JP2001514516A - Activated vitamin K-dependent blood factor and method for producing the same - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method for activating a vitamin K-dependent blood factor by treating it with a protease obtained from a plant or a prokaryote. The present invention further comprises a blood factor produced according to the invention that does not exhibit animal protease contamination, and a set for pharmaceutical preparations and medical applications.

Description

The present invention relates to an activated vitamin K-dependent blood factor and a method for producing the same. The protease / substrate complex cascade in the physiological activation of pro-factors requires proteases with high selectivity, especially in the area of blood coagulation. These proteases are present in human organisms themselves, especially in plasma. The use of other human proteases to activate pro-factors, such as digestive enzymes such as trypsin and pepsin, does not lead to the production of selective activated blood coagulation factors. Rather, it leads to non-specific degradation of the targeted substrate. Thus, for example, it is known that tryptic degradation of the X coagulation factor is initially activated, but then results in low molecular weight peptides due to non-specific degradation. It is known from AT 397 390 that certain process changes must persist in the activation of prothrombin with the aid of digestive enzymes in order to prevent further non-specific degradation of the resulting thrombus. . Factor X activation is therefore a specialty in that a selective factor X activator is available from the venom from Vipera ruselli (Russell's snake venom RVV), which can be used industrially to obtain factor Xa. It shows a simple case. However, since the recognition of the transmission of animal viruses to unrelated species (prion problem), it has been viewed as undesirable to use heterologous animal proteins as adjuvants for the manufacture of drugs. However, the use of RVV for productive factor X activation has traditionally been the standard method, since suitable human proteases are not available in industrially sufficient quantities. Proteolytic enzymes that can be isolated from prokaryotes, lower eukaryotes, such as fungi, or higher eukaryotes, such as plants, are known to have low substrate specificity. For this reason, they are mainly used for the total degradation of proteins in crude cell extracts, in order to separate or isolate other cellular components in this way, for example carbohydrates or nucleic acids. In addition, these proteases have been used for protein sequencing and characterization by degradation into small peptides. To date, the use of bacterial or plant proteases or fungal proteases as activators or inhibitors of plasma proteins or plasma protein cofactors from the group of prothrombin complex proteins is known. It is an object of the present invention to provide a method for activating pro-factors in the area of blood coagulation with the aid of highly selective proteases that are not of animal origin. The above-mentioned problem is solved according to the invention by the method described at the outset, and according to the method, the activation takes place by proteases obtained from plants or prokaryotes. The method of the invention is particularly suitable for activating vitamin K-dependent blood factors. The activation of human blood factors obtained from the group of factor I, factor VII, factor IX, factor X and protein C is particularly suitable. The following examples demonstrate the benefits obtained in activating factor X to factor Xa-β. The method of the present invention is equally suitable for obtaining natural, recombinantly produced and chemically or recombinantly modified blood factors. As regards the proteases used according to the invention, one can start with prokaryotic proteases. Here, bacterial proteases such as thermolysin from Bacillus polymyxa, clostripain proteases IX and X, and protease IX from Bacillus thermoproteolyticus are particularly mentioned. In addition, proteases from lower eukaryotes, such as fungi, in particular, protease XXIII from the mold fungus Aspergillus oricae can also be used in the method of the invention. Furthermore, proteolytic enzymes derived from higher eukaryotes such as plants are also suitable. Proteolytic enzymes belonging to the cysteine protease are particularly suitable. For example, mention is made here of bromelain (for example from Ananas comosus), papain (for example from milk or Carica papaya) or ficin (for example from Ficus carica). However, the proteolytic enzymes mentioned from prokaryotes, fungi and plants have low substrate specificity. It was therefore surprising to confirm that the use of these enzymes could activate