WO1995005836A1 - Draculin, its method of preparation and use - Google Patents

Abstract

This invention provides a novel protein, 'Draculin', having anticoagulant activity for mammalian blood. This protein was isolated from the saliva of vampire bats. This invention further provides methods for purifying Draculin, therapeutic uses thereof, isolated nucleic acid molecules encoding Draculin, and methods of preparing Draculin using recombinant DNA technology.

Description

DRACULIN, ITS METHOD OF PREPARATION AND USE

FIELD OF THE INVENTION

This invention relates to a novel protein having anticoagulant activity fo mammalian blood, methods for purifying this novel protein, and uses therefor

BACKGROUND OF THE INVENTION

When blood vessels are damaged, the bleeding which ensues is controlled by a complex array of hemostatic mechanisms. Platelets aggregat at the damaged surface, vasoconstrictive factors are released, and the blood coagulation cascade is initiated around the injured site.

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 exquisite control over the coagulation pathway and also provides the potentia for a rapidly amplifiable response to trauma. The coagulation cascade culminates in the throm bin-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. There are two major routes to the activation of Factor X, which a known as the intrinsic (contact activation) pathway and the extrinsic (tissue factor-dependent) pathway. Although these pathways have classically been described separately, they are probably highly intertwined under physiological conditions.

Tissue injury exposes the blood to 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" 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.

Whereas a properly regulated coagulation cascade is critical to hemostasis, pathologic activation of the cascade may cause occlusive thrombus formation in both arteries and veins, leading to a plethora of thrombotic complications and diseases. There is a clinical need, therefore, for potent and specific anticoagulants, which inhibit various steps of the coagulation cascade.

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.

Several naturally occurring peptides have been shown to have anticoagulant activity.

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 International Society of Hematoloαv. European and African Division, Basel- Switzerland, Thrombosis Research, 1991; Riehl-Bellon, N., Biochemistry 28:2941-2949, 1989; Courtney, M.. Seminar in Thrombosis & Haemostasis 15:288-292, 1989).

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 ("tenase") consisting of factor IXa (enzyme), Factor Villa (cofactor) on phospholipid.

Two inhibitors of Factor Xa have been obtained from natural sources. Antistasin, a reversible, slow binding inhibitor of Factor Xa, is a 119 amino acid protein isolated from the salivary gland of the leech Haementeria officinalis (Nutt, E., J. Biol. Chem. 263:10162-10167. 1988). Tick anticoagulant peptide (TAP) is a 60 amino acid peptide that also binds slowly and reversibly to Factor Xa (Waxman, L, Science 248:593-596. 1990). The equilibrium dissociation constant Kj of both inhibitors is in the range of 0.3 - 0.5 nM (Dunwiddie, C, -L Biol. Chem. 264:16694-16699. 1989; Waxman, L, Science 248:593-596. 1990). Both peptides have been expressed recombinantly (Neeper, M.P., i Biol. Chem. 265:17746-17752. 1990; Nutt, E.M., Arch. Biochem. Biophvs. 285:37-44. 1991). Both peptides show antithrombotic properties in a rabbit model (Vlasuk, G.P., Thrombosis & Haemostasis 65:257-262. 1991).

In addition to antistatin and TAP, two other FXa inhibitors have been described.

A family of five closely related proteins (molecular weight approximately

18000 daltons) has been isolated from the South American leech Haementeria αhilianii. and has been named Ghilantens. In addition to causing a dose- dependent prolongation of the prothrombin time of normal plasma, these proteins inhibit the FXa-mediated hydrolysis of the chromogenic substrate methoxycarbonyl-D-cyclohexylglycyl-glycyl-arginine-p-nitroanilide acetate.

They also show antimetastatic activity (Brankamp, R.G., et al., J. Lab. Clin. Med. 115:89-97, 1990).

Another 18000 dalton polypeptide has been isolated from the salivary glands of the black fly Simulium vittatum (Jacobs. J.W., et al., Thrombosis and Haemostasis 64:235-238. 1990). It inhibits FXa in stoichiometric quantities.

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. (Parish 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).

Three related forms of bat salivary plasminogen activator have recently been isolated from the submandibular glands of vampire bats (Gardell, S.J. et al., J. Biol. Chem. 264:17947-17952. 1989). One form, 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.

SUMMARY OF THE INVENTION

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 75-90 kD. The sensitivity of the method of determination and form of Draculin (e.g. whether it is glycosylated or not) will affect the apparent molecular weight.

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.

Other and further objects, features and advantages will be apparent from the following description of the preferred embodiments of the invention given for the purpose of disclosure when taken in conjunction with the following drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

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. β-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 defibrinated 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 polypeptide 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.

Definitions

Unless indicated otherwise herein, the following terms have the indicated meanings.

The term "polypeptide" means a linear array of amino acids connected one to the other by peptide bonds between the α-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 polypeptide 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.

The term "biologically active polypeptide" means the naturally occurring polypeptide 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. The term "biologically active polypeptide" 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.

The term "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.

As used herein the term "salts" refers to both salts of carboxy groups of the polypeptide 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. Some of the assays that can be used to measure blood coagulation are described in Example 1.

In this application, nucleotides are indicated by their bases, using the following standard one-letter abbreviations:

Guanine G

Adenine A

Thymine T

Cytosine C Uracil U

Inosine I

Unknown N

In this application, amino acid residues are indicated using the following standard one- or three-letter abbreviations:

Threonine T Thr

Valine V Val

Tryptophan W Trp

Tyrosine Y Tyr Unknown X Xaa

The term "amino acid" as used herein is meant to denote the above- recited natural amino acids and functional equivalents thereof.

The term "coding strand" is used herein to mean DNA sequences which, in the appropriate reading frame, code for the amino acids of a protein. For the purpose of the present invention, it should be understood that the synthesis or use of a coding sequence may necessarily involve synthesis or use of the corresponding complementary strand, as shown by: 5'-CGGGGAGGA-373'- GCCCCTCCT-5' which "encodes" the tripeptide NH2-Arg-Gly-Gly-Cθ2H. 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.

The term "cDNA" is used herein to mean a DNA molecule or sequence which has been enzymatically synthesized from the sequence(s) present in an mRNA template.

The term "vector" is used herein to mean a plasmid, 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.

The term "plasmid" is used herein to mean a non-chromosomal double- stranded DNA sequence comprising an intact "replicon" such that the plasmid is replicated in a host cell. When the plasmid is placed within a prokaryotic or eukaryotic host cell, the characteristics of that cell may be changed (or transformed) as a result of the DNA of the plasmid. For example, a plasmid 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 plasmid is called a "transformant."

The term "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").

The term "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, based on the deduced amino acid sequence determined in Example 12 (which assumes no glycosylation), 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. Examples of 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.

This invention provides a 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. For example, 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. In addition, 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. In one embodiment, the present invention provides a proteinaceous anticoagulant useful in the treatment of human and animal disorders characterized by abnormal blood coagulation. Such disorders include, but are not limited to, acute myocardial infarction, deep vein thrombosis, pulmonary embolism, unstable angina, transient ischemic attacks, peripheral vascular occlusion, bypass occlusion, and disseminated intravascular coagulation. Administration of 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, useful in the therapeutic method 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. For parenteral administration, 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.

Deposit of Strains Useful in Practicing the Invention

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.

Strain/Plasmid ATCC No. Deposit Date

pBSNDrac 69393 August 20. 1993

The present invention will be further understood through reference to the following examples, which are offered purely by way of illustration, and should not be taken as limiting the true scope of the invention unless otherwise specified.

Example 1

Coagulation Assays A number of clinically relevant, global coagulation assays such as described in sections 1b throjgh e bolow were util'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 1 i below) within the coagulation cascade inhibited by Draculin.

a) Preparation of blood and plasma samples: Whole human blood was obtained by venipuncture from healthy male donors. The first 1 ml to 2 ml of blood were discarded. Blood samples were then collected in 0.13 M trisodium citrate (nine parts of blood to 1 part of citrate solution).

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 10⁸ 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.

b) Human Whole Blood Coagulation Time (WBCT^ was measured using one ml samples of whole human blood. Coagulation was allowed to proceed at 37°C in glass tubes containing 50 μl of Epsilon amino caproic acid (EACA, 114 mg/ml) to inhibit fibrinolysis due to any of the plasminogen activators known to be present in bat saliva. Coagulation time was determined manually by recording the elapsed time to clot or gel formation. The coagulation time of control samples was 18'.^". seconds.

c) Activated Partial Thromboplastin Time (APTH was measured using a commercial partial thromboplastin (Activated Thrombofax, Ortho Diagnostic

Systems, Raritan, NJ, USA), which contains Elagic acid as an activator. Because the saliva and partially purified fractions (from Sephacryl S-300 columns) contain an activator of plasminogen (Cartwright, Blood. 43:318-326 (1974)), all the coagulation assays were performed in the presence of EACA . One hundred μl of citrated plasma, 25 μl of EACA (128 mg/ml), 80 μl distilled water containing various concentrations of partially purified saliva (PPS, see Example 3) or purified Draculin, and 20 μl Thrombofax were incubated for 3 minutes at 37°C, coagulation was then triggered by the addition of 100 μl of 20 mM calcium chloride, to yield a final volume of 325 μl. The time for clot or gel formation was measured by visual observation. Under these conditions, the APTT for control sample- wat: 45 ^• 47 econds.

d) Prothombin Time fPT> was measured using a commercial rabbit brain thromboplastin/calcium chloride reagent (Ortho). For the assay 100 μl of normal human citrated plasma was mixed with 50 μl of buffer containing Draculin. The mixture was maintained at 37°C for 3 minutes. At zero time, 200 μl of prewarmed thromboplastin/calcium reagent was added to initiate coagulation. The time to clot or gel formation was measured by visual observation. e) Thrombin Time CTT⁾ was assayed using commercial bovine thrombin (Sigma Chemical., St. Louis, Mo. USA). For the assay 100 μl of normal human citrated plasma was mixed with 50 μl of buffer containing Draculin. The mixture was prewarmed to 37°C. To this, 50 μl 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).

f) Factor Xa amidolvtic activit^y 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). Factor X, isolated according to the method of Fujikawa (Fujikawa, K. Biochemistry 11:4882-4891, 1972), was activated by incubation with 0.1% Factor IXa. A 10 μl sample of Factor Xa was added to 490 μl of a 16 μM solution of chromogenic substrate i; .a Duffer of 5': M 7rh;-HC!. 0.1 M NaCI, pH 7.9 containing 0.05% egg albumin and 20 mM EDTA. The reaction was followed at 405 nm in a fixed dual-wavelength photometer, and calculations were made using dedicated software developed for that instrument (Laudy, P., van der Steld, B., Kessels, H., Instrumental Development and Biochemistry Dept., Univ. of Limburg). g) Inhibition of Factor Xa activity by fractions obtained from gel filtration or Hydroxyapatite columns was measured using a microplate assay. To each well of a 96-well microplate, was sequentially added:

1. 100 μl buffer Tris-HCI, 50 mM, pH 7.35, 100 mM NaCI, containing 0.05% egg albumin.

