EP0833897A2 - Derives de prothrombine - Google Patents

Derives de prothrombine

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Publication number
EP0833897A2
EP0833897A2 EP96915903A EP96915903A EP0833897A2 EP 0833897 A2 EP0833897 A2 EP 0833897A2 EP 96915903 A EP96915903 A EP 96915903A EP 96915903 A EP96915903 A EP 96915903A EP 0833897 A2 EP0833897 A2 EP 0833897A2
Authority
EP
European Patent Office
Prior art keywords
thrombin
hirudin
prothrombin
derivative
derivatives
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96915903A
Other languages
German (de)
English (en)
Inventor
Bernhard Fischer
Uwe Schlokat
Artur Mitterer
Falko-Günter Falkner
Johann Eibl
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Baxter AG
Original Assignee
Baxter AG
Immuno AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Baxter AG, Immuno AG filed Critical Baxter AG
Publication of EP0833897A2 publication Critical patent/EP0833897A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6429—Thrombin (3.4.21.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21005—Thrombin (3.4.21.5)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/974—Thrombin

Definitions

  • the invention relates to new prothrombin mutants or derivatives thereof which can be used as antagonists of their natural function.
  • extrinsic blood coagulation begins with the release of thromboplastin and activation of factor VII.
  • Activated factor VII in turn activates factor X, followed by activation of factor V and factor II (prothrombin).
  • Factor Ha thrombin converts fibrinogen to fibrin at the end of the cascade.
  • the other route is by activating factor XII by contact and then activating factor XI factor IX and factor X in the presence of calcium and factor VIII, followed by activation of factor II to factor Ha, which is the Triggers coagulation by cleavage of fibrinogen to fibrin.
  • Factor Ha therefore plays a central role in both routes of the blood coagulation cascade. So far, intensive research has been carried out into anticoagulants which can be used in particular in the treatment of septic shock, thromboses, embolisms, arteriosclerosis and heart attacks, and also in blood transfusions or after operations.
  • One method of suppressing blood clotting is to administer substances that inhibit thrombin directly.
  • heparin or coumarin have been used as anticoagulants. However, these are relatively systemic and increase the risk of internal bleeding.
  • hirudin is extremely specific in its binding to thrombin and offers even more advantages over the other anticoagulants. It does not require endogenous cofactors, is pharmacodynamically inert, has no effect on blood cells, plasma proteins (with the exception of thrombin) or enzymes, and is not immunogenic due to its small molecular size.
  • Hirudin does not lie down into organs and is excreted unchanged in the urine.
  • Hirudin is a single chain 65 amino acid polypeptide that is naturally formed by the medicinal leech (Hirudo medicinalis) in its secretory glands. Hirudin acts as an extremely strongly binding and very specific inhibitor against the protease thrombin and prevents blood clotting.
  • the mechanism of the action of hirudin as a thrombin inhibitor has been elucidated: the C-terminal part of hirudin binds to the anion binding sites of thrombin and thus occupies the binding site of the fibrinogen chain to thrombin.
  • the N-terminal part of hirudin blocks the active center of thrombin (Szyperski et al. 1992, J. Mol. Biol.
  • hirudin in hemodialysis, as an anticoagulant during pulmonary transluminal coronary angioplasty (PTCA), for the prophylaxis of postoperative thrombosis, to prevent re-thrombosis, for microvascular surgery, as an anticoagulant in hemodialysis and with extracorporeal circulation, as an admixture to thrombolytics such as Plasmin gene activators and streptokinase, as an anticoagulant during surgery and for clinical coagulation suppression.
  • PTCA pulmonary transluminal coronary angioplasty
  • thrombolytics such as Plasmin gene activators and streptokinase
  • WO 93/15757 proposes prothrombin intermediates as antidotes to hirudin.
  • these products are burdened with the usual dangers inherent in preparations derived from plasma, e.g. B. Contamination by human pathogenic viruses.
  • thrombin inhibitors such as NAPAP (Na- (2-naphthylsulforylglycyl) - D, L-amidinophenylalanine peptide) or PPACK (D-Phe-Pro Arg-CHCl) is known.
  • NAPAP Na- (2-naphthylsulforylglycyl) - D, L-amidinophenylalanine peptide
  • PPACK D-Phe-Pro Arg-CHCl
  • modified proteins such as inactivated coagulation factors to be used directly as anticoagulants. A particular problem with this is that the modified protein could possibly be removed from the blood faster than the wild-type protein in vivo.
  • the coagulation process comprising the interaction of the intrinsic and extrinsic blood coagulation cascade and cell surface receptors, is very complex.
  • An inactivated coagulation factor that can be used in vivo for therapy or prophylaxis should therefore not differ from the natural protein in any other essential property, such as e.g., due to its greatly reduced or completely inhibited coagulation activity. Distinguish receptor binding capacity. It would be desirable to have an in vivo half-life of the inactive protein which corresponds to that of the active coagulation factor or is even increased. Since thrombin in particular has a very short in vivo half-life, an inactive coagulation factor with an extended half-life would result in the active protein, e.g. Thrombin, with a competitive inhibition, increasingly displaced by its receptor. This would have the advantage that only a relatively small dose would have to be administered for the efficient anticoagulant action of the inactive protein.
  • the present invention therefore has as its object to provide a medically applicable antagonist of hirudin which has essentially no enzymatic activity which promotes blood clotting.
  • Another object of the present invention is to provide an inactive coagulation factor which does not differ from the natural protein in its essential properties, such as, for example, receptor binding capacity, and in which the in vivo half-life may be increased.
  • prothrombin mutants or derivatives thereof which have one or more changes in the protein sequence compared to the natural protein, are either inactive or at most have an activity of about 10%, preferably at most about 0.25% , of natural protein and in which the change in the protein sequence does not affect the binding capacity to thrombin-specific ligands and receptors, such as natural and synthetic anticoagulants.
  • the prothrombin mutants according to the invention or their derivatives do not differ functionally from their naturally occurring protein except for a strongly or completely reduced coagulation activity and possibly a changed in vivo half-life.
  • mutated prothrombin mutants or derivatives thereof are understood to mean all proteins which can be derived from the protein sequence of prothrombin and which have the essential binding determinants of thrombin which are necessary for binding to the thrombin-specific, natural and synthetic anticoagulants.
  • the structure of the prothrombin mutant should therefore not be changed as much as possible by the mutations compared to the wild-type protein or to its proteolytic derivatives, so that an optimal binding to the ligands, in particular to the natural ligands, is ensured.
  • mutants and derivatives according to the invention that the change in the protein sequence does not influence the binding capacity to thrombin-specific ligands and receptors, such as natural and synthetic anticoagulants.
  • mutants or derivatives according to the invention have a binding capacity of at least 80% of the binding must have capacity of the natural thrombin, so that the binding capacity can be regarded as not influenced. Mutants or derivatives, which have a higher binding capacity than natural thrombin, naturally also fall under the present invention.
  • DAPA Dansylarginine-N- (3-ethyl-l, 5-pentanediyl) -amide
  • Pei et al., J.Biol.Chem. 266: 9598, 1991 or by examining the binding affinity to an immobilized natural and syn ⁇ theoretical anticoagulant or inhibitor.
  • the natural synthetic anticoagulant or the inhibitor is immobilized on a solid matrix, a sample containing the derivative to be examined in a certain amount is brought into contact with the natural and synthetic anticoagulant or the inhibitor, the amount of bound ⁇ ) Mutant or derivative determined and the results relativized with a parallel determination with natural thrombin.
