Field of the invention The present invention relates to novel polypeptides derived from S1 serine peptidases (other than thrombin) and their use in the treatment and prevention of inflammation. In particular, the invention provides polypeptides comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 1 to 1 13, or a fragment, variant, fusion or derivative thereof, or a fusion of said fragment, variant or derivative thereof, for use in medicine, e.g. in the treatment or prevention of inflammation and/or excessive coagulation of the blood.

Introduction

Infectious and inflammatory diseases account for millions of deaths worldwide each year and incur tremendous health care costs. The disease spectrum is broad and includes acute disease, such as erysipelas, sepsis, pneumonia and numerous other infections, having a direct association to a given pathogen, as well as chronic diseases, where microbes often cause a long-standing inflammatory state. Sepsis is an infection-induced syndrome characterized by a generalized inflammatory state and represents a frequent complication in the surgical patient, in immunocompromized patients, or in relation to burns. Severe sepsis is a common, expensive and frequently fatal condition, having a documented worldwide incidence of 1.8 million each year, but this number is confounded by a low diagnostic rate and difficulties in tracking sepsis in many countries. It is estimated that with an incidence of 3 in 1000 the true number of cases each year reaches 18 million, and with a mortality rate of almost 30% it becomes a leading cause of death worldwide. Sepsis costs on average US$22 000 per patient, and its treatment therefore has a great impact on hospitals' financial resources, with US$16.7 billion each year being spent in the USA alone. The cost of treating an ICU patient with sepsis is six times greater than that of treating a patient without sepsis. In other settings, harmful inflammatory cascades are initiated by other mechanisms than bacterial, such as during trauma, surgery, extracorporeal circulation, ischemia, burns, drug reactions, hemorrhagic shock, toxic epidermal necrolysis, transfusion reactions, leading to ARDS or SIRS. Chronic obstructive pulmonary disorder (COPD) refers to a range of chronic disorders in the airways characterized by irreversible and progressing decline in airflow to the lung capillaries. Although several factors contribute to the development of COPD, smoking and recurring infections are the most important causes. COPD predominantly develops in long-term smokers from their late-30s and progressively develops in an irreversible fashion. According to 2007 estimates by WHO, there are currently 210 million patients with COPD, and 3 million people died of COPD in 2005. WHO also predicts that COPD will become the fourth leading cause of death worldwide by 2030. Several factors are expected to contribute to this increase, including increased diagnosis rates, lack of treatments that reverse the inflammatory disease progression, and a globally ageing population burden. Microbes cause, and/or aggravate, a spectrum of diseases including bacterial conjunctivitis and keratitis, o titis, postoperative and burn wound infections, chronic leg ulcers, pneumonia, and cystic fibrosis.

New agents addressing infection are therefore needed, and there is significant interest in the potential use of AMPs as novel treatment modalities (Marr, A. K., W. J. Gooderham, et al. (2006). Curr Opin Pharmacol 6(5): 468-472). Considering the increasing resistance problems against conventional antibiotics, antimicrobial peptides have recently emerged as potential therapeutic candidates. AMPs provides a first line of defense against invading microbes in almost all organisms (Tossi, A., L. Sandri, et al. (2000). Biopolymers 55(1): 4-30; Lehrer, R. I. and T. Ganz (2002). Curr Opin Hematol 9(1): 18- 22; Zasloff, M. (2002). Nature 415(6870): 389-95; Yount, N. Y., A. S. Bayer, et al. (2006). Biopolymers 84: 435-458, Harder, J., R. Glaser, et al. (2007). J Endotoxin Res 13(6): 317-38). Ideally, AMP should display high bactericidal potency, but low toxicity against (human) eukaryotic cells. Various strategies, such as use of combinational library approaches (Blondelle, S. E. and K. Lohner (2000). Biopolymers 55(1 ): 74-87), stereoisomers composed of D-amino acids (Sajjan, U. S., L. T. Tran, et al. (2001 ). Antimicrob Agents Chemother 45(12): 3437-44) or cyclic D,L-ct-peptides ( Fernandez- Lopez, S., H. S. Kim, et al. (2001). Nature 412(6845): 452-5), high-throughput based screening assays (Hilpert, K., R. Volkmer-Engert, et al. (2005). Nat Biotechnol 23(8): 1008-12; Taboureau, O., O. H. Olsen, et al. (2006). Chem Biol Drug Pes 68(1): 48-57), quantitative structure-activity relationship (QSAR) approaches (Hilpert, K., R. Volkmer- Engert, et al. (2005). Nat Biotechnol 23(8): 1008-12; Marr, A. K., W. J. Gooderham, et al. (2006). Curr Opin Pharmacol 6(5): 468-472; Jenssen, H., T. Lejon, et al. (2007). Chem Biol Drug Pes 70(2): 134-42; Pasupuleti, M., B. Walse, et al. (2008). Biochemistry 47(35): 9057-70), and identification of endogenous peptides ( Papareddy, P., V. Rydengard, et al. PLoS Pathoq 6(4): e1000857; Nordahl, E. A., V. Rydengard, et al. (2005). J Biol Chem 280(41 ): 34832-9; Malmsten, M., M. Davoudi, et al. (2006). Matrix Biol 25(5): 294-300; Malmsten, M., M. Davoudi, et al. (2007). Growth Factors 25(1): 60- 70; Pasupuleti, M., B. Walse, et al. (2007). J Biol Chem 282(4): 2520-8) are currently employed for identifying selective and therapeutically interesting AMPs (Hancock, R. E. and H. G. Sahl (2006). Nat Biotechnol 24(12): 1551-7; Marr, A. K., W. J. Gooderham, et al. (2006). Curr Opin Pharmacol 6(5): 468-472). Despite the potential of these approaches, naturally occuring peptide epitopes may show advantages in a therapeutic setting considering low immunogenecity as well as inherent additional biological functions.

The present invention seeks to provide new polypeptide agents for use in the treatment or prevention of inflammation and/or excessive coagulation of the blood.

Summary of the invention

A first aspect of the invention provides a polypeptide comprising or consisting of an amino acid sequence from the C-terminal region of an S1 serine peptidase other than thrombin, or a fragment, variant, fusion or derivative thereof, or a fusion of said fragment, variant or derivative thereof, for use in the treatment or prevention of inflammation and/or excessive coagulation of the blood, wherein the fragment, variant, fusion or derivative exhibits an anti-inflammatory and/or anti-coagulant activity.

The invention derives from the unexpected discovery by the inventors that S1 serine peptidase enzymes comprise "cryptic peptides" within their C-terminal region, which exhibit anti-inflammatory activity. It is believed that such peptides may be 'released' by cleavage of the parent peptidase holoprotein in response to wounding and other physiological challenges. Thus, the polypeptides of the invention constitute a novel and previously undisclosed class of HDPs, which have therapeutic potential against disorders and conditions associated with inflammation.

By "S1 serine peptidase" we mean a class of enzymes that catalyse the hydrolysis of peptide bonds in proteins (EC 3.4.21.x). The peptidases of family S1 belong to the chymotrypsin family and contain the catalytic triad residues His, Asp and Ser in the active site. All of the characterised peptidases of the chymotrypsin family are endopeptidases. There are three main activity types: trypsin-like where there is cleavage of amide substrates following Arg or Lys at P1 of the substrate peptide, chymotrypsin-like where cleavage occurs following one of the hydrophobic amino acids at P1 , and elastase-like with cleavage following an Ala at position P1. Substrate specificity in family S1 is dependent only on what is in the P1 position. The majority of the peptidases of this family enter the secretory pathway and have an N-terminal signal peptide. They are synthesized as precursors with an N-terminal extension that is cleaved to form the active enzyme. Activation does not always require the propeptide to be removed; in the blood coagulation factors cleavage may be sufficient, with the propeptide remaining bound by disulfide bridges as the heavy chain.

The S1 serine peptidases all share the same structural motif where the proteins are folded into two domains, each containing an open-ended beta barrel at right angles to each other (Fig. 2e). This crossing pattern of the beta strands in the barrel has been described as a "Greek Key". The carboxy terminus of all these proteins ends with an alpha helix (Fig. 2e).

S1 serine peptidases are described in Page & Cera, 2008, Cell Mol Life Sci. 65(7- 8): 1220-36.

By "C-terminal region" we mean that the one hundred amino acids adjacent the C- terminus of the parent S1 serine peptidase. Thus, in a hypothetical S1 serine peptidase of 600 amino acids in length, wherein the amino acid sequence is specified in the conventional N-terminus to C-terminus direction, the C-terminal region corresponds to amino acid residues 501 to 600.

Thus, the polypeptides of the invention comprise or consist of a sequence of amino acids from within this C-terminal region, and preferably at least 20 contiguous amino acids from within this region.

By "anti-inflammatory activity" we mean an ability to reduce or prevent one or more biological processes associated with inflammatory events. Such anti-inflammatory activity of polypeptides may be determined using methods well known in the art, for example by measuring LPS-induced release of pro-inflammatory cytokines from macrophages (e.g. TNFa, IL-6, IF-γ), or neutrophils (see Examples below). Other relevant assays comprise effects of lipoteichoic acid, zymosan, DNA, RNA, flagellin or peptidoglycan in the above systems as well as determination of regulation at the transcriptional level (e.g. Gene-array, qPCR etc). Furthermore, dendritic cell activation or activation of thrombocytes may also be used as a measure of anti-inflammatory activity.

By "anti-coagulant activity" we mean an ability to increase the prothrombin time (PT), the thrombin clotting time (TCT) and/or the activated partial thromboplastin time (aPTT). Alternatively, peripheral blood mononuclear cells (PBMNC)s can be stimulated by E. coli LPS with or without the peptide and tissue factor and clot formation followed after addition of human plasma, or clotting times for whole blood can be measured. It will be appreciated by persons skilled in the art that the S1 serine peptidase may be from a human or non-human source. For example, the S1 serine peptidase may be derived (directly or indirectly) from a non-human mammal, such as an ape (e.g. chimpanzee, bonobo, gorilla, gibbon and orangutan), monkey (e.g. macaque, baboon and colobus), rodent (e.g. mouse, rat) or ungulates (e.g. pig, horse and cow).

In one preferred embodiment, the S1 serine peptidase is human S1 serine peptidase.

For example, the S1 serine peptidase may be selected from the group of S1 serine peptidases listed in Table A below (by reference to UniProt KB / Swiss Prot accession number):

Table A - Exemplary S1 serine peptidases

P10323 ACRO_HUMAN

P08519 APOA_HU AN

Q9GZN4 BSSP4_HUMAN

P00736 C1 R_HUMAN

Q9NZP8 C1 RL_HUMAN

P09871 C1 S_HUMAN

P20 60 CAP7_HUMAN

P0831 1 CATG_HU AN

P08217 CEL2A_HUMAN

P08218 CEL2B_HUMAN

P09093 CEL3A_HUMAN

P08861 CEL3B_HUMAN

Q9UNI1 CELA1_HUMAN

P00751 CFAB_HUMAN

P00746 CFAD_HU AN

P05156 CFAI_HUMAN

P23946 CMA1_HUMAN

P06681 C02_HUMAN

Q9Y5Q5 CORIN_HUMAN

P17538 CTRB1_HU AN

Q6GPI1 CTRB2_HUMAN

Q99895 CTRC HUMAN P40313 CTRL_HUMAN

P08246 ELNE_HUMAN

£98073 ENTK_HUMAN

P00742 FA10_HUMAN

P03951 FA11_HUMAN

P00748 FA12 HUMAN

P08709 FA7_HUMAN

P00740 FA9JHUMAN

P 12544 GRAA HUMAN

P10144 GRABJHUMAN

P20718 GRAHJHUMAN

P49863 GRAK HUMAN

P51124 GRAM_HUMAN

Q 14520 HABP2_HUMAN

P05981 HEPS HUMAN

P14210 HGF_HUMAN

QQ4756 HGFAJ-1UMAN

P26927 HGFL_HUMAN

P00738 HPT HUMAN

PQ0739 HPTRJHUMAN

P06870 KLK1_HUMAN

043240 KLK10_HUMAN

Q9UBX7 KLK11_HUMAN

Q9UKR0 KLK12_HUMAN

Q9UKR3 KLK13_HUMAN

Q9P0G3 KLK14_HUMAN

Q9H2R5 KLK15_HUMAN

P20151 KL 2_HUMAN

P07288 KLK3_HUMAN

Q9Y5K2 KLK4_HUMAN

Q9Y337 KLK5_HU AN

Q92876 KLK6_HU AN

P49862 KLK7_HUMAN

060259 KLK8_HUMAN

Q9UKQ9 KLK9_HUMAN

P03952 KLKB1_HUMAN

P48740 MASP1_HUMAN

000187 MASP2_HUMAN

Q2TV78 MSTP9 HUMAN

P56730 NETR_HUMAN

Q7RTY7 OVCH1_HUMAN

Q7RTZ1 0VCH2_HUMAN

Q6UXH9 PAMR1_HUMAN

P00747 PLMN_HUMAN

Q5K4E3 P0LS2JHUMAN

P04070 PROC_HUMAN

P22891 PROZ_HUMAN

095084 PRS23_HUMAN

Q9BQR3 PRS27_HUMAN

A6NIE9 PRS29_HUMAN

Q81VY7 PRS30_HUMAN

Q8NF86 PRS33_HUMAN

Q8N3Z0 PRS35_HUMAN

A4D T9 PRS37_HUMAN

A1L453 PRS38 HUMAN Q7RTY9 PRS41JHUMAN

Q7Z5A4 PRS42J-IUMAN

Q7RTY3 PRS45JHUMAN

Q7RTY5 PRS48_HUMAN

Q2L4Q9 PRS53_HUMAN

Q6PEW0 PRS54_HUMAN

Q6UWB4 PRS55JHUMAN

Q6UWY2 PRSL1 HUMAN

Q16651 PRSS8_HUMAN

P2 158 PRTN3_HUMAN

Q9Y5Y6 ST14_HUMAN

Q9Y6M0 TESTJHUMAN

P00734 THRB_HUMAN

Q6Z R5 TM11A_HUMAN

Q86T26 TM11 B_HUMAN

060235 TM11 DJHUMAN

Q9UL52 TM11E_HUMAN

Q6ZWK6 TM11 F_HUMAN

015393 TMPS2_HUMAN

P57727 TMPS3_HUMAN

Q9NRS4 TMPS4JHUMAN

Q9H3S3 TMPS5_HUMAN

Q8IU80 TMPS6JHUMAN

Q7RTY8 TMPS7_HUMAN

Q7Z410 TMPS9_HUMAN

Q86WS5 TMPSC_HUMAN

Q9BYE2 TMPSD HUMAN

P00750 TPAJHUMAN

P07477 TRY1_HUMAN

P07478 TRY2_HUMAN

P35030 TRY3_HUMAN

Q8NHM4 TRY6_HUMAN

P15157 TRYA1_HUMAN

Q15661 TRYB1_HUMAN

P20231 TRYB2_HUMAN

Q9BZJ3 TRYD_HUMAN

Q9NRR2 TRYG1_HUMAN

Q8IYP2 TRYX3_HUMAN

Q9UI38 TSP50_HUMAN

P00749 UROK_HUMAN

A8MTI9 YI033 HUMAN

In one embodiment, the S1 serine peptidase is selected from the group consisting of Factor X, Kallikrein 8, Hyaluronan binding protein 2, granzyme B, apolipoprotein A, Protein C and Plasminogen.

Thus, in a preferred embodiment, the S1 serine peptidase is human factor X (for example, see Swiss-Prot Accession No. P00742). In a preferred embodiment, the S1 serine peptidase is Kallikrein 8 (for example, see Swiss-Prot Accession No. 060259).

In a further preferred embodiment, the S1 serine peptidase is Hyaluronan binding protein 2 (for example, see Swiss-Prot Accession No. P05981 ).

In a further preferred embodiment, the S1 serine peptidase is granzyme B (for example, see Swiss-Prot Accession No. Ρ10Ί44). In a further preferred embodiment, the S1 serine peptidase is apolipoprotein A (for example, see Swiss-Prot Accession No. P08519).

In a further preferred embodiment, the S1 serine peptidase is Protein C (for example, see Swiss-Prot Accession No. P04070).

In a further preferred embodiment, the S1 serine peptidase is Plasminogen (for example, see Swiss-Prot Accession No. P00747)

It will be appreciated by persons skilled in the art that the invention encompasses polypeptides comprising or consisting of an amino acid sequence from the C-terminal region of an S1 serine peptidase, as well as fragments, variants, fusions and derivatives of such amino acid sequence which retain an anti-inflammatory activity. Preferably, however, the polypeptide is not a naturally occurring protein, e.g. a holoprotein (although it will, of course, be appreciated that the polypeptide may constitute an incomplete portion or fragment of a naturally occurring protein).

In one embodiment, the polypeptide comprises or consists of an amino acid sequence from the C-terminal region of an S1 serine peptidase. In another embodiment, the polypeptide comprises an alpha helix domain. By "alpha helix domain" we mean an amino acid sequence which may adopt an alpha helix configuration under physiological conditions, and having an amphipathic character. It will be appreciated by persons skilled in the art that the alpha helix domain may adopt a helix configuration when in the parent holoprotein (under physiological conditions), but may not necessarily do so in the peptide of the invention. For example, the amino acids in an alpha helix may be arranged in a right-handed helical structure where each amino acid corresponds to a 100° turn in the helix (i.e., the helix has 3.6 residues per turn), and a translation of 1.5 A (= 0.15 nm) along the helical axis. The pitch of the helix (the vertical distance between two points on the helix) is 5.4 A (= 0.54 nm) which is the product of 1.5 and 3.6. Most importantly, the N-H group of an amino acid forms a hydrogen bond with the C=0 group of the amino acid four residues earlier; this repeated i + 4 to i hydrogen bonding defines an alpha-helix. Residues in o helices typically adopt backbone (φ, ψ) dihedral angles around (-60°, -45°). More generally, they adopt dihedral angles such that the ψ dihedral angle of one residue and the φ dihedral angle of the next residue sum to roughly -105°. Consequently, alpha- helical dihedral angles generally fall on a diagonal stripe on the Ramachandran plot (of slope -1 ), ranging from (-90°, -15°) to (-35°, -70°).

In a further embodiment, the polypeptide comprises a heparin-binding domain. By "heparin-binding domain" we mean an amino acid sequence within the polypeptide which is capable of binding heparin under physiological conditions. The sequences often comprise XBBXB (as found in SEQ ID:1 ) and XBBBXXB (where B = basic residue and X = hydropathic or uncharged residue), or clusters of basic amino acids (XBX, XBBX, XBBBX). Spacing of such clusters with non-basic residues (BXB, BXXB) may also occur. Additionally, a distance of approximately 20 A between basic amino acids constitutes a prerequisite for heparin-binding.

However, in an alternative embodiment, the polypeptide does not comprise a heparin- binding domain.

One preferred embodiment of the first aspect of the invention provides polypeptides comprising or consisting of an amino acid sequence of SEQ ID NO:1

X1-X2-X3-X4-X5-X6- 7-X8-X9-X10-X1I- 12-X13- 14- l5" l6"Xl7-Xl8- l9- 20

[SEQ ID NO: 1] wherein

XL X ₁₆ and Xi ₈ independently represent any amino acid;

X ₂ represents G, F, L, P, R, S, V or Y; X ₃ represents any amino acid except C, L or Q;

X ₄ represents A, F, G, I, L, T, V or Y;

X ₅ represents A, C, F, H, I, P, R, T or Y;

X _B represents A, G, I, L, S, T or V;

X ₇ represents A, D, G, H, K, L, N, P, Q, R, S or Y

X _B represents A, C, I, L, P, S, T, V or Y;

X ₉ represents any amino acid except D or R;

X ₁₀ represents any amino acid except T;

Xn represents any amino acid except A, G or P;

X ₁₂ represents any amino acid except G or Y;

X ₁₃ represents any amino acid except F, I, M or W;

Xi ₄ represents W;

Xi5 represents I, L or V

X ₁₇ represents any amino acid except C, I, L or P;

X ₁₉ represents A, D, F, I, L, M, T, V, W or Y; and

X ₂₀ represents a sequence of any amino acids having a length of between 1 and 58 amino acids.

