WO1995005836A9 - La draculine, son procede de preparation et d'utilisation - Google Patents

La draculine, son procede de preparation et d'utilisation

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Publication number
WO1995005836A9
WO1995005836A9 PCT/US1994/009488 US9409488W WO9505836A9 WO 1995005836 A9 WO1995005836 A9 WO 1995005836A9 US 9409488 W US9409488 W US 9409488W WO 9505836 A9 WO9505836 A9 WO 9505836A9
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ala
seq
leu
gly
draculin
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PCT/US1994/009488
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English (en)
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WO1995005836A1 (fr
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Priority to AU78282/94A priority Critical patent/AU7828294A/en
Publication of WO1995005836A1 publication Critical patent/WO1995005836A1/fr
Publication of WO1995005836A9 publication Critical patent/WO1995005836A9/fr

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  • This invention relates to a novel protein having anticoagulant activity fo mammalian blood, methods for purifying this novel protein, and uses therefore
  • Blood coagulation involves a series of proteolytic reactions in which inactive enzyme precursors (factors) are converted to their catalytically active forms (activated factors). It is referred to as a cascade because the activated form of each factor catalyzes the activation of the next factor in the pathway.
  • the organization of the enzymes into a cascade of catalytic steps allows for extraordinarily control over the coagulation pathway and also provides the potential for a rapidly amplifiable response to trauma.
  • the coagulation cascade culminates in the thrombin-catalyzed conversion of soluble fibrinogen into insoluble fibrin.
  • the fibrin polymerizes and, along with activated platelets, forms a stable hemostatic plug at the site of injury.
  • Thrombin is activated by the action of Factor Xa (activated Factor X) on prothrombin.
  • Factor Xa activated Factor X
  • Tissue injury exposes the blood to tissue factor, which is normally present only in the deep subendothelial layers of blood vessels.
  • tissue factor which is normally present only in the deep subendothelial layers of blood vessels.
  • the exposed tissue factor binds circulating Factor VII and facilitates the conversion of Factor VII into its enzymatically active form, Factor Vila.
  • the Factor Vila/tissue factor complex proteolytically activates Factors IX and X to their enzymatically active forms, Factors IXa and Xa.
  • Factor IXa associates with the cofactor Villa on the surface of activated platelets and endothelial cells, constituting the enzymatic "tenase" complex which also catalyzes the activation of Factor X.
  • Factor Xa associates with cofactor Va on cell surfaces, constituting the enzymatic "prothrombinase' 1 complex, which catalyzes the formation of thrombin.
  • Thrombin acts as a feedback activator of several factors and cofactors in the coagulation cascade. The sum of these reactions leads to a burst of thrombin. This thrombin both activates platelets and catalyzes the generation of fibrin, resulting in the formation of the hemostatic plug.
  • Anticoagulants are agents that interfere with the activities of the intrinsic and/or extrinsic coagulation pathways. Anticoagulants are used in the prophylaxis and treatment of various thrombotic disorders, in medical procedures which require the circulation of blood outside the body, and in the preparation of stored blood and blood products. Many different compounds have an anticoagulant action.
  • An example is the Kunitz inhibitor which is isolated from soybeans. This inhibitor blocks the coagulation cascade by inhibition of activated Factor X, but the specificity is so low that many side effects occur, ruling out therapeutic applications. It is desirable to have anticoagulants which have low toxicity and few side effects for use in medical applications.
  • Anticoagulants can act by accelerating the inactivation of clotting factors or by inhibiting their synthesis.
  • Two types of anticoagulant therapy are in general clinical use, heparin injection and oral anticoagulation. Both types of therapy are administered in order to treat or prevent thrombosis (arterial and venous), but both are far from ideal drugs. There is a continuing search for new medicines having similar effects.
  • the heparins isolated from hog gastric mucosa or pig lung, consist of negatively-charged sulfated mucopolysaccharide polymers.
  • the heparins (and heparin-like drugs) act by enhancing the activity of natural inhibiting proteins in the plasma, including antithrombin III and heparin cofactor II, thus accelerating the inactivation of thrombin and Factors IXa, Xa, and Xlla.
  • the therapeutic use of standard heparins can, in some situations, lead to fatal hemorrhage or irreversible organ damage, so its usage is contraindicated in many patients.
  • Hirudin is a protein found in the saliva of the medicinal leech, Hirudo medicinalis. which helps keep the blood ingested by the leech from coagulating. Hirudin acts by combining rapidly and essentially irreversibly with thrombin and inactivating it. Its derivatives and fragments (Hirulogs) act in a similar way (Krstenansky, J.L., Thrombosis Research 52:137-141. 1988). Hirudin has been expressed recombinantly (Ambler, J., Satellite Symposium of the Intemational Societ y of Hematolo ⁇ y.
  • prothrombinase In order to prevent the generation of thrombin, one can also inhibit the enzyme that converts prothrombin into thrombin, prothrombinase.
  • This enzyme is a complex of Factor Xa, having the active site, and a cofactor (Factor Va), both adsorbed at a phospholipid surface.
  • Factor Xa is obtained from the proenzyme Factor X by the action of a similar enzyme complex ftenase" consisting of factor IXa (enzyme), Factor Villa (cofactor) on phospholipid.
  • Antistasin a reversible, slow binding inhibitor of Factor Xa
  • Tick anticoagulant peptide TMP
  • the equilibrium dissociation constant Kj of both inhibitors is in the range of 0.3 - 0.5 nM (Dunwiddie, C, iL Biol. Chem.
  • the anticoagulant properties of vampire bat saliva have been intuitively known for many years. Vampire bats are indigenous to the new world; they exist only from the northern part of Mexico to the northern part of Argentina. Early accounts of the anticoagulant effect of saliva from an haematophage bat were reported by Bier et al. (Bier, O.G., C. R. Soc. Biol. (Paris) 110:129-131. 1932; Romana. C. Soc. Pathol. Exot. 211:399-403. 1939).
  • the best characterized factor from vampire bat saliva is a plasminogen activator that has been named Desmokinase (Hawkey, C, Nature 211:434-435. 1966; Cartwright, T., Blood 43:318-326. 1974). Plasminogen activator exhibits modest inhibition of platelet aggregation (Hawkey, C, Brit. J. Haematol. 13:1014-1020, 1967).
  • Bat-PA(H) shows 85% amino acid homology to human tissue-type plasminogen activator (tPA).
  • Bat- PA(H) is similar to tPA in its ability to activate plasminogen, but is notable in the degree to which this activity is stimulated by the presence of a fibrin cofactor.
  • a cDNA encoding full length vampire bat salivary plasminogen activator has also been isolated.
  • the present invention relates to a novel anticoagulant protein which has been identified in and purified from the saliva of vampire bats.
  • This protein is characterized in having the following properties: (1) anticoagulant activity characterized by the prolongation of whole blood coagulation time, prolongation of activated partial thromboplastin time, prolongation of prothrombin time at concentrations that prolong activated partial thromboplastin time, the inhibition of Factor Xa activity, the inhibition of Factor IXa activity, no prolongation of thrombin time, (2) stability for at least 7 days at room temperature, after freezing at -30°C or -80°C, after repeated freezing and thawing, and after incubation for 30 minutes at pH 5.5- 9.0, (3) sensitivity to heating for 10 minutes at 80°C, but only partial inactivation after incubation at 60°C for 10 minutes and 30 minutes
  • the anticoagulant activity is not inhibited by serine protease inhibitors such as PMFS or DFP.
  • the molecular weight of Draculin ranges from approximately
  • the anticoagulant protein of the present invention (hereinafter called "Draculin”) has been substantially purified from vampire bat saliva, and the partial amino acid sequence has been determined. Draculin and its fragments have also been prepared in a recombinant DNA biosystem. The present invention provides Draculin or its fragments in essentially homogeneous form.
  • the present invention further provides replicable expression vectors incorporating a DNA sequence encoding Draculin or biologically active fragments thereof, and a self-replicating prokaryotic or eukaryotic host cell system transformed or transfected by said vectors.
  • the present invention further provides a nucleic acid probe comprising a nucleic acid of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding Draculin.
  • the anticoagulant protein of the present invention is useful to inhibit or prolong coagulation when administered to an individual in need of such treatment. Accordingly, the present invention provides pharmaceutical compositions containing Draculin as the active ingredient in a pharmaceutically acceptable carrier.
  • Figure 1 demonstrates protein and anti FXa activity profiles of a saliva run on a Sephacryl S-200 molecular sieve column.
  • Figure 2 demonstrates isoelectric focusing of purified Draculin.
  • Isoelectric point markers were: 1. trypsinogen (9.3); 2. lentil lectin (8.15); 3. horse myoglobin (6.85); 4. ⁇ -lactoglobulin A (5.2); 5. soybean trypsin inhibitor (4.55); and 6. amyloglucosidase (3.5).
