GB2153366A - Plasminogen activator - Google Patents

Abstract

A plasminogen activator PA-KYM available as a thrombolytic drug is prepared from a suspension culture medium of human rhabdomyosarcoma cells KYM-A. The PA-KYM is a plasminogen activator of the tissue plasminogen activator type.

Description

SPECIFICATION Plasminogen activator kym Field of the Invention: This invention relates to plasminogen activator KYM which activates plasminogen, a process for the preparation thereof and a pharmaceutical composition having a thrombolytic activity.

Background of the Invention: It has been a serious problem that thrombi would result in various diseases. Conventional therapeutical agents therefor include streptokinase and urokinase. However it has been known that either of these enzymes would not only effect the aimed thrombolysis but decompose clotting factors in circulating blood to thereby bring about hemorrhage since these enzymes have poor affinities for the thrombi to be lysed and are liable to decompose clotting factors in circulating blood. Therefore it has been an urgent problem to develop a thrombolytic drug which has a strong affinity for thrombi with little side effects including hemorrhage.

Under these circumstances, there have been many studies and reports on plasminogen activators (which will be referred to as PA hereinafter) capable of lysing thrombi with little side effects. With reference to PA originating from human tissues, detailed studies on enzymes obtained from normal human tissues (i.e. endothelial or uterine cells), human tumors or cultured cells thereof have been reported (cf. Wilson et al., Cancer Res., 40, 933-938 (1980) and Ricken and Colin, J. Biol. Chem., 256, 7035-7041 (1981)).

It has been known that normal human tissue cells such as endothelial or uterine cell or tumorous cells such as human melanoma or mammary carcinoma cells would produce PA.

However normal tissue cells are inavailable for the industrial production of PA since they have finite life span.

On the other hand, human tumorous cells such as melanoma or mammary carcinoma cells have been conventionally cultured in an anchorage dependent manner with the use of bottles or similar containers, which results in a poor productivity of PA and is also inavailable in an industrial scale.

It is generally believed that human PA may be classified into two types, i.e. the urokinase type having a poor affinity for fibrin and the tissue plasminogen activator (TPA) type having an excellent affinity for fibrin.

It has been well known that melanoma cells would produce PA of the TPA type. Melanoma cells have attracted a good deal of attention since they would mainly product PA of the TPA type only. On the other hand, no other tumorous cells except some mammary carcinoma cells have been reported to produce PA of the TPA type only. Rhabdomyosarcoma cells have been hitherto thought to produce only PA of the urokinase type.

Summary of the Invention: Under these circumstances, the present inventors tried to find out tumorous cells which would grow by suspended spinner culture and succeeded in isolating a variant which would grow in suspension from the KYM-1 cell cultures which had been established from human rhabdomyosarcoma cells (presented by Dr. Morimasa Sekiguchi of Institute of Medicine, the University of Tokyo). This variant was named variant KYM-A. This variant KYM-A can be subcultured by suspended spinner culture. Though the deposition thereof was not accepted by the Fermentation Research Institute, Agency of Industrial Science and Technology in Ibaraki-ken, Japan, it has now been deposited with the Institute for Fermentation (foundation), in Osaka, Japan, under IFO 50030.

Subsequent studies have revealed that the variant KYM-A might produce and accumulate a remarkable amount of PA in a medium. The PA thus attained was named PA-KYM.

No PA of the urokinase type was detected in the medium of the variant KYM-A.

Accordingly the PA-KYM of the present invention is a PA of a novel origin (i.e. human rhabdomyosarcoma cells). It is further accepted as a novel PA of the TPA type because of its physicochemical properties.

Brief Description of the Drawings: Figure 1 is a graph of the activity curve of the PA-KYM at each pH value, Figure 2 is a graph of the residual activity of the PA at each pH value, Figure 3 is a graph of the activity curve at different temperatures, Figure 4 is a graph of the residual activity of the PA after incubation at each temperature and Figure 5 is an ultraviolet spectrum thereof.

Detailed Description of the Invention: The properties of the variant KYM-A are as follows.

