CZ315799A3 - Activated blood factor dependant on vitamin K and process of its preparation - Google Patents

Abstract

A method of activating vitamin K dependent blood factor processing with a plant-derived protease or prokaryote. The solution also includes a blood factor that does not no contamination with animal proteases, as well as pharmaceutical composition and kit for medical use.

Description

The present invention relates to activated vitamin K dependent and to a method for its production of blood factor

BACKGROUND OF THE INVENTION

The complex protease / substrate cascade requires highly selective proteases for the physiological activation of profactors, particularly in the blood coagulation area. These proteases occur in the human body on their own, particularly in blood plasma. The use of other human proteases for the activation of pro-factors, such as the digestive enzymes trypsin and pepsin, therefore does not lead to selective formation of activated blood clotting factors, but to non-specific degradation of the target substrate. Thus, for example, it is known that tryptic degradation of coagulation (clotting) factor X is initially activated, but then non-specific degradation leads to low molecular weight peptides. It is known from AT-3 9 7 390 that for the activation of prothrombin by an appropriate digestive enzyme, special variants of the method must be observed in order to prevent further non-specific degradation of the resulting thrombus (blood plug).

In the case of Factor X activation, this is therefore a special case where a selective Factor X activator is available with Russelli's Viper Venom (Russell with Viper Venom, RVV), which can also be used technically to obtain Factor Xa. However, the use of xenogeneic animal proteins as excipients for the manufacture of medicines has been refused since knowledge of the transmission of animal viruses to little-related animal species (prion issues). However, since suitable human proteases are not available in technically sufficient quantities, the use of RVV for preparative activation of factor X is still standard.

Proteolytic enzymes that can be isolated from prokaryotes, from lower eukaryotes, such as fungi, or from higher eukaryotes, such as plants, are known to have low substrate specificity. For this reason, these enzymes are mainly used for total protein degradation in crude cell extracts in order to separate or isolate other cellular components such as sugars (carbohydrates) or nucleic acids in this way. In addition, these proteases are also used to rank and characterize proteins by degrading them into small peptides. To date, the use of bacterial or plant proteases or fungal proteases as plasma protein activators or plasma protein cofactors or inhibitors of the prothrombin complex protein family has not been known.

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for activating profactors in the field of blood coagulation with the aid of highly specific non-animal proteases.

This object is achieved according to the invention as described in the introduction, wherein activation is effected by means of a protease which is derived from plants or prokaryotes.

The method of the invention is particularly suitable for activating vitamin K dependent blood factors. Activation of human blood factors of Group II, VII, IX, X and Protein C is particularly useful. The following examples show the advantages of obtaining factor X to factor Xa-β activation.

The method of the invention is equally suitable for obtaining naturally occurring, genetically engineered as well as chemically or genetically engineered blood factors.

With respect to the protease used according to the invention, it is possible to start from a proteolytic enzyme from prokaryotes. Particular mention may be made of bacterial proteases, such as thermolysin, clostripain proteases IX and X from bacillus p olymyxa, and protease IX from bacillus thrombothelytic.

In addition, proteases from lower eukaryotes, such as fungi, in particular proteases XXIII from Aspergillus oricae can also be used in the method of the invention. Proteolytic enzymes from higher eukaryotes, such as plants, are also suitable. Particularly suitable are proteolytic enzymes belonging to the family of cysteine proteases. For example, bromelain (eg, from Pineapple comos), papain (eg, from milk juice from Carica papayi) or ficin (eg from Ficus carica) may be mentioned. However, said proteolytic enzymes from prokaryotes, fungi and plants have little substrate specificity. It was therefore surprising to find that by using these enzymes, blood coagulation (blood coagulation) propellers can be highly specifically activated. These enzymes are either selected natural enzymes, but also from enzymes and enzymes, respectively. enzyme derivatives, especially enzymes produced by recombinant DNA technology.

In particular, for cysteine proteases, it is known that their broad protease activity can be fully developed under reducing conditions or in the presence of activators containing sulfhydryl groups. For this reason, SH-donors are often added to the incubation buffer as effectors when incubated with these enzymes. However, the broad-acting spectrum of these proteases can be shifted to suboptimal ratios by deviating from optimal incubation conditions, i. conditions outside the optimum pH or temperature, or in the absence of partially or totally necessary cofactors or effectors, or in the presence of special effectors, such that special substrates, such as plasma proteins, are no longer fully degraded and thus their activity in terms of profactors, that must be activated does not lose. Rather, the activation is stopped at a given stage of the target substrate and the activation factors as such are retained. This is also demonstrated for the first time by the activation of factor X to factor Xa, in particular β-factor Xa.

