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J. Biol. Chem., Vol. 277, Issue 49, 47588-47595, December 6, 2002

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Coordinated Signaling through Both G_12/13 and G_i Pathways Is Sufficient to Activate GPIIb/IIIa in Human Platelets^*

Robert T. Dorsam^§¶, Soochong Kim, Jianguo Jin, and Satya P. Kunapuli^§**

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

Received for publication, August 27, 2002, and in revised form, September 11, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Activation of GPIIb/IIIa is known to require agonist-induced inside-out signaling through G_q, G_i, and G_z. Although activated by several platelet agonists, including thrombin and thromboxane A₂, the contribution of the G_12/13 signaling pathway to GPIIb/IIIa activation has not been investigated. In this study, we used selective stimulation of G protein pathways to investigate the contribution of G_12/13 activation to platelet fibrinogen receptor activation. YFLLRNP is a PAR-1-specific partial agonist that, at low concentrations (60 µM), selectively activates the G_12/13 signaling cascade resulting in platelet shape change without stimulating the G_q or G_i signaling pathways. YFLLRNP-mediated shape change was completely inhibited by the p160^ROCK inhibitor, Y-27632. At this low concentration, YFLLRNP-mediated G_12/13 signaling caused platelet aggregation and enhanced PAC-1 binding when combined with selective G_i or G_z signaling, via selective stimulation of the P2Y₁₂ receptor or _2A-adrenergic receptor, respectively. Similar data were obtained when using low dose U46619 (10 nM), a thromboxane A₂ mimetic, to activate G_12/13 in the presence of G_i signaling. These results suggest that selective activation of G_12/13 causes platelet GPIIb/IIIa activation when combined with G_i signaling. Unlike either G_12/13 or G_i activation alone, co-activation of both G_12/13 and G_i resulted in a small increase in intracellular calcium. Chelation of intracellular calcium with dimethyl BAPTA dramatically blocked G_12/13 and G_i-mediated platelet aggregation. No significant effect on aggregation was seen when using selective inhibitors for p160^ROCK, PKC, or MEKK1. PI 3-kinase inhibition lead to near abolishment of platelet aggregation induced by co-stimulation of G_q and G_i pathways, but not by G_12/13 and G_i pathways. These data demonstrate that co-stimulation of G_12/13 and G_i pathways is sufficient to activate GPIIb/IIIa in human platelets in a mechanism that involves intracellular calcium, and that PI 3-kinase is an important signaling molecule downstream of G_q but not downstream of G_12/13 pathway.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Agonists for platelet activation, though having varying efficacies for platelet dense granule secretion and fibrinogen receptor (GPIIb/IIIa; integrin _IIb3) activation, often signal through similar G-protein signaling pathways (1, 2). GPIIb/IIIa receptor activation occurs by G protein-mediated inside-out signaling stimulated by platelet agonists such as ADP, thromboxane A₂, and thrombin (3). These agonists cause GPIIb/IIIa to go from a low affinity state to a high affinity binding state that results in the binding of fibrinogen and cross-linking of platelets (3). Epinephrine binds to the _2A-adrenergic receptor and causes activation of the G_z pathway that leads to the inhibition of adenylyl cyclase (4, 5). Stimulation of the _2A-adrenergic receptor alone is insufficient to cause either dense granule secretion or GPIIb/IIIa activation in washed platelets; however, epinephrine potentiates both secretion and platelet aggregation caused by other agonists (6-9). ADP binds to the G_q¹-coupled P2Y1 and the G_i-coupled P2Y12 receptors, and signaling through both of these pathways is necessary for ADP-induced GPIIb/IIIa activation (8, 10-12), although ADP does not cause dense granule secretion in aspirin-treated human platelets (13). Thromboxane A₂ binds to the TP and TP receptor subtypes that activate both G_q (14, 15) and G_12/13 signaling (16). Thromboxane receptor stimulation causes both platelet aggregation and dense granule secretion but depends upon secreted contents to provide G_i signaling. The combined signaling from TP receptor stimulation and the G_i signaling from the secreted ADP or epinephrine causes GPIIb/IIIa activation (17). Both ADP and thromboxane A₂ require co-stimulation of G_q and G_i pathways to cause platelet aggregation (8, 17). Thrombin cleaves the N terminus of PAR-1 and PAR-4 on human platelets, uncapping a tethered ligand that activates the PAR receptors (18). Both PAR-1 and PAR-4 receptors couple to G_q and G_12/13, and cause fibrinogen receptor activation independently of G_i stimulation by secreted ADP (19).

