The isoform of PDE3A phosphorylated by Akt during thrombin activation of platelets is the 136-kDa species

The isoform of PDE3A phosphorylated by Akt during thrombin activation of platelets is the 136-kDa species. reduced thrombin-induced cAMP reduction. The combination of both reversed cAMP decrease by thrombin. Thrombin-mediated phosphorylated PDE3A was isolated by liquid chromatography, detected by a monoclonal antibody against Akt-phosphorylated substrate, and verified by immunoprecipitation study. The predominant isoform phosphorylated by Akt was the 136-kDa species. We suggest that activation/phosphorylation of PDE3A via Akt signaling pathway participates in regulating cAMP during thrombin activation of platelets. Introduction Thrombin activates human platelets by cleaving and activating protease-activated receptor 1 (PAR-1) and PAR-4. In turn, these receptors activate G proteins (Gq, G12/13, and Gi), leading to the activation of phospholipase C (PLC), phosphatidyl inositol-3 kinase (PI3K), Rho, and Rac, which, by stimulating phosphoinositide hydrolysis, raise cytosolic Ca++ concentration and lower intracellular cAMP content. Cyclic AMP (cAMP) is a control molecule in platelets that interrupts multiple signaling pathways N2-Methylguanosine and plays a significant role in down-regulating platelet activation. Synthesis of cAMP in platelets is stimulated by the binding of mediators, such as prostacyclin and adenosine, to cell-surface receptors coupled to GTP-binding proteins. G proteins mediate the interaction of agonist-occupied 7-transmembraneCspanning cell surface receptors to regulate intracellular membrane-bound enzymes or ion channel activity. Gs forms a link between purinergic or prostaglandin receptors and adenylate cyclase, leading to stimulation of the latter. On the other hand, activation of platelets by thrombin diminishes the elevated intracellular cAMP levels via a Gi-coupled receptor.1 cAMP levels are also regulated by the degradation of cAMP via the cyclic nucleotide phosphodiesterases, a group of enzymes that catalyze the hydrolysis of 3,5-cyclic nucleotides to inactive 5-nucleotides by cleaving a phosphodiesterase bond. The levels of cAMP are tightly controlled and are ultimately dependent on its rate of synthesis by adenylate cyclase and its rate of hydrolysis by cAMP-phosphodiesterases (PDEs). In vitro, intracellular cAMP levels can N2-Methylguanosine be increased by stimulating adenylate cyclase2 or by inhibiting cAMP-PDE.3 In this study, we present N2-Methylguanosine evidence that the cAMP-dependent phosphodiesterase (PDE3A) is a component of the thrombin signaling pathway in platelets. Thrombin raises PDE3A activity through phosphorylation/activation of PDE3A and activated PDE3A participates N2-Methylguanosine in regulating intracellular cAMP contents through acceleration of cAMP hydrolysis. We show that the PI3K/Akt signaling pathway is involved in thrombin-induced PDE3A activation, and we compare the contribution of this pathway to Gi-adenylate cyclase regulation of intracellular cAMP content. Knowledge of which intermediate signaling pathways are involved will allow a more complete understanding of the mechanisms of platelet activation. Materials and methods Thrombin, milrinone, forskolin, 3-isobutyl-1-methylxanthine (IBMX), cAMP, for 20 minutes to generate platelet-rich plasma. The platelet-rich plasma was incubated with 100 mol/L aspirin. The platelets were then centrifuged at 1000for 10 minutes and resuspended in Tyrode’s buffer (138 mmol/L NaCl, 2.7 mmol/L KCl, 2 mmol/L MgCl2, 0.42 mmol/L NaH2PO4, 5 mmol/L glucose, 10 mmol/L HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], and 0.2% bovine serum albumin, pH 7.4). In the experiments, thrombin was added to the washed platelets and incubated at 37C for 3 minutes without stirring. The reactions were stopped by addition of 0.5% Triton X-100, and the samples were transferred to melting ice-water bath. In the presence of inhibitors, samples were incubated with each inhibitor at 37C for 30 minutes or with Akt inhibitor VIII for 1 hour. Assay of PDE activity The washed platelets (2 108/mL) were lysed by 0.5% Triton X-100 after stimulation with thrombin or vehicle in the presence or absence of inhibitors. PDE activity, which depends on cAMP, was measured as described previously4 SMAD2 with the following modification: 100-L assay volume contained N2-Methylguanosine 50 mmol/L Tris-HCl buffer, pH 7.8, 10 mmol/L MgCl2, 2 mol/L cAMP and [3H]cAMP (40?000 cpm/assay). Reactions were started by addition of 25 L platelet lysate and incubated at 24C for 15 minutes and stopped by addition of 0.2 mL of 0.2 mol/L ZnSO4 and 0.2 mL of 0.2 mol/L Ba(OH)2. The samples were.