 2). Signaling downstream Gq involves the stimu-lation ofmembers of the� subfamily of phospholipase C (PLC),which generate the second messengers inositol 1,4,5-trisphos-phosphate (IP3) and diacylglycerol (DAG), responsible forCa2�

mobilization from intracellular stores and protein kinase C(PKC) activation, respectively (3). This pathway is necessarybut not sufficient to elicit a full platelet response, and the con-comitant activation of a Gi-coupled receptor is absolutelyman-datory (4).To fulfill this requirement, some agonists, like ADP, directly

stimulatemultipleG-protein coupled receptors, one ofwhich isassociated to Gi (5, 6), whereas others, such as TxA2, rely on theauxiliary action of secondary messengers released by activatedplatelets to stimulate a Gi-coupled receptor (7). Despite theiressential role, there are very few Gi-coupled receptors on theplatelet surface, and these include the �2-adrenergic receptorfor epinephrine and the P2Y12 receptor for ADP (8, 9). There-fore, ADP, which is also one of the major component of theplatelet releasate, is a crucial regulator of platelet function, andits receptors have gained increasing attention as potential tar-get for anti-thrombotic agents, including thienopyridines(9–11).The biochemical basis for the critical role ofGi stimulation in

platelet aggregation is not completely known. Inhibition of ad-enylyl cyclase and reduction of intracellular cAMP levels do notappear sufficient to complement Gq-dependent pathway forplatelet aggregation (12). However, binding of ADP to the Gi-coupled P2Y12 receptor regulates, through the G protein ��dimers, additional intracellular effectors, includingmembers ofthe class I phosphatidylinositol 3-kinase and the small GTPaseRap1b (13–17). Activation of phosphatidylinositol 3-kinase liesupstream stimulation of Rap1b (15–18), which in turn hasemerged as a critical regulator of integrin �IIb�3 and plateletaggregation (19–21). The phosphatidylinositol 3-kinase-Rap1bpathway has been proposed to be responsible for the Gi-medi-ated contribution to platelet aggregation (15, 18, 22–26), butmany aspects are not clearly defined.In this work we report that whereas activation of PLC is

exclusively controlled by Gq-coupled receptors, the down-stream stimulation of PKC requires the concomitant activationof the Gi-coupled P2Y12 receptor. Moreover, we show that allthe inhibitory effects of P2Y12 receptor antagonists on plateletfunction can be reversed by pharmacologic inhibition of theDAGmetabolizing enzymediacylglycerol kinase (DGK) but notby direct stimulation of PKC.These results indicate thatDAG is

* This work was supported by grants from the Ministero dell’Istruzione, Uni-versita e Ricerca Scientifica (MIUR, PRIN 2006), from the University of Pavia,and from the Consorzio Interuniversitario Biotecnologie. The costs of pub-lication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement” inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 These authors equally contributed to the work.2 To whom correspondence should be addressed. Tel.: 39-0382-987238; Fax:

39-0382-987240; E-mail: [email protected] The abbreviations used are: TxA2, thromboxane A2; DiC8, sn-1,2 dioctanoyl-

glycerol; PLC, phospholipase C; DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphosphate; PKC, protein kinase C; DGK, diacylglycerol kinase; PMA,phorbol 12-myristate 13-acetate; PIPES, 1,4-piperazinediethanesulfonic acid;PA, phosphatidic acid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 43, pp. 28795–28805, October 24, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

OCTOBER 24, 2008 • VOLUME 283 • NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 28795

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FIGURE 1. Co-stimulation of P2Y1 and P2Y12 receptors is required for ADP-induced pleckstrin phosphorylation. A, 32P-labeled platelets were leftuntreated (bas) or were stimulated with 10 �M ADP in the absence of inhibitors or in the presence of the P2Y1 receptor antagonist MRS2179 (200 �M) or theP2Y12 receptor antagonist AR-C69931MX (1 �M) as indicated on the top for times ranging from 30 s to 3 min, as indicated on the bottom. As control, plateletswere stimulated with 1 unit/ml thrombin (THR). Platelet proteins were separated by SDS-PAGE, and phosphorylated pleckstrin, indicated by the arrow on theright, was identified by autoradiography. B, autoradiographic analysis of pleckstrin phosphorylation in 32P-labeled platelets left untreated (bas) or stimulatedwith 100 nM PMA for the indicated times in the absence or in the presence of 1 �M AR-C69931MX. C, platelets were preincubated with buffer (none) or with theP2Y1 and P2Y12 receptors antagonists MRS2179 (200 �M), AR-C69931MX (1 �M), and 2MeSAMP (1 �M) as indicated on the bottom and then were left untreated(bas) or stimulated with 10 �M ADP for 1 min. PKC-directed protein phosphorylation was analyzed by immunoblotting with an anti-phospho(Ser) PKCsubstrates antibody. The position of the 47-kDa pleckstrin is indicated by the arrow on the right. D, platelets were incubated in the absence or presence of theP2Y12 receptor antagonist 2MeSAMP (1 �M) and then stimulated with the indicated doses of ADP for 1 min. Immunoblotting with an anti-phospho(Ser) PKCsubstrates antibody was used to detect phosphorylated pleckstrin, indicated by the arrow on the right. E, platelets were incubated with the P2Y12 receptorantagonists AR-C69931MX or 2MeSAMP in combination with the adenylyl cyclase inhibitor dideoxyadenosine (DDA, 100 �M) or were treated with thephosphatidylinositol 3-kinase inhibitors LY294002 (25 �M) or wortmannin (50 nM) as indicated on the bottom. Upon stimulation with 10 �M ADP, pleckstrinphosphorylation, indicated by the arrow, was analyzed by immunoblotting with the anti-phospho(Ser) PKC substrates antibody.

