In our study we could clearly demonstrate that endothelial cells play an important role in the antiaggregatory effect of SNP. Incubation with endothelial cells increased both the inhibitory effect on platelet aggregation and the duration of this effect. Levin et al. already mentioned that the supernates taken from SNP-incubated endothelial cells had an inhibiting effect on platelet aggregation. They speculated that their data could be explained by the synergy of NO and PGI2. The Levin study used supernatants of SNP-incubated endothelial cells and did not preincubate the endothelial cells with cyclooxygenase inhibitors, which may have led to different results. In contrast to their experimental setting, the platelets in our study were in direct contact with the endothelial cells during SNP incubation, which suggests possible additional interactions between platelets and endothelial cells. It has been well established that PGs of the E series and PGI2 inhibit platelet aggregation by counteracting the proaggregatory effect of thromboxane A2. This could explain the additional inhibitory affect of the endothelial cells in the presence of NO. However, this explanation seems insufficient, as the preincubation of endothelial cells with cyclooxygenase inhibitors, suppressing the PGI2 production, did not change the SNP effect as expected. In an earlier study we could show that the effect of other NO-donors, such as SNAP, is increased by the PG pathway. Induction of PGI2 via NO, following reduced platelet aggregation, could be blocked by preincubation of the endothelial cells with cyclooxygenases. The effect of SNP was not attenuated by the preincubation of the endothelial cells with a cyclooxygenase inhibitor. This is in contrast to the conclusions of Levin and coworkers. Sogo et al. could clearly demonstrate that, in the absence of endothelial cells, the effect of SNP is exclusively cGMP-dependent, which should be the case if the effect is due to NO release. In our setting the effect of SNP is not attenuated by the guanylate cyclase inhibitor ODQ, which is supposed to block the cGMP-dependent NO effect. This opposes the hypothesis that the effect of SNP is exclusively cGMP dependent in the presence of endothelial cells. These results highlight the importance of the endothelial cell in SNP metabolism and platelet interaction. Our data do not allow us to decide which pathway is responsible for the results measured. Because the effect of endothelial cells on platelet aggregation could not be reversed by incubation with a COX-inhibitor, we speculate that activation of endothelial cyclooxygenases by SNP may not be too important for the antiaggregatory effect of SNP.
Our results without endothelial cells show an effect of SNP on platelets which is in accordance with other in vitro studies[7, 26, 30].
Another interesting fact is that the addition of cyanide is able to reverse the effect, not only after immediate administration, as shown by Brune et al., but even after 4 h of incubation with SNP. This effect has not been described before in the literature. Although we do not have an explanation for this observation, we speculate that a cGMP independent pathway may be responsible for this effect.
In summary, our results show that endothelial cells play an important role in the SNP- mediated inhibition of platelet activation, which can be reversed only by cyanide. We could demonstrate that this effect is not mediated by activation of endothelial cyclooxygenases, as the inhibition of cyclooxygenases had no influence on the results. It is not dependent on cGMP, as it is not influenced by the guanylate cyclase inhibitor ODQ. Thus we suggest a cGMP independent, possibly not yet known pathway for platelet inhibition by SNP/endothelial cells.
The identification of this unknown antiaggregatory metabolite could be of great interest, since its effect on ADP-dependent platelet aggregation is long-lasting, but reversible by cyanide. We speculate that other less toxic ion metabolites could also reverse this effect, thus offering the opportunity for antidot treatment.