DK174754B1 - DNA encoding human tissue factor inhibitor - Google Patents

Abstract

DNA sequence encoding human tissue factor inhibitor (TFI) is claimed. Also claimed is human TFI having a specified aminoacid sequence.

Description

in DK 174754 B1

Human tissue factor inhibitor (TFI), binding specific antibodies thereto, method for using a TFI peptide column to purify an antibody, and methods for detecting TFI or a polypeptide comprising one or more Kunitz domains of TFI in a biological flu -5 idum

The present invention relates to a human coagulation inhibitor termed tissue factor inhibitor (TFI) and, alternatively, as a lipoprotein-associated coagulation inhibitor (LACI) as well as antibodies with binding specificity thereof. Further, the invention relates to a method for using a TFI peptide column to purify an antibody to TFI and methods for detecting TFI or a polypeptide comprising one or more Kunitz domains of TFI in a biological fluid.

BACKGROUND OF THE INVENTION

The coagulation process that occurs in mammalian blood involves two distinct systems - the so-called internal and external systems. The latter system is activated when the blood is exposed to tissue thromboplastin (factor III), hereinafter referred to as the tissue factor (TF). Tissue factor is a lipoprotein found in the plasma membrane of a whole range of cell types, with the brain and lungs particularly rich in it. Upon contact with TF, plasma factor VII or its activated form, factor VIIa, forms a calcium-dependent complex with TF, which then proteolytically activates factor X to factor Xa, and factor IX to factor IXa.

Early studies on the regulation of TF-initiated coagulation showed that incubation of TF (in crude tissue thromboplastin preparations) with serum inhibited its activity in vitro and prevented its lethal effect when infused into mice. Further studies of Hjort, Scand. J. din.

Lab. Invest 9, Suppl. 27, 76-97 (1957) confirmed and further developed previous work in this field and led to the conclusion that an inhibitory part of serum recognizes the factor VII-TF complex. In accordance with this hypothesis, the facts are that the inhibition of TF occurring in plasma requires the presence of Ca 2+ (which is also necessary for the binding of factor VII / VIIa to TF) and that the inhibition can be prevented and / or reversed by chelating divalent cations with EDTA. Recent studies have shown that not only factor VIa but also catalytically active factor Xa and an additional factor are required for the achievement of plasma TF inhibition in plasma or serum. See Broze and Miletich, Blood 69, 150-155 (1987) and Sanders et al., Ibid. 66, 204-212 (1985). This additional factor, herein defined as tissue factor inhibitor (TFI) and alternatively as lipoprotein-associated coagulation inhibitor (LACI), is present in barium-absorbed plasma and appears to be associated with lipoproteins, as TFI functional activity is secreted by the lipoprotein fraction flowing on top serum is centrifuged at a density of 1.21 g / cm 3. According to Broze and Miletic, supra, and Proc. Natl. Acad. Sci. USA 84, 1886-1890 (1987), HepG2 cells (a human hepatoma cell line) secrete an inhibitory moiety with the same properties as the TFI present in plasma.

EP Publication No. 300,988 discloses a purified tissue factor inhibitor (TFI) secreted by HepG2 cells. It was found to be available in two forms, a TFh migrating at about 37000-40000 daltons and a TFb migrating at 25000-26000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A partial N-terminal amino acid sequence for TFI was mapped as: 25 1 15 X-X-Glu-Glu-Asp-Glu-Glu-His-Thr-III-II-Thr-Asp-Thr-Glu-16 27

Leu-Pro-Pro-Leu-Lys-Leu-Met-His-Ser-Phe- (Phe) -Ala, where X-X had not been determined. The content of this application is considered to be incorporated into this specification by reference.

DK 174754 B1 3

BRIEF DESCRIPTION OF THE INVENTION

The invention is based on the provision of the complete coding sequence for a cDNA clone which essentially represents full-size tissue factor inhibitor (TFI).

First, human placental and fetal liver Agt11 cDNA libraries were screened with a polyclonal rabbit antiserum against a purified TFI. Immunologically positive clones were further screened for 125 I factor Xa binding activity. Seven clones that were immunologically and functionally active were obtained.

