WO2003091284A1 - Anticoagulants - Google Patents

Abstract

Anticoagulants derived from the salivary glands of haematophagous arthropods are provided. In particular, anticoagulants that are thrombin inhibitors are provided. The invention also provides the use of these anticoagulants in the treatment and prevention of diseases.

Description

Anticoagulants

The present invention relates to anticoagulants derived from the salivary glands of haematophagous arthropods. In particular, the invention relates to anticoagulants that are thrombin inhibitors. The invention also relates to the use of such anticoagulants in the treatment and prevention of diseases.

All documents mentioned in the text and listed at the end of this description are incorporated herein by reference.

Anticoagulants are used to treat or prevent thromboembolic diseases. Such diseases, which include coronary thrombosis, pulmonary embolism, deep vein thrombosis and stroke, account for almost half of adult deaths in the United Kingdom (Kumar et al, 1996).

Thrombosis is defined as the formation of a solid mass of blood constituents within the circulatory system during life. An embolus is a fragment of thrombus which detaches and is carried downstream where it may occlude smaller blood vessels. Two major types of thrombosis are recognised: arterial and venous. Diseases associated with arterial thrombosis include coronary artery disease, peripheral vascular disease and stroke. Venous thrombosis may lead to deep vein thrombosis and pulmonary embolism, a significant cause of post-operative death.

Arterial thrombi form when blood coagulates at the site of platelet deposition on an already damaged arterial wall. Venous thrombosis is usually the result of venous stasis during and following operation or prolonged inactivity such as air travel. The major means of prevention and treatment of arterial thrombosis are anti-platelet agents and thrombolytics. Prevention and treatment of venous thrombosis is usually by anticoagulant agents which are either anti-thrombin agents or antagonists of coagulation factors such as Factor Xa.

Other reasons for the short or long term use of anticoagulants include the permanent or temporary insertion of foreign material into the circulatory system (e.g. cardiac catheterisation, transluminal endoplasty, heart valve replacement or vascular prosthetic grafts). In addition, patients undergoing prolonged operation routinely receive prophylactic anticoagulation and there is a trend towards increased usage of low-dose anticoagulation for all patients undergoing surgery under general anaesthesia. Patients with uncontrolled atrial fibrillation, in whom embolisation from intra-cardiac thrombus is a risk, may also be treated with long term anticoagulants. Anticoagulants are also used to maintain the potency of extracorporeal circuits such as cardiopulmonary by-pass and renal dialysis. Thrombin is a central regulator of coagulation and is therefore a key target for anticoagulating agents. Heparin, one of the most widely used anticoagulants, is an indirect thrombin inhibitor which works by binding to the plasma glycoprotein AT-III, itself an inhibitor of serine protease coagulation enzymes (Brody et al, 1998). The requirement for a plasma co-factor is a disadvantage of heparin and has led to the development of direct inhibitors which bind directly to thrombin. Other disadvantages of heparin and its low molecular weight analogues include unwanted bleeding, thrombosis associated with thrombocytopaenia, allergic reactions, hyperkalaemia and osteoporosis. A further disadvantage is that the only substance that can be given to reverse the unwanted effects of heparin is protamine sulphate which is itself an anticoagulant and may promote immunogenic reactions (Esmon et al, 1987; Hirsch et al, 1992).

Thrombin inhibitors are generally proteins or peptides which need to be given parenterally. Since there is also a need for long term, prophylactic anticoagulant therapy, oral anticoagulants are also available. These agents, known as coumarins, are structurally related to Vitamin K, an essential co-factor in the synthesis of several endogenous coagulation factors including VII, IX and X. A disadvantage of coumarin therapy is that its initiation needs to be carefully controlled over a period of days or weeks making it unsuitable for short term use or in extracorporeal circuits. Coumarins also suffer from a number of unwanted side effects including unwanted bleeding, drug interactions and genetically inherited drug resistance.

