+

WO1994007515A1 - Thromboplastine tissulaire mutante depourvue d'activite activant le facteur vii - Google Patents

Thromboplastine tissulaire mutante depourvue d'activite activant le facteur vii Download PDF

Info

Publication number
WO1994007515A1
WO1994007515A1 PCT/US1993/009570 US9309570W WO9407515A1 WO 1994007515 A1 WO1994007515 A1 WO 1994007515A1 US 9309570 W US9309570 W US 9309570W WO 9407515 A1 WO9407515 A1 WO 9407515A1
Authority
WO
WIPO (PCT)
Prior art keywords
mhutf
factor
vila
mutant
seq
Prior art date
Application number
PCT/US1993/009570
Other languages
English (en)
Inventor
Wolfram Ruf
Thomas S. Edgington
Original Assignee
The Scripps Research Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Scripps Research Institute filed Critical The Scripps Research Institute
Publication of WO1994007515A1 publication Critical patent/WO1994007515A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)
    • G01N2333/96441—Serine endopeptidases (3.4.21) with definite EC number
    • G01N2333/96447—Factor VII (3.4.21.21)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
    • G01N2405/04—Phospholipids, i.e. phosphoglycerides

Definitions

  • the present invention relates to a functional mutant of human tissue factor (mhuTF) that binds Factor VII or activated Factor VII (Vila) and can proteolytically convert Factor X into activated Factor X (Xa) , but does not significantly proteolytically activate Factor VII.
  • the mutant human tissue factor is useful as a diagnostic and therapeutic reagent.
  • TF tissue factor
  • TF tissue factor
  • Vila serine protease Factor Vila
  • a mediator for the auto-activation of VII to Vila ⁇ akagaki et al., Biochemistry 30: 10819-10824 ⁇ 1991.
  • TF tissue factor
  • the assays for Factor Vila are typically a clotting assay in which patient plasma sample is added to a Factor VII deficient plasma. The addition of a thromboplastin (as a source of wild type tissue factor) and calcium drive the clotting reaction. Thereafter, a standard curve of clotting time versus "percent factor Vila" is constructed using pooled normal plasma, whereupon patient values are interpolated from the curve. Examples of typical Factor Vila assays are described by Miller et al., British J. Haematol..
  • the conventional assays have a number of significant limitations.
  • the performance of a clotting assay with standard thromboplastin is not only dependent upon Factor Vila, but is also influenced by the concentration of all other vitamin K-dependent clotting factors that might participate in the clotting assay that are present in the Factor Vila deficient plasma.
  • human tissue factor huTF contains sites defined by amino acid residues which, when modified, can alter the function of huTF to selectively inactivate the Factor VII activation activity of the huTF:VIIa complex without affecting the ability of the modified huTF to bind Factor VII or Vila or the ability of modified huTF:VIIa complex to activate Factor X.
  • the modified (mutant) human tissue factor is referred to as huTF.
  • a region of huTF around the structural loop defined by amino acid residues 157-167 (SEQ ID NO 2) is shown herein to not be required for high affinity binding to Factor Vila, whereas amino acid residues in the region are important for the proteolytic activation of Factor VII to Vila.
  • mutations in one or more of the residues in the region of huTF defined by the amino acid residues 106 to 219 shown in SEQ ID NO 2 are desirable for the introduction of the functional defect defined herein.
  • the selective elimination of the ability of huTF to activate Factor VII is advantageous for use in clinical hospital clotting assays, and in particular for assays to measure the amount of activated factor
  • the mutant huTF described herein provides particular advantages for use as a thromboplastin reagent. These advantages include the ready use in a standard coagulation assay without the complications of the other vitamin K-dependent proteins typically present because the assay biochemistry relies on the typical capacity of the mhuTF:Factor Vila complex to activate Factor X without "feedback" interference by the production of Factor Vila during the assay. The selective loss of Factor VII activation allows precision measurement in a standard curve over at least 4 logs of Factor Vila concentrations.
  • the mhuTF may be phospholipid reconstituted as described herein for use as a standard thromboplastin reagent, and may readily be adapted for use in standard analytical equipment.
  • the invention describes a mutant human tissue factor protein (mhuTF) having the capacity to bind Factor Vll/VIIa and to proteolytically hydrolyze Factor X, but being substantially free of the capacity to activate Factor VII when present in a complex of mhuTF:VIIa.
  • mhuTF human tissue factor protein
  • a related embodiment describes a composition that comprises a mutant human tissue factor protein of this invention.
  • Preferred compositions further contain liposomes, cryopreservatives, and/or detergents.
  • the invention describes a method for detecting the amount of Factor Vila in a body fluid sample comprising the steps of: a) admixing a preselected amount of the body fluid sample with a clotting assay admixture, wherein the clotting assay admixture comprises a mutant human tissue factor (mhuTF) composition of this invention and is substantially free of Factor Vila and wild type human tissue factor, to form a Factor Vila assay admixture; b) maintaining the Factor Vila assay admixture under conditions sufficient for the mhuTF to bind to any of the Factor Vila present in the sample and form a clot; and c) determine the amount of time required for the clot to form, which time is proportional to a predefined amount of Factor Vila, thereby determining the amount of Vila present in the sample.
  • mhuTF mutant human tissue factor
  • Figure 1 is a schematic representation of residues 151-172 in tissue factor (TF) . Alignment to strand C according to Bazan, Proc. Natl. Acad. Sci.. USA. 87:6934-6938 (1990) is indicated and the functionally important residues are highlighted. The single letter code for amino acids is used.
  • TF tissue factor
  • Figure 2 illustrates the VII binding to TF A161D162A163 * Specific binding of VII to cell surface TF A161D162A163 (Figure 2A) and wild-type TF ( Figure 2B) is shown. The insets give the Scatchard analysis for the same data obtained in a representative experiment. The binding assay is described in Example 3.
  • Figure 3 illustrates the amidolytic and proteolytic activity of mutant TF-VIIa complexes.
  • Figure 3A the cleavage of small peptidyl substrates was assessed with Spectrozyme FXa in the presence of 10 nM Vila and 5 nM wild-type or mutant TF.
  • the rate of hydrolysis of the peptidyl substrate was determined in a 200 ul reaction with a kinetic plate reader and is given as the increase in absorbance (mOD/min) .
  • Figure 3B the activation of Factor X (1 uM) by mutant and wild-type TF in the presence of excess Vila (5 nM) was determined at 37C.
  • the rate of Xa formation per enzymatic unit TF-VIIa was calculated based on the TF concentration in the assay. Mean and standard deviation calculated for three independent experiments are shown in both panels.
  • the amidolytic and proteolytic assays are described in Example 3.
  • Figure 4 shows the effect of Vila on the specific clotting activity of mutant and wild-type TF.
  • amino acid residue refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages.
  • the amino acid residues described herein are preferably in the "L” iso eric form.
  • residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
  • amino acid residue sequences represented herein by formulae have a left- to-right orientation in the conventional direction of amino terminus to carboxy terminus.
  • amino acid residue is broadly defined to include the amino acids listed in the Table of Correspondence and modified and unusual amino acids, such as those ⁇ listed in 37 CFR 1.822(b)(4), and incorporated herein by reference.
  • a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or a covalent bond to an amino-terminal group such as NH 2 or acetyl or to a carboxy-terminal group such as COOH.
  • rDNA Recombinant DNA
  • a recombinant DNA molecule refers to a DNA molecule produced by operatively linking two DNA segments.
  • a recombinant DNA molecule is a hybrid DNA molecule comprising at least two nucleotide sequences not normally found together in nature. rDNA's not having a common biological origin, i.e., evolutionarily different, are said to be “heterologous”.
  • Vector refers to a rDNA molecule capable of autonomous replication in a cell and to which a DNA segment, e.g., gene or polynucleotide, can be operatively linked so as to bring about replication of the attached segment.
  • Vectors capable of directing the expression of genes encoding for one or more polypeptides are referred to herein as "expression vectors". Particularly important vectors allow convenient expression of a mhuTF protein of this invention.
  • BHT butyrated hydroxytoluene
  • CHAPS 3-[ (3-cholamidopropyl)-dimethylammonio]-l-propanesulfo nate.
  • MOPS refers to 3-(N-morpholino)-propanesulfonic acid.
  • OTG refers to octyl beta-D-thioglucopyranoside.
  • Phospholipid refers to an organic molecule derived from either glycerol (most commonly) or sphingosine. Phospholipids derived from glycerol (or phosphoglycerides) comprise a glycerol backbone, two fatty acid chains esterified to the first and second carbons of the glycerol and phosphoric acid esterified to the third carbon. Optionally, an alcohol moiety is esterified to the phosphoric acid.
  • PC refers to phosphatidyl choline, an uncharged phosphoglyceride having an alcohol moiety derived from choline is esterified to the phosphoric acid.
  • PE refers to phosphatidyl ethanolamine, a positively charged phosphoglyceride, having an alcohol moiety derived from ethanolamine is esterified to the phosphoric acid.
  • PG refers to phosphatidyl glycerol, a negatively charged phosphoglyceride, having an alcohol moiety derived from glycerol is esterified to the phosphoric acid.
  • PS refers to phosphatidyl serine, a negatively charged phosphoglyceride, having an alcohol moiety derived from serine is esterified to the phosphoric acid.
  • Prothrombin time is abbreviated as PT and refers to the time interval between the addition of a thromboplastin or prothrombin time reagent and the appearance of a clot in platelet poor, citrated plasma.
  • Prothrombin ratio is abbreviated as PR and refers to the prothrombin time of an individual's plasma (either normal or abnormal) divided by the prothrombin time of pool of normal individual plasmas.
  • rTF refers to recombinant tissue factor.
  • TBS refers to 20 mM Tris (pH 7.5) containing
  • the invention describes a modified (mutant) human tissue factor protein, designated mhuTF, which has the desirable properties of:
  • mhuTF protein has the ability to bind VII or Vila at concentrations of Vll/VIIa of about 0.1 to 60 nanomolar (nM) , preferably 0.5 to 30 nM, and when bound provides a mhuTF:VIIa complex which is capable of hydrolysis of Factor X.
  • nM nanomolar
  • Vll/VIIa is used. Representative binding assays for detecting the ability of mhuTF to bind to Vll/VIIa are described in Example 3.
  • Factor VII or recombinant Vila for use in a binding assay are generally available, at least from Novo Nordisk, Inc. , (Gentofte, Denmark) .
  • Exemplary assays for measuring the capacity for hydrolysis of Factor X are described in Example 3, and can alternatively include the use of Factor X-related peptide substrates such as the chromogenic substrates Spectrozyme FXa, peptide S- 2288, and the like, available from American Diagnostica, Inc. (Greenwich, CT) .
  • the proteolytic conversion of VII to Vila when mhuTF is present in a mhuTF:VIIa complex, is considered to be insignificant if the inhibition of conversion of VII to Vila, when measured as described in Example 3, is at least 70%, preferably is at least 90%, and more preferably is about 95-100%, as compared to the rate of conversion by wild type huTF. Stated differently, mhuTF is substantially free of the ability to activate Factor VII when significant activation of Factor VII does not occur in the presence of a mhuTF:VIIa complex. Assays for measuring the proteolytic conversion of Factor VII to Vila by a mhuTF:VIIA or huTF:VIIa complex are described in Example 3.
  • the invention contemplates a mutant human tissue factor protein (mhuTF) having the capacity to bind Factor Vll/VIIa and to proteolytically hydrolyze Factor X, but being substantially free of the capacity to activate Factor VII when present in a complex of mhuTF:VIIa.
  • mhuTF human tissue factor protein
  • a preferred mhuTF protein contains one or more mutations in the amino acid residue sequence within the region of huTF at amino acid residues 106 to 219 shown in SEQ ID NO 2 of the mature huTF.
  • the complete amino acid residue sequence of mature wild type huTF is shown in SEQ ID NO 2.
  • Preferred mutations are substitutions of a native (wild type) amino acid residue for an alternate residue, thereby altering the primary amino acid residue sequence of wild type huTF.
  • a substitution is indicated by listing the substituted residue in single letter code together with the residue position number in the wild type huTF sequence.