The longest clone, placenta-derived λΡ9, was 1.4 kilobases (kb) long, while the other six had a length of 1.0 kb. Partial DNA sequencing showed that the 1.0 kb clones had sequences identical to part of the longer 1.4 kb clone. Nucleotide sequence analysis revealed that AP9 consisted of a 1432 base pair (pb) 15 cDNA insert comprising a 5 'non-coding region of 133 bp, an open reading frame of 912 bp, a stop codon and a 3' non-coding region of 384 bp.

The cDNA sequence encodes a 31,950 dalton protein of 276 amino acids, 20 comprising 18 cysteine residues and 7 methionine residues. The translated amino acid sequence shows that a signal peptide of about 28 amino acids precedes the mature TFI protein. It should be noted that the "mature" TFI is defined to include both TFI and methionyl TFI due to the translational ATG codon of the AP9 clone described herein.

25

There are three potential N-linked glycosylation sites in the TFI protein of the sequence Asn-X-Ser / Thr, wherein X may be any of the common 20 amino acids. These sites are at amino acid positions Asn 145, Asn 195 and Asn 256 when the first methionine residue after the 5 'non-coding region is designated amino acid position +1.

The translated amino acid sequence of TFI shows several distinguishable domains, including a highly negatively charged N-terminal, a highly positively charged carboxyl terminus, and an intermediate portion consisting of three homologous domains with sequences typical of Kunitz-type enzyme inhibitors. . Based on a homology study, TFI appears to be a member of the basal protea signal inhibitor gene superfamily.

5

The original source of the protein material to produce cDNA clone AP9 was human placental tissue. This tissue can be extensively obtained after birth by common surgical methods. AgtII (Iac5 nin5 c1857 S100) used herein is a well known and widely available lambda phage expression vector.

Its construction and restriction endonuclease map are described by Young and Davis, Proc. Natl. Acad. Sci. USA 80, 1194-1198 (1983).

Northern blot analysis revealed that the following liver-derived cell lines: Chang liver cell, HepG2 hepatoma and SK-HEP-1 hepatoma all contained two heads of mRNA (1.4 and 4.4 kb) hybridizing with TFi cDNA ' one.

The cloning of the cDNA for TFI and the development of its entire protein sequence and structural domains referred to herein enables detailed structural functional analyzes and provides a basis for studying its biosynthetic regulation. Thus, the invention is important to the medical science of investigating the coagulation with respect to agents capable of inhibiting factor Xa and factor VIIIa / TF enzyme complex.

The present invention particularly relates to human tissue factor inhibitor (TFI) 25 with that of FIG. 3 of the drawings, protein amino acid sequence shown.

In addition, the invention relates to an isolated and purified antibody having a binding factor for tissue factor inhibitor (TFI) having an amino acid sequence as shown in FIG. Third

30

Furthermore, the invention relates to a method of using a TFI peptide column to purify an antibody to that of FIG. 3 of the drawings, protein amino acid sequence shown.

DK 174754 B1 5

The invention further relates to methods for detecting a first polypeptide in a biological fluid, wherein said first polypeptide is selected from TFI and a polypeptide comprising one or more Kunitz domains of TFI, wherein one method of the invention comprises the following steps: a) contacting said fluid with a second polypeptide having a binding specificity for said first polypeptide, and (b) testing for the presence of said second polypeptide to determine the level of said first polypeptide; 10 and another method of the invention include the following steps: (a) contacting said fluid with an antibody having a binding specificity for said first polypeptide and a second polypeptide capable of binding said antibody and (b) testing for the presence of said second polypeptide 15 to determine the level of said first polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

The invention is now described in more detail with reference to the drawing, in which:

FIG. Figure 1 shows the screening of AgtII clones with 12S1 factor X * Cloned phage lysates (0.1 ml) were stained on a nitrocellulose paper by suction using a dot blot apparatus. The nitrocellulose paper was then probed with 125 I factor Xa and autoradiographed as described in detail. Clones that appear as dark spots are positive clones that bind 125l factor Xa. Control EgtII (lower right corner) and other clones do not bind 125l factor Xa.

FIG. 2 shows a partial restriction map and sequencing strategy for the AP9-30 insertions. The scale below indicates the nucleotide position. The thick bar represents the coding region. The thin bars represent 5 'and 3' non-coding regions. The restriction endonuclease sites were affected by degradation. The arrows show the overlapping M13 clones used to sequence the cDNA.