2. 15 μl of 60 nM FXa. (5.45 nM final concentration)

3. 30 μl of the sample being tested.

4. 20 μl of S-2222 from a 4 mg/ml stock solution.

The micro-plate was incubated at 37°C for 5 to 0 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.

h) 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 μM phospholipids (20% phosphatidylserine, 80% phosphatidylcholine). To 100 μl of this mixture was added 100 μl 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 μl of Reagent 2, which contained 1 μM purified Factor X, 3 units/ml purified Factor VIII, and 0.1 mM CaCl2. At 4 minutes, 100 μl samples were removed. The production of Factor Xa was measured in 20 mM EDTA, 175 mM NaCI, 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 østerud 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.

i) Thrombin Generation TimefrGT): L-κi-'ir.sic Thrombin generation was measured in platelet-free plasma. To 200 μl of defibrinated plasma was added 50 μl of a buffer containing 0.05 M Tris-HCI, 0.1 M NaCI pH 7.35, and 0.05% egg albumin plus Draculin or PPS. At time zero, thrombin generation was triggered by the addition of 50 μl of buffer containing 100 mM of CaCl2 and human brain thromboplastin as a trigger. At fixed intervals (10, 15, 20, 30 or 60 sees) after time zero, the amount of thrombin generated was determined by removing a 10 μl aliquot of the mixture and adding it to a disposable plastic microcuvette containing 465 μl of buffer and 25 μl 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 mi 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 μl of buffer containing 1 μM phospholipids (20% phosphatidylserine, 80% phosphatidylcholine), 100 mM CaCl2, and 25 μg/ml of kaolin.

Example 2

Collection of Crude Saliva

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 (Halothan, Hoechst), 30% nitrous oxide, in oxygen. The anesthetized animals received 20 μl of 1% pilocarpine (Isopto Carpin, Alcon Labs., Inc. Ft. Worth, TX, USA) to stimulate salivation. 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 3 Partial Purification of Crude Saliva

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 Quantitatlon 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).

Example 4

Properties of the Crude Saliva and of the Partially Purified Fractions

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.

a) Stability of the anticoagulant activity: The anticoagulant activity of crude saliva, as well as that of the partially purified fractions, was stable for more than a week at room temperature. The activity was also stable when the samples were frozen at -30°C or -80°C. Repeated thawing and freezing did not affect the anticoagulant activity.

Extensive dialysis against deionized water (MW cutoff of the membrane = 14 kD) followed by lyophilization, did not affect the anticoagulant activity.

The anticoagulant activity was completely lost after heating the samples for 10 minutes at 80°C. When the samples were heated to 60°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.

b) Effect of protease inhibitors on the anticoagulant activity: Treatment of crude or partially purified saliva with a serine protease inhibitor such as PMSF (1mM) or DFP (2 mM), followed by extensive dialysis against distilled water (to eliminate traces of PMSF or DFP which might interfere with the coagulation assay) did not inhibit the anticoagulant activity, indicating that the anticoagulant activity did not result from proteolytic activity.

c) Absence of Heparin-like components in crude or partially purified saliva: Uronic acid was not detectable in 10-fold concentrated samples of saliva, suggesting that the observed anticoagulant activity was not due to heparin-like substances.

Example 5

Isolation and Purification of Draculin

Approximately 2 ml of crude saliva was prepared as in Example 2. The cleared dialysate was diluted two-fold with 1 mM NaCI and loaded onto a hydroxyapatite column (1.2 x 4.0 cm, vol. = 4.5 ml), which had been equilibrated with 1 mM NaCI, pH 7.2. The sample was pumped through the column at the rate of 12 - 14 ml/minute During the sample loading, a slight decrease of flow rate was observed. After all the sample volume had passed through the column, the flow returned to its original rate. The column was washed with 45 ml of 1 mM NaCI, and 13.5 ml of 200 mM potassium phosphate buffer, pH 7.2. After the phosphate wash, 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 NaCI wash, or the 200 mM phosphate wash. A sharp peak of anticoagulant activity eluted at about 0.3 M potassium phosphate. These fractions were pooled and dialyzed overnight against 0.3 M potassium phosphate, pH 7.2, then concentrated approximately 4-fold by filtration through an Amicon PM30 membrane. The concentrated material was filtered through a 0.22 micron low protein-binding ultrafilter, then aliquoted and kept at -30°C until further use. SDS-polyacrylamide gel electrophoresis of the concentrated material (5 μg), under reducing and non-reducing conditions yielded a single protein band with an apparent molecular weight of approximately 80.5 kD as shown in Fig. 3.

Size exclusion HPLC of purified Draculin was performed in a Shimadzu HPLC equipment (Shimadzu Corp., Japan) using a silica based molecular sieve column (10 μm 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, β-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, β-Lactoglobulin A, Bovine Carbonic Anhydrase B, Human Carbonic Anhydrase B, Horse Myoglobin, Lentil Lectin and Trypsinogen. Isoelectric focusing of native Draculin resulted in a single polypeptide band which immobilizes at pH = 4.1 - 4.2, corresponding to its isoelectric point, pi (Figure 2). In an effort to detect possible domain structure, native Draculin was incubated for 16 hours at 37°C in the presence of plasmin, thrombin, trypsin, V8 protease, chymotrypsin, endo Lys-C or a proline-specific protease. Analysis of reduced samples on SDS gels showed that chymotrypsin produced small amounts of several 10 - 20K fragments while trypsin cleaved most of the

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.

Example 6

Effect of PPS and Draculin on coagulation assays

The coagulation time of whole blood was significantly increased in the presence of PPS. One ml of whole blood was taken directly onto 50 μl 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.

TABLE 1

PPS, μg protein/ml (nM^*) Prolongation of WBCT, seconds (fold-increase)

2.5 (31 nM) 56 (1.3-fold)

5.0 (62 nM) 126 (1.7-fold)

10.0 (125 nM) 213 (2.2-fold)

*Based on a molecular weight for Draculin of approximately 80 kD.

Clot retraction was not affected by the presence of PPS. In both control samples and samples containing 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. These observations indicated that the contractile power of the clot, a function of platelets, was not impaired by 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.

TABLE 2

"Based on a molecular weight for Draculin of approximately 80 kD.

Preincubation of plasma with Thrombofax or kaolin for 10 minutes in the presence of saliva did not affect the inhibitory effect of the saliva. Because this preincubation produces large amounts of activated Factor IX, this observation indicated that Draculin affects a step in the coagulation cascade before Factor XI activation.

Incubation of plasma with PPS at concentrations that prolonged the APTT > 10 minutes produced a prolongation of the PT of 3 seconds. This suggested that the effect of Draculin was centered around Factor IXa and /or Factor Xa.

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 μg/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. TABLE 3

Some of the assays described in this example were repeated using purified Draculin, and similar results were obtained. The addition of 4 μg/ml of purified Draculin produced a 30 second prolongation of the PT, and the addition of 1 μg/ml of purified Draculin produced a 200 second prolongation of the APTT.

Fig. 5 (curve A) 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.

Example 7

Effect of Purified Draculin on the Amidolytic Activity of Purified FXa

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 NaCI.

The time course for the amidolytic activity of FXa (20 nM) on S-2337 is shown in Fig. 6, line a. The addition of Draculin (4.3 μg protein/ml) during the course of the reaction produced, within seconds of addition, an inhibition of the catalytic splitting of the substrate by FXa (Fig. 6, line b), indicating that Draculin is a kinetically rapid inhibitor of free FXa.

As shown in Figure 7, 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 μg of Draculin/ml, respectively. Based on a molecular weight for Draculin of approximately 80 Kd; 3 and 5 μg/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.

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. These observations indicate that Draculin does not interact with FX proenzyme, and that Draculin inhibits FXa by interaction with its active site.

To test whether Draculin is able to inhibit Factor Xa in the presence of the other components of the prothombinase complex (and hence would be expected to have an effect under physiological conditions), the following experiment was performed: Factor Xa (40 nM), was mixed with Factor Va (100 nM) and 100 μM phospholipid (10% phosphatidylserine, 90% phosphatidylcholine) and Ca⁺⁺ (5 mM). Under these conditions more than 95% of the Factor Xa is assembled into the physiologically relevant prothrombinase complex. The velocity of pNA production (substrate hydrolysis) after addition of 16 mM of S-2337 in the presence and in the absence of about 20 nM Draculin was recorded. 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.

As shown in Fig. 10, 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 FlXa was achieved with 7.1 μg of Draculin/ml (89 nM).

Factor IXa (10 nM), was mixed with Factor Villa (25 nM), , 20 μM 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 μg/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 FlXa was incubated with Draculin in the absence of the other components of the tenase system. The presence of phospholipids slightly protected FlXa from inactivation by Draculin, and when both phospholipids and FVIIIa were present, the protection amounted to about 40%. These results suggest that FlXa is partially protected from inhibition by Draculin when it is assembled into the Tenase complex.

Titration of Draculin with either FlXa or FXa did not interfere with its inhibitory activity towards the other factor. Fig. 12 shows the anti-IXa activity of a Draculin-Xa complex. Fig. 13 shows the anti-Xa activity of a Draculin-IXa complex. 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. As seen in the figures, Draculin-Xa is still an efficient inhibitor of Factor IXa and vice versa. This indicates that the Draculin preparation contains independent Factor Xa and Factor IXa inhibiting sites. 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. However, the intrinsic thrombin generation (generation by components of the tenase complex) was both inhibited and retarded by Draculin as shown in Fig. 15. As shown above for the purified factors, no competition among FlXa and FXa was observed. The increase of the lag- phase seems to be brought about by Factor Xa inhibition, while the inhibition of the peak value is due to Factor IXa inhibition. In the experiment of Fig. 15, it is seen that both types of inhibitory sites (Factor Xa and Factor IXa) contribute to the inhibition of intrinsic thrombin generation. When both sites of Draculin are saturated (Drac-Xa-IXa), the inhibitory capacity is lost.

