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    • In contrast to RhoA Rac

    2022-01-15

    In contrast to RhoA, Rac1 and Rap1 which are stimulated by platelet activators and inhibited by NO/PKG, the GTPase ADP-ribosylation factor 6 (Arf6) is regulated in the reverse way. Platelet agonists like thrombin, collagen, or ADP, reduce Arf6-GTP levels, whereas NO and PGI2 oppose this reduction [147]. The GEFs and GAPs regulating Arf6 in platelets are not known. Fig. 3 shows a model of the regulation of small G-proteins in human platelets.
    Phosphoproteomic approaches and future perspectives Since the original discovery that activation of the NO/cGMP pathway inhibits ADP-induced aggregation of human platelets [17], it has been clearly established that cGMP stimulation of PKG inhibits activation of human and murine platelets by various agonists at several distinct sites (Fig. 1, Fig. 2). However, the number of well-defined platelet PKG substrates is still rather low, and a systematic approach to investigate cGMP/PKG targets in platelets was needed. Over the last few years, we developed functional assays, platelet isolation procedures, and proteomic/phosphoproteomic methods, to study basal and regulated protein phosphorylation in human platelets [57,93,[148], [149], [150], [151]]. Using the stable prostacyclin analog iloprost as a specific activator of cAMP/PKA signaling in human platelets, our first comprehensive cAMP/PKA phosphoproteome quantified more than 2700 phosphorylation sites and demonstrated almost 300 iloprost/cAMP-regulated phosphoproteins. This study confirmed many of the previously-described platelet PKA substrates, but most of the observed phosphoproteins were identified as new cAMP/PKA targets which, based on gene ontology annotation, appeared to be richly associated with the pi3k inhibitors cytoskeleton, small GTPases, degranulation, vesicle-mediated transport, cell surface receptor signaling, Ras signaling, and platelet activation [93]. These successful cAMP/PKA studies prompted new ones with the goal of establishing the sGC/cGMP/PKG phosphoproteome in human platelets. We first compared the effects of NO donors often used with human platelets (S-nitrosocysteine, sodium nitroprusside, DEA-NO) and the sGC stimulator, riociguat, using our previously established conditions. Recently, we characterized riociguat in detail as a cGMP-specific inhibitor of murine and human platelets, as discussed in section 2 [52]. Specificity was based on several observations, including that all tested riociguat effects were abolished in murine platelets obtained from platelet-specific sGC knock-out mice. Secondly, in the absence of cAMP-elevating agents, riociguat did not affect cAMP levels and PKA substrates in both murine and human platelets. Thirdly, using established PKG substrates such as VASP (S239) and PDE5A (S102) as markers, we could show that riociguat-induced phosphorylation in human platelets was strongly decreased by the PKG inhibitor Rp-8-Br-PET-cGMPS (unpublished data). Furthermore, a broad and long-term experience with pre-clinical and clinical studies demonstrated that riociguat is chemically a relatively stable and selective stimulator of the sGC/cGMP system [51,63]. Therefore, for further phosphoproteomic experiments with riociguat, we selected conditions that produced maximal increase of platelet cGMP levels and phosphorylation of the PKG substrates VASP (S239) and PDE5A (S102), and used similar conditions for NO donors. In these experiments, riociguat and the three NO donors stimulated the phosphorylation of more than 150 proteins (>1.5 fold above control) and, interestingly, decreased the phosphorylation of more than 60 proteins (<0.66 below control). Table 1 demonstrates the top list of proteins with more than 4-fold riociguat (Rio)-stimulated phosphorylation, in comparison to the fold stimulation caused by three NO donors. Although there are some quantitative differences (riociguat evoked the strongest phosphorylation response in most cases), the pattern of response of all 4 sGC stimulators was generally similar. This list of NO- or riociguat-stimulated phosphoproteins contains several established platelet PKG substrates [VASP (S239), MRVI1/IRAG (S657/S670), RAP1GAP2 (S7), RASGRP2/CalDAG-GEFI (S587), and PLCß3 (S1105) [31,32]. Other established PKG substrates, such as PDE5A (S102) and IP3 receptor/ITPR1 (S1764), were also well detected (not shown here), but the increased phosphorylation was less than 4-fold. Whereas these established PKG substrates are important positive controls, the majority of NO- or riociguat-stimulated phosphorylation sites/phosphoproteins have not been previously identified as targets of the NO/cGMP pathway. A first analysis of these proteins (with the information provided by Uniprot) indicated that some proteins are protein kinases/phosphatases and their regulators (ENSA, CASS4, CDK16), others are small GTPases and their regulators (RAP1GAP2, RAPGEF2, RASGRP2), or are intracellular membrane/ER proteins (LRMP, MRVI, SSR3). Cell or peripheral membrane-associated proteins (CLDN5, PDE3A, TJP2, PIK3R6, TEX2) are also notable. Further analysis of all NO/riociguat regulated phosphoproteins shows that these belong to a broad spectrum of cell biological and/or functional classes, as we also observed with the iloprost/cAMP phosphoproteome [93].