CA2083286C - Recombinant fibrinogenases, preparation and use thereof - Google Patents

Abstract

Glycosylated, partially glycosylated and non-glycosylated polypeptides which have the amino-acid sequences indicated in the sequence listing and in which up to 10 amino-acid residues can be replaced by residues of other natural amino acids are described. The peptides are suitable for controlling diseases.

Description

RECOMBINANT FIBRINOGENASES, PREPARATION AND USE THEREOF
The present invention relates to novel proteins with fibrinogenolytic properties, called fibrinogenases, the preparation and use thereof for the prophylaxis and therapy of diseases.
To date it has been possible to isolate from the venom of the Malayan pit viper (Agkistrodon rhodostoma) only one fibrinogen-cleaving enzyme having anticoagulant properties (Biochem. J. 131 (1973) 799). This protein is :LO called Arvin, Arvin or ancrod in the literature.
The possib:Le uses of this protein are limited because signs of resistance may appear after 6 to 8 weeks and are presumably attributable to the production of ancrod-neutralizing antibodies. Hemorrhagic complications also occur in a few cases.
We have now found, and prepared pure, other proteins with fibri.nogenolytic properties.
The present invention relates to glycosylated, partially glycosylated or non-glycosylated polypeptides with the amino-acid sequences 1, 2, 3, 4 and 5 given in the sequence listing, where Xaa and Xab are residues of natural a-amino acids, and to the allelic variants thereof which are identical in more than 95$ of the amino-acid positions to the indicated sequences.
The residue Xaa is Asn, Gln, Ser, Thr, Gly, Asp, Glu, Lys, Arg or Pro, but preferably Asn, Gln, Ser and Thr-and, in particular, Asn and Gln. Xab is preferably Phe, Tyr, Leu, Ile,. Ala, Val, Thr or Ser.
The present invention also relates to DNA sequen ces which code for the abovementioned proteins, and to vectors which contain these DNA sequences. Preferred DNA
sequences are depicted as sequences Nos . 6 to 9 in the sequence listing.
The proteins according to the invention can be prepared by known methods of genetic manipulation.
Thus, it is possible to isolate from the glandu-lar tissue of a Malayan pit viper (Agkistrodon 20$2$

- 2 - O.Z. 0050/41781 rhodostoma) mRNA and to convert it into double-stranded cDNA. -A cDNA library is set up after insertion of this cDNA into a commercial cloning vector, eg. a gt 10. The methods used for this can be found, for example, in Maniatis et al., Molecular Cloning, CSH Press (1982). The screening of such gene banks with radiolabeled oligo-nucleotide probes or radiolabeled DNA fragments is also now a widely used and described method. This method can be used to isolate and characterize a cDNA clone which has homology with the oligonucleotide probe or with radiolabeled DNA fragments, and is described in DNA
cloning, Vol. I, IRL Press, 1985.
The cDNA which has been characterized in this way can easily be obtained using restriction enzymes. The fragments resulting from this can be used, where appro priate in combination with chemically synthesized oligo-nucleotides, adaptors or gene fragments, to clone the sequences coding for the protein. The gene fragments or synthetic DNA sequences are incorporated into cloning vectors, eg. the commercial plasmids Ml3mp or pkk-223-3, in a conventional manner. The genes or gene fragments can also be provided with suitable control regions which have been chemically synthesized or isolated from bacteria, phages, eukaryotic cells or viruses thereof and which make expression of the proteins possible.
The transformation or transfection of suitable host organisms with the hybrid plasmids obtained in this way is likewise known and described in detail (M. Wigler et al., Cell 16 (1979) 777-785; F.L. Graham and A.J. van der Eb, Virology 52 (1973) 456-467). The hybrid plasmids can also be provided with appropriate signal sequences to allow the polypeptides to be secreted into the medium.
Vectors which can be used for expression in mammalian cells are those which place the gene to be expressed, in this case the cDNA which codes for one of the fibrinogenases described in the sequence listing, under the control of the mouse metallothionein or viral 2~~~~$

