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    • Ki16425 In human platelets only and

    2022-06-13

    In human platelets, only α1 and β1 isoforms of sGC are expressed in equimolar concentrations (3700 copies for β1, and 3500 copies for α1 per platelet) [57] [62]. sGC, is usually a heterodimer and contains heme as cofactor which is required for high-affinity NO binding and stimulation of its activity [63,64]. In platelets, sGC is mainly activated by endothelial cell derived NO [65], exogenously added NO donors, or direct sGC activators/stimulators [52,64]. Over many years, there was also confusing literature suggesting that platelets express NO-synthase (NOS) and produce themselves NO. As summarized in a recent review [66], the majority of publications in which platelet NOS activity and function are described, are based on unspecific detection of NOS proteins by Western blotting or proteomic techniques, and succumb to pitfalls with regard to cGMP and NOS activity measurements. However, NOS protein is not detectable in human or mouse platelets as performed by Western blotting [67,68], or by proteomic methods with human platelets [57] or murine platelets [59]. Furthermore, NOS mRNA is also not detectable in human and mouse platelets, neither by the genome-wide RNA-seq analysis [58] nor in platelet mRNA data banks (www.plateletomics.com). However, under certain conditions, sGC may be directly, NO-independently activated by various mechanisms such as protein-protein interaction with Hsp70, Hsp90, PSD95, or MyD88 (which recruits the sGC to the plasma membrane), by Ser/Thr and/or Tyr phosphorylation of sGC [67,[69], [70], [71]], by vWF [41], or by several compounds such as thrombin inhibitors [72], or gemfibrozil [73]. In platelets, sGC is not only “soluble” but can also, upon activation of platelets, be bound to plasma membranes which may affect its activity [74]. It has been well recognized that a diminished responsiveness of the NO/sGC/cGMP system is associated with many cardiovascular diseases, although the mechanisms are not fully understood [43,75,76]. It is therefore of considerable therapeutic importance that the sGC can also be NO-independently regulated by heme-independent sGC activators and heme-dependent sGC stimulators (drugs) [64]. Extensive development yielded the sGC stimulator riociguat, which has been clinically validated for therapy of pulmonary arterial Ki16425 and chronic thromboembolic pulmonary hypertension [51,77]. The vasodilating and antihypertensive effects of riociguat are mediated by stimulation of sGC and elevation of tissue cGMP levels, similar to the effects of nitrates/NO-donors.
    Regulation of platelet cGMP levels by phosphodiesterases In the human genome, twenty-one genes encode phosphodiesterases (PDEs) which belong to 11 PDE families. According to their specific activity, PDEs can be divided into three groups, one which specifically hydrolyzes only cAMP (PDEs 4, 7, and 8), one which specifically hydrolyzes cGMP (PDEs 5, 6, 9), and one which hydrolyzes both cAMP and cGMP (PDEs 1, 2, 3, 10, 11) [78]. Only PDE2A, PDE3A and PDE5A were identified in platelets by proteomic methods [57], Western blots [79], and PDE activity assays (rev. in Ref. [80]). PDE2A (cGMP-stimulated PDE) and PDE3A (cGMP-inhibited PDE) preferentially hydrolyze cAMP and are mainly responsible for the regulation of cAMP content in platelets, by exerting negative and positive effects, respectively, of cGMP on the cAMP pathway [[80], [81], [82]]. PDE2 and PDE3 appear to play a minor, if any, role in regulation of platelet cGMP content [83,84]. Decrease of cAMP content in thrombin-stimulated platelets is mediated by PKC- and PKB-dependent phosphorylation of PDE3 [85,86], but not by increase of cGMP and stimulation of PDE2 activity [87]. After stimulation of platelets with NO donors, the elevated cGMP concentration rapidly decreases to near basal level due to strong activation of PDE5, which correlates with PDE5 phosphorylation by PKG [28]. In contrast, cAMP concentration, after stimulation with the prostacyclin mimetic iloprost or forskolin, reaches a plateau and does not decrease then for the next 20 min [79]. Understanding the mechanisms of PDE5 inhibitors on platelet functions became important when selective PDE5 inhibitors such as sildenafil were established as drugs for the treatment of pulmonary hypertension and male erectile dysfunction. This is clinically relevant since there are reports of bleeding disorders after sildenafil treatment of men [88]. In NO donor-stimulated platelets, but not ones under resting conditions, PDE5 was shown to be associated with PKG and inositol-1, 4, 5-trisphosphate receptor type 1 (IP3R1). Thus, PDE5, under certain conditions, might be involved in compartmentalization of cGMP signaling [29].