A Gi-dependent Pathway Is Required for Activation of the Small GTPase Rap1B in Human Platelets
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m111803200
ISSN1083-351X
AutoresPaolo Lova, Simona Paganini, Fabiola Sinigaglia, Cesare Balduini, Mauro Torti,
Tópico(s)Diabetes Treatment and Management
ResumoStimulation of human platelets by cross-linking of the low affinity receptor for immunoglobulin, FcγRIIA, caused the rapid activation of the small GTPase Rap1B, as monitored by accumulation of the GTP-bound form of the protein. This process was totally dependent on the action of secreted ADP since it was completely prevented in the presence of either apyrase or creatine phosphate and creatine phosphokinase. Dose-dependent experiments revealed that the inhibitory effect of ADP scavengers was not related to the reduced increase of cytosolic Ca2+ concentration in stimulated platelets. Activation of Rap1B induced by clustering of FcγRIIA was totally suppressed by AR-C69931MX, a specific antagonist of the Gi-coupled ADP receptor P2Y12, but was not affected by blockade of the Gq-coupled receptor, P2Y1. Similarly, direct stimulation of platelets with ADP induced the rapid activation of Rap1B. Pharmacological blockade of the P2Y1 receptor totally prevented ADP-induced Ca2+ mobilization but did not affect activation of Rap1B. By contrast, prevention of ADP binding to the P2Y12 receptor totally suppressed activation of Rap1B without affecting Ca2+ signaling. In platelets stimulated by cross-linking of FcγRIIA, inhibition of Rap1B activation by ADP scavengers could be overcome by the simultaneous recruitment of the Gi-coupled α2A-adrenergic receptor by epinephrine. By contrast, serotonin, which binds to a Gq-coupled receptor, could not restore activation of Rap1B. When tested alone, epinephrine was found to be able to induce GTP binding to Rap1B, whereas serotonin produced only a slight effect. Finally, activation of Rap1B induced by stimulation of the Gq-coupled thromboxane A2receptor by U46619 was completely inhibited by ADP scavengers under conditions in which intracellular Ca2+ mobilization was unaffected. Inhibition of U46619-induced Rap1B activation was also observed upon blockade of the P2Y12 but not of the P2Y1 receptor for ADP. These results demonstrate that stimulation of a Gi-dependent signaling pathway by either ADP of epinephrine is necessary and sufficient to activate the small GTPase Rap1B. Stimulation of human platelets by cross-linking of the low affinity receptor for immunoglobulin, FcγRIIA, caused the rapid activation of the small GTPase Rap1B, as monitored by accumulation of the GTP-bound form of the protein. This process was totally dependent on the action of secreted ADP since it was completely prevented in the presence of either apyrase or creatine phosphate and creatine phosphokinase. Dose-dependent experiments revealed that the inhibitory effect of ADP scavengers was not related to the reduced increase of cytosolic Ca2+ concentration in stimulated platelets. Activation of Rap1B induced by clustering of FcγRIIA was totally suppressed by AR-C69931MX, a specific antagonist of the Gi-coupled ADP receptor P2Y12, but was not affected by blockade of the Gq-coupled receptor, P2Y1. Similarly, direct stimulation of platelets with ADP induced the rapid activation of Rap1B. Pharmacological blockade of the P2Y1 receptor totally prevented ADP-induced Ca2+ mobilization but did not affect activation of Rap1B. By contrast, prevention of ADP binding to the P2Y12 receptor totally suppressed activation of Rap1B without affecting Ca2+ signaling. In platelets stimulated by cross-linking of FcγRIIA, inhibition of Rap1B activation by ADP scavengers could be overcome by the simultaneous recruitment of the Gi-coupled