SK124699A3 - Activated vitamin k-dependent blood factor and method for the production thereof - 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 activated vitamin K dependent blood factor α, and at the same time to a method for its production.

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BACKGROUND OF THE INVENTION

Highly selective proteases are particularly needed where blood clots and complex protease / substrate cascade occur during physiological activation of professors. These proteases enter the human body itself, e.g. blood plasma. Other human proteases, such as e.g. digestive enzymes trypsin and pepsin, which do not lead to selective generation of activating factors of blood clots, but to non-specific degradation of the final substrate. Thus, e.g. it is known that tryptic degradation of clot factor X is initially activated, but then non-specific degradation leads to low molecular peptides. It follows from AT 397 390 that, in order to prevent further non-specific degradation of the resulting thrombus, special process variants must be followed for the activation of prothrombin by the digestive enzyme.

In the case of Factor X activation, it is therefore one particularity that Russelli's Viper Venom (RVV) has a selective Factor X 'activator which can also be used technically to obtain Factor Xa. Since it has been known that animal viruses are rarely transmitted to related animal species (Prionenproblematik), xenogeneic animal proteins have not been used as excipients in the manufacture of medicaments. However, as there are no technically sufficient human proteases available, the standard RW method is used for preparative factor X activation.

From proteolytic enzymes that can be isolated from prokaryotes, from low eukaryotes, such as e.g. from fungi or from higher eukaryotes, e.g. from plants, are known to exhibit specific substrates. For this reason, they are used for total protein degradation in crude cell extracts in order to separate or isolate other cell extracts, such as e.g. carbohydrates or nucleic acids. In addition, these proteases are also used to sequence and characterize proteins via degradation into small peptides. To date, the use of bacterial or plant proteases or fungal proteases as plasma protein activators or plasma protein - cofactors or - an inhibitor of the prothrombin complex protein family has not been known.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a process for activating a profactor in the blood clot region by means of highly specific non-animal proteases. According to the invention, the present task is solved at the outset by the aforementioned process, wherein the activation is carried out by a protease originating from plants or from prokaryontene.

According to the invention, this procedure is primarily intended to activate the dependent blood factor of vitamin K. The activation is mainly from human blood factors from the group of factors Π, VII, IX, X and protein C. The following examples demonstrate the benefits of activating factor X to factor Xa. -β.

The process according to the invention is also suitable for obtaining naturally occurring genetically produced as well as chemically or genetically modified blood factors.

Depending on which protease is used according to the invention, it can be started from a single proteolytic enzyme from prokaryontene. Bacterial proteases such as e.g. thermolysin, clostripain proteases DC and X from bacilli polymyxa and proteases DC from bacilli thermoproteolyticus.

In addition, low eukaryontene proteases such as e.g. from fungi, in particular protease XXIII from fungal Aspergillus oricae. Prothelotic enzymes belonging to the family of cysteine proteases are also suitable. Let's name eg. bromelain (e.g. from Pineapple comosus), papain (e.g. from Carica papaya milk juice) or ficin (e.g. from Ficus caricä). However, these enumerated proteolytic enzymes from prokaryotes, fungi and plants have a slight substrate specificity. Surprisingly, it has been found that by the use of these enzymes, blood clot proactivators can be highly specifically activated. These enzymes are either selected natural enzymes or also modified enzymes or enzyme derivatives, in particular enzymes produced by recombinant DNA technology.

In particular, cysteine proteases are known to fully develop their protease activity under reductive conditions or in the presence of sulfohydryl groups containing activators. Therefore, SH-donors are often added as incubators for incubation regulation when incubated with these enzymes. The broad spectrum of activity of these proteases can be shifted from optimal conditions to suboptimal relationships, i.e., conditions in terms of pH or temperature optimum are improved, or in the case of partially or totally malfunctioning necessary cofactors or effectors, or from special effectors that this may result, and thus special substrates such as e.g. plasmoproteins do not completely degrade and thus their activity in terms of activating prophakers is not lost. Much more, activation stops at the target substrate stage and the activation products are retained as such. This will then be demonstrated for the first time in the example of activation from factor X to factor Xa, in particular β-factor Xa. , ¹ i

