A new fibrinolytic enzyme from velvet antler mushroom and its preparation method and application

Abstract

The present invention provides a new plasminogen activator from velvet antler mushroom and a preparation method and application thereof, and belongs to the field of bioengineering technology. The new plasminogen activator from velvet antler mushroom provided by the present invention is composed of a single subunit, and the molecular weight of the new plasminogen activator is 30.9 kDa; the twelve amino acid sequences at the N-terminus of the new plasminogen activator are shown in SEQ ID NO.1, and after comparison with the NCBI database, it is a new plasminogen activator. The new plasminogen activator from velvet antler mushroom provided by the present invention can sequentially degrade the α, β and γ chains of human fibrinogen, and the degradation time is short; at the same time, the new plasminogen activator can also dissolve blood clots, and has a good thrombolytic effect. In addition, the plasminogen activator from velvet antler mushroom provided by the present invention has high safety, is easy to be absorbed by the human body, has low production cost, and has a prospect for development and application.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a new fibrinolytic enzyme of Pleurotus eryngii and a preparation method and application thereof. Background Art

[0002] Thrombotic diseases have been harming human health, with the incidence increasing year by year, and are one of the main causes of death in humans today. Thrombolysis is currently the main means of treating and preventing thrombotic diseases. The thrombolytic drugs currently used in clinical treatment have significant efficacy, but they still have disadvantages such as large side effects and high prices. Therefore, it is an urgent need for modern medicine to develop new thrombolytic drugs that are efficient, rapid, prevent re-embolism, and have few side effects. Developing new thrombolytic substances from various biological resources in nature is an important way to develop thrombolytic products.

[0003] Deer antler mushroom (Lyophyllum decastes) belongs to the phylum Basidiomycetes, the order Agaricales, the family Trichoderma, and the genus Lyophyllum. It is also known as "roadbed mushroom", "railway mushroom", "lotus leaf mushroom", etc. Deer antler mushroom has a crisp and smooth taste and delicious taste. Its fruiting body and mycelium have high nutritional value and are rich in dietary fiber. The fruiting body has good biological activity, and has the effects of anti-tumor, improving immune function, antibacterial effect, lowering blood sugar, and lowering blood lipids. It is a traditional medicinal and edible fungus with broad prospects for biomedical development and health food application, and is an important resource to be developed urgently. Due to the different genetic compositions of different edible fungi, not all edible fungi have fibrinolytic enzymes. At the same time, it is unpredictable what kind of medicinal effect the fibrinolytic enzymes from different edible fungi have. Moreover, since the fibrinolytic enzymes from different edible fungi have different biological properties, it is undoubtedly a great difficulty in obtaining pure enzymes. There is no report on extracting fibrinolytic enzymes from deer antler mushrooms in this field. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a new fibrinolytic enzyme from velvet antler mushroom and a preparation method and application thereof. The new fibrinolytic enzyme from velvet antler mushroom provided by the present invention has high fibrinolytic activity and stable activity at human physiological pH. It not only has a good thrombolytic effect, but also has the advantages of high safety, easy absorption by the human body, and low production cost, and has a prospect for development and application.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a new plasminogen activator from Pleurotus eryngii, which is composed of a single subunit and has a molecular weight of 30.9 kDa; the twelve amino acid sequences at the N-terminus of the new plasminogen activator are shown in SEQ ID NO. 1. Comparison with the NCBI database shows that the new plasminogen activator is a new plasminogen activator.

[0007] The present invention also provides a method for preparing the above-mentioned new fibrinolytic enzyme of Pleurotus eryngii, comprising the following steps: extracting Pleurotus eryngii fruiting bodies with PBS buffer solution to obtain an extract; after solid-liquid separation, subjecting the obtained crude enzyme solution to ammonium sulfate salting out, hydrophobic interaction chromatography 1, strong cation exchange chromatography and hydrophobic interaction chromatography 2 separation in sequence to obtain the new fibrinolytic enzyme of Pleurotus eryngii; the pH value of the PBS buffer solution used for extraction is 7.4 and the concentration is 0.02 mol / L.

