CONICET | Buscador de Institutos y Recursos Humanos

We have shown that both HNO and NO significantly inhibit human platelet aggregation induced by thrombin (Thr) probably through cGMP/cAMP accumulation. To investigate the effect provoked by HNO and NO on protein kinase activation pathway, we studied several signaling markers, including Akt Ser473, ERK1/2, and p38MAPK phosphorylation in human washed platelets (WP) stimulated with Thr. Methods: All the tests were assessed with WP. Angeli's salt (AS), sodium nitroprusside (SNP), and buffer were used as HNO, NO releasers or control respectively. The samples (n= 5) were incubated with AS,SNP, or buffer at 37°C for 2 min. Thereafter, the samples were stimulated with Thr or buffer during 1min and 10 min. Phosphorylation of ERK1/2, p38MAP Kinase and Akt were studied by inmunobloting and analyzed by densitometric scanning at both time Results: Both AS and SNP decreased p38MAPK phosphorylation 24% and 48% at 1min and 10 min, respectively. ERK1/2 phosphorylation decreased, 28% with AS and 100% with SNP only at 10min. Both donors per se have no effects on p38 and ERKs. Akt phosphorylation was increased in presence of both releasers without Thr at 1min and 10min, but the results were no conclusive post Thr activation Conclusions: SA and SNP effects on p38MAPK and ERK1/2 are in agreement with the inhibition observed on platelet recruitment. Remarkably, both releasers increased Akt phosphorylation in Ser473, which could be explained by a pathway involving activation of PKG/A. Our results showed that HNO and NO would inhibit human platelet aggregation by a decrease in protein kinase activation pathway related to cGMP/cAMP accumulation, leading to platelet integrin alphaIIb beta3 inhibition. Besides, a regulatory pathway of Akt activity is suggested.