MXPA99008147A - 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

BLOOD FACTOR DEPENDENT ON VITAMIN K, ACTIVATED AND METHOD FOR THE PRODUCTION OF THE SAME Field of the Invention The present invention relates to a blood factor dependent on activated vitamin K as well as to a method for its production.

Background of the Invention The complex substrate / protease cascade in the activation of profactors, especially in the area of blood coagulation, requires highly selective proteases. These proteases are present in the human body itself, particularly in the blood plasma. The use of other human proteases for the activation of pro-factors, such as, for example, digestive enzymes of trypsin and pepsin, does not lead to a selective generation of the activated blood coagulation factors, but rather to a non-degrading degradation. specifies the target or target substrate. Accordingly, it is already known for example that tryptic degradation of coagulation factor X is Ref.031264 active at the beginning but then leads to low molecular weight peptides due to non-specific degradation. It is already known from AT 397 390 that particular process variations must be adhered to the activation of prothrombin with the aid of a digestive enzyme to prevent additional non-specific degradation of the resulting thrombus. Therefore, the activation of factor X represents a special case in which a selective X-factor activator is available from the venom of the Vípera russellí (RW of Venom of the Viper of Russell), which can also be used industrially to obtain the factor Xa. However, due to the recognition of the transmission of the animal virus to a less related animal species (previous problem), the use of xenogenic animal proteins as adjuvants for the production of drugs has been observed with displeasure. But, since adequate human proteases are not available in industrially sufficient amounts, the use of the RW for the activation of preparative factor X is as above, a standard method. It is already known that proteolytic enzymes that can be isolated from prokaryotes, from lower eukaryotes, such as for example fungi, or from higher eukaryotes, such as for example plants, have a low substrate specificity. For this reason, they are used mainly for the total degradation of proteins in crude cell extracts to separate or to isolate other components of the cell, such as for example carbohydrates or nucleic acids, in this way. Additionally, these proteases are also used to sequence and characterize the proteins by means of degradation to small peptides. Until now, the use of bacterial or plant proteases or proteases of fungi as activators of plasma proteins or cofactors of the plasma protein or inhibitors of the group of prothrombin complex proteins has not been known.

