Coordinated Signaling through Both G12/13 and Gi Pathways Is Sufficient to Activate GPIIb/IIIa in Human Platelets
2002; Elsevier BV; Volume: 277; Issue: 49 Linguagem: Inglês
10.1074/jbc.m208778200
ISSN1083-351X
AutoresRobert T. Dorsam, Soochong Kim, Jianguo Jin, Satya P. Kunapuli,
Tópico(s)Blood disorders and treatments
ResumoActivation of GPIIb/IIIa is known to require agonist-induced inside-out signaling through Gq, Gi, and Gz. Although activated by several platelet agonists, including thrombin and thromboxane A2, the contribution of the G12/13signaling 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 G12/13 activation to platelet fibrinogen receptor activation. YFLLRNP is a PAR-1-specific partial agonist that, at low concentrations (60 μm), selectively activates the G12/13 signaling cascade resulting in platelet shape change without stimulating the Gq or Gi signaling pathways. YFLLRNP-mediated shape change was completely inhibited by the p160ROCKinhibitor, Y-27632. At this low concentration, YFLLRNP-mediated G12/13 signaling caused platelet aggregation and enhanced PAC-1 binding when combined with selective Gi or Gz signaling, via selective stimulation of the P2Y12 receptor or α2A-adrenergic receptor, respectively. Similar data were obtained when using low dose U46619 (10 nm), a thromboxane A2 mimetic, to activate G12/13 in the presence of Gi signaling. These results suggest that selective activation of G12/13 causes platelet GPIIb/IIIa activation when combined with Gisignaling. Unlike either G12/13 or Giactivation alone, co-activation of both G12/13 and Gi resulted in a small increase in intracellular calcium. Chelation of intracellular calcium with dimethyl BAPTA dramatically blocked G12/13 and Gi-mediated platelet aggregation. No significant effect on aggregation was seen when using selective inhibitors for p160ROCK, PKC, or MEKK1. PI 3-kinase inhibition lead to near abolishment of platelet aggregation induced by co-stimulation of Gq and Gipathways, but not by G12/13 and Gipathways. These data demonstrate that co-stimulation of G12/13 and Gi 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 Gq but not downstream of G12/13 pathway. Activation of GPIIb/IIIa is known to require agonist-induced inside-out signaling through Gq, Gi, and Gz. Although activated by several platelet agonists, including thrombin and thromboxane A2, the contribution of the G12/13signaling 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 G12/13 activation to platelet fibrinogen receptor activation. YFLLRNP is a PAR-1-specific partial agonist that, at low concentrations (60 μm), selectively activates the G12/13 signaling cascade resulting in platelet shape change without stimulating the Gq or Gi signaling pathways. YFLLRNP-mediated shape change was completely inhibited by the p160ROCKinhibitor, Y-27632. At this low concentration, YFLLRNP-mediated G12/13 signaling caused platelet aggregation and enhanced PAC-1 binding when combined with selective Gi or Gz signaling, via selective stimulation of the P2Y12 receptor or α2A-adrenergic receptor, respectively. Similar data were obtained when using low dose U46619 (10 nm), a thromboxane A2 mimetic, to activate G12/13 in the presence of Gi signaling. These results suggest that selective activation of G12/13 causes platelet GPIIb/IIIa activation when combined with Gisignaling. Unlike either G12/13 or Giactivation alone, co-activation of both G12/13 and Gi resulted in a small increase in intracellular calcium. Chelation of intracellular calcium with dimethyl BAPTA dramatically blocked G12/13 and Gi-mediated platelet aggregation. No significant effect on aggregation was seen when using selective inhibitors for p160ROCK, PKC, or MEKK1. PI 3-kinase inhibition lead to near abolishment of platelet aggregation induced by co-stimulation of Gq and Gipathways, but not by G12/13 and Gipathways. These data demonstrate that co-stimulation of G12/13 and Gi 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 Gq but not downstream of G12/13 pathway. Agonists for platelet activation, though having varying efficacies for platelet dense granule secretion and fibrinogen receptor (GPIIb/IIIa; integrin αIIbβ3) activation, often signal through similar G-protein signaling pathways (1Brass L.F. Manning D.R. Cichowski K. Abrams C.S. Thromb. Haemost. 