Characterization of a rabbit cationic protein (CAP18) with lipopolysaccharide-inhibitory activity.

Abstract

Cationic antibacterial proteins (CAP) were purified from rabbit granulocytes, and the effects of CAP on lipopolysaccharide (LPS)-induced tissue factor generation by murine peritoneal macrophages and human blood monocytes were studied. CAP were purified from rabbit peritoneal leukocytes by using as an assay the agglutination of erythrocytes coated with Re-LPS. Two proteins with CAP activity, CAP18 (18 kDa) and CAP7 (7 kDa), were isolated by acid extraction, ethanol precipitation, affinity chromatography, gel filtration, and reverse-phase high-pressure liquid chromatography. On the basis of protein sequencing, CAP7 was identified as the C-terminal fragment of CAP18, designated CAP18(106-142). Various forms of LPS (S-LPS, Re-LPS, and lipid A) activate murine macrophages and human blood monocytes to generate tissue factor (tissue thromboplastin). Incubation of LPS for 18 h with partially purified CAP (heparin-Sepharose fraction) inhibited the capacity of LPS to induce tissue factor; however, purified CAP18 inhibited about 75% of the activity of S-LPS after 1 h of incubation. CAP more effectively inhibited S-LPS than Re-LPS or lipid A. Synthetic CAP18(106-142) inhibited LPS-induced tissue factor generation by murine macrophages. CAP18(106-142) has greater LPS-binding and LPS-neutralizing activities than CAP18. We hypothesize that CAP18 and the derivative peptide, CAP18(106-142), bind to LPS and alter the capacity of LPS to initiate disseminated intravascular coagulation. In this regard, CAP may have therapeutic potential for sepsis and endotoxin shock.