WO1994012204A1 - Proteines anticoagulantes - Google Patents

Proteines anticoagulantes Download PDF

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Publication number
WO1994012204A1
WO1994012204A1 PCT/US1993/011154 US9311154W WO9412204A1 WO 1994012204 A1 WO1994012204 A1 WO 1994012204A1 US 9311154 W US9311154 W US 9311154W WO 9412204 A1 WO9412204 A1 WO 9412204A1
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Prior art keywords
protein
mammal
composition
dna
inhibiting
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Shi-Shan Mao
Jules A. Shafer
Jin Huang
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Merck and Co Inc
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Merck and Co Inc
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Priority to AU56704/94A priority Critical patent/AU5670494A/en
Publication of WO1994012204A1 publication Critical patent/WO1994012204A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the role of blood coagulation is to provide an insoluble fibrin matrix for consolidation and stabilization of a haemostatic plug. Formation of a cross-linked fibrin clot results from a series of biochemical interactions involving a range of well-characterized plasma proteins.
  • the interactions are divided into what are termed the "intrinsic pathway", in which all the substances necessary for fibrin formation are present in precursor form in circulating plasma, and the extrinsic pathway in which thromboplastin, derived from tissues, bypasses several steps in the process and accelerates clot formation.
  • the two pathways are highly interdependent and Factor VII, Factor IX and Factor X are mutually activated (J.C. Giddings "Molecular Genetics and Immunoanalysis in Blood Coagulation" Ellis Horwood Ltd., Chichester, England (1988), p. 17).
  • Human Factor X circulates in plasma as a two-chain glycoprotein with a molecular weight of about 67000 estimated by gel electrophoresis in the presence of sodium dodecylsulphate (Di Scipio et al., "A comparison of human prothrombin, Factor IX (Christmas factor), Factor X (Stuart factor) and protein S", Biochemistry, vol. 16, pp. 698-706). Slightly lower Mr values of about 59000 are obtained by sedimentation equilibrium analysis. The normal plasma concentration is about 7-10 mg per liter and the protein contains about 15% carbohydrate.
  • the light chain contains the ⁇ -carboxylated glutamic acid residues and shows significant homology with prothrombin fragment 1.
  • Activation of Factor X by complexes of Factor IXa and Factor Villa or by tissue factor/Factor Vila involves at least one peptide cleavage. The principal mechanism releases a small activation peptide from the heavy chain of the molecule by hydrolysis of an arginine- isoleucine bond.
  • the active product formed in this way is termed - Factor Xa.
  • ⁇ -Factor Xa This is modified further by cleavage of an arginine-glycine bond near the carboxy terminus to form ⁇ -Factor Xa.
  • ⁇ -Factor Xa and ⁇ -Factor Xa have the same coagulation activity.
  • the light chain of Factor X is unaffected by the activation process and remains linked to the heavy chain of the molecule by disulphide bridges. In this way the active substance retains the ⁇ - carboxyglutamic acid domains necessary for calcium-mediated attachment to phospholipid micelles or cellular surfaces. Fundamentally, therefore, in contrast to thrombin, Factor Xa remains associated with phospholipid and platelet membranes.
  • European Publication 419099 describes a Factor Xa inhibitor derived from Ornithodoros moubata tick extract (TAP), a method for recombinantly producing TAP in yeast, and several conservatively mutated forms of TAP which retain anticoagulant properties.
  • TAP Ornithodoros moubata tick extract
  • Vermulen et al Int. J. Biochem., Vol. 20, No. 6, pp. 621- 31 (1988), describes the relative protease inhibition activities of tick toxins isolated from R. evertsi evertsi, B. decoloratus, B. microplus, and H. truncatum. They were found to be fast-binding or slow-binding inhibitors of trypsin and chymotrypsin.
  • the S. cerevisiae ⁇ -mating factor pre-pro leader sequence has been utilized in the expression of heterologous genes as secreted products in yeast (Brake et al., Proc. Natl. Acad. Sci. USA vol. 81, pp. 4642-4646 (1984); Miyajima et al, Gene vol. 37, pp. 155-161 (1985); Vlasuk et al, J. Biol. Chem. vol. 261, pp. 4789-4796 (1986); Schultz et al, Gene vol. 54, pp. 113-123 (1987); Schultz et al, Gene vol. 61, pp. 123-133 (1987); Bayne et al, Gene, vol.
  • Proteins produced as fusion products are proteolytically processed by the Lys- Arg-cleaving endopeptidase (KEX2) encoded by the KEX2 gene and products are secreted into the culture medium.
  • KEX2 cleaves on the C-terminal side of Lys-Arg residues which are present between the ppL sequence and the heterologous gene.
  • Proteins of the invention are more potent inhibitors of Factor Xa than wild type TAP.
  • the mutant rTAP proteins dissociate from the mutant rTAPiFactor Xa complex at a significantly slower rate than does wild type TAP from the wild type TAP:Factor Xa complex.
  • the present invention includes mutant recombinant TAP proteins and variants which specifically inhibit coagulation Factor Xa.
  • the proteins are low molecular weight polypeptides having about 60 amino acids which are highly specific for Factor Xa. They do not significantly inhibit (less than 25% at 1 ⁇ M) human Factor Vila, bovine trypsin, bovine chymotrypsin, human thrombin, human high molecular weight urokinase, human plasmin, human tissue plasminogen activator, porcine pancreatic elastase, human activated protein C, human plasma kallikrein and bovine tissue kallikrein.
  • the mutant recombinant TAP proteins of the present invention contain amino acid substitutions at positions 1 and 10 of the wild type TAP sequence.
  • the proteins of the present invention can contain either a single mutation at amino acid 1, a single mutation at amino acid 10, or mutations at both amino acid 1 and amino acid 10. Mutants of the present invention containing mutations at both positions are called double mutants.
  • the present invention also encompasses mutant recombinant TAP proteins containing mutations in addition to mutations at positions 1 and 10 (e.g. triple mutants containing mutations at positions 1, 10, and another amino acid), provided that the additional mutations are conservative or do not substantially affect the Factor Xa inhibitory properties of the double mutant protein or the rate of dissociation between the double mutant protein and Factor Xa.
