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      Differential regulation of platelet inhibition by cGMP- and cAMP-dependent protein kinases

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      1 , 2 ,   1 , 3 ,
      BMC Pharmacology & Toxicology
      BioMed Central
      6th International Conference on cGMP: Generators, Effectors and Therapeutic Implications
      28-30 June 2013

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          Abstract

          Background Under normal conditions circulating platelets are maintained in a resting state by the paracrine signaling molecules, prostacyclin (PGI2) and nitric oxide (NO), released from vascular endothelial cells. PGI2 and NO trigger the synthesis of cAMP and cGMP, respectively, which in turn leads to the activation of Protein Kinase A (PKA) and Protein Kinase G (PKG). These cyclic nucleotide-dependent protein kinases phosphorylate numerous substrate proteins involved in several aspects of platelet activation. Both, PKA and PKG can phosphorylate the same substrates including VASP, LASP, RAP1GAP2, Rap1B, HSP27, and some others. Only few substrates are known to be specific for PKA (PDE3) and PKG (PDE5) [1,2]. Here we report the identification and characterization of a new PKA specific substrate, CalDAG-GEFI (calcium and diacylglycerol regulated guanine nucleotide exchange factor I), a guanine exchange factor for Rap1b in platelets. Results Using radioactive phosphate incorporation assay, we identified S587 as the major PKA phosphorylation site in CalDAG-GEFI, which was confirmed by mass spectrometry. In platelets, PKA stimulation leads to strong phosphorylation of CalDAG-GEFI and inhibition of Rap1b activation triggered by thrombin, as well as calcium ionophore, which indicates a potential role of CalDAG-GEFI phosphorylation in the inhibition of Rap1b activation. However, PKG activation did not significantly phosphorylate CalDAG-GEFI, and inhibits thrombin-stimulated Rap1b by inhibition of calcium mobilization but did not inhibit calcium ionophore-triggered Rap1b activation. In HEK293 cells transfected with CalDAG-GEFI, calcium ionophore triggered Rap1b activation and forskolin stimulated PKA phosphorylation of CalDAG-GEFI leads to inhibition of Rap1b activation. But in cells with S587A mutant, PKA activation did not inhibit Rap1b activation. The phospho-mimetic (S587D) mutant failed to activate Rap1b following calcium ionophore stimulation. Conclusion These results confirmed that PKA phosphorylation of CalDAG-GEFI inhibits Rap1b activation. We propose that this is one of the important mechanisms of platelet inhibition mediated by cAMP/PKA signaling. Presented data show that PKA and PKG could utilize similar and/or diverse intracellular mechanisms involved in platelet inhibitory pathways.

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          Novel roles of cAMP/cGMP-dependent signaling in platelets.

          Endothelial prostacyclin and nitric oxide potently inhibit platelet functions. Prostacyclin and nitric oxide actions are mediated by platelet adenylyl and guanylyl cyclases, which synthesize cyclic AMP (cAMP) and cyclic GMP (cGMP), respectively. Cyclic nucleotides stimulate cAMP-dependent protein kinase (protein kinase A [PKA]I and PKAII) and cGMP-dependent protein kinase (protein kinase G [PKG]I) to phosphorylate a broad panel of substrate proteins. Substrate phosphorylation results in the inactivation of small G-proteins of the Ras and Rho families, inhibition of the release of Ca(2+) from intracellular stores, and modulation of actin cytoskeleton dynamics. Thus, PKA/PKG substrates translate prostacyclin and nitric oxide signals into a block of platelet adhesion, granule release, and aggregation. cAMP and cGMP are degraded by phosphodiesterases, which might restrict signaling to specific subcellular compartments. An emerging principle of cyclic nucleotide signaling in platelets is the high degree of interconnection between activating and cAMP/cGMP-dependent inhibitory signaling pathways at all levels, including cAMP/cGMP synthesis and breakdown, and PKA/PKG-mediated substrate phosphorylation. Furthermore, defects in cAMP/cGMP pathways might contribute to platelet hyperreactivity in cardiovascular disease. This article focuses on recent insights into the regulation of the cAMP/cGMP signaling network and on new targets of PKA and PKG in platelets. © 2011 International Society on Thrombosis and Haemostasis.
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            cGMP and cGMP-dependent protein kinase in platelets and blood cells.

            Platelets are specialized adhesive cells that play a key role in normal and pathological hemostasis through their ability to rapidly adhere to subendothelial matrix proteins (platelet adhesion) and to other activated platelets (platelet aggregation). NO plays a crucial role in preventing platelet adhesion and aggregation. In platelets, cGMP synthesis is catalyzed by sGC, whereas PDE2, PDE3 and PDE5 are responsible for cGMP degradation. Stimulation of cGK by cGMP leads to phosphorylation of multiple target substrates. These substrates inhibit elevation of intracellular calcium, integrin activation, cytoskeletal reorganization, and platelet granule secretion, events normally associated with platelet activation. The NO/cGMP pathway also plays a significant role in many other blood cell types in addition to platelets. In leukocytes, depending on the specific cell type, cGMP signaling regulates gene expression, differentiation, migration, cytokine production, and apoptosis.
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              Author and article information

              Contributors
              Conference
              BMC Pharmacol Toxicol
              BMC Pharmacol Toxicol
              BMC Pharmacology & Toxicology
              BioMed Central
              2050-6511
              2013
              29 August 2013
              : 14
              : Suppl 1
              : P69
              Affiliations
              [1 ]Institute of Clinical Biochemistry& Pathobiochemistry, University of Wuerzburg, Wuerzburg, Germany
              [2 ]Center for Thrombosis and Hemostasis (CTH), Johannes Gutenberg University, Mainz, Germany
              [3 ]Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia
              Article
              2050-6511-14-S1-P69
              10.1186/2050-6511-14-S1-P69
              3765619
              3c90930a-8af1-4119-94a8-53e47a0023b3
              Copyright ©2013 Subramanian et al; licensee BioMed Central Ltd.

              This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

              6th International Conference on cGMP: Generators, Effectors and Therapeutic Implications
              Erfurt, Germany
              28-30 June 2013
              History
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              Toxicology
              Toxicology

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