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Costimulation of Gi- and G12/G13-mediated Signaling Pathways Induces Integrin αIIbβ3 Activation in Platelets

2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês

10.1074/jbc.m207256200

1083-351X

Bernhard Nieswandt, Valerie Schulte, Alexandra Zywietz, Marie‐Pierre Gratacap, Stefan Offermanns,

Antiplatelet Therapy and Cardiovascular Diseases

Platelet activation is a complex process induced by a variety of stimuli, which act in concert to ensure the rapid formation of a platelet plug at places of vascular injury. We show here that fibrillar collagen, which initiates platelet activation at the damaged vessel wall, activates only a small fraction of platelets in suspension directly, whereas the majority of platelets becomes activated by mediators released from collagen-activated platelets. In Gαq-deficient platelets that do not respond with activation of integrin αIIbβ3 to a variety of mediators like thromboxane A2 (TXA2), thrombin, or ADP, collagen at high concentrations was able to induce aggregation, an effect that could be blocked by antagonists of the TXA2 or P2Y12 receptors. The activation of TXA2 or P2Y12 receptors alone, which in Gαq-deficient platelets couple to G12/G13 and Gi, respectively, did not induce platelet integrin activation or aggregation. However, concomitant activation of both receptors resulted in irreversible integrin αIIbβ3-mediated aggregation of Gαq-deficient platelets. Thus, the activation of G12/G13- and Gi-mediated signaling pathways is sufficient to induce integrin αIIbβ3 activation. Although Gq-mediated signaling plays an important role in platelet activation, it is not strictly required for the activation of integrin αIIbβ3. This indicates that the efficient induction of platelet aggregation through G-protein-coupled receptors is an integrated response mediated by various converging G-protein-mediated signaling pathways involving Gq and Gi as well as G12/G13. Platelet activation is a complex process induced by a variety of stimuli, which act in concert to ensure the rapid formation of a platelet plug at places of vascular injury. We show here that fibrillar collagen, which initiates platelet activation at the damaged vessel wall, activates only a small fraction of platelets in suspension directly, whereas the majority of platelets becomes activated by mediators released from collagen-activated platelets. In Gαq-deficient platelets that do not respond with activation of integrin αIIbβ3 to a variety of mediators like thromboxane A2 (TXA2), thrombin, or ADP, collagen at high concentrations was able to induce aggregation, an effect that could be blocked by antagonists of the TXA2 or P2Y12 receptors. The activation of TXA2 or P2Y12 receptors alone, which in Gαq-deficient platelets couple to G12/G13 and Gi, respectively, did not induce platelet integrin activation or aggregation. However, concomitant activation of both receptors resulted in irreversible integrin αIIbβ3-mediated aggregation of Gαq-deficient platelets. Thus, the activation of G12/G13- and Gi-mediated signaling pathways is sufficient to induce integrin αIIbβ3 activation. Although Gq-mediated signaling plays an important role in platelet activation, it is not strictly required for the activation of integrin αIIbβ3. This indicates that the efficient induction of platelet aggregation through G-protein-coupled receptors is an integrated response mediated by various converging G-protein-mediated signaling pathways involving Gq and Gi as well as G12/G13. thromboxane A2 thromboxane B2 forward/side scatter Under normal conditions, platelets circulate freely in the blood and do not adhere to each other. At sites of vascular damage, however, platelets adhere to subendothelial surfaces and become activated. Platelet activation involves a rapid change of shape followed by degranulation and integrin αIIbβ3-mediated aggregation. The activation of platelets is induced by various extracellular stimuli and involves positive feedback loops, which in a complex process ensure the rapid formation of a platelet plug. Collagen, exposed at subendothelial surfaces at sites of vascular injury, induces platelet activation initially by binding to glycoprotein VI, which through the associated FcRγ chain signals in a G-protein-independent manner (1Watson S.P. Gibbins J. Immunol. Today. 