Inhibitory regulation of platelets by the gasotransmitter hydrogen sulfide

M Emerson1, Z Ilkan1, F Mustafa1, E Smyth1, G Apostoli1, A Solomon1, ME Wood2, M Whiteman2. 1Imperial College London, London, UK, 2University of Exeter, Exeter, UK

Introduction: Platelets are key drivers of acute cardiovascular events such as myocardial infarction. Hydrogen sulfide (H2S) is a gasotransmitter with emerging roles in cardiovascular biology although there is currently no consensus on the regulation of platelets by H2S. We aimed to define the role of H2S in regulating platelets and investigate the enzymatic source of H2S in platelets.

Methods: We determined the effect of a slow-releasing H2S compound GYY4137 (morpholin-4-ium 4 methoxyphenyl(morpholino) phosphinodithioate, Fig 1A) and synthesised and tested a “spent donor” control (Fig 1B) upon human platelet aggregation in vitro and in a mouse model of radiolabelled platelet thromboembolism which involved infusion of radiolabelled platelets into anaesthetised (urethane 25% w/v, 10 ml g-1) mice and subsequent real-time monitoring. We also assessed expression and catalytic activity of H2S-generating enzymes in human platelets.

Results: The slow releasing H2S donor GYY4137 inhibited thrombin- (Fig 1C) and collagen-induced human platelet aggregation in a concentration-dependent manner and to a significantly larger extent than the decayed control compound. In vivo, GYY4137 (50 mg/kg) significantly (P<0.05, unpaired t-test) reduced collagen-induced radiolabelled platelet aggregation from 22.3±3.5% (spent control) to 15.5±1.5%. Western blotting showed that human platelets express the H2S generating enzyme cystathionine-β-synthase (CBS) but not cystathionine-γ-lysase (CSE). In addition, CBS but not CSE was catalytically active in platelets under basal conditions whereas CSE was not. Experiments with the H2S selective probe WSP-1 showed that platelets generated H2S and that H2S production was inhibited by the CBS inhibitor 2-(aminooxy)acetic acid (1 mM).

Figure 1: Chemical structures of A) GYY4137 and B) “ spent donor” control. GYY4137 concentration-dependently inhibited C) thrombin- (0.04 U/ml) induced human platelet aggregation in vitro. Equivalent H2S concentration (0.4 nM to 4 μ M) was measured with an H2S selective microelectrode.

Discussion and conclusions: H2S, when applied to platelets in a manner that mimics slow, physiological release from enzymes, inhibits platelet aggregation through mechanisms that remain to be identified. Platelets generate H2S endogenously from CBS although the functional relevance of endogenous H2S remains to be evaluated. H2S may therefore be an important regulator of platelet activation and thrombotic events. Further research is warranted to fully evaluate the role of H2S in the negative regulation of platelets.