Time course of C5 cleavage by thrombin and C5 convertase. (A) SDS-PAGE analysis of plasma-derived C5 (400nM) cleavage with C5 convertase (CVF,Bb; 100nM) alone or combined with thrombin (20nM) or with S195A thrombin (500nM). At selected time intervals, aliquots of the reactions were subjected to SDS-PAGE under reducing conditions. Proteins were visualized by Coomassie staining. Additional lanes on right are shown to match bands to their corresponding protein. (B) Generation of the C5b α′-chain is shown over time during C5 cleavage with C5 convertase alone (CVF,Bb; 100nM; blue) or thrombin alone (IIa: 400nM; red) or with both C5 convertase (CVF,Bb; 100nM) and thrombin (IIa: 2nM; light gray, 20nM; dark gray or 400nM; black) or with inactive S195A thrombin (S195A IIa: 500nM; clear with blue border). C5b α′-chain–containing bands were quantified by scanning densitometry. C5b α′-chain generated by C5 convertase after 90 minutes was considered 100%. Data are mean ± SD of 2-5 independent experiments.