A Gi-dependent Pathway Is Required for Activation of the Small GTPase Rap1B in Human Platelets

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. 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 C. C. PubMed Google Scholar, J. J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and the P2Y12 receptor, coupled to G. G. L. A. 2001; PubMed Scopus Google Scholar, L. J. M. G. B. M. F.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of ADP to the P2Y1 receptor C Ca2+ and platelet (10.Hechler B. Léon C. Vial C. Vigne C. C. PubMed Google Scholar, J. J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google but is to aggregation the Gi-coupled P2Y12 receptor is activated J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the P2Y12 receptor is by antagonists, platelet to ADP can be by the simultaneous activation of the Gi-coupled α2A-adrenergic receptor by epinephrine J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of activation is also by many other P2Y12 receptor has been and as the of such as and G. G. L. A. 2001; PubMed Scopus Google Scholar, J. 2001; PubMed Scopus Google Scholar), as as a number of of the Sci. 16: Full Text PDF PubMed Scopus Google Scholar). Moreover, this receptor is in with a to ADP M. A. J.L. 1992; PubMed Google Scholar, C. A. J. PubMed Scopus Google Scholar). Finally, in platelets from Gq high of ADP can still induce aggregation by binding to the P2Y12 receptor A. M. S. C. 2000; PubMed Google Scholar). Several also that the for a pathway for platelet activation is not to ADP but is a of many platelet For instance, the thromboxane U46619 binds to a specific receptor on the platelet that is coupled to Gq A. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). U46619-induced platelet aggregation has been found to on the simultaneous stimulation of a Gi-dependent pathway by either secreted ADP or epinephrine J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Similarly, platelet aggregation induced by the thrombin which is a agonist than thrombin, is by ADP scavengers or by of the P2Y12 receptor C. B. M. B. C. H. C. PubMed Google Scholar). Finally, platelet stimulation is by the recruitment of receptors that are to tyrosine signaling pathways than heterotrimeric such as in the of FcγRIIA platelet on the activation of the Gi-coupled receptor P2Y12 by secreted ADP C. A. H. M. B. 2000; PubMed Google Scholar). Although the of a signaling pathway in platelet activation by many agonists is the exact mechanism of this effect is poorly understood. Several results that inhibition of by the not be and the of a still intracellular Biochem. J. PubMed Scopus Google Scholar, J.L. C. J. Haemostasis. PubMed Scopus Google Scholar, C. J. Thromb. Haemostasis. 2001; PubMed Scopus Google Scholar). receptor creatine phosphate creatine GTPase protein In this have the stimulation of Gi-dependent pathways and activation of the small GTPase Rap1B. have found that under a number of the experimental including platelet stimulation with or ADP, or by clustering of the FcγRIIA, activation of Rap1B is dependent on stimulation of a membrane receptor coupled to This results a heterotrimeric of the family and small GTPases of the family and a mechanism for the of platelet activation by Gi-coupled receptors. and the for thromboxane ADP, thrombin, and from was a from was the FcγRIIA was from Rap1B was described (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). for the binding of was by J. L. Bos of of from platelets from by on and with as described M. G. Lapetina E.G. C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). concentration was to at in an under and upon the addition of stimulated with the of FcγRIIA was by of platelets with for by the addition of platelet stimulated with ADP epinephrine serotonin thromboxane or thrombin stimulation was for When or to the platelet of Rap1B activation was as described by (8.Franke B. Akkerman J.-W.N. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (367) Google Scholar), the specific and of the binding of on to and the GTP-bound form of Rap1B from a platelet stimulation was by the addition of an of Cell was on for by at in an for at was coupled to by of the protein with of for at under In that under the was on the was to the platelet of and of GTP-bound Rap1B was by at for by and the with and with of and Rap1B was by on and to presence of Rap1B in with was by the with a specific Rap1B, at a of proteins by the are of at as described with slight was with for at by at for at and in a small of by on and with and was to of cytosolic Ca2+ was on at under in a in the presence of either or and and to the of Biochem. J. PubMed Scopus Google Scholar). was by the addition of and of whereas was by the addition of and at with