1998; 19: 260-264Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Full platelet aggregation by collagen and subsequent recruitment of platelets into a growing platelet plug, however, requires the formation and release of mediators like thromboxane A2(TXA2)1 and ADP. The transmembrane receptors of TXA2 and ADP couple to heterotrimeric G proteins, which represent central mediators of platelet activation. TXA2 receptors couple to Gq and to G12/G13 (2Offermanns S. Laugwitz K.L. Spicher K. Schultz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 504-508Crossref PubMed Scopus (398) Google Scholar, 3Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Crossref PubMed Scopus (317) Google Scholar), whereas ADP acts through two G-protein-coupled receptors, P2Y1, which couples to Gq, and P2Y12, which couples to Gi (4Gachet C. Thromb. Haemostasis. 2001; 86: 222-232Crossref PubMed Scopus (375) Google Scholar, 5Hollopeter 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 (1305) Google Scholar). Some of the roles of individual G-protein-mediated signaling pathways in platelet activation have been described during recent years. It is generally believed that Gq-mediated phospholipase Cβ activation plays an essential role in platelet activation as demonstrated by the phenotype of Gαq-deficient platelets, which fail to aggregate and secrete in response to thrombin, ADP, and the TXA2 mimetic U46619 because of a lack of agonist-induced phospholipase C activation (6Offermanns S. Toombs C.F., Hu, Y.H. Simon M.I. Nature. 1997; 389: 183-186Crossref PubMed Scopus (501) Google Scholar). Evidence for a role of Gq-independent processes in the regulation of platelet activation emerged from studies describing the mechanism of ADP-induced platelet activation. Platelets deficient in P2Y1 or in which P2Y1 was blocked pharmacologically do not aggregate in response to low and intermediate concentrations of ADP (7Hechler B. Leon C. Vial C. Vigne P. Frelin C. Cazenave J.P. Gachet C. Blood. 1998; 92: 152-159Crossref PubMed Google Scholar, 8Jin J. Kunapuli S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8070-8074Crossref PubMed Scopus (481) Google Scholar, 9Savi P. Beauverger P. Labouret C. Delfaud M. Salel V. Kaghad M. Herbert J.M. FEBS Lett. 1998; 422: 291-295Crossref PubMed Scopus (180) Google Scholar, 10Fabre J.E. Nguyen M. Latour A. Keifer J.A. Audoly L.P. Coffman T.M. Koller B.H. Nat. Med. 1999; 5: 1199-1202Crossref PubMed Scopus (396) Google Scholar, 11Leon C. Hechler B. Freund M. Eckly A. Vial C. Ohlmann P. Dierich A. LeMeur M. Cazenave J.P. Gachet C. J. Clin. Invest. 1999; 104: 1731-1737Crossref PubMed Scopus (403) Google Scholar). Aggregation could be restored by serotonin, which induces Gq-mediated phospholipase C activation but alone is not able to induce platelet aggregation (8Jin J. Kunapuli S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8070-8074Crossref PubMed Scopus (481) Google Scholar, 9Savi P. Beauverger P. Labouret C. Delfaud M. Salel V. Kaghad M. Herbert J.M. FEBS Lett. 1998; 422: 291-295Crossref PubMed Scopus (180) Google Scholar, 11Leon C. Hechler B. Freund M. Eckly A. Vial C. Ohlmann P. Dierich A. LeMeur M. Cazenave J.P. Gachet C. J. Clin. Invest. 1999; 104: 1731-1737Crossref PubMed Scopus (403) Google Scholar). Platelet activation by ADP obviously requires an additional signal, which appears to be mediated by the Gi-coupled P2Y12 receptor (5Hollopeter 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 (1305) Google Scholar). Platelets deficient in P2Y12 or in which P2Y12was blocked did not aggregate in response to ADP unless the Gi-mediated pathway was activated by adrenaline, which itself is unable to induce platelet aggregation (8Jin J. Kunapuli S.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8070-8074Crossref PubMed Scopus (481) Google Scholar, 12Foster C.J. Prosser D.M. Agans J.M. Zha I.