A further preferred embodiment of the first aspect of the invention provides polypeptides comprising or consisting of an amino acid sequence of SEQ ID NO.2

X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11- 12-X13-X14- X15-X16 [SEQ ID NO:2] wherein

Xi, 3, X7, Xg _> X10. X11 , X ₁ 2 and X ₁₃ independently represent any amino acid;

X ₂ represents P, F or Y;

X ₄ represents A, F, I, L or V;

X ₅ represents A, F or Y;

X ₆ represents A, I, T or V;

X ₈ represents I, L or V;

X ₁₄ represents W;

X ₁₅ represents I or L; and

X16 represents a sequence of any amino acids having a length of between 5 and 26 amino acids. Advantageously, the polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOS: 3 to 1 12, or a fragment, variant, fusion or derivative of said sequence, or a fusion of said fragment, variant or derivative thereof which exhibits an anti-inflammatory and/or antimicrobial and/or anti-coagulant activity:

KYGIYTKVTAFLKWIDRSMK ("FA10") [SEQ ID NO:3]

KPGVYTNICRYLDWIKKIIG ("NRPN" or " KLK8") [SEQ ID NO:4]

RPGVYTQVTKFLNWIKATIK ("HABP2") [SEQ ID NO:5]

QYGVYTKVINYIPWIENIIS ("MASP2") [SEQ ID NO:6]

GYGFYTKVLNYVDWI KEME ("C ") [SEQ ID Νθ-.η

TYGLYTRVKNYVDWIMKTMQ ("C1 S") [SEQ ID NO:8]

NYGVYTKVSRYLDWIHGHIR ("PROC") [SEQ ID NO:9]

HFGVYTRVSQYIEWLQKLMR ("FA7") [SEQ ID NO:10]

K YG I YTKVS R YVNWI KE KTK ("FA9") [SEQ ID NO:11]

FPGVYTKVANYFDWISYHVG ("CFAI") [SEQ ID NO:12]

R PGVYTNWE YVDWI LE KTQ ("FA11") [SEQ ID NO:13]

QPGVYTKVAEY DWILEKTQ ("KL B1 ") [SEQ ID NO:14]

KPGVYTDVAYYLAWIREHTV ("FA12") [SEQ ID NO:15]

KPGVYTRVANYVDWINDRIR ("HGFA") [SEQ ID NO: 16]

VPGVYTKVTNYLDWIRD MR ("TPA") [SEQ ID NO:17]

KPGVYTRVSHFLPWIRSHTK ("UROK") [SEQ ID NO:18]

SPGVYTKVSAFVPWIKSVTK ("NETR") [SEQ ID NO: 19]

TPGVYTKVSAYLNWIYNVWK ("TMPS4") [SEQ ID NO:20]

RPGVYARVSRFTEWIQSFLH ("ENTK") [SEQ ID NO:21]

KPGVYTKVSDFREWIFQAIK ("HEPS") [SEQ ID NO:22]

KPGVYTRVTSFLDWIHEQME ("TMPS3") [SEQ ID NO:23]

HPGVYAKVAEFLDWIHDTAQ ("T PS5") [SEQ ID NO:24]

KPGVYTRLPLFRDWIKENTG ("ST14") [SEQ ID NO:25]

FPGVYTRVSNFVPWIHKYVP ("TMPS7") [SEQ ID N0.26]

PGVYTRVTAYLDWIRQQTG ("TM1 1 D") [SEQ ID NO:27]

KPGVYTRVTALRDWITSKTG ("T 1 1 E") [SEQ ID NO:28]

YFGVYTRITGVISWIQQ\A/T ("TMPS6") [SEQ ID NO:29]

RPGVYARVTRLRDWILEATT ("TMPS9") [SEQ ID NO:30]

RPGVYIRVTAHHNWIHRIIP ("PRS27") [SEQ ID NO:31]

RPGIYTRVTYYLDWIHHYVP ("TRYA1 ") [SEQ ID NO:32]

RPGIYTRVTYYLDWIHHYVP ("TRYB1 ") [SEQ ID NO:33]

RPGIYTRVTYYLDWIHHYVP ("TRYB2") [SEQ ID NO:34]

GPDFFTRVALFRDWIDGVLN ("CAP7") [SEQ ID NO:35]

KPGVYTKVCHYLEWIRETMK ("KLK15") [SEQ ID NO:36]

YPDAFAPVAQFVNWIDSIIQ ("ELNE") [SEQ ID NO:37]

FPDFFTRVALYVDWIRSTLR ("PRTN3") [SEQ ID NO:38]

KPGIYTRVASYAAWIDSVLA ("CFAD") [SEQ ID NO:39]

KPPVATAVAPYVSWIRKVTG ("GRAM") [SEQ ID NO:40]

PPAVFTRISHYRPWINQILQ ("MCPT1 " or "CMA1") [SEQ ID NO:41]

PPEVFTRVSSFLPWIRTTMR ("CATG") [SEQ ID NO:42]

PPGVYIKVSHFLPWIKRTMK ("GRAH") [SEQ ID NO:43]

KPGVYVRVSRFVTWIEGVMR ("PLMN") [SEQ ID NO:44]

WPAVFTRVSVFVDWIHKVMR ("HGFL") [SEQ ID NO:45]

RPGIFVRVAYYAKWIHKIIL ("HGF") [SEQ ID NO:46]

HPAVYTQICKYMSWINKVIR ("KLK10") [SEQ ID NO:47]

I P G VYTY I C K YV D W I R M I M R ("KLK12") [SEQ ID NO:48] KPGVYTKVCKYVDWIQETMK "KLK11 ") [SEQ ID NO:49] RPAVYTSVCHYLDWIQEIME "KLK9") [SEQ ID NO:50] YPGVYTNLCKYRSWIEET R "KLK14") [SEQ ID NO:51] RPGVYTRVSRYVLWIRETIR "KLK13") [SEQ ID NO:52] KPGVYTNVCRYTNWIQKTIQ "KLK6") [SEQ ID NO:53] DPGVYTQVCKFTKWINDT K "KLK7") [SEQ ID NO:54] VPG VYTN LC KFTEWI EKTVQ "KLK4") [SEQ ID NO:55] RPGVYTNLCKFTKWIQETIQ "KLK5") [SEQ ID NO:56] KPSVAVRVLS YVKWI EDTIA "KLK1") [SEQ ID ΝΟ:5η KPAVYTKWH YRKWI KDTIA "KLK2") [SEQ ID NO:58] RPSLYTKWHYRKWIKDTIV "KLK3") [SEQ ID NO:59] KPGVYTKVYN YVKWI KNTIA "TRY1") [SEQ ID NO:60] RPGVYTKVYNYVDWI KDTIA "TRY2") [SEQ ID NO:61] RPGVYTKVYNYVDWI KDTIA "TRY3") [SEQ ID NO:62] APAVYTRVSKFSTWINQVIA "CTRL") [SEQ ID NO:63] KPWYTRVSAYIDWINEK Q "CLCR" or "CTRC") [SEQ ID NO:64] KPSVFTRVSNYIDWINSVIA "ELA2A" or "CEL2A") [SEQ ID NO:65] KPSIFTRVSNYNDWINSVIA "ELA2B" or "CEL2B") [SEQ ID NO:66] KPTVFTQVSAYISWINNVIA "ELA1 " or "CELA1") [SEQ ID NO:67] KPTVFTRVSAFIDWIEETIA "ELA3A" or "CEL3A") [SEQ ID NO:68] KPTVFTRVSAFIDWIEETIA "ELA3B" or "CEL3B") [SEQ ID NO:69] GYDFYTKVLSYVDWIKGV N "C1 RL") [SEQ ID NO:70] SRELFAAIGPEEAWISQTVG "POLS2") [SEQ ID NO:71] LSTAFTKVLPFKDWIERNMK "PAMR1 ") [SEQ ID NO:72] ARDFHINLFQVLPWLKEKLQ "CFAB") [SEQ ID NO:73] PRDFHINLFRMQPWLRQHLG "C02") [SEQ ID NO:74] VRITPLKYAQICYWIKGNYL "PRS23") [SEQ ID NO:75] VRITPLKYAQICLWIHGNDA "PRS35") [SEQ ID NO:76] DVGIYTNVYKYVSWIENTAK "TRYX2" or "PRS37") [SEQ ID NO:77] DVGIYAKIFYYIPWIENVIQ "TRYX3") [SEQ ID NO:78] EYGVYVKVTS I Q HWVQKTI A "HPTR") [SEQ ID NO:79] EYGVYVKVTSIQDWVQKTIA "HPT") [SEQ ID NO:80] NYSVYTKVSRYLDWIHGHIR "PROC" or "variant Q8J006") [SEQ ID NO:81] RYGVYSYIHHNKDvVIQRVTG "MASP1") [SEQ ID NO:82] RPAVFTALPAYEDWVSSLDW "POLS3" or "PRS53") [SEQ ID NO:83] RPGVYTQVLSYTDWIQRTLA "PRS30") [SEQ ID NO:84] APPIYLQVSSYQHWIWDCLN "TSP50") [SEQ ID NO:85] NPGVYTRITKYTKWIKKQMS "TSSP5" or "PRS45") [SEQ ID NO:86] GLFLYTKVEDYSKWITSKAE "KLBL4" or "PRS54") [SEQ ID NO:87] SPGIFTDISKVLPWIHEHIQ "OVCH2") [SEQ ID NO:88] RPGIYTATWPYLNWIASKIG "ACRO") [SEQ ID NO:89] FPGVYIGPSFYQKWLTEHFF "TMPSC") [SEQ ID NO:90] TPDVYTQVSAFVAWIWDWR "PRSL1") [SEQ ID NO:91] PPRACTKVSSFVHWIKKTMK "GRAB") [SEQ ID NO:92] PGVYILLSKKHLNWIIMTIK "GRAA") [SEQ ID NO:93] PGIYTLLTKKYQTWIKSNLV "GRAK") [SEQ ID NO:94] FPGVYTHVQIYVPWILQQVG "PRS29") [SEQ ID NO:95] KPWYTRVSAYI DWI N E KLS "CTRC" or "variant A8MTQ9") [SEQ ID NO:96] SPGVYARVTKLI PWVQKI LA "CTRB1 ") [SEQ ID NO:97] TPAVYARVTKLI PWVQKI LA "CTRB2") [SEQ ID NO:98] RPGVYTLASSYASWIQSKVT "PRSS8") [SEQ ID NO:99] RPGVYISLSAHRSWVEKIVQ "BSSP4") [SEQ ID NO:100] RPGVYTSVATYSPWIQARVS "PRS33") [SEQ ID NO:101] RPGVYTNISHHFEWIQKLMA "TEST") [SEQ ID NO:102] RPGVYTRVPAYVNWIRRHIT "TRYG1 ") [SEQ ID NO:103] YPGVYASVSYFSKWICDNIE ("MPN2" or "PRS38") [SEQ ID NO: 104]

LPGVYTNVIYYQKWINATIS ("ESSPL" or "PRS48") [SEQ ID NO:105]

DSGGRLACEYNDT VQVGIV ("TSSP2" or "PRS42") [SEQ ID NO: 106]

GPGVYSNVSYFVEWIKRQIY ("CORIN") [SEQ ID ΝΟ:10η

KPGVFARVMIFLDWIQSKIN ("OVCH1 ") [SEQ ID NO:108]

KPGVYARVSRFVTWIEGMMR ("APOA") [SEQ ID NO: 109]

KPGVYTRVTALRDWITSKTG ("T11 E2" or "TM1 1 E") [SEQ ID NO: 110]

RPGVYGNVMVFTDWIYRQMR ("TMPS2") [SEQ ID O:111]

KPGVYTKVTEVLPWIYSKME ("TMPSD") [SEQ ID NO:112]

Thus, the polypeptide may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 1 12.

In one embodiment, the polypeptides of the invention may be derived from the C-terminal region of Factor X. Thus, the polypeptide comprises or consists of an amino acid sequence of SEQ ID NO:3 or 113, or a fragment, variant, fusion or derivative thereof which retains an anti-inflammatory and/or antimicrobial and/or anti-coagulant activity of SEQ ID NO:3 or 113: KYGIYTKVTAFLKWIDRSMK [SEQ ID NO:3]

GKYGIYTKVTAFLKWIDRSMKTRGL [SEQ ID NO:113].

Alternatively, the polypeptides of the invention may alternatively be derived from the C- terminal region of Kallikrein 8. Thus, the polypeptide comprises or consists of an amino acid sequence of SEQ ID NO:4, or a fragment, variant, fusion or derivative thereof which retains an anti-inflammatory and/or antimicrobial and/or anti-coagulant activity of SEQ ID NO:4:

KPGVYTNICRYLDWIKKIIG ("NRPN") [SEQ ID NO:4]

Alternatively, the polypeptides of the invention may alternatively be derived from the C- terminal region of Hyaluronan binding protein 2. Thus, the polypeptide comprises or consists of an amino acid sequence of SEQ ID NO:5, or a fragment, variant, fusion or derivative thereof which retains an anti-inflammatory and/or antimicrobial and/or anti- coagulant activity of SEQ ID NO:5:

RPGVYTQVTKFLNWIKATIK ("HABP2") [SEQ ID NO:5]

It will be appreciated by persons skilled in the art that the term 'amino acid', as used herein, includes the standard twenty genetically-encoded amino acids and their corresponding stereoisomers in the 'D' form (as compared to the natural form), omega-amino acids other naturally-occurring amino acids, unconventional amino acids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, etc.) and chemically derivatised amino acids (see below).

When an amino acid is being specifically enumerated, such as 'alanine' or 'Ala' or Ά', the term refers to both L-alanine and D-alanine unless explicitly stated otherwise. Other unconventional amino acids may also be suitable components for polypeptides of the present invention, as long as the desired functional property is retained by the polypeptide. For the peptides shown, each encoded amino acid residue, where appropriate, is represented by a single letter designation, corresponding to the trivial name of the conventional amino acid.

In one embodiment, the polypeptides of the invention comprise or consist of L-amino acids.

Where the polypeptide comprises an amino acid sequence according to a reference sequence (for example, SEQ ID NOs: 3 to 113), it may comprise additional amino acids at its N- and/or C- terminus beyond those of the reference sequence, for example, the polypeptide may comprise additional amino acids at its N-terminus. Likewise, where the polypeptide comprises a fragment, variant or derivative of an amino acid sequence according to a reference sequence, it may comprise additional amino acids at its N- and/or C- terminus. In a further embodiment the polypeptide comprises or consists of a fragment of the amino acid sequence according to a reference sequence (for example, SEQ ID NO: 3). Thus, the polypeptide may comprise or consist of at least 5 contiguous amino acid of the reference sequence, for example at least 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acid of SEQ ID NOS: 3 to 1 13.

In one embodiment the polypeptide fragment commences at an amino acid residue selected from amino acid residues 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, and 15of SEQ ID NO: 3 . Alternatively/additionally, the polypeptide fragment may terminate at an amino acid residue selected from amino acid residues 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, and of SEQ ID NO: 3 . For example, the polypeptide fragment may comprise or consist of amino acids 4 to 19 of SEQ ID NO: 3 .

It will be appreciated by persons skilled in the art that the polypeptide of the invention may comprise or consist of a variant of the amino acid sequence according to a reference sequence (for example, SEQ ID NO: 3), or fragment of said variant. Such a variant may be a non-naturally occurring.

By 'variants' of the polypeptide we include insertions, deletions and substitutions, either conservative or non-conservative. For example, conservative substitution refers to the substitution of an amino acid within the same general class (e.g. an acidic amino acid, a basic amino acid, a non-polar amino acid, a polar amino acid or an aromatic amino acid) by another amino acid within the same class. Thus, the meaning of a conservative amino acid substitution and non-conservative amino acid substitution is well known in the art. In particular we include variants of the polypeptide which exhibit an anti-inflammatory activity.

In a further embodiment the variant has an amino acid sequence which has at least 50% identity with the amino acid sequence according to a reference sequence (for example, SEQ ID NO: 3) or a fragment thereof, for example at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity.

The percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequences have been aligned optimally.

The alignment may alternatively be carried out using the Clustal W program (as described in Thompson er a/., 1994, Nuc. Acid Res. 22:4673-4680, which is incorporated herein by reference). The parameters used may be as follows:

Fast pairwise alignment parameters: K-tuple(word) size; 1 , window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent.

Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05.

Scoring matrix: BLOSUM.

Alternatively, the BESTFIT program may be used to determine local sequence alignments.

In one embodiment, amino acids from the above reference sequences may be mutated in order to reduce proteolytic degradation of the polypeptide, for example by I, F to W modifications (see Stromstedt ef a/, Antimicrobial Agents Chemother 2009, 53, 593).

Variants may be made using the methods of protein engineering and site-directed mutagenesis well known in the art using the recombinant polynucleotides (see example, see Molecular Cloning: a Laboratory Manual, 3rd edition, Sambrook & Russell, 2000, Cold Spring Harbor Laboratory Press, which is incorporated herein by reference).

In one embodiment, the polypeptide comprises or consists of an amino acid which is a species homologue of any one of the above amino acid sequences (e.g. SEQ ID NOS: 1 to 1 13). By "species homologue" we include that the polypeptide corresponds to the same amino acid sequence within an equivalent protein from a non-human species, i.e. which polypeptide exhibits the maximum sequence identity with of any one of SEQ ID NOS: 1 to 1 13 (for example, as measured by a GAP or BLAST sequence comparison). Typically, the species homologue polypeptide will be the same length as the human reference sequence (i.e. SEQ ID NOS: 1 to 1 13).

In a still further embodiment, the polypeptide comprises or consists of a fusion protein.

By 'fusion' of a polypeptide we include an amino acid sequence corresponding to a reference sequence (for example, SEQ ID NO: 3, or a fragment or variant thereof) fused to any other polypeptide. For example, the said polypeptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said polypeptide. Examples of such fusions are well known to those skilled in the art. Similarly, the said polypeptide may be fused to an oligo-histidine tag such as His6 or to an epitope recognised by an antibody such as the well-known Myc tag epitope. Similarly, fusions comprising a hydrophobic oligopeptide end-tag may be used. Fusions to any variant or derivative of said polypeptide are also included in the scope of the invention. It will be appreciated that fusions (or variants or derivatives thereof) which retain desirable properties, such as an anti-inflammatory activity, are preferred.

The fusion may comprise a further portion which confers a desirable feature on the said polypeptide of the invention; for example, the portion may be useful in detecting or isolating the polypeptide, or promoting cellular uptake of the polypeptide. The portion may be, for example, a biotin moiety, a streptavidin moiety, a radioactive moiety, a fluorescent moiety, for example a small fluorophore or a green fluorescent protein (GFP) fluorophore, as well known to those skilled in the art. The moiety may be an immunogenic tag, for example a Myc tag, as known to those skilled in the art or may be a lipophilic molecule or polypeptide domain that is capable of promoting cellular uptake of the polypeptide, as known to those skilled in the art.

It will be appreciated by persons skilled in the art that the polypeptide of the invention may comprise one or more amino acids that are modified or derivatised, for example by PEGylation, amidation, esterification, acylation, acetylation and/or alkylation.

As appreciated in the art, pegylated proteins may exhibit a decreased renal clearance and proteolysis, reduced toxicity, reduced immunogenicity and an increased solubility [Veronese, F.M. and J.M. Harris, Adv Drug Deliv Rev, 2002. 54(4): p. 453-6., Chapman, A.P., Adv Drug Deliv Rev, 2002. 54(4): p. 531-45.]. Pegylation has been employed for several protein-based drugs including the first pegylated molecules asparaginase and adenosine deaminase [Veronese, F.M. and J.M. Harris, Adv Drug Deliv Rev, 2002. 54(4): p. 453-6., Veronese, F.M. and G. Pasut, Drug Discov Today, 2005. 10(21): p. 1451-8.].

In order to obtain a successfully pegylated protein, with a maximally increased half-life and retained biological activity, several parameters that may affect the outcome are of importance and should be taken into consideration. The PEG molecules may differ, and PEG variants that have been used for pegylation of proteins include PEG and monomethoxy-PEG. In addition, they can be either linear or branched [Wang, Y.S., et al. , Adv Drug Deliv Rev, 2002. 54(4): p. 547-70]. The size of the PEG molecules used may vary and PEG moieties ranging in size between 1 and 40 kDa have been linked to proteins [Wang, Y.S., et al., Adv Drug Deliv Rev, 2002. 54(4): p. 547-70., Sato, H„ Adv Drug Deliv Rev, 2002. 54(4): p. 487-504, Bowen, S., et al., Exp Hematol, 1999. 27(3): p. 425-32, Chapman, A.P., et al., Nat Biotechnol, 1999. 17(8): p. 780-3]. In addition, the number of PEG moieties attached to the protein may vary, and examples of between one and six PEG units being attached to proteins have been reported [Wang, Y.S., et al., Adv Drug Deliv Rev, 2002. 54(4): p. 547-70., Bowen, S., et al., Exp Hematol, 1999. 27(3): p. 425-32]. Furthermore, the presence or absence of a linker between PEG as well as various reactive groups for conjugation have been utilised. Thus, PEG may be linked to N-terminal amino groups, or to amino acid residues with reactive amino or hydroxyl groups (Lys, His, Ser, Thr and Tyr) directly or by using γ-amino butyric acid as a linker. In addition, PEG may be coupled to carboxyl (Asp, Glu, C-terminal) or sulfhydryl (Cys) groups. Finally, Gin residues may be specifically pegylated using the enzyme transglutaminase and alkylamine derivatives of PEG has been described [Sato, H., Adv Drug Deliv Rev, 2002. 54(4): p. 487-504].

It has been shown that increasing the extent of pegylation results in an increased in vivo half-life. However, it will be appreciated by persons skilled in the art that the pegylation process will need to be optimised for a particular protein on an individual basis.

PEG may be coupled at naturally occurring disulphide bonds as described in WO 2005/007197. Disulfide bonds can be stabilised through the addition of a chemical bridge which does not compromise the tertiary structure of the protein. This allows the conjugating thiol selectivity of the two sulphurs comprising a disulfide bond to be utilised to create a bridge for the site-specific attachment of PEG. Thereby, the need to engineer residues into a peptide for attachment of to target molecules is circumvented.

