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. Abbreviations: ACC: acetylCoA carboxylase; AKT: protein kinase B; AMPK: AMP-activated protein kinase; CaMKK: Ca2+/calmodulin kinase kinase; eNOS: endothelial nitric oxide synthase; LKB1: liver kinase B1; NO: nitric oxide; PHA: phaseolus vulgaris agglutinin; PI3K: phosphatidylinositol 3 kinase; PKA: protein kinase A; PKG: protein kinase G; PLC: phospholipase C; VASP: vasodilator-stimulated phosphoprotein; WGA: wheat germ agglutinin. Introduction Platelet activation is involved in both haemostasis and thrombosis. When platelets encounter matrix proteins exposed by injury to the vessel wall, they stop on the exposed subendothelial surface, become activated showing morphological alterations, secrete the content of their granules and aggregate. Inhibition of platelet aggregation can be produced by the block of membrane receptors interaction with intracellular signalling pathways, by interfering with platelet-activating messengers or by potentiating the action of physiological platelet inhibitors such as endothelium derived PGI2 and nitric oxide (NO). These compounds activate adenylyl and guanylyl cyclases leading to cAMP and cGMP increase, respectively. The elevation of these two platelet cyclic nucleotides interferes with platelet activatory signalling pathways such as the intracellular Ca2+ elevation and the reorganization of the cytoskeleton. In human platelets NO formation depends on endothelial nitric oxide synthase (eNOS) activation. Platelet eNOS is now largely considered a Ca2+-independent enzyme and the phosphorylation/ dephosphorylation of ser1177 and/or thr495 residues plays a vital role in the regulation of its activity. Phosphorylation of ser1177 residue activates eNOS, while phosphorylation of thr495 residue inhibits the activity of the enzyme [1]. Several kinases regulate eNOS *Correspondence to: Giuliana Leoncini, Department of Pharmacy, Biochemistry Lab, University of Genoa, Viale Benedetto XV 3, 16132 Genova, Italy, Tel: 39010353-8154, Fax: 390103538157; E-mail: giuliana.leoncini@libero.it