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: 1591-1598Crossref PubMed Scopus (386) Google Scholar). Thus, Gq and Gi can synergize to induce platelet aggregation, and full platelet activation in response to ADP at intermediate concentrations requires concomitant activation of both G-proteins. G-proteins of the G12 family, G12 and G13, which are activated through TXA2 and thrombin receptors (2Offermanns S. Laugwitz K.L. Spicher K. Schultz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 504-508Crossref PubMed Scopus (398) Google Scholar, 3Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Crossref PubMed Scopus (317) Google Scholar), have been involved in the induction of the platelet shape change. This is mainly based on the finding that in Gαq-deficient platelets in which TXA2receptors only couple to G12 and G13, U46619still induces a rapid shape change. This effect appears to be mediated by the Rho/Rho kinase pathway (3Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Crossref PubMed Scopus (317) Google Scholar). Here we demonstrate a role for G12/G13-mediated signaling in platelet aggregation and show that in the absence of Gαq, TXA2, and ADP or adrenaline can induce integrin αIIbβ3-dependent platelet aggregation by concomitant activation of G12/G13 through TXA2 receptors and of Gi via P2Y12 or α2-adrenergic receptors, whereas each of the stimuli given alone was without effect. Thus, G12/G13-mediated as well as Gi-mediated signaling processes can synergize to activate the platelet fibrinogen receptor. Mutant mice deficient in Gαq were produced as described previously (6Offermanns S. Toombs C.F., Hu, Y.H. Simon M.I. Nature. 1997; 389: 183-186Crossref PubMed Scopus (501) Google Scholar). Gαq-deficient and wild-type littermates, which were of 129/Sv × C57BL/6 genetic background were used for experiments. The ATP analog AR-C69931MX was a generous gift from ASTRA Charnwood (Loughborough Leics, England). ADP, adrenaline, essentially fatty acid-free bovine serum albumin, and human fibrinogen were from Sigma. The thromboxane analog U46619 was purchased from Alexis Biochemicals (Grünberg, Germany), and fibrillar type I collagen (Horm) from equine tendon was from Nycomed (Munich, Germany). The thromboxane A2 receptor antagonist SQ29548 was fromBiomol (Hamburg, Germany), and the Rho kinase inhibitor Y-27632 was kindly provided by Yoshitomi Pharmaceutical Industries, Ltd. (Saitama, Japan). JON/APE, which selectively binds to activated mouse αIIbβ3 integrin (13Bergmeier W. Schulte V. Brockhoff G. Bier U. Zirngibl H. Nieswandt B. Cytometry. 2002; 48: 80-86Crossref PubMed Scopus (123) Google Scholar), was produced and modified in our laboratory. Fluorescein isothiocyanate-conjugated anti-P-selectin and control IgG antibodies were purchased from BD Pharmingen. Unlabeled JON/A was used to block αIIbβ3 in aggregometry as described previously (13Bergmeier W. Schulte V. Brockhoff G. Bier U. Zirngibl H. Nieswandt B. Cytometry. 2002; 48: 80-86Crossref PubMed Scopus (123) Google Scholar). Mice were bled under ether anesthesia from the retroorbital plexus. Blood was collected in a tube containing 10% (v/v) 7.5 units/ml heparin, and platelet-rich plasma was obtained by centrifugation at 300 × g for 10 min at room temperature. Platelet-rich plasma was removed and centrifuged at 1570 × g for 10 min. The platelet pellet was washed twice in Tyrode's buffer (137 mmol/liter NaCl, 2 mmol/liter KCl, 12 mmol/liter NaHCO3, 0.3 mmol/liter NaH2PO4, 5.5 mmol/liter glucose, 5 mmol/liter Hepes, pH 7.3) containing 0.35% bovine serum albumin and finally resuspended at a density of 5 × 105 platelet/μl in the same buffer in the presence of 0.02 units/ml ADP scavenger apyrase, a concentration sufficient to prevent desensitization of platelet ADP receptors during storage. Platelets were kept at 37 °C throughout all experiments. To block thromboxane A2 or P2Y12 receptors, platelets were incubated with 10 μm SQ29548 or 10 μm AR-C69931MX, respectively, for 10 min at 37 °C before the start of the experiment. To inhibit Rho kinase activity, platelets were incubated with Y-27632 (10 μm) for 30 min before the start of the experiment. To determine platelet aggregation, light transmission was measured using washed platelets adjusted to a platelet concentration of 3 × 105 platelets/μl with Tyrode's buffer containing CaCl2 (1 mm) and human fibrinogen (0.2 mg/ml). Transmission was recorded on a Fibrintimer 