CFHR1 inhibits nonenzymatic steps of the terminal complement pathway. (A) Chicken erythrocytes were incubated with C5b6 complexes (5 ng/mL) and increasing concentrations of CFHR1, and nonlytic complement inactive defHP was added as a source for terminal complement components. MAC formation was assayed by lyses of erythrocytes. Hemolysis was recorded after 30 minutes by measuring the absorbance at 415 nm. Increasing concentrations of CFHR1 (25-100 μg/mL) affected MAC activity, and CFH or human serum albumin (HSA) showed no effect. Data represent mean values in percentage ± SD derived from 3 independent assays. (B) MAC formation on sheep erythrocytes was induced by incubation with C5b6, C7, C8, and C9 components and detected by hemolysis of cells (column 1). Preincubation of C5b6 with recombinant CFHR1 (50 μg/mL) or plasma-purified CFHR1 (12.5 μg/mL) inhibited hemolysis (columns 2 and 3, respectively). CFH (12.5 μg/mL) showed no effect on MAC formation (column 4), but vitronectin did (12.5 μg/mL; column 5). BSA (12.5 μg/mL) did not induce hemolysis (column 6). Data represent mean values ± SD of 3 separate experiments (except for plasma-purified CFHR1). (C) Inhibitory role of CFHR1 on MAC formation. CFHR1 purified from human plasma (0.1-0.3 μM) inhibited MAC formation on the surface of sheep erythrocytes (red triangles, red line). In addition, the established MAC inhibitor vitronectin was assayed (blue diamond, blue line). CFHR1 and vitronectin had similar activity and BSA did not affect MAC formation (black squares, black line). A representative experiment of 2 is shown.