Gi-mediated Regulation of DAG Signaling in Platelets

28796 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 43 • OCTOBER 24, 2008


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a critical messenger for platelet aggregation regulated throughGi-dependent signaling pathways.

EXPERIMENTAL PROCEDURES

Materials—ADP, U46619, thrombin, MRS2179, 2MeSAMP,phorbol 12-myristate 13-acetate (PMA), 1,2-dioleoylglycerol,prostaglandin E1, acetylsalicylic acid, sn-1,2 dioctanoylglycerol(DiC8), and apyrase were from Sigma. [32P]Orthophosphate,myo-[2-3H]inositol (16 Ci/mmol), and [5,6,8,9,11,12,14,15-3H(N)]-arachidonic acid (189 Ci/mmol) were from GE Health-care. Dideoxyadenosine, wortmannin, and LY294002 werefrom Alexis (Vinci-Biochem, Vinci, Italy). FURA-2-AM wasfrom Calbiochem. AR-C69931MX was a generous gift fromAstraZeneca (Charnwood, UK). The rabbit polyclonal antibod-ies against Rap1 (121) was from Santa Cruz Biotechnology(Tebu-Bio, Magenta, Italy). The rabbit polyclonal antibodyagainst phospho(Ser) PKC substrates was from Cell SignalingTechnology (Celbio, Pero, Italy).

Platelet Isolation—Human plate-lets were obtained from healthy vol-unteers in citric acid/citrate/dex-trose (152 mM sodium citrate, 130mM citric acid, 112 mM glucose).Whole blood was centrifuged at120 � g for 10 min at room temper-ature. Apyrase (0.2 units/ml), pros-taglandin E1 (1 �M), and acetylsali-cylic acid (1mM)were then added tothe platelet-rich plasma. Plateletswere recovered by centrifugation at720 � g for 15 min, washed with 10ml of PIPES buffer (20 mM PIPES,136 mM NaCl, pH 6.5), and finallygently resuspended inHEPES buffer(10 mM HEPES, 137 mM NaCl, 2.9mM KCl, 12 mM NaHCO3, pH 7.4).The cell count was typicallyadjusted to 0.3 � 109 platelets/mlunless otherwise stated.Platelet Stimulation and Meas-

urement of Aggregation—All theexperiments were performed with0.1–0.4-ml samples of washedplatelets placed at 37 °C in an aggre-gometer under constant stirring inthe presence of 1 mM CaCl2. Plate-lets were stimulated with 10 �MADP or 1 �M U46619 for 1 minunless otherwise stated in the figurelegends. Preincubation with specificinhibitors, activators, or receptorantagonists was as follows: 1 or 2.5nMPMA for 1min, 0.1–3.5�MDiC8for 1 min, 200 �M MRS2179, 1 �MAR-C69931MX or 2MeSAMP for 2min, 1 �M R59949 for 5 min, 50 nMwortmannin, 25 �M LY294002 for15 min, 100 �M dideoxyadenosine

for 30 min. When compounds were dissolved in DMSO, anequal volume of vehicle was added to control and samples. Foraggregation measurement, stimulation was prolonged up to 7min, and light transmission was continuously monitored. Allthe aggregation traces reported in the figures are representativeof al least three different experiments.Measurement of Pleckstrin Phosphorylation and DiC8-PA

Production Using 32P-Labeled Platelets—Platelets, at the finalconcentration of 109 cells/ml in PIPES buffer, were incubatedwith 0.1 mCi/ml 32P for 90 min at 37 °C, centrifuged at 800 � gfor 15min, and finally resuspended inHEPES buffer containing1 mM CaCl2 and 5.5 mM glucose. Samples (0.2 ml) were prein-cubatedwith different inhibitors and then stimulatedwithADPor U46619 as above indicated. The reaction was stopped by theaddition 0.1ml of SDS sample buffer 3X (37.5mMTris/HCl, pH8.3, 288 mM glycine, 6% SDS, 1.5% dithiothreitol, 30% glycerol,0.03% bromphenol blue) and by heating at 95 °C for 3 min.Identical aliquots of total platelet proteins (20 �l) were sepa-

FIGURE 2. Stimulation of the P2Y12 receptor by ADP is required for U46619-induced pleckstrin phospho-rylation. Analysis of pleckstrin phosphorylation induced by U46619 was performed both by autoradiographywith 32P-labeled platelets (panel A) and by immunoblotting with the anti-phospho(Ser) PKC substrates anti-body (panels B and C). bas, untreated. Platelets were stimulated with 1 �M U46619 for the indicated times (panelA) or for 1 min (panels B and C) in the absence or presence of receptor antagonists or 1 mM RGDS peptide, asindicated. In panel D platelets were stimulated with the indicated concentrations of U46619 in the absence orpresence of the P2Y12 receptor antagonist AR-C69931MX (1 �M) as indicated. The arrow on the right indicatesthe position of the 47-kDa protein pleckstrin. Quantification of pleckstrin phosphorylation induced by U46619was performed by densitometric analysis of seven different experiments and is reported in panel E. Pleckstrinphosphorylation induced by U46619 in the absence of inhibitors was considered as 100%. The results are themean � S.D. of seven different experiments (*, p � 0.01 versus control).




