JPH06133780A - New modified t-pa - Google Patents

Abstract

PURPOSE:To provide a new modified t-PA having lowered activity before being incised with plasmin compared with natural t-PA and exhibiting an activity comparable or superior to natural t-PA when incised with plasmin near the thrombus. CONSTITUTION:This modified t-PA contains a sequence to be recognized and incised with plasmin at the C-terminal of t-PA and has a peptide added to the C-terminal of the sequence.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel tissue plasminogen activator (hereinafter, t-PA) variant. More specifically, by adding a sequence for recognition / cleavage by plasmin to the C-terminal of t-PA and further adding a peptide to the C-terminal side thereof, plasminogen activating activity was obtained before the cleavage by plasmin. Is lower than that of natural t-PA, but exhibits a similar activity to that of natural t-PA or more by being cleaved by plasmin in the vicinity of the thrombus. .

[0002]

2. Description of the Related Art t-PA is converted to plasmin which is an active enzyme by hydrolyzing plasminogen which is an inactive enzyme precursor present in plasma. Plasmin decomposes fibrin clots (thrombus) in blood vessels caused by various causes (Reference 1). While existing thrombolytic agents such as urokinase and streptokinase have no fibrin specificity, t-PA has a relatively high fibrin specificity. Therefore, it has been attracting attention as a substance that specifically dissolves fibrin and eventually thrombus. However, when t-PA was actually used clinically, it became clear that recanalization of the occluded coronary artery requires a much higher dose than expected (Reference 2). As a result of this large dose, the effect of the fibrin specificity of t-PA is diminished, and the side effect of generalized bleeding tendency eventually occurs, which remains a problem that cannot be ignored.

[0003] t-PA is converted into a double chain by being decomposed by plasmin between the arginine at the 275th position and the isoleucine at the 276th position from the N-terminal. The N-terminal side of the converted product is called the A chain and the C-terminal side is called the B chain. The active center of t-PA exists in the B chain having a small molecular weight and has a structure similar to that of other serine enzymes such as trypsin. In the case of a general serine enzyme, a single chain is a precursor and does not have an enzymatic activity, but it is said that a specific peptide bond is hydrolyzed and becomes active only when it becomes a double chain. But t-P
In the case of A, not only double-stranded but also single-stranded t-PA has enzymatic activity (Reference 3, 4, 5). Therefore, single-stranded t-
When PA is administered, the single-chain t-PA is circulated in the circulating blood before it reaches the thrombus (“solid phase”) and is converted into the double chain by plasmin localized near the thrombus. It is believed that this also contributes to the side effect of generalized bleeding tendency, as it also activates the middle (“liquid phase”) fibrinolytic system.

Under these circumstances, attempts have been made to develop a so-called prodrug-like t-PA which is inactive in the "liquid phase" and becomes active after reaching the thrombus site. For example, in References 6 and 7, t-PA differs from other serine proteases in that salt-bri between specific amino acids.
It is presumed that the single-chain type has activity because of the occurrence of dging, and salt-b in such a single-chain state is
Attempts have been made to prevent single-chain activity by substituting amino acids involved in rigging.

[0005]

DISCLOSURE OF THE INVENTION According to the present invention, a sequence for recognition / cleavage by plasmin is added to the C-terminal of t-PA, and a peptide is added to the C-terminal side thereof to be cleaved by plasmin. Previously, i.e. in the single-stranded state, plasminogen activating activity was associated with native t-PA.
After being cleaved by plasmin, but at a lower level, ie, in the double-stranded state, it exerts an activity similar to or higher than that of natural t-PA, resulting in the concentration of plasmin at the thrombus site, in other words, concentration of plasmin. Novel t-PA that exhibits high activity under a controlled environment (Reference 8)
The purpose is to provide a variant. T-PA of the present invention
Since the modified form has a lower plasminogen activating activity in the single-chain state than natural t-PA, a side effect associated with large dose administration which was a problem when using natural t-PA, that is, systemic bleeding tendency Is expected to be reduced.

[0006]

Means for Solving the Problems As a result of investigations by the present inventors, by adding a sequence for recognition and cleavage by plasmin to the C-terminal of t-PA, and further adding a peptide to the C-terminal side, The activity in the single-stranded state is natural t-P
It was found that new t-PA variants lower than A were obtained. Furthermore, such a t-PA variant undergoes recognition and cleavage by plasmin, resulting in the natural t-PA.
It was also found to exhibit activity similar to or higher than.

That is, the gist of the present invention is that (1) the C-terminal of t-PA has a sequence added for recognition / cleavage by plasmin, and a peptide is added to the C-terminal side. T-PA variant, (2) DNA containing a nucleotide sequence encoding the t-PA variant described in (1) above, (3) transformed prokaryotic cell or eukaryotic organism A recombinant expression vector capable of expressing the DNA of (2) above in a cell; (4) a prokaryotic or eukaryotic cell transformed with the recombinant expression vector of (3); and (5) ) A therapeutic agent for thrombosis, which comprises the t-PA variant described in (1) above as an active ingredient.

In the present invention, the "C-terminal of t-PA" refers to the 527th proline residue of natural t-PA. Further, in the present invention, the “sequence that is recognized and cleaved by plasmin” is, for example, Gln-Phe-Arg-I.
A possible amino acid sequence is le-Lys-Gly-Gly (SEQ ID NO: 1). In the present invention, the “peptide added to the C-terminal side of the sequence that is recognized and cleaved by plasmin” is not particularly limited. However, since this t-PA variant is cleaved by plasmin at the thrombus site, the added peptide is released,
It is more preferable that the released peptide has some anticoagulant effect. That is, it is considered preferable to add a peptide or the like that inhibits the activity of the coagulation factor thrombin. Such a peptide is
Since thrombin activity is expected to be suppressed after release, it is expected that clinically problematic re-occlusion can be improved. Examples of such peptides include:
Specific examples thereof include the E456 domain of thrombomodulin, the C-terminal partial sequence of hirudin, or a sequence similar thereto (SEQ ID NOS: 2 and 3).

Thrombomodulin is a glycoprotein present on vascular endothelial cells, and binds thrombin generated in blood with a very high affinity to form a complex, thereby forming protein C.
Increase the rate of conversion of the enzyme to anticoagulant. That is, thrombomodulin has an action of converting thrombin from a coagulation enzyme to an anticoagulation enzyme. A human thrombomodulin cDNA has been reported, and a chemical structure diagram is provided (Reference 9). In addition, analysis of the functional site of thrombomodulin revealed that thrombin was added to the 4th to 6th repeating sequence portions (amino acid sequence portion from the N-terminal to the 345th to 462nd positions-hereafter E456 domain) of the 6 repeating sequences of the cysteine-rich domain. It has been reported that there is a function that binds to and significantly increases the activation of protein C (References 10, 11, 12, 13).

Hirudin is a hill (Hirudo media)
Cyanalis) is a polypeptide consisting of 65 amino acids purified from the head and is known to bind to thrombin and inhibit the activity of thrombin to convert fibrinogen to fibrin (Reference 14). It is said that the action of thrombin is inhibited by binding between the fibrinogen binding site of thrombin and the C-terminal side of hirudin. In the experiment in which a plurality of C-terminal analogs of hirudin were synthesized and their properties were investigated (Reference 15 , 16),
C consisting of only 12 amino acids as shown in SEQ ID NO: 4
It has been pointed out that the terminal peptide has a significant antithrombin effect. Furthermore, natural hirudin has a sulfonated tyrosine that is the 63rd amino acid, but it has been found that recombinant hirudin without this modification also has thrombin inhibitory activity (Reference 1).
7).

The t-PA variant of the present invention is prepared by t-P
It can be carried out by adding an amino acid sequence that is recognized and cleaved by plasmin to the C-terminal of A, and further adding a peptide as described above to the C-terminal side. Specifically, for example, a cDNA encoding a modified C-terminal amino acid sequence in which these amino acid sequences to be added (plasmin recognition sequence and peptide) are inserted immediately after the C-terminal of natural t-PA, or a synthetic oligonucleotide is a natural t-PA. −
It can be carried out by substituting the corresponding sequence of PA, ligating it with an appropriate expression vector, introducing this into a host, and expressing / producing a modified t-PA which is the final target substance of the present invention.

For the production of the modified t-PA of the present invention, a cDNA encoding naturally occurring t-PA can be used. The cDNA encoding native t-PA has been cloned, for example, from Bose melanoma cells by
For example, it has already been obtained as described in Reference 18, and therefore its cDNA sequence and amino acid sequence are also known. In addition, in order to modify natural t-PA, for example, modified t-PA having a long half-life (References 19, 20, etc.) c
DNA sequences and amino acid sequences are also known, and these can also be used.

T-PA cloned in a plasmid
The method of substituting a specific sequence of cDNA or inserting it into a specific portion is already in a freely available state, and in the present invention, an amino acid sequence is inserted into the C-terminal region of t-PA by a conventional method. can do. As a method of insertion, a method of inserting a cDNA sequence using a restriction enzyme site is convenient. The cDNA sequence to be inserted may be a synthetic DNA having a restriction enzyme site required for insertion at both ends, or may be a product obtained by introducing a restriction enzyme site required for insertion at both ends of a cDNA fragment and amplifying this. DNA synthesis is based on the principle of the phosphoramidite method (Reference 21), Applied Biosystems model 38.
It may be carried out using a 1A DNA synthesizer. Further purification may be performed using the OPC cartridge for purifying oligonucleotides manufactured by the same company. When the cDNA of the sequence to be inserted or its similar sequence exists naturally, it is also a simple method to use the cDNA fragment amplified by using an appropriate oligonucleotide as a primer for the insertion as described above. Let's do it. A polymerase chain reaction (PCR) method, for example, is effectively used as a method for amplifying a region sandwiched by two kinds of primers (Reference 22). That is, two kinds of oligonucleotides are used as primers, and cDNA is used as a template to amplify DNA by Taq polymerase. To perform this operation, a commercially available automatic DNA amplification device is effectively used.

Further, in order to confirm the desired substituted or inserted DNA sequence, the principle of the dideoxy method (Reference 23) can be used, for example, the method of cycle sequence using PCR method. It includes the Applied Biosystems model 373AD.
The NA sequencer is effectively used. In addition, all of the genetic manipulations such as the cleavage and deletion of DNA by restriction enzymes as described in Examples, the separation, collection, and ligation of DNA fragments produced by them by polyacrylamide gel electrophoresis are well known, and mainly Reference 24 is made.

The thus obtained cDNA encoding the modified t-PA is ligated to a well-known vector such as CDM8 (ori ⁻ ) and then introduced into an appropriate host to give the cDNA of the present invention. It is possible to express and produce the t-PA variant protein which is the final target substance. Escherichia coli strains and animal cell strains as hosts are already widespread and easily available, unless otherwise specified, and examples of animal cell hosts include COS-1 and CHO cells. To transform an appropriate host with the expression plasmid, the electric pulse method (Reference 25) may be used. The culture supernatant of the transformed cells contains the t-PA variant in such an amount that it can be used as it is for various activity measurements by appropriate dilution.

The t-PA variant of the present invention thus obtained has a plasminogen activating activity in the single-stranded state lower than that of natural t-PA, but in the double-stranded state. Since it shows an activity equivalent to that of natural t-PA, it is a natural t-PA.
It can be used as an active ingredient of a pharmaceutical composition having a reduced systemic bleeding tendency. The t-PA variant of the present invention is
It can be extremely easily obtained as a preparation by dispensing a required amount into a container and lyophilizing it by a conventional method. This lyophilizate is dissolved in a pharmaceutically acceptable carrier substance, for example saline, and administered by intravenous or intraarterial or intracardiac injection. The administration method is a continuous intravenous injection or a method in which a part of the planned dose is first intravenously injected and the rest is continuously intravenously injected.

[0017]

EXAMPLES Examples will be shown below as examples of the present invention, but the present invention is not limited thereto.

1. Construction of plasmid pUC-D01 having a sequence considered to be recognized and cleaved by plasmin in the C-terminal region of t-PA A conceptual diagram of this construction method is shown in FIGS. 1 and 2. First, reference 2
6. The plasmid pCDM8-000 (Sal ⁺ ) described in 6.
(The t-PA expression plasmid pCDM8- described in Reference 28)
000 t-PA cDNA sequence having a SalI site introduced at the 10th to 15th bases downstream of the translation termination codon) was digested with SmaI and SalI, and 55-base-long fragment SS was subjected to polyacrylamide gel electrophoresis. After purification and isolation, this and plasmid pUC118 (Takara Shuzo Co., Ltd.) were digested with SmaI and SalI and ligated, and Escherichia coli JM109 strain (Toyobo Co., Ltd.) was transformed to obtain plasmid pUCSS.
Got Next, this plasmid was digested with EcoT14I, the ends were blunted with Klenow fragment, and self-ligation was performed to obtain plasmid pUC.
SS (ET ^-) was obtained (Figure 1).

On the other hand, 6 kinds of oligonucleotide DC
(SEQ ID NO: 5), DCR (SEQ ID NO: 6), D0 (SEQ ID NO: 7), D0R (SEQ ID NO: 8), IKGG (SEQ ID NO: 9), and IKGGR (SEQ ID NO: 10) were synthesized.
The 381A type DNA synthesizer manufactured by Applied Biosystems was used for the synthesis of these oligonucleotides, and the purification was performed using the OPC cartridge of the same. DC and DCR, D0 and DOR, and IKGG and IKGGR were mixed in equimolar amounts and annealed to form a double strand. Next, these three types of double-stranded DNA
Was ligated (ligated) with DNA ligase to obtain fragment A. Thereafter, this fragment A was amplified by a PCR reaction using DC and IKGGR as primers. The amplified fragment A was digested with BstEII and SalI, and the 82-base long fragment BS was purified and separated by polyacrylamide gel electrophoresis by a standard method (FIG. 2). And this fragment BS, previous plasmid pUCSS (ET ^-) was ligated as previously digested with BstEII and SalI, which in Escherichia coli JM109 strain was transformed plasmid pUC-D
0I was obtained (Fig. 2).

2. Construction of DNA Fragments HS and HL Encoding C-Terminal Similar Sequences of Hirudin.
Two kinds of fragment DNA, fragment HS and fragment HL, having a paired end with oT14I were prepared. Fragment HS encodes an amino acid as shown in SEQ ID NO: 2. This is Leu-Gly-A at the N-terminal side of the 54th to 64th amino acid sequence (from the N terminus) of hirudin consisting of 65 amino acids in total length.
It encodes a peptide with the addition of the sn sequence. The fragment HL encodes the amino acid as shown in SEQ ID NO: 3, which encodes a peptide in which Leu is added to the N-terminal side of the amino acid sequence from the 42nd to the C-terminal (65th) of hirudin. . Specifically, the above-mentioned double-stranded fragment HS and fragment HL were prepared by synthesizing oligonucleotides and annealing them. The synthesized oligonucleotide is P
-HSM (SEQ ID NO: 11), P-HSR (SEQ ID NO: 1)
2), P-HLM (SEQ ID NO: 13), and P-HLR
(SEQ ID NO: 14).

Synthetic oligonucleotides P-HSM and P-
Fragment HS and fragment HL annealed in combination with HSR, P-HLR and P-HLR
Got Annealing was carried out by mixing 77.7 ng of each synthetic oligonucleotide, making it 7 μl with water, treating at 65 ° C. for 30 minutes, and then returning to room temperature (25 ° C.) for about 1 hour. Phosphorylation buffer [Tris.HCl (pH 7.6) 0.6
6M, ATP10mM, spermidine10m
M, MgCl ₂ 0.1 M, DTT 150 mM, gela
tin 2 mg / ml] and T4 DNA k
Add 2 μl (2 units) of inase, 37 ℃
The ones that had been reacted at 1 hour for 1 hour and were phosphorylated at the ends were used as the fragments HS and HL in the subsequent experiments.

3. A plasmid pUC- having a portion of t-PA cDNA having a peptide addition at the C-terminus.
Construction of CTM, pUC-CHS, and pUC-CHL The C-terminal of t-PA has a sequence that is considered to be recognized and cleaved by plasmin, and E4 is present at the C-terminal side.
T-PA cDNA to which the amino acid sequence of 56 domain or the amino acid sequence similar to the C-terminal region of hirudin described above was added
Construction of a plasmid having a portion of This includes Fragment SE (Reference 27), Fragment H described above.
S and the fragment HL to the plasmid pUC described above.
It was carried out by using the EcoT14I restriction site present in -D01 and inserting it downstream of the putative plasmin recognition / cleavage sequence in pUC-D01. Fragment SE
Is a PCR-amplified double-stranded DNA fragment that encodes the amino acid sequence of the E456 domain of thrombomodulin and has SpeI at the gene upstream end and EcoT14I paired end at the gene downstream end.
It is a digestion product of eI and EcoT14I. Plasmid pU
A conceptual diagram of C-CTM, pUC-CHS, and pUC-CHL construction is shown in FIG.