Heparin and coumarins have been widely used for many years. More recently, direct thrombin inhibitors have been developed that are able to target thrombin by binding specifically to it. The first direct thrombin inhibitor to be used was Hirudin, an anticoagulant isolated from leeches (for review see Salzet, 2001). However, the therapeutic use of leech derived anticoagulants has been limited by the formation of neutralising or allergenic antibodies in some patients.

Another recent approach to anticoagulation has been the development of therapeutic monoclonal antibodies such as abciximab which prevents the binding of thrombin to platelets. Apart from the requirement that it is given by injection or infusion, it is advised that abciximab is used on a once-only basis because of the possible development of antibodies (British National Formulary, Sept 2001 : 118). haematophagous animals have evolved various mechanisms that interfere with the haemostatic system of their hosts (Arocha-Pinango et al, 1999). Studies on naturally occurring anticoagulant molecules have shown that they act at different points of the coagulation cascade - they specifically inhibit certain coagulation factors which block either the formation or the effect of thrombin, or inhibit platelet aggregation (Markwardt, 1994; Salzet, 2001). Thrombin inhibitors derived from blood-sucking animals have been found to interfere with fibrin formation and modulate other bioregulatory functions of the enzyme. As noted above, Hirudin, a single-chain peptide of 65 residues that is currently the most potent naturally occurring specific inhibitor of thrombin, was isolated from the medical leech Hirudo medicinalis (Markwardt, 1985). A number of other thrombin inhibitors have been isolated from blood-sucking arthropods, for example, ixin in the hard tick Ixodes ricinus (Hoffman et al, 1991), oraithodorin in the soft tick Ornithodoros moubata (Hawkins & Hellman, 1966), Prolixin G/rhodniin, Maculatin, Triabin and Dipetalogastin in blood-sucking bugs (Markwardt & Schulz, 1960a; Hellmann & Hawkins, 1965, 1966; Noeske-Jungblut et al, 1995; Mende et al, 1999) and tabanin in the horsefly Tabanus bovinus (Markwardt & Schulz, 1960b).

The most extensively studied tick-derived anticoagulant, TAP - an inhibitor of factor Xa - was isolated from the salivary glands of the soft tick, Ornithodoros moubata (Argasidae) (Waxman et al, 1990). Proteins that act as anticoagulants via thrombin inhibition or factor Xa inhibition have also been isolated from the embryos and nymphs of the camel tick Hyalomma dromedarii (Ibrahim et al, 2000, 2001), from salivary glands of the soft tick O. savignyi (Nienaber et. al, 1999) and the bont-legged tick, Hyalomma truncatum (Joubert et al, 1995) and saliva of the cattle tick Boophilus microplus (Horn et al, 2000). Americanin, a specific thrombin inhibitor, was isolated from the salivary glands of the lone star tick Amblyomma americanum (Zhu et al, 1997). In addition, various peptides with anti- thrombin activity have been isolated from salivary glands of blackflies (Abebe et al, 1995), mosquitoes (Waidhet-Kouadio et al, 1998; Valenzuela et al, 1999), tse-tse flies (Cappello et al, 1996) and recently from the hornfly Haematobia irritans (Zhang et al, 2002). More frequent use of short-term anticoagulation during and after surgery, increased numbers of percutaneous cardiac interventions and the use of vascular prostheses all increase the requirement for anti-thrombin agents. All currently available anti-thrombin anticoagulants however suffer from disadvantages and possible side-effects which increase the need for new agents both for use in their own right and as replacements or alternatives for pre-existing therapy. In view of the importance of anticoagulants in the treatment of a wide range of diseases, there remains a need for additional anticoagulants and in particular, for anti-thrombin agents. Summary of the invention

According to a first aspect of the invention, there is provided an EV445 protein comprising the amino acid sequence given in Figure 5A or a homologue thereof, or a fragment of said EV445 protein or homologue, wherein the EV445 protein, homologue or fragment possesses anticoagulant activity. Preferably, the anticoagulant activity is anti-thrombin activity. Preferably, the anti-thrombin activity inhibits platelet aggregation.

