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J. Biol. Chem., Vol. 279, Issue 6, 4186-4195, February 6, 2004

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Akt Activation in Platelets Depends on G_i Signaling Pathways^*

Soochong Kim, Jianguo Jin, and Satya P. Kunapuli¶||

From the Department of Physiology, the Department of Pharmacology, and the ^¶Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

Received for publication, June 11, 2003 , and in revised form, November 17, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The serine-threonine kinase Akt has been established as an important signaling intermediate in regulating cell survival, cell cycle progression, as well as agonist-induced platelet activation. Stimulation of platelets with various agonists including thrombin results in Akt activation. As thrombin can stimulate multiple G protein signaling pathways, we investigated the mechanism of thrombin-induced activation of Akt. Stimulation of platelets with a PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin resulted in Thr³⁰⁸ and Ser⁴⁷³ phosphorylation of Akt, which results in its activation. This phosphorylation and activation of Akt were dramatically inhibited in the presence of AR-C69931MX, a P2Y₁₂ receptor-selective antagonist, or GF 109203X, a protein kinase C inhibitor, but Akt phosphorylation was restored by supplemental G_i or G_z signaling. Unlike wild-type mouse platelets, platelets from G_q-deficient mice failed to trigger Akt phosphorylation by thrombin and AYPGKF, whereas Akt phosphorylation was not affected by these agonists in platelets from mice that lack P2Y₁ receptor. However, ADP caused Akt phosphorylation in G_q- and P2Y₁-deficient platelets, which was completely blocked by AR-C69931MX. In contrast, ADP failed to cause Akt phosphorylation in platelets from mice treated with clopidogrel, and thrombin and AYPGKF induced minimal phosphorylation of Akt, which was not affected by AR-C69931MX in these platelets. These data demonstrate that G_i, but not G_q or G_12/13, signaling pathways are required for activation of Akt in platelets, and G_i signaling pathways, stimulated by secreted ADP, play an essential role in the activation of Akt in platelets.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Akt (also known as protein kinase B (PKB))¹ is a 57-kDa serine/threonine kinase, which is the cellular homologue of the viral oncogene v-akt of the acutely transforming retrovirus Akt8 (1). Akt plays an important role in mediating the anti-apoptotic effect of many growth factors, and it is also overexpressed in several cancer forms (2, 3). Akt contains a pleckstrin homology domain adjacent to a centrally located catalytic domain that is connected to a short C-terminal tail (4). The catalytic domain of Akt contains a Thr³⁰⁸ phosphorylation site and displays high homology to the catalytic domains of cAMP-dependent protein kinase A and protein kinase C. A second phosphorylation site, Ser⁴⁷³, is located in the C-terminal tail (4). There are at least three different Akt isoforms identified in humans, which display more than 80% sequence homology and are named Akt1 (PKB), Akt2 (PKB), and Akt3 (PKB) (5). It has been shown that Akt1 (PKB) and Akt2 (PKB) are expressed in human platelets, whereas Akt3 (PKB) is not present in human platelets (6). Both translocation of Akt to cell membranes and phosphorylation of both Thr³⁰⁸ and Ser⁴⁷³ are required for full enzyme activity. Akt is activated by various agonists including platelet-derived growth factor, epidermal growth factor, insulin, nerve growth factor, U46619 [GenBank] (a thromboxane A₂ analogue), convulxin (a glycoprotein VI agonist), and thrombin (6–8). Phosphatidylinositide (PI) 3-kinase inhibitors prevent activation of Akt (9), indicating that PI 3-kinase is an upstream regulator of Akt. PI 3-kinase products PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ trigger the simultaneous phosphorylation of Akt by phosphatidylinositol-dependent kinases (PDK) 1 and 2. Membrane attachment of Akt is a prerequisite for phosphorylation by PDK1 and 2. PDK1 has been shown to phosphorylate Thr³⁰⁸ of Akt1, and the phosphorylation of Ser⁴⁷³ is independent of Thr³⁰⁸ (10). Ser⁴⁷³ is phosphorylated by a kinase whose identity is still obscure but has been proposed as PDK2. Some researchers propose that autophosphorylation at the Ser⁴⁷³ site is the mechanism by which this serine residue is phosphorylated (11). Recently, the integrin-linked kinase has been shown to be an important regulator of Ser⁴⁷³ phosphorylation (12). In addition to the PI 3-kinase-mediated activation of Akt, a phospholipase ₂-dependent pathway via a calcium-dependent PKC subtype has been known to increase activity of Akt1 by thrombin (6). Activation of PKC results in the PI 3-kinase-independent increase in Akt1 activity by selective phosphorylation of Ser⁴⁷³, but selective phosphorylation of Ser⁴⁷³ is insufficient for full PKB activation. Yano et al. (13) have also identified Ca²⁺-dependent regulation of Akt in which Ca²⁺/calmodulin-dependent protein kinase kinase can activate Akt in vitro through phosphorylation of Thr³⁰⁸.

It is well established that ADP-induced platelet aggregation requires concomitant signaling from both the P2Y₁ and P2Y₁₂ receptors that couple to G_q and G_i, respectively (14). Thrombin mediates its cellular effects primarily through a family of G_q-coupled protease-activated receptors (PARs), and we have shown that thrombin and thrombin receptor-activating peptides cause G_i stimulation through P2Y₁₂ receptor activation by secreted ADP (15). It has been shown that stimulation of platelets with thrombin results in Akt activation (6), and Akt functions as a downstream effector of PI 3-kinase. However, the signal transduction mechanisms of Akt activation are less clear, and it is not well established whether Akt activation in platelets is stimulated by G_q- or G_i-coupled pathways.

