JP4220985B2 - Angiogenesis inhibitors containing amino acid sequence fragments obtained by using angiostatin converting enzyme with novel conversion selectivity as active ingredients - Google Patents

Description

  INDUSTRIAL APPLICABILITY The present invention relates to an angiogenesis inhibitor comprising as an active ingredient a bacterial Bacillus megaterium A9542 strain (Accession No. FERM P-18268), a novel protein (enzyme) originating from the bacterium, and a plasminogen fragment produced by the action of the enzyme. An agent (hereinafter, a plasminogen fragment effective in inhibiting angiogenesis) or a thrombolytic agent (hereinafter, a plasminogen fragment effective in thrombolysis is referred to as a miniplasminogen-like molecule), Plasma serine protease group activator comprising the enzyme as an active ingredient, and a method for producing angiostatin-like molecule and miniplasminogen-like molecule using the enzyme as a substrate for plasminogen, particularly novel conversion selection Amino acid sequence fragment obtained by using angiostatin converting enzyme The present invention relates to an angiogenesis inhibitor or a thrombolytic agent.

In recent years, angiogenesis inhibitors have attracted attention as new cancer treatment strategies. Research on angiogenesis in cancer has recently received much attention. It has been elucidated that when cancer cells proliferate to a size of 1-2 mm ³ , more oxygen and nutrients are required to grow larger. Cancer cells explode using oxygen and nutrients in the blood by releasing angiogenic factors called angiogenic factors, and creating new blood vessels from nearby blood vessels and drawing them into the tumor. The growth rate can be increased. In addition, distant metastasis via this intratumoral microvessel is possible.

JP-A-4-178328, Claims JP-A-6-234645, Claims Japanese Patent Laid-Open No. 10-81631, Claims

  Looking at the role of angiogenesis in cancer growth, it can be easily understood that inhibiting angiogenesis of cancer leads to suppression of cancer proliferation, invasion, and metastasis. In fact, many drugs that inhibit angiogenesis have been developed and proposed as antitumor agents (Patent Documents 1-3, etc.). However, no drug has been approved to date as an angiogenesis inhibitor. The development of therapeutic agents that target angiogenesis has the potential to prevent metastasis as well as increase in primary tumors. One of these, angiostatin, has been known to selectively suppress vascular endothelial cell proliferation, migration, and lumen formation, cut off oxygen and nutrient supply, and cause tumor dormancy. (American Dr. Folkman et al. Discovered Angiostatin and proposed this concept.) Angiostatin has a longer half-life in the circulating blood than angiogenesis-promoting substances, so it is thought that the inhibitor concentration is dominant in metastases far from the primary lesion and suppresses metastatic growth. . It is also known to increase endothelial cell apoptosis. Angiostatin is a peptide having a molecular weight of about 38 kDa, including plasminogen kringle 1-4, which is a fibrinolytic factor. Conventionally, angiostatin is produced in vitro by limited degradation of plasminogen by elastase, or by acting on plasminogen activator, inducing it to plasmin, and then acting on a reducing agent such as glutathione. The In vivo, angiostatin conversion is considered to occur by several types of matrix metalloproteinases. Conventional techniques for producing angiostatin can be broadly classified into (1) a method using an esterase that degrades the protein, and (2) a method of producing in E. coli using a recombinant DNA technique. However, according to the conventional production method, there are problems such as low selectivity for conversion to angiostatin, poor reproducibility of the activity of the obtained angiostatin, and difficulty in purification.

  The problem to be solved by the present invention is basically the problem of the substance that acts as angiostatin (angiostatin-like molecule), and the method for producing the substance, particularly the method for decomposing the plasminogen with an enzyme. The point is to find an enzyme that converts to angiostatin with improved substrate specificity. In order to find the improved enzyme, the present inventor searched for a protein released from various soils as a metabolite of a microorganism having the desired conversion property to angiostatin. And the bacteria which discharge | release the protein with the said substrate specificity as a metabolite from the soil of Higashimoto-cho, Kokubunji-shi, Tokyo were discovered. The inventor was entrusted on March 21, 2001, with the bacterium designated as A9542 and as the accession number FERM P-18268 at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology.

The present invention relates to an amino acid sequence fragment from Glu1 to Ser441, an amino acid sequence fragment from Phe75 to Ser441, which is obtained by limited degradation of human plasminogen with an enzyme having the amino acid sequence described in SEQ ID NOs: 1 and 2, Glu1 to Val449, And an angiogenesis inhibitor comprising all four types of amino acid sequence fragments of Phe75 to Val449 .