blood coagulation profactors with high specificity. These enzymes are selected natural enzymes, but may also be modified enzymes and / or enzyme derivatives, especially those produced by recombinant DNA technology. Cysteine proteases are particularly known to exhibit their full spectrum of protease activity under reducing conditions or in the presence of activators containing sulfhydryl groups. For this reason, the SH-donor is often added as an effector to the incubation buffer during incubation with these enzymes. However, the broad spectrum of activity of these proteases deviates from optimal incubation conditions, i.e., certain substrates, such as plasma proteins, are no longer completely degraded, while the activity of that substrate is Conditions away from optimal pH or temperature, with or without a partial or complete lack of the required cofactors or effectors, or in the presence of specific effectors, to the extent that they are not lost as far as the profactors that must be carried out Can shift to sub-optimal behavior. Rather, activation stops at the level of the target substrate, and the activation product is obtained on its own. This is also demonstrated for the first time using the example of activation of factor X to factor Xa, in particular factor Xaβ. In addition, highly specific cysteine proteases can be obtained by purifying the appropriate crude enzyme preparations from plants by further purification steps to isolate those protein fractions whose protease activity shows a limited substrate spectrum, e.g. by chromatography. It can also be obtained by subjecting to lithography. Although these methods alone may not be suitable for obtaining the desired highly specific activators, a further step in improving the specificity of the proteases, namely, the activity and activity spectrum of these proteases It is suitable in connection with a process by oxidation of these proteases, which is reduced as a result of contact with oxygen. From this it can be seen that oxidized cysteine proteases are particularly suitable for the method of the invention, whereby this is the preferred case for chromatographically enriched and / or purified cysteine proteases. . In that regard, the protease has a specific blood factor activating activity that is at least 2-fold increased compared to a crude extract from a plant or cultured cells and / or compared to a non-specific proteolytic activity. Is particularly preferred. Further, by adding a effector to the incubation buffer of the enzyme with the substrate to be activated, the substrate spectrum can be narrowed. Heavy metal ions or alkaline earth ions are specifically mentioned as effectors. Here, the addition of calcium ions is particularly preferred. Thus, in the examples below, the activation product, factor Xaβ, is obtained in high yield in the activation of factor X with calcium ions, whereby other cleavage products are produced in negligible amounts. Indicates no. However, the activation reaction proceeds strongly without the addition of calcium ions and can be adjusted with respect to the resulting activator. With appropriate combinations of experiments, factor Xaα and / or factor Xaβ can also be produced according to the present invention. For preferred industrial applications of the enzymes discussed here, these enzymes can be used in immobilized form. Thereby, simple separation of the protease from the substrate is possible, for example by filtration. Furthermore, the use of an immobilized enzyme, ie an enzyme bound to a solid phase, guarantees repeated use of the enzyme. For example, in the case of proteins that have been modified secondarily by oxidation, these proteins are irreversible to the conformation present after conversion to a highly specific activator by immobilisation, ie by covalent attachment of the enzyme to a carrier substance. Fixed. The method of the present invention is suitable for activating natural as well as recombinantly produced blood factors. In this context, the proteolytic cleavage site of blood factor as a coagulation proenzyme is then specified by a protease that does not correspond to a physiological activation mechanism only or by a defined protease that can also be activated by that protease It can be modified by an amino acid sequence that is specifically recognized and cleaved. This specifically constructed analog is, for example, an analog of a vitamin K-dependent blood factor such as factor X. Using the example of factor X, the region around Arg52-Ile53, which indicates the cleavage site for factor IXa, can be exchanged with a different amino acid sequence. For example, amino acids Arg53 and Ile53 can be replaced with the sequence Glu-Gly, which eliminates cleavage by factor IXa and activation by factor IXa, but allows proteolytic digestion