In this study, we have investigated the signaling events involved in Akt activation mediated by thrombin and thrombin receptor-activating peptides in platelets, giving special emphasis to the role of secretion and G_i-coupled receptors in regulation of Akt. In human and mouse platelets, we have found that thrombin- and thrombin receptor-activating peptide-mediated Akt activation is largely dependent on signaling through the P2Y₁₂ receptor and PI 3-kinase. We also show that thrombin- and AYPGKF-induced Akt phosphorylation is eliminated in G_q-deficient mouse platelets. Using platelets from mice lacking G_q, lacking P2Y₁ receptor, and clopidogrel-dosed mice, we demonstrate that G_i-coupled P2Y₁₂ receptor is required for agonist-stimulated Akt phosphorylation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Thrombin, MRS-2179, apyrase (type V), PMA, epinephrine, fibrinogen (type I), and bovine serum albumin (fraction V) were purchased from Sigma. Hexapeptides SFLLRN and AYPGKF were custom-synthesized at Research Genetics Inc. (Huntsville, AL). Anti-Akt and anti-phospho-Akt (Ser⁴⁷³ and Thr³⁰⁸) antibodies and Akt kinase assay kit were purchased from Cell Signaling Technology (Beverly, MA). Alkaline phosphatase-labeled secondary antibody was from Kirkegaard & Perry Laboratories (Gaithersburg, MD). CDP-Star® chemiluminescent substrates were purchased from Applied Biosystems (Foster City, CA). LY294002 was from Biomol%20Research%20Laboratories">Biomol Research Laboratories (Plymouth Meeting, PA). 5,5'-Dimethyl-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (dimethyl-BAPTA) was obtained from Molecular Probes (Eugene, OR). AR-C69931MX was a gift from Astra-Zeneca (Loughborough, UK). Bisindolylmaleimide I (GF 109203X) was from Calbiochem. Clopidogrel was from Bristol-Myers Squibb Co. All other reagents were reagent-grade, and deionized water was used throughout.

Animals—G_q-deficient mice were obtained from T. Kent Gartner (16), with permission from Stefan Offermanns, University of Heidelberg, Germany. P2Y₁-deficient mice were generated by subcontract with Lexicon Genetics Inc. (Woodlands, TX) through knockout constructs as described previously (17, 18). The phenotype of P2Y₁ receptor-deficient mouse platelets was confirmed by platelet function studies.

Preparation of Human Platelets—Human blood was obtained from a pool of healthy volunteers in a one-sixth volume of ACD (2.5 g of sodium citrate, 1.5 g of citric acid, and 2.0 g of glucose in 100 ml of H₂O). Platelet-rich plasma (PRP) was prepared by centrifugation of citrated blood at 230 x g for 20 min at room temperature (RT). Acetylsalicylic acid was added to PRP to a final concentration of 1 mM, and the preparation was incubated for 1 h at 37 °C followed by centrifugation at 1000 x g for 10 min at RT. The platelet pellet was resuspended in Tyrode's buffer (pH 7.4) containing 138 mM NaCl, 2.7 mM KCl, 2 mM MgCl₂, 0.42 mM NaH₂PO₄, 5 mM glucose, 10 mM HEPES, 0.2% bovine serum albumin, and 0.05 units/ml apyrase. This low concentration of apyrase is not enough to block responses to ADP but will prolong the responsiveness of platelets to ADP by preventing desensitization of the P2Y receptors. These conditions have been standardized in the laboratory, with a platelet count adjusted to 2 x 10⁸ cells/ml.

Preparation of Mouse Platelets—Blood was collected from the vena cava of anesthetized mice into syringes containing 1/10th blood volume of 3.8% sodium citrate as anticoagulant. For clopidogrel treatment, mice were orally dosed with clopidogrel (30 mg/kg/day) for 2 days, and blood was collected 1 h after the final dose. Red blood cells were removed by centrifugation at 100 x g for 10 min. Platelet-rich plasma was recovered, and platelets were pelleted at 400 x g for 10 min. The platelet pellet was resuspended in Tyrode's buffer (pH 7.4) containing 0.05 units/ml apyrase.

Aggregometry—Aggregation of 0.5 ml washed platelets was analyzed using a P.I.C.A. lumiaggregometer (Chrono-log Corp., Havertown, PA). Aggregation was measured using light transmission under stirring conditions (900 rpm) at 37 °C. Each sample was allowed to aggregate for at least 3 min. The chart recorder (Kipp and Zonen, Bohemia, NY) was set for 0.2 mm/s.

Measurement of Akt Phosphorylation—Platelets were stimulated with agonists under non-stirring conditions for the appropriate time, and the reaction was stopped by the addition of 3x SDS sample buffer. In some experiments, GF 109203X (10 µM), a PKC inhibitor, and dimethyl-BAPTA (10 µM), a calcium chelator, were added and incubated for 5 min at 37 °C without stirring before agonist stimulation. Platelet samples were boiled for 5 min, and proteins were separated on 10% SDS-PAGE and transferred onto polyvinylidene difluoride membrane. Nonspecific binding sites were blocked by incubation in Tris-buffered saline/Tween (TBST; 20 mM Tris, 140 mM NaCl, 0.1% (v/v) Tween 20) containing 0.5% (w/v) milk protein and 3% (w/v) bovine serum albumin (BSA) for 30 min at RT, and membranes were incubated overnight at 4 °C with primary antibody (1:1000 in TBST, 2% BSA) with gentle agitation. After three washes for 5 min each with TBST, the membranes were probed with the alkaline phosphatase-labeled goat anti-rabbit IgG (1:5000 in TBST, 2% BSA) for 1 h at RT. After additional washing steps, membranes were then incubated with a CDP-Star® chemiluminescent substrates for 10 min at RT, and immunoreactivity was detected using Fujifilm Luminescent Image Analyzer (model LAS-1000 CH, Japan).

