Synergistic modulation of cAMP and cGMP rescues hemin-induced plasma membrane fragmentation

Hemin, which gets released during erythrocyte lysis, induces concentration-dependent platelet activation, aggregation, and thrombus formation as well as plasma membrane destruction and cytoskeleton reorganization. Classical platelet antagonists do not inhibit the plasma membrane destruction induced by high concentrations of hemin. Both cyclic guanosine monophosphate (cGMP) and adenosine 3':5' -cyclic monophosphate (cAMP) are key endogenous inhibitors of platelet activation. Thus, we investigated whether they inhibit hemin-induced plasma membrane destruction, both individually and in combination. In addition to standard platelet assays, we performed cGMP and cAMP ELISAs, immunoblot analysis of vasodilator-stimulated phosphoprotein (VASP) and immunofluorescence staining. We found that pharmacological modulation of these pathways via NO donors (DEA/NO), soluble guanylyl cyclase (sGC) stimulators (riociguat) or IP receptor agonists (PGE₁), and phosphodiesterase (PDE) inhibitors (PDE-5 inhibitor: sildenafil, PDE-3 inhibitor: ibudilast), phosphorylate the downstream vasodilator-stimulated phosphoprotein (VASP) and inhibit hemin-induced platelet activation and degranulation. In particular, synergistically modulation of cGMP and cAMP and the resulted phosphorylation of VASP at Ser239 and Ser157 significantly attenuates platelet aggregation and plasma membrane destruction induced by high concentrations of hemin. Further, the riociguat NO-induced cGMP synthesis was significantly reduced in the presence of hemin. In comparison, the PGE₁-induced cAMP synthesis was enhanced in the presence of hemin. In conclusion, high concentrations of hemin change the cGMP and cAMP synthesis induced by their associated stimulators, while promoting plasma membrane destruction, which can be significantly inhibited by the simultaneous administration of riociguat DEA/NO and PGE₁.