FIG. 3 shows the nucleotide sequence and the translated amino acid sequence of 5 the human TF1 cDNA. Nucleotides are numbered on the left and amino acids on the right. The underlined sequences have been independently confirmed by amino acid sequence analysis of the purified TFI protein and two Ve protease + trypsin degraded peptides. Amino acid +1 was assigned to the first methionine residue after a stop codon in the 5 'non-coding region. Potential N-linked N-linked glycosylation sites are marked with asterisks.

FIG. 4 is a graph showing the charge distribution of the amino acid sequence in TFI. Charges are calculated from the first residue to the end residue and shown at the end residue. Thus, the value in the infinite position is the summation of all charges from the first residue to the infinite residue, and the difference in the charges between the infinite and the jth residue (j> i) is the net charge of the fragment from the end to the j-th residue.

FIG. Figure 5 is a graph showing the hydrophobicity profile of TFI. The hydrophobicity profile was analyzed by a computer program in which the hydrophobicity index of the amino acid residues is defined as the depth at which an amino acid residue is buried within a protein (from X-ray crystallographic data) [Kidera et al., J. Protein Chem. 4, 23-55 (1985)]. The hydrophobicity profile along the sequence was smoothed using the program 25 ICSSCU in the IMSL Library [IMSL Library Reference Manual, 9th Edition, Institute for Mathematical and Statistical Subroutine Library, Houston, Texas (1982)].

FIG. 6 shows a line alignment of the basal protease inhibitor domains of TFI with other basal protease inhibitors. All sequences except TFI were obtained from the National Biomedical Research Foundation Protein Sequence Database (Georgetown University, Washington, D.C., U.S.A., released June 13, 1987). 1. Bovine basal protease inhibitor precursor; 2. Bovine colostrum-try psi ni n hi bitor; 3. Bovine serum basal protease inhibitor; 4. Edible snail isoin inhibitor K; 5. Red Sea Turtle Basal Protease Inhibitor (amino acids 1-79 only indicated); 6. Western Sandworm Poison-Basal Protease Inhibitor I; 7th

DK 174754 B1 7

Ring-neck poison basal protease inhibitor II; 8. Cape cobra venom-basal protease inhibitor II; 9. Russell's venomous poison-basal protease inhibitor II; 10. Sandworm poison-basal protease inhibitor III; 11. Eastern green mamba venom-basal protease inhibitor In homologue; 12. Black mamba venom-basal protease inhibitor B; 13. Black 5 mamba venom-basal protease inhibitor E; 14. Black mamba venom-basal protease inhibitor I; 15. Black mamba venom-basal protease inhibitor K; 16. β-1-Bungarotoxin B chain (minor); 17. β-1-Bungarotoxin B chain (larger); 18. β-2-Bungarotoxin B chain; 19. Horse inter-α-trypsin inhibitor [amino acids 1-57 (1); 58-123 (2)]; 20. Pig inter-α-trypsin inhibitor [amino acids 1-57 (1); 10, 58-123 (2)]; 21. Bovine inter-α-trypsin inhibitor [amino acids 1-57 (1); 58 - 123 (2)]; 22. Human α-1 microglobulin / inter-α-trypsin inhibitor precursor [amino acids 227-283 (1); 284-352 (2)]; 23. TFI [amino acids 47-117 (1); 118-188 (2); 210-280 (3)]. Spaces were included in 16, 17, 18 for best compliance. Standard one-letter codes for amino acids are used.

FIG. Figure 7 shows Northern blot analysis of RNAs from 3 liver-derived cell lines. 10 µg of poly (A) + RNA was used per Lane. Lane 1: Chang liver cell; lane 2: SK-HEP-1 hepatoma cell; lane 3: HepG2 hepatoma cell.

