WO2009030464A2 - PRODUCTION AND USE OF VARIANTS OF HUMAN KUNITZ-TYPE PROTEASE INHIBITORS (hKTPI) - Google Patents

Abstract

The present invention relates to protease inhibitors of the Kunitz type, which were isolated from human genomic DNA as DNA fragments, to the modification and expression thereof in suitable expression systems, and to the production and potential use thereof.

Description

 Preparation and Use of Variants of Human Kunitz-Type Protease Inhibitors (hKTPi)

The present invention relates to Kunitz-type protease inhibitors which have been isolated as DNA fragments from human genomic DNA, their modification and expression in suitable expression systems, as well as their preparation and possible use.

Kunitz domains are polypeptides of 55-62 amino acids (aa) in length that inhibit a variety of serine proteases of varying potency. They usually contain three disulfide bridges, which stabilize the protein and determine its three-dimensional structure. The interaction with the respective serine protease occurs mainly via an approximately 9 aa long loop in the N-terminal region of the Kunitz domain. This loop binds to the catalytic center of the protease and thus prevents cleavage of the corresponding protease substrates (Laskowski and Kato, 1980, Bode and Huber, 1992).

Aprotinin, also referred to as bovine pancreatic trypsin inhibitor (BPTI), is considered a prototype of Kunitz domains (Fritz and Wunderer, 1983). It is a basic protein of 58 aa length that can be isolated from various bovine organs (including pancreas, lung, liver and heart). Aprotinin is stabilized by three disulfide bridges (Cys 5 - Cys 55, Cys 14 - Cys 38, Cys 30 - Cys 51) and is among others. a potent inhibitor of trypsin, plasmin and plasma kallikrein.

X-ray crystallographic analysis of the aprotinin-bovine trypsin complex has shown that the aprotinin contact region to the protease catalytic center is essentially formed by a loop consisting of amino acid residues 11 through 19 (see Bode and Huber, 1992 and references therein). Of central importance for the inhibitory action of aprotinin is the amino acid residue Lys / K 15, which is in particularly close contact with the catalytically active serine residue of the protease. The amino acid Lys / K 15 is therefore designated by definition as P1 residue (Schlechter and Berger, 1967). N-terminal of Lys / K 15 are the residues P2, P3, etc., while the amino acids C-terminal of Lys / K15 are referred to as PV, P2 ', etc. It has previously been shown that the inhibitory effect of aprotinin can be altered by targeted replacement of amino acid residues ranging from residue 11 to residue 19 (Otlewski et al., 2001, Apeler et al., 2004, Krowarsch et al., 2005). , In addition, the amino acids at position 36-39 are important for the effectiveness of aprotinin (Fritz and Wunder, 1983, Krowarsch et al., 2005).

Aprotinin is now widely used in cardiac surgery under the trade name Trasylol, after clinical studies have shown that treatment with aprotinin significantly reduces the need for transfusion in such operations and reduces rebleeding (Royston, 1992). Its clinical effect is attributed to the inhibition of fibrinolysis, the inhibition of intrinsic blood coagulation (contact activation) and the reduction of thrombin formation (Blauhut et al., 1991, Dietrich et al., 1995). Thus, for the hemostatic effect of aprotinin, the inhibition of both plasminβ and plasma kallikrein and factor XIa is important.

As a bovine protein, aprotinin in humans leads to the formation of antibodies. Repeated Trasylol may cause allergic reactions (anaphylactic shock). The risk is 2.8%, which limits the possibility of multiple use of aprotinin (Dietrich et al., 2001, Beierlein et al., 2005). There is thus a high medical need for active ingredients with a similar or better clinical effect than aprotinin, which do not cause an allergic reaction.

There are a number of genes in the human genome that contain one or more Kunitz domains (KD), e.g. Amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimers disease, Ponte et al., 1988, Oltersdorf et al., 1989), serine peptidase inhibitor-like, with Kunitz and WAP domains 1 (Eppin, Richardson et al. , 2001), serine peptidase inhibitor, Kunitz type, 2, SPINT 2 (placental bikunin, 2KD, Delaria et al., 1997), tissue factor pathway inhibitor 2 precursor (TFPI-2, 3KD, Sprecher et al., 1994 ), Tissue factor pathway inhibitor, TFPI (lipoprotein-associated coagulation inhibitor, 3KD, Wun et al., 1988, van der Logt et al., 1991), amyloid beta (A4) precursor-like protein 2, APLP2 (Yang et al., 1996), alpha-1-microglobulin / bicunin precursor, AMBP, 2KD (Vetr and Gebhard, 1990), hepatocyte growth factor activator inhibitor type, (HAI-1, SPINT 1, 2KD, Shimomura et al., 1997), collagen, type VI, alpha 3 (COL6A3, Chu et al., 1988), collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive, COL7A1, Parente et al., 1991), Kunitz et al. type protease inhibito r 3 precursor (HKIB9, SPIT3, Deloukas et al., 2001), WAP four-disulfide core domain 8 (WFDC8, Clauss et al., 2002), WAP, follistatin / kazal, immunoglobulin, kunitz and netrin domain containing (WFIKKN , 2 KD, Trexler et al., 2001), WAP, follistatin / kazal, immunoglobulin, kunitz and netrin domain containing 2 (WFIKKN2, 2 KD, Hill et al., 2003), as well as hypothetical proteins and open reading frames, respectively of the human genome analysis, such as serine peptidase inhibitor, Kunitz type 4 (SPINT 4, Corf137), papilin, proteoglycan-like sulfated glycoprotein (PAPLN, hypothetical protein), Q9BQP5-OTTHUMP00000031164 (fragment, C20orf168), LOC285929, similiar to matrilin 2 precursor (hypothetical protein).

Some of the Kunitz domains from the above genes have been shown to inhibit high potency serine proteases (Nexin II: Dennis and Lazarus, 1994, Dennis et al., 1995, Wagner et al., 1992, Navaneetham et al. , 2005, Van Nostrand et al., 1990; Spint-1: Kirchhofer et al., 2003; HAI-1: Shimomura et al., 1997, Denda et al., 2002; COL6A3: Kohlfeldt et al., 1996; WFIKKN Nagy et al., 2003), but others are not further characterized in terms of their inhibitory activity spectrum. The aim was therefore to express the Kunitz domains isolated via PCR from human genomic DNA in suitable expression systems, to test them for their inhibitory effect and to prepare variants which have properties comparable to aprotinin. Summary of the invention

The aim of the present invention is the recombinant production of Kunitz domains which have been isolated from human genomic DNA and have an action comparable to aprotinin. The desired properties were achieved by PCR and mutagenesis techniques, the mutagenesis resulting in amino acid residues that may occur in natural or non-natural Kunitz domains. This approach leads to variants of human Kunitz domains

• which can be produced recombinantly in suitable yeast and other expression systems

• capable of inhibiting serine proteases (Table 1)

• whose plasmin, plasma kallikrein and factor Xla inhibition are comparable to aprotinin (Table 1)

• show comparable effects on fibrinolysis and coagulation in functional in vitro assays on aprotinin

Detailed description of the invention

Kunitz domains for the purposes of this invention are homologs of aprotinin having 55 to 62 amino acid residues, usually containing six cysteine residues and three disulfide bridges, each between the positions Cys 5 - Cys 55, Cys 14 - Cys 38 and Cys 30 - Cys 51 (Numbering according to aprotinin, see Seq. A). In addition, up to three further arbitrary amino acids can be added at the N- or / and C-terminus. Figure 1 shows the structure of a Kunitz domain. The amino acids of a Kunitz domain are numbered so that the six cysteine residues are in positions 5, 14, 30, 38, 51 and 55. Similarly, the fourth amino acid is designated N-terminal of Cys 5 number 1. If additional amino acids are added at the N-terminus, they are given the numbers -1, -2, -3. Certain amino acids found in previously known Kunitz domains are indicated in Figure 1 accordingly. All unspecified positions may contain any amino acids.

Figure 1 shows the definition of a Kunitz domain.

Aprotinin, also referred to as bovine pancreatic trypsin inhibitor (BPTI), is considered a prototype of Kunitz domains (Fritz and Wunderer, 1983). It is a basic protein of 58 aa length containing the typical Kunitz domain cysteine residues at positions 5, 14, 30, 38, 51, and 55. The cysteine residues form three disulfide bridges (Cys 5 - Cys 55, Cys 14 - Cys 38, Cys 30 - Cys 51), which stabilize the molecule. The numbering of the amino acids of the Kunitz domains for the purposes of this invention corresponds to the numbering of aprotinin (see Seq. A). In this case, amino acid 1 is four positions N-terminal of Cys 5 and corresponds to the amino acid Arg 1 of Aprotinin. If additional amino acids are added at the N-terminus, they are given the numbers -1, 2, -3.

Seq. A: aprotinin

1 2 3 4 5

1234567890123456789012345678901234567890123456789012345678 RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGA

Kunitz inhibitors have been found in various vertebrates as well as invertebrates (Laskowski and Kato, 1980). These naturally occurring Kunitz domains are referred to in this application as "Kunitz natural domains" (Shimomura et al., 1997, Li et al., 1998, Ponte et al., 1988, Richardson et al., 2001, Wun et al., 1988, Marlor et al., 1997, Petersen et al., 1994, Vetr and Gebhard, 1990, Chun et al., 1990, Parente et al., 1991, Norris et al., 1993, Claus et al ., 2002, Trexier et al., 2001, 2002), Kunitz domains modified by genetic engineering techniques, eg, by replacement of amino acid residues and / or deletions and so not found in nature, are referred to in this application as "non- Naturally Kunitz domains "(US 6010880, US 5795865, WO 92/06111, US 5777568, US 6482798, P 0 238993, EP 0 307592 EP 0 821 007, US 5863893, US 5914315, US 7019123).

