Patent application title: Fibrinogen Preparations Enriched In Fibrinogen With An Extended Alpha Chain
Joseph Grimbergen (Lisse, NL)
Jacob Koopman (Leiderdorp, NL)
Abraham Bout (Moerkapelle, NL)
Abraham Bout (Moerkapelle, NL)
IPC8 Class: AA61K3836FI
Class name: Hydrolases (3. ) (e.g., urease, lipase, asparaginase, muramidase, etc.) acting on peptide bonds (3.4) (e.g., urokinease, etc.) serine proteinases (3.4.21) (e.g., trypsin, chymotrypsin, plasmin, thrombin, elastase, kallikrein, fibrinolysin, streptokinease, etc.)
Publication date: 2013-01-10
Patent application number: 20130011382
The present invention relates to fibrinogen preparations enriched in
α-extended fibrinogen. Compositions comprising such preparations
show improved clotting properties compared to preparations based on HMW
Fib which typically contain no or only low amounts of α-extended
fibrinogen. In particular, clot formation time and the clot strength of a
clot made by α-extended fibrinogen are improved. In addition,
plasmin-mediated degradation of α-extended fibrinogen is reduced as
compared to plasma derived fibrinogen.
1. A fibrinogen preparation which has a clot formation time which is less
than the clot formation time of a plasma-derived fibrinogen preparation
in a ROTEM® thromboelastography test under similar conditions.
2. a fibrinogen preparation according to claim 1 containing at least 95% (w/w) fibrinogen and wherein at least 10% (w/w) of the fibrinogen is in the form of Fib420.
3. A fibrinogen preparation according to claim 1, which is free of Factor XIII.
4. A pharmaceutical composition comprising a fibrinogen preparation according to claims 1 and a pharmaceutically acceptable carrier, diluents or excipient.
5. A pharmaceutical composition according to claim 4, wherein the composition is suitable for facilitating tissue adherence, improving wound healing or for intravenous administration.
6. A pharmaceutical composition according to claim 4, wherein the pharmaceutical composition is a fibrin sealant or a topical haemostat.
7. A pharmaceutical composition according to claims 4 which further comprises thrombin.
8. A pharmaceutical composition according to claims 4 in dry form.
9. A fibrinogen preparation according to claims 1 for use as a medicament.
10. A fibrinogen preparation according to claims 1 for use in a method for treating acute bleeding episodes, hyperfibrinolysis, fibrinogen deficiency or other bleeding disorders.
11. A method of using a fibrinogen preparation according to claim 1 for the preparation of a medicament for the treatment of acute bleeding episodes, hyperfibrinolysis, fibrinogen deficiency or other bleeding disorders.
12. A method for preparing a Fib420 fibrinogen preparation, which method comprises: providing an expression vector comprising a nucleotide sequence encoding an alpha extended polypeptide chain of fibrinogen; transforming a mammalian cell with the expression vector; maintaining the mammalian cell under such conditions which allow for the expression of the nucleotide sequence encoding the alpha extended polypeptide chain of fibrinogen.
13. The method according to claim 12, wherein the nucleotide sequence encoding an alpha extended polypeptide chain of fibrinogen is an optimized sequence, preferably an optimized sequence according to SEQ ID No. 5.
14. The method according to claim 12, wherein the mammalian cell is a human cell, preferably a PER.C6 cell.
15. A pharmaceutical composition according to claim 4 for use in a method for treating acute bleeding episodes, hyperfibrinolysis, fibrinogen deficiency or other bleeding disorders.
16. A method of using a pharmaceutical composition according to claim 4 for the preparation of a medicament for the treatment of acute bleeding episodes, hyperfibrinolysis, fibrinogen deficiency or other bleeding disorders.
FIELD OF THE INVENTION
 The present invention relates to fibrinogen preparations and the use of fibrinogen preparations in medical applications. In particular, it relates to fibrinogen preparations which are enriched in fibrinogen with an extended alpha chain, to methods for producing it and to its applications.
BACKGROUND OF THE INVENTION
 Fibrinogen is a soluble plasma glycoprotein which is synthesized in the human body primarily by liver parenchymal cells. It is a dimeric molecule, consisting of two pairs of three polypeptide chains designated Aα, Bβ and γ, which are connected by disulfide bridges. The three polypeptide chains are encoded by three separate genes. The predominant form (HMW fib) harbors an Aα chain which is synthesized as a 625 amino acid precursor and is present in fibrinogen found in blood plasma as a 610 amino acids polypeptide chain, the Bβ chain contains 461 and the γ chain 411 amino acids. The three polypeptides are synthesized individually from 3 mRNAs. Assembly of the three component chains (Aα, Bβ, and γ) into its final form as a six-chain dimer (Aα, Bβ, γ)2 occurs in the lumen of the endoplasmic reticulum (ER).
 Fibrinogen circulates in blood at high concentrations (1-2 g/L) and demonstrates a high degree of heterogeneity. It is estimated that in each individual about one million different fibrinogen molecules circulate. Most of these variants, which account for just a small portion of the total fibrinogen (in most cases not more than a few percents), differ in function and structure.
 Proteolysis of the carboxy-terminal part of the Aα chain results in three major circulating forms of fibrinogen having clearly different molecular weights. Fibrinogen is synthesized in the high-molecular weight form (HMW; molecular weight 340 kDa; the predominant form of Aα chains in the circulation contains 610 amino acids). The degradation of one of the Aα chains gives rise to the LMW form (MW=305 kDa); the LMW' form (270 kDa) is the variant where both Aα chains are partially degraded at the carboxy-terminus. In blood of healthy individuals, 50-70% of the fibrinogen is HMW, 20-50% is fibrinogen with one or two degraded Aα chains (de Maat and Verschuur (2005) Curr. Opin. Hematol. 12, 377). The HMW and LMW' variants show distinct differences in clotting time and fibrin polymer structure (Hasegawa N, Sasaki S. (1990) Thromb. Res. 57, 183).
 Well-known variants which are the result of alternative splicing are the so-called gamma prime (γ') variant and the α-ext Fib or Fib420 variant.
 The α-ext Fib or Fib420 variant, which has a molecular weight of 420 kDa, accounts for 1-3% of the total circulating fibrinogen (de Maat and Verschuur (2005) Curr. Opin. Hematol. 12, 377). The extended α-ext Fib isoform is distinguished from the conventional α-chain of fibrinogen by the presence of an additional 236 residue C-terminus globular domain due to alternative splicing. Contradictary data on the function and characteristics of Fib420 is given in literature. Based on studies with plasma derived α-ext Fib, Applegate et al., Blood (2000) 95: 2297, concluded that the polymerization and cross-linking properties of α-ext Fib are not grossly different from plasma derived HMW Fib. They conclude that the additional C-domain has no effect on coagulation and they suggest that the function of the domain may be to support integrin-mediated cell adhesion. In EP 1 495 051 it is suggested that Fib420 might be less sensitive to degradation and could have an effect on clot structure. However, no substantiation is given and there is no suggestion as to whether the effect may be an enhancement or deterioration in clot structure or strength. Mosesson et al. (Biophys. Chem. 112, 209: 2004) have studied the ultrastructure of clots that are based on umbilical cord plasma-derived α-ext Fib. They reported that the fibers of α-ext Fib clots are thinner and more branched than those based on HMW fibrinogen, but that they have the same periodicity that characterizes all fibrin fibers. The authors suggest that the likely function for the extended α-chain in α-ext Fib is to provide sites for interaction with cellular integrins.
 The present invention relates to a fibrinogen preparation which contains at least 95% (w/w) fibrinogen and wherein at least 10% of the fibrinogen is in the form of Fib420.
 In the field, Fib420 is also referred to as `α-ext Fib`, `fibrinogen with an extended alpha chain` or `fibrinogen420`. In the present context, these terms are used interchangeably. All these terms refer to a symmetrical molecule of the structure (Aαext, Bβ, γ), wherein both conventional fibrinogen α-chains, as found in HMW fib, have been replaced by extended alpha chains.
 In the present context, the term `fibrinogen preparation` refers to fibrinogen in isolated form, for example to a plasma isolate or cell supernatant isolate of fibrinogen. It may also refer to a synthetic preparation of fibrinogen. A fibrinogen preparation according to the invention preferably contains at least 65% w/w, at least 70% w/w, at least 75% w/w, at least 80% w/w or at least 85% fibrinogen, based on total protein. More preferably, it is a pure preparation in which substantially no contaminants, such as other proteins, are present and it contains at least 90% w/w, at least 95% w/w, at least 96% w/w, at least 97% w/w or at least 98% fibrinogen, based on total protein. Most preferably, a fibrinogen preparation according to the invention comprises at least 99% w/w or at least 99.5% w/w fibrinogen, based on total protein. Such pure fibrinogen preparations are particularly suitable to formulate compositions which are used in medical applications, such as compositions for wound therapy and surgical repair.