The effect of increasing the concentration of Factor Villa during the lag- phase of intrinsic TGT is shown in Fig. 16. Addition of Factor Villa during the lag-phase markedly shortened it, however, the effect of Draculin on the thrombin potential (the surface under the thrombin generation curve) was only slightly affected. Prolongation of the lag-phase of thrombin generation indicates that insufficient thrombin is generated for the feedback activation of Factor VIII in the plasma. This is seen in the experiment of Fig. 16, where the addition of Factor Villa (5 nM) shortened the lagtime. The inhibition of the amount of thrombin produced is not explained by this mechanism because addition of Factor Villa (2 U/ml) does not augment the peak value or the area under the curves.

The effects of Draculin and crude saliva on intrinsic thrombin generation in platelet rich plasma are shown in Fig. 17. As can be seen, Draculin (< 5 μg/ml) prolonged the lag-phase and, at the same time decreased the thrombin peak. Crude saliva also prolonged the lag-phase but produced less inhibition of the thrombin peak. It should be noted that the amounts of saliva and Draculin 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. As can be seen, under these conditions, 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.

The effect of Draculin and crude saliva on thrombin generation in non- anticoagulated whole blood is shown in Figs. 19 and 20 respectively. As observed with PRP, the effect of Draculin on the thrombin peak is more marked than that of crude saliva. Their effects on the lag-phase, however, are comparable.

Example 8

Determination of the Amino Acid Sequence of Draculin

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.

The 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.

TABLE 4

Amino acid composition of non-reduced (Native) and reduced, alkylated (SPE) Draculin*

'Results shown are single analyses, calculated to result in a molecular weight of approximately 80,000. Seven moles SPE cysteine/mole protein was recovered, but cannot be relied upon in a single analysis. Tryptophan was not determined. SPE: S-pyridylethyl.

In order to obtain more extensive amino acid sequence information, deliberate cleavage of the Draculin polypeptide 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. A summary of the peptide sequences obtained is presented in Table 5. In Table 5, upper case residues are at least 90% certain, lower case residues are less than 50% certain, and X represents an undetermined residue. A 20 μg sample of the non-reduced Draculin was cleaved with cyanogen bromide. Only one peptide was clearly seen to be released from the molecule when the digest was analyzed by reverse phase HPLC, while most of the mass was eluted late in the chromatogram, close to uncleaved Draculin. The same single peptide was also released when reduced, alkylated Draculin was cleaved; its apparent molecular weight was approx. 7,000 D as determined by SDS gel electrophoresis. This sequence [SEQ ID NO: 2] 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 μg) of the Draculin sample was reduced by incubation of the protein in 100 μl 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 1μl 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.

A portion of the material recovered from the C-18 column (20 μg) was cleaved with endoproteinase Lys-C. The alkylated material obtained after reverse phase HPLC was partially dried (volume reduction of 1/3 to 1/2); Tris-CI was added to a final concentration of 0.3 M and pH of 8.6, followed by 3 μg/mL (final concentration) of endoproteinase Lys-C. The mixture was incubated 16 hours at 37°C. The digestion products were fractionated by reverse phase HPLC, using a C-18 column equilibrated in 0.1% trifluoroacetic acid and eluted with a gradient of acetonitrile. Twenty six separable peaks, as well as a number of peaks which were considered too small to analyze were collected manually. The recovered peaks were sequenced and the peptide sequences determined are shown in Table 5 [SEQ ID NO: 5 - SEQ ID NO: 19]. Table 5 does not include sequences which were determined to be from digestion of the protease itself. The initial K (lysine) in each case is implied by the cleavage method used and was not experimentally determined.

Further definition of the amino acid sequence of Draculin was sought by cleaving reduced, alkylated 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. Subtilisin cleavage was used, but the conditions were poorly chosen, and almost all the protein was reduced to amino acids. Two peptide sequences which were obtained following cleavage with subtilisin are included in Table 5 [SEQ ID NO: 36 and SEQ ID NO: 37].

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 Draculin Peptide Sequences Arranged by Specific Cleavage

INTACT CHAIN

A R R R G V R W X T I S K P E A [SEQ ID NO: 1]

CYANOGEN BROMIDE H2- r r X G V R W X T I S K P E A A K w s l Q Q N L

[SEQ ID NO: 2] (M) X L D G g F I Y I A G K X G L [SEQ ID NO: 3]

(M) X L X F X Q T R S X N F D E F [SEQ ID NO: 4] ENPQ-LY$-C

(K) G T S G S F Q L F S S P P G q [SEQ ID NO: 5]

NH2-A R R R G V R C T I S K P E A A K C s K l Q Q N L [SEQ ID NO: 6]

(K) A S T V L E N T D G R g t E A [SEQ ID NO: 7]

(K) D G A q G F L R I P A R V D [SEQ ID NO: 8]

(K) X X A G L T w N S L R G T K [SEQ ID NO: 9]

(K) L E D F E L L C L D G T R K P V S E F [SEQ ID NO: 10]

(K) E D S I X R L [SEQ ID NO: 11]

(K) X A D A M S L D A G L V Y E A G Q D P Y R L R P V A A E V Y [SEQ ID NO: 12]

(K) c L X S S k E P Y F G Y S [SEQ ID NO: 13]

(K) S Y L E c I Q a i [SEQ ID NO: 14]

(K) X X A a E V E A X G A R V v X X A V g P E [SEQ ID NO: 15]

(K) d R V Q Y L E Q V L X D Q Q G [SEQ ID NO: 16]

(K) A E R D Q Y E X L C P D N T R K P V [SEQ ID NO: 17]

(K) X A P N S N E R Y F X Y A G A F R X L V E

[SEQ ID NO: 18]

(K) X V X g P S L S C l S R K [SEQ -ID NO: 19] CHYMOTRYPSIN

NH2-A R R R G V R [SEQ ID NO: 20]

R L R P V A A E V Y [SEQ ID NO: 21]

S G Q S X G T V T C X X A A D D E d [SEQ ID NO: 22]

E X L C P D N T R K P V D E X X Q C A L A R V P S X A V V A r S V [SEQ ID NO: 23]

C A V G P E E L R K C Q Q [SEQ ID NO: 24]

E K Y L G P E Y V A X X A N L r Q C X T [SEQ ID NO: 25]

E N L P N K A E R D Q Y [SEQ ID NO: 26]

C T I S K P E A A K C S K L Q Q N L K R V X g P S

[SEQ ID NO: 27]

F G Y S G A F K C L K D G A X D V A F V X D X v f

[SEQ ID NO: 28]

C L F Q S E T K N L L F N D N [SEQ ID NO: 29]

S N E R Y F S Y A G A F R C L V X N A G D V A F V K

[SEQ ID NO: 30]

S S P P G Q K D L L F K D X A Q G F L R I P [SEQ ID NO: 31]

G T E G A P R T X Y [SEQ ID NO: 32]

E A G Q D P Y [SEQ ID NO: 33]

C L F Q S E T K N L L F N D N T E C L A K L Q g K T t Y X K Y L g [SEQ ID NO: 34]

V L K G E A D A M S L D G G F I Y I A [SEQ ID NO: 35] SUBTILISIN-C

K E P Y F G Y [SEQ ID NO: 36]

S L D G G F I Y [SEQ ID NO: 37]

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.

TABLE 6 Combined Draculin Peptide Sequences

NH2-A R R R G V R W C T I S K P E A A K C S K L Q Q N L K R V X g P S L S C I S R K [SEQ ID NO: 38]

(K) S Y L E c I Q a i [SEQ ID NO: 14]

(K) X A D A M S L D A G L V Y E A G Q D P Y R L R P V A A E V Y [SEQ ID NO: 12]

G T E G A P R T X Y [SEQ ID NO: 32]

(K) G T S G S F Q L F S S P P G Q K D L L F K D G A Q G F L R I P X R V D [SEQ ID NO: 39]

(K) C A X S S K E P Y F G Y S G A F K C L K D G A X D V A F V X D X X v f [SEQ ID NO: 40] (K) X A P N S N E R Y F X Y A G A F R C L V E N A G D V A F V K [SEQ ID NO: 41]

(K) A S T V L E N T D G R g t E A [SEQ ID NO: 7]

(K) L E D F E L L C L D G T R K P V S E F [SEQ ID NO: 10]

(K) d R V Q Y L E Q V L X D Q Q G [SEQ ID NO: 16]

E N L P N K A E R D Q Y E X L C P D N T R K P V D E X X Q C X L A R V P S X A V V A r S V [SEQ ID NO: 42]

(K) E D S I X R L [SEQ ID NO: 11]

V L K G E A D A M S L D G G F I Y I A G K X G L

[SEQ ID NO: 43]

(K) X X A G L T w N S L R G T K [SEQ ID NO: 9]

(M) X L X F X Q T R S X N D E F [SEQ ID NO: 4]

(K) X X A a E V E A X G A R V v X X A V G P E E L R K C Q Q

[SEQ ID NO: 44]

S G Q S X G T V T C X X A A D X E D [SEQ ID NO: 45]

E K Y L G P E Y V T X X A N L^'r Q C X T [SEQ ID NO: 46]

C L F Q S E T K N L L F N D N T E C L A K L Q G K T T Y E K Y L G [SEQ ID NO: 47]

The peptides of Table 6 represent about 60% of the residues comprising a polypeptide 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).

