FR2558848A1 - PLASMINOGEN ACTIVATOR KYM, PROCESS FOR THE CULTURAL PREPARATION OF A HUMAN RHABDOMYOSARCOMA MUTANT AND PHARMACEUTICAL COMPOSITION COMPRISING THE SAME WITH THROMBOLYTIC ACTIVITY - Google Patents

Abstract

PLASMINOGEN ACTIVATOR (DESIGNATED BY PA-KYM) OF THE TISSUE PLASMINOGEN ACTIVATOR TYPE. IT IS PREPARED BY CULTURE OF KYM-A MUTANT CELLS DERIVED FROM HUMAN RHABDOMYOSARCOMA CELLS. IT HAS THE ADVANTAGE OVER UROKINASE OF ACTING ONLY ON THROMBUS, WITHOUT CAUSING BLEEDING.

Description

The present invention relates to an activator of

  plasminogen, its preparation process, as well as a

  pharmaceutical position which contains it and has a

thrombolytic activity.

  The fact that various diseases cause thrombus formation is a serious problem. In

  classical therapy, we use in these cases, especially

  streptokinase and urokinase. However, it is well known that these enzymes are not only

  to perform the desired thrombolysis, but that they decompose

  also the circulating blood clotting factors

  haemorrhage, since these enzymes have little affinity for thrombi to be lysed, and are predisposed to break down coagulation factors

  blood in circulation. That is why it is urgent to

  develop thrombolytic drug with high affinity for thrombi, with few side effects

to provoke hemorrhages.

  For this purpose, numerous reports and studies have been carried out on plasminogen activators (designated

  hereinafter PA) capable of lysing the thrombi

  bus, with few side effects. Concerning the

  PA from human tissues, detailed studies exist

  on enzymes obtained from normal human tissues, including endothelial or uterine cells, human tissue / tumors, or their cultured cells [Cf. Wilson et al., Cancer Res. 40, 933938 (1980) and Ricken and Colin,

  J. Biol. Chem., 256, 7035-7041 (1981)].

  It is known that normal human tissue cells

  such as endothelial or uterine cells, or tumor cells, such as human melanoma or

  mammary carcinoma, produce PA.

  However, normal tissue cells are unusable for the industrial production of PA,

  made of their limited life.

  On the other hand, human tumor cells, such as melanoma or breast carcinoma cells, are usually cultured by dependent attachment, with

  use of bottles or similar containers, which

  end up at low productivity of PA, and are therefore

  unusable on an industrial scale.

  It is generally thought that human PAs can

  To be cataloged in two types, that is, the uroki- type

  nase with low affinity for fibrin, and excellent tissue plasminogen activator (TPA) type

affinity for fibrin.

  It is well known that melanoma cells

  produce PA of the second type. The attention was strong-

  attracted to them because they mainly produce

  tPA type PA. On the other hand, apart from a few breast carcinoma cells, no mention has been made of other tumor cells capable of producing only TPA-type PA. Until today, it was thought that Rhabdomyosarcoma cells produced only

PA urokinase type.

  In order to overcome the disadvantages of the prior art,

  the plaintiff tried to find cells

  able to grow in rotational suspension culture.

  ve, and succeeded in isolating a mutant that believes in suspension from KYM-1 cell cultures, which have been established from human rhabdomyosarcoma cells (presented

  by Dr.Morimasa Sekigucchi of the Institute of Medicine of U-

  University of Tokyo) .This mutant was called KYM-A.I1 mutant can be subcultured in suspension suspension culture.

  that its deposit was not accepted by the Fermentation Re-

  Research Institute, the Agency of Industrial Science and Technology

  logy in Ibaraki-ken, Japan, he was registered at the Institute

  for Fermentation of Osaka, under IFO 50030. Further studies

  Later, it was found that the KYM-A mutant could produce and accumulate a marked amount of PA in a medium. The PA thus obtained was called PA-KYNo In the middle of the mutant

  KYM-A, urokinase-like PAs were not detected.

  The new product according to the invention, called

  PA-KYM, is a PA of new origin, originating from

  human rhabdomyosarcoma. It is further recognized, because of its physicochemical properties, described

  further, that it is a new AP type TPA.