The heterotrimeric G proteins G₁₂ and G₁₃ are found in human platelets (20) and are activated upon thromboxane and thrombin receptor stimulation (16). The first evidence for the role of G_12/13 in platelet shape change came from the studies with G_q knockout mice wherein thrombin and thromboxane A₂ failed to cause platelet aggregation but caused platelet shape change (21). However, ADP failed to cause shape change in these mouse platelets indicating that ADP receptors do not couple to G_12/13 pathways (21). G_12/13 activates Rho/Rho kinase, causing the phosphorylation of myosin light chain and calcium-independent shape change (22). G_12/13 signaling mediates calcium-independent platelet shape change, involving RhoA and p160^ROCK activity in human and mouse platelets (22). Y-27632, a specific inhibitor of p160^ROCK, blocks the calcium-independent shape change that occurs because of G_12/13-mediated signaling, suggesting that p160^ROCK is a key signaling molecule downstream of G_12/13 for the platelet shape change response (23, 24). Though the G_12/13 pathway has been implicated in p160^ROCK activation and subsequent shape change, this pathway remains the least characterized of the known G protein-coupled pathways in platelets.

The G_q pathway stimulates phospholipase C, which cleaves phosphatidylinositol 4,5-bisphosphate and results in cofactors that activate protein kinase C (PKC) (1). The -subunit of the heterotrimeric G protein G_i pathway inhibits the activity of adenylyl cyclase while the -subunit activates PI 3-kinase (25). Together, these pathways lead to the activation of numerous kinases including protein kinase B (PKB/Akt) (26), PKC (4), Map kinase kinase (MEKK1) (27), Src family tyrosine kinases (28), among many others.

YFLLRNP is a heptapeptide that binds to PAR-1 and causes shape change but no calcium mobilization when used at low concentrations (29). This YFLLRNP-induced platelet shape change is mediated by the G_12/13-RhoA-p160^ROCK pathway and can be completely blocked by Y-27632 (24). Similarly, low concentrations of the thromboxane mimetic, U46619, also cause activation G_12/13 pathways without activating the G_q pathways (30, 31). In this study we used these selective agonists of G_12/13 pathways, in combination with selective activation of G_i pathways, to demonstrate the contribution of G_12/13 signaling cascades to fibrinogen receptor activation in human platelets. Previously, G_q and G_i have been recognized as the G proteins that activate pathways leading to platelet aggregation (8). Our studies demonstrate that the G_12/13 pathway, in the presence of G_i signaling, can lead to GPIIb/IIIa activation in human platelets and that PI-3 kinase is an important signaling molecule downstream of G_q, but not downstream of G_12/13 pathway.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- Apyrase grade VII, human fibrinogen, and acetylsalicylic acid were obtained from Sigma. The heptapeptide YFLLRNP was synthesized by New England Biolabs (Beverly, MA), and the same peptide was also synthesized by Research Genetics (Huntsville, AL). ADP and epinephrine were purchased from Chrono-Log Corp. (Havertown, PA). Fluorescein isothiocyanate-conjugated monoclonal antibody PAC-1 was purchased from BD Pharmingen. Fura-2, AM was purchased from Molecular Probes (Eugene, OR). [2,8-³H]Adenine was purchased from PerkinElmer Life Sciences. The acetoxymethyl ester of 5,5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (dimethyl BAPTA), Y-27632, LY294002, and Ro 31-8220 were purchased from Biomol (Plymouth Meeting, PA). U0126 was purchased from Alexis Biochemicals (Lausen, Switzerland). AR-C 69931MX was a gift from Astra-Zeneca Research Laboratories-Charnwood, Loughborough, UK.