By "EV445 protein" is meant a protein comprising the sequence given in Figure 5A, or a homologue thereof. The protein having the sequence given in Figure 5A was isolated from the salivary glands of the tick Amblyomma variegatum and has been found to possess anticoagulant activity. More particularly, it has been found to possess anti-thrombin activity.

As discussed in more detail previously, there is a continuing need for effective anticoagulants and in particular for thrombin inhibitors due to the crucial role of these drugs in the treatment and prevention of many diseases and conditions. The EN445 proteins, homologues and fragments of the invention will have a wide range of medical applications, in the treatment, prevention and diagnosis of diseases and conditions, as well as being useful research tools into in the study of anticoagulation and of thrombin inhibition in particular.

No homology was found when the complete EV445 sequence was compared with sequences in the Genbank database but a short stretch of 20 amino acids in the EV445 sequence showed significant homology to a part of the Hirudin sequence.

By "anticoagulant activity" is meant that the EV445 protein, homologue or fragment prolongs the time taken for plasma coagulation to occur in the presence of a coagulating agent. By "anti-thrombin activity" is meant that the EV445 protein, homologue or fragment inhibits thrombin activity. Inhibition of thrombin activity is demonstrated by the fact that the protein inhibits the cleavage of a chromogenic substrate in the presence of thrombin. Anticoagulant activity may be caused by a variety of different mechanisms. It is believed that the anticoagulant activity of the EV445 protein having the sequence given in Figure 5A is primarily due to anti-thrombin activity. Thrombin is a potent platelet aggregating agent (Packham (1994)) and it is believed that the anticoagulant activity of the EV445 protein having the sequence given in Figure 5A is due, at least in part, to inhibition of thrombin-stimulated platelet aggregation.

The term "homologue" is meant to include reference to paralogues and orthologues of the EV445 sequence that is explicitly identified in Figure 5A, including, for example, the EV445 protein sequence from other tick species, including Rhipicephalus appendiculatus, R. sanguineus, R. bursa, A. americanum, A. cajennense, A. hebraeum, Boophilus microplus, B. annulatus, B. decoloratus, Dermacentor reticulatus, D. andersoni, D. marginatus, D. variabilis, Haemaphysalis inermis, Ha. leachii, Ha. punctata, Hyalomma anatolicum anatolicum, Hy. dromedarii, Hy. marginatum marginatum, Ixodes ricinus, I. persulcatus, I. scapularis, I. hexagonus, Argas persicus, A. reflexus, Ornithodoros erraticus, O. moubata moubata, O. m. porcinus, and O. savignyi. The term "homologue" is also meant to include the EV445 protein sequence from mosquito species, including those of the Culex, Anopheles and Aedes genera, particularly Culex quinquefasciatus, Aedes aegypti and Anopheles gambiae; flea species, such as Ctenocephalides felis (the cat flea); horseflies; sandflies; blackflies; tsetse flies; lice; mites; leeches; and flatworms. Methods for the identification of homologues of the EV445 sequence given in Figure 5A will be clear to those of skill in the art. For example, homologues may be identified by homology searching of sequence databases, both public and private. Conveniently, publicly available databases may be used, although private or commercially-available databases will be equally useful, particularly if they contain data not represented in the public databases. Primary databases are the sites of primary nucleotide or amino acid sequence data deposit and may be publicly or commercially available. Examples of publicly-available primary databases include the GenBank database (http://www.ncbi.nlm.nih.gov/), the EMBL database (http://www.ebi.ac.uk/), the DDBJ database (http://www.ddbj.nig.ac.jp/), the SWISS-PROT protein database (http://expasy.hcuge.ch/), PIR (http://pir.georgetown.edu/), TrEMBL

(http://www.ebi.ac.uk/), the TIGR databases (see http://www.tigr.org/tdb/index.html), the NRL-3D database (http://www.nbrfa.georgetown.edu), the Protein Data Base (http://www.rcsb.org/pdb), the NRDB database (ftp://ncbi.nlm.nih.gov/pub/nrdb/README), the OWL database