[0008] Preferably, the volume-to-weight ratio of the PBS buffer solution used for extraction to the fruiting body of Pleurotus eryngii is 8L-12L:1kg.

[0009] Preferably, the leaching temperature is 3°C-5°C, and the leaching time is 3h-5h.

[0010] Preferably, the ambient temperature for the ammonium sulfate salting out is 3°C-5°C, and the time for the ammonium sulfate salting out is 22h-26h.

[0011] Preferably, the chromatographic column of the hydrophobic interaction chromatography 1 is an Octyl-Sepharose FastFlow hydrophobic interaction chromatography column, the separation of the hydrophobic interaction chromatography 1 adopts linear elution, the starting buffer solution is a 0.01-0.03 mol / L PBS buffer solution containing 25% ammonium sulfate saturation, the eluent is a 0.01-0.03 mol / L PBS buffer solution, and the flow rate is 1-3 mL / min; the pH of the eluent is 7.4.

[0012] Preferably, the chromatographic column of the strong cation exchange chromatography is an SP-Sepharose High Performance ion exchange chromatography column, the separation of the strong cation exchange chromatography adopts linear elution, the starting buffer solution is a PBS buffer solution, the eluent is a PBS buffer solution containing 0.3-0.5 mol / L NaCl, and the flow rate is 1-3 mL / min; the pH of the PBS buffer solution used for the strong cation exchange chromatography is 5.2 and the concentration is 0.01-0.03 mol / L.

[0013] Preferably, the chromatographic column of the hydrophobic interaction chromatography 2 is a Source 15PHE hydrophobic interaction chromatography column, and the separation of the hydrophobic interaction chromatography 2 adopts linear elution, the starting buffer solution is a 0.01-0.03 mol / L PBS buffer solution, the eluent is a 0.01-0.03 mol / L PBS buffer solution containing 10% ammonium sulfate saturation, the flow rate is 1-3 mL / min, and the pH value of the PBS buffer solution used in the hydrophobic interaction chromatography 2 is 7.4.

[0014] The present invention also provides an application of the above-mentioned novel fibrinolytic enzyme from Pleurotus eryngii or the above-mentioned preparation method in the preparation of thrombolytic drugs.

[0015] Beneficial effects of the present invention:

[0016] The present invention obtains for the first time a new plasminogen activator from velvet antler mushroom with fibrinolytic effect, which is a new plasminogen activator after comparison with NCBI data. The new plasminogen activator from velvet antler mushroom provided by the present invention has high fibrinolytic activity and stable activity at human physiological pH, and can not only directly degrade fibrin, but also activate plasminogen to indirectly degrade fibrin. The new plasminogen activator from velvet antler mushroom provided by the present invention can sequentially degrade the α, β and γ chains of human fibrinogen, and the degradation time is short; it can also dissolve blood clots and has a good thrombolytic effect. In addition, the new plasminogen activator from velvet antler mushroom provided by the present invention has high safety, is easy to be absorbed by the human body, has low production cost, and has a prospect for development and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The activity changes of the new fibrinolytic enzyme of Pleurotus eryngii under different pH conditions;

[0018] Figure 2 The activity changes of new fibrinolytic enzyme of Pleurotus eryngii under different temperature conditions;

[0019] Figure 3 The effects of different metal ions on the activity of new fibrinolytic enzyme from Pleurotus eryngii;

[0020] Figure 4 The solubility of the new plasmin from Pilosa velutipes on fibrin;

[0021] Figure 5 This is the degradation profile of human fibrinogen by the new plasmin from Pleurotus eryngii;

[0022] Figure 6 is the degradation rate of blood clot by Pilosa velutipes new fibrinolytic enzyme. DETAILED DESCRIPTION

[0023] The invention provides a novel plasminogen activator from velvet antler mushroom, which consists of a single subunit with a molecular weight of 30.9 kDa. The twelve amino acid sequences at the N-terminus of the single subunit are shown in SEQ ID NO.1, and are compared with the NCBI database to be a novel plasminogen activator.

[0024] The relative molecular mass of the new fibrinolytic enzyme from Pleurotus eryngii provided by the present invention is about 30.9 kDa. The twelve amino acid sequences at the N-terminus of the new fibrinolytic enzyme from Pleurotus eryngii provided by the present invention are shown in SEQ ID NO.1: GAVTQCNAPWGL.