1997; 78: 581-589Google Scholar, 2Offermanns S. Biol. Chem. 2000; 381: 389-396Google Scholar). GPIIb/IIIa receptor activation occurs by G protein-mediated inside-out signaling stimulated by platelet agonists such as ADP, thromboxane A2, and thrombin (3Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Google Scholar). 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 (3Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Google Scholar). Epinephrine binds to the α2A-adrenergic receptor and causes activation of the Gz pathway that leads to the inhibition of adenylyl cyclase (4Brass L.F. Woolkalis M.J. Manning D.R. J. Biol. Chem. 1988; 263: 5348-5355Google Scholar, 5Yang J. Wu J. Kowalska M.A. Dalvi A. Prevost N. O'Brien P.J. Manning D. Poncz M. Lucki I. Blendy J.A. Brass L.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9984-9989Google Scholar). Stimulation of the α2A-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 (6Steen V.M. Holmsen H. Aarbakke G. Thromb. Haemost. 1993; 70: 506-513Google Scholar, 7Lanza F. Beretz A. Stierle A. Hanau D. Kubina M. Cazenave J.P. Am. J. Physiol. 1988; 255: H1276-H1288Google Scholar, 8Jin J. Kunapuli S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8070-8074Google Scholar, 9Dangelmaier C. Jin J. Smith J.B. Kunapuli S.P. Thromb. Haemost. 2001; 85: 341-348Google Scholar). ADP binds to the Gq 1The abbreviations used are: Gq, heterotrimeric GTP-binding protein which stimulates phospholipase C; Gi, 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; G12/13, 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 -coupled P2Y1 and the Gi-coupled P2Y12 receptors, and signaling through both of these pathways is necessary for ADP-induced GPIIb/IIIa activation (8Jin J. Kunapuli S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8070-8074Google Scholar, 10Daniel J.L. Dangelmaier C. Jin J. Ashby B. Smith J.B. Kunapuli S.P. J. Biol. Chem. 1998; 273: 2024-2029Google Scholar, 11Hollopeter J. Jantzen H.-M. Vincent D. Li G. England L. Ramakrishnan V. Yang R.-B. Nurden P. Nurden A. Julius D.J. Conley P.B. Nature. 2001; 409: 202-207Google Scholar, 12Foster C.J. Prosser D.M. Agans J.M. Zhai Y. Smith M.D. Lachowicz J.E. Zhang F.L. Gustafson E. Monsma Jr., F.J. Wiekowski M.T. Abbondanzo S.J. Cook D.N. Bayne M.L. Lira S.A. Chintala M.S. J. Clin. Invest. 2001; 107: 1591-1598Google Scholar), although ADP does not cause dense granule secretion in aspirin-treated human platelets (13Mills D.C.B. Thromb. Haemost. 1996; 76: 835-856Google Scholar). Thromboxane A2 binds to the TPα and TPβ receptor subtypes that activate both Gq (14Raychowdhury M.K. Yukawa M. Collins L.J. McGrail S.H. Kent K.C. Ware J.A. J. Biol. Chem. 1995; 270: 7011Google Scholar, 15Raychowdhury M.K. Yukawa M. Collins L.J. McGrail S.H. Kent K.C. Ware J.A. J. Biol. Chem. 1994; 269: 19256-19261Google Scholar) and G12/13 signaling (16Offermanns S. Laugwitz K.