  • the present invention also includes compositions comprising a protein of the invention which inhibits Factor Xa and prevents coagulation in patients, and methods for inhibiting blood coagulation comprising administering to the patient therapeutically effective amounts of the compositions.
  • the present invention also includes methods for recombinantly producing the proteins in yeast cells and recovering the expressed proteins, and vectors for transforming the yeast cells. DESCRIPTION OF THE FIGURE
  • Figure 1 represents double mutant recombinant TAP Y1W/D10R .
  • nucleotide bases are designated as adenine (A); thymine (T); guanine (G); and cytosine (C).
  • adenine A
  • T thymine
  • G guanine
  • C cytosine
  • Corresponding nucleotides are, for example, deoxyadenosine-5'-triphosphate (dATP).
  • dATP deoxyadenosine-5'-triphosphate
  • Wild type TAP is described in European Publication 419099.
  • the proteins of the present invention are prepared using site directed mutagenesis.
  • Single mutant rTAP proteins include such proteins having single amino acid substitutions at position 1 (where the wild type TAP amino acid is tyrosine) or position 10 (where the wild type TAP amino acid is aspartate).
  • Amino acid 1 is substituted with a highly hydrophobic amino acid, preferably one having an aromatic side chain. More preferred amino acids at position 1 are selected from the group of amino acids consisting of tryptophan, phenylalanine, or ⁇ -naphthyl- alanine. Even more preferably, the amino acid is tryptophan (this more preferred single mutant rTAP protein is "Y1W” or "Tip 1 -TAP").
  • Amino acid 10 is substituted with a highly hydrophilic amino acid, preferably basic and positively charged and having a polar side chain.
  • Preferred amino acids at amino acid position 10 are selected from the group of amino acids consisting of arginine, lysine, and histidine. More preferably, the amino acid is arginine (this more preferred single mutant rTAP protein is "DIOR” or "Arg 10 -TAP").
  • Double mutant rTAP proteins of the present invention have two specific amino acid substitution mutations. Double mutant TAP proteins of the present invention are more preferred than single mutant TAP proteins. One mutation is at amino acid 1 (where the wild type TAP amino acid is tyrosine) and the other is at amino acid 10 (where the wild type TAP amino acid is aspartate). Amino acid 1 is substituted with a highly hydrophobic amino acid having an aromatic side chain. Preferred amino acids at position 1 are selected from the group of amino acids consisting of tryptophan, phenylalanine, or ⁇ - naphthylalanine. More preferably, the amino acid is tryptophan, which is more hydrophobic than tyrosine.
  • Amino acid 10 is substituted with a hydrophilic amino acid, preferably basic and positively charged and having a polar side chain.
  • Preferred amino acids at amino acid position 10 are selected from the group of amino acids consisting of arginine, lysine, and histidine. More preferably, the amino acid is arginine.
  • the most preferred double mutant protein (referenced hereinafter as "Y1W/D10R", or "TrpiArgiO-TAP”) has a substitution combination of amino acid 1 as tryptophan (replacing wild type tyrosine) and amino acid 10 as arginine (replacing wild type aspartate).
  • TAP proteins containing mutations in addition to mutations at positions 1 and 10 e.g. triple mutants containing mutations at positions 1 , 10, and another amino acid
  • the additional mutations are conservative or do not substantially affect the Factor Xa inhibitory properties of the double mutant protein or the rate of dissociation between the double mutant protein and Factor Xa.
  • European Publication 419099 is illustrative of mutation strategies which would be expected to provide sequences that retain activity.
  • Both single mutant TAP proteins and double mutant TAP proteins of the invention are more effective Factor Xa inhibitors than wild type TAP.
  • the proteins of the present invention have a significantly slower dissociation rate than the dissociation rate corresponding to the wild type TAP.
  • the most preferred protein, Y1W/D10R, is 14 times slower.
  • proteins of the present invention can be prepared according to the recombinant procedures described below.
  • the protein can be prepared by first preparing the minus 1 mutant of wild type TAP (amino acids 2-60 of wild type TAP) or the minus 1 mutant of TAP having an amino acid substitution for amino acid 10 of wild type TAP, as described above (amino acids 2-60 of a mutant TAP having arginine, lysine, or histidine at position 10). Thereafter, an activated, protected ⁇ -naphthylalanine derivative can be attached to amino acid 2 under proper protection and pH conditions.
  • the first prepared minus 1 mutant can also have an amino acid replacement for the amino acid at position 7 of wild type TAP.
  • wild type TAP lysine (Lys ⁇ -TAP, or wild type TAP) is replaced with arginine (Arg ⁇ -TAP).
  • Amino acid 10 may optionally be changed as described above. Thereafter, an activated, protected ⁇ -naphthylalanine derivative can be more conveniently attached to amino acid 2.
  • Standard protection procedures, activation procedures, coupling procedures, and deprotection procedures necessary for attaching an amino acid such as ⁇ -naphthylalanine to amino acid 2 in minus 1 mutants of rTAP would be obvious to a person skilled in the art in view various peptide synthesis publications such as Merrifield, J. Am. Chem. Soc, 85, 2149 (1964), Rivier et al, United States Patent 4,244,946 (1982), and Stewart and Young, "Solid Phase Peptide Synthesis", Pierce Chemical Company, Rockford, IL (1984).
  • Proteins of the invention include variations on the disclosed protein sequence or sequences which conserve the activity of the disclosed sequence or sequences, including fragments or subunits, naturally occurring mutations, allelic variants, randomly generated artificial mutants, and microheterogeneous forms. Fragments or subunits refers to any portion of the sequence which contains fewer amino acids than the complete protein, e.g. partial sequences excluding portions at the N- and/or C-termini of the complete protein.
  • Microheterogeneous forms refers to a single gene product, that is a protein produced from a single gene unit of DNA, which is structurally modified following translation. These structural modifications, however, do not result in any significant alterations of the activity of the protein. The modifications may take place either in vivo or during the isolation and purification process. In vivo modification may result in, but is not limited to, acetylation at the N-terminus, proteolysis, glycosylation or phosphorylation. Proteolysis may include exoproteolysis, wherein one or more terminal amino acids are sequentially, enzymatically cleaved to produce microheterogeneous forms having fewer amino acids than the original gene product.
  • Proteolysis may also include endoproteolytic modification that results from the action of endoproteases that cleave the peptide at specific sequence locations. Similar modifications, especially proteolytic modifications, can occur during the purification process which may result in the production of microheterogeneous forms.
  • hybrid proteins such as fusion proteins or proteins resulting from the expression of multiple genes within the expression vector, and may include a polypeptide having the specific activity of a disclosed protein linked by peptide bonds to a second polypeptide.
  • bifunctional proteins having a polypeptide sequence comprising the mutant rTAP protein and included therein the tripeptide sequence arginine-glycine-aspartic acid (RGD).
  • RGD arginine-glycine-aspartic acid
  • the bifunctional molecules are particularly suited for targeting proteins of the present invention to activated platelets where there is an assembled prothrombinase complex.
  • the prothrombinase complex composed of Factor Xa, Factor Va, an appropriate phospholipid surface and prothrombin, is responsible for the Factor Xa conversion of prothrombin to thrombin in vivo.
  • the phospholipid component of this complex can be supplied by any cellular surface.
  • platelets vesicularize into microparticles which expose Factor Va binding sites. These activated platelet micro particles are extremely active in supporting the prothrombinase complex.
  • Insertion of an RGD sequence in an appropriate location of a non-adhesive protein creates a protein capable of binding to integrins which recognize this peptide sequence such as the glycoprotein ⁇ b/IIIa protein (Maeda, T. et al. (1989), J. Biol. Chem. 264, 15165-15168). Upon activation, the platelet Hb/IIIa complex becomes exposed and is critical in the subsequent fibrinogen- mediated aggregation. Insertion of an RGD sequence in an appropriate region of mutant rTAP of the present invention by, for example, site- specific mutagenesis, creates a bifunctional molecule targeted to activated platelets where the prothrombinase complex is assembled.
  • Recombinant DNA technology may be used to produce proteins of the invention.
  • This technology allows segments of genetic information, DNA, from different cells, and usually from different organisms, to be joined end-to-end outside the organisms from which the DNA was obtained and to incorporate this hybrid DNA into a cell that will allow the production of the protein for which the original DNA encodes.
  • Genetic information, DNA or mRNA is isolated and incorporated into an appropriate cloning vector, and transduced into an appropriate host cell.
  • Cloning vectors useful for this technology include a DNA sequence which accommodates specific experimental foreign DNA. The vectors are introduced into host cells that can exist in a stable manner and express the protein dictated by the experimental DNA.
  • Cloning vectors may include plasmids, bacteriophage, viruses and cosmids.
  • Expression vectors are DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. These vectors can express either procaryotic or eucaryotic genes in a variety of cells such as bacteria, yeast, insect and mammalian cells.
  • Proteins may also be expressed in a number of virus systems.
  • a suitably constructed expression vector contains an origin of replication for autonomous replication in host cells, selective markers, a limited number of useful restriction enzyme sites, a high copy number, and strong promoters. Promoters are DNA sequences that direct RNA polymerase to bind to DNA and initiate RNA synthesis; strong promoters cause such initiation at high frequency.
  • Expression vectors may include, but are not limited to cloning vectors, modified cloning vectors and specifically designed plasmids or viruses.
  • Procaryotes most frequently are represented by various strains of E. coll Other microbial strains may be used, such as bacilli, e.g. Bacillus subtilis, various species of Pseudomonas, or other bacterial strains.
  • plasmid vectors which contain replication sites and control sequences derived from a species compatible with the host are used.
  • E. coli is typically transformed using derivatives of pBR322, a plasmid derived from an E. coli species by Bolivar et al, Gene (1977) 2:95.
  • procaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the ⁇ -lactamase (penicillinase) and lactose (lac) promoter systems (Chang et al, Nature (1977) 198:1056 and the tryptophan (Tip) promoter system (Goeddel et al, Nucleic Acids Res. (1980) £:4057) and the lambda-derived PL promoter and N-gene ribosome binding site (Shimatake et al, Nature (1981) 292:128).
  • promoters as the ⁇ -lactamase (penicillinase) and lactose (lac) promoter systems (Chang et al, Nature (1977) 198:1056 and the tryptophan (Tip) promoter system (Goeddel et al, Nucleic Acids Res. (19
  • Expression systems useful in eucaryotic systems of the invention comprise promoters derived from appropriate eucaryotic genes.
  • a class of promoters useful in yeast include promoters for synthesis of glycolytic enzymes, including those for 3- phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. (1980) 255:2073).
  • Other promoters include those from the enolase gene (Holland, M.J., et al., J. Biol. Chem. (1981) 256:1385) or the Leu2 gene obtained from YEpl3 (Broach, J., et al, Gene (1978) 8:121).
  • Mutant rTAP proteins of the present invention are preferably expressed in eukaryotic systems such as transformed yeast cell expression systems, more preferably transformed Saccharomyces cerevisiae strain DMY6. More preferably, the transformed yeast cell expression system is DMY6(TAP3).
  • Oligonucleotide primers are prepared which will hybridize to different strands of the desired sequence and at relative positions along the sequence such that an extension product synthesized from one primer, when it is separated from its template (complement), can serve as a template for extension of the other primer into a nucleic acid of defined length.
  • the primers may be prepared using any suitable method, such as, for example, the phosphotriester and phosphodiester methods, described respectively in Narang, S.A., et al. Meth. Enzymol, 68, 90 (1979) and Brown, E.L. et al., Meth. Enzymol 68, 109 (1979), or automated embodiments thereof.
  • diethylphosphoramidites are used as starting materials and may be synthesized as described by Beaucage et al, Tetrahedron Letters (1981), 22: 1859-1862.
  • One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Patent No. 4,458,066. It is also possible to use a primer which has been isolated from a biological source (such as a restriction endonuclease digest).