 According to the invention, at least 10% w/w of the fibrinogen in the preparation is in the form of Fib420. Preferably, at least 15% w/w, at least 20% w/w, at least 25% w/w or at least 30% w/w of the fibrinogen is in the form of Fib420. More preferably, at least 40% w/w, at least 50% w/w, at least 60% w/w, at least 70% w/w, at least 80% w/w or at least 90% w/w is in the form of Fib420. Most preferably, at least 95% w/w, at least 99% w/w or all of the fibrinogen is in the form of Fib420.
 One advantage of the fibrinogen preparation according to the invention is that plasmin-mediated digestion of the fibrinogen preparation is slower than for plasma derived fibrinogen. Its enhanced resistance to plasmin digestion is likely to be beneficial for treatment of patients who suffer from hyperfibrinolysis which is often seen in cases of acquired coagulopathy.
 Using a fibrinogen preparation according to the invention, clots are formed faster and the clots which are formed have a higher clot firmness than clots formed with plasma derived fibrinogen or HMW fibrinogen. This means that fibrin clot stability is enhanced when using the fibrinogen preparation according to the invention and that the fibrinogen preparation according to the invention is more potent than state of the art preparations. A more potent fibrinogen preparation allows for the reduction of both the amount of fluid and the amount of therapeutical protein to be administered. This is an advantage for intravenous use to treat dilutional coagulopathy. Currently, for intravenous (i.v) injection of fibrinogen to compensate for low blood clotting activity, high dosages of fibrinogen are required (˜5 gram of fibrinogen per treatment). This is administered by direct injection at a dose of 70 mg/kg. As fibrinogen in general can be dissolved at a maximum concentration of 20 mg/ml, this means that about 250 ml of fluid has to be administered intravenously to in an adult. Lowering this dose by providing a more potent fibrinogen would be beneficial.
 Yet another advantage is that clot formation using the fibrinogen preparation of the invention is less Factor XIII dependent than preparations of plasma derived fibrinogen or HMW fibrinogen. The strength of a clot made in buffer by alpha-extended Fib (so in the absence of Factor XIII) is higher than for plasma-derived fibrinogen which will contain some Factor XIII. No Factor XIII is required to form firm clots. Therefore, in one embodiment, the fibrinogen preparation according to the invention is free of Factor XIII.
 Clotting time (CT), clot formation time (CFT) and clot firmness of α-ext rhFib and plasma derived fibrinogen can be measured using ROTEM analysis. ROTEM® (Pentapharm GmbH, Munich, Germany) stands for ROtation ThromboElastoMetry. The technique utilizes a rotating axis submerged in a (blood) sample in a disposable cuvette. Changes in elasticity under different clotting conditions result in a change in the rotation of the axis, which is visualized in a thromboelastogram, reflecting mechanical clot parameters (see e.g. Luddington R. J. (2005) Clin Lab Haematol. 2005 27(2):81).
 In a ROTEM® system test under similar conditions, a fibrinogen preparation according to the invention typically performs at least as good or better than a fibrinogen preparation or composition in which the fibrinogen variant distribution resembles the variant distribution in human plasma, viz. less than 5% w/w of the fibrinogen is of the Fib420 type. This is for example the case for fibrinogen concentrates which are based on plasma-derived fibrinogen. Such concentrates are commercially available. In particular, its clot formation time will be less than the clot formation time of a fibrinogen preparation in which less than 5% w/w of the fibrinogen is of the Fib420 type. Preferably, the clot formation time of a fibrinogen preparation according to the invention is maximally 80%, maximally 60%, maximally 50%, maximally 40%, maximally 30%, maximally 20%, maximally 10% of the clot formation time of a fibrinogen preparation in which less than 5% w/w of the fibrinogen is of the Fib420 type, such as plasma-derived fibrinogen preparations. The ROTEM experiments with α-ext rhFib presented in the Examples indicate that a fibrinogen preparation according to the invention has a clot formation time (CFT) which is less than the CFT of purified plasma derived fibrinogen produced for intravenous applications.
 In another aspect, the invention relates to a composition comprising a fibrinogen preparation according to the invention. In addition to the fibrinogen, the composition may comprise an activator, such as thrombin or a thrombin-like protein, such as reptilase. It may also comprise excipients which are suitable for use in an injectable preparation. The preparation may be in dry form and subsequently reconstituted, with e.g. buffered saline, and the like, or it may be in liquid form, either as a suspension or solution. Suitable excipient materials may include solvents and co-solvents, such as ethanol, glycerin, PEGs, oils, and the like; solubilizing, wetting, suspending, emulsifying or thickening agents, such as carboxymethylcellulose, hydrolyzed gelatin, pluronics, polysorbates, and the like; chelating agents, such as calcium EDTA, DTPA, and the like; antioxidants and reducing agents, such as BHT, ascorbic acid, sodium metabisulphite, and the like; antimicrobial preservatives, such as benzyl alcohol, phenol, parabens, and the like; buffers and pH adjusting agents, such as tromethamine, sodium phosphate, sodium acetate, sodium hydroxide, and the like; bulking agents, protectants, and tonicity adjustors, such as alanine, albumin, dextran, lactose, sorbitol, sodium chloride, histidine, and the like; special additives, such as simethicone as anti-foaming agent, trehalose for reduction of protein aggregation.
 The composition may be used in any application in which plasma-derived or recombinant fibrinogen is used. The main applications are hemostasis and to seal tissue. In one embodiment of the invention, the composition is a pharmaceutical composition. The pharmaceutical composition comprises a fibrinogen preparation according to the invention and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to a vehicle, auxiliary agent, adjuvant, diluent, excipient or carrier with which the fibrinogen preparation of the invention is administered. Examples of pharmaceutically acceptable carriers include, without limitation, water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof. Suitable pharmaceutically acceptable carriers and formulations are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995) and "Remington: The Science And Practice Of Pharmacy" by Alfonso R. Gennaro (Lippincott Williams & Wilkins, 2005).
 The pharmaceutical composition may be applied topically and may include carriers which are water-soluble, water-absorbent, water-insoluble or water-swellable. Suitable materials include saccharides such as mono-and di-saccharides, including lactose, mannitol and trehalose, or dextran and dextran polymers, like e.g. Sephadex, which are available in different particle sizes, starches, pullulan derivatives, hyaluronic acid esters, and the like. Cellulose products such as microcrystalline cellulose (Avicel range), methylcellulose, carboxymethyl cellulose, microfine cellulose or hydroxy propyl cellulose, and other materials such as cross-linked polyvinyl pyrrolidone (PVP), may be used singly or in admixture. Also, suitable carriers include polyethylene glycol (PEG), preferably having a molecular weight of about 1000; polyvinylpyrrolidone (PVP), preferably having an average molecular weight of about 50,000; poly(acrylic acid), PVA, poly(methylvinylether co-maleic anhydride), poly(ethyleneoxide), and dextran, typically having an average molecular weight of about 40,000.
 Tablet disintegrants may be included. These materials will absorb moisture from the wound, expand rapidly and thereby enhance the wettability of the hemostatic components of the powder blend. Suitable examples include sodium starch glycolate (Explotab® or Primojel®) which has an average particle size in the range of 35-55 μm. About 25% of the glucose units are carboxymethylated; cross-linked polyvinyl pyrrolidone (Polyplasdone®); alginates and alginic acid; cross-linked sodium carboxymethylcellulose (Ac-Di-Sol). Gums and gelling agents that can be used include, for example, tragacanth, karaya gum, soluble starch, gelatin, pectin, guar gum and gellan gum.
 A particularly preferred additive is Emdex®, i.e. a hydrated form of dextrates (spray crystallized dextrose containing small amounts of starch oligosaccharides). It is a highly refined product composed of white, free-flowing, spray-crystallized macroporous spheres with a median particle size of 190-220 μm.
 A most preferred additive is NON-PAREIL SEEDS®: (Sugar Spheres). These are used in multiple drug units for improved content uniformity, consistent and controlled drug release and high drug stability, size ranges from 200 to 2000 mm.
 The pharmaceutically acceptable carrier may comprise an effervescent couple. The gas produced following an effervescent reaction can expand the fibrin sealant into a `foam` and/or increase wettability of the powders comprising the fibrin sealant. As the powders are applied to a wound, the effervescent components dissolve, react and liberate, say, carbon dioxide, thereby increasing the wettability of the hemostatic components and thus enhancing time to clot formation. The fibrin sealant will appear as a stable foam once fully reacted and the clot has formed.
 The effervescent couple typically comprises citric acid or sodium hydrogen citrate and sodium bicarbonate, but other physiologically acceptable acid/alkaline or alkaline earth metal carbonate mixtures may be used, for example tartaric, adipic, fumaric or malic acids, and sodium, potassium or calcium (bi)carbonates or sodium glycine carbonate.
 In general it has been found that preferred taste characteristics are exhibited when the relative proportions of the components of the effervescent couple on a chemical molecular equivalent basis are in the range of 4:3 to 1:3, more preferably about 2:3, expressed as the ratio of molecular equivalent of the acidic component to the basic component. In terms of a preferred combination of citric acid and sodium bicarbonate these values represent on a weight basis, a range from 1:1 to 0.3:1, preferably 0.5:1 expressed as the ratio of acidic to basic component.
 The pharmaceutical composition according to the invention is suitable for facilitating tissue adherence, improving wound healing or for intravenous administration.
 In one embodiment, the pharmaceutical composition according to the invention is a fibrin sealant. The fibrin sealant according to the invention typically comprises thrombin. The sealant may be in any convenient form, be it dry or liquid. A suitable example of a dry sealant in which the fibrinogen preparation according to the invention may be used is Fibrocaps® powder sealant, which is described in WO97/44015 and which is based on micro-particles of fibrinogen and thrombin. The separate components are prepared by spray-drying, fibrinogen with trehalose and thrombin with trehalose. Each powder component has a predominant particle size of approximately up to 50 μm in diameter. The Fibrocaps® fibrin sealant, which is a blend of these components, has been demonstrated to be an easy-to-use, stable and efficacious topical haemostat. The product can be used immediately, without reconstitution and is useful in wound therapy, in surgical repair and as an extravascular stent. On contact with aqueous fluid such as blood, the exposed active thrombin converts the exposed fibrinogen into insoluble fibrin polymers. The fibrinogen preparation according to the invention would give further improved blood clotting properties.
 The composition of the invention may be applied to wounds, sutures, incisions and other openings where bleeding may occur. A wound includes damage to any tissue in a living organism. The tissue may be an internal (e.g. organ) or external tissue (e.g. eye or skin), and may be a hard tissue (e.g. bone) or a soft tissue (e.g, liver or spleen). The wound may have been caused by any agent, including infection, surgical intervention, burn or trauma. The composition of the invention may be used for surgical interventions such as in the gastrointestinal system, e.g. the oesophagus, stomach, small intestine, large intestine, rectum, on parenchymal organs such as the liver, pancreas, spleen, lungs, kidney, adrenal glands, lymph and thyroid glands; surgical interventions in the ear, nose and throat area (ENT) including dental surgery, cardiovascular surgery, aesthetic surgery, neurological surgery, lymphatic, biliary, and cerebrospinal (CSF) fistulae, air leakages during thoracic and pulmonary surgery, thoracic surgery including surgery of the trachea, bronchi and lungs, gynaecological, vascular, urological, bone (e.g. spongiosa resection), and emergency surgery.
 As an extravascular stent or support, the composition of the invention may be applied to the outside of a segment or the whole of a vein graft. A suitable extravascular stent composition is a composition of a fibrinogen preparation according to the invention and thrombin. The composition may be in any suitable for, be it liquid or dry. In one embodiment, a dry powder composition, for instance one as described above, is used as an extravascular stent. The dry powder composition polymerizes in the limited amounts of bodily fluids which are naturally present at the outside of the vessel wall, thus forming an extravascular stent.
 A vein coated with an extravascular stent composition according to the present invention is also part of the invention. The vein may be any kind of vein which needs to be protected from overextension or which needs support, for instance a varicose vein. In a preferred embodiment, the vein is a venous graft. In one embodiment, the composition is applied before the venous graft is introduced in the human or animal body. In another embodiment, the composition is applied after the venous graft has been introduced in the human or animal body.
 In yet another aspect, the present invention relates to the use of a fibrinogen preparation according to the invention as a medicament. It can be used for the preparation of a medicament for the treatment of acute bleeding episodes, hyperfibrinolysis, fibrinogen deficiency, be it acquired or congenital, or other bleeding disorders.
 In one embodiment, a composition according to the invention is used in a method for reducing bleeding at a hemorrhaging site. Preferably, a hemostatically effective amount of the composition according to the invention is used. When used as a topical haemostat, a time to hemostasis (TTH) of about 10 minutes or less, about 5 minutes or less, or about 3 minutes or less may be achieved. In the present context, TTH is the time it takes to stop a bleeding. If a pressure sheet is used, measurement of TTH typically starts when the pressure sheet is applied to the bleeding site and runs until bleeding stops by visualization of the dressing and/or no bleeding through or around the dressing is observed.
 In yet another aspect, the present invention relates to a method for preparing a fibrinogen preparation according to the invention. Such preparation may be prepared in any suitable way, using techniques available in the art. In order to produce α-ext Fib in an economically feasible way, high expression levels of intact, functional fibrinogen are required and therefore recombinant production is preferred. In the context of the present invention, fibrinogen or a fibrinogen chain is `in intact form` when the amino acid sequence contains all the amino acids which were encoded for by the nucleotide sequence, optionally without the amino acids which are removed during normal cell (secretion) processing. Therefore, alpha-ext chains having 866 or 847 amino acids are examples of an alpha chain in intact form.
 Recombinant production of fibrinogen has many advantages over the use of plasma derived materials. These include its preferred safety profile, the possibility to make pure homogeneous preparations of variants free of any other blood born contaminants and the unlimited supply. In addition, for specific applications (e.g. use of fibrinogen as an intravenous hemostat) proper post-translational modifications (e.g. glycosylation) are required. Therefore, expression in eukaryotic, in particular mammalian systems, more particular in human systems, is preferred.
 In a preferred embodiment, a fibrinogen preparation according to the invention is prepared by a method which comprises the steps of:  providing an expression vector comprising a nucleic acid sequence, which nucleic acid sequence encodes an alpha extended polypeptide chain of fibrinogen;  transforming a eukaryotic cell with the expression vector;  maintaining the transformed eukaryotic cell under such conditions which allow for the expression of the nucleic acid sequence encoding the alpha extended polypeptide chain of fibrinogen.
 Expression vectors for eukaryotic hosts are known in the art and any of the vectors conventionally used for expression in eukaryotic cells may be used. An expression vector typically contains a promoter operably linked to the nucleic acid sequence to be expressed and ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains and enhancers. In one embodiment, an expression vector for expression in mammalian cells, such as for CHO or PER.C6® cells is used. Such vectors are known in the art and suitable examples include pcDNA3.1 plasmids, GATEWAY (Invitrogen), pCMV/Bsd (Invitrogen), pFN vectors (Promega) and numerous other vector systems.
 In the present context, `an alpha extended polypeptide chain of fibrinogen` may refer to a fibrinogen alpha chain of 866 amino acids with a signal sequence or to one without a signal sequence and to any variants thereof which have arisen through genetic polymorphisms or differences in glycosylation and phosphorylation. A suitable example of an alpha extended chain amino acid sequence is given in SEQ ID No. 1. The terms `alpha chain` and `Aα chain` are used interchangeably in the context of the present invention.
 The skilled person will understand that the eukaryotic cell must also contain nucleic acid sequences which encode the beta and gamma chain of fibrinogen to be able to produce a fibrinogen molecule. Recombinant production of fibrinogen from alpha, beta and gamma chains have been described before, see for instance PCT/EP2009/058754, U.S. Pat. No. 6,037,457 or WO 95/023868.
 In the context of the present invention, the terms `beta chain` and `Bβ chain` are used interchangeably. They may refer to both wild type and variants of the beta chain, with or without signal sequence. A suitable example of a fibrinogen beta chain amino acid sequence is given in SEQ ID No. 2.
 In the context of the present invention, the term `gamma chain` and `γ chain` are used interchangeably. They may refer to both wild type and variants of the gamma chain, with or without signal sequence. Suitable examples of a fibrinogen gamma chain amino acid sequence are given in SEQ ID No. 3 and 4.
 Preferably, the nucleic acid sequences encoding a fibrinogen chain are optimized. An optimized nucleic acid sequence allows for the efficient expression of recombinant fibrinogen in intact form. Preferably, they are optimized for expression in a eukaryotic cell culture system, such as for expression in a COS cell, BHK cell, NS0 cell, Sp2/0 cell, CHO cell, a PER.C6 cell, a HEK293 cell or insect cell culture system. More preferably, they are optimized for expression in a mammalian cell culture system. Most preferably, the nucleic acid sequences are optimized for expression in a human cell culture system, such as for a PER.C6 cell or a HEK293 cell culture system. The nucleotide sequence which is optimized may be DNA or RNA. Preferably, it is cDNA.