Detailed description of the invention An object of the present invention is to provide a method for activating the profactors in the area of blood coagulation with the aid of highly specific proteases which are not of animal origin. The above problem is solved according to the invention by the method mentioned in the introduction, whereby activation occurs by means of a protease which is derived from plants or prokaryotes. The method according to the invention is especially suitable for the activation of vitamin K-dependent blood factors. The activation of the human blood factors of factor II, VII, IX, X of the group and the protein C. The following examples demonstrate the advantages as they are obtained in the activation of factor X to factor Xa-β. The method according to the invention is equally suitable for obtaining blood factors that are naturally present, recombinantly produced as well as chemically or recombinantly modified. With respect to the protease used according to the invention, it can be started from a proteolytic enzyme of the prokaryotes. Here, bacterial proteases such as for example thermolysin, clostripain proteases IX and X of Bacillus polymyxa and protease IX of Bacillus thermoproteolyticus are going to be especially mentioned. Additionally, proteases of lower eukaryotes, such as for example fungi, especially the protease XXIII of the mold fungus Aspergillus oricae, can also be used in the method according to the invention. In addition, proteolytic enzymes of higher eukaryotes, such as for example plants, are also suitable. Proteolytic enzymes are especially suitable, such as those belonging to the group of cysteine proteases. For example, those that are going to be mentioned here: bromelain (for example of Ananas comosus), papain (for example of milk of Carica papaya) or ficina (for example of Ficus carica). However, the mentioned proteolytic enzymes of prokaryotes, mold fungi and plants have a low substrate specificity. Therefore, it was surprising to dmine that the pro-factors of blood coagulation can be highly activated in a specific manner using these enzymes. These enzymes are any selected natural enzymes, but they can also be modified enzymes and / or enzyme derivatives, especially the enzymes produced by recombinant DNA technology. It is particularly known from cysteine proteases that they fully exhibit their broad protease activity under reducing conditions or in the presence of activators containing the sulfhydryl groups. For this reason, SE donors are frequently added to the incubation buffer as effectors during incubation with these enzymes. However, the broad activity spectrum of these proteases can be displaced to suboptimal behaviors by deviation from optimal incubation conditions, ie conditions away from pH or optimal temperatures or with partial or compllack of cofactors or effectors necessary, or in the presence of particular effectors, to the point that special substrates such as for example plasma proteins are no longer comply degraded and, because of this, their activity is not lost as far as the profactors are concerned. which have to be activated. Instead, activation is stopped at the target or target substrate level and activation products as such are obtained. This was also demonstrated for the first time that the example of factor X activation for factor Xa, especially factor Xa-β, is used. In addition, a higher specificity of the cysteine proteases can also be obtained by subjecting the preparations of the appropriate crude enzyme from the plants to further purification steps, for example chromatography, to isolate those fractions of the protein whose protease activity exhibits a spectrum of limited substrate. Although this measurement alone is possibly not adequate to obtain the highly desired specific activators, it is adequate in relation to an additional step to increase the specificity of the proteases, especially by the oxidation thereof, whereby the proteases are reduced in their activity and spectrum of activity as a result of contact with atmospheric oxygen. It follows from this that the oxidized cysteine proteases are particularly suitable for the method according to the invention, whereby this is preferably the case with the cysteine proteases enriched and / or purified chromatographically. Therefore, it is particularly preferred that the protease has an increased specific blood factor activation activity at least twice, compared to the crude extract of plants or cell cultures and / or compared with the non-specific proteolytic activity. In addition, the spectrum of the substrate can be narrowed because the detectors are added to the buffer solution of incubation of the enzyme with the substrates that are going to be activated. Heavy metal ions or alkaline earth ions are to be mentioned especially as detectors. The addition of calcium ions is particularly preferred here. Accordingly, it is shown in the following examples that the Xa-β factor of the activation product is obtained in the activation of factor X using calcium ions with high yield, whereby the cleavage products only arise in negligible amounts. However, the activation reaction can proceed strongly without the addition of calcium ions and can also be modulated with respect to the activated factor obtained. With suitable experimental arrangements, factor Xa-a and / or factor Xa-β can also be produced according to the invention. For a preferred industrial application of the enzymes dibed herein, these can be employed in an immobilized form. Therefore, a simple separation of the protease from the substrate is possible, for example by filtration. Additionally, the use of immobilized enzymes, ie enzymes linked to a solid phase, ensures the repeated use thereof. In the case of secondarily modified proteins, for example by oxidation, they are irreversibly fixed in their confirmation as if they were present after the conversion into a highly specific activator by immobilization, ie by the covalent attachment of the enzyme to the carrier material. The method according to the invention is suitable for activating the blood factors produced both naturally and recombinantly. Related to this, the site of proteolytic cleavage in a blood factor as a coagulation proenzyme can be modified by an amino acid sequence which is then specifically recognized and cleaved by a defined protease, such that activation only possible or also possible with a protease which does not correspond to the mechanism of physiological activation. This particularly constructed analogue, for example, is an analogue of a vitamin K-dependent blood factor such as factor X. Using the example of factor X, the region around the Arg52-Ile53 position which represents the cleavage site for Factor IXa can be exchanged for a different amino acid sequence. For example, the amino acids Arg52, Ile53 can be replaced by the Glu-Gly sequence which then excludes a FlXa cleavage and an activation by factor IXa but allows the proteolytic digestion with endoproteinase Glu C from Staphylococcus auraeus V8. Therefore, a bacterial enzyme which can be produced in a highly purified manner and does not represent the danger of contamination with an animal or human protein, can be used for the activation of factor X. Similarly, Gly-Ser can also be introduced as a segmentation sequence so that it is possible then a segmentation with the proteases of the plant, for example with ficin. It is preferred that the activated blood factor according to the invention be subjected to further purification steps to remove the proteolytic degradation products possibly formed in this way. Chromatographic purification processes or purification by filtration with a gel are particularly preferred here. The invention also comprises a pharmaceutical preparation which comprises a blood factor and a protease derived from plants or prokaryotes with a specificity for the blood factor. The protease can also be a protease derived from mammals, in particular a protease derived from humans. However, in relation to this, the coagulation factor is not used as a protease according to the invention. Particularly, plasma proteins, for example also vitamin K-dependent proteins, are contained in this pharmaceutical preparation as the blood factors. The pharmaceutical preparation comprises especially an activated vitamin K-dependent blood factor and a protease. This blood factor is preferably human and natural. The protease, especially a purified protease, is to be used as for topical use in hemostasis, whereby the vitamin K-dependent blood factor in this case originates from a bleeding wound. By applying the factor X activator in a pharmaceutical carrier material, for example in a bandage material or in a powder or an ointment, the activating or triggering effect of the coagulation of the blood of the clotting activator can be used here . Another pharmaceutical composition also comprises a blood factor which is present as a pro-protein and contains a pro-protein cleavage protease isolated from plants, fungi or prokaryotes or produced recombinantly in the cells of these species. Proteases of this type with a specificity towards pro-proteins are known, for example, as furine (Van de Ven, J. et al., Enzyme 45: 257-270, 1991) or the Basic Amino Acid Residue Segmentation Enzyme. Duplicate, PACE, (Rehemtulla, A. et al., Biochemistry 32: 11586-11590, 1993). If the mature protein resulting from the pro-protein is particularly labile in a pharmaceutical preparation, the mature protein can be produced in situ by the simultaneous administration of the pro-protein processing enzyme and is then available therapeutically. Additionally, an extra activation with an activating protease is possible since the mature protein must be converted to an active form by further proteolytic digestion. Accordingly, a set or therapeutic set consisting of the stable pro-protein and the activation enzyme and / or proteolytic processing also allows the administration of labile proteins as active ingredients for the first time. Furthermore, the invention comprises an activated vitamin K-dependent blood factor, as obtained according to the method of the invention. The human activated blood factor obtained according to the invention is distinguished in that it is not contaminated by an animal protease. The method according to the invention is illustrated more closely by the following examples. These examples refer to the activation of factor X to factor Xa whereby the different process variations according to the dependent claims find use.

