Analysis of nitric oxide-sensitive guanylyl cyclase in human platelets before and after aggregation

Abstract

Nitric oxide (NO) inhibits platelet adhesion to vascular endothelium and platelet aggregation through activation of soluble guanylyl cyclase (sGC) and a consequent increase in cGMP. The aim of the present study was to analyze NO-sensitive sGC in human platelets before and after aggregation. NO-sensitive sGC activity was tested in the cytosol and membrane fractions of native human platelets and ADP-induced platelet aggregates in the presence of 3 mM Mn²⁺ as cofactor. After ADP-induced platelet aggregation there was a significant increase of sGC activity in membranes. Western blot analysis showed a partial translocation of the enzyme to the plasma membrane. These findings support recent data that sGC is associated with cellular membranes in various tissues and cell types and that this membrane association is influenced by the activation state in human platelets (Nat Cell Biol 2002; 4: 307–11). Using 3 mM Mg²⁺ instead of Mn²⁺ as cofactor, a sharp decrease of sGC activity was apparent in the cytosol of aggregated platelets. Kinetic analysis of the cytosolic enzyme and concentration–response curves for free Mg²⁺ showed that platelet aggregation changes binding of free Mg²⁺ but not binding of the substrate complex Mg·GTP. This effect was specific for free Mg²⁺ and was not seen for free Mn²⁺. In addition, changes in free Mg²⁺ concentration in a physiological range markedly influenced NO-stimulated sGC activity. This provides a possible explanation for the increased platelet aggregability in patients with low intraplatelet Mg²⁺ levels.