Studies on the Mechanism of Inactivation of Human Complement by Plasmin and by Antigen-Antibody Aggregates
Authors: Lepow Irwin H. , Pillemer Louis , Wurz Leona 
Published: January 2023

Discussion and Summary
The experiments which have been summarized above indicate that the inactivation of human C′ by antigen-antibody aggregates occurs in two distinct stages. The first, designated the activation stage, appears to be a reaction between antigen-antibody and C′1, resulting in the formation of an “activated aggregate.” This reaction occurs almost instantaneously at 1°C. It is not affected by Ca++ and is insensitive to changes in pH within the pH-stability range of C′1. It is gradually inhibited with increasing ionic strength but still proceeds partially at ionic strength 0.5. The second stage, designated the transfer stage, involves a reaction of the activated aggregate (or a product of the combination of antigen-antibody with C′1) with C′2 and C′4, resulting in inactivation of these components. The transfer stage occurs rapidly at 37°C and more slowly at lower temperatures. In further contradistinction to the activation stage, the transfer stage is potentiated by Ca++, has a pH optimum at or near pH 7, and a sharp ionic strength optimum at or near ionic strength 0.15. The characteristics of the activation stage, therefore, are those of an adsorption reaction, while the characteristics of the transfer stage suggest and enzymatic reaction.
Evidence is not yet available on the exact nature of thee raction between antigen-antibody and C′1 in the activation stage. The active factor for the inactivation of C′2 and C′4 could be a complex of antigen-antibody and adsorbed C′1. It is also possible that C′1 (or another member of the complex) is altered in some manner during the activation stage. In this case, the altered complex or a substance dissociating from the altered complex (i.e., “activated C′1”) would be the active factor for the transfer stage. Work is in progress on the mechanism of the activation stage and will be reported later.
Similarly, the mechanism of the transfer stage remains to be elucidated. While the general characteristics of the transfer stage suggest enzymatic inactivation of C′2 and C′4, this is yet to be demonstrated in more rigorous fashion. It is also possible that the transfer stage may be more complex than it has been necessary to assume thus far. Some apparent anomalies have already presented themselves: the inactivation of C′2 shows a greater time and pH dependency than C′4; the ionic strength optima for the inactivation of C′2 and C′4 do not coincide. The significance of these observations awaits further investigation.
The existence of at least two stages of complement fixation could have been predicted from the hypothesis put forward previously that C′1 may be an enzyme precursor and that its activation by antigen-antibody or plasmin would result in inactivation of C′2 and C′4 (2). This hypothesis implied a two-stage reaction and implied further that the second stage would have the characteristics of an enzymatic reaction. The experimental verification of these predictions is not taken as proof of the hypothesis but does lend it importance as a guide for continuing investigations.
It is not yet possible to evaluate the significance of the reactions just described in the phenomenon of immune hemolysis. If it is assumed that the pattern of events is fundamentally similar in complement fixation by aggregates of soluble antigen and antibody and in the reactions of immune hemolysis preceding the lytic stage or stages, then the relation of the two phenomena would be clear. In that case, the two stages of complement fixation described here would correspond to the “first stage” of immune hemolysis in the mechanisms which have been suggested by Pillemer et al. (16), Mayer and Levine (17, 18), and Silverstein (19). However, there is no direct evidence yet available on which to base this assumption.