VWF cofactor activity in fI-mediated C3b cleavage depends on the multimeric size of VWF. (A) C3b (1 μg) was incubated with fI (0.5 μg) and pVWF (1 μg), recombinant VWF dimers (VWF-Δpro) (0.5 or 1 μg), or ULVWF (1 μg). Western blotting of the degradation products using anti-C3 antibody (upper panel) showed that pVWF and VWF-Δpro acted as cofactors for fI-mediated C3b cleavage, but ULVWF did not function as a cofactor. Lower panel shows the results of 1.7% sodium dodecyl sulfate-agarose gel electrophoresis and immunoblotting with anti-VWF antibody confirming the multimeric distribution of pVWF, ULVWF, and VWF-Δpro used in (A). (B) C3 (1 μg), proactivation complement proteins (factor B and factor D, 0.5 μg each), and fI (0.5 μg) were incubated with VWF-Δpro (1 μg) or ULVWF (1 μg). Degradation products were immunoblotted using anti-C3a, which detects C3a and any C3 fragments containing C3a (upper panel) or anti-C3 antibodies (lower panel). Although factor B in the presence of factor D and a small amount of spontaneously generated C3b (tick-over) promoted complement activation and production of C3a (upper panel) and C3b (lower panel), addition of VWF dimers helped fI to shift the balance toward complement inactivation (iC3b in the lower panel and no C3a in the upper panel). A 76-kDa band in the upper panel detected by anti-C3a antibody after incubation of C3 with VWF dimers, fI, factor B, and factor D was probably a degradation product of the α chain of C3 that was not cleaved by C3 convertase and still contained C3a but was partially cleaved by fI and lost its 43-kDa fragment. ULVWF did not enhance fI-mediated complement inhibition, and as a default pathway, C3 was directed toward generation of C3a and C3b (activation) (n = 3). (C) Plasma VWF in 0.3 mL of cryoprecipitate was fractionated on a Sephacryl S500 column (1 × 10 cm; volume, 8 mL) by using a fast protein liquid chromatograpy system. Eluted fractions (0.3 mL total) from high to low molecular weight (fractions 12 to 17) were collected in a buffer (10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 25 nM NaCl [pH 7.4]). VWF fractions were analyzed by 1.7% agarose gel electrophoresis (22 hours at 3 mA) and immunoblotted with anti-VWF antibody (1:1000 dilution). (D) Each eluted VWF fraction was used in a C3b cleavage assay. After co-incubation of C3b, fI, and eluted VWF fractions, the cleavage products of C3b were separated by SDS-PAGE and detected by immunoblotting with anti-C3 antibody (n = 2).