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

Hyperhomocysteinemia is a risk factor for neurovascular and cardiovascular disease associated to endothelial dysfunction and accelerated atherosclerosis [1], [2] and [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] and [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 (H2S), through multiple steps involving cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) [7]. To date, the influence of H2S on platelet function has been poorly explored. Here we have evaluated the involvement of H2S 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 H2S, 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/H2S pathway is involved in the increased responsivity in MTHFR++ carriers. The role played by this pathway is further supported by the finding that H2S levels were significantly higher in both platelets and plasma. To gain further insights into the downstream signaling we evaluated the involvement of thromboxaneA2 (TXA2). TXA2 levels were markedly increased in response to both NaHS or l-cysteine in platelets of healthy volunteers. Inhibition of phospholipase A2, cyclooxygenase or blockade of thromboxane receptor markedly reduced TXA2 production triggered by H2S. These results confirmed that H2S triggers the arachidonic acid cascade and that the main downstream signal is thromboxane. Since phospholipase A2 is the rate limiting enzyme of the arachidonic cascade we evaluated if H2S can activate phospholipase A2 by measuring its phosphorylation. Phospholipase A2 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 H2S generated within the platelet that boosts the arachidonic acid cascade. Therefore in MTHFR++ carriers the activation of the l-cysteine/H2S pathway within the platelets primes the cells making them more responsive to endogenous stimuli that normally do not activate healthy platelets.