Selective inhibition of collagen-induced arachidonic acid liberation by 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1, 4-diazepine hydrochloride (ML-7), a myosin light chain kinase inhibitor, in washed rabbit platelets

Abstract

Effects of myosin light chain (MLC) kinase inhibitor ML-7 [1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1, 4-diazepine hydrochloride] and protein kinase C inhibitor H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydro-chloride] on collagen-induced platelet activation were investigated in washed rabbit platelets. Upon stimulation with collagen (1μg/mL), H-7 decreased protein kinase C-mediated pleckstrin phosphorylation, but had no inhibitory effect on thromboxane (TX) A 2 formation or platelet aggregation. In contrast, ML-7 produced a concentration-dependent inhibition of the collagen-induced platelet aggregation and TXA 2 formation by preventing arachidonic acid (AA) liberation from membrane phospholipids. However, ML-7 had little effect on AA liberation induced by thrombin, Ca 2+ ionophore A-23187 or melittin, suggesting that ML-7 may affect the signal transduction pathway specific for collagen-induced AA liberation, without direct inhibition of phospholipase A 2 activity. In indomethacin-treated platelets, collagen caused MLC phosphorylation and AA liberation in the absence of a significant increase in intracellular Ca 2+ concentration ([Ca 2+] i) or protein tyrosine phosphorylation. ML-7 inhibited both MLC phosphorylation and AA liberation induced by collagen in indomethacin-treated platelets. These results demonstrate that MLC phosphorylation and AA liberation are early events detectable in collagen-stimulated platelets, and suggest that ML-7 inhibits these early steps of collagen-induced signal transduction pathway in rabbit platelets.