-L. Spicher K. Schulz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 504-508Google Scholar). Thromboxane receptor stimulation causes both platelet aggregation and dense granule secretion but depends upon secreted contents to provide Gi signaling. The combined signaling from TP receptor stimulation and the Gi signaling from the secreted ADP or epinephrine causes GPIIb/IIIa activation (17Paul B.Z.S. Jin J. Kunapuli S.P. J. Biol. Chem. 1999; 274: 29108-29114Google Scholar). Both ADP and thromboxane A2 require co-stimulation of Gq and Gi pathways to cause platelet aggregation (8Jin J. Kunapuli S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8070-8074Google Scholar, 17Paul B.Z.S. Jin J. Kunapuli S.P. J. Biol. Chem. 1999; 274: 29108-29114Google Scholar). Thrombin cleaves the N terminus of PAR-1 and PAR-4 on human platelets, uncapping a tethered ligand that activates the PAR receptors (18Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11023-11027Google Scholar). Both PAR-1 and PAR-4 receptors couple to Gq and G12/13, and cause fibrinogen receptor activation independently of Gistimulation by secreted ADP (19Kim S. Foster C. Lecchi A. Quinton T.M. Prosser D.M. Jin J. Cattaneo M. Kunapuli S.P. Blood. 2002; 99: 3629-3636Google Scholar). The heterotrimeric G proteins G12 and G13 are found in human platelets (20Milligan G. Mullaney I. Mitchell F.M. FEBS Lett. 1992; 297: 186-188Google Scholar) and are activated upon thromboxane and thrombin receptor stimulation (16Offermanns S. Laugwitz K.-L. Spicher K. Schulz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 504-508Google Scholar). The first evidence for the role of G12/13 in platelet shape change came from the studies with Gq knockout mice wherein thrombin and thromboxane A2 failed to cause platelet aggregation but caused platelet shape change (21Offermanns S. Toombs C.F. Hu Y.-H. Simon M.I. Nature. 1997; 389: 183-186Google Scholar). However, ADP failed to cause shape change in these mouse platelets indicating that ADP receptors do not couple to G12/13 pathways (21Offermanns S. Toombs C.F. Hu Y.-H. Simon M.I. Nature. 1997; 389: 183-186Google Scholar). G12/13 activates Rho/Rho kinase, causing the phosphorylation of myosin light chain and calcium-independent shape change (22Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Google Scholar). G12/13 signaling mediates calcium-independent platelet shape change, involving RhoA and p160ROCK activity in human and mouse platelets (22Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Google Scholar). Y-27632, a specific inhibitor of p160ROCK, blocks the calcium-independent shape change that occurs because of G12/13-mediated signaling, suggesting that p160ROCK is a key signaling molecule downstream of G12/13 for the platelet shape change response (23Paul B.Z.S. Daniel J.L. Kunapuli S.P. J. Biol. Chem. 1999; 274: 28293-28300Google Scholar, 24Bauer M. Retzer M. Wilde J.I. Maschberger P. Essler M. Aepfelbacher M. Watson S.P. Siess W. Blood. 1999; 94: 1665-1672Google Scholar). Though the G12/13 pathway has been implicated in p160ROCK activation and subsequent shape change, this pathway remains the least characterized of the known G protein-coupled pathways in platelets. The Gq pathway stimulates phospholipase C, which cleaves phosphatidylinositol 4,5-bisphosphate and results in cofactors that activate protein kinase C (PKC) (1Brass L.F. Manning D.R. Cichowski K. Abrams C.S. Thromb. Haemost. 1997; 78: 581-589Google Scholar). The α-subunit of the heterotrimeric G protein Gi pathway inhibits the activity of adenylyl cyclase while the βγ-subunit activates PI 3-kinase (25Hirsch E. Bosco O. Tropel P. Laffargue M. Calvez R. Altruda F. Wymann M. Montrucchio G. FASEB J. 2001; 15: 2019-2021Google Scholar). Together, these pathways lead to the activation of numerous kinases including protein kinase B (PKB/Akt) (26Banfic H. Tang X. Batty I.H. Downes C.P. Chen C. Rittenhouse S.E. J. Biol. Chem. 1998; 273: 13-16Google Scholar), PKC (4Brass L.F. Woolkalis M.J. Manning D.R. J. Biol. Chem. 1988; 263: 5348-5355Google Scholar), Map kinase kinase (MEKK1) (27Li Z. Xi X. Du X. J. Biol. Chem. 2001; 276: 42226-42232Google Scholar), Src family tyrosine kinases (28Bauer M. Maschberger P. Quek L. Briddon S.J. Dash D. Weiss M. Watson S.P. Siess W. Thromb. Haemost. 