[0025] The present invention also provides a method for preparing the above-mentioned new fibrinolytic enzyme of Pleurotus eryngii, comprising the following steps: extracting Pleurotus eryngii fruiting bodies with PBS buffer solution to obtain an extract; after solid-liquid separation, subjecting the obtained crude enzyme solution to ammonium sulfate salting out, hydrophobic interaction chromatography 1, strong cation exchange chromatography and hydrophobic interaction chromatography 2 separation in sequence to obtain the new fibrinolytic enzyme of Pleurotus eryngii; the pH value of the PBS buffer solution used for extraction is 7.4 and the concentration is 0.02 mol / L.

[0026] The present invention preferably purchases the fruiting bodies of Pleurotus eryngii from Gutian County Tianxian Agricultural Products Co., Ltd., whose address is No. 75, Fenglin Road, Shuangzhu Village, Fengdu Town, Gutian County, and the product barcode is 6951370171033. In the present invention, the fruiting bodies of Pleurotus eryngii are preferably dried and then crushed, and then extracted. The present invention has no special limitation on the crushing method, and the conventional crushing method in the art can be used.

[0027] In the present invention, the volume-to-weight ratio of the PBS buffer solution used for extraction to the fruiting body of Pleurotus eryngii is preferably 8L-12L:1kg, more preferably 10L:1kg. The extraction temperature is preferably 3°C-5°C, more preferably 4°C, and the extraction time is preferably 3h-5h, more preferably 4h. The present invention does not specifically limit the specific source of the PBS buffer solution. After the extract is obtained by extraction, solid-liquid separation is required. The present invention does not specifically limit the specific method of solid-liquid separation, and conventional solid-liquid separation methods in the art can be used. In an embodiment of the present invention, it is preferred to use a centrifugal method for solid-liquid separation, and the centrifugal speed is preferably 8000-12000r / min, more preferably 10000r / min; the centrifugal time is preferably 10-20min, more preferably 15min.

[0028] After solid-liquid separation, the obtained crude enzyme solution is subjected to ammonium sulfate salting out. In the present invention, the ambient temperature of the ammonium sulfate salting out is preferably 3°C-5°C, more preferably 4°C; the time of the ammonium sulfate salting out is preferably 22h-26h, more preferably 24h. In the present invention, the purpose of the salting out is to remove foreign proteins.

[0029] In the present invention, after the crude enzyme solution is salted out with ammonium sulfate, it is preferably necessary to perform solid-liquid separation, and the obtained precipitate is dissolved in PBS buffer and then separated by hydrophobic interaction chromatography 1. In the present invention, the chromatographic column of the hydrophobic interaction chromatography 1 is preferably an Octyl-Sepharose Fast Flow hydrophobic interaction chromatography column, and the separation of the hydrophobic interaction chromatography 1 is preferably performed by linear elution, the starting buffer solution is preferably a 0.01-0.03 mol / L PBS buffer solution containing 25% ammonium sulfate saturation, more preferably a 0.02 mol / L PBS buffer solution containing 25% ammonium sulfate saturation, the eluent is preferably a 0.01-0.03 mol / L PBS buffer solution, more preferably a 0.02 mol / L PBS buffer solution, the flow rate is preferably 1-3 mL / min, more preferably 2 mL / min, and the pH value of the eluent is preferably 7.4.

[0030] In the present invention, the active ingredient obtained by the hydrophobic interaction chromatography 1 is preferably subjected to strong cation exchange chromatography separation. The strong cation exchange chromatography is preferably carried out using a Sp-Sepharose High Performance strong cation exchange chromatography column. In the present invention, the separation of the strong cation exchange chromatography preferably uses linear elution, and the starting buffer solution is preferably a PBS buffer solution with a pH of 5.2 and a concentration of 0.01-0.03 mol / L, and more preferably a PBS buffer solution with a pH of 5.2 and a concentration of 0.02 mol / L. The eluent is preferably a PBS buffer solution containing 0.3-0.5 mol / L NaCl, and more preferably a PBS buffer solution containing 0.4 mol / L NaCl. The concentration of the PBS buffer solution used as the eluent is preferably 0.01-0.03 mol / L, and more preferably 0.02 mol / L. The pH of the PBS buffer solution used as the eluent is preferably 5.2. In the present invention, the flow rate is preferably 1-3 mL / min, and more preferably 2 mL / min.

