Thrombin Regulates Intracellular cAMP Concentration in Human Platelets through Phosphorylation/Activation of PDE3A

Thrombin-induced cAMP reduction potentates several steps in platelet activation, including Ca mobilization, cytoskeletal reorganization, and fibrinogen receptor conformation. We now reinvestigate the signaling pathways by which intracellular cAMP content is controlled following platelet activation by thrombin. When washed human platelets were stimulated with thrombin, cAMP-dependent phosphodiesterase (PDE3A) activity was significantly increased. A non-selective PDE inhibitor, 3-Isobutyl1-methylxanthine (IBMX), and the PDE3 selective inhibitors milrinone and cilostazol each suppressed thrombin-induced cAMP-dependent PDE responses, but not two different PDE2 inhibitors. Selective inhibition of PDE3A resulted in reversal of thrombin-induced cAMP reduction indicating that thrombin activated PDE3A. In synergy with inhibition of adenylate cyclase by thrombin, activated PDE3A accelerates cAMP hydrolysis and maximally reduces the cAMP content. Thrombin-induced PDE3A activation was diminished concomitantly with dephosphorylation of PDE3A by proteinphosphatase 1 (PP1). An Akt inhibitor blocked PDE3A activation and constrained thrombin-induced cAMP reduction. A P2Y12 inhibitor also 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 mAb 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. For personal use only. on October 28, 2017. by guest www.bloodjournal.org From