In Vitro Kinase Assay for Akt—Akt kinase activity was measured using a modification of the Akt kinase assay kit from Cell Signaling Technology (Beverly, MA). Briefly, platelets were stimulated with agonists under non-stirring conditions for 3 min, and the reaction was stopped by the addition of 5x ice-cold cell lysis buffer (100 mM Tris (pH 7.5), 750 mM NaCl, 5 mM EDTA, 5 mM EGTA, 5% Triton X-100, 12.5 mM sodium pyrophosphate, 5 mM -glycerol phosphate, 5 mM Na₃VO₄, 5 µg/ml leupeptin, and 5 mM phenylmethylsulfonyl fluoride). Resuspended immobilized Akt antibody slurry (20 µl) was added to 200 µl of platelet lysates to selectively immunoprecipitate Akt from the lysate with gentle rocking 3 h at 4 °C. The pellet was washed twice with 500 µl of 1x lysis buffer and twice with 500 µl of 1x kinase buffer (25 mM Tris (pH 7.5), 5 mM -glycerol phosphate, 2 mM dithiothreitol, 0.1 mM Na₃VO₄, and 10 mM MgCl₂). The immunoprecipitated pellet was then incubated with 40 µl of 1x kinase buffer supplemented with 200 µM ATP and 1 µg of GSK-3 fusion protein for 30 min at 30 °C allowing immunoprecipitated Akt to phosphorylate GSK-3. The reaction was terminated with 20 µl of 3x SDS sample buffer. Samples were boiled for 5 min and loaded on 14% SDS-PAGE gel. Akt-induced phosphorylation of GSK-3 was detected by Western blotting using phospho-GSK-3/ (Ser^21/9) antibody.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
SFLLRN, AYPGKF, and Thrombin Induce Akt Phosphorylation in a Dose- and Time-dependent Manner—It has been shown that thrombin induces phosphorylation of Akt in platelets (6, 19). To determine the kinetics of Akt phosphorylation, Ser⁴⁷³ phosphorylation was monitored over a time range of 0.5–20 min. Fig. 1A illustrates that a rapid increase in Akt phosphorylation in response to thrombin was detectable as early as 30 s after stimulation. The level of phosphorylation peaked at around 3 min and remained stable after 20 min of stimulation.

View larger version (46K): [in this window] [in a new window]   FIG. 1. Akt is phosphorylated in response to SFLLRN, AYPGKF, and thrombin in normal human platelets. A is a representative experiment of a time-dependent phosphorylation of Akt at Ser473 with thrombin. Washed platelets were stimulated at 37 °C for the time points indicated with thrombin (0.5 unit/ml) without stirring. B, washed platelets were incubated with different concentrations of SFLLRN, AYPGKF, or thrombin for 3 min at 37 °C without stirring. The reaction was stopped by the addition of 3x SDS sample buffer. Samples were separated by SDS-PAGE, transferred onto polyvinylidene difluoride membranes, and probed with anti-phospho-Akt (Ser473) antibody. The data shown are representative of three experiments.

Effect of Secretion and G_i Signaling Pathways in Akt Phosphorylation and Activation by SFLLRN, AYPGKF, and Thrombin in Human Platelets—Immunoblot analysis revealed that Akt is phosphorylated on Ser⁴⁷³ and Thr³⁰⁸ in response to SFLLRN, AYPGKF, and thrombin in human platelets (Fig. 2). SFLLRN induced a lower extent of Ser⁴⁷³ and Thr³⁰⁸ phosphorylation, whereas AYPGKF induced a similar extent of Akt phosphorylation as thrombin. These results are consistent with a previous study in which phosphorylation of Thr³⁰⁸ and Ser⁴⁷³ induced by SFLLRN in human platelets is 50% of the response induced by thrombin (6). However, they failed to activate Akt completely in response to the PAR4-activating peptide GYPGKF, which is in contrast with our data, wherein dual phosphorylation and activation of Akt occur in response to AYPGKF. These results indicate that both PAR1 and PAR4 receptors can induce concurrent phosphorylation of Ser⁴⁷³ and Thr³⁰⁸, and AYPGKF and thrombin are more potent activators of Akt phosphorylation than SFLLRN.

View larger version (72K): [in this window] [in a new window]   FIG. 2. Role of secretion and Gi signaling pathways in Akt phosphorylation in response to SFLLRN, AYPGKF, and thrombin in human platelets. Platelets preincubated with 1 µM AR-C69931MX, 25 µM LY294002, 10 µM GF 109203X, or 10 µM dimethyl-BAPTA were stimulated at 37 °C for 3 min with either SFLLRN (10 µM), AYPGKF (500 µM), or thrombin (0.5 unit/ml). The reaction was stopped by the addition of 3x SDS sample buffer. Samples were separated by SDS-PAGE, transferred onto polyvinylidene difluoride membranes, and probed with anti-phospho-Akt (Ser473 and Thr308) antibody. Results are representative of three experiments.

It has been known that phosphorylation of both Thr³⁰⁸ and Ser⁴⁷³ is required for full activation of Akt (4). Akt activity was measured by in vitro kinase assay that detects phosphorylation of GSK-3 induced by immunoprecipitated Akt. Phosphorylation of GSK-3 was measured by Western blotting using a phospho-GSK-3/ (Ser^21/9) antibody, and GSK-3/ kinase activity has been shown to be negatively regulated by Akt via phosphorylation at Ser^21/9. Our results of Akt kinase activity in human platelets (Fig. 3) correspond to Akt phosphorylation shown in Fig. 2. In the presence of dimethyl-BAPTA, activation of Akt in response to SFLLRN, AYPGKF, and thrombin was diminished but not completely abolished. Furthermore, Akt activation was nearly abolished in the presence of AR-C69931MX and GF 109203X confirming that G_i stimulation by secretion is essential for thrombin- and thrombin receptor-activating peptide-induced Akt activation.

View larger version (63K): [in this window] [in a new window]   FIG. 3. Gi- and PI 3-kinase-dependent Akt activation by SFLLRN, AYPGKF, and thrombin in human platelets. Platelets preincubated with 1 µM AR-C69931MX, 25 µM LY294002, 10 µM GF 109203X or 10 µM dimethyl-BAPTA were stimulated at 37 °C for 3 min with either SFLLRN (10 µM), AYPGKF (500 µM), or thrombin (0.5 unit/ml). The reaction was stopped by adding ice-cold lysis buffer. Platelet lysates (200 µl) were incubated for 2 h with immobilized Akt 1G1 monoclonal antibody to selectively immunoprecipitate Akt from lysates. The resulting immunoprecipitate was then incubated with GSK-3 fusion protein in the presence of ATP and Kinase Buffer to allow immunoprecipitated Akt to phosphorylate GSK-3. Phosphorylation of GSK-3 was measured by Western blotting using a phospho-GSK-3/ (Ser21/9) antibody. The data shown are representative of three experiments. Reprobes of blots for total Akt are shown in the bottom panel of each agonist.