2001; 85: 331-340Google Scholar), 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 (29Rasmussen U.B. Gachet C. Schlesinger Y. Hanau D. Ohlmann P. Van Obberghen-Schilling E. Pouyssegur J. Cazenave J.P. Pavirani A. J. Biol. Chem. 1993; 268: 14322-14328Google Scholar). This YFLLRNP-induced platelet shape change is mediated by the G12/13-RhoA-p160ROCK pathway and can be completely blocked by Y-27632 (24Bauer M. Retzer M. Wilde J.I. Maschberger P. Essler M. Aepfelbacher M. Watson S.P. Siess W. Blood. 1999; 94: 1665-1672Google Scholar). Similarly, low concentrations of the thromboxane mimetic, U46619, also cause activation G12/13 pathways without activating the Gqpathways (30Ohkubo S. Nakahata N. Ohizumi Y. Br. J. Pharmacol. 1996; 117: 1095-1104Google Scholar, 31Simpson A.W. Hallam T.J. Rink T.J. FEBS Lett. 1986; 201: 301-305Google Scholar). In this study we used these selective agonists of G12/13 pathways, in combination with selective activation of Gi pathways, to demonstrate the contribution of G12/13 signaling cascades to fibrinogen receptor activation in human platelets. Previously, Gq and Gi have been recognized as the G proteins that activate pathways leading to platelet aggregation (8Jin J. Kunapuli S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8070-8074Google Scholar). Our studies demonstrate that the G12/13 pathway, in the presence of Gisignaling, can lead to GPIIb/IIIa activation in human platelets and that PI-3 kinase is an important signaling molecule downstream of Gq, but not downstream of G12/13 pathway. 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. 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 mmMgCl2, 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 × 106platelets/ml. 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 μmdimethyl BAPTA, 10 μm Ro 31-8220, or 25 μmLY294002 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). 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. Fmin was obtained by addition of 20 mm Tris and 4 mm EGTA, and Fmax was determined by adding 0.25% Triton and saturating levels of CaCl2. Calculation of the calcium mobilization was performed as outlined previously (32Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Google Scholar). 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. Platelet-rich plasma was incubated with 2 μCi/ml [3H]adenine and aspirin (1 mm) for 1 h at 37 °C (33Kunapuli S.P. Fen Mao G. Bastepe M. Liu-Chen L.Y. Li S. Cheung P.P. DeRiel J.K. Ashby B. Biochem. J. 1994; 298: 263-267Google Scholar). 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 μmforskolin for 3 min to stimulate cAMP formation, or forskolin and agonist for measurement of Gi 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 (34Salomon Y. Adv. Cyc. Nucl. Res. 1979; 10: 35-55Google Scholar) and expressed as percentage of total [3H]adenine nucleotides. The agonists ADP, thrombin, and thromboxane A2activate multiple G protein pathways, including Gq, G12/13, and Gi, to activate platelet shape change, dense granule secretion, and GPIIb/IIIa receptor activation (1Brass L.F. Manning D.R. Cichowski K. Abrams C.S. Thromb. Haemost. 1997; 78: 581-589Google Scholar). 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 Gq and Gi pathways have been identified as regulating GPIIb/IIIa activation (8Jin J. Kunapuli S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8070-8074Google Scholar), and G12/13 signaling has been implicated in platelet shape change (22Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Google Scholar, 23Paul B.Z.S. Daniel J.L. Kunapuli S.P. J. Biol. Chem. 1999; 274: 28293-28300Google Scholar, 24Bauer M. Retzer M. Wilde J.I. Maschberger P. Essler M. Aepfelbacher M. Watson S.P. Siess W. Blood. 