4-channel aggregometer (APACT Laborgeräte und Analysensysteme, Hamburg, Germany) and is expressed as arbitrary units with 100% transmission adjusted with plasma. Agonists were added as 20–100-fold concentrates. For determination of platelet degranulation, platelets were loaded with serotonin by incubation of platelet-rich plasma for 1 h at 37 °C with 2 μCi/ml [3H]serotonin (80–130 Ci/mmol). Thereafter, platelets were washed once in Tyrode's buffer, and platelets were adjusted at 2 × 105/ml. Platelets were exposed for 3 min to the indicated stimuli and chilled. After centrifugation, the secreted serotonin was determined by scintillation counting of the supernatants. TXB2 formation was determined using an enzyme immunoassay (Cayman, Ann Arbor, MI). 2.5 × 106 washed platelets were incubated for 3 min in the presence of the indicated stimuli. After centrifugation, TXB2 levels in supernatants were determined according to manufacturer's instructions. Washed platelets were adjusted to 2 × 104 platelets/μl with Tyrode's buffer, and 50 μl of this dilution were stimulated with the indicated agonists for 5 min at 37 °C. Samples were then stained with fluorophore-labeled monoclonal antibodies for 10 min at room temperature and directly analyzed on a FACScalibur (BD Pharmingen). Platelets were gated by forward/side scatter (FSC/SSC) characteristics. We have previously shown that Gαq-deficient platelets display defective activation by collagen (6Offermanns S. Toombs C.F., Hu, Y.H. Simon M.I. Nature. 1997; 389: 183-186Crossref PubMed Scopus (501) Google Scholar) although primary signaling through the activating platelet collagen receptor, GPVI/FcRγ, is not affected by the absence of Gαq (14Nieswandt B. Bergmeier W. Eckly A. Schulte V. Ohlmann P. Cazenave J.P. Zirngibl H. Offermanns S. Gachet C. Blood. 2001; 97: 3829-3835Crossref PubMed Scopus (89) Google Scholar). This may be because of the fact that fibrillar collagen is an insoluble macromolecule and, therefore, only a small fraction of platelets comes into direct contact with collagen (15Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W. Zirngibl H. Fassler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (457) Google Scholar), whereas the majority of platelets exposed to collagen may be activated secondarily by mediators like ADP and TXA2, which are released from the collagen-responsive fraction of platelets. To test this possibility, we performed flow cytometric analysis of single platelets in diluted suspensions under experimental conditions that largely exclude the accumulation of released mediators. Only 11% of the wild-type platelets were directly activated by collagen at high concentrations (50 μg/ml) as shown by the activation of αIIbβ3 using the JON/APEantibody (13Bergmeier W. Schulte V. Brockhoff G. Bier U. Zirngibl H. Nieswandt B. Cytometry. 2002; 48: 80-86Crossref PubMed Scopus (123) Google Scholar) and by degranulation-dependent exposure of P-selectin (Fig. 1). In contrast, >99% of platelets are activated upon exposure to stimuli like ADP and TXA2 under the same conditions (data not shown). The primary activation of a subpopulation of platelets by fibrillar collagen was not impaired in Gαq-deficient platelets of which also ∼11% responded to collagen. Thus, a small fraction of platelets is activated by collagen directly and subsequently releases soluble agonists like ADP, TXA2, and other mediators, which in turn activate the majority of platelets, resulting in the full aggregation response. This mechanism mediates not only collagen-induced activation of platelets in suspension as studied here but most probably also underlies the initial activation of platelets by subendothelial collagen at the damaged vessel wall in vivo. The amplification of collagen-induced platelet activation by mediators like ADP and TXA2 is thought to be largely Gq- dependent, because both ADP and TXA2 fail to induce platelet aggregation in the absence of Gαq (6Offermanns S. Toombs C.F., Hu, Y.H. Simon M.I. Nature. 