1. Each cell is spherical in shape and highly refractile, 2. The cells, respectively, may be present as a single cell. Alternatively they may form a chain or spherical cluster together. A cluster contains approximately two to 100 cells.

3. When cultured in a plastic Petri dish or subjected to spinner culture in a tank, this variant may grow mostly in the form of suspended cells.

4. This variant may be subcultured by suspended spinner culture.

5. This variant may be converted into anchorage dependent cells by, e.g., the use of an spent culture.

The converted cells are morphologically similar to epithelial cells.

A spent culture medium may be a supernatent of cultures other than that of this variant which is prepared by culturing appropriate cells in an appropriate basal medium for one to 100 hours and removing the cells and cell debris from the medium. Examples of the available cells other than this variant are human hepatoma cells such as HuH6-C15 and HuH7 strains (cf.

Nakabayashi et al., Cancer Research, 42, 3858-3863 (1982)).

Besides using the spent culture medium, this variant may be converted into anchoragedependent cells by the use of a medium containing fibronectin. Alternatively substratum attached culture of this variant is also possible by treating the surface of a Petri dish or similar containers with proteins such as collagen, gelatin, poly-L-lysine or egg lysozyme.

6. Subcutaneous transplantation of 106 cells to a nude mouse or transplantation thereof to a cheek pouch of an ALS administered hamster would result in tumorigenesis.

7. Chromosome According to the conventional cytogenic method, the most frequent chromosome number of this variant are 46 and 47 with some distribution in the neighbourhood thereof. This fact suggests that the chromosome number of the variant KYM-A is relatively close to that of the normal human diploid (i.e. 46) inspite of its tumor cell origin.

8. This variant produces a remarkable amount of the PA-KYM.

In order to produce the PA-KYM of the present invention, the variant may be subjected either to substratum attached culture or suspension culture.

In order to produce the PA-KYM, the variant KYM-A may be allowed to adhere to the surface of plastic or glass Petri dishes, roller bottles or microcarrier beads.

However suspension culture in spinner flasks is more suitable from the viewpoint of industrial production.

In order to produce the PA-KYM, it is preferable to culture the variant KYM-A in a medium containing serum and replace it with a medium containing no serum at the initiation of a stationary phase of the growth of the cells. Preferable serum media are those containing 10% of bovine fetal serum to RPMI, MEM or F12 (products of Flow Laboratories) or a mixture thereof.

Preferable non-serum media are those prepared by adding insulin, human transferrin, monoethanolamine and selenous acid to a basic medium such as RPMI, MEM, F12 or a mixture thereof. A medium containing the above-mentioned four components will be referred to as ITEScontaining medium hereinafter.

In order to produce the PA-KYM by the variant KYM-A, the cell strain is subjected to suspension culture in a plastic Petri dish untii an appropriate number of cells are obtained. Then it was subjected to culture in a spinner flask of 100 to 8000 ml in volume. It is preferable to inoculate the cells at the density of 104 to 105 cells/ml. A stationary state is reached at the density of 5 X 105 to 2 X 106 cells/ml. The culture temperature may be from approximately 30 to 40 C, preferably approximately 37"C. The culture may be carried out in an atmosphere of 100% air, air containing 5 to 10% of carbon dioxide gas or air containing 5 to 100% of oxygen.

The culture may be carried out in a batchwise operation. Alternately, when the cells grow sufficiently, the media may be exchanged at an interval of one to four days to thereby obtain a medium containing the PA-KYM continuously for approximately one month after the initiation of the culture.

Since the PA-KYM of the variant KYM-A is an enzyme which activates plasminogen into plasmin and belongs to protein, it may be purified by any method generally used in purifying protein such as salting-out, affinity chromatography, ion-exchange chromatography, application of molecular sieve, or combinations thereof. The purification may be carried out either continuously or a batchwise operation depending on the efficiency and convenience.

The serum-free medium containing the PA-KYM is then loaded on a zinc-chelate agarose column which has been sufficiently equilibrated with a tris hydrochloride buffer solution containing sodium chloride. This buffer solution also contains Tween 80 and sodium azide.