1999; 94: 1665-1672Google Scholar), the contribution of G12/13 stimulation to platelet fibrinogen receptor activation has not been demonstrated. Thrombin-mediated cleavage of the PAR-1 receptor causes activation of both Gq and G12/13 pathways, leading to a calcium-dependent and calcium-independent shape change, respectively (16Offermanns S. Laugwitz K.-L. Spicher K. Schulz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 504-508Google Scholar, 35Hung D.T. Wong Y.H. Vu T.-K.H. Coughlin S.R. J. Biol. Chem. 1992; 267: 20831-20834Google Scholar). YFLLRNP is a partial agonist at the PAR-1 receptor that antagonizes both α-thrombin- and SFLLRNP-mediated platelet aggregation and causes platelet shape change without calcium mobilization or platelet aggregation (29Rasmussen U.B. Gachet C. Schlesinger Y. Hanau D. Ohlmann P. Van Obberghen-Schilling E. Pouyssegur J. Cazenave J.P. Pavirani A. J. Biol. Chem. 1993; 268: 14322-14328Google Scholar). 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 G12/13 but not activating Gq signaling. We noted that 60 μm YFLLRNP caused platelet shape change (Fig. 1 A) without aggregation or calcium mobilization (Fig. 1 B). Intracellular calcium mobilization occurred at 100 μm YFLLRNP or higher, suggesting that the peptide activated both Gq and G12/13 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 (29Rasmussen U.B. Gachet C. Schlesinger Y. Hanau D. Ohlmann P. Van Obberghen-Schilling E. Pouyssegur J. Cazenave J.P. Pavirani A. J. Biol. Chem. 1993; 268: 14322-14328Google Scholar), higher concentrations of YFLLRNP (100–200 μm) caused small calcium mobilization in our hands, suggesting that there is an increase in Gq coupling. This difference in potency of the peptide could be due to different quality/purity of the synthesized peptide. Thromboxane receptors and protease activated receptors couple to Gq and G12/13 pathways and this coupling is dependent on the concentration of the agonist (16Offermanns S. Laugwitz K.-L. Spicher K. Schulz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 504-508Google Scholar, 30Ohkubo S. Nakahata N. Ohizumi Y. Br. J. Pharmacol. 1996; 117: 1095-1104Google Scholar, 31Simpson A.W. Hallam T.J. Rink T.J. FEBS Lett. 1986; 201: 301-305Google Scholar). Subsequent studies revealed that G12/13-mediated platelet shape change is slow, occurs in the absence of calcium mobilization, involves p160ROCK as an important signaling molecule, and can be completely blocked by the p160ROCK inhibitor, Y-27632 (22Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Google Scholar, 23Paul B.Z.S. Daniel J.L. Kunapuli S.P. J. Biol. Chem. 1999; 274: 28293-28300Google Scholar, 24Bauer M. Retzer M. Wilde J.I. Maschberger P. Essler M. Aepfelbacher M. Watson S.P. Siess W. Blood. 1999; 94: 1665-1672Google Scholar). 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 G12/13 activation. To ensure that YFLLRNP was activating the G12/13 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. 1 A), suggesting that low dose YFLLRNP is causing only G12/13-mediated shape change without a calcium-dependent shape change component. PAR-1 can couple to the Gi pathway and cause the inhibition of adenylyl cyclase (35Hung D.T. Wong Y.H. Vu T.-K.H. Coughlin S.R. J. Biol. Chem. 1992; 267: 20831-20834Google Scholar); however, other data suggest that PAR-1 stimulation relies upon secreted ADP for Gi activation (19Kim S. Foster C. Lecchi A. Quinton T.M. Prosser D.M. Jin J. Cattaneo M. Kunapuli S.P. Blood. 