Platelet Preparation-- Whole blood was drawn from healthy, consenting human volunteers into tubes containing one-sixth volume of ACD (2.5 g of sodium citrate, 1.5 g of citric acid, and 2 g of glucose in 100 ml of deionized water). Blood was centrifuged (Eppendorf 5810R centrifuge, Hamburg, Germany) at 230 rcf for 20 min at room temperature to obtain platelet-rich plasma (PRP). PRP was incubated with 1 mM acetylsalicylic acid (Sigma) for 30 min at 37 °C, and for calcium measurement PRP was also incubated with 2 mM Fura-2, AM for 45 min at 37 °C. The PRP was then centrifuged for 10 min at 980 rcf (room temperature) to pellet the platelets. Platelets were resuspended in Tyrode's buffer (138 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 3 mM NaH₂PO₄, 5 mM glucose, 10 mM Hepes pH 7.4, 0.2% bovine serum albumin) containing 0.01 units/ml apyrase. Cells were counted using the Z1 Coulter Particle Counter and adjusted to 2 × 10⁸ platelets/ml. For flow cytometry studies, cells were adjusted to a concentration of 4.2 × 10⁶ platelets/ml.

Aggregometry-- Aggregation of 0.5 ml of washed platelets was analyzed using a P.I.C.A. lumiaggregometer (Chrono-log Corp., Havertown, PA). Aggregation was measured using light transmission under mixing conditions (900 rpm) at 37 °C. Agonists were added simultaneously for platelet stimulation; however, platelets were preincubated with each inhibitor as follows: 1 µM dimethyl BAPTA, 10 µM Ro 31-8220, or 25 µM LY294002 for 3 min at 37 °C and 10 µM Y-27362 or 10 µM U0126 for 10 min 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. All samples contained exogeneously added human fibrinogen (1 mg/ml).

Intracellular Calcium Mobilization-- Calcium mobilization was measured in platelets that were loaded with 2 mM Fura-2, AM in PRP for 45 min at 37 °C, and washed platelets were isolated as noted above and brought to a final concentration of 2 × 10⁸ platelets/ml in Tyrode's buffer. Samples of Fura-2, AM-loaded platelets (0.5 ml) were placed in a quartz cuvette with a magnetic stir bar, and incubated for 1 min at 37 °C in a temperature-controlled chamber. An Aminco Bowman Series 2 Luminescence Spectrometer was used for measurement of intracellular calcium mobilization. Two wavelengths (340 and 380 nm) were used for excitation, and the emitted light was measured at 510 nm. Samples were stimulated after 1 min of incubation at 37 °C, and all concentrations of YFLLRNP were added in a volume of 5 µl to account for dilution effects. F_min was obtained by addition of 20 mM Tris and 4 mM EGTA, and F_max was determined by adding 0.25% Triton and saturating levels of CaCl₂. Calculation of the calcium mobilization was performed as outlined previously (32).

Analysis of PAC-1 Binding-- Activation of GPIIb/IIIa was measured by PAC-1 mAb binding to washed platelets and subsequent analysis by flow cytometry. Aspirin-treated platelets were isolated by centrifugation as noted, then counted, and brought to a concentration of 4.2 × 10⁶ platelets/ml. The assay was performed considering that three compounds, each 5 µl in volume, were added to each to each tube prior to addition of the platelets. PAC-1 mAb (5 µl) was also added to each tube. Tyrode's buffer was added in samples where less than three compounds were necessary to normalize the volume. Considering that there is a 20-µl total volume of agonist/mAb added to each sample, adding 50 µl of platelets to the 20 µl of agonist/Ab resulted in a final concentration to 3 × 10⁶ platelets/ml. The platelets were added to each tube in 15-s increments to begin stimulation. The samples were stimulated for a period of 10 min in the dark, and then diluted with 450 µl of Tyrode's buffer. 450 µl of each sample was transferred to a 12 × 75 mm cuvette (Fisher Scientific, Pittsburgh, PA) and analyzed by flow cytometry, using FACSCAN (BD Biosciences), to measure an increase in fluorescence that indicates an increase in GPIIb/IIIa receptor activation. The experiment was performed three times, and data are presented as mean ± S.E.