These compounds will be added to all buffer solutions hereinafter. After the completion of loading, the column is washed with the same buffer solution and subjected to gradient elution with the foregoing buffer solution and another buffer solution containing imidazole. The imidazole gradient fractions exhibit a PA-KYM activity. The active fractions are combined, concentrated with polyethylene glycol and dialyzed against a phosphate buffer solution at 4 C for 24 hours with several changes of the external solution.

The dialysate exhibiting a PA-KYM activity is loaded on a concanavalin A Sepharose column which is sufficiently equilibrated with the same phosphate buffer solution as described above.

After the completion of loading, the column is washed with the same buffer solution and subjected to gradient elution with a buffer solution containing potassium thiocyanate and a ar- methyl-mannoside. The gradient fractions of potassium thiocyanate and a-methylmannoside exhibit a PA-KYM activity. The active fractions are combined, concentrated to an appropriate volume with polyethylene glycol and dialyzed against a physiological saline solution containing Tween 80 and azide nitride at 4'C for 24 hours with several changes of the external solution.

After the completion of the dialysis, a white precipitate in the dialysate is recovered by centrifugation. A PA-KYM activity is observed mainly in the recovered precipitate. The precipitate is dissolved in a small amount of a phosphate buffer solution containing potassium thiocyanate and centrifuged, thus removing insoluble matters and giving a supernatant which exhibits a PA-KYM activity.

The supernatant thus obtained is loaded on a Sephadex (G-200) column sufficiently washed with a phosphate buffer solution containing, potassium thiocyanate and developed with the same buffer solution as described above to give a fraction which exhibits a PA-KYM activity.

The physicochemical properties of the PA-KYM thus purified are as follows.

a. The PA-KYM is a product of a variant KYM-A originating from human rhabdomyosarcoma cells.

b. Molecular weight According to SDS-polyacrylamide gel electrophoresis, unreduced PA-KYM shows two closely migrating bands in a molecular weight range of approximately 56,000 to 62,000. On the other hand, reduced PA-KYM shows two bands at approximately 32,000 and approximately 36,000 when measured in the same manner as described above.

c. Function and substrate specificity The PA-KYM is an enzyme protein which causes fibrinolysis in the presence of plasminogen.

Since plasminogen is essential in this reaction, this enzyme protein is one of typical plasminogen activators. The PA-KYM is shown to have a much higher affinity for fibrin than that of urokinase.

d. Optimum pH value The optimum pH value thereof is approximately 8 to 1 0. Fig. 1 shows an activity curve thereof.

e. Stable pH value The stable pH value thereof is approximately 5 to 11. Fig. 2 shows the residual activity (%) thereof.

f. Range of optimum temperature for function Fig. 3 shows the enzyme activity at various temperatures. The optimum temperature for function is between 30 and 45at.

9. Thermoresistance The PA-KYM is hardly inactivated by heating to 50"C for 90 min. Its residual activity at 60"C or above is not higher than 60%. Fig. 4 shows the residual activity (%) thereof.

h. Inhibition Table 1 shows the residual activities thereof when exposed to each inhibitor in a concentration of 0.1 mM, 1mM and 10 mM.

Table 1 0.1 mM 1 mM 10 m! CaCS2 94.9 104.4 85.8 EDTA - 112.9 FeSO4 - 103.3 KCss - 99.8 MgCS2 95.5 907 89.0 AgN03 87.2 79.6 13.6 CuSO4 89.2 70.6 9.9 HgCS2 115.8 45.6 0 MnCl2 95.5 26.6 5.1 ZnS04 34.3 15.6 0 An examination on the inhibition of the PA-KYM is performed by assigning a value of 100% to the activity of the enzyme in the absence of any inhibitor.

i. Amino acid composition The PA-KYM, which is an enzyme protein, is hydrolyzed with 6 N hydrochloric acid and subjected to amino acid analysis. Table 2 shows the result wherein each amino acid composition is shown in % based on the total number of amino acid residues.

Table 2 PA-KYM ASP 10.6 THR 5.3 SER 9.7 GLU 10.9 PRO 6.3 GLY 9.5 ALA 7.2 VAL 5.0 MET 1.0 ILE 3.5 LEU 8.1 TYR 4.5 PHE 3.5 HIS 3.4 LYS 4.5 ARG 7.0 Cysteine and tryptophan were not determined.

j. Ultraviolet spectrum Fig. 5 shows an ultraviolet spectrum of an aqueous solution of the PA-KYM.

k. Solubility in solvents The PA-KYM is soluble in water or in salt solutions such as a phosphate buffer solution at a concentration up to approximately 50 g/ml. A solution of higher concentrations may be prepared in the presence of such chemicals as 1.6 M of potasium thiocyanate.

It is insoluble in organic solvents such as ethanol or ether.

I. Form The PA-KYM is in the form of a white powder when lyophilized.

m. Color reaction After subjected to SDS-polyacrylamide gel electrophoresis and to PAS reaction successively, the PA-KYM turns to pink characteristic of glycoproteins. Its affinity for a concanavalin Aagarose resin further suggests that it is a glycoprotein.

n. Isoelectric point Chromatofocusing analysis of the PA-KYM in the presence of 8 M urea has revealed that the isoelectric pH of its main component is 7.5 to 8.0 while that of the minor component is 7.0 to 7.5, suggesting that it is a mixture of weakly basic proteins.

Methods for determining the PA-KYM activity, which are similar to those conventionally employed for determining the PA activity, are as follows.

(i) Fibrin plate method (cf. Astlap et al., Arch. Biochem. Biophys., 40, 346-351 (1952)).

Thrombin is reacted in an agarose suspension containing fibrinogen and plasminogen at 37"C to thereby prepare a fibrin plate containing plasmiogen. A hole of 3 mm in diameter into which a sample is introduced is bored through the fibrin plate. Five to 10 jul of the sample is introduced into the hole and the fibrin plate is placed in an incubator for a certain period. Then it is taken out of the incubator and the diameter of the distinct fibrinolysis zone is measured.

Since the activity of the sample is correlated to the diameter of the fibrinolysis zone, the diameters of the zones are compared to determine the activity.

(ii) Method for determining the PA-KYM activity by a two stages reaction with the use of a synthetic substrate (S-2251).

Plasminogen dissolved in a 50 mM tris hydrochloride buffer solution (pH 7.4) containing 0.15 M of sodium chloride and a solution of the PA-KYM in a 50 mM tris hydrochloride buffer solution (pH 7.4) containing 0.15 M of sodium chloride are maintained at 37"C for accurately 10 min (the first reaction). Then a 0.33 M lysine solution in a 0.15 M tris hydrochloride buffer solution containing 0. 1 5 M sodium chloride is immediately added to stop the first reaction.

Subsequently a solution of a synthetic substrate prepared by dissolving 3 mg/ml of a synthetic substrate S-2251 in a 50 mM tris hydrochloride buffer solution (pH 7.4) containing 0.15 M sodium chloride is maintained at 37"C for 30 min and the plasmin formed by the first reaction is colorimetrically determined at a wavelength of 405 nm (the second reaction). After maintaining for accurately 30 min, a 2.5 M acetic acid solution is immediately added to thereby stop the second reaction. The PA-KYM activity is determined based on the absorption at 405 nm.

(iii) Method for determining the PA-KYMN activity by a one stage reaction with the use of a synthetic substrate (S-2444).

To a PA-KYM solution in a 50 mM phosphate buffer solution (pH 8.8) containing 0.15 M sodium chloride, a solution of a synthetic substrate prepared by dissolving 3 mg/ml of a synthetic substrate S-2444 in a 50 mM tris hydrochloride buffer solution (pH 8.8) containing 0.15 M sodium chloride is added and the mixture is maintained at 37'C for accurately 90 min.

Then a 2.5 M solution of acetic acid is added to the mixture to thereby stop the reaction. The PA-KYM activity is colorimetrically determined at a wavelength of 405 nm.

The PA-KYM of the present invention is effective in lying thrombi formed in blood.

