JPH05310787A - New polypeptide - Google Patents

Abstract

PURPOSE:To obtain a new polypeptide having inhibitory action on coagulation of thrombin and blood platelet aggregation and/or promoting action on protein C activation of thrombin. CONSTITUTION:In an amino acid sequence of human thrombomodulin, an amino acid residue playing an important role in bonding to thrombin when a promoting action on activation of protein C with thrombin develops, is identified to prepare a thrombomodulin having integrity of various strength. Since the polypeptide has inhibitory action on coagulation of thrombin and blood platelet aggregation and/or promoting action on protein C activation of thrombin, the polypeptide is useful as a medicine or treating and preventing diseases followed by coagulation.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel polypeptide. The present invention also relates to peptides having an inhibitory action on thrombin's blood coagulation and platelet aggregation actions and / or an action to promote the activation of thrombin protein C, and a pharmaceutical composition using these peptides. is there. Since these peptides are involved in the anticoagulant system and fibrinolytic system for blood coagulation, they are drugs, especially diseases associated with blood coagulation disorders such as myocardial infarction, thrombosis, embolism, peripheral vascular occlusion, and arteriosclerosis obliterans. It is useful as a therapeutic agent for infectious diseases, intravascular coagulation syndrome (DIC), angina, transient cerebral ischemic attack, toxemia of pregnancy and the like.

In the present specification, amino acid sequences and peptides are represented by the abbreviations adopted by the IUPAC-IUB Biochemical Nomenclature Committee (CNB) shown below. In addition,
When an amino acid or the like may have an optical isomer, it represents the L-form unless otherwise specified, and the left and right ends of the amino acid sequence of the peptide are the N-terminal and C-terminal, respectively.

Gln: Glutamine residue Asp: Aspartic acid residue Pro: Proline residue Tyr: Tyrosine residue Val: Valine residue Lys: Lysine residue Glu: Glutamic acid residue Ala: Alanine residue Asn: Asparagine residue Leu : Leucine residue Phe: Phenylalanine residue Gly: Glycine residue His: Histidine residue Ser: Serine residue Thr: Threonine residue Ile: Isoleucine residue Trp: Tryptophan residue Arg: Arginine residue Met: Methionine residue Cys : Cystine residue Gla: γ-carboxyglutamic acid residue

Polydeoxynucleotides and oligonucleotides are represented by the sequences of deoxyribonucleotides represented by the following abbreviations. A: adenine (deoxyadenylic acid) C: cytosine (deoxycytidylic acid) G: guanine (deoxyguanylic acid) T: thymine (deoxythymidylic acid) Unless otherwise specified, the left and right ends of the deoxyribonucleotide sequence are 5 ′, respectively. End and 3'end.

[0005]

2. Description of the Related Art Protein C is known as a vitamin K-dependent protein that plays an important role in the blood coagulation mechanism. In recent years, it has been reported that a substance that promotes the activation of protein C and suppresses platelet activation and fibrin formation due to the action of thrombin is present in rabbit lung, human lung, placenta, etc. Is called thrombomodulin.

Further, N. L. Esmon et al. [J. Bio
l. Chem. 257.859-864 (1982)].
Reported that the above substance purified from rabbit lung requires calcium ions for binding to thrombin and activating protein C. In addition, K. Suzuki
i et al. [Biochim. Biophys. Acta, 8
82.343-352 (1986)] regarding the above substance purified from bovine lung. H. Salem et al. [J.
Biol. Chem. , 259.122246-1225
1 (1984)] similarly report that the above substances purified from human placenta require calcium ions in activating protein C after binding with thrombin.

Furthermore, S. Kurosawa et al. [J. B
iol. Chem. , 262.2206-2212 (1
987)], a soluble peptide obtained by cleaving the above substance purified from rabbit lung with elastase showed a maximum value of activity at a calcium ion concentration of 0.3 mM upon activation of protein C, and the Gla domain was not detected. It has been reported that upon activation of the removed protein C (hereinafter abbreviated as GDPC), such calcium ion concentration dependence is not exhibited. By the way, the cDNA of the above-mentioned substance of human origin
The present inventors have previously clarified the cloning of E. coli. [S. Yamamoto et al. International Publication Number WO88
/ 05033]].

On the other hand, recent advances in genetic engineering techniques have made it possible to substitute an arbitrary amino acid in a protein with another amino acid or to delete or insert an amino acid sequence at an arbitrary site in the protein. .. As described above, studies have been conducted to modify naturally occurring proteins to create new proteins that meet specific purposes. Regarding human thrombomodulin, the present inventors have already revealed that a peptide consisting of 115 amino acids has an activity of promoting activation of protein C by thrombin.
[M. Zushi et al. Biol. Chem. 26
4.10351-10353 (1989)] Furthermore,
A mutant (E45) in which 38 amino acids at the C-terminal side of this 115-amino acid peptide was deleted was constructed, and its activity was investigated. It has become clear that [The present inventors, J. Biol. Chem.
265.20156-20159 (1990)] Furthermore,
A mutant (E456-N1) in which one amino acid (Val) on the N-terminal side of this 115 amino acid peptide was deleted was constructed, and its activity was examined. As a result, a sufficient activity was found.

[The present inventors, J. Biol. Chem.
266.19886-19889 (1991)] In the above-mentioned peptide, the present inventors further found that an amino acid sequence containing a thrombomodulin protein C-binding ability-imparting sequence and a thrombomodulin thrombin-binding site promotes thrombin protein C activation. It has been clarified that this is an essential region for the action. [See the inventors of the present application, Japanese Patent Application No. 3-282369] In addition, regarding thrombin, a region having a positive charge on the surface of the molecule is involved in binding with other protein molecules, for example, hirudin having an anticoagulant action. It is clear that there is. [Rydel et al., Science 249, 277-
280 (1990)].

[0010]

[Problems to be Solved by the Invention] As a method of administering a protein preparation in the treatment of blood coagulation disorders, intravenous injection by infusion,
Percutaneous transluminal coronary artery recanalization [PTCR (percutane
ous transluminal coronary
Localization by recanalization)] is the mainstream. Such an administration method is acceptable for short-term treatment, but is problematic for patients who require long-term treatment or patients who require prophylactic administration. This is because, since the protein has a high molecular weight, it is necessary to administer a relatively large amount of the preparation in order to obtain a certain effect, but in this case, the risk of antigenic expression may be given to the patient. In addition, a long hospital visit puts a mental and financial burden on the patient. The currently considered possible method for administering human thrombomodulin is intravenous injection.

Under these circumstances, there is a demand for the development of peptides having higher activity and lower molecular weight thrombomodulin activity with less possibility of antigenic expression.
In addition, administration methods that do not rely on intravenous injection, topical administration, etc.
Development of thrombomodulin preparations that can be administered orally or nasally is also desired. An object of the present invention is to provide a novel polypeptide having a thrombomodulin-like action which promotes the activation of protein C by thrombin, which meets the above-mentioned demand. Another object of the present invention is to provide a DNA encoding the above polypeptide. Still another object of the present invention is to provide a recombinant DNA in which a DNA encoding the above-mentioned polypeptide is incorporated into a vector. Still another object of the present invention is to provide a microorganism and an animal cell transformed with the above recombinant DNA. Still another object of the present invention is to provide a method for producing the above-mentioned polypeptide by culturing a microorganism and an animal cell transformed with the above recombinant DNA. Still another object of the present invention is to provide a pharmaceutical composition containing the above polypeptide as an active ingredient.

[0012]

As described above, the present inventors have been studying peptides that promote the activation of protein C of thrombin using a gene recombination technique. It was previously revealed that a peptide consisting of 115 amino acids in the amino acid sequence consisting of 575 has the above activity [M. Zushi et al. Biol. Chem. , 26
4.1351-10353 (1989)]. The present inventors have further identified the amino acid sequence containing the protein C-binding ability-imparting sequence of thrombomodulin and the thrombin-binding site of thrombomodulin as a region essential for the action of promoting the protein C activation of thrombin in the above peptide. Successful. [See the present inventors, Japanese Patent Application No. 3-282369].

The present inventors have found that, in the amino acid sequence containing the thrombin-binding site that is essential for the expression of the thrombin-stimulating activity of thrombomodulin for protein C activation, the amino acid residue essential for binding to thrombin remains. In order to identify the group, we focused on 11 amino acid residues having a negatively charged side chain,
When the mutant protein was purified by substituting with la, and the specific activity of the promoting activity of thrombin for protein C activation was measured, surprisingly, thrombomodulin having various strengths of thrombin binding was obtained. . It was revealed that the substitution of some amino acid residues significantly changed the activity and was involved in the binding to thrombin. The sequence A shown in the above-mentioned claims is represented by A, which is the protein C described in the above-mentioned prior art.
The binding site is indicated by C, which is the thrombin binding site identified this time. Based on this finding, it became possible to obtain thrombomodulin having various strengths of thrombin binding by modifying the residue that is the binding site for thrombin and the residues around it.

Furthermore, it was confirmed that this peptide was efficiently expressed in animal cells and various microorganisms, and the present invention was completed based on these findings. That is, the present invention provides a polypeptide having the following amino acid sequence. (A)-(B)-(C)-(D)-(C)-(E)-
(C)-(F)-(C)-(G)-(C)-(H)-
(C) (where A and C are Asp or Glu and Gla
It is an amino acid residue consisting of only one or a peptide residue, or a peptide residue consisting of a combination of at least two or more selected from the group consisting of Asp and Glu and Gla. B is an arbitrary amino acid residue or peptide residue, and its length is 0 or more and 5 or less as the number of amino acid residues.
8 or less. D, E, F, G, and H are arbitrary amino acid residues or peptide residues, and their lengths are
The number of amino acid residues is 0 or more and 25 or less.
In addition, said Gla shows (gamma) -carboxyglutamic acid residue).

The present invention also provides a polypeptide having the following amino acid sequence. (A) -Pro-Cys-Phe-Arg-Ala-A
sn-Cys-Glu-Tyr-Gln-Cys-Gl
n-Pro-Leu-Asn-Gln-Thr-Ser
-Tyr-Leu-Cys-Val-Cys-Ala-
Glu-Gly-Phe-Ala-Pro-Ile-P
ro-His-Glu-Pro-His-Arg-Cy
s-Gln-Met-Phe-Cys-Asn-Gln
-Thr-Ala-Cys-Pro-Ala-Asp-
Cys-Asp-Pro-Asn-Thr-Gln-A
la-Ser-Cys- (C)-(D)-(C)-
(E)-(C)-(F)-(C)-(G)-(C)-
(H)-(C) (where A and C are Asp or Glu and Gl
It is an amino acid residue consisting only of a or a peptide residue, or a peptide residue consisting of a combination of at least two or more selected from the group consisting of Asp and Glu and Gla. D, E, F, G, and H are arbitrary amino acid residues or peptide residues, and their lengths are 0 or more and 25 or less in terms of the number of amino acid residues. In addition, the G
la indicates a γ-carboxyglutamic acid residue).

The present invention also provides a polypeptide having the following amino acid sequence. (A) -Pro-Cys-Phe-Arg-Ala-A
sn-Cys-Glu-Tyr-Gln-Cys-Gl
n-Pro-Leu-Asn-Gln-Thr-Ser
-Tyr-Leu-Cys-Val-Cys-Ala-
Glu-Gly-Phe-Ala-Pro-Ile-P
ro-His-Glu-Pro-His-Arg-Cy
s-Gln-Met-Phe-Cys-Asn-Gln
-Thr-Ala-Cys-Pro-Ala-Asp-
Cys-Asp-Pro-Asn-Thr-Gln-A
la-Ser-Cys- (C)-(D)-(C)-
(E)-(C)-(F)-(C)-(G)-(C)-
(H)-(C) -Cys-Ile-Cys-Gly-P
ro-Asp-Ser-Ala-Leu-Val-Ar
g-His-Ile-Gly-Thr-Asp-Cys (where A and C are Asp or Glu and Gla)
It is an amino acid residue consisting of only one or a peptide residue, or a peptide residue consisting of a combination of at least two or more selected from the group consisting of Asp and Glu and Gla. D, E, F, G, and H are arbitrary amino acid residues or peptide residues, and their lengths are 0 or more and 25 or less in terms of the number of amino acid residues. The Gl
a represents a γ-carboxyglutamic acid residue).

Further, the present invention provides DNAs encoding the above three peptides and recombinant DNAs in which the DNAs are incorporated into a vector. Furthermore, the present invention provides a microorganism transformed with the recombinant DNA,
Alternatively, an animal cell is provided. Furthermore, the present invention provides a method for producing any of the above-mentioned three peptides by culturing a microorganism transformed with the above-mentioned recombinant DNA and an animal cell. Furthermore, the present invention comprises a pharmaceutical composition containing any of the above-mentioned three peptides as an active ingredient, which has an inhibitory effect on thrombin blood coagulation and platelet aggregation and / or a thrombin protein C activation activity. A composition is provided.

In the course of completing the present invention, the present inventors first identified the residues involved in the binding of human thrombomodulin to thrombin. That is, the DNA fragment encoding the 1st to 516th amino acid sequence in all amino acids (sequence listing) of human thrombomodulin, which was previously clarified by the present inventors, was subcloned into an M13 phage vector, and a known site-specific mutation was performed. Using the method of
Substitution of the DNA at the position encoding the amino acid (aspartic acid or glutamic acid) with a negatively charged side chain existing in and near the 19-amino acid sequence, which is the thrombin binding site of human thrombomodulin, was clarified earlier. Then, a DNA encoding a peptide in which the amino acid at each position was replaced with another amino acid, for example, Ala, was prepared.

These DNAs were expressed in COS-1 cells using pSV2, which is an animal cell expression vector, purified by ion exchange chromatography, and their ability to promote activation of protein C by thrombin was measured. As described in Example 1 below, it was found that the substitution of Ala for the amino acid at a specific position significantly reduced the ability of thrombin to promote protein C activation. It was revealed that the amino acid residue at that position plays an important role in the binding between thrombin and thrombomodulin. Therefore, the strength of the binding to thrombin is changed by substituting the amino acid residues involved in this binding and the residues around it with other amino acids, or by inserting or deleting the amino acid sequence, and as a result, the protein of thrombin is changed. It has become possible to obtain a mutant having an improved action of promoting C activation. Therefore, the present invention provides a novel polypeptide having an action of promoting activation of protein C of thrombin. The present invention further provides a novel polypeptide which enables non-intravenous administration such as oral administration and nasal administration as a method of administering thrombomodulin.

The peptide provided by the present invention consists essentially of the amino acids represented by the following formula (I), (II) or (III). Formula (I): (A)-(B)-(C)-(D)-(C)-(E)-
(C)-(F)-(C)-(G)-(C)-(H)-
(C) (where A and C are Asp or Glu and Gla
It is an amino acid residue consisting of only one or a peptide residue, or a peptide residue consisting of a combination of at least two or more selected from the group consisting of Asp and Glu and Gla. B is an arbitrary amino acid residue or peptide residue, and its length is 0 or more and 5 or less as the number of amino acid residues.
8 or less. D, E, F, G, and H are arbitrary amino acid residues or peptide residues, and their lengths are
The number of amino acid residues is 0 or more and 25 or less.
In addition, said Gla shows (gamma) -carboxyglutamic acid residue).

Formula (II): (A) -Pro-Cys-Phe-Arg-Ala-A
sn-Cys-Glu-Tyr-Gln-Cys-Gl
n-Pro-Leu-Asn-Gln-Thr-Ser
-Tyr-Leu-Cys-Val-Cys-Ala-
Glu-Gly-Phe-Ala-Pro-Ile-P
ro-His-Glu-Pro-His-Arg-Cy
s-Gln-Met-Phe-Cys-Asn-Gln
-Thr-Ala-Cys-Pro-Ala-Asp-
Cys-Asp-Pro-Asn-Thr-Gln-A
la-Ser-Cys- (C)-(D)-(C)-
(E)-(C)-(F)-(C)-(G)-(C)-
(H)-(C) (where A and C are Asp or Glu and Gla)
It is an amino acid residue consisting of only one or a peptide residue, or a peptide residue consisting of a combination of at least two or more selected from the group consisting of Asp and Glu and Gla. D, E, F, G, and H are arbitrary amino acid residues or peptide residues, and their lengths are 0 or more and 25 or less in terms of the number of amino acid residues. The Gl
a represents a γ-carboxyglutamic acid residue).

Formula (III): (A) -Pro-Cys-Phe-Arg-Ala-A
sn-Cys-Glu-Tyr-Gln-Cys-Gl
n-Pro-Leu-Asn-Gln-Thr-Ser
-Tyr-Leu-Cys-Val-Cys-Ala-
Glu-Gly-Phe-Ala-Pro-Ile-P
ro-His-Glu-Pro-His-Arg-Cy
s-Gln-Met-Phe-Cys-Asn-Gln
-Thr-Ala-Cys-Pro-Ala-Asp-
Cys-Asp-Pro-Asn-Thr-Gln-A
la-Ser-Cys- (C)-(D)-(C)-
(E)-(C)-(F)-(C)-(G)-(C)-
(H)-(C) -Cys-Ile-Cys-Gly-P
ro-Asp-Ser-Ala-Leu-Val-Ar
g-His-Ile-Gly-Thr-Asp-Cys (where A and C are Asp or Glu and Gla)
It is an amino acid residue consisting of only one or a peptide residue, or a peptide residue consisting of a combination of at least two or more selected from the group consisting of Asp and Glu and Gla. D, E, F, G, and H are arbitrary amino acid residues or peptide residues, and their lengths are 0 or more and 25 or less in terms of the number of amino acid residues. The Gl
a represents a γ-carboxyglutamic acid residue).

The length of the protein C binding site A in the peptide of the present invention depends on the binding partner protein C.
1-2 based on the number of Gla residues in the Gla domain of
It is within 0 residues, preferably 1 to 10 residues. The peptide of the present invention also comprises the amino acid sequence represented by the above formula (I), formula (II) or formula (III) and at least one other amino acid linked to its N-terminal and / or C-terminal. It may further contain the amino acid sequence of the peptide. Furthermore,
Since it is possible to change a part of the peptide structure by natural mutation or artificial mutation without significantly changing the activity of the peptide, the peptide of the present invention has the above amino acid sequence. It also includes a peptide having a structure corresponding to a homologous variant (homologous variant) of the peptide.

The peptide of the present invention may or may not contain at least one sugar residue. Further, according to the present invention, the above formula (I) or formula (II) or formula (II) in which at least one base is substituted or not substituted based on the degeneracy of the genetic code is used.
There is provided a deoxyribonucleic acid encoding a peptide containing the amino acid sequence represented by I). DNA of the present invention
Is a base sequence encoding the amino acid sequence represented by the above formula (I), formula (II) or formula (III) and its 5 '.
It may contain at least one other base sequence linked to the terminal and / or the 3 ′ terminal. According to the present invention, DNA complementary to the above DNA is also provided. According to the present invention, the above DNA and the DNA complementary thereto may form a double-stranded DNA in which they are bound to each other complementarily.

Since it is possible to mutate a part of the structure of DNA and the structure of the peptide deduced from it without changing the main activity by natural mutation or artificial mutation, the DNA of the present invention is It is also possible to contain a nucleotide sequence encoding a peptide having a structure corresponding to homologous variants of all the aforementioned peptides. According to the degeneracy of the genetic code, at least one base of the base sequence of the gene can be replaced with another type of base without changing the amino acid sequence of the polypeptide produced from the gene. Therefore, the DNA of the present invention also comprises a peptide comprising such an amino acid sequence, since the amino acid sequence deduced from the nucleotide sequence changed by the substitution based on the degeneracy of the genetic code matches the amino acid sequence defined above. It is also possible to contain an altered base sequence encoding

Furthermore, according to the present invention, a recombinant DN in which the above-mentioned deoxyribonucleic acid of the present invention is incorporated into a vector
A is provided. The recombinant DNA is capable of expressing the peptide of the present invention in the microorganism or cell transformed with it. Examples of suitable vectors include plasmids pBR322, pBR327, pUC1.
8, pUC19, YRp7, YEp24 (ATCC37
051), pPGACY2, pBSFAHY83, pS
V2-dhfr (ATCC37146), pBPV-1
(9-1) (ATCC37111) and the like. It is necessary to select an expression vector suitable for the microorganism or cell used as the host.

Furthermore, the present invention also provides the recombinant DNA described above.
A microorganism or cell transformed with is provided. Examples of microorganisms include Escherichia coli (Escheri)
C. coli), for example E. coli
K12 strain 294 (ATCC31446) E. coli
B strain, E. coliX1776 (ATCC3153
7), E. coli C600, E. coli JM1
05; Bacillus subtilus (Bacillus su
btilis) or Serratia mars (Serr)
Enterobacteria other than Escherichia coli such as atia marcesans; various strains of the genus Pseudomonas; and Saccharomyces cerevisiae (Sacc)
aspergillus nidrians (Aspergillus nidrians)
dulans), Acremonium chrysogenum (Ac
remonium chrysogenum) (ATC
C11550) and other fungi. Examples of cells include VERO cells (ATCCCL-81), HeLa.
Cells, Chinese hamster (CHO) cells, W13
8, animal cells such as BHK, COS-1, COS-7 and MDCK cells.

According to the method of the present invention, the above-mentioned D of the present invention is used.
A replicable recombination having the DNA by incorporating the DNA of the present invention downstream of a DNA region such as a promoter of a replicable expression vector so that NA is correctly transcribed and translation from the resulting mRNA is correctly carried out. DN
A is obtained, and a microorganism or cell is transformed with the obtained recombinant DNA to obtain a transformant containing the recombinant DNA. The resulting transformant is isolated from the microorganism or parent cell of the cultured cell according to the phenotype imparted to the recombinant DNA. The transformant thus obtained is cultured to express the genetic information contained in the deoxyribonucleic acid to produce the peptide of the present invention.

The DNA of the present invention and the recombinant DNA
It is preferable to use a host-vector system in which a prokaryotic cell is used as a host in order to clone a DNA sequence necessary for constructing E. coli, such as a promoter and an origin of replication. Examples of prokaryotic cells include Escherichia coli (E
strains of Scherichia coli), for example,
E. coli K12 strain 294 (ATCC31446)
E. coli B strain, E. coli. coli X1776 (AT
CC31537), E.I. coli C600, E. co
li C600hfl and E. coli W3110
(F-, λ-prototrophic, ATCC2737
5); Bacillus subtilus (Bacillus su
strains of the genus Bacillus, such as b.
Salmonella typhimurium (Salmonella t
Enterobacteria other than Escherichia coli, such as S. typhimurium) or Serratia marcesans; Pseudomona (Pseudomona)
s) various strains of the genus; and Saccharomyces cerevisiae.
e); Aspergillus nidulans
lus nidulans, Acremonium chrysogenum (Acremonium chrysogenu)
m) (ATCC 11550) and other fungi.
Of these cells, E. E. coli K12 strain 294 is most preferred.

When the above microorganism is used as a host,
Recombinant D is a plasmid vector suitable for these microorganisms.
It is commonly used as an NA-replicable expression vector. For example, plasmid vectors for transforming E. coli include plasmids pBR322 and pBR327.
Alternatively, pUC18, pUC19 and the like can be used.
A plasmid vector usually contains an origin of replication, a promoter,
And a marker gene which gives the recombinant DNA a phenotype useful for selecting cells transformed with the recombinant DNA. Examples of promoters include β-lactamase and lactose promoters, tryptophan promoters and the like. Examples of marker genes include ampicillin resistance gene, tetracycline resistance gene, hygromycin resistance gene and the like.

On the other hand, in order to produce the peptide of the present invention by expressing the DNA of the present invention, a host-vector system using the above-mentioned prokaryotic cells as a host and eukaryotic cells such as vertebrate cells are used as host cells. The host-vector system used as can be used. Examples of eukaryotic cells include cells such as the above-mentioned animal cell lines. In order to express the DNA of the present invention in the aforementioned eukaryotic cells, the recombinant DNA of the present invention generally has a functional sequence for controlling gene expression, for example, an origin of replication, located upstream of the DNA of the present invention. It contains the desired promoter, ribosome binding site, polyadenylation site and transcription termination sequence. DNA of the present invention
Can be used for expression in eukaryotic cells, such functional sequences can be obtained from viruses or viral substances.

For example, promoters that can be used in the present invention include adenovirus 2, polyoma virus,
It can be obtained from simian virus 40 (SV40) and the like. In particular, the adenovirus 2 major late promoter and S
The early and late promoters of V40 are preferred. Also,
A promoter naturally existing at a position upstream of a gene encoding a peptide derived from human lung, which has an action of promoting protein C activation of thrombin, is also used if it is suitable for use in the above-described host-vector system. be able to.

Regarding the origin of replication, foreign sources such as adenovirus, polyoma, SV40 vesicular stomatitis virus (VSV), bovine papilloma virus (BPV).
Replication origins derived from viruses such as can be used. When a vector having a property of being integrated into a host chromosome is used as an expression vector, the origin of replication of the host chromosome can be used. Microorganisms or cells transformed with the replicable recombinant DNA of the present invention are selected from parent cells that remain untransformed by at least one phenotype imparted to the recombinant DNA as described above. To be done. The phenotype can be conferred by inserting at least one marker gene into the recombinant DNA. Alternatively, the marker gene originally possessed by the replicable expression vector can be used.

Examples of marker genes include drug resistance genes such as neomycin resistance and genes encoding dihydrofolate reductase (hereinafter referred to as "DHFR"). In this regard, when the DHFR gene is used as a marker gene, since there are various types of DHFR, it is necessary to select a host to be used depending on the type of DHFR encoded by the marker gene used. For example, when using a gene encoding wild-type DHFR as a marker gene, it is preferable to use a DHFR-deficient strain as a host.

Since the DHFR-deficient strain requires hypoxanthine, glycine and thymidine, it cannot grow in a medium free of hypoxanthine, glycine and thymidine. However, when a DHFR-deficient strain is transformed with a recombinant DNA containing the DHFR gene, the strain no longer requires hypoxanthine, glycine and thymidine and can grow in a medium free of hypoxanthine, glycine and thymidine. it can. Therefore, the transformed cells can be easily selected from the cells remaining untransformed, based on the auxotrophy for hypoxanthine, glycine and thymidine as criteria.

On the other hand, when a gene encoding a mutant DHFR having a low affinity for methotrexate (MTX) (hereinafter referred to as "MTX-resistant DHFR gene") is used as a marker gene, the host cell is normal DH.
It may have a gene encoding FR and need not be DHFR deficient. The reason is as follows. Since normal DHFR is inhibited by MTX, a host cell containing a gene encoding normal DHFR is inhibited by MTX, and thus a host cell containing a gene encoding normal DHFR is hypoxanthine in the presence of MTX, Requires glycine and thymidine. However, if the host cell is MTX resistant DHF
When transformed with recombinant DNA containing the R gene, transformed cells no longer require hypoxanthine, glycine and thymidine in the presence of MTX. Therefore,
Transformed cells can be selected from non-transformed cells using auxotrophy for hypoxanthine, glycine and thymidine in the presence of MTX as a criterion. In this regard, since the majority of eukaryotic cells are MTX-sensitive, the MTX-resistant DHFR gene
It is convenient to use as a marker gene.

Saccharomyces cerevisiae (Sacch
Yeasts such as Aromyces cerevisiae) can also be used as hosts for expressing the DNA of the present invention. For expressing the DNA of the present invention in yeast, a replicable expression vector such as plasmid YEp2 is used.
4 can be used. The plasmid YEp24 is U
Since it contains the ra3 gene, this Ura3 gene can be used as a marker gene.