Measurement of Cyclic AMP Formation in Intact Platelets-- Platelet-rich plasma was incubated with 2 µCi/ml [³H]adenine and aspirin (1 mM) for 1 h at 37 °C (33). Platelets were isolated from plasma by centrifugation at 980 × g for 10 min and resuspended in Tyrode's buffer. Platelet preparations were incubated with 20 µM forskolin for 3 min to stimulate cAMP formation, or forskolin and agonist for measurement of G_i signaling stimulated by the agonist. Reactions were stopped with 1 M HCl and 4000 dpm of [¹⁴C]cAMP as recovery standard. Cyclic AMP was determined by the method of Salomon (34) and expressed as percentage of total [³H]adenine nucleotides.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The agonists ADP, thrombin, and thromboxane A₂ activate multiple G protein pathways, including G_q, G_12/13, and G_i, to activate platelet shape change, dense granule secretion, and GPIIb/IIIa receptor activation (1). Each agonist has a distinct mechanism to achieve full platelet activation and much work has been focused on identifying signaling molecules and determining the roles of each pathway in platelet activation. Whereas G_q and G_i pathways have been identified as regulating GPIIb/IIIa activation (8), and G_12/13 signaling has been implicated in platelet shape change (22-24), the contribution of G_12/13 stimulation to platelet fibrinogen receptor activation has not been demonstrated.

Determination of the Functional Coupling Specificity of YFLLRNP-- Thrombin-mediated cleavage of the PAR-1 receptor causes activation of both G_q and G_12/13 pathways, leading to a calcium-dependent and calcium-independent shape change, respectively (16, 35). YFLLRNP is a partial agonist at the PAR-1 receptor that antagonizes both -thrombin- and SFLLRNP-mediated platelet aggregation and causes platelet shape change without calcium mobilization or platelet aggregation (29). We first evaluated the concentration-dependent activation of G proteins by YFLLRNP ranging from 50 to 200 µM to identify the proper concentration of peptide that is activating G_12/13 but not activating G_q signaling. We noted that 60 µM YFLLRNP caused platelet shape change (Fig. 1A) without aggregation or calcium mobilization (Fig. 1B). Intracellular calcium mobilization occurred at 100 µM YFLLRNP or higher, suggesting that the peptide activated both G_q and G_12/13 at higher concentrations. The same peptide synthesized from a different source provided similar results (data not shown). While other studies used up to 300 µM YFLLRNP without calcium mobilization (29), higher concentrations of YFLLRNP (100-200 µM) caused small calcium mobilization in our hands, suggesting that there is an increase in G_q coupling. This difference in potency of the peptide could be due to different quality/purity of the synthesized peptide.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Characterization of YFLLRNP-mediated human platelet responses. A, platelet shape change induced by YFLLRNP was measured in a washed human platelet system using lumi aggregometer. The sample was incubated with 10 µM Y-27632 for 10 min at 37 °C before addition of agonist. The additions are indicated by arrows. Data are representative of tracings obtained from three different donors. B, calcium mobilization in Fura-2-loaded washed platelets. Arrows are representative of addition of agonist. Tracings are representative of data obtained from three separate donors. C, cyclic AMP formation was measured after stimulation with 20 µM forskolin and either 10 µM ADP or 60 µM YFLLRNP. Data are expressed as percent of total [3H]adenine nucleotides and are the means ± S.E. of three separate experiments performed on different donors.

Thromboxane receptors and protease activated receptors couple to G_q and G_12/13 pathways and this coupling is dependent on the concentration of the agonist (16, 30, 31). Subsequent studies revealed that G_12/13-mediated platelet shape change is slow, occurs in the absence of calcium mobilization, involves p160^ROCK as an important signaling molecule, and can be completely blocked by the p160^ROCK inhibitor, Y-27632 (22-24). Thus, the slow platelet shape change in the absence of intracellular calcium mobilization that can be blocked by Y-27632 can be taken as a measure of G_12/13 activation.

To ensure that YFLLRNP was activating the G_12/13 pathway specifically, we measured YFLLRNP-mediated platelet shape change in the presence or absence of 10 µM Y-27632. As expected, 10 µM Y-27632 completely inhibited platelet shape change caused by 60 µM YFLLRNP (Fig. 1A), suggesting that low dose YFLLRNP is causing only G_12/13-mediated shape change without a calcium-dependent shape change component.