platelets from different of platelets in an under and with and for cell the GTP-bound form of Rap1B was with and by a specific that clustering of FcγRIIA caused a strong and rapid activation of Rap1B that was of activation of Rap1B clearly platelet of Rap1B was found to in with the of platelet aggregation Although platelets stimulated in the presence of activation of Rap1B was found to be of extracellular since it was also observed was Moreover, it did not the of thromboxane since it was only affected by the treatment of platelets with has been that platelet activation is dependent on the action of secreted ADP C. A. H. M. B. 2000; PubMed Google Scholar, J. A. PubMed Google Scholar). the of ADP on Rap1B cross-linking of FcγRIIA was in the presence of two ADP apyrase and that in the presence of ADP activation of Rap1B was completely effect of apyrase and was found to be specific and since both scavengers cell and did not affect activation of Rap1B Therefore, activation of Rap1B was totally dependent on the action of secreted ADP. In studies, activation of Rap1B has been to be mediated by an increase of the intracellular (8.Franke B. Akkerman J.-W.N. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (367) Google Scholar). Moreover, the ADP has been found to Ca2+ mobilization from induced by clustering of FcγRIIA C. A. H. M. B. 2000; PubMed Google Scholar). Therefore, inhibition of Rap1B activation by ADP scavengers was a consequence of the reduced cytosolic Ca2+ platelets, in the presence of extracellular both apyrase and reduced the increase of Ca2+ concentration by clustering of FcγRIIA two scavengers found to be differently since of cytosolic Ca2+ increase was with than with apyrase ADP as found that both apyrase and totally prevented cytosolic Ca2+ that they in ADP not Moreover, the addition of P2Y1 and P2Y12 receptor to platelets did not in of intracellular induced by clustering of FcγRIIA not that the ADP is able to completely ADP at the of the platelet cell These results secreted ADP was a increase of cytosolic Ca2+ in platelets stimulated by cross-linking of platelets with of that treatment of platelets with caused an increase of cytosolic Ca2+ to that observed in platelets stimulated with in the presence of and still than that in the presence of In studies, activation of Rap1B was found to at of as low as These results that inhibition of activation of Rap1B by ADP scavengers is not to the reduced increase of intracellular of ADP scavengers on increase of cytosolic Ca2+ FcγRIIA FcγRIIA platelets at in the presence of and without or with apyrase or and for stimulated by clustering of FcγRIIA by addition of and cytosolic concentration of Ca2+ was as described under and in a platelets at in the presence of and without or with apyrase or and for stimulated by clustering of FcγRIIA by addition of and cytosolic concentration of Ca2+ was as described under and ADP binds to two different receptors on the platelet P2Y1 and P2Y12, which are coupled to Gq and PubMed Scopus Google Scholar). the of of receptors on activation of Rap1B induced by clustering of FcγRIIA, experiments with that of platelets with a specific antagonist of the Gq-coupled P2Y1 receptor, did not affect activation of Rap1B. By contrast, AR-C69931MX, an antagonist of the P2Y12 receptor, totally suppressed activation of Rap1B induced by clustering of These results clearly that the action of secreted ADP on Rap1B activation was totally mediated by its binding to the Gi-coupled receptor on the platelet the activation of Rap1B by ADP. When platelets stimulated with ADP, a rapid and activation of Rap1B was observed Experiments with of P2Y1 and P2Y12 receptors revealed that the of ADP to activate Rap1B was mediated by agonist binding to the Gi-coupled receptor as it was completely inhibited by but by platelets, under experimental ADP-induced cytosolic Ca2+ increase was totally suppressed by the P2Y1 receptor antagonist but was by Therefore, ADP was to to the P2Y12 receptor in the presence of Rap1B was cytosolic Ca2+ was not By contrast, the binding of ADP to the P2Y1 receptor in the presence of did not in Rap1B intracellular Ca2+ These results that a Gi-dependent than a cytosolic Ca2+ is for activation of Rap1B. the of a pathway for Rap1B 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. that in the presence of Rap1B activation by FcγRIIA cross-linking could be by the simultaneous addition of epinephrine. By contrast, the addition of serotonin, which binds to a membrane Gq-coupled receptor, did not in of Rap1B activation. that stimulation of the Gi-coupled P2Y12 receptor by ADP is sufficient for activation of Rap1B, to the of epinephrine to stimulate binding of GTP to Rap1B. in treatment of platelets with epinephrine caused a rapid and activation of Rap1B in the of other stimulation of platelets with serotonin, which not through produced only a slight activation of Rap1B platelets, in with studies, that epinephrine did not Ca2+ not By contrast, platelet stimulation with serotonin caused a small but increase of the intracellular concentration of to results Rap1B activation to a Gi-dependent signaling pathway than to cytosolic Ca2+ the of the Gi-dependent pathway on Rap1B activation by the effect of the thromboxane has been that U46619 binds to a Gq-coupled receptor, intracellular Ca2+ through activation of C, but totally on secreted ADP to platelet aggregation A. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that U46619 was a strong of Rap1B. activation of Rap1B was totally suppressed stimulation was in the presence of the ADP scavengers apyrase or has also been that cytosolic Ca2+ increase induced was not reduced was prevented by inhibition of protein kinase C J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In with found that ADP scavengers did not affect the of intracellular Ca2+ in platelets stimulated with U46619 Therefore, secreted ADP is Rap1B activation is the of intracellular By the of of membrane ADP found that U46619-induced activation of Rap1B was dependent on stimulation of the Gi-coupled P2Y12 receptor Finally, that but not serotonin, could overcome the inhibitory effect of apyrase and on Rap1B activation induced by Rap1 proteins are expressed small GTPases involved in cellular processes, including cell and (6.Bos J.L. de Rooij J. Reedquist K.A. Nature Rev. Mol. Cell Biol. 2001; 2: 369-377Crossref PubMed Scopus (512) Google Scholar). platelets high of the Rap1 protein Rap1B, which is rapidly activated upon stimulation with extracellular agonists (1.Torti M. Lapetina E.G. Thromb. Haemostasis. 1994; 71: 533-543Crossref PubMed Scopus (49) Google Scholar, 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). In the have the underlying Rap1B activation in human platelets, and have that this process is by a signaling pathway by members of the family of heterotrimeric In the most recent studies, it has that activation of is a of almost platelet agonists for and of the platelet activation of a Gi-dependent pathway be through different For instance, thrombin receptors are coupled to heterotrimeric including Gq and J. Biol. Chem. 1992; Full Text PDF PubMed Google ADP binds to at two different coupled to Gq and the other coupled to (10.Hechler B. Léon C. Vial C. Vigne C. C. PubMed Google Scholar, J. J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. G. L. A. 2001; PubMed Scopus Google Scholar, L. J. M. G. B. M. F.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In the of agonists receptors are coupled to such as the thromboxane or to tyrosine such as the FcγRIIA, recruitment of a Gi-coupled receptor is through the action of secreted ADP J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. A. H. M. B. 2000; PubMed Google Scholar). the prevention of activation of platelet aggregation in to extracellular it is that the activation of a Gi-coupled receptor is not sufficient to platelet such as Ca2+ and In of platelets are stimulated with which binds to the Gi-coupled α2A-adrenergic receptor, or with ADP in the presence of of the Gq-coupled P2Y1 Therefore, it is that stimulation of to activation of or more intracellular which are not sufficient to platelet aggregation but are for the of platelet is to and to the intracellular of a mounting body of evidence that inhibition of is not for the of platelet activation Biochem. J. PubMed Scopus Google Scholar, J.L. C. J. Haemostasis. PubMed Scopus Google Scholar, C. J. Thromb. Haemostasis. 2001; PubMed Scopus Google Scholar). Therefore, it is that a different activated by is study differently from other of platelet such as Ca2+ and which are but not by activation of the small GTPase Rap1B is by the stimulation of a Gi-dependent In found that the binding of ADP to the Gi-coupled P2Y12 receptor is sufficient to Rap1B activation and that binding to the Gq-coupled P2Y1 receptor not to this Moreover, which binds to the Gi-coupled α2A-adrenergic receptor, activate Rap1B. epinephrine is not a platelet but its to platelet aggregation induced by many agonists has been for Lapetina E.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, H. Biochem. J. PubMed Scopus Google Scholar, Lapetina E.