α2A-adrenergic receptor by epinephrine. By contrast, serotonin, which binds to a Gq-coupled receptor, could not restore activation of Rap1B. When tested alone, epinephrine was found to be able to induce GTP binding to Rap1B, whereas serotonin produced only a slight effect. Finally, activation of Rap1B induced by stimulation of the Gq-coupled thromboxane A2receptor by U46619 was completely inhibited by ADP scavengers under conditions in which intracellular Ca2+ mobilization was unaffected. Inhibition of U46619-induced Rap1B activation was also observed upon blockade of the P2Y12 but not of the P2Y1 receptor for ADP. These results demonstrate that stimulation of a Gi-dependent signaling pathway by either ADP of epinephrine is necessary and sufficient to activate the small GTPase Rap1B. Rap1 proteins are members of a family of small GTPases highly related to the product of the ras protooncogene. Two isoforms of Rap1 are known, Rap1A and Rap1B: they share more than 90% sequence homology but are differently expressed in different cell types. For instance, in human platelets, expression of Rap1B is particularly high as it accounts for about 0.1% of the total cellular proteins, whereas Rap1A is almost undetectable (1.Torti M. Lapetina E.G. Thromb. Haemostasis. 1994; 71: 533-543Crossref PubMed Scopus (49) Google Scholar, 2.Klinz F.J. Seifert R. Schwaner I. Gaussephol H. Frank R. Schultz G. Eur. J. Biochem. 1992; 207: 207-213Crossref PubMed Scopus (31) Google Scholar). For this reason, human platelets represent an excellent experimental model to study the biological and functional properties of Rap1B. In resting platelets, Rap1B is mainly located at the membrane as a consequence of post-translational modifications, including isoprenylation, proteolysis, and carboxylmethylation (3.Winegar D.A. Ohmstede C.A. Chu L. Reep B.R. Lapetina E.G. J. Biol. Chem. 1991; 266: 4375-4380Abstract Full Text PDF PubMed Google Scholar). Upon stimulation of platelets with extracellular agonists, Rap1B associates with the actin-based cytoskeleton (4.Fischer T.H. Gatling M.N. Lacal J.C. White G.C. J. Biol. Chem. 1990; 265: 19405-19408Abstract Full Text PDF PubMed Google Scholar), whereas upon platelet treatment with antagonists, such as prostacyclin, Rap1B undergoes cAMP-dependent phosphorylation and translocates from the membrane to the cytosol (5.Lapetina E.G. Lacal J.C. Reep B.R. Molina y Vedia L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3131-3134Crossref PubMed Scopus (113) Google Scholar). Although Rap1B has been suggested to be involved in a number of cellular processes, its exact function in human platelets is still poorly understood. Like other GTPases, Rap1B is activated by binding of GTP. Several factors able to stimulate the exchange of GDP for GTP on Rap1 proteins have been discovered (6.Bos J.L. de Rooij J. Reedquist K.A. Nature Rev. Mol. Cell Biol. 2001; 2: 369-377Crossref PubMed Scopus (512) Google Scholar). These exchange factors can be activated by different intracellular messengers, including Ca2+, cAMP, protein kinase C, and tyrosine kinases (6.Bos J.L. de Rooij J. Reedquist K.A. Nature Rev. Mol. Cell Biol. 2001; 2: 369-377Crossref PubMed Scopus (512) Google Scholar). In human platelets, Rap1B is rapidly activated by thrombin, the most potent extracellular agonist (7.Torti M. Lapetina E.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7796-7800Crossref PubMed Scopus (64) Google Scholar, 8.Franke B. Akkerman J.-W.N. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (367) Google Scholar). Experiments with cell-permeable Ca2+-chelating agents and with Ca2+ ionophores have suggested that the rapid activation of Rap1B induced by thrombin is mediated by the increase of cytosolic Ca2+ concentration (8.Franke B. Akkerman J.