The PA-KYM of the present invention is preferably formulated into pharmaceutical compositions suitable for intravenous administration. These compositions may contain various additives including stabilizers such as mannitol, albumin, gelatin or sodium bisulfite; pH regulators such as sodium hydroxide or sodium phosphate; and isotonizing agents such as sodium chloride, mannitol or glucose.

The PA-KYM of the present invention may be clinically administered by venoclycis, intravenous drip, instillation or subconjuctival or retrobulbar injection in the form of a solution containing 100 to 100,000 IU thereof depending on the age, body weight and condition of a patient.

To further illustrate the present invention, the following examples will be given.

Example 1: Culture In order to culture the variant KYM-A, IFO 50030, a medium RPMI-1640, MEM or F12 (products of Flow Laboratories, USA) containing 2.0 g of sodium bicarbonate, 1.1 92 9 of N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid and 60 mg of kanamycin sulfate per liter of the medium and further containing decomplemented bovine fetal serum (a product of KC Biological, USA) to a final concentration of 10%, or a mixture thereof at various ratios may be used.

A plastic Petri dish was inoculated with the KYM-A cells. When approximately 100 ml of a cell suspension was obtained, suspension culture was initiated with the use of a spinner flask.

Suspension culture in a volume of up to 81 is able to be performed by gradually scaling up the culture. The initial inoculation ratio in each stage was 104 to 10 cells/ml. When it reached 5 x 105 to 2 x 106 cells/ml, the cells were inoculated into another flask.

Example 2: Collection When the cell concentration reached 5 x 105 to 2 X 106 cells/ml, the medium was replaced with a serum free medium comprising a medium -RPMI-1640, MEM, F12 or a mixture thereof containing 2.0 g of sodium bicarbonate, 1.192 9 of N-hydroxyethylpiperazine-N1-2-ethanesul- fonic acid, 60 mg of kanamycin sulfate, 8.5 mg of insulin, 1 mg of human transferrin (a product of Sigma), 4.6 mg of ethanolamine and 1 3 jig of selenous acid per liter and further containing 20 KlU/ml of Aprotinin (a product of Sigma). The strain was subjected to suspension culture in this serum free medium for one to four days. After repeating it four to 10 times, the medium was filtered and the obtained filtrate was subjected to purification.

Example 3: Purification The filtrates recovered in Example 2 were combined and Aprotinin, Tween 80 and sodium azide were added thereto to give a final concentration of 20 KlU/ml, 0.01% and 0.02%, respectively. These three substances were added to all buffer solutions as described hereinafter.

The sample thus obtained was purified in the following manner.

(a) 30 l of the filtrate was loaded on a zinc chelate-agarose column (9 cm O x 21 cm) which had been adjusted according to the method reported by Porath et al. (cf. Nature, 258, 958-959 (1975)) and sufficiently washed with a 20 mM tris hydrochloride buffer solution (pH 7.05) containing 1 M NaCI. After the completion of the loading, the column was washed with 3000 ml of the same buffer solution as described above. The filtrate and the washing passed through the column exhibited no PA-KYM activity. Then the column was subjected to gradient eluion with 1 500 ml of the same buffer solution as described above and another buffer solution (pH 7.3) prepared by adding 1 50 mM imidazole to the abovementioned buffer solution to give fractions of 15-ml portions.PA-KYM activity determination on each fraction revealed that fractions in the neighbourhood of fraction No. 1 25 exhibited a remarkable PA-KYM activity.

These active fractions were combined, introduced into a dialysis tube, dusted with dry polyethylene glycol 20,000 powder and concentrated at 4"C. Subsequently it was dialyzed against a 0.01 M phosphate buffer solution (pH 6.7) at 4"C for 24 hours with several changes of the external solution. After the completion of the dialysis, the solution in the tube was centrifuged at 10,000 rpm for 10 min to give a supernatant.