by endoprotease Glu C from Staphylococcus auraeus V8. it can. Thus, bacterial enzymes that can be produced by highly purified methods and that do not present the risk of contamination by animal or human proteins can be used for activating factor X. Similarly, Gly-Ser, which can then be cleaved by a plant protease such as ficin, can be introduced as a cleavage sequence. The activated blood factor of the present invention is preferably subjected to a further purification step in order to remove protein hydrolysates that may be formed in this method. Here, a purification process by chromatography or purification by gel filtration is particularly preferred. The invention also includes a pharmaceutical preparation containing a blood factor and a protease specific for that blood factor obtained from a plant or prokaryote. The protease may be an animal-derived protease, especially a human-derived protease. However, in this context, clotting factors are not used as proteases according to the invention. In particular, plasma proteins such as vitamin K-dependent proteins are likewise included as blood factors in this pharmaceutical preparation. The pharmaceutical preparation specifically contains activated vitamin K-dependent blood factors and proteases. The blood factor is preferably human and natural. Proteases, especially purified proteases, use themselves for topical use in hemostasis, whereby vitamin K-dependent blood factors arise from bleeding wounds. The blood coagulation-inducing effect of a coagulation activator can be used here by applying the factor X in a pharmaceutical carrier substance, such as a wound dressing, powder or ointment. Another pharmaceutical composition also contains blood factors present as proproteins and cleaves proproteins isolated from plants, fungi or prokaryotes, or produced recombinantly in cells of these species Containing protease. Proteases of this type specific for proproteins are, for example, furin (Van de ven, WJ, et al., Enzyme 45: 257-270, 1991) or Paired Basic Amino Acid Residue Cleaving Enzyme, PACE, (Pehemtulla, A. et al., Biochemistry 32: 11586-11590, 1993). If the mature protein derived from the proprotein is particularly unstable in a pharmaceutical preparation, co-administration of the proprotein processing enzyme can result in the mature protein being made therapeutically available in situ. Furthermore, further activation by activator protease is possible, as long as the mature protein has to be converted into the active form by further proteolysis. Thus, therapeutic kits consisting of stable proproteins and proteolytic processing and / or activating enzymes also allow, for the first time, the administration of unstable proteins as active ingredients. Furthermore, the present invention includes an activated vitamin K-dependent blood factor obtained according to the method of the present invention. The human activated blood factor obtained according to the invention is characterized in that it is not contaminated by animal proteases. The following examples illustrate the method of the invention in more detail. These examples relate to the activation of factor X to factor Xa, which allows variations of the various manufacturing methods described in the dependent claims. Example 1 Production of Substrate X Factor Brummelhuis (Brummelhuis HGJ, Preparation of the prothrombin complex. In: Methods of plasmid Protein Fractionation, Curling, JM Ed. York: Academic Press, 1980, pp 117-128). Factor X was purified from the prothrombin complex factor preparation prepared according to the method described above and heat-treated according to EP 0 159 311 for virus inactivation. The lyophilized product containing the prothrombin complex factor was dissolved in distilled water so as to have an activity of 50,000 UX factor X / L, and the pH was adjusted to 7.0. After addition of 12% (v / v) TWEEN 80, it is stirred for 1 hour at room temperature, then diluted to 5 volumes with distilled water and mixed with trisodium citrate dihydrate (7 g / L) PH was adjusted to 7.0. Next, this was added to 30 g / L Ca _Three (PO _Four ) _Two And stirred at room temperature for 1 hour. The solid phase is then separated by centrifugation, and by resuspension and in each case a new centrifugation, using 25 mL / g calcium phosphate in 20 mmol / L Tris-HCl buffer (pH 7.0) containing 10% ammonium sulfate. Each was washed twice. Thereafter, a third re-suspension and subsequent centrifugation was performed using 25 mL / g calcium phosphate in 20 mmol / L Tris-HCl buffer (pH 7.0) containing 150 mmol / L NaCl, for the third time. Was. For elution of the adsorbed Factor X, the pellet was stirred with 1 mmol / L sodium phosphate buffer pH 7.0 for 1 hour at room temperature (using 25 mL eluate / g calcium phosphate). Thereafter, the