Furthermore, a higher specificity of cysteine proteases can also be achieved by subjecting the purified crude enzyme preparations from plants to further purification measures, eg chromatography, to isolate the protein fraction whose protease activity exhibits a limited substrate spectrum. While this measure alone may not yet be appropriate to obtain the desired highly specific activators, it is advisable in conjunction with the next step to increase the specificity of proteases, namely their oxidation, whereby proteases are reduced by contact with air oxygen in the proteases. their activity and their activity spectrum. Accordingly, oxidized cysteine proteases are particularly suitable for the process of the present invention, with preference being given to chromatography with enriched or purified cysteine proteases. It is particularly preferred that the protease in question has at least a two-fold increased specific activity for activating blood factor against crude plant or cell culture extract and / or non-specific proteolytic activity.

Further, the substrate spectrum can be narrowed by adding appropriate defectors to the enzyme incubation buffer with the substrates to be activated. In particular, heavy metal ions or alkaline earth ions may be mentioned as defectors. Particularly preferred here is the addition of calcium ions. Thus, for example, the following examples show that activation of factor X with the addition of calcium ions yields the β-factor Xa activator product with high yield, while other cleavage products are produced only in negligible amounts.

However, the activation reaction can proceed successfully without the addition of calcium ions and can also be modulated with respect to the activated factor obtained. However, α-factor Xa and / or β-factor Xa can also be produced according to the invention in suitable experimental arrangements.

· * * *.............

For the preferred technical applicability of the enzymes of interest herein, these enzymes may be used in immobilized form. This allows a simple separation of the protease from the substrate, e.g. by filtration. In addition, it guarantees the use of immobilized, ie. solid phase bound enzymes their re-deposition. In the case of secondary modified proteins, e.g., by oxidation, such immobilization, i.e., by covalent binding of the enzyme to the support material, irreversibly fixes the material in its spatial arrangement as it exists after conversion to a highly specific activator.

The method of the invention is suitable for activating natural as well as blood factors produced by appropriate recombination. In this case, the blood factor as a coagulation proenzyme can be modified by a proteolytic cleavage site by a series of amino acids, which is then targeted and cleaved by a defined protease, so that activation is now enabled by only a few or even with a single protease . This specially designed analogue is, for example, a vitamin K-dependent blood factor analogue as factor X.

In the example of factor X, the region around the Arg52Ile53 position, which represents the factor IXa cleavage site, could be replaced by another sequence or amino acid sequence. E.g. the amino acids Arg52, Ile53 could be replaced by a series of Glu-Glv, which then excludes FIXa cleavage and activation by factor IXa, but allows proteolytic digestion with Glu C endoproteinase from Staphyloccoc auraeu V8. Thus, a bacterial enzyme, which can be produced in high purity and does not present any risk of contamination with animal or human protein, can be used to activate factor X. ficinem.

It is preferred that the blood factor activated according to the invention be subjected to further purification measures in order to eliminate any proteolytic degradation products produced in this way. Particularly preferred herein are chromatographic purification procedures or gel filtration purification.

The invention also encompasses a pharmaceutical composition comprising a blood factor and a plant-derived protease or prokaryote with an appropriate furnace for a given blood factor. This protease can also be derived from mammals, in particular a protease of human origin. However, no coagulation factor is used as protease according to the invention. In particular, plasma proteins, e.g. also vitamin K-dependent proteins, are included as blood factors in this pharmaceutical composition. In particular, the pharmaceutical composition comprises an activated vitamin K-dependent blood factor and a given protease. This blood factor is preferably human and natural.

A topical protease, in particular a purified protease, may also be used as such for topical (topical) use to stop bleeding, in which case the vitamin K-dependent blood factor is derived from a bleeding injury or wound. By placing the Factor X activator in a pharmaceutical carrier material, e.g.

in a dressing or powder; as an ointment, the effect of a coagulation activator that triggers blood clotting can be utilized. Another pharmaceutical composition also includes a blood factor, which is present as a pro-protein and comprises protease-cleaving protease cleavage from plants, fungi or prokaryotes isolated or genetically engineered or recombinantly produced by cells of these species. Protease-specific proteases of this type are known, for example, as furin (Van de Ven, W.J. et al., Enzyme 45: 257-270, 1991) or Paired Basic Amino Acid Residue

Cleaving Enzyme, (a paired basic enzyme that cleaves amino acid residues), PACE, (Rehemtulla, A. et al., Biochemistry 32: 1 1586-1 1590, 1993). If a given protein is particularly labile from a given protein, the protein may be produced in situ by simultaneous administration of the protein processing enzyme and therapeutically available. In addition, further activation by the protease activator is possible if the finished (mature) protein must be converted to the active form by further proteolytic digestion or cleavage. The treatment set, consisting of a stable proprotein and a processing or activating proteolytic enzyme, therefore allows for the first time to provide or administer also labile proteins as active ingredients.

Further, the invention includes an activated vitamin K dependent blood factor as obtained by the method of the invention. The human activated blood factor obtained according to the invention is characterized in that it is not contaminated with an animal protease.

• »· ·

The process according to the invention is illustrated by the following examples. These examples relate to the activation of factor X to factor Xa, using various variants of the method according to the subclaims.

DETAILED DESCRIPTION OF THE INVENTION

EXAMPLE 1

Production of substrate factor X

Purification of factor X was performed from a prothrombin complex factor preparation containing FII, FIX, FX, protein C and protein S, and produced according to the von Brummelhuise method (Brummelhuis HGJ, Protein Protein Fractionation Preparation. Curieing, JM, New York: Academic Press, 1980, pp. 117-128) and was heat treated for virus inactivation according to EP 0 159 311. The respective lyophilisate containing prothrombin complex factors was dissolved according to the activity of 50,000 units of factor X (E FX) / L in distilled water and pH was adjusted to 7.0. After the addition of 12% (v / v) TWEEN 80, the solution was stirred for 1 hour at room temperature, then diluted to 5 times with distilled water, mixed with trisodium citrate dihydrate (7 g / l) and adjusted to pH 7.0. Next, the solution was mixed with 30 g / l Ca ₃ (PO ₄ ) ₂ and stirred for 1 hour at room temperature. The solid phase was then separated by centrifugation and washed twice with 25 ml / g of calcium phosphate buffer from 20 mM tris-HCl, pH 7.0, containing 10% ammonium sulfate, re-suspension and / or re-centrifugation. Then a third was carried out • · ·

Washing with 25 ml / g calcium phosphate buffer 20 mM trisHCl containing 150 mM NaCl, pH 7.0, a new resuspension and subsequent centrifugation. To elute the adsorbed Factor X, the pellet or tablet was mixed with 1 mmol / l sodium phosphate buffer, pH 7.0 (25 ml of elution solution / g of calcium phosphate used) for 1 hour at room temperature. The solid phase was then separated by centrifugation and the trap (floating surface) containing factor X was further purified by chromatography through DEAE Sepharose® Fast Flow, Fa. Pharmacia. For this purpose, a column filled with swollen, washed DEAE Sepharose® Fast Flow gel and having a configuration having an internal column diameter: gel bed height = 1: 8.3 ratio was used. The chromatographic material was loaded with 10 E FX / ml gel. Previously, the FX (Factor X) containing solution was gel-buffered against a buffer containing 25 mM trisodium citrate dihydrate, 100 mM NaCl, pH 6.0. This factor X solution was adsorbed on DEAE Sepharose® Fast Flow at a flow rate of 2 ml / min and the column was washed at a flow rate of 2 ml / min with 1.7 times the gel volume, 100 mmol / l NaCl in 25 mmol / l trisodium citrate dihydrate. pH 6.0 followed by 2.6 times the gel volume, 250 mmol / l NaCl, 25 mmol / l trisodium citrate dihydrate, pH 6.0. The elution of factor X was performed at 5.2 times the gel volume of citrate buffer, pH 6.0, containing 280 mmol / l NaCl, pH 6.0. Fractions were collected and eluted by standard coagulation assays for their prothrombin complex protein content (Factor X, Protein C, Factor IX, and Factor II). Factor X containing fractions that were poor in content of other prothrombin complex proteins were pooled and then further purified via monoclonal antibody. * -s »purified from ascites directed against factor X and immobilized on Actigel ALD (Sterogene, Bioseparations, Arcadia, CA). The FX-containing protein solution was adsorbed onto a gel packed with 10 E FX / ml gel. It was then further washed with a 5-fold column volume of 20 mM Tris-HCl, pH 7.4. Elution was performed with a 10-fold column volume of buffer containing 100 mmol of 3 - [(3-cholamidopropyl) dimethylammonium] -1-propanesulfonate, 25 mmol / L NaCl, pH 10.5. This buffer eluted protein fraction was collected and then concentrated by ultrafiltration through a membrane with an exclusion limit of 10,000 daltons to 1/20 of the initial volume. Then, the factor X-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 proteins, in particular monoclonal antibodies based on an immunoaffinity chromatography column, by adsorption to Phenylsepharose® High Performance, PharmaciaBiotech. For this purpose, the FX-containing solution after diafiltration was adjusted to 1.8 mmol / l NaCl and pH 7.4. The hydrophobic interaction chromatography gel was maintained in equilibrium in a 20 mmol / l tris-HCl, 2 mol / L NaCl, pH 7.4 chromatography column, and the adjusted factor X solution loaded with 30 E FX / ml gel was applied to that column. The flow from the column containing factor X was collected and then ultrafiltrated through a 10,000 dalton exclusion limit membrane concentrated to 1/20 of the initial volume and further diafiltered against a buffer containing 20 mmol / l tris-HCl, 150 mmol / l. NaCl, pH 7.4. The highly purified Factor X solution thus obtained had a specific activity of about 100 E / mg protein.

