Protein Kinase C-mediated Phosphorylation and Activation of PDE3A Regulate cAMP Levels in Human Platelets

Abstract

The elevation of [cAMP] i is an important mechanism of platelet inhibition and is regulated by the opposing activity of adenylyl cyclase and phosphodiesterase (PDE). In this study, we demonstrate that a variety of platelet agonists, including thrombin, significantly enhance the activity of PDE3A in a phosphorylation-dependent manner. Upon vascular injury, platelets adhere to the newly exposed subintimal collagen and undergo activation leading to platelet spreading to cover the damaged region and release of thrombogenic factors such as ADP and thromboxane A 2 . In addition, platelets are activated by thrombin, which is generated as a result of activation of the coagulation pathway, and stimulates platelets by cleaving the protease-activated receptors (PAR), 2 PAR-1 and PAR-4. The final common pathway is the exposure of fibrinogen binding sites on integrin ␣ IIb ␤ 3 resulting in platelet aggregation and thrombus formation.Thrombin-mediated cleavage of PARs leads to activation of phospholipase C ␤ (PLC), hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate and a subsequent increase in [Ca 2ϩ ] i and activation of protein kinase C (PKC). Protein kinase C contributes to platelet activation both directly, through affinity regulation of the fibrinogen receptor, integrin ␣ IIb ␤ 3 (1), and indirectly by enhancing degranulation (2). Thrombin also stimulates activation of PI 3-kinases and subsequent generation of PI (3, 4, 5) trisphosphate and PI (3, 4) bisphosphate (3), which recruit protein kinase B (PKB) to the plasma membrane where it becomes phosphorylated and activated.Platelet activation is opposed by agents that raise intracellular 3Ј-5Ј-cyclic adenosine monophosphate ([cAMP] i ). cAMP is a powerful inhibitory second messenger that down-regulates platelet function by interfering with Ca 2ϩ homeostasis, degranulation and integrin activation (4). Synthesis of cAMP is stimulated by mediators such as prostaglandin I 2 (PGI 2 ), which bind to G s -coupled receptors leading to activation of adenylate cyclase (AC). This inhibitory pathway is opposed by thrombin, which inhibits the elevation of cAMP indirectly via autocrine activation of the G i -coupled ADP receptor P2Y 12 . cAMP signaling is terminated by hydrolysis to biologically inert 5Ј-AMP by 3Ј-phosphodiesterases. Platelets express two cAMP phosphodiesterase isoforms, cGMP-stimulated PDE2 and cGMP-inhibited PDE3A. PDE3A is the most abundant isoform in platelets and has a ϳ250-fold lower K m for cAMP than PDE2 (4). As a consequence of these properties, PDE3A exerts a greater influence on cAMP homeostasis, particularly at resting levels. The importance of PDE3A in platelet function is further emphasized by the finding that the PDE3A inhibitors cilostamide and milrinone raise basal cAMP levels and strongly inhibit thrombin-induced platelet activation (5). Furthermore, PDE3A

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.…”

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

“…These data indicate that activating pathways rapidly phosphorylate and stimulate PDE3A to reduce cAMP levels below an inhibitory threshold whereas inhibitory PKA pathways may use PDE3A in a negative feedback loop. 14‐3‐3 binding to PDE3A might help to discriminate between activating and inhibitory pathways 71. Modelling of cyclic nucleotide levels confirms that PDEs are highly regulated exhibiting very low basal catalytic activity 72.…”

“…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. Stimulation of PDE3A by thrombin is mediated by protein kinase C (PKC) which phosphorylates serines 312, 428, 438, 465, and 492 of PDE3A 71. A similar phosphorylation pattern is induced by treatment of platelets with collagen‐related peptide or by U46619.…”

Translocation of PKC by Yessotoxin in an in Vitro Model of Alzheimer’s Disease with Improvement of Tau and β-Amyloid Pathology

“…However, later reports described that PDE inhibition induced by YTX, measured by electrochemical and colorimetric methods, 15 had no effect on cAMP levels in cardiomyocytes, 16 pointing to different effects depending on cell type and bringing up doubts about the specific role of YTX in PDE signaling. In platelets, it has been shown that PKC was engaged in the activation of PDE3 as happens with other human cellular models, 17,18 showing a link between these two enzymes. PDE modulators have been studied for their possible therapeutic effect in neurodegener-ative diseases.…”