-W.N. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (367) Google Scholar). Moreover, a second, delayed phase of Rap1B activation has been observed and found to be regulated by protein kinase C (9.Franke B. van Triest M. de Bruijn K.M.T. van Willigen G. Nieuwenhuis H.K. Negrier C. Akkerman J.-W.N. Bos J.L. Mol. Cell. Biol. 2000; 20: 779-785Crossref PubMed Scopus (85) Google Scholar). Therefore, multiple pathways for Rap1B activation clearly exist in human platelets. In addition to thrombin, a number of other strong and weak platelet agonists, including ADP, collagen, and PAF, have been described to induce binding of GTP to Rap1B (8.Franke B. Akkerman J.-W.N. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (367) Google Scholar), but the biochemical mechanism underlying their action has not been characterized as yet. In the most recent studies, a mounting body of evidence indicates that platelet aggregation induced by many different agonists results from concomitant signaling through both Gq- and Gi-coupled receptors. This concept has been initially developed upon studies on ADP-induced platelet activation. ADP binds to two different membrane receptors coupled to heterotrimeric G-proteins: the P2Y1 receptor, coupled to Gq (10.Hechler B. Léon C. Vial C. Vigne P. Frelin C. Cazenave J.-P. Gachet C. Blood. 1998; 92: 152-159Crossref PubMed Google Scholar, 11.Jin J. Daniel J.L. Kunapuli S.P. J. Biol. Chem. 1998; 273: 2030-2034Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar), and the recently cloned P2Y12 receptor, coupled to Gi (12.Holloperter G. Jantzen H.-M. Vincent D. Li G. England L. Ramakrishnan V. Yang R.-B. Nurden P. Nurden A. Julius D. Conley P.B. Nature. 2001; 409: 202-207Crossref PubMed Scopus (1300) Google Scholar, 13.Zhang F.L. Luo L. Gustafson E. Lachowicz J. Smith M. Qiao X. Liu Y.H. Chen G. Pramanik B. Laz T.M. Palmer K. Bayne M. Monsma Jr., F.J. J. Biol. Chem. 2001; 276: 8608-8615Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Binding of ADP to the P2Y1 receptor induces phospholipase C activation, Ca2+ mobilization, and platelet shape change (10.Hechler B. Léon C. Vial C. Vigne P. Frelin C. Cazenave J.-P. Gachet C. Blood. 1998; 92: 152-159Crossref PubMed Google Scholar, 11.Jin J. Daniel J.L. Kunapuli S.P. J. Biol. Chem. 1998; 273: 2030-2034Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar) but is unable to trigger aggregation unless the Gi-coupled P2Y12 receptor is concomitantly activated (14.Jin J. Kunapuli S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8070-8074Crossref PubMed Scopus (476) Google Scholar). Interestingly, when the P2Y12 receptor is blocked by selective antagonists, full platelet response to ADP can be restored by the simultaneous activation of the Gi-coupled α2A-adrenergic receptor by epinephrine (14.Jin J. Kunapuli S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8070-8074Crossref PubMed Scopus (476) Google Scholar). The critical role of Gi activation is also supported by many other findings. The P2Y12 receptor has been recently cloned and recognized as the target of antithrombotic drugs, such as clopidogrel and ticlopidine (12.Holloperter G. Jantzen H.-M. Vincent D. Li G. England L. Ramakrishnan V. Yang R.-B. Nurden P. Nurden A. Julius D. Conley P.B. Nature. 2001; 409: 202-207Crossref PubMed Scopus (1300) Google Scholar, 15.Foster C.J. Prosser D.M. Agans J.M. Zhai Y. Smith M.D. Lachowicz J.E. Zhang F.L. Gustafson E. Monsma F.J.Jr. Wiekowski M.T. Abbondanzo S.J. Cook D.N. Bayne M.L. Lira S.A. Chintala M.S. J. Clin. Invest. 2001; 107: 1591-1598Crossref PubMed Scopus (385) Google Scholar), as well as a number of ATP analogues of the AR-C series (16.Humphries R.G. Robertson M.J. Leff P. Trends Pharmacol. Sci. 1995; 16: 179-181Abstract Full Text PDF PubMed Scopus (78) Google Scholar). Moreover, this receptor is defective in patients with a selective congenital impaired response to ADP (17.Cattaneo M. Lecchi A. Randi A.M. McGregor J.L. Mannucci P.M. Blood. 