Effects of the cAMP-elevating agents cilostamide, cilostazol and forskolin on the phosphorylation of Akt and GSK-3β in platelets

Summary

Elevating intracellular cAMP has been shown to inhibit platelet function. cAMP interferes with platelet-activating signals which lead to aggregation inhibition, but the precise mechanism is unclear.The present study examined if cAMP-elevating agents inhibited phosphatidylinositol 3-kinase (PI3-kinase) signaling in rat platelets by immunoblotting. Akt is one of the key molecules downstream of PI3K, and is phosphorylated by collagen stimulation. The phosphodiesterase-3 (PDE3) inhibitors cilostamide and cilostazol, and the adenylate cyclase activator forskolin, inhibited collagen-induced Akt phosphorylation at Ser473.The inhibitory effects of these cAMP-elevating agents on Akt phosphorylation were unchanged in the presence of the PKA (cyclic AMP-dependent protein kinase) inhibitor H-89. These effects were consistent with inhibition of platelet aggregation. It is known that inhibition of Akt phosphorylation leads to inhibition of phosphorylation of glycogen synthase kinase 3-beta (GSK-3β), which is an effector of Akt, but cAMP-elevating agents stimulated GSK-3βphosphorylation at Ser9.The PKA inhibitor H-89 attenuated GSK-3βphosphorylation.The cAMP-elevating agents cilostamide, cilostazol and forskolin did not directly affect the enzyme activity of PI3-kinase.These results suggested that cAMP-elevating agents have two effects on PI3K signalling: inhibition of Akt phosphorylation independent of PKA; and stimulation of GSK-3β phosphorylation dependent on PKA. Our results provide new insights into the inhibitory effect of cAMPelevating agents on platelet function.