1997; 389: 183-186Crossref PubMed Scopus (501) Google Scholar). To test this hypothesis, we studied aggregation of Gαq-deficient platelets in response to increasing concentrations of fibrillar collagen. Although low concentrations of collagen did not induce aggregation of platelets in the absence of Gαq, collagen at concentrations of >10 μg/ml induced robust aggregation of Gαq-deficient platelets, which occurred with a pronounced shape change (as shown by the decrease in light transmission before the start of aggregation) (Fig.2 A). Whereas collagen was much less potent in Gαq-deficient platelets compared with wild-type platelets, the maximal efficacy appeared to be only moderately affected by the absence of Gq-mediated signaling. The aggregation of Gαq-deficient platelets in response to high collagen concentrations was accompanied by serotonin release and the formation of TXA2 (Fig. 2, B andC), whereas collagen at low concentrations was unable to induce significant release or formation of mediators in the absence of Gαq. These results suggested that soluble agonists released from collagen-activated platelets can induce shape change and aggregation in the absence of Gαq. In Gαq-deficient platelets, the receptors for ADP and TXA2 couple to Gi and G12/G13, respectively. Therefore, we studied the role of these signaling pathways in the overall process of platelet activation/aggregation. To test the significance of TXA2receptor-mediated activation of G12/G13 for the observed shape change and aggregation response in collagen-stimulated Gαq-deficient platelets, we inhibited the platelet TXA2 receptor by the specific antagonist SQ29548 (16Hedberg A. Hall S.E. Ogletree M.L. Harris D.N. Liu E.C. J. Pharmacol. Exp. Ther. 1988; 245: 786-792PubMed Google Scholar). As evaluated by light transmission, this treatment completely abolished the collagen-induced shape change of Gαq-deficient platelets (Fig. 2 D). Thus, the activation of G12/G13 through TXA2receptors plays a major role in this process. Unexpectedly, the inhibition of the TXA2 receptor also markedly reduced the aggregation response, suggesting that G12/G13stimulation is involved in the activation of the platelet integrin αIIbβ3. On the other hand, the inhibition of the Gi-coupled P2Y12 receptor by the specific antagonist AR-C69931MX (10 μm) had no influence on the shape change but also abolished the aggregation response (Fig.2 D). The finding that both TXA2 and ADP are required for collagen-induced platelet aggregation in the absence of Gαq suggests that G12/G13- and Gi-mediated signaling pathways act in concert to activate integrin αIIbβ3. To study the role of G12/G13 in platelet integrin activation, we used the TXA2 mimetic U46619, which is known to specifically stimulate Gq and G12/G13 through the TXA2 receptor. As shown previously, U46619 induced a rapid shape change but no aggregation in Gαq-deficient platelets at concentrations up to 30 μm, whereas robust aggregation was already seen at 0.3 μm in wild-type platelets (Fig.3 A), confirming the crucial role of Gq in this process. U46619-induced aggregation of wild-type platelets has been shown to largely depend on released ADP acting on the Gi-coupled P2Y12 receptor (17Paul B.Z. Jin J. Kunapuli S.P. J. Biol. Chem. 1999; 274: 29108-29114Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). This finding suggests that the activation of Gq and G12/G13 is not sufficient to stimulate full integrin activation and, possibly, degranulation in platelets unless Gi is concomitantly activated. This was confirmed when we examined U46619-stimulated wild-type platelets by flow cytometry for activated αIIbβ3 integrin and P-selectin expression. U46619 at concentrations of up to 30 μminduced only very low levels of αIIbβ3activation and P-selectin expression in wild-type platelets (Fig.3 B). Thus, the activation of Gq and G12/G13 is indeed not sufficient for efficient integrin activation and granule release in platelets. However, the addition of low concentrations of exogenous ADP (5 μm) resulted in profound αIIbβ3 activation and P-selectin expression. At this concentration, ADP alone is known to induce only low levels of αIIbβ3 activation and no P-selectin expression in wild-type platelets, resulting in reversible platelet aggregation (Fig. 3 B) (4Gachet C. Thromb. Haemostasis. 