Epinephrine-mediated protein kinase C and Rap1b activation requires the co-stimulation of Gz-, Gq-, and Gi-coupled receptors

Summary

We have recently shown that ADP-induced activation of protein kinase C (PKC) requires the co-stimulation of both P2Y1 and P2Y12 receptors. In this work, we show that inhibition of ADP-mediated phosphorylation of pleckstrin, the main PKC substrate, caused by antagonists of the P2Y12 receptor can be reversed by stimulation of the α2-adrenergic receptor by epinephrine. However, we also observed that addition of epinephrine alone caused a marked phosphorylation of pleckstrin. This effect occurred in the absence of Gq stimulation, as it was not associated to intracellular Ca²⁺ release. Epinephrine-induced pleckstrin phosphorylation was time- and dose-dependent, and was inhibited by the α2-adrenergic antagonist yohimbin. Phosphorylation of pleckstrin did not occur when platelet stimulation with epinephrine was performed in the presence of the ADP scavenger apyrase, and was suppressed by antagonists of both P2Y1 and P2Y12 ADP receptors. Importantly, no release of dense granules was measured in epinephrine-treated platelets. Addition of epinephrine to platelets was also able to stimulate Rap1b activation. Similarly to pleckstrin phosphorylation, however, this effect was prevented in the presence of apyrase or upon pharmacologic blockade of either P2Y1 or P2Y12 receptors. These results indicate that sub-threshold amounts of ADP in the medium are essential to allow epinephrine stimulation of α2-adrenergic receptor to elicit platelet responses, and reveal a novel synergism among strong stimulation of Gz and sub-threshold stimulation of both Gq and Gi, able to dissociate PKC activation from intracellular Ca²⁺ mobilisation.

Keywords

Signal transduction - epinephrine - protein kinase C - Rap1b - ADP receptors

^* These authors contributed equally to this work.

