Hydrogen sulphide enhances human platelet aggregation through phospholipase A2 activation

E Mitidieri1, R d\'Emmanuele di Villa Bianca1, N Kirkby3,2, TD Warner2, G Cirino1, R Sorrentino1. 1University of Naples, Dept. Experimental Pharmacology, 80131, Italy, 2Bart\'s and The London SMD, William Harvey Reserch Institute, EC1M 6BQ, UK, 3Imperial College, National Heart and Lung Institute, SW7 2AZ, UK

Hydrogen sulphide (H2S) is the third gaseous mediator alongside with nitric oxide and carbon monoxide. It is endogenously produced in various mammalian tissues from L-cysteine (L-cys) by the action of cysthationine-β-synthase (CBS) and/or cysthationine-γ-lyase (CSE), which are expressed in tissue specific fashion1. As a biologically diffusible mediator, H2S exerts physiological and pathological effects in many organs and systems. Several evidences show the H2S involvement in the regulation of vascular homeostasis2, including vascular smooth muscle cell relaxation and constriction3, pro- and anti-inflammatory actions4, modification of apoptosis5, promotion of angiogenesis6. To date, evidences for the role(s) of H2S in platelets is still lacking. Therefore, we evaluated the effect of H2S on human platelets investigating its underlying mechanism. Human washed platelets were collected from healthy volunteers. The expression of both CBS and CSE was evaluated by western blot analysis. H2S levels were measured by a colorimetric assay for CBS and CSE activity. Traditional light transmission aggregometry technique was used to analyse platelet aggregation; H2S-induced effect was evaluated using both an exogenous source of H2S (NaHS, 0.1 µM–1 mM) and the metabolic precursor (L-cys, 0.1 µM–1 mM) on thrombin receptor activator peptide 6 amide (TRAP-6, 2 µM) stimulus. In order to assess the H2S mechanism(s) we operated a pharmacological modulation by using specific inhibitor of arachidonic acid cascade i.e. 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. In addition, thromboxane (TXA2) and cAMP levels were evaluated. Our results clearly showed that human washed platelets possess both CBS and CSE and generate detectable amounts of H2S, significantly increased after incubation with L-cys (n=6, 1,43c0,09 vs 0,40±0,04 nmol/mg protein/min, p<0.001). Neither L-cys nor NaHS (0.1 µM–1 mM) affected human washed platelets in resting conditions (n=10) but, both significantly increased aggregation induced by TRAP-6 [n=15, p<0.001 L-cys 0.1 mM (62.06±1%) and NaHS 0.1 mM (61,7±1.0%) NaHS 10 µM (59,5±1.3%) and p<0.01 NaHS 1 µM (57,2±1,6%) and p<0.05 for L-cys 10 µM (56,9±1,8%).vs TRAP 6 (50,9±1%). H2S did not affect platelet cAMP levels (n=5). On the other hand, INDO, AACOCF3 and SQ29548 blocked the potentiating effect of H2S on platelet aggregation (n=8). Interestingly, both NaHS (0.1mM) and L-cys (0.01mM) induced an increase in TXA2 production compared to TRAP-6 (n=10, p<0.01, 60,5±6,9ng/ml and 60,5±10,0 ng/ml respectively vs 24,2±10,01 ng/ml). One way anova or t-student tests were performed as needed. In conclusion, our data suggest that H2S is endogenously produced within human platelets and it is involved in platelet aggregation through PLA2 activation. These findings may highlight new targets for the development of interventions in platelet dependent disorders.

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