  • a 157 a mhuTF protein having a substitution of alanine (A) for tyrosine (Y) at residue position number 157 is designated as A 157 .
  • Particularly preferred are the mhuTF proteins described herein at Example 3, where specific amino acid substitutions were made in the wild type huTF to produce a modified huTF (mhuTF) having the properties described herein.
  • tissue factor from species other than human are highly related both structurally and in terms of primary sequence
  • the invention also contemplates mutant tissue factor having the characteristics of mhuTF which are derived from other mammals, including cow, rat, rabbit, mouse, pig, primates, and the like.
  • the primary amino acid residue sequence of non-human tissue factor is known for a variety of the recited mammalian species, including rabbit, mouse and cow. See, for example, Hartzell et al., Mol. Cell. Biol.. 9:2567-2573, 1989; Andrews et al., Gene. 98:265-269, 1991; and Takayeniki et al., Biochem. Biophys. Res. Co m.. 181:1145-1150, 1991.
  • the amino acid residue sequence of a protein or polypeptide is directly related via the genetic code to the deoxyribonucleic acid (DNA) sequence of the structural gene that codes for the protein.
  • a structural gene or DNA segment can be defined in terms of the amino acid residue sequence, i.e., protein or polypeptide, for which it codes.
  • An important and well known feature of the genetic code is its redundancy. That is, for most of the amino acids used to make proteins, more than one coding nucleotide triplet (codon) can code for or designate a particular amino acid residue. Therefore, a number of different nucleotide sequences may code for a particular amino acid residue sequence.
  • the DNA segments of the present invention are characterized as including a DNA sequence that encodes a mutated human tissue factor heavy chain protein (mhuTFh) according to the present invention. That is, the DNA segments of the present invention are characterized by the presence of a mhuTF structural gene. Preferably the gene is present as an uninterrupted linear series of codons where each codon codes for an amino acid residue found in the mhuTFh protein, i.e., a gene free of introns.
  • mhuTFh mutated human tissue factor heavy chain protein
  • One preferred embodiment is a DNA segment that codes an amino acid residue sequence that defines a mhuTF protein corresponding in sequence to a wild type huTF protein except that the amino acid residue sequence has at least one of the substitutions selected from the group consisting of R 158 G 160 , A 159 ,
  • a preferred DNA segment codes for an amino acid residue sequence consisting essentially of the sequence shown in SEQ ID NO 2 except that the sequence shown contains at least one of the substitutions selected from the group consisting of
  • Homologous DNA and RNA sequences that encode the above mhuTF are also contemplated.
  • DNA segments i.e., synthetic oligonucleotides
  • DNA segments that encode mhuTF proteins
  • chemical techniques for example, the phosphotriester method of Matteucci, et al. , (J. Am. Chem. Soc.. 103:3185-3191, 1981) or using automated synthesis methods.
  • larger DNA segments can readily be prepared by well known methods, such as synthesis of a group of oligonucleotides that define the DNA segment, followed by hybridization and ligation of oligonucleotides to build the complete segment.
  • DNA segments consisting essentially of structural genes encoding a mhuTF protein can be obtained from recombinant DNA molecules containing a gene that defines huTF, and can be subsequently modified, as by site directed mutagenesis, to introduce the desired substitutions.
  • Site-specific primer-directed mutagenesis is now standard in the art, and is conducted using a primer synthetic oligonucleotide complementary to a single- stranded 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 that harbor the phage.
  • any of the described DNA segments that code a mhuTF described herein by starting, for example, with the expression vector shown in SEQ ID NO 1 that codes and expresses wild type huTF, and mutating selected nucleotides, as described herein, to form one or more of the DNA segments that code a mhuTF of this invention.
  • the invention contemplates a recombinant DNA molecule (rDNA) containing a DNA segment of this invention.
  • rDNA recombinant DNA molecule
  • a rDNA can be produced by operatively linking a vector to a DNA segment of the present invention.
  • a vector refers to a DNA molecule capable of autonomous replication in a cell and to which another DNA segment can be operatively linked so as to bring about replication of the attached segment.
  • a vector capable of directing the expression of a mhuTF gene is referred to herein as an "expression vector".
  • a recombinant DNA molecule is a hybrid DNA molecule comprising at least two nucleotide sequences not normally found together in nature.
  • a vector contemplated by the present invention is at least capable of directing the replication, and preferably also expression, of the mhuTF structural gene included in DNA segments to which it is operatively linked.
  • a vector contemplated by the present invention includes a procaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a procaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
  • procaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
  • procaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant
  • Those vectors that include a procaryotic replicon can also include a procaryotic promoter capable of directing the expression (transcription and translation) of the mhuTF gene in a bacterial host cell, such as E. coli. transformed therewith.
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
  • vector plasmids Typical of such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA) and pPL and pKK223 available from Pharmacia, Piscataway, N.J.
  • Expression vectors compatible with eucaryotic cells preferably those compatible with vertebrate cells, can also be used to form the recombinant DNA molecules of the present invention.
  • Eucaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment.
  • the eucaryotic cell expression vectors used to construct the recombinant DNA molecules of the present invention include a selection marker that is effective in an eucaryotic cell, preferably a drug resistance selection marker.
  • a preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (neo) gene.
  • the selectable marker can be present on a separate plasmid, and the two vectors are introduced by co-transfection of the host cell, and selected by culturing in the appropriate drug for the selectable marker. Exemplary is the co-transfection described in the Examples.
  • the invention also contemplates a host cell transformed with a recombinant DNA molecule of the present invention.
  • the host cell can be either procaryotic or eucaryotic, although eucaryotic cells are preferred.
  • Eucaryotic cells useful for expression of a mhuTF protein are not limited, so long as the cell line is compatible with cell culture methods and compatible with the propagation of the expression vector and expression of the mhuTF gene product.
  • Preferred eucaryotic host cells include yeast and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line.
  • Preferred eucaryotic host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, baby hamster kidney cells (BHK) , and the like eucaryotic tissue culture cell lines.
  • CHO-K1 cell line is particularly preferred and exemplary. Transformation of appropriate cell hosts with a recombinant DNA molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of procaryotic host cells, see, for example, Cohen et al., Proc. Natl. Acad. Sci. USA. 69:2110 (1972); and Maniatis et al., Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982) . With regard to transformation of vertebrate cells with vectors containing rDNAs, see, for example, Graham et al., Virol.. 52:456 (1973); Wigler et al., Proc.
  • Successfully transformed cells i.e., cells that contain a rDNA molecule of the present invention
  • cells resulting from the introduction of an rDNA of the present invention can be cloned to produce monoclonal colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using a method such as that described by Southern, J. Mol. Biol.. 98:503 (1975) or Berent et al., Biotech.. 3:208 (1985).
  • successful transformation can be confirmed by well known immunological methods when the rDNA is capable of directing the expression of mhuTF, or by the detection of the biological activity of mhuTF.
  • cells successfully transformed with an expression vector produce proteins displaying mhuTFh antigenicity or biological activity.
  • Samples of cells suspected of being transformed are harvested and assayed for either mhuTFh biological activity or antigenicity.
  • the present invention also contemplates a culture of those cells, preferably a monoclonal
  • the culture also contains a protein displaying mhuTF antigenicity or biologically activity.
  • Nutrient media useful for culturing transformed host cells are well known in the art and can be obtained from several commercial sources.
  • a "serum-free" medium can be used. Preferred is the culturing conditions described herein.
  • Mutant human tissue factor (mhuTF) of this invention can be produced by a variety of means, and such production means are not to be considered as limiting.
  • Preparation of a mhuTF typically comprises the steps of: providing a DNA segment that codes a mhuTF protein of this invention; introduction of the provided DNA segment into an expression vector; introduction of the vector into a compatible host cell; culturing the host cell under conditions sufficient for expression of the mhuTF protein; and harvesting the expressed mhuTF protein from the host cell.
  • the harvested mhuTF is reconstituted into phospholipids as described herein to form a composition containing mhuTF.
  • the purification of mhuTF can be conducted by a variety of art-recognized procedures for preparing purified huTF from cell culture. See, in particular, the purification procedure described herein.
  • a mhuTF protein is prepared using a DNA segment as described herein.
  • additional substitutions (mutations) other than those described specifically herein can be readily designed to form a mhuTF having the disclosed biological activities.
  • a mutant human tissue factor protein (mhuTF) of the invention is typically provided in one or more of a variety of compositional forms suitable for the contemplated use.
  • mhuTF retains its biological activity in a variety of buffers and solutions, it is preferred to be formulated in a mild detergent or phospholipid composition.
  • Particularly preferred are phospholipid compositions which afford maximum stability and biological activity of the mhuTF in the composition.
  • Such phospholipid compositions are preferably formulated to form liposome compositions, as are generally well known in the art.
  • the composition contains an amount of biologically active mhuTF suitable for its contemplated use.
  • the phospholipid composition comprises liposo es having mhuTF associated with the lipid bilayer of the liposomes, such that the mhuTF is inserted through the lipid bilayer.
  • the lipid bilayer of the liposomes comprises phospholipids, preferably, phosphoglycerides.
  • mhuTF compositions which comprise phospholipid micelle compositions which have mhuTF associated with phospholipid micelles such that the mhuTF factor is inserted into the micelle.
  • the mhuTF compositions of the present invention comprise about 0.1 mg to about 3 mg of mhuTF per mg of phospholipid mixture.
  • the ratio of mhuTF to phospholipid mixture may determine the sensitivity of the resulting reagent.
  • use of a ratio of about 1 to 2 mg mhuTF per mg phospholipid mixture may be suitable for a mhuTF reagent having a International Sensitivity Index ("ISI") of about 1.0.
  • ISI International Sensitivity Index
  • Use of a ratio of about 0.25 to about 0.5 mg mhuTF per mg phospholipid mixture may be suitable to prepare a composition having an ISI of about 1.6 to about 2.0.
  • the reagent preferably includes a cryopreservative, preferably a carbohydrate preservative, most preferably trehalose.
  • Suitable phospholipids for use in the liposome compositions of the present invention include those which contain fatty acids having twelve to twenty carbon atoms; said fatty acids may be either saturated or unsaturated.
  • Preferred phospholipids for use according to the present invention include phosphatidylcholine (PC) , phosphatidylethanolamine (PE) , phosphatidylglycerol (PG) and phosphatidylserine (PS) . These phospholipids may come from any natural source and the phospholipids, as such, may be comprised of molecules with differing fatty acids. Phospholipid mixtures comprising phospholipids from different sources may be used.
  • PC, PG and PE may be obtained from egg yolk; PS may be obtained from animal brain or spinal chord. These phospholipids may come from synthetic sources as well. Phospholipid (PL) mixtures having a varied ratio of individual PLs may be used. Suitable PL mixtures comprise (a) from about 20 to about 95 mole percent PC; (b) from about 2.5 to about 50 mole percent PE;