2001; 86: 222-232Crossref PubMed Scopus (375) Google Scholar). The potentiating effect of ADP was dependent on stimulation of the Gi-coupled P2Y12 receptor, because it was abrogated in the presence of the highly specific P2Y12 antagonist AR-C69931MX (10 μm). This finding suggested that the failure of Gαq-deficient platelets to aggregate in response to U46619 is not only because of the absence of the Gq-mediated pathway leading to inside-out activation of integrin αIIbβ3 but is also based on defective release of stored ADP and subsequent Giactivation. To test this hypothesis, we analyzed αIIbβ3 activation on U46619-stimulated Gαq-deficient platelets in the absence or presence of exogenously added ADP. In these platelets, which do not degranulate in response to TXA2 receptor activation, U46619 alone did not induce αIIbβ3 activation at concentrations up to 30 μm (Fig. 3 C). Strikingly, however, weak but consistent activation of αIIbβ3was detectable upon the addition of 5 μm ADP, which by itself had no effect because of the lack of P2Y1-mediated calcium mobilization (18Ohlmann P. Eckly A. Freund M. Cazenave J.-P. Offermanns S. Gachet C Blood. 2000; 96: 2134-2139Crossref PubMed Google Scholar). Similar to wild-type platelets, the potentiating effect of ADP was inhibited by the P2Y12antagonist AR-C69931MX (10 μm) (Fig. 3 C). The relatively low levels of αIIbβ3activation induced by U46619 and ADP in Gαq-deficient platelets were sufficient to mediate robust and irreversible aggregation that was inhibited by a blocking antibody against αIIbβ3 integrin (Fig. 3 D). Importantly, aggregation was also blocked in the presence of AR-C69931MX, confirming the critical role of the P2Y12receptor-mediated Gi activation in this process (Fig.3 D). Similar results were obtained when G12/G13 activation was induced by thrombin instead of U46619. Although thrombin (0.2 units/ml) alone induced shape change but no aggregation of Gαq-deficient platelets, robust αIIbβ3-dependent aggregation occurred in the presence of exogenously added ADP (5 μm). This potentiating effect of ADP was again inhibited by AR-C69931MX (10 μm) (data not shown). It is known that Gαq-deficient platelets do not aggregate in response to U46619 or ADP because of the lack of intracellular calcium mobilization (6Offermanns S. Toombs C.F., Hu, Y.H. Simon M.I. Nature. 1997; 389: 183-186Crossref PubMed Scopus (501) Google Scholar). To test whether the combination of both agonists would be able to bypass this defect by undefined mechanisms, we measured intracellular calcium levels in Gαq-deficient platelets in response to increasing concentrations of U46619 and ADP. However, although wild-type platelets responded with robust calcium mobilization to a combination of 1 μmU46619 and 5 μm ADP, no response was seen in Gαq-deficient platelets, even at 10 μmU46619 plus 50 μm ADP (data not shown). These results suggested that concomitant stimulation of G12/G13 and Gi results in integrin activation in platelets and that Gq is not necessarily required to induce platelet activation through G-protein-coupled receptors. To test this hypothesis further, we stimulated wild-type and Gαq-deficient platelets with U46619 in the absence or presence of the Gi-selective agonist, adrenaline. Adrenaline alone is unable to promote platelet shape change or aggregation (14Nieswandt B. Bergmeier W. Eckly A. Schulte V. Ohlmann P. Cazenave J.P. Zirngibl H. Offermanns S. Gachet C. Blood. 2001; 97: 3829-3835Crossref PubMed Scopus (89) Google Scholar, 19Lanza F. Cazenave J.-P. Thromb. Haemostasis. 1985; 54: 402-408Crossref PubMed Scopus (36) Google Scholar) but can potentiate various platelet responses through Gi and the Gi family member Gz (20Yang 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: 9984-9989Crossref PubMed Scopus (171) Google Scholar). Neither wild-type nor Gαq-deficient platelets responded to adrenaline (10 μm) with αIIbβ3 activation (Fig.4, A and C) or P-selectin expression (Fig. 4 C and data not shown), and consequently, no aggregation occurred (Fig. 4 B and data not shown). Adrenaline (like ADP) potentiated the effect of U46619 in both mouse strains as shown by αIIbβ3 activation and P-selectin expression in wild-type (Fig. 4 C) and αIIbβ3 activation in Gαq-deficient platelets (Fig. 4 A). The αIIbβ3 activation observed in Gαq-deficient platelets in