DE19710190A1 - Activated vitamin K-dependent blood factor and method for its production - Google Patents

Abstract

The invention relates to a method for activating a vitamin K-dependent blood factor by treatment with a protease derived from plants or procaryotes. The invention also comprises the blood factor produced according to the invention, which shows no animal protease contamination, as well as a pharmaceutical preparation and a set for medical application.

Description

The present invention relates to an activated vitamin K-dependent blood factor and a method to its Production.

The complex protease / substrate cascade at the physiological activation of profactors, especially in the Area of blood clotting, requires highly selective proteases. These proteases come in the human organism itself, especially in the blood plasma, before. The application of others human proteases for activating Profaktoren such. B. the digestive enzymes trypsin and pepsin, therefore, does not lead to a selective generation activated Blood clotting factors, but to a nonspecific degradation of the target substrate. So z. B. known that the tryptic Degradation of coagulation factor X is initially activated, but then by non-specific degradation to low-molecular Peptides leads. From AT-397 390 it is known that for the Activation of prothrombin by means of a digestive enzyme special process variants must be adhered to a further unspecific degradation of the resulting thrombus to prevent.

In the case of the activation of factor X, it therefore represents one Peculiarity that with the Venom from Vipera Russelli (Russell's Viper Venom, RVV) a selective factor  X-activator is available, which is also technically to Obtaining Factor Xa can be used. Since knowledge the transmission of animal viruses to unrelated However, animal species (prion problematic) will be the use xenogenic animal proteins as adjuvants for the Production of medicines refused. But because suitable human proteases are not available in sufficient quantities Available, the use of RVV is preparative Factor X activation still standard method.

Of proteolytic enzymes derived from prokaryotes low eukaryotes, such. As mushrooms, or from higher Eukaryotes, such. B. plants, can be isolated is known to have a low substrate specificity. For this reason, they are mainly for the total dismantling of Proteins used in crude cell extracts to access these Do other cell components, such. As carbohydrates or Nucleic acids, to separate or isolate. In addition, will these proteases also for sequencing and characterization of proteins by way of degradation to small peptides used. So far, the application was bacterial or plant proteases or proteases from fungi as Activators of plasma proteins or plasma protein cofactors or inhibitors from the group of prothromes bin complex proteins are unknown.

The object of the present invention is to provide a method for Activation of profactors in the area of blood coagulation with Help high-specific proteases that are not animal Origin are to provide.

The above object is achieved according to the invention by the initially mentioned method, wherein the activation by means of a protease derived from plants or Prokaryotes is derived.  

The inventive method is in particular for Activation of vitamin K-dependent blood factors suitable. Particularly suitable is the activation of human Blood factors from the group Factor II, VII, IX, X and protein C. The following examples show the advantages as they obtained in the activation of factor X to factor Xa-β become.

The inventive method is equally suitable for the recovery of naturally occurring, genetically engineered produced, as well as chemically or genetically modified blood factors.

With respect to the protease used in the invention can assumed a proteolytic enzyme from prokaryotes become. Here are in particular the bacterial proteases too call, such. B. thermolysin, clostripain proteases IX and X. from Bacillus polymyxa and protease IX from Bacillus thermoproteolyticus.

In addition, proteases can also be made from low levels Eukaryotes, such. As fungi, especially protease XXIII the fungus Aspergillus oricae, in which use method according to the invention. In addition, suitable proteolytic enzymes from higher eukaryotes, such. B. Plants. In particular, proteolytic enzymes are suitable, belonging to the group of cysteine proteases. Here are for example: bromelain (eg from pineapple comosus), papain (eg from the milk juice of Carica papaya) or ficin (eg from Ficus carica). The mentioned proteolytic enzymes from prokaryotes, molds and However, plants have a low substrate specificity on. It was therefore surprising to note that by Use of these enzymes profactors of blood clotting high can be activated specifically. These enzymes are either selected native enzymes, but also modified ones  Enzymes or enzyme derivatives, in particular by enzymes produced by recombinant DNA technology.

