Induction of the paroxysmal nocturnal hemoglobinuria phenotype in normal human erythrocytes: effects of 2-aminoethylisothiouronium bromide on membrane proteins that regulate complement

Abstract

To investigate the mechanism by which treatment of normal human erythrocytes with the sulfhydryl reagent 2-aminoethylisothiouronium bromide (AET) induces susceptibility to complement mediated lysis, the effects of AET on the structural and functional integrity of decay accelerating factor (DAF), membrane inhibitor of reactive lysis (MIRL), and complement receptor type 1 (CR1) were examined. Following treatment with AET, erythrocyte MIRL and CR1 were no longer recognized in situ by antibodies, and antibody binding to DAF was diminished by approximately 50%. These studies indicated that the structural integrity of the three complement regulatory proteins was either partially (DAF) or completely (MIRL and CR1) disrupted by AET. Subsequent experiments showed that functional inactivation paralleled the structural disruption. Treatment of normal erythrocytes with AET induced susceptibility to cobra venom factor-initiated hemolysis, indicating that the functional activity of MIRL had been destroyed. The capacity of erythrocyte CR1 to serve as a cofactor for factor I-mediated cleavage of iC3b to C3c and C3dg was lost following treatment with AET. C3 convertase activity increase markedly following treatment of erythrocytes with AET, but convertase activity on AET cells was approximately 50% less than that observed when DAF function on normal cells was completely inhibited by antibody. Susceptibility of AET cells to acidified serum lysis was shown to be due primarily to inactivation of MIRL. Unexpectedly, in acidified serum the activity of the amplification C3 convertase of the APC was found to be controlled by MIRL as well as by DAF. These studies show that AET induces susceptibility to complement-mediated lysis by disrupting the structural and functional integrity of membrane constituents that regulate the activity of both the C3 convertases and the membrane attack complex of complement.

Complement Evasion Mediated by Enhancement of Captured Factor H: Implications for Protection of Self-Surfaces from Complement

“…The absence of these regulators is linked to complement-mediated lysis of erythrocytes in these patients. FH and FH-inspired candidate therapeutics protect erythrocytes from PNH patients ( 25 ), as well as erythrocytes that have been stripped of GPI-linked proteins and rendered “PNH-like” by treatment with 2-aminoethylisothiouronium bromide ( 39 ). We demonstrated ( Fig.…”

“…Human erythrocytes lacking GPI-linked proteins (PNH-like) were prepared according to a modified version of the method of Ezzell et al ( 39 ). Whole human blood was obtained by phlebotomy and stored in Alsever’s solution (4°C).…”

Immune Adherence–mediated Opsonophagocytosis: The Mechanism of Leishmania Infection

“…The involvement of C3 suggested that erythrocyte CR1 participates in the IA reaction. To test this, erythrocytes were treated with AET (29) or trypsin (30), which destroy or inactivate CR1. In previous studies we confirmed (data not shown), that erythrocyte CR1 recognition by rabbit anti-CR1–specific antiserum (R137) decays in parallel to erythrocyte incubation time with AET, and that AET-treated erythrocytes no longer bind opsonized promastigotes; again, after AET or trypsin treatment, no IA reaction was observed (Table I).…”

Anti‐T haemolysins: the effects of sialic acid removal and 2‐aminoethylisothiouronium bromide treatment of erythrocytes on immune lysis

“…In the present report, we evaluated the potency of anti-T haemolysins in vitro by comparing the levels of haemolysis produced by anti-T antibodies against T-activated RBCs, with those produced by anti-A and anti-B antibodies against group A1 and group B RBCs, respectively. To improve the detection of haemolysins, we used RBCs treated with 2-aminoethylisothiouronium bromide (AET), a sulphydryl reagent that partially or completely inactivates membrane complement regulatory proteins CD55 and CD59, respectively [12]. Using this sensitive haemolysis assay, anti-T haemolysins were found in sera of most blood donors but with lower activity compared with anti-A and anti-B antibodies.…”