Molecular mechanisms of von Willebrand-factor (VWF)-stimulated, NO-independent acitivation of soluble guanylyl cyclase (sGC) in human platelets

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Platelets, specialized adhesive cells, play a key role in normal and pathological haemostasis through their ability to rapidly adhere to subendothelial matrix proteins (adhesion) and to other activated platelets (aggregation), functions inhibited by NO/cGMP/PKG signalling pathways. Recently, we addressed a controversial issue in this field. We discovered that human and murine platelets do not express any type of functional NOS proteins and that platelet soluble guanylyl cyclase (sGC) is NOS-independently activated by von Willebrand factor (VWF). This may represent a new mechanism of feedback inhibition. The main goal of this new project is to prove our hypothesis that NO-independent sGC activation by VWF and GPIb-IX-V signalling represents a novel inhibitory platelet feedback mechanism. We also want to elucidate the molecular mechanisms and posttranslational modifications involved in platelet sGC regulation. Using our recently established quantitative phosphoproteomic approach and immunoprecipitation (IP) of sGC we will elucidate the phosphorylation status of sGC and characterize the sGC protein complexes upon platelet stimulation by VWF. Generation of GC mutants will help to analyze the role of specific phosphorylation sites in regulation of sGC activity in model systems. Finally, we will use different flow models to establish conditions for maximal NO-independent activation of platelet sGC.