It is known in particular for cysteine proteases that they under reductive conditions or in the presence of Sulfhydrilgruppen containing activators their broad Protease activity can fully develop. For this reason become common during incubation with these enzymes SH donors added as effectors to the incubation buffer. The However, broad spectrum of action of these proteases can by Departing from the optimal incubation conditions towards suboptimal conditions, d. H. Conditions away from the pH or temperature optimum or partial or complete absence of necessary cofactors or effectors, or in the presence of special effectors to that effect be moved that special substrates such. B. Plasma proteins are no longer completely degraded and hence their activity, if it is to be activated Profaktoren acts, not lose. Rather, the Activation stopped at the stage of the target substrate and it the activation products are obtained as such. Also this is shown below using the example of activation of factor X to factor Xa, in particular β-factor Xa, for the first time demonstrated.

Furthermore, a higher specificity of cysteine proteases also be achieved by taking the plants purified crude enzyme preparations further Subject cleaning measures, z. B. a chromatography, to isolate those protein fractions whose Protease activity shows a limited substrate spectrum. While this measure may not be alone is appropriate to the desired high-specific Activators can be used in conjunction with Another measure to increase the specificity of Proteases, namely by oxidation of the same, wherein the  Proteases due to contact with atmospheric oxygen in their Activity and its spectrum of activity can be reduced. It follows that for the inventive method particularly oxidized cysteine proteases are suitable, wherein this is preferred in chromatographically enriched or purified cysteine proteases is the case. Especially it is preferred that the protease has at least one Twofold increased specific blood factor activation activity compared to the crude extract from plants or cell cultures or towards the non-specific proteolytic activity or towards non-specific proteolytic activity having.

Furthermore, the substrate spectrum can thereby be narrowed be added to the incubation buffer of the enzymes with the activating substrates are added to defectors. When Defectors are in particular heavy metal ions or To call alkaline earth ions. Particularly preferred here is the Addition of calcium ions. So z. B. in the following Examples have shown that in the activation of factor X. with the addition of high-yield calcium ions Activation product β-factor Xa is obtained, with others Fission products arise only in negligible amount.

However, the activation reaction without addition of Calcium ions increasingly run off and also in terms of modulated activated factor. at However, suitable experimental arrangements are also α-factor Xa and / or β-factor Xa according to the invention.

For a preferred technical applicability of here in question These enzymes can be immobilized be used. This will be a simple separation of Protease from the substrate, e.g. B. by filtration enabled. In addition, the use of immobilized, d. H. the enzymes bound to a solid phase  Use of the same. In the case of the secondarily modified Proteins, e.g. B. by oxidation, is characterized by the Immobilization, d. H. by a covalent bond of the enzyme to a support material the same in its conformation as after converting it into a high-specific activator present, irreversibly fixed.

The inventive method is suitable for activation natural as well as recombinant blood factors.

It is preferred that the invention activated Blood Factor is subjected to further cleaning measures in this way about resulting proteolytic degradation products to remove. Particularly preferred are here chromatographic purification processes or a purification by gel filtration.

The invention also includes a pharmaceutical preparation, the one blood factor and one of plants or prokaryotes derived protease with specificity for the blood factor contains. The protease may also be one of mammals, in particular, be a human-derived protease. However, according to the invention, no protease is used Coagulation factor used. As blood factors are especially plasma proteins, e.g. B. also vitamin K-dependent Proteins contained in this pharmaceutical preparation. In particular, the pharmaceutical preparation comprises a activated vitamin K-dependent blood factor and the protease. This blood factor is preferably human and native.

For topical application for haemostasis is also the Protease, in particular a purified protease, as such in which the vitamin K-dependent blood factor in this case comes from a bleeding wound. By Applying the Factor X Activator in a Pharmaceutical Carrier material, for. B. in a bandage or in a  Powder or as an ointment, here can the blood clotting triggering effect of the coagulation activator can be availed. Another pharmaceutical preparation also includes one Blood factor, which is present as pro-protein, and one out Plants, fungi or prokaryotes isolated or in cells this species genetically engineered pro-protein containing cleavage protease. Proteases of this type with a Specificity for pro-proteins are e.g. B. as Furin (Van de Ven, W.J. et al, Enzyme 45: 257-270, 1991) or Paired Basic Amino Acid Residue Cleaving Enzyme, PACE, (Rehemtulla, A et al, Biochemistry 32: 11586-11590, 1993). If that is out of the Pro-protein resulting mature protein in one pharmaceutical preparation is particularly unstable, can through simultaneous administration of pro-protein processing Enzyme's mature protein is displayed in situ and stands then therapeutically available. Furthermore, one is further activation with an activator protease, if that mature protein by further proteolytic digestion only in an active form must be transferred, possible. On therapeutic set consisting of the stable pro-protein and the processing or activating proteolytic Enzyme, therefore, for the first time allows the administration as well labile proteins as active ingredients.

Furthermore, the invention comprises an activated vitamin K-dependent blood factor, as he after the invention Procedure is obtained. The inventively obtained Human, activated blood factor is characterized in that he is not contaminated by an animal protease.

The inventive method is characterized by the following Examples explained. These examples concern the Activation of factor X to factor Xa, where the various process variants according to the subclaims Find application.

EXAMPLE 1 Production of the substrate factor X

Factor X was purified from a prothrombin complex factor preparation containing the factors FII, FIX, FX, protein C and protein S and purified by the method of Brummelhuis, (Brummelhuis HGJ, Preparation of the prothrombin complex. In: Methods off Plasma Protein Fractionation , edited by Curling, JM, New York: Academic Press, 1980, pages 117-128) and heat-treated for virus inactivation according to EP 0 159 311. A lyophilizate containing the prothrombin complex factors was dissolved in distilled water according to an activity of 50,000 E FX / l and adjusted to pH 7.0. After addition of 12% (v / v) TWEEN 80 was stirred for 1 hour at room temperature, then diluted with distilled water to 5 times the volume, mixed with tri-sodium citrate dihydrate (7 g / l) and adjusted to pH 7.0 set. Subsequently, it was mixed with 30 g / l of Ca ₃ (PO ₄ ) ₂ and stirred for 1 hour at room temperature. Thereafter, the solid phase was separated by centrifugation and washed twice with in each case 25 ml / g calcium phosphate of a buffer 20 mmol / l Tris-HCl, pH 7.0, containing 10% ammonium sulfate, by resuspension and again centrifuged off. Thereafter, a third wash with 25 ml / g calcium phosphate of a buffer 20 mmol / l Tris-HCl containing 150 mmol / l NaCl, pH 7.0, was performed by re-resuspending and subsequent centrifugation. To elute the adsorbed factor X, the pellet was stirred with 1 mmol / l sodium phosphate buffer, pH 7.0 (25 ml elution solution / g calcium phosphate used) for 1 hour at room temperature. Thereafter, the solid phase was separated by centrifugation and the factor X-containing supernatant by chromatography on DEAE Sepharose® Fast Flow, Fa. Pharmacia, further purified. For this, a column filled with swollen, washed DEAE Sepharose® Fast Flow gel and a configuration of column inner diameter: gel bed height = 1: 8.3 was used. The chromatography material was loaded with 10 U FX / ml gel. Previously, the FX-containing solution was buffered by gel filtration against a buffer, 25 mmol / l trisodium citrate dihydrate, 100 mmol / l NaCl, pH 6.0. This Factor X solution was adsorbed to DEAE Sepharose® Fast Flow at a flow rate of 2 ml / min and the column was flown at 2 ml / min with 1.7 times gel volume, 100 mmol / l NaCl in 25 mmol / l trisodium citrate dihydrate, pH 6.0, and further washed with 2.6 times the volume of gel, 250 mmol / l NaCl, 25 mmol / l tri-sodium citrate dihydrate, pH 6.0 , Elution of Factor X was done with 5.2 times the gel volume of a citrate buffer, pH 6.0, containing 280 mmol / L NaCl, pH 6.0. The elution collected fractions that were assayed for the content of the prothrombin complex proteins Factor X, Protein C, Factor IX and Factor II, using standard coagulation tests. The fractions containing Factor X, which were poor in other prothrombin complex proteins, were pooled and then immobilized on ascites-purified monoclonal antibody directed against Factor X and immobilized on Actigel ALD (Sterogens, Bioseparations, Arcadia, CA) , further cleaned. The FX-containing protein solution was adsorbed to the gel with a loading of 10 U FX / ml gel. The mixture was then washed with 5 times the volume of a column of 20 mmol / l Tris-HCl, pH 7.4. The elution was carried out with 10 times the column volume of a buffer containing 100 mmol of 3 - [(3-cholamidopropyl) dimethyl ammonium] -1-propanesulfonate, 25 mmol / l NaCl, pH 10.5. The protein fraction eluting with this buffer was collected and then concentrated by ultrafiltration through a membrane with an exclusion limit of 10,000 daltons to 1/20 of the starting volume. Subsequently, the FX-containing solution was diafiltered against a buffer containing 4 g / l trisodium citrate dihydrate, 8 g / l NaCl, pH 7.0. This factor X preparation was freed from traces of contaminating protein, especially from the immunoaffinity chromatography column, by bleeding monoclonal antibody by adsorption on Phenylsepharose® High Performance, Pharmacia Biotech. For this purpose, the FX-containing solution was adjusted to 1.8 mmol / l NaCl and a pH of 7.4 after diafiltration. The hydrophobic interaction chromatography bar was equilibrated in a chromatography column with 20 mmol / l Tris-HCl, 2 mol / l NaCl, 7.4, and the adjusted factor X solution was loaded onto the column at a loading of 30 U FX / ml gel. The FX-containing flow from the column was collected and subsequently concentrated by ultrafiltration over a membrane with an exclusion limit of 10,000 Daltons to _^1/20 of the starting volume and then against a buffer containing 20 mmol / l Tris-HCl, 150 mmol / l NaCl pH 7.4, diafiltered. The highly purified factor X solution thus obtained had a specific activity of about 100 U / mg protein.

EXAMPLE 2 Quantitative determination of factor Xa

Test principle:
The chromogenic peptide substrate CH ₃

OCO-D-CHA-Gly-Arg-pNA is hydrolyzed by Factor Xa, where CH ₃

OCO-D-CHA-Gly-Arg-OH and paranitroaniline are formed. The kinetics of the increase of paranitroaniline is measured spectrophotometrically at 405 nm. The increase in optical density (OD) is proportional to the content of factor Xa in the sample to be quantified.
reagents:
Diluent:
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
Substrate Solution:
1.3 mmol / l CH ₃

OCO-D-CHA-Gly-Arg-pNA in dilution buffer
Method:
50 μl of a sample containing Factor Xa are mixed with 50 μl of dilution buffer and incubated for 90 seconds at 37 ° C. Then, 100 μl of the substrate solution is added and the increase in OD per minute at 37 ° C at 405 nm is determined. The increase in OD must remain constant linear over the measurement period.

The default preparation is to create a reference curve used by bovine factor Xa "NIBSC Reagent 75/595", wherein one ampoule after reconstitution with 1 ml A.dest. 1 E FXa contains (see Datasheet, National Institute for Biological Standards and Controls).