Example 9 Assays of Anticoagulant Activities of Lactoferrin

Because of the unexpected sequence homology found between Draculin and lactoferrin, the anticoagulant activity of lactoferrin was measured in order to determine whether these two proteins share functional as well as structural similarities. 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 Lack of Inhibition of Factor Xa activity by lactoferrin:

A 90 μM stock solution of lactoferrin was prepared by dissolving 7.4 mg of lactoferrin (molecular weight 85,000; 90% pure) in 870 μl water. Dilutions of the lactoferrin stock were made in buffer containing 50 mM Tris-HCI, 227 mM NaCI, pH 8.3, and assayed using a modified FXa assay. To 25 μl of buffer (50 mM Tris-HCI, 227 mM NaCI, pH 8.3) was added 15 μl of diluted FXa (final concentration of 4.4 nM) and 135 μl of lactoferrin (to final concentrations as indicated in Table 7). After incubation for 20 minutes at room temperature, 25 μl of S-2222 substrate (2.67 mM) were added. The samples were mixed gently then incubated for three minutes at room temperature. The reaction was stopped by the addition of 25 μl of 20% acetic acid. The O.D. (405 nm) was then measured. As can be seen in Table 7, essentially iron-free lactoferrin had only a very low level of FXa inhibitory activity, yielding 28% inhibition at a lactoferrin to FXa molar ratio of 13,800 to 1. In contrast, Draculin completely inhibited the enzymatic activity of FXa at a molar ratio of 2:1 as demonstrated in Example 7 and Figure 7. TABLE 7

b) Lack of Inhibition of Factor IXa activity bv lactoferrin:

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 μM without inhibiting the Factor IXa activity. This result suggests that essentially iron-free lactoferrin does not inhibit Factor IXa activity even at concentrations representing a 350-fold molar excess of lactoferrin to FlXa. In contrast, Draculin completely inhibited the activity of FlXa at almost stoichiometric molar ratios of 2:1 as demonstrated in Example 7 and Figure 10. Although different assay protocols were used in Example 7 and the above described experiment, the difference in assay conditions could not account for the magnitude of the differences in inhibiting molar ratios observed between Draculin and lactoferrin.

Taken together, these examples demonstrate that despite the unexpected sequence homology between Draculin and lactoferrin, lactoferrin does not possess the anticoagulant activities exhibited by Draculin.

Example 10

Isolation of a Draculin cDNA

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.

a) Preparation of mRNA from the salivary gland tissue of Desmodus rotundus (vampire bat):

Total 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 per ml.

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 Clonino: A Laborator^y Manual. 1982). A 500 μl (1 mg) sample of the total RNA isolated above was precipitated by the addition of 75 μl 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 NaCI, 0.2 M Tris pH 7.5, 15 mM MgCl2, 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 μl of 5M NaCI 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 NaCI, 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 NaCI) 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 mRNA, eluted from the oligo (dT) cellulose with 400 μl of elution buffer (10 mM Tris pH 7.5), was precipitated by the addition of 0.15 volumes of 2M sodium acetate and 2.5 volumes of ethanol. 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 μl of elution buffer at a concentration of 0.22 μg/μl.

b) Synthesis of cDNA from salivary gland mRNA:

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 μl) of mRNA were used in the first-strand synthesis reaction, which also included 4 μl of buffer I (250 mM Tris pH 8.5, 40 mM MgCl2, 150 mM KCI, 0.5 mM DTT), 1 μl (10 units) of human placental RNase inhibitor, 2 μl of 10 mM dNTP mix (10 mM each of dATP, dCTP, dTTP, and dGTP), 2 μl of oligo dT-|5 primer (133 picomoles/μl), and 2 μl (40 units) of avian myeloblastosis virus (AMV) reverse transcriptase. 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 μl of buffer II (80 mM Tris, pH 7.5, 240 mM KCI, 10 mM MgCl2, 130 μg/ml BSA), 1 μl (1μCi) of alpha ³²P-dCTP, 1μl (1 unit) of RNase H, 10 μl (50 units) of E. coli DNA polymerase I, 4 μl (4 units) of Klenow fragment of E. coli DNA polymerase I, and 24 μl of distilled water. 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 μl 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 μl of 3M sodium acetate and 825 μl 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).

c Design of olioonucleotide primers for amplification bv the polymerase chain reaction (PCR) of a 1.7 Kbp fragment of Draculin cDNA:

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 #1

[SEQ ID NO : 48 ] 5 ' -CTG CGA CCT GTA GCG GCG GAA GTC TAC GGG ACC-3 ' L R P V A A E V Y G T

[SEQ ID NO : 49]

Primer #2 [SEQ ID NO : 50]

3 ' -G GAA GAC AAG TTA CTG TTG TGA CTC ACA GAC CGG-5 ' L L F N D N T E C L A

[SEQ ID NO: 51]

The codon usage for the primers was based on that from the homologous regions of the human lactoferrin cDNA sequence (GenBank accession #M83202). Primer #1 represents the sense strand of the predicted cDNA sequence, and Primer #2 represents the antisense strand. d) PCR amplification of a Draculin cDNA fragment:

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). To 10 μl of cDNA was added 10 μl of 10 X Hot Tub Reaction Buffer (500 mM Tris, pH 9.0, 15 mM MgCl2, 200 mM (NH4)2Sθ4), 2.5 μl of a 2 mM dNTP mixture (2 mM each of dATP, dTTP, dCTP, and dGTP), 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 μl. This reaction mixture was overlaid with 100 μl 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 μl) was analyzed by agarose gel electrophoresis. The major product of the reaction was a DNA fragment with electrophoretic mobility corresponding to approximately 1700 bp.

e) Cloning of the 1700 bp PCR product:

A 40 μl 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. The reaction was incubated for 60 minutes, placed on ice for 15 minutes, then centrifuged at 13000 X g for 15 minutes at 4°C. 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 μl (final buffer concentration: 50 mM Tris-HCI pH 7.5, 10 mM MgCl2, 5 mM dithiothreitol, 0.5 mM ATP, and 0.1 μg/μl 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 μl 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 phenokchloroform 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: re.cAI, endA1, gyrA96, thil, hsdR17, supE44, relA1, lac, [F¹ proAB, Laclq, 2ΔM15, 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.

Example 11

Isolation of Additional Draculin cDNAs

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 5'-ends of the Draculin coding sequence using the PCR and vampire bat salivary gland RNA. a) Isolation of a Draculin 3'-cDNA:

A clone encompassing the 3'-end of Draculin cDNA was isolated using a kit (Gibco-BRL, Grand Island, NY) based on the 3' RACE (Rapid Amplification of cDNA Ends) technique (Frohman, M. A. et al, Proc. Natl. Acad. Sci. USA £§18998-9003, 1988). Total RNA (1 μg) 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 μg/ml BSA, 10 mM Dithiothreitol, and 500 μM 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

Adapter Primer furnished with the 3' RACE kit [SEQ ID NO: 54] and a 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 μl) of the reverse transcriptase reaction was used in the PCR amplification. Buffer and reagents were added to give a final composition (in 100 μl total volume) of 20 picomoles of each primer, 200 μM each dNTP, 1X Hot Tub Reaction Buffer (Amersham), and 3 units Hot Tub DNA polymerase (Amersham). This reaction mixture was overlaid with 70 μl 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 μl 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 μl 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 phenohchloroform (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).

b) Isolation of a Draculin 5'-cDNA:

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 μg of total bat salivary gland RNA and a Draculin-specific primer (Primer #2 from Example 10, [SEQ ID NO: 50]). After the first-strand synthesis reaction was complete, 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 μl of sterile deionized water the eluent was lyophilized then resuspended in 13 μl of water.

The purified single-stranded cDNA was tailed by incubating it with dCTP for 10 minutes at 37°C in a 20 μl reaction mixture consisting of 10 mM Tris-HCI, pH 8.4, 25 mM KCI, 1.25 mM MgCl2, 50 μg/ml BSA, 200 μM 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.

PCR amplification of the tailed Draculin cDNA was performed using the Anchor Primer supplied with the 5' RACE kit [SEQ ID NO: 57] and a 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. This was combined with 40 picomoles of each primer, 10 μl of 10X Hot Tub reaction buffer, 200 μM each of the four dNTPs, and 3 units of Hot Tub DNA polymerase in a total reaction volume of 100 μl. The mix was overlaid with 70 μl of mineral oil and subjected to 40 cycles of amplification consisting of the sequential steps of 1 minute at 94°C, 1 minute at 57°C, and 2 minutes at 72°C. Twenty μl of the reaction mix was analyzed on a 0.8% agarose gel, and the major amplification product was found to be approximately 1.2 kilobases in length.

A 50 μl 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. coH 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 sequence of this 5'-clone was compared with the Draculin protein sequences determined in Example 8. This comparison suggested that the cDNA clone contains a deletion of approximately 165 bp (encoding approximately 55 amino acids) from the Draculin coding region. An inspection of the murine lactoferrin gene structure (GenBank accession #M64423) shows that exon 2 has a size and location similar to that of the missing region in the 5'- Draculin clone. It is possible, therefore, that the 5-Draculin clone isolated in this example represents an alternate splicing of the mRNA for Draculin.

Example 12

Isolation of a cDNA encoding a complete Draculin protein

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, XBAIδ'Drac [SEQ ID NO: 60], was designed to contain sequences immediately upstream of the putative translational initiation site for Draculin, as determined from the δ'-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 μl 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 μl sample of the amplification reaction was purified by use of a

Magic PCR Clean Up column (Promega) and 40% of the recovered DNA was digested with the restriction enzymes Xba I and Sal I. The restricted DNA was extracted with one volume of phenol, with one volume of phenol:chloroform (1 : 1), with one volume of chloroform:isoamyl alcohol (24: 1), and then precipitated with ethanol. The precipitated DNA was resuspended in TE (10 mM Tris pH 7.0, 1 mM EDTA) buffer and inserted into Sal I and Xba l-digested M13mp18 using T4 DNA ligase. Products of the ligation reaction were transformed into £. coli cells and the resulting recombinants were screened by restriction analysis. Seven of nine clones analyzed had the expected insert, and two of these were randomly chosen for partial sequence analysis. A partial sequence analysis showed that these clones contained the expected 5'- and 3'-ends of a Draculin cDNA. These clones contained the coding region which appeared to be absent in the 5'-clone isolated in Example 11b, but which was predicted to exist based on the protein sequences obtained in Example 8.

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. After 20 cycles, 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 (i. e. amino acid residues 20-708 of SEQ ID NO: 62) is approximately 75 kD. This calculation assumes that the polypeptide 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.

One skilled in the art will readily appreciate the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The peptides, polynucleotides, methods, procedures and techniques described herein are presented as representative of the preferred embodiments, or intended to be exemplary and not intended as limitations on the scope of the present invention. Changes therein and other uses will occur to those of skill in the art which are encompassed within the spirit of the invention or defined by the scope of the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Hem er, H. Coenraad Apitz-Castrσ, Rafael Bequin, Suzette Holt, John C. Lynch, Kevin J.