Further, a higher specification of cysteine proteases can also be achieved by subjecting the purified crude enzyme preparation to other purification processes, e.g. chromatography to isolate those protein reactions whose protease activity has a limited substrate spectrum. This procedure alone does not yet allow the necessary highly specific activators to be achieved, but is useful in conjunction with other procedures to increase the specificity of proteases, e.g. oxidation, whereby proteases are reduced by contact with air oxygen in activity and spectrum by activation. Accordingly, according to the invention, the oxidizing cysteine proteases are primarily intended for this process, and in this case, chromatically achieved or purified protease cysteines are preferred. In this case, it is preferred that the proteases have at least a 2-fold increase in the specific activity of the activating blood factor over the crude plant or cell culture extract, or non-specific proteolytic activity.

The substrate spectrum can also be further limited by adding activating defector substrates for enzyme incubation. The defectors are mainly heavy metal ions or alkaline earth ions. But above all, calcium ions are preferred here. In the following examples, e.g. shows that when calcium ion is added with high yield when activating factor X, the activating product β-factor Xa is retained, with the other Steppe products being produced only in negligible amounts.

However, the activation reaction may also be enhanced without the addition of calcium ions, and may also be modulated with respect to the activating factor involved. With a suitable experimental design, α-factor Xa and / or β-factor Xa can be produced according to the invention.

For technical applicability, preference is given here to those enzymes which can be used in immobilized form. Thus, it is quite easy to separate proteases from substrates e.g. filtration. This also guarantees immobilized use, i.e. the re-use of solid phase bound enzymes. In the case of secondary modified proteins, e.g. oxidation, the enzyme irreversibly immobilizing 'I ¹ * * * are fixed, i.e., the covalent coupling is transmitted to a highly specific activator to the support material in its conformation.

The process according to the invention is suitable not only for activation of natural, but also recombination of blood factors produced. In one blood factor, as a clot of a proenzyme, a proteolytic cut-off site can be identified, identified and cleaved by the amino acid sequence, which is then targeted by a definable protease, so that activation is possible with one or more proteases that do not correspond to a physiological activation mechanism. This specially assembled analogue is e.g. an analog dependent blood factor vitamin K as factor X.

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For example, Factor X could be replaced by another amino acid sequence around the Arg52-Ile53-position, which represents the cut-off site for Factor IXa. E.g. amino acids Arg52, Ile53 could be used instead of the Glu-Gly sequence, which excludes cleavage of FIXa and activation by factor IXa, but allows proteolytic association with the endoprotein Glu C from Staphylococcus aureus V8. Thus, a bacterial enzyme can be used for activation from factor X, which can be presented as pure and does not present any contaminating hazard to animal or human proteins. Thus, analogously, it can be introduced as a Gly-Ser cleavage sequence, allowing cleavage by plant proteases, e.g. Ficino.

It is preferred that the activating blood factor according to the invention be subjected to further purification procedures to remove the resulting proteolytic degradation products. Here, chromatographic purification methods or gel filtration purification are preferred.

The invention also encompasses a pharmaceutical preparation comprising a blood factor or a protease with a blood factor specification that is derived from plants or prokaryotes. This protease can also be derived from mammals or humans. However, no clotting factor is used as protease according to the invention. In particular, plasma proteins, e.g. also a vitamin K dependent protein in this pharmaceutical preparation. The pharmaceutical preparation includes activating dependent blood factor vitamin K and protease. This factor is mostly human and natural.

For external use to stop bleeding, the protease itself is used, in particular a purifying protease, wherein the vitamin K dependent blood factor in this case originates from a bleeding wound. By applying Factor X activator to a pharmaceutical carrier material, e.g. into a dressing or powder or ointment, there may be a blood clot-releasing effect. Another pharmaceutical treatment also includes a blood factor, which is referred to as a pro-protein, another is from plants, fungi, or prokaryotes, or is isolated in the cells of these admixtures, and contains genetically engineered protease cleavage proteins. Protease-specific proteases of this type are e.g. known as furin (Van de Ven, WJ 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 ). When the resulting mature protein in the pharmaceutical preparation is particularly labile from the pro-proteins, then the mature therapeutic protein is available. Activity with another activating protease is possible as long as the mature protein is further proteolytically transferred to the active form. The therapeutic kit, which consists of a stable pro-protein and a processing or activating proteolytic enzyme, also enables the administration of labile proteins as active ingredients.