EXAMPLE 3 Activation of factor X

Activation of purified Factor X from Example 1 was performed with the enzymes, Clostripain (Calbiochem, La Jolla, CA) 10 U / ml, Thermolysin (Calbiochem, La Jolla, CA) 200 U / ml, Papain (Boehringer Mannheim, FRG) 400 μg / ml and ficin (Sigma Chemical Co., St. Louis, MO) 20 μg / ml, in a buffer containing 20 mmol / l Tris-HCl, 150 mmol / l NaCl, 5 mmol / l CaCl ₂ , pH 7, 4, performed at 37 ° C and incubated for several hours. The concentration of factor X was 3.2 U / ml. At 5 minutes, 30 minutes, 1 hour, 2 hours and 19 hours, samples were taken from the respective incubation mixtures and assayed for factor Xa activity as described in Example 2. The results are shown in Fig. 1. It was found that activation of factor X was possible with all the enzymes used and led to the highest yield of factor Xa for thermolysin after 2 hours. Except for incubation with ficin, the activation phase, which reached a maximum of between 30 minutes and 2 hours depending on the enzyme, was followed by inactivation of the resulting factor Xa. Upon activation of ficin, steady activation with stable factor Xa activity was also detectable after 19 hours.

EXAMPLE 4 Activation of factor X Comparing Ficin and Russell's Viper Venom

From the literature factor X activator is from Vipera russellii (RVV, Pentapharm AG, Basel, CH) as a non-plasmatic Factor X-activator known with a high selectivity for in vitro activations of factor X commonly is used.

In this example, the activation of highly purified Factor X from Example 1 with RVV was examined as compared to activation with the plant Factor X activator, Ficin. For this purpose, the highly purified Factor X at a concentration of 4 U / ml in a buffer containing 20 mmol / l Tris-HCl, 150 mmol / l NaCl, 5 mmol / l CaCl ₂ , pH 7.4 was used and treated with either 2.7 μg / ml RVV (Pentapharm AG, Basel, CH) or 20 μg / ml Ficin (Sigma Chemical Co., St. Louis, MO) at 37 ° C. At various times samples were taken from the incubation mixtures within 22 hours and assayed for factor X activity as described in Example 2. The result of the investigation is shown in Fig. 2. It was found that the incubation of factor X with the factor X activator from ficin resulted in a factor Xa activity comparable to that which could be achieved by incubation with RVV. The activation products of factor X, which could be achieved after incubation with the two activators after 22 hours, were also examined by SDS-polyacrylamide gel electrophoresis in gradient gels 8-18% under non-reducing conditions. After electrophoretic separation factor X before activation, factor X after activation with RVV and after activation with ficin, and subsequent blot on nitrocellulose membranes and detection with an anti-factor X polyclonal antibody and immunostaining according to standard methods were analyzed. The result is shown in Fig. 3. It was found that the homogeneous factor X was cleaved before activation by both the activator RVV and the activator ficin into several factor X-specific protein fragments smaller molecular weight than the unactivated factor X, wherein after activation with RVV a mixture of alpha- and Beta factor Xa was formed, these two activation products of factor X being contained in approximately the same amount, and at least three further activation products could be identified. In contrast, the activation by Ficin showed that the main product beta-factor Xa was obtained (see arrow), where as with the activation with RVV also three other activation products could be detected, however, unlike the activation with RVV a larger molecular weight difference to the main product beta Had factor Xa, so that these by simple other methods, eg. As gel filtration, could be separated.

EXAMPLE 5 Influence of effectors on factor X activation with ficin

The incubation conditions for proteolytic degradation with ficin indicated in the literature comprise buffer systems, which mostly contain cysteine as the SH reagent and activator for the protease. Furthermore, the addition of a metal ion complexing agent, for. As ethylenediaminetetraacetic acid (EDTA), because it is known that such SH-dependent proteases, in particular by heavy metal ions are inactivated. In this example, Factor X at a concentration of 4 U / ml in a buffer containing 20 mmol / l Tris-HCl, 150 mmol / l NaCl, pH 7.4, with 2 μm ficin / ml was analogous to Example 4 at 37 ° C incubated for 24 hours, the buffer medium (1) 2 mmol / l CaCl ₂ , (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 _{2 was} added. After 24 hours of incubation, the factor Xa activity was determined according to Example 2. The results are shown in Table 1.