solid phase was separated by centrifugation, and the factor X-containing supernatant was further purified by chromatography using DEAE Sepharose® Fast Flow (Pharmacia). For this, the column was packed with swollen and washed DEAE Sepharose® Fast Flow Gel and the setting of column inner diameter: gel-bed height = 1: 8.3 was used. Chromatographic material was loaded at 10 U X Factor / mL. The solution containing factor X was previously rebuffered against a buffer of 25 mmol / L trisodium citrate dihydrate, 100 mmol / L NaCl (pH 6.0). The Factor X solution was adsorbed on DEAE Sepharose® Fast Flow at a flow rate of 2 mL / min, and the column was then allowed to flow at a flow rate of 2 mL / min with 1.7 gel volumes of 25 mmol / L trisodium citrate disodium. Washed with 100 mmol / L NaCl in hydrate (pH 6.0) and 250 mmol / L NaCl in 2.6 gel volume of 25 mmol / L trisodium citrate dihydrate (pH 6.0). Elution of factor X was performed with 5.2 gel volumes of citrate buffer (pH 6.0) containing 280 mmol / L NaCl (pH 6.0). Fractions were collected while eluting and tested for the content of prothrombin complex protein factor X, protein C, factor IX and factor II by standard coagulation tests. Fractions containing factor X and low in other prothrombin complex proteins were combined and further purified by a monoclonal antibody purified from ascites against factor X, immobilized on Acrigel ALD (Sterogene, Bioseparations, Acradia CA). . The protein solution containing factor X was adsorbed on the gel at a concentration of 10 U factor X / mL gel. Next, it was washed with 5 column volumes of 20 mmol / L Tris-HCl buffer (pH 7.4). Elution was performed with 10 column volumes of buffer (pH 10.5) containing 100 mmol of 3-[(3-cholamidopropyl) dimethyl-ammonium] -1-propanesulfonate, 25 mmol / L NaCl. After the protein fraction eluted with this buffer was collected, it was concentrated to 1/20 of the initial volume by ultrafiltration using a 10,000 exclusion limit membrane. Next, the solution containing factor X was diafiltrated against a buffer (pH 7.0) containing 4 g / L trisodium citrate dihydrate, 8 g / L NaCl. This Factor X preparation is free of traces of contaminating proteins, particularly monoclonal antibodies, which have flowed out of the immunoaffinity chromatography column due to adsorption on Phenyl Sepharose® High Performance (Pharmacia-Biotech). For this, after diafiltration, the factor X-containing solution was adjusted to 1.8 mmol / L NaCl and the pH value to 7.4. The hydrophobic interaction chromatography gel was equilibrated with 20 mmol / L Tris-HCl, 2 mol / L NaCl (pH 7.4) in a chromatography column, and the adjusted factor X solution was applied to the column at a concentration of 30 U X factor / mL gel. Flowed away. The factor X-containing solution passed through the column was collected, concentrated to 1/20 of the initial volume by ultrafiltration using a 10,000 dalton exclusion membrane, and then 20 mmol / L Tris-HCl and 150 mmol / L NaCl were added. Diafiltration was performed on the buffer solution (pH 7.4). The high-purity factor X solution thus obtained had a specific activity of about 100 U / mg protein. Example 2 Principle of Quantitative Measurement of Factor Xa Test: Chromogenic peptide substrate CH _Three OCO-D-CHA-Gly-Arg-pNA is hydrolyzed by Factor Xa, thereby _Three OCO-D-CHA-Gly-Arg-OH and p-nitroaniline are formed. The kinetics of the increase in p-nitroaniline is measured at 405 nm on a spectrophotometer. The increase in optical density (OD) is proportional to the content of factor Xa in the sample being quantified. Reagents: Dilution buffer 3.7 g / L Tris- (hydroxymethyl) aminomethane 2.1 g / L Imidazole 18.0 g / L NaCl 1.0 g / L Human albumin pH 8.4 1.3 mmol in substrate solution dilution buffer / L CH _Three OCO-D-CHA-Gly-Arg-pNA Method: Mix 50 μL of the sample containing factor Xa with 50 μL of dilution buffer and incubate at 37 ° C. for 90 minutes. Then 100 μL of substrate solution is added and the increase in OD at 37 ° C. per minute is measured at 405 nm. The increase in OD must be linearly constant during the measurement. For the generation of the reference curve, a standard preparation of bovine factor Xa "NIBSC reagent 75/595" is used. The ampoule contains 1 U of Factor Xa after reconstitution with 1 mL of distilled water (see Datasheet, National Institute for Biological Standards and Controls). Example 3 Activation of Factor X Activation of the purified Factor X obtained from the example was performed at 20 mmol / L Tris-HCl, 150 mmol / L NaCl, 5 mmol / L CaCl. _Two 10 U / mL of the enzyme clostripain (Calbiochem, La Jolla, CA), 200 U / mL of thermolysin (Calbiochem, La Jolla, CA), 400 μg of papain (Boehringer Mannheim, Germany) in a buffer solution (pH 7.4) containing / ML and 20 μg / mL of ficin (Sigma