Aprotinin is a potent inhibitor of plasmin, plasma kallikrein and factor XIa, the clinical effect of which is attributed to the inhibition of fibrinolysis, the inhibition of intrinsic blood coagulation (contact activation) and the reduction of thrombin generation (Blauhut et al., 1991, Dietrich et al. , 1995).

As a bovine protein, aprotinin in humans leads to the formation of antibodies. Repeated doses of trasylol may cause allergic reactions (anaphylactic shock), thereby limiting the possibility of multiple use of aprotinin (Dietrich et al., 2001, Beierlein et al., 2005).

The aim of the present invention is therefore to identify Kunitz domains of human origin or variants of these Kunitz domains which have a comparable or improved aprotinin functional activity. Kunitz domains according to the invention inhibit both plasmin and plasma kallikrein and factor XIa with high potency. They are also potent inhibitors of fibrinolysis and intrinsic coagulation.

Kunitz domains according to the invention and their variants inhibit plasmin with an IC50 <2 μM, but better with an IC50 <100 nM. Plasma kallikrein is inhibited by Kunitz domains according to the invention and their variants with an IC50 <2 μM, but better with an IC50 <100 nM. Furthermore, Kunitz domains according to the invention and their variants inhibit factor XIa with an IC50 <2 μM, but better with an IC50 <100 nM. The following are examples of human Kunitz domains according to the invention with an effect comparable or improved to aprotinin (see Table 1):

Seq. No. 38 (APP4) Seq. No. 50 (APLP-2) Seq. No. 54 (AMBP-D2)

It has also been found that various human Kunitz domains inhibit the serine proteases plasmin, plasma kallikrein and / or factor XIa only with low potency or not at all. The following are examples of such human Kunitz domains (see Table 1):

Seq. No. 42 (TFPH -D2) Seq. No. 48 (TFPI2-D3) Seq. No. 66 (WFI-2-D2) Seq. No. 63 (WFI-1-D2) Seq. No. 27 (COL6A3-6His)

Surprisingly, it has been found that the inhibitory effect of such human Kunitz domains against plasmin, plasma kallikrein and factor XIa can be improved by the targeted introduction of mutations, so that the resulting variants have a comparable or improved aprotinin effect.

Variants of human Kunitz domains according to the invention have the numbered according to aprotinin

Positions the following amino acids:

X10 = E, X11 = T, X15 = R, X16 = A, X17 = A, X18 = H X19 = P, X20 = R, X21 = W, X22 = W.

The remaining positions contain any amino acids of human Kunitz domains.

Further variants according to the invention of human Kunitz domains have the following amino acids at the positions numbered according to aprotinin:

X10 = V, X11 = T, X15 = R or K, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F.

The remaining positions contain any amino acids of human Kunitz domains.

Further variants according to the invention of human Kunitz domains have the following amino acids at the positions numbered according to aprotinin:

X10 = E, X11 = T, X15 = R or K, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F.

The remaining positions contain any amino acids of human Kunitz domains.

Further variants according to the invention of human Kunitz domains have the following amino acids at the positions numbered according to aprotinin:

X10 = V, X11 = T, X15 = R, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F. The remaining positions contain any amino acids of human Kunitz domains.

Further variants according to the invention of human Kunitz domains have the following amino acids at the positions numbered according to aprotinin:

X10 = E, X11 = T, XI S = R ₁ XIe = A ₁ XI y = A ₁ XIS = M ₁ XIQ = K, X20 = R, X21 = F.

The remaining positions contain any amino acids of human Kunitz domains.

Particularly preferred are the following variants of human Kunitz domains:

Seq. No. 60d (COL6A3-mut4) Seq. No. 6Oe (COL6A3-mut5) Seq. No.63a (WFI1-D2-mut1) Seq. No. 63c (WFI1-D2-mut3) Seq. No.63d (WFI1-D2-mut4)

Human Kunitz domains according to the invention and their variants also inhibit fibrinolysis and intrinsic coagulation in human plasma with aprotinin comparable or improved efficacy (FIGS. 1 and 2)

The described and with aprotinin comparable properties possessing human Kunitz domains and their variants are suitable for the treatment of the following disease states: Blood loss in operations with increased risk of bleeding; Therapy of thromboembolic conditions (eg after surgery, accidents), shock, polytrauma, sepsis, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), unstable angina, heart attack, stroke, embolism, deep vein thrombosis, inflammatory diseases (eg rheumatism, Asthma), invasive tumor growth and metastasis, pain and edema therapy (cerebral edema, spinal cord edema), prevention of activation of hemostasis in dialysis treatment, treatment of skin aging symptoms (elastosis, atrophy, wrinkling, vascular changes, pigment changes, actinic keratosis, blackheads, cysts), Wound healing, skin cancer, treatment of skin cancer symptoms (actinic keratosis, basal cell carcinoma, squamous cell carcinoma, malignant melanoma), multiple sclerosis, fibrosis, cerebral hemorrhage, inflammation of the brain and spinal cord, brain infections, tendinopathy.

The invention relates to an isolated protein or polypeptide which contains a Kunitz domain and is characterized in that the domain has the following amino acids at the positions numbered according to aprotinin:

X10 = E, X11 = T, X15 = R, X16 = A, X17 = A, X18 = H X19 = P, X20 = R, X21 = W, X22 = W, the remaining positions containing any amino acids of human Kunitz domains ; or

characterized in that the domain follows the positions numbered according to aprotinin

Has amino acids:

X10 = V, X11 = T, XIS = R OdBr K ₁ XIe = A ₁ XIT = A ₁ XIe = M ₁ XIg = K, X20 = R ₁ X21 = F, where the remaining positions contain any amino acids of human Kunitz domains;

or

characterized in that the domain has the following amino acids at the positions numbered according to aprotinin:

X10 = E, X11 = T, X15 = R or K, X16 = A, X17 = A, X18 = M ₁ X19 = K, X20 = R ₁ X21 = F, the remaining positions containing any amino acids of human Kunitz domains;

or

characterized in that the domain follows the positions numbered according to aprotinin

Has amino acids:

X10 = V, X11 = T, X15 = R, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F ₁ the remaining positions containing any amino acids of human Kunitz domains;

or

characterized in that the domain follows the positions numbered according to aprotinin

Has amino acids:

XIO = E ₁ XH = T, X15 = R, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F, the remaining positions containing any amino acids of human Kunitz domains.

According to the invention, an isolated protein is as described above, wherein the remaining positions of the Kunitz domain contain amino acids of a particular human Kunitz domain.

Particularly preferred is an isolated protein or polypeptide containing an amino acid sequence selected from the group consisting of Seq. No. 6Od (COL6A3-mut4, SEQ ID NO: 121), Seq. No. 6Oe (COL6A3-mut5, SEQ ID NO: 122), Seq. No. 63a (WFH-D2 mut1, SEQ ID NO: 126), Seq. No. 63c (WFI1-D2-mut3, SEQ ID NO: 128) and Seq. No. 63d (WFI1-D2-mut4, SEQ ID NO: 129).

The invention further relates to a fragment of one of the above-described proteins or polypeptides, characterized in that the fragment has an inhibitory activity against plasmin, plasma kallikrein, or factor XIa, which is comparable to or better than the action of aprotinin with the action of aprotinin. Also according to the invention is a nucleic acid which encodes a polypeptide or protein as described above. The nucleic acid may contain heterologous vector sequences or a heterologous promoter sequence 5 'to the coding region.

The invention also includes a cell or a non-human organism containing a nucleic acid as described above.

The invention also relates to a method for isolating a polypeptide or protein according to the invention, characterized in that a cell which contains a nucleic acid coding for a protein or polypeptide according to the invention is cultured and the protein is purified.

The invention also relates to a monoclonal or polyclonal antibody specific for a protein of the invention.

The invention further relates to the use of a protein or polypeptide of the invention or a nucleic acid of the invention for the manufacture of a medicament for the treatment of conditions or diseases selected from the group consisting of blood loss in operations with increased risk of bleeding; thrombotic bolsic states, shock, polytrauma, sepsis, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), unstable angina, myocardial infarction, stroke, embolism, deep vein thrombosis, inflammatory diseases (eg rheumatism, asthma), invasive tumor growth and metastasis, pain and edema therapy (cerebral edema, spinal cord edema), prevention of activation of hemostasis in dialysis treatment, treatment of skin aging symptoms (elastosis, atrophy, wrinkles, vascular changes, pigment changes, actinic keratosis, blackheads, cysts), wound healing, skin cancer, treatment of skin cancer symptoms ( actinic keratosis, basal cell carcinoma, squamous cell carcinoma, malignant melanoma), multiple sclerosis, fibrosis, cerebral hemorrhage, inflammation of the brain and spinal cord, brain infections and tendinopathy.

Particularly preferred is the use of a protein or polypeptide of the invention or a nucleic acid of the invention for the manufacture of a medicament for the treatment of conditions or diseases selected from the group consisting of blood loss in operations with increased risk of bleeding; thromboembolic conditions, shock, sepsis, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), myocardial infarction, stroke, embolism, deep venous thrombosis, and wound healing.

Brief description of the figures

Figure 1 shows the definition of a Kunitz domain.

FIG. 2 describes the antifibrinolytic activity of WFI1-D2-mut4 in human plasma.

FIG. 3 describes the anticoagulant activity of WFI1-D2-mut4 in human plasma. embodiments

Molecular Biology, Cell Biology and Biochemical Techniques

Routine cloning work was done according to Sambrook et al. (Molecular Cloning Co., Spring Harbor, 1989). For the isolation of plasmid DNA from E. coli (so-called mini- and midipreps) Qiagen-tips (Qiagen) were used. As a host organism for transformations of the E. coli strain DH5α (Stratagene) was used. The extraction of DNA fragments from agarose gels was performed using the Qiagen gel extraction kit according to the manufacturer (Qiagen). Oligonucleotides for site directed mutagenesis experiments, primers for PCR and sequencing reactions were purchased from the company Operon, synthetic genes (optimized for S. cerevisiae coden-usage) from Geneart.