1992; 80: 2787-2796Crossref PubMed Google Scholar, 18.Nurden P. Savi P. Heilmann E. Bihour C. Herbert J.M. Maffrand J.P. Nurden A. J. Clin. Invest. 1995; 95: 1612-1622Crossref PubMed Scopus (190) Google Scholar). Finally, in platelets from Gq knockout mice, high concentrations of ADP can still induce partial aggregation by binding to the P2Y12 receptor (19.Ohlmann P. Eckly A. Freund M. Cazenave J.-P. Offermanns S. Gachet C. Blood. 2000; 96: 2134-2139Crossref PubMed Google Scholar). Several findings also indicate that the requirement for a Gi pathway for full platelet activation is not restricted to ADP but is a general feature of many platelet agonists. For instance, the thromboxane A2 analogue U46619 binds to a specific receptor on the platelet surface that is coupled to Gq (20.Habib A. FitzGerald G.A. Maclouf J. J. Biol. Chem. 1999; 274: 2645-2651Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). U46619-induced platelet aggregation has been found to rely on the simultaneous stimulation of a Gi-dependent pathway by either secreted ADP or epinephrine (21.Paul B.Z.S. Jin J. Kunapuli S.P. J. Biol. Chem. 1999; 274: 29108-29114Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Similarly, platelet aggregation induced by the thrombin receptor-activating peptide, TRAP, which is a much weaker agonist than thrombin, is reversed by ADP scavengers or by selective antagonists of the P2Y12 receptor (22.Trumel C. Payrastre B. Plantavid M. Hechler B. Viala C. Presek P. Martinson E.A. Cazenave J.-P. Chap H. Gachet C. Blood. 1999; 94: 4156-4165Crossref PubMed Google Scholar). Finally, even when platelet stimulation is promoted by the recruitment of receptors that are linked to tyrosine kinase-based signaling pathways rather than heterotrimeric G-proteins, such as in the case of FcγRIIA 1The abbreviations used are: FcγRIIAFcγIIA receptormAbmonoclonal antibodyCPcreatine phosphateCPKcreatine phosphokinaseGAPGTPase activating proteinRBDrap-binding domainGSTglutathione S-transferaseBAPTA-AM1,2-bis (o-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid tetra (acetoxy-methyl) ester cross-linking, platelet responses largely depend on the activation of the Gi-coupled receptor P2Y12 by secreted ADP (23.Gratacap M.-P. Hérault J.-P. Viala C. Ragab A. Savi P. Herbert J.-M. Chap H. Plantavid M. Payrastre B. Blood. 2000; 96: 3439-3446Crossref PubMed Google Scholar). Although the essential role of a Gi-mediated signaling pathway in potentiating platelet activation by many agonists is very well documented, the exact mechanism of this effect is poorly understood. Several results indicate that inhibition of adenylyl cyclase by the Gi α-subunit may not be relevant, and therefore, suggest the involvement of a still unidentified intracellular effector (24.Haslam R.J. Davidson M.M. Desjardins J.V. Biochem. J. 1978; 176: 83-95Crossref PubMed Scopus (174) Google Scholar, 25.Daniel J.L. Dangelmaier C. Jin J. Kim Y.B. Kunapuli S.P. Throm. Haemostasis. 1999; 82: 1322-1326Crossref PubMed Scopus (124) Google Scholar, 26.Dangelmaier C. Jin J. Smith J.B. Kunapuli S.P. Thromb. Haemostasis. 