(ii) TITLE OF INVENTION: Draculin, Its Method of Preparation and

Use (iii) NUMBER OF SEQUENCES: 63

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Rhone-Poulenc Rorer Legal Department

(B) STREET: 500 Arcola Road (C) CITY: Collegeville

(D) STATE: PA

(E) COUNTRY: USA

(F) ZIP: 19426 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: Macintosh

(C) OPERATING SYSTEM: System 7

(D) SOFTWARE: Word 5.0 (PatentIn)

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Goodman, Rosanne

(B) REGISTRATION NUMBER: 32,534

(C) REFERENCE/DOCKET NUMBER: A0927-US

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (215) 454-3817

(B) TELEFAX: (215) 454-3808

(2) INFORMATION FOR SEQ ID N0:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE: (A) ORGANISM: Desmodus rotundus

(F) TISSUE TYPE: saliva (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:

Ala Arg Arg Arg Gly Val Arg Trp Xaa Thr lie Ser Lys Pro Glu Ala 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Ala Arg Arg Xaa Gly Val Arg Trp Xaa Thr lie Ser Lys Pro Glu Ala 1 5 10 15

Ala Lys Trp Ser Leu Gin Gin Asn Leu 20 25 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Met Xaa Leu Asp Gly Gly Phe lie Tyr lie Ala Gly Lys Xaa Gly Leu 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Xaa Leu Xaa Phe Xaa Gin Thr Arg Ser Xaa Asn Phe Asp Glu Phe 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Lys Gly Thr Ser Gly Ser Phe Gin Leu Phe Ser Ser Pro Pro Gly Gin 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE: (A) ORGANISM: Desmodus rotundus

(F) TISSUE TYPE: saliva (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Ala Arg Arg Arg Gly Val Arg Trp Cys Thr lie Ser Lys Pro Glu Ala 1 5 10 15

Ala Lys Cys Ser Lys Leu Gin Gin Asn Leu 20 25

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iϋ) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE: (A) ORGANISM: Desmodus rotundus

(F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Lys Ala Ser Thr Val Leu Glu Asn Thr Asp Gly Arg Gly Thr Glu Ala 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Lys Asp Gly Ala Gin Gly Phe Leu Arg lie Pro Ala Arg Val Asp 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Lys Xaa Xaa Ala Gly Leu Thr Trp Asn Ser Leu Arg Gly Thr Lys 1 5 10 15 (2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: Lys Leu Glu Asp Phe Glu Leu Leu Cys Leu Asp Gly Thr Arg Lys Pro 1 5 10 15

Val Ser Glu Phe 20

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE: (A) ORGANISM: Desmodus rotundus

(F) TISSUE TYPE: saliva (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Lys Glu Asp Ser lie Xaa Arg Leu 1 5

(2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Lys Xaa Ala Asp Ala Met Ser Leu Asp Ala Gly Leu Val Tyr Glu Ala 1 5 10 15

Gly Gin Asp Pro Tyr Arg Leu Arg Pro Val Ala Ala Glu Val Tyr 20 25 30

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Lys Cys Leu Xaa Ser Ser Lys Glu Pro Tyr Phe Gly Tyr Ser 1 5 10

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE: (A) ORGANISM: Desmodus rotundus

(F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Lys Ser Tyr Leu Glu Cys He Gin Ala He 1 5 10

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Lys Xaa Xaa Ala Ala Glu Val Glu Ala Xaa Gly Ala Arg Val Val Xaa 1 5 10 15

Xaa Ala Val Gly Pro Glu 20 (2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Lys Asp Arg Val Gin Tyr Leu Glu Gin Val Leu Xaa Asp Gin Gin Gly 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17;

Lys Ala Glu Arg Asp Gin Tyr Glu Xaa Leu Cys Pro Asp Asn Thr Arg 1 5 10 15

Lys Pro Val

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Lys Xaa Ala Pro Asn Ser Asn Glu Arg Tyr Phe Xaa Tyr Ala Gly Ala 1 5 10 15

Phe Arg Xaa Leu Val Glu 20 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Lys Xaa Val Xaa Gly Pro Ser Leu Ser Cys He Ser Arg Lys 1 5 10

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

Ala Arg Arg Arg Gly Val Arg 1 5 (2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva (xi) SEQUENCE DESCRIPTION: SEQ ID NO.-21:

Arg Leu Arg Pro Val Ala Ala Glu Val Tyr 1 5 10

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

Ser Gly Gin Ser Xaa Gly Thr Val Thr Cys Xaa Xaa Ala Ala Asp Asp 1 5 10 15 Glu Asp

(2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

Glu Xaa Leu Cys Pro Asp Asn Thr Arg Lys Pro Val Asp Glu Xaa Xaa 1 5 10 15

Gin Cys Ala Leu Ala Arg Val Pro Ser Xaa Ala Val Val Ala Arg Ser 20 25 30

Val (2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE: (A) ORGANISM: Desmodus rotundus

(F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

Cys Ala Val Gly Pro Glu Glu Leu Arg Lys Cys Gin Gin 1 5 10

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

Glu Lys Tyr Leu Gly Pro Glu Tyr Val Ala Xaa Xaa Ala Asn Leu Arg 1 5 10 15

Gin Cys Xaa Thr 20 (2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

Glu Asn Leu Pro Asn Lys Ala Glu Arg Asp Gin Tyr 1 5 10

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

Cys Thr He 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

20 25

(2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

Phe Gly Tyr Ser Gly Ala Phe Lys Cys Leu Lys Asp Gly Ala Xaa Asp 1 5 10 15 Val Ala Phe Val Xaa Asp Xaa His Val Phe 20 25

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

Cys Leu Phe Gin Ser Glu Thr Lys Asn Leu Leu Phe Asn Asp Asn 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

Ser Asn Glu Arg Tyr Phe Ser Tyr Ala Gly Ala Phe Arg Cys Leu Val 1 5 10 15 Xaa Asn Ala Gly Asp Val Ala Phe Val Lys

20 25

(2) INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

Ser Ser Pro Pro Gly Gin Lys Asp Leu Leu Phe Lys Asp Xaa Ala Gin 1 5 10 15 Gly Phe Leu Arg He Pro

20

(2) INFORMATION FOR SEQ ID NO:32: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

Gly Thr Glu Gly Ala Pro Arg Thr Xaa Tyr 1 5 10

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE: (A) ORGANISM: Desmodus rotundus

(F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: Glu Ala Gly Gin Asp Pro Tyr 1 5

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

Cys Leu Phe Gin Ser Glu Thr Lys Asn Leu Leu Phe Asn Asp Asn Thr 1 5 10 15

Glu Cys Leu Ala Lys Leu Gin Gly Lys Thr Thr Tyr Xaa Lys Tyr Leu 20 25 30 Gly

(2) INFORMATION FOR SEQ ID NO:35: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

Val Leu Lys Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Phe He 1 5 10 15

Tyr He Ala

(2) INFORMATION FOR SEQ ID NO:36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

Lys Glu Pro Tyr Phe Gly Tyr 1 5

(2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iϋ) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE: (A) ORGANISM: Desmodus rotundus

(F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

Ser Leu Asp Gly Gly Phe He Tyr 1 5

(2) INFORMATION FOR SEQ ID NO:38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

Ala Arg Arg Arg Gly Val Arg Trp Cys Thr He Ser Lys Pro Glu Ala 1 5 10 15

Ala Lys Cys Ser Lys Leu Gin Gin Asn Leu Lys Arg Val Xaa Gly Pro 20 25 30

Ser Leu Ser Cys He Ser Arg Lys 35 40

(2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

Lys Gly Thr Ser Gly Ser Phe Gin Leu Phe Ser Ser Pro Pro Gly Gin 1 5 10 15 Lys Asp Leu Leu Phe Lys Asp Gly Ala Gin Gly Phe Leu Arg He Pro

20 25 30

Xaa Arg Val Asp 35

(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iϋ) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE: (A) ORGANISM: Desmodus rotundus

(F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: Lys Cys Ala Xaa Ser Ser Lys Glu Pro Tyr Phe Gly Tyr Ser Gly Ala 1 5 10 15 Phe Lys Cys Leu Lys Asp Gly Ala Xaa Asp Val Ala Phe Val Xaa Asp 20 25 . 30 Xaa Xaa Val Phe 35

(2) INFORMATION FOR SEQ ID NO:41: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:

Lys Xaa Ala Pro Asn Ser Asn Glu Arg Tyr Phe Xaa Tyr Ala Gly Ala 1 5 10 15

Phe Arg Cys Leu Val Glu Asn Ala Gly Asp Val Ala Phe Val Lys 20 25 30

(2) INFORMATION FOR SEQ ID NO:42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:

Glu Asn Leu Pro Asn Lys Ala Glu Arg Asp Gin Tyr Glu Xaa Leu Cys 1 5 10 15

Pro Asp Asn Thr Arg Lys Pro Val Asp Glu Xaa Xaa Gin Cys Xaa Leu 20 25 30 Ala Arg Val Pro Ser Xaa Ala Val Val Ala Arg Ser Val

35 40 45 .2) INFORMATION FOR SEQ ID NO:43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:

Val Leu Lys Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Phe He 1 5 10 15 Tyr He Ala Gly Lys Xaa Gly Leu

20

(2) INFORMATION FOR SEQ ID NO:44: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

Lys Xaa Xaa Ala Ala Glu Val Glu Ala Xaa Gly Ala Arg Val Val Xaa 1 5 10 15

Xaa Ala Val Gly Pro Glu Glu Leu Arg Lys Cys Gin Gin 20 25

(2) INFORMATION FOR SEQ ID NO:45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:

Ser Gly Gin Ser Xaa Gly Thr Val Thr Cys Xaa Xaa Ala Ala Asp Xaa 1 5 10 15

Glu Asp

(2) INFORMATION FOR SEQ ID NO:46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:

Glu Lys Tyr Leu Gly Pro Glu Tyr Val Thr Xaa Xaa Ala Asn Leu Arg 1 5 10 15

Gin Cys Xaa Thr 20 (2) INFORMATION FOR SEQ ID NO:47: -

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: saliva (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:

Cys Leu Phe Gin Ser Glu Thr Lys Asn Leu Leu Phe Asn Asp Asn Thr 1 5 10 15

Glu Cys Leu Ala Lys Leu Gin Gly Lys Thr Thr Tyr Glu Lys Tyr Leu 20 25 30 Gly

(2) INFORMATION FOR SEQ ID NO:48: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:

CTGCGACCTG TAGCGGCGGA AGTCTACGGG ACC 3

(2) INFORMATION FOR SEQ ID NO:49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