As an effect of the invention, by using the novel enzyme, plasminogen is selectively converted into an amino acid sequence fragment that functions as an angiogenesis inhibitor or a thrombolytic agent, and the angiogenesis inhibitor comprises the amino acid sequence as an active ingredient. It can also be mentioned that a thrombolytic agent can be obtained.

The present invention will be described in more detail. A. The bacterium of the present invention was obtained by isolating from soil collected in Higashimoto-cho, Kokubunji, Tokyo, and was cultured under the culture conditions described in the examples, from the culture solution to the angiostatin-like fragment of plasminogen. A microorganism producing an enzyme that catalyzes the conversion was found and identified as follows. B, producing the enzymes described in SEQ ID NOs: 1 and 2 as mycological characteristics 1, metabolite. 2. It is a gonococcus from microscopic observation. 3. The size of the bacteria is about 3-6 × 0.8-1.0 μm. 4. Gram-stained by gram staining and gram-positive. 5. From spore staining to spore-forming bacteria. 6. From the catalase test, it is positive for catalase. 7. It is aerobic because it cannot grow under anaerobic conditions. 8. Produced from sugar (D-(+)-glucose, L-(+)-arabinose, D-(+)-xylose, D-(-)-mannitol) on an agar medium. From the above characteristics, the A9542 strain is consistent with Bacillus megaterium. Therefore, this bacterium was designated as Bacillus megaterium A9542 strain.

9. The gene of this bacterium showed 97% homology with the Bacillus megaterium nprM gene. This bacterium produces a protein having an amino acid sequence (as shown in SEQ ID NO: 2) in which a difference of 11 amino acids is observed with respect to the nprM gene product (NprM) at the translation product level. Therefore, the translation product of the A9542 strain was designated as basilolysin MA.

C, Growth conditions of the bacterium 1, medium composition: Brain heart infusion (Nissui 05508), cow brain extract powder 21%, peptone 28.6%, heart extract powder 23%, glucose 5.7%, NaCl 14.3 %, Hydrogen phosphate-potassium 7.7% 2, medium pH: 7.0, 3, medium sterilization conditions 121 ° C., 15 minutes, 4, culture temperature: 28 ° C.
D, the fungus of the present invention is entrusted to the Biological Deposit Center of the National Institute of Advanced Industrial Science and Technology (at the time of entrustment, the Patent Biological Deposit Center of the Biotechnology Institute of the National Institute of Advanced Industrial Science and Technology) as the deposit number (FERM P-18268). ing.

Separation and culture of strain 1, isolation 1 g of soil collected in Higashimoto-cho, Kokubunji, Tokyo, added to 5 mL of sterilized water, stirred well, further diluted to 10 ^-4 (w / v), and cultured by the following operation The present invention was conducted by searching for microorganisms that release the activity of producing angiostatin-like fragments from plasminogen as metabolites.

Starch-casein-agar medium (corn starch 1%, casein 0.03%, KNO ₃ 0.2%, NaCl 0.2%, K ₂ HPO ₄ 0.2%, MgSO4 · 7H _{2 O 0.005%, CaCO 3 0.002} %, FeSO 4 · 7H 2 O 0.001%, agar L.5%, nystatin (nystatin) 0.005%), was used. The diluted solution (0.1 mL) was applied on the medium in the petri dish and cultured at 28 ° C. for 6 days. The resulting colonies were picked and cultured on a storage slant composed of starch-casein-agar medium at 28 ° C. until appropriate growth, and then stored at 4 ° C.

2. The following medium was used for liquid culture of the fungus selected for liquid culture. 1% glucose, 3% corn starch, 1% soy bean meal, 0.5% peptone, 0.5% yeast extract, 0.2% CaCO ₃ , 0.01% CB442, pH 7. 0. A test tube (21 × 210 mm) containing 10 mL of a liquid medium was inoculated from above the storage slant using a platinum loop, and cultured at 28 ° C. for 6 days with shaking (220 strokes / min).