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rated by SDS-PAGE on a 5–15% acrylamide gradient gel andstained with Coomassie Blue. Gels were then dried, and phos-phorylation of pleckstrin was visualized upon autoradiographyfor about 18 h at �80 °C.For measurement of DiC8 conversion to DiC8-PA, samples

of 32P-labeled platelets (0.2 ml) were treated with 500 nM DiC8in the presence or in the indicated concentrations of ADP plus200 �MMRS2179, 1 �M AR-C69931MX, or 1 mM R59949 for 1min. The reaction was stopped by the addition of 0.275 ml ofchloroform and 0.55 ml of methanol. Phase partition wasobtained through the addition of 0.275 ml of chloroform and0.44 ml of 1 M HCl. Upon centrifugation at 800 � g for 10 min,the lipids recovered in the lower phasewere spotted on silica gelTLC plates and eluted in ethyl acetate/iso-octane/acetic acid/water (45:20:12:6). Spots on the TLC plates were visualized byautoradiography for about 18 h at �80 °C, and DiC8-derivedPA production was quantified by analysis through ImageJsoftware.Evaluation of PKC-dependent Protein Phosphorylation Using

an Anti-phospho(Ser) PKC Substrate Antibody—Platelet stim-ulation on 0.1-ml samples (see above) was stopped by the addi-tion of 0.05 ml of SDS sample buffer 3X. Proteins were sepa-rated by SDS-PAGE on a 5–15% acrylamide gradient gel,transferred to nitrocellulose, and probed by immunoblottingusing an anti-phospho(Ser) PKC substrates antibody diluted1:1000, as previously described (24). Immunoreactive bandswere visualized by enhanced chemiluminescence reaction.Quantification of pleckstrin phosphorylationwas performed bydensitometric analysis. All the reported figures are representa-tive of at least three different experiments.Measurement of Cytosolic Ca2� Concentration—Intracellu-

lar Ca2� concentration was measured in Fura-2-AM-loadedplatelets essentially as previously described (24). For theseexperiments, the final platelet concentration was 2 � 108 cells/ml, and preincubation and stimulation was performed on0.4-ml samples prewarmed at 37 °C under gentle stirring in aPerkinElmer Life Sciences LS3 spectrofluorometer in the pres-ence of 1 mM EGTA. All determinations were repeated at leastfour times with platelets from different donors.Measurement of [3H]Inositol Phosphate Production—Plate-

lets in PIPES buffer (2 � 109 cells/ml) were labeled with 0.125mCi/ml myo-[2-3H]inositol for 3 h at 37 °C, centrifuged, andfinally resuspended in HEPES buffer containing 5.5 mM glu-cose, 0.5 mM RGDS, 1 mM CaCl2, and 10 mM LiCl at the con-centration of 109 cells/ml. Platelet samples (0.4 ml) were incu-bated in the presence or absence of 1 �M AR-C69931MX andstimulated under constant stirring with 1 �M U46619. Thereaction was stopped by adding 0.55 ml of chloroform and 1.1ml of methanol. Samples were placed on ice, and phase parti-tion was obtained by the addition of 0.55 ml of chloroform and0.88 ml of 1 M HCl. The upper phase (2 ml), collected uponcentrifugation at 800 � g for 10 min, was neutralized with 1 mlof 2 M ammonium acetate, and [3H]inositol phosphates wereseparated by ion exchange chromatography on 1 ml of AG 1-Xresin, as previously described (27) and quantified by scintilla-tion counting in the gel phase.Measurement of DAG Accumulation—Platelets in PIPES

buffer (109 cells/ml) were labeled with 2.5 �Ci/ml [3H]arachi-

donic acid for 30 min at 37 °C and then resuspended in HEPESbuffer containing 5.5 mM glucose, 0.5 mM RGDS, and 1 mMCaCl2. Incubation and stimulation of platelet samples (0.2 ml)as well as phase partition was performed as described for theinositol phosphates measurement; however, the lower phases(0.4 ml) were collected and dried under N2 flux. Lipids wereresuspended with 20 �l of methanol:chloroform (1:1). Eachsample was mixed with 1 �g of standard purified DAG andspotted on silica gel TLC plates. Elution was performed withdiethyl ether:exane:acetic acid (70:30:1), and DAG spots werevisualizedwith iodine vapors. The spots corresponding toDAGwere scraped, and the radioactivity was measured by scintilla-tion counting.Rap1b Activation Assay—Activation of Rap1b in platelet

samples (0.2 ml) was evaluated by a pulldown assay using theGST-tagged Rap binding domain of RalGDS (GST-RalGDS-RBD) essentially as previously reported (28). Quantification of

FIGURE 3. Analysis of phospholipase C activation and intracellular Ca2�

mobilization. A, platelets were preincubated with buffer (black bars) or with1 �M AR-C69931MX (white bars) and then stimulated with 1 �M U46619 for 1min. Accumulation of DAG was measured using [3H]arachidonic acid-labeledplatelets, whereas myo-[3H]inositol-labeled platelets were used to measureagonist-induced accumulation of soluble inositol phosphates (IP). Results arereported as raw measured DPM after subtraction of the radioactivity of non-stimulated samples and are the means � S.D. of three different experiments.B, intracellular Ca2� concentration in Fura-2-loaded platelets stimulated with1 �M U46619 in the absence (none) or presence of apyrase or antagonists ofthe P2Y12 receptor as indicated. In resting platelets, the cytosolic concentra-tion of Ca2� was 50 � 9 nM (n � 46). Results are the means � S.D. of 5– 6different experiments.




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Rap1b activation was performed by densitometric analysis ofthe immunoblots.

RESULTS

Stimulation of theGi-coupled P2Y12Receptor Is Necessary forADP- or U46619-induced PKC Activation—The possible cross-talk between P2Y1 and P2Y12 receptors in the regulation of plate-let PKC was evaluated by measuring the phosphorylation of the47-kDa protein pleckstrin, themain PKC substrate, in 32P-labeledcells (29). Autoradiographic analysis allowed the detection of aclear, albeit faint, phosphorylation of pleckstrin in ADP-stimu-lated platelets which was rapid and transient (Fig. 1A). Asexpected, pleckstrin phosphorylationwas totally suppressed uponblockade of the Gq-coupled P2Y1 receptor byMRS2179. Surpris-ingly, however, we observed that ADP-induced pleckstrin phos-

phorylation was also completely abrogated upon platelet incuba-tion with AR-C69931MX, a specific antagonist of the Gi-coupledP2Y12 receptor (Fig. 1A), which is not supposed to contribute toPLC regulation. By contrast, AR-C69931MX did not alter pleck-strin phosphorylation induced by the phorbol ester PMA (Fig. 1B)or by thrombin (data not shown).Because our analysis with 32P-labeled platelets revealed

only a rather weak metabolic labeling of the PKC substrates,we consolidated our observations with a different, immuno-logical approach using a phosphospecific antibody againstphosphorylated PKC substrates. This anti-phospho(Ser)PKC substrates antibody detected a number of proteins inresting platelets whose reactivity was clearly increased uponstimulation with ADP (Fig. 1C). Such an increase was partic-ularly evident for a 47-kDa band, which was identified as

FIGURE 4. Subthreshold concentrations of PMA restore agonist-induced pleckstrin phosphorylation but not platelet aggregation. A, analysis ofpleckstrin phosphorylation evaluated by immunoblotting with the anti-phospho(Ser) PKC substrates antibody upon incubation with 1 nM or 2.5 nM PMA in theabsence or presence of 1 �M AR-C69931MX as indicated. Stimulation was with 1 �M U46619 for 1 min. bas, untreated. B, analysis of platelet aggregation inducedby 1 or 2.5 nM PMA. C, platelet were stimulated with 1 �M U46619 in the absence (none) or in the presence of 1 �M AR-C69931MX alone or in combination with1 or 2.5 nM PMA as indicated on the right. The aggregation traces reported are representative of at least three different experiments.




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pleckstrin based on immunoblotting analysis performedwith 32P-labeled platelets (data not shown). None of thebands depicted in Fig. 1C was detected when platelets werepreincubated with the PKC inhibitor Ro31-8220 (data notshown), confirming the specificity of the antibody. Usingthis alternative and more sensitive approach, we confirmedand extended our observations. ADP-induced pleckstrinphosphorylation as well as phosphorylation of other uniden-tified PKC substrates was completely suppressed not only byMRS2179 but also by AR-C69931MX (Fig. 1C). In addition,we found that a different P2Y12 antagonist, 2MeSAMP, wasas efficient as AR-C69931MX in preventing ADP-inducedactivation of PKC (Fig. 1C). In a dose-dependent analysis, wefound that even when platelets were stimulated with doses ofADP as high as 50 �M, pleckstrin phosphorylation was com-pletely prevented by blockade of the P2Y12 receptor (Fig.1D). These results demonstrate that co-stimulation of bothP2Y1 and P2Y12 receptors by ADP is absolutely required foractivation of PKC.

In platelets, the Gi-coupledP2Y12 receptor is responsible forinhibition of adenylyl cyclase andfor activation of phosphatidylinosi-tol 3-kinase (12–16). Fig. 1E showsthat when P2Y12 receptor wasblocked by AR-C69931MX or2MeSAMP, direct inhibition of ad-enylyl cyclase by dideoxyadenosinedid not restore ADP-induced pleck-strin phosphorylation. By contrast,PKC-directed protein phosphoryla-tion in ADP-stimulated plateletswas prevented by the phosphati-dylinositol 3-kinase inhibitorsLY294002 and wortmannin (Fig.1E). These results indicate thatthe P2Y12 receptor contributes tothe regulation of PKC through thephosphatidylinositol 3-kinase-de-pendent pathway.Similarly to the P2Y1 receptor for

ADP, the platelet TxA2 receptor,TP�, is coupled to Gq and is able tostimulate PLC (1, 2). Nevertheless,platelet aggregation by TxA2 or byits stable analogue U46619 requiresconcomitant stimulation of the Gi-coupled P2Y12 receptor by secretedADP (7, 22). We investigatedwhether the P2Y12 receptor partic-ipates in PKC regulation inU46619-activated platelets. In 32P-labeledcells, U46619 induced a strongerand more sustained phosphoryla-tion of pleckstrin than ADP, which,however, was severally impairedwhen the P2Y12 receptor wasblocked by AR-C69931MX (Fig.