2002; 99: 3629-3636Google Scholar). To investigate whether YFLLRNP can activate the Gipathway, we measured cAMP formation in YFLLRNP-stimulated platelets. YFLLRNP (60 μm) did not cause significant inhibition of forskolin-stimulated adenylyl cyclase (Fig. 1 C), indicating that at this concentration YFLLRNP does not activate Gisignaling pathways. Selective activation of Gq pathways by ADP results only in shape change, while supplementing Gq signaling with Gi activation, through P2Y12 receptor activation or α2A receptor activation, results in platelet aggregation (8Jin J. Kunapuli S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8070-8074Google Scholar, 36Jin J. Daniel J.L. Kunapuli S.P. J. Biol. Chem. 1998; 273: 2030-2034Google Scholar). As selective activation of G12/13 pathways with YFLLRNP (60 μm) resulted only in shape change (Fig. 1 A), we investigated the effect of supplementing this pathway with Gi signaling cascade on platelet fibrinogen receptor activation. ADP causes platelet aggregation by stimulating both the Gq-coupled P2Y1 receptor and the Gi-coupled P2Y12 receptor (8Jin J. Kunapuli S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8070-8074Google Scholar). We used A3P5P, a P2Y1-selective antagonist to block ADP signaling through the Gq-coupled P2Y1 receptor. Addition of 10 μm ADP in the presence of 1 mm A3P5P results in selective stimulation of the Gi-coupled P2Y12 receptor, and is evident by the loss of ADP-induced shape change and aggregation (8Jin J. Kunapuli S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8070-8074Google Scholar). 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). Though we have demonstrated that platelet aggregation can occur in the presence of G12/13 and Gi signaling, we wanted to directly correlate concomitant G12/13 and Gisignaling 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 (3Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Google Scholar). The PAC-1 mAb is directed against the active conformation of the GPIIb/IIIa receptor (37Shattil S.J. Hoxie J.A. Cunningham M. Brass L.F. J. Biol. Chem. 1985; 260: 11107-11114Google Scholar). 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 Gi 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 G12/13 or Gi signaling alone cannot cause GPIIb/IIIa receptor activation, co-stimulation of G12/13and Gi signaling pathways can result in GPIIb/IIIa activation. The thromboxane receptor couples to Gq and G12/13 in human platelets (16Offermanns S. Laugwitz K.-L. Spicher K. Schulz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 504-508Google Scholar, 38Shenker A. Goldsmith P. Unson C.G. Spiegel A.M. J. Biol. Chem. 1991; 266: 9309-9313Google Scholar). 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 G12/13 pathway (30Ohkubo S. Nakahata N. Ohizumi Y. Br. J. Pharmacol. 1996; 117: 1095-1104Google Scholar, 31Simpson A.W. Hallam T.J. Rink T.J. FEBS Lett. 1986; 201: 301-305Google Scholar). Thus, a low concentration of U46619 provides an alternative to low dose of YFLLRNP to stimulate G12/13 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. 4 A) and calcium-dependent shape change that is not inhibited by Y-27632 (Fig. 4 B). Simultaneous addition of either 10 μm epinephrine or 10 μm ADP in the presence of 1 mm A3P5P to 10 nmU46619-stimulated platelets lead to both shape change and platelet aggregation (Fig. 4 C). This illustrates that either P2Y12 receptor or α2A-adrenergic receptor stimulation is capable of causing platelet aggregation when combined with G12/13 signaling from the TP receptor. When we were finalizing the article, Nieswandt et al. (39Nieswandt B. Schulte V. Zywietz A. Gratacap M.P. Offermanns S. J. Biol. Chem. 2002; Google Scholar) reported that stimulation of G12/13 and Gi is sufficient to cause fibrinogen receptor activation in mouse platelets using mice-deficient in Gαq. 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 Gq pathways and does not activate the G12/13 pathways, whereas both thromboxane A2and thrombin do activate this pathway. Since either Gq or G12/13 can synergize with Gi to result in the activation of GPIIb/IIIa, thrombin and thromboxane A2, activating both Gq and G12/13, could additionally synergize with Gi and thereby cause more robust platelet aggregation. Calcium plays an important role in the platelet function, including the activation of GPIIb/IIIa (1Brass L.F. Manning D.R. Cichowski K. Abrams C.S. Thromb. Haemost. 