Carbonic anhydrases are producers of S-nitrosothiols from inorganic nitrite and modulators of soluble guanylyl cyclase in human platelets

Nitric oxide (NO), S-nitrosoglutathione (GSNO) and S-nitrosocysteine are highly potent signaling molecules, acting both by cGMP-dependent and cGMP-independent mechanisms. The NO metabolite nitrite (NO2 (-)) is a major NO reservoir. Hemoglobin, xanthine oxidoreductase and carbonic anhydrase (CA) have been reported to reduce/convert nitrite to NO. We evaluated the role and the physiological importance of CA for an extra-platelet CA/nitrite/NO/cGMP pathway in human platelets. Authentic NO was analyzed by an NO-sensitive electrode. GSNO and GS(15)NO were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). cGMP was determined by LC-MS/MS or RIA. In reduced glutathione (GSH) containing aqueous buffer (pH 7.4), human and bovine erythrocytic CAII-mediated formation of GSNO from nitrite and GS(15)NO from N-15-nitrite. In the presence of l-cysteine and GSH, this reaction was accompanied by NO release. Incubation of nitrite with bovine erythrocytic CAII and recombinant soluble guanylyl cyclase resulted in cGMP formation. Upon incubation of nitrite with bovine erythrocytic CAII and washed human platelets, cGMP and P-VASP(S239) were formed in the platelets. This study provides the first evidence that extra-platelet nitrite and erythrocytic CAII may modulate platelet function in a cGMP-dependent manner. The new nitrite-dependent CA activity may be a general principle and explain the cardioprotective effects of inorganic nitrite in the vasculature. We propose that nitrous acid (ONOH) is the primary CA-catalyzed reaction product of nitrite.