  • DNA coding for mutant rTAP proteins may be obtained using polymerase chain reaction (PCR) amplification techniques as described in Mullis et al, U.S. Patent No. 4,800,159.
  • PCR polymerase chain reaction
  • the nucleic acid sequence strands are heated until they separate, in the presence of oligonucleotide primers that bind to their complementary strand at a particular site of the template.
  • the primer template complexes act as substrate for DNA polymerase which, in performing its replication function, extends the primers.
  • the region in common with both primer extensions, upon denaturation, serves as template for a repeated primer extension.
  • This process is continued with a series of heating and cooling cycles, heating to separate strands, and cooling to reanneal and extend the sequences. More and more copies of the strands are generated as the cycle is repeated.
  • the coding domain and any additional primer-encoded information such as restriction sites or translation signals (signal sequences, start codons and/or stop codons) is obtained.
  • PCR polymerase chain reaction
  • Non-terminal location mutations can be generated at residue positions 9 and 23, resulting in replacement of arginine with asparagine.
  • Primers are prepared to amplify the TAP coding sequences and incorporate the mutations as before. The primers extend far enough to include restriction site sequences present in the TAP gene. Another set of primers is used to amplify remaining sequences of gene and the same restriction site. The resulting fragments are digested with that enzyme and ligated, reforming the TAP gene complete with the mutation.
  • Mutants can be prepared using a modification of the Kunkel method in which single strand p276-2E plasmid DNA was annealed with a complementary primer containing the desired mutagenic sequences.
  • the Kunkel method was specifically employed to obtain a wild type TAP mutation at residue position 27 which results in replacement of arginine by asparagine.
  • This complex was extended by DNA polymerase, covalently linked with DNA ligase and the product used to transform E. coli DH5 cells (Bethesda Research Laboratories). Mutation recovery was improved by selection against the parental strand using E. coli host CAG629 (dut-ung-BioRad) to prepare the single strand template.
  • This method is employed using the expression vector as the template for the mutagenesis reaction.
  • Plasmids containing the desired coding and control sequences employs standard ligation and restriction techniques which are well understood in the art. Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and religated in the form desired.
  • Site specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g. New England Biolabs, Product Catalog.
  • the suitable restriction enzyme or enzymes
  • about 1 ⁇ g of plasmid or DNA sequence is cleaved by one unit of enzyme in about 20 ⁇ l of buffer solution.
  • an excess of restriction enzyme is used to ensure complete digestion of the DNA substrate. Incubation times of about 1 to 2 hours at about 37°C are workable, although variations can be tolerated.
  • protein is removed by extraction with phenol/chloroform, and may be followed by running over a Sephadex® G-50 spin column.
  • size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is formed in Methods in Enzymology (1980) : 499-560.
  • Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates (dNTPs) using incubation times of about 15 to 25 minutes at 20 to 25°C in 50 mM Tris, pH 7.6, 50 mM NaCl, 6mM MgCl2, 6mM DTT and 5-
  • oligonucleotides may be prepared by the triester method of Matteucci, et al.(J. Am. Chem. Soc. (1981) 703:3185) or using commercially available automated oligonucleotide synthesizers.
  • kinasing of single strands prior to annealing or for labelling is achieved using an excess, e.g., approximately 10 units of polynucleotide kinase to 0.1 nmol substrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgCl2, 5 mM dithiothreitol, 1-2 mM ATP, 1.7 pmoles 32p-ATP(2.9 mCi/mmole), 0.1 mM spermidine, 0.1 mM EDTA.
  • an excess e.g., approximately 10 units of polynucleotide kinase to 0.1 nmol substrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgCl2, 5 mM dithiothreitol, 1-2 mM ATP, 1.7 pmoles 32p-ATP(2.9 mCi/mmole), 0.1 mM spermidine, 0.1 mM EDTA.
  • Ligations are performed in 15-30 ⁇ l volumes under the following standard conditions and temperatures: 20 mM Tris-HCl, pH 7.5, 10 mM MgC_2, 10 mM DTT, 33 ⁇ g/ml BSA, 10 mM-50 mM NaCl, and 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14°C (for "blunt end” ligation).
  • Intermolecular "sticky end” ligations are usually performed at 33-100 ⁇ g/ml total DNA concentrations (5-100 nM total end concentration).
  • Intermolecular blunt end ligations (usually employing a 10-30 fold molar excess of linkers) are performed at 1 ⁇ M total ends concentration.
  • vector construction employing "vector fragments,” the vector fragment is commonly treated with bacterial alkaline phosphatase (BAP) or calf intestinal phosphatase (CIP) in order to remove the 5' phosphate and prevent self-ligation of the vector.
  • BAP digestions are conducted at pH 8 in approximately 150 mM Tris, in the presence of Na+ and Mg2+ using about 1 unit of BAP per ⁇ g of vector at 60°C for about 1 hour.
  • the preparation is extracted with phenol/chloroform and ethanol precipitated and desalted by application to a Sephadex® G-50 spin column.
  • self-ligation can be prevented in vectors which have been double digested by-additional restriction enzyme digestion of the unwanted fragments.
  • Site specific primer directed mutagenesis is conducted using a primer synthetic oligonucleotide complementary to a single stranded plasmid or phage DNA to be mutagenized except for limited mismatching, representing the desired mutation.
  • the synthetic oligonucleotide is used as a primer to direct synthesis of a strand complementary to the phage, and the resulting double-stranded DNA is transformed into a phage-supporting host bacterium. Cultures of the transformed bacteria are plated in top agar, permitting plaque formation from single cells which harbor the phage.
  • 50% of the new plaques will contain the phage having, as a single strand, the mutated form; 50% will have the original sequence.
  • the resulting plaques are hybridized with kinased synthetic primer at a temperature which permits hybridization of an exact match, but at which the mismatches with the original strand are sufficient to prevent hybridization. Plaques which hybridize with the probe are then picked, cultured, and the DNA recovered.
  • transformation is done using standard techniques appropriate to such cells.
