JP2553425B2 - Thrombin-binding substance and method for producing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel thrombin-binding substance, a DNA fragment encoding the amino acid sequence of the thrombin-binding substance, a recombinant vector containing the DNA fragment, and a transformation carrying the recombinant vector. The present invention relates to somatic cells, an anticoagulant having a thrombin-binding substance as an active ingredient and having an inhibitory effect on platelet aggregation, and a method for producing the thrombin-binding substance.

[0002]

2. Description of the Related Art Various studies have been conducted on the role of thrombin as a proteolytic enzyme in the blood coagulation system, and the mechanism of the coagulation system has been clarified.

Thrombin activates protein C, which is said to act on the fibrinolytic system and anticoagulant system in vivo, and the factor that acts as a coenzyme on the mechanism is present in rabbit lung tissue extracts. Has been reported,
This factor is designated thrombomodulin [N.
L. Esmon et al., J. Biol. Chem., 257, (2), 859-864.
(1982)].

Aoki et al., A human with a molecular weight of about 71,000 (non-reducing state) having the same properties as isolated from human placenta.
Thrombomodulin has been reported [Thromb. Res., 3
7, 353-364 (1985)].

Further, I. Maruyama et al. Compared the activity of human thrombomodulin having a molecular weight of about 75,000 isolated from human placenta with the above-mentioned rabbit thrombomodulin, and reported that both activities were equal [J. Clin. Invest.,
75, 987-991 (1985)].

Also, H. Ishii et al. Have a substance having the same activity as thrombomodulin in human plasma and urine, and have a molecular weight of about 63,000 and about 5 in plasma.
Reported to be 4,000 [J. Clin. Invest., 7
6, 2178-2181 (1985)].

Furthermore, the present inventors found two kinds of thrombin-binding substances having a small molecular weight different from the above substances in human urine (molecular weights in non-reduced state of about 39,000 and about 31,000) and applied for a patent ( JP-A-63-146898).

Furthermore, the present inventors differed from the above substances in human urine and in tissue culture medium of human tissue-derived cells.
Separating the thrombin-binding substances (A) and (B) of the species and applying for a patent in advance (European Patent Publication No. 455,681),
Further, a method for producing a stable and large-scale production of the thrombin-binding substance obtained from this urine by using a gene recombination technique was developed and a patent application was filed (Japanese Patent Application No. 3-54446, the substance obtained here -Called UTM).

[0009]

On the other hand, rabbit lung thrombomodulin is known to enhance the activity of antithrombin III [KT Preissner et al., J. B.
iol. Chem., 265, 4915-4922 (1990)]. However,
This effect is not found in bovine thrombomodulin [HV Jakubowski et al., J. Biol. Chem., 261, 38.
76 (1986)], and human placental thrombomodulin inhibits the activity of antithrombin III [K. Hirahara et al.
al., Thromb. Res., 57, 117-126 (1990)].

It is known that there are two types of soluble thrombomodulin produced by genetic engineering techniques, one having antithrombin III enhancing activity and the other not having it [K. Nawa et al., Biochem. .Biophys.
Res. Commun., 171, 729-737 (1990)]. However,
It is known that these thrombomodulins suppress thrombin aggregation but not ADP aggregation on platelets that play an important role in the blood coagulation system [NL Esmon, J. Biol. Chem. ., 258, 12238-12242 (19
83)].

Therefore, by means of genetic engineering techniques,
It has been desired to add an action of enhancing the activity of antithrombin III and an action of inhibiting platelet aggregation to thrombomodulin and other thrombin-binding substances.

[0012]

Under such circumstances, the present inventors have conducted extensive studies to solve the above-mentioned problems, and as a result, a DNA encoding a human urine-derived thrombin-binding substance has been obtained.
D obtained by ligating a specific DNA fragment to the 3'end of the fragment
A human urine-derived thrombin-binding substance having antithrombin III activity-enhancing activity and platelet aggregation-suppressing activity is produced by transforming a host cell with a recombinant vector incorporating an NA fragment and expressing the DNA. The inventors have found out what can be done and have completed the present invention.

That is, the present invention relates to a thrombin-binding substance having the amino acid sequence shown in FIG. 1, a DNA fragment having a base sequence capable of encoding the amino acid sequence, a recombinant vector comprising the DNA fragment and a replicable vector, and the set. It is intended to provide a transformant cell carrying a recombinant vector, an anticoagulant having the thrombin-binding substance as an active ingredient and having a platelet aggregation inhibitory action, and a method for producing the thrombin-binding substance.

The thrombin-binding substance of the present invention is produced, for example, as follows. First, human placenta genomic DNA is cleaved with an appropriate restriction enzyme and used as a template DNA. Then, the nucleotide sequence of a known human thrombomodulin gene [Shirai, T. et al., J. Biochem., 103, 281-285 (198
8)] is used as a probe to screen the template DNA. The obtained DNA is cleaved with an appropriate restriction enzyme, and the generated DNA fragment is ligated with a cloning vector to transform a microorganism. By extracting plasmid DNA from the obtained transformant and treating it with a restriction enzyme, a DNA fragment containing 1404 bases encoding a thrombin-binding substance derived from human urine can be obtained. This DNA fragment contains the amino acid sequence X ₁ X ₂ Y ₁ SerGlySerGlyY
By inserting an oligonucleotide having a nucleotide sequence capable of encoding ₂ , a DNA fragment containing the DNA fragment of the present invention can be obtained. Representative examples of the nucleotide sequence of the DNA fragment of the present invention include those shown in SEQ ID NOS: 3 and 4. However, the present invention
The nucleotide sequence of the DNA fragment is the amino acid sequence constituting the peptide of the thrombin-binding substance of interest in the present invention [Fig.
(Preferably SEQ ID NO: 1 or 2)] is not limited to the above sequences.

To construct a recombinant vector containing the DNA fragment of the present invention, a replicable expression vector may be connected to the DNA fragment of the present invention. The expression vector may be any vector such as those derived from prokaryotes such as Escherichia coli, yeast, insect virus, vertebrate virus, etc., so long as they can be replicated.

However, in order to efficiently produce a thrombin-binding substance, the following (1) to (7) are sequentially provided in the downstream direction of transcription.
(1) A base sequence that acts as a promoter (2) A base sequence that is a ribosome binding site (3) A base sequence that is a start codon (4) A base sequence that encodes a signal peptide (5) Figure 1 (preferably It is preferable to construct an expression recombinant vector containing a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or 2), (6) a nucleotide sequence that is a stop codon, and (7) a nucleotide sequence that acts as a poly A addition signal.