  • mutants or derivatives according to the invention should preferably be completely inactive, i.e. they should have no thrombin or thrombin-analogous activity.
  • derivatives with minor activity can also be used successfully according to the invention, since an activity of at most about 10%, in particular at most 0.25%, of the natural thrombin does not generally lead to undesirable side effects, e.g. Tendency to coagulate.
  • the mutants or derivatives according to the invention are further distinguished in that they can form a complex with hirudin and are therefore able to neutralize hirudin. Furthermore, they can dissociate a complex consisting of plasmatic or recombinant wt thrombin with hirudin, and that thereby freeing up hirudin complex. This also means that the released plasmatic or recombinant wild-type (wt) thrombin is active again and can perform its function in blood coagulation. According to the invention, this is also a necessary parameter for the therapeutic use of the thrombin derivatives.
  • Preferred embodiments of the mutants or derivatives according to the invention have an in vivo half-life of more than one hour.
  • the change in the amino acid sequence can consist of the exchange of one or more amino acids, but it can also consist of a deletion, preferably a deletion, which corresponds to the processing procedure when activating prothrombin, or an insertion if these changes result in the parameters essential to the invention an activity of at most about 10%, in particular of at most 0.25%, of the natural thrombin and a deletion binding to thrombin ligands and receptors are fulfilled.
  • the term "derivative" is intended to encompass both the mutated and the processed mutated proteins.
  • the most suitable amino acids to be introduced are those which influence the spatial structure of the protein as little as possible. These are either very small amino acids, such as alanine, or amino acids that are very similar to the original amino acid and differ from it only by a functional group, for example asparagine and aspartic acid.
  • the parameters according to the invention make the mutants or derivatives mentioned ideal thrombin inhibitor antagonists since they do not have the disadvantages mentioned in the prior art, namely undesired coagulation activity, toxicity or a lack of efficiency or specificity.
  • mutants or derivatives according to the invention are inactive or at most an activity of about 10%, in particular of at most about 0.25% of the natural thrombin (as a result of which the in vivo thrombin activity of the mutants or derivatives is still somewhat below this about 0.25% ), these cannot lead to undesirable coagulation effects even if they are administered in an overdose.
  • mutants or derivatives according to the invention are highly efficient and highly specific as antagonists, since their binding determinants are essentially unchanged from the natural and synthetic inhibitors and correspond to those of the natural thrombin.
  • Preferred changes in the protein sequence relate to amino acids from the active center of the prothrombin, meizothrombin or thrombin molecule, in particular the amino acids His-363 and Asp-419, based on the amino acid numbering in human prosthrombin according to FIG. 1 (the numbering of the amino acids
  • the cDNA sequence and the amino acid sequence of prothrombin are shown in Fig. 1.
  • the cleavage sites of factor Xa are indicated in the cDNA sequence so that the cDNA and amino acid sequence of the thrombin can be derived
  • the numbering begins with the 1st amino acid of the mature prothrombin after the leader sequence and the propeptide have been split off
  • the cDNA sequence of the prothrombin is in SEQ. ID.NO. 8, the amino acid sequence in SEQ. ID.NO 9 reproduced.).
  • amino acid aspartic acid 419 (Asp-419) has no close contact with the bound hirudin, which is why the exchange of this amino acid is particularly preferred in the context of the present invention.
  • cysteine residues Cys-293 and Cys-439 are related to amino acid numbering in prothrombin according to Fig.l, also preferred. These mutations enable the formation of a single-chain thrombin derivative (since the sulfur bridge bond between the B chain and the A chain is prevented) which, despite binding to hirudin, ultimately has no enzymatic activity (since the A chain is absent). In this case, the amino acids serine and alanine offer themselves as exchange partners.
  • the invention therefore preferably relates to prothrombin mutants or derivatives in which at least one amino acid selected from His-363 or Asp-419 and optionally Cys-293 or Cys-439 has been changed, in particular Asp-419 mutants.
  • a very particularly preferred embodiment of the mutants or derivatives according to the invention relates to mutants or derivatives in which the amino acid Asp-419 is replaced by Asn.
  • this variant is inactive, even against the artificial substrate AcOH-HD-CHG-Ala-Arg-pNA, only has a residual activity of about 0.25%, so that no coagulation-active side effects are to be expected .
  • the binding capacity of this derivative for example with respect to hirudin, cannot be distinguished from that of natural thrombin, since the structural change which the exchange Asp for Asn entails is very small and, moreover, the binding to the natural and synthetic ones Inhibitors, in particular hirudin, not in the region of the protein concerned.
  • prothrombins Although mutated prothrombins have been described in the prior art, derivatives which the claimed claims have been described Have properties have not yet been disclosed. However, it is precisely these properties which make the use of the prothrombin, meizothrombin and thrombin derivatives according to the invention so advantageous.
  • the serine 528 in the active center of the bovine prothrombin (synonymous with the serine 525 in the corresponding human prothrombin) was mutated to an alanine.
  • Basic science experiments were then carried out with such a mutated prothrombin in order to study the influence of this mutation on the expression, ⁇ -carboxylation and activation of prothrombin.
  • the structural analysis of the thrombin-hirudin complex has shown that amino acids from the active center of thrombin also make a weak contribution to the formation of the complex.
  • Ser-525 can form hydrogen bonds in human prothrombin to the N-terminal amino acid of hirudin and be located within a radius of 3.2 A from the N-terminus of hirudin. Ser-525 thus apparently contributes to the binding of hirudin (Rydel et al., Science 249: 277, 1990).
  • the bovine Ser 528 variant only has a 74% binding capacity compared to DAPA, compared to natural thrombin. This confirmed the assumption that this serine residue lies directly in the DAPA or hirudin binding determinant. Therefore, mutations which only affect the Ser 528 site in bovine prothrombin or the Ser 525 site in human prothrombin do not meet the requirement of sufficient binding capacity to the inhibitor.
  • thrombin fragments with larger deletions were produced (Gan et al., Arch. Biochem. Biophys. 1993: 301, 228).
  • a degradation product of thrombin, ⁇ -thrombin was obtained which has amino acids 469 to 579 of the ⁇ -thrombin sequence.
  • the amino acids arginine-517 (against glutamine), aspartic acid-519 (against glutamine) and serine-525 (against alanine) were mutated and less activity was found in the individual mutants than in wild-type thrombin.
  • the ability to bind hirudin was only partially preserved with some ⁇ -thrombins.
  • the Ser-525-Ala mutant showed the lowest enzymatic activity and the best results in terms of hirudin binding, but the binding capacity in these tests was significantly below that of natural thrombin. Although it was shown that the thrombin fragments compete with thrombin-hirudin binding to varying degrees in competitive binding studies and an absolute indication of the binding ability of the fragments to hirudin was not made, the results showed, however, that the binding capacity to hirudin was significantly reduced by the mutation has been.
  • ⁇ -thrombins are therefore not suitable for the object on which the invention is based: they are very greatly changed in comparison to the wild-type thrombins, and optimum binding to the natural ligands cannot be guaranteed.
  • prothrombin or thrombin derivatives described in the prior art.
  • prothrombin mutants derivatives
  • ⁇ -thrombin fragments there is also no information about a therapeutic or diagnostic application of these prothrombin mutants (derivatives) or ⁇ -thrombin fragments.