  • PL mixtures comprising from about 5 to 15 mole percent PE, from about 3 to about 20 mole percent PS, from about 10 to about 25 mole percent PG; and the remainder PC, preferably from about 50 to about 90 mole percent PC.
  • PL mixture ⁇ comprising from about 8 to about 12 mole percent PE, from about 3 to about 10 mole percent PS, from about 14 to about 20 mole percent PG and from about 58 to about 75 mole percent PC.
  • the phospholipids may be used in varied ratios, mixtures of phospholipids having preselected amounts of individual phospholipids result in mhuTF compositions having advantageous activity and stability of activity.
  • a certain level of PS in the total phospholipid composition is preferred.
  • the amount of PS that is preferably present to some extent is determined by the remaining components of the PL mixture and their relative amounts as part of the total PL mixture. For example, use of high amounts of PG, another negatively charged phospholipid, (on the order of about 10% or more) allow use of lower levels of PS, on the order of about 3%.
  • the phospholipids are conveniently combined in the appropriate ratios to provide the PL mixture for use in preparing the mhuTF composition of the present invention.
  • the PL mixture may comprise PC, PG, PE and PS in the mole ratio of 67: 16: 10: 7, respectively.
  • the PL mixture may comprise PC, PG, PE and PS in the mole ratio of 7.5: 0: 1: 1, respectively.
  • Recombinant mhuTF may prepared by recombinant technology using methods and expression systems known to the art. See, e.g., Morrissey, J.H. , et al., Cell 50: 129-135 (1987); Summers, M.D. , "A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures," Texas Agricultural Experiment
  • Mutant human tissue factor may also be purified by immuno-affinity chromatography or other chromatographic methods designed to separate a specific protein from other protein contaminants.
  • mhuTF composition will be lyophilized prior to storage for later use, it is preferred to include a carbohydrate or carbohydrates as cryopreservative(s) to protect the integrity of liposomes in the resulting liposome composition during lyophilization and subsequent rehydration.
  • Cryopreservation relates to preserving the integrity of delicate substances when liquids containing them are frozen and dehydrated.
  • the use of a carbohydrate as a cryopreservative of liposome integrity upon freezing and subsequent lyophilization has been reported.
  • Racker, E. Membrane Biol.. 10: 221-235 (1972); Sreter, F. et al., Biochim. Biophys. Acta.. 203: 254-257 (1970); Crowe et al. , Biochem. J. , 242: 1-10 (1987); Crowe et al., Biochim. Biophys. Acta.. 987: 367-384 (1988).
  • Suitable carbohydrate cryopreservatives include trehalose, maltose, lactose, glucose and mannitol.
  • trehalose is included in aqueous buffer solution used in the preparation of the mhuTF composition of the present invention (prior to lyophilization) , preferably at a concentration in the range of about 50 mM to about 250 mM.
  • glycine is included as an additional component of a mhuTF composition.
  • a preferred mhuTF composition further comprises from about 0.5 percent to about 1.5 percent (w:v) glycine, and more preferably comprises from about 0.6 to about 1.2 percent glycine.
  • the phospholipids which may be obtained from the manufacturer in an organic solvent, are mixed together in the appropriate ratios to yield the specified composition.
  • An antioxidant can also be added to reduce alkyl chain peroxidation of the fatty acid portions of the phospholipids, and the organic solvent, if present, is removed by evaporation.
  • One suitable antioxidant is butyrated hydroxy toluene. Preferably about 0.1% (by weight) of antioxidant is used.
  • the dried (evaporated) phospholipid mixture is then redissolved with an aqueous detergent solution.
  • Suitable detergents include those which have a relatively high critical micelle concentration (CMC) . Womack et al., Biochim. Biophys. Acta. 733: 210 (1983) .
  • Such detergents include detergents having a
  • the detergent solution may include other components.
  • These components may include buffer salts such as HEPES, Tris, phosphate, and the like; various other salts such as NaCl, KC1, and the like; a carbohydrate cryopreservative such as trehalose, maltose, glucose, and the like; and glycine.
  • buffer salts such as HEPES, Tris, phosphate, and the like
  • various other salts such as NaCl, KC1, and the like
  • a carbohydrate cryopreservative such as trehalose, maltose, glucose, and the like
  • glycine glycine
  • the detergent solution comprises 20 mM Tris, pH 7.5, 150 mM NaCl, (TBS) containing 100 mM CHAPS, 150 mM trehalose and 0.8% glycine.
  • the phospholipids are redissolved in this solution to give a final concentration of about 20 mg/ml.
  • mhuTF Expressed and purified mhuTF, together with carrier protein, are combined with the redissolved phospholipids and the volume of the resulting mixture is adjusted with a buffer as described above, preferably containing cryopreservative (most preferably trehalose) and glycine but no detergent.
  • mhuTF is admixed with carrier protein, such as bovine gamma globulin, and sufficient buffer is added to adjust the final concentrations of tissue factor to 10 mg/ml, bovine gamma globulin to 1 mg/ml, phospholipid to 4 mg/ml and detergent to 20 mM.
  • Suitable buffers include TBS containing 150 mM trehalose and 0.8% glycine.
  • the resulting clear, colorless solution requires no vortexing or sonicating to ensure co-solubilization.
  • the detergent in the phospholipid-mhuTF admixture can be removed by a number of methods resulting in a stable liposome composition having mhuTF associated with and inserted through the lipid bilayer. Suitable methods of removal of detergent include dialysis, tangential flow diafiltration, cross flow hollow fiber filtration, treatment with hydrophobic chromatography resin, and simple dilution.
  • One preferred method of detergent removal from the phospholipid-mhuTF admixture utilizes dialysis for at least 30 hours at room temperature in dialysis membrane tubing against a buffer such as TBS containing 150 mM trehalose, 0.8% glycine and 0.05% NaN 3 to remove the detergent.
  • Another preferred method of detergent removal utilizes resin treatment. Suitable resins include hydrophobic chromatographic resins such as Amberlite XAD-2 (Rohm and Haas Co. in Philadelphia, Pennsylvania) or Bio-Beads SM-2 (BioRad in Richmond, California) . The resins may be used to remove the detergent, either by direct contact with the phospholipid-mhuTF solution admixture or separated from it by a dialysis membrane.
  • the rate of removal of detergent from the phospholipid-mhuTF admixture is proportional to the weight ratio of the detergent in solution and the chromatographic resin beads.
  • the liposome solution resulting from the detergent removal step is then made to 5 mM CdCl-,.
  • the liposome composition which contains the fully active mhuTF is diluted to a concentration 50 mM Tris, pH 7.5, 75 mM trehalose, 0.8% glycine and 10 to 15 mM CaCl 2 before use.
  • the diluted reagent may be lyophilized for long term preservation of its biological performance characteristics and then later reconstituted by suspension in water before use.
  • detergent solubilized phospholipid compositions containing mhuTF are diluted into a buffer without detergent to produce mixed micelles containing mhuTF which remain capable of being fully activated by CdCl 2 .
  • phospholipids are dissolved to 20 mg/ml in a buffer containing detergent, preferably an alkyl glucopyranoside.
  • a suitable buffer-detergent solution comprises 20 mM HEPES (pH 6) containing 50 mM octyl beta-D-thioglucopyranoside
  • carrier protein mhuTF
  • CdCl 2 150 mM NaCl.
  • buffer without detergent such as 20 mM HEPES (pH 6) containing 150 mM NaCl, to yield final concentrations of mhuTF at 10 mg/ml, carrier protein (bovine gamma globulin) at 1 mg/ml, CdCl 2 at 5mM, phospholipids at 4 mg/ml, and OTG at 10 mM.
  • the reagent may be lyophilized for storage as described above, or diluted as described above before use.
  • this reagent may be prepared by following methods for the preparation of vesicles and detergent-phospholipid mixed micelles from phospholipids by methods based on mechanical means, by removal of organic solvents, by detergent removal, and by size transformation as has been described by Lichtenberg, D. and Barenholz, Y., Methods of Biochemical Analysis. 33: 337-462 (1988), and the disclosures of which are incorporated herein by reference.
  • Therapeutic Compositions Insofar as the present invention also contemplates therapeutic uses of a mhuTf protein of this invention, therapeutic compositions useful for practicing the therapeutic methods are also contemplated.
  • Therapeutic compositions of the present invention contain a physiologically tolerable carrier together with at least one species of mhuTF as described herein, dissolved or dispersed therein as an active ingredient. In a preferred embodiment, the therapeutic composition is not immunogenic when administered to a human patient for therapeutic purposes.
  • compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
  • compositions that contains active ingredients dissolved or dispersed therein are well understood in the art.
  • compositions are prepared as sterile injectables either as liquid solutions or suspensions, aqueous or non-aqueous, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared.
  • the preparation can also be emulsified.
  • Particularly preferred are phospholipid and liposome compositions as described herein.
  • a therapeutic amount of mhuTF can be present in a ointment or on a diffusible patch, such as a bandage, as to afford local delivery of the agent.
  • the active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.
  • the therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein.
  • Pharmaceutically acceptable salts include the acid addition salts
  • salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.
  • Physiologically tolerable carriers are well known in the art.
  • Exemplary of liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline.
  • aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, propylene glycol, polyethylene glycol and other solutes.
  • Liquid compositions can also contain liquid phases in addition to and to the exclusion of water, as described herein.
  • additional liquid phases are glycerin, vegetable oils such as cottonseed oil, organic esters such as ethyl oleate, and water-oil emulsions, particularly the liposome compositions described earlier.
  • a therapeutic composition contains an effective amount of mhuTF of the present invention, typically an amount of at least 0.1 weight percent of active protein per weight of total therapeutic composition.
  • a weight percent is a ratio by weight of mhuTF protein to total composition.
  • 0.1 weight percent is 0.1 grams of mhuTF per 100 grams of total composition.
  • the present invention contemplates various assay methods for analyzing components of the huTF- mediated coagulation cascade through the use of a mhuTF composition of this invention. Particularly preferred are assays for measuring Factor Vila in a body fluid sample such as plasma or blood.
  • the methods generally involves the use of mhuTF in place of huTF in a clotting time assay.
  • the method comprises admixture of the reagents as described in the Examples with a body fluid sample believed to contain Vila, incubating (maintaining) the admixture for a preselected time under conditions sufficient to allow the clotting reaction to occur, and measuring the amount of time passed, thereby indicating the amount of Vila present in the sample.
  • a mhuTF protein of this invention is substantially free of Factor Vila activation activity, the induction of coagulation activity (i.e., as measured by clotting time) of plasma is proportional to the plasma concentration of Vila. There is no "feedback" by the production of Factor Vila from Factor VII present during the assay reactions.
  • the amount of time for a clot to form is proportional to the time of clotting for a preselected amount of Factor Vila, and is determined by preparation of a standard curve, as is well known.
  • the invention contemplates a method for detecting the presence, and preferably the amount, of Factor Vila in a body fluid sample comprising the steps of: a) admixing a preselected amount of said body fluid sample having Factor Vila with a clotting assay admixture, wherein said clotting assay admixture comprises a mutant human tissue factor (mhuTF) composition according to claim 5 and is substantially free of Factor Vila and wild type human tissue factor, to form a Factor Vila assay admixture; b) maintaining said Factor Vila assay admixture under conditions and a time period sufficient for said mhuTF to bind to any of said Factor Vila in said sample and catalyze the formation of a clot; and c) determining the amount of time required for the clot to form, which time is proportional to a predefined amount of Factor Vila, thereby determining the presence, and preferably the amount, of Vila present in said sample.
  • a clotting assay admixture can vary widely so long as the addition of purified Factor Vila, or Vila present in the body fluid sample, to the admixture is sufficient to initiate a coagulation cascade and form a clot, typically according to conventional clotting time assays as is well known and also as described herein.