In-vitro mutagenesis was performed using the Quick-change IL XL site-directed mutagenesis kit according to the manufacturer (Stratagene). For PCR experiments, kits from Qiagen (Hot Star Mastermix), Stratagene (PfuUltra Hotstart DNA Polymerase) or Novagen (KOD HiFi, Hot Start and XL DNA Polymerases) were used according to the manufacturer's instructions; PCR was used to purify PCR fragments Purification Kit used by the company Qiagen. For cell-free transcription and translation experiments, the Rapid Translation System (RTS) pIVEX HIS tag, 2 ^nd Generation Vector Set was used according to the manufacturer (Roche). All vector constructs and mutagenesis were confirmed by cycle DNA sequencing with fluorescently-labeled terminators (Big Dye Terminator, Version 1.1, Applied Biosystems) on a sequencer (3100 Avant Genetic Analyzer, Applied Biosystem).

example 1

Cloning of Kunitz-type protease inhibitor domains from human genomic DNA by PCR

For cloning via PCR, corresponding PCR primers (1a, b-23a, b) were derived for the following human genes which contain protease inhibitor domains of the Kunitz type (referred to below as human Kunitz domains) in their coding sequences:

Amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease)

SEQ ID NO: 1: 1a App4-up 5 '-GTTCGAGAGGTGTGCTCTGAACAAGCCGAG-3' SEQ ID NO: 2: 1b App4-down 5 '-GGCGCTGCCACACACGGCCATGCA-3'

Serine peptidase inhibitor-like, with Kunitz and WAP domains 1 (Eppin)

SEQ ID NO: 3: 2a Eppi-up 5'-TAGATCTCAAACAAGATGTATGCGAAATGCCAA-B '

SEQ ID NO: 4: 2b Eppi-down 5 '-TTTATTCTTGCAGGTGTTCAGGCA-3' Tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor, TFPI-1), domains 2 and 3

SEQ ID NO: 5: 3a TFPI-1 d2-up 5'-GAAAAGCCAGATTTCTGCTTTTTG-3 '

SEQ ID NO: 6: 3b TFPI-1 d2-down 5'-ACCATCTTCACAAATGTTCTTGCA-3 '

SEQ ID NO: 7: 4a TFPI-1 -d3-up 5'TTTCACGGTCCCTCATGGTGTCTC-3 '

SEQ ID NO: 8: 4b TFPI-1-d3-down 5'-ACCTTTTTTACATGCCCTCAGACA-3 '

• Tissue factor pathway inhibitor 2 (TFPI-2), domains 2 and 3

SEQ ID NO: 9: 5a TFPI-2-d2-up 5 '-GTTCCCAAAGTTTGCCGGCTGCAA-3'

SEQ ID NO: 10: 5b TFPI-2-d2-down 5'-CTTTGGTGCGCAGAAGCCCATACA-3 '

SEQ ID NO: 11: 6a TFPI-2-d3-up 5'-ATTCCATCATTTTGCTACAGTCCA-3 '

SEQ ID NO: 12: 6b TFPI-2-d3-down 5'-AGCTTTTGCACATGCACGTTTGCA-3 '

Amyloid beta (A4) precursor-like protein 2 (APLP 2)

SEQ ID NO: 13: 7a Aplp2-up 5 '-GTCAAAGCTGTCTGCTCCCAGGAGGCGATG-3'

SEQ ID NO: 14: 7b Aplp2-down 5'-CATCGCTTTACACACAGCCATACA-3 '

Alpha-1-microglobulin / bicunin precursor (AMBP), domains 1 and 2

SEQ ID NO: 15: 8a Ambp-d1-up 5'-AAGAAGATTCCTGCCAGCTGGGCTACTCG-3 '

SEQ ID NO: 16: 8b Ambp-d1 -dwn 5'-CACAGTTCGGCAGGTCTGCAGACA-3 '

SEQ ID NO: 17: 9a Ambp-d2-up 5'-ACTGTGGCGGCCTGCAATCTCCCCATAGTC-3 '

SEQ ID NO: 18: 9b Ambp-d2-dwn 5'-AGGGACACCGCAGTACTCTCTGCA-3 '

Serine peptidase inhibitor, Kunitz type 1 (SPINT 1, HAI-1), domains 1 and 2

SEQ ID NO: 19: 10a Hai1-d1 -up 5'-ACAGAAGACTACTGCCTCGCATCCAACAAG-S '

SEQ ID NO: 20: 10b Hai1 -d1 -dwn 5 '-CACACCCCGACAGGCTAGAATGCA-3'

SEQ ID NO: 21: 11a Hai1 -d2-up 5'-GACAAAGGGCACTGCGTGGACCTGCCAGAC-3 '

SEQ ID NO: 22: 11b Hai1 -d2-dwn 5 '-GGAGATGCCGCGACAAGACTCGAG-3'

• Collagen, type VI, alpha 3 (COL6A3)

SEQ ID NO: 23: 12a Col6a3-up 5'-GAAACAGATATATGCAAGTTGCCGAAAGAC-3 '

SEQ ID NO: 24: 12b Col6a3-dwn 5'CACAGGAGCGCAAACCTTTTCACATTC-3 '

• Collagen, type VII, alpha 1 (COL7A1)

SEQ ID NO: 25: 13a Col7a1-up 5'-GACCCCTGTTCCCTGCCACTGGAT-3 '

SEQ ID NO: 26: 13b Col7a1 -dwn 5'-CCGGGGTGGGCAGCGGCGCTCGCA-3 '

WAP four-disulfide core domain 8 (WFDC 8, WAP 8)

SEQ ID NO: 27: 14a Wap8-up 5 '-TTTCAAGAACCCTGCATGCTACCTGTGAGG-3'

SEQ ID NO: 28: 14b Wapδ-dwn 5'-ATTAACATGCAGGCCGTTCTGCA-3 '

WFIKKN1 WAP, follistatin / kazal, immunoglobulin, kunitz and netrin domain containing (WFI-1), domains 1 and 2 (NNM 75575) SEQ ID NO: 29: 15a Wfi1-d1-up 5'-CCGGCGGCCGAGTGCCTGAAGCCC-3 '

SEQ ID NO: 30: 15b Wfi1-d1-dwn 5'-CCCGCTCÄTGCAGGCCAGCATGCA-3 '

SEQ ID NO: 31: 16a Wfi1 -d2-up 5 '-GGGCCGCTGGCCGCGTGCAGCCTG-3'

SEQ ID NO: 32: 16b Wfi1 -d2-dwn 5 '-GGGGAAGGGGCACGACTCCTCACA-3'

WAP, follistatin / kazal, immunoglobulin, kunitz and netrin domain containing (WFI-2), domains 1 and 2 (NM_053284)

SEQ ID NO: 33: 17a Wfi2-d1-up 5'-GCCCCGGCCGAGTGCCTGCCGGAT-3 '

SEQ ID NO: 34: 17b Wfi2-d1-dwn 5'-GCCGCGGGCACAGGCCTGCTGGCAT-3 '

SEQ ID NO: 35: 18a Wfi2-d2-up 5'-CCCGGCGACGCCTGCGTGCTGCCT-3 '

SEQ ID NO: 36: 18b Wfi2-d2-dwn 5'CGGCACGGGGCAGGCATCCTCGCA-3 '

Serine peptidase inhibitor, Kunitz type 4 (SPINT 4, C20orf137)

SEQ ID NO: 37: 19a spint4-up 5 '-CTCAAAGATCCCTGCAAATTGGACATGAAT-3'

SEQ ID NO: 38: 19b spint4-dwn 5 '-TTTTGCAACACAGGCTACTTCACG-3'

Papilin, proteoglycan-like sulfated glycoprotein (PAPLN, Hypothetical Protein - hypro)

SEQ ID NO: 39: 20a Hypro-up 5'-TACCCCGTGCGGTGCCTGCTGCCC-3 '

SEQ ID NO: 40: 20b Hypro-dwn 5'-AGATCCCTGGCAGCTGCTCATGCA-3 '

Q9BQP5_HUMAN (c20orf168)

SEQ ID NO: 41: 21a c20orf168-up 5'-AGTAGTAAAGAAAACCTGACTCTGGA-S '

SEQ ID NO: 42: 21b c20orf 168-dwn 5'-ATCCAGAGACTTACTTTTCAGTAATAC-3 '

Homo sapiens simitar to matrilin 2 precursor, transcript variant 2 (LOC285929)

SEQ ID NO: 43: 22a LOC285929-up 5'-TAGATCCTAGATGTTTGGAAGCC-3 'SEQ ID NO: 44: 22b l_OC285929-dwn 5'-TCCTTGAATGCAGGTTTCTTGACA-3'

• Similar to Kunitz-type protease inhibitor 3 precursor (HKIB9, SPIT 3, P49223)

SEQ ID NO: 45: 23a spit3-up 5'-ATTCTCACTCTCTGCCTAGAGCTT-3 '

SEQ ID NO: 46: 23b spit3-dwn 5 '-GGTGAACTTGCAGAATTTCTCACA-3'

The PCR reactions each contained approximately 100 ng of human genomic DNA, 10 pmol gene-specific primer-up, 10 pmol gene-specific primer-down (dwn), 1 mM dNTPs, IxPCR reaction buffer (Stratagene), 2.5 U PfuUltrahotstart DNA polymerase (Stratagene) in one Total volume of 50μl. The 'cycle' conditions were 5 min. at 94 ° C, 35 cycles of 1 min each. at 94 ° C, 1 min. at 50 ^° C., 1 min. at 72 ° C and subsequent incubation for 10 min at 72 ° C. The individual PCR reactions were purified with a purification kit (Qiagen), subcloned into the plasmid vector pCR-blunt (invitrogen) and each sequence was checked and confirmed by cycle DNA sequencing.

