PHOSPHORYLATION OF COMPLEMENT COMPONENT C3 AND C3 FRAGMENTS BY A HUMAN PLATELET PROTEIN-KINASE - INHIBITION OF FACTOR I-MEDIATED CLEAVAGE OF C3B

Phosphorylation of C3 in vitro has been shown previously to lead to si gnificantly altered function of the protein. Platelets are known to co ntain and release considerable amounts of protein kinases and ATP, whi ch are prerequisites for protein phosphorylation. The aim of the prese nt study was to investigate whether C3 is phosphorylated extracellular ly by human platelets. Platelet-rich plasma was stimulated by human ag gregated gamma-globulin or ADP. The remaining cells were removed by ce ntrifugation, and the plasma was incubated with [gamma-P-32]ATP. After precipitation with Sepharose-bound Abs to C3c followed by SDS-PAGE, i t was shown that C3 was phosphorylated in the cr-chain by a protein ki nase dependent on Mn2+, Ca2+, or Mg2+ ions. The supernatant from washe d, activated platelets was incubated with purified C3 or soluble or ac tivated thiol Sepharose-bound C3b, together with [gamma-P-32]ATP. Phos phorylation was seen in the ct-chain of C3, and to the same extent in the alpha'-chain of both C3b preparations. The analysis of acid hydrol ysate demonstrated that C3 contained P-32-labeled Thr and P-32-labeled Ser. After extensive proteolysis with trypsin, the major phosphorylat ion site was located to a peptide of 3 to 4 kDa that was bound to the activated thiol Sepharose via the free sulphydryl group in the C3d fra gment. Incubation of phosphorylated C3b with factors I and H showed th at phosphorylation inhibited the cleavage of the alpha'-chain of C3b. The results in this study suggest that phosphorylation is a regulator of C3 during platelet activation induced, for example, by immune compl exes.