  • Figure 3 demonstrates the PAGE electrophoresis of crude saliva and purified Draculin under reducing and non-reducing conditions.
  • Figure 4 demonstrates HPLC of purified Draculin on a Protein Pak 300sw column.
  • Figure 5 demonstrates the effect of partially purified saliva on the amidolytic activity of Factor Xa.
  • Figure 6 demonstrates influence of Draculin on the splitting of the chromogenic substrate S-2337 by Factor Xa.
  • Figure 7 demonstrates the titration of Factor Xa with Draculin.
  • Figure 8 demonstrates the effect of Draculin on Factor Xa bound in prothrombinase.
  • Figure 9 demonstrates the influence of Draculin on the splitting of S2337 by free Factor Xa.
  • Figure 10 demonstrates the titration of Factor IXa with Draculin.
  • Figure 11 demonstrates the influence of Draculin on Factor IXa activity.
  • Figure 12 demonstrates the anti-IXa activity of a Draculin-Xa complex.
  • Figure 13 demonstrates the anti-Xa activity of a Draculin-IXa complex.
  • Figure 14 demonstrates The effect of Draculin on extrinsic thrombin generation of def ibrinated plasma.
  • Figure 15 demonstrates the influence of Draculin on intrinsic thrombin generation.
  • Figure 16 demonstrates the effect of increasing the concentration of Factor Villa during the lag-phase of intrinsic TGT
  • Figure 17 demonstrates the effect of Draculin and crude saliva on intrinsic thrombin generation in platelet rich plasma.
  • Figure 18 demonstrates the effect of Draculin and crude saliva on intrinsic thrombin generation in platelet poor plasma.
  • Figure 19 demonstrates the effect of Draculin on thrombin generation in non-anticoagulated whole blood.
  • Figure 20 demonstrates the effect of crude saliva on thrombin generation in non-anticoagulated whole blood.
  • Figure 21 demonstrates the homology between Draculin sequences and human lactoferrin.
  • Line 1 shows the amino acid sequence of human lactoferrin.
  • Line 2 shows the amino acid sequence of Draculin peptides, as determined in Example 8.
  • Line 3 shows the amino sequence deduced from the cDNA sequences determined in Examples 10-12. Identical residues are shaded. Vertical bars separate distinct peptides or segments of peptides. The boundary between the N- and C- lobes of lactoferrin is indicated by I— . DETAILED DESCRIPTION OF THE INVENTION
  • the present invention provides a biologically active anticoagulant poiypeptide isolated from the saliva of vampire bats in substantially homogeneous form.
  • the invention encompasses this naturally occurring anticoagulant protein in partially purified as well as substantially homogeneous form, as well as synthetically produced anticoagulant protein, anticoagulant protein produced by a recombinant biosystem, biologically active fragments of the anticoagulant protein, biologically active sequence analogues of the anticoagulant protein, and pharmaceutically acceptable salts and derivatives thereof.
  • aminopeptide means a linear array of amino acids connected one to the other by peptide bonds between the ⁇ -amino and carboxy groups of adjacent amino acids.
  • substantially purified is used herein to mean “substantially homogeneous", which is defined as a proteinaceous material which is substantially free of compounds normally associated with it in its natural state (e.g., other proteins or peptides, carbohydrates, lipids). Most preferably, it means a poiypeptide which may be glycosylated or non-glycosylated and which is characterized by a reproducible single molecular weight and/or multiple set of molecular weights, chromatographic response and elution profiles, amino acid composition and sequence and biological activity. "Substantially purified” is not meant to exclude artificial or synthetic mixtures with other compounds. The term is also not meant to exclude the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilizers, or compounding with a pharmaceutically acceptable preparation.
  • biologically active poiypeptide means the naturally occurring poiypeptide per se. as well as biologically active analogues thereof, including synthetically produced polypeptides and analogues thereof, and natural and pharmaceutically acceptable salts and pharmaceutically acceptable derivatives thereof.
  • biologically active poiypeptide also encompasses biologically active fragments, as well as “biologically active sequence analogues” thereof. Different forms of the peptide may exist in nature. These variations may be characterized by differences in the nucleotide sequence of the structural gene coding for proteins of identical biological function.
  • biologically active sequence analogue includes non-naturally occurring analogues having single or multiple amino acid substitutions, deletions, additions, or replacements. All such allelic variations, modifications, and analogues resulting in derivatives which retain one or more of the native biologically active properties are included within the scope of this invention.
  • salts refers to both salts of carboxy groups of the poiypeptide or protein chain and to acid addition salts of amino groups of the poly-peptide chain.
  • Salts of the carboxy group may be formed with either inorganic or organic bases by means known in the art per se.
  • Inorganic salts include, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like.
  • Salts with organic bases include those formed, for example, with amines such as triethanolamine, arginine, lysine, piperidine, caffeine, procaine and the like.
  • Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids such as, for example, acetic acid or oxalic acid.
  • Derivatives may also be prepared from the functional groups which occur at side chains on the residues of the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain diagnostically or therapeutically acceptable.
  • Both the salts and the derivatives encompassed by the invention are those which are therapeutically or diagnostically acceptable, i.e., those which do not eliminate the biological activity.
  • Therapeutically useful salts and derivatives are further non-toxic to the human or other animal patient in the appropriate dosage utilized in treatment.
  • the term "specific activity" refers to the activity in assays described in this application and other assays known in the art that measure blood coagulation. This term is related to the amount of biologically active protein by weight in a sample and more precisely understood to be a measure of purity of active protein/total sample protein calculated without considering the presence of intentionally added protein materials such as albumin.
  • nucleotides are indicated by their bases, using the following standard one-letter abbreviations:
  • amino acid residues are indicated using the following standard one- or three-letter abbreviations:
  • amino acid as used herein is meant to denote the above- recited natural amino acids and functional equivalents thereof.
  • coding strand is used herein to mean DNA sequences which, in the appropriate reading frame, code for the amino acids of a protein.
  • synthesis or use of a coding sequence may necessarily involve synthesis or use of the corresponding complementary strand, as shown by: ⁇ '-CGGGGAGGA-S'/S'- GCCCCTCCT-5' which "encodes" the tripeptide NH2-Arg-Gly-Gly-C02H.
  • a discussion of or claim to one strand is deemed to refer to or to claim the other strand and the double stranded counterpart thereof as is appropriate, useful or necessary in the practice of the art.
  • cDNA is used herein to mean a DNA molecule or sequence which has been enzymatically synthesized from the sequence(s) present in an m RNA template.
  • vector is used herein to mean a piasmid, phage DNA or other DNA sequence which is able to replicate in a host cell, typically characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion for the insertion of heterologous DNA without attendant loss of an essential biological function of the DNA, e.g., replication, production of coat proteins or loss of expression control regions such as promoters or binding sites, and which may contain a selectable gene marker suitable for use in the identification of host cells transformed therewith, e.g., tetracycline resistance or ampicillin resistance.
  • piasmid is used herein to mean a non-chromosomal double- stranded DNA sequence comprising an intact "replicon" such that the piasmid is replicated in a host cell.
  • the characteristics of that cell may be changed (or transformed) as a result of the DNA of the piasmid.
  • a piasmid carrying the gene for tetracycline resistance (TetR) transforms a cell previously sensitive to tetracycline into one which is resistant to it.
  • a cell transformed by a piasmid is called a "transformant."
  • phage or bacteriophage is used herein to mean a bacterial virus, many of which consist of DNA sequences encapsulated in a protein envelope or coat ("capsid").
  • promoter is used herein to mean the DNA sequences upstream from a gene which promote its transcription.
  • the biologically active anticoagulant protein of the present invention (hereinafter also referred to as "Draculin”) has been isolated from vampire bat saliva. It can also be prepared by chemical synthesis or in a recombinant DNA biosystem. Biologically active fragments of Draculin can also be prepared using synthetic or recombinant technologies which are known in the art.
  • Draculin has been substantially purified using the methods of dialysis, hydroxyapatite chromatography, and ultrafiltration. It is characterized as having an anticoagulant activity which is stable for at least seven days at room temperature, and which is stable after repeated freezing and thawing. It has an apparent molecular weight of approximately 80.5 kD as measured by polyacrylamide gel electrophoresis under both reducing and nonreducing conditions (Fig. 3). Purified Draculin elutes at a position corresponding to a molecular mass of approximately 88.5 kDa from a silica based molecular sieve HPLC column (Fig. 4).
  • the calculated molecular weight of Draculin is approximately 75 kD. Differences between the apparent molecular weights which are estimated for Draculin using these different methods may reflect the relative sensitivities or limitations of the methods themselves, whether the Draculin is glycosylated or not and/or other post- translational processing of native Draculin, or a combination of these factors. One of ordinary skill in the art will appreciate that the molecular weight determinations are subject to interpretation in light of these factors. Draculin is distinct from known anticoagulants in that it inhibits both Factors IX and X of the blood coagulation cascade. A partial amino acid sequence has been determined for Draculin and this is shown in Example 8. The amino acid sequence of Draculin shows partial sequence homology to the transferrin family of proteins, particularly to lactoferrin.