In this disclosure with claims, standard biochemical nomenclature is used wherein the nucleotide bases are designated as adenine (A); thymine (T); guanine (G); and cytosine (C). Corresponding nucleotides are, for example, deoxyguanosine 5'-triphosphate (dGTP). As is common for convenience in the structural analysis of a DNA nucleotide sequence, only one strand is shown, where A on one strand represents T on its complementary strand, and G represents C. Amino acids are shown either at three letters. or one-letter abbreviations as follows: 30 Abbreviated designation ___Amino Acid_ A Ala Alanine C Cys Cysteine D Asp Aspartic Acid E Glu Glutamic Acid 35 F Phe Phenylalanine 8 DK 174754 B1 G Gly Glycine H His Histidine I Ile Isoleucine K Lys Lysine 5 L Leu Lein N Asn Asparagine P Pro Proline Q Gin Glutamine 10 R Arg Arginine S Ser Serine T Thr Threonine V Val Valin W Trp Tryptophan 15 Y_Tyr_Tyrosin_

Commonly available restriction endonucleases described herein have the following sequences and (indicated by arrows) cleavage patterns:

EcoR1 GAATTC

GTTAAG

T

25 i

Ssp1 AATAAT

TTATAA

T

30 1

Cla1 ATCGAT

TAGCTA

T

35 i

Alu1 AGCT

TCGA

T

40 i

Stu1 AGGCCT

TCCGGA

T

DK 174754 B1 9

In order to elucidate specific preferred embodiments of the invention, the following examples of preparatory laboratory work are set forth.

EXAMPLE 1 5

materials

Human placental and fetal liver cDNA libraries were obtained from Clonetech. Protoblot immuno-screening equipment was purchased from Promega Biotech. Restriction enzymes were from New England Biolabs. Calf intestinal alkaline phosphatase, T4 DNA ligase, DNA polymerase I (Klenow), exonuclease III and S1 nuclease were from Boehringer Mannheim. dNTP was from P.L. Biochemicals. 5 '- [α-35S] -thio-dATP (600 Ci / mml) was from Amersham. Sequencing (Sequenase) equipment was from United States Biochemicals. Chang liver cells (ATCC CCL 15 13) and HepG2 hepatoma cells (ATCC HB 8065) were obtained from the Ameri can Type Culture Collection. SK-HEP-1 hepatoma cells were originally derived from a liver tract adenocarcinoma of G. Trempe of the Sloan-Kettering Institute for Cancer Research in 1971 and are now widespread and readily available.

20 125l factor Xa was prepared by radiolabelling using lodo gene. The specific activity was 2000 ppm / ng. More than 97% of the radioactivity could be precipitated with 10% trichloroacetic acid (TCA). The iodinated protein retained> 80% of their catalytic activity against Spectrozyme Xa (American Diagnostica Product).

25

An anti-TFI-Ig "Sepharose® 4B" column was prepared as follows: A peptide (designated TFI peptide) containing a sequence corresponding to the amino acid sequence 3-25 of the mature TF1 was synthesized using the solid phase of Biosystems -peptidsyntese system. The TFI peptide (5 mg) was conjugated to 10 mg of Keyhole elbow peel hemocyanin by glutaraldehyde. Two New Zealand breed white rabbits were immunized by intradermal injection of a homogenate containing 1 ml Freund's complete adjuvant and 1 ml conjugate (200 µg TFI peptide). One month later, the rabbits were re-injected with a homogenate containing 1 ml of Freund's incomplete adjuvant and 1 ml of conjugate (100 µg conjugate). Antiserum was collected weekly for three months and additional injections were performed once a month. To isolate spiked antibodies to the TFI peptide, antiserum was chromatographed on a TFI peptide MSepharose 4B "column. The column was washed with 10 volumes of PBS (0.4 M NaCl-0.1 M benzamidine). -1% "Triton © X-100"), and the same solution without "Triton X-100" The antibody was eluted with 0.1 M glycine / HCl, pH 2.2, and then immediately neutralized by the addition of 1 / 10 volumes of Tris-OH and dialyzed against saline solution The isolated antibody was coupled to cyanobromide activated "Sepharose 4B" following the manufacturer's (Pharmacia) instructions and used to isolate TFI from the cell culture substrate.

Chang liver cells were grown by the method previously described by Broze and Miletich, Proc. Natl. Acad. Sci. USA 84, 1886-1890 (1987). The conditioned substrate was chromatographed on the anti-TFI-Ig "Sepharose 4B" column.