A variety of alternative block copolymers may also be covalently conjugated as described in WO 2003/059973. Therapeutic polymeric conjugates can exhibit improved thermal properties, crystallisation, adhesion, swelling, coating, pH dependent conformation and biodistribution. Furthermore, they can achieve prolonged circulation, release of the bioactive in the proteolytic and acidic environment of the secondary lysosome after cellular uptake of the conjugate by pinocytosis and more favourable physicochemical properties due to the characteristics of large molecules {e.g. increased drug solubility in biological fluids). Block copolymers, comprising hydrophilic and hydrophobic blocks, form polymeric micelles in solution. Upon micelle disassociation, the individual block copolymer molecules are safely excreted. Chemical derivatives of one or more amino acids may also be achieved by reaction with a functional side group. Such derivatised molecules include, for example, those molecules in which free amino groups have been derivatised to form amine hydrochlorides, p-toluene sulphonyl groups, carboxybenzoxy groups, f-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatised to form salts, methyl and ethyl esters or other types of esters and hydrazides. Free hydroxyl groups may be derivatised to form O-acyl or O-alkyl derivatives. Also included as chemical derivatives are those peptides which contain naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine and ornithine for lysine. Derivatives also include peptides containing one or more additions or deletions as long as the requisite activity is maintained. Other included modifications are amidation, amino terminal acylation (e.g. acetylation or thioglycolic acid amidation), terminal carboxylamidation (e.g. with ammonia or methylamine), and the like terminal modifications.

It will be further appreciated by persons skilled in the art that peptidomimetic compounds may also be useful. Thus, by 'polypeptide' we include peptidomimetic compounds which have an anti-inflammatory activity. The term 'peptidomimetic' refers to a compound that mimics the conformation and desirable features of a particular peptide as a therapeutic agent. For example, the polypeptides of the invention include not only molecules in which amino acid residues are joined by peptide (-CO-NH-) linkages but also molecules in which the peptide bond is reversed. Such retro-inverso peptidomimetics may be made using methods known in the art, for example such as those described in Meziere ef a/. (1997) J. Immunol. 159, 3230-3237, which is incorporated herein by reference. This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Retro-inverse peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis. Alternatively, the polypeptide of the invention may be a peptidomimetic compound wherein one or more of the amino acid residues are linked by a -y(CH ₂ NH)- bond in place of the conventional amide linkage. In a further alternative, the peptide bond may be dispensed with altogether provided that an appropriate linker moiety which retains the spacing between the carbon atoms of the amino acid residues is used; it may be advantageous for the linker moiety to have substantially the same charge distribution and substantially the same planarity as a peptide bond.

It will be appreciated that the polypeptide may conveniently be blocked at its N- or C- terminal region so as to help reduce susceptibility to exoproteolytic digestion. A variety of uncoded or modified amino acids such as D-amino acids and N-methyl amino acids have also been used to modify mammalian peptides. In addition, a presumed bioactive conformation may be stabilised by a covalent modification, such as cyclisation or by incorporation of lactam or other types of bridges, for example see Veber ef a/., 1978, Proc. Natl. Acad. Sci. USA 75:2636 and Thursell ef a/., 1983, Biochem. Biophys. Res. Comm. 111:166, which are incorporated herein by reference.

A common theme among many of the synthetic strategies has been the introduction of some cyclic moiety into a peptide-based framework. The cyclic moiety restricts the conformational space of the peptide structure and this frequently results in an increased specificity of the peptide for a particular biological receptor. An added advantage of this strategy is that the introduction of a cyclic moiety into a peptide may also result in the peptide having a diminished sensitivity to cellular peptidases.

Thus, exemplary polypeptides of the invention comprise terminal cysteine amino acids. Such a polypeptide may exist in a heterodetic cyclic form by disulphide bond formation of the mercaptide groups in the terminal cysteine amino acids or in a homodetic form by amide peptide bond formation between the terminal amino acids. As indicated above, cyclising small peptides through disulphide or amide bonds between the N- and C- terminal region cysteines may circumvent problems of specificity and half-life sometime observed with linear peptides, by decreasing proteolysis and also increasing the rigidity of the structure, which may yield higher specificity compounds. Polypeptides cyclised by disulphide bonds have free amino and carboxy-termini which still may be susceptible to proteolytic degradation, while peptides cyclised by formation of an amide bond between the N-terminal amine and C-terminal carboxyl and hence no longer contain free amino or carboxy termini. Thus, the peptides of the present invention can be linked either by a C- N linkage or a disulphide linkage. The present invention is not limited in any way by the method of cyclisation of peptides, but encompasses peptides whose cyclic structure may be achieved by any suitable method of synthesis. Thus, heterodetic linkages may include, but are not limited to formation via disulphide, alkylene or sulphide bridges. Methods of synthesis of cyclic homodetic peptides and cyclic heterodetic peptides, including disulphide, sulphide and alkylene bridges, are disclosed in US 5,643,872, which is incorporated herein by reference. Other examples of cyclisation methods includes cyclization through click chemistry, epoxides, aldehyde-amine reactions, as well as and the methods disclosed in US 6,008,058, which is incorporated herein by reference.

A further approach to the synthesis of cyclic stabilised peptidomimetic compounds is ring-closing metathesis (RCM). This method involves steps of synthesising a peptide precursor and contacting it with an RCM catalyst to yield a conformational^ restricted peptide. Suitable peptide precursors may contain two or more unsaturated C-C bonds. The method may be carried out using solid-phase-peptide-synthesis techniques. In this embodiment, the precursor, which is anchored to a solid support, is contacted with a RCM catalyst and the product is then cleaved from the solid support to yield a conformationally restricted peptide. Another approach, disclosed by D. H. Rich in Protease Inhibitors, Barrett and Selveson, eds., Elsevier (1986) , which is incorporated herein by reference, has been to design peptide mimics through the application of the transition state analogue concept in enzyme inhibitor design. For example, it is known that the secondary alcohol of staline mimics the tetrahedral transition state of the scissile amide bond of the pepsin substrate.

In summary, terminal modifications are useful, as is well known, to reduce susceptibility by proteinase digestion and therefore to prolong the half-life of the peptides in solutions, particularly in biological fluids where proteases may be present. Polypeptide cyclisation is also a useful modification because of the stable structures formed by cyclisation and in view of the biological activities observed for cyclic peptides.

Thus, in one embodiment the polypeptide of the first aspect of the invention is linear. However, in an alternative embodiment, the polypeptide is cyclic. It will be appreciated by persons skilled in the art that the polypeptides of the invention may be of various lengths. Typically, however, the polypeptide is between 10 and 200 amino acids in length, for example between 20 and 150, 20 and 100, 20 and 50, 20 and 30, 22 and 28, or 24 and 26 amino acids in length. For example, the polypeptide may be at least 20 amino acids in length.

As stated at the outset, anti-inflammatory activity is a feature common to the polypeptides of the invention. In one embodiment, the polypeptides are capable of inhibiting the release of one or more pro-inflammatory cytokines from human monocyte- derived macrophages, such as monocyte-derived macrophages, including macrophage inhibitory factor, TNF-alpha, HMGB1 , C5a, IL-17, IL-8, CP-1 , IFN-gamma, II-6, IL-1 b, IL-12. Antiinflammatory IL-10 may be unaffected or transiently increased.

Other markers may also be affected: These include tissue factor on monocytes and endothelial cells, procalcitonin, CRP, reactive oxygen species, bradykinin, nitric oxide, PGE1 , platelet activating factor, arachidonic acid metabolites, MAP kinase activation. In particular, the polypeptide may exhibit anti-inflammatory activity in one or more of the following models: in vitro macrophage models using LPS, LTA, zymosan, flaggelin, dust mites, triacyl lipopeptides, glycolipids, human, viral or bacterial DNA or RNA, , host derived glycosaminoglycan fragments, or bacterial peptidoglycan as stimulants; in vivo mouse models of endotoxin shock; and/or in vivo infection models, either in combination with antimicrobial therapy, given alone.

In a further embodiment of the invention, the polypeptide exhibits anticoagulant activity.

By "anti-coagulant activity" we mean an ability to reduce or prevent coagulation {i.e. the clotting of blood) or an associated signal or effect. Such activity may be determined by methods well known in the art, for example using the activated partial thromboplastin time (aPTT) test, prothrombin time (PT) test or the thrombin clotting time (TCT) test. Furthermore, specific measurements of prekallikrein activation or the activity of Factor X and other coagulation factors may be performed.

In a still further embodiment of the invention, the polypeptide exhibits Toll-like receptor (TLR) blocking activity. Such receptor blocking activity can be measured using methods well known in the art, for example by analysis of suitable down-stream effectors, such as iNOS, nuclear factor kappa B and cytokines.

By virtue of possessing an anti-inflammatory activity, the polypeptides of the first aspect of the invention are intended for use in the treatment or prevention of inflammation.

By "treatment or prevention of inflammation" we mean that the polypeptide of the invention is capable of preventing or inhibiting (at least in part) one or more symptom, signal or effect constituting or associated with inflammation.

It will be appreciated by persons skilled in the art that inhibition of inflammation may be in whole or in part. In a preferred embodiment, the polypeptide is capable of inhibiting one or more markers of inflammation by 20% or more compared to cells or individuals which have not been exposed to the polypeptide, for example by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

In one embodiment, the polypeptides of the invention are capable of treating or preventing inflammation selectively. By 'selectively' we mean that the polypeptide inhibits or prevents inflammation to a greater extent than it modulates other biological functions. For example, the polypeptide or fragment, variant, fusion or derivative thereof may inhibit or prevent inflammation only.

However, in a further embodiment, the polypeptide also (or alternatively) inhibits or prevents coagulation of the blood. As above, it will be appreciated by persons skilled in the art that inhibition of coagulation may be in whole or in part. In a preferred embodiment, the polypeptide is capable of inhibiting one or more measures and. or markers of coagulation by 20% or more compared to cells or individuals which have not been exposed to the polypeptide, for example by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

In one embodiment, the polypeptides are for use in the treatment or prevention of inflammation associated with (i.e. caused by or merely co-presenting with) an infection. In preferred but non-limiting embodiments of the invention, the polypeptides are for use in the treatment or prevention of a disease, condition or indication selected from the following: i) Acute systemic inflammatory disease, with or without an infective component, such as systemic inflammatory response syndrome (SIRS), ARDS, sepsis, severe sepsis, and septic shock. Other generalized or localized invasive infective and inflammatory disease, including erysipelas, meningitis, arthritis, toxic shock syndrome, diverticulitis, appendicitis, pancreatitis, cholecystitis, colitis, cellulitis, burn wound infections, pneumonia, urinary tract infections, postoperative infections, and peritonitis. ii) Chronic inflammatory and or infective diseases, including cystic fibrosis, COPD and other pulmonary diseases, gastrointestinal disease including chronic skin and stomach ulcerations, other epithelial inflammatory and or infective disease such as atopic dermatitis, oral ulcerations (aphtous ulcers), genital ulcerations and inflammatory changes, parodontitis, eye inflammations including conjunctivitis and keratitis, external otitis, mediaotitis, genitourinary inflammations. iii) Postoperative inflammation. Inflammatory and coagulative disorders including thrombosis, DIC, postoperative coagulation disorders, and coagulative disorders related to contact with foreign material, including extracorporeal circulation, and use of biomaterials. Furthermore, vasculitis related inflammatory disease, as well as allergy, including allergic rhinitis and asthma.. iv) Excessive contact activation and/or coagulation in relation to, but not limited to, stroke. v) Excessive inflammation in combination with antimicrobial treatment. The antimicrobial agents used may be administred by various routes; intravenous (iv), intraarterial, intravitreai, subcutaneous (sc), intramuscular (im), intraperitoneal (ip), intravesical, intratechal, epidural, enteral (including oral, rectal, gastric, and other enteral routes), or topically, (including dermal, nasal application, application in the eye or ear, eg by drops, and pulmonary inhalation). Examples of agents are penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides, and fluoroquinolones. Antiseptic agents include iodine, silver, copper, clorhexidine, polyhexanide and other biguanides, chitosan, acetic acid, and hydrogen peroxide.

For example, the polypeptides may be for use in the treatment or prevention of an acute inflammation, sepsis, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), cystic fibrosis, wounds, asthma, allergic and other types of rhinitis, cutaneous and systemic vasculitis, thrombosis and/or disseminated intravascular coagulation (DIC).

In one embodiment, the polypeptide exhibits both anti-inflammatory and anti-coagulant activity and may be used in the concomitant treatment or prevention of inflammation and coagulation. Such polypeptides may be particularly suited to the treatment and prevention of conditions where the combined inhibition of both inflammatory and coagulant processes is desirable, such as sepsis, chronic obstructive pulmonary disorder (COPD), thrombosis, DIC and acute respiratory distress syndrome (ARDS). Furthermore, other diseases associated with excessive inflammation and coagulation changes may benefit from treatment by the polypeptides, such as cystic fibrosis, asthma, allergic and other types of rhinitis, cutaneous and systemic vasculitis.

In a further embodiment, the polypeptides of the invention are for use in combination with one or more additional therapeutic agent. For example, the polypeptides of the invention may be administered in combination with antibiotic agents, anti-inflammatory agents, immunosuppressive agents and/or antiseptic agents, as well as vasoactive agents and/or receptor-blockers or receptor agonists. The antimicrobial agents used may be applied iv, sc, im, intratechal, per os, or topically. Examples of agents are penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides, and fluoroquinolones. Antiseptic agents include iodine, silver, copper, clorhexidine, polyhexanide and other biguanides, chitosan, acetic acid, and hydrogen peroxide. For example, the peptides of the invention may serve as adjuvants to antiseptic treatments, for example silver/PHMB treatment of wounds to quench LPS effects. Thus, the peptides of the invention may serve as adjuvants (for blocking inflammation) to complement antibiotic, antiseptic and/or antifungal treatments of internal and external infections (such as erysipelas, lung infections including fungal infections, sepsis, COPD, wounds, and other epithelial infections). Likewise, the peptides of the invention may serve as adjuvants to antiseptic treatments, for example silver/PHMB treatment of wounds to quench LPS effects.

In one embodiment, the polypeptides of the invention are for use in combination with a steroid, for example a glucocorticoid (such as dexamethasone).

A second, related aspect of the invention provides an isolated polypeptide comprising or consisting of an amino acid sequence of SEQ ID NO: 1 or 2 or a fragment, variant, fusion or derivative thereof, or a fusion of said fragment, variant or derivative thereof, which exhibits an anti-inflammatory and/or antimicrobial and/or anti-coagulant activity, with the proviso that the polypeptide is not a naturally occurring protein (e.g. holoprotein).

By "naturally occurring protein" in this context we mean that the polypeptide is synthesized de novo. However, fragments of such naturally occurring holoproteins generated in vivo are not excluded.

It will be appreciated by persons skilled in the art that terms such as fragment, variant, fusion or derivative should be construed as discussed above in relation to the first aspect of the invention.

In one embodiment, the polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOS: 3 to 1 13, or a fragment, variant, fusion or derivative of said sequence, or a fusion of said fragment, variant or derivative thereof. For example, the polypeptide may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NOS: 3 to 1 13.

In a particularly preferred embodiment, the polypeptide comprises or consists of an amino acid sequence of SEQ ID NO:3, or a fragment, variant, fusion or derivative thereof which retains an anti-inflammatory and/or antimicrobial and/or anti-coagulant activity of SEQ ID NO:3. For example, the polypeptide may comprise or consist of an amino acid sequence of SEQ ID NO:3. It will be appreciated by persons skilled in the art that the optional features discussed above in relation to the polypeptides of the first aspect of the invention are also of relevance to the related polypeptides of the second aspect of the invention. For example, in one preferred embodiment the polypeptide is capable of inhibiting the release of one or more pro-inflammatory cytokines from human monocyte-derived macrophages (such as IL-6, IFN-gamma, TNF-alpha, IL-12, IL-1 and/or IL-18).

In another preferred embodiment, the polypeptide exhibits anticoagulant activity.

The present invention also includes pharmaceutically acceptable acid or base addition salts of the above described polypeptides. The acids which are used to prepare the pharmaceutically acceptable acid addition salts of the aforementioned base compounds useful in this invention are those which form non-toxic acid addition salts, i.e. salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate and pamoate [i.e. 1 ,1 '-methylene-bis-(2- hydroxy-3 naphthoate)] salts, among others.

Pharmaceutically acceptable base addition salts may also be used to produce pharmaceutically acceptable salt forms of the polypeptides. The chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of the present compounds that are acidic in nature are those that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to those derived from such pharmacologically acceptable cations such as alkali metal cations (e.g. potassium and sodium) and alkaline earth metal cations (e.g. calcium and magnesium), ammonium or water-soluble amine addition salts such as N-methylglucamine- (meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines, among others.

It will be appreciated that the polypeptides of the invention may be lyophilised for storage and reconstituted in a suitable carrier prior to use, e.g. through freeze drying, spray drying, spray cooling, or through use of particle formation (precipitation) from supercritical carbon dioxide. Any suitable lyophilisation method (e.g. freeze drying, spray drying, cake drying) and/or reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of activity loss and that use levels may have to be adjusted upward to compensate. Preferably, the lyophilised (freeze dried) polypeptide loses no more than about 1 % of its activity (prior to lyophilisation) when rehydrated, or no more than about 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or no more than about 50% of its activity (prior to lyophilisation) when rehydrated.

Methods for the production of polypeptides of the invention are well known in the art. Conveniently, the polypeptide is or comprises a recombinant polypeptide. Suitable methods for the production of such recombinant polypeptides are well known in the art, such as expression in prokaryotic or eukaryotic hosts cells (for example, see Sambrook & Russell, 2000, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor, New York, the relevant disclosures in which document are hereby incorporated by reference).

Polypeptides of the invention can also be produced using a commercially available in vitro translation system, such as rabbit reticulocyte lysate or wheatgerm lysate (available from Promega). Preferably, the translation system is rabbit reticulocyte lysate. Conveniently, the translation system may be coupled to a transcription system, such as the TNT transcription-translation system (Promega). This system has the advantage of producing suitable mRNA transcript from an encoding DNA polynucleotide in the same reaction as the translation. It will be appreciated by persons skilled in the art that polypeptides of the invention may alternatively be synthesised artificially, for example using well known liquid-phase or solid phase synthesis techniques (such as ί-Boc or Fmoc solid-phase peptide synthesis).

Thus, included within the scope of the present invention are the following:

(a) a third aspect of the invention provides an isolated nucleic acid molecule which encodes a polypeptide according to the second aspect of the invention;

(b) a fourth aspect of the invention provides a vector (such as an expression vector) comprising a nucleic acid molecule according to the third aspect of the invention;

(c) a fifth aspect of the invention provides a host cell comprising a nucleic acid molecule according to the third aspect of the invention or a vector according to the fourth aspect of the invention; and

(d) a sixth aspect of the invention provides a method of making a polypeptide according to the second aspect of the invention comprising culturing a population of host cells according to the fifth aspect of the invention under conditions in which said polypeptide is expressed, and isolating the polypeptide therefrom.

A seventh aspect of the invention provides a pharmaceutical composition comprising a polypeptide according to the first aspect of the invention together with a pharmaceutically acceptable excipient, diluent or carrier.

As used herein, 'pharmaceutical composition' means a therapeutically effective formulation for use in the treatment or prevention of disorders and conditions associated with inflammation.

Additional compounds may also be included in the pharmaceutical compositions, such as other peptides, low molecular weight immunomodulating agents and antimicrobial agents. Other examples include chelating agents such as EDTA, citrate, EGTA or glutathione.

The pharmaceutical compositions may be prepared in a manner known in the art that is sufficiently storage stable and suitable for administration to humans and animals. The pharmaceutical compositions may be lyophilised, e.g. through freeze drying, spray drying, spray cooling, or through use of particle formation from supercritical particle formation.

By "pharmaceutically acceptable" we mean a non-toxic material that does not decrease the effectiveness of the biological activity of the active ingredients, i.e. the antiinflammatory polypeptide(s). Such pharmaceutically acceptable buffers, carriers or excipients are well-known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed ., Pharmaceutical Press (2000).

The term "buffer" is intended to mean an aqueous solution containing an acid-base mixture with the purpose of stabilising pH. Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES. The term "diluent" is intended to mean an aqueous or non-aqueous solution with the purpose of diluting the peptide in the pharmaceutical preparation. The diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil). The term "adjuvant" is intended to mean any compound added to the formulation to increase the biological effect of the peptide. The adjuvant may be one or more of colloidal silver or gold, or of zinc, copper or silver salts with different anions, for example, but not limited to fluoride, chloride, bromide, iodide, tiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetates of different acyl composition. The adjuvant may also be cationic polymers such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as polyvinyl imidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.

The excipient may be one or more of carbohydrates, polymers, lipids and minerals. Examples of carbohydrates include lactose, sucrose, mannitol, and cyclodextrines, which are added to the composition, e.g., for facilitating lyophilisation. Examples of polymers are starch, cellulose ethers, cellulose, carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, ethyl cellulose, methyl cellulose, propyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polysulphonate, polyethylenglycol/polyethylene oxide, polyethyleneoxide/ polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, poly(lactic acid), poly(glycholic acid) or copolymers thereof with various composition, and polyvinylpyrrolidone, all of different molecular weight, which are added to the composition, e.g. for viscosity control, for achieving bioadhesion, or for protecting the active ingredient (applies to A-C as well) from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di-, and triglycerides, ceramides, sphingolipids and glycolipids, all of different acyl chain length and saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are added to the composition for reasons similar to those for polymers. Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduction of liquid accumulation or advantageous pigment properties.

