CN107760660B - A tissue-type plasminogen activator mutant and its application - Google Patents

Abstract

The present invention provides a tissue-type plasminogen activator mutant and application thereof. The mutant comprises that the A146 site is a completely new mutation site, and at least one or more amino acid residues including A146 are substituted, deleted or Add to. The mutant of the present invention has the ability to significantly resist the inhibition by its endogenous inhibitor (PAI-1) and the enhanced ability to activate plasminogen. It can be applied to prepare medicines for treating thrombotic diseases, including acute myocardial infarction, acute pulmonary embolism, cerebral apoplexy, venous thrombosis and other diseases.

Description

A tissue-type plasminogen activator mutant and its application

technical field

The invention belongs to the field of biomedicine, in particular to a tissue-type plasminogen activator mutant and its application.

Background technique

With the rapid development of society and economy, people's quality of life is improving day by day. Among them, high-sugar, high-protein and high-fat foods account for an increasing proportion of people's daily nutritional intake, which leads to the formation of diseases such as hypertension, hyperlipidemia, coronary heart disease, myocardial infarction, cerebral infarction, etc. Health poses a huge threat. At present, cardiovascular disease has become the number one killer that threatens human health, especially thromboembolic disease. It mainly includes three categories: (1) coronary thrombosis, mainly referring to acute myocardial infarction (AMI); (2) cerebrovascular thrombosis, namely acute ischemic stroke; (3) venous thrombosis, such as acute pulmonary embolism. According to statistics, about 900,000 people suffer from AMI every year in the United States, and 225,000 of them die. Due to the rapid time from onset to death, about 125,000 of them have died without timely treatment. In my country, according to statistics, 3 million people need thrombolytic drug treatment every year. Furthermore, most acute myocardial infarctions result from insufficient blood flow and myocardial death due to closed coronary artery embolization. Thrombolytic therapy is an important method for the treatment of thrombotic diseases. Thrombolytic drugs are used to dissolve thrombus, increase myocardial blood flow, improve oxygen supply, reduce infarct size, and improve brain nerve cell damage in ischemic area, so that clinical treatment can be greatly improved. a substantial increase. Several international large-scale clinical trials have confirmed that thrombolytic therapy can reduce the mortality rate of AMI by more than 30%.

Tissue plasminogen activator (tPA) is a physiological activator of the fibrinolytic system in the blood. Because tPA has a strong specific affinity with the thrombus matrix fibrin, it can effectively activate plasminogen to plasmin locally in the thrombus to dissolve the thrombus. Compared with thrombolytic agents such as streptokinase and urokinase, tPA has the advantages of fast action, no systemic fibrinolysis, and high treatment. Natural tPA is composed of 527 amino acids, including five domains: 1. The finger region (F region, amino acids 4-49) is related to fibrin binding. 2. The epidermal growth factor region (E region, amino acids 50-86) is related to the binding of cell membrane receptors. 3. kringle 1 (K1 region, 87-175 amino acids) is the binding site of the liver receptor. 4. kringle 2 (K2 region, 176-261 amino acids) contains a fibrin binding site. 5. The hydrolase domain region (SPD region, amino acids 262-527) catalyzes the conversion of plasminogen into plasmin, and is the binding site for PAI-1.

Tissue plasminogen activator (tPA) was approved by the US FDA in 1987 as the first genetically engineered recombinant thrombolytic drug for the treatment of acute myocardial infarction. In 1990, the US FDA approved it for the treatment of acute pulmonary embolism. In 1999, the FDA approved it again for the treatment of acute ischemic stroke. And it is currently the only thrombolytic drug for acute ischemic stroke.