  • This invention also provides an isolated nucleic acid molecule encoding Draculin and having a coding sequence comprising the sequence shown in SEQ ID NO: 61.
  • This invention provides a vector comprising an isolated nucleic acid molecule such as DNA, RNA, or cDNA encoding Draculin.
  • vectors are viruses such as bacteriophages (such as phage lambda), cosmids, plasmids (such as pUC18, available from Pharmacia, Piscataway, N.J.), and other vectors.
  • Nucleic acid molecules are inserted into vector genomes by methods well known in the art. For example, insert and vector DNA can both be digested with a restriction enzyme to create complementary ends on both molecules which are then ligated together using a ligase. Alternatively, linkers can be ligated to the insert DNA which correspond to a restriction site in the vector DNA, which is then digested with the restriction enzyme which cuts at that site.
  • Expression vectors refer to vectors which are capable of transcribing and translating DNA sequences contained therein, where such sequences are linked to regulatory sequences capable of affecting their expression. These expression vectors are capable of replicating in the host organism either as episomes or as an integrated part of the genomic DNA.
  • This invention provides a method of preparing Draculin which comprises inserting a nucleic acid encoding Draculin in a suitable vector, inserting the resulting vector in a suitable host cell, recovering the Draculin produced by the resulting cell, and purifying the Draculin so recovered.
  • This method for preparing Draculin uses recombinant DNA technology methods which are well known in the art and are exemplified in Examples 10-12.
  • nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding Draculin, for example with a coding sequence included within the sequence shown in SEQ ID NO: 61.
  • Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary greatly in length and may be labeled with a detectable label, such as a radioisotope or fluorescent dye, to facilitate detection of the probe.
  • DNA probe molecules are produced by insertion of a DNA molecule which encodes Draculin into suitable vectors, such as plasmids or bacteriophages, followed by insertion into suitable bacterial host cells and replication and preparation of the DNA probes, all using methods well known in the art.
  • the DNA may be extracted from a cell lysate using phenol and ethanol, digested with restriction enzymes corresponding to the insertion sites of the DNA into the vector, electrophoresed, and cut out of the resulting gel, then labeled with a radioisotope or a fluorescent dye.
  • Such DNA probes are useful for the isolation of Draculin genes by homology screening of genomic or cDNA libraries, or by the use of amplification techniques such as the Polymerase Chain Reaction.
  • synthesized oligonucleotides produced by a DNA synthesizer
  • complementary to the sequence of DNA molecule which encodes Draculin are useful as probes for these genes, for their associated mRNA, or for the isolation of related genes.
  • the invention also encompasses compositions comprising Draculin, such as pharmaceutical and diagnostic compositions, and methods of using these Factors In the treatment and diagnosis of coagulation disorders.
  • the biologically active proteinaceous factor of the present invention is able to prolong whole blood coagulation time, prolong activated partial thromboplastin time, prolong prothrombin time at concentrations that prolong activated partial thromboplastin time, inhibit Factor Xa activity, and inhibit Factor IXa activity, but not prolong thrombin time.
  • the present invention provides a proteinaceous anticoagulant useful in the treatment of human and animal disorders characterized by abnormal blood coagulation.
  • Draculin may be by parenteral, intravenous, intra ⁇ muscular, subcutaneous, rectal, transdermal or any other suitable means.
  • the dosage administered may be dependent upon the age, weight, kind of concurrent treatment, if any, and nature of the pathology being treated.
  • the biologically active anticoagulant protein of the present invention may be employed in such forms as liquid solutions, suspensions, elixirs, or sterile liquid forms such as solutions or suspensions.
  • Suitable carriers include diluents or fillers, sterile aqueous media and various non-toxic organic solvents.
  • the compositions may be formulated in the form of powders, aqueous suspensions, or solutions, injectable solutions, elixirs, syrups and the like and may contain one or more stabilization agents such as human serum albumin, sugar or amino acid, antibacterial, and preserving agents in order to provide a pharmaceutically acceptable preparation.
  • Any inert carrier is preferably used, such as saline, or phosphate-buffered saline, or any such carrier in which the factors used in the method of the present invention have suitable solubility properties for use in the method of the present invention.
  • the particular carrier and the ratio of active compound to carrier are determined by the solubility and chemical properties of the proteinaceous factors, the particular mode of administration and standard pharmaceutical practice.
  • solutions or suspensions of these Factors In aqueous alcoholic media or in sesame or peanut oil or aqueous solutions of the soluble pharmaceutically acceptable salts can be employed.
  • the dosage regimen in carrying out the methods of this invention is that which insures maximum therapeutic response until improvement is obtained and thereafter the minimum effective level which gives relief.
  • Doses may vary, depending on the age, severity, body weight and other conditions of the patients but are ordinarily in the area of about 0.01 mg/kg to about 100 mg/kg, preferably about 0.1 mg/kg to about 50 mg/kg, and most preferably about 1 mg/kg to about 10 mg/kg of body weight in injectable form; such may, of course, be given in divided doses. With other forms of administration equivalent or adjusted doses will be administered depending on the route of administration.
  • the compounds of this invention may be administered in combination with thrombolytic agents, anti-platelet agents, or other antithrombotic agents.
  • the compounds of the invention may be administered as frequently as is necessary to achieve and sustain the desired therapeutic response. Some patients may respond quickly to a relatively large or small dose and require little or no maintenance dosage. On the other hand, other patients may require sustained dosing to prolong the desired response.
  • a deposit of biologically pure culture of the following strain was made with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, the accession number indicated was assigned after successful viability testing, and the requisite fees were paid. Access to said culture will be available during pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 C.F.R. ⁇ 1.14 and 35 U.S.C. ⁇ 122. All restriction on availability of said culture to the public will be irrevocably removed upon the granting of a patent based upon the application and said culture will remain permanently available for a term of at least five years after the most recent request for the furnishing of a sample and in any case for a period of at least 30 years after the date of the deposit. Should the culture become nonviable or be inadvertently destroyed, it will be replaced with a viable culture(s) of the same taxonomic description.
  • Coagulation Assays A number of clinically relevant, global coagulation assays such as described in sections 1b through 1 ⁇ b low were i-til'zeri in order to characterize the anticoagulant properties of partially purified and purified Draculin. In addition, in vitro coagulation assays consisting of reconstituted, purified components were utilized to identify the specific proteases (assays 1f through 1i below) within the coagulation cascade inhibited by Draculin.
  • Platelet rich plasma was obtained by centrifugation (900 x g, 15 minutes, 15° C) of freshly drawn citrated blood. The platelet count was adjusted to 3 X 10 ⁇ cells/ml with homologous platelet poor plasma.
  • Platelet poor plasma was obtained by centrifuging platelet rich plasma once for 15 minutes at 15° C at 10,000 x g; and again at 4° C for 60 minutes at 23,000 x g.
  • WBCT Human Whole Blood Coagulation Time
  • APTH Activated Partial Thromboplastin Time
  • PT Prothombin Time
  • TT Thrombin Time
  • the mixture was prewarmed to 37°C. To this, 50 ⁇ l of a thrombin solution was added and the time to clot or gel formation was measured by visual observation. The thrombin concentration was chosen so as to obtain a clotting time of 30 seconds when no Draculin was added (control TT).
  • Factor Xa amidolvtic activity was assayed spectrophotometrically by monitoring the release of paranitroaniline (pNA) from the chromogenic substrate S-2222 (N-benzoyl-L-isoleucyl-L-glutamyl-glycyl-L-arginine-pNA, Kabi Diagnostica, Sweden) or S-2337 (N-benzoyl-L-isoleucyl-L-glutamyl- (gamma piperidyl)-glycyl-L-arginine-pNA, Kabi Diagnostica, Sweden).
  • pNA paranitroaniline
  • the micro-plate was incubated at 37° for 5 to IC minutes, then read at 405 nm in a micro-plate reader.
  • Control wells contained buffer as the sample. Positive fractions (containing anti-FXa activity) were those where the yellow color due to splitting of the substrate was diminished compared to the controls. This was shown as a decrease in O.D. at 405 nm. Fractions yielding O.D. values below 70% of the control value were typically pooled.
  • Factor IXa activity was assayed using the method of Wagenvoord et al. (Haemostasis 20:276-288. 1990).
  • Reagent 1 contained 300 nM thrombin (to activate Factor VIII), 15 mM CaCl2, and 60 ⁇ M phospholipids (20% phosphatidylserine, 80% phosphatidylcholine).
  • thrombin to activate Factor VIII
  • CaCl2 mM
  • phospholipids (20% phosphatidylserine, 80% phosphatidylcholine).
  • To 100 ⁇ l of this mixture was added 100 ⁇ l of the sample (Factor IXa at a maximal concentration of 0.5 nM, with or without the addition of Draculin).