• References

• 1 Woulfe DS. Platelet G protein-coupled receptors in hemostasis and thrombosis. J Thromb Haemost 2004; 3: 2193-2200.
• 2 Dorsam RT, Kunapuli SP. Central role of the P2Y12 receptor in platelet activation. J Clin Invest 2004; 113: 340-345.
• 3 Offermanns S, Toombs CF, Hu YH. et al. Defective platelet activation in G alpha(q)-deficient mice. Nature 1997; 389: 183-186.
• 4 Rhee SG. Regulation of phosphoinositide-specific phospholipase C. Annu Rev Biochem 2001; 70: 281-312.
• 5 Offermanns S, Laugwitz KL, Spicher K. et al. G proteins of the G12 family are activated via thromboxane A2 and thrombin receptors in human platelets. Proc Natl Acad Sci. USA 1994; 91: 504-508.
• 6 Jin J, Kunapuli SP. Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation. Proc Natl Acad Sci USA 1998; 95: 8070-8074.
• 7 Eckly A, Gendrault JL, Hechler B. et al. Differential involvement of the P2Y1 and P2YT receptors in the morphological changes of platelet aggregation. Thromb Haemost 2001; 85: 694-701.
• 8 Paul BZ, Jin J, Kunapuli SP. Molecular mechanism of thromboxane A(2)-induced platelet aggregation. Essential role for p2t(ac) and alpha(2a) receptors. J Biol Chem 1999; 274: 29108-29114.
• 9 Andre P, Delaney SM, LaRocca T. et al. P2Y12 regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries. J Clin Invest 2003; 112: 398-406.
• 10 Daniel JL, Dangelmaier C, Jin J. et al. Role of intracellular signaling events in ADP-induced platelet aggregation. Thromb Haemost 1999; 82: 1322-1326.
• 11 Zurn CS, Geisler T, Gawaz M. ADP-receptor blockade: a case for personalized pharmacology?. Thromb Haemost 2010; 103: 496-506.
• 12 Lova P, Paganini S, Sinigaglia F. et al. A Gi-dependent pathway is required for activation of the small GTPase Rap1B in human platelets. J Biol Chem 2002; 277: 12009-12015.
• 13 Hirsch E, Bosco O, Tropel P. et al. Resistance to thromboembolism in PI3Kgamma-deficient mice. FASEB J 2001; 15: 2019-2021.
• 14 Jackson SP, Schoenwaelder SM, Goncalves I. et al. PI 3-kinase p110beta: a new target for antithrombotic therapy. Nat Med 2005; 11: 507-514.
• 15 Woulfe D, Jiang H, Mortensen R. et al. Activation of Rap1B by G(i) family members in platelets. J Biol Chem 2002; 277: 23382-23390.
• 16 Lova P, Paganini S, Hirsch E. et al. A selective role for phosphatidylinositol 3,4,5-trisphosphate in the Gi-dependent activation of platelet Rap1B. J Biol Chem 2003; 278: 131-138.
• 17 Bertoni A, Tadokoro S, Eto K. et al. Relationships between Rap1b, affinity modulation of integrin αIIbβ3, and the actin cytoskeleton. J Biol Chem 2002; 277: 25715-25721.
• 18 Chrzanowska-Wodnicka M, Smyth SS, Schoenwaelder SM. et al. Rap1b is required for normal platelet function and hemostasis in mice. J Clin Invest 2005; 115: 680-687.
• 19 Bernardi B, Guidetti GF, Campus F. et al. The small GTPase Rap1b regulates the cross talk between platelet integrin alpha2beta1 and integrin alphaIIbbeta3. Blood 2006; 107: 2728-2735.
• 20 Guidetti GF, Lova P, Bernardi B. et al. The Gi-coupled P2Y12 receptor regulates diacylglycerol-mediated signaling in human platelets. J Biol Chem 2008; 283: 28795-28805.
• 21 Yang J, Wu J, Kowalska MA. et al. Loss of signaling through the G protein, Gz, results in abnormal platelet activation and altered responses to psychoactive drugs. Proc Natl Acad Sci USA 2000; 97: 9984-9989.
• 22 Lanza F, Beretz A, Stierle A. et al. Epinephrine potentiates human platelet activation but is not an aggregating agent. Am J Physiol 1988; 255: 1276-1286.
• 23 Steen VM, Holmsen H, Aarbakke G. The platelet-stimulating effect of adrenaline through α2-adrenergic receptors requires simultaneous activation by a true stimulatory platelet agonist. Evidence that adrenaline per se does not induce human platelet activation in vitro. Thromb Haemost 1993; 70: 506-513.
• 24 Lalau Keraly C, Kinlough-Rathbone RL, Packham MA. et al. Conditions affecting the responses of human platelets to epinephrine. Thromb Haemost 1988; 60: 209-216.
• 25 Ghilotti M, Lova P, Balduini C. et al. Epinephrine induces intracellular Ca2+ mobilization in thrombin-desensitized platelets: a role for GPIb-IX-V. Platelets 2007; 18: 135-142.
• 26 Banga HS, Simons ER, Brass LF. et al. Activation of phospholipases A and C in human platelets exposed to epinephrine: Role of glycoproteins IIb/IIIa and dual role of epinephrine. Proc Natl Acad Sci USA 1986; 83: 9197-9201.
• 27 Marti KB, Lapetina EG. Epinephrine suppresses rap1B. GAP-activated GTPase activity in human platelets. Proc Natl Acad Sci USA 1992; 89: 2784-2788.
• 28 Siess W, Weber PC, Lapetina EG. Activation of phospholipase C is dissociated from arachidonate metabolism during platelet shape change induced by thrombin or platelet activating factor. Epinephrine does not induce phospholipase C activation or platelet shape change. J Biol Chem 1984; 259: 8286-8292.
• 29 Nash CA, Severin S, Dawood BB. et al. Src family kinases are essential for primary aggregation by Gi-coupled receptors. J Thromb Haemost 2010; 8: 2273-2282.
• 30 Cifuni SM, Wagner DD, Bergmeier W. CalDAG-GEFI and protein kinase C represent alternative pathways leading to activation of integrin αIIbβ3 in platelets. Blood 2008; 112: 1696-1703.
• 31 Brass LF, Hoxie JA, Kieber-Emmons T. et al. Agonist receptors and G proteins as mediators of platelet activation. Adv Exp Med Biol 1993; 344: 17-36.