Modulation Of Platelet Function In Vivo And In Vitro By Sildenafil

Georgina Apostoli, Antonia Solomon, Michael Emerson. Imperial College London, London, UK

Thrombosis is a major cause of cardiovascular disease with life-threatening implications such as myocardial infarction and stroke. It is characterised by the over activity of platelets aggregating together and forming a thrombus. Previously, nitric oxide (NO) has been shown to negatively regulate platelets by activating cyclic guanosine monophosphate (cGMP) signalling and inhibiting many platelet activation pathways. Sildenafil, a phosphodiesterase V (PDE 5) inhibitor, has been shown to enhance NO signalling in platelets in vitro and reduce the incidence of aggregation in the presence of the NO donor sodium nitroprusside (SNP) (Gudmundsdóttir et al., 2005). The aim of this study is to further investigate sildenafil’s antithrombotic potential and the mechanisms behind its action.

Aggregation responses to sildenafil (10nM, 100nM and 1μM), SNP (100nM) and the additive effect when used together were investigated in collagen-induced (5μg/mL) washed human platelet optical aggregometry experiments (Born, 1962). The effects of sildenafil (50μg/kg administered i.v) on collagen-induced (50μg/kg) platelet aggregation were investigated in vivo in W.T C57 B/6 male mice (20-25g) and eNOS knockout mice (C57 B/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 (10mL/kg of 25% (w/v)) and procedures were non-recovery. In vitro the drugs were incubated for 10 minutes before stimulation and in vivo 5 minutes.

SNP (control 94±3, 100nM 84±5, % aggregation expressed mean±S.E.M, n = 7) and sildenafil (control 96±3, 10nM 88±6, 100nM 70±8, 1µM 63±9, % aggregation expressed mean±S.E.M, n = 8) had a non-cumulative concentration dependent inhibition on platelet aggregation in vitro. The inhibitory effect of SNP was enhanced by sildenafil (P<0.05 using a Friedmans statistical test, n = 7). Sildenafil significantly inhibited platelet aggregation by 53±11% in vivo (P<0.05 using a Mann-Whitney statistical test, n = 6). The inhibitory effect of sildenafil on platelet aggregation was not seen in vivo in eNOS knockout mice (n = 3).

Sildenafil is able to reduce platelet aggregation in vitro and in vivo. Sildenafil has no effect on collagen-induced platelet aggregation in eNOS knock out mice which suggests that sildenafil acts in the presence of NO produced by eNOS. The enhancing effect of sildenafil upon SNP in platelets suggests that sildenafil acts via potentiating the cGMP pathway. Future pharmacological experiments will confirm whether sildenafil acts solely by this pathway and assess the involvement of cGMP-independent pathways. This study will be extended to models of vascular dysfunction to further investigate sildenafil’s effect and mechanism of action on platelets and provide insight into its value as an antithrombotic therapy.

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

Gudmundsdóttir IJ et al. (2005) Biochem Biophys Res Commun, 337, 382-385.

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