Further, the invention encompasses activating vitamin K dependent blood factor and how it is obtained according to the method of the invention. The human activating blood factor obtained according to the invention is characterized in that it is not contaminated with any animal protease.

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The process according to the invention will be explained in more detail in the following examples. These examples relate to activation from factor X to factor Xa, wherein various process variants find use according to the subclaims.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Production of factor X substrate ₍

Purification of factor X occurred from a prothrombin complex factor preparation containing FD, FIX, FX, protein C and protein S, which was produced according to the Brummelhuis method (Brummelhuis HGJ, Preparation of the Prothrombin Complex. In: Methods of Plasma Protein Fractionation, Curling, JM) (ed.), New York: Academic Press, 1980, pp. 117-128) and was heat treated for viral inactivation according to EP 0 159 311. The prothrombin factor complex, including the lyophilisate, was dissolved in distilled water to correspond to activity from 50,000 E FX / L and was adjusted to pH 7.0. After addition of 12% (v / v) TWEEN 80, it was stirred for one hour at room temperature and then diluted to five times the volume with distilled water, supplemented with trisodium citrate dihydrate (7 g / l) and adjusted to pH 7.0. 30 g / L Ca 3 (PO 4) 2 was added and stirred for one hour at room temperature. The solid phase was then separated by centrifugation and washed twice with a resuspender containing 25 mg / g of 20 mM Tris-HCl regulator, pH 7.0 and 10% ammonium sulfate, and then centrifuged again. Next, a third wash was performed again with a resuspender with 25 ml / g calcium phosphate of a 20 mmol / l Tris-HCl regulator containing 150 mmol / l NaCl, pH 7.0 and subsequent centrifugation. For the elution of absorbing factor X, the pellet was mixed with 1 mmol / l sodium phosphate regulator, pH 7.0 (25 mL elution solvent / g of calcium phosphate used) for one hour at room temperature. The solid phase was then separated by centrifugation and the Factor X-containing overlap was further purified by DEAE Sepharose ^R Fast Flow, Fa. Pharmacia. One column was filled with a swelling, washed DEAE Sepharose ^R Fast flow-Gel and a configuration from the inside diameter of the columns was used: gel bed height = 1: 8.3. The chromatographic material was coated with a 10 E FX / ml gel. First, the FX-holding solvent was regulated by gel filtration, instead of the regulator with 25 mmol / l tri-sodium citrate dihydrate, 100 mmol / NaCl, pH 6.0. This Factor X-solvent was adsorbed in a liquid state of 2 ml / min on DEAE Sepharose ^R Fast Flow and a 2 ml / min flow column: 1.7 times the gel volume, 100 mmol / NaCl in 25 mmol / l tri-sodium citrate dihydrate followed by a 2.6-fold gel content, 250 mmol / l NaCl, 25 mmol / l trisodium citrate dihydrate, pH 6.0. Elution from factor X was followed by a 5.2-fold * 1 * volume gel gel citrate regulator, pH 6.0, containing 280 mmol / l NaCl, pH 6.0. This elution produced fractions in which, by standard assays, the contents of prothrombin complex proteins Factor X, Protein C, Factor DC and Factor Π were detected. Fractions containing factor X but lacking other prothrombin complex proteins were pooled and further purified by monoclonal protiteles, which were placed against factor X and immobilized on Actigel ALD (Sterogene, Bioseparations, Arcadia, CA). The FX protein diluent was adsorbed with a gel containing a 10 E FX / ml gel. It was then washed again with a 5-column volume of the regulator of 20 mmol / l Tris-HCl, pH 7.4. Elution followed by a 10-column volume of the regulator containing 100 mmol of 3 [(cholamidopropyl) dimethylammonium] -1-propanesulfonate, 25 mmol / l. NaCl, pH 10.5. The eluted protein fraction was pooled with this regulator and then concentrated by ultrafiltration through a membrane at an exclusion value of 10,000 daltons to 1/2 of the starting volume. Accordingly, the contained FX solvent was diafiltered against a regulator containing 4 g / l tri-sodium citrate dihydrate, 8 g / l NaCl, pH 7.0. This factor X preparation no longer contains protein impurities, in particular due to the adsorption of the immune final chromatographic column of the bleeding monoclonal protiteles on the phenolic separation of ^R High Performance, Pharmacia Biotech. After diafiltration, diluent FX- containing 1.8 mmol / l NaCl and pH 7.4 was used. The hydrophobic internally active chromatographic gel was equilibrated into a 20 mM Tris-HCl, 2 mol / L NaCl, pH 7.4 chromatography column and loaded onto the set factor of diluent X with a 30 E FX / ml gel per column. The course comprising FX was retained in the column and was then concentrated from 10,000 daltons to _1/2 of the starting volume by ultrafiltration through an exclusion membrane. Diafiltered against a regulator containing 20 mM Tris-HCl 1,150 mM NaCl, pH 7.4. The highly purified Factor X diluent thus obtained had a specific activity of about 100 E / mg protein.