Examples of promoters of expression vectors for yeast expression include 3-phosphoglycerate kinase, or elanose, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-. Promoters of genes of enzymes involved in glycolysis such as phosphate isomerase and glucokinase, and genes of enzymes involved in utilization of alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, enzymes related to nitrogen metabolism, galactose, maltose and lactose The promoter of is mentioned. Of these,
Alcohol dehydrogenase 2, isocytochrome C,
Promoters of acid phosphatase, enzymes related to nitrogen metabolism, glyceraldehyde-3-phosphate dehydrogenase, enzymes related to utilization of galactose, maltose and lactose can control transcription by these promoters by changing host culture conditions. Therefore, it is advantageous. As the origin of replication, stop codon and other DNA sequences for controlling transcription and translation in yeast cells, known DNA sequences that are generally known and suitable for yeast cells can be used.

Aspergillus nidulans (Asper
gillus nidulans) and Acremonium chrysogenum (Acremonium chrys)
filamentous fungi such as ogenum) (ATCC 11550) can also be used as a host for expressing the DNA of the present invention. To express the DNA of the present invention in filamentous fungi, expression vectors such as pPGACY2 and pBSFAH are used.
Y83 and the like can be obtained by the method described in Matsuda et al. (Japanese Patent Application No. 2-166566).

Examples of the promoter and terminator of the expression vector for Acremonium chrysogenum include, for example, phosphoglycerate kinase (PGK),
Examples thereof include promoters and terminators of genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPD) and actin. DNA fragments containing these promoters and terminators are, for example,
It can be obtained from the chromosome library of Acremonium chrysogenum according to the method described in [Matsuda et al., Japanese Patent Application No. 2-166566].

The transformed microorganism or cell can be cultured in an ordinary nutrient medium by an ordinary known method to express the DNA encoding the peptide of the present invention to obtain the peptide of the present invention. After culturing, the peptide of the present invention can be isolated from the transformant culture by a known method,
For example, it can be isolated using column chromatography or the like. The peptide thus obtained may contain at least one sugar chain of various types and lengths. Whether or not the obtained peptide contains a sugar chain,
It depends on the type of host used. Further, when the peptide contains a sugar chain, the kind and length of the sugar chain also differ depending on the kind of host used.

It is generally known that a peptide translated from the translation initiation signal ATG undergoes processing to become a mature protein when secreted from a host cell. The peptide of the present invention may also undergo such processing. The site where the peptide is processed may vary depending on the host or culture conditions. For example, when the peptide of the present invention is produced in a transformed cell in an unprocessed immature form containing the peptide represented by the above formula (I) or (II) or (III) and a leader sequence, The immature peptide may be processed to a mature form with the leader sequence deleted. However, as described above, the position where the immature peptide is processed varies depending on the type of host used and the culture conditions of the host, and thus the above processing does not always occur.

The peptides of the present invention can also be expressed using leader sequences of other proteins. Furthermore, if a leader sequence of a specific protein is used, the amino acid of the peptide that follows can be modified. For example, if a leader sequence such as prothrombin, blood coagulation factor IX, blood coagulation factor X, blood coagulation factor VII, protein C, protein S is used, glutamic acid near the N-terminal portion in the subsequent peptide is converted into γ-carboxyl. Can be converted. [B. Furie et al., Blood, 75, 9,
1753-1762 (1990)] As mentioned above, the peptide of the present invention can be produced by a method using recombinant DNA technology. The peptide of the present invention can also be produced by an organic synthesis according to a commonly known method, for example, using a commercially available automatic peptide synthesizer.

The peptide of the present invention inhibits thrombin from blood coagulation and platelet aggregation, and / or
It has an action of promoting activation of protein C of thrombin.
Protein C is a vitamin K-dependent protein that plays an important role in the blood coagulation / fibrinolysis mechanism, and is activated by the action of thrombin. Active protein C is
It is known that it is involved in the production of plasminogen activator, which inactivates the active factor V and the active factor VIII of the coenzyme of the blood coagulation system in vivo, and also has a thrombolytic action. There is. [Koji Suzuki, Medical History, 12th
5, p. 901, (1983)] Therefore, the peptide of the present invention greatly contributes to anticoagulation and thrombolysis in a living body.

As described above, the peptide of the present invention has anticoagulant activity, platelet aggregation inhibitory activity and thrombolytic activity. , Blood vessel coagulation syndrome (DI
C), angina, transient cerebral ischemic attack, pregnancy toxemia and the like can be used for treatment and prevention. When the peptide of the present invention is used for the treatment and prevention of the above-mentioned diseases, it can be mixed with a pharmaceutically usable carrier. That is, an effective amount of the peptide of the present invention for treating or preventing the above-mentioned diseases may be mixed with an appropriate amount of a carrier to prepare a pharmaceutical composition suitable for effective administration to a patient. it can.

The peptide of the present invention can be used not only as an injection but also as a composition which can be administered orally or mucosally, for example, via the nasal mucosa. The dose of the peptide of the present invention per adult varies depending on age, sex, weight, symptoms, etc., but is generally about 0.1 to 200 mg, and is administered once a day or several times as needed. To do.

[0047]

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Example 1 Identification of Amino Acid Residues that Bind Human Thrombomodulin to Thrombin (1) Construction of pSV2TMD1 pSV2TMJ2 (pSV2TMJ2 ( AT
CC Deposit No. 67283) was completely digested with NcoI and the digested ends were digested with E. The ends were made blunt using E. coli DNA polymerase. Then, it was completely digested with HindIII to obtain a DNA fragment of about 1900 bp. Obtained DN
The A fragment was designated TMJ3. Meanwhile, phage M13mp
19 (Takara Shuzo, Catalog No. 3119) to HindIII
A vector was prepared by digesting with HindII. The DNA fragment TMJ3 was inserted into this vector to obtain a recombinant plasmid M13mp19TMJ3.

In addition, a separate nucleotide sequence: 5'-GGAGG
CCGCTCAGCCCGAATGCACG-3 '(2
A deletion DNA probe (hereinafter referred to as "deleter") TMd1 having 5 mer) was organically synthesized. Using the deleter TMd1 produced as described above, a method in enzymology (Method in En
Zymology), 100, 468, (198).
3 years), Academic Press (Academic Pr)
recombinant plasmid M13m obtained as described above by the method of site-directed mutagenesis according to the method described in Ess).
The nucleotide sequence consisting of 177 bp of p19TMJ3 was deleted. That is, 25pmol deleter TMd1
And 10 pmol of M13 primer M3 (Takara Shuzo, Catalog No. 3831) were phosphorylated at the 5'end with T4 kinase, and then 0.5 pmol of recombinant plasmid M13mp19TMJ3 single-stranded DNA
Was added, the mixture was heated at 95 C for 5 minutes, and then cooled to room temperature.

Then, 5 units of E. coIi DNA polymerase I (Klenow Fragment), and 10 units of T4 DNA ligase were added to the mixture at 37 ° C.
The recombinant plasmid was generated in the mixture by incubating for 30 minutes at. The resulting mixture was E. coliJ
M105 (Pharmacia, Catalog No. 27-155
0) with recombinant plasmid E. co
li was transfected. The plaques on the agar medium produced by culturing overnight at 37 ° C. were transferred onto a nitrocellulose filter, heated at 80 ° C. for 2 hours, and then prehybridized. Pre-hybridization was performed with 6 × SET (0.9 M NaCl, 180 mM Tris buffer (pH 8.0), 6 mM EDTA), 5 × De.
nharts' (0.1% (w / v) polyvinylpyrrolidone, 0.1% (w / v) bovine serum albumin (BS
A)), 0.1% SDS, 100 μg / ml denatured salmon sperm DNA was heated in a solution at 55 ° C. for 2 hours. Then denatured salmon sperm DN in the above solution
The hybridization reaction was carried out at 55 ° C. for 2 hours using a solution containing TMd1 labeled with ³² P instead of A.

Then, 6 × SSC (0.9M NaCl
, 0.09 M trisodium citrate in water) was used to wash the nitrocellulose filter. Washing was performed twice at room temperature for 5 minutes, then gradually increased to 55 ° C., 65 ° C. and 75 ° C., and washed twice for 5 minutes each. When X-ray film XAR-5 (Eastman Kodak) was brought into close contact with the obtained nitrocellulose filter and exposed overnight at −80 ° C., several 10 black spots strongly exposed to the X-ray film were detected. Each spot corresponds to a clone infected with the recombinant plasmid. Of these, 6 clones were selected, and recombinant plasmids of each clone were isolated and subjected to restriction enzyme analysis and nucleotide sequence analysis. As a result, the recombinant plasmids possessed by these clones were found to have restriction enzyme sites and bases. The sequences were found to be identical.

Further, it was found that this DNA fragment had a nucleotide sequence containing a nucleotide sequence encoding a peptide consisting of the 516th amino acid from the start codon (ATG) in the sequence listing. This DNA fragment was called TMD1, and the recombinant plasmid containing this DNA fragment was called M13TMD1. Fig. 1 shows recombinant plasmid M13mp19TM
DN by hybridizing J3 and Deleter TMd1
A part of the DNA region corresponding to the A fragment TMJ3 is deleted. This recombinant plasmid M13TMD
The DNA of 1 was completely digested with HindIII and BamHI, and an approximately 1700 bp DNA fragment of TMD1 was isolated.
On the other hand, the plasmid pSV2-dhfr (ATCC 37
146) was completely digested with HindIII and BglII to obtain a vector, and this vector and the above 1700 bp DNA fragment were ligated together using T4 DNA ligase to obtain plasmid pSV2TMD1.