View larger version (44K): [in this window] [in a new window]   FIG. 4. Role of secretion and Gi signaling pathways in Akt phosphorylation in response to AYPGKF and thrombin in mouse platelets. Mouse platelets preincubated with 1 µM AR-C69931MX, 25 µM LY294002, 10 µM GF 109203X, or 10 µM dimethyl-BAPTA were stimulated at 37 °C for 3 min with either AYPGKF (500 µM) or thrombin (0.5 unit/ml). The reaction was stopped by the addition of 3x SDS sample buffer. Samples were separated by SDS-PAGE, transferred onto polyvinylidene difluoride membranes, and probed with anti-phospho-Akt (Ser473 and Thr308) antibody. The data shown are representative of three experiments.

View larger version (35K): [in this window] [in a new window]   FIG. 5. Akt phosphorylation in response to ADP, AYPGKF, and thrombin in Gq(–/–) mice. A, wild-type platelets and Gq-deficient platelets preincubated in the absence and presence of 1 µM AR-C69931MX were stimulated at 37 °C for 3 min with either AYPGKF (500 µM) or thrombin (0.5 unit/ml). B, platelets preincubated in the absence or presence of 1 µM AR-C69931MX or 10 µM MRS-2179 were stimulated at 37 °C for 3 min with 2-MeSADP (1 µM). The reaction was stopped by the addition of 3x SDS sample buffer. Equal amounts of proteins were analyzed by Western blot analysis with anti-Thr(P)308, anti-Ser(P)473, or anti-Akt antibody. The Western analysis shown is a representative of three independent experiments.

View larger version (25K): [in this window] [in a new window]   FIG. 6. Platelet aggregation and Akt phosphorylation (Ser473) in P2Y1 –/– mice. Platelets were isolated from P2Y1 –/– mice or their wild-type littermates. A, platelets were stimulated with 10 µM ADP, and platelet aggregation was measured as described under "Experimental Procedures." B, platelets preincubated in the absence or presence of 1 µM AR-C69931MX or 10 µM MRS-2179 were stimulated at 37 °C for 3 min without stirring with 2-MeSADP (1 µM), AYPGKF (500 µM), or thrombin (0.5 unit/ml). Samples were separated by SDS-PAGE, Western-blotted, and probed with anti-phospho-Akt (Ser473) or anti-Akt antibody. Results are representative of three experiments.

View larger version (25K): [in this window] [in a new window]   FIG. 7. Platelet aggregation and Akt phosphorylation (Ser473) in mice dosed with clopidogrel. Platelets were isolated from control and clopidogrel-dosed mice (30 mg/kg/day for 2 days). A, platelet aggregation induced by 10 µM ADP was measured as described previously. B, platelets were stimulated for 3 min at 37 °C with 2-MeSADP (1 µM), AYPGKF (500 µM), or thrombin (0.5 unit/ml) in the absence or presence of 1 µM AR-C69931MX or 10 µM MRS-2179. Samples were run on SDS-PAGE and probed with anti-Ser(P)473 or anti-Akt antibody. The data shown are representative of three experiments.

View larger version (35K): [in this window] [in a new window]   FIG. 8. Phosphorylation of Akt induced by direct activation of PKC with PMA. Platelets preincubated with 1 µM AR-C69931MX, 10 µM GF 109203X, or 25 µM LY294002 were stimulated at 37 °C for 3 min with PMA (0.5 µM). The addition of epinephrine (20 µM) was made as indicated. The reaction was stopped by the addition of 3x SDS sample buffer. Samples were separated by SDS-PAGE, Western-blotted, and probed with anti-phospho-Akt (Ser473 and Thr308) antibody. The data shown are representative of three independent experiments.

View larger version (58K): [in this window] [in a new window]   FIG. 9. Restoration of Akt phosphorylation by selective stimulation of either Gi- or Gz-coupled receptor. A, platelets preincubated in the absence and presence of 10 µM GF 109203X were stimulated at 37 °C for 3 min with either SFLLRN (10 µM), AYPGKF (500 µM), or thrombin (0.5 unit/ml). The addition of 2-MeSADP (1 µM) or epinephrine (20 µM) was made as indicated. B, platelets preincubated with 1 µM AR-C69931MX were stimulated at 37 °C for 3 min with either SFLLRN (10 µM), AYPGKF (500 µM), or thrombin (0.5 unit/ml) in the absence and presence of 20 µM epinephrine. The reaction was stopped by the addition of 3x SDS sample buffer. Samples were separated by SDS-PAGE, Western-blotted, and probed with anti-phospho-Akt (Ser473) antibody. Results are representative of three experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our experiments reveal that Akt was phosphorylated on Ser⁴⁷³ and Thr³⁰⁸ in response to SFLLRN, AYPGKF, and thrombin, and this response was found to be time-dependent (Fig. 1) confirming dual phosphorylation of Akt in platelets by these agonists. Kroner et al. (6) reported that phosphorylation of Thr³⁰⁸ and Ser⁴⁷³ induced by SFLLRN is 50% of the response induced by thrombin, whereas GYPGKF fails to activate Akt completely. This observation with GYPGKF is in contrast with our data, which shows dual phosphorylation of Akt in response to AYPGKF. This seems due to the difference in PAR4-activating peptides wherein AYPGKF has shown to be a selective and more potent PAR4-activating peptide (31). Akt activity in stirred platelets stimulated with thrombin has been shown to be biphasic, which mirrors the biphasic activation of PI 3-kinase, where PtdIns(3,4,5)P₃ is generated upon initial stimulation and PtdIns(3,4)P₂ is generated later in platelet aggregation (19). Because platelets were prevented from aggregating during kinetic studies of Akt phosphorylation, a biphasic activation pattern of Akt was not observed in our study.