Example 2

Quantitative determination from factor Xa

Test principle:

• I

The chromogenic peptide substrate CH ₃ OCO-D-Gly-Arg-pNA is hydrolyzed with Factor Xa to form CHsOCO-D-CHA-GGly-ARG-OH and paranitroaniline. The kinetics of paranitroaniline uptake are measured spectrophotometrically at 405 nm. In the quantification assay, the optical density (OD) uptake is proportional to the factor Xa content.

reaction:

Dilution controller:

3.7 g / l Tris (hydromethyl) aminomethane

2.1 g / l Imidazole

18.0 g / l NaCl

1.0 g / l Humanalbium pH 8.4

Substrate thinner:

1.3 mmol / l CH ₃ OCO-D-CHA-Gly-Ar-pNA in dilution regulator

method:

μΐ of Factor Xa in-progress was supplemented with 50 μΐ of dissolution regulator and incubated for 90 seconds at 37 ° C. Then 100 μΐ of substrate diluent was added and after a minute OD was added at 37 ° C at 405 nm. The addition of OD must remain linearly constant during the measurement period.

A standard preparation of bovine factor Xa NIBSC - Reagent 75/595 is used to determine the reference curve, one ampoule containing 1 ml of A.dest after reconstitution. 1 E Fxa (see Datasheet, National Institute of Biological Standards and Controls).

Example 3

Activation of factor X

Activation of purified Factor X of Example 1 was performed with enzymes, clostripaines (Calbiochem, La Jolla, CA) at 10 E / ml, thermolysin (Calbiochem, La Jolla, CA) at 200 E / ml, papain (Boehringer, Mannheim, BRD) 400 µg / ml and after a few hours incubation at 37 ° C with a regulator containing 20 mmol / l Tris-HCl, 150 mmol / l NaCl, 5 mmol / l CaCl 2, pH 7.4. The concentration of factor X was 3.2 E / ml. As described in Example 2, the activity of Factor Xa on assay incubation mixtures was examined at time intervals of 5 minutes, 30 minutes, 1 hour, 2 hours and 19 hours. The results can be seen in Figure 1. It was shown that activation of Factor X was possible with all the enzymes used and after two hours thermolysin was converted to the highest peak for Factor Xa. From the inactivation of the resulting factor Xa, the activation phase following incubation with ficin reached its maximum, depending on the enzyme used between 30 minutes and 2 hours. Upon activation from ficin, stable activity with stable factor Xa activation even after 19 hours could be demonstrated.

Example 4

Activation from factor X

Comparison of ficin and Vipera Russeli venom

The factor X-activator from Vipera russellii (RW, Pentapharm AGG, Basel, CH) is not known from the literature as a plasma selectivity factor X-activator used in vitro - activation from factor X.