  • the present invention relates to the use of prothrombin mutants or derivatives thereof as medicaments, in particular for the production of a medicinal preparation for preventing side effects in an anticoagulation treatment, or as diagnostics.
  • This use of the mutants or derivatives according to the invention is particularly preferred for anticoagulation treatments with hirudin, heparin, antithrombin III and / or their derivatives, and also synthetic inhibitors.
  • the medical treatment according to the invention therefore comprises the administration of an effective dose of the prothrombin mutants or derivatives thereof to a patient, preferably by intravenous administration.
  • the effective dose depends on each individual case and should preferably be optimized using the results obtained in a thrombin and / or hirudin determination.
  • the prothrombin mutants or derivatives with the properties according to the invention with regard to insufficient thrombin activity and sufficient binding capacity are of course preferably used, but under certain conditions known derivatives can also be used, in particular those which are largely inactive, such as e.g. an analog to the bovine Ser-528 mutant described above (or its thrombin derivative), but one must accept the lack of the deteriorated binding capacity.
  • the in vivo half-life of the proteins in the blood circulation is influenced by the glycosylation.
  • Proteins from mammalian cells can be glycosylated via amino acid side chains of asparagine (N-glycosylation) and serine / threonine (O-glycosylation) located on the protein surface.
  • the glycosylation of circulating proteins delays excretion the blood circulation, ie prolongation of the half-life.
  • Recombinant proteins, produced by manipulating mammalian cells are naturally provided with the natural glycosylations which are usual for mammals and thus correspond to the surface structure of the corresponding human proteins.
  • amino acids located on the surface of a protein e.g. Asparagine (Asn) or serine (Ser) or threonine (Thr) into another amino acid, or by deleting one of these amino acids e.g. the native glycosylation can be abolished. It is known that weakly or non-glycosylated proteins are eliminated from the circulation much faster, i.e. that their half-life is shortened.
  • mutation and amino acid exchange of individual amino acids located on the protein surface in e.g. Asparagine increases the number of glycosylation sites of a protein molecule and thus also extends the in vivo half-life. Depending on the number of mutated, deleted or additionally introduced asparagine residues in the protein, the half-life can be varied.
  • Mutants in which the half-life of the protein is shortened by mutation are particularly suitable for the use according to the invention of the prothrombin mutants or derivatives thereof as antagonists against thrombin inhibitors. Therefore, mutants which have a half-life of at most 10 minutes are preferably used as antagonists.
  • the medical use according to the invention of the mutated prosthrombin mutants or derivatives also includes their use as anticoagulants by competitive inhibition of thrombin or as antagonists of their natural functions. This enables medicine to control blood coagulation with an almost nature-identical product.
  • prothrombin mutants or their derivatives are particularly suitable as Anticoagulants in vivo.
  • prothrombin mutants or derivatives thereof for the use according to the invention of the prothrombin mutants or derivatives thereof as anticoagulants, those mutants in which the half-life of the protein is increased by targeted amino acid exchange are particularly suitable. Those inactive mutants which have a half-life of more than 1 hour are therefore preferably used as anticoagulants.
  • prothrombin mutants according to the invention When used as anticoagulants, after application, corresponding to the natural protein, they are processed in vivo to inactive thrombin, which is then able to displace active thrombin occurring in the blood from its receptors.
  • the prothrombin mutant can optionally also be activated in vitro to the corresponding thrombin or meizothrombin mutant and the activated form can be used directly for administration to the patient.
  • blood coagulation can be slowed down or completely stopped in vivo.
  • prothrombin mutants or derivatives thereof which are characterized by an increased in vivo half-life, show the particular advantage that they circulate in the blood much longer than their natural proteins and can therefore effectively influence blood coagulation.
  • the amount of therapeutically used protein can also be reduced accordingly, if necessary.
  • the mutated prothrombin derivatives according to the invention are preferably produced using recombinant DNA technology.
  • the invention therefore also relates to a method for producing the prothrombin mutants or derivatives according to the invention, in which the genetic information of prothrombin is mutated, preferably point-mutated, and in a eukaryo- is expressed expression system and then the expressed derivative is obtained.
  • the human sequences are preferably used here.
  • expression in eukaryotic systems has the advantage that post-translational modifications such as glycosylation and carboxylation are also carried out and thus make the expressed protein more suitable for use in humans.
  • the mutated sequence parts of thrombin were expressed in E. coli and the recombinant peptides artificially provided with sulfur bridges in vitro.
  • the yield of expressed and suitable for experiments thrombin-like structures was accordingly very low.
  • the loss of thrombin activity can be attributed to the lack of large parts of the thrombin sequence as well as to the mutations introduced.
  • the cDNA sequence of the human prothrombin or the cDNA sequence of the human thrombin is preferably point-mutated, which results in an exchange of at least one amino acid in the amino acid sequence.
  • the mutation place according to the invention in the region of the prothrombin sequence which lies in the thrombin sequence after activation of the prothrombin.
  • the mutated prothrombin derivatives are preferably expressed in CHO-DUXS B1 cells (Urlaub & Chasin, Proc. Natl. Acad. Sci. USA 77: 4216, 1980) under the control of the SV40 promoter.
  • expression can be carried out using any conventional expression system, such as yeast, permanent cell lines or viral expression systems, and using any cell line which ensures that the protein is processed correctly and secreted in its functional form.
  • Proper processing of the derivatives includes not only complete glycosylation but also complete ⁇ -carboxylation.
  • the common eukaryotic expression systems include yeast, permanent cell lines (which are either created by stable integration of the foreign DNA into the host cell chromosomes, eg Vero, MRC5, CHO, BHK, 293, Sk-Hepl, in particular liver and kidney cells, or but through the use of a vector which is permanently inherited in the episomal state, for example vectors which are derived from papilloma viruses and which grow in C-127 cells, for example), or viral expression systems such as vaccinia virus, baculovirus or retroviral systems. Vero, MRC5, CHO, BHK, 293, Sk-Hepl, in particular liver and kidney cells, can generally be used as cell lines.
  • prothrombin mutants or derivatives Following the extraction of the expressed derivatives, further processing steps can then be carried out.
  • One possibility in the further processing of prothrombin mutants or derivatives is a process step in which the prothrombin derivative is cleaved into meizothrombin analogs by means of a snake venom protease (e.g. Venom protease).
  • a snake venom protease e.g. Venom protease
  • These meizothrombin analogs can then also be used as antagonists to the natural functions of thrombin, but show no enzymatic thrombin activity. All methods known from the literature can be used.
  • a prothrombin derivative obtained can be converted into the thrombin derivative using trypsin, preferably immobilized trypsin.
  • trypsin preferably immobilized trypsin.
  • any common method for cleaving prothrombin into thrombin can of course be used, including those that make use of other suitable proteases, for example with the snake venom from E. carinatue (Ecarin) or from 0. scvutellatus.
  • the derivatives according to the invention are either prepared with a physiological saline solution and optionally lyophilized, or lyophilized in distilled water and reconstituted with a physiological saline solution before administration.
  • the preparations can also be kept ready for use in other customary solutions and / or with a pharmaceutical carrier or auxiliary.
  • the preparations are in one for parenteral administration, i.e. form suitable for subcutaneous, intramuscular or intravenous administration.
  • preparations according to the invention are free from contamination by viruses.
  • preparations Before being released for medical use, the preparations can also indicate possible contamination by residual nucleic acids of the expression cell line using an extremely sensitive PCR method (for example, described in Austrian patent application A 1830/94) examined and if necessary cleaned again.
  • the derivatives according to the invention must be checked for their ability to bind their natural ligands.
  • a test system was developed for this, in which the binding capacity of the (pro) thrombin derivatives to hirudin or hirudin derivatives is analyzed qualitatively and quantitatively in a simple and reproductive manner.
  • This test system consists of a solid matrix to which natural or recombinant hirudin, derivatives or peptides thereof are bound.
  • the derivative according to the invention is finally bound to this immobilized hirudin and can by a subsequent detection reaction can be detected.
  • the invention therefore also relates to a solid matrix to which natural or recombinant hirudin, derivatives or peptides thereof are bound, and their use in the determination of thrombin or thrombin derivatives.
  • the determination can include both the quantification and the determination of the binding capacity of the thrombin or thrombin derivative.
  • a solid matrix means any solid phase on which the natural and synthetic inhibitor can be effectively immobilized, for example natural polymers, such as cellulose, starch, dextran, alginates, agarose, collagen, in particular the Sepharose or Cellulose materials, synthetic polymers such as polyacrylamide, polyvinyl alcohol, methyl acrylate, nylon or oxiranes, which can easily be shaped into user-friendly devices, such as, for example Microtiter plates, and finally inorganic materials, such as porous glasses, silica gel, etc. (see also Römpp Lexicon of Biotechnology, pages 385 ff.).
  • natural polymers such as cellulose, starch, dextran, alginates, agarose, collagen, in particular the Sepharose or Cellulose materials
  • synthetic polymers such as polyacrylamide, polyvinyl alcohol, methyl acrylate, nylon or oxiranes, which can easily be shaped into user-friendly devices, such as, for example Microtiter plates, and finally inorganic materials,
  • the device according to the invention With the device according to the invention, a simple and precise determination of the thrombin or thrombin derivative concentration can be carried out, whereby not only the active thrombin itself can be determined, but also enzymatically inactive or only weakly active prothrombin or thrombin and derivatives thereof. Furthermore, due to its user-friendly design, the device according to the invention can also be used indirectly for determining the concentration of any thrombin-binding substances, such as thrombin inhibitors, but especially hirudin. In addition, a determination of the binding strength of thrombin or thrombin derivatives to the respectively examined natural and synthetic inhibitors can also be determined with the device according to the invention.
  • thrombin or thrombin derivatives are understood to mean all proteins which can be derived from the protein sequence of the prothrombin, in particular those described above. wrote mutant thrombin, meizothrombin or prothromine derivatives. The derivative can also be changed in the binding determinants, as long as these changes do not rule out binding to the natural and synthetic inhibitors.
  • the thrombin derivatives can differ from natural thrombin in one or more point deletion or insertion mutations.
  • Prothrombin derivatives, meizothrombin and their derivatives can also be determined using the device according to the invention and - insofar as they relate to the determination thereof - are also to be regarded as thrombin derivatives within the scope of the present invention.
  • a test kit for the actual quantification of thrombin, thrombin derivatives and / or hirudin or hirudin derivatives, a test kit is provided according to the invention which contains the device according to the invention and one or more containers with reagents for a specific detection reaction, preferably a thrombin derivative-specific detection reaction.
  • Specific detection reaction should be understood to mean any suitable detection reaction, in particular those reactions which work with dyes (peroxidase, alkaline phosphatase, luminescence reactions, biotin, avidin or biotin-streptavidin (as amplification systems)) or radioactive determination methods.
  • the color reaction which is easier to handle, is preferably preferred for radioactive determination to determine the concentration.
  • peroxidase-labeled sheep anti-thrombin antibodies are used for the invention and the substrate solutions customary for the peroxidase reaction are used for the color reaction.
  • the test kit according to the invention furthermore contains a container with a physiological buffer solution containing a carrier protein, whereby the reproducibility of the quantification is considerably improved.
  • the specific detection reaction in the context of the test kit according to the invention is preferably a labeled thrombin-binding substance, since the determination of thrombin is common in the clinic is of outstanding importance compared to the other determinable components.
  • a large number of labeled thrombin-binding substances are known in the prior art.
  • a dye-labeled polyclonal or monoclonal antibody against thrombin is preferably used. Detection by means of chromogenic substances is often preferred to radioactive determination methods, since the dye reactions do not involve any radioactive contamination and the strict safety measures when working with radioactive material often make the radioactive determination method very impractical.
  • the detection process can proceed according to the process steps common in protein chemistry.
  • a thrombin solution is incubated with the hirudin-coupled solid matrix for 15 minutes to 16 hours, preferably between 45 minutes and 4 hours.
  • the reaction usually takes place in a physiological buffer, preferably in a Tris-HCl buffer. It is particularly advantageous if the physiological salt buffer contains a carrier protein, e.g. Albumin is added.
  • test kit according to the invention further comprises a reference solution containing thrombin, which allows the establishment of a reliable calibration line in the test system.
  • the invention relates to a method for quantifying thrombin or thrombin derivatives, which is characterized by the following steps:
  • the specific detection reaction can either be carried out in the context of the test kit according to the invention with the reagents for a specific detection reaction or directly by a measuring device on the solid matrix itself, for example with a sensor chip with a connected measuring system.
  • the method according to the invention can be carried out in a simple manner, it being particularly suitable for rapid and uncomplicated use in the clinical field.
  • a preferred embodiment of the method according to the invention relates to a method in which the specific detection reaction is a color reaction, the concentration of thrombin or thrombin derivative being determined by correlation with the intensity of the color reaction.
  • the method according to the invention is also suitable for the quantification of hirudin or hirudin derivatives, such a method being characterized by the following steps:
  • the present invention relates to the use of the device according to the invention or the test kit according to the invention for the quantification of thrombin, thrombin, Binder derivatives and / or hirudin or hirudin derivatives and for determining the binding strength of thrombin or thrombin derivatives to hirudin or hirudin derivatives.
  • this test kit can also be used for the first time to determine the binding strength of thrombin or thrombin derivatives to hirudin or other thrombin-preventing substances.
  • the binding strength of thrombin to hirudin is particularly interesting when thrombin derivatives are present, the binding properties of which are unknown to hirudin.
  • test kit can also be used for the functional analysis of hirudin antagonists.
  • the method can also be used when testing hirudin peptides or hirudin derivatives as effective anticoagulants.