Chemicals Co., St. Louis, Mo.) at 37 ° C. for several hours. Factor X concentration was 3.2 U / mL. At 5 minutes, 30 minutes, 1 hour, 2 hours and 19 hours, samples were removed from the individual incubation mixtures and examined for Factor Xa activity as described in Example 2. The results are shown in FIG. Activation of Factor X was possible with all of the enzymes used, indicating that after 2 hours thermolysin resulted in the highest yield of Factor Xa. Except for incubation with ficin, the phase of enzyme-dependent activation is maximal between 30 minutes and 2 hours, followed by inactivation of the generated factor Xa. Continued activation by stable factor Xa activity was measured 19 hours after activation by ficin. Example 4 Comparison of Factor X Activated Ficin and Russell Snake Venom The Factor X activator from Vipera russellii (RVV, Pentapharm AG, Basel, CH 2) is from the literature non-plasma factor X activity with high specificity Known as activator, it is currently used in the in vitro activation of factor X. In this example, the activation of the highly purified factor X obtained according to example 1 by RVV was investigated in comparison with the activation by the plant X activator ficin. Therefore, 20 mmol / L Tris-HCl, 150 mmol / L NaCl, 5 mmol / L CaCl _Two Using highly purified factor X at a concentration of 4 U / mL in a buffer solution (pH 7.4) containing 2.7 μg / mL RVV (Pentapharm AG, Basel, CH) or 20 μg at 37 ° C. / ML of ficin (Sigma Chemicals Co., St. Louis, MO). Samples were taken from the incubation mixture at various time points within 22 hours and examined for Factor X activity as described in Example 2. FIG. 2 shows the results of the test. Incubation of factor X with the factor X activator ficin has been shown to have factor Xa activity comparable to that obtained by incubation with RVV. The activation product of Factor X obtained after incubation with both activators after 22 hours was further tested by SDS-polyacrylamide gel electrophoresis under non-reducing conditions on an 8-18% gradient gel. . In this context, factor X before activation, RVV and factor X after activation with ficin are separated by electrophoresis, blotting on nitrocellulose membrane, detection with anti-factor X polyclonal antibody and standard methods. Analyzed after immunostaining. The results are shown in FIG. The homogeneous factor X before activation is cleaved by the activator RVV as well as by the activator ficin into several factor X-specific protein fragments of smaller molecular weight than the non-activated factor X, whereby Xaα and β factor Have been shown to occur after activation by RVV. In this mixture, these two activation products of factor X were obtained in approximately equal amounts, and at least three additional activation products were identified. In contrast, activation by ficin indicated that factor Xaβ was obtained as the major product (see arrows), indicating that three additional activation products were detected as well as activation by RVV. In contrast to activation by RVV, these fragments had a large difference in molecular weight from the main product, factor Xaβ, so that they could be purified by further simple methods, such as gel filtration. Example 5 Effector Effect on Factor X Activation by Ficin The incubation conditions described in the literature for proteolysis by ficin use a buffer system containing at least cysteine as SH reagent and an activator for protease. Since such SH-dependent proteases are known to be particularly inactivated by heavy metal ions, the addition of metal ion complexing agents such as ethylenediaminetetraacetic acid (EDTA) has also been described. In this example, in a buffer solution (pH 7.4) containing 20 mmol / L Tris-HCl and 150 mmol / L NaCl, (1) 2 mmol / L CaCl 2 was used. _Two (2) 1 mmol / L cysteine (3) 1 mmol / L cysteine and 2 mmol / L EDTA, and (4) 1 mmol / L cysteine and 2 mmol / L CaCl _Two Was added to the buffer medium, and Factor X was incubated at 4 U / mL at 2 ° g / L ficin / mL for 24 hours at 37 ° C. as in Example 4. After incubation for 24 hours, factor Xa activity was measured according to Example 2. Table 1 shows the results. Table 1: Factor X activation by ficin, effector effect The addition of calcium chloride yields a clearly higher yield of factor Xa, whereas under standard conditions (cysteine ± EDTA) the yield of factor X is very small. It was shown that only The batch was also examined for activation product composition by SDS-polyacrylamide gel electrophoresis as described above. FIG. 4 shows the results of the electrophoretic test after protein staining according to the silver staining method and after specific immunostaining with anti-factor X antibody. The addition of calcium ions to the incubation medium was shown to result in a