  • a typical clotting assay admixture contains mhuTF and any buffered solution sufficient to support a coagulation cascade dependent upon Factor V, Factor IX, Factor X, Ca++, prothrombin, fibrinogen, and phospholipids as is well known.
  • a clotting assay admixture can be mhuTF and buffer alone where the sample is plasma or blood providing the other reagents necessary for Factor Vila-dependent coagulation.
  • a clotting assay admixture is substantially free of both wild type human tissue factor and Factor Vila.
  • substantially free in the context of Factor Vila is meant that background levels of clotting are observed when the clotting assay admixture is combined with a plasma sample that contains no detectable Factor Vila.
  • substantially free in the context of wild type human tissue factor is meant that background levels of clotting are observed when the clotting assay admixture is combined with a plasma sample that contains Factor VII but no detectable Factor Vila.
  • a preferred clotting assay admixture is described in the Examples.
  • the present invention also describes a diagnostic system, preferably in kit form, for assaying for the presence and/or amount of one or more of the members of a huTF-mediated coagulation cascade in a sample according to the diagnostic methods described herein.
  • a diagnostic system includes, in an amount sufficient to perform at least one assay, a subject mhuTF composition, as a separately packaged reagent.
  • Instructions for use of the packaged reagent are also typically included.
  • Instructions for use typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like.
  • a diagnostic system of the present invention can also include a one or more of the other reagents used in the preparation of a clotting time assay as described herein, in an amount sufficient for at least one assay.
  • the reagent species of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g. in lyophilized form.
  • a solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system.
  • packaging materials discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems, and can be formulated for single assay use, multiple assay use, manual or automated assay protocols, and the like.
  • a package refers to a solid matrix or material such as glass, plastic (e.g., polyethylene, polypropylene and polycarbonate) , paper, foil and the like capable of holding within fixed limits a diagnostic reagent such as a mhuTF composition of the present invention.
  • a package can be a bottle, vial, plastic and plastic-foil laminated envelope or the like container used to contain a contemplated diagnostic reagent.
  • kits may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method.
  • container means such as vials, tubes, and the like
  • each of the container means comprising one of the separate elements to be used in the method.
  • one of the container means may comprise a mhuTF composition of the invention.
  • the kit may also have containers containing any of the other above- recited immunochemical reagents used to practice the diagnostic methods.
  • mhuTF of this invention can be used therapeutically to prevent the activation of Factor VII. Inhibition of Factor VII activation is desirable where the reduction of Factor VIIA-dependent coagulation is indicated.
  • the method comprises contacting, in vivo or in vitro, Factor VII of Factor Vila with an molar excess of mhuTF present in a therapeutic composition of this invention.
  • the contacting in vivo is accomplished by administering a therapeutically effective amount of a physiologically tolerable composition containing mhuTF of this invention to a patient, thereby contacting the Factor Vll/VIIa present in the patient.
  • the present invention describes in one embodiment a method for inhibiting Factor Vila- dependent coagulation in a human comprising administering to the human an immunotherapeutically effective amount of the mhuTF of this invention.
  • a representative patient for practicing the present methods is any human at risk for coagulation.
  • a therapeutically effective amount of a mhuTF is a predetermined amount calculated to achieve the desired effect, i.e., to bind Factor Vll/VIIa present in the patient, and thereby decrease the likelihood of coagulation in the patient.
  • an effective amount can be measured by improvements in one or more symptoms associated with Factor Vila-dependent coagulation.
  • the dosage ranges for the administration of a mhuTF of the invention are those large enough to produce the desired effect in which the symptoms of coagulated are ameliorated or the likelihood of coagulation are decreased.
  • the dosage should not be so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art.
  • a therapeutically effective amount of an mhuTF of this invention is typically an amount such that when administered in a physiologically tolerable composition is sufficient to achieve a plasma or local concentration of from about 100 picomolar (pM) to 100 nanomolar (nM) , preferably about 1 to 50 nM, and most preferably about 10 to 30 nM.
  • the mhuTF of the invention can be administered parenterally by injection or by gradual infusion over time.
  • the mhuTF of the invention can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, dermally, and can be delivered by peristaltic means.
  • the therapeutic compositions containing a mhuTF of this invention are conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • quantity to be administered depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgement of the practitioner and are peculiar to each individual.
  • suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.
  • Tissue Factor Mutants The cell surface receptor tissue factor (TF) initiates coagulation by supporting the proteolytic activation of Factors X and IX as well as VII to active serine proteases.
  • TF cell surface receptor tissue factor
  • Architectural similarity of TF to the cytokine receptor family suggests a strand-loop-strand structure for TF amino acid residues 151-174 (SEQ ID NO 2).
  • Site-directed mutagenesis as described herein of native human TF resulted in the production of the mutant human TF
  • Lys 159 to Ala substitution was compatible with efficient activation of Factor X, whereas the Tyr 157 to Ala exchange and mutations in the carboxyl aspect of the predicted loop resulted in diminished activation of Factor X.
  • the specific plasma procoagulant activity of all functionally deficient mutants increased 7- to 200-fold upon the supplementation of Vila suggesting that TF residues 157-167 also provide important interactions that accelerate the activation of VII to Vila.
  • the pCDM8 vector contained the following elements: a simian virus 40 (SV40) derived origin of replication; the eucaryotic transcription regulatory elements splice and poly(A)+; a bacterial episome origin of replication; and a procaryotic genetic marker (supF, suppressor tRNA gene) ; a polyoma origin of replication, bacteriophage M13 origin of replication, and the cytomegalovirus promoter (CMV) .
  • SV40 simian virus 40
  • the 775 base pair (bp) EcoR I fragment containing the cDNA (nucleotides 1 to 775) encoding amino acid residues 1-215 of TF was excised from the plasmid CTF545 prepared as described by Morissey et al.. Cell. 50:129-135 (1987), the disclosure of which is hereby incorporated by reference. This resulting fragment was ligated into the 505 bp EcoR I-Hind III fragment of pCTF439 consisting of nucleotides 776 to 1280 that encode the amino acid residues of TF from 216-263 and the concatenated DNA was then cloned into pUC19 to yield the construct pCTF553 as described by Rehemtulla et al..
  • Thrombosis and Haemostasis 65:521-527, (1991) , the disclosure of which is hereby incorporated by reference.
  • the resulting fragment was then cloned into pUC18 using BamH I linkers to form pCTF1200.
  • the sequence of this construct confirmed the presence of the entire coding region of native human TF in addition to 360 bp of untranslated 3' region and 38 bp of untranslated 5' sequence.
  • the BamH I insert from pCTF1200 was excised, blunt-ended using Klenow fragment of DNA polymerase and ligated into the vector pCDM8 prepared as described above that had been digested with Xho I and blunt-ended with Klenow.
  • the resultant construct, designated pETF1773, contained the TF cDNA in an orientation that allowed transcription under the control of the strong CMV promoter of pCDM8.
  • the complete nucleotide sequence of the pCDM ⁇ vector containing the TF cDNA insert is listed in SEQ ID NO 1.
  • nucleotide sequence encoding the signal peptide of TF begins at nucleotide position 2267 and ends at 2362 followed by the nucleotide sequence encoding TF beginning at 2363 and ending at 3154.
  • the encoded 263 amino acid residue sequence of the nonmutagenized native human TF is listed in SEQ ID NO 2.
  • Other expression vectors having equivalent elements are contemplated for use in this invention.
  • Oligonucleotide-directed mutagenesis was performed using the uracil substitution method according to Kunkel, Proc. Natl. Acad. Sci.. USA. 82:488-492 (1985) and also described in Ausebel et al., Current Protocols in Molecular Biology. Unit 8, Wiley and Sons, New York, (1990) .
  • the following modifications to the basic site-directed mutagenesis procedure were performed to adapt the procedure for use with the pCDM8 expression vector as described by Rehemtulla et al., J. Biol. Chem.. 266:10294-10299 (1991) .
  • Phosphorylated mutagenic oligonucleotides (10 nanograms (ng) ) were separately annealed to single-stranded template (100 ng) which was isolated from the strain CJ236/p3 (Invitrogen, San Diego, CA) in 20 mM Tris-HCl at pH 7.4, 2 mM MgCl 2 , 50 mM NaCl at 70 degrees Celsius (70C) and cooled to room temperature.
  • the second strand was synthesized using T4 DNA polymerase and T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN) in 10 mM Tris-HCl at pH 7.5, 5 mM MgCl 2 , 2 mM dithiothreitol, 5 mM deoxynucleotide triphosphates and 10 mM ATP for 5 minutes at 4C, 5 minutes at room temperature, and 90 minutes at 37C.
  • T4 DNA polymerase and T4 DNA ligase Boehringer Mannheim, Indianapolis, IN
  • TF A157A159 was generated with the oligonucleotide TATACACTTTACGCGTGGGCATCTTCAAGT (SEQ ID NO 3) ; TF A 1 6 1 D16 2 A163 with TTGGAAATCTGCAGATGCAGGAAAGAAA (SEQ ID NO 4) ; TF A161 with GGAAATCTGCCTCGAGTGGAAAGAA (SEQ ID NO 5) ; TF A162 with AATCTTCAGCCTCGGGAAAGAA (SEQ ID NO 6) ; TF A163 with TGGAAATCCTCGAGTGCAGGAAA (SEQ ID NO 7) ; TF A1M With TATTGGAAATCCTCGAGTTCAGCAAAGAAAACA (SEQ ID NO 8) ; TF A167 with TCTTCAAGCTCAGGAAAGAAAGCAGCCAAA (SEQ ID NO 9); TF T162 with TGGAAATCTTCAACCTCGGGAAAGAAA (SEQ ID NO 10) ; TF D16
  • GCCAAAGCAAACGCTAATGCGTTTTTGATCGATGTG SEQ ID NO 15
  • TF R158G160 with CTTTATTATCGAAAAGGTTCAAGTTCA SEQ ID NO 16
  • TF A159 with TATTATTGGGCATCCTCGAGTTCAGGA SEQ ID NO 17
  • TF R158A159G160 with CTTTATTATCGCGCAGGTTCAAGTTC SEQ ID NO 18
  • TF G152n54 ⁇ 156 with GGCAAGGACCTCGGGTATATACTTACTTATTGGAAA
  • CHO-K1 Chinese Hamster Ovary cells (CHO-K1) having an ATCC Accession No CCL61 were grown in Dulbecco's modified Eagles medium (DMEM) , 10% newborn calf serum (HyClone Laboratories, Logan UT) , 2 mM 1-glutamine, 0.1 mM proline, 100 U/ml penicillin and 100 ug/ml streptomycin.
  • DMEM Dulbecco's modified Eagles medium
  • the prepared cells were transfected using the calcium phosphate precipitation method and stable cell lines were generated by separately cotransfecting 1 ug of a neomycin resistance gene for selection purposes (pMAMneo, Clontech Laboratories,
  • Example IB encoding the mutations described in Example IB.
  • the DNA was first diluted in 250 mM CaCl 2 then precipitated by drop-wise addition of 2X Hepes buffered saline (0.25 M NaCl, 40 mM Hepes, 0.7 mM Na 2 HP0 4 at pH 7.05 with 0.5 M NaOH) followed by vigorous vortexing and then incubation at room temperature for 20 minutes. The precipitate was added to a monolayer to CHO-K1 cells.
  • the cells were treated with 2 ml of 10% glycerol in medium for 3 minutes and then washed three times with PBS (10 mM sodium phosphate at pH 7.4 and 0.15 M NaCl). The washed cells were then maintained with fresh medium for 36 hours in transient transfection experiments.
  • PBS 10 mM sodium phosphate at pH 7.4 and 0.15 M NaCl
  • the cultures were suspended and diluted to a density of 10 4 cells/100 mm petri dish.
  • the medium was supplemented with 600 ug/ml G418 (Geneticin, Gibco, Gaithersburg, MD) and the cells were replated in petri dishes. Fresh medium was added to the cells after 7 days and G418 resistant colonies appeared after 10-14 days. Single colonies were picked using cloning cylinders and grown in large cultures for analysis. Tunicamycin (Sigma, St. Louis, MO) treatment of the cells was performed at 1 ug/ml for 48 hours.
  • the stable cell lines expressing mutant TF prepared in Example 1C were then grown to allow for purification of the recombinant mutant TF.
  • the stable cells lines were separately grown in 2 liter spinner flasks in Excell 301 (JR Scientific, Woodland, CA) , 10% newborn calf serum (Hanna Biologicals) , 2 mM L-glutamine, 0.1 mM proline, 100 U/ml penicillin and 100 ug/ml streptomycin. Cells were harvested at maximum density and lysed in 200 ml of 1% Triton X-100 in TBS.
  • insoluble debris was pelleted at 10,000 X g at 4C for 20 minutes.
