Complement-driven anemia: more than just paroxysmal nocturnal hemoglobinuria

Abstract

Atypical hemolytic uremic syndrome (aHUS); hemolysis, elevated liver function tests, and low platelets syndrome; and transplant-associated thrombotic microangiopathy are related conditions, in that many patients harbor germline heterozygous mutations in genes that regulate the alternative pathway of complement (APC). Penetrance is variable because development of clinically significant disease appears to require supervention of a process such as inflammation. Complement activation on the endothelial surfaces leads to endothelial damage, platelet consumption, microthrombi, and a mechanical hemolytic anemia with schistocytes. Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic disease caused by expansion of a stem cell that harbors a somatic mutation in PIGA PIGA mutant blood cells are deficient in the complement regulator proteins CD55 and CD59, making them susceptible to intravascular hemolysis due to a failure to regulate the APC on erythrocytes. Eculizumab is a monoclonal antibody that binds to C5 and inhibits terminal complement by interfering with the cleavage of C5 by the C5 convertases. The drug is approved by the US Food and Drug Administration for the treatment of aHUS and PNH; however, a new generation of complement inhibitors that block C5 and other components of the complement cascade is showing promise in preclinical and clinical trials.

Figure 1.

Discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria. Schematic diagram of the complement cascade. The 3 primary routes for activation of complement are the lectin pathway (LP), the classical pathway (CP), and the alternative pathway (AP). The LP and CP are activated when specific triggers are recognized by host pattern-recognition receptors. The AP is constitutively active. Initial activation through the LP or CP generates a shared C3 convertase (C4b·C2a). In the AP, C3b pairs with factor B (FB) to form the AP proconvertase (C3b·B), which is processed by factor D (FD) to form the AP C3 convertase (C3b·Bb·P). Both types of C3 convertases cleave C3 to generate C3a and C3b. C3a is a weak anaphylatoxin, a substance that promotes an inflammatory response. Nascent C3b binds covalently to cell surface proteins, forming the nidus of the C3 convertase. C3b that binds to the surface of a healthy host cell is quickly inactivated; C3b that attaches to the surface of a pathogen or altered host cell triggers a rapid amplification loop to generate more C3b, resulting in opsonization. C3b also complexes with the C3 convertases to form the C5 convertases (C4b·C2a·C3b and C3b·Bb·C3b·P). In the terminal complement cascade, C5 convertases cleave C5 into C5a (a strong anaphylatoxin) and C5b. C5b combines with C6-9 to form the membrane attack complex (MAC), also referred to as the terminal complement complex. Regulatory factors act at various stages of the cascade to control complement activation via their decay accelerating activity and/or cofactor activity. MASPs, mannose-binding lectin-associated serine proteases; MBL, mannose-binding lectin; PAMPs, pathogen-associated molecular patterns.

Figure 2.

Complement cascade with inhibitors. CR1, membrane cofactor protein (MCP/CD46), factor I (FI), factor H (FH), and CD55 are the major proteins that inhibit the APC. Of these, FH is the most critical. FH is a fluid-phase protein that can bind to cells via its interaction with cell surface glycosaminoglycan and sialic acid residues. It inhibits the APC in the fluid phase and on the cell surface. It competes with factor B for C3b attachment, thereby limiting formation of the C3 convertase, and it accelerates decay of the C3 convertase. Deficiency or impaired function of FH leaves healthy host cells and tissues highly vulnerable to complement-mediated attack. Complement FI functions as a complement inhibitor in the presence of its cofactors (FH, MCP/CD46, and CR1), to cleave and inactivate C3b (in the alternative pathway) and C4b (in the classical pathway and lectin pathway), blocking C3 convertase formation. CD55 (also known as a decay-accelerating factor) is a cell surface glycosylphosphatidylinositol-anchored protein that inhibits the classical and alternative pathway C3 convertases and accelerates their decay. CD59 blocks terminal complement by preventing C9 from oligomerizing with the C5b-8 complex.

Figure 3.

mHam test results and next-generation sequencing in TMAs. The mHam test is a cell-based in vitro viability assay that tests the ability of a PIGA mutant (CD55 and CD59–deficient) cell line to regulate complement. Each participant is represented by a symbol. A black circle indicates that no rare variants in APC-regulatory genes were detected; a green circle indicates that a rare variant was detected in an APC-regulatory gene, and an open triangle indicates that next-generation sequencing was not performed. A positive mHam test is defined as cell killing > 20.5% (dotted horizontal line) after exposure to patient serum. The median value of cell kill is depicted for each group. Patients with partial HELLP fit some, but not all, of the clinical criteria for HELLP.