The column was washed with 10 volumes of PBS-1% Triton X-100 and PBS. The bound TFI was eluted with 0.1 M glycine HCl, pH 2.2. Immunoaffinity-isolated TFI was further purified by preparative sodium dodecyl sulfate-polyacrylamide gel. Electrophoresis (Savant's apparatus) Amino acid analysis of the final product showed the same amino-terminal sequence as the TFI isolated from HepG2 cells as described in the co-pending Danish patent application PA 1988 04135. The isolated Chang liver cell TFI was then used to The immunized serum obtained had a titer of about 100 µg / ml in the double immunodifusion assay, which was used in the immuno-screening of Agt11 cDNA libraries.

Methods

Isolation of cDNA clones

Methods for scanning placental and fetal liver cDNA libraries with antibody, plaque purification and preparation of λ-phag lysate and DNA were as described by Wun and Kretzmer, FEBS Lett. 1, 11-16 (1987). The antiserum was pre-adsorbed with BNN97 Agt11 lysate and diluted 1/500 to screen the library.

DK 174754 B1 11

Screening for factor Xa binding activity

Recombinant proteins induced with isopropyl-β-thio-galactoside from immunopositive λ-phag isolates or from control AgtII were screened for factor Xa-5 binding activity, the λ-phag lysates (0.1 ml) were filtered through a nitrocellulose paper under using a dot-blot apparatus (Bio Rad). Subsequently, the nitrocellulose paper was immersed and stirred in phosphate buffered saline containing 5 mg / ml bovine serum albumin and 2.5 mg / ml bovine gamma globulin at room temperature for 1 hour. The solution was replaced with 125 I factor Xa (1.0 x 10 6 cmp / ml) dissolved in the same solution added with 0.1 mg / ml heparin and stirring was continued for an additional 1 hour. Then the nitrocellulose paper was washed with phosphate buffered saline containing 0.05% "Tween® 20". The wash buffer was replaced every five minutes, four times. Then, the nitrocellulose paper was air dried and prepared for autoradiography using "Kodak 15 XR5" film. The film was developed after a week of exposure.

Preparation of poly (A) * RNA and Northern blotting

Total RNAs were prepared from cultured C-hanging liver cell, HepG2 hepatoma cell and SK-HEP-1 hepatoma cell using the sodium perchlorate extraction method described by Lizardi and Engelberg, Anal. Biochem. 98, 116-122 (1979). Poly (A) + RNA preparations were isolated by batch adsorption on oligo (dT) cellulose (P-L Biochemical, type 77F) using the manufacturer's recommended procedure. For Northern blot analysis, 10 25 µg of each poly (A) + RNA was treated with glyoxal [Thomas, Methods Enzymol.

100, 255-266 (1983)] and subjected to agarose gel electrophoresis in a buffer containing 10 mM sodium phosphate, pH 7.0. Bethesda Research Laboratory's RNA ladder was used as a molecular weight marker. The RNAs were dipped onto a nitrocellulose paper which was then heated for 2 hours at 80 ° C.

The 30 insertion DNA of the AP9 clone was radiolabelled with 32P by notch translation and used as a probe [Maniatis et al., Molecular Cloning: A Laboratory Model, Cold Spring Laboratory, Cold Spring Harbor, N.Y. (1982)]. The immersion was hybridized with 5 x 10 6 cpm of the probe in 5 ml of a solution containing 50% formamide, 5X SSC, 50 mM sodium phosphate, pH 7.0, 35 250 pg / ml denatured salmon sperm DNA and 1X Denhardt's solution at 42

° C for 16 hours. The filter was washed in 0.1% sodium dodecyl sulfate (SDS), 2X SSC

At room temperature 3 times, each time for 5 minutes, and in 0.1% SDS, 0.2 X SSC at 50 ° C twice, each time for 5 minutes. The nitrocellulose paper was air dried and then autoradiographed for 3 days at -70 ° C using Kodak XAR-5 film and intensifier screen.

5

Other recombinant DNA methods

Preparation of cloned λgt11 DNA, subcloning into pUC19 plasmid and M13mp18 vector, generation of deletion by exonuclease I II degradation and DNA-10 sequencing by the dideoxy method [Sanger et al., Proc. Natl. Acad. Sci. United States 83, 6776-6780 (1977)] was carried out as described by Wun and Kretzmer, supra.

The FASTP program written by Lipman and Pearson, Science 227, 1435-1441 (1985) was used to identify homologous families of proteins from the National Biomedical Research Foundation Sequence Data Bank (released June 13, 1987) and to align the sequences with the homologous family.

