Title

 

Authors

E. Mitidieri^1,2, R. d’Emmanuele di Villa Bianca¹_, N. Kirkby^2,3_, G. Cirino¹, R. Sorrentino¹, T.D. Warner<sup>2

1</sup>Dept of Experimental Pharmacology, University of Naples “Federico II”, Italy, ²Bart's and the London School of Medicine and Dentistry, William Harvey Research Institute, London, United Kingdom, ³National Heart and Lung Institute, Imperial College, London, United Kingdom

 

Abstract

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 PLA_­2 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.