  • the reaction was initiated by the addition of 100 ⁇ l of Reagent 2, which contained 1 ⁇ M purified Factor X, 3 units/ml purified Factor VIII, and 0.1 mM CaCl2- At 4 minutes, 100 ⁇ l samples were removed.
  • the production of Factor Xa was measured in 20 mM EDTA, 175 mM NaCl, 50 mM Tris-HCI) using a chromogenic substrate (S-2222 or S- 2337).
  • the production of Factor Xa in this assay has been shown by Wagenvoord et al. to be proportional to the concentration of Factor IX.
  • Factor IX was isolated using the method of Miletich (Methods Enzvmol. 80:221-229. 1981 ).
  • Factor IX was activated with 0.1 % of Factor Xla.
  • Factor Xla was isolated as described by ⁇ sterud and Rapaport (Proc. Natl. Acad. Sci. USA 12:5260- 5264, 1974). The concentrations of Draculin added to the sample of Factor IXa did not significantly inhibit the amount of Factor Xa formed.
  • Thrombin Generation Time(TGT) .xi- p.sic Thrombin generation was measured in platelet-free plasma.
  • TGT Thrombin Generation Time
  • the amount of thrombin generated was determined by removing a 10 ⁇ l aliquot of the mixture and adding it to a disposable plastic microcuvette containing 465 ⁇ l of buffer and 25 ⁇ l of a 4 mM solution of the chromogenic substrate HD gly-pro-arg-pNA. After 120 seconds the reaction in the cuvette was stopped by adding 0.3 ml of concentrated (98%) acetic acid, the cuvette was again thoroughly mixed then kept at room temperature until reading. The samples were read at 405 nM in a dual wavelength photometer. The generation of p-Nitroaniline was linear in time up to an O.D. of 0.900 and directly proportional to the amount of thrombin generated in the sample.
  • Intrinsic thrombin generation was measured similarly in defibrinated plasma. In this case, thrombin generation was triggered by the addition of 50 ⁇ l of buffer containing 1 ⁇ M phospholipids (20% phosphatidylserine, 80% phosphatidylcholine), 100 mM CaCl2, and 25 ⁇ g/ml of kaolin.
  • Vampire bats were captured from wild colonies living in a cave in the northwest part of Venezuela (State of Falcon). They were kept in individual metabolic cages under controlled light and temperature conditions. The animals were maintained on bovine blood anticoagulated with sodium citrate (0.32%). Food was given every 24 hours, in the late afternoon. Water was given ad libitum. Vampire bats were anesthetized with a mixture of 2.5% 2- bromo-2-cloro, 1,1,1-trifluorethane (Haiothan, Hoechst), 30% nitrous oxide, in oxygen. The anesthetized animals received 20 ⁇ l of 1% piiocarpine (Isopto Carpin, Alcon Labs., Inc. Ft.
  • Saliva was collected in plastic microcentrifuge tubes kept on ice. The collection period was 30 to 40 minutes for each animal, with a yield of about 1 ml saliva per animal. Individual saliva samples were kept at -30°C until further use. Approximately 13 ml of saliva were thawed, and centrifuged at 12000 x g for 5 minutes The samples were pooled and dialyzed overnight against 8 liters of double distilled water at 4°C. The dialysate was centrifuged at 48000 xg for 20 minutes and the insoluble material was discarded. The dialysate was lyophilized and redissolved with sonication in about 4.8 ml of double distilled water.
  • Example 2 The crude saliva sample obtained in Example 2 was loaded on a Sephacryl S-200 column (58 x 4.5 cm) and eluted with water. Two ml fractions were collected. The protein content of each fraction was assayed using the Bradford technique (Bradford, M.M., A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem., (1976) 72: 248-254). Two protein peaks were observed ( Figure 1). The second protein peak was found to contain anti- Xa activity. Fractions corresponding to this peak were pooled, lyophilized, and stored at -30°C until further use. These fractions are referred to as partially purified saliva (PPS).
  • PPS partially purified saliva
  • the anticoagulant activity of crude saliva and of the partially purified fractions was measured under a variety of conditions using the APTT assay, as described in Example 1.
  • the anticoagulant activity was completely lost after heating the samples for 10 minutes at 80°C.
  • 40% of the activity was lost after 10 minutes and 80% of the activity was lost after 30 minutes.
  • Incubation of the samples for 30 minutes at pHs in the range of 5.5 to 9.0 did not affect the anticoagulant activity.
  • the column was eluted with a 22.5 ml gradient from 0.25 M to 1 M potassium phosphate, pH 6.8. Fractions of 1-2 ml were collected. Protein content of the fractions was assayed by the Bradford dye binding assay, and anticoagulant activity was measured using the microplate Factor Xa assay as described in Example 1. The bulk of the proteins from the saliva samples eluted with the sample buffer, the NaCl wash, or the 200 mM phosphate wash. A sharp peak of anticoagulant activity eluted at about 0.3 M potassium phosphate.
  • Size exclusion HPLC of purified ' Draculin was performed in a Shimadzu HPLC equipment (Shimadzu Corp., Japan) using a silica based molecular sieve column (10 ⁇ m Protein Pak 300 sw, Waters, Millipore Corp., USA). Equilibration of the column and elution of the sample was done with 250 mM potassium phosphate, pH 6.8. Calibration of this column was done with molecular weight markers from Pharmacia (Sweden).
  • HPLC of the purified native Draculin showed a single, symmetric, protein peak eluting at a position corresponding to a molecular mass of 88.5 kDa (Fig. 4). This result was obtained using 0.25 M phosphate for elution of the column. Several trials using lower ionic strength eluents resulted in apparent binding of the protein to the column matrix, which markedly retarded elution of the protein.
  • Isoelectric focusing was performed on an agarose-based gel in a Multiphor LKB (Sweden) electrophoresis chamber as described by Vesterberg et al., (Vesterberg, O. and Gramstrump-Christensen, B., Sensitive silver staining of proteins after isoelectric focusing in agarose gels. Electrophoresis 5:282-285, 1984) with minor modifications. Ampholine (pH 3.5-10, LKB, Sweden) was added to obtain a final concentration of 5%.
  • Markers for calibration were obtained from Pharmacia (Isoelectric Focusing Calibration Kit in the range of :a) pH 2.5 - 6.5 containing Pepsinogen, Amiloglucosidase, Glucose oxidase, Soybean trypsin inhibitor, ⁇ -Lactoglobulin A, Bovine Carbonic Anhydrase B, Human Carbonic Anhydrase B and Methyl Red dye; b) in the range of pH 3-10, containing Amiloglucosidase, Soybean Trypsin Inhibitor, ⁇ -Lactoglobulin A, Bovine Carbonic Anhydrase B, Human Carbonic Anhydrase B, Horse Myoglobin, Lentil Lectin and Trypsinogen.
  • Draculin into large fragments 60 - 70K. None of the other enzymes produced detectable cleavage, suggesting that Draculin does not consist of multiple distinct structural domains connected by easily cleavable hinge regions. Draculin, like many other proteins is probably folded into a complex three- dimensional structure.
  • the coagulation time of whole blood was significantly increased in the presence of PPS.
  • One ml of whole blood was taken directly onto 50 ⁇ l of EACA (114 mg/ml), with or without various amounts of PPS.
  • the mean coagulation time of the control (no PPS added) was 180 seconds. Table 1 shows the prolongation over the control time in the presence of PPS.
  • Clot retraction was not affected by the presence of PPS.
  • the clot was firm and had withdrawn from the side of the tube after one hour.
  • the amount of serum expressed spontaneously from the clot after two hours at 37°C was also not diminished by the presence of PPS.
  • PPS prolonged the APTT over control values (46 seconds) in a dose-dependent manner as shown in Table 2. Increasing the incubation time of plasma and saliva prior to activation did not influence this inhibitory effect, showing that the anticoagulant activity of PPS was not simply due to proteolytic destruction of coagulation factors, consistent with the results described in Example 4b.
  • Draculin ⁇ ased on a molecular weight for Draculin of approximately 80 kD.
  • Thrombin time was not affected by PPS even at concentrations that produced an almost infinite prolongation of the APTT (> 60 minutes). This is in agreement with preliminary experiments using the chromogenic substrate S- 2238, which showed no effect of PPS on the amidolytic activity of thrombin.
  • Table 3 shows the effect of PPS (5 ⁇ g/ml protein) on plasma coagulation triggered by diluted thromboplastin. Values were obtained for PT at various rabbit brain thromboplastin (Ortho) dilutions. PT increased with increasing thromboplastin dilution.
  • Thrombo ⁇ plastin 0 1/10 1/100 1/500 1/1000 dilution
  • Fig. 5 shows the progress of color generation due to the amidolytic activity of FXa on the chromogenic substrate S-2337.
  • Curves B and C correspond to color generation, under the same conditions, in the presence of two concentrations of partially purified saliva. The concentrations were arbitrarily chosen to produce approximately 50% and 70% inhibition of the control FXa activity.