First, the plasmid pUC-D01 was transformed into E.
After digestion with coT14I, the 5'end was dephosphorylated. Fragments SE, HS, and HL are each
Since it has ends that pair with the EcoT14I site of pUC-D01 at both ends (the ends of SpeI and EcoT14I pair with each other), EcoT14 of plasmid pUC-D01 is present.
The I digest and the fragments SE, HS, and HL were ligated, respectively, and E. coli JM109 strain was transformed with this. It was confirmed that each fragment was inserted in the same orientation as the t-PA cDNA, and these were designated as plasmids pUC-CTM (inserted fragment SE), pUC-CHS, and pUC-CHL, respectively (Fig. 3).

4. Expression plasmids pCDM8-CTM, pCDM8 encoding t-PA containing a modified C-terminal region
-CHS and construction of pCDM8-CHL Expression plasmid pCD containing all the translation regions of t-PA
M8-000 (Sal ⁺ ) is t-P described in Reference 28.
T-PAcDN of A expression plasmid pCDM8-000
A SalI site was introduced at the 10th to 15th bases (CCCGAC) downstream of the translation stop codon (TGA) on the A sequence (Reference 26). This pCDM8-000
Expression plasmids pCDM8-CTM, pCD encoding t-PA containing a modified C-terminal region using (Sal ⁺ )
Construction of M8-CHS and pCDM8-CHL was performed. pUC-CTM, pUC-CHS, and pUC-
Since the region of CHL other than the C-terminal insertion region has exactly the same structure, the following operations can be performed in the same manner for these three types of constructions. As an example of the construction, a conceptual diagram of the construction method of pCDM8-CTM is shown in FIG.

First, the plasmid pCDM8-000 (S
al ⁺ ) was digested with BstEII and SalI and 5 ′
The ends were dephosphorylated. On the other hand, the plasmid pUC-
CTM, pUC-CHS, and pUC-CHL to Bs
t-PAcD after digestion with tEII and SalI
The BstEII / SalI fragment containing the modified C-terminal region of NA was purified and separated by polyacrylamide gel electrophoresis. Bs of plasmid pCDM8-000 (Sal ⁺ )
tEII / SalI digestion product, pUC-CTM, pUC
-CHS, and BstEII / Sa of pUC-CHL
The E. coli MC1061 / p3 strain (manufactured by Funakoshi Yakuhin Co., Ltd.) was ligated with each of the II digested fragments and the expression plasmids pCDM8-CTM, pCDM8- encoding t-PA containing the modified C-terminal region.
CHS and pCDM8-CHL were obtained (FIG. 4). These three types of plasmids contained the entire region of t-PA structural gene, and recognized plasmin at the C-terminal of t-PA.
Cleavage deduced amino acid sequence Gln-Phe-Arg-Ile
-Lys-Gly-Gly followed by E45
The 6-domain sequence or the hirudin-like amino acid sequence shown in SEQ ID NO: 2 or 3 is added.

5. COS of thrombolytic protein of the invention
-1 Expression and secretion in cells pCDM expressing native t-PA as a control
8-000 (Sal ⁺ ), and pCDM8-CTM,
COS-1 cells were transformed with pCDM8-CHS and pCDM8-CHL by the electric pulse method described above. Fetal bovine serum 10% and aprotinin 10 μg / m
After culturing in a DMEM medium containing 1 for 24 hours, the medium was replaced with a serum-free and aprotinin-free DMEM medium, and the culturing was continued for another 96 hours. The culture solution was centrifuged and the supernatant was recovered, and 1/9 volume of 1% bovine serum albumin (BSA) -containing DMEM medium was added to the culture solution and stored frozen. This cryopreserved supernatant was called a t-PA sample and used for the subsequent evaluation.