clearly higher yield of factor Xaβ among the factor X cleavage products. Factor Xaβ obtained in this way can easily remove residual ficin still contained in the mixture by conventional chromatographic purification methods. In particular there are simple gel filtration, ion exchange chromatography or substrate affinity chromatography using immobilized benzamidines which can, for example, selectively bind factor Xa. Example 6 Isolation of Factor X Activator from Plant Latex 10 mg of fine powdered latex from Ficus glabrata containing 2 mg of protein was added to 5 mmol / L sodium phosphate buffer (pH 5.5) containing 1 mmol / L EDTA 2. It was suspended in 5 mL and mixed with 100 μL of a 50 mmol / L cysteine solution for activation of cysteine protease. Free cysteine was then removed by gel filtration using Sephadex® G50 and further diluted with the same phosphate buffer 1 + 3. Next, the whole amount was passed through a MonoSHR5 / 5 cation exchange chromatography column (Pharmacia) at a flow rate of 1 mL / min. This was then washed with 45 column volumes of the same phosphate buffer, and gradient elution was continued over 55 column volumes by continuously increasing the phosphate concentration to 185 mmol / L in the same buffer composition. went. The results are shown in FIG. The protein mixture separated into several individual proteins as can be seen from the elution profile due to absorption (protein) at 280 nm. The individual fractions were then examined for Factor X activating enzyme activity by the Factor Xa assay described in Example 2, eluting between the protein peak eluted at an elution volume of 49 mL and the elution volumes between 68 and 69 mL. The protein peak was shown to have the highest factor X activator activity (-×-). In a similar manner, fractions were examined for protease activity on non-specific protease substrates. For this purpose, the chromogenic protease substrate benzoyl-DL-arginine-p-nitroanilide used according to their method under the conditions of Englund et al., Biochemistry 7: 163-175 (1968) was used in a photometric test. did. As can be seen from FIG. 5, considerable protease activity was found in the fraction passing through the column as well as in all subsequent fractions (Δ). Here, the peak with an elution volume of 49 mL and further with an elution volume of 68 to 69 mL had the strongest activity. However, apart from this, considerable protease activity was further measured in the protein fraction with an elution volume of 53 mL and an elution volume of 74-83 mL. It is noteworthy that the factor X activator activity was significantly higher than the non-specific protease activity in the fractions eluted from the 68-69 mL column, indicating that factor X was derived from crude plant extracts. This is due to the separation of the activator. The fraction containing the highest factor X activator activity in terms of protein content was then incubated for 15 hours at 4 ° C. under the influence of atmospheric oxygen. This further increased factor X activator activity in relation to non-specific protease activity. Example 7 Immobilization of Plant Factor X Activator Crystallized ficin (Sigma) in a crystal suspension was diluted to a concentration of 2.5 mg / mL and Sephadex® G50 (Pharmacia) was added. The buffer was rebuffered against a buffer (pH 7.4) containing 20 mmol / L Tris-HCl, 150 mmol / L, NaCl by gel filtration used. The pre-activated gel Actigel ALD Superflow (Sterogene Bioseparations, Arcadia, CA) is washed with a buffer solution (pH 7.4) containing 20 mmol / L Tris-HCl and 150 mmol / L NaCl, and contains ficin to be immobilized. The solution was mixed at a 1 + 1 ratio and incubated with 1/15 volume of coupling solution (Sterogene Biosparation, Arcadia, CA) for 3 hours at room temperature. Then, the immobilized product was separated by a sintering nutchet, and a buffer containing 20 mmol / L Tris-HCl and 150 mmol / L NaCl (pH 7.4) and a buffer containing 20 mmol / L Tris-HCl and 2 mmol / L NaCl (pH 7) were used. Washing was alternately and thoroughly performed in .4) to remove unbound ficin and coupling reagent. Next, the immobilized ficin was used for activating factor X as follows. The highly purified Factor X obtained in Example 1 was adjusted to 2 U X Factor / mL in a buffer (pH 7.4) containing 20 mmol / L Tris-HCl, 150 mmol / L NaCl, and It was mixed with the wet ficin immobilization. It was then incubated at 37 ° C. for 60 minutes with continued thorough mixing, after which factor Xa activity was measured as described in Example 2. Thereby, factor Xa activity of 4 U / mL was measured. This can lead to the fact that ficin also maintains factor X activator activity in a form bound to a solid phase and is therefore suitable as an immobilizate for producing factor Xa. Example 8 An industrial