Kunitz-type protease inhibitor domains amplified by PCR from human genomic DNA had the following deduced amino acid sequences: Seq.Nr. 1 APP4, SEQ ID NO: 47:

VREVCSEQAETGPCRAMISRWYFDVTEGKCAPFFYGGCGGNRNNFDTEEYCMAVCGSA

SEQ.2 EPPI, SEQ ID NO: 48:

DLKQDVCEMPKETGPCLAYFLRWWYDKKDNTCSMFVYGGCQGNNNNFQSKANCLNTCKNG

Seq.No.3 TFPI-1-D2, SEQ ID NO: 49:

EKDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLEECKNICEDG

Seq.No.4 TFPI-1-D3, SEQ ID NO: 50:

FHGSWCLTPADRGLCRANENRFYYNSVIGKCRPFKYSGCGGNENNFTSKQECLRACKKG

SEQ.ID.5 TFPI-2-D2, SEQ ID NO: 51:

VPKVCRLQVSVDDQCEGSTEKYFFNLSSMTCEKFFSGGCHRNRIENRFPDEATCMGFCAPK

Seq.No.6 TFPI-2-D3, SEQ ID NO: 52:

IPSFCYSPKDEGLCSANVTRYYFNPRYRTCDAFTYTGCGGNDNNFVSREDCKRACAKA

SEQ ID NO: 7 APLP2, SEQ ID NO: 53:

VKAVCSQEAMTGPCRAVMPRWYFDLSKGKCVRFIYGGCGGNRNNFESEDYCMAVCKAM

SEQ. 8 AMBP-D1, SEQ ID NO: 54:

KEDSCQLGYSAGPCMGMTSRYFYNGTSMACETFQYGGCMGNGNNFVTEKECLQTCRTV

SEQ.No.9 AMBP-D2, SEQ ID NO: 55:

TVAACNLPIVRGPCRAFIQLWAFDAVKGKCVLFPYGGCQGNGNKFYSEKECREYCGVP

SEQ ID NO: 10 HAI-1-D1, SEQ ID NO: 56:

TEDYCLASNKVGRCRGSFPRWYYDPTEQICKSFVYGGCLGNKNNYLREEECILACRGV

Seq.No.11 HAI-1-D2, SEQ ID NO: 57:

DKGHCVDLPDTGLCKESIPRWYYNPFSEHCARFTYGGCYGNKNNFEEEQQCLESCRGIF

Seq.No.12 COL6A3, SEQ ID NO: 58:

ETDICKLPKDEGTCRDFILKWYYAPNTKSCARFWYGGCGGNENKFGSQKECEKVCAPV

Seq.Nr.13 C0L7A1, SEQ ID NO: 59:

DPCSLPLDEGSCTAYTLRWYHRAVTGSTEACHPFVYGGCGGNANRFGTREACERRCPPR Seq.No.14 WAP8, SEQ ID NO: 60:

FQEPCMLPVRHGNCNHEAQRWHFDFKNYRCTPFKYRGCEGNANNFLNEDACRTACMLI

SEQ.ID.15 WFI-1-D1, SEQ ID NO: 61:

AECLKPPDSEDCGEEQTRWHFDAQANNCLTFTFGHCHRNLNHFETYEACMLACMSEG

Seq.Nr.16 WFI-1-D2, SEQ ID NO: 62:

GPLAACSLPALQGPCKAYAPRWAYNSQTGQCQSFVYGGCEGNGNNFESREACEESCPFPS

Seq.Nr.17 WFI-2-D1, SEQ ID NO: 63:

APAECLPDVQACTGPTSPHLVLWHYDPQRGGCMTFPARGCDGAARGFETYEACQQACARG

Seq.Nr.18 WFI-2-D2, SEQ ID NO: 64:

PGDACVLPAVQGPCRGWEPRWAYSPLLQQCHPFVYGGCEGNGNNFHSRESCEDACPVP

SEQ.19 SPINT4, SEQ ID NO: 65:

LKDPCKLDMNFGSCYEVHFRYFYNRTSKRCETFVFSSCNGNLNNFKLKIEREVACVAK

Seq.No.20 PAPLN / HyPro, SEQ ID NO: 66:

YPVRCLLPSAHGSCADWAARWYFVASVGQCNRFWYGGCHGNANNFASEQECMSSCQGS

Seq.Nr.21 c20orf168, SEQ ID NO: 67:

SSKENLTLEFSFTEYCNYPLKKGTCNSYLTRFYYNTLTFLCEPFVFSGCGGNRNFKQ KYFCEKMCITEK

Seq.No.22 LOC285, SEQ ID NO: 68:

DPRCLEALKPGNCGEYVVRWYYDKQVNSCARFWFSGCNGSGNRFNSEKECQETCIQG

SEQ.2 SPIT3, SEQ ID NO: 69:

ILTLCLELRSELARDTIKDLLPNVCAFPMEKGPCQTYMTRWFFNFETGECELFAYGG CGGNSNNFSRKEKCEKFCKFT

Example 2

Cloning of human Kunitz domains into the Vecker plVEX2.3d

For cell-free transcription and tranlation experiments, human Kunitz domains were cloned into the vector plVEX2.3d by PCR and appropriate restriction enzymes. By way of example, the cloning of the human Kunitz domain APP4 (amyloid beta-A4 precursor protein, peptidase nexin-II) is shown:

SEQ ID NO: 70: 24a APP4-iveX-Up 5'-tataccatgggtGTTCGAGAGGTGTGCTCTGAACAAGCCGAG-3 ' SEQ ID NO: 71: 24b APP4-ivβX-dwn 5'-cccccccgggGGCGCTGCCACACACGGCCATGCA-3 '

Flanking and restriction sites comprising sequences (24a: Ncol: ccatgg, 24b: Xmal: cccggg) are shown in lower case, recognition sequences for restriction enzymes are underlined, for human Kunitz domains coding sequences are shown in capital letters. The PCR was purified with a Purification Kit (Qiagen), cut with the restriction enzymes NcoI and XaII and ligated into the vector PlVEX2.3d also cut with NcoI and XmaI. E.coli DH5α cells were transformed with the ligation mixture. From the resulting clone (designated APP4.plVEX), the sequence was determined by DNA sequence analysis. The coding region of the clone APP4.plVEX comprises 212 bp (scanned from 2 stop codons), encodes a protein (APP4.pep) of 71 amino acids in total and has the following sequence:

Seq. No. 24 APP4.pep, SEQ ID NO: 72:

MGVREVCSEQAETGPCRAMISRWYFDVTEGKCAPFFYGGCGGNRNNFDTEEYCMAVCGSAPGGGSHHHHHH * *

Amino acids (aa) 1 and 2 at the N-terminus are from the vector construct, aa 3-60 represent the human Kunitz domain, aa 61-71 are left and one histidine part (6xHis). The protein APP4 was used in a cell-free transcription and translation system (RTS, Roche) and successfully produced a protein of the expected size (8 kDa). Analogously to the above-described example APP4, the following human Kunitz domains were each cloned into the vector plVEX2.3d:

Seq.Nr. 25 AMBP-D1.pep (71aa), SEQ ID NO: 73:

MGKEDSCQLGYSAGPCMGMTSRYFYNGTSMACETFQYGGCMGNGNNFVTEKECLQTCRTVPGGGSHHHHHH * *

Seq.Nr. 26 HAI1-D1.pep (71aa), SEQ ID NO: 74:

MGTEDYCLASNKVGRCRGSFPRWYYDPTEQICKSFVYGGCLGNKNNYLREEECILACRGVPGGGSHHHHHH * *

Seq.Nr. 27 COL6A3.pep (71aa), SEQ ID NO: 75: MGETDICIOiPKDEGTCRDFILKWYYDPNTKSCARFWYGGCGGNENKFGSQKECEKVCAPVPGGGSHHHHHH * *

Seq.Nr. 28 EPPI.pep (74aa), SEQ ID NO: 76:

MGLDLKQDVCEMPKETGPCLAYFLRVWYDKKDNTCSMFVYGGCQGNNNNFQSKANCLigTCKNKPGGGSHHHHHH * *

Seq.Nr. 29 APLP2.pep (71aa), SEQ ID NO: 77:

MGVKAVCSQEAMTGPCRAVMPRWYFDLSKGKCVRFIYGGCGGNRNNFESEDYCMAVCKAMPGGGSHHHHHH * *

Seq.Nr. 30 AMBP-D2.pep (71aa), SEQ ID NO: 78:

MGTVAACNLPIVRGPCRAFIQLWAFDAVKGKCVLFPYGGCQGNGNKFYSEKECREYCGVPPGGGSHHHHHH * * Seq.Nr. 31 WFI2-D2.pep (71 aa), SEQ ID NO: 79:

MGPGDACVIiPAVQGPCRGWEPRWAYRPLLQQCHPFVYGGCEGNGNNFHSRESCEDACPVPPGGGSHHHHHH * *

Seq.Nr. 32 HAI1-D2.pep (72 aa), SEQ ID NO: 80:

MGDKGHCVDLPDTGLCKESIPRWYYNPFSEHCARFTYGGCYGNKNNFEEEQQCLESCRGISPGGGSHHHHHH * *

Seq.Nr. 33 TFPI1-D2.pep (71aa), SEQ ID NO: 81:

MGKPDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLEECKNICEDGPGGGSHHHHHH * *

Seq.Nr. 34 TFPI1-D3.pep (71 aa), SEQ ID NO: 82:

MGGPSWCLTPADRGLCRANENRFYYNSVIGKCRPFKYSGCGGNENNFTSKQECLRACKKGPGGGSHHHHHH * *

Seq.Nr. 35 TFPI2-D2.pep (74aa), SEQ ID NO: 83:

MGVPKVCRLQVSVDDQCEGSTEKYFFNLSSMTCEKFFSGGCHRNRIENRFPDEATCMGFCAPKPGGGSHHHHHH * *

Seq.Nr. 36 TFPI2-D3.pep (71 aa), SEQ ID NO: 84:

MGIPSFCYSPKDEGLCSANVTRYYFNPRYRTCDAFTYTGCGGNDNNFVSREDCKRACAKAPGGGSHHHHHH * *

As synthetic genes, optimized for S. cerevisiae codon usage, the following genes were used (mutated sequences are underlined):

Seq.Nr. 27a COL6A3-mut1 (71aa), SEQ ID NO: 85:

MGETDICKLPKDEGTCRDAILKWYYAPNTKSCARFWYGGCGGNENKFGSQKECEKVCAPVPGGGSHHHHHH * *

Seq.Nr. 27b COL6A3-mut2 (71 aa), SEQ ID NO: 86:

MGETDICKLPKDEGPCRDAILKWYYAPNTKSCARFWYGGCGGNENKFGSQKECEKVCAPVPGGGSHHHHHH * *

Seq.Nr. 27c COL6A3-mut3 (71aa), SEQ ID NO: 87:

MGETDICKLPKVTGTCRAAMKRFYYAPNTKSCARFWYGGCGGNENKFGSQKECEKVCAPVPGGGSHHHHHH * *

Seq.Nr. 27d COL6A3-mut4 (71 aa), SEQ ID NO: 88:

MGETDICKLPKETGTCRAAMKRFYYAPNTKSCARFWYGGCGGNENKFGSQKECEKVCAPVPGGGSHHHHHH * *

Seq.Nr. 27e COL6A3-mut5 (71 aa), SEQ ID NO: 89:

MGETDICKLPKETGTCRAAHPRWWYAPNTKSCARFWYGGCGGNENKFGSQKECEKVCAPVPGGGSHHHHHH * * Example 3

Cloning of human Kunitz domains into yeast secretion vectors plU10.10.W and plU3.12.M

The commercially available E. coli / S. cerevisiae shuttle vector pYES2 (Invitrogen) was modified and used as starting material for the construction of the yeast secretion vectors plU10.10.W and plU3.12.M (Apeler, 2005).

By means of PCR and corresponding primers with suitable restriction sites, the human Kunitz domains from the vector plVEX2.3d were recloned into the yeast secretion vectors plU10.10.W and plU3.12.M.

By way of example, the cloning of the human Kunitz domain APP4 (amyloid beta-A4 precursor protein, peptidase nexin-II) is shown.

The cloning into the yeast secretion vector plU10.10.W took place via the restriction sites

BsaBI (GAT tccc ATC in primer 25a), and Xhol (CTCGAG in primer 25b), for cloning in plU3.12.M, the restriction sites HindIII (AAGCTT in primer 26a) and BamHI (GGATCC in primer 26b) were used. Used as a template in the respective PCR reactions Seq. Number 1.

The primers had the following sequences:

Primer 25a (plU10-1), SEQ ID NO: 90:

5 '-tgagattcccatctattttcactgctgtcttgttcgctgcttcttctgctttggctGTTCGAGAGGTG TGCTCTGAACAAGCCGAG- 3'

Primer 25b (plU10-2), SEQ ID NO: 91:

5 '-atgctcgagctattaGGCGCTGCCACACACGGCCATGCA-3'

Primer 26a (plU312-1), SEQ ID NO: 92:

5'-ggtaagcttggataaaagaGTTCGAGAGGTGTGCTCTGAACAAGCCGAG-3 '

Primer 26b (plU312-2), SEQ ID NO: 93:

5'-cgaaggatccctaGGCGCTGCCACACACGGCCATGCA-3 '

The PCR reactions were purified with a purification kit (Qiagen), cut with the restriction enzymes BsaBI and Xhol for cloning into plU10.10.W or HindIII and BamHI for cloning into plU3.12.M and into the corresponding vectors ligated. E.coli DH5α cells were transformed with the ligation mixtures and the DNA sequence was determined from the resulting clones by sequence analysis.

Derived amino acid sequence of the human Kunitz domain APP4 cloned into yeast secretion vector plU10.10.W (the pre-sequence of MFa mating factor α - is underlined, the amino acid sequence of APP4 is in bold):

Seq.Nr. 37, SEQ ID NO: 94:

MRFPSIFTAVLFAASSALAVREVCSEQAETGPCRAMISRWYFDVTEGKCAPFFYGGCGGNRMNFDTEEYCMA VCGSA Derived amino acid sequence of the human Kunitz domain APP4 cloned into yeast secretion vector plU3.12.M (the pre-sequence of the MFa mating factor α - is underlined, the MFα-Pro sequence is italic, the amino acid sequence of the human Kunitz domain APP4 is in bold):

Seq.Nr. 38, SEQ ID NO: 95:

MRFPSIFΎAVLFΆASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAK BEGVSLDKRVTLLI ^ CSEQAETGPCRJ ^ ISRWYFDVTEGKCAPFFYGGCGGNRNNFDTEEYCMAVCGSA

Both expression constructs (APP4 in yeast secretion vector plU10.10.W and plU3.12.M) were successfully expressed in baker's yeast.

Analogous to the example of the human Kunitz domain APP4 shown, other human Kunitz domains were cloned into the yeast secretion vectors plU10.10.W and plU3.12.M. All constructs in the yeast secretion vector plU10.10.W each contain the pre-sequence of the mating factor α (MFa) in front of the human Kunitz domain coding sequence (see SEQ ID NO: 37), constructs in the yeast secretion vector plU3.12. M each contain the pre-pro sequence of the mating factor α (see seq. 38). As synthetic genes were used Seq.No. 63a - Seq. 63d and Seq. 64a - Seq. 64d (mutated sequences are underlined). The deduced amino acid sequences are as follows:

Seq.Nr. 39 EPPI-plU10.10W, SEQ ID NO: 96:

LDLKQDVCEMPKETGPCLAYFLRWWYDKKDNTCSMFVYGGCQGNNNNFQSKANCLNTCKNG

Seq.Nr. 40 EPPI-plU3.12.M, SEQ ID NO: 97:

LDLKQDVCEMPKETGPCLAYFLRWWYDKKDNTCSMFVYGGCQGNNNNFQSKANCLNTCKNG

Seq.Nr. 41 TFPM -D2-plU 10.1 OW, SEQ ID NO: 98:

KPDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLEECKNICEDG

Seq.Nr. 42 TFPI1-D2-plU3.12.M, SEQ ID NO: 99:

EKDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLEECKNICEDG

Seq.Nr. 43 TFPI1-D3-plU10.10W, SEQ ID NO: 100:

GPGSWCLTPADRGLCRANENRFYYNSVIGKCRPFKYSGCGGNENNFTSKQECLRACKKG

Seq.Nr. 44 TFPI1-D3-plU3.12.M, SEQ ID NO: 101:

HGSWCLTPADRGLCRANENRFYYNSVIGKCRPFKYSGCGGNENNFTSKQECLRACKKG

Seq.Nr. 45 TFPI2-D2-plU10.10W, SEQ ID NO: 102:

VPKVCRLQVSVDDQCEGSTEKYFFNLSSMTCEKFFSGGCHRNRIENRFPDEATCMGFCAPK Seq.Nr. 46 TFPI2-D2-plU3.12.M, SEQ ID NO: 103:

KVKVCRLQVSVDDQCEGSTEKYFFNLSSMTCEKFFSGGCHRNRIENRFPDEATCMGFCAPK

Seq. No. 47 TFPI2-D3-plU10.10W, SEQ ID NO: 104:

IPSFCYSPKDEGLCSANVTRYYFNPRYRTCDAFTYTGCGGNDNNFVSREDCKRACAKA

Seq. No. 48 TFPI2-D3-plU3.12.M, SEQ ID NO: 105:

KISFCYSPKDEGLCSANVTRYYFNPRYRTCDAFTYTGCGGNDNNFVSREDCKRACAKA

Seq.Nr. 49 APLP2-plU10.10W, SEQ ID NO: 106:

VKAVCSQEAMTGPCRAVMPRWYFDLSKGKCVRFIYGGCGGNRNNFESEDYCMAVCKAM

Seq.Nr. 50 APLP2-plU3.12.M, SEQ ID NO: 107:

VKAVCSQEAMTGPCRAVMPRWYFDLSKGKCVRFIYGGCGGNRNNFESEDYCMAVCKAM

Seq.Nr. 51 AMBP-D1-plU10.10W, SEQ ID NO: 108:

KEDSCQLGYSAGPCMGMTSRYFYNGTSMACETFQYGGCMGNGNNFVTEKECLQTCRTV

Seq.Nr. 52 AMBP-D1-plU3.12.M, SEQ ID NO: 109:

KEDSCQLGYSAGPCMGMTSRYFYNGTSMACETFQYGGCMGNGNNFVTEKECLQTCRTV

Seq.Nr. 53 AMBP-D2-plU10.10W, SEQ ID NO: 110:

TVAACNLPIVRGPCRAFIQLWAFDAVKGKCVLFPYGGCQGNGNKFYSEKECREYCGVP

Seq.Nr. 54 AMBP-D2-plU3.12.M, SEQ ID N0: 111:

TVAACNLPIVRGPCRAFIQLWAFDAVKGKCVLFPYGGCQGNGNKFYSEKECREYCGVP

Seq.Nr. 55 HAI1-D1-plU10.10W, SEQ ID NO: 112:

TEDYCLASNKVGRCRGSFPRWYYDPTEQICKSFVYGGCLGNKNNYLREEECILACRGV

Seq.Nr. 56 HAI1-D1-plU3.12.M, SEQ ID NO: 113:

