Inhibition of complement by covalent attachment of rosmarinic acid to activated C3b

Abstract

Rosmarinic acid has been reported to inhibit complement activation in vivo as well as in vitro. Previous studies suggested that the inhibitory effect was due to inhibition of C3/C5 convertases, but inhibition of C3b attachment would yield the same results. Recent work in our laboratory demonstrated that compounds with polyhydroxylated phenyl rings are highly reactive with the thioester bond in nascent C3b. These compounds block complement activation by preventing attachment of C3b to the activating surface. Because rosmarinic acid contains two 3,4-dihydroxyphenyl groups, the current study was undertaken to re-examine the mechanism of inhibition by analyzing the effect of rosmarinic acid on C3b attachment. In assays using purified complement proteins, rosmarinic acid inhibited covalent attachment of C3b to cells with an ic50 = 34 μM. Inhibition of C5 convertase activity required 1500 μM rosmarinic acid, and no significant inhibition of the C3 convertase enzyme, which produces C3b from C3, was observed at 10,000 μM. In hemolytic assays using human serum, rosmarinic acid was shown to inhibit activation of both the classical (ic50 = 180 μM) and the alternative (ic50 = 160 μM) pathways of complement. Rosmarinic acid concentrations up to 10,000 μM did not cause direct inactivation of C3. Radioiodination of rosmarinic acid was used to demonstrate covalent activation-dependent incorporation of rosmarinic acid specifically into the thioester-containing α′-chain of nascent C3b. These findings indicate that inhibition of complement activation by rosmarinic acid is due to the reaction of rosmarinic acid with the activated thioester of metastable C3b, resulting in covalent attachment of the inhibitor to the protein.