(b) 1 30 ml of the solution containing the PA-KYM thus obtained was loaded on a concanavalin A Sepharose column (1 X 20 cm; a product of Pharmacia Fine Chemicals) which had been sufficiently washed with a 0.01 M phosphate buffer solution (pH 6.7). After the completion of the loading, the column was washed with the same buffer solution as described above and subjected to gradient elution with 1 50 ml of the abovementioned buffer solution and 1 50 ml of another buffer solution prepared by adding 0.6 M sodium thiocyanate and 3 M amethylmammoside to the above buffer solution to give fractions of 3.5-ml portions. Fractions in the neighborhood of fraction No. 32 exhibited a PA-KYM activity.

The active fractions were combined and concentrated to a volume of 5 ml with the use of polyethylene glycol 20,000. The concentrate was dialyzed against a physiological saline solution at 4"C for 24 hours with several changes of the external solution. During the dialysis, the enzyme solution became turbid. The dialysate was centrifuged at 15,000 rpm for 30 min, and the precipitate exhibited a PA-KYM activity. At.this stage, protein contaminants were removed which mainly comprising proteins of higher molecular weights than PA-KYM by gel filtration.

The precipitate obtained by the centrifugation was dissolved in 2 ml of 0.01 M phosphate buffer solution (pH 6.7) containing 1.6 N potassium thiocyanate. Insoluble residue was removed by centrifugation to give a supernatant showing a PA-KYM activity. The solution thus obtained was loaded on a Sephadex G-200 (a product of Pharmacia Fine Chemicals) column (1.6 X 85 cm) which had been sufficiently washed with 0.01 M phosphate buffer solution (pH 6.7) containing 1.6 N potassium thiocyanate and developed with the same buffer solution as described above to give fractions of 2.4-mI portions. The fractions in the neighborhood of fraction No. 35 exhibited a PA-KYM activity.

As described above, a purified specimen of the PA-KYM was obtained by the process comprising four stages including fractional precipitation. Table 3 shows the degree of purification at each stage.

Table 3 Enzyme con- Protein con- Specific Degree of centration centration activity purification (x 10 ) Medium 3.56 350 10.17 1.0 Eluate from zinc chelate column 54.27 122 445 44 Eluate from Con A Sepharose column 273.6 124 2206 217 Eluate from Sephadex column 369.0 22.5 16400 1613 Note to the Table: A color reaction is carried out at 37"C for 90 min with the use of an appropriate amount (a ml) of the enzyme solution and a synthetic substrate S-2444. When the degree of coloration at 405 nm (OD405) is b, the enzyme concentration (C) can be determined in the following manner: C=bX 1.0/a.

3 Protein concentration is determined by Lowry's method and represented in ,ug/ml.

The specific activity is given by /.

The degree of purification is represented by an increase in the specific activity at each stage by assigning the value of 1.0 to the case of the medium.

Example 4: Molecular weight and purity The molecular weight and purity of the purified specimen as obtained in Example 3 were examined.

An unreduced specimen was subjected to electrophoresis on SDS-polyacrylamide gels containing 10% of acrylamide at room temperature with a current of 5 mA for 1 8 hours according to the method reported by Laemmli (cf. Nature, 227, 680 (1970)). The gel thus obtained was washed with a 2.5% aqueous solution of Triton X-1 00 for one hour to remove the SDS. Then it was overlaid on an agarose plate containing fibrin and plasminogen, which had been prepared according to the method reported by Granelli Piperno and Reich (J. Exp. Med., 148m, 223-234 (1978)), and maintained at 37"C for one to five hours. Subsequent observation revealed that there was no fibrinolysis zone except two closely migrating zones in a molecular weight range of 56,000 to 62,000.On the other hand, no fibrinolysis zone was observed when the same procedure was followed except that no plasminogen was added to the agarose gel. These results suggested that the fibrinolysis would depend on plasminogen. When 20 jug/ml of an IgG fraction of antiurokinase antiserum was added to the fibrin plate, similar fibrinolysis zones to those obtained in the absence of IgG were observed. Therefore the two bands in the molecular weight range of 56,000 to 62,000 should be assigned not to a plasminogen activator of the urokinase type but to that of the TPA type.