  • test kit according to the invention is therefore suitable for answering all questions relating to concentration, binding strength and functionality which arise in connection with thrombin, hirudin and blood coagulation. It should be particularly emphasized that an extremely precise result can be achieved due to the specificity of the binding of hirudin to thrombin. Contamination by other blood factors or proteins cannot falsify the result. The presence of prothrombin does not interfere with the analyzes either, since prothrombin does not bind to hirudin.
  • hirudin is coupled to the matrix in a buffer system.
  • any buffer which is free of amino groups such as phosphate buffer, citrate buffer or preferably carbonate buffer, is suitable as the buffer system.
  • the pH value of the buffer system should ge are between 6 and 10, preferably at pH 9.3 to 9.7.
  • the coupling reaction of hirudin to the solid support is incubated between one and 48 hours, preferably between one and 16 hours.
  • the incubation time depends essentially on the incubation temperature, preferably in the case of a coupling reaction in the cold (4 ° C.) for 16 h, at room temperature for two to three hours and at 37 ° C. for one hour.
  • the excess, unbound hirudin is removed according to the invention with a washing buffer from a physiological salt solution, preferably from a Tris-HCl buffer.
  • a detergent preferably Tween 20, can be added to this washing buffer, the detergent concentration being between 0.01 and 1%, preferably 0.1%.
  • concentrations of thrombin or thrombin derivatives in the range from 0.1 pg / ml to 100 mg / ml thrombin, preferably in the range from 0.1 ng / ml to 200 ng / ml thrombin, can be determined.
  • test kit according to the invention is suitable for distinguishing thrombins with recombinantly designed, targeted mutations, deletions or insertions, it being possible to test whether the binding ability to hirudin has been retained regardless of the enzymatic activity.
  • This test according to the invention or the test kit according to the invention can be used in particular if the thrombin level in the blood is to be determined for a medical question in order to prevent thrombosis with a precisely metered dose of hirudin.
  • This test also has the particular advantage that thrombin can also be determined which is not functionally active and which is therefore not detectable in the tests which detect the enzymatic activity of thrombin. This is the case, for example, with genetic defects when physiologically inactive forms of thrombin are present.
  • the invention is explained in more detail with the aid of the following examples and associated drawing figures, to which, however, it should not be limited.
  • 1 shows the coding part of the cDNA sequence of recombinant human prothrombin and the amino acid sequence which can be derived therefrom, the physiological cleavage sites for processing the protein and the cleavage sites of factor Xa for activating the prothrombin Thrombin are shown; 2 shows the sequence listing; 3 shows a summary of the point mutation of a preferred prothrombin derivative in comparison to wt prothrombin, the underlined amino acid / nucleotides having been replaced; 4A shows the flow diagram of the cloning of the prothrombin Asn419; 4B shows a western blot for comparing plasmatic prothrombin, recombinant wt prothrombin and prothrombin Asn419; 5: the denaturing electrophoresis of individual purification stages of recombinant prophrombin derivative (A: cell culture supernatant; B: eluate 3; C: eluate 4; D: molecular weight marker); 6: the denatur
  • FIG. 12 shows the molecular structure of the catalytic Center in the thrombin-hirudin complex (comparison of human thrombin and recombinant thrombin derivative), with arrows pointing to the structural change caused by the mutation Asp ⁇ Asn and Ser, His and Asp or Asn the position of the amino acids of the catalytic center in the Thrombin molecule and Ile denote the N-terminal amino acid of hirudin.
  • Example 1 uses the example of prothrombin Asn419 to show how a point-mutated prothrombin can be obtained.
  • Example 2 demonstrates the purification and functional analysis of the prothrombin derivative.
  • Example 3 shows the recovery and functional analysis of the thrombin derivative.
  • Example 4 quantifies the binding activity of the thrombin derivative to hirudin;
  • Example 5 checks the prothrombin derivative for its ability to act as an antagonist of hirudin.
  • Example 6 shows that hirudin can be neutralized by the thrombin derivative.
  • Example 7 shows that the thrombin derivative can reactivate thrombin from a thrombin-hirudin complex;
  • Example 8 shows that the thrombin derivative is also effective in plasma and
  • Example 9 shows the recovery and functional analysis of a meizothrombin derivative.
  • Example 1 Construction of pSV-FIIwt and pSV-FII-Asn419 (Asp to Asn)
  • the plasmid pSVß (Nucl. Acids Res. 17: 2365; 1989) was cut with NotI to remove the internal ⁇ -galactosidase gene fragment. The remaining vector was religated and called pSV.
  • pSV was cut with HindII and Xbal. After removing the small polylinker fragment, the vector ends were filled in with Klenow enzyme and religated. The resulting plasmid was called pSV ⁇ .
  • the MCS was chemically synthesized in the form of two complementary oligonucleotides:
  • the two oligonucleotides were annealed and set in pSV ⁇ . Since the MCS insert had Xhol-compatible, "sticky" ends, but no complete Xhol sites, the ligation reaction was cut with Xhol. Constructs that could not be cut represented the desired plasmid, which was called pSV-MCS III.
  • a DNA fragment with the complete, human wt prothrombin cDNA was excised from plasmid pTKemc-PT2 (WO 91/11519) by means of partial Ncol and complete Smal restriction digestion.
  • This fragment was placed in the vector pSV-MCS III after it was also fully opened via partial Ncol and complete Smal digestion.
  • the resulting plasmid was named pSV-FIIwt and expresses wt prothrombin as evidenced by transient expression in COS cells and stable expression in CHO cells; the order of the functional elements on pSV-FIIwt is SV40 promoter / enhancer (of the early genes), SV40-5 'UTR, wt prothromine bin cDNA, SV40-16s / 19s intron, SV40 polyadenylation site, and pUC 19 sequences (with bacterial origin of replication and ampicillin resistance gene).
  • pSV-FIIwt was mutated: the codon coding for said aspartic acid is located on an EcoRV-DralII restriction fragment. Both restriction sites are unique in pSV-FIIwt.
  • the intended mutagenesis was carried out by means of a polymerase chain reaction with the primer pair 2104/2066 (Seq.ID.No.3 and 4), as a result of which the wt-prothrombin-EcoRV-Swalll fragment by the PCR containing the mutation Ecll36lI-DraIII fragment was substituted.
  • the two oligonucleotides were chemically synthesized:
  • Primer 2104 (5'-TAACTGACGG TCCTTGAGCT CCATGTTGGA AAAGATCTAC ATC-3 ') (Seq.ID.No.3) as a 5' primer; after the polymerase chain reaction, the Ecll36ll 'half site' is ligated onto the EcoRV 'half site' of the vector, whereby some nucleotides of the wt prothrombin have been changed at the DNA level, but the amino acid sequence as in the wt prothrombin preserved.
  • Primer 2066 (5'-GCAGACACAC AGGGTGAATG TAGTCACTGA AGGCAACAGG CTTCTTCAGC TTCATCAGGG CAATATTCCG GTCCAGGTTC TCCCGC-3 ') (Seq.ID. No.4) as 3' primer; this primer mutates the aspartic acid into asparagine at the DNA level, an Sspl restriction site is introduced and a Neil site is lost.
  • the PC reaction was carried out under standard conditions at an annealing temperature of 55 ° C.
  • the resulting plasmid pSV-FIIAsn419 which contains the Asp ⁇ Asn mutation, was identified by its restriction pattern with EcoRV, Dralll, Sspl, and Neil in comparison with pSV-FIIwt.
  • the expected nucleotide sequence of the Ecll36II-DraIII insert in pSV-FIIAsn419 was determined by subsequent sequencing with the 5 'and 3' primers 2197 (5'-CATAAGCCTG AAATCAACTC-3 ') (Seq.ID. No.5) and . 2198 (5'-CTTCGGAGCG TGGAGTCATC-3 ') (Seq.ID.No.6) confirmed.
  • Dihydrofolate reductase gene-deficient CHO-DUKS B1 grow routinely in "full medium” (DMEM / Ham's F12 1: 1 medium, supplemented with 2 mM glutamine, 0.075% bicarbonate, 100 IU penicillin and 100 mg streptomycin / ml, 10% fetalem Calf serum, and 10 mg deoxyadenosine, adenosine, and thymidine per ml).
  • DMEM / Ham's F12 1 1 medium, supplemented with 2 mM glutamine, 0.075% bicarbonate, 100 IU penicillin and 100 mg streptomycin / ml, 10% fetalem Calf serum, and 10 mg deoxyadenosine, adenosine, and thymidine per ml.
