Thrombin regulates intracellular cyclic AMP concentration in human platelets through phosphorylation/activation of phosphodiesterase 3A

Abstract

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, cAMPdependent 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. Thrombininduced PDE3A activation was diminished concomitantly with dephosphorylation of PDE3A by protein phosphatase 1 (PP1). An Akt inhibitor blocked PDE3A activation and constrained thrombininduced 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.

Thrombin and Collagen Induce a Feedback Inhibitory Signaling Pathway in Platelets Involving Dissociation of the Catalytic Subunit of Protein Kinase A from an NFκB-IκB Complex

“…For example, ADP and epinephrine induce G i -protein-dependent inhibition of adenylyl cyclases. Thrombin stimulation of platelets has been reported to decrease cAMP by PKB-and PKC-dependent phosphorylation and activation of PDE3A (25)(26). Our experiments also confirmed that thrombin decreases cAMP levels in platelets (Fig.…”

“…Increase of cGMP in thrombin-and collagen-stimulated platelets has been described in several publications (reviewed in Ref. 24), whereas cAMP has been shown to decrease in thrombin-stimulated platelets (25)(26). In our experiments, we could not detect any increase of cGMP in thrombin-or collagen-stimulated platelets (Fig.…”

Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin

“…Protease-activated receptor activation by thrombin both inhibits adenylate cyclase via G i (35) and activates PDE3, a cGMP-inhibited cAMP phosphodiesterase (36,37). The resulting decrease in cAMP impairs the function of signaling pathways involving both protein kinase A and Epac1.…”

Cyclic nucleotide‐dependent inhibitory signaling interweaves with activating pathways to determine platelet responses

“…PDE3A inhibition by cilostazol or JF959602 leads to increased cAMP levels and inhibits the initial accumulation of platelets at sites of injury as well as attachment and detachment of platelets from growing thrombi in vivo 70. Thrombin stimulates the enzymatic function of PDE3A resulting in reduced cAMP levels 11. Thus, costimulation of Gq and Gi during platelet activation leads to PDE3A activation and AC inhibition resulting in a pronounced decrease in intracellular cAMP levels.…”

“…For example, to prevent platelet activation, inhibitory signaling targets key nodes in activating pathways 7. On the other hand, platelet activation requires active blockage of endothelium‐dependent inhibitory pathways,8, 9, 10, 11 including ADP‐mediated inhibition of cAMP production via P2Y12 and thrombin‐mediated cAMP degradation via PDE type 3A. Interestingly, both P2Y12 and PDE3A are targets of multiple clinically used drugs which reduce thrombosis 12.…”