2A). Moreover, inhibition of U46619-induced pleckstrin phos-phorylation in AR-C69931MX- or 2MeSAMP-treated plateletswas confirmed by the immunoblotting analysis with the anti-phospho(Ser) PKC substrates antibody (Fig. 2B). Incidentally,we noticed that the analysis of pleckstrin phosphorylation byimmunoblotting with anti-phospho(Ser) PKC substrates anti-body, which certainly generates stronger signals than the auto-radiography approach, does not allow a reliable comparison ofthe strength of the response to different agonists. Although thisobservation has no impact on the present study, it certainlyshould be taken into considerationwhen choosing to adopt thistechnique. On stimulation with U46619, but not with ADP,washed platelets may form aggregates, and outside-in signalingthrough integrin �IIb�3 may contribute to PLC activation andPKC stimulation. To rule out the possibility that the inhibitoryeffect of P2Y12 antagonists on U46619-induced pleckstrinphosphorylation was a consequence of the inhibition of aggre-gation, comparative analysis were performed in the absenceand presence of the integrin antagonist RGDS. Fig. 2C shows

FIGURE 5. Inhibition of pleckstrin phosphorylation and platelet aggregation by P2Y12 receptor antag-onists is overcome by inhibition of diacylglycerol kinase. A, accumulation of DAG in platelets treated with1 �M R59949 for 10 min. The levels of DAG were measured using [3H]arachidonic acid-labeled platelets. Theresults are the mean � S.D. of three different experiments (*, p � 0.05 versus control). B, analysis of pleckstrinphosphorylation in platelets incubated with 1 �M of the DGK inhibitor R59949 for increasing times. As control,pleckstrin phosphorylation induced by 1 �M U46619 is reported. C, platelets were incubated without or with 1�M R59949, as indicated, left untreated (bas), or stimulated with 1 �M U46619 for 1 min in the absence orpresence of the P2Y12 antagonists AR-C69931MX or 2MeSAMP (1 �M each), as indicated. Phosphorylation ofpleckstrin was evaluated by immunoblotting with the anti-phospho(PKC) substrates antibody. D, plateletswere treated with buffer (none) or with the P2Y12 antagonists AR-C69931MX or 2MeSAMP alone or in combi-nation with the DGK inhibitor R59949 in a lumiaggregometer, and aggregation was initiated by the addition of1 �M U46619 as indicated by the arrow. The figure reports aggregation traces representative of at least threedifferent experiments.




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that even under non-aggregatingconditions U46619-induced pleck-strin phosphorylation was inhibitedby AR-C69931MX. A dose-depend-ent analysis revealed that inhibitionof pleckstrin phosphorylation byblockade of the P2Y12 receptor wasstill evident when platelets werestimulated with higher doses ofU46619 (Fig. 2D). In all the experi-ments performed, we noticed thatwhereas inhibition of pleckstrinphosphorylation was constantlycomplete upon stimulation withADP, some relevant variability wasobserved on stimulation withU46619 (see for instance, Figs. 2, Band C). However, an accurate quan-titative analysis of several differentexperiments revealed that theinhibitory effects of the antagonistsof the P2Y12 receptor on U46619-induced pleckstrin phosphorylationwas relevant, as it accounted forabout 70–80% and was statisticallysignificant (Fig. 2E).TheP2Y12ReceptorDoesNot Influ-

ence Gq-mediated Activation of Phos-pholipase C Induced by U46619—The effect of P2Y12 antagonists onthe PKC activity could reflect across-talk between Gi and Gq at thelevel of PLC activation. AlthoughADP is clearly able to induce PLCactivation under our experimentalconditions, as revealed by measure-ment of PKC activation (Fig. 1)and intracellular Ca2� mobiliza-tion (data not shown), we have beenunable to reliably measure agonist-induced accumulation of inositolphosphates or DAG, probablybecause of the weakness of theresponse (data not shown), as sug-gested by early studies (30). There-fore, we took advantage from theevidence that the P2Y12 receptor isalso implicated in PKC activationinduced by U46619, which is astronger activator of PLC. Using[3H]inositol- or [3H]arachidonicacid-labeled platelets, we found thatneither accumulation of inositolphosphates nor production of DAGwas affected by AR-C69931MX inU46619-treated platelets (Fig. 3A).Signals downstream of PLC branchinto IP3-Ca2� and DAG-PKC path-

FIGURE 6. Inhibition of U46619-induced Rap1b activation by the P2Y12 receptor antagonists isreversed by R59949 but not by PMA. Rap1b activation induced by stimulation of platelets with 1 �M

U46619 for 1 min was analyzed by the pulldown assay with GST-RalGDS-RBD followed by immunoblottingwith anti Rap1 antibody. In panel A platelets were preincubated with either 1 �M AR-C69931MX or 1 �M

2MeSAMP, and the effect of 1 �M R59949 was analyzed. TOT, total. In panel B some samples were incubatedwith AR-C69931MX, and the effect of 1 nM PMA is reported. The upper panels show the active form ofRap1b (GTP-Rap1b), whereas the lower panels report the level of total Rap1b present in the platelet lysates.The levels of Rap1b activation were quantified by densitomeric analysis of the immunoblots, and theresults are summarized in panel C. The amount of active Rap1b in U46619-stimulated platelets in theabsence of any inhibitor was taken as 100%. Data are the mean � S.D. of eight different experiments. Therecovery of Rap1b activity caused by inhibition of DGK with R59949, albeit partial, was found statisticallysignificant.




