OP13 The hydrogen sulfide pathway contributes to the enhanced human platelet aggregation in hyperhomocysteinemia

Abstract

Hyperhomocysteinemia is a risk factor for neurovascular and cardiovascular disease associated to endothelial dysfunction and accelerated atherosclerosis [1] , [2] , [3] . Many clinical and epidemiological studies have demonstrated a positive correlation among homocysteine (Hcy) plasma levels and cardiovascular disorders [4] leading to the general conclusion that Hcy is a pro-thrombotic factor [4] , [5] . Hcy is metabolized to methionine by the action of 5,10 methylenetetrahydrofolate reductase (MTHFR) [6] . Alternatively, by the transulfuration pathway, homocysteine is transformed to hydrogen sulfide (H 2 S), through multiple steps involving cystathionine β -synthase (CBS) and cystathionine γ -lyase (CSE) [7] . To date, the influence of H 2 S on platelet function has been poorly explored. Here we have evaluated the involvement of H 2 S in platelet reactivity by using platelets harvested from healthy volunteers or patients firstly diagnosed with hyperhomocysteinemia (MTHFR++ carriers). Sodium hydrogen sulfide (NaHS) or l -cysteine were used as exogenous or endogenous source of H 2 S, respectively. NaHS (0.1–100 μM) or l -cysteine (0.1–100 μM) did not cause per se platelet aggregation. However both NaHS and l -cysteine significantly increased platelet aggregation induced by the thrombin receptor activator peptide-6 amide (TRAP-6, 2 μM) in a concentration-dependent manner in platelets harvested from healthy volunteers. When platelets harvested from MTHFR++ carriers were used platelet aggregation was further potentiated. The potentiating effect present in this latter group was reverted by the inhibition of either CBS or CSE. Therefore the l -cysteine/H 2 S pathway is involved in the increased responsivity in MTHFR++ carriers. The role played by this pathway is further supported by the finding that H 2 S levels were significantly higher in both platelets and plasma. To gain further insights into the downstream signaling we evaluated the involvement of thromboxaneA 2 (TXA 2 ). TXA 2 levels were markedly increased in response to both NaHS or l -cysteine in platelets of healthy volunteers. Inhibition of phospholipase A 2 , cyclooxygenase or blockade of thromboxane receptor markedly reduced TXA 2 production triggered by H 2 S. These results confirmed that H 2 S triggers the arachidonic acid cascade and that the main downstream signal is thromboxane. Since phospholipase A 2 is the rate limiting enzyme of the arachidonic cascade we evaluated if H 2 S can activate phospholipase A 2 by measuring its phosphorylation. Phospholipase A 2 phosphorylation was significantly higher in MTHFR++ carriers when compared to healthy volunteers. Our data suggest that in MTHFR++ carriers there is an increase in platelet activity dependent upon H 2 S generated within the platelet that boosts the arachidonic acid cascade. Therefore in MTHFR++ carriers the activation of the l -cysteine/H 2 S pathway within the platelets primes the cells making them more responsive to endogenous stimuli that normally do not activate healthy platelets.