In this case, the activity from the highly purified Factor X of Example 1 s was investigated

RW and was compared to activation with plant factor X-activator, ficin. For this purpose, highly purified Factor X was used at a concentration of 4 E / ml regulator containing 20 mmol / l Tris-Cl, 150 mmol / l NaCl, 5 mmol / l CaCl 2, pH 7.4 and incubated with 2, 7 gg / ml RW (Pentapharm AG, Basel, CH) or with 20 gg / ml ficin ($ igma Chemicals Co., St. Louis, MO) at 37 ° C. At different time ¹¹ 'intervals, incubations on factor X-activation as described in Example 2 were detected over a 22 hour period. The result of the survey is shown in Figure 2. It was shown that incubation from factor X with factor X-activator Ficin activates Factor Xa, which can be compared to an incubation that could be achieved by incubation with RW. Factor X activation products that could be achieved after incubation with both activators were examined by SDS polyacrylamide-gel electrophoresis in grading gels of 8 to 18% under non-reducing conditions. Factor X was analyzed after electrophoretic separation after activation with RVV and after activation with ficin, followed by blood on nitrocellulose membranes and detection with antifactor X - polyclonal protithelium and immune staining according to standard methods. The result is shown in Figure 3. It has been shown that the homogeneous factor X was cleaved prior to activation not only by the RW activator but also by the ficin activator into more factor X-specific protein fragments of less molecular than the non-activating factor X. and both of these Factor X activation products were maintained in approximately the same amount, and at least three other activation products could be identified.

Activation with ficin showed that beta-factor Xa could be produced as the main product (see arrow), and as with RW activation, three other activation products could also be detected, but in contrast to activation with RW, the molar amount of k the major beta-factor Xa product, so by these other simple methods, e.g. gel filtration, they could be separated.

Example 5

Effect of effectors on activation of factor X with ficin

The literature indicates that regulator systems containing at least cysteine as well as SH-Reagents and protease activators are required to generate proteolytic incubation conditions with ficin. An additive which forms complex metal ions, e.g. acetic acid ethylenediaminetetrate (EDTA), since these SH-dependent proteases are inactivated mainly by heavy metal ions. In this case, Factor X was incubated analogously at a concentration of 4 E / ml into a regulator containing 20 mmol / l Tris-HCl, 150 mmol / l NaCl, pH 7.4 with 2 gg of ficin / ml to Example 4 at 37 ° C. C in 24 hours and 2 mM CaCl 2, (2) 1 mM cysteine, (3) 1 mM cysteine and 2 mM EDTA and (4) 1 mM cysteine were added to the regulator medium (1). and 2 mmol / l CaCl 2. After 24 hours, factor Xa activity was determined according to Example 2. The results are shown in Table 1.

Table 1: Factor X - activation with ficin, effector effect

additive FX- activation after 24 hours (U / ml) 2 mM CaCl ₂ 42.1 1 mM Cysteine 17.5 1 mM Cysteine, 2 mM EDTA 10.3 1 mM Cysteine, 2 mM CaCl ₂ 45.1

It was shown that the resulting factor Xa was also increased by calcium chloride, whereas under standard conditions (cysteine ± EDTA) only a minor factor X was realized. found out the composition of the activation products. Figure 4 shows the result of electrophoretic examination of protein staining, not only after the silvery staining method, but also after specific immunostaining with anti-factor X-protitelesis. It has been shown that when calcium is added to the incubation medium, the proportion of beta-factor Xa over the factor X separation products is substantially increased. The beta-factor Xa thus obtained can be easily released by conventional chromatographic purification methods from a mixture which still contains tannin residues. In order to selectively attach factor Xa, e.g. immobilized benzamidine which allows simple gel filtration, ion exchange chromatography, or chromatographic substrate affinity.