 An optimized nucleotide sequence encoding a fibrinogen alpha extended, beta or gamma chain shows at least 70% identity to its respective non-optimized counterpart. In one embodiment, an optimized nucleotide sequence encoding a fibrinogen α-ext Fib, Bβ and γ chain shows 70-80% identity to its respective non-optimized sequences. Preferably, the optimized nucleotide sequences encoding a fibrinogen alpha extended, beta or gamma chain contain no cis-acting sites, such as splice sites and poly(A) signals.
 An optimized nucleotide sequence which is used in the method according to the invention and which encodes a fibrinogen alpha extended chain contains no 39 basepair direct repeat sequences which are normally present in the gene encoding the alpha chain of human fibrinogen. The repeating sequence must be changed without changing the encoded protein sequence.
 In a preferred embodiment, an optimised nucleotide sequence according to SEQ ID NO. 5 or the part of this sequence without the signal (nucleotides 60-2598) is used for expressing the alpha extended chain.
 In another preferred embodiment, an optimised nucleotide sequence according to SEQ ID NO. 6 or the part of this sequence without the signal sequence (nucleotides 93-1473) is used for expressing the beta chain.
 In another preferred embodiment, an optimised nucleotide sequence according to SEQ ID NO. 7 or 8 or the part of these sequences without a signal sequence (nucleotides 51-1311 of SEQ ID NO. 7 and nucleotides 51-1359 of SEQ ID NO. 8) is used for expressing the gamma chain.
 Nucleic acid sequences according to the invention may be encoding any type of fibrinogen chains. Preferably they are encoding mammalian fibrinogen chains, more preferably they are encoding primate fibrinogen chains, most preferably they are encoding human fibrinogen chains. Also combinations are possible, such as for example one or two mammalian fibrinogen chains combined with two or one rodent fibrinogen chains. Recombinant expression according to the method of the present invention allows for expression levels of Fib420 which are similar to those of recombinant HMW Fib.
BRIEF DESCRIPTION OF THE DRAWINGS
 FIG. 1 Western blot analysis. Lane 1 is a control containing plasma derived wild-type fibrinogen (FIB3, Enzyme Research Laboratories). Lane 2 contains culture supernatant of clone W115, a PER.C6 clones expressing α-ext rhFib. The molecular weight marker (MW) is indicated at the left.
 FIG. 2 ROTEM analysis. Clotting time, clot formation time and clot firmness were determined by ROTEM analysis. The left panel displays the fibrinogen preparations in buffer, the right panel displays plasma mixed 1:1 with the fibrinogen preparations:
 2A: Plasma derived fibrinogen (Haemocomplettan, CSL Behring, Marburg, Germany) in buffer;
 2B: PER.C6 derived recombinant HMW fibrinogen (α-625) in buffer;
 2C: PER.C6 derived rhFib-ext fibrinogen (α-847) in buffer;
 2D: Plasma derived fibrinogen (Haemocomplettan, CSL Behring, Marburg, Germany) mixed with plasma;
 2E: PER.C6 derived recombinant HMW fibrinogen (α-625) mixed with plasma;
 2F: PER.C6 derived rhFib-ext fibrinogen (α-847) mixed with plasma.
 FIG. 3 The picture shows a Coomassie stained protein gel loaded with material from plasmin degraded plasma derived material (ERL FIB3; upper panel, and α-ext Fib (lower panel). The conditions are indicated on top of the gel; the time of incubation (0, 1, 5, 30, 60 and 120 minutes and o/n (overnight)) is shown as well. At the right of the gel the MW marker is displayed.
Preparation of Optimized cDNA Constructs
 cDNAs coding for human fibrinogen polypeptide chains of α-ext Fib (Fib420), Bβ and γ, were synthesized in codon optimized format by GeneArt (Regensburg, Germany): (i) cis-acting sites (splice sites, poly(A) signals) were removed; (ii) repeat sequence of Aα chain was modified; (iii) GC content was increased for prolonged mRNA half life; (iv) Codon usage was adapted to CHO (codon adaption index--CAI→0.95).
 The codon optimized cDNAs for α-ext Fib (SEQ ID NO.5), Bβ (SEQ ID No. 6), and γ (SEQ ID NO. 6) chain were subcloned in pcDNA3.1 deriviates. Aα-extended (Fib420) in pcDNA3.1(+)neo, Bβ chains in pcDNA3.1(+)hygro and γ chain in pcDNA3.1(-)hygro (Invitrogen, Carlsbad, USA).
PER.C6 Cell Lines Expressing Recombinant Human α-ext Fib
 The generation of PER.C6 cell lines producing recombinant human fibrinogen is similar as described before in PCT/EP2009/058754. In summary, cells were cultured in suspension in MAb medium and transfected using the AMAXA nucleofection device (program A-27) and using Nucleofector kit T with three vectors encoding the three different chains of the human fibrinogen protein (Aα-ext, Bβ, and γ chain) and containing the optimized cDNA chains (SEQ ID no.5, SEQ ID no. 6, and SEQ ID no.7, respectively).
 After transfection and plating in 96-well plates, 325 clones were transferred and screened in 48-well plates. At the end of the expansion path, 24 clones were transferred to shaker flasks, of which 8 were selected for stability and expression analysis in continued batch culture testing.
 Yields in batch culture were similar to yields obtained with cell lines that express the Aα-chain in 610 or 625 amino acid format, indicating that the extension of the Aα-chain does not impair expression levels. This was not expected on forehand, as plasma derived fibrinogen only contains 1-3% of extended Aα-chain as compared to 610/625 Aα-chain containing fibrinogen. Protein analysis using SDS-PAGE and Western blotting analysis indicate that the recombinant fibrinogen is produced in intact format, with the α-chain having the expected size (FIG. 1).
Purification of α-Extended Fibrinogen from PER.C6 Cell-Culture Medium
 Recombinant human α-extended fibrinogen was purified from cell culture supernatant according to standard methods. Briefly, (NH4)2SO4 was added to the culture supernatant to 40% saturation and the precipitate was collected by centrifugation. Subsequently, the precipitate was dissolved in TMAE loading buffer (5 mM Tris-HCl pH 8.5, 0.01% Tween-20) followed by dialysis to the same buffer. The protein solution was then loaded on a Fractogel EMD TMAE (m) 40-90 μm (3 ml) (Merck KGaA, Darmstadt, Germany) Ion Exchange Column. Recombinant human α-extended fibrinogen was subsequently eluted using a continuous salt gradient of 0-1 M NaCl in 20 column volumes. Recombinant human fibrinogen in the peak fractions was precipitated again by adding (NH4)2SO4 to 40% saturation and collected by centrifugation. Finally the material was dissolved in TBS (50mM Tris-HCl, pH7.4, 100mM NaCl) and dialysed against TBS to remove any remaining (NH4)2SO4.
 Clotting time (CT), clot formation time (CFT) and clot firmness of α-ext rhFib and plasma derived fibrinogen were measured using ROTEM analysis. ROTEM® (Pentapharm GmbH, Munich, Germany) stands for ROtation ThromboElastoMetry. The technique utilizes a rotating axis submerged in a (blood) sample in a disposable cuvette. Changes in elasticity under different clotting conditions result in a change in the rotation of the axis, which is visualized in a thromboelastogram, reflecting mechanical clot parameters (see e.g. Luddington R. J. (2005) Clin Lab Haematol. 2005 27(2):81).
 Pooled normal (citrate) plasma was mixed 1:1 with plasma-derived fibrinogen (Haemocomplettan, CSL Behring GmbH, Marburg, Germany) or PER.C6 fibrinogen (HMW rhFib or α-ext rhFib) all at 2 mg/ml in TBST (TBS+0.001% Tween-20). Also the fibrinogen preparations (at 2 mg/ml in TBST) were used directly. CaCl2 was added to a final concentration of 17 mM (measurement in plasma) or 1.7 mM (measurement in buffer). To start clotting, α-thrombin was added to a final concentration of 1 IU/ml. ROTEM® analysis graphs for the fibrinogen preparations in buffer or mixed 1:1 with plasma are shown in FIG. 2. A10, A20, CFT and MCF values (mm) are shown in Table 1. A10 and A20 reflect the firmness of the clot at 10 and 20 minutes post α-thrombin addition. CFT reflects the time from initiaton of clotting until a clot firmness of 20 mm is detected. MCF reflects maximum clot firmness.