The DNA used as the vector is preferably a plasmid. For example, a plasmid that can be amplified using Escherichia coli as a host and that can express an inserted gene by transforming mammalian cells is preferable. Such a plasmid
DNA has a nucleotide sequence necessary for the growth of the plasmid in E. coli cells, for example, a nucleotide sequence of the origin of replication of ColE1 type plasmid, and further functions as a promoter in mammalian cells, a selectable marker for transformed E. coli. And a gene serving as a selectable marker for transformed mammalian cells, and more preferably a replication origin base sequence such as SV40 ori, polyoma ori, HSV ori that functions in mammalian cells. Examples of promoters include cytomegalovirus, SV40, polyoma virus, bovine papilloma virus, adenovirus promoters and MMTV.
Examples include the retrovirus LTR and the promoter of the metallothionein gene. Examples of E. coli selectable markers include ampicillin resistance gene, kanamycin resistance gene, tetracycline resistance gene, chloramphenicol resistance gene and the like. Examples of selection markers for mammalian cells include neomycin resistance gene,
Examples thereof include a hygromycin B resistance gene, a thymidine kinase gene, a dihydrofolate reductase gene, a xanthine guanine phosphoribosyl transferase gene, and one or more of these genes are used.

To incorporate the DNA fragment of the present invention into the above vector, the DNA containing this is cleaved with an appropriate restriction enzyme, and if necessary, an appropriate linker is added, and then the vector is cleaved with an appropriate restriction enzyme. It is performed by binding. Examples of restriction enzymes used include EcoRI and Sph
I, PstI, HindIII, BamHI, XhoI, XbaI, BanIII, Sma
I, NcoI and the like. Also, exonuclease I
II, Ba131, Sl nuclease, exonuclease VII,
Nucleic acid modifying enzymes such as Mung bean nuclease and DNA polymerase I can also be used. Examples of the linker used include EcoRI linker, SmaI linker, NcoI linker, Bam
HI linker, XhoI linker, HindIII linker, PstI linker, SphI linker, XbaI linker and the like can be used.

The obtained recombinant vector for expression is introduced into a host cell by the competent cell method, protoplast method, calcium phosphate coprecipitation method, electroporation method, DEAE dextran method, lipofectin method, etc. A transformant cell having the ability to efficiently produce the recombinant vector and / or the thrombin-binding substance is obtained. Host cells for obtaining such transformant cells include bacteria, unicellular microorganisms such as yeast, or cultured insect cells,
Cultured vertebrate cells and the like are preferable. When E. coli is used as a host, various mutant strains of the E. coli K12 strain, such as HB10
1, C600K, JM101, JM103, JM105, JM109, MV1034, MV11
84, MC1061 / P3, etc. can be used. When cultured mammalian cells are used as hosts, COS cells, CHO cells, L cells, C127 cells, NIH3T3 cells, HeLa cells and the like can be used.

The thrombin-binding substance is produced by culturing the obtained transformant cells and extracting and separating from the cultured cells and / or the culture solution. In culturing the transformant cells, various natural media and synthetic media are used. The medium preferably contains carbon sources such as sugars, alcohols and organic acid salts; nitrogen sources such as protein mixtures, amino acids and ammonium salts; and inorganic salts. Furthermore,
It is desirable to add vitamins and antibiotics corresponding to the selectable marker gene. If the vector can control the expression, it is necessary to add an operation for inducing gene expression during the culture. After culturing, centrifugation is performed to separate the culture solution and the cultured cells. When the thrombin-binding substance accumulates in cultured cells, for example, freeze-thawing, sonication, French press, enzyme treatment, after disrupting the cells using a homogenizer, for example, EDTA, a surfactant,
It is necessary to solubilize the thrombin-binding substance with urea, guanidine hydrochloride or the like.

The purified thrombin-binding substance can be obtained by subjecting the obtained culture solution or cultured cell extract containing the thrombin-binding substance to various column chromatography. As column chromatography, ion exchange chromatography, affinity chromatography (for example, using the monoclonal antibody described in JP-A-64-45398), gel filtration chromatography and the like can be used alone or in combination.

Among the thrombin-binding substances of the present invention thus obtained, the substance having the amino acid sequence of SEQ ID NO: 1 or 2 has the following properties. (1) Amino acid sequence From the nucleotide sequence of the DNA fragment of the present invention, the amino acid sequence of the thrombin-binding substance of the present invention is judged to be as shown in SEQ ID NOS: 1 and 2. (2) Molecular weight 55,000 to 100,000 (SDS-polyacrylamide gel electrophoresis, non-reducing state) (3) Isoelectric point (isoelectric focusing using amphorite) pH 3 to 4 (4) Sugar analysis Multiple from molecular weight It is thought that the acid saccharide is added to Ser (474) from the amino acid sequence. (5) Action a. Has antithrombin activity. B. Enhances the activity of antithrombin III. C. It has an inhibitory effect on platelet aggregation.

The dosage form of the thrombin-binding substance of the present invention used as an active ingredient of an anticoagulant is, for example, an injection. The lyophilized powder is preferably dissolved in distilled water for injection, physiological saline or the like for administration before use. A suitable site for administration is intravenous.

Although the dose varies depending on the severity of the disease, the weight of the patient, etc., it is usually preferably 10 μg to 10 mg / kg / day. It should be noted that the thrombin-binding substance of the present invention is safe since no abnormalities were observed within the above dose range.

[0025]