  • the resultant supernatant was applied to an immunoaffinity column as described by Morissey et al., Cell. 50:129-135 (1987), the disclosure of which is hereby incorporated by reference.
  • the unbound material was washed from the column using TBS, 0.1% Triton X-100 and followed by 0.1 M glycine at pH 4.5, containing 0.1% Triton X-100.
  • the TF mutant proteins were separately eluted with 0.1 M glycine at pH 2.5 containing 0.1% Triton X-100. Fractions containing the eluted mutant TF proteins were immediately neutralized to pH greater than 5.5 and rapidly dialyzed against 0.01% Triton X-100 in TBS for storage at -70C. Concentration of the eluted proteins was determined by immunoassay and by direct protein using the BCA protein assay (Pierce, Rockford, IL) . All assays were standardized with purified natural human TF quantitated by amino acid composition based on a protein mass of 29,593 as described by Morissey et al., supra. Expression of mutant TF ranged from 0 to 970 ng per 10 6 cells equivalent to that seen with normal TF as described by Rehemtulla et al., Thrombosis and Haemostasis. 65:521-527 (1991).
  • Coagulation factor deficient plasmas were purchased from George King Bio-medical.
  • TF antigen in a detergent (CHAPS) cell lysate was determined by immunoassay using two non-overlapping monoclonal antibodies or, alternatively, polyvalent antibody purified by affinity for immobilized TF as capture antibody followed by detection with monoclonal antibody.
  • the purification and assays were performed as described by Ruf et al., J. Biol. Chem.. 266:2158-2166 (1991), the disclosure of which is hereby incorporated by reference.
  • the assay was calibrated with recombinant human TF prepared as described for mutant TF in Example 1.
  • Initiation of coagulation by wild-type and mutant TF in recalcified plasma was determined after lysis of cell pellets from 2 X 10° cells/ml with 15 mM octyl-glucopyranoside in HBS forl ⁇ minutes at 37C followed by 3-fold dilution according to Rehemtulla et al., Biochem. J.. 282:737-740 (1992).
  • clotting times were determined for the cell lysates in a one stage clotting assay containing equal volumes of sample, plasma, lysate and 20 mM CaCl 2 and converted to units based on a calibration curve established with purified TF reconstituted in phospholipid vesicles (70% phosphatidylcholine, 30% phosphatidylserine) using detergent solubilization and dialysis, as described in detail by Ruf et al., Thrombosis and Heamostasis.
  • the results of the assays are presented in Table 1.
  • the specific functional activity was calculated based on the determinations of the TF antigen by ELISA and for functional activity in the one stage clotting assay. The mean and standard deviation were calculated for the indicated number (n) of duplicate determinations is given.
  • the specific functional activity relative to wild-type TF is given as % of wild-type.
  • Tyr 157 and Lys 159 have previously been identified as functionally important and are flanked in linear sequence b the non-critical residues Tyr 156 and Ser 160 as described by Rehemtulla et al., Biochem. J.. 282:737-740 (1992).
  • Ala replacements for Tyr 157 or Lys 159 resulted in a 87% or 92% respective loss of specific functional activity by plasma coagulation assay. Replacement of both residues in one mutant reduced the functional activity by 98% as shown in Table 1 which may indicate an additive effect of the two mutations.
  • Ala replacements for Ser 161 and Ser 162 did not result in significant loss of function.
  • Ala substitution for Ser 163 reduced specific functional activity by 89%, indicating importance of the Ser 163 side chain.
  • Gly 164 appears to be necessary for function of the 157-167 region, since the Gly 164 to Ala substitution resulte in very low specific functional activity (Table 1) .
  • Gly residues are often found in reverse turns as described by Creighton et al.. Proteins. W.H. Freemann and Company, New York (1984) , because of the lack of a C—atom, the increased flexibility of their backbone and their more favorable phi and psi angles.
  • the functional defect resulting from the Gly 164 to Ala exchange is likely to reflect local perturbation of the orientation of adjacent functionally important residues.
  • Thr 167 could be replaced by Ala without alterations in the functional properties of TF.
  • the TF mutant R 158 G 160 exhibited overall specific functional activity of 41 +/- 15.
  • Dysfunction of a TF mutant could follow from reduced affinity for its ligand Vll/VIIa.
  • the binding characteristics of VII and Vila to cell surface expressed mutant or wild type TF were determined essentially as described by Fair et al., J. Biol. Chem.. 262:11692-11698 (1987) and further described by Rehemtulla et al., J. Biol. Chem.. 266:10294-10299 (1991). Briefly, stable cell lines produced in Example 1 expressing mutant TF as well as a control normal TF were seeded at equal densities into 24-well tissue culture dishes (Costar) .
  • the cell monolayers were washed three times and maintained with plasma derived 125 I-VII/VIIa, in the presence of 5 mM CaCl 2 , 0.5% BSA in 10 mM Hepes, 150 mM NaCl, 4 mM KC1, 11 mM glucose, pH 7.4.
  • Nonspecific binding was determined in the presence of 50-fold molar excess of a monoclonal antibody against TF (TF9-6B4) which completely blocks the binding of VII to TF.
  • Bound radioactivity was determined after rapidly washing the cells and solubilizing the monolayer. Duplicate determinations from at least three experiments were used for Scatchard analysis which was performed using the LIGAND program, as described by Rehemtulla et al., J.
  • Lys 166 do not contribute significantly to the binding energy required for assembly of the TF-VIIa complex. Since remova of charged side chains, as in the TF A165A166 mutant, may be tolerated more readily than the addition of an oppositely charged or bulkier side chain in the same region, the VII binding characteristics of TF A161D162A163 . This mutant was chosen because it exhibited the greatest loss of function, when mutants at the Ser 162 and Ser 163 position were compared Radioligand binding analysis with Vll/VIIa resulted in similar binding profiles for wild-type TF and TF A161D162A163 as shown in Figure 2 specific binding of VII to cell surface
  • TF A161D162A163 A and wild-type TF (B) is shown.
  • the insets give the Scatchard analysis for the same data obtained in a representative experiment.
  • the dissociation constant derived from Scatchard analysis demonstrated high affinity binding of VII by the mutant TF (Table 2) .
  • Table 2 The binding analysis for TF A ⁇ 61D ⁇ 62A163 may be taken as representative for the TF A163 mutant and mutants with other substitutions for Ser 162 which exhibited less profound decreases in specific functional activity. This provides additional evidence that the carboxyl aspect of the putative 157-167 loop in TF is not required for binding of VII.
  • mutant TF proteins of this invention cleavage of small peptidyl substrates by the TF-VIIa complex was analyzed using lysates of cell lines stably expressing wild-type or mutant TF. Cells were lysed with 4 mM CHAPS dissolved in TBS and this lysate was diluted two-fold in the final reaction (200 ul) which contained Vila (10 nM) , CaCl 2 (5 mM) and Spectrozyme FXa (1.25 mM) . The rate of Spectrozyme hydrolysis was determined at ambient temperature in a kinetic plate reader (Molecular Devices, Mountain View, CA) .
  • Spectrozyme FXa was observed as shown in Figure 3 which shows the amidolytic and proteolytic activity of mutant TF-VIIa complexes.
  • Figure 3A the cleavage of small peptidyl substrates was assessed with Spectrozyme FXa in th presence of 10 nM Vila and 5 nM wild-type or mutant TF. Th rate of hydrolysis of the peptidyl substrate was determined in a 200 ul reaction with a kinetic plate reader and is given as the increase in absorbance (mOD/min) .
  • the mutants shown in Figure 3 shows the mutant of the mutants.
  • TFR 158G16fJ exhibited similar activity having a mean amidolytic activity of 7 +/- 1.3.
  • the data further supports the binding analysis that all TF mutants in the 157-167 region form equivalent complexes with Vila. This analysis also excludes the notion that a significant fraction of the mutant TF is misfolded and non-interacting with Vila.
  • these data demonstrate that the catalytic functiono of Vila towards small peptidyl substrates is normal indicating a fully functional catalytic triad in Vila when complexed with the mutant TFs.
  • mutant TF proteins of this invention were further analyzed by their ability to activate Factor X whic is a property mediated by normal TF.
  • X activation was analyzed by incubating a freshly prepared octyl-glucopyranoside cell lysate (0.03 to 0.12 nM TF) with excess Vila (5 nM) at 5 mM CaCl 2 for 5 minutes at 37C followed by addition of Factor X (1 uM) . Samples were removed from the reaction and quenched in 100 mM EDTA in TBS (20 mM Tris-HCl, 140 mM NaCl, pH 7.4).
  • Xa in the quenched reaction was determined with Spectrozyme FXa and the rate of Xa generation was calculated for several points in the initial linear portion of the progress curve as described by Ruf et al., J. Biol. Chem.. 266:2158-2166 (1991).
  • TF A161D162A163 ' TF A157 o TF A165A166 demonstrated rates of X activation which were reduced by 85%, 55%, and 59%, respectively as shown in Figure 3B.
  • all dysfunctional mutants with the exception of TF A159 formed catalytic complexes with Vila that exhibited some loss of proteolytic activation of the natural protein substrate.
  • Amidolytic activity of the mutant TF-VIIa complexes was indistinguishable from that of the wild-type TF-VIIa complex suggesting that the mutants have a selective defect either in extended recognition and hydrolysis of protein substrates, or in the release of Xa, the cleaved product.
  • the TF R158G160 mutant exhibited similar proteolytic characteristics having a value of 1.33 +/- 0.27.
  • the inability of the mutant TF proteins of this invention to convert the substrate VII to Vila is the critical factor for the function of the proteins in a standard hospital clotting assay where it is essential to only measure the amount of Vila currently present in the plasma and not the amount of Vila converted from VII as the clotting assay progresses.
  • Clotting activity of TF mutants with or without added Vila was evaluated with freshly prepared octyl-glucopyranoside lysates. Cell lysate (100 ul) , normal or coagulation factor deficient plasma (50 ul) and 500 nM Vila or buffer (50 ul) were equilibrated at 37C for 1 minute followed by initiation of the reaction by adding 20 mM CaCl 2 (100 ul) .
  • the Vila concentration was chosen to provide a 50 to 100-fold excess over VII in the plasma. Control experiments with a 10-fold lower concentration of Vila gave similar results. Further, preincubation of wild-type and mutant TF with Vila in the presence of CaCl 2 followed by the addition of plasma did not reveal differences compared to the assay where the reaction was started by the addition of Ca + . This suggests that a slower assembly of Vila with the TF mutants does not contribute to the functional defect. Functional activity was derived from double logarithmic calibration curves of serial dilutions of purified and phospholipid reconstituted TF versus the clotting times in normal or factor IX (IX) deficient plasma.
  • the concentration of TF which produced a 50 seconds (s) clotting time in normal or IX deficient plasma was set to lU/ml of TF activity. Specific functional activity was based on the TF antigen concentration of the cells determined by ELISA. Several dilutions of mutant TF (30 to 200 pM) were used to establish the functional activities for each experimental condition, and mean and standard deviation were calculated for three independent experiments.
  • Lys 159 is important for the conversion of VII to Vila.
  • this residue may be critical for assembly of Xa with the TF-VII complex during activation of the bound VII, or Lys 159 may be important for recognition and hydrolysis of the substrate VII by the TF-VIIa complex.
  • the TF mutants described here may aid in elucidating the specific contribution of cofactor residues to the auto-activation of VII by Vila.
  • the G152YILT mutant (TF G1S2I154T156 ) is not sensitive for Vila but the addition of the R158KG160 mutation (TF R158G160 ) renders the mutant TF Vila sensitive and results in a mutant with the desired function lacking the ability to convert VII to Vila while having the functions of amidolytic and proteolytic activity as described herein.
  • the most preferred mutant TF proteins of this invention contained the mutation at amino acid position 158 where a tryptophan has been changed to an arginine and at amino acid position 160 where a serine has been changed to a glycine.
  • the prothrombin time assay measures coagulation factors of the extrinsic pathway. These include Factors VII, X, V, II and I. Some of these factors are affected by oral anticoagulant drugs. The prothrombin time assay was thus useful for monitoring oral anticoagulant therapy.
  • an operator of a typical coagulation instrument distributed by commercial entities such as Ortho Diagnostics only has to pipette 0.1 ml of patient plasma sample into an assay cuvette well and place the cuvette into the instrument. As the automatic carousel indexes through the incubation plate, the samples are warmed to 37.5C.