 The results indicate that clotting of α-ext rhFib in buffer results in a stronger clot (A10=12 mm) than what is observed for HMW rhFib (A10=4 mm). Plasma derived fibrinogen has an A10 of 15 mm; this stronger clot formation in buffer can be explained by the activity of co-purified blood derived FXIII which will (partially) cross-link the fibrin monomers. In purified recombinant fibrinogen, no FXIII is present and hence no cross-linking can occur. This can be compensated for by running the experiment in diluted plasma (which contains FXIII). In this case, stronger clots are formed. Interestingly, in this situation, α-ext rhFib forms a stronger clot and forms it faster than HMW rhFib or plasma derived Fib, with A20 values of 21, 18 and 17 mm, resp.
TABLE-US-00001 TABLE 1 CT A10 A20 MCF CFT Sample (sec) (mm) (mm) (mm) (sec) Plasma-derived 47 10 10 10 -- fbg in buffer Plasma-derived 50 15 17 18 4696 fbg in plasma PER.C6 α-625 fbg 70 4 4 4 -- in buffer PER.C6 α-625 fbg 61 17 18 20 3368 in plasma PER.C6 α-847 fbg 49 12 12 12 -- in buffer PER.C6 α-847 fbg 62 18 21 21 950 in plasma
Plasmin Digestion of α-ext rhFib and Plasma Derived Fibrinogen
 Fibrinogenolysis of purified recombinant human α-ext rhFib was tested by incubation with plasmin. Briefly, fibrinogen was diluted in TBST (50 mM Tris-HCl, pH7.4, 100 mM NaCl, 0.01% Tween-20), CaCl2 or EDTA was added (5 mM final concentration) and plasmin was added (10 nM final concentration), followed by an incubation at 37° C. At several points in time samples were taken and mixed immediately with SDS-PAGE sample buffer (NuPAGE LDS sample buffer, Invitrogen, cat #NP0007). Samples were then subjected to size separation on a non-reduced SDS-PAGE gel (NuPAGE 3-8% Tris-Acetate, Invitrogen, cat #WG1602). Protein was visualized by Coomassie staining (SimplyBlue SafeStain, Invitrogen, cat #LC6060).
 The results, as shown in FIG. 4, indicate that the plasmin mediated digestion of α-ext rhFib is significantly slower than for plasma derived fibrinogen. For example, in the presence of Ca2+, after 60 minutes all of the plasma derived fibrinogen is degraded down to fragment D and E species. For α-ext rhFib this takes more than 120 minutes and only the overnight incubation shows substantial amounts of fragment E generation, which are already present in the digest of the plasma derived fibrinogen after 30 minutes.
81866PRTHomo sapiens 1Met Phe Ser Met Arg Ile Val Cys Leu Val Leu Ser Val Val Gly Thr 1 5 10 15 Ala Trp Thr Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly 20 25 30 Gly Val Arg Gly Pro Arg Val Val Glu Arg His Gln Ser Ala Cys Lys 35 40 45 Asp Ser Asp Trp Pro Phe Cys Ser Asp Glu Asp Trp Asn Tyr Lys Cys 50 55 60 Pro Ser Gly Cys Arg Met Lys Gly Leu Ile Asp Glu Val Asn Gln Asp 65 70 75 80 Phe Thr Asn Arg Ile Asn Lys Leu Lys Asn Ser Leu Phe Glu Tyr Gln 85 90 95 Lys Asn Asn Lys Asp Ser His Ser Leu Thr Thr Asn Ile Met Glu Ile 100 105 110 Leu Arg Gly Asp Phe Ser Ser Ala Asn Asn Arg Asp Asn Thr Tyr Asn 115 120 125 Arg Val Ser Glu Asp Leu Arg Ser Arg Ile Glu Val Leu Lys Arg Lys 130 135 140 Val Ile Glu Lys Val Gln His Ile Gln Leu Leu Gln Lys Asn Val Arg 145 150 155 160 Ala Gln Leu Val Asp Met Lys Arg Leu Glu Val Asp Ile Asp Ile Lys 165 170 175 Ile Arg Ser Cys Arg Gly Ser Cys Ser Arg Ala Leu Ala Arg Glu Val 180 185 190 Asp Leu Lys Asp Tyr Glu Asp Gln Gln Lys Gln Leu Glu Gln Val Ile 195 200 205 Ala Lys Asp Leu Leu Pro Ser Arg Asp Arg Gln His Leu Pro Leu Ile 210 215 220 Lys Met Lys Pro Val Pro Asp Leu Val Pro Gly Asn Phe Lys Ser Gln 225 230 235 240 Leu Gln Lys Val Pro Pro Glu Trp Lys Ala Leu Thr Asp Met Pro Gln 245 250 255 Met Arg Met Glu Leu Glu Arg Pro Gly Gly Asn Glu Ile Thr Arg Gly 260 265 270 Gly Ser Thr Ser Tyr Gly Thr Gly Ser Glu Thr Glu Ser Pro Arg Asn 275 280 285 Pro Ser Ser Ala Gly Ser Trp Asn Ser Gly Ser Ser Gly Pro Gly Ser 290 295 300 Thr Gly Asn Arg Asn Pro Gly Ser Ser Gly Thr Gly Gly Thr Ala Thr 305 310 315 320 Trp Lys Pro Gly Ser Ser Gly Pro Gly Ser Thr Gly Ser Trp Asn Ser 325 330 335 Gly Ser Ser Gly Thr Gly Ser Thr Gly Asn Gln Asn Pro Gly Ser Pro 340 345 350 Arg Pro Gly Ser Thr Gly Thr Trp Asn Pro Gly Ser Ser Glu Arg Gly 355 360 365 Ser Ala Gly His Trp Thr Ser Glu Ser Ser Val Ser Gly Ser Thr Gly 370 375 380 Gln Trp His Ser Glu Ser Gly Ser Phe Arg Pro Asp Ser Pro Gly Ser 385 390 395 400 Gly Asn Ala Arg Pro Asn Asn Pro Asp Trp Gly Thr Phe Glu Glu Val 405 410 415 Ser Gly Asn Val Ser Pro Gly Thr Arg Arg Glu Tyr His Thr Glu Lys 420 425 430 Leu Val Thr Ser Lys Gly Asp Lys Glu Leu Arg Thr Gly Lys Glu Lys 435 440 445 Val Thr Ser Gly Ser Thr Thr Thr Thr Arg Arg Ser Cys Ser Lys Thr 450 455 460 Val Thr Lys Thr Val Ile Gly Pro Asp Gly His Lys Glu Val Thr Lys 465 470 475 480 Glu Val Val Thr Ser Glu Asp Gly Ser Asp Cys Pro Glu Ala Met Asp 485 490 495 Leu Gly Thr Leu Ser Gly Ile Gly Thr Leu Asp Gly Phe Arg His Arg 500 505 510 His Pro Asp Glu Ala Ala Phe Phe Asp Thr Ala Ser Thr Gly Lys Thr 515 520 525 Phe Pro Gly Phe Phe Ser Pro Met Leu Gly Glu Phe Val Ser Glu Thr 530 535 540 Glu Ser Arg Gly Ser Glu Ser Gly Ile Phe Thr Asn Thr Lys Glu Ser 545 550 555 560 Ser Ser His His Pro Gly Ile Ala Glu Phe Pro Ser Arg Gly Lys Ser 565 570 575 Ser Ser Tyr Ser Lys Gln Phe Thr Ser Ser Thr Ser Tyr Asn Arg Gly 580 585 590 Asp Ser Thr Phe Glu Ser Lys Ser Tyr Lys Met Ala Asp Glu Ala Gly 595 600 605 Ser Glu Ala Asp His Glu Gly Thr His Ser Thr Lys Arg Gly His Ala 610 615 620 Lys Ser Arg