The unreduced purified specimen of the PA-KYM (approximately 0.1 pg) obtained in Example 3 was separated, either as such or after reduced by adding 5% of 2-mercaptoethanol, by SDSpolyacrylamide gel electrophoresis in the same manner as described above. Proteins contained in the gel thus obtained were stained according to the method reported by Oakly et al. (cf. Anal.

Biochem., 105, 361-363 (1980)). Consequently it was found that the unreduced purified specimen exhibited two bands in a molecular weight range of 56,000 to 62,000 while the reduced specimen exhibited two bands at 32,000 and 36,000. No remarkable band other than those as described above was observed, which suggested that the specimen was purified almost completely.

The following standard proteins of known molecular weights were employed in the determination of the molecular weight; phosphorylase b (94000), bovine serum albumin (67000), egg albumin (43000) and carbonic anhydrase (30,000).

Example 5: Examination on the affinity for fibrin 10 9 of CNBr-activated Sepharose 4B (a product of Pharmacia Fine Chemicals) was swollen with two liters of 1 mM HCI and washed. 1 g of fibrinogen (a product of Kabi) dissolved in 200 ml of a coupling buffer solution of 0.5 m NaCl/O.1 M NaHCO3 (pH 8.3) was then mixed with the resin slurry and allowed to stand at room temperature for 10 hours to be adsorbed by the swollen resin. The resulting resin was introduced into 100 ml of a 0.05 M phosphate buffer solution (pH 7.5) containing two units of thrombin (bovine thrombin; a product of Mochida Pharmaceutical Co., Ltd.) and maintained at 37'C for 10 min, thus giving a fibrin-Sepharose resin.The resin thus obtained was packed into a minicolumn (6 ml) and equilibrated with a 0.05 M phosphate buffer solution (pH 7.5) containing 0.01 % of Tween 80. 1 ml of a PA-KYM solution or a urokinase solution was loaded on the column and the column was washed with 1 5 ml of a 0.05 M phosphate buffer solution (pH 7.5) to collect fractions of 1-ml portions. Then the column was developed with 25 ml of the same buffer solution as described above containing 1.6 M potassium thiocyanate to collect fractions of 1-ml portions. The enzyme activities of the washing and the potassium thiocyanate solution were determined with the use of a synthetic substrate S-2444 to determine the recovery. As shown in Table 4, the washing contained no PA-KYM and the PA-KYM required potassium thiocyanate for elution, which suggested a strong affinity thereof for fibrin. On the other hand, urokinase exhibited a poor affinity and the washing exhibited the activity.

Table 1 0.1 mM 1 mM 10d CaCl2 94.9 104.4 85.8 EDTA - 112.9 FeSO4 - 103.3 KCl - 99.8 MgCl2 95.5 90.7 89,0 AgNO3 87.2 79.6 13.6 CuS04 89,2 70.6 9.9 HgCl2 115,8 45.6 0 MnCl2 95.5 26.6 5.1 ZnS04 34.3 15.6 0 An examination on the inhibition of the present substance is performed by assigning a value of 100% to the activity of the enzyme in the absence of any inhibitor; amino acid composition The present substance, which is an enzyme protein, is hydrolyzed with 6N hydrochloric acid and subjected to amino acid analysis; Table 2 shows the result wherein each amino acid composition is shown in % based on the total number of amino acid residues;

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (4)

**WARNING** start of CLMS field may overlap end of DESC **. Table 1 0.1 mM 1 mM 10d CaCl2 94.9 104.4 85.8 EDTA - 112.9 FeSO4 - 103.3 KCl - 99.8 MgCl2 95.5 90.7 89,0 AgNO3 87.2 79.6 13.6 CuS04 89,2 70.6 9.9 HgCl2 115,8 45.6 0 MnCl2 95.5 26.6 5.1 ZnS04 34.3 15.6 0 An examination on the inhibition of the present substance is performed by assigning a value of 100% to the activity of the enzyme in the absence of any inhibitor; amino acid composition The present substance, which is an enzyme protein, is hydrolyzed with 6N hydrochloric acid and subjected to amino acid analysis; Table 2 shows the result wherein each amino acid composition is shown in % based on the total number of amino acid residues; Table 4 PA-KYM Urokinase - * Loaded sample 1.218 0. 970 Washing 0 0.690 Potassium thiocyanate 1.072 0 eluate Recovery 88 % 71 96 A A color reaction was carried out with the use of an appropriate amount (i.e. 0.001 to 0.3 ml) of the enzyme solution and a synthetic medium S-2444 at 37"C for 90 min. The indicated value represents the degree of coloration at 405 nm calculated by assuming that the whole enzyme solution is used based on the absorbance measured at 405 nm obtained with aliquots of collected fractions. For example, when 1 ml of a sample was loaded in total and 0.1 ml thereof is used in the color reaction at 37"C for 90 min to give an absorbance (OD405) of 01218, the indicated value was calculated in the following manner: 0.128 X 1.0/0.1 = 1.218 CLAIMS