- 3 - O.Z. 0050/41781 SV40 promoter {J. Page Martin, Gene, 37 (1985) 139-144).
The presence of the methionine start codon and of the leader/prosequence of the gene for the appropriate protein is necessary for expression. Clones which contain copies of these vectors as episomes or integrated in the genome are then isolated. Integration and expression of the foreign gene on the basis of the bovine papilloma virus are particularly advantageous. It is possible to construct shuttle vectors in conjunction with prokaryotic sequences which code for replication in bacterial cells and for antibiotic resistance. The plasmid is initially constructed and multiplied in bacterial cells and is then transferred into the eukaryotic cells, eg. into the mouse fibroblast cell line c127.
It is also possible to use other cell systems, eg. yeast and other fungi, insect cells and animal and human cells such as CHO, COS, L and 293 cells, in con-junction with suitable expression vectors for the expres-sion of the cloned cDNA.
These eukaryotic expression systems have the advantage that they are able to secrete their products efficiently and usually in native form. They also have the ability to carry out post-translational modification on their products.
Thus, on expression in eukaryotic cells, the described fibrinogenases acquire glycoside side-chains.
These side-chains are absent in the polypeptides produced in bacteria. The glycoside side-chains can also be removed completely or partially using appropriate glyco-sidases. Most eukaryotic proteins expressed in bacteria, result as denatured inclusion bodies in the cell and must be renatured by appropriate methods. In addition, bac-teria are often incapable of eliminating the initiator amino acid methionine from the finished protein. These difficulties can be avoided by using secretion systems (Donald Oliver, Ann. Rev. Microbiol. 39, (1985) 615-48;
John Ghrayeb et al. The EMBO Journal 3 {1984) 2437-2442.

- O.Z. 0050/41781 However, because of the degeneracy of the genetic code, it is also possible to use other DNA sequences, eg.
chemically synthesized genes with different DNA sequen-ces, for the expression of the described fibrinogenases.
Application of established methods of mutagenesis to the cloned genes allows production of variants of these fibrinogenases with a similar action.
The resulting polypeptides are purified from the culture medium by chromatography, eg. affinity chromato graphy on arginine-Sepharose~, Matrex-RedA-Sepharose , heparin-Sepharose or ion exchange materials in a conven-tional manner (Lit.: Guide to Protein Purification, Murray P. Deutscher [ed), Academic Press 1990).
The purification of the fibrinogenases can likewise be purified [sic) directly from the venom of A.
rhodostoma by a combination of suitable chromatographic methods, preferably using Matrex-redA-Sepharose , heparin Sepharose~, arginine-Sepharose , conA-Sepharose , Q
Sepharose , S-Sepharose and chromatofocussing as described in Example 5. Particularly suitable for the final purification are HPLC methods.
The present invention also relates to drugs which contain the proteins prepared according to the invention, where appropriate in a pharmaceutically tolerated carrier or excipient. The drugs can also contain combinations of the proteins prepared according to the invention with other pharmacologically active substances such as thrombolytics (tPA, streptokinase), hirudin or throm-boxane receptor antagonists.
Further embodiments of the invention are des-cribed in detail in the Examples.
For methods of genetic manipulation, reference may be made to, for example, the handbook by Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1982 or DNA cloning Vol. I - III, IRI [sic] Press 1985 -87, edited by D.M. Glover.
The polypeptides according to the invention are - 5 - O.Z. 0050/41781 suitable for the treatment of glomerulonephritis, myo-cardial infarct, non-ischemic stroke, disturbances of peripheral arterial blood flow (especially athero-sclerosis obliterans, thrombangitis obliterans, diabetic microangiopathy and Raynaud's disease), unstable angina pectoris, deep vein thrombosis and other thromboses, rethrombosis after thrombolysis or vascular surgery, such as angioplasty, and for preventing thromboses in extra-corporeal circulations.

Isolation of a fibrinogenase cDNA clone from the Malayan pit viper (Agkistrodon rhodostoma) 1 g of venom gland tissue from a 5-year old snake of the genus [sic] Agkistrodon rhodostoma was disrupted in 6 M guanidinium thiocyanate, 5 mM sodium citrate (pH
7.0), 0.1 M 2-mercaptoethanol, 0.5~ sarkosyl in an ULTRA-TURRAX~. Large cell detritus was removed by centrifugation at 3000 rpm. The RNA was removed by centrifugation through a 5.7 M CsCl cushion at 45,000 rpm overnight. The polyA+-containing RNA fraction was then isolated by affinity chromatography on oligo(dT)-cellulose.
The polyA+ RNA was converted into single-stranded cDNA using AMV reverse transcriptase and oligo(dT)12-18 as primer. The second strand was synthesized using E.coli DNA polymerase I. An EcoRI adaptor of the sequence 5'AATT
CCATGG ATG CATGC 3' was attached to the double-stranded cDNA using T4-DNA ligase. The commercial phage vector a gt 10 (Fig. la, lb) was linearized with the restriction '''~f enzyme EcoRI. The two DNAs were ligated together and packaged with the commercial packaging extract to give infectious phages. The recombinant phages were plated out with E.coli C 600 Hfl on NZYDT plates and incubated at 37°C overnight. The resulting cDNA library contained 2x106 independent clones. Amplification of the cDNA library by conventional methods was followed by plating out of 500,000 phages with C 600 Hfl cells. The phages were transferred to nitrocellulose filters, lyzed with 0.5 N

- 6 - O.Z. 0050/41781 NaOH/1.5 M NaCl, and the denatured DNA was firmly bound to the filter by baking at 80°C for 2 hours. The filters were prehybridized in 6 x SET buffer (1 x SET = 0.15 M
NaCl, 15 mM tris/HC1, pH 7.4, 1 mM EDTA), 0.1% SDS and 5 x Denhardt's solution (100 x Denhardt = 1 g of Ficoll, 1 g of polyvinylpyrrolidone, 1 g of 8SA per 50 ml) at 68°C for 4 h.
Hybridization was carried out with a nick-trans-lated cDNA (Fig. 2) which codes for ancrod protein.
The filters were incubated in a solution which contained 2 x SET, 0.1% SDS, 30% formamide, 5 x Denhardt's and 10% dextran sulfate at 42°C overnight while shaking gently. They were then washed several times with 2 x SET/0.1% SDS at 42°C, dried and exposed to an X-ray film. Clones which gave a radioactive response in the screening were isolated and cultured further in order to obtain the corresponding phage DNA.
Phage DNA was prepared by incubating the purified phages with protenase [sic] K (ad 60 ~g/ml) at 55°C for 1 h and subsequent phenol/chloroform extraction. Addition of 3 volumes of ethanol (-20°C) resulted in precipitation of the phage DNA, which was transferred with a sterile injection needle into 70% ethanol, washed and briefly sedimented. The pellet was briefly dried in air and then suspended in TE buffer.
The purified phage DNA was transferred to nitro-cellulose filters, renatured, reneutralized, baked and prehybridized as described above. Hybridization was then carried out under stringent conditions, using a radio-labeled oligonucleotide probe which was homologous to the ancrod-encoded [sic] cDNA:
5' GTC TAC GAT TAT CGT GAC TGG GTC AA 3' The filters were incubated in a solution which contained 2 x SET, 0.1% SDS, 30% formamide, 5 x Denhardt's and 10% dextran sulfate at 42°C overnight while shaking gently. They were then washed several times in 2 x SET/0.1% SDS at 60°C, dried and exposed to an - 7 - O.Z. 0050/41781 X-ray film.
The DNA which did not hybridize under these conditions was subconed [sic] in the single-stranded phage MB for further analysis.

