DAPHNORETIN, A NEW-PROTEIN KINASE-C ACTIVATOR ISOLATED FROM WIKSTROEMIA-INDICA CA MEY

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 muM, 20.6 +/- 2.1 nM and 38.6 +/- 1.7 muM respectively. Platelet aggregation induced by daphnoretin and PDBu was not inhibited by indomethacin, BN52021 or sodium nitroprusside. ADP-scavenging systems, apyrase and phospho-creatine/creatine kinase, showed weak inhibition of the aggregation, and EGTA, triflavin, verapamil and prostaglandin E, 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 muM)-, PDBu (100 nM)- and OAG (50 muM)-induced aggregation were 37.7 +/- 8.3, 52.2 +/- 6.3 and 42.8 +/- 8.9 nM respectively. Daphnoretin did not cause significant thromboxane B2 formation in rabbit platelets. Neither daphnoretin nor PDBu caused [H-3]inositol monophosphate formation or an increase in intracellular Ca2+ concentration in myo-[H-3]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 muM and 18.7 +/- 1.4 nM respectively. Membrane-associated protein kinase C activity was increased by either daphnoretin or PDBu. [H-3]PDBu binding to washed rabbit platelets was inhibited by daphnoretin in a concentration-dependent manner with an IC50 value of 45.2 +/- 5.2 muM. These results indicate that daphnoretin is a protein kinase C activator in rabbit platelets,