Example 1 Production of substrate factor X The purification of factor X was carried out from a preparation of the prothrombin complex factor which contained the factors FII, FIX, FX, protein C and protein S and which were produced according to the Brummelhuis method (Brummelhuis HGJ, Prothrombin complex preparation, In: Methods of Plasma Protein Fractionation, edited by Curling, JM, New York: Academic Press, 1980, p.127-128) and heat treated for inactivation of the virus in accordance with EP 0 159 311. A lyophilisate containing the prothrombin complex factors is dissolved in distilled water corresponding to an activity of 50,000 U / L / 1 and adjusted to pH 7.0. After addition of 12% (v / v) of TWEEN 80, this is stirred for 1 hour at room temperature, subsequently diluted with distilled water to the volume of 5 times, mixed with the trisodium citrate dihydrate (7). g / 1) and adjusted to pH 7.0. Subsequently, this is mixed with 30 g / 1 of Ca 3 (P0) 2 and stirred for 1 hour at room temperature. After this, the solid phase is separated by centrifugation and washed twice each with 25 ml / g calcium phosphate of a buffer solution of 20 mmol / l Tris-HCl, pH 7.0, containing 10% sulphate of ammonium by resuspension and respective renewed centrifugation. After this, a third wash is carried out with 25 ml / g of calcium phosphate of a buffer solution of '20 mmoles / 1 of Tris-HCl, pH 7.0, containing 150 mmoles / 1 of NaCl by renewed resuspension and subsequent centrifugation. For elution of adsorbed factor X, the microspheres are shaken with 1 mmol / 1 of sodium phosphate buffer, pH 7.0, (25 ml of the elution solution / g of calcium phosphate used) for 1 hour at room temperature. After this, the solid phase is separated by centrifugation and the supernatant containing factor X is further purified by chromatography on DEAE Sepharose® Fast Flow, Pharmacia. For this, a column filled with the washed, swollen DEAE Sepharose® Fast Flow gel, and a configuration of the internal diameter of the column: gel bed height = 1: 8.3, was used. The chromatography material is loaded with 10 U FX / ml. The solution containing the FX is again treated with the buffer solution in advance against another buffer solution, 25 mmol / l of trisodium citrate dihydrate, 100 mmol / l of NaCl, pH 6.0. This factor X solution was adsorbed at a flow rate of 2 ml / min in the DEAE Sepharose® Fast Flow and the column was subsequently washed at a flow rate of 2 ml / min with 1.7 times the gel volume of 100 mmol / l. of NaCl in 25 mmoles / 1 of trisodium citrate dihydrate, pH 6.0, and also with 2.6 times the gel volume of 250 mmoles / 1 of NaCl, 25 mmoles / 1 of trisodium citrate dihydrate, pH 6.0. Elution of factor X occurred with 5.2 times the gel volumes of a citrate buffer solution, pH 6.0, containing 280 mmoles / 1 NaCl, pH 6.0. Fractions were collected during the elution, which were examined with standard coagulation tests to verify their content of factor X, protein C, factor IX and factor II of the prothrombin complex proteins. Fractions containing factor X, which were poor in other prothrombin complex proteins, were combined and further purified on a purified monoclonal antibody from the ascifos which were directed against factor X and immobilized with respect to Actigel. ALD (Sterogene, Bioseparations, Acradia CA). The protein solution containing the FX was adsorbed to the gel at a concentration of 10 U FX / ml of the gel.