Preparation of single-stranded DNA which codes for ancrod The starting point were [ sic ] the phage DNA which did not hybridize with the ancrod-specific oligonucleotide as described in Example 1. They were each separately cut preparatively with the restriction enzyme Eco RI. The Eco RI fragments which contained the cDNA
inserts were eluted from the gel by electrophoresis. 30 ng of each of these fragments were ligated at 4°C for 12 h with 100 ng of the commercial cloning vector M13mp18 or M13mp19 (Fig. 3) which had been cut with Eco RI. The volume of the ligation mixture was 10 ~1. Ligation was stopped by heating at 80°C for 5 min.
1/10 of the volume of each ligation mixture was employed to transform 100 ~l of competent SR 101 cells.
After the transformation was complete, 60 ~1 of 0.2 M
IPTG solution and 120 ~sl of XGal (20 mg/ml) were added to the transformation mixture. The resulting mixture was plated out in NZYDT top agar on NZYDT agar plates con-taining 200 ~1 of SR 101 cells (ODsoo-1)~ The NZYDT medium is commercially available (GIBCO-BRL). Clones which contained cDNA inserts were identifiable because the plaques were not stained blue. DNA sequence analysis (Sanger et al., Proc. Natl. Acad. Sci. USA 74, (1977) 5463-67) was used to elucidate the sequence of this cDNA
insert (sequence listing, Nos. 6 to 9).

Construction of vectors for the expression of ancrod in eukaryotic cells SV40 DNA was cut with the restriction enzymes BamHI and BclI, and the 0.24 kb fragment was prepared by gel electrophoresis (Fig. 4). The ends were filled in '~---' with the Klenow fragment in the presence of the four _ ~~~33~'~~
- 8 - O.Z. 0050/41781 deoxynucleotide triphosphates dATP, dCTP, dGTP and dTTP.
XhoI linkers were then ligated on.
In parallel the commercial vector pUCl8 was linearized with SmaI. XhoI linkers were then likewise attached. The DNA of this vector (puCl8Xho) was linear ized with Xhol, treated with alkaline phosphatase and ligated to the 0.24 kb XholI [sic] SV40 fragment (see above). The result was pSVpA.
pSVpA DNA was cleaved preparatively with XhoI and incubated with Klenow polymerase in the presence of the four dNTPs as above. The 0.24 kb fragment was isolated from the gel.
At the same time, the eukaryotic expression vec tor CL28XhoBPV, produced by ligation of CL28x and pB2-2 (Reddy et al. DNA 6, (1987) 461-72) was partially cut with the restriction enzyme XbaI, ie. the incubation time was restricted so as to result in molecules cleaved at only one of the two XbaI recognition sequences, ie.
linearized (Fig. 5) . The mixture was then reacted with Klenow polymerase and dNTPs as described. The linear molecules were subsequently isolated by gel electro-phoresis.
The linear pCL28XhoBPV fragments were then ligated with the pretreated 0.24 kb SV40 fragment. After transformation and screening of minilysates, a clone which carried the SV40 fragment in the former XbaI site located about 0.15 kB [sic] 3' of the XhoI site was isolated; this DNA (pCL28XhoBPV-SVpolyA) carried the SV40 transcription stop signals of the early genes.
Plasmid DNA from pCL28XhoBPV-SVpolyA was linear-ized with Xhol and treated with alkaline phosphatase. At the same time, the cDNA inserts which did not hybridize with the Ancrod-specific oligonucleotide as described in Example 2 were provided with Xho linkers using T4 ligase.
The two fragments were connected together using T4 ligase. After transformation and analysis of minilysates, a clone which contained the cDNA inserts singly and in ~~8'~~g~b O.Z. 0050/41781 the correct orientation was isolated: pCL28BPV-fibro-genase [sic] I-IV.