PAR-1 can couple to the G_i pathway and cause the inhibition of adenylyl cyclase (35); however, other data suggest that PAR-1 stimulation relies upon secreted ADP for G_i activation (19). To investigate whether YFLLRNP can activate the G_i pathway, we measured cAMP formation in YFLLRNP-stimulated platelets. YFLLRNP (60 µM) did not cause significant inhibition of forskolin-stimulated adenylyl cyclase (Fig. 1C), indicating that at this concentration YFLLRNP does not activate G_i signaling pathways.

Contribution of G_12/13 Signaling to Platelet Aggregation and GPIIb/IIIa Receptor Activation-- Selective activation of G_q pathways by ADP results only in shape change, while supplementing G_q signaling with G_i activation, through P2Y₁₂ receptor activation or _2A receptor activation, results in platelet aggregation (8, 36). As selective activation of G_12/13 pathways with YFLLRNP (60 µM) resulted only in shape change (Fig. 1A), we investigated the effect of supplementing this pathway with G_i signaling cascade on platelet fibrinogen receptor activation.

ADP causes platelet aggregation by stimulating both the G_q-coupled P2Y1 receptor and the G_i-coupled P2Y₁₂ receptor (8). We used A3P5P, a P2Y₁-selective antagonist to block ADP signaling through the G_q-coupled P2Y₁ receptor. Addition of 10 µM ADP in the presence of 1 mM A3P5P results in selective stimulation of the G_i-coupled P2Y₁₂ receptor, and is evident by the loss of ADP-induced shape change and aggregation (8). YFLLRNP (60 µM) in the presence of P2Y₁₂-selective stimulation caused platelet aggregation (Fig. 2). Whereas epinephrine alone does not cause aggregation, simultaneous addition of epinephrine with YFLLRNP caused platelet aggregation (Fig. 2). We also noted that addition of 10 µM epinephrine immediately subsequent to the addition of YFLLRNP caused platelet aggregation (data not shown).

View larger version (10K): [in this window] [in a new window]   Fig. 2.   The effect of combined G12/13 and Gi signaling on human platelet aggregation. Samples (0.5 ml) of aspirin-treated and washed human platelets were placed in a cuvette in the presence of 1 mg/ml human fibrinogen. In cases of multiple agonists, either 60 µM YFLLRNP + 10 µM epinephrine or 60 µM YFLLRNP + 10 µM ADP were added simultaneously. The P2Y1 antagonist 1 mM A3P5P was added to samples prior to stimulation with YFLLRNP + ADP. Tracings are representative of three experiments.

Though we have demonstrated that platelet aggregation can occur in the presence of G_12/13 and G_i signaling, we wanted to directly correlate concomitant G_12/13 and G_i signaling with GPIIb/IIIa activation. The GPIIb/IIIa receptor shifts from a low affinity state to a high affinity state upon platelet stimulation with agonists such as thrombin, ADP, or thromboxane A₂ (3). The PAC-1 mAb is directed against the active conformation of the GPIIb/IIIa receptor (37). YFLLRNP-stimulated platelets bound similar levels of PAC-1 mAb compared with unstimulated platelets (Fig. 3). Platelets treated with either 10 µM epinephrine or ADP and A3P5P bound background levels of PAC-1 Ab confirming that G_i signaling alone was insufficient to cause significant GPIIb/IIIa activation. ADP (10 µM) caused a similar magnitude of PAC-1 mAb binding compared with YFLLRNP plus epinephrine. Also, platelets stimulated simultaneously with YFLLRNP and selective P2Y₁₂ stimulation bound levels of PAC-1 mAb similar to ADP-stimulated cells (Fig. 3). These results suggest that while activation of either G_12/13 or G_i signaling alone cannot cause GPIIb/IIIa receptor activation, co-stimulation of G_12/13 and G_i signaling pathways can result in GPIIb/IIIa activation.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   The effect of combined G12/13 and Gi signaling on PAC-1 mAb binding. Aspirin-treated and washed human platelets were added to tubes containing 5 µl of PAC-1 mAb and the agonists noted. Platelets were stimulated for 10 min each, diluted with Tyrode's and immediately analyzed on a FACSCAN flow cytometer for increases in fluorescence that correlate with GPIIb/IIIa activation. Data was calculated as median fluorescence by multiplying the median point of the cell population with the percentage of the cell population in the marker. Each bar is the average of three experiments ± S.E. from three donors. Asterisk denotes p < 0.05. NS, statistically not significant.