Subsequently, this was washed with 5 times the volume of the column of a buffer solution of 20 mmoles / 1 of Tris-HCl, pH 7.4. Elution occurred with 10 times the volume of the column of a buffer solution containing 100 mmoles of 3- [(3-colamidopropiDdimethyl-ammonium] -1-propansulfonate, 25 mmol / 1 NaCl, pH 10.5. The protein fraction eluting with this buffer is collected and subsequently concentrated to 1/20 of the starting volume by ultrafiltration on a membrane with an exclusion limit of 10,000 daltons. Subsequently, the solution containing the FX was diafiltered against a buffer solution containing 4 g / 1 of trisodium citrate dihydrate, 8 g / 1 NaCl, pH 7.0. This preparation of factor X was freed from traces of contaminating proteins, especially monoclonal antibodies that are purged from the immunoaffinity chromatography column, by adsorption to Phenylsepharose® High Performance, Pharmacia-Biotech. For this, the solution containing the FX is adjusted to 1.8 mmoles / 1 of NaCl and a pH value of 7.4 after the diafiltration. The hydrophobic interaction chromatography gel was equilibrated on a chromatography column with 20 mmoles / liter of Tris-HCl, 2 mol / l of NaCl, pH 7.4, and the adjusted factor X solution is applied to the column at a concentration of 30 U FX / ml gel. The material containing the FX and passing through the column is collected and concentrated to 1/20 of the starting volume by ultrafiltration on a membrane with an exclusion limit of 10,000 daltons and then diafiltered against a buffer solution containing 20 mmoles / 1 Tris-HCl, 150 mmoles / 1 NaCl, pH 7.4. The highly pure factor X solution, obtained in this way, had a specific activity of approximately 100 U / mg of the protein.

Example 2 Quantitative determination of factor Xa Test Principle: The chromogenic peptide substrate CH3OCO-D-CHA-Gly-Arg-pNA was hydrolyzed by factor Xa, resulting in CH3OCO-D-CHA-Gly-Arg-OH and paranitroaniline. The kinetic characteristics of the paranitroaniline increase are determined spectrophotometrically at 405 nm. The increase in optical density (OD) is proportional to the content of factor Xa in the sample that is to be quantified.