(2) Point mutation in thrombin binding site (a) Construction of plasmid pSV2TB1 Instead of the deleter TMd1 using the site-directed mutation method, the base sequence: 5'-CTTCAGGGCACG
A DNA probe for mutation having CACAGCTAGCCTG-3 ′ (25mer) (hereinafter, “mutator”)
Example 1- (1) except that tb1 is used.
In a manner substantially similar to the above, by modifying a part of the recombinant plasmid M13TMD1 obtained in Example 1- (1),
Glu426 was converted to Ala to obtain a recombinant plasmid M13TB1 containing a DNA fragment called TB1.

This TB1 is a start codon (AT
G) to the 516th amino acid, but 426
The second Glu had a nucleotide sequence containing a nucleotide sequence encoding a peptide converted to Ala. FIG. 2 shows the recombinant plasmid M13TMD1 and the mutator tb.
1 hybridizes to Glu426 and Ala426.
It is converted to. After confirming that no mutation has occurred except for Glu426 by a conventional method, the DNA of this recombinant plasmid M13TB1 was completely digested with HindIII and BamHI,
An approximately 1700 bp DNA fragment of TB1 was isolated. On the other hand, a plasmid pSV2-dhfr (ATCC37146) was completely digested with HindIII and BglII to obtain a vector, and this vector and the above 1700 bp DNA fragment were treated with T
The plasmid p was ligated using 4DNA ligase.
SV2TB1 was obtained.

(B) Construction of plasmid pSV2TB2 Using the site-directed mutagenesis method, instead of the deleter TMd1, the nucleotide sequence: 5'-GGATGTAGCCTG
Example 1 above except that the mutator tb2 having CAGGGCACTCACA-3 '(25mer) is used.
-In a manner substantially similar to (1), a part of the recombinant plasmid M13TMD1 obtained in Example 1- (1) was modified to convert Glu429 into Ala, and TB
Recombinant plasmid M13 containing a DNA fragment designated 2
TB2 was obtained. This TB2 is a start codon (A
TG) to the 516th amino acid, but 42
The 9th Glu had a nucleotide sequence containing a nucleotide sequence encoding a peptide in which Alu was converted to Ala.

FIG. 3 shows the recombinant plasmid M13TMD1.
And mutator tb2 hybridize to Glu4
29 is converted to Ala429.
After confirming that no mutation has occurred except for Glu429 by sequencing, the DNA of this recombinant plasmid M13TB2 was analyzed with HindIII and Ba.
Approximately 1700 bp DNA of TB2 after complete digestion with mHI
The fragment was isolated. On the other hand, the plasmid pSV2-dhfr
(ATCC37146) was completely digested with HindIII and BglII to obtain a vector.
The 0 bp DNA fragment was ligated with T4 DNA ligase to obtain plasmid pSV2TB2.

(C) Construction of plasmid pSV2TB3 Using the site-directed mutagenesis method, instead of the deleter TMd1, the nucleotide sequence: 5'-GCAGATGAAACC
GGCGGGCCAGGATGTAGCC-3 '(30m
Example 1 in the substantially same manner as in Example 1- (1) above except that the mutator tb3 having er) is used.
-Recombinant plasmid M13TMD1 obtained in (1)
Of Asp434 and Asp435 by modifying a part of
After conversion into a, recombinant plasmid M13TB3 containing a DNA fragment called TB3 was obtained. This TB3
Consists of amino acids from the start codon (ATG) to the 516th position in the sequence listing, but has a base sequence containing a base sequence encoding a peptide in which the 434th Asp and the 435th Asp are converted to Ala. It was

FIG. 4 shows the recombinant plasmid M13TMD1.
And mutator tb3 hybridize to Asp4
34 and Asp435 are Ala434 and Ala, respectively.
It is shown being converted to 435. After confirming by the standard method that no mutations other than Asp434 and Asp435 have occurred, the DNA of this recombinant plasmid M13TB3 was analyzed with HindIII and Bam.
A complete 1700 bp DNA fragment of TB3 was isolated by complete digestion with HI. On the other hand, the plasmid pSV2-dhfr
(ATCC37146) was completely digested with HindIII and BglII to obtain a vector.
The 0 bp DNA fragment was ligated with T4 DNA ligase to obtain plasmid pSV2TB3.

(D) Construction of plasmid pSV2TB4 Using the site-directed mutagenesis method, instead of the deleter TMd1, the nucleotide sequence: 5'-GCACTCGTCCGAT
GGCCGTGCAGATGAA-3 '(27mer)
Example 1-in substantially the same manner as in Example 1- (1) above, except that the mutator tb4 having
A part of the recombinant plasmid M13TMD1 obtained in (1) was modified to convert Asp441 to Ala to obtain a recombinant plasmid M13TB4 containing a DNA fragment called TB4. This TB4 was composed of amino acids from the start codon (ATG) to the 516th position in the sequence listing, but had a base sequence containing a base sequence encoding a peptide in which Asp at the 441st position was converted to Ala.

FIG. 5 shows the recombinant plasmid M13TMD1.
And mutator tb4 hybridize and Asp4
41 is converted to Ala441.
After confirming that no mutations other than Asp441 have occurred by a conventional method, the DNA of this recombinant plasmid M13TB4 was analyzed with HindIII and Bam.
A complete 1700 bp DNA fragment of TB4 was isolated by complete digestion with HI. On the other hand, the plasmid pSV2-dhfr
(ATCC37146) was completely digested with HindIII and BglII to obtain a vector.
The 0 bp DNA fragment was ligated with T4 DNA ligase to obtain plasmid pSV2TB4.

(E) Construction of plasmid pSV2TB5 Using the site-directed mutagenesis method, instead of the deleter TMd1, the nucleotide sequence: 5'-GCCGTTTTCGCA
CGCGGCGATGTCCGTGCA-3 '(30m
Example 1 in substantially the same manner as in Example 1- (1) above except that the mutator tb5 having er) is used.
-Recombinant plasmid M13TMD1 obtained in (1)
Of Asp443 and Glu444 by modifying a part of
After conversion to a, a recombinant plasmid M13TB5 containing a DNA fragment called TB5 was obtained. This TB5
Consists of amino acids from the start codon (ATG) to the 516th position in the sequence listing, and has a base sequence containing a base sequence encoding a peptide in which the 443th Asp and the 444th Glu are converted to Ala. It was

FIG. 6 shows the recombinant plasmid M13TMD1.
And mutator tb5 hybridize to Asp4
43 and Glu444 are Ala443 and Ala, respectively.
It is shown being converted to 444. After confirming that no mutation has occurred except for Asp443 and Glu444 by a conventional method, the DNA of this recombinant plasmid M13TB5 was digested with HindIII and Bam.
Completely digested with HI, an approximately 1700 bp DNA fragment of TB5 was isolated. On the other hand, the plasmid pSV2-dhfr
(ATCC37146) was completely digested with HindIII and BglII to obtain a vector.
The 0 bp DNA fragment was ligated with T4 DNA ligase to obtain plasmid pSV2TB5.

(F) Construction of plasmid pSV2TB6 Using the site-directed mutagenesis method, instead of the deleter TMd1, the nucleotide sequence: 5'-GCCGCCGTTTGC
The recombinant plasmid M13TMD1 obtained in Example 1- (1) was prepared in substantially the same manner as in Example 1- (1) above, except that the mutator tb6 having GCACTCGT-3 '(20mer) was used. Partly modified, G
Conversion of lu446 to Ala was performed to obtain a recombinant plasmid M13TB6 containing a DNA fragment called TB6. This TB6 consists of amino acids from the start codon (ATG) to the 516th amino acid in the sequence listing.
It had a base sequence containing a base sequence encoding a peptide in which lu was converted to Ala.

FIG. 7 shows the recombinant plasmid M13TMD1.
And mutator tb6 hybridize to Glu4
It shows that 46 is converted into Ala441. G
After confirming that no mutation has occurred except for lu446 by a conventional method, the DNA of this recombinant plasmid M13TB6 was analyzed with HindIII and BamH.
After complete digestion with I, an approximately 1700 bp DNA fragment of TB6 was isolated. On the other hand, the plasmid pSV2-dhfr (AT
CC37146) was completely digested with HindIII and BglII to obtain a vector.
The DNA fragment was ligated with T4 DNA ligase to obtain plasmid pSV2TB6.

(G) Construction of plasmid pSV2TB7 Instead of the deleter TMd1 using the site-directed mutagenesis method, the nucleotide sequence: 5'-CAGATGCACGCG was used.
The recombinant plasmid M13TMD1 obtained in Example 1- (1) was obtained in substantially the same manner as in Example 1- (1) above except that the mutator tb7 having AAGGTACC-3 ′ (20mer) was used. Partly modified, G
Conversion of lu463 to Ala was performed to obtain a recombinant plasmid M13TB7 containing a DNA fragment called TB7. This TB7 consists of the amino acid from the start codon (ATG) to the 516th amino acid in the sequence listing.
It had a base sequence containing a base sequence encoding a peptide in which lu was converted to Ala.

FIG. 8 shows the recombinant plasmid M13TMD1.
And mutator tb7 hybridize to Glu4
It shows that 63 is converted to Ala463. G
After confirming that no mutation has occurred except for lu463 by a conventional method, the DNA of this recombinant plasmid M13TB7 was analyzed with HindIII and BamH.
Completely digested with I, an approximately 1700 bp DNA fragment of TB7 was isolated. On the other hand, the plasmid pSV2-dhfr (AT
CC37146) was completely digested with HindIII and BglII to obtain a vector.
The DNA fragment was ligated with T4 DNA ligase to obtain plasmid pSV2TB7.