TEDYCLASNKVGRCRGSFPRWYYDPTEQICKSFVYGGCLGNKNNYLREEECILACRGV

Seq.Nr. 57 HAI1-D2-plU10.10W, SEQ ID NO: 114:

DKGHCVDLPDTGLCKESIPRWYYNPFSEHCARFTYGGCYGNKNNFEEEQQCLESCRGIS

Seq.Nr. 58 HAI1-D2-plU3.12.M, SEQ ID NO: 115:

DKGHCVDLPDTGLCKESIPRWYYNPFSEHCARFTYGGCYGNKNNFEEEQQCLESCRGIS Seq.Nr. 59 COL6A3-plU10.10W, SEQ ID NO: 116:

ETDICKLPKDEGTCRDFILKWYYAPNTKSCARFWYGGCGGNENKFGSQKECEKVCAPV

Seq.Nr. 60 COL6A3-plU3.12.M, SEQ ID NO: 117:

ETDICKLPKDEGTCRDFILKWYYAPNTKSCARFWYGGCGGNENKFGSQKECEKVCAPV

Seq.Nr. 60a COL6A3-mut1-plU3.12.M, SEQ ID NO: 118:

ETDICKLPKDEGTCRDAILKWYYAPNTKSCARFWYGGCGGNENKFGSQKECEKVCAPV

Seq.Nr. 60b COL6A3-mut2-plU3.12.M, SEQ ID NO: 119:

ETDICKLPKDEGPCRDAILKWYYAPNTKSCARFWYGGCGGNENKFGSQKECEKVCAPV

Seq.Nr. 60c COL6A3-mut3-plU3.12.M, SEQ ID NO: 120:

ETDICKLPKVTGTCRAAMKRFYYAPNTKSCARFWYGGCGGNENKFGSQKECEKVCAPV

Seq.Nr. 6Od COL6A3-mut4-plU3.12.M, SEQ ID NO: 121:

ETDICKLPKETGTCRAAMKRFYYAPNTKSCARFWYGGCGGNENKFGSQKECEKVCAPV

Seq.Nr. 6Oe COL6A3-mut5-plU3.12.M, SEQ ID NO: 122:

ETDICKLPKETGTCRAAHPRWWYAPNTKSCARFWYGGCGGNENKFGSQKECEKVCAPV

Seq.Nr. 61 COL7A1.plU10.10W, SEQ ID NO: 123:

DPCSLPLDEGSCTAYTLRWYHRAVTGSTEACHPFVYGGCGGNANRFGTREACERRCPPR

Seq.Nr. 62 COL7A1-plU3.12.M, SEQ ID NO: 124:

DCSLPLDEGSCTAYTLRWYHRAVTGSTEACHPFVYGGCGGNANRFGTREACERRCPPR

Seq.Nr. 63 WFI1-D2-plU10.10W, SEQ ID NO: 125:

GPLAACSLPALQGPCKAYAPRWAYNSQTGQCQSFVYGGCEGNGNNFESREACEESCPFPS

Seq.Nr. 63a WFI1-D2-mut 1-plU10.10W, SEQ ID NO: 126:

GPLAACSLPALQGPCRAAAPRWAYNSQTGQCQSFVYGGCEGNGNNFESREACEESCPFP

Seq.Nr. 63b WFI1-D2-mut 2-plU10.10W, SEQ ID NO: 127:

GPLAACSLPAVTGPCRAAMKRFAYNSQTGQCQSFVYGGCEGNGNNFESREACEESCPFP

Seq.Nr. 63c WFI1-D2-mut 3-plU10.10W, SEQ ID NO: 128:

GPLAACSLPAETGPCRAAMKRFAYNSQTGQCQSFVYGGCEGNGNNFESREACEESCPFP

Seq.Nr. 63d WFI1-D2-mut 4-plU10.10W, SEQ ID NO: 129: GPLAACSLPAETGPCRAAHPRWWYNSQTGQCQSFVYGGCEGNGNNFESREACEESCPFP

SEQ ID NO: 64 WFI1-D2-plU3.12.M, SEQ ID NO: 130:

GLAACSLPALQGPCKAYAPRWAYNSQTGQCQSFVYGGCEGNGNNFESREACEESCPFPS

SEQ ID NO: 64a WFI1-D2-mut 1-plU3.12.M, SEQ ID NO: 131:

GLAACSLPALQGPCRAAAPRWAYNSQTGQCQSFVYGGCEGNGNNFESREACEESCPFP

SEQ ID NO: 64b WFI1-D2-mut 2-plU3.12.M, SEQ ID NO: 132:

GLAACSLPAVTGPCRAAMKRFAYNSQTGQCQSFVYGGCEGNGNNFESREACEESCPFP

Seq.Nr. 64c WFI1-D2-mut 3-plU3.12.M, SEQ ID NO: 133:

GLAACSLPAETGPCRAAMKRFAYNSQTGQCQSFVYGGCEGNGNNFESREACEESCPFP

Seq.Nr. 64d WFI1-D2-mut 4-plU3.12.M, SEQ ID NO: 134:

GLAACSLPAETGPCRAAHPRWWYNSQTGQCQSFVYGGCEGNGNNFESREACEESCPFP

Seq.Nr. 65 WFI2-D2-plU10.10W, SEQ ID NO.135:

PGDACVLPAVQGPCRGWEPRWAYSPLLQQCHPFVYGGCEGNGNNFHSRESCEDACPVP

Seq.Nr. 66 WFI2-D2-plU3.12.WI, SEQ ID NO: 136:

GDACVLPAVQGPCRGWEPRWAYSPLLQQCHPFVYGGCEGNGNNFHSRESCEDACPVP

Seq.Nr. 67 SPINT4-plU10.10W, SEQ ID NO: 137:

GDACVLPAVQGPCRGWEPRWAYSPLLQQCHPFVYGGCEGNGNNFHSRESCEDACPVP

Seq.Nr. 68 SPINT4-plU3.12.M, SEQ ID NO: 138:

GDACVLPAVQGPCRGWEPRWAYSPLLQQCHPFVYGGCEGNGNNFHSRESCEDACPVP

Seq.Nr. 69 PAPLN / HyPro-plU10.10W, SEQ ID NO: 139:

GDACVLPAVQGPCRGWEPRWAYSPLLQQCHPFVYGGCEGNGNNFHSRESCEDACPVP

Seq.Nr. 70 PAPLN / HyPro-plU3.12.M, SEQ ID NO: 140:

YVRCLLPSAHGSCADWAARWYFVASVGQCNRFWYGGCHGNANNFASEQECMSSCQGS

Seq.Nr. 71 SPIT3-plU10.10W, SEQ ID NO: 141:

CAFPMEKGPCQTYMTRWFFNFETGECELFAYGGCGGNSNNFSRKEKCEKFCKFT

Seq.Nr. 72 SPIT3-plU3.12.M, SEQ ID NO: 142:

CAFPMEKGPCQTYMTRWFFNFETGECELFAYGGCGGNSNNFSRKEKCEKFCKFT Seq.Nr. 73 SPIT3 desPro-plU3.12.M, SEQ ID NO: 143:

LNVCAFPMEKGPCQTYMTRWFFNFETGECELFAYGGCGGNSNNFSRKEKCEKFCKFT

Seq.Nr. 73a SPIT3 of Pro-mut1-plU3.12.M, SEQ ID NO: 144:

LNVCAFPMEKGPCRTYMTRWFFNFETGECELFAYGGCGGNSNNFSRKEKCEKFCKFT

Seq. A aprotinin (58aa), SEQ ID NO: 145:

RPDFCLEPPYTGPCKARI IRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGA

Seq.Nr. 64e WFI1-D2-mut5-plU3.12.M, SEQ ID NO: 146:

GLAACSLPAETGPCRAAHKRFAYNSQTGQCQSFVYGGCEGNGNNFESREACEESCPFP

Example 4

Transformation of Saccharomyces cerevisiae

Yeast cells, eg strain JC34.4D (MATa ₁ ura3-52, suc2) were grown in 10 ml of YEPD (2% glucose, 2% peptone, 1% Difco yeast extract) and harvested at ODOQQ of 0.6 to 0.8 , The cells were washed with 5 ml of solution A (1 M sorbitol, 10 mM bicin pH 8.35, 3% ethylene glycol), in 0.2 ml

Resuspended solution A and stored at -70 ⁰ C.

Plasmid DNA (5 μg) and carrier DNA (50 μg of herring sperm DNA) were added to the frozen ones

Given cells. The cells were then thawed by shaking at 37 ^° C. for 5 min. After addition of 1.5 ml of solution B (40% PEG 1000; 200 mM bicin pH 8.35), the cells were allowed to stand for 60 min

30 ⁰ C, washed after pelleting with 1, 5 ml of solution C (0.15 M NaCl, 10 mM bicine pH 8.35) and resuspended in 100 ul of solution C. The Ausplattierung took place on a

Selection medium with 2% agar. Transformants were obtained after incubation for 3 days at 30 ^° C.

Example 5

Fermentation of the yeast cells

nutrient solutions

For the fermentation of yeast cells for the expression of aprotinin or variants modified with KTPI

Amino acid sequence, the following nutrient solutions were used

SL4 solution:

Titriplex III (Merck 8418) 5 g

FeSO ₄ ^• 7H ₂ O (Merck 3965) 2 g

ZnSO ₄ ^• 7H ₂ O (Merck 8883) 0.1 g

MnCl ₂ ^• 4H ₂ O (Merck 5927) 30 mg

H ₃ BO ₃ (Merck 165) 0.3 g

CoCl ₂ ^• 6H ₂ O (Merck 2533) 0.2 g

CuCl ₂ ^• 2H ₂ O (Merck 2733) 10 mg

NiCl ₂ ^■ 6H ₂ O (Merck 6717) 20 mg

Na ₂ MoO ₄ ^.2H ₂ O (Merck 6521) 30 mg

(Dissolve 1000 ml in distilled H ₂ O, adjust the pH to 3 - 4 with NaOH, divide the batch into a maximum of 12 containers and store at -18 ° C., thawing the individual containers for a maximum of 1 month.)