Factor X activation with ficin, influence of effectors approach FXa activity after 24 h (E / ml) 2mM CaCl ₂ 42.1 1 mM cysteine 17.5 1mM cysteine, 2mM EDTA 10.3 1 mM cysteine, 2 mM CaCl ₂ 45.1

It was found that by the addition of calcium chloride, a significantly higher yield of factor Xa could be achieved, while under standard conditions (cysteine ± EDTA) only lower factor X yields could be realized. The mixtures were also analyzed for the composition of the activation products by SDS-polyacrylamide gel electrophoresis as described above. The result of the electrophoretic examination after staining of the proteins according to the silver staining method as well as after specific immunostaining with an anti-factor X antibody is shown in FIG . It has been found that the addition of calcium ions to the incubation medium leads to a significantly higher proportion of beta-factor Xa among the factor X cleavage products. The thus obtained beta-factor Xa can now be easily freed by conventional chromatographic purification methods of the mixture still contained in the mixture of ficin. These include simple gel filtration, ion exchange chromatography or substrate affinity chromatography, for example on immobilized benzamidine, which is capable of selectively binding factor Xa.

EXAMPLE 6 Isolation of Factor X Activator from Plant Latex

10 mg of a Ficus glabrata powdered latex containing 2 mg of protein was suspended in 2.5 ml of a 5 mmol / l sodium phosphate buffer containing 1 mmol / l EDTA, pH 5.5, and with 100 μl of 50 mmol / l cysteine solution for activation the cysteine protease is added. Subsequently, the free cysteine was removed by gel filtration over Sephadex® G50 and further diluted with the same phosphate buffer 1 + 3. The total amount was then applied to a Mono S HR 5/5 cation exchange chromatography column, Pharmacia, at a flow rate of 1 ml / min. Subsequently, washing was carried out with 45 column volumes of the same phosphate buffer and then a gradient elution was carried out, wherein the phosphate concentration was continuously increased to 185 mmol / l in the same buffer composition over 55 column volumes. The result is shown in Fig. 5. By elution, the protein mixture was separated into several individual proteins. As can be seen in the elution profile at the absorption at 280 nm (- protein). Subsequently, the individual fractions were assayed for FX activating enzyme activity by the Factor Xa assay described in Example 2, and it was found that a protein peak eluting at 49 ml elution volume and a protein peak eluting between 68 and 69 ml elution volumes, had the highest factor X activator activity (- x - -). Similarly, the fractions were assayed for protease activity with a non-specific protease substrate. For this purpose, the chromogenic protease substrate, benzoyl-DL-arginine p-nitroanilide hydrochloride, which was used under the conditions and by the method of Eglund et al., Biochemistry 7: 163-175 (1968), was used in a photometric test , As can be seen from Fig. 5, significant protease activity (Δ) could be found both in the column pass and in all other fractions, with the peaks with elution volume 49 ml and elution volume 68-69 ml also showing the strongest activities here. In addition, however, 53 ml and 74-83 ml significant protease activities were also detected in the protein fractions with elution volume. Remarkably, in the fraction which eluted from the column at 68-69 ml, Factor X activator activity was significantly higher than non-specific protease activity due to separation of the factor X activator from the crude plant extract. 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 action of atmospheric oxygen. As a result, the factor X activator activity could be further increased in relation to the non-specific protease activity.