The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1 Cloning of thrombin-binding substance gene: nucleotide sequence of human thrombomodulin gene [Shirai, T. et a
l., J. Biochem., 103, 281-285 (1988)] as a reference.
Primer # 1 shown in SEQ ID NO: 5 and primer # 2 shown in SEQ ID NO: 6 were synthesized using NA synthesizer (ABI model 381A). Next, human placenta genomic DNA (Clontech)
Was cleaved with BamHI, and gene amplification was attempted using the following reaction solution using this as a template DNA. Gene amplification is Quick Thermo Sys
tem (MODEL QTS-10M, made by Nippon Genetics Co., Ltd.)
It was carried out by repeating 30 cycles of 4 ° C. for 2 minutes, 50 ° C. for 3 minutes, and 72 ° C. for 4 minutes. After the reaction was completed, a part of the reaction solution was taken and confirmed by agarose gel electrophoresis that a DNA band of a desired size was amplified. Reaction solution Distilled water 71 μl Buffer ^*) 10 μl dNTP mixture (2.5 mM) 8 μl Primer # 1 (20 μM) 5 μl Primer # 2 (20 μM) 5 μl Template DNA (1 μg / μl) 1 μl AmpliTaq (5 units / μl) 0.5 μl *) : Buffer 0.1M Potassium chloride 0.1M Tris-HCl (pH8.3) 0.1% Gelatin 15mM Magnesium chloride The DNA was recovered from the reaction solution by ethanol precipitation, and XhoI and K
Digested with pnI and agarose gel electrophoresis for 1.57kb Xh
The oI-KpnI fragment was prepared. Separately, a cloning vector pUC118 (Vieira, J. and Messing, J., Method
S Enzymol., 153, 3-11 (1987)] was cut with HindII and Xho
After connecting the I linker, it was further digested with XhoI and KpnI, and a vector fragment was prepared by agarose gel electrophoresis.
After ligating the above vector fragment, the 1.57 kb XhoI-KpnI fragment, E. coli MV1304 [Vieira, J. and Messi
ng, J., Methods Enzymol., 153, 3-11 (1987)]. Plasmid DNA was extracted from the obtained transformant and cut with a restriction enzyme. As a result, the target human thrombomodulin gene-derived 1.57 kb XhoI-KpnI
Six strains were selected for the clones carrying the plasmid in which the fragment had been inserted. When the nucleotide sequences of the above-mentioned 6 clones of the inserted fragments were determined, it was found that each had a mutation at 1 to 3 positions. Therefore, 0 of clone 2 where no mutation was observed.
The 31 kb XhoI-SmaI fragment, the 0.65 kb SmaI-MluI fragment of clone 1 and the 0.62 kb MluI-KpnI fragment of clone 4 were religated with the above vector fragment, and a plasmid pUCTM / XHO containing an insert fragment having the correct human thrombomodulin gene sequence. -KP
Constructed N.

Example 2 Construction of a vector for expressing a thrombin-binding substance: A C-terminal Asp determined by amino acid analysis of a human urine-derived thrombin-binding substance is newly expressed by binding a glycosaminoglycan addition site. For this purpose, linkers $ 1 to $ 6 shown in SEQ ID NOS: 7 to 12 containing a stop codon were synthesized, and the 5 ′ end was phosphorylated. Cut pUCTM / XHO-KPN with XhoI and KpnI,
A 1.57 kb XhoI-KpnI fragment derived from the human thrombomodulin gene was prepared by agarose gel electrophoresis. Book 1.57
The kb fragment was ligated with the linkers $ 1, $ 2, $ 3 and $ 4 to an animal cell expression vector CDM8 (manufactured by Invitrogen) which was dephosphorylated after digestion with XhoI. In addition, this 1.57 kb fragment was
It was cut with I and ligated with vector CDM8 which was dephosphorylated after XhoI digestion with linkers $ 1, $ 2, $ 5 and $ 6.
E. coli MC1061 / P3 with these ligated DNAs
(Seed, B. and Aruffo, A., Proc. Natl. Acad. Sci.
USA., 84, 3365-3369 (1987)]. Extracting the plasmid DNA from the obtained transformant,
The restriction enzyme digestion was performed to confirm the insertion direction and the inserted fragment. Clone 8 showing correct insertion direction and restriction map
A 1.68 kb fragment containing the DNA fragment of the present invention was cut out from the strain by XhoI cleavage, and the nucleotide sequence was confirmed. All clones showed the nucleotide sequence shown in SEQ ID NO: 13 or 14,
It was confirmed that the expression vector of the present invention was constructed correctly. The expression vector of the present invention constructed as described above was transformed into pC
DM-GAG-UTM1 and pCDM-GAG-UTM2 (Fig. 2) and transformants carrying the vector of the present invention were respectively transformed into E. coli MC1061 / P3.
(PCDM-GAG-UTM1) and E. coli MC1061 / P3 (pCDM-GAG-U
It was named TM2).

Example 3 Expression of thrombin-binding substance by cultured animal cells: pCDM
-DEG-Dextran method (Seed, B. and Aruffo, A. Proc. Natl. Acad. Sci. U) using -GAG-UTM1 and pCDM-GAG-UTM2
SA. 84, 3365-3369 (1987)] was used to transfect COS7 cells. 5 × 10 ⁵ C in a 60 mm culture dish
OS7 cells were seeded, and the next day, the culture solution was aspirated to 10% Nu-seru.
The medium was replaced with 2 ml of Dulbecco's modified minimal essential medium (DMEM) containing m (Collaborative Research Inc.). DEAE-Dextran (average molecular weight 5 × 10 ⁵ ; dissolved in PBS at a concentration of 10 mg / ml;
(Pharmacia) pCDM-GAG-UTM1 or pCD in 100 μl of solution
Add 10 μg of M-GAG-UTM2 (1 μg / μl) and add 10 μl of 20
It was added to the cell culture medium together with mM chloroquine. After culturing at 37 ℃ for 4 hours, suck the culture solution and 10 ml DMSO (dissolved in PBS) 2 ml
Was added and the mixture was allowed to stand at room temperature for 2 minutes. After removing the DMSO solution by suction, 3 ml of DMEM containing 10% FCS was added and the mixture was cultured at 37 ° C. for 24 hours. The culture solution was changed to serum-free DMEM, and the culture was further continued for 48 hours, and then the culture supernatant was collected. The obtained culture solution was passed through a column of 1 ml of Sepharose 4B (2 mg IgG / ml resin) to which monoclonal antibody A-73 (Japanese Patent Laid-Open No. 64-45398) was bound. The column was (1) 2 ml of 0.02 M Tris-HCl buffer (pH 7.4) containing 0.1 M sodium chloride, (2) 1 M sodium chloride and 0.1 ml.
20% 0.02 M Tris-HCl buffer (pH 7.4) with 05% Tween20,
(3) 0.02 M Tris-HCl buffer (pH 7.
4) Sequentially wash with 5 ml, 2M sodium thiocyanate and 5m
Elution was performed with 5 ml of 0.02 M Tris-HCl buffer (pH 7.4) containing 1 M sodium chloride containing M EDTA. The eluate was dialyzed against 50 mM acetate buffer (pH 4.5) containing 0.1 M sodium chloride to obtain Mono Q
Attached to the column. After washing the column with the same buffer,
Purified thrombin-binding substances (r-GAG-UTM1 and r-GAG-UTM2) were obtained by elution by a linear concentration gradient method with a 50 mM acetate buffer containing 0.1 to 2 M sodium chloride.

Example 4 Expression of thrombin-binding substance by cultured animal cells: pCDM
-Calcium phosphate precipitation method using GAG-UTM1 (Gorman,
C., "DNA cloning", IRL Press, England, 1985, vol.
2, P.143-190) was used to transfect CHO / K1 cells (RCB285). 5 × 10 ⁵ CHO ・ K1 on a 10 cm dish
The cells were seeded, and the next day, the culture medium (10% FCS-added Ham F12 medium; hereinafter referred to as medium) was exchanged, and 4 hours after that, a coprecipitate of DNA and calcium phosphate was added. The coprecipitate was prepared as follows. 20 μg pCDM-GAG-UTM1 and 10
Dissolve 0 ng of neomycin resistance gene in 450 μl of 1 mM Tris-HCl (pH8.0) /0.1 mM EDTA, and add 2.5M calcium chloride 5
After mixing 0 μl, 500 μl of 50 mM HEPES (pH 7.12) / 280 mM sodium chloride / 1.5 mM sodium hydrogen phosphate solution was added dropwise. After leaving it at room temperature for 30 minutes, add it to the above cell culture medium,
It was cultured for 24 hours. Then, the medium was replaced with a fresh medium, and after further culturing for 24 hours, the medium was replaced with a selective medium containing 400 μg / ml G418.
After 2 weeks, the resulting colonies were transferred to a 24-well culture plate and further cultured until they became confluent. The culture supernatant was collected, the amount of secreted thrombin-binding substance (r-GAG-UTM1) was quantified, and a high-producing clone was selected. The selected clone was further cloned by the limiting dilution method. The resulting transformed cell line was named CHO-GUTM 1-8,
It was deposited in the Institute of Microbial Technology, Ministry of International Trade and Industry, as a micro-machine research article No. 3260. Transformed cell line CHO-GUTM 1
-8 using 1% FCS-added UC202 (Nissui Pharmaceutical) medium
50 ml after culturing in a 5 cm ² flask until confluent
The medium was replaced with UC202 medium containing no FCS. After 1 week, the culture broth was collected, and then the same amount of serum-free medium was newly added, followed by culturing for another 1 week to collect the culture broth. The thrombin-binding substance was secreted in the culture medium in an amount of 3 to 4 μg / ml. Thereafter, the same treatment as in the latter half of Example 3 was carried out to obtain a purified thrombin-binding substance (r-GAG-UTM1).

Example 5 Physical properties of thrombin-binding substance: Purified thrombin-binding substance was subjected to SDS-P according to the method of Laemmli (Nature, 227 , 680-685).
I did AGE. The gel is prepared according to the method of Matudaira [J. Biol. Che.
m., 262 (21), 10035-10038]]. PVDF membrane then contains 0.1% bovine serum albumin
0.05M Tris-HCl buffer (TBS) with 0.1M sodium chloride
The reaction was carried out in the solution at room temperature for 2 hours. Discard the solution, 0.05
After thorough washing with TBS containing 20% Tween, contains monoclonal antibody A-60 labeled with horseradish peroxidase.
The reaction was carried out in TBS containing 0.05% Tween 20 at room temperature for 1 hour. After discarding the solution and thoroughly washing with 0.05% Tween20-added TBS, 5-amino-9-ethylcarbazole 5 mg and 30% hydrogen peroxide solution 25
When the sample was placed in 50 ml of 50 mM acetate buffer (pH 5.0) containing μl for color development, a broad band peculiar to the glycosaminoglycan adduct was confirmed.

Example 6 r-UTM, thrombin-binding substance of the present invention r-GAG-UTM1 and rG
AG-UTM2 (0.1 μg / ml) was reacted with 5 μl of chondroitinase ABC (10 mU; manufactured by Seikagaku Corporation) at 37 ° C. for 40 minutes, and immunoblotting was performed in the same manner as in Example 5. Inventive thrombin-binding substance was confirmed to be covalently bound to chondroitin sulfate type glycosaminoglycan.

Example 7 Anticoagulant activity: r-UTM, r-GAG-UTM1 or r-GAG-UTM2 (2.5 μg
/ ml), human fibrinogen (2.5 mg / ml), human antithrombin III (0 or 250 μg / ml) and calcium chloride
Bovine thrombin (0.5 U / ml) was added to the (5 mM) solution, and the coagulation time was measured. The results are shown in Table 1.

[0033]

[Table 1]

As is clear from Table 1, the thrombin-binding substance of the present invention binds with thrombin to prolong blood coagulation time, and its anticoagulant action is antithrombin III.
It is significantly enhanced by coexistence with.

Example 8 Anticoagulant activity: r-UTM (9 to 90 nM), thrombin-binding substance of the present invention r-GAG-UTM1 or r-GAG-UTM2 (9 to 90 nM) and bovine fibrinogen (1 mg / ml) Of 0.15M sodium chloride added 20
Bovine thrombin (18 mM) was added to the mM tris-hydrochloric acid buffer solution (pH 7.4) solution, and the coagulation time was measured. The 50% inhibition rate (IC ₅₀ ) was determined from a calibration curve prepared using bovine thrombin at various concentrations in advance. Table 2 shows the results.

[0036]

[Table 2]

Example 9 Anticoagulant activity: substance of the present invention (17 nM) or r-UTM (17 nM), bovine fibrinogen (1 mg / ml) and human antithrombin III (125 μg / ml) in 0.15 M sodium chloride 20 mM Tris
-Bovine thrombin (18 nM) was added to a hydrochloric acid buffer pH 7.4 solution, and coagulation time was measured. The results are shown in Table 3.

[0038]

[Table 3]

Example 10 Inhibitory action on platelet aggregation: 200 μl of PRP (platelet-rich plasma) prepared from blood collected from rabbit / ear artery, ⁸ μl of the substance of the present invention (10 ⁻⁶ to 10 ⁻⁸ M) in ADP (Adenosine diphosphate) 2 μM was added and platelet aggregation was measured. R-GAG-U obtained from a calibration curve prepared beforehand using various concentrations of ADP
The 50% inhibitory concentration of TM1 and r-GAG-UTM2 against ADP aggregation was
They were 2 × 10 ⁻⁷ M and 2.1 × 10 ⁻⁷ M, respectively. Note that rU
The TM had no aggregation inhibitory effect at the concentration used (10 ⁻⁶ to 10 ⁻⁸ M).

Example 11 A rat (Wister strain, male) was anesthetized and a catheter was inserted into the right femoral vein, and the test drug (GAG-
UTM1 or r-UTM) was rapidly administered to 1 mg / ml / kg. Before administration and at 1, 3, 6, 10, 20, 30, 60, 120 minutes after administration
Blood was collected in an amount of 0.1 ml, mixed with heparin to obtain plasma, and used for blood concentration measurement. The blood concentration was measured by sandwich ELISA using an antithrombin-binding substance monoclonal antibody. As a result, both drugs have one-compertment mo
It was possible to analyze with del. The results are shown in Table 4.

[0041]

[Table 4]

[0042]

INDUSTRIAL APPLICABILITY As described above, the thrombin-binding substance of the present invention has an antithrombin III activity-enhancing action and a platelet aggregation-inhibiting action, and also has an antithrombin action by itself. It is useful as an active ingredient.
Further, the thrombin-binding substance of the present invention can be produced in large quantities and at low cost.

[0043]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 476 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu His Asp 5 10 15 Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gln 20 25 30 Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45 Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys Gly Asp 65 70 75 80 Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr Gly Asp 85 90 95 Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110 Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125 Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160 Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr Tyr Gly 165 170 175 Thr Pro Phe Ala Ala Arg Gly Ala Asp Ph e Gln Ala Leu Pro Val Gly 180 185 190 Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys Thr Ala 195 200 205 Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220 Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240 Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln 245 250 255 Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp 260 265 270 Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser Tyr 275 280 285 Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His Arg 290 295 300 Cys Glu Asp Val Asp Asp Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro Gln 305 310 315 320 Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro Asn 325 330 335 Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe 340 345 350 Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr 355 360 365 Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu Pro His 370 375 380 Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cy s Pro Ala Asp Cys Asp 385 390 395 400 Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile Leu Asp 405 410 415 Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly Gly Phe 420 425 430 Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys Ile Cys 435 440 445 Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr Asp Cys Asp Ser 450 455 460 Gly Lys Val Asp Glu Asp Tyr Ser Gly Ser Gly Glu 465 470 475

SEQ ID NO: 2 Sequence length: 476 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu His Asp 5 10 15 Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gln 20 25 30 Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45 Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys Gly Asp 65 70 75 80 Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr Gly Asp 85 90 95 Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110 Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125 Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160 Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr Tyr Gly 165 170 175 Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro Val Gly 180 185 190 Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys Thr Ala 195 200 205 Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220 Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240 Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln 245 250 255 Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp 260 265 270 Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser Tyr 275 280 285 Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His Arg 290 295 300 Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro Gln 305 310 315 320 Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro Asn 325 330 335 Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe 340 345 350 Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr 355 360 365 Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu Pro His 370 375 380 Arg Cys Gln Met Phe CysAsn Gln Thr Ala Cys Pro Ala Asp Cys Asp 385 390 395 400 Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile Leu Asp 405 410 415 Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly Gly Phe 420 425 430 Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys Ile Cys 435 440 445 Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr Asp Cys Asp Ser 450 455 460 Gly Lys Val Asp Asp Glu Ala Ser Gly Ser Gly Asp 465 470 475

SEQ ID NO: 3 Sequence length: 1428 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA to mRNA Sequence GCACCCGCAG AGCCGCAGCC GGGTGGCAGC CAGTGCGTCG AGCACGACTG CTTCGCGCGCTC 60 TACCCGGGCC CCGCGACCTT CCTCAATGCC AGTCAGAG GCGGGGCCAC 120 CTAATGACAG TGCGCTCCTC GGTGGCTGCC GATGTCATTT CCTTGCTACT GAACGGCGAC 180 GGCGGCGTTG GCCGCCGGCG CCTCTGGATC GGCCTGCAGC TGCCACCCGG CTGCGGCGAC 240 CCCAAGCGCC TCGGGCCCCT GCGCGGCTTC CAGTGGGTTA CGGGAGACAA CAACACCAGC 300 TATAGCAGGT GGGCACGGCT CGACCTCAAT GGGGCTCCCC TCTGCGGCCC GTTGTGCGTC 360 GCTGTCTCCG CTGCTGAGGC CACTGTGCCC AGCGAGCCGA TCTGGGAGGA GCAGCAGTGC 420 GAAGTGAAGG CCGATGGCTT CCTCTGCGAG TTCCACTTCC CAGCCACCTG CAGGCCACTG 480 GCTGTGGAGC CCGGCGCCGC GGCTGCCGCC GTCTCGATCA CCTACGGCAC CCCGTTCGCG 540 GCCCGCGGAG CGGACTTCCA GGCGCTGCCG GTGGGCAGCT CCGCCGCGGT GGCTCCCCTC 600 GGCTTACAGC TAATGTGCAC CGCGCCGCCC GGAGCGGTCC AGGGGCACTG GGCCAGGGAG 660 GCGCCGGGCG CTTGGGACTG CAGCGTGGAG AACGGCGGCT GCGAGCACGT CG 720 ATCCCTGGGG CTCCCCGCTG CCAGTGCCCA GCCGGCGCCG CCCTGCAGGC AGACGGGCGC 780 TCCTGCACCG CATCCGCGAC GCAGTCCTGC AACGACCTCT GCGAGCACTT CTGCGTTCCC 840 AACCCCGACC AGCCGGGCTC CTACTCGTGC ATGTGCGAGA CCGGCTACCG GCTGGCGGCC 900 GACCAACACC GGTGCGAGGA CGTGGATGAC TGCATACTGG AGCCCAGTCC GTGTCCGCAG 960 CGCTGTGTCA ACACACAGGG TGGCTTCGAG TGCCACTGCT ACCCTAACTA CGACCTGGTG 1020 GACGGCGAGT GTGTGGAGCC CGTGGACCCG TGCTTCAGAG CCAACTGCGA GTACCAGTGC 1080 CAGCCCCTGA ACCAAACTAG CTACCTCTGC GTCTGCGCCG AGGGCTTCGC GCCCATTCCC 1140 CACGAGCCGC ACAGGTGCCA GATGTTTTGC AACCAGACTG CCTGTCCAGC CGACTGCGAC 1200 CCCAACACCC AGGCTAGCTG TGAGTGCCCT GAAGGCTACA TCCTGGACGA CGGTTTCATC 1260 TGCACGGACA TCGACGAGTG CGAAAACGGC GGCTTCTGCT CCGGGGTGTG CCACAACCTC 1320 CCCGGTACCT TCGAGTGCAT CTGCGGGCCC GACTCGGCCC TTGTCCGCCA CATTGGCACC 1380 GACTGTGACT CCGGCAAGGT GGACGAGGAC TATAGCGGCT CTGGCGAG 1428

SEQ ID NO: 4 Sequence length: 1428 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence GCACCCGCAG AGCCGCAGCC GGGTGGCAGC CAGTGCGTCG AGCACGACTG CTTCGCGCTC 60 TACCCGGGCC CCGCGACCTT CCTCAATGCC AGTCAGATCT GCGGGGCCAC 120 CTAATGACAG TGCGCTCCTC GGTGGCTGCC GATGTCATTT CCTTGCTACT GAACGGCGAC 180 GGCGGCGTTG GCCGCCGGCG CCTCTGGATC GGCCTGCAGC TGCCACCCGG CTGCGGCGAC 240 CCCAAGCGCC TCGGGCCCCT GCGCGGCTTC CAGTGGGTTA CGGGAGACAA CAACACCAGC 300 TATAGCAGGT GGGCACGGCT CGACCTCAAT GGGGCTCCCC TCTGCGGCCC GTTGTGCGTC 360 GCTGTCTCCG CTGCTGAGGC CACTGTGCCC AGCGAGCCGA TCTGGGAGGA GCAGCAGTGC 420 GAAGTGAAGG CCGATGGCTT CCTCTGCGAG TTCCACTTCC CAGCCACCTG CAGGCCACTG 480 GCTGTGGAGC CCGGCGCCGC GGCTGCCGCC GTCTCGATCA CCTACGGCAC CCCGTTCGCG 540 GCCCGCGGAG CGGACTTCCA GGCGCTGCCG GTGGGCAGCT CCGCCGCGGT GGCTCCCCTC 600 GGCTTACAGC TAATGTGCAC CGCGCCGCCC GGAGCGGTCC AGGGGCACTG GGCCAGGGAG 660 GCGCCGGGCG CTTGGGACTG CAGCGTGGAG AACGGCGGCT GCGAGCACGT TGCG 720 ATCCCTGGGG CTCCCCGCTG CCAGTGCCCA GCCGGCGCCG CCCTGCAGGC AGACGGGCGC 780 TCCTGCACCG CATCCGCGAC GCAGTCCTGC AACGACCTCT GCGAGCACTT CTGCGTTCCC 840 AACCCCGACC AGCCGGGCTC CTACTCGTGC ATGTGCGAGA CCGGCTACCG GCTGGCGGCC 900 GACCAACACC GGTGCGAGGA CGTGGATGAC TGCATACTGG AGCCCAGTCC GTGTCCGCAG 960 CGCTGTGTCA ACACACAGGG TGGCTTCGAG TGCCACTGCT ACCCTAACTA CGACCTGGTG 1020 GACGGCGAGT GTGTGGAGCC CGTGGACCCG TGCTTCAGAG CCAACTGCGA GTACCAGTGC 1080 CAGCCCCTGA ACCAAACTAG CTACCTCTGC GTCTGCGCCG AGGGCTTCGC GCCCATTCCC 1140 CACGAGCCGC ACAGGTGCCA GATGTTTTGC AACCAGACTG CCTGTCCAGC CGACTGCGAC 1200 CCCAACACCC AGGCTAGCTG TGAGTGCCCT GAAGGCTACA TCCTGGACGA CGGTTTCATC 1260 TGCACGGACA TCGACGAGTG CGAAAACGGC GGCTTCTGCT CCGGGGTGTG CCACAACCTC 1320 CCCGGTACCT TCGAGTGCAT CTGCGGGCCC GACTCGGCCC TTGTCCGCCA CATTGGCACC 1380 GACTGTGACT CCGGCAAGGT CGACGACGAG GCCAGCGGCT CTGGCGAC 1428

SEQ ID NO: 5 Sequence Length: 21 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence AGGGCCGGGC ACTTATAAAC T 21

SEQ ID NO: 6 Sequence Length: 21 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence CCCAGTGGTC CAGTGACGTC A 21

SEQ ID NO: 7 Sequence length: 39 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CTTCGAGTGC ATCTGCGGGC CCGACTCGGC CCTTGTCCG 39

SEQ ID NO: 8 Sequence length: 49 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ATGTGGCGGA CAAGGGCCGA GTCGGGCCCG CAGATGCACT CGAAGGTAC 49

SEQ ID NO: 9 Sequence length: 65 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCACATTGGC ACCGACTGTG ACTCCGGCAA GGTGGACGAG GACTATAGCG GCTCTGGCGA 60 GTGAC 65

SEQ ID NO: 10 Sequence length: 63 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TCGAGTCACT CGCCAGAGCC GCTATAGTCC TCGTCCACCT TGCCGGAGTC ACAGTCGGTG 60 CCA 63

SEQ ID NO: 11 Sequence length: 65 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCACATTGGC ACCGACTGTG ACTCCGGCAA GGTCGACGAC GAGGCCAGCG GCTCTGGCGA 60 CTGAC 65

SEQ ID NO: 12 Sequence length: 63 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TCGAGTCAGT CGCCAGAGCC GCTGGCCTCG TCGTCGACCT TGCCGGAGTC ACAGTCGGTG 60 CCA 63