  • the results of these standard clotting assays demonstrate that Draculin dose-dependently inhibits one or several enzymes in the coagulation cascade, prior to the formation of thrombin.
  • the inhibition of Factor Xa catalyzed substrate hydrolysis indicates that FXa represents at least one of the coagulation enzymes targeted by the anticoagulation protein Draculin.
  • Draculin was isolated as in Example 5. The sample had a protein concentration of 60 mg/ml. In those experiments where calcium was a participant in the reaction, Draculin was extensively dialyzed against 150 mM NaCl.
  • Draculin was able to titrate FXa amidolytic activity.
  • the term "titration" is used here to indicate how much Draculin, in molar terms, is needed to completely inhibit a constant amount of coagulation factor (Xa here), also expressed in molarity.
  • the FXa assay was as described in Example 1 , using a constant concentration of FXa and variable concentrations of Draculin. The effect is expressed as the % of inhibition of the amidolytic reaction in terms of O.D. per minute
  • the amidolytic activity of FXa at 17.0 and 29.0 nM was completely titrated by 3 and 5 ⁇ g of Draculin/ml, respectively.
  • Draculin Based on a molecular weight for Draculin of approximately 80 Kd; 3 and 5 ⁇ g/ml correspond to about 37 and 62 nM Draculin. This suggests that the stoichiometry of the interaction between Draculin and Factor Xa is 2:1 , that is, two molecules of approximately 80 Kd Draculin interact with one molecule of Factor Xa.
  • Draculin The anti-Xa activity of Draculin was not affected by 10 nM Factor X or of the derivative dansyl-Glu-Gly-Arg-Chloromethyl Ketone-[FXa] at equimolar concentration with FXa.
  • Draculin inhibits Factor Xa in the prothrombinase system as shown in Fig. 8.
  • the rate of inactivation of bound-FXa was slower than that of free Xa as shown in Fig. 9.
  • Full inhibition of bound Factor Xa occurred in about 60 seconds whereas free Factor Xa was inhibited in 5 seconds or less. This suggests that Draculin interacts with the free fraction of Factor Xa and thereby displaces Factor Xa from the prothrombinase complex.
  • Draculin also inhibits the catalytic activation of FX by Factor IXa as assessed in the Factor IXa determination described by Wagenvoord. Increasing amounts of Draculin were added to a fixed amount (31.3 nM) of Factor IXa. The Factor IXa activity decreased linearly with the amount of Draculin added. Complete inhibition of 31.3 nM FIXa was achieved with 7.1 ⁇ g of Draculin/ml (89 nM).
  • Factor IXa (10 nM), was mixed with Factor Villa (25 nM), , 20 ⁇ M phospholipid (10% phosphatidylserine, 90% phosphatidylcholine) and 5 mM Ca ++ . Under these conditions more than 95% of the Factor IXa is bound.
  • the Factor X activating capacity of this mixture was assessed, using the method of Wagenvoord, in the presence and absence of about 1 ⁇ g/ml Draculin. It was observed that Draculin inhibits bound Factor IXa completely and with similar stoichiometry to free Factor IXa.
  • the anti-IXa activity of Draculin was dependent on the order of addition with regard to the other components of the system (phospholipids and FVIIIa), as shown in Fig. 11.
  • the inhibitory effect was maximal if FIXa was incubated with Draculin in the absence of the other components of the tenase system.
  • the presence of phospholipids slightly protected FIXa from inactivation by Draculin, and when both phospholipids and FVIIIa were present, the protection amounted to about 40%.
  • Draculin-Xa is Draculin to which so much Factor Xa is added that its activity to inhibit further Factor Xa is lost.
  • Draculin-IXa is Draculin to which so much Factor IXa is added that its activity to inhibit further Factor IXa is lost.
  • Draculin-Xa is still an efficient inhibitor of Factor IXa and vice versa.
  • Draculin preparation contains independent Factor Xa and Factor IXa inhibiting sites.
  • extrinsic thrombin generation generation by thromboplastin
  • Fig. 14 The effect of Draculir. on extrinsic thrombin generation (generation by thromboplastin) of defibrinated plasma is shown in Fig. 14 where it can be seen that Draculin only slightly affected thrombin generation triggered by thromboplastin.
  • the intrinsic thrombin generation was both inhibited and retarded by Draculin as shown in Fig. 15. As shown above for the purified factors, no competition among FIXa and FXa was observed.
  • Draculin and crude saliva were equivalent on the basis of their anti-Xa activity.
  • Figure 18 shows the effect of Draculin and crude saliva on intrinsic thrombin generation in platelet poor plasma.
  • the effect of Draculin on lag-phase and thrombin peak was clearly maintained, however, in contrast to the result obtained in platelet rich plasma, in platelet poor plasma the crude saliva showed only a slightly inhibitory effect on the thrombin peak.
  • Draculin was isolated as in Example 5. Further purification prior to protein chemistry was accomplished by reverse phase HPLC on a C-18 column (Vydac 201TP, The Separations Group, Hesperia CA, or Asahipak ODP-50, from Anspec Co., Ann Arbor, Ml) equilibrated in 0.1% trifluoroacetic acid and developed with a gradient (0.8% per minute) of acetonitrile. Each column yielded a single peak. Automated NH2-terminal sequencing of this non-reduced sample revealed a single amino acid sequence (16 residues were defined) [SEQ ID NO: 1]. All evidence therefore pointed towards a single homogeneous protein species.
  • amino acid composition of such a desalted sample was determined by derivatization with phenyl isothiocyanate (PITC) after vapor phase acid hydrolysis for 16 hours at 110°C. ("Picotag” methodology of Milligen Corp., Waters Division). The results obtained are shown in Table 4. The composition of human lactoferrin is shown for comparison.
  • PITC phenyl isothiocyanate
  • Draculin poiypeptide chain In order to obtain more extensive amino acid sequence information, deliberate cleavage of the Draculin poiypeptide chain by enzymatic and chemical methods was performed. Chemical cleavage was achieved at methionine residues with cyanogen bromide.
  • the purified Draculin was also proteolytically digested with endoproteinase Lys-C (which cleaves after lysine residues), chymotrypsin in the presence of 0.5% w/w sodium dodecyl sulfate (which cleaves mainly after aromatic residues), and subtilisin (which cleaves without recognizable specificity) as described below.
  • endoproteinase Lys-C which cleaves after lysine residues
  • chymotrypsin in the presence of 0.5% w/w sodium dodecyl sulfate
  • subtilisin subtilisin
  • This sequence contained the 16 amino acid sequence from intact Draculin [SEQ ID NO: 1], indicating that it represented the amino-terminus of the molecule.
  • Two internal sequences of Draculin were also detected in the late eluting material [SEQ ID NO: 3 and SEQ ID NO: 4].
  • Draculin was reduced and S-pyridylethylated prior to cleavage in order to ensure that the cleavages would be as complete as possible.
  • a portion (40 ⁇ g) of the Draculin sample was reduced by incubation of the protein in 100 ⁇ l 6M guanidine hydrochloride, 0.1 M Tris-CI, pH 8.6, 5 mM EDTA, 3 mM dithiothreitol under a blanket of argon and protected from light for 1 hour at 24°C.
  • Alkylation of free sulfhydryl groups was carried out by adding 1 ⁇ l vinyl pyridine to the above mixture, and continuing the incubation under the same conditions for a further period of 1 hour.
  • the protein was recovered from the reaction mixture by reverse-phase HPLC, which was carried out on a C-18 column in 0.1% trifluoroacetic acid, developed with a gradient of 0.8% per minute acetonitrile. A single peak was observed.
  • Draculin Further definition of the amino acid sequence of Draculin was sought by cleaving reduced, aikylated Draculin in other ways. Since a limited amount of material was available, conditions for cleavage could not be optimized for this particular protein; rather, it was necessary to choose one, generically reasonable condition.
  • the size of the Draculin chain also indicated that, after reduction, alkylation and desalting by reverse phase HPLC, it would likely only be solubilized under fairly harsh conditions, where many useful enzymes (e.g., V8 protease, endo Asp-N, endo Asn-C) are inactive. Stronger enzymes, on the other hand, are mostly less specific so that many pieces, each in low yield, are produced.
  • Draculin was cleaved with chymotrypsin (10% w/w of protein substrate) in 0.25% w/v SDS, 0.2 M Tris HCI, pH 7.9, 5 mM CaCl2, for 16 hours at 37°C. Fractionation of the digest as described for endo Lys-C resulted in 19 peptide fractions. These were sequenced and the results (16 distinct peptide sequences) are shown in Table 5 [SEQ ID NO: 20 - SEQ ID NO: 35].
  • Table 5 The peptide sequences shown in Table 5 were examined for overlaps and redundancies. Overlapping sequences were combined into longer combination peptide sequences. The peptides were also compared to the amino acid sequences deduced from the cDNAs isolated in Examples 10-12. The primary experimental data for each sequence was then reexamined. Table 6 shows the Draculin peptides of Table 5 after the overlapping peptides were combined and the primary sequences were reexamined. In Table 6, upper case residues are at least 90% certain, lower case residues are less than 50% certain, and X represents an undetermined residue.
  • the peptides of Table 6 represent about 60% of the residues comprising a poiypeptide chain having a molecular weight of approximately 80 kD.
  • the peptide sequences were compared for homology to known proteins in the PIR database of protein sequences (Release 29). Partial homology was detected between the peptides and several serum transferrins and lactoferrins.
  • Figure 21 shows the homology between Draculin peptides of Table 6 and human lactoferrin (PIR Accession No. S10324, SEQ ID NO: 63).
  • Lactoferrin was obtained from Sigma (St. Louis, Cat. #L- 0520). This preparation is essentially iron-free, having an iron content of 0.036% (equivalent to the iron content of 0.1-0.24% of iron-loaded lactoferrin).
  • a 90 ⁇ M stock solution of lactoferrin was prepared by dissolving 7.4 mg of lactoferrin (molecular weight 85,000; 90% pure) in 870 ⁇ l water. Dilutions of the lactoferrin stock were made in buffer containing 50 mM Tris-HCI, 227 mM NaCl, pH 8.3, and assayed using a modified FXa assay. To 25 ⁇ l of buffer (50 mM Tris-HCI, 227 mM NaCl, pH 8.3) was added 15 ⁇ l of diluted FXa (final concentration of 4.4 nM) and 135 ⁇ l of lactoferrin (to final concentrations as indicated in Table 7).
  • Purified Factor IXa (3.2 U/ml; 280 nM final concentration) was mixed with DNBA (5,5' dithiobis 2-nitrobenzoic acid) at a final concentration of 0.6 mM. The reaction was incubated at 37°C for 3 minutes and Z-Lys-S-Bzl (thiobenzyl benzyloxycarboxyl-L-lysinate) was added to a final concentration 0.6 mM. The change in absorbance at 405 nm was measured over time, and was directly proportional to the Factor IXa activity. Lactoferrin was added to the assay at concentrations up to 100 ⁇ M without inhibiting the Factor IXa activity.
  • Draculin The amino acid sequence of Draculin, as determined in Example 8, was used to design oligonucleotide primers which would hybridize to DNA sequences encoding Draculin. These primers were used in the polymerase chain reaction (PCR) to amplify a Draculin cDNA fragment using cDNA prepared from vampire bat salivary gland mRNA as a template.
  • PCR polymerase chain reaction
  • RNA from bat salivary gland tissue was prepared by the method described by Chomczynski and Sacchi (Anal. Biochem. 162:156-159. 1987), using a RNAzol B kit (Tel-Test, Inc., Friendswood TX). Salivary gland tissue was homogenized with a glass-teflon homogenizer in the presence of 2 ml per 100 mg of tissue of RNAzol B (a mixture of guanidine isothiocyanate and phenol as described by Chomczynski and Sacchi).
  • the sample was extracted by the addition of 0.2 ml chloroform per 2 ml of homogenate, shaken vigorously for 15 seconds, incubated for 5 minutes on ice, and centrifuged for 15 minutes at 12000 X g.
  • the upper aqueous layer containing the RNA was transferred to a fresh tube, an equal volume of ethanol was added, and the sample was chilled for 15 minutes at 4°C.
  • the sample was centrifuged at 12000 X g for 15 minutes.
  • the supernatant was removed, and the RNA pellet was washed once by the addition of 1 ml of 75% ethanol, followed by vortexing and centrifuging for 8 minutes at 7500 X g.
  • the supernatant was removed and the pellet was dried briefly under vacuum.
  • the RNA was dissolved in diethylpyrocarbonate (DEPC)-treated, RNase-free TE (10 mM Tris pH 7.0, 1 mM EDTA) at a concentration of 2 mg RNA
  • Polyadenylated mRNA was isolated from the total RNA by the Fast Track mRNA Isolation System (Invitrogen, San Diego, CA), which uses an affinity column packed with oligo(dT) cellulose (Maniatis, et al., ed. in Molecular Cloning: A Laborator y Manual. 1982).
  • a 500 ⁇ l (1 mg) sample of the total RNA isolated above was precipitated by the addition of 75 ⁇ l of 3M sodium acetate and 1.2 ml of ethanol, followed by incubation on dry ice for 15 minutes. The sample was centrifuged for 20 minutes at 13000 X g. The supernatant was removed, and the pellet was rinsed with 80% ethanol.
  • the pellet was dissolved in 1 ml of lysis buffer (0.2 M NaCl, 0.2 M Tris pH 7.5, 15 mM MgC-2, 2% SDS) and the volume was adjusted to 10 ml with lysis buffer.
  • the sample was incubated at 45°C for 60 minutes.
  • Six hundred twenty ⁇ l of 5M NaCl and one oligo(dT) cellulose tablet (200 mg, containing 75 mg of active oligo(dT) ) were then added to the sample.
  • the oligo(dT) cellulose was allowed to swell for 2 minutes, then the mixture was incubated for 90 minutes at room temperature with gentle rocking.
  • the oligo(dT) cellulose-mRNA was centrifuged at 4000 X g for five minutes.
  • the supernatant was removed and the pellet was suspended in 20 ml of binding buffer (0.5 M NaCl, 10 mM Tris pH 7.5), then centrifuged at 4000 X g for five minutes.
  • the pellet was resuspended in 10 ml binding buffer, and again centrifuged at 4000 X g for five minutes.
  • the pellet was then washed three times by resuspension in 10 ml of low salt wash buffer (0.25 M NaCl) followed by centrifugation at 4000 X g for five minutes. After the final centrifugation, the pellet was resuspended in 0.8 ml low salt wash buffer.
  • the sample was transferred to a microfuge tube spin column and the oligo(dT) cellulose-mRNA was washed three times with 0.4 ml low salt wash buffer.
  • the RNA was chilled at -70°C overnight.
  • the mRNA was centrifuged at 13000 X g at 4°C for 15 minutes, and the pellet was washed with 80% ethanol.
  • the RNA was dried briefly under vacuum and resuspended in 50 ⁇ l of elution buffer at a concentration of 0.22 ⁇ g/ ⁇ l.
  • the mRNA isolated above was converted to double stranded cDNA by reverse transcription, using essentially the method of Gubler and Hoffmann (Gene 25: 263-269, 1983) with a cDNA Synthesis Kit from Boehringer Mannheim (Indianapolis, IN) .
  • Two micrograms (9.1 ⁇ l) of mRNA were used in the first-strand synthesis reaction, which also included 4 ⁇ l of buffer I (250 mM Tris pH 8.5, 40 mM MgCl2, 150 mM KCI, 0.5 mM DTT), 1 ⁇ l (10 units) of human placental RNase inhibitor, 2 ⁇ l of 10 mM dNTP mix (10 mM each of dATP, dCTP, dTTP, and dGTP), 2 ⁇ l of oligo dT-
  • buffer I 250 mM Tris pH 8.5, 40 mM MgCl2, 150 mM KCI, 0.5 mM DTT
  • 1 ⁇ l (10 units) of human placental RNase inhibitor 2 ⁇ l of 10 mM dNTP mix (10 m
  • This reaction mixture was incubated for 60 minutes at 42°C, then placed on ice.
  • the following components of the second-strand synthesis reaction were then added: 40 ⁇ l of buffer II (80 mM Tris, pH 7.5, 240 mM KCI, 10 mM MgCl2, 130 ⁇ g/ml BSA), 1 ⁇ l (1 ⁇ Ci) of alpha 32p_dCTP, 1 ⁇ l (1 unit) of RNase H, 10 ⁇ l (50 units) of E. coli DNA polymerase I, 4 ⁇ l (4 units) of Klenow fragment of E. coli DNA polymerase I, and 24 ⁇ l of distilled water.
  • buffer II 80 mM Tris, pH 7.5, 240 mM KCI, 10 mM MgCl2, 130 ⁇ g/ml BSA
  • 1 ⁇ l (1 ⁇ Ci) of alpha 32p_dCTP 1 ⁇ l (1 unit) of RNase H
  • This mixture was incubated sequentially at 12°C for 60 minutes, at 22°C for 60 minutes, and at 65°C for 10 minutes, then placed on ice.
  • the volume of the sample was adjusted to 300 ⁇ l by the addition of water.
  • the sample was extracted with one volume of phenol: chloroform (1:1) then one volume of chloroform: isoamyl alcohol (24:1).
  • the cDNA was precipitated by the addition of 33 ⁇ l of 3M sodium acetate and 825 ⁇ l of ethanol and incubated on ice for 30 minutes, then centrifuged at 13000 X g for 20 minutes The pellet was washed with ice cold 70% ethanol, dried briefly under vacuum, and suspended in TE buffer (10 mM Tris, pH 7.0, 1 mM EDTA).