(http://www.biochem.ucl.ac.ulc/bsm/dbbrowser/OWL/) and the secondary databases PRO SITE (http ://expasy .hcuge. ch sprot/prosite .html),

PRINTS (http://iupab.leeds.ac.uk/bmb5dp/prints.html), Profiles (http://ulrec3.unil.ch/software/PFSCAN_form.html), Pfam

(http://www.sanger.ac.uk/software/pfam), Identify (http://dna.stanford.edu identify/) and Blocks (http://www.blocks.fhcrc.org) databases. Examples of commercially-available databases or private databases include PathoGenome (Genome Therapeutics Inc.) and PathoSeq (Incyte Pharmaceuticals Inc.). Typically, greater than 30% identity between two polypeptides (preferably, over a specified region) is considered to be an indication of functional equivalence and thus an indication that two proteins are homologous. Preferably, proteins that are homologues have a degree of sequence identity with the EV445 protein sequence identified in Figure 5 A of greater than 30%. More preferred homologues have degrees of identity of greater than 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, respectively with the EV445 protein sequence given in Figure 5A. Percentage identity, as referred to herein, is as determined using BLAST version 2.1.3 using the default parameters specified by the NCBI (the National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/) [Blosum 62 matrix; gap open penalty=l 1 and gap extension penalty=l]. Homologues of the EV445 protein sequence given in Figure 5A include mutants containing amino acid substitutions, insertions or deletions from the wild type sequence, provided that the anticoagulant activity and preferably the anti-thrombin activity of the wild type protein sequence is retained. Mutants thus include proteins containing conservative amino acid substitutions that do not affect the function or activity of the protein in an adverse manner. This term is also intended to include natural biological variants (e.g. allelic variants or geographical variations within the species from which the tissue cement proteins are derived). Mutants with improved anticoagulant activity compared to that of the wild type protein sequence may also be designed through the systematic or directed mutation of specific residues in the protein sequence. Preferably, such mutants have improved anti-thrombin activity compared to that of the wild type protein sequence.

An example of a homologue of the EV445 protein identified in Figure 5A is a protein comprising the sequence in Figure 5B. This sequence is identical to the sequence in Figure 5 A except for one amino acid residue. It was isolated from the salivary glands of the tick Amblyomma variegatum and has been found to have anticoagulant activity and in particular anti-thrombin activity.

Further examples of homologues of the EV445 protein identified in Figure 5 A include the EV460 protein comprising the amino acid sequence given in Figure 6A and the EV634 protein comprising the amino acid sequence given in Figure 6B. Two possible amino acid residues are suggested at a number of positions in the amino acid sequences for EV460 and EV634 given in Figures 6 A and 6B. The invention includes all possible amino acid sequences given in Figure 6A and 6B. These proteins were isolated from the salivary glands of the tick Amblyomma variegatum and have been found to have anticoagulant activity and in particular anti-thrombin activity. EV634 and EV460 are believed to be variants of the EV445 protein having the sequence given in Figure 5A as they show significant homology with this sequence.

Fragments of the EV445 protein and of homologues of the EV445 protein are also provided by the invention. Included as such fragments are not only fragments of the Ambyϊomma variegatum EV445 protein that is explicitly identified herein in Figure 5A, but also fragments of homologues of this protein, as described above (such as fragments of the EV460 and EV634 proteins). Such homologous fragments will typically possess greater than 65% identity with the EV445 protein sequence in Figure 5A, although more preferred homologues will display degrees of identity of greater than 70%, 80%, 90%, 95%, 98% or 99%, respectively with the fragments of the EV445 protein sequence in Figure 5A. Fragments of the EV445 protein comprising the sequence in Figure 5A and homologues thereof preferably possess anticoagulant activity, more preferably anti-thrombin activity. Fragments with anti-thrombin activity preferably inhibit thrombin-stimulated platelet aggregation. Fragments with improved anticoagulant activity and in particular improved anti-thrombin activity may, of course, be rationally designed by the systematic mutation or fragmentation of the wild type sequence followed by appropriate activity assays. Preferably, fragments with improved anti-thrombin activity possess improved inhibition of thrombin-stimulated platelet aggregation. It is considered that the EV445 proteins, homologues and fragments of the invention may be prepared in recombinant form by expression in a host cell. Such expression methods are well known to those of skill in the art and many are described in detail by Sambrook et al (2000) and Fernandez & Hoeffler (1998).

The proteins and fragments of the present invention can also be prepared using conventional techniques of protein chemistry. For example, protein fragments may be prepared by chemical synthesis.

According to a further embodiment, the invention provides an antibody which binds to an EV445 protein, homologue or fragment thereof as described above. Antisera and monoclonal antibodies can be made by standard protocols using the EV445 protein, homologue or fragment as an immunogen (see, for example, Antibodies: A Laboratory Manual ed. By Harlow and Lane, Cold Spring Harbor Press, 1988). As used herein, the term "antibody" includes fragments of antibodies which also bind specifically to an EV445 protein, homologue or fragment. The term "antibody" further includes chimeric and humanised antibody molecules having specificity for the EV445 proteins, homologues and fragments of the invention. In some cases, it will be desirable to attach a label group to the antibody in order to facilitate detection. Preferably, the label is an enzyme, a radiolabel or a fluorescent tag.

Derivatives of the EV445 protein and homologues and fragments thereof described above are also included as embodiments of the invention. Such derivatives include a fusion protein comprising an EV445 protein, homologue or fragment that is genetically or chemically fused to one or more peptides or polypeptides. The purpose of the additional peptide or polypeptide may be to aid detection, expression, separation or purification of the protein or it may lend the protein additional properties as desired. Examples of potential fusion partners include beta-galactosidase, glutathione-S-transferase, luciferase, a polyhistidine tag, a T7 polymerase fragment and a secretion signal peptide. Such fusion proteins will have medical applications. Since the EV445 protein, homologues and fragments are able to bind thrombin, it can be used as a means of conveying a therapeutic molecule to the site of a fibrin or platelet thrombus. The additional polypeptide may therefore be a therapeutic molecule that is useful in the treatment of a fibrin or a platelet thrombus. Preferably, such a therapeutic molecule is an anti-inflammatory agent or a thrombolytic agent.

The EV445 protein, homologue or fragment may also be fused to a marker domain. Preferably, the marker domain is a fluorescent tag, an epitope tag that allows purification by affinity binding, an enzyme tag that allows histochemical or fluorescent labelling, or a radiochemical tag. In a preferred embodiment, the marker domain is a radiochemical tag. Such fusion proteins will be useful as diagnostic tools. For example, since the EV445 protein is able to bind to thrombin, it can be used as a means of imaging a fibrin or platelet thrombus when linked to a suitable marker domain, such as a suitable radiochemical tag.

Methods for the generation of fusion proteins are standard in the art and will be known to the skilled reader. For example, most general molecular biology, microbiology, recombinant DNA technology and immunological techniques can be found in Sambrook et al. (2000) or Ausubel et al. (1991). Generally, fusion proteins may be most conveniently generated recombinantly from nucleic acid molecules in which two nucleic acid sequences are fused together in frame. These fusion proteins will be encoded by nucleic acid molecules that contain the relevant coding sequence of the fusion protein in question.

According to a further aspect of the invention, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding an EV445 protein, homologue or fragment according to the above-described aspects of the invention. Such molecules include single- or double-stranded DNA, cDNA and RNA, as well as synthetic nucleic acid species. Preferably, the nucleic acid sequences comprise DNA.

The invention also includes cloning and expression vectors comprising the nucleic acid molecules of this aspect of the invention. Such expression vectors may incorporate the appropriate transcriptional and translational control sequences, for example enhancer elements, promoter-operator regions, termination stop sequences, mRNA stability sequences, start and stop codons or ribosomal binding sites, linked in frame with the nucleic acid molecules of the invention.