Example 6

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Isolation of Factor X Activator from Vegetable mg Latexs of Ficus glabrata powder latex containing 2 mg protein was added to 2.5 ml of one 5 mmol / l calcium phosphate powder containing 1 mmoVl EDTA, pH 5.5 and supplemented with 100 μΐ 50 mmol a cysteine diluent to activate cysteine proteases. The pure cysteine was then separated by gel filtration through sephadec ^R G50 and further diluted with the same 1 + 3 phosphate regulator. The total amount was now transferred to a Mono S HR 5/5 cation exchange chromatography column, Fa. Pharmacia, with an impact group of 1 ml / min. It was then washed with a 45 column content of the same phosphate regulator and undergoing gradual elution, continuously increasing the phosphate concentration to 185 mmol / l with the same regulator composition above 55 column contents. The result of this is Figure 5. The protein mixture was eluted into multiple proteins. As known in the elution profile for adsorption at 280 nm (- - protein). The individual fractions in Example 2 were then examined by the described factor Xa-Assay for FX-activating enzyme activities and the protein peak eluting at 49 ml elution volume was shown and the protein peak eluting between 68 and 69 elution volume showed the highest factor X- activator activation (- -x- -).

Fractions with non-specific protease substrate for protease activity were investigated analogously. Chromogenic protease substrates, benzoyl-DL-arginine-pnitroanilide hydrochloride, were used in accordance with the conditions of Eglung et al., Biochemistry 7: 163-175 (1968) in a photometric assay. As can be seen from Figure 5, the listed protease activities (Δ) could be discovered not only in the column run, but also in other fractions, where the peak of the 49 ml elution volume and the 68 to 69 ml elution volume also showed the strongest activity. In addition, said protease activities were also found in protein fractions at a volume of 53 ml and 74-83 ml. Remarkably, in the fraction eluting from the column at 68-69 ml, the activation factor X was significantly higher than the non-specific protease activity, which in turn showed the separation of factor X-activator from the crude plant extract.

The fraction containing the highest factor X-activator activator, depending on the protein content, was then incubated for 15 hours at 4 ° C under the action of atmospheric oxygen. Here, the factor X-activator activity could be further increased relative to the non-specific protease activity.

Example 7

Immobilization from plant factor X-activator

Crystallized ficin (Sigma) in the crystal suspension was diluted to a concentration of 2.5 mg / l and re-regulated by gel filtration through Sephadex ^R G650 (Pharmacia) with a regulator containing 20 mmol / l Tris-HCl, 150 mmol / l NaCl, pH 7 '4. A pre-activating gel (Actigel ALD Superflow Sterogene Bioseparations, Arcadia, CA) was washed with a diluent-containing regulator and incubated for 3 hours at room temperature with 1/15 dilution binding content (Sterogene Bioseparations, Arcadia, Ca). Thereafter, the immobilisate was separated into sintered nucleate and, by excessive washing, alternately released the controller reactions from still unlinked ficin with 20 mmol / l Tris-HCl, 150 mmol / l, pH 7.4. The ficin mobilisate was further used for activation from Factor X as follows.

The highly purified Factor X of Example 1 was adjusted to 20 E FX / ml by a regulator containing 20 mmol / l Tri-HCl, 150 mmol / l NaCl, pH 7.4, and was supplemented with 19 μΐ / ml wet ficin immobilizer. It was then incubated with stirring at 37 ° C for 60 minutes, and then the activity of factor Xa was measured as described in Example 2. The activity from factor Xa was determined from 4 E / ml. From this, it can be concluded that when ficin is combined with the solid phase form of factor X activator activity, it is also predisposed to immobilize factor Xa.

Example 8

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The technical preparation of ficin from Ficus glabrata latex was purified according to the conditions and method of Englund et al., Biochemistry 7: 163-175 (1986), collecting each fraction that showed the highest specific activator X activity, comparable to the non-specific protease activity against benzoyl- argitin DL-p-nitroanilide hydrochloride. The chromatographic purification preparations were subjected to a 15 minute activation with 10 mM cysteine at 37 ° C and the activation mixture was deprotected by group separation by gel filtration through Sephadex ^R G-25. Activation was performed in an incubation mixture containing 4 units / ml Factor X and 3 µg / ml activating ficin preparation in powder from 20 mmol / l Tris-HCl, 150 mmol / l sodium chloride, pH 7.4 in the absence or presence of calcium and manganese. The deposits contained either 2 mmol / l calcium ions, 1 mmol / l manganese ions, or a combination of two metal ions. After 5 hours of incubation at 37 ° C, the deposition of the activating products was determined by SDS-polyacrylamide-gel electrophoresis. The electrophoretic separation was visible by the silver staining method and the intensity of the separated beams was evaluated densitometrically. The result of this evaluation can be seen in the following table.