The pharmaceutical composition may also contain one or more mono- or di-sacharides such as xylitol, sorbitol, mannitol, lactitiol, isomalt, maltitol or xylosides, and/or monoacylglycerols, such as monolaurin. The characteristics of the carrier are dependent on the route of administration. One route of administration is topical administration. For example, for topical administrations, a preferred carrier is an emulsified cream comprising the active peptide, but other common carriers such as certain petrolatum/mineral-based and vegetable-based ointments can be used, as well as polymer gels, liquid crystalline phases and microemulsions.

It will be appreciated that the pharmaceutical compositions may comprise one or more polypeptides of the invention, for example one, two, three or four different peptides. By using a combination of different peptides the anti-inflammatory effect may be increased.

As discussed above, the polypeptide may be provided as a salt, for example an acid adduct with inorganic acids, such as hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, phosphoric acid, perchloric acid, thiocyanic acid, boric acid etc. or with organic acid such as formic acid, acetic acid, haloacetic acid, propionic acid, glycolic acid, citric acid, tartaric acid, succinic acid, gluconic acid, lactic acid, malonic acid, fumaric acid, anthranilic acid, benzoic acid, cinnamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, sulfanilic acid etc. Inorganic salts such as monovalent sodium, potassium or divalent zinc, magnesium, copper calcium, all with a corresponding anion, may be added to improve the biological activity of the antimicrobial composition.

The pharmaceutical compositions of the invention may also be in the form of a liposome, in which the polypeptide is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids, which exist in aggregated forms as micelles, insoluble monolayers and liquid crystals. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Suitable lipids also include the lipids above modified by poly(ethylene glycol) in the polar headgroup for prolonging bloodstream circulation time. Preparation of such liposomal formulations is can be found in for example US4,235,871. The pharmaceutical compositions of the invention may also be in the form of biodegradable microspheres. Aliphatic polyesters, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) or poly(carprolactone) (PCL), and polyanhydrides have been widely used as biodegradable polymers in the production of microshperes. Preparations of such microspheres can be found in US 5,851 ,451 and in EP 213 303.

The pharmaceutical compositions of the invention may also be formulated with micellar systems formed by surfactants and block copolymers, preferably those containing poly(ethylene oxide) moieties for prolonging bloodstream circulation time.

The pharmaceutical compositions of the invention may also be in the form of polymer gels, where polymers such as starch, cellulose ethers, cellulose carboxymethy!cellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, ethyl cellulose, methyl cellulose, propyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polyvinyl imidazole, polysulphonate, polyethylenglycol/polyethylene oxide, polyethylene-oxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone are used for thickening of the solution containing the peptide. The polymers may also comprise gelatin or collagen.

Alternatively, the polypeptides of the invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth gum, and/or various buffers. The pharmaceutical composition may also include ions and a defined pH for potentiation of action of anti-inflammatory polypeptides. The compositions of the invention may be subjected to conventional pharmaceutical operations such as sterilisation and/or may contain conventional adjuvants such as preservatives, stabilisers, wetting agents, emulsifiers, buffers, fillers, etc., e.g., as disclosed elsewhere herein.

It will be appreciated by persons skilled in the art that the pharmaceutical compositions of the invention may be administered locally or systemically. Routes of administration include topical, ocular, nasal, pulmonary, buccal, parenteral (intravenous, subcutaneous, and intramuscular), oral, vaginal and rectal. Also administration from implants is possible. Suitable preparation forms are, for example granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, microemulsions, defined as optically isotropic thermodynamically stable systems consisting of water, oil and surfactant, liquid crystalline phases, defined as systems characterised by long-range order but short-range disorder (examples include lamellar, hexagonal and cubic phases, either water- or oil continuous), or their dispersed counterparts, gels, ointments, dispersions, suspensions, creams, aerosols, droplets or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients, diluents, adjuvants or carriers are customarily used as described above. The pharmaceutical composition may also be provided in bandages, plasters or in sutures or the like.

In preferred embodiments, the pharmaceutical composition is suitable for parenteral administration or topical administration.

In alternative preferred embodiments, the pharmaceutical composition is suitable for pulmonary administration or nasal administration.

For example, the pharmaceutical compositions of the invention can be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoro-methane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2- tetrafluoroethane (HFA 134A3 or 1 ,1 ,1 ,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a polypeptide of the invention and a suitable powder base such as lactose or starch. Aerosol or dry powder formulations are preferably arranged so that each metered dose or 'puff' contains at least 0.1 mg of a polypeptide of the invention for delivery to the patient. It will be appreciated that the overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.

The pharmaceutical compositions will be administered to a patient in a pharmaceutically effective dose. By "pharmaceutically effective dose" is meant a dose that is sufficient to produce the desired effects in relation to the condition for which it is administered. The exact dose is dependent on the, activity of the compound, manner of administration, nature and severity of the disorder, age and body weight of the patient different doses may be needed. The administration of the dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administrations of subdivided doses at specific intervals. The pharmaceutical compositions of the invention may be administered alone or in combination with other therapeutic agents, such as additional antibiotic, antiinflammatory, immunosuppressive, vasoactive and/or antiseptic agents (such as antibacterial agents, anti-fungicides, anti-viral agents, and anti-parasitic agents). Examples of suitable antibiotic agents include penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines, macroiides, and fluoroquinolones. Antiseptic agents include iodine, silver, copper, clorhexidine, polyhexanide and other biguanides, chitosan, acetic acid, and hydrogen peroxide. Likewise, the pharmaceutical compositions may also contain additional anti-inflammatory drugs, such as steroids and macrolactam derivatives.

In one embodiment, the pharmaceutical compositions of the invention are administered in combination with a steroid, for example a glucocorticoid (such as dexamethasone). It will be appreciated by persons skilled in the art that the additional therapeutic agents may be incorporated as part of the same pharmaceutical composition or may be administered separately. In one embodiment of the seventh aspect of the invention, the pharmaceutical composition is associated with' a device or material to be used in medicine (either externally or internally). By 'associated with' we include a device or material which is coated, impregnated, covalently bound to or otherwise admixed with a pharmaceutical composition of the invention (or polypeptide thereof).

For example, the composition may be coated to a surface of a device that comes into contact with the human body or component thereof (e.g. blood), such as a device used in by-pass surgery, extracorporeal circulation, wound care and/or dialysis. Thus, the composition may be coated, painted, sprayed or otherwise applied to or admixed with a suture, prosthesis, implant, wound dressing, catheter, lens, skin graft, skin substitute, fibrin glue or bandage, etc. In so doing, the composition may impart improved antiinflammatory and/or anti-coagulant properties to the device or material.

Preferably, the device or material is coated with the pharmaceutical composition of the invention (or the polypeptide component thereof). By 'coated' we mean that the pharmaceutical composition is applied to the surface of the device or material. Thus, the device or material may be painted or sprayed with a solution comprising a pharmaceutical composition of the invention (or polypeptide thereof). Alternatively, the device or material may be dipped in a reservoir of a solution comprising a polypeptide of the invention.

Advantageously, the device or material is impregnated with a pharmaceutical composition of the invention (or polypeptide thereof). By 'impregnated' we mean that the pharmaceutical composition is incorporated or otherwise mixed with the device or material such that it is distributed throughout.

For example, the device or material may be incubated overnight at 4°C in a solution comprising a polypeptide of the invention. Alternatively, a pharmaceutical composition of the invention (or polypeptide thereof) may be immobilised on the device or material surface by evaporation or by incubation at room temperature.

In an alternative embodiment, a polypeptide of the invention is covalently linked to the device or material, e.g. at the external surface of the device or material. Thus, a covalent bond is formed between an appropriate functional group on the polypeptide and a functional group on the device or material. For example, methods for covalent bonding of polypeptides to polymer supports include covalent linking via a diazonium intermediate, by formation of peptide links, by alkylation of phenolic, amine and sulphydryl groups on the binding protein, by using a poly functional intermediate e.g. glutardialdehyde, and other miscellaneous methods e.g. using silylated glass or quartz where the reaction of trialkoxysilanes permits derivatisation of the glass surface with many different functional groups. For details, see Enzyme immobilisation by Griffin, ., Hammonds, E.J. and Leach, C.K. (1993) In Technological Applications of Biocatalysts (BIOTOL SERIES), pp. 75-1 18, Butterworth-Heinemann. See also the review article entitled 'Biomaterials in Tissue Engineering' by Hubbell, J.A. (1995) Science 13:565-576.

In a preferred embodiment, the device or material comprise or consists of a polymer. The polymer may be selected from the group consisting of polyesters (e.g. polylactic acid, polyglycolic acid or poly lactic acid-glycolic acid copolymers of various compositions), polyorthoesters, polyacetals, polyureas, polycarbonates, polyurethanes, polyamides) and polysaccharide materials (e.g. cross-linked alginates, hyaluronic acid, carageenans, gelatines, starch, cellulose derivatives).

Alternatively, or in addition, the device or material may comprise or consists of metals (e.g. titanium, stainless steel, gold, titanium), metal oxides (silicon oxide, titanium oxide) and/or ceramics (apatite, hydroxyapatite).

Such materials may be in the form of macroscopic solids/monoliths, as chemically or physicochemically cross-linked gels, as porous materials, or as particles. Thus, the present invention additionally provides devices and materials to be used in medicine, to which have been applied a polypeptide of the invention or pharmaceutical composition comprising the same.

Such devices and materials may be made using methods well known in the art.

An eighth aspect of the invention provides polypeptide according to the second aspect of the invention or a pharmaceutical composition according to the seventh aspect of the invention for use in medicine, for example in the treatment or prevention of inflammation and/or excessive coagulation.

In preferred embodiments, the polypeptide according to the second aspect of the invention or the pharmaceutical composition according to the seventh aspect of the invention are for use in the treatment and/prevention of a disease, condition or indication selected from the following: i) Acute systemic inflammatory disease, with or without an infective component, such as systemic inflammatory response syndrome (SIRS), ARDS, sepsis, severe sepsis, and septic shock. Other generalized or localized invasive infective and inflammatory disease, including erysipelas, meningitis, arthritis, toxic shock syndrome, diverticulitis, appendicitis, pancreatitis, cholecystitis, colitis, cellulitis, burn wound infections, pneumonia, urinary tract infections, postoperative infections, and peritonitis. ii) Chronic inflammatory and or infective diseases, including cystic fibrosis, COPD and other pulmonary diseases, gastrointestinal disease including chronic skin and stomach ulcerations, other epithelial inflammatory and or infective disease such as atopic dermatitis, oral ulcerations (aphtous ulcers), genital ulcerations and inflammatory changes, parodontitis, eye inflammations including conjunctivitis and keratitis, external otitis, mediaotit ^' is, genitourinary inflammations. iii) Postoperative inflammation. Inflammatory and coagulative disorders including thrombosis, DIC, postoperative coagulation disorders, and coagulative disorders related to contact with foreign material, including extracorporeal circulation, and use of biomaterials. Furthermore, vasculitis related inflammatory disease, as well as allergy, including allergic rhinitis and asthma.. iv) Excessive contact activation and/or coagulation in relation to, but not limited to, stroke. v) Excessive inflammation in combination with antimicrobial treatment. The antimicrobial agents used may be administred by various routes; intravenous (iv), intraarterial, intravitreal, subcutaneous (sc), intramuscular (im), intraperitoneal (ip), intravesical, intratechal, epidural, enteral (including oral, rectal, gastric, and other enteral routes), or topically, (including dermal, nasal application, application in the eye or ear, eg by drops, and pulmonary inhalation). Examples of agents are penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides, and fluoroquinolones. Antiseptic agents include iodine, silver, copper, clorhexidine, polyhexanide and other biguanides, chitosan, acetic acid, and hydrogen peroxide.

For example, the polypeptides may be for use in the treatment or prevention of an acute inflammation, sepsis, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), cystic fibrosis, wounds, asthma, allergic and other types of rhinitis, cutaneous and systemic vasculitis, thrombosis and/or disseminated intravascular coagulation (DIC).

In particularly preferred embodiments, a polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 1 13 (such as SEQ ID NO: 3, 4, 5, 9, 44, 92 or 109), or a fragment, variant, fusion or derivative of said sequence, or a fusion of said fragment, variant or derivative thereof which retains the anti-inflammatory and/or anti-coagulant activity thereof, is for use in the treatment or prevention of bacterial sepsis (e.g. P. aeruginosa sepsis) and/or endotoxin-mediated shock. Optionally, the polypeptide may be used in combination with a conventional antibiotic agent (such as those discussed above).

A related ninth aspect of the invention provides the use of a polypeptide according to the second aspect of the invention or a pharmaceutical composition according to the seventh aspect of the invention in the preparation of a medicament for the treatment or prevention of inflammation and/or excessive coagulation (as described above).

A tenth aspect of the invention provides a method for treating or preventing inflammation and/or coagulation in a patient, the method comprising administering to the patient a therapeutically-effective amount of a polypeptide according to the second aspect of the invention or a pharmaceutical composition according to the seventh aspect of the invention (as described above). . In preferred but non-limiting embodiments, the method is for the treatment or prevention of disease, condition or indication as listed above, for example an acute inflammation, sepsis, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), cystic fibrosis, asthma, allergic and other types of rhinitis, cutaneous and systemic vasculitis, thrombosis and/or disseminated intravascular coagulation (DIC). Persons skilled in the art will further appreciate that the uses and methods of the present invention have utility in both the medical and veterinary fields. Thus, the polypeptide medicaments may be used in the treatment of both human and non-human animals (such as horses, dogs and cats). Advantageously, however, the patient is human.

Preferred aspects of the invention are described in the following non-limiting examples, with reference to the following figures: Figure 1 : Generation of antimicrobial peptides by degradation of prothrombin and thrombin

Degradation of the proteins was performed at 37°C for the indicated time periods. RDA was performed in low-salt conditions with E. coli as test organism. Each 4 mm-diameter well was loaded with 6 μΙ of the solution. The bar diagrams indicate the zones of clearance obtained (in mm). The inset visualizes the results obtained with prothrombin. C, buffer; NE, neutrophil elastase only. LL-37 (100 μΜ) was included for comparison (b) Intact prothrombin (PT) and thrombin (T), and cleavage products from the different incubations with neutrophil elastase (NE, indicated above) were analyzed by SDS-PAGE (16.5% Tris-Tricine gel). The gels were overloaded in order to visualize generated fragments, and the purity of the proteins is better assessed in the lanes loaded with 2 pg of protein (rightmost two lanes).

Figure 2: Activities of peptides derived from prothrombin

(a) Sequence of prothrombin and overlapping peptides. The boxes indicate antimicrobial regions. In addition to the regular overlapping peptides, peptide regions of high net charge, and/or content of predicted helical regions (Agadir; http://agadir.crg.es) were selected, (b) Overlapping peptides of prothrombin were analysed for antimicrobial activities against E. coli. The inhibitory zones, relative hydrophobic moment (μΙ-lrel) as well as net charge of respective peptides (only active peptides are numbered) are indicated in the 3-D graph. For determination of antibacterial activities, E. coli (4 x 10 ⁶ cfu) was inoculated in 0.1 % TSB agarose gel. Each 4 mm-diameter well was loaded with 6 μΙ of peptide (at 100 μΜ). The zones of clearance correspond to the inhibitory effect of each peptide after incubation at 37 °C for 18-24 h (mean values are presented, n=3). (c) Helical wheel representation of the C-terminal peptide VFR17. The amino acids are indicated, (d) LPS-binding activity of the prothrombin-derived peptide sequences. Peptides (5 pg) were applied to nitrocellulose membranes followed by incubation in PBS (containing 2% bovine serum albumin) with iodinated ( ¹²⁵ I)-LPS. Only peptides from the C-terminal part of prothrombin bound to LPS. (e) Molecular model of thrombin. The peptides GKY25 (highlighted, indicated by * in Fig. 2a) and VFR17 (highlighted, peptide 48 in Fig. 2a) are indicated in the crystal structure of human thrombin (PDB code 1 C5L). (f) Activities of prothrombin (PT), thrombin (T), GKY25 and VFR17 on E. coli ATCC 25922. In viable count assays GKY25 and VFR17 displayed antibacterial activities. 2 x 10 ⁶ cfu/ml of bacteria were incubated in 50 μΙ with proteins and peptides at a concentration of 3 and 6 μΜ, respectively.

Figure 3: Identification of antibacterial regions of thrombin and prothrombin (a) RP-HPLC separation of thrombin digested with neutrophil elastase. The bars indicate the antibacterial activity of the fractions in low (gray) as well as high salt conditions (black). Fraction 30 (lower left) contained two peaks of masses 2034.78 and 2270.88, perfectly matching the indicated sequences obtained after ESI-MS/MS analysis. Fraction 38 was analysed by MALDI-MS, and subsequently by ESI-MS. The ESI-MS analysis identified a dominant mass of 1 1041 corresponding to the 96-amino acid long peptide N527-E622 with two intact disulphide bridges (indicated by ^** in Fig 2a). A minor mass corresponding to V528-E622 was also detected by ESI-MS/MS. N- and C-terminal sequencing yielded NLPI and EGFQ, respectively. The rightmost insets illustrate the ~1 1 kDa peptide analysed by SDS-PAGE and stained for protein (stain), or after immunoblot (blot), and below, the peptide (F38) was analyzed by gel-overlay for detection of antibacterial activity. The peptide activity was identical to the one identified in elastase- digested thrombin (T+NE). Peptides of fractions 20-21 were predicted using the FINDPEPT tool (www.expasy.org/tools/findpept.html) (Table 1 ). (b) Degradation of thrombin by neutrophil supernatants generates antibacterial activity in RDA (upper inset). RDA was performed in low-salt conditions. E. coli (4 x 10 ⁶ cfu) was used as test organism. Each 4 mm-diameter well was loaded with 6 μΙ of material (C, supernatant only; T, thrombin only; T+NS; thrombin incubated for 30 and 180 min respectively, with neutrophil supernatants). The digests were analysed by SDS-PAGE (16.5 % Tris-Tricine gels) and immunoblotting with antibodies against VFR17 (lower panel), (c) Prothrombin was digested with the enzymes as indicated for 3 h, and analysed by SDS-PAGE (16.5 % Tris-Tricine gels) and immunoblotting using antibodies against VFR17 (NE, neutrophil elastase; CG, cathepsin G; PAE, P. aeruginosa elastase). (d) RDA results of prothrombin digested with cathepsin G (CG) and P. aeruginosa elastase (PAE) for different time periods. VFR17 and LL-37 ( 0 μΜ) are shown for comparison. Figure 4: Thrombin-derived C-terminal peptides, their presence and antimicrobial effects ex vivo and in vivo

(a) Fibrin clots were produced from human plasma and incubated with neutrophil elastase for the indicated time periods (Fibrin), or obtained from a patient with a venous, non-infected, chronic ulcers (PF), extracted, and analyzed by Western blot using polyclonal antibodies against the thrombin C-terminal peptide VFR17. (b) Human plasma, incubated with neutrophil elastase for the indicated time periods (Plasma, left panel), acute wound fluid (patients 1-2, AWF, middle panel), or wound fluid from patients with chronic ulcers (patients 1-6, CWF, right panel) was analysed by Western blot using polyclonal antibodies against the thrombin C-terminal peptide VFR17. (c) Viable count analysis of the thrombin C-terminal peptide GKY25 in 10 mM Tris, pH 7.4 containing 0.15 M NaCI (buffer) only, or in presence of 20% human or murine plasma, (d) The thrombin C-terminal peptide GKY25 significantly increases survival. Mice were i.p. injected with P. aeruginosa bacteria, followed by subcutaneous injection of GKY25 or buffer only, after 1 h and then with intervals of 24 h for the three following days. Treatment with the peptide significantly increased survival (n=10 for controls and treated, p=0.002). (e) G Y25 suppresses bacterial dissemination to the spleen and kidney. Mice were infected as above, GKY25 was administrated subcutaneously after 1 h, and the cfu of P. aeruginosa in spleen and kidney was determined after a time period of 8 h (n=10 for controls and treated, P<0.05 for spleen and kidney. Horizontal line indicates median value).

Figure 5: Mode of action of thrombin-derived C-terminai peptides(a) Electron microscopy analysis. P. aeruginosa and S. aureus bacteria was incubated for 2 h at 37°C with 30 μΜ of GKY25 and LL-37 and analysed with electron microscopy. Scale bar represents 1 μητι. Control; Buffer control, (b) Permeabilizing effects of peptides on E. coli. Bacteria were incubated with the indicated peptides at 30 μΜ and permeabilization was assessed using the impermeant probe FITC. (c) Helical content of the thrombin-derived C-terminal peptides GKY25 and VFR17 in presence of negatively charged liposomes (PA). The two peptides showed a marked helix induction upon addition of the liposomes. (d) CD spectra of GKY25 and VFR17 in Tris-buffer and in presence of LPS. For control, CD spectra for buffer and LPS alone are also presented, (e) Effects of the indicated peptides on liposome leakage. The membrane permeabilizing effect was recorded by measuring fluorescence release of carboxyfluorescein from DOPE/DOPG (negatively charged) liposomes. The experiments were performed in 10 mM Tris-buffer, in absence and presence of 0.15 M NaCI. Values represents mean of triplicate samples, (f) Activities of corresponding C-terminal peptides of the indicated coagulation factors. Peptides were tested in RDA against the indicated bacteria. Bacteria (4 x 10 ⁶ cfu) was inoculated in 0.1 % TSB agarose gels. Each 4 mm-diameter well was loaded with 6 μΙ of peptide at 100 μΜ. The zones of clearance correspond to the inhibitory effect of each peptide after incubation at 37 °C for 18-24 h. (g) Overlay 3D-model showing the three coagulation factors thrombin, and factor X and IX. The C-terminal parts are indicated.