(H) Construction of plasmid pSV2TB8 Instead of the deleter TMd1 using the site-directed mutagenesis method, the nucleotide sequence: 5'-AGGGCCGAGGCG
The recombinant plasmid M13TMD1 obtained in Example 1- (1) was obtained in substantially the same manner as in Example 1- (1) above, except that the mutator tb8 having GGCCCCGCA-3 ′ (20mer) was used. Partially modified to A
By converting sp469 into Ala, a recombinant plasmid M13TB8 containing a DNA fragment called TB8 was obtained. This TB8 consists of amino acids from the start codon (ATG) to the 516th amino acid in the sequence listing,
It had a base sequence containing a base sequence encoding a peptide in which sp was converted to Ala.

FIG. 9 shows the recombinant plasmid M13TMD1.
And mutator tb8 hybridize and Asp4
69 shows conversion to Ala469. A
After confirming that no mutation has occurred except for sp469 by a conventional method, the DNA of this recombinant plasmid M13TB8 was cloned into HindIII and BamH.
Completely digested with I, an approximately 1700 bp DNA fragment of TB8 was isolated. On the other hand, the plasmid pSV2-dhfr (AT
CC37146) was completely digested with HindIII and BglII to obtain a vector.
The DNA fragment was ligated with T4 DNA ligase to obtain plasmid pSV2TB8.

(I) Construction of plasmid pSV2TB9 The nucleotide sequence: 5'-GAGTCACAGGCG was used instead of the deleter TMd1 using the site-directed mutagenesis method.
Except for using the mutator tb9 having GTGCCAAT-3 ′ (20mer), the recombinant plasmid M13TMD1 obtained in Example 1- (1) was prepared in substantially the same manner as in Example 1- (1) above. Partially modified to A
Conversion of sp479 to Ala was performed to obtain a recombinant plasmid M13TB9 containing a DNA fragment called TB9. This TB9 consists of amino acids from the start codon (ATG) to the 516th amino acid in the sequence listing,
It had a base sequence containing a base sequence encoding a peptide in which sp was converted to Ala.

FIG. 10 shows the recombinant plasmid M13TMD.
1 and mutator tb8 hybridize and Asp
It shows that 479 is converted to Ala479.
After confirming that no mutation other than Asp479 has occurred by a conventional method, the DNA of this recombinant plasmid M13TB9 was analyzed with HindIII and Bam.
A complete digestion with HI isolated the approximately 1700 bp DNA fragment of TB9. On the other hand, the plasmid pSV2-dhfr
(ATCC37146) was completely digested with HindIII and BglII to obtain a vector.
The 0 bp DNA fragment was ligated with T4 DNA ligase to obtain plasmid pSV2TB9.

(3) Transfection of COS-1 cells COS-1 cells (ATCC CRL1650) were placed in a tissue culture dish at 10% (v / v) fetal calf serum (hereinafter referred to as FCS; Gibco). After culturing to a confluent state using Dulbecco's minimum essential medium (hereinafter referred to as DMEM) (Catalog No. 10-311, manufactured by Flow Laboratories), the trypsin solution (0.
The plate was peeled off using 25% trypsin, 0.02% EDTA-containing PBS), and electroporation buffer (272 mM saccharose, 1 mM MgCl ₂ ) was used.
500 μl of the resulting cell suspension was suspended in 7 mM phosphate buffer (pH 7.4) to a concentration of 8 × 10 ⁶ cells / ml.
Were placed in an electroporation cuvette (Bio-Rad, Catalog No. 165-2085).

Next, the plasmids pSV2TMD1, pSV2TB1 and pSV2TB constructed in Example 1- (1) were used.
2, pSV2TB3, pSV2TB4, pSV2TB
5, pSV2TB6, pSV2TB7, pSV2TB
DNA of 8 and pSV2TB9 were suspended in distilled water to 4 μg / μl each, and 20 μg of plasmid DN was added.
5 μl of the suspension containing A was charged with COS-in the above cuvette.
One cell suspension was added and left at 4 ° C. for 5 minutes. After 5 minutes, transfer the cuvette to an electroporation device (Bio-Rad catalog number 165-2075),
Two pulses were given for 30 seconds under the condition of 450V.
Then, after leaving it at 4 ° C for 5 minutes, the cell suspension was treated with 10% FC.
Diameter 10 with 10 ml of DMEM plus S (v / v)
The cells were transferred to a cell culture dish of cm and cultured at 37 ° C. for 24 hours in a carbon dioxide gas incubator. After 24 hours, the medium was replaced with 10 ml of DMEM without FCS for another 48 hours.
After culturing for a time, the culture solution was collected.

(4) Purification of peptide 0.15 M of each of the culture solutions obtained in the above-mentioned Examples
Adsorb on a 0.5 ml Q sepharose column equilibrated with 20 mM Tris buffer (pH 7.4) containing NaCl.
20 mM Tris buffer containing 0.18 M NaCl (pH
After washing with 7.4), elution was carried out with 20 mM Tris-acid buffer (pH 7.4) containing 0.3 M NaCl.

(5) Measurement of activity of peptide for promoting protein C activation by thrombin A solution containing the peptide purified in the above Example 1- (3) was used as a TM dilution buffer (0.1 M NaCl, 0.1%). Lub
roll PX (Sigma), 0.1% NaN ₃ , 0.0
5 μl of a solution containing 01% bovine serum albumin, 0.02 M Tris-HCl buffer (pH 7.5) and appropriately diluted, thrombin (Sigma, Catalog No. T-6759, 20 ng /
μl) 3 μl, 10 × assay buffer (1M NaCl
, Containing 30 mM CaCl ₂ , 1% bovine serum albumin,
5 μl of 0.5 M Tris-HCl buffer (pH 8.5) and 29.5 μl of distilled water were mixed and allowed to stand at 37 ° C. for 5 minutes, followed by addition of 7.5 μl of protein C (cattle-derived, 0.2 μg / μl) The reaction was carried out at 30 ° C. for 30 minutes. The reaction was stopped by adding 6.25 μl of stop solution (100 mM NaCl, 0.3 A ₂₈₀ antithrombin III, 100 μg / ml heparin-containing 20 mM Tris-HCl buffer pH 7.5).

The activity of activated protein C is as follows:
Eu-Ser-Thr-Arg-MCA (Peptide Institute, 5 mg / ml) 10 μl, 5M CsCl 5 μl Substrate reaction buffer (50 mM Tris-HCl buffer containing 100 mM NaCl) 495 μl, reacted at 37 ° C. for 20 minutes, and acetic acid After stopping the reaction by adding 55 μl, the AM released
The C (7-amino-7-methyl-coumarin) concentration was measured by a fluorescence spectrophotometer (Shimadzu RF-540) at an excitation wavelength of 380 nm and an emission wavelength of 440 nm. The obtained fluorescence intensity was compared with the fluorescence intensity of AMC of known concentration to determine the amount of free AMC. The value obtained by subtracting the AMC amount when an aqueous solution containing no peptide of the present invention was added from this AMC amount shows the strength of promoting protein C activation by thrombin in the sample. The activity of producing 1 nM of activated protein C per 1 ml of the reaction solution per minute was defined as 1 u.

(6) Quantification of Peptide (a) The amount of the peptide in the solution containing the peptide purified in Example 1- (3) was determined by the enzyme immunoassay (hereinafter referred to as ELISA) using an anti-human thrombomodulin monoclonal antibody. (Abbreviated). Details are given below. Preparation of anti-TM monoclonal antibody is performed by Mar
uyama et al. [J. Biol. Chem. 26
0, 15432 (1985)]. That is, Balb / C mice were immunized several times with TM purified from placenta as an antigen, spleen cell fluid of the mice was prepared, and mouse myeloma cells from a suitable line and a fusion accelerator such as polyethylene glycol were used. , Cell fusion.

Mouse myeloma cells used for cell fusion are, for example, P3-X63-Ag8-U1 cells (P3-U1).
[Yelton et al., Current. Topics i
nMicrobiology and Immunol
, 81, 1 (1978)] and the like are used. The fused cells are selected using an appropriate selection medium, for example, HAT (hypoxanthine aminopterin thymidine) medium. After the hybridoma cells are detected in this manner, the culture supernatant is collected and screened for an antibody against TM by ELISA (enzyme-linked immunosorbent assay) using TM as a solid phase antigen. Hybridoma cells producing antibodies against TM are cloned by a suitable method, such as the limiting dilution method. As a result, two types of anti-TM monoclonal antibodies were obtained, which were named anti-TM monoclonal antibodies 1 and 2, respectively.

(B) Plasmid M13mp19TMD3
Construction of the base sequence: 5′-GGAGGCCGCTCA instead of the deleter TMd1 using a site-directed mutagenesis method
Example 1 above except that the deleter TMd3 having ACAGTCGGTGCCA-3 '(25mer) was used.
The nucleotide sequence consisting of 285 bp of the recombinant plasmid M13mp19TMJ3 obtained in Example 1- (1) was deleted in substantially the same manner as in (1). This D
The NA fragment is 480 from the start codon (ATG) in the sequence listing.
It was found to have a base sequence containing a base sequence encoding a peptide consisting of amino acids up to. This DN
The A fragment was called TMD3, and the recombinant plasmid containing this DNA fragment was called M13TMD3.

