Nitrate And Nitrite Exert Inhibitory Effects On Platelet Function In Vitro And In Vivo

G Apostoli, A Solomon, M Emerson. Imperial College London, National Heart and Lung Institute, Molecular Medicine, Platelet Biology Group, London, SW7 2AZ, UK

Arterial thrombosis is characterised by the over activity of platelets leading to the formation of a thrombus. Thrombi may restrict blood flow to vital organs including the heart and brain causing life-threatening ischaemic conditions such as myocardial infarction and stroke. The trigger of these events may be vascular endothelial damage involving reduced endothelial nitric oxide synthase (eNOS) activity and impaired nitric oxide (NO) signalling. A healthy diet high in nitrates/nitrites (NO3/NO2) may provide additional NO sources following their reduction to NO. Nitrate/nitrite has been shown to lower blood pressure but their effect on platelet function is unclear.

The aims of this study were to investigate the functional effects of NaNO3/NaNO2 on platelets in vitro and in vivo.

Aggregation responses to NaNO2 (10 nM-100 μM), sildenafil citrate (phosphodiesterase 5 inhibitor, 10 nM) and 1H-​[1,​2,​4]oxadiazolo[4,​3-​a]quinoxalin-​1-​one (ODQ - soluble guanylyl cyclase (sGC) inhibitor, 10 μM) were investigated in collagen-induced (5 μg mL-1) washed human platelet optical aggregometry experiments (Born, 1962). The effects of NaNO3 (1 mmol kg-1 administered i.p) on collagen-induced (50 μg kg-1) platelet aggregation were investigated in vivo in wild-type C57bl/6 male mice (20-25g) and eNOS-/- mice (C57bl/6 background) by measuring radiolabelled platelet thromboembolism in real-time via external scintillation probes connected to a spectrometer (Tymvios et al., 2008). All animals were anaesthetised using urethane (10 mL kg-1 of 25% (w/v)) and procedures were non-recovery. In vitro, drugs were incubated for 10 minutes before stimulation and in vivo 1 hour.

NaNO2 alone did not significantly affect collagen-induced platelet aggregation in vitro, however in combination with a fixed concentration of sildenafil, NaNO2 caused a concentration-dependent inhibition (control 98±3, 1nM 76±6, 10nM 80±5, 100nM 69.8±9, % aggregation expressed mean±S.E.M, n=6, P<0.05, unpaired t-test). This effect was reversed in the presence of ODQ which showed that this inhibitory effect was sGC/cGMP dependent. In vivo, NaNO3 was associated with a non-significant trend towards inhibition of platelet aggregation in wild-type (reduced collagen response by 15%, n=6, P>0.05) and significantly inhibited aggregation in eNOS-/- mice (reduced collagen response by 26%, n=3, P<0.05, unpaired t-test).

In conclusion, sildenafil is able to amplify the NO signal from nitrite in platelets in vitro suggesting potential therapeutic uses of this compound directed towards platelet-driven disorders. Nitrate exerted an inhibitory effect upon platelet aggregation in vivo when endogenous NO production was impaired suggesting a potentially important contribution of this pathway in limiting platelet hyper-activity in states of vascular dysfunction associated with deficient eNOS activity. Nitrate and nitrite, including those from dietary sources, may therefore be beneficial in reducing platelet-driven cardiovascular disorders and as targets for therapeutic intervention to reduce conditions such as myocardial infarction.

References:-

Born G (1962) Nature, 194(4832), 927-929.

Tymvios C et al. (2008) Thromb Haemost, 99(2), 435-440.