Identifying the soluble guanylyl cyclase-dependent and independent action of nitric oxide on platelets

Nicola Truss, Tim Warner. William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, EC1M 6BQ, London, United Kingdom.

Platelets are important in the maintenance of vascular integrity and are closely regulated by a range of mediators. Nitric oxide (NO) is an important inhibitor of platelet action which, in other systems, has been shown to predominantly act through stimulation of soluble guanylyl cyclase (sGC). Studies have suggested that the inhibitory action of NO in platelets is not mediated purely by an action on sGC, but also by sGC-independent effects (1). We show here variations in the degree of both sGC-dependent and independent effects of NO on platelet aggregation dependent upon which particular platelet agonists are used.

Blood was collected from healthy human volunteers into 3.2% sodium citrate (w/v) and centrifuged to obtain platelet rich plasma (PRP). The PRP was then incubated for 20mins with the sGC inhibitor ODQ (10µM) and then for 5mins with the NO donor DEA-NONOate (1µM) before the addition of agonist and measurement of aggregation by light transmission in a 96-well plate reader. A one sample t-test used to compare the data to the hypothetical value of 0% inhibition.

Stimulation of aggregation by 3µM U46619 was inhibited 79±5% by DEA (P<0.05 vs 0%, n = 4), while in the presence of ODQ aggregation was only inhibited 24±8% (P>0.05 vs. 0%, n = 4). DEA inhibited aggregation induced by 3µg/ml collagen by 80±3% (P<0.05 vs 0%, n = 4), which was reduced to 33±9% (P<0.05 vs 0%, n = 4) by ODQ. 3µM ADP stimulated aggregation was inhibited 61±13% by DEA (P<0.05 vs 0%, n = 4), but by only 12±14% (P>0.05 vs. 0%, n = 4) in the presence of ODQ.

We have shown that under the experimental conditions reported here the inhibition of platelet aggregation by NO is mediated through both sGC dependent and independent mechanisms, as defined by the use of ODQ, an inhibitor of sGC. Interestingly, the proportions of NO inhibition which were sGC-dependent and sGC-independent, varied depending on the agonist used. In particular, we found that the sGC-independent effects were more pronounced when studying NO inhibition of collagen stimulated aggregation than inhibition of U46619 or ADP induced aggregation which displayed little sGC independent inhibition. The sGC-dependent mechanisms leading to inhibition of platelet aggregation are predominantly exerted through the phosphorylation of receptors involved in calcium signalling. The sGC independent mechanisms have not been fully elucidated, but may be through NO causing tyrosine nitration or having actions on other heme containing proteins within the platelet.

1. Sogo, N et. al., (2000). Biochem Biophys Res Comm., 279, 412-9.