EFFECT OF ANTICOAGULANTS ON BINDING AND NEUTRALIZATION OF LIPOPOLYSACCHARIDE BY THE PEPTIDE IMMUNOGLOBULIN CONJUGATE CAP18(106-138)-IMMUNOGLOBULIN-G IN WHOLE-BLOOD

The 18-kDa cationic protein CAP18 is an antimicrobial protein isolated from rabbit granulocytes that binds lipopolysaccharide (LPS) and inhi bits many of its biological activities. We covalently coupled a synthe tic peptide representing amino acids 106 to 138 of CAP18 to human immu noglobulin G (IgG) by using the hetero-bifunctional linker N-succinimi dyl-3-(2-pyridyldithio)propionate. The ability of CAP18(106-138)-IgG t o bind and neutralize LPS in whole blood in the presence and absence o f anticoagulants was studied. Both CAP18(106-138) and CAP18(106-138)-I gG significantly suppressed LPS-induced tumor necrosis factor (TNF) pr oduction in whole blood in the absence of anticoagulants. EDTA potenti ated the ability of CAP18(106-138) and CAP18(106-138)-IgG to decrease LPS-induced TNF production in a dose-dependent manner. In contrast, he parin inhibited the ability of CAP18(106-138) and CAP18(106-138)-IgG t o suppress LPS-induced TNF production, EDTA also enhanced LPS capture in a fluid-phase binding assay that utilizes magnetic anti-IgG beads t o capture CAP18(106-138)-IgG (and bound [H-3] 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 effi cacy in animal models are warranted. Caution should be used in interpr eting assays that measure the binding and neutralization of LPS in who le blood in the presence of calcium-binding anticoagulants or heparin.