CFHR1 regulates C5 convertase activity, binds to C5 and C5b6, and inhibits binding of C5b6 to the cell surfaces. (A) Sheep erythrocytes were incubated in complement active CFH/CFHR1-depleted HP (HP_ΔCFH) in the presence or absence of CFHR1 (18-300 μg/mL). Lysis was recorded after 30 minutes. In addition, the concentration of C3a (gray squares) and C5a (red triangles) was assayed in the supernatant. Data represent mean values of 3 separate experiments and SDs are indicated. *P < .05, **P < .005 versus control. (B) Effect of CFHR1 on C3b (solid line) and C5/C5b (stippled line) deposition on the surface of sheep erythrocytes. Sheep erythrocytes were incubated in HP_ΔCFH-ΔC8 plasma, and deposition of C3b and C5b was assayed in the presence of increasing concentrations of CFHR1 (10, 20, and 40 μg/mL, red solid and stippled lines) and CFH (10, 20, and 40 μg/mL, blue solid and stippled lines) by flow cytometry. Data represent mean values ± SDs of 3 separate experiments. (C) CFHR1 inhibits C5/C5b deposition on sheep erythrocytes (v) that were incubated in human CFH- and C8-depleted plasma (HP_ΔCFH-ΔC8). CFHR1 does not effect C3b deposition (ii). Factor H (CFH) inhibits both C3b and C5/C5b deposition (iii and vi). Bars represent 10 μm. (D) CFHR1 inhibits C5 convertase activity. Recombinant CFHR1 (column 3) as well as plasma-purified CFHR1 (column 4) inhibit the C5 convertase and cleavage of C5. The C5 convertase was generated on the surface of sheep erythrocytes using purified C3, factor B, and factor D in the presence of Ni²⁺ and properdin. Cleavage and C5a generation was determined by ELISA. CFH also affected C5a generation (column 5) in contrast to BSA (column 6). Data represent mean values of 3 separate experiments and SDs are indicated. *P < .05, **P < .005 versus BSA.