Figure 6: Activities of peptides (RDA) of prothrombin-derived peptides against P. aeruginosa in absence and presence of 0.1 M NaCI, and against E. coli in 0.1 M NaCI

Each 4 mm-diameter well was loaded with 6 μΙ of the solution. The bar diagrams indicate the zones of clearance obtained (in mm).

Figure 7: Effects on eukaryotic cells

(a) Hemolytic effects of GKY25 in blood (EDTA-blood made 50% with PBS) were investigated. The cells were incubated with different concentrations of the peptide or LL-37. 2% Triton X-100 (Sigma-Aldrich) served as positive control. The absorbance of hemoglobin release was measured at λ 540 nm and is expressed as % of Triton X-100 induced hemolysis (note the scale of the y-axis). (b) Upper panel; HaCaT keratinocytes were subjected to GKY25 and LL-37 in presence of 20% human serum. Cell permeabilizing effects were measured by the LDH based TOX- 7 kit. LDH release from the cells was monitored at λ 490 nm and was plotted as % of total LDH release. Lower panel; The MTT-assay was used to measure viability of HaCaT keratinocytes in the presence of the indicated peptides. In the assay, MTT is modified into a dye, blue formazan, by enzymes associated with metabolic activity. The absorbance of the dye was measured at λ 550 nm.

Figure 8: TNF-a release is inhibited by GKY25

RAW264.7 macrophages were stimulated with LPS from E. coli, in presence of GKY25 at the indicated concentrations. LL-37 is presented for comparison. Figure 9: Kinetics of GKY25 action

E. coli bacteria were grown to mid-logarithmic phase in Todd-Hewitt (TH) medium. They were then washed and diluted in 10 mM Tris, pH 7.4 containing 5 mM glucose. Following this, bacteria (50 μΙ; 2 x 106 cfu/ml) were incubated, at 37°C for 5, 10,- 20, 40, 60 and 120 min with GKY25 at 6 μ in presence of 10 mM Tris, 0.15 M NaCI, pH 7.4. To quantify the bactericidal activity, serial dilutions of the incubation mixtures were plated on TH agar, followed by incubation at 37°C overnight and the number of colony-forming units was determined. 100% survival was defined as total survival of bacteria in the same buffer and under the same condition in the absence of peptide.

Figure 10: γ-core motif of TCP

Cartoon representation of the part corresponding to the C-terminal 96 amino acids of the crystal structure of thrombin (PDB code: 1 C5L, amino acids 527-622). The region containing the proposed γ-core motif is highlighted. Cysteines and glycines are also highlighted. The motif corrsponds to the levomeric isoform 1 described by Yount and Yeaman (Yount, N.Y. & Yeaman, M.R. Multidimensional signatures in antimicrobial peptides. Proc Natl Acad Sci U S A W , 7363-7368 (2004)); (NH ₂ ...[C]-[X ₁₃ ]-[CXG]- [X ₂ ]- P...COOH), and is quite similar to the γ-core motif found in kinocidins (Yeaman, M.R & Yount, N.Y. Unifying themes in host defence effector polypeptides. Nat Rev Microbiol. 5, 727-740 (2007). Figure 11 : Alignment of human TCP with related thrombin sequences

The conserved cysteine residues, as well as two CXG motifs are indicated. Arrow indicates the N-terminus of the 96 amino acid peptide generated by neutrophil elastase.

Figure 12.

GKY25 binds heparin and LPS. 2 and 5 pg GKY25 were applied to nitrocellulose membranes. These membranes were then blocked in PBS (containing 2% bovine serum albumin) for 1 h at room temperature and incubated in PBS-with iodinated ( ¹²⁵ l) heparin or LPS. Unlabeled heparin (6 mg/ml) (+) was added for competition of binding. LL-37 was used for comparison. The membranes were washed (3 x 10 min in PBS). A Bas 2000 radioimaging system (Fuji) was used for visualization of radioactivity.

Figure 13.

GKY25 inhibits NO production. RAW264.7 macrophages were stimulated with LPS from E. coli and P. aeruginosa, in presence of GKY25 at the indicated concentrations. LL-37 is presented for comparison.

Figure 14.

GKY25 significantly increases survival in LPS-induced shock. Mice were injected with LPS followed by intraperitoneal administration of GKY25 (200 pg). Survival was followed for 7 days. (n=9 for controls, n=10 for treated animals, P<0.001 ). Figure 15. GKY25 attenuates proinflammatory cytokines. In a separate experiment, mice were sacrificed 6 hours after i.p. injection of LPS followed by treatment with GKY25 (200 pg) or buffer, and the indicated cytokines were analysed in blood (n=9 for controls, n=10 for treated animals, the P values for the respective cytokines are IL-6, 0.001 ; IFN- Y=0.009; TNF, 0.001 ; IL-12p70, 0.001. IL-10 was not significant.).

Figure 16.

Lungs were analyzed by scanning electron microscopy 20 h after LPS injection i.p. followed by treatment with GKY25 (200 pg) or buffer. Treatment with the peptides blocked leakage of proteins and erythrocytes (see inset) (n=3 in both groups, and a representative lung section is shown).

Figure 17: Anti-inflammatory effects of GKY25

GKY25 blocks NO production of RAW264.7 macrophages stimulated with various microbial products. Cells were subjected to the indicated concentrations of E.coli LPS, liipoteichoic acid (LTA) and peptidoglycan (PGN) from S. aureus as well as zymosan A from Saccharosmyces cerevisiae. NO production with or without 10 μΜ G Y25 was determined by using the Griess reagent. Figure 18; Anti-inflammatory effects of TCP-variants

Truncated variants of the peptide GKY25 were analysed for NO-blocking activity. RAW macrophages were stimulated with 10 ng/ml E. coli LPS, and peptides (10 uM) were added. The production of NO was measured using Griess reagent. (A) N-terminally truncated TCPs (B) C-terminal truncations, (C) N and C-terminal truncations.

Figure 19: Anti-inflammatory effects of evolutionary related thrombin peptides

Related thrombin peptides of various organisms were analysed for NO-blocking activity. RAW macrophages were stimulated with 10 ng/ml E. coli LPS, and peptides (10 uM) were added. The production of NO was measured using Griess reagent.

Figure 20 Anti-inflammatory effects of evolutionary TCP related sequences of S1 peptidases

(A) and (B) Related S1 peptide sequences were analysed for NO-blocking activity. RAW macrophages were stimulated with 10 ng/ml E. coli LPS, and peptides (30 uM) were added. The production of NO was measured using Griess reagent. Figure 21 : C-terminal peptide of thrombin blocks coagulation

GKY25 impairs the intrinsic pathway of coagulation in normal human plasma. This was determined by measuring the activated partial thromboplastin time (aPTT). Other parts of the coagulation system, as judged by the prothrombin time (PT) monitoring the extrinsic pathway of coagulation, and the thrombin clotting time (TCT), measuring thrombin induced fibrin network formation, were not significantly affected.

Figure 22. Activities of C-terminal S1 peptides comprising the pattern sequence.

(A) Indicated are peptide sequences, antimicrobial activity against E. coli ATCC 25922 in 10 mM Tris-buffer (with or without additional 0.15 M NaCI), and hemolysis (in %). For determination of antimicrobial activities, E. coli (4 x 10 ⁶ cfu) was inoculated in 0.1% TSB agarose gel. Each 4 mm-diameter well was loaded with 6 μΙ of peptide (at 100 μΜ). The zones of clearance (in mm) correspond to the inhibitory effect of each peptide after incubation at 37 °C for 18-24 h (mean values are presented, n=3). (S) Activities of S1- derived peptides against P. aeruginosa ATCC 27853, S. aureus ATCC 29213, and Candida albicans ATCC 90028. The graphs show the relation between activities against E. coli (x-axis) and the respective bacteria (y-axis). For detailed information see Supplementary Figure 1. (C) Effects of S1-derived peptides on NO production by macrophages. RAW264.7 mouse macrophages were incubated with LPS from E. coli or zymosan in presence of S1 -peptides at 10 μΜ. The indicated four peptides blocked NO responses. (D) List of peptide sequences from part (A) of figure.

Figure 23. Structure-function analyses on S-derived peptides.

(A) Graph illustrating the relation between inhibitory zones in RDA using E. coli and a salt strength of 0.15 NaCI, relative hydrophobic moment ( Hrel) as well as net charge of respective S1 -peptides. (B) Corresponding graph illustrating the correspondence to NO- blocking activity. Active peptides are are indicated in the 3-D graph. (C) QSAR analyses. QSAR models for the experimental versus predicted values for the set of S1 -peptides with respect to activity agains E. coli. E. coli, (D) Scatter plot illustrating NO-blocking activity.

Figure 24. 3D graphs showing activity of active peptides

3D graphs showing activity of active peptides from FN, FIX, FX, plasminogen, protein C, ApoA, HABP2, granzyme B, and H, kallikreins 5, 8, and 10, as well as negative controls: F1 1 , C1 r, KLK9. (A) Antimicrobial activity (as % inhibition) in 10 mM Tris and in 0.15 M NaCI. (B) LPS blocking activity (as % inhibition). Color coding using net charge x uHrel. (C) Survival curves with FX, HABP2, KLK8 and FXI. (D) Cytokine analyses Figure 25. Structural modelling and biophysical aspects.

(A) 3D models of Fll, FX, HABP2, KLK8, and FXI. (8) 3D models of the 20-mer sequence (complying with the pattern sequence) illustrating peptides of Fll, FX, HABP2, KLK8 (NRPN), and FXI. Dark shading: negative charge, light shading: positive charge.

Figure 26

(A) Activities of C-terminal S1 peptides comprising the pattern sequence. For determination of antimicrobial activities, P. aeruginosa ATCC 27853, S. aureus ATCC 29213, and Candida albicans C. albicans ATCC 90028 (1 x 10 ⁵ cfu) was inoculated in 0.1 % TSB agarose gel. Each 4 mm-diameter well was loaded with 6 μΙ of peptide (at 100 μΜ). The zones of clearance (in mm) correspond to the inhibitory effect of each peptide after incubation at 37 °C for 18-24 h (mean values are presented, n=3). (6) Effects of S1 - derived peptides on NO production by macrophages. RAW264.7 mouse macrophages were incubated with LPS from E. coli or zymosan in presence of S1-peptides at 10 μΜ.

Figure 27 Activities of identified C-terminal S1 peptides comprising the pattern sequence.

[ ] Antimicrobial activity against E. coli ATCC 25922 in 10 m Tris-buffer only, or 10 mM Tris supplemented with 0.15 M NaCI, and hemolysis (in %). For determination of antimicrobial activities, E. coli (4 x 10 ⁶ cfu) was inoculated in 0.1 % TSB agarose gel.

Each 4 mm-diameter well was loaded with 6 μΙ of peptide (at 100 μ ). The zones of clearance (in mm) correspond to the inhibitory effect of each peptide after incubation at

37 °C for 18-24 h (mean values are presented, n=3). RAW264.7 mouse macrophages were incubated with LPS from E. coli in presence of S1-peptides at 10 μΜ. (B) QSAR diagram illustrating activities for E. coli. Second set of peptides; grey. Previous set; black.

(C) Scatter diagram for LPS blocking activity. Second set of peptides; grey. Previous set; black. (D) List of peptide sequences from part (A) of figure. Figure 28.

Effect of exemplary polypeptides on the invention on animal weight. EXAMPLES

Example A - Proteolysis of human thrombin generates novel host defense peptides

Introduction

In the following experiments, C-terminal peptides of thrombin are demonstrated to constitute a novel class of host defense peptides, released upon proteolysis of thrombin in vitro, and detected in human wounds in vivo. In vitro, these peptides exert antimicrobial effects against Gram-positive and Gram-negative bacteria, mediated by membrane lysis, as well as immunomodulatory functions, by inhibiting macrophage responses to bacterial lipopolysaccharide. In mice, they are protective against P. aeruginosa sepsis, as well as lipopolysaccharide-induced shock. Moreover, the thrombin-derived C-terminal peptides exhibit helical structures upon binding to bacterial lipopolysaccharide, and permeabilize liposomes, features typical of "classical" helical antimicrobial peptides. These findings provide a novel link between the coagulation system and host-defense peptides, two fundamental biological systems activated in response to injury and microbial invasion.

The exemplary C-terminal peptides of thrombin, designated "GYK25" herein, corresponds to the amino acid of SEQ ID NO: 126.

Materials & Methods

Peptides and proteins

Prothrombin and thrombin were from Innovative Research, USA. The coagulation factor- derived peptides (Table 3) and omiganan (ILRWPWWPWRRK-amide [SEQ IS NO:114]) were synthesized by Biopeptide Co. The purity (>95%) and molecular weight of these peptides was confirmed by mass spectral analysis (MALDI.TOF Voyager). LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES [SEQ IS NO:115]) was from Innovagen AB. 20mer peptides corresponding to various overlapping regions of prothrombin (Fig. 2) were from Sigma (PEPscreen®, Custom Peptide Libraries, SigmaGenosys). Biological materials

Wound fluids (100-600 μΙ) from patients with chronic venous leg ulcers were collected under a Tegaderm dressing for 2 h as previously described ²⁵ . Sterile wound fluids were obtained from surgical drainages after mastectomy. Collection was for 24 h, 24 to 48 h after operation. Wound fluids were centrifuged, aliquoted and stored at -20°C. The use of human wound fluids was approved by the Ethics Committee at Lund University (LU 708-01 , LU 509-01 ). Microorganisms

Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, Bacillus subtilis ATCC 6633 bacterial isolates, and the fungal isolate Candida albicans ATCC 90028 were from the Department of Bacteriology, Lund University Hospital.

Radial diffusion assay

Essentially as described earlier ^{36, 37} , bacteria were grown to mid-logarithmic phase in 10 ml of full-strength (3% w/v) trypticase soy broth (TSB) (Becton-Dickinson). The microorganisms were then washed once with 10 mM Tris, pH 7.4. Subsequently, 4x10 ⁶ cfu were added to 15 ml of the underlay agarose gel, consisting of 0.03% (w/v) TSB, 1 % (w/v) low electroendosmosis type (EEO) agarose (Sigma-Aldrich) and 0.02% (v/v) Tween 20 (Sigma-Aldrich). The underlay was poured into a 0 144 mm petri dish. After agarose solidification, 4 mm-diameter wells were punched and 6 μΙ peptide solution of required concentration added to each well. Plates were incubated at 37°C for 3 h to allow peptide diffusion. The underlay gel was then covered with 15 ml of molten overlay (6% TSB and 1 % Low-EEO agarose in distilled H ₂ 0). Antimicrobial activity of a peptide was visualized as a zone of clearing around each well after 18-24 h of incubation at 37°C. Viable-count analysis

E. coli ATCC 25922 bacteria were grown to mid-logarithmic phase in Todd-Hewitt (TH) medium. Bacteria were washed and diluted in 10 mM Tris, pH 7.4 containing 5 mM glucose. £ coli ATCC 25922 (50 μΙ; 2 x 10 ⁶ cfu/ml) were incubated, at 37°C for 2 h with prothrombin, thrombin, GKY25, or VFR17 at 3 and 6 μΜ. Other experiments with GKY25 and LL-37 were performed in 10 mM Tris, pH 7.4, containing also 0.15 M NaCI, with or without 20% citrate-plasma. Serial dilutions of the incubation mixture were plated on TH agar, followed by incubation at 37°C overnight and cfu determination.

Slot-blot assay

LPS-binding ability of the peptides was examined by a slot-blot assay. Peptides (2 and 5 μg) were bound to nitrocellulose membranes (Hybond-C, GE Healthcare Biosciences), pre-soaked in PBS. Membranes were then blocked by 2 wt% BSA in PBS, pH 7.4, for 1 h at RT, and subsequently incubated with ¹²⁵ l-labelled LPS (40 μg/ml; 0.13* 10 ⁶ cpm/pg) for 1 h at RT in PBS. After incubation, the membranes were washed 3 times, 10 min each time, in PBS and visualized for radioactivity on Bas 2000 radioimaging system (Fuji). Unlabeled heparin (6 mg/ml) was added for competition of binding.

Liposome preparation and leakage assay

Dry lipid films were prepared by dissolving either dioleoylphosphatidylethanolamine (Avanti Polar Lipids, Alabaster, AL) (75 mol%) and dioleoylphosphatidylglycerol (25 mol%) in chloroform, and then removing the solvent by evaporation under vacuum overnight. Subsequently, buffer solution containing 10 mM Tris, pH 7.4, either with or without additional 150 mM NaCI, was added together with 0.1 M carboxyfluorescein (CF) (Sigma). After hydration, the lipid mixture was subjected to eight freeze-thaw cycles consisting of freezing in liquid nitrogen and heating to 60°C. Unilamellar liposomes with a diameter of about 130 nm (as found with cryo-TEM; results not shown) were generated by multiple extrusions through polycarbonate filters (pore size 100 nm) mounted in a LipoFast miniextruder (Avestin). Untrapped carboxyfluorescein was then removed by filtration through two subsequent Sephadex G-50 columns with the relevant Tris buffer as eluent. Both extrusion and filtration was performed at 22°C. The CF release was monitored by fluorescence at 520 nm from a liposome dispersion (10mM lipid in 10 mM Tris pH 7.4). An absolute leakage scale is obtained by disrupting the liposomes at the end of the experiment through addition of 0.8 mM Triton X100 (Sigma), thereby causing 100% release and dequenching of CF. A SPEX-fluorolog 1650 0.22-m double spectrometer (SPEX Industries) was used for the liposome leakage assay. CD-spectroscopy

Circular dichroism (CD) spectra were measured by a Jasco J-810 Spectropolarimeter (Jasco, Easton, USA). The measurements were performed in triplicate at 37°C in a 10 mm quartz cuvette under stirring with a peptide concentration of 10 μΜ. The effect on peptide secondary structure of liposomes at a lipid concentration of 100 μΜ, and of E. coli LPS at a concentration of 0.02 wt%, was monitored in the range 200-250 nm. To account for instrumental differences between measurements, the background value (detected at 250 nm, where no peptide signal is present) was subtracted. Signals from the bulk solution were also corrected for.

Fluorescence microscopy

For study of membrane permeabilization using the impermeant probe FITC, E. coli ATCC 25922 bacteria were grown to mid-logarithmic phase in TSB medium. The bacteria were washed and resuspended in either 10 mM Tris, pH 7.4, 10 mM glucose, to yield a suspension of 1x10 ⁷ cfu/ml. 100 μΙ of the bacterial suspension was incubated with 30 μΜ of the respective peptides at 30°C for 30 min. Microorganisms were then immobilized on poly (L-lysine)-coated glass slides by incubation for 45 min at 30°C, followed by addition onto the slides of 200 μΙ of FITC (6 μg/ml) in the appropriate buffers and incubated for 30 min at 30°C. The slides were washed and bacteria fixed by incubation, first on ice for 15 min, then in room temperature for 45 min in 4% paraformaldehyde. The glass slides were subsequently mounted on slides using Prolong Gold antifade reagent mounting medium (Invitrogen). For fluorescence analysis, bacteria and fungi were visualized using a Nikon Eclipse TE300 (Nikon, Melville, NY) inverted fluorescence microscope equipped with a Hamamatsu C4742-95 cooled CCD camera (Hamamatsu) and a Plan Apochromat *100 objective (Olympus, Orangeburg, NY). Differential interference contrast (Nomarski) imaging was used for visualization of the microbes themselves.

Electron Microscopy

For transmission electron microscopy, P. aeruginosa ATCC 27853 and S. aureus ATCC 29213 (1-2 x 10 ⁶ cfu/sample) were incubated for 2 h at 37°C with the peptide GKY25 at 30 μΜ. LL-37 (30 μΜ) was included as a control. Samples of P. aeruginosa and S. aureus suspensions were adsorbed onto carbon-coated copper grids for 2 min, washed briefly on two drops of water, and negatively stained on two drops of 0.75 % uranyl formate. The grids were rendered hydrophilic by glow discharge at low pressure in air. Specimens were observed in a Jeol JEM 1230 electron microscope operated at 60 kV accelerating voltage. Images were recorded with a Gatan Multiscan 791 CCD camera. For scanning electron microscopy of lung tissues, specimens were washed with cacodylate buffer, and dehydrated with an ascending ethanol series from 50% (v/v) to absolute ethanol (10 min per step). The specimens were then subjected to critical-point drying in carbon dioxide, with absolute ethanol as intermediate solvent, mounted on aluminium holders, sputtered with 30 nm palladium/gold, and examined in a JEOL JSM- 350 scanning electron microscope.