Reagents: Dilution buffer solution 3.7 g / 1 Tris- (hydroxymethyl) -aminomethane 2.1 g / 1 imidazole 18.0 g / 1 NaCl 1.0 g / 1 human albumin pH 8.4 Substrate solution 1.3 mmoles / 1 of CH3OCO-D-CHA-Gly-Arg-pNA in the dilution buffer Method: 50 μl of a sample containing factor Xa are mixed with 50 μl of the buffer for dilution 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 of the DO must remain linearly constant during the time period of the measurement. For the generation of a reference curve, the standard preparation of bovine factor Xa "NIBSC reagent 75/595" is used, for which an ampoule containing 1 U FXa is used after reconstitution with 1 ml of distilled water ( see Datasheet, National Institute for Biological Standards and Controls).

Example 3 Factor X activation The activation of the purified factor X of example 1 was carried out with the clostripain enzymes (Calbioche, La Jolla, CA) 10 U / ml, thermolysin (Calbiochem, La Jolla, CA) 200 U / ml, papain (Boehringer Mannheim, Germany) 400 μg / ml and ficin (Sigma Chemicals Co., St. Louis, MO) 20 μg / ml, in a buffer solution containing 20 mmoles / 1 Tris-HCl, 150 mmoles / 1 NaCl, 5 mmoles / 1 CaCl 2, pH 7.4, at 37 ° C and incubation for several hours. The concentration of factor X was 3.2 U / ml. At the instants of the time of 5 minutes, 30 minutes, 1 hour, 2 hours and 19 hours, the samples were taken from the respective incubation mixtures and examined to verify the activity of factor Xa as described in example 2. The results they will be taken from Figure 1. It was shown that an activation of factor X was possible with all the enzymes used and led to higher yields of factor Xa with thermolysin after 2 hours. Except for ficin incubation, the activation phase which, depending on the enzyme, was obtained at a maximum of between 30 minutes and 2 hours, was followed by an inactivation of the resulting factor Xa. A continuous activation with a stable factor Xa activity could also be determined after 19 hours when the activation is carried out with the ficin.

Example 4 Activation of Factor X Comparison of Ficina and Russell's Vibora Venom The activator of Factor X of Vípera russellii (RW, Pentapharm AG, Basel, CH) is known from the literature as a non-plasma factor X activator with a high selectivity, which is commonly used for in vitro activations of factor X. In this example, factor activation The highly purified X of Example 1 with the RW was examined in comparison with the activation with the factor activating factor X of the plant. For this purpose, highly purified X factor was used at a concentration of 4 U / ml in a buffer containing 20 mmoles / 1 Tris-HCl, 150 mmoles / 1 NaCl, 5 mmoles / 1 CaCl 2, pH 7.4 , and incubated at 37 ° C with either 2.7 μg / ml of RW (Pentapharm AG, Basel, CH) or 20 μg / ml of ficin (Sigma Chemicals Co., St. Louis, MO). Samples were taken at different time points from the incubation mixtures within the course of 22 hours and examined to verify factor X activity as described in example 2. The result of the examination is presented in Figure 2 It was shown that the incubation of Factor X with the factor X activator of ficin led to a factor Xa activity which was comparable to that which could be obtained by incubation with RW. The factor X activation products which could be obtained after incubation with both activators after 22 hours, are also examined by electrophoresis of the polyacrylamide-SDS gel in gradual gels of 8-18% under non-reducing conditions. In relation to this, the 'X factor before the activation, the X factor after the activation with RW and after the activation with ficin, were analyzed after the electrophoretic separation and the subsequent staining to the nitro-cellulose membranes and detection with a polyclonal anti-factor X antibody and immunostaining according to standard methods. The result is to be taken from Figure 3. It was shown that the homogeneous factor X before activation was segmented by the RW of the activator as well as by the activating factor in several fragments of the specific protein of factor X of smaller molecular mass. that factor X not activated, whereby a mixture of the factors Xa a and ß led after the activation with RW where these 2 activation products of factor X were obtained in approximately the same amount and at least 3 activation products additional items could be identified. In contrast, activation by ficin showed that factor Xa-ß was obtained as the main product (see arrow), whereby three additional activation products could be detected as with activation with RW. In contrast to activation with RW, these fragments had a larger molecular weight difference with respect to the Xa-β factor of the main product such that these could be separated by simple additional methods, for example gel filtration.