However, the clinical application of tPA also shows some limitations. Among them, a large amount of plasminogen activator inhibitor (PAI-1) is mainly enriched in the lesion site, which leads to the irreversible inhibition of the therapeutic drug tPA by PAI-1, which rapidly inactivates it. In clinical practice, in order to ensure the therapeutic effect, a dose of up to 100 mg/50 kg is usually injected into the patient. High doses of tPA not only pose a risk of fatal intracranial hemorrhage, but also lead to neurotoxic side effects, thereby increasing patient mortality. In addition, during thrombus formation, a large number of platelets are contained in the thrombus. Accompanied by the stimulation of thrombin, platelets are continuously activated, releasing a large amount of PAI-1, which also leads to a great decrease in the titer of tPA drugs in the blood. Although a TNK-tPA mutant with resistance to PAI-1 has been developed. However, the KHRR ^296-299 mutation contained in the TNK-tPA mutant to AAAA is not sufficient for resistance to PAI-1 and does not show enhanced resistance to PAI-1. Ability to activate plasminogen. Therefore, clinically, there is still a huge need to develop new tPA drugs, which can be used to treat acute myocardial infarction, cerebral thrombosis, pulmonary embolism and other diseases, and further improve their application value in medicine.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a tissue-type plasminogen activator mutant and its application, which have the ability to significantly resist the inhibition by its endogenous inhibitor (PAI-1) and the enhanced ability to activate plasminogen .

To achieve the above object, the present invention adopts the following technical solutions:

The mutation point is located in the hydrolase domain A146 of tPA (amino acid nomenclature is Chymotrypsinogennumbering). The tPA mutant includes substitution, deletion or addition of at least one or more amino acid residues including A146.

Based on the hydrolase domain of tPA, A146 is mutated to Y as an example (ie; tPA(A146Y)), wherein the amino acid sequence of SEQ ID NO. 1 is: IKGGLF ADIAS HPWQA AIFAK HRRSP GERFL CGGIL ISSCW ILSAAHCFQE RFPPH HLTVI LGRTY RVVPG EEEQK FEVEK YIVHK EFDDD TYDND IALLQ LKSDS SRCAQESSVV RTVCLPPAD LQLPD WTECE LSGYG KHEYL SPFYS ERLKE AHVRL YPSSR CTSQH LLNRTVTD NMLCA GDTRS GGPQA NLHDA CQGDS GGPLV CLNDG RMTLV GIISW GLGCG QKDVP GVYTKVTNYL DWIRD NMRP.

The obtained tPA(A146Y) mutant recombinant protein improved the PAI-1 resistance by 75% and the plasminogen activation ability by 5 times compared with the wild type.

The above-mentioned amino acid at position 146 is Ala and can be substituted to include Tyr and Gly, Val, Leu, Met, IIe, Ser, Thr, Pro, Asn, Gln, Phe, Trp, Lys, Arg, His, Asp, Glu any of the . In the examples of the present invention, it has been proved that the substitution of the amino acid at position 46 of Ala to Tyr can not only significantly improve the ability to resist PAI-1, but also enhance the ability to activate plasminogen.

The advantages of the present invention are:

The present invention is a mutant of tissue-type plasminogen activator (tPA), which has remarkable resistance to inhibition by its endogenous inhibitor (PAI-1) and enhanced ability to activate plasminogen. This mutant includes the A146 (amino acid nomenclature as Chymotrypsinogen numbering) site, a completely new mutation site that has never been reported before. We confirmed through the experiments of the examples that the tPA(A146Y) mutant recombinant protein not only significantly improved the resistance to PAI-1 by 75%, but also exhibited a new function, that is, the ability to activate plasminogen by 5 times. The mutant can be used to prepare drugs for treating thrombotic diseases, including acute myocardial infarction, acute pulmonary embolism, stroke, and venous thrombosis.

Description of drawings

Figure 1: SDS-PAGE electropherogram after protein purification, where M is marker, 1 is tPA protein, and 2 is tPA(A146Y) mutant protein.

Figure 2: Detection of tPA (A146Y) mutant enzyme activity by chemiluminescent substrate assay.

Figure 3: Evaluation of tPA(A146Y) mutants for their ability to activate plasminogen.

Figure 4: Detection of PAI-1 resistance of tPA(A146Y) mutants.

Figure 5: In vitro thrombolysis assay. A is that in normal blood, tPA (A146Y) has a significantly stronger and faster thrombolytic capacity than tPA. B is that in the case of adding exogenous active PAI-1, the thrombolytic ability of tPA (A146Y) was not affected at all, while the activity of tPA-SPD was completely inhibited. This indicated that the tPA(A146Y) mutant protein had very strong PAI-1 resistance to promote the dissolution of thrombus.

Detailed ways

The method and advantages of the present invention will be further described below with reference to the accompanying drawings and embodiments, but these embodiments do not limit the scope of the claims of the present invention. Without departing from the scope of the main features of the present invention, the present invention can also have various changes and improvements, and these changes and improvements all fall within the protection scope of the present invention.

