Endotoxin-binding synthetic peptides with endotoxin-neutralizing, antibacterial and anticoagulant activities.
Endotoxin(lipopolysaccharide = LPS), cell wall component of gram-negative bacteria, activates monocytes and macrophages to release cytokines, reactive nitrogen intermediates (RNI), and to generate tissue factor(TF) which initiate coagulation. We have purified 7kDa and 18kDa cationic antibacterial proteins (CAP-7 and CAP-18) with LPS-binding and LPS-neutralizing activities from rabbit granulocytes using as an assay the agglutination of erythrocytes coated with Re-LPS. From protein sequencing, CAP-7 was identified as the C-terminal 37 amino acid fragment of CAP-18. Synthetic peptide #197 (identical sequence to CAP-7, Gly1-Try37) and #36-1 (a truncation of CAP consisting of 32 amino acid residues, Gly1-Ala32) showed LPS-binding activity. Each peptide inhibited LPS-induced tissue factor(TF) generation by murine peritoneal macrophages, even added 1-3 hours after stimulation of cells with LPS. C57BL/6 mice treated with #197 were significantly protected from lethal LPS challenge. Peptide #36 also blocked the LPS-induced lethality. These peptides had antibacterial activity to gram-negative bacteria, such as E.coli, S.typhimurium, K.pneumonia, Ps.aeruginosa and also to gram-positive S.aureus (Methicillin sensitive and resistant strains). Both peptides inhibited TF- and Xa-induced plasma clotting. Using synthetic chromotogenic substrates, both CAP7 peptides blocked the coagulation cascade at two sites, activation of factor X to Xa and conversion of Factor II (prothrombin) to factor IIa (thrombin). In vivo treatment of peptide #197 prevented acute lethality in mice injected with tissue factor (rabbit brain thromboplastin). Two other peptides, #32(Gly1-Phe9) and #50(Ile13-Typ37) failed to demonstrate LPS-binding, LPS-neutralizing, antibacterial and anticoagulant activities. The active peptides but not the inactive peptide maintain a putative heparin binding domain at their N-termini. This heparin binding domain is participate in the LPS-binding, LPS neutralizing, antibacterial and anticoagulant activities of CAP7. These active peptides may have a therapeutic potential for treatment for DIC due to sepsis and endotoxin shock.
Hirata
M
M
Department of Bacteriology, School of Medicine, Iwate Medical University, Morioka, Japan.
Shimomura
Y
Y
Yoshida
M
M
Wright
S C
SC
Larrick
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides; D001777:Blood Coagulation / Q000187:drug effects; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D054804:Cathelicidins; D004731:Endotoxins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D005170:Factor X / Q000037:antagonists & inhibitors; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000633:toxicity; D051379:Mice; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010455:Peptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D013925:Thromboplastin / Q000037:antagonists & inhibitors
D000900:Anti-Bacterial Agents; D000925:Anticoagulants; D023181:Antimicrobial Cationic Peptides; D002352:Carrier Proteins; D054804:Cathelicidins; D004731:Endotoxins; D008070:Lipopolysaccharides; D010455:Peptides; D005170:Factor X; D013925:Thromboplastin