Leu Arg Pro Val Ala Ala Glu Val Tyr Gly Thr 1 5 10 (2) INFORMATION FOR SEQ ID NO:50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: GGCCAGACAC TCAGTGTTGT CATTGAACAG AAGG 3

(2) INFORMATION FOR SEQ ID NO:51: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:

Leu Leu Phe Asn Asp Asn Thr Glu Cys Leu Ala 1 5 10

(2) INFORMATION FOR SEQ ID NO:52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 372 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: Salivary gland

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:

GGCCAGACAC TCAGTGTTGT CATTGAACAG AAGGTTCTTG GTTTCAGACT GGAACAAGCA 60 GAACTTGCCT GGGCAGAGGG TGCCATTTCT TCCAAACTTG CCCTGTTGGT CCAGCAGCAC 120

CTGCTCCAGG TATTGTACCC TATCTTTCCG AGATACCACA CCGTGACTCG GGGCCCTGGC 180

NNNGTGGCAG GTCTCAAACT CAGACACAGG CTTCCGGGTG CCATCAAGGC ACAAGAGCTC 240 AAAGTCCTCC AGCTTCAGAT CCTTAGCCCA TGCTTCGGTG CCCCTTCCAT CCGTGTTCTC 300

CAAGACAGTG GAAGCTTTCA CAAAGGCCAC GTCTCCAGCA TTCTCGACCA GGCACCTGAA 360

AGCCCCAGCG TA 372 (2) INFORMATION FOR SEQ ID NO:53:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: GGCCACGCGT CGACTAGTAC TTTTTTTTTT TTTTTTT 3

(2) INFORMATION FOR SEQ ID NO:54:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: CUACUACUAC UAGGCCACGC GTCGACTAGT AC 3

(2) INFORMATION FOR SEQ ID NO:55:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: TCCAGTCGAC AGCAACTTGC AACTGAGC 2

(2) INFORMATION FOR SEQ ID NO:56: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 371 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(i.i) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO ( iv) ANTI-SENSE : NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: Salivary gland

( i) SEQUENCE DESCRIPTION: SEQ ID NO:56: CTCCTGTTCA ATGACAACAC CGAGTGTCTG GCCAAACTCC AGGGCAAAAC AACGTATGAG 6 AAATATTTGG GACCAGAGTA TGTCACAGCG GTTGCTAATC TGAGGCAATG CTCCACCTCC 12 CCACTTCTGG AAGCCTGTAC CTTCCTGAGG AATTGAAACC AAGAAGGTGG CCCAGCCCCC 18 TGCCACCCCC ACCACCCCAA AGCTGCAGCC GCCACTGCCC TGGCCCCATC CCCAGGCCCG 24

CTGGGGCCTG CTGCTCCCTT CTCGGGGGGC TGCTTACTAG TCACACCTAT TTTCACAATT 30

CCCTGCTGTC ATCTCAACAA GAAATAAAAC CGCAAATGCC ATTGATTTTC AAAAAAAAAA 36 AAAAAAAAAA A 37

(2) INFORMATION FOR SEQ ID NO:57:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 48 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid

(ix) FEATURE:

(A) NAME/KEY: modified_base (B) LOCATION: 36

(D) OTHER INFORMATION: /mod_base= i

(ix) FEATURE:

(A) NAME/KEY: modified_base (B) LOCATION: 37

(D) OTHER INFORMATION: /mod_base= i

(ix) FEATURE:

(A) NAME/KEY: modified_base (B) LOCATION: 41

(D) OTHER INFORMATION: /mod_base= i

(ix) FEATURE:

(A) NAME/KEY: modified_base (B) LOCATION: 42

(D) OTHER INFORMATION: /mod_base= i

(ix) FEATURE:

(A) NAME/KEY: modified_base (B) LOCATION: 46

(D) OTHER INFORMATION: /mod_base= i

(ix) FEATURE:

(A) NAME/KEY: modified_base (B) LOCATION: 47

(D) OTHER INFORMATION: /mod_base= i

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:

CUACUACUAC UAGGCCACGC GTCGACTAGT ACGGGNNGGG NNGGGNNG 4

(2) INFORMATION FOR SEQ ID NO:58: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: ATCCAAACTC ATGGCATCAG CTTCTCCTTT CAGCAC 36

(2) INFORMATION FOR SEQ ID NO:59: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 151 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO ( iv) ANTI-SENSE : NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: Salivary gland

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: AGCCTTCGCT GGCTGGAGTC TCCTTGGGAC CTCAGACATG AAGCTCCTCT TCCTTGCACT 60 GCTGTCCCTC CTGGCCCTCG GCGAAAAGGC AGATGCCATG AGCCTTGACG CAGGTCTGGT 120 GTACGAGGCA GGACAGGACC CGTACAGACT G 151 (2) INFORMATION FOR SEQ ID NO:60:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: GCTCTAGACA TGAAGCTCCT CTTCCTTGCA CTG 33

(2) INFORMATION FOR SEQ ID NO:61:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2347 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE:

(A) ORGANISM: Desmodus rotundus (F) TISSUE TYPE: Salivary gland

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION: 1..2127

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:

ATG AAG CTC CTC TTC CTT GCA CTG CTG TCC CTC CTG GCC CTC GGG CCG 4 Met Lys Leu Leu Phe Leu Ala Leu Leu Ser Leu Leu Ala Leu Gly Pro 1 5 10 15 AGT CTG GCT GCC CGC AGG AGA GGT GTT CGA TGG TGC ACC ATA TCA AAA 9 Ser Leu Ala Ala Arg Arg Arg Gly Val Arg Trp Cys Thr He Ser Lys 20 25 30

CCA GAG GCA GCA AAA TGC TCT AAA CTG CAA CAG AAT CTA AAA CGA GTG 14 Pro Glu Ala Ala Lys Cys Ser Lys Leu Gin Gin Asn Leu Lys Arg Val 35 40 45

CGT GGC CCC TCT CTC TCC TGC ATA AGC AGA AAG TCC TAC CTG GAA TGT 19 Arg Gly Pro Ser Leu Ser Cys He Ser Arg Lys Ser Tyr Leu Glu Cys 50 55 60

ATC CAG GCC ATC GCG GCG AAA AGG GCA GAT GCC ATG AGC CTT GAT GCA 24 He Gin Ala He Ala Ala Lys Arg Ala Asp Ala Met Ser Leu Asp Ala 65 70 75 80

GGT CTG GTG TAC GAG GCA GGA CAG GAC CCG TAC AGA TTG CGG CCT GTG 28 Gly Leu Val Tyr Glu Ala Gly Gin Asp Pro Tyr Arg Leu Arg Pro Val 85 90 95 GCA GCA GAG GTC TAC GGG ACC GAG GGG GCA CCG CGG ACG CAC TAT TAC 33 Ala Ala Glu Val Tyr Gly Thr Glu Gly Ala Pro Arg Thr His Tyr Tyr 100 105 110

GCT GTG GCC CTG GTG AAA AAG GAC AGC AAC TTG CAA CTG AGC CAG CTG 38 Ala Val Ala Leu Val Lys Lys Asp Ser Asn Leu Gin Leu Ser Gin Leu 115 120 125

CAA GGC GTG AGG TCC TGC CAC ACT GGC CTC AAC AGG TCC GCC GGG TGG 43 Gin Gly Val Arg Ser Cys His Thr Gly Leu Asn Arg Ser Ala Gly Trp 130 135 140

AAA ATC CCT GTG GGC ACG CTC CGT CCG TAC CTG GGC TGG GCA GGG CCA 48 Lys He Pro Val Gly Thr Leu Arg Pro Tyr Leu Gly Trp Ala Gly Pro 145 150 155 160

CCT CGA CCC CTC CAG GAA GCT GTG GCC AAC TTC TTC TCC GCT AGC TGT 52 Pro Arg Pro Leu Gin Glu Ala Val Ala Asn Phe Phe Ser Ala Ser Cys 165 170 175 GTT CCC TGT GCA GAT GGC AAC CAG TAC CCC AAC CTG TGT CGC TTG TGT 576 Val Pro Cys Ala Asp Gly Asn Gin Tyr Pro Asn Leu Cys Arg Leu Cys 180 185 190 GCG GGG ACA GGG GCA GAT AAA TGT GCC TGC TCC TCC AAG GAA CCG TAC 624 Ala Gly Thr Gly Ala Asp Lys Cys Ala Cys Ser Ser Lys Glu Pro Tyr 195 200 205

TTT GGC TAC TCC GGT GCC TTC AAG TGT CTG AAA GAT GGG GCT GGA GAC 672 Phe Gly Tyr Ser Gly Ala Phe Lys Cys Leu Lys Asp Gly Ala Gly Asp 210 215 220

GTG GCT TTT GTC AAG GAC AGT ACG GTG TTT GAG AAC CTG CCA AAC AAG 720 Val Ala Phe Val Lys Asp Ser Thr Val Phe Glu Asn Leu Pro Asn Lys 225 230 235 240

GCC GAG AGA GAC CAG TAT GAG CTG CTC TGC CCA GAC AAC ACC CGA AAG 768 Ala Glu Arg Asp Gin Tyr Glu Leu Leu Cys Pro Asp Asn Thr Arg Lys 245 250 255

CCG GTG GAT GAG TTT GAG CAG TGC CAC CTG GCC CGG GTC CCT TCT CAT 816

Pro Val Asp Glu Phe Glu Gin Cys His Leu Ala Arg Val Pro Ser His

260 265 270 GCA GTT GTG GCC CGA AGC GTG GGT GGC AAG GAG GAC TCG ATC TGG AGG 864

Ala Val Val Ala Arg Ser Val Gly Gly Lys Glu Asp Ser He Trp Arg

275 280 285

CTT CTC AGC AAG GCA CAG GAG AAG TTT GGA AAA GGC ACG TCA GGG AGC 912 Leu Leu Ser Lys Ala Gin Glu Lys Phe Gly Lys Gly Thr Ser Gly Ser 290 295 300

TTC CAG CTC TTC AGC TCC CCT CCT GGG CAG AAG GAC CTG CTT TTC AAA 960

Phe Gin Leu Phe Ser Ser Pro Pro Gly Gin Lys Asp Leu Leu Phe Lys 305 310 315 320

GAT GGA GCC CAA GGG TTT TTG AGG ATC CCC TCA AGG GTG GAC GCT GAG 1008

Asp Gly Ala Gin Gly Phe Leu Arg He Pro Ser Arg Val Asp Ala Glu 325 330 335

CTG TAC CTC GGT CCC AGC TAC CTC ACC GTC ATC AAG AAC CTG AAG GAA 1056 Leu Tyr Leu Gly Pro Ser Tyr Leu Thr Val He Lys Asn Leu Lys Glu 340 345 350 TCG GCA GCA GAG GTG GAG GCC CGG GGG GCC CGG GTT GTG TGG TGC GCG 1104 Ser Ala Ala Glu Val Glu Ala Arg Gly Ala Arg Val Val Trp Cys Ala 355 360 365

GTG GGC CCA GAG GAG CTG CGC AAG TGC CAG CAG TGG AGT GGC CAG AGC 1152 Val Gly Pro Glu Glu Leu Arg Lys Cys Gin Gin Trp Ser Gly Gin Ser 370 375 380

AAT GGG ACA GTG ACG TGC ACA ACA GCC GCT GAC ACA GAG GAC TGC ATC 1200 Asn Gly Thr Val Thr Cys Thr Thr Ala Ala Asp Thr Glu Asp Cys He 385 390 395 400

GCC CTG GTG CTG AAA GGA GAA GCC GAT GCC ATG AGT CTG GAC GGA GGG 1248 Ala Leu Val Leu Lys Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly 405 410 415 TTC ATC TAT ATC GCC GGC AAA TGT GGT TTG GCG CCT GTG CTG GCA GAG 1296 Phe He Tyr He Ala Gly Lys Cys Gly Leu Ala Pro Val Leu Ala Glu 420 425 430 AGC CAA AGA TCC GAA GGA GGC AGT AAC TTG GAT TGT GTG AAT AGA CCA 1344 Ser Gin Arg Ser Glu Gly Gly Ser Asn Leu Asp Cys Val Asn Arg Pro 435 440 445

CTG GAA GGG TAT CGT GCT GTG GCG GTT GTC AGG AAA TCA AGT GCT GGC 1392 Leu Glu Gly Tyr Arg Ala Val Ala Val Val Arg Lys Ser Ser Ala Gly 450 455 460

CTC ACC TGG AAC TCC CTG AGG GGC ACG AAG TCC TGC CAC ACC GCT GTG 1440 Leu Thr Trp Asn Ser Leu Arg Gly Thr Lys Ser Cys His Thr Ala Val 465 470 475 480

GGC AGG ACA GCA GGC TGG AAC ATC CCC ATG GGT CTG CTC TTC AAC CAG 1488 Gly Arg Thr Ala Gly Trp Asn He Pro Met Gly Leu Leu Phe Asn Gin 485 490 495

ACA CGC TCC TGC AAC TTT GAT GAA TTC TTC AGT CAA ACG TGC GCC CCT 1536 Thr Arg Ser Cys Asn Phe Asp Glu Phe Phe Ser Gin Thr Cys Ala Pro 500 505 510 GGA GCA GAC CCG AAC TCC AAC CTC TGC GCC CTG TGC GTC GGC AAT GAG 1584 Gly Ala Asp Pro Asn Ser Asn Leu Cys Ala Leu Cys Val Gly Asn Glu 515 520 525

CAG GGC CAG GAC AAG TGC GCT CCC AAC AGC AAC GAG AGG TAC TTC AGC 1632 Gin Gly Gin Asp Lys Cys Ala Pro Asn Ser Asn Glu Arg Tyr Phe Ser 530 535 540

TAC GCT GGG GCT TTC AGG TGC CTG GTC GAG AAT GCT GGA GAC GTG GCC 1680 Tyr Ala Gly Ala Phe Arg Cys Leu Val Glu Asn Ala Gly Asp Val Ala 545 550 555 560

TTT GTG AAA GCT TCC ACT GTC TTG GAG AAC ACG GAT GGA AGG GGC ACC 1728 Phe Val Lys Ala Ser Thr Val Leu Glu Asn Thr Asp Gly Arg Gly Thr 565 570 575

GAA GCA TGG GCT AAG GAT CTG AAG CTG GAG GAC TTT GAG CTC TTG TGC 1776 Glu Ala Trp Ala Lys Asp Leu Lys Leu Glu Asp Phe Glu Leu Leu Cys 580 585 590 CTT GAT GGC ACC CGG AAG CCT GTG TCT GAG TTT GAG ACC TGC CAC CTG 1824 Leu Asp Gly Thr Arg Lys Pro Val Ser Glu Phe Glu Thr Cys His Leu 595 600 605

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

TAC CTG GAG CAG GTG CTG CTG GAC CAA CAG GGC AAG TTT GGA AGA AAT 1920 Tyr Leu Glu Gin Val Leu Leu Asp Gin Gin Gly Lys Phe Gly Arg Asn 625 630 635 640

GGC ACC CTC TGC CCA GGC AAG TTC TGC TTG TTC CAG TCT GAA ACC AAG 1968 Gly Thr Leu Cys Pro Gly Lys Phe Cys Leu Phe Gin Ser Glu Thr Lys 645 650 655 AAC CTC CTG TTC AAT GAC AAC ACC GAG TGT CTG GCC AAA CTC CAG GGC 2016 Asn Leu Leu Phe Asn Asp Asn Thr Glu Cys Leu Ala Lys Leu Gin Gly 660 665 670 AAA ACA ACG TAT GAG AAA TAT TTG GGA CCA GAG TAT GTC ACA GCG GTT 2064 Lys Thr Thr Tyr Glu Lys Tyr Leu Gly Pro Glu Tyr Val Thr Ala Val 675 680 685

GCT AAT CTG AGG CAA TGC TCC ACC TCC CCA CTT CTG GAA GCC TGT ACC 2112 Ala Asn Leu Arg Gin Cys Ser Thr Ser Pro Leu Leu Glu Ala Cys Thr 690 695 700

TTC CTG AGG AAT TGAAACCAAG AAGGTGGCCC AGCCCCCTGC CACCCCCACC 2164

Phe Leu Arg Asn 705

ACCCCAAAGC TGCAGCCGCC ACTGCCCTGG CCCCATCCCC AGGCCCGCGG GGGCCTGCTG 2224

CTCCCTTCTC GGGGGGCAGC TTACTAGTCA CACCTATTTT CACAATTCCC TGCTGTCATC 2284

TCAACAAGAA ATAAAACCGC AAATGCCATT GATTTTCATT CCTTAAAAAA AAAAAAAAAA 2344

AAA 2347

[2) INFORMATION FOR SEQ ID NO:62:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 708 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:

Met Lys Leu Leu Phe Leu Ala Leu Leu Ser Leu Leu Ala Leu Gly Pro 1 5 10 15 Ser Leu Ala Ala Arg Arg Arg Gly Val Arg Trp Cys Thr He Ser Lys

20 25 30

Pro Glu Ala Ala Lys Cys Ser Lys Leu Gin Gin Asn Leu Lys Arg Val 35 40 45

Arg Gly Pro Ser Leu Ser Cys He Ser Arg Lys Ser Tyr Leu Glu Cys 50 55 60

He Gin Ala He Ala Ala Lys Arg Ala Asp Ala Met Ser Leu Asp Ala 65 70 75 80

Gly Leu Val Tyr Glu Ala Gly Gin Asp Pro Tyr Arg Leu Arg Pro Val 85 90 95 Ala Ala Glu Val Tyr Gly Thr Glu Gly Ala Pro Arg Thr His Tyr Tyr 100 105 110

Ala Val Ala Leu Val Lys Lys Asp Ser Asn Leu Gin Leu Ser Gin Leu 115 120 125 Gin Gly Val Arg Ser Cys His Thr Gly Leu Asn Arg Ser Ala Gly Trp 130 135 140

Lys He Pro Val Gly Thr Leu Arg Pro Tyr Leu Gly Trp Ala Gly Pro

145 150 155 160

Pro Arg Pro Leu Gin Glu Ala Val Ala Asn Phe Phe Ser Ala Ser Cys

165 170 175 Val Pro Cys Ala Asp Gly Asn Gin Tyr Pro Asn Leu Cys Arg Leu Cys 180 185 190

Ala Gly Thr Gly Ala Asp Lys Cys Ala Cys Ser Ser Lys Glu Pro Tyr 195 200 205

Phe Gly Tyr Ser Gly Ala Phe Lys Cys Leu Lys Asp Gly Ala Gly Asp 210 215 220

Val Ala Phe Val Lys Asp Ser Thr Val Phe Glu Asn Leu Pro Asn Lys 225 230 235 240

Ala Glu Arg Asp Gin Tyr Glu Leu Leu Cys Pro Asp Asn Thr Arg Lys

245 250 255 Pro Val Asp Glu Phe Glu Gin Cys His Leu Ala Arg Val Pro Ser His

260 265 270

Ala Val Val Ala Arg Ser Val Gly Gly Lys Glu Asp Ser He Trp Arg 275 280 285

Leu Leu Ser Lys Ala Gin Glu Lys Phe Gly Lys Gly Thr Ser Gly Ser 290 295 300

Phe Gin Leu Phe Ser Ser Pro Pro Gly Gin Lys Asp Leu Leu Phe Lys 305 310 315 320

Asp Gly Ala Gin Gly Phe Leu Arg He Pro Ser Arg Val Asp Ala Glu

325 330 335 Leu Tyr Leu Gly Pro Ser Tyr Leu Thr Val He Lys Asn Leu Lys Glu

340 345 350

Ser Ala Ala Glu Val Glu Ala Arg Gly Ala Arg Val Val Trp Cys Ala 355 360 365

Val Gly Pro Glu Glu Leu Arg Lys Cys Gin Gin Trp Ser Gly Gin Ser 370 375 380

Asn Gly Thr Val Thr Cys Thr Thr Ala Ala Asp Thr Glu Asp Cys He 385 390 395 400

Ala Leu Val Leu Lys Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly 405 410 415 Phe He Tyr He Ala Gly Lys Cys Gly Leu Ala Pro Val Leu Ala Glu 420 425 430

Ser Gin Arg Ser Glu Gly Gly Ser Asn Leu Asp Cys Val Asn Arg Pro 435 440 445 Leu Glu Gly Tyr Arg Ala Val Ala Val Val Arg Lys Ser Ser Ala Gly 450 455 460

Leu Thr Trp Asn Ser Leu Arg Gly Thr Lys Ser Cys His Thr Ala Val

465 470 475 480

Gly Arg Thr Ala Gly Trp Asn He Pro Met Gly Leu Leu Phe Asn Gin

485 490 495 Thr Arg Ser Cys Asn Phe Asp Glu Phe Phe Ser Gin Thr Cys Ala Pro 500 505 510