  • the calcium treatment employing calcium chloride, as described by Cohen, S.N., Proc. Natl. Acad. Sci. USA (1972) 69:21 10, or the RbCl method described in Maniatis et al, "Molecular Cloning: A Laboratory Manual” (1982) Cold Spring Harbor Press, p.254 is used for procaryotes or other cells which contain substantial cell wall barriers.
  • Infection with Agrobacterium tumefaciens (Shaw, C.H., et al, Gene (1983) 23:315) is used for certain plant cells.
  • Example 4 describes a procedure for expressing wild type recombinant TAP in yeast which involves synthesizing and constructing a recombinant gene encoding wild type TAP (described therein as "TAP-1 ”) based on the amino acid sequence of wild type TAP.
  • the synthetic gene was inserted into a yeast expression vector that allows for secretory expression. Yeast cells were transformed with the vector containing the synthetic gene.
  • the described synthetic gene was inserted into pKH4 to form pKH4 » TAP.
  • pKH4 Construction of pKH4 is described in Schultz, et al, Gene 61 (1987) 123-133, which is incorporated by reference.
  • the plasmid pJC197 (Schultz et al. Gene 54 (1987) 113-123) is an E. coli - S. cerevisiae shuttle vector which contains a unique BamHl cloning site between the yeast MF l pre-pro leader and transcriptional terminator, originally derived in Kurjan and Herskowitz (1982) ibid.
  • pJC197 was digested with EcoRl + Pstl, and the 0.7-kb Pstl-EcoRl fragment containing a portion of the MFal pre-pro-leader, a three-frame translational terminator, and MFal transcriptional terminator, was gel- purified.
  • GALlOp was isolated from YEP51 by digestion with Sau3A, flush-ending with Pollk, ligating with octameric BamHl linkers, and digestion with Sail.
  • the resulting 0.5-kb BamH -Sal fragment bearing GALlOp was gel purified and ligated to a 35-bp Sa ⁇ -Pstl synthetic oligodeoxynucleotide adapter encoding the first 11 bp of the MFal nontranslated leader plus the ATG and first 8 aa of the MFal pre-pro- leader.
  • the resulting 0.5-kb fragment was digested with BamHl, gel- purified, and ligated together with the aforementioned 0.7-kb Pstl- EcoRl fragment plus the 4.0kb EcoRl- BamHl vector fragment derived from pBR322.
  • the resulting plasmid, pKH207-l contains GALlOp fused to the MFal pre-pro-leader plus BamHl cloning site, translational termination codons, and MFal transcriptional terminator.
  • an expression cassette of GALlOp fused to the yeast MFal pre-pro-leader, a unique BamHl cloning site, translational termination codons in all three reading frames, and MFal transcriptional terminator sequence was inserted into the yeast shuttle vector pCl/1 (Rosenberg et al Nature 312 (1984) 77-80) which contains the yeast 2 ⁇ DNA sequence for stable propagation of the plasmid in yeast at high copy number, to form pKH4.
  • a 213-bp BamHl-Psil fragment encoding aa 9-79 of the ppL was prepared from the plasmid pa2 (Bayne et al, Gene 66 (1988) 235-244).
  • the plasmid p 2 contains a portion of the yeast MFal pre- pro sequence (79aa) modified at codons 80 and 81 to create a BamHl site 6 aa upstream from the KEX2 processing site.
  • the region corresponding to codon 9 (Pstl) of the ppL to the BamHl site of pKH4 was removed from pKH4 after digestion with BamHl followed by partial digestion with Pstl.
  • Plasmid pKH4 2 contains the yeast GAL10 promoter, a portion of the MFal pre-pro leader (79 aa), a three-frame translational terminator and MFal transcriptional terminator, the yeast LEU2 gene, yeast 2 ⁇ sequences, pBR322 derived sequences, including the ApR gene and the origin of DNA replication (on).
  • Fusion products produced from pKH4»TAP are proteolytically processed by the Lys- Arg-cleaving endopeptidase (KEX2) encoded by the KEX2 gene and products are secreted into culture medium.
  • KEX2 cleaves on the C- terminal side of Lys-Arg residues.
  • Diploid yeast strain DMY6 (Schultz, L.D. (1987) Gene 61: 123-133) was transformed with pKH4»TAP using standard protocols (Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75: 1929-1933).
  • One isolate was chosen and designated S. cerevisiae MY 2030 9718P281-3. The isolate was deposited with the American Type Culture Collection and identified ATCC No. 20984.
  • Cells were grown by inoculating a frozen stock culture of 281-3 on leucme-minus agar media for 3 days at 28°C prior to the inoculation of 500 ml of selective medium (5 x CM Leu ⁇ ).
  • the 500 ml seed culture was grown in a 2-1 Ehrlenmeyer flask for 16-18 hr at 28°C using a rotary shaker at 300 m prior to its inoculation into 2.5 1 of 5 x CM Leu ⁇ broth containing 4% (w/v) galactose in a 1.5 1 Bio-Flo HI fermentor (New Brunswick Scientific).
  • the fermentor was operated at 28°C, 900 ⁇ m, 2.5 1/min air for 110-120 hr.
  • Yeast cells were separated from broth (8 liters containing 1.69 gm. wild type recombinant TAP) of yeast culture (strain 281 -3) secreting wild type recombinant TAP by diafiltering the liquid through a 500,000 MWCO hollow fiber cartridge (Romicon, PM500, 5ft) using an Amicon DC 10 unit. The clarified broth was then diafiltered through a 100,000 MWCO hollow fiber cartridge (Amicon, H5P 100-43) until approximately 500 mL of liquid remained in the reservoir. The retained liquid was diafiltered with 8 liters of 20mM BIS-TRIS, pH 6.0. (Buffer A).
  • the effluent of the 100,000 MWCO fibers was adjusted to pH 6.0 and diluted with 63 liters of cold buffer A.
  • the diluted broth contained 1.67 gm of wild type recombinant TAP .
  • the diluted fermentation broth was divided into two 40 liter portions and wach was pumped at 2 liters/hr onto separate columns of Q-Sepharose Fast FlowTM (Pharmacia: 14 cm x 10 cm I.D.) that had been equilibrated with buffer A. After all the diluted broth had been pumped onto the columns they were each washed with 8 liters of Buffer A to finish eluting unbound materials.