  • the cells were treated with 10 ⁇ g pSV-FIIwt or pSV-FIIAsn-419 and 1 ⁇ g pSV-dhfr (Fischer et al., FEBS Lett . 351: 345, 1994) co-transfected: 25 ml of 2.5M CaCl 2 were added to the DNA in 250 ml 1mM Tris pH 8.0.0.0mM EDTA. Then 250 ml of 280mM NaCl, 45mM Hepes, 2.8mM Na 2 HP0 4 , pH 7.12 were added. After 10 minutes, the resulting DNA coprecipitate was added to the subconfluent cells.
  • the medium was suctioned off and the cells were overlaid with 15% glycerol in PBS.
  • the glycerin was aspirated, the cells were washed with PBS, and the cells were provided with fresh "complete medium".
  • DMEM / F12 1 1 medium without hypoxanthine, glycine and thymidine; supplemented with 2 mM glutamine, 100 IU penicillin and 100 mg streptomycin / ml, and 10 % dialyzed fetal calf serum with an exclusion volume of 10OOOOd.
  • selection medium DMEM / F12 1: 1 medium without hypoxanthine, glycine and thymidine; supplemented with 2 mM glutamine, 100 IU penicillin and 100 mg streptomycin / ml, and 10 % dialyzed fetal calf serum with an exclusion volume of 10OOOOd.
  • Example 2 Purification and activity determination of recombinant wt prothrombin and prothrombin derivatives
  • buffer D 50 mM Tris / HCl buffer pH 7.4, 180 mM NaCl (buffer B) 50 mM Tris / HCl buffer pH 7.4, 300 mM NaCl (buffer C) 50 mM Tris / HCl buffer pH 7.4, 160 mM NaCl , 10 mM Ca acetate (buffer D)
  • the recombinant wt prothrombin and the prothrombin derivative from the cell culture supernatant were purified by Liquid chromatography. During the chromatography, the course was followed in the usual way by absorption measurement at 280 nm. The content of prothrombin or prothrombin derivatives of the individual fractions and eluates was determined in the usual way by means of ELISA using commercially available prothrombin preparation as standard.
  • the total protein concentration was determined by the method of Bradford, M. (Anal. Biochem. 72, 248 (1976)).
  • the anion exchange column was equilibrated with buffer A and then 970 ml of cell culture supernatant (prothrombin content (ELISA) 20 ⁇ g / ml; protein concentration 2.7 mg / ml) at a rate of 4 ml / Minute applied. Material not bound to the exchange gel was removed by rinsing the column with buffer A (eluate 1: 1030 ml: 1.2 mg / ml). Proteins weakly bound to the column were then removed by rinsing the column with buffer B (eluate 2: 20 ml; prothrombin content (ELISA) 2 ⁇ g / ml; total protein content 10.0 mg / ml).
  • ELISA cell culture supernatant
  • the column was then eluted with buffer C and protein bound to the column was obtained in the eluate (eluate 3: 30 ml; prothrombin content (ELISA) 355 ⁇ g / ml; total protein content 16 mg / ml).
  • the column was then regenerated by washing with 1 M NaCl solution and equilibrated with buffer D. 28 ml of eluate 3 were diluted 1.9 times with buffer A and Ca acetate was added to the final concentration of 10 mM. This solution was again filtered through the anion exchange column and rinsed with buffer D, whereby unbound protein in the eluate (eluate 4: 60 ml; prothrombin content (ELISA) 170 ⁇ g / ml) was obtained.
  • Example 3 Extraction, analysis and activity determination of wt-thrombin and thrombin-Asn99
  • Thrombin-Asn99 was obtained analogously to the method described in EP-A-0 565 512 by cleaving the prosthrombin Asn419 using immobilized trypsin.
  • the eluate obtained after the activation was examined by means of denaturing SDS-PAGE (FIG. 6).
  • the results of SDS-PAGE show that recombinant prothrombin derivative was converted into a thrombin derivative (thrombin-Asn99) with a molecular weight of 33,000 (heavy chain).
  • the N-terminal amino acid sequence analysis showed the following two sequences: (A) Thr-Ala-Thr-Ser-Glu-Tyr-Gln-Thr-Phe-Phe-Asn-Pro-Arg-Thr-Phe; (B) Ile-Val-Glu-Ser-Asp-Glu-Ile-Gly-Met-Ser-Pro Trp-Gln.
  • the sequences thus show that the recombinant thrombin derivative by proteolysis at the authentic cleavage sites of prothrombin (Arg271-Thr272 and Arg320-Ile321) as a double-chain molecule with an ⁇ -thrombin structure.
  • Figure 12 shows the molecular structure of the catalytic center.
  • FIG. 12 shows the comparison of human thrombin and the recombinant thrombin derivative Asn99.
  • the thrombin activity was determined using a chromogenic substrate at 25 ° C. in 50 mM Tris / HCl buffer, 150 mM NaCl, 0.1% PEG 6000, pH 8.0, with a concentration of the synthetic chromogenic substrate of 0. 2mM AcOH-DH-CHG-Ala-Arg-pNA (TH-1, pentapharm) in a volume of 1 ml. The absorption at 410 nm was determined as a function of time. Thrombin standard with defined activity (Immuno AG) was used as a reference The samples were diluted in the test buffer with the addition of 1% Prionex (collagen hydrolyzate, Pentapharm).
  • the activity determination revealed an activity of 0.24 nmol / min .mu.g protein for the recombinant thrombin derivative thrombin-Asn99.
  • Thrombin-Asn99 thus only has an activity of 0.24% in the chromogenic assay against human plasma thrombin.
  • Table 1 Determination of thrombin activity using a chromogenic substrate
  • Thrombin derivative specific activity ___ (nmol / min ⁇ g protein)
  • thrombin derivatives were examined for their thrombin activity using a thrombin standard (Immuno AG) defined activity. In this activity determination, no activity was found for thrombin-Asn99 (Table 2).
  • Thrombin derivative activity (IU / mg protein)
  • the active center of the thrombin derivatives was titrated by the method of M.F. Doyle and P.E. Haley (Methods in Enzymology (1993), 222, 299-312), using p-nitrophenyl-p'-guanidinobenzoate as substrate and an extinction coefficient of 16,595 M cm at 410 nm.
  • Thrombin derivative concentration of active thrombin (nmol / mg protein)
  • thrombin-Asn99 shows extremely low thrombin activity in only one of three test methods, which corresponds to approximately 1/400 of the native thrombin activity.
  • Recombinant wt thrombin and human plasma thrombin show very similar activity patterns.
  • hirudin from the thrombin derivative thrombin-Asn99 was examined by means of an ELISA test and compared with human plasma thrombin and recombinant wt thrombin.
  • This ELISA test is based on the use of immobilized hirudin.
  • thrombin is bound to hirudin, which is immobilized on microtiter plates, and detected via antibodies with a subsequent color reaction. This test is independent of the enzymatic activity of the thrombin.
  • Recombinant hirudin variant 1 (variant 1; Rhein Biotech company, FRG; 2 ⁇ g / ml, 100 ⁇ l) is bound to microtitration plates to produce the ELISA plates. After washing, recombinant wt thrombin, thrombin Asn99 or human plasma thrombin (100 ⁇ l of a solution with concentrations according to FIG. 7) were added and incubated for one hour. Non-bound thrombin was removed and bound thrombin using per- oxidase-labeled anti-thrombin immunoglobulin (Sheep anti-human thrombin;. Enzyme Research Lab Inc., Indiana USA; ul 100 a, 00 "dilution) were detected (Fig. 7). The measurement of the tion took place at 450 nm.
  • the binding constant of thrombin to hirudin was determined using fluorescence emissions using the PC program ENZFITTER (RJ. Leatherbarrow, Elsevier-Biosoft, 1987) using a binding model with a common binding site.
  • the intrinsic fluorescence of aromatic amino acids of the thrombin derivatives was determined in 50 mM Tris / HCl buffer, 150 mM NaCl, 0.1% PEG 6000, pH 7.4. The excitation took place at 280 nm (slit width 2.5 nm), the emission was registered between 300 nm and 400 nm (slit width 5 nm).
  • the intrinsic fluorescence of tryptophan in the thrombin molecule was excited at 280 nm and the emission was measured between 300 nm and 400 nm without adding hirudin or in the presence of hirudin.
  • the fluorescence at 341 nm (excitation 280 nm) of 390 nM thrombin-Asn99, 326 nM recombinant wt-thrombin and 350 nM human plasmatic thrombin was determined as a function of the hirudin concentration.
  • thrombin derivative Thrombin-Asn99 is compared with recombinant wt thrombin and human plasma thrombin. It can be seen from the results that in the presence of hirudin for all three thrombin derivatives, the fluorescence of tryptophan in the thrombin molecule (hirudin has no tryptophan) increases significantly (FIG. 8). This is obviously due to the formation of a hirudin-thrombin complex.
  • Trp 51, Trp 148 and Trp 227 from thrombin in particular come into contact with the inhibitor by hirudin binding.
  • FIG. 8 shows the dependence of the thrombin fluorescence on the hirudin concentration. Very similar bindings of hirudin to thrombin were obtained for all three thrombin derivatives. The binding of hirudin to all three thrombin derivatives corresponds to saturation and results in one binding site per thrombin molecule.
  • Example 5 Recombinant prothrombin as a hirudin antagonist
