Effect of anticoagulants on binding and neutralization of lipopolysaccharide by the peptide immunoglobulin conjugate CAP18(106-138)-immunoglobulin G in whole blood

Abstract

The 18-kDa cationic protein CAP18 is an antimicrobial protein isolated from rabbit granulocytes that binds lipopolysaccharide (LPS) and inhibits many of its biological activities. We covalently coupled a synthetic peptide representing amino acids 106 to 138 of CAP18 to human immunoglobulin G (IgG) by using the heterobifunctional linker N-succinimidyl-3-(2-pyridyldithio)propionate. The ability of CAP18 106-138-IgG to bind and neutralize LPS in whole blood in the presence and absence of anticoagulants was studied. Both CAP18 106-138 and CAP18 106-138-IgG significantly suppressed LPS-induced tumor necrosis factor (TNF) production in whole blood in the absence of anticoagulants. EDTA potentiated the ability of CAP18 106-138 and CAP18 106-138-IgG to decrease LPS-induced TNF production in a dose-dependent manner. In contrast, heparin inhibited the ability of CAP18 106-138 and CAP18 106-138-IgG to suppress LPS-induced TNF production. EDTA also enhanced LPS capture in a fluid-phase binding assay that utilizes magnetic anti-IgG beads to capture CAP18 106-138-IgG (and bound [ 3 H]LPS) in whole blood. In contrast, heparin inhibited the binding dose dependently. We conclude that CAP18 106-138-IgG binds to and neutralizes LPS in whole blood in the absence of anticoagulants. Further studies of its protective efficacy in animal models are warranted. Caution should be used in interpreting assays that measure the binding and neutralization of LPS in whole blood in the presence of calcium-binding anticoagulants or heparin.

Endotoxin‐neutralizing effects of histidine‐rich peptides

“…Because we were investigating the possible effects of Hn on LPS‐induced inflammatory responses in whole blood, heparin was precluded as an anticoagulant because it strongly enhances LPS responses [11]. EDTA, on the other hand, suppresses LPS‐induced cytokine production [18].…”

Influence of EDTA and heparin on lipopolysaccharide binding and cell activation, evaluated at single‐cell level in whole blood

“…This difference is connected to the differential effect of both anticoagulants on the neutrophil‐derived proteins CAP18 (18‐kDa cationic antimicrobial protein) and heparin binding protein (HBP/CAP37), which enhance (15) and abrogate (16) the LPS‐induced cellular activation, respectively. The inhibitory effect of CAP18 is enhanced in the presence of EDTA and removed in the presence of heparin (17). On the other hand, EDTA abrogates the enhancement of LPS‐induced TNF‐α production by HBP (18).…”

Neutralization of Endotoxin In Vitro and In Vivo by a Human Lactoferrin-Derived Peptide

“…Thus, in the cell assay, LBP in the serum can counteract the endotoxin-neutralizing activity of other serum LPS-binding molecules such as lipoproteins. In addition, since EDTA potentiates and divalent cations (Mg 2ϩ and Ca 2ϩ ) reduce the endotoxinneutralizing capacity of human serum (25,42), the presence of divalent cations in the incubation medium in the cell assay should further decrease the endotoxin-neutralizing capacity of the serum. a TNF-␣ secretion induced by endotoxin at 10 ng/ml.…”