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ways. Although DAG-mediated activation of PKC was almostcompletely suppressed by the antagonists of P2Y12 receptor(Fig. 2), we found that, under the same conditions, IP3-medi-ated mobilization of intracellular Ca2� was not affected (Fig.3B). Altogether our results indicate that blockade of the P2Y12receptor does not likely affect Gq-mediated PLC activation inU46619-stimulated platelets.Restoration of PKCActivityDoesNotOvercome the Inhibition of

Platelet Aggregation Induced by P2Y12 Receptor Antagonists—It has been previously shown that both ADP- and U46619-in-duced platelet aggregation can be abrogated by blockade of theGi-coupled P2Y12 receptor (5, 7). We addressed the possibilitythat this effect could be a consequence of the impaired PKCactivation here described, and we analyzed whether restorationof PKC activity by subthreshold doses of PMA could overcomethe inhibitory effect of AR-C69931MX. Using U46619-stimu-lated platelets like a model, we found that treatment with 1 nMPMA did not result in either pleckstrin phosphorylation orplatelets aggregation,whereas 2.5 nMPMA induced aweak acti-vation of PKC, associated to a very modest platelet aggregation(Figs. 4A and 4B). Fig. 4A shows that both 1 and 2.5 nM PMAwere able to synergize with U46619 and restored normal PKCactivation upon blockade of the P2Y12 receptor byAR-C69931MX.However, inhibition ofU46619-induced plate-let aggregation byAR-C69931MXwas not overcomeby either 1or 2.5 nM PMA (Fig. 4C) despite normal PKC activation.Inhibition of Diacylglycerol Kinase Bypasses the Need of

P2Y12 Stimulation for Agonist-induced PKC Activation andPlatelet Aggregation—The endogenous PKC activator DAG istypicallymetabolized andneutralized through phosphorylationby DGK, which, therefore, may indirectly regulate PKC func-tion. To investigate whether DGK could be targeted by P2Y12,we analyzed the effect of a cell-permeable DGK inhibitor,R59949, on AR-C69931MX- and 2MeSAMP-mediated inhibi-tion of platelet aggregation and pleckstrin phosphorylationinduced by U46619. R59949 alone promoted only a small, butstatistically significant increase of the levels of endogenousDAG, and caused a weak activation of PKC after 10 min (Fig. 5,A and B) but did not cause any detectable platelet aggregation(data not shown). Preincubation of platelets with R59949 for 5min before stimulation with U46619 resulted in a very smallincrease of pleckstrin phosphorylation and in a modest poten-tiation of aggregation (Fig. 5,C andD). However, Fig. 5C showsthat inhibition of PKC-directed protein phosphorylation byAR-C69931MX or 2MeSAMP in U46619-stimulated plateletswas completely overcome by R59949. Similar results were alsoobtained when platelets were stimulated with ADP (data notshown). Interestingly, in the presence of R59949, U46619

induced evident platelet aggregation despite the inhibition ofGisignaling by AR-C69931MX or 2MeSAMP (Fig. 5D). Theseresults indicate that the need of P2Y12 stimulation for Gq-ini-tiated platelet aggregation can be bypassed by the inhibition ofDGK rather than by activation of PKC.Beside activating PKC, PLC-generated DAG regulates other

intracellular effectors, including CalDAG-GEFI, a nucleotideexchange factor for the small GTPase Rap1 (20, 21), which isimportant for integrin �IIb�3 activation and platelet aggrega-tion (19). As previously reported (13), we confirmed thatU46619-induced activation of Rap1b was prevented byAR-C69931MX and 2MeSAMP (Fig. 6, A and B). However, wealso found that the DGK inhibitor R59949, but not the PKCactivator PMA, was able to partially overcome the effect of theP2Y12 receptor blockade on agonist-induced activation ofRap1 (Fig. 6, A and B). Because the recovery of Rap1b activitywas not complete in the presence of R59949, we performedan accurate quantitative analysis of immunoblots from eightdifferent experiments. The results are reported in Fig. 6Cand show that in the presence of the P2Y12 receptor antag-onists, U466619-induced activation of Rap1b was restoredby about 50% by R59949 and that this effect resulted statis-tically significant.Finally we investigated the interplay between Gi-dependent