  The properties of the KYM-A mutant are the following:

  lowing. 1. Each cell is spherical in shape and highly retractile, 2. Cells can stand alone

  can also come together as a chain or cluster.

  A cluster contains about 2 to 100 cells.

  3. When grown in a petri dish by hand

  plastic material, or transplant it to a rotating culture in a container, this mutant can grow mainly

  in the form of cells in suspension.

  4. This mutant can be subcultured by hanging culture.

rotational pressure.

  5. It can be converted into fixing cells de-

  pendent, for example by use of a used culture.

  Converted cells are morphologically similar to epithelial cells. A cultured community can

  be constituted by the supernatant part of cultures,

  This cell is prepared by culturing appropriate cells in a suitable basal medium for 1 to 100 hours, followed by removal of cells and cell debris from this medium. Suitable cells, for example, other than this mutant, are human hepatoma cells, such as the HuH6-C15 and HuH7 strains [cf. Nakabayashi

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

  This mutant can also be converted into

  dependent fixation, thanks to the use of a

  closing of fibronectin. Or, a culture attached to the substrate of this mutant is also possible by treatment

  surface of a petri dish, or

  with proteins such as collagen, gelatin, poly-

L-lysine or egg lysozyme.

  6. The subcutaneous transplantation of 106 cells to

  a nude mouse, or the transplantation to a jaw of hams-

  ter receiving ALS, leads to tumorigenesis.

7. Chromosome.

  According to the classical cytogenetic procedure, the most frequent number of chromosomes of this mutant is 46 to 47, with some distribution in the neighborhood. This fact suggests that the chromosome number of the KYM-A mutant is relatively close to that of the normal human diploid

  (ie 46) despite its tumoral origin.

  8. This mutant produces a remarkable amount of PA-KYM.

  In order to obtain the PA-KYM according to the present invention, the mutant can be subjected to a fixed culture.

  on the substrate or in a suspension culture.

  During the production of PA-KYM, the mutant KYM-A can be adhered to the surface of a plastic material or glass of Petri dishes, cylindrical bottles

or micro-support beads.

  However, the suspension culture in rotating flasks is better suited from the point of view of the production

industrial.

  To produce PA-KYM, it is better to cultivate

  strain the KYM-A mutant in serum-containing medium and replace it with serum-free medium at the beginning of a stationary phase of cell growth. Preferred serum media are those containing 10% fetal bovine serum in RPMI, MEM or F12 (Flow Laboratories products), or a mixture thereof. Preferred serum-free media are those prepared by the addition of insulin, human transferrin,

  monoethanolamine and selenious acid, has a basic

  such as RPMI, MEM, F12 or a mixture thereof. A half

  place containing the four constituents mentioned above.

  above shall be hereinafter referred to as "medium containing

ITES ".

  During the production of PA-KYM using

  mutant KYM-A, the cell strain is subjected to

  suspended in a Petri dish in plastic material.

  tick until an appropriate number of cells are obtained.

  It is then subjected to culture in a rotating flask of 8000 ml volume. It is preferable to seed the cells at a density of 104 to 105 cells / ml. A steady state is reached for a density of 5 x 105 to 2 x 106 cells / ml. The temperature of the culture may be of the order of 30 to 40 C, preferably close to 370C. This culture can be carried out in an atmosphere with 100% air, air containing 5 to 10% of carbon dioxide

  or air containing 5 to 100% oxygen.

  The culture can be carried out batchwise. Or

  well, when the cells grow sufficiently, the

  places can be exchanged at intervals of 1 to 4 days, to obtain continuously for about 1 month, after the beginning of the culture, a medium containing

PA-KYM.

  Since the KYM-A mutant PA-KYM is an enzyme

  which activates plasminogen in plasmin, and as it

  protein, it can be purified by any method generally used for the purification of proteins,

  as release, affinity chromatography, chromatography,

  ion exchange, application of molecular sieves, or combinations thereof. The purification can be carried out in continuous or discontinuous operation, depending on the efficiency

and convenience.

