The Ca2+/calmodulin kinase/AMP-activated protein kinase pathway regulates the lectin phaseolus vulgaris agglutinin induced NO production in human platelets

Previously it was shown that wheat germ agglutinin (WGA) and, at a minor extent, phaseolus vulgaris agglutinin (PHA), are able to induce platelet activation. Since the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO)/soluble guanylyl cyclase/cGMP/cGMP-dependent protein kinase (PKG) pathway is one of the major antiaggregating mechanism present in platelets, we tested the WGA or PHA effect on this pathway. It has been shown that platelets treated with WGA did not produce NO, while PHA stimulated NO production in a dose and time dependent manner. It has been found that the increased NO formation induced by PHA was dependent on eNOS phosphorylation/activation. The Ca2+/calmodulin-dependent kinase kinase/AMP activated protein kinase pathway seems to be greatly involved as STO-609 and Compound C, Ca2+/calmodulin protein kinase kinase/AMP kinase inhibitors respectively, cancelled eNOS phosphorylation induced by PHA. One crucial effect of NO and cGMP elevation is the activation of PKG, that can phosphorylate vasodilator-stimulated phosphoprotein (VASP). It was found that NO and cGMP elevation and VASP phosphorylation both on ser239 and thr278 were greatly stimulated by PHA and strongly inhibited by STO-609 and Compound C and by the eNOS inhibitor L-NAME. Thus, the CaMKK/AMPK pathway activated by PHA can regulate platelet activation stimulating the eNOS/NO/cGMP/PKG signalling pathway