Example 5 Influence of effectors on the activation of factor X with ficin The incubation conditions given in the literature for proteolytic degradation with ficin have buffer systems which contain at least cysteine as an SH reagent and the activator for the protease. In addition, the addition of a metal ion complex former, for example ethylenediaminetetraacetic acid (EDTA), is also described because it is known that such SH-dependent proteases are inactivated particularly by heavy metal ions. In this example, factor X was incubated at a concentration of 4 U / ml in a buffer solution containing 20 mmoles / 1 Tris-HCl, 150 mmoles / 1 NaCl, pH 7.4, with 2 μg ficin / ml of analogous to example 4 at 37 ° C for 24 hours, whereby (1) 2 mmoles / 1 CaCl 2, (2) 1 mmol / 1 cysteine, (3) 1 mmol / L of cysteine and 2 mmol / 1 of EDTAA and (4) 1 mmol / 1 of cysteine and 2 mmol / L of CaCl2 were added to the buffer medium. The activity of factor Xa was determined according to Example 2 after 24 hours of incubation. The results are going to be taken from Table 1.

Table 1: Activation of factor X with ficin, influence of effectors It was shown that a clearly higher yield of factor Xa could be obtained by the addition of calcium chloride, while under standard conditions (cysteine + EDTA), only smaller yields of factor X could be obtained. The lots were also examined to verify the composition of the activation products by means of polyacrylamide gel electrophoresis-SDS as described above. The result of the electrophoretic examination after the dyeing of the proteins according to the silver-staining method as well as after the specific immunostaining with an anti-factor X antibody, is to be taken from Figure 4. It was shown that the addition of calcium ions to the incubation medium led to a clearly higher portion of factor Xa-ß among the cleavage products of Factor X. The factor Xa-ß obtained in this way can be easily released from the residual ficin still contained in the mixing by conventional chromatography purification methods. Among these are gel filtration, simple, ion exchange chromatography or affinity chromatography of the substrate, for example, on the immobilized benzamidine which is capable of selectively binding to factor Xa.

Example 6 Plant latex factor X activator isolation mg of a powdered Ficus glabrata latex containing 2 mg of protein were suspended in 2.5 ml of a 5 mMol / 1 sodium phosphate buffer solution containing 1 mmol / l EDTA, pH 5.5, suspended and mixed with 100 μl 50 mmoles / 1 of cysteine solution for the activation of the cysteine protease. Subsequently, the free cysteine was removed by filtration with a gel on Sephadex® G50 and further diluted with the same 1 + 3 phosphate buffer solution. The entire amount was then applied to a Mono S HR 5 cation exchange chromatography column. / 5, Pharmacia, at a flow rate of 1 ml / min. Subsequently, this was washed with 45 column volumes of the same phosphate buffer and a gradual elution was then carried out, whereby the phosphate concentration was continuously increased above 55 column volumes at 185 mmol / 1. in the same buffer composition. The result is taken from Figure 5. The mixture of the protein is separated into several individual proteins, as can be recognized in the elution profile by absorption at 280 nm (- proteins). Subsequently, the individual fractions were examined to verify the activity of the enzymes in the activation of the FX with the factor Xa assay described in example 2 and it is shown that a protein peak which eluted to 49 ml of the elution volumes and a protein peak which eluted between 68 and 69 ml of the elution volumes, had the highest factor X activator activity (- -x- -). In an analogous manner, the fractions were examined to verify the activity of the protease with a non-specific protease substrate. For this, the crcmogenic protease substrate of benzoyl-DL-arginine-p-nitroanilide hydrochloride was used, which was used in a photometric test under the conditions and according to the method of Engíund et al., Biochemistry 7: 163 -175 (1968). As recognized in Figure 5, considerable protaase activity (?) Could be found in the material passing through the column as well as in all of the additional fractions, hence the peaks at the elution volumes of 49 ml. and elution volumes of 68-69 mi also had the strongest activities here, however, apart from this, considerable proteinase activities were also determined in the protein fractions at the elution volumes of 53 ml and 74-83 ml. It is notable that the activity of the factor X activator was clearly higher than the activity of the non-specific protease in the fraction which eluted from the column at 68-69 ml which was due to the separation of the factor X activator of the raw plant extract The fraction containing the highest factor X activator activity with respect to the protein content was subsequently incubated for 15 hours at 4 ° C. luence of atmospheric oxygen. The activity of the activator of factor X could be further increased by it in relation to the activity of the non-specific protease.