2001; 85: 341-348Crossref PubMed Scopus (91) Google Scholar). FcγIIA receptor monoclonal antibody creatine phosphate creatine phosphokinase GTPase activating protein rap-binding domain glutathione S-transferase 1,2-bis (o-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid tetra (acetoxy-methyl) ester In this work, we have investigated the possible link between stimulation of Gi-dependent pathways and activation of the small GTPase Rap1B. We have found that under a number of the experimental models examined, including platelet stimulation with U46619, epinephrine, or ADP, or by clustering of the FcγRIIA, activation of Rap1B is absolutely dependent on stimulation of a membrane receptor coupled to Gi. This results reveal a new link between heterotrimeric G-proteins of the Gi family and small GTPases of the Rap family and suggest a potential mechanism responsible for the potentiation of platelet activation by Gi-coupled receptors. Sepharose CL-2B, GSH-Sepharose 2B, and the enhanced chemiluminescence substrate were for Amersham Biosciences, Inc. The thromboxane A2 analogue U46619, ADP, thrombin, sheep anti-mouse F(ab′)2 fragments, acetylsalicylic acid, and A3P5PS were from Sigma. AR-C69931MX was a generous gift from AstraZeneca R&D, Charnwood, UK. Fura-2/AM was fromCalbiochem. The monoclonal antibody IV.3 against the FcγRIIA was obtained from Medarex. The rabbit polyclonal antiserum against Rap1B was described previously (3.Winegar D.A. Ohmstede C.A. Chu L. Reep B.R. Lapetina E.G. J. Biol. Chem. 1991; 266: 4375-4380Abstract Full Text PDF PubMed Google Scholar). The cDNA for the rap binding domain (RBD) of ralGDS was kindly provided by Dr. J. L. Bos (Department of Physiological Chemistry, University of Utrecht, The Netherlands). Peroxidase-conjugated goat anti-rabbit IgG were from Bio-Rad. Human platelets from healthy donors were prepared by gel filtration on Sepharose CL-2B and eluted with Hepes buffer (10 mm HEPES, 137 mmNaCl, 2.9 mm KCl, 12 mm NaHCO3, pH 7.4) as described previously (27.Torti M. Ramaschi G. Sinigaglia F. Lapetina E.G. Balduini C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7553-7557Crossref PubMed Scopus (40) Google Scholar). Platelet concentration was adjusted to 0.35 × 109 platelets/ml. Platelet samples (0.5 ml) were incubated at 37 °C in an aggregometer under constant stirring, and upon the addition of 1 mm CaCl2, stimulated with the indicated agonists. Cross-linking of FcγRIIA was obtained by incubation of platelets with 2 μg/ml monoclonal antibody IV.3 for 2 min followed by the addition of 30 μg/ml sheep anti-mouse F(ab′)2 fragments. Other platelet samples were stimulated with ADP (10 μm), epinephrine (1 μm), serotonin (5 μm), thromboxane A2 analogueU46619 (10 μm), or thrombin (0.6 units/ml). Platelet stimulation was typically performed for 1 min unless otherwise stated. When indicated, 1 unit/ml apyrase, 5 mm CP, 40 units/ml CPK, 500 μm A3P5PS, or 100 nmAR-C69931MX were added to the platelet samples 2 min before stimulation. Measurement of Rap1B activation was performed essentially as described by Franke et al. (8.Franke B. Akkerman J.-W.N. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (367) Google Scholar), exploiting the specific and selective ability of the GST-tagged rap binding domain (GST·RBD) of ralGDS immobilized on GSH-Sepharose to bind and precipitate the active, GTP-bound form of Rap1B from a platelet lysate. Platelet stimulation was stopped by the addition of an equal volume of ice-cold modified 2× RIPA buffer (100 mm Tris/HCl, pH 7.4, 400 mm NaCl, 5 mm MgCl2, 2% Nonidet P-40, 20% glycerol, 2 mm phenylmethylsulfonyl fluoride, 2 μmleupeptin, 0.2 μm aprotinin, 0.2 mmNa3VO4). Cell lysis was performed on ice for 10 min. Lysates were clarified by centrifugation at 13,000 rpm in an Eppendorf microcentrifuge for 10 min at 4 °C. Recombinant purified GST·RBD was coupled to GSH-Sepharose by incubating 200 μg of the protein with 100 μl of GSH-Sepharose (75% slurry) for 2 h at room temperature under constant tumbling. In preliminary experiments, we determined that under these conditions, all the added GST·RBD was immobilized on the resin. GSH-Sepharose-coupled GST·RBD was added to the cleared platelet lysates (20 μg of GST·RBD/sample), and