ficin preparation from Ficus glabrata latex was purified according to their method under the conditions of Englund et al., Biochemistry 7: 163-175 (1968), whereby benzoyl-DL-arginine-p Those fractions with the highest specific factor X activator activity compared to the non-specific protease activity on nitroanilide were collected. The pooled preparation from the chromatographic purification was subjected to activation with 10 mM cysteine at 37 ° C. for 15 minutes, and the excess from the activation mixture was removed by a series of separations by gel filtration using Sephadex® G-25 Superfine. Activators were removed. Calcium and manganese ions in an incubation mixture containing 4 U / mL of factor X and 3 μg / mL of activated ficin preparation in a buffer system (pH 7.4) of 20 mmol / L Tris-HCl, 150 mmol / L sodium chloride. Activation was performed in the presence and in the presence. The batch contained either 2 mmol / L calcium ion, 1 mmol / L manganese (II) ion or a combination of two metal ions. After incubation at 37 ° C. for 5 hours, the batch was checked for the composition of the activated product by SDS-polyacrylamide gel electrophoresis. The results of the separation by electrophoresis were visualized according to a silver staining method, and the intensities of the separated bands were analyzed with a densitometer. The results of this analysis are shown in the table below. Activation by purified, activated ficin preparations has been shown to result in increased degradation of factor Xa formed without the addition of two metal ions. Ca ²⁺ And / or Mn ²⁺ The addition of ions instead reduced the production of various cleavage products, but the combination of both ions almost reversed the ratio of activated factor X to its degradation products. In addition, Ca ²⁺ And Mn ²⁺ It was shown that under ion addition, an activated factor X resulted which did not undergo any further degradation processes after the activation process. To clarify this evidence, an activation batch similar to the conditions described above was performed with the addition of 2 mmol / L calcium and 1 mmol / L manganese (II) ions, and in this experiment the activation kinetics was Samples were taken from the incubation mixture at some point for recording. Factor Xa activity was measured according to Example 2, and the fraction of factor Xa degraded cleavage products was measured in the same manner as described above. The results of these experiments are summarized in the following table: The further purification of the obtained factor Xa can then be carried out according to example 5.

[Procedure for Amendment] Article 184-8, Paragraph 1 of the Patent Act [Date of Submission] June 14, 1999 (June 14, 1999) [Details of Amendment] Claims 1. Wherein the protease is derived from a plant, fungus or prokaryote and has a specific blood factor activating activity that is at least 2-fold increased compared to a crude extract derived from the plant or cultured cells. A method for activating a K-dependent blood factor. 2. The method according to claim 1, wherein the blood factor is selected from the group of human factor II, factor VII, factor IX, factor X and protein C. 3. 3. The method according to claim 2, wherein the factor Xa- [beta] is obtained after activation of human factor X. 4. 4. The method according to claim 1, wherein the protease is selected from the group consisting of ficin, bromelain, papain, thermolysin and clostripain. 5. 5. A method according to any one or more of the preceding claims, characterized in that the treatment is carried out under suboptimal conditions for the protease. 6. The method according to any one or more of the preceding claims, wherein the protease is an oxidized cysteine protease. 7. 7. The method according to any one of claims 1 to 6, wherein the protease is purified by chromatography. 8. The method according to claim 1, wherein the activation is performed in the presence of a defector. 9. 9. The method according to claim 1, wherein the activation is carried out in the presence of heavy metal ions or alkaline earth ions. 10. The method according to claim 9, wherein the activation is performed in the presence of calcium ions. 11. The method according to any one of claims 1 to 10, wherein the protease is immobilized on a solid carrier. 12. The method according to any one or more of the preceding claims, wherein the protease is produced by recombinant DNA technology. 13. The method according to claim 1, wherein the recombinant blood factor is activated. 14． 15. The method of claim 14, wherein a blood factor analog having a protease specific proteolytic cleavage site is activated. 15. 16. The method according to claim 15, wherein the proteolytic cleavage site is modified and activation is performed by a protease that does not correspond to a physiological activation mechanism. 