br cAMP cGMP effector systems
cAMP/cGMP effector systems in human platelets (PKA, PKG, PDEs) In eukaryotic cells, the effects of intracellular cAMP are mediated by two types of cAMP-dependent protein kinases (PKA type I and II) and cAMP-regulated guanine nucleotide exchange factors (EPAC/cAMP-GEF) which activate the RAS GTPases Rap1 and Rap2  . Whereas platelet EPAC has not been detected so far, both major types of PKA with their catalytic (α,β) and regulatory subunits (Iα, Iβ, IIα, IIβ) are well expressed in human platelets, as quantitatively assessed by our proteomic studies . In comparison, the effects of cGMP are mediated in eukaryotes by cGMP-dependent protein kinases (PKG) I (α, β) and II, cGMP-gated cation channels and cGMP-regulated PDEs [19,27,31,91]. Human platelets express high levels of PKG Iß [57,92] and the cGMP-regulated/cGMP-binding phosphodiesterases PDE2A, PDE3A, and PDE5A [27,57,80]. The effects of cyclic nucleotides in human platelets are primarily mediated by cAMP- and cGMP-dependent protein kinases with an additional modulation of cAMP levels/PKA activity by cGMP via PDEs [31,32]. Elevation of platelet cAMP levels and PKA activation results in the inactivation of small G-proteins of the Ras and Rho families, inhibition of the release of Ca2+ from intracellular stores, and modulation of CHC mg cytoskeleton dynamics, which all lead to the inhibition of platelet adhesion, granule release and aggregation. Although several platelet PKA substrates such as GPIb, filamin-A, VASP, LASP, IP3 receptors, RGS18, and Rap1b have been well established [32,33] a recent phosphoproteomic approach indicated that there are many more PKA substrates (in fact more than 100) with a broad range of functions which need further validation . With respect to cGMP, functional studies with human and murine platelets which lacked cGMP-dependent protein kinases strongly support the concept that the major effects of cGMP in platelets are mediated by PKG [94,95]. Therefore, considerable efforts were undertaken to identify specific PKG substrates which may be important for mediating the effects of cGMP-elevating agents. As with the cAMP/PKA system, novel phosphoproteomic approaches permit a new depth of investigation (see below). But first, the present state of three selected areas with established cGMP-regulated functions in human platelets will be discussed.
Regulation of G-protein coupled receptor (GPCR) signaling by the sGC/cGMP/PKG pathway In platelets, thrombin, ADP, and TXA2 are the main agonists that activate G-protein coupled receptors (GPCRs) which initiate GPCR signaling by the exchange of GDP to GTP on Gα subunits. Regulators of G-protein signaling (RGS) act as GAPs by accelerating subsequent hydrolysis of GTP which facilitates reassociation of Gα to Gβγ and terminates the signaling . Among GPCRs in platelets, only the TXA2 receptor (TP) was shown to be phosphorylated by PKG, later also by PKA, at sites S331 and S329, respectively, which appeared to be involved in heterologous receptor desensitization in cell culture [, , , ]. In our recent iloprost/cAMP study with human platelets, we found that TP is phosphorylated by PKA at S329 , and in our ongoing studies with NO donors and riociguat, TP phosphorylation at both S329 and S331 was detected. Although this is potentially of considerable importance, further work is required to understand the significance. PKG is involved in inhibition of GPRC signaling by direct phosphorylation of RGS18. Of 37 genes encoding RGS proteins, human platelets express predominantly RGS10 and RGS18 [57,101]. Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein that turns off Gq signaling in platelets stimulated by thrombin, TXA2 or ADP. These platelet agonists stimulate the interaction of RGS18 and 14-3-3 by increasing the phosphorylation of S49 RGS18. 14-3-3 proteins are dimeric phospho-serine/threonine-binding adapter proteins and 14-3-3γ was shown to bind to the phosphorylated S49 and S218 sites of RGS18 . Increased binding of 14-3-3γ to RGS18 leads to attenuated RGS function and higher levels of active GTP-bound Gαq. Activation of PKA and PKG inhibits the interaction of RGS18 and 14-3-3γ by increasing RGS S216 phosphorylation, and concomitantly decreasing phosphorylation of the 14-3-3γ-binding sites S49 and S218. This results in the removal of 14-3-3γ, activating RGS18 to turn off Gq signaling, thus contributing to platelet inhibition as shown in Fig. 2 . Dephosphorylation of S49 and S218 may be mediated by protein phosphatase 1 (PP1) which is linked to RGS18 by the regulatory subunit PPP1R9B (spinophilin) [102,103].