Pro Val Arg Asp Cys Asp Asp Val Leu Gln Thr His Pro 625 630 635 640 Ser Gly Thr Gln Ser Gly Ile Phe Asn Ile Lys Leu Pro Gly Ser Ser 645 650 655 Lys Ile Phe Ser Val Tyr Cys Asp Gln Glu Thr Ser Leu Gly Gly Trp 660 665 670 Leu Leu Ile Gln Gln Arg Met Asp Gly Ser Leu Asn Phe Asn Arg Thr 675 680 685 Trp Gln Asp Tyr Lys Arg Gly Phe Gly Ser Leu Asn Asp Glu Gly Glu 690 695 700 Gly Glu Phe Trp Leu Gly Asn Asp Tyr Leu His Leu Leu Thr Gln Arg 705 710 715 720 Gly Ser Val Leu Arg Val Glu Leu Glu Asp Trp Ala Gly Asn Glu Ala 725 730 735 Tyr Ala Glu Tyr His Phe Arg Val Gly Ser Glu Ala Glu Gly Tyr Ala 740 745 750 Leu Gln Val Ser Ser Tyr Glu Gly Thr Ala Gly Asp Ala Leu Ile Glu 755 760 765 Gly Ser Val Glu Glu Gly Ala Glu Tyr Thr Ser His Asn Asn Met Gln 770 775 780 Phe Ser Thr Phe Asp Arg Asp Ala Asp Gln Trp Glu Glu Asn Cys Ala 785 790 795 800 Glu Val Tyr Gly Gly Gly Trp Trp Tyr Asn Asn Cys Gln Ala Ala Asn 805 810 815 Leu Asn Gly Ile Tyr Tyr Pro Gly Gly Ser Tyr Asp Pro Arg Asn Asn 820 825 830 Ser Pro Tyr Glu Ile Glu Asn Gly Val Val Trp Val Ser Phe Arg Gly 835 840 845 Ala Asp Tyr Ser Leu Arg Ala Val Arg Met Lys Ile Arg Pro Leu Val 850 855 860 Thr Gln 865 2491PRTHomo sapiens 2Met Lys Arg Met Val Ser Trp Ser Phe His Lys Leu Lys Thr Met Lys 1 5 10 15 His Leu Leu Leu Leu Leu Leu Cys Val Phe Leu Val Lys Ser Gln Gly 20 25 30 Val Asn Asp Asn Glu Glu Gly Phe Phe Ser Ala Arg Gly His Arg Pro 35 40 45 Leu Asp Lys Lys Arg Glu Glu Ala Pro Ser Leu Arg Pro Ala Pro Pro 50 55 60 Pro Ile Ser Gly Gly Gly Tyr Arg Ala Arg Pro Ala Lys Ala Ala Ala 65 70 75 80 Thr Gln Lys Lys Val Glu Arg Lys Ala Pro Asp Ala Gly Gly Cys Leu 85 90 95 His Ala Asp Pro Asp Leu Gly Val Leu Cys Pro Thr Gly Cys Gln Leu 100 105 110 Gln Glu Ala Leu Leu Gln Gln Glu Arg Pro Ile Arg Asn Ser Val Asp 115 120 125 Glu Leu Asn Asn Asn Val Glu Ala Val Ser Gln Thr Ser Ser Ser Ser 130 135 140 Phe Gln Tyr Met Tyr Leu Leu Lys Asp Leu Trp Gln Lys Arg Gln Lys 145 150 155 160 Gln Val Lys Asp Asn Glu Asn Val Val Asn Glu Tyr Ser Ser Glu Leu 165 170 175 Glu Lys His Gln Leu Tyr Ile Asp Glu Thr Val Asn Ser Asn Ile Pro 180 185 190 Thr Asn Leu Arg Val Leu Arg Ser Ile Leu Glu Asn Leu Arg Ser Lys 195 200 205 Ile Gln Lys Leu Glu Ser Asp Val Ser Ala Gln Met Glu Tyr Cys Arg 210 215 220 Thr Pro Cys Thr Val Ser Cys Asn Ile Pro Val Val Ser Gly Lys Glu 225 230 235 240 Cys Glu Glu Ile Ile Arg Lys Gly Gly Glu Thr Ser Glu Met Tyr Leu 245 250 255 Ile Gln Pro Asp Ser Ser Val Lys Pro Tyr Arg Val Tyr Cys Asp Met 260 265 270 Asn Thr Glu Asn Gly Gly Trp Thr Val Ile Gln Asn Arg Gln Asp Gly 275 280 285 Ser Val Asp Phe Gly Arg Lys Trp Asp Pro Tyr Lys Gln Gly Phe Gly 290 295 300 Asn Val Ala Thr Asn Thr Asp Gly Lys Asn Tyr Cys Gly Leu Pro Gly 305 310 315 320 Glu Tyr Trp Leu Gly Asn Asp Lys Ile Ser Gln Leu Thr Arg Met Gly 325 330 335 Pro Thr Glu Leu Leu Ile Glu Met Glu Asp Trp Lys Gly Asp Lys Val 340 345 350 Lys Ala His Tyr Gly Gly Phe Thr Val Gln Asn Glu Ala Asn Lys Tyr 355 360 365 Gln Ile Ser Val Asn Lys Tyr Arg Gly Thr Ala Gly Asn Ala Leu Met 370 375 380 Asp Gly Ala Ser Gln Leu Met Gly Glu Asn Arg Thr Met Thr Ile His 385 390 395 400 Asn Gly Met Phe Phe Ser Thr Tyr Asp Arg Asp Asn Asp Gly Trp Leu 405 410 415 Thr Ser Asp Pro Arg Lys Gln Cys Ser Lys Glu Asp Gly Gly Gly Trp 420 425 430 Trp Tyr Asn Arg Cys His Ala Ala Asn Pro Asn Gly Arg Tyr Tyr Trp 435 440 445 Gly Gly Gln Tyr Thr Trp Asp Met Ala Lys His Gly Thr Asp Asp Gly 450 455 460 Val Val Trp Met Asn Trp Lys Gly Ser Trp Tyr Ser Met Arg Lys Met 465 470 475 480 Ser Met Lys Ile Arg Pro Phe Phe Pro Gln Gln 485 490 3437PRTHomo sapiens 3Met Ser Trp Ser Leu His Pro Arg Asn Leu Ile Leu Tyr Phe Tyr Ala 1 5 10 15 Leu Leu Phe Leu Ser Ser Thr Cys Val Ala Tyr Val Ala Thr Arg Asp 20 25 30 Asn Cys Cys Ile Leu Asp Glu Arg Phe Gly Ser Tyr Cys Pro Thr Thr 35 40 45 Cys Gly Ile Ala Asp Phe Leu Ser Thr Tyr Gln Thr Lys Val Asp Lys 50 55 60 Asp Leu Gln Ser Leu Glu Asp Ile Leu His Gln Val Glu Asn Lys Thr 65 70 75 80 Ser Glu Val Lys Gln Leu Ile Lys Ala Ile Gln Leu Thr Tyr Asn Pro 85 90 95 Asp Glu Ser Ser Lys Pro Asn Met Ile Asp Ala Ala Thr Leu Lys Ser 100 105 110 Arg Lys Met Leu Glu Glu Ile Met Lys Tyr Glu Ala Ser Ile Leu Thr 115 120 125 His Asp Ser Ser Ile Arg Tyr Leu Gln Glu Ile Tyr Asn Ser Asn Asn 130 135 140 Gln Lys Ile Val Asn Leu Lys Glu Lys Val Ala Gln Leu Glu Ala Gln 145 150 155 160 Cys Gln Glu Pro Cys Lys Asp Thr Val Gln Ile His Asp Ile Thr Gly 165 170 175 Lys Asp Cys Gln Asp Ile Ala Asn Lys Gly Ala Lys Gln Ser Gly Leu 180 185 190 Tyr Phe Ile Lys Pro Leu Lys Ala Asn Gln Gln Phe Leu Val Tyr Cys 195 200 205 Glu Ile Asp Gly Ser Gly Asn Gly Trp Thr Val Phe Gln Lys Arg Leu 210 215 220 Asp Gly Ser Val Asp Phe Lys Lys Asn Trp Ile Gln Tyr Lys Glu Gly 225 230 235 240 Phe Gly His Leu Ser Pro Thr Gly Thr Thr Glu Phe Trp Leu Gly Asn 245 250 255 Glu Lys Ile His Leu Ile Ser Thr Gln Ser Ala Ile Pro Tyr Ala Leu 260 265 270 Arg Val Glu Leu Glu Asp Trp Asn Gly Arg Thr Ser Thr Ala Asp Tyr 275 280 285 Ala Met Phe Lys Val Gly Pro Glu Ala Asp Lys Tyr Arg Leu Thr Tyr 290 295 300 Ala Tyr Phe Ala Gly Gly Asp Ala Gly Asp Ala Phe Asp Gly Phe Asp 305 310 315 320 Phe Gly Asp Asp Pro Ser Asp Lys Phe Phe Thr Ser His Asn Gly Met 325 330 335 Gln Phe Ser Thr Trp Asp Asn Asp Asn Asp Lys Phe Glu Gly Asn Cys 340 345 350 Ala Glu Gln Asp Gly Ser Gly Trp Trp Met Asn Lys Cys His Ala Gly 355 360 365 His Leu Asn Gly Val Tyr Tyr Gln Gly Gly Thr Tyr Ser Lys Ala Ser 370 375 380 Thr Pro Asn Gly Tyr Asp Asn Gly