4D, F and H) are presented. The D501N mutant did not degrade C4b (Fig. 4B) or C3b (Fig. 4D, F and H), even at the highest concentration used (30 μg/mL FI). This mutant was impaired irrespective of which cofactor was used (C4BP, FH, CR1 and MCP). P32A showed impaired function towards degradation of the α′-chains of C4b at the two highest concentrations and of the α′-chain of C3b at the highest concentration when FH was used as

cofactor. P32A did not show significant impairment when CR1 and MCP were used as cofactors (Fig. 4E and G). At some Wnt inhibitor concentrations, M120V and H165R could cleave the α′-chains of C4b and C3b more efficiently than WT FI in the presence of C4BP and FH as cofactors. The M120V mutant cleaved C3b more efficiently also in the presence of MCP (Fig. 4H). The kinetics of degradation of C4b and C3b were analyzed by incubating WT or mutant

FI with C4b/C3b, C4BP/FH and I125-labeled Quizartinib nmr C4b/C3b for different times. The intensities of the α′-chain band for C4b are shown in Fig. 5A and B and for C3b in Fig. 5C and D. Consistent with the above results, the D501N mutant was not able to degrade the α′-chains of C4b and C3b. The P32A mutant was able to cleave the α′-chain of C4b as efficiently as WT FI, but the cleavage of the α′-chain of C3b was impaired. The remaining mutants (M120V, H165R, A222G and R299W) cleaved the α′-chains of C4b and C3b as efficiently as WT FI. The ability of FI WT and mutants to cleave surface-bound C3b was elucidated using two approaches: a modified hemolytic assay and flow cytometry. For the hemolytic assay, sheep erythrocytes were coated with C3b, incubated with WT or mutant FI and C4BP and the amounts of membrane attack complex formed were measured by lysis of erythrocytes. If the FI is functional, less C3 convertase should be formed, resulting in diminished lysis. The D501N mutant showed no ability to degrade opsonized C3b (Fig. 6A). Also, the P32A and A222G mutants had impaired function,

whereas the M120V had enhanced function compared with WT FI. The two remaining mutants, H165R and R299W, both showed similar cleavage activities to WT FI (Fig. 6A). In the flow Etomidate cytometry assay, C3b opsonized sheep erythrocytes were incubated with WT or mutant FI and FH and the cleavage products, iC3b and C3d, were detected with Ab. A histogram shows the amounts of iC3b and C3d when C3b is degraded using WT and D501N FI (Fig. 6B). A high ratio of iC3b:C3d indicates degradation by FI. Flow cytometry results showed that the function of the D501N mutant was abolished and that P32A and A222G were less active than WT FI (Fig. 6C). The M120V, H165R and R299W mutants showed similar cleavage activities to WT FI (Fig. 6C), but the M120V and H165R mutants showed higher activities than WT, albeit only at 0.5 μg/mL. aHUS patients appear to have impaired regulation of complement activity on endothelial cells in the kidney.