Example 7 Immobilization of the factor X activator of the plant The crystallized ficin (Sigma) in the crystalline suspension is diluted to a concentration of 2.5 mg / ml and re-treated with a buffer solution against a buffer solution containing 20 mmoles / 1 Tris-HCl, 150 mmoles / 1 NaCl , pH 7.4, by filtration with a gel on Sephadex® G50 (Pharmacia). A preactivated gel, Actigel ALD Superflow (Sterogene Bioseparations, Arcadia, CA), was washed with a buffer containing 20 mmoles / 1 Tris-HCl, 150 mmoles / 1 NaCl, pH 7.4, and subsequently mixed in a ratio 1 + 1 with the solution containing ficin to be immobilized and incubated with 1/15 of the volume of the coupling or contact solution (Sterogene Bioseparation, Arcadia, CA) for 3 hours at room temperature. Subsequently, the immobilized material was separated on sintering charcoal and freed of the unbonded ficin and the coupling or contact reagent by an alternative excessive wash with a buffer solution of 20 mmoles / 1 Tris-HCl, 150 mmoles / 1 NaCl, pH 7.4, and a buffer solution of 20 mmoles / 1 of Tris-HCl, 2 mmoles / 1 of NaCl, pH 7.4. The immobilized ficin substance is subsequently employed for the activation of factor X as follows. The highly purified factor X of Example 1 is adjusted to 2 U FX / ml in a buffer solution containing 20 mmoles / 1 Tris-HCl, 150 mmoles / 1 NaCl, pH 7.4, and mixed with μl / ml mist of the immobilized substance of ficin. Subsequently, it is incubated for 60 minutes at 37 ° C under continuous complete mixing and, after. this, the factor Xa activity is measured as described in Example 2. By this, a factor Xa activity of 4 U / ml could be determined. It can be derived from this that ficin also maintains the activity of the activator of factor X in a form bound to the solid phase and, therefore, is suitable as an immobilized substance to produce factor Xa.

Example 8 An industrial ficin preparation of Ficus glabrata latex was purified according to the conditions and method of Englund et al., Biochemistry 7: 163-175 (1968), whereby these fractions were collected, which had the most activity. Elevation of the activator of specific factor X, compared to the activity of the non-specific protease against the hydrochloride of benzoyl-DL-arginine-p-nitroanilide. The accumulation of the preparation from the chromatographic purification was subjected to a 15 minute activation with 10 mM cysteine at 37 ° C and the activation mixture was subsequently freed from the excess activator by a separation of the group by means of the filtration with a gel on Sephadex® G-25 Superfine. The activation was carried out in incubation mixtures containing 4 U / ml of factor X and 3 μg / ml of the activated ficin preparation in a buffer system of 20 mmoles / 1 of Tris-HCl, 150 mmoles / 1 of Sodium chloride, pH 7.4, in the absence and presence of calcium and manganese ions. The batches contained either 2 mmol / 1 calcium ion, 1 mmol / 1 manganese ion (II) or a combination of the two metal ions. After 5 hours of incubation at 37 ° C, the batches were examined by electrophoresis of the SDS-polyacrylamide gel to verify the composition of the activation products. The result of the electrophoretic separation became visible according to the silver dyeing method and the intensity of the separated bands was analyzed densitometrically. The result of this analysis will be taken from the following Table.