  • the bound proteins were eluted with 16 liters linear gradient of 0-500 mM NaCl in Buffer A. Fractions of 400 mL were collected and monitored for A28O -Md inhibition of human Factor Xa. Recombinant wild type TAP eluted at 175 mM NaCl.
  • the fractions that contained wild type recombinant TAP were combined and diluted with four volumes of 50 mM sodium acetate, pH 4.0 (Buffer B) before being pumped at 2 liters /hr onto a column of S-Sepharose Fast FlowTM (Pharmacia; 24.5 cm x 7.5 cm I.D.) that had been equilibrated with Buffer B.
  • Buffer B 50 mM sodium acetate, pH 4.0
  • the fractions were monitored for A28O and human Factor Xa inhibitory activity and wild type recombinant TAP eluted at 190 mM NaCl.
  • the amount of wild type recombinant TAP eluted from the column was 1.58 gm.
  • the pooled fractions containing inhibitory activity were loaded directly onto a 4.5 x 30 cm prep-pak Ci HPLC column (Waters Associates) equilibrated with aqueous 0.1 % (v/v) CF3COOH at 50 ml/min using a Delta-Prep preparative HPLC system (Waters Assoc).
  • the purified recombinant polypeptide prepared by either method was found to be greater than 99% pure by analytical reverse phase Cjg HPLC, quantitative amino acid analysis and automated amino-terminal sequence analysis.
  • a typical specific content of the recombinant inhibitor was between 1 15 and 120 nmole/mg with a homogeneous ammo-terminus beginning with tyrosine.
  • the electrophoretic mobility of wild type recombinant TAP was identical to the native inhibitor as assessed sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions.
  • the Factor Xa inhibitory activity of the wild type recombinant TAP was similar to natural TAP with a K ⁇ determined to be 0.2 nM.
  • This work includes the construction of a chemically synthesized TAP gene from selected oligonucleotides and cloning of the gene in a suitable plasmid vector.
  • a mutant recombinant TAP gene (Sequence ID No. 1) was then constructed by digestion of the coding sequences with selected restriction enzymes to afford desired DNA fragments. The fragments were isolated and ligated with selected synthetic oligonucleotides and introduced into an appropriate plasmid vector for cloning and subsequent expression of a double mutant recombinant TAP having the protein sequence represented by T iArgiO-TAP (see Figure 1, and Sequence ID No. 2).
  • the DTAP open reading frame isolated from the yeast expression vector pKH4 «TAP was inserted into the vector pKH4-3B (Carty et al, Bitechnol. Lett. 1990 12, 879-994) in the following manner, to generate the plasmid p3B-TAP.
  • the pKH4-3B vector was digested with Sphl and the 3'- extensions were made flush-ended with T4-DNA polymerase. Following digestion with Bglll, the blunt-_5g/ ⁇ pKH4-3B vector was agarose gel purified, The wild type TAP gene was amplified by polymerase chain reaction with DNA oligomers that inco ⁇ orated a BamHl site downstream of the wild type TAP gene on the 3'-end and a 5'-blunt end beginning with the DNA sequence encoding the first amino acid of wild type TAP. The wild type TAP gene was agarose gel purified and ligated with the pKH4-3B vector to yield p3B-TAP.
  • the wild type TAP clone was used to transform E. coli strain CJ 236 [dut-, ung", thi", rel A-, pDJ105 (Cm r )] to produce single- stranded DNA template in the presence of helper phage M13K07 (Bio- Rad).
  • the single-stranded DNA served as the template for annealing of mutagenic primers.
  • Site-directed mutation of TAP was performed by using the Muta-gene Ml 3 in vitro mutagenesis kit (Bio-Rad) according to the Kunkel method.
  • the template was annealed with an oligonucleotide, 5'-GTC-GAT-CCA-GCG-ACG- CGG-TTT, extended by T4 DNA polymerase, and ligated.
  • the double strand DNA was used to transform E. coli strain DH5 (dut + , ung + ) in which the mutant sequence was propagated due to selective degradation of the uracil -containing parental strand.
  • the double mutant designated Y1W/D10R, which has a second mutation at the first amino acid (tyrosine) to tryptophan was produced by polymerase chain reaction (PCR) using the newly generated DIOR mutant DNA as template.
  • PCR polymerase chain reaction
  • the PCR product was digested with BamHl to generate a cohesive 3'-end and ligated into the Sph I (blunted by T4 DNA polymerase) and BglU sites of the vector pKH4- 3B.
  • the double mutant DNA was propagated in the E. coli strain DH5 similar to mutant DIOR.
  • the sequences of the double-stranded DNA of DIOR and Y1W/D10R produced by DH5 were confirmed using SequenaseTM (U.S. Biochemical Co ⁇ .).
  • the double-stranded DNA of Y1W/D10R was used to transform the Saccharomyces cerevisiae strain DMY6 (Mata/a, ade 1 , ura3-52, his3::GAL10p-GAL4-ura3, leu2-2, 112/leu2-04, cirYcir 0 ) according to the procedure of Hinnen et al (Proc. Natl Acad. Sci. US A 1978 75, 1929-1933) to form S. cerevisiae strain DMY6(TAP3). Transformation was selected on leu- minimal medium agar plates, and confirmed with colony hybridization using 3*2p_labeled, random primed TAP gene probe.
  • the combined solution was adjusted to pH 4.0 with glacial acetic acid, and loaded onto a column (2.5 x 6 cm) of SP-Sepharose fast flow resin (Pharmacia) that had been pre- equilibrated with the same buffer in a cold room.
  • the column was washed with 150 mL of acetate buffer, and then eluted with a 200 mL linear gradient from 0 to 1.2 M sodium chloride in acetate buffer, and fractions of 4 mL were collected. The fractions were assayed for Factor Xa inhibitory activity, and fractions containing rTAP double mutant were pooled.
  • the collected solution was concentrated to 5-10 mL using Amicon with a YM3 membrane.
  • the final purification was completed using reverse phase-HPLC with a semiprep C-18 peptide column (Vydac).