1997; 78: 581-589Google Scholar, 3Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Google Scholar). Although the βγ subunits of Gi are known to increase intracellular calcium by the activation of phospholipase C in other cells (40Clapham D.E. Neer E.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Google Scholar), selective activation of Gi in platelets through either P2Y12 or α2A receptors does not mobilize intracellular calcium (8Jin J. Kunapuli S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8070-8074Google Scholar,36Jin J. Daniel J.L. Kunapuli S.P. J. Biol. Chem. 1998; 273: 2030-2034Google Scholar). 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.5 A). As stimulation of G12/13 or Gi 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. 5 A). Hence, we used ADP (300 nm) in the presence of AR-C 69931MX, a selective P2Y12 receptor antagonist, to selectively activate the Gq pathway and increase a small and comparable intracellular calcium (Fig.5 A), and evaluated the effect of epinephrine on platelet aggregation. As shown in Fig. 5 B, although selective activation of P2Y1 receptor alone did not cause any aggregation, co-stimulation of P2Y1 and α2A-adrenergic receptors led to comparable extent of aggregation as the combined G12/13 and Gistimulation (Fig. 5 B). These data indicate that co-stimulation of G12/13 and Gi 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. (39Nieswandt B. Schulte V. Zywietz A. Gratacap M.P. Offermanns S. J. Biol. Chem. 2002; Google Scholar) did not observe any intracellular calcium mobilization with the combined G12/13 and Gi 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. 5 C, 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 G12/13 and Gi stimulation, play an important role in the activation of GPIIb/IIIa 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 (4Brass L.F. Woolkalis M.J. Manning D.R. J. Biol. Chem. 1988; 263: 5348-5355Google Scholar), MEKK1 (41McNicol A. Philpott C.L. Shibou T.S. Israels S.J. Biochem. Pharmacol. 1998; 55: 1759-1767Google Scholar), PI 3-kinase (25Hirsch E. Bosco O. Tropel P. Laffargue M. Calvez R. Altruda F. Wymann M. Montrucchio G. FASEB J. 2001; 15: 2019-2021Google Scholar, 26Banfic H. Tang X. Batty I.H. Downes C.P. Chen C. Rittenhouse S.E. J. Biol. Chem. 1998; 273: 13-16Google Scholar), and p160ROCK (23Paul B.Z.S. Daniel J.L. Kunapuli S.P. J. Biol. Chem. 1999; 274: 28293-28300Google Scholar, 24Bauer M. Retzer M. Wilde J.I. Maschberger P. Essler M. Aepfelbacher M. Watson S.P. Siess W. Blood. 1999; 94: 1665-1672Google Scholar), among many others (3Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Google Scholar). We measured the effects of selective inhibitors for these molecules on platelet aggregation stimulated by combined G12/13 and Gi signaling. We then compared the effects of these inhibitors on concomitant Gq- and Gi-mediated platelet aggregation (8Jin J. Kunapuli S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8070-8074Google Scholar), using ADP as the agonist. PKC inhibition with Ro 31-8220, an inhibitor of novel and conventional PKC isoforms (42Wilkinson S.E. Parker P.J. Nixon J.S. Biochem. J. 1993; 294: 335-337Google Scholar), had no effect on the aggregation caused by concomitant G12/13 and Gi signaling or Gq and Gi 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 (43Quinton T.M. Kim S. Dangelmaier C. Dorsam R.T. Jin J. Daniel J.L. Kunapuli S.P. Biochem. J. 2002; (in press)Google Scholar). U0126, a MEKK1 inhibitor (44Rosado J.A. Sage S.O. J. Biol. Chem. 2001; 276: 15659-15665Google Scholar), also had no