P44 l-Cysteine/H2S pathway involvement in human platelet aggregation

Abstract

H2S is endogenously produced from l-cysteine (L-cys) by the action of three key enzymes cysthationine β-synthase (CBS), cysthationine-γ-lyase (CSE) and the newly discovered 3-mercaptopyruvate sulfurtransferase (3-MST) [1], [2]. It is present in human blood at micromolar concentrations [3] and it is involved in the maintenance of cardiovascular homeostasis [4]. To date, the influence of H2S on platelets, which play a central role in blood homeostasis, has been poorly explored. Therefore, we aimed to evaluate the effect of H2S signaling in human platelets. Human washed platelets were collected from 15 healthy volunteers. The expression of both CBS, CSE and 3-MST was evaluated by Western blot analysis. The enzymes activity was evaluated through H2S measurement by a colorimetric assay [5]. Light transmission aggregometry technique was used to analyze platelet aggregation. H2S-induced effect was evaluated using both an exogenous source of H2S, sodium hydrogen sulphide (NaHS, 0.1 μM–10 mM) and the metabolic precursor, L-cys (0.1 μM–10 mM) on thrombin receptor activator peptide 6 amide (TRAP-6, 2 μM) stimulus. We operated a pharmacological modulation by using specific inhibitors of arachidonic acid cascade, the main pathway involved in platelet function. Indomethacin (INDO, 10 μM, 15 min), a COX inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3, 1 μM, 15 min), a phospholipase A2 (PLA2) inhibitor or SQ29548 (1 μM), a thromboxane receptor antagonist were used. In addition, thromboxane (TXA2) and cAMP levels were evaluated. Human washed platelets expressed CBS, CSE and 3-MST and generated detectable amounts of H2S. Incubation with L-cys significantly increased H2S release (p < 0.001). Neither L-cys nor NaHS (0.1 μM–10 mM) affected human washed platelets in resting conditions, but both significantly increased TRAP-6-induced aggregation (p < 0.001 for L-cys 0.1 mM and NaHS 0.1 mM and 10 μM; p < 0.01 for NaHS 1 μM and p < 0.05 for L-cys 10 μM). Besides, H2S did not modify platelet cAMP levels. Conversely, INDO, AACOCF3 and SQ29548 blocked the potentiating effect of H2S on platelet aggregation induced by TRAP-6. Interestingly, both NaHS and L-cys induced a significantly increase in TXA2production (p < 0.01). Our data imply that the H2S endogenously produced within human platelets is involved in platelet aggregation through PLA2 activation. These findings may open a new pharmacological approache in platelet dependent disorders.