Gly Ala Asp Pro Asn Ser Asn Leu Cys Ala Leu Cys Val Gly Asn Glu 515 520 525

Gin Gly Gin Asp Lys Cys Ala Pro Asn Ser Asn Glu Arg Tyr Phe Ser 530 535 540

Tyr Ala Gly Ala Phe Arg Cys Leu Val Glu Asn Ala Gly Asp Val Ala 545 550 555 560

Phe Val Lys Ala Ser Thr Val Leu Glu Asn Thr Asp Gly Arg Gly Thr 565 570 575 Glu Ala Trp Ala Lys Asp Leu Lys Leu Glu Asp Phe Glu Leu Leu Cys 580 585 590

Leu Asp Gly Thr Arg Lys Pro Val Ser Glu Phe Glu Thr Cys His Leu 595 600 605

Ala Arg Ala Pro Ser His Gly Val Val Ser Arg Lys Asp Arg Val Gin 610 615 620

Tyr Leu Glu Gin Val Leu Leu Asp Gin Gin Gly Lys Phe Gly Arg Asn 625 630 635 640

Gly Thr Leu Cys Pro Gly Lys Phe Cys Leu Phe Gin Ser Glu Thr Lys

645 650 655 Asn Leu Leu Phe Asn Asp Asn Thr Glu Cys Leu Ala Lys Leu Gin Gly 660 665 670

Lys Thr Thr Tyr Glu Lys Tyr Leu Gly Pro Glu Tyr Val Thr Ala Val 675 680 685

Ala Asn Leu Arg Gin Cys Ser Thr Ser Pro Leu Leu Glu Ala Cys Thr 690 695 700

Phe Leu Arg Asn 705

(2) INFORMATION FOR SEQ ID NO:63: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 710 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:

Leu Val Phe Leu Val Leu Leu Phe Leu Gly Ala Leu Gly Leu Cys Leu 1 5 10 15

Ala Gly Arg Arg Arg Arg Ser Val Gin Trp Cys Ala Val Ser Gin Pro 20 25 30 Glu Ala Thr Lys Cys Phe Gin Trp Gin Arg Asn Met Arg Lys Val Arg

35 40 45

Gly Pro Pro Val Ser Cys He Lys Arg Asp Ser Pro He Gin Cys He 50 55 60

Gin Ala He Ala Glu Asn Arg Ala Asp Ala Val Thr Leu Asp Gly Gly 65 70 75 80

Phe He Tyr Glu Ala Gly Leu Ala Pro Tyr Lys Leu Arg Pro Val Ala 85 90 95

Ala Glu Val Tyr Gly Thr Glu Arg Gin Pro Arg Thr His Tyr Tyr Ala 100 105 110

Val Ala Val Val Lys Lys Gly Gly Ser Phe Gin Leu Asn Glu Leu Gin 115 120 125

Gly Leu Lys Ser Cys His Thr Gly Leu Arg Arg Thr Ala Gly Trp Asn 130 135 140

Val Pro He Gly Thr Leu Arg Pro Phe Leu Asn Trp Thr Gly Pro Pro

145 150 155 160

Glu Pro He Glu Ala Ala Val Ala Arg Phe Phe Ser Ala Ser Cys Val 165 170 175

Pro Gly Ala Asp Lys Gly Gin Phe Pro Asn Leu Cys Arg Leu Cys Ala 180 185 190 Gly Thr Gly Glu Asn Lys Cys Ala Phe Ser Ser Gin Glu Pro Tyr Phe

195 200 205

Ser Tyr Ser Gly Ala Phe Lys Cys Leu Arg Asp Gly Ala Gly Asp Val 210 215 220

Ala Phe He Arg Glu Ser Thr Val Phe Glu Asp Leu Ser Asp Glu Ala 225 230 235 240

Glu Arg Asp Glu Tyr Glu Leu Leu Cys Pro Asp Asn Thr Arg Lys Pro 245 250 255

Val Asp Lys Phe Lys Asp Cys His Leu Ala Arg Val Pro Ser His Ala

260 265 270 Val Val Ala Arg Ser Val Asn Gly Lys Glu Asp Ala He Trp Asn Leu

275 280 285 Leu Arg Gin Ala Gin Glu Lys Phe Gly Lys Asp Lys Ser Pro Lys Phe 290 295 300

Gin Leu Phe Gly Ser Pro Ser Gly Gin Lys Asp Leu Leu Phe Lys Asp 305 310 315 320

Ser Ala He Gly Phe Ser Arg Val Pro Pro Arg He Asp Ser Gly Leu 325 330 335

Tyr Leu Gly Ser Gly Tyr Phe Thr Ala He Gin Asn Leu Arg Lys Ser 340 345 350

Glu Glu Glu Val Ala Ala Arg Arg Ala Arg Val Val Trp Cys Ala Val 355 360 365

Gly Glu Gin Glu Leu Arg Lys Cys Asn Gin Trp Ser Gly Leu Ser Glu 370 375 380

Gly Ser Val Thr Cys Ser Ser Ala Ser Thr Thr Glu Asp Cys He Ala 385 390 395 400

Leu Val Leu Lys Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Tyr 405 410 415

Val Tyr Thr Ala Gly Lys Cys Gly Leu Val Pro Val Leu Ala Glu Asn 420 425 430

Tyr Lys Ser Gin Gin Ser Ser Asp Pro Asp Pro Asn Cys Val Asp Arg 435 440 445

Pro Val Glu Gly Tyr Leu Ala Val Ala Val Val Arg Arg Ser Asp Thr 450 455 460 Ser Leu Thr Trp Asn Ser Val Lys Gly Lys Lys Ser Cys His Thr Ala 465 470 475 480

Val Asp Arg Thr Ala Gly Trp Asn He Pro Met Gly Leu Leu Phe Asn 485 490 495

Gin Thr Gly Ser Cys Lys Phe Asp Glu Tyr Phe Ser Gin Ser Cys Ala 500 505 510

Pro Gly Ser Asp Pro Arg Ser Asn Leu Cys Ala Leu Cys He Gly Asp 515 520 525

Glu Gin Gly Glu Asn Lys Cys Val Pro Asn Ser Asn Glu Arg Tyr Tyr 530 535 540 Gly Tyr Thr Gly Ala Phe Arg Cys Leu Ala Glu Asn Ala Gly Asp Val

545 550 555 560

Ala Phe Val Lys Asp Val Thr Val Leu Gin Asn Thr Asp Gly Asn Asn 565 570 575

Asn Glu Ala Trp Ala Lys Asp Leu Lys Leu Ala Asp Phe Ala Leu Leu 580 585 590

Cys Leu Asp Gly Lys Arg Lys Pro Val Thr Glu Ala Arg Ser Cys His 595 600 605 Leu Ala Met Ala Pro Asn His Ala Val Val Ser Arg Met Asp Lys Val 610 615 620

Glu Arg Leu Lys Gin Val Leu Leu His Gin Gin Ala Lys Phe Gly Arg 625 630 635 640

Asn Gly Ser Asp Cys Pro Asp Lys Phe Cys Leu Phe Gin Ser Glu Thr 645 650 655 Lys Asn Leu Leu Phe Asn Asp Asn Thr Glu Cys Leu Ala Arg Leu His

660 665 670

Gly Lys Thr Thr Tyr Glu Lys Tyr Leu Gly Pro Gin Tyr Val Ala Gly 675 680 685

He Thr Asn Leu Lys Lys Cys Ser Thr Ser Pro Leu Leu Glu Ala Cys 690 695 700

Glu Phe Leu Arg Lys Leu 705 710

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule \3bis)

For International Bureau use only

I This sheet was received by the Internationa! Bureau on:

ot cer

Form PCT/RO/134 (Jul 1992

Claims

What is claimed is:

1. A novel anticoagulant polypeptide comprising the amino acid sequence of SEQ ID NO: 62 or biologically active fragments thereof.

2. The polypeptide of claim 1 which inhibits Factor Xa activity and Factor IXa activity.

3. A DNA sequence comprising the DNA of SEQ ID NO: 61 substantially free of other DNA of bat origin.

4. A DNA sequence encoding a novel anticoagulant polypeptide comprising the amino acid sequence of SEQ ID NO: 62 or biologically active fragments thereof said DNA sequence being substantially free of other DNA of bat origin.

5. A method of providing anticoagulant therapy comprising administration of a therapeutically effective amount of the factor of claim 1 to an individual in need of said therapy.

6. A composition of matter having the following properties:

(1) anticoagulant activity stable for at least 7 days at room temperature, after freezing at about -30°C or about -80°C, and after repeated freezing and thawing, and after incubation for about 30 minutes at pH 5.5- 9.0,

(2) said activity is destroyed after heating for about 10 minutes at about 80°C, but only partially destroyed after incubation at about 60°C for about 10 minutes and about 30 minutes,

(3) said activity characterized by the prolongation of whole blood coagulation time, activated partial thromboplastin time, prothrombin time at concentrations that prolong activated partial thromboplastin time, the inhibition of Factor Xa activity, the inhibition of Factor IXa activity, but not the prolongation of thrombin time,

(4) said activity is not inhibited by PMFS or DFP.

7. A substantially purified anticoagulant polypeptide which inhibits Factor Xa activity and Factor IXa activity.

8. The polypeptide of claim 6 wherein said polypeptide is encoded by the DNA of SEQ ID NO: 61.

9. The anticoagulant of claim 1 produced recombinantly.

10. A nucleotide sequence encoding a novel anticoagulant polypeptide selected from the group consisting of:

(a) nucleotide sequences of SEQ ID NO: 61 or its complementary strand;

(b) nucleotide sequences which hybridize under stringent conditions to the protein coding regions of the nucleotide sequences defined in (a) or fragments thereof; and (c) nucleotide sequences which, but for the degeneracy of the genetic code, would hybridize to the nucleotide sequences defined in (a) or (b); wherein said nucleotide sequence is substantially free of other nucleotide sequences of bat origin.

11. The nucleotide sequence of claim 10 wherein said nucleotide is a

DNA sequence.

12. A vector comprising the DNA of claim 11.

13. The vector of claim 12 designated pBSNDrac and deposited under ATCC Accession No. 69393.

14. A cell comprising the vector of claim 12.

15. A nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with the DNA sequence according to claim 11.