signaling and DAG-mediated effects on platelet activationusing the cell-permeable DAG analogue, DiC8. A weak plateletaggregation was observed upon treatment of platelets with 3.5�M but not with 2 �MDiC8 (Fig. 7A). Concomitant stimulationof P2Y12 receptor through the addition of ADP in the presenceof MRS2179 significantly potentiated the effect of DiC8 onplatelet aggregation (Fig. 7A). DiC8-induced PKC activationwas dose-dependent and occurred in a range of concentrationsmuch lower than those required for aggregation (Fig. 7B) Wefound that the faint pleckstrin phosphorylation detectable at100 nM DiC8 was clearly potentiated by the DGK inhibitorR59949 (Fig. 7Bi). TLC analysis of the lipids extracted from32P-labeled platelets revealed that exogenous DiC8was activelyphosphorylated to DiC8-PA by a DGK activity in intact cellsand that this process was inhibited upon platelet incubationwith R59949 (Fig. 7C). Similarly to R59949, we found that stim-ulation of the P2Y12 receptor by ADP in the presence ofMRS2179 resulted in a potentiation of pleckstrin phosphoryla-tion induced by exogenous DiC8 at all the doses analyzed (Fig.7Bii). Concomitantly, an evident inhibition of DiC8 conversionto DiC8-PA in intact platelets was observed upon stimulationwith ADP. This effect of ADP in 32P-labeled cells was dose-de-pendent and was prevented by AR-C69931MX but not byMRS2179, indicating that it was mediated by activation of the

FIGURE 7. Potentiation of DiC8-mediated platelet activation by stimulation of P2Y12 receptor. A, platelet aggregation was measured upon addition of 2�M (i) or 3.5 �M (ii) DiC8 in the presence of buffer (none) or 10 �M ADP and 200 �M MRS2179, as indicated on the right. The reported traces are representativeof at least three different experiments producing comparable results. B, platelets were stimulated for 1 min with 0 –500 nM DiC8, as indicated at the bottom, inthe absence or presence of 1 �M R59949 (i) or 10 �M ADP and 200 �M MRS2179 (ii). PKC-directed protein phosphorylation was analyzed by immunoblottingwith an anti-phospho(Ser) PKC substrates antibody. The position of the 47-kDa pleckstrin is indicated by the arrow on the right. C, 32P-labeled platelets weretreated with 500 nM DiC8 or with an equivalent volume of DMSO, as indicated on the top of the panel (i), and then left untreated (none) or stimulated with theindicated doses of ADP for 1 min in the absence or in the presence of 200 �M MRS2179, 1 �M AR-C69931MX, or 1 �M R59949, as indicated. Lipids were extracted,separated by TLC, and visualized by autoradiography. A representative image is reported in panel i. Accumulation of DiC8-derived PA was quantified bydensitometric analysis of the autoradiographies, and the results are reported in panel (ii). The production of DiC8-PA in the presence of ADP or ADP plusantagonists is reported as percentage of that observed in samples treated with DiC8 alone. Results are the means � S.D. of four different experiments. *, p �0.01 versus non-stimulated platelets.




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P2Y12 receptor (Fig. 7C). These results support the hypothesisthat Gi-dependent signaling potentiates the effect of endoge-nous DAG by limiting its conversion to PA.

DISCUSSION

In this work we have documented a novel interplay betweenGq andGi stimulation in human platelets as we have shown thatactivation of the Gi-coupled P2Y12 receptor is essential forcomplete PKC activation in response to stimulation of Gq-cou-pled receptors by ADP or U46619. The cross-talk between Gqand Gi is most likely to occur at the level of DAG metabolism,which plays amore crucial role than PKC itself in the regulationof platelet aggregation.Gq-coupled receptors are able to stimulate PLC� isoforms,

which hydrolyze phosphatidylinositol 4,5-bisphosphate, andproduce two intracellular messengers, IP3 and DAG, whichmediate the release of Ca2� from internal stores and the acti-vation of PKC, respectively (3). According to this paradigm, wehave found that blockade of ADP binding to the Gq-coupledP2Y1 receptor abolished PKC-mediated protein phosphoryla-tion. Unexpectedly, however, we have found that antagonists ofthe Gi-coupled P2Y12 receptor totally prevented PKC activa-tion as well. Therefore, stimulation of the Gq is necessary butnot sufficient for ADP to induce PKC activation, and the con-comitant stimulation of Gi is also required. Importantly, Gi-mediated regulation of PKC is not limited to ADP-stimulatedplatelets but is a common event when Gq is stimulated. It hasbeen previously shown that stimulation of the Gq-coupled TP�receptor by the TxA2 analogue U46619 activates PLC� but isnot sufficient to trigger platelet aggregation unless activation ofa Gi-dependent pathway by binding of secreted ADP to theP2Y12 receptor occurs (2, 7, 22). In this context we have foundthat, similarly to what observed in ADP-stimulated platelets,the P2Y12 antagonists AR-C69931MX and 2MeSAMP pre-vented PKC-mediated protein phosphorylation induced byU46619. Therefore, even in this alternative experimentalmodel, PKC activation is regulated by concomitant signalsthrough Gq- and Gi-coupled receptors. Direct measurement ofinositol phosphate accumulation DAG production in U46619-stimulated platelets indicates that although Gi regulates PKCactivation, PLC is probably not affected by blockade of theP2Y12 receptor. Moreover, we also found that U46619-in-duced, IP3-dependent Ca2� mobilization from intracellularstores was not affected by ADP scavengers or antagonists (Fig.3B and Ref. 11) Previous works reported that ADP-inducedCa2� mobilization is reduced by blockade of the P2Y12 recep-tor, suggesting a cross-talk between Gq and Gi at the level ofPLC (31, 32). Using ADP as platelet agonist, we observed a com-parable partial inhibition of Ca2� release by AR-C69931MX(data not shown), but importantly, under the same conditions,pleckstrin phosphorylation was completely suppressed, indi-cating that Gi signaling exerts an additional specific control ontheDAG-PKC pathway.Moreover, it is of note thatmost of ourstudy was performed with U46619-strimulated platelets underconditions in which PLC activation and Ca2� mobilizationwere found to occur normally.We have also addressed the fundamental question as to the