Thrombin mediates its cellular effects primarily through a family of G_q-coupled PARs, whereas the G_i pathway is activated by the released ADP through activation of the P2Y₁₂ receptor (15). However, it has not been clear whether activation of Akt by thrombin is through G_q or G_i pathways. Akt has been shown to become activated in response to mutationally activated G_q and G_i in COS cells (32). Recently, G_q-coupled receptors have been shown to activate Akt in astrocytoma cells, which involves phospholipase C- and Ca²⁺-mediated PI 3-kinase activation (33). Others reported that G_i-coupled receptors can activate Akt by G protein heterodimers in a PI 3-kinase-dependent manner, whereas activated G_q has a prominent inhibitory effect on Akt activation in COS and HEK-293 cells (34). A recent report (35) indicates that activated G_q inhibits p110 PI 3-kinase activity and blocks Akt activation in HEK-293 cells. To determine whether thrombin and thrombin receptor-activating peptides can activate Akt independently of secreted ADP and G_i signaling pathways, we have used GF 109203X, a selective inhibitor of PKC isoforms, and AR-C69931MX, an antagonist at the G_i-coupled P2Y₁₂ receptor. PKC has been shown to play a major role in the induction of platelet secretion. In the presence of GF 109203X, Akt activation in response to SFLLRN, AYPGKF, and thrombin was dramatically blocked, suggesting that these agonists depend on secretion to activate Akt. Inhibition of Akt activation by AR-C69931MX also indicates an essential role of G_i-coupled P2Y₁₂ receptor by secreted ADP in PAR1- and PAR4-induced Akt activation. Consistent with our observations, Tilton et al. (36) demonstrated that G_i-coupled receptors can activate Akt by PI 3-kinase in human phagocytes. The observation that activation of Akt was inhibited in the presence of dimethyl-BAPTA suggests the involvement of the calcium-dependent PKC subtypes. Previous reports show calcium-dependent regulation of Akt by Ca²⁺/calmodulin-dependent protein kinase kinase on the Thr³⁰⁸ phosphorylation (13) or by calcium-dependent PKC on the level of Ser⁴⁷³ phosphorylation (6). The latter observation is in contrast with our data, which show the inhibition of both Thr³⁰⁸ and Ser⁴⁷³ phosphorylation by dimethyl-BAPTA. Because it is known that intracellular calcium increase has a role in platelet secretion (37), it is conceivable that dimethyl-BAPTA interferes with platelet secretion resulting in the inhibition of G_i stimulation by secreted ADP and inhibits both Thr³⁰⁸ and Ser⁴⁷³ phosphorylation. The role of calcium on Akt activation is further defined in the experiment by using platelets from G_q- and P2Y₁-deficient mice.

Some degree of thrombin-induced Akt phosphorylation occurs even in the absence of P2Y₁₂ signaling. There are two possibilities. First, platelet granules contain several other agonists, such as chemokines, that could activate G_i pathways (29). Thus, thrombin could activate G_i pathways even in the absence of P2Y₁₂ receptor by these other G_i-activating agonists. This could result in a small Akt phosphorylation by thrombin even in the absence of the P2Y₁₂ receptor. The second possibility is that thrombin could phosphorylate Akt to a very small extent independently of G_i pathways. Our experiments do not distinguish these two scenarios.

Even though the role of PI 3-kinase in Akt activation is well known (9, 30), several reports have suggested a PI 3-kinase-independent activation of Akt. Stimulation of ₃-adrenergic receptor, which couples to both G_s and G_i proteins (38), has been shown to cause the stimulation of Akt which is not dependent on PI 3-kinase in rat adipose cells (39). It is also shown that cAMP-elevating agents or cellular stress such as heat shock and hyperosmolarity can increase the activity of Akt through a pathway that is independent of PI 3-kinase (40, 41). Our study reveals that PI 3-kinase activity is necessary for thrombin- and thrombin receptor-activating peptide-mediated activation of Akt in platelets. Our data are also consistent with other studies, where thrombin-induced Akt activation is dependent on PI 3-kinase activity in platelets (6), airway smooth muscle cells (42), and IIC9 cells (43). It is conceivable that the alternative pathways that stimulate PI 3-kinase-independent activation of Akt are present in some other cell preparations, which are activated by agonists linked to G_i-coupled receptors.

Our data using knockout mouse platelets demonstrate that ADP induces Akt phosphorylation in P2Y₁- or G_q-deficient platelets. However, ADP-induced Akt phosphorylation was completely abolished in the presence of AR-C69931MX in these platelets further indicating that G_q is not required for Akt activation in platelets, whereas G_i-coupled P2Y₁₂ receptor is necessary and sufficient for activation of Akt (Figs. 5B and 6B). It is known that thrombin receptors couple to G_q and G_12/13 pathways (44). However, AYPGKF and thrombin fail to induce Akt phosphorylation in G_q-deficient platelets (Fig. 5A), suggesting that G_12/13 pathways do not have any role in Akt activation in platelets. It also has been shown that platelets from G_q-deficient mice fail to release their granule contents in response to thrombin (25), and we have shown that thrombin fails to directly couple to G_i in the absence of secretion (15). Thus, the complete inhibition of Akt phosphorylation by AYPGKF and thrombin in G_q-deficient platelets may not be due to the direct inhibitory effect of G_q but due to the absence of secretion to stimulate G_i, supporting the idea that thrombin-induced Akt activation depends on secretion/G_i pathways.