  The serum-free medium, containing PA-KYM, is then loaded onto a zinc-chelated agarose column, which has been sufficiently equilibrated with a buffer solution.

  tris hydrochloride, containing sodium chloride. This-

  the buffer solution also contains Tween 80 and

  sodium zide. These compounds are added to all the

  buffers below. When the loading is complete, the column is rinsed with the same buffer solution and is subjected to gradient elution with the buffer solution

  preceding and another buffer solution containing imitation

  imidazol. The imidazole gradient fractions exhibit PAKYM activity. The active fractions are combined,

  concentrated with polyethylene glycol, and dialysed

  be a phosphate buffer solution at 4 C for 24 hours,

  with several changes of the external solution.

  The dialysate, having a PA-KYM activity, is

  loaded on a concanavalin A Sepharose column, suffi-

  balanced with the phosphate buffer solution

  above. When the loading is complete, the column is rinsed with the same buffer solution and subjected to gradient elution with a buffer solution containing

  potassium thiocyanate and alpha-methyl mannoside.

  The active fractions are combined, concentrated to one

  lume with polyethylene glycol and dialysed against saline solution containing Tween 80 and sodium azide at 4 ° C for 24 hours, with several changes of the external solution. When the dialysis is complete, a white precipitate is recovered by centrifugation of the dialysate. We observe mainly,

  in the recovered precipitate, a PA-KYM activity. This precis

  is dissolved in a small amount of a

  potassium phosphate, and centrifuged to remove insoluble substances and thereby obtain

  a supernatant that has PA-KYM activity.

  The supernatant part, thus obtained, is charged

  on a column of Sephadex (G-200), which is rinsed sufficiently

  with a phosphate buffer solution containing thio-

  potassium cyanate, before developing it with the same buffer solution, as described above, to collect

  a fraction that exhibits PA-KYM activity.

  The physicochemical properties of PA-KYM, thus purified, are as follows. at. PA-KYM is a product of KYM-A, a mutant from

  of human rhabdomyosarcoma cells.

  b. Molecular weight, According to the gel electrophoresis of SDSpolyacrylamide, the

  PA-KYM non-reduced has two migrating bands very close to

  within a range of molecular weight

  approximately 56,000 to 62,000. On the other hand, PA-KYM4

  Reduced has two bands at about 32,000 and 36,000,

  as described previously.

  c. Functioning and specificity of the substrate, PA-KYM is an enzymatic protein which, in the presence of

  plasminogen, causes fibrinolysis. Like plasminogen

  is not necessary for this reaction, this enzyme protein

  is one of the specific activators of plasma

  nogène. It turns out that PA-KYM has a lot more affinity

for fibrin as urokinase.

  d. Optimum pH value; Its optimum pH value is around 8 to 10. A curve

  activity is shown in Figure 1.

e. PH value of stability.

  The product is stable at pH between 5 and 11. The figure

  2 indicates the percentage of residual activity.

  f. Optimum temperature range of operation

is lying.

  Figure 3 represents the percentage of enzymatic activity

  only depending on the temperature. The optimal temperature

  operating temperature is between 30 and 45 C.

  g. Heat-resistance PA-KYM is barely inactivated by heating at 50 ° C for 90 minutes. Its residual activity at 60 C or more is not

  greater than 60%. This is shown in Figure 4.

h. Inhibition.

  Table 1 shows the residual activities of the product exposed to the action of each inhibitor at a concentration of 0.1 mM, 1 mM and 10 mM.

TABLE 1

  0.1 mM 1 mM 10 mM CaC 2 94.9 104.4 85.8

EDTA - 112.9 -

FeSO4 - 103.3 -

KCt - 99.8 -

  MgCl 2 95.5 90.7 89.0 AgNO 3 87.2 79.6 13.6 CuSO 4 89.2 70.6 9.9 HgCl 2 115.8 45.6 0 MnCl 2 95.5 26.6 5.1 ZnSO 4 34 , 3 15.6 0 The inhibition of PA-KYM is examined by assigning a value of 100% to the activity of the enzyme, in the absence

of any inhibitor.

i. Amino acid composition.

