Heparin and polyP increase the rate of complex formation between C1-INH and C1s. For panels A-D, proteins were separated by 10% SDS-PAGE and gels were stained with Coomassie blue R-250. Concentrations of polyP reflect the concentration of the monomer. (A) 1 μM C1-INH was reacted at 4°C with 1 μM recombinant C1s (C1s SP domain), in the presence of increasing concentrations of polyP₁₃₀ (P130) for the indicated times. Purified C1-INH is shown in the lane between the 1 and 5 minute panels. (B) 1 μM C1-INH was reacted at 4°C for 0 to 30 minutes with 1 μM recombinant C1s (C1s SP domain) in the absence (C, control) or presence of either 50 μg/mL heparin or 160 μM polyP₁₃₀ (left). Densitometry performed on 3 gels (right). The percent C1-INH complex is based on C1-INH alone being assigned as 100%. (C) 1 μM C1-INH was reacted at 4°C for 0 to 120 minutes with 1 μM plasma C1r in the absence (C) or presence of either 50 μg/mL heparin or 166 μM polyP₁₃₀ (left). Densitometry performed on 3 gels (right). The percent C1-INH complex is based on C1-INH at t = 0 being assigned as 100%. (D) 10 nM C1-INH was reacted at 4°C for varying periods of time as shown, with 10 nM C1s (CCP12SP), 1 μM C4, and either buffer alone (C), 50 μg/mL heparin, or 166 μM polyP₁₃₀ (left). Densitometry performed on 3 gels (right). The percent C4 α chain is based on C4α at 5 hours being assigned as 100%. CCP, complement control protein-like domain; H, heparin; P, polyP₁₃₀.