Thrombin regulates intracellular cyclic AMP concentration in human platelets through phosphorylation/activation of phosphodiesterase 3A.
Thrombin-induced cyclic AMP (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 after platelet activation by thrombin. When washed human platelets were stimulated with thrombin, cAMP-dependent phosphodiesterase (PDE3A) activity was significantly increased. A nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the PDE3 selective inhibitors milrinone and cilostazol each suppressed thrombin-induced cAMP-dependent PDE responses, but not 2 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 protein phosphatase 1 (PP1). An Akt inhibitor blocked PDE3A activation and constrained thrombin-induced cAMP reduction. A P2Y(12) 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 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.
Zhang
Wei
W
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Colman
Robert W
RW
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000067956:Adenylyl Cyclase Inhibitors; D001792:Blood Platelets / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology; D000077407:Cilostazol; D000242:Cyclic AMP / Q000378:metabolism*; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D003599:Cytoskeleton / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006490:Hemostatics / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D020105:Milrinone / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D015539:Platelet Activation / Q000187:drug effects / Q000502:physiology; D054645:Protein Phosphatase 1; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058925:Receptors, Purinergic P2Y12; D013777:Tetrazoles / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*
D000067956:Adenylyl Cyclase Inhibitors; D006490:Hemostatics; C548576:P2RY12 protein, human; D010726:Phosphodiesterase Inhibitors; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12; D013777:Tetrazoles; D000242:Cyclic AMP; D051057:Proto-Oncogene Proteins c-akt; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517287:PDE3A protein, human; D013917:Thrombin; D020105:Milrinone; D000077407:Cilostazol; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine