Time-course analysis of C3a and C5a quantifies the coupling between the upper and terminal Complement pathways in vitro

• Serum C3a and C5a concentrations were assayed as markers of upper and terminal Complement activation.
• Upper Complement activation peaked as 39% of C3 molecules were converted to C3a.
• More than 31% upper Complement activation triggered significant activation of the terminal pathway.
• The molar ratio of C3a:C5a decreased from ~ 1000:1 to 17:1 once the terminal pathway was activated.
• Upper Complement (opsonisation) is 49-fold more active than the terminal pathway (cell lysis).

An in vitro zymosan-activation of the Complement system, through the lectin and alternative pathways, was performed in pooled human serum over a 24 h time-course. Activation was quantitatively monitored by measuring the concentration of the upper Complement pathway fragment, C3a and the terminal pathway fragment, C5a. Upper Complement showed a maximum activation of 39% and the time-to-maximum activation reduced 8-fold, as a highly non-linear function of the zymosan dose. The C3a:C5a molar ratio rose to a maximum of 1100:1, before terminal pathway activation was initiated; indicating a flux threshold. This threshold appears to be exceeded once more than 31% of C3 molecules are activated. Above this threshold, significant activation of terminal pathway was observed; reducing the molar ratio to 17:1. The C5a/C3a molar ratio was used to determine the terminal pathway activation relative to total Complement activation and ranged from 0.1–0.8%. This depicts upper Complement activation to be 49-fold larger than terminal activation, a figure consistent with the observed density of the membrane attack complex in the membrane of cells. Our results thus indicate that the relative activity of opsonisation is ~ 50-fold greater than membrane attack complex formation, in vitro, in the pooled serum phenotype. The results suggest a potential clinical application, where an in vitro analysis of a patient on admission, or prior to a surgical procedure, would indicate their upper Complement activation capacity, with activation of C3 measured thereafter, or post-operatively. A patient with an exhausted upper Complement capacity may be vulnerable to infections and complications, such as sepsis.

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