  • the reagent arm then dispenses 0.2 ml prewarmed mutant TF reagent of this invention and clotting times are measured. The remainder of the assay is automated as is the data reduction.
  • the replacement of normal TF (thromboplastin) with the mutant TF proteins of this invention along with corresponding instrument software changes would support data reduction to yield the accurate concentration of Factor Vila present in the blood without any confounding by the amount of Factor Vila resulting from the conversion of VII effected by normal TF.
  • Residues He 152 , Thr 154 and Tyr 156 may form the hydrophilic side of the beta-strand and hydrophilic substitutions for He 152 and Tyr 156 are found in the TF sequence of other species as described by Andrews et al.. Gene. 98:265-269 (1991).
  • the hydrophobic residues Leu 151 , Tyr 153 and Leu 155 were each replaced by Ala and the triple mutant TF was transiently expressed.
  • TF A151A153A155 was expressed at levels one tenth or less of a wild-type TF control transfected in parallel. This suggested diminished efficiency of cellular processing which may indicate alteration of the protein fold. See, Bass et al., Proc. Natl. Acad.
  • the sequence 168-174 in TF which corresponds to the predicted D beta-strand is hydrophilic.
  • the region of TF which includes residues 151-174 has been predicted to adopt a strand-loop-strand structure, from sequence based secondary structure predictive algorithms as described by Bazan et al., Proc. Natl. Acad. Sci.. USA. 87:6934-6938 (1990) and apparent homology to the growth hormone receptor structure as described by De Vos et al.. Science. 255:306-312 (1992).
  • the previously demonstrated predominant beta-strand secondary structure of TF as described by Ruf et al. , Proc. Natl. Acad. Sci.. USA. 88:8430-8434 (1991) in conjunction with the mutations of residues 151-174 of this invention are consistent with this hypothesized immunoglobulin-like fold of TF.
  • Residues Tyr 157 , Lys 159 , Ser 163 , Gly 164 , Lys 165 and Lys 166 were identified as important for function, either directly or indirectly through maintenance of a functional structure in the predicted 157-167 loop.
  • Replacement of Gly 164 with the larger and more rigid Ala resulted in severe loss of function consistent with location of Gly 164 in a turn which may be required for the proper conformation of the putative 157-167 loop.
  • All dysfunctional mutants in the 157-167 region were characterized by high affinity binding of Vll/VIIa and the ability to form mutant TF-VIIa complexes which efficiently hydrolyzed small peptidyl substrates. These data are consistent with expression of mutant proteins with proper overall fold.
  • the functionally defective mutants displayed two phenotypes. Whereas removal of the Lys 159 side chain only affected the conversion of VII to Vila, the other mutants formed catalytic binary complexes with selectively reduced proteolytic activity for Factor X and a suggested consecutive defect in VII activation.
  • the Tyr 157 to Ala mutation in the amino-terminal aspect of the predicted loop resulted in a functional phenotype similar to the charge modifying mutations in the carboxyl aspect. It must be considered whether Tyr 157 may be important for stabilization of the loop by providing a hydrophobic center with its aromatic side chain.
  • Tyr 157 to Phe substitution has been shown to be compatible with full functional activity of TF as described by Rehemtulla et al., Biochem J.. 282:737-740 (1992).
  • a supporting role for the structure of the 157-167 loop can also be considered for residues Ser 162 and Ser 163 .
  • functionally important interactions for the Ser 163 side chain cannot be excluded, loss of function due to side chain capping (Ser 163 ) or introduction of additional side chain atoms (Ser 162 ) would also be consistent with a perturbation of the structural integrity of the 157-167 loop resulting in loss of proper alignment of adjacent functionally important residues.
  • TF-VIIa complex for protein substrates which is composed entirely or in part by TF residues. It appears that these residues contribute differently to the activation of X and VII.
  • the human TF mutants characterized here thus help to define the molecular structures which mediate the well documented roles of TF as an enhancer of VII activation as described by Nakagaki et al., Biochem..
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PS phosphatidylserine
  • PG phosphatidylglycerol
  • Phospholipids are prepared for resolubilization in the following manner.
  • PC, PE, PS, and PG are warmed to room temperature and combined in a suitable tube or flask at the specified mole ratios.
  • the antioxidant butyrated hydroxytoluene (BHT)
  • BHT butyrated hydroxytoluene
  • Organic solvent is removed by evaporation under a stream of dry nitrogen or under reduced pressure in a rotary evaporator. Residual organic solvent is eliminated by pumping an additional 1 hour at room temperature with a vacuum pump at a pressure of 10 mm or less.
  • the mixture of phospholipids is redissolved to 20 mg/ml in 20 mM Tris-HCl at pH 7.5, 150 mM NaCl (TBS) containing 100 mM CHAPS
  • tissue factor mutants prepared in Example 2 are then separately admixed with carrier protein and are then combined with the redissolved phospholipids prepared above.
  • the volume of the resulting mixture is adjusted with a buffer as described above, preferably containing cryopreservative (most preferably trehalose) and glycine but no detergent.
  • cryopreservative most preferably trehalose
  • glycine glycine but no detergent
  • Phospholipids are combined at the specified mole ratios of PC, PE, PS, and PG, then resolubilized as described above.
  • the resolubilized phospholipids are combined with the mutant TF proteins of this invention and bovine gamma globulin.
  • Additional TBS containing 150 mM trehalose is added to yield final concentrations of 4 mg/ml total phospholipid, 10 mg/ml mhuTF, 1 mg/ml bovine gamma globulin and 20 mM CHAPS.
  • This clear and colorless solution is placed in a dialysis membrane tubing (Spectrapore", Spectrum Medical Industries, molecular weight cutoff of 12,000 to 14,000) and dialyzed for at least 30 hours at room temperature against TBS containing 150 mM trehalose and 0.05% NaN 3 . After dialysis the volume of the dialysate is determined and adjusted back to the original volume, if required, with dialysis buffer. CdCl 2 is added to a final concentration of 5 mM and the solution is incubated at 37C for 2 hours. The solution is frozen on dry ice, then lyophilized using a cycle beginning at -40C and ending at room temperature, over a 48 hour period.
  • a dialysis membrane tubing Spectrum Medical Industries, molecular weight cutoff of 12,000 to 14,000
  • the liposomes are then reconstituted to a working concentration with 0.1 M Tris-HCl at pH 7.5, 150 mM trehalose to yield a solution containing mhuTF at approximately 1 to 2 mg/ml, phospholipids at approximately 400 to 800 mg/ml, and bovine gamma globulin at 50 to 100 mg/ml.
  • Phospholipids are prepared for resolubilization in the following manner.
  • PC, PE, and PS are warmed to room temperature and combined in a suitable tube or flask at a mole ratio of 7.5:1:1 of PC, PE, and PS, respectively.
  • the antioxidant, butyrated hydroxytoluene (BHT) is dissolved in chloroform and added to the mixture of phospholipids at a weight ratio of 0.1% (BHT:total phospholipids).
  • Organic solvent is removed by evaporation under a stream of dry nitrogen or under reduced pressure in a rotary evaporator. Residual organic solvent is eliminated by pumping an additional 1 hour at room temperature with a vacuum pump at a pressure of 10 mm or less.
  • the mixture of phospholipids is redissolved in
  • octyl beta-D-thioglucopyranoside 50 mM octyl beta-D-thioglucopyranoside (OTG) in 20 mM HEPES (pH 6) , 150 mM NaCl to a final concentration of 4 mg/ml.
  • the mutant TF (mhuTF) proteins from Example 2 and bovine gamma globulin are mixed with the resolubilized phospholipids. Enough 20 mM HEPES (pH 6) , 150 mM NaCl is added to adjust the final concentrations to 10 mg/ml mhuTF, 1 mg/ml bovine gamma globulin, 4 mg/ml phospholipids, and 10 mM OTG.
  • CdCl 2 is added to a final concentration of 5 mM to activate the mhuTF.
  • the resulting mixed micelles comprised of mhuTF, OTG, and phospholipids are diluted with 20 mM HEPES, pH 6, 150 mM NaCl to yield a solution containing mhuTF at approximately 0.5 to 1 mg/ml, phospholipids at approximately 500 to 700 mg/ml, and bovine gamma globulin at 25 to 50 mg/ml to give mhuTF PT reagent.
  • the detergent (CHAPS) is removed by tangential flow diafiltration using, a Pyrostart or Ultrastart filter unit (Sartorius Corp., Bohemia, NY, molecular weight cutoff of 20,000) and TBS containing 150 mM trehalose as the dialysis buffer. Approximately 95 to 100% of the CHAPS can be removed by passing 10 volumes of dialysis buffer through the device. After diafiltration the volume of the dialysate is determined and adjusted back to the original volume (if required) with TBS containing 150 mM trehalose and 0.05% NaN 3 . CdCl 2 is added to a final concentration of 5 mM and the solution was incubated at 37C for 2 hours.
  • the solution may be frozen on dry ice, then lyophilized using a cycle beginning at -40C and ending at room temperature, over a 48 hour period.
  • the resulting reagent may be reconstituted to working concentration with the addition of 0.1 M Tris-HCl at pH 7.5, 150 mM trehalose to yield a solution containing mhuTF at approximately 1 to 2 mg/ml, phospholipids at approximately 400 to 800 mg/ml, and bovine gamma globulin at 50 to 100 mg/ml.
  • Phospholipids are combined at mole ratio of 67: 16: 10: 7 (PC: PG: PE: PS), dried to remove organic solvent, then resolubilized as described above.
  • the resolubilized phospholipids at 15 mg/ml in TBS containing 100 mM CHAPS and 0.8% glycine are combined mutant TF proteins prepared in Example 2 and bovine gamma globulin.
  • Additional TBS containing 150 mM trehalose and 0.8% glycine is added to yield final concentrations of 3 mg/ml phospholipid, 4.5 mg/ml mhuTF, 1 mg/ml bovine gamma globulin and 20 mM CHAPS.
  • Hydrophobic chromatographic resins such as Amberlite XAD-2 (Rohm and Haas Co., Philadelphia, Pa) or Bio-Beads SM-2 (BioRad, Richmond, Ca) can also be used to remove the detergent (CHAPS) , either in direct contact with the phospholipid solution or separated from it by a dialysis membrane. The rate of removal is proportional to the weight ratio of the detergent in solution and the chromatographic resin beads.
  • the rate of removal is proportional to both the amount of resin added and the rate of addition.
  • the amount required to remove all of the detergent is calculated from the capacity of the resin (provided by the manufacturer) and the total mass of detergent to be removed. Moreover, 99.9% removal of the detergent may be achieved either in 1 hour or in 24 hours, at 30C depending upon the rate at which this amount of resin is added. CdCl 2 was added to a final concentration of 5 mM and the solution was incubated at 37C for 2 hours.
  • the liposomes are then diluted to a working concentration with 50 mM Tris-HCl at pH 7.5, 75 mM trehalose, 15 mM CaCl 2 , 0.8% glycine, 1% maltose, and 0.05% NaN 3 to yield a solution containing mhuTF at approximately 0.04 to 0.20 mg/ml, phospholipids at approximately 40 to 150 mg/ml, and bovine gamma globulin at 50 to 100 mg/ml.
  • the solution is frozen on dry ice, then lyophilized using a cycle beginning at -40C and ending at room temperature, over a 48 hour period.
  • the lyophilized reagent was reconstituted with distilled water prior to use.
  • the resultant phospholipid-reconstituted tissue factor mutants of this invention can then be used in clinical clotting assays where the accurate determination of the concentration of plasma factor Vila is required without the confounding of the amount of Vila produced by the rapid conversion of VII to Vila by normal tissue factor.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FEATURE FEATURE
  • GAGCCCCCGA TTTAGAGCTT GACGGGGAAA GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA 960
  • ATCAGCCATA TAGCCCCCGC TGTTCGACTT ACAAACACAG GCACAGTACT GACAAACCCA 4601
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:3: TATACACTTT ACGCGTGGGC ATCTTCAAGT 30
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:16: CTTTATTATC GAAAAGGTTC AAGTTCA 27
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI - SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention se rapporte à une protéine de thromboplastine tissulaire humaine mutante qui lie le facteur fonctionnel VII/VIIa et qui active protéolytiquement le facteur X, mais qui est sensiblement dépourvue d'activité protéolytique par rapport à l'activation du facteur VII. L'invention se rapporte également à des compositions contenant la protéine mutante, ainsi qu'à des procédés diagnostiques consistant à utiliser la protéine de thromboplastine tissulaire humaine mutante pour la détection du facteur VIIa, et à des vecteurs d'ADN recombinés permettant d'exprimer la protéine.
PCT/US1993/009570 1992-10-06 1993-10-06 Thromboplastine tissulaire mutante depourvue d'activite activant le facteur vii WO1994007515A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US95798592A 1992-10-06 1992-10-06
US07/957,985 1992-10-06