6. Quantification of protein amount The protein amount of the t-PA sample in each culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (manufactured by Biopool).

7. Assay (Assay) Method The plasminogen activating activity of the thrombolytic protein of the present invention is based on an indirect chromogenic assay (Reference 29) using a synthetic chromogenic substrate S-2251, which is mold specific to plasmin (Kabi). Synthetic tripeptide chromogen substrate S-2251 (HD-Val-Leu manufactured by K.K.)
Was measured quantitatively in _{-Lys-pNA · 2HCl · H 2} O) following method used. Further, in the above-mentioned Example 5. The t-PA sample obtained in 1. exists as single-chain t-PA. The double-stranded activity was measured by using a double-stranded t-PA sample double-stranded by the following method. That is, 10 kinds of various single-stranded t-PA samples (final 50 ng / ml) were used.
What was incubated at 37 ° C. for 30 minutes in the presence of nM plasmin was used as a double-stranded t-PA sample below.

1 of each variant diluted to 50 ng / ml
200 μl of 20 μl of double-stranded and double-stranded t-PA samples
1 reaction mixture [plasminogen 125 nM, S-2
251 350 μM, fibrin fragment 0.3 m
g / ml, Tween800.1%, Tris · HCl
(PH 7.8) 0.15 M, and gelatin 5 mg / m
1] and incubated at 37 ° C., and the absorbance at 405 nm was measured with time using a plate reader (manufactured by Molecular Device). Data analysis was performed using Softmax, the analysis software of the same company, to determine the activity per constant amount of protein. The results are shown in Table 1.

[Table 1]

Reference 1) European Patent Application Publication No. 0041766 A2 2) The TIMI study group: N. Engl. J. Med., 320, 6
18, 1989 3) DC Rijiken et al .: J. Biol. Chem., 256, 703
5, 1981. 4) M. Ranby et al .: Thromb. Res., 27, 175, 1982. 5) L. Haggroth et al .: Thromb. Res., 42, 585, 198
6.6) P. Wallen et al .: Eur. J. Biochem., 132, 681,
1983 7) European Patent Application Publication No. 89307194.4 8) B. Wiman et al .: Nature, 272, 549, 1978. 9) K. Suzuki et al .: EMBO J., 6, 1891, 1987. 10) CT Esmon et al .: J. Biol. Chem., 6, 3352,
1989. 11) Japanese Patent Laid-Open No. 2-19399, 12) M. Zushi et al .: J. Biol. Chem., 26, 10351,
1989. 13) Japanese Patent Laid-Open No. 2-255699 14) F. Markwardt: Method Enzymol., 19, 924, 197
0.15) JM Maraganore et al .; J. Biol. Chem., 264,
8682, 1989. 16) JA Jakubowski et al .: Blood, 72, 326A, 198.
8 (abstr). 17) E. Degryse et al .: Protein Eng., 2, 459, 198.
9. 18) Pennica et al .: Nature, 301, 214, 1983. 19) Special Table No. 63-501335 20) S. Yoshitake et al .: Thromb. Haemostasis, 6
2, 542, 1982. 21) SL Beaucage et al .: Tetrahedron Letters-22
(20), 1859, 1981. 22) HA Erlich et al .: PCR Technology, 1990. 23) F. Sanger et al .: Proc. Natl. Acad. Sci. US
A, 74, 5463, 1977. 24) T. Maniatis et al .: Molecular Cloning, A Lab
oratory Manual, Cold Spring Harbor Laboratory. 198
2 25) Shinichiro Takayama, Cell Engineering 6, 771, 1987. 26) Japanese Patent Application No. 4-125595 27) Japanese Patent Application No. 3-29624 28) Japanese Patent Application No. 2-87005 29) JH Verheijen et al .: Thromb Haemostasis,
48 (3), 266, 1982.

