apexbt dilution calculator br All platelet agonists acting t

All platelet agonists, acting through specific receptors and triggering different signaling pathways, activate phospholipases (PLCß or PLCγ) and the subsequent messengers inositol 1,4,5-trisphosphate (IP3) and 1,2 diacylglycerol which activate inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) or protein kinases C (PKC), respectively [[11], [12], [13], [14]]. IP3 receptors increase intracellular Ca2+-concentration [iCa2+] by release of Ca2+ from intracellular stores. Regulation of platelet [iCa2+] is a multifunctional process that includes systems responsible for the influx of Ca2+ inside the cell, extrusion of Ca2+ through plasma membrane, release of Ca2+ from intracellular stores, and back transport to those stores. Two major sources of [iCa2+] increase are Ca2+ release from intracellular stores and influx through the plasma membrane. Conversely, two counteracting systems, including sarcoplasmic/endoplasmic Ca2+ ATPases (SERCAs), which pump Ca2+ back into intracellular stores, and plasma membrane Ca2+ ATPases (PMCAs) which pump it out of cells, have been identified in platelets [104]. It has been known for a long time that cGMP-elevating agents inhibit platelet agonist (thrombin, TXA2)-activation of phospholipase Cß, generation of IP3 and 1,2 diacylglycerol, and subsequent PKC activation and rise of [iCa2+] [19,105,106], although the mechanisms remained unclear for many years. It now seems likely that cGMP-elevating agents inhibit platelet action at several sites, as also suggested by our ongoing phosphoproteomic studies. The possible inhibitory effects via apexbt dilution calculator of the TXA2 receptor (S329/S331) and the RGS18 protein (↓pS49, ↓pS218; ↑pS216) were already mentioned (see above). cGMP inhibition of Gq signaling via RGS18 is an important pathway which blocks the activation of PLCß by three important platelet agonists. Regulation (inhibition) and phosphorylation of PLCß3 at S1105 by PKA and PKG has been reported in vitro [107,108], but this has not been followed up in more detail. In our studies, significant PLCß3 phosphorylation at S1105 was observed. Overall, platelet agonist-stimulated PLCß activation is strongly inhibited by the cGMP pathway. Activation of both PKA and PKG in human platelets strongly inhibited release of Ca2+ from intracellular stores [109,110]. The inhibitory effects of cGMP, but not cAMP, on Ca2+-release were specifically abolished in PKG-deficient human platelets [94]. This indicated that the intracellular membrane system (endoplasmic reticulum-derived dense tubular system (DTS) in platelets) is a possible PKG target. The inositol 1, 4, 5-trisphosphate (IP3) receptors (IP3R) of this complex were then identified as substrates for both PKA and PKG [111,112], and this phosphorylation inhibited IP3R-induced Ca2+-release from intracellular stores in platelets [113]. IP3 receptor-associated cGK I substrate protein (IRAG) was the second identified PKG substrate in platelet Ca2+ signaling. IRAG copurified together with PKG I and the IP3 receptor from smooth muscle cells and was phosphorylated by PKG at S664 and S677 [114]. Further studies with IRAG-deficient murine platelets indicated that phosphorylation of IRAG by PKG inhibits agonist-induced Ca2+ mobilization and platelet activation [115,116]. The IP3 receptor not only forms a complex with IRAG, PKG Iß [117], and PDE5 [29], but also serves as a binding partner for many other regulatory proteins [118], forming a “signaling hub” at an essential site of intracellular regulation. It is remarkable that this signaling hub is phosphorylated at multiple sites on multiple proteins by the NO/cGMP pathway in human platelets. Ca2+-release from intracellular stores stimulates store-operated Ca2+ entry (SOCE) through the plasma membrane. Several proteins including stromal interaction molecule 1 (STIM1), Ca2+-release activated calcium modulator 1 (CRACM1 or Orai1), and members of transient receptor potential channels (TRPC) are involved in Ca2+-entry in platelets through the plasma membrane [104]. To date there is no indication in the literature that functions of STIM1 or Orai1 could be modulated by PKG activation, whereas TRPCs are established PKG substrates with identified phosphorylation sites at T11 and S263 [119]. However, the question of whether phosphorylation of TRPC by PKG in platelets is involved in regulation of Ca2+-transport through the plasma membrane still remains open [104,120]. Activity of another well-established Ca2+-channel, the ATP receptor P2X1, is most probably also not regulated by PKG. At least PKG activation does not inhibit P2X1-mediated platelet shape change triggered by the activation of this Ca2+-channel [52]. There are also no data concerning regulation of SERCAs and PMCAs functions in platelets by PKG.