Publications (1)

Publication Number Publication Date
WO1994007515A1 true WO1994007515A1 (fr) 1994-04-14

Family

ID=25500449

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/009570 WO1994007515A1 (fr) 1992-10-06 1993-10-06 Thromboplastine tissulaire mutante depourvue d'activite activant le facteur vii

Country Status (1)

Country Link
WO (1) WO1994007515A1 (fr)

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997019357A1 (fr) * 1995-11-22 1997-05-29 Oklahoma Medical Research Foundation Essai de dosage pour les facteurs de coagulation sanguine viii et ix
US5877289A (en) * 1992-03-05 1999-03-02 The Scripps Research Institute Tissue factor compositions and ligands for the specific coagulation of vasculature
US6004555A (en) * 1992-03-05 1999-12-21 Board Of Regents, The University Of Texas System Methods for the specific coagulation of vasculature
WO2000004148A1 (fr) * 1998-07-15 2000-01-27 Genentech, Inc. Variantes de proteines de facteurs tissulaires presentant une affinite accrue pour le facteur de coagulation fvii/fviia
US6036955A (en) * 1992-03-05 2000-03-14 The Scripps Research Institute Kits and methods for the specific coagulation of vasculature
US6093399A (en) * 1992-03-05 2000-07-25 Board Of Regents, The University Of Texas System Methods and compositions for the specific coagulation of vasculature
US6132730A (en) * 1997-01-22 2000-10-17 Board Of Regents, The University Of Texas System Combined tissue factor and factor VIIa methods and compositions for coagulation and tumor treatment
WO2004006847A2 (fr) 2002-07-15 2004-01-22 Board Of Regents, The University Of Texas System Anticorps et peptides de duramycine selectionnes se fixant a des phospholipides et des aminophospholipides anioniques et leur utilisation dans le traitement d'infections virales et du cancer
US6749853B1 (en) 1992-03-05 2004-06-15 Board Of Regents, The University Of Texas System Combined methods and compositions for coagulation and tumor treatment
US6773896B2 (en) 2001-05-08 2004-08-10 Warner-Lambert Company Method for direct measurement of coagulation factor VIIa activity in plasma
US6905683B2 (en) 2000-05-03 2005-06-14 Novo Nordisk Healthcare A/G Human coagulation factor VII variants
US6911323B2 (en) 2002-09-25 2005-06-28 Novo Nordisk Healthcare A/G Human coagulation factor VII polypeptides
US6960657B2 (en) 2001-11-02 2005-11-01 Novo Nordisk Healthcare A/G Human coagulation factor VII polypeptides
US7026524B2 (en) 2000-09-13 2006-04-11 Novo Nordisk Healthcare A/G Human coagulation factor VII variants
US7052868B2 (en) 2001-09-27 2006-05-30 Novo Nordisk Healthcare A/G Human coagulation factor VII polypeptides
US7173000B2 (en) 2000-11-09 2007-02-06 The Scripps Research Institute Modified factor VIIa
US7399743B2 (en) 2001-10-26 2008-07-15 The Scripps Research Institute Targeted thrombosis
EP2386312A2 (fr) 2002-05-08 2011-11-16 TERMAN, David S. Superantigènes intrathécaux et intratumoraux servant a traiter des maladies malignes
EP2614837A1 (fr) 2007-11-09 2013-07-17 Affitech Research AS Compositions d'anticorps anti-VEGF et procédés
US8674074B2 (en) 2003-09-09 2014-03-18 Novo Nordisk Healthcare Ag Coagulation factor VII polypeptides
EP3225991A1 (fr) * 2016-03-30 2017-10-04 Sysmex Corporation Réactif pour la mesure de temps de prothrombine, son procédé de production et procédé de mesure de temps de prothrombine
US10775393B2 (en) 2016-03-30 2020-09-15 Sysmex Corporation Reagent for prothrombin time measurement, method for production thereof, and method for measurement of prothrombin time

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992018870A1 (fr) * 1991-04-10 1992-10-29 Oklahoma Medical Research Foundation Titrage quantitatif de coagulation pour mesurer le facteur vii active

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992018870A1 (fr) * 1991-04-10 1992-10-29 Oklahoma Medical Research Foundation Titrage quantitatif de coagulation pour mesurer le facteur vii active

