APC activation on PNH erythrocytes. (A) The hemolytic anemia of PNH is mediated by the antibody-independent alternative pathway of complement (APC). In vivo, the APC is in a state of continuous activation as a consequence of low-grade, fluid-phase hydrolysis of the internal thioester bond of C3 (C3·H₂O) in a process called the tick-over phenomenon. Nascent C3·H₂O can bind factor B, and upon cleavage of factor B by factor D, an unstable C3 convertase (H₂O·C3Bb) is formed. This complex can convert a small amount of C3 to C3b before it decays, and if an APC activator surface is in close proximity, the nascent C3b can bind covalently and form the nidus for the cell-bound APC C3 convertase, consisting of activated C3 (C3b), activated factor B (Bb, the enzymatic subunit of the complex), and factor P (a protein that stabilizes the complex, formally called properdin). The C5 convertase has the same components as the C3 convertase except that 2 C3b molecules are required to bind and position C5 for cleavage by activated factor B (Bb). C3a and C5a are bioactive peptides that are generated by cleavage of C3 and C5, respectively, by their specific activation convertases. The surface-bound C3 and C5 convertases greatly amplify complement activation by cleaving multiple substrate molecules (C3* and C5*). The membrane attack complex (MAC) consists of activated C5 (C5b), C6, C7, C8, and multiple molecules of C9 (C9_n). The MAC is the cytolytic unit of the complement system. On normal erythrocytes, the GPI-anchored complement regulatory protein CD55 restricts formation and stability of both the C3 and the C5 amplification convertases by destabilizing the interaction between activated factor B (Bb) and C3b, whereas GPI-anchored CD59 blocks formation of the MAC by inhibiting the binding of C9 to the C5b-8 complex. These 2 membrane proteins are deficient in PNH, allowing unregulated activation of the APC and leading to MAC formation and hemolysis. Inhibition of MAC formation by the humanized monoclonal anti-C5 antibody eculizumab (arrow) ameliorates the intravascular hemolysis of PNH. The current studies show that hemolysis of PNH erythrocytes can also be inhibited by mAb H17/3E7. These antibodies bind to C3·H₂O and C3b (arrows) and inhibit the tick-over phenomenon and block formation of the APC C3 convertase. (B) Untreated, PNH erythrocytes are lysed when the APC is activated on the membrane surface. Treatment with eculizumab blocks direct (intravascular) hemolysis of PNH erythrocytes by inhibiting formation of the MAC, but the cells become opsonized with activation and degradation products of C3 (C3b, iC3b, and C3dg) because eculizumab has no effect on the APC C3 convertase. The opsonized PNH erythrocytes are recognized by specific receptors on reticuloendothelial cells, resulting in extravascular hemolysis. mAb H17/3E7 blocks formation of the APC C3 convertase by binding to activated C3b, thereby preventing both opsonization and hemolysis of PNH erythrocytes in vitro.