Degradation of prothrombin and thrombin

Prothrombin and thrombin (Innovative Research) (27 0.6 mg/ml) was incubated at 37°C with human neutrophil elastase (NE) (0.6 pg, 20 units/mg) (Sigma) and prothrombin also with cathepsin G (0.5 pg, 2 units/mg) (BioCol GmbH) or P. aeruginosa elastase (PAE) (30 mU, a generous gift from Dr. H. Maeda, Kumamoto University, Japan) in a total volume of 45 μΙ 10 mM Tris, pH 7.4 for different time periods as indicated in the figures. Neutrophils were prepared by routine procedures (Polymorphprep) from blood obtained from healthy human donors. The cells were disrupted by freeze-thawing and addition of 0.3% Tween 20. Neutrophil extracts (corresponding to 4.8 * 10 ⁷ cells) were incubated at 37° with thrombin (27 μ9, 0.6 mg/ml) for 180 min. The reaction was stopped by boiling at 95°C for 3 min. 6 μΙ of the material was analysed by RDA and 20 μΙ fractions analysed by SDS-PAGE using 16.5% precast Tris-tricine gels (Bio-rad), run under non-reducing or reducing conditions. The gels were stained with Coomassie brilliant blue and destained. Definition of cleavage products of thrombin

Peptide fragments of thrombin, digested by neutrophil elastase for 30 min, were separated by hplc (PerkinElmer Series 200) on a reversed phase column (Vydac 218TPC18, 250 x 4.6 mm, 5 μηη) (Dalco chromtech AB). After injection, samples were eluted with a gradient of acetonitrile in 0.1 % aqueous trifluroacetic acid at 1 ml per minute. Fractions were collected and stored at -80°C. Samples were freeze-dried, redissolved in water, and analyzed by RDA, SDS-PAGE, immunoblotting and gel-overlay assay (Fig. 4). Active fractions were analysed by combinations of MALDI-TOF MS, ESI- MS/MS, N- and C-terminal sequencing at the Karolinska Institutet Protein Analysis Center (PAC Stockholm). See legend to Figure 3 for additional information.

SDS-PAGE and immunoblotting

Prothrombin and thrombin, either intact or subjected to enzymes, were analyzed by SDS- PAGE on 16.5% Tris-tricine gels (Bio-Rad). Proteins and peptides were transferred to nitrocellulose membranes (Hybond-C). Membranes were blocked by 3% (w/v) skimmed milk, washed, and incubated for 1 h with rabbit polyclonal VFR 7 antibodies (1 :800) (Innovagen AB), washed three times for 10 min, and subsequently incubated (1 h) with HRP-conjugated secondary antibodies (1 :2000) (Dako), and then washed again three times, each time for 10 min. C-terminal thrombin peptides were visualized by an enhanced chemiluminesent substrate (LumiGLO®) developing system (Upstate cell signaling solutions). For identification of TCPs in human fibrin, normal citrate-plasma was supplemented with 10 mM Ca ²⁺ in eppendorf tubes at 37°C overnight. The clots formed were washed three times with PBS and incubated with human neutrophil elastase (20 units/mg) for 0, 1, and 3 h at 37°C. Samples were centrifuged at 10000 RPM for 10 min, after which supernatants and pellets were separated. Samples were freeze-dried and then redissolved in 60 % acetonitrile and 0.1 % aqueous TFA. Pooled samples were freeze-dried, redissolved in water and analysed by SDS-PAGE followed by immunoblotting as above. For identification of TCPs in human citrate plasma, 1.5 μΙ of citrate-plasma or patient would fluids were analysed by SDS-PAGE under reducing conditions, followed by immunoblotting as above.

Gel-overlay assay

Gel overlay assay was performed essentially as described previosly ³⁷ . Briefly, duplicate samples were run on non-denaturing acid urea (AU-PAGE) gels in 5% acetic acid at 100 V for 1 h 15 min. Bacteria were grown overnight in TH broth, inoculated, and grown until the OD was 0.4. The bacteria were washed and resuspended in 10 mM Tris, pH 7.4. Bacteria (4 x 0 ⁶ ) were added to 12 ml melted underlay agarose (10 mM Tris, pH 7.4, 0.03% TH broth, 1% agarose type 1 (Sigma-Aldrich)) and poured into a square petri dish. One AU gel was stained with Coomassie brilliant blue and one AU gel was washed three times for 4 min in 10 mM Tris, pH 7.4 and then placed on top of the agarose gel and incubated for 3 h at 37°C. The AU gel was then removed and an overlay agarose (6% TH broth, 1% agarose type 1) was poured on top of the underlay and incubated overnight at 37°C. To make the clearing zones more visible, the agarose was stained with Coomassie brilliant blue and then destained with water.

Animal infection model

Animals were housed under standard conditions of light and temperature and had free access to standard laboratory chow and water. P. aeruginosa 15159 bacteria were grown to early logarithmic phase (ODg20~0.35), harvested, washed in 10 mM Tris, pH

7.4, diluted in the same buffer to 2 x 10 ⁶ cfu/ml, and kept on ice until injection. Five hundred microliter of the bacterial suspension were injected intraperitoneally (ip) into female BALB/c mice. Sixty minutes after the bacterial injection, 0.5 mg GKY25 or buffer alone was injected sc into the mice. This was repeated after 24 hours. In this Pseudomonas infection model, infected mice develop severe signs of sepsis within 1-2 days and usually do not recover from the infection. In order to study bacterial dissemination to target organs, the mice were infected as previously described and after a time period of 8 hours, spleen and kidney were harvested, placed on ice, homogenized, and colony-forming units determined. The P-value was determined using the Mann-Whitney U-test. The Laboratory Animal Ethics Committee of Malmo/Lund has approved the animal experiments.

LPS effects on macrophages in vitro 3.5x10 ⁵ cells were seeded in 96-well tissue culture plates (Nunc, 167008) in phenol red- free DMEM (Gibco) supplemented with 10% FBS and antibiotics. Following 6 hours of incubation to permit adherence, cells were stimulated with 100 or 10 ng/mL E. coli (011 1 :B4) or P. aeruginosa LPS (Sigma), with and without peptide of various doses. The levels of NO in culture supernatants were determined after 24 hours from stimulation using the Griess reaction ³⁸ . Briefly, nitrite, a stable product of NO degradation, was measured by mixing 50 μΙ of culture supernatants with the same volume of Griess reagent (Sigma, G4410) and reading absorbance at 550 nm after 15 min. Phenol-red free DMEM with FBS and antibiotics were used as a blank. A standard curve was prepared using 0-80 μΜ sodium nitrite solutions in ddH20. LPS model in vivo

C57BL/6 mice (8-10 weeks, 22 +/- 5g), divided into weight and sex matched groups, were injected intrapentoneally with 18 mg £ coli 011 1 :B4 LPS (Sigma) per kg of body weight. Thirty minutes after LPS injection, 0.2 mg GKY25 or buffer alone was intrapentoneally into the mice. Survival and status was followed during seven days. For the cytokine assay, mice were sacrificed 6 hours post LPS challenge, and blood was collected by cardiac puncture. For SEM, mice were sacrificed 20 h after LPS challenge, and lungs were removed and fixed. The Laboratory Animal Ethics Committee of Malmo/Lund has approved the animal experiments.

Cytokine assay

The cytokines IL-6, IL-10, MCP-1 , INF-γ, TNF, and IL-12p70 were measured in plasma from LPS-infected mice (with or without GKY25 treatment) using the Cytometric bead array; mouse inflammation kit (Becton Dickinson AB) according to the manufacturer's instructions.

Statistical analysis

Bar diagrams (RDA, VCA) are presented as mean and standard deviation, from at least three independent experiments. Animal data are presented as dot plots, with mean for normally distributed data, or median for data, which do not meet the criteria for normal distribution. Outliers were not excluded from the statistical analysis. Differences with P<0.05 were considered statistically significant

MIC, hemolysis, MTT, and LDH assay

MIC assay was carried out by a microtiter broth dilution method as previously described in the NCSLA guidelines ³⁹ .

Minimal inhibitory concentration (MIC) determination

MIC assay was carried out by a microtiter broth dilution method as previously described in the NCSLA guidelines (Wiegand, I., Hilpert, K. & Hancock, R.E. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 3, 163-175 (2008)). In brief, fresh overnight colonies were suspended to a turbidity of 0.5 units and further diluted in Mueller-Hinton broth (Becton Dickinson). For determination of MIC, peptides were dissolved in water at concentration 10 times higher than the required range by serial dilutions from a stock solution. Ten μΙ of each concentration was added to each corresponding well of a 96-well microtiter plate (polypropylene, Costar Corp.) and 90 μΙ of bacteria (1x105) in MH medium added. The plate was incubated at 37 °C for 16-18 h. MIC was taken as the lowest concentration where no visual growth of bacteria was detected.

Hemolysis assay

EDTA-blood was diluted (1 : 1 ) with PBS. The cells were then incubated with end-over- end rotation for 1 h at 37°C in the presence of peptides (60 and 120 μΜ). 2% Triton X- 100 (Sigma-Aldrich) served as positive control. The samples were then centrifuged at 800 g for 10 min. The absorbance of hemoglobin release was measured at λ 540 nm and is in the plot expressed as % of TritonX-100 induced hemolysis.

Lactate dehydrogenase (LDH) assay

HaCaT keratinocytes were grown in 96 well plates (3000 cells/well) in serum free keratinocyte medium (SFM) supplemented with bovine pituitary extract and recombinant EGF (BPE-rEGF) (Invitrogen, USA) to confluency. The medium was then removed, and 100 μΙ of the peptides investigated (at 3, 6, 30 and 60 μΜ, diluted in SFM/BPE-rEGF with 20% human serum), were added in triplicates to different wells of the plate, and incubations were performed for 16 h. The LDH based TOX-7 kit (Sigma-Aldrich, St Louis, USA) was used for quantification of LDH release from the cells. Results given represent mean values from triplicate - 1 - measurements. Results are given as fractional LDH release compared to the positive control consisting of 1 % Triton X-100 (yielding 100% LDH release). MTT assay

Sterile filtered MTT (3-(4,5-dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide; Sigma-Aldrich) solution (5 mg/ml in PBS) was stored protected from light at - 20°C until usage. HaCaT keratinocytes, 3000 cells/well, were seeded in 96 well plates and grown in keratinocyte-SFM/BPE-rEGF medium to confluency. Peptides were then added at the concentrations indicated in the figure (in the same medium supplemented with 20% human serum). After incubation for 16 h, 20 μΙ of the MTT solution was added to each well and the plates incubated for 1 h in C02 at 37°C. The MTT containing medium was then removed by aspiration. The blue formazan product generated was dissolved by the addition of 100 μΙ of 100% DMSO per well. The plates were then gently swirled for 10 min at room temperature to dissolve the precipitate. The absorbance was monitored at 550 nm, and results given represent mean values from triplicate measurements.

Alignment of TCPs

The prothrombin amino acid sequence was retrieved from the NCBI site. Each sequence was analyzed with Psi-Blast (NCBI) to find the ortholog and paralog sequences. Sequences that showed structural homology >70% were selected. These sequences were aligned using ClustalW using Blosum 69 protein weight matrix settings. Internal adjustments were made taking the structural alignment into account utilizing the ClustalW interface. The level of consistency of each position within the alignment was estimated by using the alignment-evaluating software Tcoffee.

Results

Proteolysis of prothrombin and thrombin generates antimicrobial activity

To test the hypothesis that prothrombin or its activated forms may generate antimicrobial peptides upon fragmentation, we incubated human prothrombin and thrombin with neutrophil elastase, a major neutral protease released by leukocytes during blood coagulation and inflammation or in response to bacterial products such as endotoxins. Earlier studies have shown that neutrophil elastase acts on proteinase-sensitive regions in human thrombin, generating smaller fragments ²¹ . As judged by the RDA assays (Fig. 1 a), digestion of the proteins yielded antimicrobial activity already after 5 min of incubation with the enzyme. In contrast, the intact mother proteins were inactive. The activity following proteolysis was still observed after several hours of incubation, suggesting the presence of relatively stable intermediates. It is noteworthy that the observed inhibition zones were similar to those generated by the classical antimicrobial peptide LL-37. Analysis by SDS-PAGE (Fig. 1 b) showed that the degradation generated several low molecular weight fragments in the range of 5-15 kDa. In spite of the known amidolytic properties of thrombin, no detectable antimicrobial activity was detected after prolonged incubation of the enzyme form alone (not shown). The observation that the zymogen as well as the activated forms generated similar activities, suggests that the antimicrobial epitopes localize to regions in the active enzyme after R271 (prothrombin numbering).

Structure-based screening for identification of antimicrobial epitopes

In order to identify possible antibacterial peptide regions of prothrombin/thrombin, overlapping peptide sequences comprising 20mers (Fig. 2a) were synthesized and screened for antibacterial activities against the two test bacteria E. coli and P. aeruginosa. Properties common for most antimicrobial peptides include minimum levels of cationicity, amphipathicity, and hydrophobicity ⁵ . Taking these structural prerequisites into account, additional peptides comprising regions of high net charge and/or presence of amphipathic helical regions, such as those encompassing the C- terminal region, were selected and synthesized (Fig. 2a). The experiments showed that particularly peptides derived from the C-terminal region (peptides 45-48) were antimicrobial, although other active peptides were also identified (e.g., 9 and 31) (Fig. 2b). However, at high ionic strength (0.1 M NaCI), only the C-terminal peptides retained their antimicrobial activity against E. coli as well as P. aeruginosa (Fig. 6) demonstrating that only this region, characterized by a high relative hydrophobicity (pHrel), positive net charge (Fig. 2b) and amphipathicity (Fig. 2c), features typical of classical antimicrobial peptides ^1-4 , may generate peptides active against bacteria at physiologic conditions. Corresponding to the antimicrobial activities observed above, only peptides derived from the C-terminal part bound to E. coli LPS (Fig. 2d). Since the absence of activity of the holoproteins in RDA could possibly be attributed to their high molecular weight (compromising diffusion during the assay), the antibacterial results above were further substantiated by matrix-free viable count assays. The results demonstrated that in contrast to the holoproteins, the selected model peptides VFR17 and GKY25 from the C- terminal part of the enzyme (highlighted in Fig. 2e) demonstrated significant antibacterial activity (Fig. 2f), thus corroborating the RDA assays above (Fig. 1 a and Fig. 2 b). In conclusion, LPS-binding and antimicrobial data, combined with structural and biophysical considerations clearly indicate a pivotal role of Thrombin-derived C-terminal Peptides, in the following text denoted "TCP", for mediating the antimicrobial activity.

Definition of low molecular weight fragments generated by degradation of thrombin In parallel to the above structure-based screening approach, studies were undertaken to identify active fragments generated after subjection of thrombin to neutrophil elastase. RP-HPLC separation of elastase-digested thrombin, followed by antibacterial assays using E. coli identified several antibacterial peptide fractions (Fig. 3a). Combined analyses using aldi-TOF, ESI- S/MS, and N- and C-terminal sequencing of fraction no. 38, which contained the majority of the activity comprising peptides active in high salt, unambiguously identified a major 1 1041 Da fragment comprising the C-terminal 96 amino acids of thrombin (predicted pi 8.4, see Fig. 2a for sequence). Correspondingly, SDS-PAGE identified one single peptide of ~1 1 kDa, that contained the C-terminal epitope, as shown by immunoblot analysis using antibodies against the C-terminal peptide VFR 7 (Fig. 3a). Gel overlay assays demonstrated that the major antimicrobial peptide of fraction 38 corresponded to one major active peptide, also identified in neutrophil elastase-digested thrombin (Fig. 3a), showing significantly lower mobility when compared to the C-terminal peptide GKY25 (not shown). Interestingly, Maldi-TOF and ESI-MS/MS of fraction no. 30 identified the peptide HVF18, described in the previous in vitro screening experiments (Fig. 2a, b), as well as a shorter 16 aa long variant, both peptides from the C-terminal region of thrombin. The analyses of the less hydrophobic material (fractions 20 and 21) yielded several low molecular weight fragments corresponding to internal, and cationic, sequences of low hydrophobicity and amphipathicity, matching antibacterial regions identified by the previous 20-mer screening (Table 1 and Fig. 2a). Taken together, these results showed that neutrophil elastase generates antimicrobial TCPs, of which the major forms comprise a ~1 1 kDa fragment of 96 amino acids, but also smaller fragments from the distal helical and amphipathic terminus.

Thrombin-derived C-terminal fragments are generated by human and bacterial proteinases

During inflammation, neutrophils release a multitude of enzymes, which could have activity on either thrombin or its proform. Therefore, thrombin was incubated with supernatants from activated neutrophils and the material analysed for antimicrobial activity and generation of TCPs. Indeed, antimicrobial activity was found upon proteolysis, while immunoblotting identified several TCPs of similar molecular weights as those generated by neutrophil elastase alone (Fig. 3b). Similar fragments (Fig. 3c) and corresponding antimicrobial activity (Fig. 3d) were also detected when prothrombin was subjected to neutrophil elastase, cathepsin G as well as the bacterial thermolysin-like proteinase of Pseudomonas aeruginosa, lasB (also denoted P. aeruginosa elastase) ²² . Interestingly, low molecular weight peptides (~3-4 kDa), generated by the latter P. aeruginosa enzyme, co-migrated with the model peptide GKY25 (Fig 3c). These results demonstrated that both human and bacterial enzymes may generate TCPs, irrespective of the activation state of prothrombin.

C-terminal thrombin peptides are generated ex vivo and in vivo and are protective against infection

Prothrombin, as many other proteins in plasma, is under meticulous control by antiproteinases in the normal state, preventing its activation and/or degradation. Therefore, we hypothesized that favorable environments promoting TCP formation should comprise i) localisation as well as concentration of thrombin, and ii) local release of enzymes, such as neutrophil elastase or bacterial proteinases. These environments are typical of sites of injury and infection, such as skin wounds, comprising thrombin activation, fibrin formation, bacterial colonisation or infection, and subsequent neutrophil influx. Earlier studies have shown that thrombin binds to fibrin clots, and that fibrin acts as a reservoir for active thrombin ²³ . Furthermore, human neutrophils release elastase during clotting, and neutrophils also penetrate fibrin ²¹ . Bacteria, such as S. aureus and P. aeruginosa frequently colonize and infect skin wounds, accompanied by excessive proteolysis and activation of neutrophils ^{24, 25} . Given this, the production of TCPs in fibrin, as well as in sterile or infected biological fluids was investigated. The results showed that TCPs were formed when fibrin clots were subjected to neutrophil elastase in vitro (Fig. 4a). Furthermore, a similar ~1 1 kDa fragment was detected in fibrin "slough" from a patient with a non-infected chronic venous leg ulcer, indicating that TCPs can be found in fibrin in vivo (Fig. 4a). Analogous results, showing rapid formation of TCPs, were obtained using human plasma subjected to proteolysis by neutrophil elastase, thus simulating the high elastase activity observed during wounding (Fig. 4b). Importantly, TCPs were also identified in wound fluid from patients post-surgery, as well as in wound fluid from patients with chronic (non-infected) venous leg ulcers (Fig. 4b). The latter wounds are always colonised by bacteria such as S. aureus and P. aeruginosa ² *. Notably, there was a variation in the occurrence of the ~1 1 kDa TCP in this latter patient group. Experiments with the model C-terminal peptide GKY25 (see Fig. 2a for sequence), were employed in order to determine effects at physiological conditions. GKY25 was bactericidal in physiological salt buffer and in presence of human plasma (Fig. 4c). The efficacy was similar to the human cathelicidin peptide LL-37 (although the latter peptide was inactive in murine plasma) (Fig. 4c). Notably, the MIC-levels of GKY25, according to standard NCSLA-protocols, were comparable to, and in some cases lower than, those observed for LL-37 and omiganan (Table 2). Since the latter is a highly active and broad-spectrum designed antimicrobial peptide now in Phase III clinical studies, the data on GKY25 also indicated a therapeutic role for TCPs. Initial studies revealed no significant permeabilizing effects of GKY25 on human erythrocytes (60-120 μΜ peptide) as well as keratinocytes (up to 60 μΜ peptide) in plasma and serum conditions, respectively (Fig. 7). In order to investigate a possible in vivo function of GKY25, we therefore injected this peptide subcutaneously into mice infected intraperitoneally with P. aeruginosa. Compared to the controls, treatment with GKY25 yielded a significant increase in survival and significantly lower bacterial numbers in the spleen and kidney of the animals (Fig. 4d, e). Taken together, these results demonstrate that TCPs constitute a previously undisclosed neo-structure of thrombin, formed in vitro as well as in vivo in plasma, but also in wound fluid and fibrin, exerting activities in physiological conditions similar to "classic" HDPs such as LL-37, and finally, showing significant therapeutic potential.