Clopidogrel has been shown to selectively target the P2Y₁₂ receptor (27). To confirm the role of secreted ADP and G_i stimulation in Akt activation, we have used clopidogrel-dosed mouse platelets. In these platelets, ADP fails to induce Akt phosphorylation confirming that G_i-coupled P2Y₁₂ receptor is required for Akt activation in platelets. Furthermore, AYPGKF and thrombin fail to cause significant Akt phosphorylation. Thus, AYPGKF and thrombin cannot cause Akt stimulation independently of secreted ADP. These findings correlate with previous findings and provide further evidence that G_i stimulation by secreted ADP is required for thrombin- and thrombin receptor-activating peptide-induced Akt activation in platelets.

The fact that ADP can cause Akt phosphorylation and, in the absence of secreted ADP, thrombin cannot cause (strong) Akt phosphorylation is surprising, as thrombin is a more potent agonist of platelets than ADP. A number of studies have shown that PI 3-kinases can be activated by thrombin downstream of PARs (6, 15, 45). As PI 3-kinases are known to be upstream of Akt, one would expect Akt phosphorylation to occur downstream of thrombin, even in the absence of ADP. But thrombin causes a very small Akt phosphorylation in the absence of the P2Y₁₂. Thrombin might activate several PI 3-kinases (, , and ), whereas ADP might activate only PI 3-kinase . Activation of PI 3-kinase may not immediately result in Akt phosphorylation without some events regulated by G_i stimulation. Thus G_i pathways might be regulating some signaling events downstream of PI 3-kinases and upstream of Akt. Consistent with our conclusions, Hirsch et al. (46) showed that ADP fails to cause Akt phosphorylation in PI 3-kinase knockout mouse platelets, but thrombin causes Akt phosphorylation in these platelets. Thus, in PI 3-kinase knockout mouse platelets, phosphatidylinositol 1,4,5-trisphosphate accumulation downstream of thrombin can lead to Akt phosphorylation as G_i signaling events regulating Akt activation are unaffected. However, ADP-induced phosphatidylinositol 1,4,5-trisphosphate formation is affected in these platelets as ADP causes phosphatidylinositol 1,4,5-trisphosphate formation through PI 3-kinase , which is missing in these platelets.

ADP causes much smaller extent of Akt phosphorylation than thrombin or TRAPs. This could be due to a potentiating effect of G_q pathways on G_i-mediated Akt phosphorylation. PARs, through strong stimulation of G_q pathways, could potentiate Akt phosphorylation mediated by G_i pathways and hence cause much stronger Akt phosphorylation than ADP. Akt phosphorylation occurs biphasically, first through inside-out signaling and then through outside-in signaling (19). In our conditions, we eliminated the contribution of outside-in signaling and its contribution to Akt phosphorylation. Thus, when aggregation is allowed to occur, thrombin-induced Akt phosphorylation could be stronger because thrombin can cause aggregation to a greater extent than ADP and hence could cause stronger Akt phosphorylation. However, under our conditions, the contribution of outside-in signaling is eliminated, and still thrombin causes more Akt phosphorylation than ADP. Hence, there might be some potentiating contribution of G_q to Akt phosphorylation mediated by G_i. The second possibility is that the downstream events of G_i regulate Akt phosphorylation, and the actual Akt activation depends on the extent of PI3K activation. Thus, if thrombin causes activation of multiple PI 3-kinases and ADP causes only one (PI 3-kinase ), it may translate into more phosphorylation of Akt by thrombin than ADP. This scenario is evaluated by selectively supplementing G_i or G_z signaling pathway stimulation to thrombin or PAR-activating peptide-induced Akt phosphorylation under the conditions of secretion blockade (Fig. 9A) or P2Y₁₂ receptor blockade (Fig. 9B). Supplemental G_i/G_z signaling restored Akt phosphorylation to the levels achieved by thrombin stimulation. These results further confirm that G_i signaling regulates Akt phosphorylation, and epinephrine, through G_z signaling cascade, can substitute for the P2Y₁₂ receptor-mediated effects.

The activation of PI 3-kinase downstream of G_i pathways is implicated in irreversible platelet aggregation (15, 45). It is also suggested that PI 3-kinase exerts a significant role in ADP-induced platelet aggregation (46). Even though the important role of PI 3-kinase in platelet activation in well known, the functional significance of Akt in platelet activation is not well established. Because a key enzyme in Akt activation is PI 3-kinase, we can speculate that Akt activation downstream of G_i/PI 3-kinase may play an important role in platelet aggregation. Recently two compounds, 1-L-6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecylcarbonate (47) and 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (48), have been shown to inhibit Akt activity. When platelets were treated with 1-L-6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecylcarbonate or 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride, platelet aggregation induced by SFLLRN became reversible and mimicked the effects of AR-C66096 or LY294002, whereas the aggregation induced by high concentrations of AYPGKF and thrombin was not affected by these inhibitors (data not shown). The addition of Akt inhibitors caused the reversion of aggregation in low concentrations of AYPGKF and thrombin in which these agonists depend on secretion for irreversible aggregation (data not shown). The signaling pathway used by platelets treated with a high concentration of AYPGKF and thrombin causes secretion-independent irreversible aggregation, whereas SFLLRN-induced irreversible aggregation depends exclusively on secretion of ADP that stimulates G_i-coupled receptors via the late activation of a PI 3-kinase. Furthermore, the absolute requirement of G_i pathways for Akt activation suggests that Akt is not required for thrombin- and thrombin receptor-activating peptide-mediated platelet fibrinogen receptor activation, and yet Akt is important in potentiation of thrombin-induced fibrinogen receptor activation and plays an important role in SFLLRN-induced irreversible aggregation. However, it is difficult to address the precise role of Akt in platelet aggregation with the promiscuous nature of the present Akt inhibitors, thus the precise role of different Akt isoforms in platelet signal transduction remains to be further elucidated.

In conclusion, we demonstrate that ADP depends on G_i-coupled P2Y₁₂ receptor to activate Akt and that ADP causes Akt phosphorylation independently of G_q stimulation. Furthermore, we find that SFLLRN-, AYPGKF-, and thrombin-induced Akt activation depends predominantly on G_i stimulation through P2Y₁₂ receptor activation by secreted ADP. In addition, G_q or G_12/13 signaling pathways downstream of PARs might have a potentiating effect on the Akt phosphorylation. Finally, the regulation of Akt phosphorylation by the P2Y₁₂ receptor can be substituted by epinephrine, which activates G_z signaling cascade.

