Endothelial cell-derived prostacyclin and nitric oxide (NO) represent major platelet inhibitors, which increase the level of the platelet second messengers cAMP and cGMP, respectively. Elevated cAMP and cGMP levels regulate specific effector systems in platelets such as certain phosphodiesterases (PDEs) and cAMP- and cGMP-dependent protein kinases A (PKA) and G (PKG), which phosphorylate multiple substrates involved in platelet inhibition [1, 2]. However, the signaling and functional targets of cAMP/PKA and cGMP/PKG pathways in platelets are incompletely understood. 1) In collaboration with the ISAS in Dortmund, a first quantitative phosphoproteome after selective cGMP/PKG stimulation in human platelets in comparison to the cAMP/PKA phosphoproteome has been established and will be submitted in time for publication with Elena Kumm as first author (shared). Here, some new interesting targets of PKG and PKA have been identified in resting platelets and in response to convulxin (GPVI) and thrombin (PAR1/4, GPIbα) by mass spectrometry, which are aimed to be validated by immunoblotting/phostag technology and functional analysis (WP1). These validation experiments are important and expected to complete the revision of this paper for acceptance. 2) Within my PhD study and a subsequent BMBF-funded CTH-TRP project, I characterized the cAMP-regulated phosphoproteins of the ARPP19 family, α-endosulfine (ENSA) and ARPP19, as new PKA/PKG targets in human platelets. In nucleated cells these proteins are well-known to control the cell cycle together with the Ser/Thr kinase MAST(L) and the Ser/Thr phosphatase PP2A via inhibition of PP2A [3, 4]. In anucleate platelets without cell cycle I identified the MAST(L)-ENSA/ARPP19-PP2A pathway as novel signaling check-point . Treatment of human platelets with cAMP- and cGMP-elevating agents on the one hand induced phosphorylation of the regulatory B56δ-subunit of PP2A, suggesting increased PP2A activity and protein dephosphorylation, and on the other hand increased ENSA S109/ARPP19 S104 phosphorylation, which might be involved in down-regulation of another very important phosphorylation site of ENSA S67/ARPP19 S62, thereby inhibiting platelet PP2A. Global PP2A inhibition in human platelets by low dose okadaic acid increased ENSA S67/ARPP19 S62 phosphorylation, indicating the presence of MASTL or a related protein kinase in human platelets. Furthermore, okadaic acid increased phosphorylation of platelet PP2A downstream effectors such as VASP, Akt and the MAP kinases p38 and Erk1/2, but diminished thrombin-induced platelet activation. Interestingly, while only pS62 ARPP19 inhibited platelet PP2A, both recombinant pS62 as well as pS104 ARPP19 served as substrates for PP2A in platelet lysates, which is in line with the concept of ‘inhibition by unfair competition’. Preliminary results of in vitro studies with mouse platelets lacking PP2A-B56δ (one important PP2A subtype of at least 14 different PP2As) revealed increased platelet adhesion, aggregation and thrombus formation on a collagen surface under arterial flow (collaboration with J. Heemskerk, CARIM, Maastricht) and enhanced platelet integrin αIIbβ3 activation and α-granule release in response to thrombin compared to wild-type platelets. These results indicate that PP2A-B56δ plays a role in the regulation of platelet reactivity. Using PP2A-B56δ-deficient and wild-type platelets, we aim in WP2 to elucidate whether 2PP2A-B56δ modulates the phosphorylation pattern of recombinant ENSA/ARPP19 in mouse platelets by immunoblotting in vitro. To investigate the impact of PP2A-B56δ on platelet adhesion, aggregation and thrombus formation in vivo, the carotid artery thrombosis model will be analyzed in PP2A-B56δ-deficient vs. wild-type mice in collaboration with C. Reinhardt (CTH-platform ‘Intravital Microscopy’). These data will be important to strengthen a planned paper addressing the role of the PP2A-B56δ pathway in mouse platelets and crosstalk with cAMP/PKA and cGMP/PKG signaling, which will be essential to obtain future grant support for this research area.