Daphnoretin, a new protein kinase C activator isolated from Wikstroemia indica C.A. Mey

Abstract

Daphnoretin, a biologically active principle isolated from Wikstroemia indica C.A. Mey., caused platelet aggregation in washed rabbit platelets, platelet-rich plasma and whole blood. The aggregation of and ATP release from platelets induced by daphnoretin were similar to phorbol ester- and diacylglycerol-induced aggregation and release. The EC50 values of daphnoretin-, phorbol 12,13-dibutyrate (PDBu)- and 1-oleoyl-2-acetylglycerol (OAG)-induced platelet aggregation in washed rabbit platelets were 17.2 ± 2.8 μM, 20.6 ± 2.1 nM and 38.6 ± 1.7 μM respectively. Platelet aggregation induced by daphnoretin and PDBu was not inhibited by indomethacin, BN52021 or sodium nitroprusside. ADP-scavenging systems, apyrase and phosphocreatine/creatine kinase, showed weak inhibition of the aggregation, and EGTA, trifiavin, verapamil and prostaglandin El markedly inhibited the aggregation. Staurosporine, a potent protein kinase C inhibitor, suppressed daphnoretin-, PDBu- and OAG-induced aggregation and ATP release in a concentration dependent manner. The IC50 values of staurosporine on daphnoretin (50 μM)-, PDBu (100 nM)- and OAG (50,μM)-induced aggregation were 37.7 ± 8.3, 52.2 ± 6.3 and 42.8 ± 8.9 nM respectively. Daphnoretin did not cause significant thromboxane Ba formation in rabbit platelets. Neither daphnoretin nor PDBu caused [3H]inositol monophosphate formation or an increase in intracellular Ca2+ concentration in myo-[3H]inositol-labelled and Fura-2-loaded platelets. Platelet cytosolic protein kinase C was activated by daphnoretin and PDBu in a concentration-dependent manner with an EC50 of 12.4 ± 1.2 μM and 18.7 ± 1.4 nM respectively. Membrane-associated protein kinase C activity was increased by either daphnoretin or PDBu. [3H]PDBu binding to washed rabbit platelets was inhibited by daphnoretin in a concentration-dependent manner with an IC50 value of 45.2 ± 5.2 μM. These results indicate that daphnoretin is a protein kinase C activator in rabbit platelets.