  • the following two oligonucleotide primers were designed and synthesized based on the amino acid sequence of Draculin. The portion of the Draculin amino acid sequence from which each primer was designed is shown under each oligonucleotide sequence.
  • Primer #2 [SEQ ID NO : 50 ]
  • Approximately 0.8 nanograms of double stranded salivary gland cDNA was used in a PCR amplification using a thermostable DNA polymerase (Hot Tub DNA polymerase, Amersham Corp., Arlington Heights, IL).
  • a thermostable DNA polymerase Hot Tub DNA polymerase, Amersham Corp., Arlington Heights, IL.
  • 10 X Hot Tub Reaction Buffer 500 mM Tris, pH 9.0, 15 mM MgC-2, 200 mM (NH4)2S ⁇ 4
  • 100 picomoles of oligonucleotide Primer #1, 100 picomoles of oligonucleotide Primer #2, and 3 units of Hot Tub DNA polymerase in a total volume of 100 ⁇ l.
  • This reaction mixture was overlaid with 100 ⁇ l of light mineral oil.
  • Amplification by the polymerase chain reaction consisted of 35 cycles (1 cycle: incubation at 94°C for 90 seconds, at 55°C for 2 minutes, and at 72°C for 2 minutes) in a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT). An aliquot of the reaction product (10 ⁇ l) was analyzed by agarose gel electrophoresis. The major product of the reaction was a DNA fragment with electrophoretic mobility corresponding to approximately 1700 bp.
  • a 40 ⁇ l sample of the PCR reaction mixture was run on a 1% low melting point agarose gel and a section of the gel containing the DNA band was excised, transferred to a microcentrifuge tube, and melted at 70°C.
  • the melted agarose was cooled to 40°C and 0.1 volume of 10X beta-agarase buffer (100 mM Bis-Tris, pH 6.5 at 40°C, 10 mM EDTA) and 10 units of beta-agarase I (New England Biolabs, Beverly, MA) were added.
  • 10X beta-agarase buffer 100 mM Bis-Tris, pH 6.5 at 40°C, 10 mM EDTA
  • 10 units of beta-agarase I New England Biolabs, Beverly, MA
  • the supernatant was extracted twice with one volume of phenol, then once with one volume of chloroform: isoamyl alcohol (24: 1).
  • the DNA was precipitated at -20°C for 30 minutes using 0.1 volume 3 M sodium acetate pH 5.2 and 2 volumes of isopropanol.
  • the precipitate was centrifuged, rinsed with cold 70% isopropanol, dried briefly under vacuum, and suspended in TE buffer.
  • the ends of the DNA fragments were phosphorylated by treatment with 20 units of T4 polynucleotide kinase in a final volume of 20 ⁇ l (final buffer concentration: 50 mM Tris-HCI pH 7.5, 10 mM MgC.2, 5 mM dithiothreitol, 0.5 mM ATP, and 0.1 ⁇ g/ ⁇ l bovine serum albumin) for 60 minutes at 37°C.
  • the ends of the DNA fragments were made blunt by incubation with two units of T4 DNA polymerase and 2.5 ⁇ l of dNTP mix (2 mM each of dATP, dGTP, dCTP, and dTTP) for 20 minutes at 14°C.
  • the reaction was stopped by extraction with one volume of phenol:chloroform followed by extraction with one volume of chloroform: isoamyl alcohol (24: 1).
  • the DNA was further purified by passing it through an Elutip-D reversed-phase resin DNA purification column (Schleicher and Schuell, Inc., Keene, NH) according to the manufacturer's instructions.
  • the phosphorylated, blunt-ended PCR product was inserted into the dephosphorylated Sma I site of the phage M13mp18 by incubation with T4 DNA ligase overnight at 16°C.
  • the ligation mix was diluted 10-fold with sterile water, and 10 ng were used to transform competent XL-1 Blue strain E. coli cells (genotype: recA1 , endA1, gyrA96, thil , hsdR17, supE44, relA1 , lac, [F 1 proAB, Laclq, 2 ⁇ M15, Tn10 (tet r )], Stratagene, Inc., La Jolla, CA).
  • the resulting recombinant phage were isolated and screened for insert size by restriction enzyme analysis. Seven of thirteen recombinant phage screened had inserts of 1700 bp as estimated by agarose gel electrophoresis.
  • the partial sequences of two of these clones were determined using a Sequenase version 2.0 DNA Sequencing Kit (United States Biochemical, Cleveland, OH), based on the Sanger chain termination sequencing method (Sanger, F. et al., Proc. Natl. Acad. Sci. USA 74:5463-5467. 1977). The sequences of the two clones were identical [SEQ ID NO: 52]. This sequence comprises Primer #2 [SEQ ID NO: 50], which was used in the PCR amplification.
  • This sequence is predicted to be the noncoding strand of the cDNA for Draculin. Due to the high error rate of thermostable DNA polymerases, clones isolated using PCR may contain some nucleotide differences as compared to the coding regions of genomic DNA or mRNA.
  • Draculin 3'-cDNA The DNA sequence of the Draculin clone isolated in Example 10a was used to design additional oligonucleotide primers which would hybridize to nucleic acid sequences encoding Draculin. These oligonucleotides were used to isolate cDNA clones containing the 3'- and ⁇ '-e ⁇ ds of the Draculin coding sequence using the PCR and vampire bat salivary gland RNA. a) Isolation of a Draculin 3'-cDNA:
  • RNA (1 ⁇ g) from bat salivary glands, prepared in Example 10 was annealed with 10 picomoles of an oligo dT-containing Adapter Primer [SEQ ID NO: 53]. This annealed mixture was incubated for 10 minutes at 70°C then chilled on ice for 2 minutes.
  • Buffer and reagents were added to give a final composition of 20 mM Tris-HCI, pH 8.4, 50 mM KCI, 2.5 mM MgCl2, 100 ⁇ g/ml BSA, 10 mM Dithiothreitol, and 500 ⁇ M each dNTP.
  • This mixture was preincubated for 2 minutes at 42°C, then 200 units of Moloney murine leukemia virus reverse transcriptase lacking RNAse H activity (Superscript reverse transcriptase, Gibco-BRL) were added. The mixture was incubated for 10 minutes at 42°C. Two units of RNase H were added to the reaction and incubation was continued at 42°C for 30 additional minutes. The reaction was then placed on ice.
  • the cDNA for Draculin was amplified using the nonspecific Universal
  • Draculin-specific primer designed from the sequence of the 1.7 Kb Draculin cDNA clone isolated in Example 10e.
  • This Draculin-specific primer SALI368 [SEQ ID NO: 55] was designed to contain a restriction site for the enzyme Sal I to facilitate cloning of the amplification product.
  • Ten percent (2 ⁇ l) of the reverse transcriptase reaction was used in the PCR amplification.
  • Buffer and reagents were added to give a final composition (in 100 ⁇ l total volume) of 20 picomoles of each primer, 200 ⁇ M each dNTP, 1X Hot Tub Reaction Buffer (Amersham), and 3 units Hot Tub DNA polymerase (Amersham).
  • This reaction mixture was overlaid with 70 ⁇ l of light mineral oil and incubated sequentially at 94°C for 1 minute, at 57°C for 1 minute, and at 72°C for 2 minutes (one cycle). This was repeated for a total of 40 cycles. On the final cycle, the 72°C incubation was extended for 15 minutes. A 20 ⁇ l aliquot of the final reaction product was analyzed on a 0.8% agarose gel.
  • the major product was a DNA fragment with a mobility corresponding to approximately 2.0 Kbp in length.
  • a 60 ⁇ l sample of the PCR amplification reaction was purified using a DNA purification kit containing a DNA binding resin (Magic PCR Clean-up reagents, Promega Corp., Madison, Wl) according to the manufacturer's instructions.
  • One-half of the purified DNA was digested with the restriction enzyme Sal I for 2 hours at 37°C, then extracted sequentially with one volume of phenol, with one volume of phenokchloroform (1: 1), and with one volume of chloroform:isoamyl alcohol (24: 1).
  • the DNA was then precipitated with two volumes of ethanol.
  • the restricted Draculin cDNA was inserted into the dephosphorylated Sal I site of the phage M13mp18 RF DNA by incubation with T4 DNA ligase overnight at 16°C.
  • the ligation mixture was diluted 10-fold with sterile water and 10 ng were used to transform competent XL-1 Blue E. coli cells (Stratagene, Inc., La Jolla, CA).
  • the resulting recombinant phage were screened for insert size using restriction enzyme digestion analysis.
  • Four of fifteen clones had an insert of the expected size of approximately 2 Kbp., as estimated by agarose gel electrophoresis.
  • a partial DNA sequence [SEQ ID NO: 56] of one of these clones was determined using the Sanger chain termination method and a Sequenase V 2.0 kit (United States Biochemical).