Ile Ile Trp Ala Thr Trp Lys Thr 385 390 395 400 Arg Trp Tyr Ser Met Lys Lys Thr Thr Met Lys Ile Ile Pro Phe Asn 405 410 415 Arg Leu Thr Ile Gly Glu Gly Gln Gln His His Leu Gly Gly Ala Lys 420 425 430 Gln Ala Gly Asp Val 435 4453PRTHomo sapiens 4Met Ser Trp Ser Leu His Pro Arg Asn Leu Ile Leu Tyr Phe Tyr Ala 1 5 10 15 Leu Leu Phe Leu Ser Ser Thr Cys Val Ala Tyr Val Ala Thr Arg Asp 20 25 30 Asn Cys Cys Ile Leu Asp Glu Arg Phe Gly Ser Tyr Cys Pro Thr Thr 35 40 45 Cys Gly Ile Ala Asp Phe Leu Ser Thr Tyr Gln Thr Lys Val Asp Lys 50 55 60 Asp Leu Gln Ser Leu Glu Asp Ile Leu His Gln Val Glu Asn Lys Thr 65 70 75 80 Ser Glu Val Lys Gln Leu Ile Lys Ala Ile Gln Leu Thr Tyr Asn Pro 85 90 95 Asp Glu Ser Ser Lys Pro Asn Met Ile Asp Ala Ala Thr Leu Lys Ser 100 105 110 Arg Lys Met Leu Glu Glu Ile Met Lys Tyr Glu Ala Ser Ile Leu Thr 115 120 125 His Asp Ser Ser Ile Arg Tyr Leu Gln Glu Ile Tyr Asn Ser Asn Asn 130 135 140 Gln Lys Ile Val Asn Leu Lys Glu Lys Val Ala Gln Leu Glu Ala Gln 145 150 155 160 Cys Gln Glu Pro Cys Lys Asp Thr Val Gln Ile His Asp Ile Thr Gly 165 170 175 Lys Asp Cys Gln Asp Ile Ala Asn Lys Gly Ala Lys Gln Ser Gly Leu 180 185 190 Tyr Phe Ile Lys Pro Leu Lys Ala Asn Gln Gln Phe Leu Val Tyr Cys 195 200 205 Glu Ile Asp Gly Ser Gly Asn Gly Trp Thr Val Phe Gln Lys Arg Leu 210 215 220 Asp Gly Ser Val Asp Phe Lys Lys Asn Trp Ile Gln Tyr Lys Glu Gly 225 230 235 240 Phe Gly His Leu Ser Pro Thr Gly Thr Thr Glu Phe Trp Leu Gly Asn 245 250 255 Glu Lys Ile His Leu Ile Ser Thr Gln Ser Ala Ile Pro Tyr Ala Leu 260 265 270 Arg Val Glu Leu Glu Asp Trp Asn Gly Arg Thr Ser Thr Ala Asp Tyr 275 280 285 Ala Met Phe Lys Val Gly Pro Glu Ala Asp Lys Tyr Arg Leu Thr Tyr 290 295 300 Ala Tyr Phe Ala Gly Gly Asp Ala Gly Asp Ala Phe Asp Gly Phe Asp 305 310 315 320 Phe Gly Asp Asp Pro Ser Asp Lys Phe Phe Thr Ser His Asn Gly Met 325 330 335 Gln Phe Ser Thr Trp Asp Asn Asp Asn Asp Lys Phe Glu Gly Asn Cys 340 345 350 Ala Glu Gln Asp Gly Ser Gly Trp Trp Met Asn Lys Cys His Ala Gly 355 360 365 His Leu Asn Gly Val Tyr Tyr Gln Gly Gly Thr Tyr Ser Lys Ala Ser 370 375 380 Thr Pro Asn Gly Tyr Asp Asn Gly Ile Ile Trp Ala Thr Trp Lys Thr 385 390 395 400 Arg Trp Tyr Ser Met Lys Lys Thr Thr Met Lys Ile Ile Pro Phe Asn 405 410 415 Arg Leu Thr Ile Gly Glu Gly Gln Gln His His Leu Gly Gly Ala Lys 420 425 430 Gln Val Arg Pro Glu His Pro Ala Glu Thr Glu Tyr Asp Ser Leu Tyr 435 440 445 Pro Glu Asp Asp Leu 450 52598DNAHomo sapiens 5atgttcagca tgaggatcgt gtgcctggtg ctgtccgtgg tgggcaccgc ctggaccgcc 60gacagcggcg agggcgactt cctggccgag ggcggtggcg tgaggggccc cagggtggtg 120gagaggcacc agagcgcctg caaggacagc gactggccct tctgcagcga cgaggactgg 180aactacaagt gccccagcgg ctgcaggatg aagggcctga tcgacgaggt gaaccaggac 240ttcaccaaca ggatcaacaa gctgaagaac agcctgttcg agtaccagaa gaacaacaag
300gacagccaca gcctgaccac caacatcatg gaaatcctga ggggcgattt ctctagcgcc 360aacaacaggg acaacaccta caacagggtg tccgaggacc tgaggtccag gatcgaggtg 420ctgaagagga aggtgatcga gaaggtgcag cacatccagc tgctgcagaa gaacgtcagg 480gcccagctgg tcgacatgaa gaggctggaa gtggacatcg acatcaagat caggtcctgc 540aggggcagct gcagccgggc tctggctaga gaggtggacc tgaaggacta cgaggaccag 600cagaaacagc tggaacaggt gatcgccaag gacctgctgc ccagcaggga caggcagcac 660ctgcccctga tcaagatgaa gcccgtgccc gacctggtgc ccggcaactt caagagccag 720ctgcagaaag tgccccccga gtggaaggcc ctgaccgaca tgccccagat gaggatggaa 780ctggaaaggc caggcggcaa cgagatcacc aggggcggca gcaccagcta cggcaccggc 840agcgagaccg agagccccag gaaccccagc agcgccggca gctggaactc cggcagcagc 900ggcccaggct ccaccggcaa caggaacccc ggctccagcg gcaccggcgg cacagccacc 960tggaagcccg gcagctccgg ccctggcagc accggctctt ggaacagcgg cagctctggc 1020accgggagca caggcaacca gaacccaggc agccccaggc ctggctctac cgggacctgg 1080aacccaggct cctccgagag gggctctgcc ggccactgga ccagcgagag cagcgtgagc 1140ggcagcacag gccagtggca cagcgagtcc ggcagcttca ggcccgacag ccccggcagc 1200ggcaacgcca ggcccaacaa ccccgactgg ggcaccttcg aggaagtgag cggcaacgtg 1260agccccggca ccaggcggga gtaccacacc gagaagctgg tgaccagcaa gggcgacaaa 1320gagctgagga ccggcaaaga aaaggtgacc agcggctcta ccaccaccac caggcggagc 1380tgcagcaaga ccgtgaccaa gacagtgatc ggccccgacg gccacaaaga ggtgaccaaa 1440gaagtcgtga ccagcgagga cggcagcgac tgccccgagg ccatggacct gggcaccctg 1500agcggcatcg gcaccctgga cggcttcagg cacaggcacc ccgacgaggc cgccttcttc 1560gacaccgcca gcaccggcaa gaccttcccc ggcttcttca gccccatgct gggcgagttc 1620gtgtccgaga ccgagtcccg cggcagcgag agcggcatct tcaccaacac caaagagtcc 1680agcagccacc atcccggcat cgctgagttc cccagcaggg gcaagagcag ctcctacagc 1740aagcagttca ccagcagcac cagctacaac aggggcgaca gcaccttcga gagcaagagc 1800tacaagatgg ccgacgaggc cggctctgag gccgaccacg agggcaccca cagcaccaag 1860aggggccacg ccaagagcag gcccgtgagg gactgcgacg acgtgctgca gacccacccc 1920agcggcaccc agtctggcat cttcaacatc aagctgcccg gcagcagcaa gatcttcagc 1980gtgtactgcg accaggaaac cagcctgggc ggctggctgc tgatccagca gaggatggac 2040ggcagcctga acttcaacag gacctggcag gactacaaga ggggcttcgg ctccctgaac 2100gacgagggcg agggcgagtt ctggctgggc aacgactacc tgcacctgct gacccagagg 2160ggatctgtcc tgagggtcga gctggaagat tgggccggca acgaggccta cgccgagtac 2220cacttcagag tgggcagcga ggccgagggc tacgctctgc aggtgtccag ctacgagggc 2280acagccggcg acgccctgat cgagggcagc gtggaagagg gcgccgagta caccagccac 2340aacaacatgc agttctccac cttcgacagg gacgccgacc agtgggagga aaactgcgcc 2400gaggtgtacg gcggagggtg gtggtacaac aactgccagg ccgccaacct gaacggcatc 2460tactacccag gcggcagcta cgaccccagg aacaacagcc cctacgagat cgagaacggc 2520gtggtgtggg tgtccttcag aggcgccgac tacagcctga