  PA-KYM, which is an enzyme protein, is hydrolysed with 6N hydrochloric acid and subjected to amino acid analysis. Table 2 shows the results: there are entered the percentages of each amino acid,

  in relation to the total number of amino acid residues

of.

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

3.5 IS

LEU 8.1

TYR 4,5

PHE 3.5

HIS - 3.4

LYS 4,5

ARG 7.0

  * Cysteine and tryptophan are not dosed.

  j. Ultra violet spectrum Figure 5 is the representation of an ultra violet spectrum

an aqueous solution of PA-KYM.

  k. Solubility in solvents This product is soluble in water or salt solutions

  as a phosphate buffer solution at a concentration

  up to about 50 g / ml. A solution to concentration

  tions can be prepared in the presence of

  chemical feeds such as 1,6 M potassium thiocyanate

  It is insoluble in organic solvents such as ethanol.

nol or ether.

1. Form

  After lyophilization, it is in the form of a

white dre.

  m. Color reaction After being successively subjected to SDS-polyacrylamide gel electrophoresis, and to a PAS reaction,

  PA-KYM turns pink characteristic of glycoproteins.

  Its affinity, for a concanavalin A-agarose resin,

  also suggests that it is a glycoprotein.

  not. Isoelectric point The analysis with chromatographic concentration of PA-KYM in the presence of 8M urea reveals that the isoelectric pH of its main constituent goes from 7.5 to 8, whereas that of its minor constituent goes from 7 to 7.5, which suggests that it consists of a mixture of weakly basic proteins. PAKYM activity assay methods, which are similar to those conventionally used

  for the assay of PA activity, are as follows.

  1) Process of the fibrin plate [Cf. Astlap et al., Arch. Biochem. Biophys., 40, 346-351 (1952)]

  Thrombin is reacted in an agar suspension.

  rose containing fibrinogen and plasminogen at 37 C,

  to prepare a fibrin plate containing the

  minogène. A cavity 3 mm in diameter is drilled in this plate into which a sample is introduced. After

  introduction of this sample of 5 to 10 Al in the cavi-

  the fibrin plate is placed in an incubator

  during a given period. Then the plate is

  taken from the oven and the diameter of the net fibrinolysis zone is measured. As the activity of the sample is related to the diameter of the fibrinolysis zone, the

  Diameters of the areas are compared to measure the activity.

  2) A method for assaying the activity of PA-KYM by a two-stage reaction, using a synthetic substrate (S-2251). Plasminogen dissolved in 50 mM

  Tris hydrochloride buffer solution (pH 7.4) contains

  0.15 M sodium chloride, and a solution of PA-

  KYM in 50 mM of the same buffer solution containing 0.15 M

  sodium chloride, are maintained at 37 ° C for

  1 10 minutes (this is the first reaction). Then, to stop this first reaction, a 0.33 M solution of lysine in a buffer solution of

  0.15 M tris hydrochloride containing 0.15 M chlo-

  sodium chloride. Subsequently, a solution of a synthetic substrate,

  prepared by dissolving 3 mg / ml of a synthetic substrate

  than S-2251 in a 50 mM buffer solution of chlorhydrin

  tris (pH 7.4) containing 0.15 M sodium chloride

  dium, is maintained at 37 ° C. for 30 minutes, and the plasma

  mine formed during the first reaction is dosed co-

  lorimetrically at a wavelength of 405 nm (second

  reaction). After maintaining this reaction for exactly

  30 minutes, a solution of a-

  2.5M acetic acid to stop it. The dosage of the activity

  PA-KYM is based on absorption at 405 nm.

  3) Method for assaying the activity of PA-KYM by

  a single-stage reaction, using a sub-

synthetic stratum (S-2444).

  To a solution of PA-KYM in a solution of

  50 mM phosphate buffer (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 buffer solution of tris hydrochloride (pH 8.8) containing 0.15 M sodium chloride, and

  maintains this mixture at 37 C for exactly 90 minutes.

* Then a solution of 2.5 M acetic acid is added, so that

  to stop the reaction. The activity of PA-KYM is dosed

  rimetrically at a wavelength of 405 nm.