Table 2

Activating sediment Factor Xa ratio densitometrically% Share of grafts, densitometrically% regulator 15.3 84.7 2 nM Ca ²⁺ 44.4 55.6 1 nM Mn ²⁺ 30.3 69.7 2 mM Ca ²⁺ + 1 nm Mn ²⁺ 65.5 34.5

Activation by pure, activating ficin preparation without the addition of both ions has been shown to lead to degradation of the factor Xa formed. Addition of Ca ²⁺ ions - possibly Mn ^{2+ 1} , I ₍ reduces the formation of cleavage products, while the combination of both ions reverses the relationship between activating factor X and its degradation products.

In addition, it could be shown that the activation with the addition of Ca ²⁺ and Mn ²⁺ ions exhibits an activation factor X which is not subject to any further degradation process after the activation process.

In order to confirm this evidence and to understand the activation kinetics, deposits were taken from the test incubation mixture at certain time intervals in which activation was carried out analogously to the same earlier conditions, with the addition of 2 mmol calcium ions and 1 mmol manganese ions. (Π). Thus, the activity of factor Xa was determined

According to Example 2, as well as the proportion of factor Xa cleavage products, analogously to the method described above. The results of this survey are grouped in the following table.

Table 3

Test after Activation fak. Xa amydolitic units / ml Fact share. X densitometric% Share of grafts. prod. densitometric% 1 hour 18.5 84.4 15.6 3 hrs 30.0 65.1 34.9 5 hours 34.0 65.4 34.5 26 hours 33.6 64.1 35.9

Further purification of the obtained Factor Xa can only be carried out according to Example 5.

Claims (20)

NEW PATENT CLAIMS 1 TO 20 «t A method for activating vitamin K dependent blood factor by protease treatment, characterized in that the protease is derived from plants, fungi or prokaryontenes and has at least twice the specific activating blood factor activity relative to the crude plant or cell culture extract. Method according to claim 1, characterized in that the blood factor is selected from the group of human factors Π, VII, IX, X and protein C. The method of claim 2, wherein after activation from human factor X, factor Xa-β is obtained. Process according to claims 1 to 3, characterized in that the protease is selected from the group of ficin, bromelain, papain, thermolysin and clostripain. Method according to one or more of claims 1 to 4, characterized in that the treatment is carried out under conditions which are suboptimal for the protease. Method according to one or more of claims 1 to 5, characterized by tm. that the protease is an oxidizing cysteine protease. »* ¹ I Method according to one or more of claims 1 to 6, characterized by tm. that the protease is chromatographically purified. Process according to one or more of Claims 1 to 7, characterized in that the activation is carried out in the presence of defectors. Method according to one or more of claims 1 to 8, characterized by tm. that the activation is carried out in the presence of heavy metal ions or alkaline earth ions. Process according to claim 9, characterized in that the activation follows in the presence of calcium ions. Process according to one or more of claims 1 to 10, characterized in that the protease is t immobilized on a solid support. Process according to one or more of claims 1 to 11, characterized in that the protease is produced by recombinant DNA technology. The method of claim 1, wherein the recombinant blood factor is activated. Method according to claim 13, characterized by tm. that the blood factor analog is activated with a proteolytic cut-off site specifically for proteases. Process according to claim 14, characterized in that the proteolytic cut site is modified and the activation is carried out by a protease which does not correspond to a physiological activating mechanism. Pharmaceutical preparations comprising a blood factor and a blood-specific protease which is derived from plants, fungi or prokaryotes. Pharmaceutical preparations according to claim 16, characterized in that t. that the blood factor is an activating vitamin K activating protein. »< Pharmaceutical preparations containing a blood-purified protease specifically characterized in that it is formulated for external use and the protease is derived from plants, fungi or prokaryontenes. Set for medical use, containing (a) a proteolytic enzyme which is derived from plants, fungi or prokaryontenes with a specificity for the pro-form of a protein; and b) a pro-form of a protein. Vitamin K activating dependent blood factor, characterized in that it has been obtained according to the process of claims 1 to 15.