Antimicrobial antithrombin III peptide 1 Antimicrobial effects of helix D-derived peptides of human antithrombin III*

Antithrombin III (ATIII) is a key antiproteinase involved in blood coagulation. Previous investigations have shown that ATIII is degraded by Staphylococcus aureus V8 protease, leading to release of heparin binding fragments derived from its D helix. As heparin binding and antimicrobial activity of peptides frequently overlap, we here set out to explore possible antibacterial effects of intact and degraded ATIII. In contrast to intact ATIII, the results showed that extensive degradation of the molecule yielded fragments with antimicrobial activity. Correspondingly, the heparin-binding, helix D-derived, peptide FFFAKLNCRLYRKANKSSKLV (FFF21) of human ATIII, was found to be antimicrobial against particularly the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. Fluorescence microscopy and electron microscopy studies demonstrated that FFF21 binds to, and permeabilizes, bacterial membranes. Analogously, FFF21 was found to induce membrane leakage of model anionic liposomes. In vivo, FFF21 significantly reduced P. aeruginosa infection in mice. Additionally, FFF21 displayed anti-endotoxic effects in vitro. Taken together, our results suggest novel roles for ATIII-derived peptide fragments in host defense. Antithrombin III (ATIII) is a glycoprotein which is synthesized in the liver and circulates in plasma at a concentration of about 290 μg/ml (5 μM). ATIII is a serine protease inhibitor and inactivates thrombin and other serine proteases of the coagulation cascade (1). Patients with septic shock and disseminated intravascular coagulation (DIC) present reduced ATIII levels in plasma (2,3), mainly due to consumption during coagulation and extravascular leakage (1,4,5). In addition to the anticoagulant effects, ATIII exerts anti-inflammatory properties, including inhibition of nuclear factor κB activation in human monocytes and vascular endothelial cells (6), reduction of leukocyteendothelial interactions (7), and prevention of microvascular leakage (8-12). ATIII may also compete with bacterial toxins for binding on endothelial cell proteoglycans (13), thereby reducing the inflammatory response after Antimicrobial antithrombin III peptide 2 bacterial challenge (14). Interestingly, plasmaderived ATIII has been found to inhibit bacterial outgrowth and limited the inflammatory response, neutrophil influx and histopathological changes in Streptococcus pneumoniae pneumonia (15), although it was unclear whether these beneficial effects were due to prostacyclin formation, interference with bacterial toxins such as pneumolysin, or reduced coagulation and related modulation of extracellular proteins and peptides. Due to its therapeutical potential in inflammation and coagulation, ATIII has been evaluated in clinical investigations targeting sepsis (1,3,14-18), but not found to significantly affect mortality in patients with sepsis in a larger phase III clinical trial (19). Inhibition of thrombin by ATIII increases dramatically in the presence of heparin, and involves specific and complex allosteric mechanisms based on minute conformational changes leading to an increased affinity for the negatively charged polysaccharide (9,20-22). As such, ATIII displays similar features as HCII, a related serpin recently shown to exert antibacterial effects in vitro and in vivo (23). Hence, proteolytic cleavage of HCII induced a conformational change, thereby inducing endotoxin-binding and antimicrobial properties. Analyses employing representative peptide epitopes mapped these effects to helices A and D. Mice deficient in HCII showed increased susceptibility to invasive infection by Pseudomonas aeruginosa. Thus proteaseinduced uncovering of cryptic epitopes by scission of the N-terminal anionic region in HCII transforms the molecule into a host defense factor. Like in HCII, the region comprising helix D in ATIII constitutes a major heparin binding site (24). Notable are also the findings that ATIII may be degraded by neutrophil S. aureus V8 protease in vitro, generating heparin-binding peptides derived from the D-helix of ATIII (5). Taken together, the relatedness to HCII, the observation that ATIII contains a heparin binding region represented by helix D, the multifunctionality of ATIII, as well as the above mentioned suppressive effects on pneumococcal infections, prompted us to investigate possible antimicrobial effects exerted by ATIII or peptides derived from its helix D. EXPERIMENTAL PROCEDURES Materials. KTS43 kTSDQIHFFFAKLNCRLYRKANKSSKLVSA NRLFGDKSLTFNET, FFF21 (FFFAKLNCRLYRKANKSSKLVS), AKL22 (AKLNCRLYRKANKSSKLVSANR) and LL37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRN LVPRTES) were synthesized by Innovagen AB (Lund, Sweden). The purity (>95%?) and molecular weight was confirmed by MALDITOF MS analysis (Voyager, Applied