Selection of microorganisms that produce metabolites that produce angiostatin-like fragments from plasminogen For the search for microorganisms that produce the activity to produce angiostatin-like fragments from plasminogen, sodium dodecyl sulfate (SDS) -polyacrylamide gel Electrophoresis (SDS-PAGE) [Nature 227, 680-685 (1979)] was used. 5 μL of plasminogen ( ¹²⁵ I-Glu-Plg) (final concentration 100 nM, 20000 cpm) radiolabeled on 1 μL of 90% methanol extract of microbial metabolites, uPA (urokinase type plasminogen activator) ) (Final concentration 200 units / mL) was added in 5 μL aliquots, and TBS / T buffer (50 mM Tris-HCl, 100 mM NaCl, 0.01% (w / v) Tween 80, pH 7.4) at 37 ° C. for 30 minutes. Incubated. Thereafter, 10 μL of sample buffer (3.6% SDS, 3.6% mercaptoethanol, 0.08% bromophenol blue, 900 mg / mL urea) was added, and the mixture was treated in a hot water bath at 60 ° C. for 30 minutes. The gel was applied to% gel, fixed, dried and subjected to autoradiography to observe the band pattern. A sample obtained by reacting only plasminogen and uPA was used as a target sample. As a result of screening about 1,500 strains of actinomycetes, bacteria and fungi isolated from the soil by this method, strong activity was observed in the culture extract of Bacillus megaterium A9542 (2. in FIG. 1). FIG. 1 shows the results obtained in the reaction (control) without the microorganism culture extract, 2 the results obtained with the culture extract of Bacillus megaterium A9542 strain, and 3 in the present invention. The result obtained by reaction containing the culture extract of some fungi other than a strain is shown.

Purification of a protein having the amino acid sequences of SEQ ID NOs: 1 and 2 (Basilolysin MA: BLMA) from the culture solution of Bacillus megaterium A9542 strain In a 500 mL Erlenmeyer flask containing 100 mL of the above liquid medium, Bacillus megaterium MA A9542 strain After shaking culture for 1 day, 3 L of the culture broth was filtered using Celite (auxiliary for facilitating passage of the filtrate), 1 L of the filtrate was diluted to 5 L with H ₂ O, and isopropyl alcohol was diluted to a final concentration of 5 % (V / v) and then 400 mL of carboxymethylcellulose gel equilibrated with 20 mM MES (2- [N-Morpholino] ethanolsulfonic acid) -NaOH (pH 6.5) / 5% isopropyl alcohol. CM-Cellulose, Biochemical Engineer Applied to the column at a flow rate of 15 mL / min. After washing with 600 mL of the same buffer, elution was performed with 20 mM MES-NaOH (pH 6.5) / 5% isopropyl alcohol / 0.2 M NaCl. The eluted fractions were fractionated 60 mL each, and active fractions were collected. The purity was confirmed by SDS-PAGE (FIG. 2), and 90 mg of purified product was obtained. BLMA undergoes limited degradation when secreted from this bacterium, and the enzyme protein obtained by this production method is a molecule having the sequence of amino acid numbers Val ¹ to Gln ³¹⁷ described in SEQ ID NO: 1. However, the BLMA according to the present invention may be a molecule further comprising any part from Pro-1 to Gln-254 of the sequence set forth in SEQ ID NO: 1.

Generation of angiostatin-like and miniplasminogen-like fragments from plasminogen by BLMA (Figure 3)

Limited degradation by BLMA using plasminogen (Glu-Plg) as a substrate was observed (measurement: SDS-PAGE). 6 μL of Glu-Plg (final concentration 2 μM), 6 μL of BLMA (final concentration 0, 3.7, 37 nM) were incubated in TBS / T buffer containing CaCl ₂ (final concentration 1 mM) at 37 ° C. for 60 minutes. Non-reducing SDS sample buffer (x4) was added, 15 μL of which was applied to SDS-10% gel. After completion of electrophoresis, it was stained with Coomassie brilliant blue R-250 and dried. As a result, plasminogen was cleaved in a BLMA concentration-dependent manner (55% at BLMA 3.7 nM and 86.5% at 37 nM plasminogen was cleaved (FIG. 3A)). In addition, plasminogen cleavage by BLMA was performed under the conditions of serum 50% and BL concentration 0-1000 nM. 2 μL of 125I-Glu-Plg, 3 μL of BLMA (final concentration 0, 10, 100, 1000 nM), 5 μL of human serum were added, incubated at 37 ° C. for 60 minutes, and 90 μL of water was added thereto. 5 μL was taken from there, and 5 μL of reduced SDS sample buffer (x2) was added thereto. 5 μL of that was applied to 12.5% gel and electrophoresed. After completion of the electrophoresis, the film was subjected to autoradiography overnight, and then the film was developed. Again, it was found that cleavage progresses in a BL concentration-dependent manner to produce angiostatin-like fragments [FIG. 3B, BLMA concentrations are 1 nM, 2 is 10 nM, 3 is 100 nM, and 4 is 1000 nM. ]].

Next, the N-terminal amino acid sequences of the cleaved fragments (fragments 1-5) of FIG. 3A were identified, and the results are shown in Table 1. From this result, BLMA cuts Ser ^441- Val ⁴⁴² (FIG. 3C, arrow 3), Leu ^74- Phe ⁷⁵ (FIG. 3C, arrow 1), and Va1 ^449- Leu ⁴⁵⁰ (FIG. 3C, arrow 2) of Glu-P1g. Fragments of plasminogen molecule, Glu ¹ -Ser ⁴⁴¹ , Glu ¹ -Val ⁴⁴⁹ , Phe ⁷⁵ -Ser ⁴⁴¹ , Phe ⁷⁵ -Val ⁴⁴⁹ (above angiostatin-like fragment) and Val ⁴⁴² -Asn ⁷⁹¹ , Leu ⁴⁵⁰ -Asn ⁷⁹¹ (mini-plasminogen-like fragment) was found to be generated.

Production of angiostatin-like and miniplasminogen-like fragments of plasminogen produced by BLMA 2 mL Glu-PLG (final concentration 2 μM) in 2 mL CaCl ₂ (final concentration 1 mM) and 4 mL BLMA (final concentration 36. 8 nM) was added, incubated in TBS / T buffer at 37 ° C. for 60 minutes, and 220 μL of ethylenediaminetetraacetic acid (EDTA) (final concentration 5 mM) was added to stop the reaction. The reaction solution was applied to a lysine sepharose column (4.6 × 50 mm) and then eluted with 1 mL of 50 mM sodium phosphate buffer. Miniplasminogen-like fragments were collected in this fraction. The column was further washed with 1 mL of 50 mM sodium phosphate buffer containing 0.5 M NaCl, and then eluted with 10 mL 0.2 M epsilon aminocaproic acid (EACA). The elution fraction was collected at 1 mL each. This fraction was confirmed for angiostatin-like fragments by SDS-PAGE analysis, and the fractions in which the desired fragment was detected were collected and dialyzed overnight against PBS (20 mM sodium phosphate, 150 mM NaCl, pH 7.4). And lyophilized. This gave 75 μg of angiostatin-like fragment. The volume of the reaction, the size of the lysine sepharose column, and the volume of the eluate can be appropriately changed, and the present invention is not limited to these.

Inhibition of Vascular Endothelial Cell Proliferation by Angiostatin-like Fragment of Plasminogen Produced by BLMA (FIG. 4) Bovine capillary endothelial cells (BCE cells) were transferred to a 24-well tissue culture plastic plate at 1.25 × 10 4 / mL per well. Inoculated with 0.5 mL of MEM medium containing 1 mM sodium pyruvate, 1% non-essential amino acid mixture and 10% fetal calf serum (FCS) at a density, and cultured in a CO ₂ incubator for 24 hours. After the medium was replaced with MEM medium containing 5% FCS, 10 μL of angiostatin-like fragment dissolved in PBS was added so that the final concentrations were 1 μg / mL and 10 μg / mL. After incubation for 30 minutes in a CO ₂ incubator, 6 μL of PBS or 100 ng / ml basic fibroblast growth factor (bFGF) was added, and the cells were cultured for 72 hours in a CO ₂ incubator. After incubation, remove the medium and wash twice with 0.5 mL of PBS. Add 100 μL of PBS containing 0.05% trypsin and 0.02% EDTA, incubate at 37 ° C. for 5 minutes, and then add 10 μL of FCS and trypsin. The reaction was stopped. The number of cells in this cell suspension was counted with a hemocytometer. When bFGF was not added, the number of cells was 2681 compared to 3712 in the presence of bFGF. When 10 μg / mL of angiostatin-like fragment was added, the number of cells was 2337 even in the presence of bFGF. That is, it was shown that an angiostatin-like fragment markedly inhibits the proliferation of vascular endothelial cells dependent on bFGF (FIG. 4).

Efficient conversion of plasminogen miniplasminogen-like fragments produced by BLMA into the thrombolytic enzyme plasmin (Figure 5) 25 μL plasminogen (final concentration 100 nM) or 25 μL uPA (miniplasminogen-like fragment) Final concentration 200 units / mL), 25 μL of BLMA (final concentration 0-37 nM) and 25 μL of plasmin substrate S2251 (final concentration 100 μM) are added, at 37 ° C. in TBS / T buffer containing CaCl ₂ (final concentration 1 mM), Absorbance at 405 nm was measured every 3 minutes from 0 to 60 minutes. In this reaction, the miniplasminogen-like fragment (●) produced plasmin at a rate more than 5 times that of plasminogen (◯) (FIG. 5). From this result, it can be seen that the miniplasminogen-like fragment exhibits better thrombolytic properties than plasminogen.

Reference example 1

Activation of prourokinase (pro-uPA) by BLMA (FIG. 6) The limited degradation of pro-uPA by BLMA and the accompanying activation were observed as follows. First, in order to investigate what kind of molecule pro-uPA is cleaved by BLMA, the cleavage pattern and the resulting fragment were identified. 6 μL of pro-uPA (final concentration 2 μM), 6 μL of BLMA (final concentration 0, 3.7, 110 nM) were incubated in TBS / T buffer containing CaCl ₂ (final concentration 1 mM) at 37 ° C. for 60 minutes. Non-reducing SDS sample buffer (x4) was added, 15 μL of which was applied to SDS-10% gel. After completion of the electrophoresis, it was stained with Coomassie brilliant blue R-250 and dried. In addition, the N-terminal amino acid sequence of the resulting fragment was identified. The protein was fractionated by SDS-PAGE and transferred to a PVDF membrane (PALLBIOSSUPPORT GROUP FLUOROTRANS). The membrane was stained with Coomassie brilliant blue R-250, decolorized with methanol, and then the band of interest was cut out and analyzed with a 476A protein sequencer (Applied Biosystems). In BLMA 3.7 nM, cleavage between Lys ^{158 and} Ile ^159, which is the activation cleavage site of pro-uPA, occurred (arrow 1 in FIG. 6C), and A chain (Ser ¹ -Lys ¹⁵⁸ ) and B chain (Ile ¹⁵⁹ -Leu). ⁴¹¹ ) occurred. In BLMA110nM, all of pro-uPA was cleaved, and the A chain was cleaved between Tyr ²⁴ -Phe ²⁵ (arrow 2 in FIG. 6C), resulting in new fragments Ser ¹ -Tyr ²⁴ and Phe ²⁵ -Lys ¹⁵⁸ . (FIGS. 6A, C and Table 2). The molecules generated by these cleavages are linked to each other by disulfide bonds.

The same experiment was performed in the presence of 50% serum. Add 2 μL ¹²⁵ I-pro-uPA (100,000 cpm), 3 μL BLMA (FIG. 6B, 1 is 0 nM, 2 is 10 nM, 3 is 100 nM, 4 is 1000 nM, all final concentrations), 5 μL human serum is added, Incubate at 37 ° C. for 60 minutes, to which 90 μL of H ₂ O was added. 5 μL was taken from there, and 5 μL reduced SDS sample buffer (x2) was added thereto. 5 μL of this was applied to 12.5% gel and electrophoresed. After completion of the electrophoresis, the film was subjected to autoradiography overnight, and then the film was developed. As a result, the same result as in the absence of serum was obtained (FIG. 6B).

Activation of prourokinase (pro-uPA) by BLMA 10 μL pro-uPA (final concentration 20 nM), 10 μL Spectrozyme UK (specific substrate for activated urokinase) (final concentration 100 μM), 10 μL CaCl ₂ (final concentration 1 mM) ), 20 μL of BLMA (final concentration 0-190 nM) was added, and the absorbance at 405 nm was measured in TBS / T buffer at 37 ° C. every 3 minutes until 0-60 minutes using a microplate reader (BIORAD). As a result, as shown in FIG. 6D, it was shown that BLMA leads to activation of pro-uPA.