FIG. 11 shows the recombinant plasmid M13mp1.
9 shows that TMJ3 and deleter TMd3 hybridize and a part of the DNA region corresponding to DNA fragment TMJ3 is deleted. Furthermore, using the site-directed mutagenesis method, instead of the deleter TMd1, the nucleotide sequence:
5'-GAAGCACGGGTCCGGGGAACCCC
Deleter TM with AGG-3 '(25mer)
In a manner substantially similar to that in Example 1- (1) above except that d5 was used, a part of the recombinant plasmid M13TMD3 was deleted to delete 1044 bases, and a set containing a DNA fragment called TMD7. Recombinant plasmid M13TM
D7 was obtained. This TMD7 is a start codon (A
TG) to the 18th amino acid, and 367 to the 4th
It had a base sequence containing a base sequence encoding a peptide consisting of the 80th amino acid.

FIG. 12 shows the recombinant plasmid M13TMD.
3 and the dilator TMd5 hybridize to DNA
A part of the DNA region corresponding to the fragment TMD3 is deleted. Furthermore, this recombinant plasmid M13
TMD7 DNA was digested to completion with HindIII and BamHI to isolate an approximately 580 bp DNA fragment of TMD7. On the other hand, the plasmid pSV2-dhfr (ATCC
37146) was completely digested with HindIII and BglII to obtain a vector.
The fragment was ligated with T4 DNA ligase to obtain plasmid pSV2TMD7.

(C) Transfection of COS-1 cells CO of pSV2TMD1 obtained in Example 1- (1) above and pSV2TMD7 obtained in Example 7- (a) above
Transfection of S-1 cells was performed according to the method of Example 1- (3) described above. The transfection of pSV2TMD1 was repeated 30 times to obtain about 300 ml of culture medium. Transfection of pSV2TMD7 was carried out once to obtain 10 ml of culture medium. The peptide produced by COS cells transfected with pSV2TMD1 was designated as D12.
3, the peptide produced by COS cells transfected with pSV2TMD7 was named E456.

(D) Measurement of activity for promoting protein C activation by thrombin Promotion of protein C activation by thrombin of the peptide contained in the culture solution obtained in the above Example 1- (7)-(b) The activity measured was according to the method of Example 1- (5) described above. The results are shown in Table 1. Strong activity was detected in all the culture solutions. No activity was detected in the culture supernatant of COS cells not transfected with the plasmid.

[0082]

[Table 1]

(E) Determination of epitope Anti-human TM obtained in the above-mentioned Example 1- (6)-(a)
The epitope of the monoclonal antibodies 1 and 2 was determined by the following method. The two types of culture solutions obtained in the above-mentioned Examples 1- (6)-(c) were diluted 2-fold and 4-fold with 0.1 M sodium bicarbonate buffer (pH 9.2), respectively, and then diluted to 50 μm.
Dispense into a 96-well flat-bottom ELISA microtiter plate (DYANATEC Co.) so that l / hole. After 3 hours, the wells of the plate were washed with 0.1 M sodium bicarbonate buffer (pH 9.2), and PBS containing 1% BSA was added.
To 100 μl / well and block overnight at 4 ° C. Plate each hole with 0.05% Tween
After washing 3 times with 20-containing PBS, 5 times the culture supernatant of the hybridoma containing anti-TM monoclonal antibody 1 or 2 was added.
Add 0 μl / well to react at 25 ° C. for 2 hours. Plate each well with 0.05% Tween in PBS
After washing three times with HRP-labeled anti-mouse IgG antibody (VEC
TOR LABORATORIES, INC. PI-2
000) is diluted 1000 times with PBS containing 1% BSA,
100 μl / well was added to each well and reacted at 25 ° C. for 1 hour.

Each well of the plate was washed 5 times with PBS containing 0.05% Tween, and then 100 μl of color developing solution (30 mg orthophenylenediamine added to 20 ml citrate phosphate buffer and 70 μl 30% hydrogen peroxide solution added). And add
When an appropriate color was obtained, 50 μl of a 4.1 M sulfuric acid solution was added to stop the reaction, and measurement was carried out at a measurement wavelength of 492 nm. The results are shown in Table 2. Color development was observed in the holes containing the culture solution containing D123 for both anti-TM monoclonal antibodies 1 and 2, but no color formation was observed in the holes containing the culture solution containing E456. It was No color was observed when a medium containing no monoclonal antibody was added. From the above results, it was revealed that the epitopes of anti-TM monoclonal antibodies 1 and 2 are portions other than E456, which is the active domain of TM.

[0085]

[Table 2]

(F) Preparation of Monoclonal Antibody Column Anti-TM monoclonal antibody 2 was subjected to salting out, ion exchange chromatography, and protein from ascites obtained by growing hybridomas in the abdominal cavity of histocompatibility animals, nude mice, etc. It was purified by a separation and purification operation such as A-column.
The anti-TM monoclonal antibody thus purified was
CNBr-activated Sepharo
se4B (Pharmacia, 52-1153-00-A
I) Pharmacia Manual (Affinity)
Chromatographinciples
& Methods) to produce a monoclonal antibody column.

The column thus prepared is anti-TM.
It was designated as Monoclonal Antibody Column 2. Example 1-
300 ml of the culture solution containing peptide TMD123 obtained in (7)-(b) was adsorbed on a Q Sepharose column equilibrated with 20 mM Tris-HCl buffer (pH 7.4) containing 0.15 M NaCl, and 20 mM Tris containing 0.18 M NaCl was added. After washing with hydrochloric acid buffer (pH 7.4), 0.3M NaC
Elution was carried out with 5 mM phosphate buffer (pH 7.4) containing l.
NaCl was added to this fraction to a final concentration of 0.5 M, and then the anti-TM monoclonal antibody column 2 prepared in the above step was equilibrated with 20 mM phosphate buffer (pH 7.4) containing 0.5 M NaCl. After that, the active fraction was adsorbed through this fraction, washed with the buffer used for equilibration, and then eluted with 0.2 M glycine / hydrochloric acid buffer (pH 4.0) containing 0.5 M NaCl to activate the active fraction. Fractions were obtained.

The molecular extinction coefficient of this purified product is 10.0 (E ^1% _{1 cm} · 280) in accordance with the molecular extinction coefficient of a general protein.
nm = 10.0), and the amount of the refined product based on it was calculated to be about 200 μg. In addition, the purified product was subjected to electrophoresis using SDS-polyacrylamide gel (SDS-PAG plate manufactured by Daiichi Pure Chemicals Co., Ltd.) having a polyacrylamide gel concentration of 10%, and CBB (Coomassie Brilliant Blue) staining was performed to observe bands. A single band with a molecular weight of 85 kDa was observed.

(G) Quantification by ELISA For the purified products of the peptides obtained in the above-mentioned Example 1- (4), the anti-TM monoclonal antibodies 1 and 2 obtained in Example 1- (6)-(a) were used. Quantitation was performed by the ELISA used. As the standard product of peptide D123, the purified product obtained in the above-mentioned Example 1- (6)-(f) was used.
That is, the purified product of anti-TM monoclonal antibody 1 obtained in Example 1- (6)-(a) was diluted with 0.1 M sodium bicarbonate buffer (pH 9.6) to 200 μg / ml, 96-well flat bottom ELISA with 50 μl / hole
Microtiter plate for A (DYNATECH)
Dispense into. After incubating at room temperature for 2 hours, plate the holes in 20
Wash 5 times with 0 μl PBS (Japanese water).

After that, 10% PBS containing 0.5% BSA was added.
Add 0 μl / well and let stand at room temperature for 2 hours.
Plate each well with 0.05% Tween80 in PBS
After washing 5 times with, the purified peptide obtained in the above-mentioned Example 1- (4) is diluted with PBS containing 0.05% Tween 80, 100 μl / well is added, and the mixture is reacted at 25 ° C. for 2 hours. Each well of the plate was washed 5 times with PBS containing 0.05% Tween, and then biotinylated anti-mouse IgG antibody (VECTOR LABORATORIES, INC.
BA-2000) was diluted 1000-fold with PBS containing 1% BSA, added to each well at 100 μl / well, and reacted at room temperature for 1 hour.

After washing each well of the plate 5 times with PBS containing 0.05% Tween, avidin D (Vector,
A-2004) was diluted 10000 times with PBS containing 1% BSA, added to each well at 100 μl / well, and reacted at room temperature for 1 hour. After washing each well of the plate 5 times with 0.05% Tween-containing PBS, 100 μl of coloring solution (30 mg ortho-phenylenediamine in 20 ml citrate phosphate buffer plus 70 μl 30% hydrogen peroxide solution) was added, When an appropriate color was obtained, 50 μl of a 4.1 M sulfuric acid solution was added to stop the reaction, and measurement was carried out at a measurement wavelength of 492 nm. E
The specific activity was calculated from the results of quantification by LISA and the results of activity measurement of Example 1- (5). The results are shown in Table 3.

TB1, T compared to unmutated D123
B2, TB3, TB4, TB5, TB7 showed a decrease in specific activity, and amino acid residues at these positions were protein C.
It is involved in the binding to thrombin in the process of promoting activation, and among them, particularly for TB3, TB4, and TB5, a marked decrease in the specific activity is observed, and thus the amino acid residues at these positions are essential for the binding. It became clear.

[0093]

[Table 3]

[0094]

According to the present invention, thrombomodulin having various strengths of thrombin binding can be obtained. As a result, the above-mentioned novel peptide having an inhibitory action on the blood coagulation and platelet aggregation action of thrombin and / or an action of promoting the protein C activation of thrombin was obtained.