functional implication of PKC regulation throughP2Y12 recep-

tor on platelet aggregation using the membrane-permeablePKC activator PMA. Subthreshold doses of PMA were actuallyable to restore normal PKC activity inU46619 (andADP)-stim-ulated platelets despite P2Y12 receptor inhibition. However,under these conditions platelets were still unable to aggregate.Therefore, blockade of P2Y12 receptor still prevents U46619-induced aggregation even if PKC is normally activated. Thisindicates that although PKC activity is regulated by Gi, this isnot the element of Gi-dependent pathway critical for plateletaggregation. A different effector similarly targeted by P2Y12receptor must be responsible for the independent but concom-itant regulation of both PKC and platelet aggregation. We pro-vide here evidence that this target may be the PKC upstreamregulator DAG. DAG is generated by activated PLC but is sub-sequently phosphorylated to PA by the enzyme DGK, whichtherefore turns off the effects ofDAGas secondmessenger (33).In this work we document that pharmacological inhibition ofDGK restores the agonist-induced phosphorylation of PKCsubstrates prevented by antagonists of the P2Y12 receptor and,in parallel, allows platelets to undergo aggregation. Impor-tantly, even AR-C69931MX-promoted inhibition of Rap1 acti-vation is overcome by pharmacologic inhibition of DGK. Thisfinding is consistent with the notion that the main nucleotideexchange factor responsible for the activation of Rap1expressed in platelets is the CAlDAG-GEFI, which is directlyactivated by DAG independently of PKC and is required forplatelet aggregation (20, 21). Our results demonstrate that theneed for Gi stimulation to trigger full platelet response can bebypassed by direct inhibition of DGK with R59949. Moreover,we have also directly demonstrated that stimulation of Gi ham-pers the conversion of exogenous DAG to PA, thus extendingand potentiating its effects on platelets. It is, therefore, possiblethat whereas DAG is generated by PLC activated through theGq-dependent pathway, its effective accumulation requiresinhibition of DGK through Gi.

Although DGK family enzymes have been known for a longtime, their biology has recently gained renewed interest (33,34). At least 10 different DGK isoforms have been identifiedwhich in many cell types are essential for proliferative andmigratory signaling (34, 35). In platelets, early works hadreported that inhibitors of DGK are able to potentiate agonist-induced secretion and aggregation, but the mechanism of thisaction has never been investigated in detail (36–38). Unfortu-nately, despite all the efforts, we have been unable to demon-strate a direct regulation of endogenous DGK by Gi in agonist-stimulated platelets. Platelets express multiple DGK isoforms(33, 39), but neither specific antibodies of acceptable qualitynor selective inhibitors for the different isoforms are commer-cially available.Wehave tried tomeasure the enzymatic activityof immunoprecipitated DGK�, one of the R59946-sensitiveDGK isoforms expressed in platelets, but we have been unableto reveal any difference upon cell stimulation (data not shown).Similarly, we have been unable to detect any stimulation-de-pendent difference in the total DGK activity measured either inwhole cell lysates, in membranes, or in cytosolic fractions (datanot shown).However, itmust be considered that all thesemeas-urements provide a value of the global amount of these metab-olites in the cell, as it results from the comprehensive action of




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all the expressed isoforms, and they are neither indicative of thecontribution of single isoforms nor informative as to the possi-bly different subcellular distribution of DAG. An increasingnumber of evidence in nucleated cells has indicated that DKGisoforms undergo complex but crucial spatial regulation (33–35). This may also be relevant in platelets, and the P2Y12-me-diated inhibition of a particular DGK isoform may transientlyoccur in specific subcellular microdomains, allowing a localincrease of DAG sufficient to efficiently trigger cellularresponses. Nevertheless, we cannot rule out the possibility thatGi-dependent pathways target intracellular effectors differentfrom DGK, producing effects equivalent to those produced byDKG inhibition and, thus, reverted by R59949. This possibilityas well as the possible involvement and contribution of selectedisoforms of DGK is a challenging task that certainly deservesfurther investigation but is pending on the future availability ofsuitable pharmacologic and genetic tools.In conclusion, our results shed new light into the signaling

pathway activated downstream of the P2Y12 receptor for ADPand required for integration of the Gq-dependent signals forplatelet aggregation, reveal a novel role for Gi in the regulationof PKC activity, and point to the importance of DAG-metabo-lizing enzymes such as DGK in the regulation of plateletactivation.

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Andrea Graziani, Cesare Balduini and Mauro TortiGianni F. Guidetti, Paolo Lova, Bruno Bernardi, Francesca Campus, Gianluca Baldanzi,

Human Platelets-coupled P2Y12 Receptor Regulates Diacylglycerol-mediated Signaling iniThe G

doi: 10.1074/jbc.M801588200 originally published online August 28, 20082008, 283:28795-28805.J. Biol. Chem.

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