[0031] In the present invention, the active components obtained by the strong cation exchange chromatography separation are further subjected to hydrophobic interaction chromatography 2 separation. In the present invention, the separation column used for the hydrophobic interaction chromatography 2 separation is preferably Source15PHE hydrophobic interaction chromatography column; the separation of the hydrophobic interaction chromatography 2 is preferably carried out in a linear elution manner. The starting buffer solution is preferably a 0.01-0.03 mol / L PBS buffer solution, more preferably a 0.02 mol / L PBS buffer solution, the eluent is preferably a 0.01-0.03 mol / L PBS buffer solution containing an ammonium sulfate saturation of 10%, more preferably a 0.02 mol / L PBS buffer solution containing an ammonium sulfate saturation of 10%, the flow rate is preferably 1-3 mL / min, more preferably 2 mL / min. In the present invention, the pH value of the PBS buffer solution used in the hydrophobic interaction chromatography 2 is preferably 7.4.

[0032] The present invention also provides an application of the above-mentioned novel fibrinolytic enzyme from Pleurotus eryngii or the above-mentioned preparation method in the preparation of thrombolytic drugs.

[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0035] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0036] Example 1

[0037] (1) Preparation of crude enzyme solution: The fruiting bodies of Pleurotus eryngii purchased from Gutian Tianxian Agricultural Products Co., Ltd. (product barcode: 6951370171033) were dried and crushed, and the powder of the fruiting bodies of Pleurotus eryngii was added to 0.02 mol / L PBS (pH 7.4) buffer solution at a ratio of 1 kg:10 L, and extracted at 4°C for 4 h, centrifuged at 4°C and 10,000 r / min for 15 min, and the supernatant was collected for later use;

[0038] (2) Add ammonium sulfate to the supernatant until the saturation of ammonium sulfate is 75%, salt out at 4°C for 24 h, centrifuge at 4°C and 10,000 r / min for 15 min, discard the supernatant, dissolve the precipitate with 0.02 mol / L PBS (pH 7.4) buffer solution, and store at 4°C for later use;

[0039] (3) Hydrophobic interaction chromatography 1 separation: The enzyme solution after salting out was separated by an Octyl-Sepharose Fast Flow hydrophobic interaction chromatography column, the starting buffer solution was a 0.02 mol / L PBS buffer solution containing 25% ammonium sulfate saturation, and the eluent was a 0.02 mol / L PBS buffer solution; the pH value of the buffer solution was 7.4; the flow rate was 2 mL / min; and the active components were collected;

[0040] (4) Strong cation exchange chromatography separation: The active components obtained in (3) above were separated by SP-Sepharose High Performance strong cation exchange chromatography column, the starting buffer solution was 0.02 mol / L PBS (pH 5.2) buffer solution, the eluent was 0.02 mol / L PBS (pH 5.2) containing 0.3 mol / L NaCl, and then linear elution was performed in the direction of increasing NaCl concentration with 3 column volumes of starting buffer solution and 3 column volumes of eluent, at a flow rate of 2 mL / min, and the active components were collected;

[0041] (5) Hydrophobic interaction chromatography 2 separation: The active components obtained in (4) above were separated by Source15PHE hydrophobic interaction chromatography column, using a linear elution method, the starting buffer solution was a 0.02 mol / L PBS (pH 7.4) buffer solution containing 10% ammonium sulfate saturation, and the eluent was a 0.02 mol / L PBS (pH 7.4) buffer solution. After loading, 3 column volumes of the starting buffer solution and 3 column volumes of the eluent were used for linear elution according to the ammonium sulfate saturation of 10%-0, and the active components were collected to obtain the new plasminogen activator from Pleurotus eryngii. The N-terminal twelve amino acid sequences of the plasminogen activator subunits of Pleurotus eryngii were measured by Edman degradation method, respectively: GAVTQCNAPWGL. After comparison with the NCBI database, the plasminogen activator was a new plasminogen activator.