Example 1 Construction, expression and purification of tPA(A146Y) mutant

The tPA(A146Y) here is a mutation of A146Y based on the hydrolase domain of tPA (tPA-SPD), also referred to as tPA(A146Y), or tPA-SPD(A146Y) (amino acid naming is Chymotrypsinogennumbering):

(1) Construction of tPA-SPD-pPICZαA plasmid.

Using human hepatocyte cDNA as template, the tPA-SPD gene fragment was amplified by PCR method. The tPA-SPD fragment was cut with restriction enzymes XhoI and SacI, and the pPICZαA plasmid (pPICZαA plasmid was purchased from Invitrogen) was cut with the same endonucleases XhoI and SacI, and the tPA-SPD fragment was ligated into pPICZαA plasmid with T4 ligase middle. The enzyme-linked product was transformed into Escherichia coli DH5α by thermal excitation at 42°C, plated on a plate, single colonies were picked, and gene sequencing was performed. OMEGA) to extract the tPA-SPD-pPICZαA plasmid for use in the following experiments.

(2) Construction and protein expression of tPA(A146Y)-pPICZαA mutant plasmid.

The template for PCR performed here was the tPA-SPD-pPICZαA plasmid obtained in (1) above.

Primer design: Sense: 5'-CAAGCATGAGTACTTGTCTCCTTTCTATTC-3';

Antisense: 5'-GAATAGAAAGGAGACAAGTACTCATGCTTG-3'.

1 µL of DpnI (Takara) was added to the above PCR product and incubated overnight at 37°C. PCR products were recovered by gel using the ZDNA gel recovery kit (OMEGA). Transform into Escherichia coli DH5α by heat excitation at 42°C, spread the plates, pick single clones for sequencing, and store the strains containing the correct mutation in 15% glycerol in a -80°C refrigerator until use.

The above-preserved glycerol bacteria were picked for activation and expansion in LB medium, and the tPA-A146Y-pPicZαA plasmid was extracted by ZDNA plasmid mini-pump kit (OMEGA). Linearize the extracted plasmid.

37°C overnight. Ethanol precipitation recovery.

The recovered DNA fragments were electroporated into Pichia pastoris X-33 strain: 1.5KV, 0.6S. Spread on YPD (containing 100 µg/ml Zeocin) plate, pick single colonies, and verify the expression in small quantities.

A small amount of successfully expressed Pichia X-33 was inoculated into YPD (containing 100 µg/ml Zeocin) for 1 day at 28°C, inoculated into BMGY medium at 1:10, expanded at 28°C for 1 day, and cultured at 1:4 Inoculated in BMMY medium to induce expression, and continued to culture for 3 days, supplemented with 1% methanol every day. The bacteria were collected, centrifuged at 10,000 rpm for 30 min, and then the supernatant was filtered through a filter membrane with a pore size of 0.45 μm to obtain a suction filtrate. The suction filtrate was passed through a Ni-NTA column (GE Company, flow rate 5 ml/min, column volume 25 ml) that had been equilibrated with equilibration solution (20 mM Tris-HCl pH 7.4, 500 mM NaCl), and the suction filtrate contained Six His-tagged tPA (A146Y) proteins bind to the Ni-NTA column. The Ni-NTA column was further washed to remove impurities with five more column volumes of equilibration solution (20 mM Tris-HCl pH 7.4, 500 mM NaCl). Finally, the protein was eluted with eluent (20 mM Tris-HCl pH 7.4, 500 mM NaCl, 500 mM imidazole) and the collected protein was eluted with dialysate (20 mM Tris-HCl pH 7.4, 150 mM NaCl) Dialysis was performed twice for at least 2 hours each. After dialysis, the pellet was removed by centrifugation (20,000 rpm, Hitachi Koki CR22N high-speed refrigerated centrifuge, R20A2 rotor) for 20 minutes. The target protein was identified by SDS-PAGE electrophoresis. tPA(A146Y) has a molecular weight of 28KDa, as shown in Figure 1, and the sequence obtained by sequencing is shown in SEQ ID NO.1. The protein solution obtained from the analysis was concentrated with Millipore ultrafiltration tube (10000Da), then divided into packages and stored at -80°C for later use.

(3) The rest of the tPA-based mutants are based on the above example, on the basis of tPA (A146Y), design appropriate primers, change the denaturation temperature and annealing time during the PCR process, transformation and expression, purification process such as (2) conduct. At the same time, the tPA protein was obtained through the same transformation, expression and purification methods described above.

Example 2 Activity detection of tPA(A146Y) mutant enzyme

Enzyme activity assays were performed using a reported chromogenic assay [Gorlatova NV (2003). Mapping of a conformational epitope on plasminogen activator inhibitor-1 by randommutagenesis. Implications for serpin function. J Biol Chem. ]. The reaction principle is as follows. A certain concentration of tPA protein is added to the reaction system with a volume of 200 µL. Then add the luminescent substrate S2288 (Chromogenix), tPA can specifically recognize the enzyme cleavage site and cut off its chromophore - p-nitroaniline (pNA), and finally detect the absorbance value at 405nm by a microplate reader. The activity of the tPA enzyme can be measured.

The specific measurement process:

(a) Materials

tPA obtained by the above Example 1, tPA(A146Y), tPA substrate S-2288.

Buffer: 20mM Tris-HCl pH7.4, 150mM NaCl, 0.2% BSA. Filtration with 0.22μm pore size filter.

(b) Step

The mass concentration of each protein was measured with BCA protein quantification kit, and then converted into molar concentration. tPA and tPA(A146Y) were diluted to 200 nM, and 320 μM S-2288 was formulated. Add to the 200 μl reaction system as needed to prepare the final concentration required for the experiment. The samples were added in the following order:

①180μl buffer+10μl tPA+10μl 320μM S-2288

②180μl buffer +10μl tPA(A146Y)+10μl 320μM S-2288

The luminescent substrate S2288 (Chromogenix) was added at the end and immediately placed in a BioTek Synergy 4 microplate reader for detection at 405 nm, 30 s/read for 30 min. Each test was repeated at least 3 times. The average value was used for linear fitting of the reaction using GraphPad Prism 5 software, and PBS was used as a blank control.

The results are shown in Figure 2. The results show that the cleavage reaction rates of the mutant tPA (A146Y) and tPA to the luminescent substrate S2288 are basically the same, indicating that the mutant tPA (A146Y) and tPA have the same enzymatic activity, and it also indicates that the point mutation of A146Y , and did not affect the activity of tPA enzyme.

Example 3 Detection of the activating ability of tPA(A146Y) on natural substrate plasminogen

The assay was performed using a reported chromogenic assay [Gorlatova NV (2003). Mapping of a conformational epitope on plasminogen activator inhibitor-1 by randommutagenesis. Implications for serpin function. J Biol Chem. ]. The reaction principle is as follows. In a reaction system with a volume of 200 µL, a certain concentration of tPA and plasminogen (PLG) are added, and tPA activates PLG to generate plasmin (plasmin, abbreviated as Pn), and then adds Pn-specific The luminescent substrate S-2403, Pn can specifically recognize the enzyme cleavage site and cleave its chromophore -p-nitroaniline (pNA), while tPA does not cleavage the luminescent substrate S2403. Finally, the ability of tPA to activate PLG can be determined by detecting the absorbance value at 405nm by a microplate reader.

The specific measurement process:

(a) Materials

tPA obtained in Example 1 above, tPA (A146Y), and PLG, Pn substrate S-2403.

Buffer: 20mM Tris-HCl pH7.4, 150mM NaCl, 0.2% BSA. Filtration with 0.22μm pore size filter.

(b) Step

The mass concentration of each protein was measured with BCA protein quantification kit, and then converted into molar concentration. tPA-SPD and tPA-SPD(A146Y) were diluted to 200 nM to make 1 μM PLG, 320 μM S-2403. Add to the 200 μl reaction system as needed to prepare the final concentration required for the experiment. The samples were added in the following order:

①170μl buffer+10μl PLG+10μl tPA+10μl 320μM S-2403;

②170μl buffer+10μl PLG+10μl tPA(A146Y)+10μl 320μM S-2403;

The luminescent substrate S2403 (Chromogenix) was added at the end and immediately placed in a BioTek Synergy 4 microplate reader for detection at 405 nm, 30 s/read for 30 min. Each test was repeated at least 3 times. Data processing was performed using GraphPad Prism 5 software with PBS as blank control.

The results are shown in Figure 3. The results show that the activating ability of tPA(A146Y) on PLG is increased by 5 times, indicating that the A146Y mutation significantly enhances the activating ability of tPA on the natural substrate PLG.

Example 4 Detection of tPA(A146Y) resistance to PAI-1

The assay was performed using a reported chromogenic assay [Gorlatova NV (2003). Mapping of a conformational epitope on plasminogen activator inhibitor-1 by randommutagenesis. Implications for serpin function. J Biol Chem. ]. The reaction principle is as follows. In a reaction system with a volume of 200µL, tPA is added to react with different concentrations of PAI-1 for 10 minutes, tPA will be inhibited by PAI-1, and then S-2288 is added, and tPA that is not bound to PAI-1 will be digested S-2288 and release the chromophore-p-nitroaniline (pNA), and finally detect the absorbance value at 405nm by a microplate reader to determine the enzymatic activity of the residual tPA, and indirectly calculate the resistance of tPA to PAI-1 ability.

The specific measurement process:

(a) Materials

tPA obtained in Example 1 above, tPA(A146Y), and PAI-1, tPA substrate S-2444.

Buffer: 20mM Tris-HCl pH7.4, 150mM NaCl, 0.2% BSA. Filtration with 0.22μm pore size filter.

(b) Step

The mass concentration of each protein was measured with BCA protein quantification kit, and then converted into molar concentration. tPA and tPA(A146Y) were diluted to 200 nM, and 320 μM S-2288 was formulated. Eight concentrations of PAI-1 were used; 40nM, 80nM, 120nM, 160nM, 200nM, 4800nM, 560nM, and 600nM. Add to the 200 μl reaction system as needed to prepare the final concentration required for the experiment. The samples were added in the following order:

①180μl buffer+10μl tPA+10μl 320μM S-2288;

②180μl buffer +10μl tPA(A146Y)+10μl 320μM S-2288;

③170μl buffer+10μl tPA+10μl PAI-1(40nM--600nM)+10μl 320μM S-2288;

④170μl buffer +10μl tPA(A146Y) +10μl PAI-1(40nM--600nM)+10μl 320μMS-2288;

Among them, tPA or tPA(A146Y) and PAI-1 were pre-incubated for 10 min, then mixed thoroughly and avoid generating air bubbles as much as possible, and finally added the luminescent substrate S2288 (Chromogenix) and immediately put it into the BioTek Synergy 4 microplate reader at 405nm, 30s/read for detection for 30min. Each test was repeated at least 3 times. Data processing was performed using GraphPad Prism5 software.

The results are shown in Figure 4. The results show that when tPA is completely inhibited by PAI-1, tPA(A146Y) still has 75% activity, indicating that the A146Y mutation significantly improves the resistance of tPA to PAI-1.

Example 5 tPA-SPD(A146Y) improves clot lysis by resisting the inhibition of PAI-1

Thromb lysis ability was tested using a reported clot lysis assay [Peng SZ (2017). A long-acting PAI-1 inhibitor reduces thrombus formation. Thromb Haemost. ]. Since the addition of Ca ²⁺ will activate the blood coagulation reaction and form a thrombus, thereby increasing the turbidity of the reaction system, the absorbance value at 405nm has the largest change. The reaction principle is as follows. In a reaction system with a volume of 200 µL, 30% human serum and a certain concentration of tPA are added, and then a certain amount of PAI-1 is added to react for 10 minutes, and then Ca ²⁺ is added. Finally, the thrombus-dissolving ability of tPA was evaluated by detecting the change of the absorbance value at 405 nm by a microplate reader.

The specific measurement process:

(a) Materials

tPA, tPA(A146Y), and PAI-1, CaCl ₂ solutions obtained in Example 1 above.

Human serum (abbreviation: PPP) was obtained from blood from healthy volunteers by centrifugation at 3500 rpm for 10 min.

Buffer: 20mM Tris-HCl pH7.4, 150mM NaCl. Filtration with 0.22μm pore size filter.

(b) Step

The mass concentration of each protein was measured with BCA protein quantification kit, and then converted into molar concentration. tPA and tPA(A146Y) were diluted to 100 nM, and a 100 mM CaCl ₂ solution was prepared. PAI-1 was used at a concentration of 120 nM. Add to 100 μl reaction system as needed to prepare the final concentration required for the experiment. The samples were added in the following order:

①65μl buffer+30μl PPP+5μl 100mM CaCl ₂ ;

②55μl buffer+30μl PPP+10μl tPA+5μl 100mM CaCl ₂ ;

③55μl buffer+30μl PPP+10μl tPA(A146Y)+5μl 100mM CaCl ₂ ;

④45μl buffer+30μl PPP+10μl tPA+10μl PAI-1+5μl 100mM CaCl ₂ ;

⑤45μl buffer+30μl PPP+10μl tPA(A146Y)+10μl PAI-1+5μl 100mM CaCl ₂ ;

④⑤Incubate tPA/tPA(A146Y) with PAI-1 for 10min, then mix thoroughly and try to avoid bubbles, finally add Ca ²⁺ , mix well, put it into BioTek Synergy 4 microplate reader at 405nm, 30s/read for detection for 60min. Each test was repeated at least 3 times and the average value was taken. Data processing was performed using GraphPad Prism 5 software.

The results are shown in Fig. 5. The results show that in the condition of no exogenous PAI-1 added in Fig. 5A, tPA(A146Y) obviously has a faster and stronger ability to dissolve thrombus than tPA. Under the condition of adding exogenous PAI-1 in Figure 5B, the ability of tPA(A146Y) to dissolve thrombus was not affected at all, but tPA was completely inhibited, indicating that tPA(A146Y) had significant PAI-1 resistance.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

SEQUENCE LISTING

<110> Fuzhou University

<120> A tissue-type plasminogen activator mutant and its application

<130> 1

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 252

<212> PRT

<213> Artificial sequences

<400> 1

Ile Lys Gly Gly Leu Phe Ala Asp Ile Ala Ser His Pro Trp Gln Ala

1 5 10 15

Ala Ile Phe Ala Lys His Arg Arg Ser Pro Gly Glu Arg Phe Leu Cys

20 25 30

Gly Gly Ile Leu Ile Ser Ser Cys Trp Ile Leu Ser Ala Ala His Cys

35 40 45

Phe Gln Glu Arg Phe Pro Pro His His Leu Thr Val Ile Leu Gly Arg

50 55 60

Thr Tyr Arg Val Val Pro Gly Glu Glu Glu Gln Lys Phe Glu Val Glu

65 70 75 80

Lys Tyr Ile Val His Lys Glu Phe Asp Asp Asp Thr Tyr Asp Asn Asp

85 90 95

Ile Ala Leu Leu Gln Leu Lys Ser Asp Ser Ser Arg Cys Ala Gln Glu

100 105 110

Ser Ser Val Val Arg Thr Val Cys Leu Pro Pro Ala Asp Leu Gln Leu

115 120 125

Pro Asp Trp Thr Glu Cys Glu Leu Ser Gly Tyr Gly Lys His Glu Tyr

130 135 140

Leu Ser Pro Phe Tyr Ser Glu Arg Leu Lys Glu Ala His Val Arg Leu

145 150 155 160

Tyr Pro Ser Ser Arg Cys Thr Ser Gln His Leu Leu Asn Arg Thr Val

165 170 175

Thr Asp Asn Met Leu Cys Ala Gly Asp Thr Arg Ser Gly Gly Pro Gln

180 185 190

Ala Asn Leu His Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu Val

195 200 205

Cys Leu Asn Asp Gly Arg Met Thr Leu Val Gly Ile Ile Ser Trp Gly

210 215 220

Leu Gly Cys Gly Gln Lys Asp Val Pro Gly Val Tyr Thr Lys Val Thr

225 230 235 240

Asn Tyr Leu Asp Trp Ile Arg Asp Asn Met Arg Pro

245 250

Claims (2)

1. A tissue-type plasminogen activator mutant, characterized in that: the mutant site of the mutant is located at the hydrolase structural domain Ala146 site of tPA, and the amino acid sequence of the mutant is such as SEQ ID NO. 1 shown. 2. The application of a tissue-type plasminogen activator mutant as claimed in claim 1 in the preparation of a medicament for treating thrombotic diseases.

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