Molecular basis of the enhanced susceptibility of the erythrocytes of paroxysmal nocturnal hemoglobinuria to hemolysis in acidified serum

Abstract

When incubated in acidified serum, the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) are hemolyzed through activation of the alternative pathway of complement (APC), but normal erythrocytes are resistant to this process. PNH cells are deficient in decay- accelerating factor (DAF), a complement regulatory protein that inhibits the activity of both the classical and the alternative pathways. However, deficiency of DAF alone does not account entirely for the aberrant effects of acidified serum on PNH cells. Recently, we have shown that PNH erythrocytes are also deficient in another complement control protein called membrane inhibitor of reactive lysis (MIRL) that restricts complement-mediated lysis by blocking formation of the membrane attack complex (MAC). To determine the effects of the DAF and MIRL on susceptibility to acidified serum lysis, PNH cells were repleted with the purified proteins. DAF partially inhibited acidified serum lysis by blocking the activity of the amplification C3 convertase. MIRL inhibited acidified serum lysis both by blocking the activity of the MAC and by inhibiting the activity the C3 convertase. When DAF function was blocked with antibody, normal erythrocytes became partially susceptible to acidified serum lysis. By blocking MIRL, cells were made completely susceptible to lysis, and control of C3 convertase activity was partially lost. When both DAF and MIRL were blocked, the capacity of normal erythrocytes to control the activity of the APC and the MAC was destroyed, and the cells hemolyzed even in unacidified serum. These studies demonstrate that DAF and MIRL act in concert to control susceptibility to acidified serum lysis; of the two proteins, MIRL is the more important. In addition to its regulatory effects on the MAC, MIRL also influences the activity of the C3 convertase of the APC. Further, in the absence of DAF and MIRL, the plasma regulators (factor H and factor I) lack the capacity to control membrane- associated activation of the APC.

GPI-anchor and GPI-anchored protein expression in PMM2-CDG patients

“…We evaluated potential qualitative rather than quantitative defects on the erythrocyte GPI-linked complement inhibitor, CD59, which might contribute to the thrombotic phenotype of PMM2-CDG patients. For this purpose, we assayed A) whether the red blood cells (RBCs) in patients were lysed by complement when normal serum was acidified by the acidified-serum lysis test (Ham test), and B) if reduced ionic strength of serum by addition of an iso-osmotic solution of sucrose could activate the classic complement pathway, and complement-sensitive cells would then be lysed by the sucrose test [ 19 , 20 ].…”

Initiation and Regulation of Complement during Hemolytic Transfusion Reactions

“…Given the evolutionary ancient history of complement, many inhibitory pathways exist that facilitate complement regulation. For example, constitutively expressed cell surface proteins, such as CD35 (CR1), CD46 (MCP), CD55, or CD59, provide various inhibitory activities to inhibit off-target complement activation [ 40 , 51 – 53 ]. Importantly, each of these factors displays unique regulatory activities.…”

“…For example, CD35 and CD46 bind C3b and C4b preventing complex formation with Bb and 2b, respectively, [ 40 , 51 , 54 ], thereby inhibiting active C3 convertase formation. Similarly, CD55 (decay accelerating factor or DAF) along with CD35 actively dissociates C3 convertase complexes once formed by displacing Bb and 2b [ 51 , 53 , 55 ]. Furthermore, CD59 (protectin) binds the C5, C6, C7, and C8 complex and inhibits C9 binding and formation of the membrane attack complex [ 56 – 58 ].…”

Elevated circulating endothelial membrane microparticles in paroxysmal nocturnal haemoglobinuria

“…PNH is characterized by a lack of GPI-anchored membrane proteins on peripheral blood cells derived from the mutant haematopoietic stem cell and is clinically manifested by intravascular haemolysis, venous thrombosis, and bone marrow failure (Parker, 2002). Haemolysis is explained by a deficiency of complement regulatory proteins: the decay-accelerating factor (CD55) and especially the membrane inhibitor of reactive lysis (CD59) (Wilcox et al, 1991). Thrombosis is the major cause of morbidity and mortality in PNH (Rosse & Nishimura, 2003).…”