  The PA-KYM of the present invention is effective

  cace to perform lysis of thrombi formed in the blood.

  It is preferably in the form of

  pharmaceutical products intended for administration by

  intravenous. These compositions may contain different

  additives, including stabilizers such as mannitol,

  albumin, gelatin or sodium bisulphite; regula-

  pH meters as soda or sodium phosphate; and isotonic promoting agents such as sodium chloride, mannitol or glucose. The PA-KYM of the present invention can be

  clinically administered by massive intravenous injection

  ve, intravenous drip, instillation or injection

  subconjunctival or retrobulbar, in the form of a solution containing 100 to 100,000 IU, depending on age,

  body weight and the condition of the patient.

  The following examples are illustrative

non-limiting embodiment of the invention.

  In the accompanying drawings: FIG. 1 represents the percentage of PA PA activity as a function of the pH value; Figure 2 shows the percentage of PA residual activity as a function of the pH value; Figure 3 shows the percentage of PA activity as a function of temperature; Figure 4 shows the percentage of PA residual activity after incubation at each temperature; and FIG. 5 represents an ultraviolet spectrum of

PA-KYM.

EXAMPLE 1

  Culture To cultivate the KYM-A mutant, IFO N 50030, it is possible to use RPMI-1640, MEM or F12 medium containing 2 g of sodium bicarbonate, 1.192 g of N- (2-hydroxyethyl) piperazine acid. N'-ethanesulfonic and 60 mg of kanamycin sulfate per liter of medium, and additionally containing bovine fetal serum, free of complement (a product of KC Biological, USA) at a final concentration of 10%, or

  a mixture in different proportions of these products.

  A petri dish made of plastic is sown with

  KYM-A cells. When we have obtained approximate

  100 ml of cell suspension, suspension culture is initiated with a rotating flask. This culture, suspended in a volume of up to 8 liters, can be achieved by gradual removal of the crop gradually. The initial seeding rate at each stage is from 104 to 105 cells / ml. When this level reaches x 105 at 2 × 10 6 cells / ml, the cells are inoculated.

in another bottle.

EXAMPLE 2

  Collection When the cell concentration reaches x105 at 2 x 10 6 cells / ml, the culture is replaced with serum-free medium, comprising RPMI-1640, MEM, F12 or a mixture thereof, containing 2 g of bicarbonate

  of sodium, 1.192 g of N-hydroxyethyl piperazine

  ethanesulfonic-2, 60 mg of kanamrycin sulfate, 8.5 mg

  insulin, 1 mg human transferrin (produced by Sig-

  ma), 4.6 mg of ethanolamine and 13 g of selenious acid,

  liter, and containing in addition 20 KIU / ml of aprotinin (

  duit by Sigma). The strain is subjected to suspension culture in this serum-free medium for 1-4 days. After repeating this operation 4 to 10 times, the medium is filtered to collect the filtrate which is

purify later.

EXAMPLE 3

  Purification The filtrates collected in Example 2 are combined, and Aprotinin, Tween 80 and

  sodium azide, so as to obtain concentra-

  final concentrations of 20 KIU / ml, 0.01% and 0.02% respectively.

  These three substances are added to all solutions

  buffers as described below. The sample thus obtained

  naked is purified in the following manner.

  a) 30 liters of filtrate are loaded on a column of zinc chelated agarose (9 cm diameter x 21 cm), which

  was adjusted according to the method reported by Porath et al.

  in Nature, 258, 958-959 (1975), and sufficiently rinsed with a 20 mM Tris hydrochloride buffer solution (pH 7.05) containing 1M NaCl. When the loading is complete, the column is rinsed with 3 liters of the same buffer solution. The filtrate and the rinsing waters of the column show no PA-KYM activity. Then we

  submits the column to a gradient elution with 1.5

  of the buffer solution used above and one

  buffer solution (pH 7.3) prepared by addition of 150

  mM of imidazole to the buffer solution mentioned above.

  to obtain 15 ml fractions. The assay of the PAKYM activity of each fraction reveals that the fractions in the vicinity of the N 125 fraction show remarkable PAKYM activity. These active fractions are combined, introduced into a dialysis tube, sprinkled with dry powder of polyethylene glycol 20,000, and concentrated at 40C. It is then dialyzed against a 0.01M phosphate buffer solution (pH 6.7) at 4 ° C.

  for 24 hours, with several changes in the

  external organization. At the end of the dialysis, the solution is centrifuged.

  in the tube at 10,000 rpm for 10 minutes.

  you, and we collect the supernatant part.

  b) 130 ml of PA-KYM solution thus obtained are loaded on a concanavalin A Sepharose column (1 x 20 cm, product of Pharmacia Fine Chemicals), which has been sufficiently rinsed with a phosphate buffer solution.

  0.01M (pH 6.7). When the loading is completed, the

  lonne is rinsed with the same buffer solution, and is often

  gradient elution with 150 ml of this solution.

  buffer and 150 ml of another buffer solution, prepa-

  by adding, to the first buffer solution, thio-

  0.6 M sodium cyanate and 3M o-methylmannoside, to obtain 3.5 ml fractions. Fractions at

255'8848

  the neighborhood of fraction N 32 exhibit a

PA-KYM.

  The active fractions are combined and concentrated to a volume of 5 ml, using polyethylene glycol 20,000. The concentrate is dialyzed against physiological saline solution at 4 ° C. for 24 hours, with several changes of the external solution. . During dialysis, the enzyme solution becomes cloudy. The dialysate is centrifuged at

  000 rpm for 30 minutes, and the precipitate ob-

  held has PA-KYM activity. At this stage, we eliminate

  impurities, which mainly include

  higher molecular weight teins than PA-KYM,

  by gel filtration. The precipitate obtained by the centrifuge

  fugation, is dissolved in 2 ml of phos-

  0.01 M phate (pH 6.7) containing potassium thiocyanate

  1.6 N Sium. The insoluble residue is removed by centrifugation

  leaving a supernatant party with a

  of PA-KYM. The solution thus obtained is loaded onto a 1.6 x 85 cm column of Sephadex G-200 (product of Pharmacia Fine Chemicals) which has been sufficiently rinsed.

  with a 0.01 M phosphate buffer solution (pH 6.7) containing

  of 1.6 N potassium thiocyanate, which is

  with an identical buffer solution to give

  2.4 ml fractions. Fractions, situtating in the neighborhood

  of the N 35 fraction, exhibit

  KYM. As described above, a purified specimen is obtained.

  relied on PA-KYM in a 4-step process involving fractional precipitation. Table 3 shows the degree of

purification at each stage.

TABLE 3

  Concentration Concentration Activity Degree of ejzyme in specific protein purification

O

  Eluate from the chelated zinc column 54,27 122,445 44 Eluate from the column of Con A Sepharose 273,6 124 2206 217 Eluate from the Sephadex column 369.0 22.5 16400 1613 wc, to Ln Note concerning Table 3; A color reaction is carried out for 90 minutes at 37 ° C., using a suitable amount (a ml) of enzyme solution and a synthetic substrate S-2444. If the degree of staining at 405 nm (OD405) is b, the concentration of enzyme C can be determined as follows:

C = b x 1 / a.

    The protein concentration is determined by the procedure

  Lowry die, and is indicated in gg / ml.

  The specific activity is given by the ratio O / O

  The degree of purification is represented by an increase

  the specific activity at each stage, with

  setting of the value 1 in the case of the medium.

EXAMPLE 4

  Molecular weight and purity The molecular weight and purity of the

purified specimen of Example 3.

  A non-reduced specimen is electrophoresed on SD-polyacrylamide gels, containing 10% acrylamide, at room temperature, under a current of 5 mA, for 18 hours, according to the method reported by Laemmli, Nature, 227, 680 (1970). ). The gel thus obtained is rinsed with a solution

  2.5% aqueous Triton X-100, for 1 hour, in order to

  eliminate SDS. An agarose plate containing fibrin and plasminogen, which has been prepared according to

  the process indicated by Granelli Piperno and Reich (Jo Exp.