gggccgtgag gatgaagatc 2580aggcccctgg tgacccag 259861473DNAHomo sapiens 6atgaagagga tggtgtcctg gtccttccac aagctgaaaa caatgaagca cctgctcctc 60ctcctcctct gcgtgttcct ggtgaagagc cagggcgtga acgacaacga agagggcttc 120ttcagcgcca gaggacaccg ccccctggac aagaagagag aagaggcccc cagcctgaga 180cccgccccac ccccaatcag cggcggaggg tacagagcca ggcccgccaa ggctgccgcc 240acccagaaga aggtcgaacg gaaggctccc gacgccggag gatgcctgca cgccgacccc 300gacctgggcg tgctgtgccc caccggctgc cagctgcagg aagctctgct ccagcaggaa 360aggcccatca gaaacagcgt ggacgagctg aacaacaacg tggaggccgt gagccagacc 420agcagcagca gcttccagta catgtacctg ctgaaggacc tgtggcagaa gaggcagaag 480caggtcaaag acaacgagaa cgtggtgaac gagtacagca gcgagctgga gaagcaccag 540ctgtacatcg acgagaccgt gaacagcaat atcccaacca acctgagggt gctgagaagc 600atcctggaga acctgaggtc caagatccag aagctggaga gcgacgtcag cgcccagatg 660gagtactgca ggaccccctg caccgtgtcc tgcaacatcc cagtggtgtc cggcaaggaa 720tgcgaggaaa tcatcaggaa gggcggcgag accagcgaga tgtacctgat ccagcccgac 780agcagcgtga agccctacag ggtgtactgc gacatgaaca ccgagaatgg gggctggacc 840gtcatccaga acaggcagga cggcagcgtg gacttcggca ggaagtggga cccctacaag 900cagggcttcg gcaacgtggc caccaacacc gacggcaaga actactgcgg cctgcctggc 960gagtattggc tgggaaacga caagatcagc cagctgacca ggatgggccc aaccgagctg 1020ctgatcgaga tggaggactg gaagggcgac aaggtgaaag cccactacgg cggcttcacc 1080gtgcagaacg aggccaacaa gtaccagatc agcgtgaaca agtacagggg caccgccggc 1140aacgccctga tggacggcgc ctcccagctg atgggcgaga acaggaccat gaccatccac 1200aacggcatgt tcttcagcac ctacgacagg gacaacgacg gctggctgac cagcgacccc 1260agaaagcagt gcagcaagga agatggcgga ggatggtggt acaacaggtg ccacgccgcc 1320aaccccaacg gcaggtacta ctggggcgga cagtacacct gggacatggc caagcacggc 1380accgacgacg gcgtggtgtg gatgaactgg aaggggtcct ggtacagcat gaggaagatg 1440agcatgaaga tcaggccatt ctttccacag cag 147371311DNAHomo sapiens 7atgagctggt ccctgcaccc caggaacctg atcctgtact tctacgccct gctgttcctg 60agcagcacat gcgtcgccta tgtggctacc agggacaact gctgcatcct ggacgagagg 120ttcggcagct actgccccac cacctgcggc atcgccgact ttctgagcac ctaccagacc 180aaggtggaca aggacctgca gagcctggag gacatcctgc accaggtgga gaacaagacc 240agcgaggtga agcagctgat caaggccatc cagctgacct acaaccccga cgagagcagc 300aagcccaaca tgatcgacgc cgccaccctg aagagcagga agatgctgga ggaaatcatg 360aagtacgagg ccagcatcct gacccacgac agcagcatca gatacctgca ggaaatctac 420aacagcaaca accagaagat cgtcaacctg aaggaaaagg tcgcccagct ggaagcccag 480tgccaggaac cctgcaagga caccgtgcag atccacgaca tcaccggcaa ggactgccag 540gacatcgcca acaagggcgc caagcagagc ggcctgtact tcatcaagcc cctgaaggcc 600aaccagcagt tcctggtgta ctgcgagatc gacggcagcg gcaacggctg gaccgtgttc 660cagaagaggc tggacggcag cgtggacttc aagaagaact ggattcagta caaggaaggc 720ttcggccacc tgagccccac cggcaccacc gagttctggc tgggcaacga gaagatccac 780ctgatcagca cccagagcgc catcccatac gccctgaggg tggagctgga ggactggaac 840ggcaggacca gcaccgccga ctacgccatg ttcaaagtgg gacccgaggc cgacaagtac 900aggctgacct acgcctactt tgccggaggg gacgctggcg acgccttcga cggcttcgac 960ttcggcgacg accccagcga caagttcttc accagccaca acggcatgca gttcagcacc 1020tgggacaacg acaacgacaa gttcgagggc aactgcgccg agcaggacgg ctccgggtgg 1080tggatgaaca agtgccacgc cgggcacctg aacggcgtgt actaccaggg cggcacctac 1140agcaaggcca gcacccccaa cggctacgac aacggcatca tctgggccac ctggaaaacc 1200aggtggtaca gcatgaaaaa aaccaccatg aagatcatcc cattcaacag actgaccatc 1260ggcgagggcc agcagcacca cctgggcgga gccaagcagg ctggcgacgt g 131181359DNAHomo sapiens 8atgagctggt ccctgcaccc caggaacctg atcctgtact tctacgccct gctgttcctg 60agcagcacat gcgtcgccta tgtggctacc agggacaact gctgcatcct ggacgagagg 120ttcggcagct actgccccac cacctgcggc atcgccgact ttctgagcac ctaccagacc 180aaggtggaca aggacctgca gagcctggag gacatcctgc accaggtgga gaacaagacc 240agcgaggtga agcagctgat caaggccatc cagctgacct acaaccccga cgagagcagc 300aagcccaaca tgatcgacgc cgccaccctg aagagcagga agatgctgga ggaaatcatg 360aagtacgagg ccagcatcct gacccacgac agcagcatca gatacctgca ggaaatctac 420aacagcaaca accagaagat cgtcaacctg aaggaaaagg tcgcccagct ggaagcccag 480tgccaggaac cctgcaagga caccgtgcag atccacgaca tcaccggcaa ggactgccag 540gacatcgcca acaagggcgc caagcagagc ggcctgtact tcatcaagcc cctgaaggcc 600aaccagcagt tcctggtgta ctgcgagatc gacggcagcg gcaacggctg gaccgtgttc 660cagaagaggc tggacggcag cgtggacttc aagaagaact ggattcagta caaggaaggc 720ttcggccacc tgagccccac cggcaccacc gagttctggc tgggcaacga gaagatccac 780ctgatcagca cccagagcgc catcccatac gccctgaggg tggagctgga ggactggaac 840ggcaggacca gcaccgccga ctacgccatg ttcaaagtgg gacccgaggc cgacaagtac 900aggctgacct acgcctactt tgccggaggg gacgctggcg acgccttcga cggcttcgac 960ttcggcgacg accccagcga caagttcttc accagccaca acggcatgca gttcagcacc 1020tgggacaacg acaacgacaa gttcgagggc aactgcgccg agcaggacgg ctccgggtgg 1080tggatgaaca agtgccacgc cgggcacctg aacggcgtgt actaccaggg cggcacctac 1140agcaaggcca gcacccccaa cggctacgac aacggcatca tctgggccac ctggaaaacc 1200aggtggtaca gcatgaaaaa aaccaccatg aagatcatcc cattcaacag actgaccatc 1260ggcgagggcc agcagcacca cctgggcgga gccaagcagg tgcggccaga gcaccccgcc 1320gagacagagt acgacagcct gtaccccgag gacgacctg 1359
Patent applications by Abraham Bout, Moerkapelle NL
Patent applications by Jacob Koopman, Leiderdorp NL
Patent applications by PROFIBRIX BV
Patent applications in class Serine proteinases (3.4.21) (e.g., trypsin, chymotrypsin, plasmin, thrombin, elastase, kallikrein, fibrinolysin, streptokinease, etc.)
Patent applications in all subclasses Serine proteinases (3.4.21) (e.g., trypsin, chymotrypsin, plasmin, thrombin, elastase, kallikrein, fibrinolysin, streptokinease, etc.)