* «Β · ·« · » ¢, • · 4 4 4 4 4 4 4

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 to form CH ₃ OCO-D-CH AGly-Arg-OH and paranitroaniline. The kinetics of paranitroaniline increase is measured spectrophotometrically at 405 nm. The optical density increase (OD) is proportional to the content of factor Xa in the measured sample.

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

Substrate solution:

1.3 mmol / l CH ₃ OCO-D-CH A-Gly-Arg-pNA in dilution buffer

Way:

μΐ of the sample containing factor Xa is mixed with 50 μΐ of dilution buffer and incubated for 90 seconds at 37 ° C. Then, 100 µl of substrate solution was added and the OD (optical density) increase per minute at 37 ° C at 405 nm was determined. The OD increase must remain constant linear throughout the measurement.

• 9 • 9 •

·· ··· · &

··· · · · «» * * * · · · ·

A standard preparation of bovine factor Xa "NIBSC reagent 75/595" is used to generate the reference curve, with one ampoule containing 1 E FXa after reconstitution with 1 ml distilled water (see National Institute for Biological Standards and Controls data sheet).

EXAMPLE 3

Activation of factor X

Activation of purified Factor X from Example 1 was performed with enzymes costripripine (Cal biochem, La Jolla, CA) 10 E / mol, thermolvsin (Calbiochem, La Jolla, CA) 200 E / ml, papain (Boehringer Mannheim, Germany) 400 pg / ml and ficin (Sigma Chemicals Co., St. Louis, MO) 20 gg / ml, in a buffer containing 20 mmol / l tris-HCl, 150 mmol / l NaCl, 5 mmol / l CaCl ₂ , pH 7.4, at 37 ° C and incubation for several hours. The factor X concentration was 3.2 E / 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 can be read from Figure 1. that activation of factor X was possible with all the enzymes used and the greatest yield of factor Xa was obtained with thermolvsin after 2 hours.

In addition to incubation with ficin, the activation phase, which, according to the enzyme, reached a maximum between 30 minutes and 2 hours, was followed by inactivation of the resulting factor Xa. When activated with ficin, continuous activation with stable factor Xa activity could be demonstrated even after 19 hours.

• · · · «F« · ······

EXAMPLE 4

Activation of factor X

Comparison of Ficin and Russel Viper Venom (RusselUs Viper Venom)

Factor X activator from Chipera russellii (RVV, Pentapharm AG, Basel, Switzerland) is known from the literature as a non-plasma activator of factor X with high selectivity, which is commonly used in in vitro (in a tube) activation of factor X.

In this example, the activation of the highly pure Factor X of Example 1 with RVV was compared to that with the plant Factor X activator, ficin. High purity Factor X at 4 E / ml in a buffer containing 20 mmol / l tris-HCl, 150 mmol / l NaCl, 5 mmol / l CaCl ₂ , pH 7.4 was used for this purpose and was incubated with either 2 , 7 µg / ml RVV (Pentapharm AG, Basel, Switzerland) or 20 µg / ml ficin (Sigma Chemicals Co., St. Louis, MO) at 37 ° C. At different times, samples were taken from incubation mixtures within 22 hours and examined as described in Example 2 for Factor X activity. The results of the study are shown in Figure 2. It has been shown that incubation of Factor X with Factor X activator Ficin resulted in Factor Xa activity comparable to that obtained by incubation with RVV. Factor X activation products, which were achieved after incubation with both activators after 22 hours, were also examined by SDS-polyvacrylamide gel electrophoresis in 8-1894 gradient gels under non-reducing conditions. In this context, factor X before activation, factor X after activation with RVV and after activation with ficin were analyzed by standard methods after electrophoretic separation and subsequent staining on nitrocellulose membranes and detection with a semi-clonal anti-factor X antibody and immune staining according to standard ways. The result can be seen in Figure 3. It was shown that homogeneous factor X prior to activation by both the RVV activator and the ficin activator was cleaved into several factor X specific protein fragments of less molecular weight than the unactivated factor X, resulting in RVV activation a mixture of α- and β-factor Xa, both of which the factor X activation products were present in approximately the same amount and at least three additional activation products were identified. In contrast, activation by ficin showed that β-factor Xa was obtained as the main product (see arrow), and as with RVV activation, three other activation products could also be detected, but showed a larger molecular weight difference to the main product compared to RVV activation. β-factor Xa, so that these fragments could be separated by other simple methods, eg gel filtration.

EXAMPLE 5

Effect of effectors on factor X activation by ficin

Incubation conditions reported in the literature for proteolytic degradation with ficin exhibit buffer systems which mostly contain cysteine as SH-reagent and activator for the protease. The addition of a complexing agent in the form of metal ions, such as ethylenediaminetetracetic acid (EDTA), is also described, as these SH-dependent proteases are known to be inactivated, especially heavy metal ions. In this example,

• • • · · · • · · • · · «> ·> · • · · • «* - »· 16 • · · · · · ·· «· · · factor was incubated X at a concentration of 4 E / ml in buffer containing 20 mmol / l tris-HCl, 150 mmol / l NaCl, pH 7.4, p 2 µg ficin / ml

analogously to Example 4 at 37 ° C for 24 hours, with (1) 2 mmol / L CaCl 2, (2) 1 mmol / L cysteine, (3) 1 mmol / L cysteine and 2 mmol to the buffer medium. (1) EDTA and (4) 1 mmol / l cysteine and 2 mmol / l C aCl ₂ After 24 hours incubation, the factor Xa activity of Example 2 was determined. The results can be taken from Table 1.

D á vka FXa activity after 24 hours (E / m 1) 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

Table 1: Activation of factor X with ficin, effect of effectors

It turned out that the addition of calcium chloride resulted in a significantly higher factor Xa yield, while only small yields of factor X could be realized under standard conditions (cysteine ± EDTA). These doses were also examined for the composition of activation products as described above by gel SDS-polyacrylamide electrophoresis. The result of electrophoretic investigation after protein staining by silver staining as well as after specific immunostaining with Antifactor X antibody can be taken from Figure 4. the β-factor Xa obtained can now easily be freed from conventional ficin residues still present in the mixture by conventional chromium atographic purification procedures. These procedures include simple gel filtration, ion exchange chromatography, or substrate affinity chromatography (eg, substrate affinity chromatography), e.g., immobilized benzamidine, which is capable of selectively binding factor Xa.

EXAMPLE 6

Isolation of Factor X Activator from plant latex mg powdered Fice glabrata latex containing 2 mg protein was suspended in 2.5 ml sodium phosphate buffer (5 mmol / l) containing 1 mmol / l EDTA, pH 5.5, and mixed with 100 μΐ 50 mmol / l cysteine solution to activate cysteine protease. The free cysteine was then removed by gel filtration through Sephadex® G50, which was further diluted with the same 1 + 3 phosphate buffer. The entire amount was now loaded onto a Mono S HR 5/5 cation exchange chromatography column, Pharmacia, at a flow rate of 1 ml / min. This was further washed with 45 column volumes of the same phosphate buffer and then a gradient elution was performed, the phosphate concentration being continuously increased through 55 column volumes to 185 mmol / Τ in the same buffer composition. The result can be seen in Fig. 5. The protein mixture was eluted into several individual proteins by elution as seen in the elution profile at 280 nm absorption (—protein). Finally, the individual fractions were examined using a Factor Xa sample described in Example 2 for Factor X activating enzyme activity and it was shown that the protein peak (peak value) eluted at 49 ° C.

1 8 • · · • · · · · · • · · · • · · · · · · · • · · elution volumes, and protein peak (top), which was eluted between 68 and 69 elution volumes. reported the highest activity

of factor X activator (- -x- -). By analogy, protease activity of fractions with a non-specific transferase substrate was examined. For this, a chromogenic protease substrate, benzoyl-DL-arginine-p-nitroanilide hydrochloride, was used under the conditions and according to the method of von Eglund et al. (Biochemistry 7: 163-175 (1968)) employed in a photometric assay. As shown in Fig. 5, significant protease activity (Δ) was found both in the column and in all other fractions, and the peaks reported here at 49 ml elution volume and 6869 ml elution volumes also showed the strongest activities. In addition, significant protease activities were also found in protein fractions at elution volumes of 53 ml and 74-83 ml, respectively. Remarkably, in the fraction eluted at 68-69 ml of a given column, the activity of Factor X activator is significantly higher than that of the non-specific protease, suggesting the separation of Factor X activator from the crude plant extract. The fraction containing the highest factor X activator activity with respect to protein content was then incubated for 15 hours at 4 ° C under the action of atmospheric oxygen. As a result, factor X activator activity could be further increased in relation to nonspecific protease activity.

EXAMPLE 7

Immobilization of plant factor X activator

Crystalline ficin (Sigma) in the crystal suspension was diluted to a concentration of 2.5 mg / ml and then gel filtered through Sephadex® G50 (Pharmacia) buffered to (counter) buffer containing 20 mmol / l tris-HCl, 150 mmol / l NaCl pH 7.4 via Sephadex® G50 (Pharmacia). The 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 mixed in a 1 +1 ratio with the solution containing mobilized ficin and then incubated with 1/15 volume of binding solution (Sterogene Bioseparations, Arcadia, CA) for 3 hours at room temperature. Thereafter, the immobilisate was separated on a sintered suction and over-washed alternately with a buffer of 20 mmol / l tris-HCl, 150 mmol / l NaCl, pH 7.4, and a buffer with 20 mmol / l tris-HCl, 2 mmol / l NaCl, pH 7.4 was freed of unbound ficin and coupling reagents. The castin immobilisate was then further used to activate Factor X according to the following.

The high purity Factor X of Example 1 was adjusted to 2 E FX / ml in a buffer containing 20 mmol / l tris-HCl, 150 mmol / l NaCl, pH 7.4 and mixed with 10 μΐ / m wet Ficin Immobilisate. It was then incubated for 60 minutes at 37 ° C with continuous agitation, after which factor Xa activity was measured as described in Example 2. A factor Xa activity of 4 E / ml was determined. It can be concluded that ficin also retains the activity of factor X activator in a solid phase bound form and is therefore suitable as an immobilisate for the formation of factor Xa.

EXAMPLE 8

The technical ficin preparation of latex Ficus glabrata was purified according to the conditions and method of von Eglund et al. (Biochemistry 7:

· · * * «« «« «« «« «« «« «« «« «« «

163-175 (1986)), collecting those fractions having the highest specific factor X activator activity compared to unspecified protease activity against benzoyl DL-arginine-p-nitroanilide hydrochloride. The preparation collected from chromatographic purification was subjected to 15-minute activation with 10 mM cysteine at 37 ° C, and the activation mixture was then deprived of excess activator by gel filtration through Sephadex® G-25 Superfine. Activation was performed in incubation mixtures containing 4 units / ml Factor X and 3μβ / ηη1 activated ficin preparation in a 20 mmol / l tris-HCl buffer system. 150 mmol / l sodium chloride, pH 7.4, in the presence and absence of calcium and manganese ions. The batch or batch contained either 2 mmol / l calcium ions, 1 mmol / l manganese ions, or a combination of both metal ions. After 5 hours incubation at 37 ° C, the batches were examined by SDS-acrylamide gel electrophoresis for the composition of the activation products. The result of electrophoretic separation was visualized by the silver staining method and the intensity of the separated bands was evaluated on the day of the t-shirt. The result of this evaluation can be seen in the following table.