  Med. 148; 223-234 (1978)], and maintained at 37 ° C. for 1 to 4 hours. Subsequent observation reveals that

  There is no fibrinolysis zone except for 2

  very similar crops in a range of molecular weights

  between 56 000 and 62 000. On the other hand, we can not observe

  ve no fibrinolysis zone when applying the same procedure, but in the absence of plasminogen in the agarose gel. These results suggest that fibrinolysis

  depends on the plasminogen. When one adds to the

  20 g / ml of an IgG fraction of anti-serum antiserum

  identical zones of fibrinolysis are observed that are obtained in the absence of IgG. Therefore, the two bands within the molecular weight range of 56,000 to 62,000 should be assigned, not to a urokinase plasminogen activator, but to an activator of

TPA type.

  The unreduced, purified specimen of PA-KYM (approximately 0.1 g) obtained in Example 3 is separated, either as such, or after reduction with additiole 5% of 2-mercaptoethanol,

  by SDS-polyacrylamide gel electrophoresis of the

  described above. The proteins, contained in the gel thus obtained, are tinted according to the method reported byOakly et al. ZAnal. Biochem., 105, 361-163 (1980)]. In

  Consequently, it is discovered that the unreduced, purified specimen

  has two bands in the range of molecular weights

  from 56 000 to 62 000, while the specimen

  Reduced has two bands at 32,000 and 36,000.

  serve no other remarkable band, except those that are

  described above, suggesting that the specimen is

  rified almost completely. For weight determination

  molecular weight, the standard proteins of

  the following known molecules: phosphorylase b (94 000),

  serum bovine serum (67,000), egg albumin (43,000)

and carbonic anhydrase (30,000).

EXAMPLE 5

  Investigation of the affinity for CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals) fibrin product was swelled with 2 liters of 1 mM HCl, and rinsed. 1 g of fibrinogen (produced by Kabi) dissolved in 200 ml of 0.5M NaCl coupling buffer solution / 0.1 M NaHCO 3 (pH 8.3) are then mixed with this resin slurry and allowed to stir. stand at room temperature for 10 hours for absorption by the swollen resin. The resulting resin is introduced into 100 ml of 0.05 M phosphate buffer solution (pH 7.5) containing two units of thrombin (bovine thrombin).

  from Mochida Pharmaceutical Co., Ltd.) and it is

  held at 37 C for 10 minutes, thus obtaining a fibrin-Sepharose resin. The resin thus obtained is loaded into a minicolumn (6 ml) and equilibrated with a 0.05 M phosphate buffer solution (pH 7.5) containing 0.01%

  Tween 80. The column is then loaded with 1 ml of

  PA-KYM or urokinase solution, and rinsed with 15 ml of 0.05 M phosphate buffer solution (pH 7.5) to collect 1 ml fractions. Then the column is

  developed with 25 ml of the same buffer solution, contains

  mantle of 1.6 M potassium thiocyanate in order to collect

  fractions of 1 ml. The enzymatic activities of the

  rinse and thiocyanate

  of potassium, are determined using a synthetic substrate.

  than S-2444, to determine the recovery rate. As

  it can be seen from Table 4, the solution of

  does not contain PA-KYM and this requires

  potency of potassium thiocyanate for its elution, which

  suggests a strong affinity of this product for fibrin.

  On the other hand, in the case of urokinase, the latter has a low affinity, the activity being found in

the rinsing solution.

TABLE 4

  PA-KYM Urokinase Sample loaded 1,218 * 0,970 Rinsing 0 0,690 Potassium eluate with thiocyanate 1,072 0 Recovery 88% 71%

  * A color reaction is performed using a quantitative

  suitable (ie, 0.001 to 0.3 ml) of the enzyme solution and S-2444 synthetic medium at 37 ° C

  90 minutes. The indicated value represents the degree of color

  at 405 nm, calculated assuming that the entire

  the enzyme solution is used, depending on the ab-

  sorption measured at 405 nm, obtained with portions of

collected fractions.

  For example, when a total of 1 ml sample is loaded, and 0.1 mm is used for the color reaction at 37 ° C. for 90 minutes, giving an absorption (OD 05) of 0.1218, the indicated value is calculated as follows:

0.1128 x 1 / 0.1 = 1.218.