1. A plasminogen activator KYM which has the following physicochemical properties: a. The present substance is a product of a variant KYM-A originating from human rhabdomyosarcoma cells; b.Molecular weight According to SDS-polyacrylamide gel electrophoresis, the unreduced type of the present substance shows two closely migrating bands in a molecular weight range of approximately 56,000 to 62,000; On the other hand, the reduced type thereof shows two bands at approximately 32,000 and approximately 36,000 when measured in the same manner as described above; c. Function and substrate specificity The present substance is an enzyme protein which causes fibrinolysis in the presence of plasminogen; Since plasminogen is essential in this reaction, this enzyme protein is one of typical plasminogen activators; The PA-KYM has a higher affinity for fibrin than that of urokinase as determined by affinity chromatography on fibrin-Sepharose resin; d. Optimum pH value The optimum pH value thereof is approximately 8 to 10; Fig. 1 shows a function curve thereof; e.Stable pH value The stable pH value thereof is approximately 5 to 11. Fig. 2 shows the residual activity (%) thereof; f. Range of optimum function temperature Fig. 3 shows a function temperature curve thereof at 30 to 45"C; g. Thermoresistance The present substance is hardly inactivated. by heating to 50"C for 90 min; Its residual activity at 60"C or above is not higher than 60%. Fig. 4 shows the residual activity (%) thereof; h. Inhibition Table 1 shows the residual activities thereof when exposed to each inhibitor in a concentration of 0.1 mM, 1 mM and 10 mM; Table 2 PA-KYM ASP 10.6 THR 5.3 SER 9.7 GLU 10.9 PRO 6.3 GLY 9.5 ALA 7.2 VAL 5.0 MET 1.0 ILE 3.5 LEU 8.1 TYR 4.5 PHE 3.5 HIS 3.4 LYS 4.5 ARG 7.0 Cysteine and tryp.

tophan were not determined j. Ultraviolet spectrum Fig. 5 shows an ultraviolet spectrum of an aqueous solution of the present substance; k. Solubility in solvents The present substance is soluble in water or in salt solutions such as a phosphate buffer solution at concentrations up to approximately 50,ug/ml; A solution of higher concentrations may be obtained in the presence of such chemicals as 1.6 M of potassium thiocyanate; It is insoluble in organic solvents such as ethanol or ether; I. Form The present substance is in the form of a white powder when lyophilized; m. Color reaction After subjected to SDS-polyacrylamide gel electrophoresis and to PAS reaction successively, the present substance turns to pink characteristic of glycoproteins; Its affinity for a concanavalin A-agarose resin further suggests that it is a glycoprotein; n. Isoelectric point Chromatofocusing analysis of the present substance in the presence of 8 M urea has revealed that the isoelectric pH of its main component 7.5 to 8.0 while that of the minor component is 7.0 to 7.5, suggesting that it is a mixture of weakly basic proteins.

2. A process for the preparation of the plasminogen activator KYM which comprises culturing a human rhabdomysarcoma cell variant KYM-A and collecting said plasminogen activator from the medium thus obtained.

3. A pharmaceutical composition which contains an effective amount of the plasminogen activator KYM and has a thrombolytic activity.

4. A process for the preparation of the plasminogen activator KYM as set forth in Claim 2, wherein said culture is suspension culture or substratum-attached culture.