The starting materials were prepared in demineralized water and the pH was adjusted to pH 5.5. Sterilization was carried out at 121 ° C. for 20 min. Glucose was dissolved in 1/5 of the required volume in demineralized water, sterilized separately and, after cooling, added to the remaining nutrient solution.

strain stocks

Stocks of all yeast transformants were prepared by mixing 1 ml aliquots of a

Preculture with 1 ml of 80% glycerol solution and storage at -140 ° C applied. precultures

The preculture fermentations were carried out in 50 ml (for main cultures in the small volume) or 1 ltr. Shake flasks (for main cultures in the medium volume) filled with 10 or 100 ml SD2 nutrient solution. The inoculation took place with a trunk preserve or with a single colony from an SD2 agar plate. The cultures were incubated with constant shaking (240 U / min) for 2 - 3 days at 28 - 30 ⁰ C.

Main culture fermentations

Small-scale main culture fermentations were carried out using 1 L_tr. Shake flasks filled with 100 ml SC5 broth. The inoculation was usually carried out with 3 ml of the preculture described above. Subsequently, the cultures were shaken continuously (240 rpm) for

Incubated for 4 days at 28-30 ° C.

In the case of the main culture fermentation on a medium or large scale, the bioreactor

System used by Wave Biotech (Tageiswangen, CH). In detail, 1000 or

Inoculated 10000 ml of SC5 medium supplemented with 30 or 300 ml pre-culture and for 4 days at 30 ⁰ C in Wavebag at a bounce frequency of 32 / min incubated (angle: 10 °; air supply: 0.25 Ltr./min). The pH of the

Cultures were checked on day 1 to 3 and adjusted to pH 5-6 with 5 N NaOH as necessary. The

Cultures were fed on days 1 to 3 with 1 ml each of 50% yeast extract solution and 4 ml of 4

M glucose solution in the case of 100 ml cultures. In the case of the 1000 and 10000 ml cultures, the addition of the 10 or 100 times the volume of feed solutions was carried out correspondingly continuously over the day.

For growth control the OD _{60O of} the cultures could be determined at different times.

After 4 days, the cell-free supernatants were harvested by centrifugation (15 min at 6000 rpm in the JA14 rotor).

Example 6

Purification of human Kunitz domains from supernatants of fermented yeast cells

The human Kunitz domain produced in the main culture fermentation with the Seq. No. 35 containing cell-free supernatants were added with 1 M NaOH until the pH was 7.8. Suspended particulates suspended in the supernatant were sedimented by centrifugation at 2,000 rpm at 4 ° C (15 minutes, Beckman-Allegra 6KR). The supernatant was applied at 1 ml / min to a 10 ml trypsin agarose column (Sigma-T1763). The column was then washed with 70 ml of 50 mM Tris pH 7.8, 250 mM NaCl and 50 ml of 50 mM Tris pH 7.8, 600 mM NaCl. It was then eluted with 180 ml of 50 mM KCl / 10 mM HCl pH 2.0 and with 100 ml of 50 mM KCl / 250 mM HCl pH 0.9. The 2 ml fractions were collected in tubes each containing 500 μl of 200 mM Tris pH 7.6, 2 M NaCl to neutralize. Human Kunitz domains containing Sequence No. 35 were identified by the trypsin inhibition assay described below. Trypsin-inhibiting fractions were pooled, mixed with the same volume of 0.1% TFA and applied to a Source 15 RPC column (GE Healthcare). The column was treated with 6 ml of 0.1% TFA (buffer HPLC-A), and then the human Kunitz domain was digested with Seq. No. 35 with a 25 ml gradient on 50% buffer HPLC-B (0.1% TFA, 60% acetonitrile) and another 5 ml gradient on 100% buffer HPLC-B eluted. The Seq. No. 35 were lyophilized and the lyophilizate was taken up in 250 μl of 50 mM Tris pH 7.5 per fraction.

Example 7

Production of Human Kunitz Domains by In Vitro Translation and Purification via Ni-NTA Agarose

The Kunitz domain with the Seq. No. 27e was expressed using the plasmid plVEX2.3d by in vitro translation with the RTS-500 E. coli disulfide kit (Roche Diagnostics) according to the manufacturer's instructions.

After completion of the expression, both solutions (reaction solution and supply solution) were mixed and admixed with 1 ml of 0.1% Tween-20. Insolubles were removed by centrifugation at 3,000 rpm (4 ° C, 30 min). Subsequently, the clear supernatant was mixed with 300 μl of Ni-NTA superflow (Qiagen) and the resulting suspension was incubated with constant agitation for 90 min at 4 ° C. The affinity matrix was sedimented by centrifugation and the supernatant discarded. Ni-NTA superflow was washed three times by incubation with 1 ml each of 50 mM Tris pH 7.5, 100 mM NaCl, 10 mM imidazole and in each case sedimented by centrifugation. Subsequently, the Kunitz domain bound to Ni-NTA superflow with the Seq. No. 27e was eluted by incubating the affinity matrix three times with 900 μl each of 50 mM Tris pH 7.5, 100 mM NaCl, 250 mM imidazole. The eluates were pooled and rebuffered to 10 mM Tris pH 7.5 by desalting with NAP-25 columns (GE Healthcare). After lyophilization, the Kunitz domain was digested with Seq. No. 27e in water and stored at -80 ° C.

Example 8

Characterization of APP4 by Tryptic Peptides and Molecular Weight

For molecular weight determination of APP4, the purified protein was diluted to 2 pmol / μl with a 0.1% formic acid solution and acidified at the same time. After separation, this sample was analyzed by mass spectrometry using a GromSil 120 ODS-4 HE (3 μm, 250 × 0.2 mm). Based on the multiply charged molecular ions, the molecular weight of APP4 could be detected.

For more precise determination of the amino acid sequence, the protein was reduced after denaturing with guanidinium hydrochloride with dithiothreitol, derivatized by means of iodoacetamide and then cleaved by tryptic. The resulting gap peptides were then analyzed by mass spectrometry and the sequence coverage of APP4 was determined on the basis of the detected peptide masses and MS / MS spectra. In this case, the entire amino acid sequence could be detected. VREVCSEQAETGPCRAMISRWYFDVTEGKCAPFFYGGCGGNRNNFDTEEYCMAVCGSA

[M + H] ⁺¹ 1777.8: VREVCSEQAETGPCR [M + H] ⁺¹ 2336.1: VREVCSEQAETGPCRAMISR [M + H] ⁺¹ 926.6: AMISRWY [M + H] ⁺¹ 1702.8: AMISRWYFDVTEGK [M + H] ⁺¹ 1144.6: WYFDVTEGK [M + H] ⁺¹ 1462.6: CAPFFYGGCGGNR [M + H] ⁺¹ 3311.1: CAPFFYGGCGGNRNNFDTEEYCMAVCGSA [M + H] ⁺¹ 2836.0: FYGGCGGNRNNFDTEEYCMAVCGSA

Example 9

Determination of the inhibitory potency of human KTPI muteins against trypsin, plasmin, FXIa and plasma kallikrein

The inhibitory potency of human KTPI muteins against the enzymatic activities of trypsin, plasmin, FXIa and plasma kallikrein were determined in biochemical assays in white 384-well microtiter plates using fluorogenic substrates. The assay buffer was composed of 50 mM Tris / Cl, pH 7.4, 100 mM NaCl, 5 mM CaCl ₂ , 0.08% (w / v) BSA. The test conditions were as follows:

Per hole, 10 μl of a serial dilution were initially charged and preincubated with 20 μl of enzyme for 5 min at RT. Subsequently, the reaction was started by addition of 20 .mu.l of substrate. The measurement was carried out after 60-90 min in a Tecan reader at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. Dose-response curves and half-maximal inhibitory constants (IC50 values) were determined using GraphPad Prism software (version 4.02). determined. Table 1: IC50 values of some Kunitz domains for the inhibition of human trypsin, plasmin, factor XIa and plasma kallikrein

Example 10

Inhibition of fibrinolysis by Seq. No. 63d (WFI-1-D2-mut-4)

The effect of WFI-1-D2-mut-4 was tested in an in vitro fibrinolytic model and compared to the effect of trasylol (aprotinin). Human citrated plasma was spiked with 0.13 pM tissue factor (TF) and 164 U / ml tissue plasminogen activator (tPA) and WFI-1-D2 mut-4 or aprotinin at various concentrations (0.1 μM to 10 μM) and 37 min at 37 min ° C incubated. As a control, physiological saline was used in place of the Kunitz domains. Clot formation by TF and clot lysis by tPA were determined by optical density measurements (OD _40S nm) with a Tecan Satire. Fibrinolysis was defined as the relative decrease in OD after clot formation. Inhibition of the relative decrease in OD is the measure of antifibrinolytic activity. WFI-1-D2-mut-4 inhibited fibrinolysis with an IC ₅₀ of 2.0 + 0.2 μM. Trasylol (aprotinin) inhibited fibrinolysis in this model with an IC ₅₀ of 1.2 ± 0.2 μM. FIG. 2 describes the antifibrinolytic activity of WFIII-D2-mut4 in human plasma. Example 11

Inhibition of intrinsic coagulation by Seq. No. 63d (WFI-1-D2-mut-4)

The effect of WFI-1-D2-mut-4 was tested in an in vitro coagulation model and compared to the effect of trasylol (aprotinin). Human citrated plasma was spiked with 12 mM CaCl ₂ for coagulation and WFI-1-D2-mut-4 or aprotinin in various concentrations (0.1 μM to 30 μM). As a control, physiological saline was used in place of the Kunitz domains. During incubation for 90 min at 37 ° C, the increase in OD at 405 nm was determined as a measure of coagulation. From this, the half-maximal coagulation time was calculated. An extension of the half-maximal coagulation time means inhibition of coagulation. WFI-1-D2-mut-4 inhibited coagulation with a CT2 (doubling the half-maximal coagulation time) of 0.8 ± 0.02 μM. The CT2 for trasylol (aprotinin) was 12 ± 0.2 μM

Fig. 3 describes the anticoagulant activity of WFI1-D2-mut4 in human plasma.