precipitation of GTP-bound Rap1B was performed by incubation at 4 °C for 45 min. The precipitates were collected by brief centrifugation, and the beads were washed three times with modified 1× RIPA buffer and finally resuspended with 25 μl of SDS sample buffer (25 mm Tris, 192 mm glycine, pH 8.3, 4% SDS, 1% dithiothreitol, 20% glycerol, and 0.02% bromphenol blue). Precipitated Rap1B was separated by SDS-PAGE on 10–20% acrylamide gradient gels and transferred to nitrocellulose. The presence of active Rap1B in precipitates with GST·RBD was evaluated by staining the nitrocellulose filters with a specific polyclonal antiserum directed against Rap1B, used at a final dilution of 1:1000. Reactive proteins were detected by enhanced chemiluminescence reaction. All the presented figures are representative of at least three separate experiments. Platelets were prepared as described above with slight modifications. Platelet-rich plasma was incubated with 3 μm Fura-2/AM for 30 min at 37 °C. Platelets were recovered by centrifugation at 300 × g for 10 min at room temperature and resuspended in a small volume (0.5–1 ml) of autologous plasma. Platelets were then isolated by gel filtration on Sepharose CL-2B and eluted with Hepes buffer containing 0.5% bovine serum albumin and 5.5 mm glucose. Platelet count was then adjusted to 2 × 108 platelets/ml. Measurement of cytosolic Ca2+ was performed on 0.4-ml samples prewarmed at 37 °C under gentle stirring in a Perkin-Elmer LS3 spectrofluorimeter in the presence of either 1 mmCaCl2 or 1 mm EGTA. The fluorescence excitation and emission wavelengths were 340 and 510 nm, respectively. Fura-2 fluorescence signals were calibrated according to the method of Pollock et al. (28.Pollock W.K. Rink T.J. Irvine R.F. Biochem. J. 1986; 235: 869-877Crossref PubMed Scopus (217) Google Scholar). Fmax was determined by the addition of 2% Triton X-100 and saturating concentrations of CaCl2, whereas Fmin was determined by the addition of 2 mm EGTA and 20 mm Tris base. All determinations were repeated at least three times with platelets from different donors. Samples of gel-filtered platelets were placed in an aggregometer under constant stirring and treated with 2 μg/ml anti-FcγRIIA mAb IV.3 and 30 μg/ml sheep anti-mouse F(ab′)2 fragments for increasing times. After cell lysis, the active, GTP-bound form of Rap1B was selectively precipitated with GST·RBD and identified by immunoblotting using a specific polyclonal antiserum. Fig. 1A shows that clustering of FcγRIIA caused a strong and rapid activation of Rap1B that was already maximal after 30 s of stimulation. The activation of Rap1B clearly preceded platelet aggregation. The amount of active Rap1B was found to decrease progressively after prolonged stimulation, in parallel with the progression of platelet aggregation (Fig. 1A). Although platelets were stimulated in the presence of 1 mm CaCl2, activation of Rap1B was found to be independent of extracellular calcium since it was also observed when 1 mm EGTA was present (Fig. 1B). Moreover, it did not require the production of thromboxane A2 since it was only minimally affected by the treatment of platelets with acetylsalicylic (Fig. 1B). It has been recently shown that FcγRIIA-mediated platelet activation is largely dependent on the action of secreted ADP (23.Gratacap M.-P. Hérault J.-P. Viala C. Ragab A. Savi P. Herbert J.-M. Chap H. Plantavid M. Payrastre B. Blood. 2000; 96: 3439-3446Crossref PubMed Google Scholar, 29.Polgar J. Eichler P. Greinacher A. Clemetson K.J. Blood. 