title: Time-course analysis of C3a and C5a quantifies the coupling between the upper and terminal Complement pathways in vitro.
abstract: An in vitro zymosan-activation of the Complement system, through the lectin and alternative pathways, was performed in pooled human serum over a 24h 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.
authors.affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK.
authors.forename: Hassan O J
authors.initials: HO
authors.lastname: Morad
authors.affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK.
authors.forename: Samuel C
authors.initials: SC
authors.lastname: Belete
authors.affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK.
authors.forename: Thomas
authors.initials: T
authors.lastname: Read
authors.affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK. Electronic address: Andrew.m.shaw@exeter.ac.uk.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Shaw
mesh_terms: D003167:Complement Activation / Q000502:physiology*; D015926:Complement C3a / Q000378:metabolism*; D015936:Complement C5a / Q000378:metabolism*; D037582:Complement Pathway, Mannose-Binding Lectin / Q000502:physiology*; D006801:Humans; D011863:Radioimmunoassay; D013997:Time Factors
chemical_list: D015926:Complement C3a; D015936:Complement C5a
keywords: C3a; C5a; Complement; In vitro; Lower cascade; Upper cascade