  • Coagulometer KC 10 (Amel Institute GmbH, Germany) Prothrombin-free normal plasma (Immuno AG, Vienna) Prothrombin concentration standard (Immuno AG, Vienna) Recombinant hirudin (Rhein Biotech, Germany)
  • prothrombin time test In a conventional laboratory method, the time required after activation of the factors involved in blood coagulation to determine normal plasma to coagulate was determined using a prothrombin time test. In this test, by adding Ca ions to the mixture of 1. Prothrombin-free Normal plasma (which however contains all other coagulation factors) and 2nd prothrombin concentration standard (prothrombin with defined activity) the coagulation factor Xa is formed, which then converts prothrombin (factor II) into thrombin (factor Ha). Thrombin then causes soluble fibrinogen to convert to insoluble fibrin. This leads to the formation of blood clots. The time interval between activation by adding the Ca ions and the formation of the blood clot is automatically determined by the coagulometer.
  • the duration of the blood coagulation depends on the concentration of the prothrombin or the concentration of the active thrombin formed. The higher the thrombin concentration in the reaction mixture, the shorter the clotting time.
  • a thrombin inhibitor such as hirudin
  • an inactive thrombin-hirudin complex is formed after the conversion of prothrombin to thrombin, so that the thrombin bound in this complex can no longer participate in the conversion of fibrinogen to fibrin.
  • the clotting time increases due to the reduced amount of active thrombin. With an excess of inhibitor over thrombin, there is complete inhibition of blood clotting.
  • Prothrombin leads to a rapid formation of the blood clot.
  • Hirudin inhibits blood clotting.
  • the recombinant prothrombin derivative does not cause blood to clot.
  • the recombinant prothrombin derivative has no influence on blood coagulation by natural prothrombin.
  • thrombin-Asn99 can neutralize hirudin and thus the inhibition of active thrombin is removed.
  • 50 ⁇ l of hirudin (44 nM, 4 ATU / ml) with different concentrations of thrombin-Asn99 were incubated for 1 minute.
  • 50 ⁇ l thrombin standard (3.9 IU / ml) and also an homogenous substrate in the measuring buffer (0.2 mM substrate according to Example 3c in 50 mM Tris / HCl buffer, 150 mM NaCl, 0.1% PEG 6000) , pH 8.0) added and the enzyme activity determined at 25 ° C.
  • Thrombin activity was determined photometrically at 410 nm.
  • hirudin is neutralized by thrombin-Asn99, and thus the inhibitory effect of hirudin on active thrombin is eliminated.
  • thrombin inhibition is neutralized at a ratio of 1 mol thrombin-Asn99 to 1 mol hirudin.
  • Example 7 Reactivation of the thrombin-hirudin complex by thrombin-Asn99
  • the aim of the experiment was to determine whether the thrombin activity can be recovered by adding thrombin-Asn99 to the thrombin-hirudin complex, ie whether thrombin-Asn99 is able to extract hirudin from the thrombin-hirudin complex neutralize.
  • the activity of thrombin (final concentration 0.1 IU / ml) was determined continuously using a chromogenic substrate. After 3 minutes, hirudin (final concentration 0.1 ATU / ml) was added and the reaction was continued for a further 4 minutes. Then different concentrations of thrombin-Asn99 (final concentrations 0.2 ⁇ g / ml, 0.4 ⁇ g / ml and 1 ⁇ g / ml) were added and the reaction was monitored photometrically (FIG. 7).
  • Fig. 10 shows that adding hirudin to thrombin inhibits its activity. From the results it can further be seen that, with the addition of increasing concentration of thrombin-Asn99, the inhibitory effect of hirudin on thrombin can be eliminated.
  • hirudin neutralization is time-dependent; it takes about 1 minute for hirudin to be neutralized by Thrombin-Asn99. This is due to the very high bin the constant of hirudin due to thrombin, the equilibrium of which is therefore shifted over time in favor of free thrombin and the formation of a hirudin-thrombin-Asn99 complex.
  • thrombin-Asn99 is also able to neutralize hirudin in plasma and thus to cancel an inhibitory effect of hirudin on thrombin.
  • hirudin is also neutralized in plasma by thrombin-Asn99, and thus the inhibition of hirudin on plasmatic thrombin is eliminated.
  • Prothrombin-Asn419 from Example 1 was used to obtain recombinant meizothrombin-Asn419.
  • Prothrombin-Asn419 was converted into meizothrombin-Asn419 by incubation with the Venom protease Ecarin.
  • Prothrombin-Asn419 was dissolved at 0.2 mg / ml in 20 mM Tris / HCl buffer, pH 7.4, 150 mM NaCl, 5 mM CaCl 2 , and 20 ng Ecarin (1 ⁇ g each of Prothrombin-Asn419 Pentapharm product) added. The activation took place at 4 ° C for 4 hours.
  • the resulting meizothrombin-Asn419 was purified and isolated in analogy to the purification of thrombin-Asn99 (Example 3) by affinity chromatography on the peptide gel.
  • Meizothrombin-Asn419 produced in this way has the identical molecular weight of prothrombin-Asn419 of 72,000 and consists of the prothrombin-Fl / F2 / A chain (molecular weight 52,000, N-terminal amino acid sequence Ala-Asn-Thr-Phe-leu -Gla- Gla-) and the B chain (molecular weight 32,000, N-terminal amino acid sequence Ile-Val-Glu-Ser-Asp-Ala-Glu-Ile).
  • meizothrombin-Asn419 that it neutralizes hirudin and thus cancels the inhibition of thrombin.
  • the thrombin inhibition is neutralized at a ratio of 1 mol of meizothrombin-Asn419 to 1 mol of hirudin.
  • meizothrombin-Asn419 that by adding meizothrombin-Asn419 to the thrombin-hirudin complex, the hirudin can be released again from the complex, and thus the thrombin regains its activity.
  • the data obtained correspond to those of Thrombin-Asn99.
  • Example 10 Characterization of thrombin-Asn99 and meizothrombin-Asn99 in vivo
  • hirudin neutralizing effects of thrombin-Asn99 and meizothrombin-Asn99 were examined in an animal model: 3 min after intravenous administration of a hirudin dose of 0.5 mg per kg body weight (200 ⁇ l) or 200 ⁇ l saline solution to NMRI mice ( 20 g body weight; each test group comprised 10 mice) 2.5 mg thrombin-Asn99 / kg body weight and 5.0 mg meizothrombin-Asn99 (200 ⁇ l each) were injected. After a further 3 minutes, blood was withdrawn from the anesthetized mice by heart puncture.
  • the citrated plasma obtained was examined for partial thromboplastin time (PTT), thrombin time (TT), anti-thrombin potential (aPT) and plasma concentration of thrombin-Asn99 and meizothrombin-Asn99, each measurement being carried out in triplicate.
  • PTT partial thromboplastin time
  • TT thrombin time
  • aPT anti-thrombin potential
  • plasma concentration of thrombin-Asn99 and meizothrombin-Asn99 each measurement being carried out in triplicate.
  • PTT 50 ⁇ l of citrated mouse plasma were mixed with 50 ⁇ l of factor II-deficient citrate plasma and 100 ⁇ l of partial thromboplastin reagent at 37 ° C. for 3 min. The coagulation was started by adding 100 ⁇ l of 25 mM CaCl 2 . To measure the TT, 50 ⁇ l of citrated mouse plasma were mixed with 150 ⁇ l of factor II-deficient citrate plasma at 37 ° C. for 1 min. The coagulation was started by adding 100 ul thrombin standard (7 units / ml).
  • the TT of all mice in groups 1 to 8 was determined using a calibration curve for the coagulation times of different thrombin standard concentrations (1 unit / ml to 10 units / ml, which gives the effective thrombin concentration in the individual TT
  • the differences which differed in the effective thrombin concentration in the tests with the mouse plasma from test groups 1 and 5 to the effective thrombin concentrations in the tests with the mouse plasma from test groups 2 to 4 and 4, respectively 6 to 8 resulted in the anti-thrombin potential, a difference in 1 thrombin unit / ml being defined as an anti-thrombin unit.
  • the plasma concentrations of thrombin-Asn99 and meizothrombin-Asn99 were determined by adding serial plasma dilutions to immobilized hirudin, thrombin-Asn99 and meizothrombin-Asn 9 being detected using sheep-anti-thrombin-IgG-peroxidase conjugate. Calibration lines with thrombin-Asn99 and meizothrombin-Asn99 concentrations of 3 ng / ml to 100 ng / ml were created for analysis.
  • test group 3 The sole administration of thrombin-Asn99 (test group 3) and meizothrombin-Asn99 (test group 7) showed no significant change in the coagulation parameters compared to test groups 1 and 5, however, both proteins could be detected in mouse plasma.
  • Hirudin-complexed forms of thrombin-Asn99 and meizothrom-bin-Asn99 are less reactive towards immobilized hirudin, which is why lower concentrations of thrombin-Asn99 and meizothrombin-Asn99 were found in plasma.