EXAMPLE 7 Immobilization of plant factor X activator

Crystallized ficin (Sigma) in crystal suspension was diluted to a concentration of 2.5 mg / ml and added to a buffer containing 20 mmol / l Tris-HCl, 150 mmol / l NaCl, pH 714, by gel filtration over Sephadex® G50 ( Pharmacia). A pre-activated gel, Actigel ALD Superflow (Sterogene Bioseparations, Arcadia, CA) was washed with a buffer containing 20 mmol / l Tris-HCl, 150 mmol / l NaCl, pH 7.4, and then in the ratio 1 + 1 with the offset to be immobilized ficin-containing solution and incubated for 3 hours at room temperature at _^1/15 of the volume of the coupling solution (Sterogene Bioseparations, Arcadia, CA). Subsequently, the immobilizate was separated on a sintered slush and washed by excessive washing alternately with a 20 mmol / l Tris-HCl, 150 mmol / l NaCl, pH 7.4, buffer and a 20 mmol / l Tris-HCl, 2 mmol / l NaCl , pH 7.4, freed buffer from unbound ficin and the coupling reagents. The ficin immobilizate was subsequently used to activate Factor X as follows.

Highly purified factor X from example 1 was in a buffer containing 20 mmol / l Tris-HCl, 150 mmol / l NaCl, pH 7.4, to 2 E FX / ml and wet with 10 μl / ml Ficinimmobilisat added. This was followed by 60 minutes Incubated at 37 ° C with constant mixing and then the Factor Xa activity as described in Example 2, measured. A factor Xa activity of 4 U / ml was found become. From this it can be deduced that Ficin is also used in a solid phase bound form the factor X acti vator activity and therefore as Immobilisat representation of factor Xa is suitable.

Claims (18)

A method for activating a vitamin K-dependent blood factor by treatment with a protease, characterized in that the protease is derived from plants, fungi or prokaryotes. 2. The method according to claim 1, characterized in that the blood factor from the group of human factors II, VII, IX, X and protein C is selected. 3. The method according to claim 2, characterized in that after activation of human factor X, factor Xa-β is won. 4. The method according to claim 1 to 3, characterized that the protease is selected from the group Ficin, Bromelain, papain, thermolysin and clostripain. 5. The method according to one or more of claims 1 to 4, characterized in that the treatment under Conditions are performed for the protease are suboptimal. 6. The method according to one or more of claims 1 to 5, characterized in that the protease oxidizes one Cysteine protease is. 7. The method according to one or more of claims 1 to 6, characterized in that the protease is purified by chromatography. 8. The method according to claim 7, characterized in that the protease has an at least 2-fold increased specificity Blood factor activation activity over the crude extract from plants or cell cultures.   9. The method according to one or more of claims 1 to 8, characterized in that the activation in the presence performed by defectors. 10. The method according to one or more of claims 1 to 9, characterized in that the activation in the presence performed by heavy metal ions or alkaline earth ions becomes. 11. The method according to claim 10, characterized in that the activation takes place in the presence of calcium ions. 12. The method according to one or more of claims 1 to 11, characterized in that the protease on a solid support is immobilized. 13. The method according to one or more of claims 1 to 12, characterized in that the protease by recombinant DNA technology is produced. 14. Pharmaceutical preparation containing a blood factor and a blood factor specific protease, preferably one of plants, fungi or prokaryotes derived protease. 15. Pharmaceutical preparation according to claim 14, characterized characterized in that the blood factor is a plasma protein, preferably a vitamin K-dependent protein, in particular an activated vitamin K-dependent protein is. 16. A pharmaceutical preparation containing one for one Blood factor specific protease, especially one purified protease, characterized in that it belongs to topical application is formulated and the protease  preferably from plants, fungi or prokaryotes is derived. 17. Set for medical use, containing

• a) a proteolytic enzyme, in particular an enzyme derived from plants, fungi or prokaryotes, having a specificity for a proform of a protein, and
• b) the proform of a protein.

18. Activated vitamin K-dependent blood factor, thereby characterized in that it according to the method of Claims 1 to 13 was won.