Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04

    • Of special interest is that riociguat and NO donors

    2021-10-16

    Of special interest is that riociguat and NO donors decreased the phosphorylation of many proteins in human platelets. This aspect is presently under further investigation but a few comments can be made already here. Of the extensive spectrum and diversity of human protein kinases and protein phosphatases [152] essentially all major p38 inhibitor of both protein kinases and phosphatases are well expressed in human platelets [57]. With both kinases and phosphatases present, the steady-state phosphorylation of proteins principally represents an equilibrium of phosphorylation (mediated by responsible protein kinases) and dephosphorylation (mediated by protein phosphatases). NO/riociguat could decrease the phosphorylation of certain proteins via inhibition at the level of the responsible protein kinase(s) or inhibition of pathways which activate this/these kinases (for example protein kinase C, Ca2+/calmodulin-dependent protein kinases). As discussed in sections 6 Regulation of G-protein coupled receptor (GPCR) signaling by the sGC/cGMP/PKG pathway, 7 sGC/cGMP/PKG pathway in platelet Ca, the NO/ricociguat/cGMP system is a powerful inhibitory pathway for the platelet-agonist-induced increase of IP3, 1,2 diacylglycerol and [iCa2+] which are required for increased PKC and MLCK (gene name MYLK) activity. Inhibition of PKC and MLCK activation by cGMP in platelets was originally observed many years ago [105,106] and has been discussed in recent reviews [31,32]. In addition to this indirect inhibition of protein kinases by cGMP/PKG, MLCK was also shown to be directly phosphorylated by PKG (and also PKA) resulting in MLCK inhibition [153]. In our recent full riociguat phosphoproteome study with human platelets, MLCK phosphorylation at several distinct kinase sites known to be important for kinase inhibition, was observed (data to be published). Both serine/threonine and tyrosine protein phosphatases are also well expressed in human platelets [57], but in comparison to protein kinases, much less is known about their specific function and regulation there [23,32]. In our previous cAMP/PKA phosphoproteomic studies [93,150] and our present riociguat/cGMP data (see Table 1), we detected α-endosulfine (ENSA) as a substrate (S109) for both PKA and PKG in human platelets. ENSA, when phosphorylated at another site (S67), is a potent inhibitor of protein phosphatase 2a (PP2A) in other systems [154]. Since PP2A has a broad and important spectrum of substrates [155], both PP2A and its endogenous modulators such as ENSA most likely have an important regulatory role in platelets. This needs to be addressed in future investigations. As already briefly mentioned in section 8, a complex stimulatory role of cGMP/PKG for the myosin light chain phosphatase (MLCP) has been reported for human platelets [124]. Phosphorylation of RhoA by PKG prevented its association with Rho kinase (ROCK), resulting in activation of myosin light chain phosphatase (MLCP), decrease of MLC phosphorylation, and inhibition of platelet shape change. These two examples (PP2A/ENSA and MLC/MLCP/MLCK) illustrate the important role of protein phosphatases in the regulation of major signaling pathways in platelets, and also identify protein phosphatases as targets of cGMP/PKG action. In human platelets, more than 10 PP2A subunits are expressed, 2 distinct catalytic subunits C, 1 scaffolding subunit A and more than 7 different regulatory subunits B [57]. Due to the structural and functional diversity of protein phosphatases, our detailed understanding of their intracellular regulation is still limited. Currently, we are completing the full analysis of this riociguat/cGMP phosphoproteome of human platelets, which will also be compared with the cAMP phosphoproteome evoked by iloprost. Published data, and our ongoing phosphoproteomic studies discussed here, show that the sGC stimulators (NO donors and riociguat) affect multiple proteins and pathways via cGMP-regulated phosphorylation in human platelets. In particular membrane proteins, G-proteins and their regulators, signaling molecules, protein kinases, and proteins involved in Ca2+ regulation are sGC/cGMP targets, which often interact with or regulate additional platelet proteins. It appears likely that the sGC/cGMP pathway regulates, and primarily inhibits, a variety of platelet functions by targeting networks of multiple proteins, rather than a few isolated PKG substrates.