Temporal analysis of thrombin-mediated cleavage of plasma-derived C5. (A-B) C5 (400nM) was incubated with plasma-derived thrombin (IIa; 400nM, A; 20nM, B) in the presence and absence of hirudin (4μM, A; 200nM, B) in HBS-M7 at 37°C. Aliquots from each reaction were removed at selected time intervals for analysis. For comparison, thrombin and C5 alone are shown in lanes 1 and 2, respectively. Molecular weight (MW) markers on the left are in kDa. (C) Schematic of C5 proteolysis by thrombin and C5 convertase is shown with the number and position of relevant amino acid residues. Thrombin-derived cleavage products were determined by mass spectrometry after in-gel trypsin digestion. C5 convertases cleave only at R751 generating C5a and C5b, the latter comprising the β-chain and a disulfide linked 104-kDa α′-chain.