Transfection and establishment of cell lines c127I cells (J. Virol. 26 (1978) 292; ATCC
catalog of cell lines and hybridomas 5th edition, 1985, p.142) were transfected with BPV expression plasmids using the calcium phosphate coprecipitation method (Virology 52 (1973), 456, DNA cloning; Volume II, ed.
D.M. Glover IRL Press, (1985) 143ff and 213).
DMEM (Dulbeccos~s Modified Eagles Medium) + 10~
FCS (fetal calf serum) in 60 mm Petri dishes was inocu-lated with 5 x 105 C127I cells. The next day the medium was changed to MEM (Modified Eagles Medium) containing 25 nM Hepes + 10$ FCS. A Ca phosphate coprecipitate was formed with 10-5 g of CsCl-purified plasmid DNA and was cautiously placed on the C172I cells. The cells were incubated at 37°C, 7~ C02 for 4 h. A subsequent glycerol shock treatment considerably increased the efficiency of transfection. For this, 4 h after addition of the preci-pitate the medium was aspirated off from the cells. The cells were incubated with 2 ml each [sic] of 15~
glycerol/HBS (DNA cloning Vol. II, page 152) in a 60 mm Petri dish at room temperature for 3 min. The glycerol/HBS solution was aspirated off and the cell lawn was washed with 3 ml of DMEM + 10~ FCS. The cells were incubated with DMEM + 10 ~ FCS at 3 7 ° C , 7 ~ COZ . The DMEM +
10% FCS was aspirated off and replaced by fresh three times a week. After 2 - 3 weeks, the transfected cells which contain the BPV genome were evident as collections of transformed cells, called foci.
After the foci had been subcloned, the medium supernatants from the individual subclones were tested for fibrinogen-cleaving activity by conventional methods.
For production, after the cell lines had reached confluence they were maintained in serum-free DMEM. The novel fibrinogenases can be purified by conventional - 10 - O.Z. 0050/41781 methods from the serum-free cell culture supernatant obtained in this way and used for pharmacological and chemical analyses.

Isolation and purification of a fibrinogen-cleaving enzyme (sequence listing, No. 5) from the venom of Agkistrodon rhodostoma 550 mg of crude venom (dry substance) from Agkistrodon rhodostoma were taken up in 20 ml of 20 mM
Na2HP0,,, 0.01 Tweeri 80, 500 mM NaCl, pH 7.0 {= buffer A).
a) Chromatography on Matrex Red A-Sepharose A chromatography column {diameter 2.5 cm, length 5.1 cm) was packed with 25 ml of Matrex red A-Sepharose (from Amicon). The column was equilibrated with 100 ml of buffer A and then loaded with the dissolved crude venom. The column was washed with 45 ml of buffer A (flow rate 120 ml/h) and then eluted with 85 ml of buffer B, which was composed of 20 mM
Na2HP04, 2 M NaCl, 0.01 Tween, pH 7Ø The W-active fraction (280 nm) was collected.
The eluate was dialyzed twice against 2.5 1 of 20 mM
NaZHP04, 0.01$ Tweeri 80, pH 7.0 (= buffer C) in a dialysis tube (Visking size 8.32/32) for 2 h each time. The conductivity of the dialyzed tubes [sic] was about 2.2 mS/cm (4°C).
b) Chromatography on arginine-Sepharose A chromatography column (diameter 2.5 cm, length 10 cm) was packed with arginine-Sepharose (from Pharmacia) and equilibrated with 200 ml of buffer C.
The dialyzed eluate (vol. about 140 ml) from the Matrex red A-Sepharose was loaded on the column with a flow rate of 120 ml/h.

- 11 - O.Z. 0050/41781 The flow-through from the column (about 180 ml) was collected and processed further. Still bound to the column was, inter alia, ancrod which can be obtained by elution with arginine salts.
c) Chromatofocussing A chromatography column (diameter 0.5 cm, length 5 cm) was packed with 1 ml of PBE~ 94 gel material (from Pharmacia). The column was equilibrated with 5 column volumes of 20 mM tris/HC1, 0.01 Tweeri 80, pH 8.0 (= buffer D).
The column was loaded with 20 ml of the flow-through from the arginine-Sepharose .
The chromatography was carried out with a linear gradient from buffer D to 20 mM acetic acid/HC1, 0.01 Tweeri 80, pH 2.0 (= buffer E) in 25 min with a flow rate of 1 ml/min. After this time, the column was eluted with buffer E for a further 13 min. The W-active fraction (280 nm) eluted during this was collected. About 1 ml of a protein solution which contained, according to protein determination (method:
Anal. Biochem. 153, 267-271), about 0.04 mg/ml was obtained.
d) Characterization of the purified fibrinogenase V
dl) SDS gel electrophoresis Comparing with standard proteins, the protein solution showed a main band {about 70 to 90$) at 42000 Dalton.
d2) N-terminal sequencing The N-terminal sequence of the purified protein solution was determined (see sequence listing, sequence No. 5).