The thromboxane receptor couples to G_q and G_12/13 in human platelets (16, 38). We used a stable thromboxane A₂ mimetic, U46619, for stimulation of the TP receptor. At low doses of U46619 (10 nM), the receptor couples only to the G_12/13 pathway (30, 31). Thus, a low concentration of U46619 provides an alternative to low dose of YFLLRNP to stimulate G_12/13 pathways through TP receptors. Stimulation of the platelets with this concentration of U46619 resulted in platelet shape change, but not in calcium mobilization or in platelet aggregation (Fig. 4). However, higher concentration of U46619 (100 nM) causes calcium mobilization (Fig. 4A) and calcium-dependent shape change that is not inhibited by Y-27632 (Fig. 4B). Simultaneous addition of either 10 µM epinephrine or 10 µM ADP in the presence of 1 mM A3P5P to 10 nM U46619-stimulated platelets lead to both shape change and platelet aggregation (Fig. 4C). This illustrates that either P2Y₁₂ receptor or _2A-adrenergic receptor stimulation is capable of causing platelet aggregation when combined with G_12/13 signaling from the TP receptor. When we were finalizing the article, Nieswandt et al. (39) reported that stimulation of G_12/13 and G_i is sufficient to cause fibrinogen receptor activation in mouse platelets using mice-deficient in G_q. Their results, obtained by a complementary approach, support our conclusions and extend the observations to mouse platelets. These results may also explain why ADP is weaker agonist than thromboxane A₂ and thrombin. ADP activates only G_q pathways and does not activate the G_12/13 pathways, whereas both thromboxane A₂ and thrombin do activate this pathway. Since either G_q or G_12/13 can synergize with G_i to result in the activation of GPIIb/IIIa, thrombin and thromboxane A₂, activating both G_q and G_12/13, could additionally synergize with G_i and thereby cause more robust platelet aggregation.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Selective stimulation of the G12/13 pathway via TP receptor causes aggregation when combined with Gi signaling. Platelet aggregation was measured as described under "Experimental Procedures." The arrows indicate the addition of agonists. Addition of multiple agonists was done simultaneously. The P2Y1 antagonist 1 mM A3P5P was added to samples prior to stimulation with 10 nM U46619 + 10 µM ADP. Tracings are representative of three experiments from three different donors.

Role of Intracellular Calcium in G_12/13 and G_i-mediated Human Platelet Aggregation-- Calcium plays an important role in the platelet function, including the activation of GPIIb/IIIa (1, 3). Although the subunits of G_i are known to increase intracellular calcium by the activation of phospholipase C in other cells (40), selective activation of G_i in platelets through either P2Y₁₂ or _2A receptors does not mobilize intracellular calcium (8, 36). Although neither epinephrine nor YFLLRNP (60 µM) caused any increases in intracellular calcium, together they mobilized a small amount of calcium (15 ± 4 nM) from the intracellular stores (Fig. 5A). As stimulation of G_12/13 or G_i alone does not cause increases in intracellular calcium, it is surprising to see this small increase with co-stimulation of these two pathways. ADP (300 nM) caused similar increases in intracellular calcium as YFLLRNP and epinephrine together (Fig. 5A). Hence, we used ADP (300 nM) in the presence of AR-C 69931MX, a selective P2Y₁₂ receptor antagonist, to selectively activate the G_q pathway and increase a small and comparable intracellular calcium (Fig. 5A), and evaluated the effect of epinephrine on platelet aggregation. As shown in Fig. 5B, although selective activation of P2Y₁ receptor alone did not cause any aggregation, co-stimulation of P2Y₁ and _2A-adrenergic receptors led to comparable extent of aggregation as the combined G_12/13 and G_i stimulation (Fig. 5B). These data indicate that co-stimulation of G_12/13 and G_i results in a small increase in intracellular calcium which may play an important role in the activation of GPIIb/IIIa. Contrary to our results, Nieswandt et al. (39) did not observe any intracellular calcium mobilization with the combined G_12/13 and G_i signaling in mouse platelets. Hence, we investigated the role of this small amount of intracellular calcium in the platelet fibrinogen receptor activation using an intracellular calcium chelator, dimethyl BAPTA. As shown in Fig. 5C, preincubation of platelets with dimethyl BAPTA (1 µM) dramatically blocked the aggregation, but not shape change, induced by YFLLRNP and epinephrine. These results indicate that the small increases in intracellular calcium, as a result of combined G_12/13 and G_i stimulation, play an important role in the activation of GPIIb/IIIa in human platelets.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   The role of calcium in platelet aggregation caused by combined G12/13 and Gi stimulation. A, intracellular calcium mobilization. The tracings are representative of each concentration of agonist-mediated calcium mobilization of three experiments. Data are compared with a single concentration of ADP (3 µM). B, platelet aggregation caused by selective activation of Gq and Gi pathways with small increase in intracellular calcium. Platelets stimulated with agonists as noted. C, effect of dimethyl BAPTA. Aspirin-treated, washed human platelets were preincubated with 1 µM dimethyl BAPTA for 3 min at 37 °C. After preincubation, samples were stimulated with G12/13 and Gi signaling via 60 µM YFLLRNP + 10 µM epinephrine. Tracings are representative of three experiments from three different donors.