effect on the aggregation induced by co-activation of either G12/13and Gi or Gq and Gi signaling. Thus, although Erk2 has been implicated in the GP1b-IX-mediated platelet fibrinogen receptor activation (27Li Z. Xi X. Du X. J. Biol. Chem. 2001; 276: 42226-42232Google Scholar), the MEKK-Erk pathway does not play any significant role in either G12/13- and Gi- or Gq- and Gi-mediated GPIIb/IIIa activation in human platelets. PI 3-kinase has been known to be involved in platelet activation (3Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Google Scholar), and knockout studies show that PI 3-kinase γ-deficient mice have decreased aggregation responses to ADP and collagen (25Hirsch E. Bosco O. Tropel P. Laffargue M. Calvez R. Altruda F. Wymann M. Montrucchio G. FASEB J. 2001; 15: 2019-2021Google Scholar). LY294002, a PI 3-kinase inhibitor (45Pasquet J.M. Noury M. Nurden A.T. Thromb. Haemost. 2002; 88: 115-122Google Scholar), caused a slight decrease in the extent of combined G12/13- and Gi-mediated aggregation; however, aggregation and shape change were still significant in the presence of PI 3-kinase inhibitor (Fig. 6 A). This effect was comparable to the decrease in ADP-induced platelet aggregation in PI 3-kinase γ-deficient mice versus wild type mice (25Hirsch E. Bosco O. Tropel P. Laffargue M. Calvez R. Altruda F. Wymann M. Montrucchio G. FASEB J. 2001; 15: 2019-2021Google Scholar). While there was a decrease in aggregation, it is unlikely that PI 3-kinase is a key signaling molecule downstream of G12/13signaling. Rather, LY 294002 is mediating its effects through decreasing the P2Y12- or α2A-adrenergic-stimulated Gi and PI 3-kinase γ signaling pathways (46Woulfe D. Jiang H. Mortensen R. Yang J. Brass L.F. J. Biol. Chem. 2002; 277: 23382-23390Google Scholar) (depicted in Fig. 7). Conversely, concomitant Gq- and Gi-mediated platelet aggregation was nearly abolished by the PI 3-kinase inhibitor (Fig. 6 B). These results indicate that PI 3-kinase is a key signaling molecule in the combined Gq and Gi pathway. By comparison, PI 3-kinase appears to be a key molecule in the Gqsignaling cascade, but not in G12/13 mediated signaling pathway, leading to the fibrinogen receptor activation (Fig. 7). p160ROCK has been identified as a key signaling molecule downstream of G12/13 activation (23Paul B.Z.S. Daniel J.L. Kunapuli S.P. J. Biol. Chem. 1999; 274: 28293-28300Google Scholar, 24Bauer M. Retzer M. Wilde J.I. Maschberger P. Essler M. Aepfelbacher M. Watson S.P. Siess W. Blood. 1999; 94: 1665-1672Google Scholar). Using the p160ROCK inhibitor Y-27632, we expected that platelet aggregation caused by concomitant G12/13 and Gisignaling would be inhibited. Interestingly, Y-27632 did not block aggregation caused by simultaneous G12/13 and Gi signaling (Fig. 6 A), suggesting that there is a divergent pathway downstream of G12/13 stimulation. Thus, G12/13 signals through at least two separate pathways, one of which involves p160ROCK and shape change, and the other that contributes to GPIIb/IIIa activation. As expected, combined Gq- and Gi-mediated platelet aggregation was also unaffected by the p160ROCK inhibitor (Fig.6 B), 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 G12/13 and Gi 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 Gq- but not G12/13-mediated activation of GPIIb/IIIa. Co-stimulation of G12/13 and Gi pathways appears to increase intracellular calcium, independently of Gq activation, which plays an important role in the fibrinogen receptor activation in human platelets. The mechanisms of increase in intracellular calcium by G12/13and Gi pathways are under investigation. We thank Drs. James L. Daniel, Barrie Ashby, and Todd M. Quinton, Temple University Medical School, for critically reading the paper.
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