Nitrous anhydrase activity of carbonic anhydrase II: cysteine is required for nitric oxide (NO) dependent phosphorylation of VASP in human platelets

Abstract

The carbonic anhydrase (CA) family does not only catalyse the reversible hydration of CO 2 to bicarbonate, but it also possesses esterase and phosphatase activity. Recently, bovine CA II and human CA II have been reported to convert inorganic nitrite (O¼N-O À ) to nitric oxide (NO) and nitrous anhydride (N 2 O 3 ). Given the ability of NO to mediate vasodilation and inhibit platelet aggregation, this CA II activity would represent a bioactivation of nitrite. There are contradictory reports in the literature and the physiological role of CA II nitrite bioactivation is still disputed. Here, we provide new experimental data in support of the nitrous anhydrase activity of CA II and the key role L-cysteine in the bioactivation of nitrite by CA II. Using washed human platelets and by measuring VASP phosphorylation we provide evidence that exogenous nitrite (10 mM) is bioactivated to NO in a manner strongly depending on L-cysteine (100 and 200 mM). The process is not inhibitable by acetazolamide, a potent CA inhibitor. The contradictory results of recently published studies in this area are thoroughly discussed.

Extra-platelet low-molecular-mass thiols mediate the inhibitory action of S-nitrosoalbumin on human platelet aggregation via S-transnitrosylation of the platelet surface

“…CysSH, GSH and Cu 2+ are of particular importance in the Wilson’s disease; treatment with the synthetic thiol penicillamine (chelator) and exogenous Zn 2+ (substitution of Cu 2+ from its stores) have favorable effects on this disease (Farinati et al 2003 ). CysSH, GSH, Zn 2+ and Cu 2+ also play a key role in the nitrous anhydrase activity of carbonic anhydrase which is associated with formation of N 2 O 3 , GSNO, CysSNO and NO (Hanff et al 2016 , 2018 ; Zinke et al 2016 ; Tsikas 2021 ; Tsikas and Gambaryan 2021 ). Intra-platelet carbonic anhydrase seems to be of significance in the pharmacological nitrite-dependent inhibition of platelet aggregation via S -transnitrosylation reactions (Tsikas and Gambaryan 2021 ).…”

“…CysSH, GSH, Zn 2+ and Cu 2+ also play a key role in the nitrous anhydrase activity of carbonic anhydrase which is associated with formation of N 2 O 3 , GSNO, CysSNO and NO (Hanff et al 2016 , 2018 ; Zinke et al 2016 ; Tsikas 2021 ; Tsikas and Gambaryan 2021 ). Intra-platelet carbonic anhydrase seems to be of significance in the pharmacological nitrite-dependent inhibition of platelet aggregation via S -transnitrosylation reactions (Tsikas and Gambaryan 2021 ). Yet, involvement and importance of carbonic anhydrase and its PTM including S -transnitrosylation in the Wilson’s disease have been sparingly investigated thus far (Di Fiore et al 2020 ).…”

The Role of NO/sGC/cGMP/PKG Signaling Pathway in Regulation of Platelet Function

“…sGC is strongly activated in washed human platelets incubated with recombinant bovine CA2 together with nitrite [ 83 ], whereas others showed that human, bovine, or mouse CA2 does not regulate nitrite-dependent NO formation in blood [ 85 ]. Recently in experiments with washed human platelets, we showed that cysteine is required for nitrite-dependent sGC activation, which was independent from CA inhibitor [ 86 ], but this assumption needs future experimental confirmations. Certainly, different experimental conditions including washed platelets, whole blood, or animal/human experiments could lead to different and even controversial results, but this does not call into question that nitrate/nitrite/NO pathway plays a beneficial role in the cardiovascular system and in regulation of platelet reactivity.…”

Exploring the carbonic anhydrase-mimetic [(PMDTA)2ZnII2(OH−)2]2+ for nitric oxide monooxygenation

“…1 Recent reports on the CA enzyme showed its nitrous anhydrase activity in converting NO 2 − to NO and NO 2 ˙ via N 2 O 3 formation. 26 Besides CA-induced NO 2 − reduction, Warren and coworkers reported the reduction of Zn II -bound NO 2 − -incorporating thiol. 27 Kundu and coworkers recently reported the reduction of M–NO 2 − (M = Co or Zn) species to NO upon reaction with an ene-diol.…”