	FOOTNOTES

 
^* This work was supported by Research Grants HL60683 and HL64943 from the National Institutes of Health (to S. P. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Dept. of Physiology, Temple University School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140. Tel.: 215-707-4615; Fax: 215-707-4003; E-mail: spk{at}temple.edu.

¹ The abbreviations used are: PKB, protein kinase B; G_q, heterotrimeric GTP-binding protein which stimulates phospholipase C; G_i, heterotrimeric GTP-binding protein which inhibits adenylyl cyclase; G_12/13, heterotrimeric GTP-binding proteins 12 and 13; P2Y₁₂, platelet ADP receptor coupled to inhibition of adenylyl cyclase; P2Y₁, platelet ADP receptor coupled to stimulation of phospholipase C; PAR, protease-activated receptor; PKC, protein kinase C; PI 3-kinase, phosphatidylinositol 3-kinase; GSK-3, glycogen synthase kinase-3; PDK, phosphatidylinositol-dependent kinase; PRP, plateletrich plasma; dimethyl-BAPTA, 5,5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; RT, room temperature; BSA, bovine serum albumin; PMA, phorbol 12-myristate 13-acetate; PtdIns, phosphatidylinositol; 2-MeSADP, 2-methylthio-ADP.

	ACKNOWLEDGMENTS

 
We thank Drs. Kent Gartner, University of Memphis, Memphis, TN, and Stefan Offermanns, University of Heidelberg, Heidelberg, Germany, for providing the G_q-deficient mice. We thank Drs. Todd M. Quinton and Robert T. Dorsam for critically reviewing the manuscript, and Dr. Fujio Sekiya, National Institutes of Health, for valuable advice.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Bellacosa, A., Testa, J. R., Staal, S. P., and Tsichlis, P. N. (1991) Science 254, 274–277[Abstract/Free Full Text]
2. Cheng, J. Q., Godwin, A. K., Bellacosa, A., Taguchi, T., Franke, T. F., Hamilton, T. C., Tsichlis, P. N., and Testa, J. R. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9267–9271[Abstract/Free Full Text]
3. Nakatani, K., Thompson, D. A., Barthel, A., Sakaue, H., Liu, W., Weigel, R. J., and Roth, R. A. (1999) J. Biol. Chem. 274, 21528–21532[Abstract/Free Full Text]
4. Alessi, D. R., Andjelkovic, M., Caudwell, B., Cron, P., Morrice, N., Cohen, P., and Hemmings, B. A. (1996) EMBO J. 15, 6541–6551[Medline] [Order article via Infotrieve]
5. Jones, P. F., Jakubowicz, T., Pitossi, F. J., Maurer, F., and Hemmings, B. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4171–4175[Abstract/Free Full Text]
6. Kroner, C., Eybrechts, K., and Akkerman, J. W. (2000) J. Biol. Chem. 275, 27790–27798[Abstract/Free Full Text]
7. Barry, F. A., and Gibbins, J. M. (2002) J. Biol. Chem. 277, 12874–12878[Abstract/Free Full Text]
8. Cho, M. J., Pestina, T. I., Steward, S. A., Lowell, C. A., Jackson, C. W., and Gartner, T. K. (2002) Blood 99, 2442–2447[Abstract/Free Full Text]
9. Burgering, B. M., and Coffer, P. J. (1995) Nature 376, 599–602[CrossRef][Medline] [Order article via Infotrieve]
10. Stokoe, D., Stephens, L. R., Copeland, T., Gaffney, P. R., Reese, C. B., Painter, G. F., Holmes, A. B., McCormick, F., and Hawkins, P. T. (1997) Science 277, 567–570[Abstract/Free Full Text]
11. Toker, A., and Newton, A. C. (2000) J. Biol. Chem. 275, 8271–8274[Abstract/Free Full Text]
12. Troussard, A. A., Mawji, N. N., Ong, C., Mui, A., St. Arnaud, R., and Dedhar, S. (2003) J. Biol. Chem. 278, 22374–22378[Abstract/Free Full Text]
13. Yano, S., Tokumitsu, H., and Soderling, T. R. (1998) Nature 396, 584–587[CrossRef][Medline] [Order article via Infotrieve]
14. Jin, J., and Kunapuli, S. P. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8070–8074[Abstract/Free Full Text]
15. Kim, S., Foster, C., Lecchi, A., Quinton, T. M., Prosser, D. M., Jin, J., Cattaneo, M., and Kunapuli, S. P. (2002) Blood 99, 3629–3636[Abstract/Free Full Text]
16. Cho, M. J., Liu, J., Pestina, T. I., Steward, S. A., Thomas, D. W., Coffman, T. M., Wang, D., Jackson, C. W., and Gartner, T. K. (2003) Blood 101, 2646–2651[Abstract/Free Full Text]
17. Fabre, J. E., Nguyen, M., Latour, A., Keifer, J. A., Audoly, L. P., Coffman, T. M., and Koller, B. H. (1999) Nat. Med. 5, 1199–1202[CrossRef][Medline] [Order article via Infotrieve]
18. Leon, C., Hechler, B., Freund, M., Eckly, A., Vial, C., Ohlmann, P., Dierich, A., LeMeur, M., Cazenave, J. P., and Gachet, C. (1999) J. Clin. Investig. 104, 1731–1737[Medline] [Order article via Infotrieve]
19. Banfic, H., Downes, C. P., and Rittenhouse, S. E. (1998) J. Biol. Chem. 273, 11630–11637[Abstract/Free Full Text]
20. Sano, K., Takai, Y., Yamanishi, J., and Nishizuka, Y. (1983) J. Biol. Chem. 258, 2010–2013[Abstract/Free Full Text]