1998; 91: 549-554Crossref PubMed Google Scholar). To evaluate the impact of ADP on Rap1B activation, cross-linking of FcγRIIA was performed in the presence of two unrelated ADP scavengers, apyrase and CP-CPK. Fig. 2 shows that in the presence of these ADP scavengers, FcγRIIA-mediated activation of Rap1B was completely inhibited. The effect of apyrase and CP-CPK was found to be specific and selective since both scavengers were inactive when added immediately after cell lysis, and CP-CPK did not affect thrombin-induced activation of Rap1B (Fig. 2). Therefore, FcγRIIA-mediated activation of Rap1B was totally dependent on the action of secreted ADP. In previous studies, activation of Rap1B has been reported to be mediated by an increase of the intracellular Ca2+concentration (8.Franke B. Akkerman J.-W.N. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (367) Google Scholar). Moreover, the ADP scavenger CP-CPK has been found to inhibit Ca2+ mobilization from internal stores induced by clustering of FcγRIIA (23.Gratacap M.-P. Hérault J.-P. Viala C. Ragab A. Savi P. Herbert J.-M. Chap H. Plantavid M. Payrastre B. Blood. 2000; 96: 3439-3446Crossref PubMed Google Scholar). Therefore, we verified whether inhibition of Rap1B activation by ADP scavengers was a consequence of the reduced cytosolic Ca2+ increase. Using Fura-2-loaded platelets, we actually confirmed that, even in the presence of extracellular CaCl2, both apyrase and CP-CPK reduced the increase of Ca2+ concentration triggered by clustering of FcγRIIA (Table I). The two scavengers were constantly found to be differently effective since reduction of cytosolic Ca2+ increase was greater with CP-CPK (about 60%) than with apyrase (about 20%). Using ADP as agonist, we found that both apyrase and CP-CPK totally prevented cytosolic Ca2+ increase, indicating that they were equally effective in neutralizing ADP (data not shown). Moreover, the addition of P2Y1 and P2Y12 receptor antagonists to apyrase-treated platelets did not result in any further reduction of intracellular Ca2+increase induced by clustering of FcγRIIA (data not shown), indicating that the ADP scavenger is actually able to completely neutralize released ADP at the microenvironment of the platelet cell surface. These results indicate that, even when secreted ADP was neutralized, a residual albeit variable increase of cytosolic Ca2+ persisted in platelets stimulated by cross-linking of FcγRIIA. We thus treated platelets with decreasing concentrations of anti-FcγRIIA monoclonal antibody IV.3. Fig. 3A shows that treatment of platelets with 0.2 μg/ml IV.3 caused an increase of cytosolic Ca2+ similar to that observed in platelets stimulated with 2 μg/ml IV.3 in the presence of CP-CPK and still lower than that measured in the presence of apyrase. In similar dose-dependent studies, activation of Rap1B was found to occur at concentrations of IV.3 as low as 0.1 μg/ml (Fig. 3B). These results indicate that inhibition of FcγRIIA-induced activation of Rap1B by ADP scavengers is not due to the reduced increase of intracellular Ca2+.Table IEffect of ADP scavengers on FcγRIIA-mediated increase of cytosolic Ca2+ concentrationTreatment[Ca2+]inmNone45 ± 12 (n = 13)FcγRIIA clustering397 ± 58 (n = 6)Apyrase + FcγRIIA clustering335 ± 41 (n = 3)CP-CPK + FcγRIIA clustering159 ± 28 (n = 4)Fura-2-loaded platelets were prewarmed at 37 °C in the presence of 1 mm CaCl2, and incubated without or with 1 unit/ml apyrase or 5 mm CP and 40 unit/ml CPK for 2 min. Platelets were then stimulated by clustering of FcγRIIA by addition of 2 μg/ml mAb IV.3 and 30 μg/ml sheep anti-mouse F(ab′)2fragments. The cytosolic concentration of Ca2+ was calculated as described under "Material and Methods." Open table in a new tab Fura-2-loaded platelets were prewarmed at 37 °C in the presence of 1 mm CaCl2, and incubated without or with 1 unit/ml apyrase or 5 mm CP and 40 unit/ml CPK for 2 min. Platelets were then stimulated by clustering of FcγRIIA by addition of 2 μg/ml mAb IV.3 and 30 μg/ml sheep anti-mouse F(ab′)2fragments. The cytosolic concentration of Ca2+ was calculated as described under "Material and Methods." ADP binds to two different purinergic receptors on the platelet surface, P2Y1 and P2Y12, which are coupled to Gq and Gi, respectively (30.Kunapuli S.P. Platelets (Edinburgh). 