[0031]

[Sequence list]

 SEQ ID NO: 1 (plasmin recognition sequence) Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gln Phe Arg Ile Lys Gly Gly

SEQ ID NO: 2 (sequence encoded by fragment HS) Sequence length: 14 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Leu Gly Asn Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu

SEQ ID NO: 3 (sequence encoded by fragment HL) Sequence length: 25 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Leu Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn Asp Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Gln

SEQ ID NO: 4 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence

[Chemical 1]

SEQ ID NO: 5 (Oligonucleotide DC) Sequence Length: 15 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Antisense: No Sequence AAGGTTACCA ACTAC 15

SEQ ID NO: 6 (Oligonucleotide DCR) Sequence Length: 21 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Antisense: Yes Sequence GTCTAGGTAG TTGGTAACCT T 21

SEQ ID NO: 7 (Oligonucleotide D0) Sequence Length: 39 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Antisense: No Sequence CTAGACTGGA TTCGTGACAA CATGCGACCG CAGTTTCGC 39

SEQ ID NO: 8 (Oligonucleotide D0R) Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Antisense: Yes Sequence CGGTCGCATG TTGTCACGAA TCCA 24

SEQ ID NO: 9 (Oligonucleotide IKGG) Sequence Length: 38 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Antisense: No Sequence ATCAAAGGAG GCCTAGGATG ACCAGGAACA GTCGACTC 38

SEQ ID NO: 10 (oligonucleotide IKGGR) Sequence length: 47 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence GAGTCGACTG TTCCTGGTCA TCCTAGGCCT CCTTTGATGC GAAACTG 47

SEQ ID NO: 11 (primer P-HSM) Sequence length: 46 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No sequence CTA GGC AAC GGC GAT TTC GAA GAA ATT CCA GAA GAA TAT CTA TAG C 46

SEQ ID NO: 12 (primer P-HSR) Sequence length: 46 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence CTAGG CTA TAG ATA TTC TTC TGG AAT TTC TTC GAA ATC GCC GTT GC 46

SEQ ID NO: 13 (primer P-HLM) Sequence length: 79 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No sequence CTA GGA GAA GGA ACA CCA AAA CCA CAA TCA CAC AAC GAT GGA GAT TTC 48 GAA GAA ATT CCA GAA GAA TAT TTA CAA TAG C 79

SEQ ID NO: 14 (primer P-HLR) Sequence length: 79 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence CTAGG CTA TTG TAA ATA TTC TTC TGG AAT TTC TTC GAA ATC TCC ATC GTT 50 GTG TGA TTG TGG TTT TGG TGT TCC TTC TC 79

[Brief description of drawings]

FIG. 1 shows a part of a schematic assembly diagram of a plasmid pUC-D01 having a sequence considered to be recognized and cleaved by plasmin in the C-terminal region of t-PA.

FIG. 2 shows a part of the schematic assembly diagram of plasmid pUC-D01 having a sequence considered to be recognized and cleaved by plasmin in the C-terminal region of t-PA.

FIG. 3: Plasmids pUC-CTM, pUC-CHS, pUC having a peptide addition at the C-terminus of t-PA.
The assembly schematic diagram of -CHL is shown.

FIG. 4 shows a schematic diagram of assembly of the expression plasmid pCDM8-CTM.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12N 1/21 7236-4B 5/10 // C12P 21/02 H 8214-4B (C12N 1/21 (C12R 1:19) (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:19) (C12P 21/02 C12R 1:91)

Claims (8)

[Claims] 1. A t-PA characterized in that a tissue plasminogen activator has a sequence at the C-terminal which is recognized and cleaved by plasmin, and a peptide is further added at the C-terminal side. A variant. 2. The modified t-PA according to claim 1, wherein the peptide added to the C-terminus is a polypeptide consisting of 150 amino acid residues or less. 3. The modified t-PA according to claim 2, wherein the peptide added to the C-terminus has a sequence known to suppress thrombin activity. 4. The t-PA variant according to claim 3, wherein the peptide added to the C-terminus is the E456 domain of thrombomodulin, or a C-terminal partial sequence of hirudin or a sequence similar thereto. 5. A D containing a nucleotide sequence encoding the t-PA variant according to any one of claims 1 to 4.
NA. 6. A recombinant expression vector capable of expressing the DNA according to claim 5 in a transformed prokaryotic cell or eukaryotic cell. 7. A prokaryotic cell or a eukaryotic cell transformed with the recombinant expression vector according to claim 6. 8. A therapeutic agent for thrombosis, which comprises the modified t-PA according to any one of claims 1 to 4 as an active ingredient.