VWF is a cofactor for fI-mediated cleavage of C3b. (A) C3b (1 μg) was incubated with either fI (Factor I; 0.5 μg) and fH (Factor H; 1 μg) or with fI and different concentrations of pVWF (1 or 2 μg/mL). After western blotting with anti-C3 antibody (upper panel), α′ and β chains of C3b (110 and 75 kDa) and iC3b (68 and 43 kDa) were detected by their molecular weight. The 68- and 43-kDa bands were the products of cleavage of the α′ chain of C3b by fI. C3dg (41 kDa) is the result of cleavage of the 68-kDa band of iC3b by fI. Lower panel shows Coomassie blue staining of the same polyacrylamide gel. (B) To identify whether VWF changes the substrate specificity of fI, C3 (1 μg), C3b (1 μg), or iC3b (1 μg) was incubated with VWF and fI. Immunoblotting by using anti-C3 antibody (upper panel) showed that similar to the fI/fH-mediated cleavage, fI/VWF cleaves mainly C3b. α and β chains of C3 (120 and 75 kDa), α′ and β chains of C3b (110 and 75 kDa), and iC3b (68 and 43 kDa) were detected by their molecular weights. Lower panel shows Coomassie blue staining of the same polyacrylamide gel (n = 3).