SEQ ID NO: 13 Sequence length: 1680 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence features Characteristic symbols: sig peptide Location: 190..243 Characterized method: S Characteristic symbol: mat peptide Location: 244..1671 Characterized method: S sequence CTCGAGCCCT GGCCGATCCG CATGTCAGAG GCTGCCTCGC AGGGGCTGCG CGCAGCGGCA GCTGGCTGCCCCCCGTCCTCCCCCCCGTCCTGCCCCGTCCTGCCCCGGCTGTCCTGCGGCCTGTGGCCTGCGCT CGCTTTCCCC GGCGCCTGCA CGCGGCGCGC 180 CTGGGTAAC ATG CTT GGG GTC CTG GTC CTT GGC GCG CTG GCC CTG GCC GGC 231 Met Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu Ala Gly -18 -15 -10 -5 CTG GGG CCC GCA TCC CCC GCA CCG CAG CCG GGT GGC AGC CAG TGC 279 Leu Gly Phe Pro Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys 1 5 10 GTC GAG CAC GAC TGC TTC GCG CTC TAC CCG GGC CCC GCG ACC TTC CTC 327 Val Glu His Asp Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu 15 20 25 AAT GCC AGT CAG ATC TGC GAC GGA CTG CGG GGC CAC CTA ATG ACA GTG 375 Asn Ala Ser Gln Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val 30 35 40 CGC TCC TCG GTG GCT GCC GAT GTC ATT TCC TTG CTA CTG AAC GGC GAC 423 Arg Ser Ser Val Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp 45 50 55 60 GGC GGC GTT GGC CGC CGG CGC CTC TGG ATC GGC CTG CAG CTG CCA CCC 471 Gly Gly Val Gly Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro 65 70 75 GGC TGC GGC GAC CCC AAG CGC CTC GGG CCC CTG CGC GGC TTC CAG TGG 519 Gly Cys Gly Asp Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp 80 85 90 GTT ACG GGA GAC AAC AAC ACC AGC TAT AGC AGG TGG GCA CGG CTC GAC 567 Val Thr Gly Asp Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp 95 100 105 CTC AAT GGG GCT CCC CTC TGC GGC CCG TTG TGC GTC GCT GTC TCC GCT 615 Leu Asn Gly Ala Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala 110 115 120 GCT GAG GCC ACT GTG CCC AGC GAG CCG ATC TGG GAG GAG CAG CAG TGC 663 Ala Glu Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys 125 130 13 5 140 GAA GTG AAG GCC GAT GGC TTC CTC TGC GAG TTC CAC TTC CCA GCC ACC 711 Glu Val Lys Ala Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr 145 150 155 TGC AGG CCA CTG GCT GTG GAG CCC GGC GCC GCG GCT GCC GCC GTC TCG 759 Cys Arg Pro Leu Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser 160 165 170 ATC ACC TAC GGC ACC CCG TTC GCG GCC CGC GGA GCG GAC TTC CAG GCG 807 Ile Thr Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala 175 180 185 CTG CCG GTG GGC AGC TCC GCC GCG GTG GCT CCC CTC GGC TTA CAG CTA 855 Leu Pro Val Gly Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu 190 195 200 ATG TGC ACC GCG CCG CCC GGA GCG GTC CAG GGG CAC TGG GCC AGG GAG 903 Met Cys Thr Ala Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu 205 210 215 220 GCG CCG GGC GCT TGG GAC TGC AGC GTG GAG AAC GGC GGC TGC GAG CAC 951 Ala Pro Gly Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His 225 230 235 GCG TGC AAT GCG ATC CCT GGG GCT CCC CGC TGC CAG TGC CCA GCC GGC 999 Ala Cys Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Al a Gly 240 245 250 GCC GCC CTG CAG GCA GAC GGG CGC TCC TGC ACC GCA TCC GCG ACG CAG 1047 Ala Ala Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln 255 260 265 TCC TGC AAC GAC CTC TGC GAG CAC TTC TGC GTT CCC AAC CCC GAC CAG 1095 Ser Cys Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln 270 275 280 CCG GGC TCC TAC TCG TGC ATG TGC GAG ACC GGC TAC CGG CTG GCG GCC 1143 Pro Gly Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala 285 290 295 300 GAC CAA CAC CGG TGC GAG GAC GTG GAT GAC TGC ATA CTG GAG CCC AGT 1191 Asp Gln His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser 305 310 315 CCG TGT CCG CAG CGC TGT GTC AAC ACA CAG GGT GGC TTC GAG TGC CAC 1239 Pro Cys Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His 320 325 330 TGC TAC CCT AAC TAC GAC CTG GTG GAC GGC GAG TGT GTG GAG CCC GTG 1287 Cys Tyr Pro Asn Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val 335 340 345 GAC CCG TGC TTC AGA GCC AAC TGC GAG TAC CAG TGC CAG CCC CTG AAC 1335 Asp Pro Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn 350 355 360 CAA ACT AGC TAC CTC TGC GTC TGC GCC GAG GGC TTC GCG CCC ATT CCC 1383 Gln Thr Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro 365 370 375 380 CAC GAG CCG CAC AGG TGC CAG ATG TTT TGC AAC CAG ACT GCC TGT CCA 1431 His Glu Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro 385 390 395 GCC GAC TGC GAC CCC AAC ACC CAG GCT AGC TGT GAG TGC CCT GAA GGC 1479 Ala Asp Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly 400 405 410 TAC ATC CTG GAC GAC GGT TTC ATC TGC ACG GAC ATC GAC GAG TGC GAA 1527 Tyr Ile Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu 415 420 425 AAC GGC GGC TTC TGC TCC GGG GTG TGC CAC AAC CTC CCC GGT ACC TTC 1575 Asn Gly Gly Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe 430 435 440 GAG TGC ATC TGC GGG CCC GAC TCG GCC CTT GTC CGC CAC ATT GGC ACC 1623 Glu Cys Ile Cys Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr 445 450 455 460 GAC TGT GAC TCC GGC AAG GTG GAC GAG GAC TAT AGC GGC TCT GGC GAG 1671 Asp Cys Asp Ser Gly Lys Val Asp Glu Asp Tyr Ser Gly Ser Gly Glu 465 470 475 TGACTCGAG 1680