_ ~os~~~~
- 12 - O.Z. 0050/41781 d3) Fibrinogenase assay Fibrinogenase activities were determined by converting fibrinogen with the enzyme to be assayed into deAA fibrinogen.
This reaction was associated with an increase in turbidity which was followed by photometry (DD
[sic] 340 nm).
The activity was quantified by calibration with an ancrod standard (Arwiri) of 3000 U/mg.
The fibrinogenase activity of the purified enzyme was about 500 U/mg.

2~8~~~
- 13 - O.Z. 0050/41781 Sequence listing Sequence No. 1: 234 amino-acid sequence Val Ile Gly Gly Asp Glu Cys Asn Ile Asn Glu His Arg Phe Leu Val Ala Leu Tyr Asp Ser Thr Thr Arg Asn Phe Leu Cys Gly Gly Val Leu Ile His Pro Glu Trp Val Ile Thr Ala Lys His Cys Asn Lys Lys Ser Met Val Leu Tyr Leu Gly Lys His Lys Gln Ser Val Lys Phe Asp Asp Glu Gln Glu Arg Phe Pro Lys Glu Lys His Phe Ile Arg Cys Asn Lys Pro Arg Thr Arg Trp Gly Glu Asp Ile Met Leu Ile Arg Leu Asn Lys Pro Val Xaa Asn Ser Glu His Ile Ala Pro Leu Ser Leu Pro Ser Gly Pro Pro Ile Val Gly Ser Val Cys Arg Val Met Gly Trp Gly Ser Ile Asn Lys Tyr Ile Asp Val Leu Pro Asp Glu Pro Arg Cys Ala Asn Ile Asn Leu Tyr Xaa Tyr Thr Val Cys Arg Gly Val Phe Pro Arg Ile Gly Lys Lys Ser Lys Ile Leu Cys Ala Gly Asp Leu Gln Gly Arg Leu Asp Ser Cys His Cys Asp Ser Gly Gly Pro Leu Ile Cys Ser Glu Glu Phe His Gly Ile Val Tyr Arg Gly Pro Asn Pro Cys Ala Gln Pro Asp Lys Pro Ala Leu Tyr Thr Asn Ile Phe Asp His Leu His Trp Ile Leu Ser Ile Val Ala Gly Xaa Ala Thr Cys Tyr Pro 2~~~~~
- 14 - O.Z. 0050/41781 Sequence listing Sequence No. 2: 236 amino-acid sequence Val Val Gly Gly Asp Glu Cys Asn Ile Asn Glu His Arg Phe Leu Ala Leu Val Tyr Ile Thr Ser Gly Phe Leu Cys Gly Gly Thr Leu Xab His Pro Glu Trp Val Val Ser Ala Ala His Cys Ala Arg Gly Glu Ile Glu Val Phe Phe Gly Val His Ser Leu Lys Asp Ile Arg Thr Asn Lys Asp Val Gln Lys Arg Val Ala Lys Glu Met Phe Phe Cys Leu Ser Ser Lys 65 70 75 g0 Xaa Tyr Thr Lys Trp Asp Lys Asp Ile Met Leu Ile Lys Leu Asp Ser Pro Val Xaa Asn Ser Thr His Ile Ala Pro Ile Ser Leu Pro Ser Ser Pro Pro Ser Val Gly Ser Val Cys Arg Val Met Gly Trp Gly Val Thr Thr Ser Pro Xaa Gly Thr Xab Pro Ser Val Pro His Cys Ala Asn Ile Asn Ile Leu Asp Tyr Xab Val Cys Arg Ala Ala Arg Pro Lys Leu Pro Ala Lys Ser Arg Thr Leu Cys Ala Gly Ile Leu Glu Gly Gly Lys Ser Ala Cys Asp Gly Asp Ser Gly Gly Pro Leu Asn Cys Asn Gly Glu Ile Gln Gly Ile Val Ser Trp Gly Gly Asn Ile Cys Ala Gln Pro Arg Lys Pro Ala His Tyr Xab Lys Val Ala Asp Tyr Thr Asp Trp Ile Lys Ser Ile Ile Ala Gly Xaa Thr Thr Ala Thr Cys Pro Pro ~~i832~~