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US 6,010,880 A

US 5,795,865 A

WO 92/06111 A1

US Pat. No. 6,482,798 B2

EP 0 238 993 A2

EP 0 307 592 A2

EP 0 821 007 A2

US 5,863,893 A.

US 5,914,315 A

US 7,019,123 B2

Claims

claims

1. An isolated protein or polypeptide containing a Kunitz domain,

characterized in that the domain has the following amino acids at the positions numbered according to aprotinin:

X10 = E, X11 = T, X15 = R, X16 = A, X17 = A, X18 = H X19 = P, X20 = R, X21 = W, X22 = W, the remaining positions containing any amino acids of human Kunitz domains ;

or

characterized in that the domain has the following amino acids at the positions numbered according to aprotinin:

X10 = V, X11 = T, X15 = R or K, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F, the remaining positions containing any amino acids of human Kunitz domains;

or

characterized in that the domain has the following amino acids at the positions numbered according to aprotinin:

X10 = E, X11 = T, X15 = R or K, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F, the remaining positions containing any amino acids of human Kunitz domains;

or

characterized in that the domain has the following amino acids at the positions numbered according to aprotinin:

X10 = V, X11 = T, X15 = R, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F, the remaining positions containing any amino acids of human Kunitz domains;

or

characterized in that the domain has the following amino acids at the positions numbered according to aprotinin:

X10 = E, X11 = T, X15 = R, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F, the remaining positions containing any amino acids of human Kunitz domains.

2. An isolated protein according to claim 1, wherein the remaining positions of the Kunitz domain contain amino acids of a particular human Kunitz domain.

3. An isolated protein or polypeptide containing an amino acid sequence selected from the group consisting of Seq. No. 6Od (COL6A3-mut4, SEQ ID NO: 121), Seq. No. 6Oe (COL6A3-mut5, SEQ ID NO: 122), Seq. No. 63a (WFH-D2 mut1, SEQ ID NO: 126), Seq. No. 63c (WFI1-D2-mut3, SEQ ID NO: 128), Seq. No. 63d (WFI1-D2-mut4, SEQ ID NO: 129) and SEQ. NO. 64e (WFI1 -D2-mut5-plU3.12.M, SEQ ID NO: 146).

4. A fragment of a protein or polypeptide according to claims 1-3, characterized in that the fragment has an inhibitory effect on plasmin, plasma kallikrein, or factor XIa, which is comparable to the action of aprotinin or better than the action of aprotinin.

5. A nucleic acid encoding a polypeptide or protein according to claims 1-4.

6. The nucleic acid according to claim 5, containing heterologous vector sequences or a heterologous promoter sequence 5 'to the coding region.

7. cell containing a nucleic acid according to claim 6.

8. Non-human organism containing a nucleic acid according to claim 6.

9. A method for isolating a polypeptide or protein according to claims 1-4, characterized in that a cell according to claim 7 is cultivated and the protein is purified.

10. A monoclonal or polyclonal antibody specific for a protein according to claims 1-4.

11. A protein or polypeptide according to claims 1-4 or a nucleic acid according to claim 5 as a medicament.

12. A pharmaceutical composition containing a protein according to claims 1-4 or a nucleic acid according to claim 5.

13. A protein or polypeptide according to claims 1-4 or a nucleic acid according to claim 5 as a medicament for the treatment of conditions or diseases selected from the group consisting of blood loss in operations with increased risk of bleeding; thrombotic bolsic states, shock, polytrauma, sepsis, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), unstable angina, myocardial infarction, stroke, embolism, deep vein thrombosis, inflammatory diseases (eg rheumatism, asthma), invasive tumor growth and metastasis, pain and edema therapy (cerebral edema, spinal cord edema), prevention of activation of hemostasis in dialysis treatment, treatment of skin aging symptoms (elastosis, atrophy, wrinkling, vascular changes, pigment changes, actinic keratosis, blackheads, cysts), wound healing, skin cancer, treatment of skin cancer symptoms ( actinic keratosis, basal cell carcinoma, squamous cell carcinoma, malignant melanoma), multiple Sclerosis, fibrosis, cerebral hemorrhage, inflammation of the brain and spinal cord, infections of the brain and tendinopathy.

14. A protein or polypeptide according to claims 1-4 or a nucleic acid according to claim 5 as a medicament for the treatment of conditions or diseases selected from the group consisting of blood loss in operations with increased risk of bleeding; thromboembolic conditions, shock, sepsis, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), myocardial infarction, stroke, embolism, deep venous thrombosis, and wound healing.

Use of a protein or polypeptide according to claims 1-4 or a nucleic acid according to claim 5 for the manufacture of a medicament for the treatment of conditions or diseases selected from the group consisting of blood loss in operations with increased bleeding risk; thromboembolic conditions, shock, polytrauma, sepsis, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), unstable angina, heart attack, stroke, embolism, deep vein thrombosis, inflammatory diseases (eg rheumatism, asthma), invasive tumor growth and metastasis, pain and Edema therapy (cerebral edema, spinal cord edema), prevention of activation of hemostasis in dialysis treatment, treatment of skin aging symptoms (elastosis, atrophy, wrinkles, vascular changes, pigment changes, actinic keratosis, blackheads, cysts), wound healing, skin cancer, treatment of skin cancer symptoms (actinic Keratosis, basal cell carcinoma, squamous cell carcinoma, malignant melanoma), multiple sclerosis, fibrosis, cerebral hemorrhage, inflammation of the brain and spinal cord, infections of the brain and tendinopathy.

16. Use of a protein or polypeptide according to claims 1-4 or a nucleic acid according to claim 5 for the manufacture of a medicament for the treatment of conditions or diseases selected from the group consisting of blood loss in operations with increased risk of bleeding; thromboembolic conditions, shock, sepsis, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), myocardial infarction, stroke, embolism, deep venous thrombosis, and wound healing.

17. A protein containing a polypeptide selected from the group consisting of Seq. No. 38 (SEQ ID NO: 95, APP4), Seq. No. 50 (SEQ ID NO: 107, APLP-2), and Seq. No. 54 (SEQ ID NO: AMBP-D2) or a nucleic acid encoding such proteins as a drug.

18. A pharmaceutical composition comprising a protein containing a polypeptide selected from the group consisting of Seq. No. 38 (SEQ ID NO: 95, APP4), Seq. No. 50 (SEQ ID NO: 107, APLP-2), and Seq. No. 54 (SEQ ID NO: AMBP-D2) or a nucleic acid encoding such proteins.

19. A protein containing a polypeptide selected from the group consisting of Seq. No. 38 (SEQ ID NO: 95, APP4), Seq. No. 50 (SEQ ID NO: 107, APLP-2), and Seq. No. 54 (SEQ ID NO: AMBP-D2) or a nucleic acid encoding such proteins as a medicament for the treatment of conditions or diseases selected from the group consisting of blood loss in operations at increased risk of bleeding; thromboembolic conditions, shock, polytrauma, sepsis, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), unstable angina, heart attack, stroke, embolism, deep vein thrombosis, inflammatory diseases (eg rheumatism, asthma), invasive tumor growth and metastasis, pain and Edema therapy (cerebral edema, spinal cord edema), prevention of activation of hemostasis in dialysis treatment, treatment of skin aging symptoms (elastosis, atrophy, wrinkles, vascular changes, pigment changes, actinic keratosis, blackheads, cysts), wound healing, skin cancer, treatment of skin cancer symptoms (actinic Keratosis, basal cell carcinoma, squamous cell carcinoma, malignant melanoma), multiple sclerosis, fibrosis, cerebral hemorrhage, inflammation of the brain and spinal cord, brain infections and tendinopathy.

20. A pharmaceutical composition comprising a protein containing a polypeptide selected from the group consisting of Seq. No. 38 (SEQ ID NO: 95, APP4), Seq. No. 50 (SEQ ID NO: 107, APLP-2), and Seq. No. 54 (SEQ ID NO: AMBP-D2) or a nucleic acid encoding such proteins for the treatment of conditions or diseases selected from the group consisting of blood loss in operations with increased risk of bleeding; thromboembolic conditions, shock, polytrauma, sepsis, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), unstable angina, heart attack, stroke, embolism, deep vein thrombosis, inflammatory diseases (eg rheumatism, asthma), invasive tumor growth and metastasis, pain and Edema therapy (cerebral edema, spinal cord edema), prevention of activation of hemostasis in dialysis treatment, treatment of skin aging symptoms (elastosis, atrophy, wrinkles, vascular changes, pigment changes, actinic keratosis, blackheads, cysts), wound healing, skin cancer, treatment of skin cancer symptoms (actinic Keratosis, basal cell carcinoma, squamous cell carcinoma, malignant melanoma), multiple sclerosis, fibrosis, cerebral hemorrhage, inflammation of the brain and spinal cord, infections of the brain and tendinopathy.

21. A protein containing a polypeptide selected from the group consisting of Seq. No. 38 (SEQ ID NO: 95, APP4), Seq. No. 50 (SEQ ID NO: 107, APLP-2), and Seq. No. 54 (SEQ ID NO: AMBP-D2) or a nucleic acid encoding such proteins as a medicament for the treatment of conditions or diseases selected from the group consisting of blood loss in operations at increased risk of bleeding; thromboembolic conditions, shock, sepsis, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), myocardial infarction, stroke, embolism, deep venous thrombosis, and wound healing.