SEQ ID NO: 14 Sequence length: 1680 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence features Characteristic symbols: sig peptide Location: 190..243 Characterized method: S Characteristic symbol: mat peptide Location: 244..1671 Characterized method: S sequence CTCGAGCCCT GGCCGATCCG CATGTCAGAG GCTGCCTCGC AGGGGCTGCG CGCAGCGGCA GCTGGCTGCCCCCCGTCCTCCCCCCCGTCCTGCCCCGTCCTGCCCCGGCTGTCCTGCGGCCTGTGGCCTGCGCT CGCTTTCCCC GGCGCCTGCA CGCGGCGCGC 180 CTGGGTAAC ATG CTT GGG GTC CTG GTC CTT GGC GCG CTG GCC CTG GCC GGC 231 Met Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu Ala Gly -18 -15 -10 -5 CTG GGG CCC GCA TCC CCC GCA CCG CAG CCG GGT GGC AGC CAG TGC 279 Leu Gly Phe Pro Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys 1 5 10 GTC GAG CAC GAC TGC TTC GCG CTC TAC CCG GGC CCC GCG ACC TTC CTC 327 Val Glu His Asp Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu 15 20 25 AAT GCC AGT CAG ATC TGC GAC GGA CTG CGG GGC CAC CTA ATG ACA GTG 375 Asn Ala Ser Gln Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val 30 35 40 CGC TCC TCG GTG GCT GCC GAT GTC ATT TCC TTG CTA CTG AAC GGC GAC 423 Arg Ser Ser Val Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp 45 50 55 60 GGC GGC GTT GGC CGC CGG CGC CTC TGG ATC GGC CTG CAG CTG CCA CCC 471 Gly Gly Val Gly Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro 65 70 75 GGC TGC GGC GAC CCC AAG CGC CTC GGG CCC CTG CGC GGC TTC CAG TGG 519 Gly Cys Gly Asp Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp 80 85 90 GTT ACG GGA GAC AAC AAC ACC AGC TAT AGC AGG TGG GCA CGG CTC GAC 567 Val Thr Gly Asp Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp 95 100 105 CTC AAT GGG GCT CCC CTC TGC GGC CCG TTG TGC GTC GCT GTC TCC GCT 615 Leu Asn Gly Ala Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala 110 115 120 GCT GAG GCC ACT GTG CCC AGC GAG CCG ATC TGG GAG GAG CAG CAG TGC 663 Ala Glu Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys 125 130 13 5 140 GAA GTG AAG GCC GAT GGC TTC CTC TGC GAG TTC CAC TTC CCA GCC ACC 711 Glu Val Lys Ala Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr 145 150 155 TGC AGG CCA CTG GCT GTG GAG CCC GGC GCC GCG GCT GCC GCC GTC TCG 759 Cys Arg Pro Leu Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser 160 165 170 ATC ACC TAC GGC ACC CCG TTC GCG GCC CGC GGA GCG GAC TTC CAG GCG 807 Ile Thr Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala 175 180 185 CTG CCG GTG GGC AGC TCC GCC GCG GTG GCT CCC CTC GGC TTA CAG CTA 855 Leu Pro Val Gly Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu 190 195 200 ATG TGC ACC GCG CCG CCC GGA GCG GTC CAG GGG CAC TGG GCC AGG GAG 903 Met Cys Thr Ala Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu 205 210 215 220 GCG CCG GGC GCT TGG GAC TGC AGC GTG GAG AAC GGC GGC TGC GAG CAC 951 Ala Pro Gly Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His 225 230 235 GCG TGC AAT GCG ATC CCT GGG GCT CCC CGC TGC CAG TGC CCA GCC GGC 999 Ala Cys Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Al a Gly 240 245 250 GCC GCC CTG CAG GCA GAC GGG CGC TCC TGC ACC GCA TCC GCG ACG CAG 1047 Ala Ala Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln 255 260 265 TCC TGC AAC GAC CTC TGC GAG CAC TTC TGC GTT CCC AAC CCC GAC CAG 1095 Ser Cys Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln 270 275 280 CCG GGC TCC TAC TCG TGC ATG TGC GAG ACC GGC TAC CGG CTG GCG GCC 1143 Pro Gly Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala 285 290 295 300 GAC CAA CAC CGG TGC GAG GAC GTG GAT GAC TGC ATA CTG GAG CCC AGT 1191 Asp Gln His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser 305 310 315 CCG TGT CCG CAG CGC TGT GTC AAC ACA CAG GGT GGC TTC GAG TGC CAC 1239 Pro Cys Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His 320 325 330 TGC TAC CCT AAC TAC GAC CTG GTG GAC GGC GAG TGT GTG GAG CCC GTG 1287 Cys Tyr Pro Asn Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val 335 340 345 GAC CCG TGC TTC AGA GCC AAC TGC GAG TAC CAG TGC CAG CCC CTG AAC 1335 Asp Pro Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn 350 355 360 CAA ACT AGC TAC CTC TGC GTC TGC GCC GAG GGC TTC GCG CCC ATT CCC 1383 Gln Thr Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro 365 370 375 380 CAC GAG CCG CAC AGG TGC CAG ATG TTT TGC AAC CAG ACT GCC TGT CCA 1431 His Glu Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro 385 390 395 GCC GAC TGC GAC CCC AAC ACC CAG GCT AGC TGT GAG TGC CCT GAA GGC 1479 Ala Asp Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly 400 405 410 TAC ATC CTG GAC GAC GGT TTC ATC TGC ACG GAC ATC GAC GAG TGC GAA 1527 Tyr Ile Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu 415 420 425 AAC GGC GGC TTC TGC TCC GGG GTG TGC CAC AAC CTC CCC GGT ACC TTC 1575 Asn Gly Gly Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe 430 435 440 GAG TGC ATC TGC GGG CCC GAC TCG GCC CTT GTC CGC CAC ATT GGC ACC 1623 Glu Cys Ile Cys Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr 445 450 455 460 GAC TGT GAC TCC GGC AAG GTC GAC GAC GAG GCC AGC GGC TCT GGC GAC 1671 Asp Cys Asp Ser Gly Lys Val Asp Asp Glu Ala Ser Gly Ser Gly Asp 465 470 475 TGACTCGAG 1680

[Brief description of drawings]

FIG. 1 is a drawing showing an amino acid sequence of a thrombin-binding substance of the present invention.

FIG. 2 Expression vectors pCDM-GAG-UTM1 and pCDM- of the present invention
It is explanatory drawing which shows the structure of GAG-UTM2.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12P 21/02 9162-4B C12N 15/00 // (C12N 1/21 C12R 1:19) (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:91) (C12P 21/02 C12R 1:19)

Claims (11)

(57) [Claims] 1. A thrombin-binding substance having the amino acid sequence shown below, in which glycosaminoglycan is added to Ser at the 474th position from at least the N-terminal side. (In the sequence, X1 and X2 represent acidic amino acids, and Y1 and Y
2 represents an arbitrary amino acid. ) 2. In the amino acid sequence, (a) X1 and Y2 are glutamic acid (Glu), and X2 is aspartic acid (As).
The thrombin-binding substance according to claim 1, wherein p), Y1 is tyrosine (Tyr), or (b) X1 and Y2 are aspartic acid (Asp), X2 is glutamic acid, and Y1 is alanine (Ala). 3. A DNA fragment having a base sequence capable of encoding the amino acid sequence according to claim 1. 4. A DNA fragment having a base sequence capable of encoding the amino acid sequence (a) or (b) of claim 2. 5. The DNA fragment according to claim 4, which has a nucleotide sequence shown in (A) or (B) below. (A) Sequence Length: 1428 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: cDNA to mRNA (B) Sequence Length: 1428 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: cDNA to mRNA 6. A recombinant vector comprising a DNA fragment having a nucleotide sequence encoding the amino acid sequence of claim 1 and a replicable vector. 7. The following base sequences (1) to (7) and (1) a base sequence acting as a promoter in the downstream direction of transcription (2) a base sequence which is a ribosome binding site (3) a base which is a start codon Sequence (4) A base sequence encoding a signal peptide (5) A base sequence encoding the amino acid sequence according to claim 1 (6) A base sequence which is a stop codon (7) A base sequence which acts as a poly A addition signal Recombinant vector. 8. A transformant cell carrying the recombinant vector according to claim 6 or 7. 9. An anticoagulant comprising the thrombin-binding substance according to claim 1 or 2 as an active ingredient. 10. A platelet aggregation inhibitor comprising the thrombin-binding substance according to claim 1 or 2 as an active ingredient. 11. The method for producing a thrombin-binding substance according to claim 1, which comprises culturing the transformant cell according to claim 8 and collecting a polypeptide produced from the culture.