- 15 - O.Z. 0050/41781 Sequence listing Sequence No. 3: 236 amino-acid sequence Val Ile Gly Gly Ala Glu Cys Asn Val Asn Glu His Arg Phe Leu Val Ala Leu Tyr Asp Xaa Leu Thr Gly Thr Leu Gln Cys Gly Gly Thr Leu Ile His Pro Glu Trp Val Leu Thr Ala Ala His Cys Asp Arg Lys Ser Met Val Ile Tyr Leu Gly Met His Xaa Lys Ser Val Asn Asn Asp Asp Gln Gln Arg Arg Ser Ala Lys Glu Lys Tyr Phe Phe Ser Cys Ser Lys Ser Ile Ala Ala Trp Glu Lys Asp Ile Met Leu Ile Arg Leu Asp Ser Pro Val Xaa Asn Ser Thr His Ile Ala Pro Leu Ser Leu Pro Ser Arg Pro Pro Thr Val Gly Ser Val Cys Arg Val Met Gly Trp Gly Ala Ile Thr Ser Pro Lys Glu Thr Tyr Pro Glu Val Pro His Cys Thr Asp Ile Asn Leu Leu Xaa Tyr Ser Glu Cys His Gly Asp Phe Pro Arg Leu Arg Ala Thr Ser Arg Ile Leu Cys Ala Gly Val Leu Gln Gly Gly Ile Asp Thr Cys Asn His Asp Ser Gly Gly Pro Leu Ile Cys Asp Glu Gln Phe Gln Gly Ile Val Ser Trp Gly Pro Tyr Pro Cys Ala Gln Pro Arg Asn Ala Ala Ile Tyr Thr Lys Val Phe Asn Tyr Leu Val Trp Val Trp Ser Thr Ile Ala Gly Xaa Thr Thr Val Thr Cys Pro Pro w - 16 - O.Z. 0050/41781 Sequence listing Sequence No. 4: 234 amino-acid sequence Val Val Asn Gly Ile Gly Asn Asn Glu Glu His Cys Arg Phe Leu V
l 5 a Ala Ile Phe Xab Phe Val Cys Ala Gly Thr Leu Ile His Ser Thr Gly Pro Glu Trp Val Val Thr Ala His Cys Glu Ser Thr Asp Leu Lys Ala Met Lys Phe Gly Met His Ser Lys Lys Val Gln Asn Glu Asp Glu Gln Thr Arg Asn Ala Lys Glu Lys Phe Ile Cys Pro Asn Lys Lys Asn Asp Glu Val Leu Asp Lys Asp Ile Met Leu Ile Lys Leu Asn His Pro Val Ser Asn Ser Glu His Ile Ala Pro Leu Ser Leu Pro Ser Ser Pro Pro Ser Val Gly Ser Phe Cys His Ile Met Gly Trp Gly Ser Ile Thr Pro Val Lys Val Thr Phe Pro Asp Val Pro His Cys Ala Asn Ile Asn Leu Leu Glu Glu Ala Glu Cys His Ala Gly Tyr Pro Glu Val Leu Ala Glu Tyr Arg Thr Leu Cys Ala Gly Ile Val Gln Gly Gly Lys Asp Thr Cys Met Tyr Asp Ser Gly Gly Pro Leu Ile Cys Asn Glu Gln Val Gln Gly Ile Val Ser Tyr Gly Ala His Pro Cys Gly Gln Pro Leu Lys Pro Gly 200 , 205 Ile Tyr Thr Arg Leu His Asp Tyr Asn Asp Trp Ile Asn Ser Ile Met Ala Gly Asn Thr Ala Val Thr Pro Pro Cys 2~~3~~~
- 17 - O.Z. 0050/41781 Sequence listing Sequence No. 5: 25 amino-acid sequence Val Ile Gly Gly Asp Glu Cys Asn Ile Asn Glu His Pro Phe Leu Val Ala Val Tyr Glu Glu Thr Ala Gly Ala ~0~32~~
- 18 - O.Z. 0050/41781 Sequence listing Sequence No. 6: 1096 nucleotide sequence corresponding to amino-acid sequence No. 1 Strandedness : double-stranded Topology: linear Molecule type . cDNA to mRNA
Original source: Agkistrodon rhodostoma The region coding for the protein of sequence No. 1 starts at base 144 and terminates at base 841.