Signaling Events Downstream of Concomitant Activation of G Proteins in Human Platelets-- The signaling events that occur downstream of platelet receptor stimulation has been the subject of intense study in several laboratories. Major signaling molecules lying downstream of G protein activation include PKC (4), MEKK1 (41), PI 3-kinase (25, 26), and p160^ROCK (23, 24), among many others (3). We measured the effects of selective inhibitors for these molecules on platelet aggregation stimulated by combined G_12/13 and G_i signaling. We then compared the effects of these inhibitors on concomitant G_q- and G_i-mediated platelet aggregation (8), using ADP as the agonist.

PKC inhibition with Ro 31-8220, an inhibitor of novel and conventional PKC isoforms (42), had no effect on the aggregation caused by concomitant G_12/13 and G_i signaling or G_q and G_i signaling (Fig. 6, A and B). These results are consistent with our previous observations, that the PKC pathway is important, but not essential, in the activation of GPIIb/IIIa (43). U0126, a MEKK1 inhibitor (44), also had no effect on the aggregation induced by co-activation of either G_12/13 and G_i or G_q and G_i signaling. Thus, although Erk2 has been implicated in the GP1b-IX-mediated platelet fibrinogen receptor activation (27), the MEKK-Erk pathway does not play any significant role in either G_12/13- and G_i- or G_q- and G_i-mediated GPIIb/IIIa activation in human platelets.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   The effect of protein kinase inhibitors on platelet aggregation caused by combined G protein stimulation. Aspirin-treated, washed human platelets were preincubated with the inhibitors as follows: 3-min preincubation with 10 µM Ro31-8220 or 25 µM LY 294002, at 37 °C, 10-min preincubation with 10 µM U0126, or 10 µM Y-27632 at 37 °C. After preincubation, samples were stimulated with G12/13 and Gi signaling via 60 µM YFLLRNP + 10 µM epinephrine (A) or Gq and Gi signaling via ADP (10 µM) (B). Tracings are representative of three experiments from three different donors. Addition of agonist(s) is indicated by an arrow.

PI 3-kinase has been known to be involved in platelet activation (3), and knockout studies show that PI 3-kinase -deficient mice have decreased aggregation responses to ADP and collagen (25). LY294002, a PI 3-kinase inhibitor (45), caused a slight decrease in the extent of combined G_12/13- and G_i-mediated aggregation; however, aggregation and shape change were still significant in the presence of PI 3-kinase inhibitor (Fig. 6A). This effect was comparable to the decrease in ADP-induced platelet aggregation in PI 3-kinase -deficient mice versus wild type mice (25). While there was a decrease in aggregation, it is unlikely that PI 3-kinase is a key signaling molecule downstream of G_12/13 signaling. Rather, LY 294002 is mediating its effects through decreasing the P2Y12- or _2A-adrenergic-stimulated G_i and PI 3-kinase  signaling pathways (46) (depicted in Fig. 7). Conversely, concomitant G_q- and G_i-mediated platelet aggregation was nearly abolished by the PI 3-kinase inhibitor (Fig. 6B). These results indicate that PI 3-kinase is a key signaling molecule in the combined G_q and G_i pathway. By comparison, PI 3-kinase appears to be a key molecule in the G_q signaling cascade, but not in G_12/13 mediated signaling pathway, leading to the fibrinogen receptor activation (Fig. 7).