1998; 9: 343-351Crossref PubMed Scopus (34) Google Scholar). To investigate the relative contribution of each one of these receptors on activation of Rap1B induced by clustering of FcγRIIA, we performed experiments with selective antagonists. Fig. 4 shows that pretreatment of platelets with A3P5PS, a specific antagonist of the Gq-coupled P2Y1 receptor, did not significantly affect FcγRIIA-mediated activation of Rap1B. By contrast, AR-C69931MX, an antagonist of the P2Y12 receptor, totally suppressed activation of Rap1B induced by clustering of FcγRIIA. These results clearly indicate that the action of secreted ADP on Rap1B activation was totally mediated by its binding to the Gi-coupled receptor on the platelet surface. To confirm these results, we investigated the activation of Rap1B triggered by exogenous ADP. When platelets were stimulated with 10 μm ADP, a rapid and sustained activation of Rap1B was observed (Fig. 5A). Experiments with selective inhibitors of P2Y1 and P2Y12 receptors revealed that the ability of exogenous ADP to activate Rap1B was exclusively mediated by agonist binding to the Gi-coupled receptor as it was completely inhibited by AR-C69931MX but unaffected by A3P5PS (Fig. 5B). Interestingly, using Fura-2-loaded platelets, we confirmed that, even under our experimental conditions, ADP-induced cytosolic Ca2+ increase was totally suppressed by the P2Y1 receptor antagonist A3P5PS but was unaffected by AR-C69931MX (Fig. 5C). Therefore, when ADP was allowed to bind exclusively to the P2Y12 receptor (i.e. in the presence of A3P5PS), Rap1B was activated, although cytosolic Ca2+ was not increased. By contrast, the sole binding of ADP to the P2Y1 receptor (i.e. in the presence of AR-C69931MX) did not result in Rap1B activation, although intracellular Ca2+ rose normally. These results indicate that a Gi-dependent pathway, rather than a cytosolic Ca2+ increase, is essential for activation of Rap1B. To confirm the essential role of a Gi pathway for Rap1B activation, we investigated whether, upon clustering of FcγRIIA, the inhibitory effect of ADP scavengers could be overcome by the simultaneous stimulation of the Gi-coupled α2A-adrenergic receptor by epinephrine. Fig. 6A actually shows that in the presence of apyrase, Rap1B activation triggered by FcγRIIA cross-linking could be restored by the simultaneous addition of epinephrine. By contrast, the addition of serotonin, which binds to a membrane Gq-coupled receptor, did not result in restoration of Rap1B activation. Our previous findings, indicating that stimulation of the Gi-coupled P2Y12 receptor by ADP is sufficient for activation of Rap1B, prompted us to investigate the ability of epinephrine alone to stimulate binding of GTP to Rap1B. As shown in Fig. 6B, treatment of platelets with 1 μm epinephrine caused a rapid and significant activation of Rap1B even in the absence of any other stimulus. Interestingly, stimulation of platelets with serotonin, which does not signal through Gi, produced only a slight activation of Rap1B (Fig. 6B). Using Fura-2-loaded platelets, we confirmed, in agreement with previous studies, that epinephrine did not cause any detectable Ca2+ movement (data not shown). By contrast, platelet stimulation with serotonin caused a small but significant increase of the intracellular concentration of Ca2+(from 50 ± 9 nm to 88 ± 16 nm, n = 6). Once again, these results cor
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