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  • Although it has been observed in numerous studies

    2022-08-04

    Although it has been observed in numerous studies and by many investigators that intraplatelet cGMP elevation results in platelet inhibition, there are a few papers which proposed a platelet stimulatory role for cGMP when elevated in response to NO donors or PDE5 inhibitors in mouse and human platelets [[37], [38], [39]]. However, these findings could not be confirmed by others [29,[40], [41], [42]]. Importantly, many cGMP-elevating drugs, including nitrovasodilators and PDE5-inhibitors such as dipyridamole and sildenafil, which ras inhibitor all stimulate the cGMP/PKG pathway, have been intensely studied and widely used clinically for treatment of different diseases [[43], [44], [45]]. Many of these clinical investigations also established that nitrates, in in vivo applications, inhibit platelet activation and elevate platelet cGMP levels [[46], [47], [48], [49]], although vasodilation is most likely the clinically predominant effect. A large meta-analysis of clinical trials with nitrates for the treatment of stable ras inhibitor documented the clinically known adverse effects of nitrates/NO-donors (e.g. headaches, postural hypotension, syncope), but an increase of prothrombotic events was not found [50]. We recently evaluated in vitro effects of riociguat on platelets in detail since a large body of pre-clinical and clinical studies established that the cellular effects of riociguat are mediated by sGC and cGMP [51]. With both human and murine platelets, riociguat alone specifically stimulated the sGC/cGMP pathway, including phosphorylation of the PKG substrate VASP, without any effect on cAMP levels. Furthermore, the riociguat-induced effects were absent in murine platelets from platelet-specific sGC knock-out animals. Under these conditions, riociguat specifically inhibited ADP-induced shape change, aggregation, secretion, and adhesion under flow conditions. However, functional antiplatelet effects of riociguat required higher concentrations in whole blood than with washed platelets. Such high riociguat plasma concentrations are not reached in riociguat-treated patients, suggesting that antiplatelet effects of riociguat under clinical conditions are therefore unlikely [52]. Our mechanistic studies with washed human platelets and platelet-rich plasma (PRP) established that the antiplatelet and phosphorylation effects of riociguat were mediated by sGC and cGMP [52]. Of particular interest was the clear demonstration that ADP-stimulated shape change, as quantitatively assessed by low-angle scattering, was inhibited by the sGC/cGMP pathway both in human and murine platelets [52,53]. Shape change is the first detectable response of platelets to agonists and is controlled by actin-myosin interactions, in particular by the balance of myosin light chain (MLC) phosphorylation, catalyzed by myosin light chain kinase (MLCK), and dephosphorylation, catalyzed by myosin light chain phosphatase (MLCP) [54,55]. Both MLC phosphorylation and dephosphorylation are targets of the NO/cGMP/PKG system with the net result of decreased myosin light chain phosphorylation and inhibition of myosin-actin dynamics which are crucial for shape change, integrin activation and granule secretion (Fig. 1).
    Regulation of platelet cGMP levels by guanylate cyclases In most mammalian cells, two enzyme families are responsible for cGMP synthesis, soluble guanylate cyclases (sGC) and particulate guanylate cyclases (pGCs). Whereas sGCs are heme-containing NO receptors (see below), pGCs are receptors for the natriuretic peptides ANP, BNP, and CNP [56]. In our recently published comprehensive and quantitative human platelet proteome [57], as well as in the mouse transcriptome and proteome reported by others [58,59], neither protein nor mRNA of any natriuretic peptide receptor were found. The absence of natriuretic peptide receptors in human platelets, as well as in the mouse platelet transcriptome and proteome [58,59], was also validated by further molecular and functional analyses [60]. Thus, sGC is therefore the only enzyme responsible for cGMP synthesis in human and murine platelets, which is also supported by studies of sGC-deficient cells [61].