response to U46619 and adrenaline was sufficient to mediate irreversible platelet aggregation that was inhibited by a blocking antibody against the integrin (Fig.4 B). This finding demonstrates the critical involvement of both G12/G13 and Gi in full integrin activation and granule release in response to the TXA2 mimetic U46619 and indicates that activation of G12/G13- and Gi-mediated pathways is sufficient for platelet activation. Recent evidence suggests that the G12/G13-mediated platelet shape change response is mediated by the Rho/Rho kinase pathway (3Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Crossref PubMed Scopus (317) Google Scholar, 21Paul B.Z. Daniel J.L. Kunapuli S.P. J. Biol. Chem. 1999; 274: 28293-28300Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 22Bauer M. Retzer M. Wilde J.I. Maschberger P. Essler M. Aepfelbacher M. Watson S.P. Siess W. Blood. 1999; 94: 1665-1672Crossref PubMed Google Scholar). In agreement with this finding, the TXA2-dependent shape change of Gαq-deficient platelets in response to high collagen concentrations was abolished in the presence of the Rho kinase inhibitor Y-27632 (10 μm) (23Ishizaki T. Uehata M. Tamechika I. Keel J. Nonomura K. Maekawa M. Narumiya S. Mol. Pharmacol. 2000; 57: 976-983PubMed Google Scholar). Strikingly, however, the inhibition of Rho kinase had no significant influence on the aggregation response, indicating that G12/G13activation contributes to integrin activation by regulating a Rho kinase-independent mechanism (Fig.5 A). To test this hypothesis, we activated Gαq-deficient platelets with U46619 alone or in combination with ADP or adrenaline in the absence or presence of Y-27632 (10 μm). Indeed, whereas the inhibition of Rho kinase abrogated the shape change induced by U46619, it had no effect on αIIbβ3 activation (Fig. 5 B) and platelet aggregation (Fig. 5 C). Similarly, inhibitors of non-receptor tyrosine kinases like src, which have been shown to be activated through G12/G13 in platelets (3Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Crossref PubMed Scopus (317) Google Scholar), had no effect on the activation of Gαq-deficient platelets in response to U46619 and ADP (data not shown). The mechanisms linking the G12/G13-mediated signaling pathway to integrin αIIbβ3 activation in platelets remains unclear. To test the significance of the synergy between G12/G13- and Gi-mediated signaling in normal platelets, we stimulated wild-type platelets with 0.03 μmU46619 in the presence or absence of adrenaline. At this concentration, U46619 alone is known to induce a rapid shape change through G12/G13 activation but no aggregation due to insufficient Gq signaling (3Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Crossref PubMed Scopus (317) Google Scholar, 24Simpson A.W. Hallam T.J. Rink T.J. FEBS Lett. 1986; 201: 301-305Crossref PubMed Scopus (32) Google Scholar, 25Ohkubo S. Nakahata N. Ohizumi Y. Br. J. Pharmacol. 1996; 117: 1095-1104Crossref PubMed Scopus (53) Google Scholar) (Fig. 5 D). However, the concomitant stimulation of Gi by the addition of adrenaline (10 μm) induced irreversible integrin αIIbβ3-dependent aggregation (Fig. 5 D). These results demonstrate that the contribution of G12/G13 signaling to platelet activation also plays a significant role in wild-type platelets, at least at low agonist concentrations. This may reflect the in vivosituation where a variety of stimuli, which are present at submaximally active concentration, synergistically induce platelet activation during thrombus formation. Taken together, our data show that the activation of G12/G13- and Gi-mediated signaling pathways is sufficient to induce integrin αIIbβ3 activation. Although, Gq-mediated signaling processes are essential for calcium mobilization and thus play an important role in platelet activation, they are not absolutely required for the activation of integrin αIIbβ3. These data indicate that the activation of platelets through G-protein-coupled receptors is a highly integrated process, which in order to occur with high efficiency requires the activation of at least three G-protein-mediated pathways involving Gq, Gi, and G12/13. We thank Barbara Wallenwein for excellent technical assistance.