C5 cleavage during tissue factor-induced clot formation. (A) Hemolytic activity of biotinylated C5 was confirmed. Reactions containing C6 (600nM) and either C5 (400nM) or C5-biotin (400nM) were incubated with C5 convertase (CVF,Bb; 100nM) for 90 minutes at 37°C. Aliquots of the reaction were subjected to SDS-PAGE followed by Coomassie staining to assess activation cleavage or diluted 160 000-fold for the terminal pathway (TP) assay to measure erythrocyte lytic activity. Lack of enzyme with C6 and either C5 or C5-biotin resulted in minimal background lysis. A standard curve (left side) was generated by inducing chicken erythrocyte lysis with purified C5b,6 and the addition of C7, C8, and C9. (B) C5 cleavage in plasma during tissue factor (TF)–dependent clot initiation. Thrombin (IIa) generation was initiated in citrated plasma by the addition of TF, phospholipid vesicles, and CaCl₂ in the presence of C5-biotin, and reactions proceeded for 1 hour at 37°C. Coimmunoprecipitation was performed on the clot fluid phase using avidin agarose to isolate all biotinylated C5-containing fragments. Reactions were analyzed on a 4%-20% Tris-HCl gel under reducing conditions and detected by Western blotting using IRDye 800CW Streptavidin. No C5a was detected, but a 35-kDa fragment was generated that was absent when thrombin was inhibited with hirudin. (C-D) Cleavage of C5-biotin (400nM) was assessed in vitro by incubating it with or without 20nM thrombin for 1.5 hours at 37°C. Reaction products were detected by Western blotting using IRDye 800CW Streptavidin (C) or a polyclonal rabbit antibody against C5a and secondary goat anti–rabbit IgG (H + L) IRDye 680 (D), both of which revealed a novel C5-derived 35-kDa fragment. An area of the blot (dotted box) corresponding to the location of C5a was overexposed and depicted below to show the presence of tiny amounts of C5a formed by thrombin in the in vitro cleavage reaction.