Hydrogen sulphide enhances human platelet aggregation through PLA2 activation

Hydrogen sulphide (H2S) is emerging as an important endogenous mediator produced in various mammalian tissues from L-Cysteine by the actions of cysthationine β-synthase (CBS) and/or cysthationine-γ-lyase (CSE), which are expressed in tissue specific fashion (Zhao et al. 2001). H2S exerts physiological and pathological effects in many organs and systems, but attention has been focused mainly on the cardiovascular system. It is now well established that H2S is involved in vascular homeostasis, for example regulating blood pressureand producing biphasic effects in the regulation of vascular tone (Yang et al., 2008; d’Emmanuele di Villa Bianca et al., 2011; Lim et al. 2008). Evidence for the role(s) of H2S in platelets is still lacking. Here, we first evaluated the effect of H2S on human platelets and then we investigated the possible pathways involved. We evaluated the presence of both CBS and CSE by western blot analysis, and used an H2S quantification assay to assess CBS and CSE activity. In order to look at influences on platelet reactivity, human washed platelets were collected from healthy volunteers; thrombin receptor activator peptide 6 amide (TRAP-6, 2µM) was used in presence or absence of sodium hydrogen sulphide (NaHS, 0.1 µM – 10 mM), as an H2S donor, or L-Cysteine, as an H2S precursor (0.1 µM – 10 mM). D-Cysteine was used as negative control. These experiments were repeated in the presence of aminoxyacetic acid (AOAA, 1 mM, 15 min) or propargylglycine (PGG, 1 mM, 15 min), CBS and CSE inhibitors, respectively. In order to assess possible mechanism(s) underlying the effects of H2S, we measured cAMP levels and evaluated the effects of either indomethacin (INDO, 10 µM, 15 min),a COX inhibitor, or arachidonyl trifluoromethyl ketone (AACOCF3, 1 µM, 15 min), a PLA­2 inhibitor. Western blot demonstrated the presence of both CBS and CSE in human platelets. L-Cysteine, but not D-Cysteine, significantly increased H2S production (p<0.001), and this effect was significantly reduced by either PGG or AOAA (p<0.001 and p<0.01, respectively). Neither L-Cysteine nor NaHS (both 0.1 µM – 10 mM) affected human washed platelets in resting conditions, but both significantly increased aggregations induced by TRAP-6 amide (p<0.001 for L-Cys 0.1 mM and NaHS 0.1 mM and 10 µM; p<0.05 for L-Cys 10 µM and NaHS 1 µM), effects that were completely abolished by pre-treatment with PGG or AOAA. H2S did not affect platelet cAMP levels. On the other hand , both INDO and AACOCF3 blocked the potentiating effects of H2S on platelet aggregation. In conclusion, our data suggest that H2S is endogenously produced within human platelets and is involved in platelet aggregation through PLA2activation. These findings may highlight new targets for the development of interventions in platelet dependent disorders. d’Emmanuele di Villa Bianca (2011) J Pharmacol Exp Ther (in press). Lim (2008)Am J Physiol Cell Physiol. 295,C1261-70. Yang (2008) Science 322, 587-90. Zhao (2001)The EMBO Journal20, 6008-16.