Keywords

Akt - cAMP - GSK - phosphorylation - platelet aggregation

• References

• 1 Barrett NE, Holbrook L, Jones S. et al. Future innovations in anti-platelet therapies. Br J Pharmacol 2008; 154: 918-939.
• 2 Ikeda Y. Antiplatelet therapy using cilostazol, a specific PDE3 inhibitor. Thromb Haemost 1999; 82: 435-438.
• 3 Goto S. Cilostazol: potential mechanism of action for antithrombotic effects accompanied by a low rate of bleeding. Atheroscler Suppl 2005; 06: 3-11.
• 4 Ikeda Y, Sudo T, Kimura Y. Cilostazol. In: Platelet. 2nd ed. Elsevier: 2007. pp. 1181-1191.
• 5 Harbeck B, Hüttelmaier S, Schluter K. et al. Phosphorylation of the vasodilator-stimulated phosphoprotein regulates its interaction with actin. J Biol Chem 2000; 275: 30817-30825.
• 6 Horstrup K, Jablonka B, Hönig-Liedl P. et al. Phosphorylation of focal adhesion vasodilator-stimulated phosphoprotein at Ser157 in intact human platelets correlates with fibrinogen receptor inhibition. Eur J Biochem 1994; 225: 21-27.
• 7 Sudo T, Ito H, Kimura Y. Phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) by the anti-platelet drug, cilostazol, in platelets. Platelets 2003; 14: 381-390.
• 8 Russo I, Doronzo G, Mattiello L. et al. The activity of constitutive nitric oxide synthase is increased by the pathway cAMP/cAMP-activated protein kinase in human platelets. New insights into the antiaggregating effects of cAMP-elevating agents. Thromb Res 2004; 114: 265-273.
• 9 Burgering BM, Coffer PJ. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 1995; 376: 599-602.
• 10 Li Z, Zhang G, Le Breton GC, Gao X. et al. Two waves of platelet secretion induced by thromboxane A2 receptor and a critical role for phosphoinositide 3-kinases. J Biol Chem 2003; 278: 30725-30731.
• 11 Kroner C, Eybrechts K, Akkerman JW. @Dual regulation of platelet protein kinase B. J Biol Chem 2000; 275: 27790-27798.
• 12 Barry FA, Gibbins JM. Protein kinase B is regulated in platelets by the collagen receptor glycoprotein VI. J Biol Chem 2002; 277: 12874-12878.
• 13 Kim S, Jin J, Kunapuli SP. Akt activation in platelets depends on Gi signaling pathways. J Biol Chem 2004; 279: 4186-4195.
• 14 Barry FA, Graham GJ, Fry MJ. et al. Regulation of glycogen synthase kinase 3 in human platelets: a possible role in platelet function?. FEBS Lett 2003; 553: 173-178.
• 15 Stojanovic A, Marjanovic JA, Brovkovych VM. et al. A phosphoinositide 3-kinase-AKT-nitric oxidecGMP signaling pathway in stimulating platelet secretion and aggregation. J Biol Chem 2006; 281: 16333-16339.
• 16 Kim S, Jee K, Kim D. et al. Cyclic AMP inhibits Akt activity by blocking the membrane localization of PDK1. J Biol Chem 2001; 276: 12864-12870.
• 17 Filippa N, Sable CL, Filloux C. et al. Mechanism of protein kinase B activation by cyclic AMP-dependent protein kinase. Mol Cell Biol 1999; 19: 4989-5000.
• 18 Sudo T, Ito H, Hayashi H. et al. Genetic strain differences in platelet aggregation and thrombus formation of laboratory rats. Thromb Haemost 2007; 97: 665-672.
• 19 Schwarz UR, Walter U, Eigenthaler M. Taming platelets with cyclic nucleotides. Biochem Pharmacol 2001; 62: 1153-1161.
• 20 Rittenhouse SE. Phosphoinositide 3-kinase activation and platelet function. Blood 1996; 88: 4401-4414.
• 21 Vlahos CJ, Matter WF, Hui KY. et al. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 1994; 269: 5241-5248.
• 22 Watanabe N, Nakajima H, Suzuki H. et al. Functional phenotype of phosphoinositide 3-kinase p85alpha-null platelets characterized by an impaired response to GP VI stimulation. Blood 2003; 102: 541-548.
• 23 Hirsch E, Bosco O, Tropel P. et al. Resistance to thromboembolism in PI3Kgamma-deficient mice. FASEB J 2001; 15: 2019-2021.
• 24 Jackson SP, Schoenwaelder SM, Goncales I. et al. PI3K beta: a new target for antithrombotic therapy. Nat Med 2005; 11: 507-514.
• 25 Andreas S, Hans PW, Klaus D. et al. Selective inhibition of the platelet phosphoinositide-3-kinase p110βas promising new strategy for platelet protection during extracorporeal circulation. Thromb Haemost 2008; 99: 609-615.
• 26 Woulfe D, Jiang H, Morgans A. et al. Defects in secretion, aggregation, and thrombus formation in platelets from mice lacking Akt2. J Clin Invest 2004; 113: 441-450.
• 27 Chen J, De S, Damron DS. et al. Impaired platelet responses to thrombin and collagen in AKT-1-deficient mice. Blood 2004; 104: 1703-1710.
• 28 Umekawa H, Tanaka T, Kimura Y. et al. Purification of cyclic adenosine monophosphate phosphodiesterase from human platelets using new-inhibitor Sepharose chromatography. Biochem Pharmacol 1984; 33: 3339-3344.
• 29 Sudo T, Tachibana K, Toga K. et al. Potent effects of novel anti-platelet aggregatory cilostamide analogues on recombinant cyclic nucleotide phosphodiesterase isozyme activity. Biochem Pharmacol 2000; 59: 347-356.
• 30 Han SJ, Vaccari S, Nedachi T. et al. Protein kinase B/Akt phosphorylation of PDE3A and its role in mammalian oocyte maturation. EMBO J 2006; 25: 5716-5725.
• 31 Zhang W, Colman RW. Thrombin regulates intracellular cyclic AMP concentration in human platelets through phosphorylation/activation of phosphodiesterase 3A. Blood 2007; 110: 1475-1482.
• 32 Li D, August S, Woulfe DS. GSK3beta is a negative regulator of platelet function and thrombosis. Blood 2008; 111: 3522-3530.
• 33 van Weeren PC, de Bruyn KM, de Vries-Smits AM. et al. Essential role for protein kinase B (PKB) in insulin-induced glycogen synthase kinase 3 inactivation. Characterization of dominant-negative mutant of PKB. J Biol Chem 1998; 273: 13150-13156.
• 34 Cone J, Wang S, Tandon N. et al. Comparison of the effects of cilostazol and milrinone on intracellular cAMP levels and cellular function in platelet and cardiac cells. J Cardiovasc Pharmacol 1999; 34: 497-504.
• 35 Manns JM, Brennan KJ, Colman RW. et al. Differential regulation of human platelet responses by cGMP inhibited and stimulated cAMP phosphodiesterases. Thromb Haemost 2002; 87: 873-879.
• 36 Fang X, Yu SX, Lu Y. et al. Phosphorylation and inactivation of glycogen synthase kinase 3 by protein kinase A. Proc Natl Acad Sci U S A 2000; 97: 11960-11965.
• 37 Li M, Wang X, Meintzer MK. et al. Cyclic AMP promotes neuronal survival by phosphorylation of glycogen synthase kinase 3beta. Mol Cell Biol 2000; 20: 9356-9363.