  • a clone encompassing the 5'-end of Draculin cDNA was isolated using a kit (GIBCO-BRL) based on the 5' RACE technique (Frohman, M. A. in PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds., Academic Press, San Diego, pp. 28-38, 1990). Single-stranded cDNA was synthesized in a reverse transcriptase reaction as described above, using 1 ⁇ g of total bat salivary gland RNA and a Draculin-specific primer (Primer #2 from Example 10, [SEQ ID NO: 50]).
  • the products were purified by passing them over a GlassMAX DNA isolation spin cartridge (GIBCO-BRL), based on the method of Vogelstein and Gillespie (Proc. Natl. Acad. Sci. USA 76:615-619, 1979). After the DNA was eluted from the column with 50 ⁇ l of sterile deionized water the eluent was lyophilized then resuspended in 13 ⁇ l of water.
  • GEBCO-BRL GlassMAX DNA isolation spin cartridge
  • the purified single-stranded cDNA was tailed by incubating it with dCTP for 10 minutes at 37°C in a 20 ⁇ l reaction mixture consisting of 10 mM Tris-HCI, pH 8.4, 25 mM KCI, 1.25 mM MgCl2, 50 ⁇ g/ml BSA, 200 ⁇ M dCTP, and 10 units of terminal deoxynucleotidyl transferase. Following incubation, the enzyme was inactivated by heating at 70°C for 10 minutes. The mixture was then chilled on ice.
  • Draculin-specific primer which was designed from the sequence obtained from the 1.7 Kb Draculin cDNA clone isolated in Example 10.
  • This primer, DRAC1217A [SEQ ID NO: 58] anneals to a region of the cDNA approximately 1.2 Kb downstream from the translation start site.
  • One-fourth of the cDNA recovered from the tailing reaction was used in the amplification reaction.
  • a 50 ⁇ l sample of the amplification reaction was purified using Magic PCR clean up columns (Promega).
  • the ends of the DNAs were phosphorylated with T4 polynucleotide kinase, blunt ended by treatment with T4 DNA polymerase in the presence of dNTPs, extracted with one volume of phenol: chloroform (1:1), and with one volume of chloroform: isoamyl alcohol (24:1), then precipitated with two volumes of ethanol.
  • the fragments were inserted into the dephosphorylated Sma I site of the phage vector M13mp18 RF DNA using T4 DNA ligase. A portion of the ligation mix was used to transform competent XL-1 Blue E.
  • coK cells and the resulting transformants were screened for recombinant phage.
  • One of fifteen phage screened was found to have an insert of the expected 1.2 Kb size, as judged by restriction enzyme analysis.
  • a partial DNA sequence of the clone [SEQ ID NO: 59] was obtained using the Sanger sequencing method.
  • the DNA sequences of the Draculin clones isolated in Example 11 were used to design an additional oligonucleotide primer which would hybridize to nucleic acid sequences encoding Draculin.
  • This oligonucleotide was used to isolate a Draculin cDNA containing coding information for the complete protein, using reagents from the 3' RACE kit (GIBCO-BRL).
  • the gene-specific primer used in the amplification reaction, XBAI ⁇ 'Drac [SEQ ID NO: 60] was designed to contain sequences immediately upstream of the putative translational initiation site for Draculin, as determined from the ⁇ '-clone isolated and partially sequenced in Example 11b. An Xba I restriction site was added to this primer to facilitate cloning of the amplification product.
  • Amplification conditions were as described in Example 11a, using 20 picomoles of the Universal Adapter Primer [SEQ ID NO: 54] and of the XBAI5ORAC primer [SEQ ID NO: 60]. A 20 ⁇ l aliquot of the amplification reaction was analyzed by gel electrophoresis, and the predominant product was found to be approximately 2.3 Kbp in size.
  • a 40 ⁇ l sample of the amplification reaction was purified by use of a
  • a second PCR designed to generate a cDNA encoding a complete Draculin protein, was also performed.
  • the oligonucleotide primers and reaction conditions were as described above, except that the reaction mixture was subjected to only 20 cycles of amplification. This was in order to minimize the possibility of mutations being introduced into the product during the PCR.
  • the DNA was purified, then digested with Sal I and Xba I.
  • the resulting Xba l-Sal I fragment was ligated into the vector pBluescript SK (+) (Stratagene, La Jolla, CA, Genebank Accession #52325), which had also been digested with Sal I and Xba I.
  • the ligation mixture was used to transform XL-1 Blue E. coli cells. Clones containing the expected Xba l-Sal I insert were identified by restriction enzyme analysis. One such clone was designated pBSNDrac, and was deposited with the ATCC (ATCC No. 69393).
  • DNA sequence analysis of the clones isolated in Examples 10-12 gave consensus DNA [SEQ ID NO: 61] and deduced amino acid [SEQ ID NO: 62] sequences, as shown in the Sequence Listing. These consensus sequences are a compilation of sequence information from several clones generated by PCR amplification.
  • Figure 21 shows the homology between the Draculin deduced amino acid sequence (SEQ ID NO: 62, the first two deduced amino acids of SEQ ID NO: 62 are not included on Figure 21), the Draculin peptide sequences determined in Example 8, and human lactoferrin (PIR Accession No. S10324, SEQ ID NO: 63). Based on the amino acid sequence analyses of Example 8, the mature Draculin protein is believed to begin with the sequence A R R R G V R W X T I S K P E A [SEQ ID NO: 1].
  • the deduced amino acids which are located upstream of this putative N-terminus i. e. the first 19 amino acids of SEQ ID NO: 62] may represent a signal peptide.
  • the molecular weight of a protein having the deduced amino acid sequence which is shown for mature Draculin is approximately 75 kD. This calculation assumes that the poiypeptide is unglycosylated. It is not known whether or not Draculin is glycosylated in its native state. Lactoferrin as well as other members of the transferrin family are thought to be glycosylated. Glycosylation would be expected to increase the molecular weight of the native protein.
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • FRAGMENT TYPE N-terminal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • FRAGMENT TYPE internal
  • Cys Thr lie Ser Lys Pro Glu Ala Ala Lys Cys Ser Lys Leu Gin Gin 1 5 10 15 Asn Leu Lys Arg Val Xaa Gly Pro Ser
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • FRAGMENT TYPE N-terminal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORIGINAL SOURCE
  • ORGANISM Desmodus rotundus
  • F TISSUE TYPE: Salivary gland
  • CAAGACAGTG GAAGCTTTCA CAAAGGCCAC GTCTCCAGCA TTCTCGACCA GGCACCTGAA 360
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:53: GGCCACGCGT CGACTAGTAC TTTTTTTTTTTT TTTTTTT 37
  • ORGANISM Desmodus rotundus
  • F TISSUE TYPE: Salivary gland
  • ORGANISM Desmodus rotundus
  • F TISSUE TYPE: Salivary gland
  • ORGANISM Desmodus rotundus
  • F TISSUE TYPE: Salivary gland
  • ATC CAG GCC ATC GCG GCG AAA AGG GCA GAT GCC ATG AGC CTT GAT GCA 240 lie Gin Ala He Ala Ala Lys Arg Ala Asp Ala Met Ser Leu Asp Ala 65 70 75 80
  • AAA ATC CCT GTG GGC ACG CTC CGT CCG TAC CTG GGC TGG GCA GGG CCA 480 Lys He Pro Val Gly Thr Leu Arg Pro Tyr Leu Gly Trp Ala Gly Pro 145 150 155 160
  • GCC AGG GCC CCG AGT CAC GGT GTG GTA TCT CGG AAA GAT AGG GTA CAA 1872 Ala Arg Ala Pro Ser His Gly Val Val Ser Arg Lys Asp Arg Val Gin 610 615 620
  • MOLECULE TYPE protein

Abstract

L'invention se rapporte à une nouvelle protéine, la 'draculine', dotée d'une activité anticoagulante pour le sang humain. Cette protéine a été isolée à partir de la salive des vampires. L'invention se rapporte en outre à des procédés de purification de la draculine, à des utilisations thérapeutiques de celle-ci, à des molécules d'acide nucléique isolées codant la draculine ainsi qu'à des procédés de préparation de la draculine utilisant la technologie de l'ADN recombinant.
PCT/US1994/009488 1993-08-20 1994-08-22 La draculine, son procede de preparation et d'utilisation WO1995005836A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU78282/94A AU7828294A (en) 1993-08-20 1994-08-22 Draculin, its method of preparation and use

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US10980793A 1993-08-20 1993-08-20
US08/109,807 1993-08-20

Publications (2)

Publication Number Publication Date
WO1995005836A1 WO1995005836A1 (fr) 1995-03-02
WO1995005836A9 true WO1995005836A9 (fr) 1995-03-30

Family

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Application Number Title Priority Date Filing Date
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Country Status (2)

Country Link
AU (1) AU7828294A (fr)
WO (1) WO1995005836A1 (fr)

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