  • the HPLC column was eluted with 0.1% trifluoroacetic acid (TFA) at a flow rate of 2 mL/min for 5 minutes, then a linear gradient from 0 to 25% 0.1 % TFA in acetonitrile in 15 minutes, followed by another linear gradient from 25 to 40% TFA in acetonitrile in 30 minutes.
  • the eluant was monitored at 280 and 220 nm using a diode array UV/Visible detector.
  • Fractions containing rTAP double mutant protein were collected, concentrated, and subjected to a second HPLC purification.
  • the final purified rTAP mutant was lyophilized to dryness and dissolved in HPLC grade water.
  • the structure of the double mutant Yl W/D10R was confirmed by amino acid sequencing analysis.
  • the concentration of Y1W/D10R was determined by absorbance at 280 nm using an extinction coefficient of 22.2 mM-lcm ⁇ l calculated from the method of Gill et al. (Anal. Biochem. 1989, 182, 319-326) or quantitative amino acid analysis with norleucine as the internal standard.
  • Human Factor Xa was activated from human Factor X (purchased from Haematologic Technologies Inc.) by Russell's viper venom Factor X activator by the procedure developed by Bock et al. (Arch. Biochem. Biophys. 1989 273, 375-388).
  • the concentration of Factor Xa was determined by absorbance at 280nm using an extinction coefficient of 1.16 (mg/mL)"l and molecular weight of 46,000 (DiScipio et al, Biochemistry 1977 16, 698-706) and confirmed by active-site titration (Bock et al 1989).
  • Table I lists inhibition constants (Ki) for the action of the double mutant and wild-type rTAP on human Factor Xa.
  • the inhibition constant for wild-type rTAP is similar to the literature value (Jordan et al. 1992).
  • the double mutant shows an enhanced inhibitory potency relative to that of wild-type rTAP.
  • both wild-type and double mutant rTAP exhibited the same association rate constant, k 0 n> (Table II). Therefore the increased inhibition potency indicates a slower dissociation rate for the YlW/D10R:Factor Xa complex.
  • the mutant rTAP of the invention can be used to inhibit blood coagulation inhibition by suitable administration to a patient in need of such treatment.
  • the amount of mutant rTAP which would normally be administered is primarily dependent upon the physical characteristics of the recipient and the severity of the condition.
  • the amount to be administered must be an effective amount, that is, an amount which is medically beneficial but does not present toxic effects which overweigh the advantages which accompany its use.
  • the preferable route of administration is parenteral, especially intravenous. Intravenous administration of the mutant rTAP in solution with normal physiologic saline is illustrative.
  • the protein inhibits coagulation by inhibiting the Factor Xa pathway.
  • Factor Xa pathway inhibition is achieved by administering the protein, either by continuous intravenous administration or bo.lus administration, in combination with a suitable pharmaceutical composition carrier e.g. saline, at a suitable pH, e.g. 7.4, such that the composition achieves the desired effect of preventing Factor Xa from inducing formation of thrombin from prothrombin.
  • the proteins form pharmaceutically acceptable salts with any non-toxic, organic or inorganic acid.
  • inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulphuric and phosphoric acid and acid metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate.
  • organic acids which form suitable salts include the mono, di and tricarboxylic acids.
  • Illustrative of such acids are, for example, acetic, glycolic, lactic, pyruvic, malonic, succinic, trifluroacetic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicylic, 2-phenoxybenzoic and sulfonic acids such as methane sulfonic acid and 2-hydroxyethane sulfonic acid.
  • Salts of the carboxy terminal amino acid moiety include the non-toxic carboxylic acid salts formed with any suitable inorganic or organic bases.
  • these salts include those of alkali metals, as for example, sodium and potassium; alkaline earth metals, such as calcium and magnesium; light metals of Group IILA including aluminium; and organic primary, secondary and tertiary amines, as for example, trialkylamines, including triethylamine, procaine, dibenzylamine, 1-ethenamine; N,N'-dibenzylethylenediamine, dihydroabietylamine, N-(lower)alkylpiperidine, and any other suitable amine.
  • alkali metals as for example, sodium and potassium
  • alkaline earth metals such as calcium and magnesium
  • light metals of Group IILA including aluminium and organic primary, secondary and tertiary amines, as for example, trialkylamines, including triethylamine, procaine, dibenzylamine, 1-ethenamine; N,N'-dibenzylethylenediamine, dihydroabietylamine, N-(lower
  • the anticoagulant dose of the proteins is from 0.01 mg/kg to 50 mg/kg of patient body weight per day depending on, for example, the patient, and the severity of the thrombotic condition to be treated.
  • the suitable dose for a particular patient can be readily determined.
  • Preferably from 1 to 4 daily doses would be administered typically with from 0.70 mg to 3.5 g of active compound per dose.
  • the concentration of the proteinaceous substance of this invention having Factor X a inhibition activity required to inhibit Factor X a when used to inhibit blood coagulation or Factor X a in a medium such as stored blood can be readily determined by those skilled in the art.
  • Anticoagulant therapy is indicated for the treatment and prevention of a variety of thrombotic conditions, particularly coronary artery and cerebrovascular disease. Those experienced in this field are readily aware of the circumstances requiring anticoagulant therapy.
  • the term "patient” used herein is taken to mean mammals such as primates, including humans, sheep, horses, cattle, pigs, dogs, cats, rats, and mice.
  • Inhibition of Factor Xa is useful not only in the anticoagulant therapy of individuals having thrombotic conditions, but is useful whenever inhibition of blood coagulation is required such as to prevent coagulation of stored whole blood and to prevent coagulation in other biological samples for testing or storage.
  • the proteinaceous substance of this invention having Factor Xa inhibition activity can be added to or contacted with any medium containing or suspected of containing Factor Xa and in which it is desired that blood coagulation be inhibited.
  • the proteinaceous substance of this invention having Factor Xa inhibition activity may survive passage through the gut following oral administration, applicants prefer non-oral administration, for example, subcutaneous, intravenous, intramuscular or intraperitoneal administration by depot injection, or by implant preparation.