[Sequence list]

SEQ ID NO: 1 Sequence length: 575 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu Ala Gly Leu Gly 1 5 10 15 Phe Pro Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu 20 25 30 His Asp Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala 35 40 45 Ser Gln Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser 50 55 60 Ser Val Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly 65 70 75 80 Val Gly Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys 85 90 95 Gly Asp Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr 100 105 110 Gly Asp Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn 115 120 125 Gly Ala Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu 130 135 140 Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val 145 150 155 160 Lys Ala Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg 165 170 175 Pro Leu Ala Va l Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr 180 185 190 Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro 195 200 205 Val Gly Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys 210 215 220 Thr Ala Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro 225 230 235 240 Gly Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys 245 250 255 Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala 260 265 270 Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys 275 280 285 Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly 290 295 300 Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln 305 310 315 320 His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys 325 330 335 Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr 340 345 350 Pro Asn Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro 355 360 365 Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr 370 375 380 Ser Tyr Leu Cys Va l Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu 385 390 395 400 Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp 405 410 415 Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile 420 425 430 Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly 435 440 445 Gly Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys 450 455 460 Ile Cys Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr Asp Cys 465 470 475 480 Asp Ser Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro 485 490 495 Pro Ser Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu 500 505 510 Val His Ser Gly Leu Leu Ile Gly Ile Ser Ile Ala Ser Leu Cys Leu 515 520 525 Val Val Ala Leu Leu Ala Leu Leu Cys His Leu Arg Lys Lys Gln Gly 530 535 540 Ala Ala Arg Ala Lys Met Glu Tyr Lys Cys Ala Ala Pro Ser Lys Glu 545 550 555 560 Val Val Leu Gln His Val Arg Thr Glu Arg Thr Pro Gln Arg Leu 565 570 575

[Brief description of drawings]

FIG. 1 shows that the recombinant plasmid M13mp19TMJ3 obtained in Example 1- (1) is hybridized with the deleter TMd1 in a complementary manner. The surrounding base sequence and the amino acid sequence encoded thereby are shown.

FIG. 2 shows that the mutator tb1 complementarily hybridizes with the recombinant plasmid M13TMD1 obtained in Example 1- (1), in which the mutator hybridizes with the plasmid to substitute amino acids. The nucleotide sequence around the portion where is occurring and the amino acid sequence encoded thereby are shown.

FIG. 3 shows that the mutator tb2 is complementary hybridized to the recombinant plasmid M13TMD1 obtained in Example 1- (1), in which the mutator hybridizes to the plasmid to substitute amino acids. The nucleotide sequence around the portion where is occurring and the amino acid sequence encoded thereby are shown.

FIG. 4 shows that the mutator tb3 is complementary hybridized to the recombinant plasmid M13TMD1 obtained in Example 1- (1), in which the mutator hybridizes to the plasmid to substitute amino acids. The nucleotide sequence around the portion where is occurring and the amino acid sequence encoded thereby are shown.

FIG. 5 shows that the mutator tb4 complementarily hybridizes to the recombinant plasmid M13TMD1 obtained in Example 1- (1), in which the mutator hybridizes to the plasmid and amino acid substitution occurs. The nucleotide sequence around the portion where is occurring and the amino acid sequence encoded thereby are shown.

FIG. 6 shows that the mutator tb5 complementarily hybridizes to the recombinant plasmid M13TMD1 obtained in Example 1- (1), in which the mutator hybridizes to the plasmid to substitute amino acids. The nucleotide sequence around the portion where is occurring and the amino acid sequence encoded thereby are shown.

FIG. 7 shows that the mutator tb6 complementarily hybridizes with the recombinant plasmid M13TMD1 obtained in Example 1- (1), in which the mutator hybridizes with the plasmid and amino acid substitution occurs. The nucleotide sequence around the portion where is occurring and the amino acid sequence encoded thereby are shown.

FIG. 8 shows that the mutator tb7 is complementary hybridized to the recombinant plasmid M13TMD1 obtained in Example 1- (1), in which the mutator hybridizes to the plasmid and amino acid substitution occurs. The nucleotide sequence around the portion where is occurring and the amino acid sequence encoded thereby are shown.

FIG. 9 shows that the mutator tb8 complementarily hybridizes to the recombinant plasmid M13TMD1 obtained in Example 1- (1), in which the mutator hybridizes to the plasmid and amino acid substitution occurs. The nucleotide sequence around the portion where is occurring and the amino acid sequence encoded thereby are shown.

FIG. 10 shows that the mutator tb9 complementarily hybridizes to the recombinant plasmid M13TMD1 obtained in Example 1- (1), in which the mutator hybridizes to the plasmid to substitute amino acids. The nucleotide sequence around the portion where is occurring and the amino acid sequence encoded thereby are shown.

FIG. 11 shows that the recombinant plasmid M13mpTMJ3 obtained in Example 1- (1) is complementarily hybridized with the deleter TMd3.
The nucleotide sequence around the portion where the deleter hybridizes with the plasmid and the amino acid sequence encoded thereby are shown.

FIG. 12 shows that the recombinant plasmid M13TMD3 obtained in Example 1- (6) is hybridized with the deleter TMd5 in a complementary manner. The surrounding base sequence and the amino acid sequence encoded thereby are shown.

Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12N 1/15 7236-4B 5/10 15/12 C12P 21/02 C 8214-4B // (C12N 1/15 C12R 1: 645) (C12P 21/02 C12R 1:91) (C12P 21/02 C12R 1: 645) C07K 99:00

Claims (10)

[Claims] 1. A polypeptide (A)-(B)-(C)-(D)-(C)-(E)-comprising the following amino acid sequence:
(C)-(F)-(C)-(G)-(C)-(H)-
(C) (where A and C are Asp or Glu and Gla
It is an amino acid residue consisting of only one or a peptide residue, or a peptide residue consisting of a combination of at least two or more selected from the group consisting of Asp and Glu and Gla. B is an arbitrary amino acid residue or peptide residue, and its length is 0 or more and 5 or less as the number of amino acid residues.
8 or less. D, E, F, G, and H are arbitrary amino acid residues or peptide residues, and their lengths are
The number of amino acid residues is 0 or more and 25 or less.
In addition, said Gla shows (gamma) -carboxyglutamic acid residue). 2. A polypeptide (A) -Pro-Cys-Phe-Arg-Ala-A having the following amino acid sequence:
sn-Cys-Glu-Tyr-Gln-Cys-Gl
n-Pro-Leu-Asn-Gln-Thr-Ser
-Tyr-Leu-Cys-Val-Cys-Ala-
Glu-Gly-Phe-Ala-Pro-Ile-P
ro-His-Glu-Pro-His-Arg-Cy
s-Gln-Met-Phe-Cys-Asn-Gln
-Thr-Ala-Cys-Pro-Ala-Asp-
Cys-Asp-Pro-Asn-Thr-Gln-A
la-Ser-Cys- (C)-(D)-(C)-
(E)-(C)-(F)-(C)-(G)-(C)-
(H)-(C) (where A and C are Asp or Glu and Gl
It is an amino acid residue consisting only of a or a peptide residue, or a peptide residue consisting of a combination of at least two or more selected from the group consisting of Asp and Glu and Gla. D, E, F, G, and H are arbitrary amino acid residues or peptide residues, and their lengths are 0 or more and 25 or less in terms of the number of amino acid residues. In addition, the G
la indicates a γ-carboxyglutamic acid residue). 3. A polypeptide (A) -Pro-Cys-Phe-Arg-Ala-A having the following amino acid sequence:
sn-Cys-Glu-Tyr-Gln-Cys-Gl
n-Pro-Leu-Asn-Gln-Thr-Ser
-Tyr-Leu-Cys-Val-Cys-Ala-
Glu-Gly-Phe-Ala-Pro-Ile-P
ro-His-Glu-Pro-His-Arg-Cy
s-Gln-Met-Phe-Cys-Asn-Gln
-Thr-Ala-Cys-Pro-Ala-Asp-
Cys-Asp-Pro-Asn-Thr-Gln-A
la-Ser-Cys- (C)-(D)-(C)-
(E)-(C)-(F)-(C)-(G)-(C)-
(H)-(C) -Cys-Ile-Cys-Gly-P
ro-Asp-Ser-Ala-Leu-Val-Ar
g-His-Ile-Gly-Thr-Asp-Cys (where A and C are Asp or Glu and Gla)
It is an amino acid residue consisting of only one or a peptide residue, or a peptide residue consisting of a combination of at least two or more selected from the group consisting of Asp and Glu and Gla. D, E, F, G, and H are arbitrary amino acid residues or peptide residues, and their lengths are 0 or more and 25 or less in terms of the number of amino acid residues. The Gl
a represents a γ-carboxyglutamic acid residue). 4. A DNA encoding the polypeptide according to claim 1, 2 or 3 and a recombinant DNA incorporating the DNA. 5. A microorganism or animal cell transformed with the recombinant DNA according to claim 4. 6. A microorganism belonging to a filamentous fungus among the microorganisms transformed with the recombinant DNA according to claim 4. 7. An Acremonium among microorganisms transformed with the recombinant DNA according to claim 4.
A microorganism belonging to chrysogenum. 8. A method for producing the polypeptide according to claim 1, 2 or 3 by culturing a microorganism or animal cell transformed with the recombinant DNA according to claim 4. 9. The culture broth obtained according to claim 8. 10. The polypeptide produced in claim 8, and at least one pharmaceutically administrable carrier, diluent or excipient, which inhibits thrombin's blood coagulation and platelet aggregation effects. And / or a pharmaceutical composition characterized by having an action of promoting protein C activation of thrombin.