[0042] Example 2

[0043] PBS buffer solutions with different pH values ​​were prepared, and the new plasminogen activator obtained in Example 1 was mixed with the buffer solution in equal proportions. The enzyme activity of the new plasminogen activator from the fruiting body of Pleurotus eryngii was determined by the fibrin plate method; 10 μL of the plasminogen activator sample was spotted on the fibrin plate, and placed in a 37°C incubator for reaction for 6 hours, and the diameter of the dissolution zone was measured and substituted into the equation y=0.0506e5.4385x, where y represents the enzyme activity (U / mL) and x represents the diameter of the dissolution zone (cm).

[0044] Calculate the relative enzyme activity.

[0045]

[0046] The pH of the control group was 7.4. Figure 1 and Figure 2 As shown. The experiment found that the activity change trend of Pleurotus eryngii fibrinolytic enzyme was the same under different temperatures and different pH conditions (so a graph is shown here as the result). Take 10μL of enzyme solution and spot it on the fibrin plate, place it under different temperature conditions for reaction for 6 hours, measure the diameter of the dissolution circle, take 37°C as the control group, calculate the relative enzyme activity, and obtain the optimal action temperature of 45°C. From the above results, it can be seen that the new Pleurotus eryngii fibrinolytic enzyme provided by the present invention can maintain good activity at the physiological pH of the human body, and is more stable in a neutral alkaline environment than in an acidic environment.

[0047] Example 3

[0048] Prepare 0.5mmol / L, 1mmol / L, 2.5mmol / L, 5mmol / L, and 50mmol / L of Cu 2+ , Na + , K + ,Mg 2+ , Zn 2+ , Al 3+ , Fe 2+ , Fe 3+ , Mn 2+ , Ca 2+ , Ba 2+ The solution was prepared by mixing the velvet antler mushroom fibrinolytic enzyme obtained in Example 1 with the ion solutions of each concentration in a volume ratio of 1:1, and incubated at 37°C for 10 hours. Then, 10 μL of the mixture was taken on a fibrin plate, and the residual fibrinolytic enzyme activity was detected after incubation at 37°C for 6 hours. The blank control was prepared by mixing the velvet antler mushroom fibrinolytic enzyme obtained in Example 1 with an equal volume of pure water, and the other operations were the same as above. The results are shown in Figure 2. Figure 3 As shown. Low concentration Fe 3+ It has a significant protective effect on the enzyme, while high concentrations of Fe 3+ Completely inhibited plasmin activity; with increasing concentration, Al 3+ , Cu 2+ The inhibitory effect on Pleurotus eryngii fibrinolytic enzyme is becoming more and more obvious.

[0049] Example 4

[0050] The fibrin dissolving ability of the velvet antler mushroom fibrinolytic enzyme obtained in Example 1 was tested by the fibrin plate method.

[0051] The fibrin plate contains fibrinogen (commercially available fibrinogen may contain fibrin) and thrombin. Soluble fibrinogen forms fibrin monomers under the action of thrombin. The fibrin monomers spontaneously form and polymerize to form a visible fibrin gel. The velvet antler mushroom fibrinolytic enzyme solution obtained in Example 1 is added to the surface of the plate gel. After a short period of incubation, the velvet antler mushroom fibrinolytic enzyme can dissolve the fibrin and form a transparent circle visible to the naked eye on the surface of the plate gel, as shown in FIG. Figure 4 shown.

[0052] Example 5

[0053] The velvet antler mushroom fibrinolytic enzyme prepared in Example 1 and human fibrinogen were mixed in equal volumes and reacted in a water bath at 37°C. The degradation of human fibrinogen by velvet antler mushroom fibrinolytic enzyme was detected by SDS-PAGE. The degradation profile is shown in Figure 5 From left to right, the lanes are human fibrinogen, velvet antler plasmin and fibrinogen mixed and acted for 1min, 3min, 5min, 10min, 20min, 30min, 1h, 1.5h, 2h, 3h, and 4h. Figure 5 It can be seen that the α chain is completely degraded after 1 minute, the β chain is completely degraded after 10 minutes, and the γ chain is completely degraded after 2 hours. This degradation order is the same as the degradation of fibrinogen by human plasmin, indicating that the velvet antler plasmin prepared by the present invention can dissolve fibrin and can sequentially degrade the α, β and γ chains of human fibrinogen.