RZVENDICATTONS

  1. New plasminogen activator (PA-KYM), characterized

  rised by the following physochemical properties:

  at. PA-KYM is a product of KYM-A, a mutant

  of human rhabdomyosarcoma cells.

  b. Molecular weight, According to the gel electrophoresis of SDSpolyacrylamide, the

  PA-KYM non-reduced has two migrating bands very close to

  within a range of molecular weight

  approximately 56,000 to 62,000. On the other hand, PA-KYM

  Reduced has two bands at about 32,000 and 36,000,

  as described previously.

  c. Functioning and specificity of the substrate PA-KYM is an enzymatic protein which, in the presence of

  plasminogen, causes fibrinolysis. Like plasmino

  gene is required for this reaction, this enzyme protein is one of the specific plasminogen activators. It turns out that PA-KYM has a lot more af =

  finite for fibrin as urokinase.

d. Optimum pH value.

  Its optimum pH value is about 8 to 10.

e. PH value of stability,

  The product is stable at pH between 5 and 11.

  f. Optimum temperature range of operation

  is lying. The optimum operating temperature is

is between 30 and 45 C.

g. Heat resistance.

  PA-KYM is barely inactivated by heating at 50 C for minutes. Its residual activity at 60 C or more is not

not more than 60%.

  h. Inhibition Table 1 indicates the residual activities of the product exposed to the action of each inhibitor at a concentration

0.1 mM, 1 mM and 10 mM.

TABLE 1

  0.1 mM 1 mM 10 mM CaCe2 94.9 104.4 85.8

EDTA - 112.9 -

FeSO4 - 103.3 -

KCz - 99.8 -

  MgCl2 95.5 90.7 89.0 AgNO3 87.2 79.6 13.6 CuSO4 89.2 70.6 9.9 HgC22 115.8 45.6 0 MncQ2 95.5 26.6 5.1 ZnSO4 34 , 3 15.6 0

  The examination of the inhibition of PA-KYM is carried out in

  a value of 100% to the activity of the enzyme in the ab-

presence of any inhibitor.

  i. PA-KYM amino acid composition, which is an enzyme protein, is hydrolysed with 6N hydrochloric acid and subjected to amino acid analysis. Table 2 shows the results:

  the percentages of each amino acid,

  brought to 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

3.5 IS

LEU 8.1

TYR 4,5

TABLE 2 (continued)

PA-KYM

PHE 3.5

HIS 3,4

LYS 4,5

ARG 7.0

  * Cysteine and tryptophan are not dosed.

  j. Ultraviolet spectrum Figure 5 is the representation of an ultra-violet spectrum

an aqueous solution of PA-KYM.

  k. Solubility in solvents This product is soluble in water or salt solutions

  as a phosphate buffer solution at a concentration

  up to about 50 g / ml. Concentrated solution

  can be prepared in the presence of

  such as 1.6 M potassium thiocyanate

  It is insoluble in organic solvents such as ethanol.

nol or ether.

  1. Form After lyophilization, it is in the form of a white powder. m. Colored Reaction After Successful Electrophoresis

  on SDS-polyacrylamide gel, and a PAS reaction,

  KYM turns pink characteristic of glycoproteins. His affair

  finite for a concanavalin A-agarose resin also suggests

  if it is a glycoprotein.

  not. Isoelectric point The "chromatofocusing" analysis of PA-KYM in the presence of urea

  8M, reveals that the isoelectric pH of its primary constituent

  average of 7.5 to 8, while that of its constituent half

  neur ranges from 7 to 7.5, suggesting that it is constituted by

  a mixture of weakly basic proteins.

  2. Process for the preparation of PA-KYM according to the claim

  cation 1, which consists in culturing a KYM-A cell mutant

the human rhabdomyosarcoma.

  3. Method according to claim 2, characterized in

  what is used in suspension culture.

  4. Method according to claim 2, characterized in that a culture is used fixed on a substrate.

  5. Pharmaceutical composition which contains a quantity

  efficacy of PA-KYM and has a thrombolytic action

tick.