Phosphorylation of C3 by a casein kinase released from activated human platelets increases opsonization of immune complexes and binding to complement receptor type 1.
We have previously demonstrated that complement component C3 is phosphorylated both in vitro and in vivo by a casein kinase released from activated human platelets. In vitro, the studies have shown that cleavage of C3b by factor I is decreased, and binding to various target surfaces is enhanced by affecting the thiol ester. In the present study we have examined the effect of phosphorylation on the binding of C3b to complement receptor 1 (CR1, CD35). Upon phosphorylation by platelet casein kinase, C3b covalently bound to activated thiol Sepharose bound higher amounts of soluble recombinant CR1. Similar effects were demonstrated with two ELISA systems in which microtiter plates were coated with phosphorylated or unphosphorylated purified C3b or with C3 activated by the alternative pathway convertase. Phosphorylated C3b was also four times more efficient than unphosphorylated C3b in inhibiting the binding of complement-opsonized human aggregated gammaglobulin to erythrocytes. A similar increase in binding was found at low serum concentrations when the C3 activation occurred in C3-deficient serum reconstituted with phosphorylated or unphosphorylated C3. In this serum system, using a monoclonal antibody specific for iC3b, we also demonstrated that the phosphorylated C3b was protected against cleavage to iC3b. Corresponding experiments using factor H showed a decrease in binding of both fluid-phase and bound C3b to factor H. We postulate that phosphorylation of C3 by activated platelets amplifies the complement-mediated binding of immune complexes to CR1 by three different mechanisms: decreased cleavage of C3b to iC3b, increased deposition of C3b to immune complexes, and increased binding of C3b to CR1.