Immunomodulatory roles of TCPs

As mentioned above, recent evidence shows that HDPs trigger a range of immunomodulatory responses. In fact, since antimicrobial activities of HDPs such as LL- 37 are antagonized by plasma (see eg. Fig. 4c, right panel), direct killing of microbes has been questioned for various antimicrobial peptides ²⁶ . The observation of LPS-binding of TCPs (Fig. 2d), prompted us to investigate possible endotoxin-neutralizing effects of the model peptide GKY25. Slot-binding experiments showed that the peptides bound heparin as well as LPS from E. coli and P. aeruginosa (Fig. 12). In a mouse macrophage model, GKY25 significantly inhibited NO-release of LPS-stimulated macrophages (Fig. 14), as well as release of TNF-a at concentrations below 2 μΜ (Fig. 8). Similar effects on TNF-a were noted using human monocyte-derived macrophages (not shown). In a mouse model of LPS-induced shock, GKY25 displayed a dramatic improvement on survival (Fig. 14). Analyses of cytokines 6 hours after LPS injection, showed significant reductions of proinflammatory IL-6, IFN-γ, TNF-a, and IL-12p70, whereas IL-10 remained unchanged (Fig. 15). SEM analyses of lungs from LPS-treated animals demonstrated pulmonary leakage of protein and red blood cells (see inset in Fig. 16), an effect completely blocked by GKY25 (Fig. 16). The results thus demonstrate that GKY25, like many HDPs, is multifunctional; in addition to its antimicrobial activity it also exerts potent anti-endotoxic and immunomodulatory effects. Functional and structural studies of thrombin-derived C-terminal peptides

To examine possible peptide-induced permeabilization of bacterial plasma membranes, P. aeruginosa and S. aureus was incubated with GKY25 at concentrations yielding complete bacterial killing (30 μΜ), and analyzed by electron microscopy (Fig. 5a). Clear differences in morphology between peptide-treated bacteria and the control were demonstrated. The peptide caused local perturbations and breaks along P. aeruginosa and S. aureus plasma membranes, and intracellular material was found extracellularly. These findings were similar to those seen after treatment with LL-37 (Fig. 5a). The data suggest that GKY25 acts on bacterial membranes, but do not demonstrate the exact mechanistic events following peptide addition to bacteria, as secondary metabolic effects on bacteria may also trigger bacterial death and membrane destabilization. However, analogous results were also obtained using the impermeant dye FITC and E. coli as test bacterium (Fig. 5b) demonstrating membrane permeabilisation after exposure to GKY25. Kinetic studies showed that GKY25 killed >90% of bacteria after 10 minutes, compatible with a direct action on bacterial membranes (Fig. 9). Furthermore, circular dichroism (CD) spectroscopy was used to study the structure and the organization of the peptides GKY25 and VFR17 in solution and on interaction with negatively charged (bacteria-like) liposomes as well as E. coli LPS. Neither GKY25 nor VFR17 adopted an ordered conformation in aqueous solution, however the CD spectra revealed significant structural change, largely induction of helicity, taking place in the presence of negatively charged liposomes (Fig. 5c), and E. coli LPS (Fig. 5d). Compatible with earlier results, LL-37 displayed some helicity also in buffer solution ²⁷ . Similarly to LL-37, the two thrombin- derived peptides induced leakage of liposomes, also at high ionic strength (Fig. 5e). Kinetic analysis showed that ~80% of the maximum leakage was reached within ~200 seconds for the two thrombin-derived peptides (at 1 μΜ) (not shown). Considering the above results with GKY25 and VFR17, both containing the crucial helical (and antimicrobial) epitope, the results therefore indicate that TCPs function like most helical AMPs such as LL-37 ^{5, 28} , by interactions with both the lipid membrane and LPS (possibly also peptidoglycan) at bacterial surfaces, leading to induction of an a-helical conformation, which in turn facilitates membrane interactions, membrane destabilization, and bacterial killing.

The TCP structure complies with a γ-core motif and is evolutionary conserved

Recently, a multidimensional signature, the γ-core motif, was identified in multiple classes of cystein-containing AMPs ²⁹ . Analysis showed that the 96aa TCP is closely related to this fundamental motif so common in various HDPs (Fig. 10). Furthermore, this region of thrombin is highly conserved in various species (Fig. 1 1 ). Next, we compared the antibacterial activities of C-terminal peptide GKY25 of thrombin with corresponding peptides from other closely related human coagulation factors (Fig. 5f, for sequences see Table 3). Whereas the peptide from thrombin (Factor II), as well as peptides from factors X and IX were antimicrobial against Gram-negative P. aeruginosa and E. coli, Gram- positive S. aureus and B. subtilis, as well as the fungus C. albicans (Fig. 5f), corresponding peptides from factor XI and kallikrein were inactive against these microbes (not shown). As seen in the 3D model (Fig. 5g), the coagulation factors (II, X, IX) share a similar overall structure with a helical C-terminal "tail". Indeed, C-terminals of these factors, as well as factor XI and kallikrein, contain a pattern sequence {DS}-X- [PFY]-G-[FIV]-Y-T-X-V-{C}-[AEQRY]-X-{R}-X-W-[IL]-X-{H}-X(4,2 4), [SEQ ID NO:131] which describes an amphipathic structure. However, only factors II, X, IX have a positive net charge (+3 or more) in this region (Table 3), thus in perfect agreement with the data obtained on the antimicrobial activity (Fig. 5f). Taken together, these analyses show that the TCP molecule represents a novel neo-structure, which is related to other cysteine- linked HDPs containing the γ-core motif, and also found in closely related coagulation factors.

Discussion

The key finding in this report is the discovery of a novel function of thrombin-derived C-terminal peptides in host defense, involving an inhibitory effect on inflammatory and coagulation pathways. The findings expand the field of innate immunity to thrombin and the coagulation system. From an evolutionary perspective, this function of thrombin is logical, since injury and infection both represent situations necessitating an optimised innate immune system. Hence, from the perspective of wounding, thrombin, after fulfilling its primary function in generating a first line of defense, the fibrin clot, further functionaiises this natural physical shield by subsequent generation of antimicrobial and anti-endotoxic HDPs upon proteolysis.

The significant and curative effect of a thrombin-derived peptide in a model of LPS- induced shock underscores the anti-inflammatory role this novel peptide, and contrasts to the pro-inflammatory actions of other HDPs, such as the anaphylatoxin C3a and chemotactic defensins ^{14, 30} . Thus, during injury and infection, different pathways are activated, employing HDPs with multiple and sometimes opposite roles, all balancing and fine-tuning inflammation while counteracting microbial invasion. Recent evidence showing a significant cross-talk between the coagulation and complement systems ³¹ further adds biological relevance to the simultaneous generation of C3a and various TCPs. TCPs further add to the increasingly recognized redundancy of host defense mechanisms, enforcing optimised control of the microbial flora by minimising the risk for resistance development against one particular HDP, as well as protecting against detrimental effects due to specific HDP deficiencies. Notably, the observation of proteolytic formation of multiple TCP fragments of different lengths parallel previous findings on LL-37 and C3a ^{14, 32} , showing that these molecules are further processed while retaining their antimicrobial activity. Efficiency of the C-termina! peptide GKY25 in plasma, under standard NCSLA conditions, as well as in an animal model of P. aeruginosa sepsis, indicate an in vivo role for released TCPs. These findings, in concert with the anti-endotoxic and immunomodulatory effects of the peptides, indicate a therapeutic application for TCPs in treatment of local and systemic infections, as well as sepsis. Recent evidence, showing a higher susceptibility to infection in mice rendered thrombin-deficient ^{33, 34} , is also compatible with the new role of thrombin-derived C- terminal peptides revealed here. Of relevance is also the increased susceptibility to infection after inhibition of the contact system, linked to abrogated release of kininogen- derived HDPs ⁶ , but possibly also due to a reduced capability to form TCPs and other antimicrobial molecules associated with fibrin networks. Of particular clinical relevance is the finding that TCPs are detected in wound fluids from patients with acute surgical wounds, as well as in patients with non-healing wounds. The latter patient group is characterised by an excessive bacterial colonization e.g. by P. aeruginosa, extensive proteolysis and inflammation ²⁴ . Furthermore, the noted absence of TCPs in some patients is indicative of a defective host-defense and diminished control of released endotoxins (local or systemic). Thus, apart from therapeutical possibilities, the present findings also provides a diagnostic marker for inflammation.

Table 1 : Peptide sequences of fraction 20-21

Masses were obtained by MALDI-MS analysis, and possible peptide sequences from the prothrombin sequence were deduced using the FINDPEPT tool (www.expasy.org/tools/findpept.html)

Table 2: Minimal inhibitory concentrations (MIC) of GKY25, LL-37 and omiganan against various bacterial isolates

The analysis was performed as described in Wiegand et al. and according to NCSLA guidelines. Additional clinical isolates were obtained from the Department of Bacteriology, Lund University Hospital. P. aeruginosa, E. coli and E. faecalis isolates were initially derived from patients with chronic ulcers, S. aureus from patients with atopic dermatitis. The S. pyogenes strain AP1 was from the WHO Collaborating Center for References and Research on Streptococci (Prague, Czech Republic).

Table 3: Sequences of coagulation factor-derived peptides

Example B - Anti-inflammatory and anti-coagulant properties of exemplary C- terminal peptides from S1 serine peptidases

Introduction

Sepsis

Sepsis is an infection-induced syndrome characterized by a generalized inflammatory state and represents a frequent complication in the surgical patient, or in relation to burns. Severe sepsis is a common, expensive and frequently fatal condition, having an estimated incidence of 176 per 100,000 of the population. The death rate varies from 20% to 80%. The normal reaction to infection involves complex immunologic cascade in response to microbial invasion in humans. Septic shock develops in a number of patients as a consequence of excessive or poorly regulated immune response to the invading microbe (Gram-negative or Gram-positive bacteria, fungi, viruses, or microbial toxins). A series of inflammatory molecules are then released and cells and defense systems activated, including the release of cytokines, the activation of neutrophils, monocytes, and microvascular endothelial cells, as well as the activation of neuroendocrine reflexes and plasma protein cascade systems, such as the complement system, the intrinsic (contact system) and extrinsic pathways of coagulation, and the fibrinolytic system. Thus, systemic activation of proteolytic host cascades, such as the complement, coagulation, and contact systems, plays an important role in sepsis, together with a massive release of pro-inflammatory cytokines. COPD

Chronic obstructive pulmonary disorder (COPD) refers to a range of chronic disorders in the airways characterized by irreversible and progressing decline in airflow to the lung capillaries. Although several factors contribute to the development of COPD, smoking is the most important cause. COPD predominantly develops in long-term smokers from their late-30s and progressively develops in an irreversible fashion. According to 2007 estimates by WHO, there are currently 210 million patients with COPD, and 3 million people died of COPD in 2005. WHO also predicts that COPD will become the fourth leading cause of death worldwide by 2030. Several factors are expected to contribute to this increase, including increased diagnosis rates, lack of treatments that reverse the inflammatory disease progression, and a globally ageing population burden. Also in this condition, activation of proteolytic host cascades, such as the complement, coagulation, and contact systems, plays an important role.

Treatments Current treatment of severe sepsis involves both treatment of the infection with appropriate early antibiotic therapy against the identified or presumed organisms plus surgical drainage where necessary, and supportive treatment according to patients' symptoms and signs. FDA has approved recombinant human activated protein C (APC; Xigris®) for use in people with severe sepsis. In August 2002 the European Agency for Evaluation of Medicinal Products approved the addition of the drug to best standard care, (based on sepsis-related multiple organ failure (SOFA) as a measure of disease activity for treatment of adult patients with severe sepsis and multiple organ failure. In a recent Cochrane report however, the authors found no evidence suggesting that APC should be used for treating patients with severe sepsis or septic shock. Additionally, APC was found to be associated with a higher risk of bleeding. It was concluded that additional RCTs must provide further evidence of a treatment effect if APC treatment is to be continued. Unless additional RCTs provide evidence of a treatment effect, the use of APC should not be promoted (Marti-Carvajal AJ, Salanti G, Cardona-Zorrilla AF. Human recombinant activated protein C for severe sepsis. Cochrane Database of Systematic Reviews 2008, Issue 1. Art. No.: CD004388. DOI: 10.1002/14651858.CD004388.pub3.)

Some reports show that therapy with polyclonal intravenous immunoglobulin (ivlg) reduces mortality among critically ill adults with severe sepsis and septic shock. One meta-analysis demonstrated an overall reduction in mortality with the use of ivlg for the adjunctive treatment of severe sepsis and septic shock in adults, although significant heterogeneity was found to exist among the trials and this result was not confirmed when only high-quality studies were analyzed (Laupland KB, Kirkpatrick AW, Delaney A, Polyclonal intravenous immunoglobulin for the treatment of severe sepsis and septic shock in critically ill adults: A systematic review and meta-analysis. Crit Care Med. 2007 Oct 23).

Materials & Methods Peptides

20mer peptides corresponding to various overlapping regions of prothrombin as well as the S1 peptidases, and the 6-25 aa peptides based on the original sequence GKY25 [SEQ IQ NO:126] were obtained from Sigma (PEPscreen®, Custom Peptide Libraries, SigmaGenosys).

Definition of S1 peptidase sequences

In order to find analogues to the described peptides sequence patterns were defined for the specific regions. Human proteins in the Swiss-Prot protein database (http://www.expasv.org/sprot/) were searched using the ScanProsite search tool (http://www.expasy.org/tools/scanprosite/).

The ScanProsite tool allows to scan protein sequence(s) (either from UniProt Knowledgebase (Swiss-Prot/TrEMBL) or PDB or provided by the user) for the occurrence of patterns, profiles and rules (motifs) stored in the PROSITE database, or to search protein database(s) for hits by specific motif(s)

Patterns for the conserved regions were constructed according to the following syntax: Pattern syntax used in the PROSITE database:

1. The standard lUPAC one-letter codes for the amino acids are used.

2. The symbol ^' x' is used for a position where any amino acid is accepted.

3. Ambiguities are indicated by listing the acceptable amino acids for a given position, between square brackets ^' [ ]'. For example: [ALT] stands for Ala or Leu or Thr.

4. Ambiguities are also indicated by listing between a pair of curly brackets ^' { }' the amino acids that are not accepted at a given position. For example: {AM} stands for any amino acid except Ala and Met.

5. Each element in a pattern is separated from its neighbor by a

6. Repetition of an element of the pattern can be indicated by following that element with a numerical value or, if it is a gap ('χ'), by a numerical range between parentheses.

Examples: x(3) corresponds to x-x-x , x(2,4) corresponds to x-x or x-x-x or x-x-x-x and A(3) corresponds to A-A-A

Note: You can only use a range with 'χ', i.e. A(2,4) is not a valid pattern element.

7. When a pattern is restricted to either the N- or C-terminal of a sequence, that pattern either starts with a ^' <' symbol or respectively ends with a ^' >' symbol. In some rare cases (e.g. PS00267 or PS00539), '>' can also occur inside square brackets for the C-terminal element. 'F-[GSTV]-P-R-L-[G>]' means that either 'F- [GSTVj-P-R-L-G' or 'F-[GSTV]-P-R-L>' are considered.

Sequence motif: The motif denoted by SEQ ID NO: 132 below was used to identify human S1 peptidases corresponding to the GKY25 peptide sequence from thrombin [i.e. SEQ ID NO: 126]:

X(2)-[PFY]-X(2)-[AFY]-[AITV]-X-[ILV]-X(5)-W-[IL]-X(5,30)& gt; [SEQ ID NO:132], Electron Microscopy

For scanning electron microscopy of lung tissues, specimens were washed with cacodylate buffer, and dehydrated with an ascending ethanol series from 50% (v/v) to absolute ethanol (10 min per step). The specimens were then subjected to critical-point drying in carbon dioxide, with absolute ethanol as intermediate solvent, mounted on aluminium holders, sputtered with 30 nm palladium/gold, and examined in a JEOL JSM- 350 scanning electron microscope.

LPS model in vivo

C57BL/6 mice (8-10 weeks, 22 +/- 5g), divided into weight and sex matched groups, were injected intraperitoneally with 18 mg £ coli 01 11 :B4 LPS (Sigma) per kg of body weight. Thirty minutes after LPS injection, 0.2 mg GKY25 or buffer alone was intraperitoneally into the mice. Survival and status was followed during seven days. For the cytokine assay, mice were sacrificed 6 hours post LPS challenge, and blood was collected by cardiac puncture. For SEM, mice were sacrificed 20 h after LPS challenge, and lungs were removed and fixed. The Laboratory Animal Ethics Committee of almo/Lund has approved the animal experiments. Cytokine assay

The cytokines lL-6, IL-10, MCP-1 , INF-γ, TNF, and IL-12p70 were measured in plasma from LPS-infected mice (with or without GKY25 treatment) using the Cytometric bead array; mouse inflammation kit (Becton Dickinson AB) according to the manufacturer's instructions. Slot-blot assay

LPS-binding ability of the peptides was examined by a slot-blot assay. Peptides (2 and 5 μς) were bound to nitrocellulose membranes (Hybond-C, GE Healthcare Biosciences), pre-soaked in PBS. Membranes were then blocked by 2 wt% BSA in PBS, pH 7.4, for 1 h at RT, and subsequently incubated with ¹²⁵ l-labelled LPS (40 μg/ml; 0.13* 10 ⁶ cpm/pg) for 1 h at RT in PBS. After incubation, the membranes were washed 3 times, 10 min each time, in PBS and visualized for radioactivity on Bas 2000 radioimaging system (Fuji). Unlabeled heparin (6 mg/ml) was added for competition of binding.

Effects of various microbial products on macrophages in vitro and anti-inflammatory effects by various peptides

3.5* 10 ⁵ cells were seeded in 96-well tissue culture plates (Nunc, 167008) in phenol red- free DMEM (Gibco) supplemented with 10% FBS and antibiotics. Following 6 hours of incubation to permit adherence, cells were stimulated with 10 ng/mL E. coli (0 11 :B4) LPS (Sigma), lipoteichoic acid, peptioglycan, or zymosan, with and without peptides at the indicated doses (se figure legends and figures). The levels of NO in culture supernatants were determined after 24 hours from stimulation using the Griess reaction ³⁸ . Briefly, nitrite, a stable product of NO degradation, was measured by mixing 50 μΙ of culture supernatants with the same volume of Griess reagent (Sigma, G4410) and reading absorbance at 550 nm after 15 min. Phenol-red free DMEM with FBS and antibiotics were used as a blank. A standard curve was prepared using 0-80 μΜ sodium nitrite solutions in ddH20.

Clotting assays

All clotting times were measured using an Amelung coagulometer. Activated partial thromboplastin time (aPTT) was measured by incubating GKY25 diluted in sterile water at the indicated concentrations , with 100 pL citrated human plasma for 1 minute followed by the addition of 100 pL aPTT reagent (aPTT Automate, Diagnostica Stago) for 60 seconds at 37°C. Clotting was initiated by the addition of 100 pL of a 25-mM CaCI ₂ solution. In the prothombrin time assay (PT), clotting was initiated by the addition of 100 pL Thrombomax with calcium (PT reagent; Sigma-Aldrich). For measuring the thrombin clotting time (TCT), clotting was initiated by the addition of 100 pL Accuclot thrombin time reagent (TCT reagent; Sigma-Aldrich). Statistical analysis

Bar diagrams (RDA, VCA) are presented as mean and standard deviation, from at least three independent experiments. Animal data are presented as dot plots, with mean for normally distributed data, or median for data, which do not meet the criteria for normal distribution. Outliers were not excluded from the statistical analysis. Differences with P<0.05 were considered statistically significant.

Results

Peptides structurally and evolutionary related to exemplary peptide GKY25 (SEQ ID NO: 126) were analysed for antiinflammatory activity. The results showed that truncated variants containing the helical core region and comprising at least 20 amino acids were particularly active with respect to anti-inflammatory activity. Likewise, closely related peptides from different S1 peptidases, as well as evolutionary related peptides were anti-inflammatory. Furthermore, the results showed that the peptides depicted in SEQ ID NO:126 induced an increase of the aPTT and to a lesser extent PT. The effect of the SEQ ID NO: 126 peptide, as depicted was dose dependent. In contrast, the TCT, measuring thrombin-induced fibrin-network formation, was not affected in human plasma. The results demonstrate that the peptide targets the intrinsic and to a lesser extent the extrinsic pathway of coagulation. Hence, the results indicate that this, as well as related peptides have a potential as modulators of coagulation. Antiinflammatory effects of GKY25 and truncated variants.

Figure 18A-C shows that truncated variants of GKY25 exhibit anti-inflammatory effects. In general, a peptide length of minimum 20 amino acids is required to achieve the antiinflammatory effect.

Antiinflammatory effects of peptides evolutionary related to thrombin

Figure 19 shows that evolutionary close peptides related to GKY25 exhibit antiinflammatory effects. Antiinflammatory effects of peptides of S1 -peptidases

The coagulation factors thrombin (factor II), factor VII, factor IX, factor X, factor XI, factor XII, protein C and plasma kallikrein belong to the peptidase S1 family originating from the superfamily of trypsin-like serine proteases. They all share the same structural motif where the proteins are folded into two domains each arranged as a six-stranded antiparallel β barrel (Fig. 25). Thrombin, which is expressed by the liver and secreted in plasma cleaves bonds after Arg and Lys and converts fibrinogen to fibrin and activates factors V, VII, VIII, XIII, and, in complex with thrombomodulin, protein C. The carboxy terminus of all these proteins ends with an alpha helix. Work described in this application has shown that peptides derived from this part of the proteins, including thrombin (Fll), and factor X possess antibacterial properties as well as antiinflammatory activities (Fig 20A and B). Using concentrations of peptides (30 uM) there was a clear correlation between net charge and relative hydrophobic moment of the peptides, and anti- inflammatory activity.

Anticoagulative effects of GKY25

Figure 21 shows that incubation of plasma with GKY25 led to a increase of the aPTT and to a lesser extent PT.. The effect of GKY25, as depicted was dose dependent. In contrast, the TCT, measuring thrombin-induced fibrin-network formation, was not affected in human plasma. The results demonstrate that GKY25 targets the intrinsic and to a lesser extent the extrinsic pathway of coagulation.

Example C - The C-terminal sequence encodes host defense functions in serine proteases

Abbreviations: CFU, colony forming units; CF, carboxyfluorescein; ip, intraperitoneally; HDP, host defense peptide; LPS, lipopolysaccharide; MIC, minimum inhibitory concentration; RDA, radial diffusion assay; TH, Todd Hewitt.