It is shown that activation by the purified, purified ficin preparation led to an increased degradation of the factor Xa formed without the addition of the two metal ions. The alternative addition of Ca2 + and / or Mn2 + ions reduced the production of several segmentation products, while the combination of both ions almost reversed the relationship between activated factor X and its degradation products. Additionally, it could be shown that the activation under the addition of the Ca2 + and Mn2 + ions led to an activated factor X which is not subject to any further degradation process after the activation process. To distinguish this evidence, a batch of activation analogous to the conditions given previously was obtained under the addition of 2 mmoles / 1 of calcium ions and 1 mmol / 1 of manganese (II) ions in which the samples were taken in certain time points from the incubation mixture to record the kinetic characteristics of the activation. The activity of factor Xa was determined according to Example 2 and the portion of the degraded segmentation products of factor Xa were determined in a manner analogous to the techniques described above. The results of these experiments are summarized in the following Table: The additional purification of the factor Xa obtained can then be carried out according to Example 5.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates.

Having described the invention as above, property is claimed as contained in the following

Claims (20)

1. A method for the activation of a vitamin K-dependent blood factor by treatment with a protease, characterized in that the protease is derived from plants, fungi or prokaryotes and has a specific blood factor activation activity, increased at least twice, when compared to the crude extract of the plants or cell cultures.

2. The method according to claim 1, characterized in that the blood factor is selected from the group of human factors II, VII, IX, X and protein C.

3. The method according to claim 2, characterized in that factor Xa-ß is obtained after activation of human factor X.

4. The method according to claims 1 to 3, characterized in that the protease is selected from the group of ficin, bromelain, papain, thermolysin and clostripain.

5. The method according to one or more of claims 1 to 4, characterized in that the treatment is carried out under conditions which are sub-optimal for the protease.

6. The method according to one or more of claims 1 to 5, characterized in that the protease is an oxidized cysteine protease.

7. The method according to one or more of claims 1 to 6, characterized in that the protease is purified chromatographically.

8. The method according to one or more of claims 1 to 7, characterized in that the activation is carried out in the presence of detectors.

9. The 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 according to claim 9, characterized in that the activation occurs in the presence of calcium ions.

11. The method according to one or more of claims 1 to 10, characterized in that the protease is immobilized on a solid carrier.

12. The method according to one or more of claims 1 to 11, characterized in that the protease is produced by recombinant DNA technology.

13. The method according to claim 1, characterized in that a recombinant blood factor is activated.

14. The method according to claim 13, characterized in that a blood factor analogue with a proteolytic cleavage site specific for the protease is activated.

15. The method according to claim 14, characterized in that the proteolytic cleavage site is modified and the activation is carried out with the protease which does not correspond to the mechanism of physiological activation.

16. A pharmaceutical preparation, characterized in that it comprises a blood factor and a specific protease for the blood factor which is derived from plants, fungi or prokaryotes.

17. The pharmaceutical preparation according to claim 16, characterized in that the blood factor is an activated vitamin K-dependent protein.

18. A pharmaceutical preparation comprising a purified protease specific for a blood factor, characterized in that it is formulated for topical use and the protease is derived from plants, fungi or prokaryotes.

19. A set or set for medical use, characterized in that it comprises: a) a proteolytic enzyme derived from plants, fungi or prokaryotes with a specificity for a proforma of a protein, and b) the proforma of a protein.

20. A blood factor dependent on vitamin K, activated, characterized in that it is obtained according to the method of claims 1 to 15.