Activation charge The ratio of factor Xa Proportion of fission products (densitometrically) (densitometrically) Buffer 15.3 84.7 2 mM Ca ⁺ 44.4 5 5,6 1 mM Mn ²⁺ 30.3 69.7 2 mM Ca ⁺ + ^and 1 mM Μn ^'+ 65.5 34.5

• 4 ································································

Activation with a purified, activated ficin formulation without the addition of both metal ions has been shown to result in enhanced degradation of the factor Xa formed. The addition of C and ^{2 +} or Mn ^{2 +} ions optionally reduces the formation of various fission products, while the combination of both ions almost reversed the ratio between activated factor X and its degradation products.

In addition, it could be shown that activation with the addition of Ca ²⁺ and Mn ²⁺ ions resulted in activated factor X, which was not subjected to any further degradation process after the activation process. In order to elucidate this evidence, an activation batch was performed analogously to the above conditions with the addition of 2 mmol / l calcium ions and 1 mmol / l manganese ions, at which time appropriate samples were taken from the incubation mixture to record the kinetics activation. Factor Xa activities according to Example 2 were determined as well as the fraction of degraded Factor Xa cleavage products determined analogously to the methodology described above. RESULTS OF X ^of these experiments are summarized in the following table:

Sampling After Factor activity Xa (amidolytically) (units / ml) Ratio of factor Xa densitome t-shirts Share of Stepne products densitome t-shirts 1 h 18.5 84.4 15.6 3 h 3 0,0 65.1 34.9 5 h 3 4,0 65.4 3 4,5 24 h 33.6 64.1 35.9

Further purification of the obtained Factor Xa can now proceed according to Example 5.

PCT /; 3/01364

I m πι η. with.

• ·

At 'Atents

• · · · · · • · · · · · • · ···· ·· · ··· 73χ249 • * · c m ^ / bk 14.6 ^^ Claims -l tti 2 0-

Claims (20)

A method of activating a vitamin K dependent blood factor by treatment with a protease, characterized in that the protease is derived from plants, fungi or prokaryotes and exhibits at least a 2-fold increased activity to activate specific blood factor over crude plant or cell extract. The method according to claim 1, wherein the blood factor is selected from the group of human factors II, VII, IX, X and protein C. The method of claim 2, wherein after activation of human factor X, factor Xa-β is obtained. The method of claims 1 to 3, wherein 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 said processing is carried out according to conditions which are suboptimal for said protease. Method according to one or more of claims 1 to 5, characterized in that the protease is an oxidized cysteine protease. Method according to one or more of claims 1 to 6, characterized in that the protease is chromatographically purified. "CHANGED SHEET’ • · - ............ Method 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 in that the activation is carried out in the presence of heavy metal ions or alkaline earth ions. 10. The method of claim 9, wherein the activation occurs in the presence of calcium ions. Method according to one or more of claims 1 to 10, characterized in that the protease is immobilized on a solid support. Method 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. 14. The method of claim 13, wherein the blood factor analogue is activated with a proteolytic cleavage site specifically for the protease. The method of claim 14, wherein the proteolytic cleavage site is modified and activation is performed with a protease that does not correspond to a physiological activation mechanism. “CHANGED SHEET’ • · * ·· · · ············ A pharmaceutical composition comprising a blood factor and a blood-specific protease derived from plants, fungi or prokaryotes. 17. The pharmaceutical composition of claim 16, wherein the blood factor is an activated vitamin K-dependent protein. A pharmaceutical composition comprising a purified blood factor-specific protease, characterized in that it is formulated for topical application and that said protease is derived from plants, fungi or prokaryotes. A file for medical use containing (a) a proteolytic enzyme derived from plants, fungi or prokaryotes with specific activity for the pro-form of the protein concerned; and b) a given pro-form of the protein. 20. Activated vitamin K-dependent blood factor, characterized in that it has been obtained according to the method of claims 1