RESULTS

20

Screening of cDNA libraries

A number of cell lines were screened for the presence of TFI in the conditioned substrate and it was found that several liver-derived cell lines, Chang-25 liver cell, HepG2 hepatoma and SK-HEP-1 hepatoma, secrete TFI in culture. Initially, an antiserum against TFI was used to screen a human fetal-liver AgtII cDNA library (106 plaque-forming units) and 15 immunologically positive clones were obtained. Then, the same method was used to screen a placental AgtII cDNA library. Out of 10® plaque-forming units, 10 immunologically positive clones were obtained. These clones were plaque purified and the lysates of the purified clones tested for TFI functional activity. The isopropylthiogalactoside-induced phage lysates were absorbed onto the nitrocellulose paper and screened for the 125 I factor Xa binding activity. FIG. 1 shows that some of these immunologically positive clones 35 showed the ability to bind 125 I factor Xg on the nitrocellulose paper, in total three out of 15 immunologically positive fetal liver clones, and 4 out of 10 immunologically * 13 DK 174754 B1 positive placental clones 125 I factor Xa binding activity. These immunologically and functionally positive clones were digested with EcoR1 and the size of the insertions was determined by gel electrophoresis. One clone from the placental library (AP9) had an insertion of approximately 1.4 kb, while all the other 5 clones contained insertions of approximately 1.0 kb. Partial DNA sequencing has shown that the 1.0 kb clones contain sequences identical to part of the longer 1.4 kb placental clone (λΡ9). λΡ9 was therefore selected for complete sequencing.

10 Nucleotide sequence and predicted protein sequence of the TFI cDNA isolate AP9 clone were subjected to restriction mapping, M13 subcloning and sequencing as indicated in FIG. 2. The entire sequence was determined on both strands by the exonuclease III deletion method [Henikofff, Ge-ne 28, 351-359 (1984)] and was found to consist of 1432 bases in length. The sequence is given in FIG. 3. It contains a 5 'non-coding region of 133 bases, an open reading frame of 912 nucleotides, and a 3' non-coding region of 387 nucleotides. The first ATG occurs at nucleotide 134 in the sequence TAGATGA, which was closely followed by a second ATG at nucleotide 146 in the sequence ACAATGA. These may be the start sequences, although different from the proposed consensus sequence for the start of eukaryotic ribosome, ACCATGG [Kozak, Cell 44, 283-292 (1986)]. 28 amino acids precede a sequence corresponding to the N-terminus of the mature protein. The length and composition of the hydrophobic portion of these 28 amino acids are typical of 25 signal sequences [Von Heijne, Eur. J. Biochem. 133, 17-21 (1983); J. Mol.

Biol. 184, 99-105 (1985)]. A signal peptidase may cleave at Ala2e-Asp29 to form a mature protein. The predicted sequence for mature TFI consists of 276 amino acids containing 18 cysteine residues and 7 methionine residues. The calculated mass of 31,950 daltons based on the deduced protein sequence for mature TFI is somewhat lower than the 37-40 kDa assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isolated protein, and the difference probably reflects the glycosylation contribution to the mobility of the natural protein. The deduced protein sequence corresponding to the mature protein contains three potential N-linked glycosylation sites with the sequence Asn-X-Thr / Ser (amino acid positions 145, 195 and 256). Amino acid sequence analysis of purified whole TFI and two isolated proteolytic fragments exactly matches the protein sequence deduced from the cDNA sequence (Figure 3, underlined), indicating that the isolated cDNA clone encodes TFI. The 3 'non-coding region is A + T rich (70% A + T). Neither the consensus polyadenylation signal, AATAAA [Proudfoot and Brownlee, Nature 252, 359-362 (1981)], nor the poly A tail was found in this clone, possibly due to artificially lost part of the 3 'terminal. part during the construction of the library.

Charge distribution, hydrophobicity / hydrophilicity and internal homology 10

The translated amino acid sequence of TFI contains 27 lysine residues, 17 arginine residues, 11 aspartic acid residues and 25 glutamic acid residues. The charge distribution along the protein is at most uneven as indicated in FIG. 4. The signal peptide region contains two positively charged lysine residues with 26 neutral residues. The 15 amino-terminal region of the mature protein contains a high negatively charged stretch. Six of the first 7 residues are either aspartic acid or glutamic acid, which is closely followed by two more negatively charged amino acids below, before a positively charged lysine residue appears. The center portion of the molecule is generally negatively charged. At the carboxy terminal there is a high positively charged portion. Amino acids 265-293 of TFI contain 14 positively charged amino acid residues, including a sequence of 6 consecutive arginine-lysine residues.