  • parenteral administration the proteinaceous substance of this invention having Factor Xa inhibition activity may be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier which can be a sterile liquid such as water and oils with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants.
  • oils which can be employed in these preparations are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil.
  • water, saline, aqueous dextrose and related sugar solutions, ethanol and glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • the proteinaceous substance of this invention having Factor Xa inhibition activity can be administered in the form of a depot injection or implant preparation which may be formulated in such a manner as to permit a sustained release of the active ingredient.
  • the active ingredient can be compressed into pellets or small cylinders and implanted subcutaneously or intramuscularly as depot injections or implants.
  • Implants may employ inert materials such as biodegradable polymers or synthetic silicones, for example, Silastic, silicone rubber or other polymers manufactured by the Dow-Corning Co ⁇ oration.
  • mutant rTAP enhances the efficiency of thrombolytic agents and mediated thrombolytic reperfusion.
  • thrombolytic agents include, for example, tissue plasminogen activator, streptokinase, urokinase, and anisoylated plasminogen-streptokinase activator complex.
  • Anisoylated plasminogen-streptokinase activator complex is commercially available from SmithKline Beecham as Anistreplase®.
  • Mutant rTAP may be administered first following thrombus formation, and the thromboltyic agent is administered thereafter.
  • the deposit was made December 1 , 1992 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Pu ⁇ oses of Patent Procedure and the Regulations thereunder (Budapest Treaty). Maintenance of a viable culture is assured for 30 years from date of deposit.
  • the organisms will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Applicants and ATCC which assures unrestricted availability upon issuance of the pertinent United States patent. Availability of the deposited strains is not to be construed as a license to practice the invention in contravention rights granted under the authority of any government in accordance with its patent laws.
  • Table III describes the sequences listed on the following pages.
  • the shorthand notation references changes at amino acid positions 1 and 10 of the wild type TAP sequence.
  • the sequence of wild type TAP is illustrated in Figure 1 , with the exception that Figure 1 shows tryptophan at position 1 (instead of wild type tyrosine) and arginine at position 10 (instead of wild type asparate). Wild type TAP is also described in European Publication 419099.
  • Xaa a nonnaturally occurring amino acid, represents ⁇ -naphthyl alanine.
  • MOLECULE TYPE DNA (genomic)
  • Cys Asp Ser Phe Trp lie Cys Pro Glu Asp His Thr Gly Ala Asp Tyr 35 40 45
  • MOLECULE TYPE protein
  • Cys Asp Ser Phe Trp lie Cys Pro Glu Asp His Thr Gly Ala Asp Tyr 35 40 45
  • Cys Asp Ser Phe Trp lie Cys Pro Glu Asp His Thr Gly Ala Asp Tyr 35 40 45
  • MOLECULE TYPE protein
  • Cys Asp Ser Phe Trp lie Cys Pro Glu Asp His Thr Gly Ala Asp Tyr 35 40 45
  • Cys Asp Ser Phe Trp lie Cys Pro Glu Asp His Thr Gly Ala Asp Tyr 35 40 45
  • MOLECULE TYPE protein
  • Cys Asp Ser Phe Trp lie Cys Pro Glu Asp His Thr Gly Ala Asp Tyr 35 40 45
  • Cys Asp Ser Phe Trp lie Cys Pro Glu Asp His Thr Gly Ala Asp Tyr 35 40 45
  • MOLECULE TYPE protein
  • Cys Asp Ser Phe Trp lie Cys Pro Glu Asp His Thr Gly Ala Asp Tyr 35 40 45
  • Cys Asp Ser Phe Trp lie Cys Pro Glu Asp His Thr Gly Ala Asp Tyr 35 40 45

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Abstract

Protéines de TAP mutantes recombinées inhibant le Facteur Xa et la formation de thrombus. Ces protéines sont des protéines mutantes dirigées sur le site du TAP de type sauvage, qui est un agent inhibiteur du facteur Xa présent dans l'extrait de tique Ornithodoros moubata.
PCT/US1993/011154 1992-12-01 1993-11-17 Proteines anticoagulantes Ceased WO1994012204A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000053264A1 (fr) * 1999-03-11 2000-09-14 Du Pont Pharmaceuticals Company Traitement de la thrombose au moyen d'une combinaison d'un inhibiteur du facteur xa et d'aspirine, d'un activateur tissulaire du plasminogene (tpa), d'un antagoniste de gpiib/iiia, d'heparine a faible masse moleculaire ou d'heparine
US6794412B1 (en) 1999-03-11 2004-09-21 Bristol-Myers Squibb Pharma Company Treatment of thrombosis by combined use of a factor Xa inhibitor and aspirin

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0419099A1 (fr) * 1989-09-07 1991-03-27 Merck & Co. Inc. Protéines ayant des propriétés anticoagulantes
US5189019A (en) * 1990-04-23 1993-02-23 Merck & Co., Inc. Antistasin derived anticoagulant protein

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0419099A1 (fr) * 1989-09-07 1991-03-27 Merck & Co. Inc. Protéines ayant des propriétés anticoagulantes
US5189019A (en) * 1990-04-23 1993-02-23 Merck & Co., Inc. Antistasin derived anticoagulant protein

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF MOLECULAR BIOLOGY, Volume 188, issued 1986, W.R. TAYLOR, "Identifiction of Protein Sequence Homology by Consensus Template Alignment", pages 233-258. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 265, Number 29, issued October 1990, NEEPER et al., "Characterization of Recombinant Tick Anticoagulant Peptide", pages 17746-17752. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000053264A1 (fr) * 1999-03-11 2000-09-14 Du Pont Pharmaceuticals Company Traitement de la thrombose au moyen d'une combinaison d'un inhibiteur du facteur xa et d'aspirine, d'un activateur tissulaire du plasminogene (tpa), d'un antagoniste de gpiib/iiia, d'heparine a faible masse moleculaire ou d'heparine
US6794412B1 (en) 1999-03-11 2004-09-21 Bristol-Myers Squibb Pharma Company Treatment of thrombosis by combined use of a factor Xa inhibitor and aspirin

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