Reference example 2

Activation of blood coagulation factor X by BLMA (FIG. 7) Cleavage of blood coagulation factor X by BLMA (B in FIG. 7) was examined as follows. Add 5 μL of blood coagulation factor X (final concentration 2 μM), 5 μL of BLMA (final concentration 0, 30, 100, 300 nM), and in TBS / T buffer containing CaCl ₂ (final concentration 1 mM) at 37 ° C. for 120 minutes. Incubated. Then 3.3 μL of reduced SDS-sample buffer (x4) was added, 12 μL of which was applied to 12.5% gel. After completion of the electrophoresis, staining and drying were performed (FIG. 7A). Furthermore, the N-terminal amino acid sequence of the generated fragment was identified by the same method as in Example 7 (Table 3). BLMA is concentration dependently between Asp ³⁴ -Leu ³⁵ of the B chain of blood coagulation factor X (arrow 1 in FIG. 7B) and between Lys ⁵² -Ile ⁵³ of the B chain which is the activation cleavage site (in FIG. 7B). Resulted in the cleavage of arrows 2) (FIGS. 7A, B and Table 3). As a result, a molecule in which the Ser ¹ -Asp ³⁴ fragment of the B chain and the Leu ³⁵ -Lys ²⁵⁴ fragment of the A chain and the B chain were linked by a disulfide bond was generated.

Activation of blood coagulation factor X by BLMA 10 μL blood coagulation factor X (final concentration 20 nM), 10 μL Spectrozyme Xa (specific substrate for activated blood coagulation factor X) (final concentration 100 μM), 10 μL CaCl ₂ ( Final concentration 1 mM) and 20 μL of BLMA (final concentration 0-100 nM) were added, and absorbance at 405 nm was measured in TBS / T buffer at 37 ° C. every 6 minutes until 0-120 minutes. As a result, BLMA resulted in the activation of blood coagulation factor X as shown in FIG. 7C.

Reference example 3

Activation of prothrombin by BLMA (FIG. 8) Activation of prothrombin by BLMA was measured by the following method. Add 12.5 μL prothrombin (final concentration 20 nM), 12.5 μL Spectrozyme TH (final concentration 100 μM), 12.5 μL CaCl ₂ (final concentration 1 mM), 12.5 μL BLMA (final concentration 0-400 nM), Absorbance at 405 nm was measured in TBS / T buffer at 37 ° C. every 6 minutes from 0 to 120 minutes. As shown in FIG. 8, BLMA activated prothrombin in a concentration-dependent manner.

Reference example 4

Activation of protein C by BLMA (FIG. 9) 12.5 μL human protein C (final concentration 20 nM), 12.5 μL BLMA (final concentration 0-40 nM), 12.5 μL S2336 (specific for active protein C) The substrate was added (final concentration 100 μM), 12.5 μL of CaCl ₂ (final concentration 1 mM), and the absorbance at 405 nm was measured in TBS / T buffer at 37 ° C. every 6 minutes until 0-120 minutes. As a result, as shown in FIG. 9, BLMA caused protein C activation in a concentration-dependent manner.

As an application example of the present invention, the enzyme BLMA produced by the bacterium of the present invention is limitedly decomposed with good selectivity using plasminogen as a substrate, and an angiostatin-like fragment having a neovascular inhibitory effect, and an excellent thrombolytic action. Efficiently produces the indicated miniplasminogen-like fragment. BLMA also activates the plasma serine protease group and has an excellent effect on the production of active plasma serine proteases such as active blood coagulation factor X and active protein C. This characteristic is useful in the industrial field. Applicable as technology.

Selection of microorganisms by searching for microbial metabolites by SDS-PAGE Purification of basilolysin MA by carboxymethylcellulose chromatography Generation of angiostatin-like and miniplasminogen-like fragments from plasminogen by basilolysin MA Inhibition of vascular endothelial cell proliferation by angiostatin-like fragments of plasminogen Efficiency of conversion of plasminogen miniplasminogen-like fragment into thrombolytic enzyme plasmin Activation of prourokinase (pro-uPA) by basilolysin MA Cleavage and activation of blood coagulation factor X by basilolysin MA Activation of prothrombin by basilolysin MA Activation of protein C by basilolysin MA

Claims (1)

An amino acid sequence fragment from Glu1 to Ser441, an amino acid sequence fragment from Phe75 to Ser441, and an amino acid sequence from Glu1 to Val449 obtained by limited degradation of human plasminogen with an enzyme having the amino acid sequence shown in SEQ ID NOs: 1 and 2 An angiogenesis inhibitor comprising a fragment and all four fragments of amino acid sequence fragments from Phe75 to Val449.

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