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL JOURNAL, Volume 282, issued 15 March 1992, A. REHEMTULLA et al., "The Third Trp-Lys-Ser (WKS) Tripeptide Motif in Tissue Factor is Associated with a Function Site", pages 737-740. *
BIOCHEMISTRY, Volume 25, issued 1986, R. BACH et al., "Factor VII Binding to Tissue Factor is Reconstituted Phospholipid Vesicles: Induction of Cooperativity by Phosphatidylserine", pages 4007-4020. *
GENE, Volume 98, issued 15 February 1991, B.S. ANDREWS et al., "Conservation of Tissue Factor Primary Sequence Among Three Mammalian Species", pages 265-269. *
JOURNAL OF PARENTERAL SCIENCE AND TECHNOLOGY, Volume 42, No. 2S, Y.J. WANG et al., "Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers", pages S3-S26. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 266, No. 32, issued 15 November 1991, S. ROY et al., "Lysine Residues 165 and 166 are Essential for the Cofactor Function of Tissue Factor", pages 22063-22066. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 266, No. 4, issued 05 February 1991, W. RUF et al., "Phospholipid-Independent and -Dependent Interactions Required for Tissue Factor Receptor and Cofactor Function", pages 2158-2166. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 267, No. 4, issued 25 March 1992, W. RUF et al., "Cofactor Residues Lysine 165 and 166 are Critical for Substrate Recognition by the Tissue Factor-Factor VIIa Protease Complex", pages 6375-6381. *
TRENDS IN BIOCHEMICAL SCIENCE, Volume 16, issued January 1991, B.S. ANDREWS, "Is the WKS Motif the Tissue-Factor Binding Site for Coagulation Factor VII?", pages 31-36. *

Cited By (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6749853B1 (en) 1992-03-05 2004-06-15 Board Of Regents, The University Of Texas System Combined methods and compositions for coagulation and tumor treatment
US5877289A (en) * 1992-03-05 1999-03-02 The Scripps Research Institute Tissue factor compositions and ligands for the specific coagulation of vasculature
US6004555A (en) * 1992-03-05 1999-12-21 Board Of Regents, The University Of Texas System Methods for the specific coagulation of vasculature
US6036955A (en) * 1992-03-05 2000-03-14 The Scripps Research Institute Kits and methods for the specific coagulation of vasculature
US6093399A (en) * 1992-03-05 2000-07-25 Board Of Regents, The University Of Texas System Methods and compositions for the specific coagulation of vasculature
WO1997019357A1 (fr) * 1995-11-22 1997-05-29 Oklahoma Medical Research Foundation Essai de dosage pour les facteurs de coagulation sanguine viii et ix
AU721720B2 (en) * 1995-11-22 2000-07-13 Oklahoma Medical Research Foundation Assay for blood clotting factors VIII and IX
US6156321A (en) * 1997-01-22 2000-12-05 Board Of Regents, The University Of Texas System Tissue factor methods and compositions for coagulation and tumor treatment
US6132730A (en) * 1997-01-22 2000-10-17 Board Of Regents, The University Of Texas System Combined tissue factor and factor VIIa methods and compositions for coagulation and tumor treatment
US6132729A (en) * 1997-01-22 2000-10-17 Board Of Regents, The University Of Texas System Combined tissue factor and chemotherapeutic methods and compositions for coagulation and tumor treatment
US7951356B1 (en) 1997-01-22 2011-05-31 Board Of Regents, The University Of Texas System Tissue factor compositions and kits for coagulation and tumor treatment
US7101557B2 (en) 1997-01-22 2006-09-05 Board Of Regents The University Of Texas System Combined tissue factor methods for coagulation and tumor treatment
US6596847B2 (en) 1998-07-15 2003-07-22 Genentech, Inc. Tissue factor protein variants
WO2000004148A1 (fr) * 1998-07-15 2000-01-27 Genentech, Inc. Variantes de proteines de facteurs tissulaires presentant une affinite accrue pour le facteur de coagulation fvii/fviia
US7416860B2 (en) 2000-05-03 2008-08-26 Novo Nordisk Helathcare A/G Nucleic acids encoding human coagulation factor VII variants
US6905683B2 (en) 2000-05-03 2005-06-14 Novo Nordisk Healthcare A/G Human coagulation factor VII variants
US7026524B2 (en) 2000-09-13 2006-04-11 Novo Nordisk Healthcare A/G Human coagulation factor VII variants
US7173000B2 (en) 2000-11-09 2007-02-06 The Scripps Research Institute Modified factor VIIa
US6773896B2 (en) 2001-05-08 2004-08-10 Warner-Lambert Company Method for direct measurement of coagulation factor VIIa activity in plasma
US7052868B2 (en) 2001-09-27 2006-05-30 Novo Nordisk Healthcare A/G Human coagulation factor VII polypeptides
US7416861B2 (en) 2001-09-27 2008-08-26 Novo Nordisk Healthcare A/G Human coagulation factor VII polypeptides
US7399743B2 (en) 2001-10-26 2008-07-15 The Scripps Research Institute Targeted thrombosis
US6960657B2 (en) 2001-11-02 2005-11-01 Novo Nordisk Healthcare A/G Human coagulation factor VII polypeptides
US7419803B2 (en) 2001-11-02 2008-09-02 Novo Nordisk A/S Nucleic acids encoding human coagulation Factor VII polypeptides
EP2386312A2 (fr) 2002-05-08 2011-11-16 TERMAN, David S. Superantigènes intrathécaux et intratumoraux servant a traiter des maladies malignes
EP2283868A2 (fr) 2002-07-15 2011-02-16 Board of Regents, The University of Texas System Anticorps et peptides de duramycine sélectionnés se liant à des phospholipides et aminophospholipides anioniques et leur utilisation dans le traitement d'infections virales et du cancer
EP2269656A2 (fr) 2002-07-15 2011-01-05 Board Of Regents, The University Of Texas Anticorps et peptides de duramycine selectionnes se fixant a des phospholipides et des inophospholipides anioniques et leur utilisation dans le traitement d'infections virales et du cancer
EP2281578A2 (fr) 2002-07-15 2011-02-09 Board of Regents, The University of Texas System Anticorps et peptides de duramycine sélectionnés se fixant à des phospholipides et des aminophospholipides anioniques et leur utilisation dans le traitement d'infections virales et du cancer
EP2283869A2 (fr) 2002-07-15 2011-02-16 Board of Regents, The University of Texas System Anticorps et peptides de duramycin sélectionnés se liant à des phospholipides et aminophospholipides anioniques et leur utilisation dans le traitement d'infections virales et du cancer
EP2263697A2 (fr) 2002-07-15 2010-12-22 Board of Regents, The University of Texas System Conjugés comprenant un peptide de duramycine se fixant à des phospholipides et des aminophospholipides anioniques et leur utilisation dans le traitement d'infections virales
WO2004006847A2 (fr) 2002-07-15 2004-01-22 Board Of Regents, The University Of Texas System Anticorps et peptides de duramycine selectionnes se fixant a des phospholipides et des aminophospholipides anioniques et leur utilisation dans le traitement d'infections virales et du cancer
EP2357009A1 (fr) 2002-07-15 2011-08-17 Board of Regents, The University of Texas System Conjugés comprenant un peptide de duramycine se fixant à des phospholipides et des aminophospholipides anioniques et leur utilisation dans le traitement d'infections virales
EP2266624A2 (fr) 2002-07-15 2010-12-29 Board of Regents, The University of Texas System Conjugés comprenant un peptide de duramycine se fixant à des phospholipides et des aminophospholipides anioniques et leur utilisation dans le traitement d'infections virales
US6911323B2 (en) 2002-09-25 2005-06-28 Novo Nordisk Healthcare A/G Human coagulation factor VII polypeptides
US8674074B2 (en) 2003-09-09 2014-03-18 Novo Nordisk Healthcare Ag Coagulation factor VII polypeptides
EP2614837A1 (fr) 2007-11-09 2013-07-17 Affitech Research AS Compositions d'anticorps anti-VEGF et procédés
EP3225991A1 (fr) * 2016-03-30 2017-10-04 Sysmex Corporation Réactif pour la mesure de temps de prothrombine, son procédé de production et procédé de mesure de temps de prothrombine
EP3225992A1 (fr) * 2016-03-30 2017-10-04 Sysmex Corporation Réactif pour la mesure de temps de prothrombine et son procédé de production
JP2017181265A (ja) * 2016-03-30 2017-10-05 シスメックス株式会社 プロトロンビン時間測定用試薬、その製造方法およびプロトロンビン時間の測定方法
CN107271698A (zh) * 2016-03-30 2017-10-20 希森美康株式会社 凝血酶原时间的测定方法、测定用试剂及其制造方法
US10640805B2 (en) 2016-03-30 2020-05-05 Sysmex Corporation Reagent for prothrombin time measurement, method for production thereof, and method for measurement of prothrombin time
US10775393B2 (en) 2016-03-30 2020-09-15 Sysmex Corporation Reagent for prothrombin time measurement, method for production thereof, and method for measurement of prothrombin time
CN107271698B (zh) * 2016-03-30 2021-04-13 希森美康株式会社 凝血酶原时间的测定方法、测定用试剂及其制造方法

Similar Documents

Publication Publication Date Title
WO1994028017A1 (fr) Compositions a base de facteur tissulaire mutant humain utiles comme antagonistes du facteur tissulaire
WO1994007515A1 (fr) Thromboplastine tissulaire mutante depourvue d'activite activant le facteur vii
JP4317976B2 (ja) 修飾されたプロテアーゼ切断部位を有するｘ因子類似体
Matthews et al. A survey of furin substrate specificity using substrate phage display
KR101623602B1 (ko) 변형된 제ⅶ 인자 폴리펩티드 및 그의 용도
EP0278776B2 (fr) Méthodes et acide desoxyribonucléique pour la préparation de la protéine du facteur tissulaire
KR20100016462A (ko) 변형 제ｖｉｉ 인자 폴리펩타이드 및 이의 용도
CZ292703B6 (cs) Mutantní proteiny lidského interleukinu-4
JP2002542831A (ja) 改変型ビタミンｋ依存性ポリペプチド
JPH0771496B2 (ja) ヒト第ｖｉｉｉ：ｃ因子活性を有する組換えタンパク複合体の生産
CA2276091A1 (fr) Medicaments diriges vers la membrane cellulaire
PL191778B1 (pl) Analog czynnika X Δ , rekombinowany kwas nukleinowy, wektor, komórka, kompozycje i ich zastosowania oraz sposób wytwarzania kompozycji
US5994296A (en) Human mutant tissue factor compositions useful as tissue factor antagonists
US6322962B1 (en) Sterol-regulated Site-1 protease and assays of modulators thereof
KR100232683B1 (ko) 콜라겐에 의해 유도되는 혈소판 응집 억제제
US6958322B1 (en) Factor X analog with an improved ability to be activated
CZ402097A3 (cs) Deriváty prothrombinu
JP2004527452A (ja) 医薬用途のためのトロンボモジュリン
JP2010533684A (ja) 血液凝固異常の治療又は予防のための変異アンチトロンビンの使用
US5405946A (en) Recombinant protein S variants deficient in C4BP binding activity, compositions and therapeutic methods
AU2002235073B2 (en) Protein C variants
AU741798B2 (en) Recombinant protein C and protein S variants
AU2002235073A1 (en) Protein C variants
WO1989009818A1 (fr) Procede de purification de phospholipase a2 et de production de polypeptides semblables a la phospholipase a2
US20150337284A1 (en) Factor ix variants

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载