[0054] Example 6

[0055] The velvet antler mushroom fibrinolytic enzyme prepared in Example 1 was respectively prepared into 60 U / mL and 100 U / mL and added to the weighed blood clot, the weight of the blood clot was recorded as X0, and the mixture was placed in a 37°C water bath for insulation, and centrifuged at 20 min, 40 min, 1 h, 1.5 h, and 2 h, respectively. After the supernatant was aspirated, the weight of the remaining blood clot was weighed as X, and the dissolution of the blood clot by the velvet antler mushroom fibrinolytic enzyme was observed. The dissolution rate was calculated as follows:

[0056]

[0057] The results are as follows Figure 6 As shown, the degradation rate of blood clots by velvet antler mushroom fibrinolytic enzyme (60 U / mL) reached 91% in 2 hours, and the degradation rate of blood clots by velvet antler mushroom fibrinolytic enzyme (100 U / mL) reached 93% in 1.5 hours, indicating that the velvet antler mushroom fibrinolytic enzyme prepared by the present invention has a certain thrombolytic ability.

[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A novel fibrinolytic enzyme from Porcupine antler, characterized in that: The new plasminogen activator from Pleurotus eryngii is composed of a single subunit, and the molecular weight of the new plasminogen activator is 30.9 kDa; the twelve amino acid sequence at the N-terminus of the new plasminogen activator is shown in SEQ ID NO.1; The preparation method of the new fibrinolytic enzyme of velvet antler mushroom comprises the following steps: extracting velvet antler mushroom fruiting bodies with PBS buffer solution to obtain an extract; after solid-liquid separation, sequentially subjecting the obtained crude enzyme solution to ammonium sulfate salting out, hydrophobic interaction chromatography 1, strong cation exchange chromatography and hydrophobic interaction chromatography 2 separation to obtain the new fibrinolytic enzyme of velvet antler mushroom; the pH of the PBS buffer solution used for extraction is 7.4 and the concentration is 0.02 mol / L; The volume weight ratio of the PBS buffer solution used for extraction to the fruiting body of Pleurotus eryngii is 10L:1kg; The leaching temperature is 4°C and the leaching time is 4h; The ambient temperature of the ammonium sulfate salting out is 4°C, and the time of the ammonium sulfate salting out is 24h; The chromatographic column of the hydrophobic interaction chromatography 1 is an Octyl-Sepharose Fast Flow hydrophobic interaction chromatography column. The separation of the hydrophobic interaction chromatography 1 adopts linear elution. The starting buffer solution is a 0.02 mol / L PBS buffer solution containing 25% ammonium sulfate saturation. The eluent is a 0.02 mol / L PBS buffer solution. The flow rate is 2 mL / min. The pH of the eluent is 7.

4. The chromatographic column of the strong cation exchange chromatography is an SP-Sepharose HighPerformance ion exchange chromatography column. The separation of the strong cation exchange chromatography adopts linear elution, the starting buffer solution is a PBS buffer solution, the eluent is a PBS buffer solution containing 0.3 mol / L NaCl, and the flow rate is 2 mL / min; the pH of the PBS buffer solution used for the strong cation exchange chromatography is 5.2 and the concentration is 0.02 mol / L; The chromatographic column of the hydrophobic interaction chromatography 2 is a Source 15PHE hydrophobic interaction chromatography column. The separation of the hydrophobic interaction chromatography 2 adopts linear elution. The starting buffer solution is a 0.02 mol / L PBS buffer solution. The eluent is a 0.02 mol / L PBS buffer solution containing 10% ammonium sulfate saturation. The flow rate is 2 mL / min. The pH value of the PBS buffer solution used in the hydrophobic interaction chromatography 2 is 7.

4.

2. Use of the new fibrinolytic enzyme from Pleurotus eryngii according to claim 1 in the preparation of thrombolytic drugs.

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