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Abstract

L'invention concerne de nouveaux mutants ou dérivés de prothrombine qui présentent, par rapport à la protéine naturelle, une ou plusieurs modifications dans la séquence de protéine, sont soit inactifs, soit présentent une activité d'environ 10 % maximum, de préférence environ 0,25 % maximum de la protéine naturelle, et possèdent une capacité de liaison par rapport aux ligands naturels (anticoagulants naturels ou artificiels), laquelle correspond sensiblement à celle de la protéine naturelle. L'invention décrit également l'utilisation de mutants ou de dérivés de prothrombine mutés comme préparations pharmaceutiques.
EP96915903A 1995-06-13 1996-06-12 Derives de prothrombine Withdrawn EP0833897A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AT100695 1995-06-13
AT0100695A AT404357B (de) 1995-06-13 1995-06-13 Prothrombin-derivate
PCT/AT1996/000105 WO1996041868A2 (fr) 1995-06-13 1996-06-12 Derives de prothrombine

Publications (1)

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EP0833897A2 true EP0833897A2 (fr) 1998-04-08

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ID=3504770

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EP96915903A Withdrawn EP0833897A2 (fr) 1995-06-13 1996-06-12 Derives de prothrombine

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Country Link
US (1) US6086871A (fr)
EP (1) EP0833897A2 (fr)
JP (1) JPH11507542A (fr)
AT (1) AT404357B (fr)
AU (1) AU700631B2 (fr)
CA (1) CA2224634A1 (fr)
CZ (1) CZ402097A3 (fr)
HU (1) HUP9900506A3 (fr)
WO (1) WO1996041868A2 (fr)

Families Citing this family (16)

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Publication number Priority date Publication date Assignee Title
AU2008202376B2 (en) * 2001-07-06 2011-02-10 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Process for producing human thrombin by gene modification technique
ATE423199T1 (de) * 2001-07-06 2009-03-15 Chemo Sero Therapeut Res Inst Verfahren zur herstelllung von menschlichem thrombin mittels genmodifikationstechnik
US20030099957A1 (en) * 2001-09-28 2003-05-29 Vitivity, Inc. Diagnosis and treatment of vascular disease
WO2006104398A1 (fr) * 2005-03-26 2006-10-05 Protemix Corporation Limited Compositions antagonistes du cuivre
AU2007320353A1 (en) 2006-11-15 2008-05-22 Fujimori Kogyo Co., Ltd. Thrombin mutant
CN102559644B (zh) 2007-09-28 2015-03-25 普托拉制药有限公司 用于因子xa抑制剂的解毒剂和其使用方法
AU2009320153B2 (en) * 2008-10-27 2016-07-14 Trustees Of Tufts College Nucleic acids encoding peptides for treating wounds, anti-angiogenic compounds, and uses thereof
NZ592837A (en) 2008-11-14 2012-10-26 Portola Pharm Inc Antidotes for factor xa inhibitors and methods of using the same in combination with blood coagulating agents
EP2414517B1 (fr) 2009-03-30 2016-09-21 Portola Pharmaceuticals, Inc. Antidotes pour les inhibiteurs du facteur xa et leur procédé d'utilisation
JP6163304B2 (ja) 2009-07-15 2017-07-12 ポートラ ファーマシューティカルズ， インコーポレイテッド 第Ｘａ因子インヒビターの解毒剤の単位用量処方物およびその使用方法
TWI513466B (zh) 2010-01-20 2015-12-21 Boehringer Ingelheim Int 抗凝血劑解毒劑
EP2471945A1 (fr) * 2010-12-30 2012-07-04 Siemens Healthcare Diagnostics Products GmbH Procédé de détermination d'inhibiteurs de coagulation
AP2013007046A0 (en) 2011-03-30 2013-08-31 Boehringer Ingelheim Int Anticoagulant antidotes
CA2870211A1 (fr) * 2012-04-17 2013-10-24 Aarhus Universitet Sorcs1 utilisable dans le traitement de l'obesite ou de la surcharge ponderale
GB2504499A (en) * 2012-07-31 2014-02-05 Baxter Healthcare Sa Selective measurement of active human protease coagulation factors
JP6640198B2 (ja) 2014-05-26 2020-02-05 アカデ−ミッシュ ズィーケンハウス ライデンＡｃａｄｅｍｉｓｃｈ Ｚｉｅｋｅｎｈｕｉｓ Ｌｅｉｄｅｎ 出血治療のための止血促進タンパク質

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5167960A (en) * 1988-08-03 1992-12-01 New England Deaconess Hospital Corporation Hirudin-coated biocompatible substance
US5112615A (en) * 1988-08-03 1992-05-12 New England Deaconess Hospital Corporation Soluble hirudin conjugates
CA2000887A1 (fr) * 1988-11-01 1990-05-01 Cecilia S.L. Ku Materiaux thromboresistants et methode de production
ES2069726T5 (es) * 1989-01-25 1999-09-16 Novartis Ag Anticuerpos monoclonales especificos para hirudina.
JP2549224B2 (ja) * 1990-01-26 1996-10-30 イムノ・アクチェンゲゼルシャフト 組換えにより産生される血液因子及びその血液因子の発現方法並びにその方法に使用されるワクシニアウイルス組換え体
US5688768A (en) * 1991-02-19 1997-11-18 Cor Therapeutics, Inc. Recombinant thrombin receptor and related pharmaceuticals
DE4203965A1 (de) * 1992-02-11 1993-08-12 Max Planck Gesellschaft Antidot fuer hirudin und synthetische thrombininhibitoren
EP0728210B1 (fr) * 1993-11-12 2005-04-13 Gilead Sciences, Inc. Mutants de thrombine
AT401270B (de) * 1994-09-26 1996-07-25 Immuno Ag Verfahren zur quantifizierung von genomischer dna

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9641868A2 *

Also Published As

Publication number Publication date
ATA100695A (de) 1998-03-15
AT404357B (de) 1998-11-25
JPH11507542A (ja) 1999-07-06
HUP9900506A2 (hu) 1999-06-28
HUP9900506A3 (en) 2001-10-29
US6086871A (en) 2000-07-11
WO1996041868A2 (fr) 1996-12-27
AU5887196A (en) 1997-01-09
AU700631B2 (en) 1999-01-07
CA2224634A1 (fr) 1996-12-27
WO1996041868A3 (fr) 1997-04-10
CZ402097A3 (cs) 1998-04-15

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