The predicted hydrophilicity / hydrophobicity in the profile of I of the TFI protein is given in FIG. 5. The signal peptide contains a high hydrophobic region as expected. Rather, residues of the molecule appear to be hydrophilic.

The translated amino acid sequence of TFI contains several special domains. In addition to the highly negatively charged N-terminal domain and the highly negatively charged C-terminal domain, the center portion consists of three homologous 30 domains having the typical sequences for Kunitz-type inhibitors (see below).

Homology to other proteins

35 By searching the National Biomedical Research Foundation sequence database, it was found that the N-terminal domain and the C-terminal domain of TFI

DK 174754 B1 15 does not show significant homology to other known proteins. However, the three internal homologous domains are separately homologous to sequences of other basal protease inhibitors, including basal bovine pancreatic protease inhibitor (aprotinin), basal venom protease inhibitors, and inter-α-trypsin-5 inhibitors (Fig. 6). Remarkably, disulfide bond structure is highly conserved in all three of these inhibitors. Based on these homologies, it is clear that TFI belongs to the basal protease inhibitor gene superfamily.

Northern blotting 10

Poly (A) + RNAs were purified from TFI-producing liver-derived cell lines, Chang liver cell, HepG2 hepatoma cells, and SK-HEP-1 hepatoma cells. The poly (A) + RNAs were resolved by denaturing agarose gel electrophoresis, duplicated on a nitrocellulose paper, and probed with 32P-labeled 15 TFI cDNA (AP9). As shown in FIG. 7, two major hybridization bands corresponding to mRNAs of 1.4 kb and 4.4 kb were observed in all three cell lines tested. Several other cell lines were examined which did not produce detectable amounts of TFI and in which no hybridization was found with the probe (data not shown).

20

Various other examples, after reading this description, will be apparent to those skilled in the art without departing from the scope of the invention. Such additional examples are also to be considered included in the invention as defined by the following claims.

Claims (16)

1. Human tissue factor inhibitor (TFI) with that of FIG. 3 of the drawings, protein amino acid sequence shown. The human tissue factor inhibitor of claim 1 which is unglycosylated. The human tissue factor inhibitor of claim 1 which is glycosylated. 4. Isolated and purified antibody having a binding factor for tissue factor inhibitor (TFI) with an amino acid sequence as shown in FIG. Third The antibody of claim 4 which is conjugated to an immunoaffinity matrix. The antibody of claim 5, wherein said immunoaffinity matrix is "Sepharose® 4B". 7. A method of using a TFI peptide column to purify an antibody to that of FIG. 3 of the drawings, protein amino acid sequence shown. A method for detecting a first polypeptide in a biological fluid, wherein said first polypeptide is selected from TFI and a polypeptide comprising one or more Kunitz domains of TFI, the method comprising the steps of: (a) bringing said fluid in contact with a second polypeptide having a binding specificity for said first polypeptide, and (b) testing for the presence of said second polypeptide to determine the level of said first polypeptide. The method of claim 8, wherein said second polypeptide is an antibody. 2 5 The method of claim 8, wherein said second polypeptide is factor Xa. DK 174754 B1 The method of claim 9 or 10, wherein said second polypeptide is radiolabelled. The method of claim 10, wherein said second polypeptide is 125 I-labeled factor Xa. A method for detecting a first polypeptide in a biological fluid, wherein said first polypeptide is selected from TFI and a polypeptide comprising one or more Kunitz domains of TFI, which method comprises the following steps: (a) bringing said fluid in contact with an antibody having a binding specificity for said first polypeptide, and a second polypeptide capable of binding said antibody, and (b) testing for the presence of said second polypeptide to determine the level of said antibody. said first polypeptide. The method of claim 13, wherein said second polypeptide is an antibody. The method of claim 13, wherein said second polypeptide is radiolabelled. The method of claim 8 or 13, wherein said biological fluid is conditioned cell culture medium.