Summary Serine proteases of the S1 family have maintained a common structure over an evolutionary span of more than one billion years, and evolved a variety of substrate specificities and diverse biological roles, involving digestion and degradation, blood clotting, fibrinolysis, and epithelial homeostasis. We here show that a wide range of C- terminal sequences of serine proteases share characteristics common with classical antimicrobial peptides of innate immunity. Under physiological conditions, these peptides exert antimicrobial effects as well as immunomodulatory functions, by inhibiting macrophage responses to bacterial lipopolysaccharide and fungal zymosan. In mice, selected peptides are protective against lipopolysaccharide-induced shock. Moreover, these S1 -derived host defense peptides exhibit helical structures upon binding to lipopolysaccharide, and also permeabilize liposomes. The results uncover new and fundamental aspects on host-defense functions of serine proteases present in particularly blood and epithelia, as well as provide predictive tools for the efficient identification of novel therapeutically interesting anti-inflammatory molecules. Introduction

The innate immune system provides a first line of defense against invading microbes (1- 5). Its effectors, such as the classic antimicrobial peptides defensins and cathelicidins, exert direct bactericidal effects, but also mediate various immunomodulatory functions including anti-endotoxic effects, chemotaxis and angiogenesis (6-8), thus motivating the broader definition host defense peptides (HDP) for these members of the innate immune system. The recent discovery that C-terminal peptides of human thrombin, a key enzyme in the coagulation cascade, constitute a novel class of host defense peptides with bactericidal and anti-inflammatory properties, has defined new HDPs and expanded the field of innate immunity to thrombin and the coagulation system (9). From an evolutionary perspective, this additional role of thrombin is logical, since injury and infection both represent situations necessitating an optimized innate immune system. Structurally, analyses show that the major thrombin-derived C-terminal peptide of 96 amino acids represents a novel structural entity, which is related to other cysteine-linked HDPs, including defensins, containing the γ-core motif (9, 10). C-terminals of thrombin, as well as other related coagulation factors, comply with a pattern sequence X-[PFY]-X-[AFILV]-[AFY]-[AITV]-X-[ILV]-X(5)-W-[IL]-X(5,26) [SEQ ID NO:2], found not only in these proteases of the coagulation system, but also present in the vast and diverse family of S1 peptidases. The observation that this sequence contains interspersed conserved hydrophobic residues, and given an a-helical conformation (which characterizes all structurally determined S1 peptidases), consequently describes a potential amphipathic structure, together with the fact that all S1 peptidases known contain a the γ-core motif, prompted us to investigate whether the concept of HDPs of coagulation factors could be extended to the family of S1 peptidases.

Materials and Methods Peptides

The S1 -derived peptides underlying Fig. 3 and 4, as well as Supplementary Fig. 2, were synthesized by Biopeptide Co. The purity (>95%) of these peptides was confirmed by mass spectral analysis (MALDI.TOF Voyager). LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES [SEQ ID NO:115]) was from Innovagen AB. 20mer peptides described in Fig. 22 and Supplementary Fig. 26 were from Sigma (Custom Peptide Libraries, SigmaGenosys).

Biological materials

Fibrin slough was collected from chronic venous leg ulcers (chronic wound slough/surface) with a sterile spatula and immediately fixed for electron microscopy. The research project was approved by the Ethics committee, Lund University Hospital. Written consent was obtained from the patients.

Microorganisms

Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa 15159, Staphylococcus aureus ATCC 29213, and the fungal isolate Candida albicans ATCC 90028 were from the Department of Bacteriology, Lund University Hospital.

Radial diffusion assay

Essentially as described earlier (1 1 , 12), bacteria were grown to mid-logarithmic phase in 10 ml of full-strength (3% w/v) trypticase soy broth (TSB) (Becton-Dickinson). The microorganisms were then washed once with 10 mM Tris, pH 7.4. Subsequently, 4x10 ⁶ cfu were added to 15 ml of the underlay agarose gel, consisting of 0.03% (w/v) TSB, 1 % (w/v) low electroendosmosis type (EEO) agarose (Sigma-Aldrich) and 0.02% (v/v) Tween 20 (Sigma-Aldrich). The underlay was poured into a 0 144 mm petri dish. After agarose solidification, 4 mm-diameter wells were punched and 6 μΙ peptide solution of required concentration added to each well. Plates were incubated at 37°C for 3 h to allow peptide diffusion. The underlay gel was then covered with 15 ml of molten overlay (6% TSB and 1 % Low-EEO agarose in distilled H ₂ 0). Antimicrobial activity of a peptide was visualized as a zone of clearing around each well after 18-24 h of incubation at 37°C.

Hemolysis assay EDTA-blood was centrifuged at 800 g for 10 min, whereafter plasma and buffy coat were removed. The erythrocytes were washed three times and resuspended in PBS, pH 7.4 to get a 5% suspension. The cells were then incubated with end-over-end rotation for 60 min at 37°C in the presence of peptides (60 μΜ). 2% Triton X-100 (Sigma-Aldrich) served as positive control. The samples were then centrifuged at 800 g for 10 min and the supernatant was transferred to a 96 well microtiter plate. The absorbance of hemoglobin released was measured at 540 nm and expressed as % of Triton X-100 induced hemolysis.

Viable count analysis

E. coli strains were grown to mid-logarithmic phase in Todd-Hewitt (TH) medium. P. aeruginosa strains were grown in TH overnight. Bacteria were washed and diluted in 10 mM Tris, pH 7.4 containing 5 mM glucose containing 0.15 M NaCI, alone or with 20% normal citrate-plasma. 2 x 10 ⁶ cfu/ml bacteria were incubated in 50 μΙ, at 37°C for 2 h with the S1-derived peptides at the indicated concentrations. Serial dilutions of the incubation mixture were plated on TH agar, followed by incubation at 37°C overnight and cfu determination. Liposome preparation and leakage assay

Dry lipid films were prepared by dissolving dioleoylphosphatidylethanolamine (Avanti Polar Lipids, Alabaster, AL) (70 mol%) and dioleoylphosphatidylglycerol (30 mol%) in chloroform, and then removing the solvent by evaporation under vacuum overnight. Subsequently, buffer solution containing 10 mM Tris, pH 7.4, either with or without additional 150 mM NaCI, was added together with 0.1 M carboxyfluorescein (CF) (Sigma). After hydration, the lipid mixture was subjected to eight freeze-thaw cycles consisting of freezing in liquid nitrogen and heating to 60°C. Unilamellar liposomes with a diameter of about 140 nm were generated by multiple extrusions through polycarbonate filters (pore size 100 nm) mounted in a LipoFast miniextruder (Avestin). Untrapped carboxyfluorescein was then removed by filtration through two subsequent Sephadex G- 50 columns with the relevant Tris buffer as eluent. Both extrusion and filtration was performed at 22°C. The CF release was monitored by fluorescence at 520 nm from a liposome dispersion (10 DM lipid in 10 mM Tris pH 7.4). An absolute leakage scale is obtained by disrupting the liposomes at the end of the experiment through addition of 0.8 mM Triton X100 (Sigma), thereby causing 100% release and dequenching of CF. A SPEX-fluorolog 1650 0.22-m double spectrometer (SPEX Industries) was used for the liposome leakage assay. Measurements were performed in triplicate at 37°C.

CD-spectroscopy

Circular dichroism (CD) spectra were recorded by a Jasco J-810 Spectropolarimeter (Jasco, Easton, USA). The measurements were performed in triplicate at 37°C in a 10 mm quartz cuvette under stirring with a peptide concentration of 10 ΠΜ. The effect on peptide secondary structure of liposomes at a lipid concentration of 100 ΠΜ, and of E. coli LPS at a concentration of 0.02 wt%, was monitored in the range 200-250 nm. To account for instrumental differences between measurements, the background value (detected at 250 nm, where no peptide signal is present) was subtracted. Signals from the bulk solution were also corrected for.

Electron Microscopy For transmission electron microscopy and visualization of peptide effects on bacteria, P. aeruginosa ATCC 27853 and S. aureus ATCC 29213 (1 -2 x 10 ⁶ cfu/sample) were incubated for 2 h at 37°C with S1 peptides (30 μΜ). LL-37 was included as a control. Samples of P. aeruginosa and S. aureus suspensions were adsorbed onto carbon- coated copper grids for 2 min, washed briefly by two drops of water, and negatively stained by two drops of 0.75 % uranyl formate. The grids were rendered hydrophilic by glow discharge at low pressure in air. For analysis of fibrin slough from patients with chronic venous ulcers, the material was fixed (1.5% PFA, 0.5% GA in 0.1 M phosphate buffer, pH 7.4) for 1 hour at room temperature, followed by washing with 0.1 M phosphate buffer, pH 7.4. The fixed and washed samples were subsequently dehydrated in ethanol and further processed for Lowicryl embedding (13). Sections were cut with a LKB ultratome and mounted on gold grids. For immunostaining, grids were floated on top of drops of immune reagents displayed on a sheet of parafilm. Free aldehyde groups were blocked with 50 mM glycine, and the grids were then incubated with 5% (vol/vol) goat serum in incubation buffer (0.2% BSA-c in PBS, pH 7.6) for 15 minutes. This blocking procedure was followed by overnight incubation at 4°C with 1 μg ml of polyclonal antibodies against the C-terminus of Factor X. Controls without these primary antibodies were included as well. After washing the grids in a large volume (200 ml) of incubation buffer, floating on drops containing the gold conjugate reagents, 1 μg/ml EM goat antiRabbit IgG 10nm Au (BBI) in incubation buffer was performed for 2 h at 4°C. After further washes by an excess volume of incubation buffer, the sections were postfixed in 2% glutaraldehyde. Finally, sections were washed with distilled water and poststained with 2% uranyl acetate and lead citrate. All samples were examined with a Jeol JEM 1230 electron microscope operated at 80 kV accelerating voltage. Images were recorded with a Gatan Multiscan 791 charge-coupled device camera.

LPS and zymosan effects on macrophages in vitro

3.5x 10 ⁵ RAW264.7 macrophage cells were seeded in 96-well tissue culture plates (Nunc, 167008) in phenol red-free DMEM (Gibco) supplemented with 10% FBS containing 1 % Anti-Anti (Invitrogen). Following 20 hours of incubation to permit adherence, cells were washed and stimulated with 10 ng/ml E. coli (01 1 1 :B4) with and without the S1 -derived peptides of various doses. The levels of NO in culture supernatants were determined after 24 hours from stimulation using the Griess reaction (14). Briefly, nitrite, a stable product of NO degradation, was measured by mixing 50 μΙ of culture supernatants with the same volume of Griess reagent (Sigma, G4410) and reading absorbance at 550 nm after 15 min. Phenol-red free DMEM with FBS and antibiotics were used as a blank. A standard curve was prepared using 0-80 μΜ sodium nitrite solutions in ddH20. LPS model in vivo

Male C57BL/6 mice (8-10 weeks, 22 +/- 5g), were injected intraperitoneal^ with 18 mg E. coli 01 11 :B4 LPS (Sigma) per kg of body weight. Thirty minutes after LPS injection, 0.5 mg of the indicated S1 -peptides or buffer alone was injected intraperitoneally into the mice. Survival and status was followed during seven days. For blood collection and histochemistry, mice were sacrificed 20 h after LPS challenge, and lungs were removed and fixed. These experiments were approved by the Laboratory Animal Ethics Committee of Malmo/Lund.

Cytokine assay

The cytokines IL-6, IL-10, MCP-1 , INF-γ, and TNF-a were measured in plasma from mice subjected to LPS (with or without peptide treatment) using the Cytometric bead array; mouse inflammation kit (Becton Dickinson AB) according to the manufacturer's instructions.

Phyloqenetic analyses of S1 sequences The sequences for S1 peptidases were retrieved from the NCBI site. Each sequence was analyzed with Psi-Blast (NCBI) to find the ortholog and paralog sequences. Sequences that showed structural homology >70% were selected. These sequences were aligned using ClustalW using Blosum 69 protein weight matrix settings. Internal adjustments were made taking the structural alignment into account utilizing the ClustalW interface. The level of consistency of each position within the alignment was estimated by using the alignment-evaluating software Tcoffee.

Results Application of the pattern sequence captured 68 peptide sequences, which were synthesized and screened for antimicrobial effects against the Gram-negative Escherichia coli and Pseudomonas aeruginosa, the Gram-positive Staphylococcus aureus and the fungus Candida albicans. In a first screening round, the results indeed showed that a significant proportion of the sequences, here grouped according to an evolutionary tree generated for the protease domain (15), displayed antimicrobial activities in low salt as well as in physiological salt conditions. In line with previous results, peptides derived from coagulation factors II, IX, and X were particularly active (9), demonstrating retained effects in high salt, an indicator of potential effects at physiological conditions. Additional peptides showing activity in 0.15 M NaCI comprised sequences from the plasma-derived peptidases, HABP2, protein C, plasminogen, as well as several epithelial derived peptidases of the kallikrein family. Hemolytic activities were analyzed in parallel, in order to get information on potential activities on eukaryotic (cholesterol-rich) membranes. A few of the peptides (Fig. 22A) displayed hemolytic activities in the same range as those generated by the thrombin peptides (and similar to LL-37). A significant correlation, particularly between killing of E. coli and effects on P. aeruginosa and Candida, was noted, and a subset of the peptides was also active against S. aureus, illustrating the broad spectrum activity of the peptides (Fig. 226, Fig.22A). As mentioned above, recent evidence shows that HDPs may trigger a range of immunomodulatory responses. In a mouse macrophage model (and using 10 μΜ of peptide), four of the peptides significantly inhibited NO-release of LPS- as well as zymosan-stimulated macrophages (Fig. 22C, Fig. 26B), indicating that these peptides inhibited TLR-4 and -2 mediated pathways, respectively. As mentioned above, properties common for most antimicrobial peptides include minimum levels of cationicity, amphipathicity, and hydrophobicity (1-5), and consequently peptides characterized by a high relative hydrophobicity (pHrel) and a positive net charge (eg. z _ne t≥ +2) retained their antimicrobial activity against E. coli at physiologic conditions (Fig. 23Λ). A similar, but even more marked correspondence was observed for the anti-endotoxic activity (Fig. 23S). Sequence-dependent QSAR models based on the data for peptides 1-68 were developed for antimicrobial effect on E. coli, as well as peptide-induced NO-blocking using the software ProPHECY™ (Figure 23C and D, respectively). The squared correlation for the fitted data, r ² , the cross-validated data, q ² , and the number of PLS components, A, are given for each model (see legend). A moderate q ² equal to x was obtained for E, coli. As seen, the scatter plot defining NO-blocking capacity was in good agreement with the experimental data. Evaluation of the QSAR parameters showed that net charge, Hrel , and helical propensity were critical for the observed effects and could be used to describe the obtained data, although several others descriptors further refined the analysis (Fig. 23C).

In order to test whether the obtained information could be utilized also predict antimicrobial and/or anti-inflammatory activities of additional C-terminal S1 peptides, new sequences not included in the first S1 dataset (and deposited during the course of the study), as well as sequences identified by a variant pattern sequence capturing additional S1 peptide sequences, X-[GFPRY]-X-[AFILVY]-[ACFHITY]-[AGILSTV]- [ADHKLNQRSY]-[ILS ]-{R}-X(4)-W-[ILV]-X(3)-[AILMTVW]-X(1 ,48)> [SEQ ID NO: 133] , were analyzed. The QSAR predictions, utilizing the above biophysical and QSAR data, and based on all known and predicted S1 peptide sequences (Fig. 27) identified a few potential antimicrobial S1 peptides, and two additional antimicrobial and anti-endotoxic ones (one derived from granzyme B), results which were subsequently verified experimentally (Figure 27). In order to further validate and extend the findings into a relevant physiologic environment, highly purified HDPs, all meeting the following criteria; Hrel >0.4, z _ne , =≥+2, and bactericidal activity in 0.15 M NaCI, were synthesized and analyzed in matrix-free viable count assays in the presence of salt and human plasma. The results demonstrated that in contrast to the control peptides from FXI and kallikrein 9, peptides derived from the coagulation associated proteins FN, IX, and X, plasminogen, protein C, the plasma proteins ApoA and HABP2, the neutrophilic granzyme B, and H, and epithelial associated kallikreins 5, 8, and 10, all demonstrated significant antibacterial activity (Fig. 2AA), thus corroborating the above RDA screening assays. In concordance with previous results, as well as predictions, peptides from thrombin, FX, HABP2, kallikrein 8, and granzyme B were particularly anti-endotoxic (Fig. 2ΛΒ). In addition, it was noted that the other peptides were inhibitory at higher concentrations (exceeding the initially used screening dose of 10 μΜ). The color coding illustrates the major concept derived from the biophysical analyses; given a high hydrophobicity and preserved pattern sequence, charge is a major determinant for the antimicrobial activity as well as anti-endotoxic activity of this family of peptides. In a mouse model of LPS- induced shock, selected peptides from FX, HABP2, and kallikrein 8 displayed a dramatic improvement on survival (Fig. 24C)., in parallel with animal weight recovery (Figure 28). Analyses of cytokines 6 hours after LPS injection showed significant reductions of proinflammatory IL-6, IFN-γ, TNF-a, and MCP-1 , whereas anti-inflammatory IL-10 remained unchanged (Fig. 24D). SEM analyses of lungs from LPS-treated animals demonstrated pulmonary leakage of protein and red blood cells (see inset in Fig. 24£), an effect completely blocked by the above peptides. In contrast, the control peptides showed no effects in these models. The results thus demonstrate a potential therapeutic application of these novel S1 -derived HDPs in settings of inflammation and endotoxic shock. The models presented in Figure 25 illustrate the cationicity as well as amphipathicity of the novel HDPs, situated in their holoproteins, or as 20mers. In contrast to the active peptides, non-active peptides which all show an helical structure in their holoproteins as well as in an hydrophobic environment (and also, like the active ones, conform to the amphipathic pattern sequence), clearly fail to induce an helical conformation in the presence of bacterial lipopolysaccharide (Fig. 25), thus not meeting the primary prerequisite of initial bacterial LPS-interactions, facilitating subsequent bacterial permeabilization and/or anti-endotoxic effects. At the structural level, high activity seems to be associated with not only a high net charge, but also the ability to form a helix stabilizing N-cap motif of the C-terminal helix. The side chain of H230 in thrombin (H7 in KYG20) makes a hydrogen bond to the backbone amide three residues downstream (R233 or R10). Most of the active peptides have an amino acid type that allows N-cap formation in this position. Most common are Asn or Gin in for example the peptides of HABP2, and kallikrein 8.

Discussion Taken together, the present results uncover previously unknown structure-function relationships of C-terminal sequences of serine proteases and suggest novel important innate immune functions for peptides of several proteases present in blood and epithelia. Serine proteases of the S1 family have maintained a common structure over an evolutionary span of more than one billion years, and evolved a variety of substrate specificities and diverse biological roles involving digestion and degradation, blood clotting, fibrinolysis, and epithelial homeostasis (15). Of importance is the observation that the C-terminal function of the sequence in the protease domain has a dominant role in evolutionary decisions, accounting S1 functional diversity, and containing critical information governing interactions of S1 proteins with substrates and also modulating enzyme activity (15). Thus, it may be argued that the current findings represent a parallel phenomenon, illustrating a mere structural and functional overlap between HDPs and sequence-encoded properties needed for S1 substrate recognition, and not as proposed here, defining novel functional roles for S1 peptides. As illustrated in Table 4, however, evidence is mounting speaking in favour of the latter possibility. Thus, in addition to previously identified thrombin-derived C-terminal HDPs, additional findings on the plasmin-mediated release of similar fragments of FX and FIX lend further support to the concept (16, 17). Furthermore, cathepsin G related C-terminal peptides (encompassing the 20 aa sequence described here)(18), as well as C-terminal peptides of HABP2 (of about ~8 kDa)(19) have indeed been identified. Finally, neutrophil elastase degrades protein C, generating low molecular weight fragments (<14 kDa) (20). Although no information is available on the identity of these peptides, it is noteworthy that activated protein C, currently approved for sepsis treatment, not only targets coagulation, but also induces significant anti-inflammatory effects (21) and inhibits LPS-mediated lung injury (22).

From an evolutionary and functional point of view, it may be argued that a coherent (and parallel) evolution of substrate recognition of S1 sequences (situated in the holoprotein), as well as as a host-defense function (of released HDPs) is both logical as well as energetically cost-effective, minimizing the need for de novo synthesis of HDPs, and providing a fast, localized, and protease-dependent innate immune response. The existence of such general serine-protease networks induced in response to a "danger" situation, and characterized by multiple cross-activations, generating a primary proinflammatory enzyme-mediated cascade (as illustrated by the coagulation and kallikrein systems), and a secondary, anti-inflammatory response mediated by HDPs (generating additional "protease-peptide patterns"), is elegantly illustrated by thrombin and the coagulation system (9). Here, selective and highly localized proteolytic activations mediate initial defense functions (including PAR activations and fibrin clot formation) (23), followed by the generation of HDPs by the subsequent proteolytic action of neutrophil elastase, cathepsin G, and possibly other enzymes such as plasmin (see Table 4 and references). In this context it is highly interesting that kallikreins (e.g., kallikrein 5 and 8) are up-regulated and activated during inflammation in skin (24), but it remains to be investigated whether further proteolysis releases anti-inflammatory kallikrein-derived HDPs in skin. It is also interesting, and supporting the above reasoning on a function of S1 peptides in innate immunity, that the here identified HDPs are derived from enzymes involved in coagulation and epithelial homeostasis, both of crucial relevance for host defense.

Table 4

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