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Model depicting GPIIb/IIIa activation caused by co-stimulation of the G12/13 and Gi pathways. The G12/13-coupled receptor (on left) represents either the TP receptor, which is stimulated by thromboxane A2, or PAR-1 receptor, which is stimulated by thrombin and YFLLRNP. The Gi, Gz-coupled receptor (center) represents either the 2A-adrenergic receptor, which is stimulated by epinephrine, or the P2Y12 receptor, which is stimulated by ADP. The Gq-coupled receptor (on the right) represents the TP receptor, PAR-1 or the P2Y1 receptor, which is stimulated by ADP. The double bars represent the inhibitory activity of Y-27632 on p160ROCK activity.

p160^ROCK has been identified as a key signaling molecule downstream of G_12/13 activation (23, 24). Using the p160^ROCK inhibitor Y-27632, we expected that platelet aggregation caused by concomitant G_12/13 and G_i signaling would be inhibited. Interestingly, Y-27632 did not block aggregation caused by simultaneous G_12/13 and G_i signaling (Fig. 6A), suggesting that there is a divergent pathway downstream of G_12/13 stimulation. Thus, G_12/13 signals through at least two separate pathways, one of which involves p160^ROCK and shape change, and the other that contributes to GPIIb/IIIa activation. As expected, combined G_q- and G_i-mediated platelet aggregation was also unaffected by the p160^ROCK inhibitor (Fig. 6B), indicating that this signaling molecule does not play any significant role in the activation of fibrinogen receptor (Fig. 7).

In conclusion, we have demonstrated that coordinated signaling between G_12/13 and G_i pathways is a sufficient and redundant mechanism for the activation of fibrinogen receptor in human platelets. PI 3-kinase appears to be an important signaling molecule downstream of G_q- but not G_12/13-mediated activation of GPIIb/IIIa. Co-stimulation of G_12/13 and G_i pathways appears to increase intracellular calcium, independently of G_q activation, which plays an important role in the fibrinogen receptor activation in human platelets. The mechanisms of increase in intracellular calcium by G_12/13 and G_i pathways are under investigation.

	ACKNOWLEDGEMENTS

We thank Drs. James L. Daniel, Barrie Ashby, and Todd M. Quinton, Temple University Medical School, for critically reading the paper.

	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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by Training Grant T32 HL07777 from the National Institutes of Health.

^** To whom correspondence should be addressed: Dept. of Physiology, Temple University, Rm. 224, OMS, 3420 N. Broad St., Philadelphia, PA 19140. Tel.: 215-707-4615; Fax: 215-707-4003; E-mail: kunapuli@ nimbus.temple.edu.

Published, JBC Papers in Press, September 23, 2002, DOI 10.1074/jbc.M208778200

	ABBREVIATIONS

The abbreviations used are: G_q, heterotrimeric GTP-binding protein which stimulates phospholipase C; G_i, heterotrimeric GTP-binding protein which inhibits adenylyl cyclase; PKC, protein kinase C; TP receptor, thromboxane A₂ receptor; U46619, 15(S)-hydroxy-11,9-epoxymethano-prosta-5Z,13E-dienoic acid; 5, 5'-dimethyl BAPTA, 5,5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; P2Y₁₂, platelet ADP receptor coupled to inhibition of adenylyl cyclase; P2Y₁, platelet ADP receptor coupled to stimulation of phospholipase C; G_12/13, heterotrimeric GTP-binding proteins 12 and 13; ROCK, Rho-associated coiled-coil forming kinase; MEKK, mitogen-activated protein kinase kinase; mAb, monoclonal antibody; PI, phosphatidylinositol; PRP, platelet-rich plasma.

	REFERENCES

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