16. A pharmaceutical preparation comprising a blood factor and a protease specific for that blood factor from plants, fungi or prokaryotes. 17． 17. The pharmaceutical preparation according to claim 16, wherein the blood factor is an activated vitamin K-dependent protein. 18. A pharmaceutical preparation containing a purified protease specific for blood factors, characterized in that it is formulated for topical use and is characterized in that the protease is obtained from a plant, fungus or prokaryote. 19. A kit for medical use comprising: a) a protein hydrolase from a plant, fungus or prokaryote having specificity for a pro-form of a protein, and b) a pro-form of the protein. 20. Activated vitamin K-dependent blood factor, characterized in that it is obtained according to the method of claims 1 to 15. FIG. FIG. 2 FIG. 3 FIG. 4 FIG. 5

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, M W, SD, SZ, UG, ZW), EA (AM, AZ, BY) , KG, KZ, MD, RU, TJ, TM), AL, AM , AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, E S, FI, GB, GE, GH, GM, GW, HU, ID , IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, M G, MK, MN, MW, MX, NO, NZ, PL, PT , RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, V N, YU, ZW (72) Inventor Filapichu, Anton             Austria, Ahr-2490 Ebenfurt,             Hans-Kudrich-Gasse 5 (72) Inventor Schwarz, Hans-Peter             Austria, Ar-1180 Vienna, Singh             Dragasse 32 (72) Inventor Ible, Johan             Austria, Ar-1180 Vienna, Gus             Tuff-Zellmarkgasse No. 2

Claims (1)

[Claims] 1. A protease, wherein the protease is derived from a plant, fungus or prokaryote. A method for activating a vitamin K-dependent blood factor by treatment with a Rotase. 2. The blood factor is human factor II, factor VII, factor IX, factor X and protein. The method of claim 1, wherein the method is selected from the group of 3. Wherein the factor Xa-β is obtained after activation of human factor X. Item 3. The method according to Item 2. 4. Proteases are ficin, bromelain, papain, thermolysin and The method according to claims 1 to 3, characterized in that it is selected from the group of Lostripain. Law. 5. Wherein the treatment is performed under suboptimal conditions for the protease, A method according to any one or more of the preceding claims. 6. Characterized in that the protease is an oxidized cysteine protease A method as claimed in any one or more of the preceding claims. 7. A protease, wherein the protease is purified by chromatography; 7. A method as claimed in any one or more of claims 1 to 6. 8. Protease is at least as compared to a crude extract from a plant or cultured cells. 8. The method according to claim 7, which has a two-fold increased specific blood factor activating activity. The method described. 9. 9. The method according to claim 1, wherein the activation is carried out in the presence of the effector. A method according to any one or more of the preceding claims. 10. Activation takes place in the presence of heavy metal ions or alkaline earth ions The method according to any one or more of claims 1 to 9, characterized in that: The method described. 11. The activation is carried out in the presence of calcium ions. 10. The method according to 10. 12. Wherein the protease is immobilized on a solid carrier. 12. The method according to any one of claims 1 to 11, or more. 13. Characterized in that the protease is produced by recombinant DNA technology, 13. A method as claimed in any one or more of the preceding claims. 14． 2. The method according to claim 1, wherein the recombinant blood factor is activated. 15. Blood factor with a protease-specific proteolytic cleavage site 15. The method of claim 14, wherein the analog is activated. 16. Modification of proteolytic cleavage sites to accommodate physiological activation mechanisms The activation is carried out by a non-protease. the method of. 17． Blood factor and a protease specific to that blood factor, preferably a plant, A pharmaceutical composition comprising a fungal or prokaryotic protease. 18. If blood factors are plasma proteins, preferably vitamin K-dependent proteins, 2. A highly activated vitamin K-dependent protein. 8. The pharmaceutical composition according to 7. 19. Formulated for topical use, the protease is preferably plant, fungal or Or a blood factor-specific protease obtained from a prokaryote Pharmaceutical compositions containing, especially purified proteases. 20. a) Proteolytic enzymes with specificity for the pro form of the protein Especially enzymes from plants, fungi or prokaryotes, and b) Pro form of the protein A kit for medical use containing 21. An activity obtained according to the method of claim 1. Sexualized vitamin K-dependent blood factor.