21. Walker, T. R., and Watson, S. P. (1993) Biochem. J. 289, 277–282[Medline] [Order article via Infotrieve]
22. Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., and Loriolle, F. (1991) J. Biol. Chem. 266, 15771–15781[Abstract/Free Full Text]
23. Ingall, A. H., Dixon, J., Bailey, A., Coombs, M. E., Cox, D., McInally, J. I., Hunt, S. F., Kindon, N. D., Teobald, B. J., Willis, P. A., Humphries, R. G., Leff, P., Clegg, J. A., Smith, J. A., and Tomlinson, W. (1999) J. Med. Chem. 42, 213–220[CrossRef][Medline] [Order article via Infotrieve]
24. Quinton, T. M., Ozdener, F., Dangelmaier, C., Daniel, J. L., and Kunapuli, S. P. (2002) Blood 99, 3228–3234[Abstract/Free Full Text]
25. Offermanns, S., Toombs, C. F., Hu, Y. H., and Simon, M. I. (1997) Nature 389, 183–186[CrossRef][Medline] [Order article via Infotrieve]
26. Baurand, A., Raboisson, P., Freund, M., Leon, C., Cazenave, J. P., Bourguignon, J. J., and Gachet, C. (2001) Eur. J. Pharmacol. 412, 213–221[CrossRef][Medline] [Order article via Infotrieve]
27. Geiger, J., Brich, J., Honig-Liedl, P., Eigenthaler, M., Schanzenbacher, P., Herbert, J. M., and Walter, U. (1999) Arterioscler. Thromb. Vasc. Biol. 19, 2007–2011[Abstract/Free Full Text]
28. Savi, P., Laplace, M. C., Maffrand, J. P., and Herbert, J. M. (1994) J. Pharmacol. Exp. Ther. 269, 772–777[Abstract/Free Full Text]
29. Kowalska, M. A., Ratajczak, M. Z., Majka, M., Jin, J., Kunapuli, S., Brass, L., and Poncz, M. (2000) Blood 96, 50–57[Abstract/Free Full Text]
30. Franke, T. F., Yang, S. I., Chan, T. O., Datta, K., Kazlauskas, A., Morrison, D. K., Kaplan, D. R., and Tsichlis, P. N. (1995) Cell 81, 727–736[CrossRef][Medline] [Order article via Infotrieve]
31. Faruqi, T. R., Weiss, E. J., Shapiro, M. J., Huang, W., and Coughlin, S. R. (2000) J. Biol. Chem. 275, 19728–19734[Abstract/Free Full Text]
32. Murga, C., Laguinge, L., Wetzker, R., Cuadrado, A., and Gutkind, J. S. (1998) J. Biol. Chem. 273, 19080–19085[Abstract/Free Full Text]
33. Tang, X., Batty, I. H., and Downes, C. P. (2002) J. Biol. Chem. 277, 338–344[Abstract/Free Full Text]
34. Bommakanti, R. K., Vinayak, S., and Simonds, W. F. (2000) J. Biol. Chem. 275, 38870–38876[Abstract/Free Full Text]
35. Ballou, L. M., Lin, H. Y., Fan, G., Jiang, Y. P., and Lin, R. Z. (2003) J. Biol. Chem. 278, 23472–23479[Abstract/Free Full Text]
36. Tilton, B., Andjelkovic, M., Didichenko, S. A., Hemmings, B. A., and Thelen, M. (1997) J. Biol. Chem. 272, 28096–28101[Abstract/Free Full Text]
37. Quinton, T. M., Kim, S., Dangelmaier, C., Dorsam, R. T., Jin, J., Daniel, J. L., and Kunapuli, S. P. (2002) Biochem. J. 368, 535–543[CrossRef][Medline] [Order article via Infotrieve]
38. Chaudhry, A., MacKenzie, R. G., Georgic, L. M., and Granneman, J. G. (1994) Cell. Signal. 6, 457–465[CrossRef][Medline] [Order article via Infotrieve]
39. Moule, S. K., Welsh, G. I., Edgell, N. J., Foulstone, E. J., Proud, C. G., and Denton, R. M. (1997) J. Biol. Chem. 272, 7713–7719[Abstract/Free Full Text]
40. Sable, C. L., Filippa, N., Hemmings, B., and Van Obberghen, E. (1997) FEBS Lett. 409, 253–257[CrossRef][Medline] [Order article via Infotrieve]
41. Konishi, H., Matsuzaki, H., Tanaka, M., Ono, Y., Tokunaga, C., Kuroda, S., and Kikkawa, U. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7639–7643[Abstract/Free Full Text]
42. Walker, T. R., Moore, S. M., Lawson, M. F., Panettieri, R. A., Jr., and Chilvers, E. R. (1998) Mol. Pharmacol. 54, 1007–1015[Abstract/Free Full Text]
43. Phillips-Mason, P. J., Raben, D. M., and Baldassare, J. J. (2000) J. Biol. Chem. 275, 18046–18053[Abstract/Free Full Text]
44. Offermanns, S., Laugwitz, K. L., Spicher, K., and Schultz, G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 504–508[Abstract/Free Full Text]
45. Trumel, C., Payrastre, B., Plantavid, M., Hechler, B., Viala, C., Presek, P., Martinson, E. A., Cazenave, J. P., Chap, H., and Gachet, C. (1999) Blood 94, 4156–4165[Abstract/Free Full Text]
46. Hirsch, E., Bosco, O., Tropel, P., Laffargue, M., Calvez, R., Altruda, F., Wymann, M., and Montrucchio, G. (2001) FASEB J. 15, 2019–2021[Free Full Text]
47. Hu, Y., Qiao, L., Wang, S., Rong, S. B., Meuillet, E. J., Berggren, M., Gallegos, A., Powis, G., and Kozikowski, A. P. (2000) J. Med. Chem. 43, 3045–3051[CrossRef][Medline] [Order article via Infotrieve]
48. Smith, U., Carvalho, E., Mosialou, E., Beguinot, F., Formisano, P., and Rondinone, C. (2000) Biochem. Biophys. Res. Commun. 268, 315–320[CrossRef][Medline] [Order article via Infotrieve]

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