The Initiation and Enhancement of Human Red Cell Lysis by Activators of the First Component of Complement and by First Component Esterase; Studies Using Normal Red Cells and Red Cells from Patients with Paroxysmal Nocturnal Hemoglobinuria*

The Hemolysis of Red Cells from Patients with Paroxysmal Nocturnal Hemoglobinuria by Partially Purified Subcomponents of the Third Complement Component*

“…The need for participation of fluid phase early C' components in PNHE lysis in acid serum is attested to by the dependence of acid hemolysis on Mg++ (2), implying that at least C'2 is involved in fluid phase events. Taken in the framework of our current knowledge of the role of C'2 in C'-dependent lysis, it seems reasonable to conclude that these C'2-mediated fluid phase events are preceded by action of C'1 (21) (and possibly C'4) and that they are concerned with rendering C'3a capable of attachment (possibly by activation) to PNHE (4,22,23). One can "drive" the entire sequence of early phase events and increase resultant PNHE lysis by adding to the system C'1 activators, C'la, or C'1 esterase (4).…”

“…The susceptibility of the PNHE hemolytic system to dilution implies that the fluid phase activation steps involved are not very efficient, and the lack of significant C' consumption during PNHE lysis confirms this impression (19). In addition, PNHE, lacking a C'-activating mechanism (antibody coat), are deprived of the ability to exert any direct influence on the initiation and localization of C' action (4,20). The need for participation of fluid phase early C' components in PNHE lysis in acid serum is attested to by the dependence of acid hemolysis on Mg++ (2), implying that at least C'2 is involved in fluid phase events.…”

“…One can "drive" the entire sequence of early phase events and increase resultant PNHE lysis by adding to the system C'1 activators, C'la, or C'1 esterase (4). That a delicate balance exists in this fluid phase mechanism is shown by the inhibition of PNHE lysis by an excess of these "driving" materials (4). This inhibition is probably due to inactivation of C'2 (possibly C'4) or C'3a or both.…”

“…The following have all been described previously: the preparation of barbital-buffered saline (BBS) at various pH's (3); the preparation of Na2MgEDTA-BBS containing 1.5 or 2.6 X 10-2 M Na2MgEDTA (3,5); the collection of guinea pig and human blood and the isolation and storage of their respective sera (3,4); the preparation and storage of sera deficient in the various components of C' and RP (4) ; the preparation of sensitized sheep red cells both with and without the use of Na3HEDTA-BBS (3); the preparation of EAhuC'1,4,2 and EAgpC'1,4,2 (3); the collection and storage of NHE and PNHE (4); the adjustment of serum pH (4); the preparation of serum containing EDTA salts (4); the estimation of in vitro acid hemolysis (4) ; the assay for C'1 esterase activity (4). Preparation of C'3a by TEAE chromatography C'3a was prepared as outlined by Mfiller-Eberhard, Nilsson, and Aronsson (6).…”

Studies of Paroxysmal Nocturnal Hemoglobinuria Erythrocytes: Increased Lysis and Lipid Peroxide Formation by Hydrogen Peroxide*

“…Although the disease is usually considered to be acquired, an abnormality intrinsic to the red cell is responsible for hemolysis (2)(3)(4)(5)(6). Extensive studies have helped to define the role of complement components in lysis of PNH red cells (7)(8)(9)(10)(11), but the nature of the intracorpuscular defect has not been clarified.…”