TAAATGGACCATTCCC

._ ~~~~~s~
- 19 - O.Z. /41781 Sequence listing Sequence No. 7: 1333 nucleotide sequence corresponding to amino-acid sequence No. 2 Strandedness: double-stranded Topology: linear Molecule type: cDNA to mRNA
Original source: Agkistrodon rhodostoma The region coding for the protein of sequence No. 2 starts at base 231 and terminates at base 935.

ATCCATGGAA TTC

~~s~~s~
- 20 - O.Z. 0050/41781 Sequence listing Sequence No. 8: 988 nucleotide sequence corresponding to amino-acid sequence No. 3 Strandedness: double-stranded Topology: linear Molecule type: cDNA to mRNA
Original source: Agkistrodon rhodostoma The region coding for the protein of sequence No. 3 starts at base 197 and terminates at base 904.

TGGTAAAAAAAAAAAAAAAA A~~~AAAAA

._.. - 21 - 0. Z . 00~
Sequence listing Sequence No. 9: 957 nucleotide sequence corresponding to amino-acid sequence No. 4 Strandedness: double-stranded Topology: linear Molecule type: cDNA to mRNA
Original source: Agkistrodon rhodostoma The region coding for the protein of sequence No. 4 starts at base 210 and terminates at base 911.
CTNAATTNNA CAAAAAAAGT GCTGCTTGGT CAAGAGGTNC TCCGCTTCGG TTATCTGATT

AGATTGATAC GTATCTCAAG TATAAGTTTG GGACTGGGAT CTTACAGGAA AACAGCTTTC

CGTGCAGAGT TGAAGTTATG GTACTGATCA GAGTGCTAGC AAACCTTCTG ATACTACAGC

CTTGCCCCCC GTGAAAACTT TAGTATCAGA AGGTTTGCTG CATGCATCCA TGAATTC

Claims (11)

We claim:

1. A glycosylated, partially glycosylated or non-glycosylated polypeptide with amino-acid sequence 1, 2, 3 or 4, which is indicated in the sequence listing and in which Xaa and Xab are each residues of natural a-amino acids, as well as the allelic variants thereof which are identical to the stated sequences in more than 95% of the amino-acid positions.

2. A polypeptide obtainable from the crude venom of Agkistrodon rhodostoma by chromatography on matrex red A-Sepharose R and arginine-Sepharose R and subsequent chromatofocusing with the gel material PBE R 94, which exhibits a main band with a molecular weight of about 42,000 dalton, has a fibrinogenase activity of about 500 U/mg and has the N-terminal amino-acid sequence 5.

3. A DNA sequence which codes for a polypeptide as claimed in claim 1 or 2.

4. A recombinant DNA which contains a DNA sequence as claimed in claim 8.

5. A recombinant DNA molecule as claimed in claim 4, which contains a DNA sequence as claimed in claim 3, which is functionally connected to an expression control system which makes expression possible in suitable host systems.

6. A recombinant DNA molecule as claimed in claim 5, wherein the expression control sequence is an E.-coli promoter system, a promoter system of an E.-coli bac-teriophage, a yeast or fungus expression control sequence or another eukaryotic expression control sequence.

7. A host cell which contains at least one recombinant DNA molecule as claimed in claim 4, 5 or 6.

8. A host cell as claimed in claim 7, which is a bacterium, a yeast, a fungus, an animal or a human cell.

9. A genetic engineering process for preparing a polypeptide as claimed in claim 1 or 2, which comprises bringing about the expression in a suitable host cell of DNA sequences which code for peptide sequences as claimed in claim 1.

10. A polypeptide as claimed in claim 1 or 2 for use for preventing and controlling diseases.

11. The use of a polypeptide as claimed in claim 1 or 2 for producing drugs for the treatment and prophylaxis of thromboembolic disorders and glomerulonephritis.