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). mechanism underlying this effect of epinephrine is still but its to activate Rap1B, in the that this small GTPase an Finally, study that stimulation of a by of or of a tyrosine by of cross-linking of FcγRIIA, is not sufficient to Rap1B activation a Gi-dependent pathway is activated through binding of secreted ADP to the P2Y12 other agonists tested in this thrombin was found to induce activation of Rap1B through a pathway that is to ADP Moreover, have also found that the P2Y12 receptor antagonist did not affect Rap1B activation induced by this agonist not it is that thrombin receptors on the platelet are coupled to both Gq and and it is that the direct stimulation of a Gi-dependent pathway by thrombin to activation of Rap1B. ADP is to be for the of platelet aggregation induced by many extracellular agonists, and this effect is mediated by its binding to the P2Y12 receptor J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. A. H. M. B. 2000; PubMed Google Scholar). Moreover, platelets from with a of the P2Y12 receptor have been to aggregation in to ADP as as other agonists M. A. J.L. 1992; PubMed Google Scholar, C. A. J. PubMed Scopus Google Scholar, M. C. Thromb. Biol. PubMed Scopus Google Scholar). aggregation is a of cell by binding to Rap1 proteins have been in the of cell and in the of function K.A. van M. Bos J.L. J. Cell Biol. 2000; PubMed Scopus Google Scholar, A. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In the of results Rap1B as a intracellular activation of a pathway to the of platelet that Rap1B can be activated by stimulation of a mechanism this small studies, on the of the cell-permeable Ca2+-chelating as as the of Ca2+ that Rap1B can be activated by a mechanism in stimulated platelets (8.Franke B. Akkerman J.-W.N. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (367) Google Scholar). the Ca2+ also found that an increase of intracellular Ca2+ concentration can to GTP binding to Rap1B not in this have in the experimental the of the Gi-dependent pathway for Rap1B activation is more than that of intracellular For instance, in platelets stimulated with epinephrine or with ADP in the presence of the P2Y1 receptor antagonist activation of Rap1B in the of a increase of cytosolic By contrast, serotonin an increase of Ca2+ in platelets, which only a small activation of Rap1B. Moreover, in the presence of ADP Rap1B activation induced by U46619 is totally prevented under conditions in which intracellular Ca2+ increase is not Finally, platelets are stimulated by cross-linking of FcγRIIA, ADP scavengers can the intracellular concentration of experiments clearly at this Ca2+ Rap1B can be activated as as secreted ADP can to the P2Y12 Therefore, the pathway and the Gi-dependent pathway for Rap1B activation described are most to represent of intracellular Ca2+ in activation of Rap1B from the observed inhibitory effect of In caused a total prevention of Rap1B activation Ca2+ such as in platelets stimulated with epinephrine or with ADP in the presence of not These the of the of this to activation of Rap1B. Although not have a for the inhibitory of in the of Ca2+ it be in that this also which is for binding to Rap1B. it is also that the low of Ca2+ in resting platelets, which are upon with are for the of exchange factors for Rap1B. of platelets with of did not restore activation of Rap1B induced by epinephrine not Therefore, the exact mechanism for the effect of to be can activation of to binding of GTP to results that inhibition of is not S. C. and M. different be of the family of heterotrimeric the of factors the binding to Rap1B, such as exchange factors or In this it is to that the of epinephrine to in platelets has been Lapetina E.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: PubMed Scopus Google Scholar). Moreover, a and functional and has been described J. J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and has been to be to the α2A-adrenergic receptor in platelets J. J. A. M. I. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). Activation of Rap1B by a Gi-dependent pathway also tyrosine kinases since an exchange for Rap1 proteins, is activated by tyrosine phosphorylation (6.Bos J.L. de Rooij J. Reedquist K.A. Nature Rev. Mol. Cell Biol. 2001; 2: 369-377Crossref PubMed Scopus (512) Google Scholar). protein tyrosine phosphorylation has been described in platelets from a with a of ADP-induced platelet aggregation that could be to P2Y12 S. J. C. G. Thromb. Haemostasis. PubMed Scopus Google Scholar). Moreover, it has been that proteins can activate members of the family of tyrosine kinases J. S. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), and and has been described in platelets M. Lapetina E.G. Biochem. 1992; PubMed Scopus Google Scholar). Finally, a of which or the of exchange factors for Rap1B, be In this the of the pathways and Rap1B In the a direct members of the family of heterotrimeric and the small GTPase Rap1B. Rap1B is as a of and the that it could be involved in the Gi-dependent of platelet L. Bos of for the and for the