Role of Endotoxin-binding proteins in Non-specific Protection to Infection

内毒素是革兰氏阴性菌的细胞壁成分，能激活单核细胞和巨噬细胞释放细胞因子、反应性氮中间产物(RNI)，并产生组织因子(TF)，从而启动凝血。用Re-LPS包被的红细胞凝集法，从兔粒细胞中纯化了具有内毒素结合和中和活性的7 kDa和18 kDa阳离子抗菌蛋白(CAP-7和CAP-18)。蛋白质序列分析表明，CAP-7是CAP-18的37个氨基酸残基。合成肽#197(与CAP-7，Gly1-Try37序列相同)和#36-1(CAP的32个氨基酸残基的截短，Gly1-Ala32)具有与内毒素结合的活性。每种多肽均能抑制脂多糖诱导的小鼠腹腔巨噬细胞产生组织因子(TF)，甚至在LPS刺激细胞1h后加入。#197处理的C57BL/6小鼠对致死性LPS攻击有明显的保护作用。肽#36也叫…更多的是对内毒素诱导的致命性的担忧。这些多肽对革兰氏阴性菌如大肠杆菌、鼠伤寒沙门氏菌、肺炎克雷伯菌、铜绿假单胞菌以及革兰氏阳性金黄色葡萄球菌(甲氧西林敏感和耐药菌株)具有抗菌活性。两种多肽均能抑制Tf和Xa诱导的血浆凝血。利用合成的显色底物，这两个CAP7肽都在两个位置阻断了凝血级联反应，即X因子到Xa的激活和II因子(凝血酶原)到IIa因子(凝血酶原)的转化。多肽#197的活体治疗预防了注射组织因子(兔脑凝血活酶)的小鼠的急性死亡。另外两个多肽#32(Gly1-Phe9)和#50(Lle13-Typ37)未能显示出与内毒素结合、中和内毒素、抗菌和抗凝的活性。活性多肽，而不是非活性多肽，在它们的N端维持着一个假定的肝素结合域。肝素结合区参与内毒素的结合、中和、抗菌和抗菌作用，这些活性多肽可能对脓毒症和内毒素休克所致的DIC有治疗作用。较少

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 hour after stimulation of cells with LPS.C57BL/6 mice treated with #197 were significantly protected from lethal LPS challenge. Peptide #36 also blo … More cked 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(Methicllin sensitive and resistant strains). Both peptides inhibited TF-and Xa-induced plasma clotting. Using synthetic chromogenic 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(prothrombin). 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(lle13-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 anticoaThese active peptides may have a therapeutic potential for treatment for DIC due to sepsis and endotoxin shock. Less