Additionally, it may be convenient to cause a recombinant protein to be secreted from certain hosts. Accordingly, further components of such vectors may include nucleic acid sequences encoding secretion, signalling and processing sequences.

Vectors according to the invention include plasmids and viruses (including both bacteriophage and eukaryotic viruses), as well as other linear or circular DNA carriers, such as those employing transposable elements or homologous recombination technology. Many such vectors and expression systems are known and documented in the art (Fernandez & Hoeffler, 1998). Particularly suitable viral vectors include baculovirus-, adenovirus- and vaccinia virus- based vectors. Suitable hosts for recombinant expression include commonly used prokaryotic species, such as E. coli, or eukaryotic yeasts that can be made to express high levels of recombinant proteins and that can easily be grown in large quantities. Mammalian cell lines grown in vitro are also suitable, particularly when using virus-driven expression systems. Another suitable expression system is the baculovirus expression system that involves the use of insect cells as hosts. An expression system may also constitute host cells that have the DNA incoφorated into their genome. Proteins, or protein fragments may also be expressed in vivo, for example in insect larvae or in mammalian tissues.

A variety of techniques may be used to introduce the vectors according to the present invention into prokaryotic or eukaryotic cells. Suitable transformation or transfection techniques are well described in the literature (Sambrook et al, 1989; Ausubel et al, 1991; Spector, Goldman & Leinwald, 1998). In eukaryotic cells, expression systems may either be transient (e.g. episomal) or permanent (chromosomal integration) according to the needs of the system. The invention also provides antisense nucleic acid molecules which hybridise under high stringency hybridisation conditions to the nucleic acid molecules encoding the ΕV445 proteins, homologues or fragments. High stringency hybridisation conditions are defined herein as overnight incubation at 42° C in a solution comprising 50% formamide, 5XSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5xDenhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at approximately 65 °C.

In a preferred embodiment, a label capable of being detected is attached to these antisense nucleic acid molecules. Preferably, the label is selected from the group consisting of radioisotopes, fluorescent compounds and enzymes. The invention also includes transformed or transfected prokaryotic or eukaryotic host cells comprising a nucleic acid molecule, an antisense nucleic acid molecule or a vector as defined above. Preferably, the host cells are prokaryotic cells, preferably E.coli cells.

A further aspect of the invention provides a method for preparing an EV445 protein, homologue or fragment, as defined above, which comprises culturing a host cell containing a nucleic acid molecule according to the invention under conditions whereby the protein is expressed and recovering the protein thus produced. According to a further aspect of the invention there is provided a composition comprising an EV445 protein, homologue or fragment, a fusion protein comprising an EV445 protein, homologue or fragment, or a nucleic acid molecule comprising a nucleic acid sequence encoding an EV445 protein, homologue or fragment, according to the above-described aspects of the invention, in conjunction with a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable carrier", as used herein, includes genes, polypeptides, antibodies, liposomes, polysaccharides, polylactic acids, polyglycolic acids and inactive virus particles or indeed any other agent provided that the excipient does not itself induce toxicity effects or cause the production of antibodies that are harmful to the individual receiving the pharmaceutical composition. Pharmaceutically acceptable carriers may additionally contain liquids such as water, saline, glycerol, ethanol or auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like. Excipients may enable the pharmaceutical compositions to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions to aid intake by the patient. A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ. 1991).

According to a further aspect, the present invention provides an EV445 protein, homologue or fragment, a fusion protein comprising an EV445 protein, homologue or fragment, or a nucleic acid molecule comprising a nucleotide sequence encoding an EV445 protein, homologue or fragment, as described above, for use in therapy.

The invention also provides a method of treating an animal suffering from a coagulopathy or preventing an animal developing a coagulopathy comprising administering to said animal an EV445 protein, homologue or fragment, a fusion protein comprising an EV445 protein, homologue or fragment, a nucleic acid molecule comprising a nucleotide sequence encoding an EV445 protein, homologue or fragment, or a pharmaceutical composition according to the above-described aspects of the invention in a therapeutically or prophylactically effective amount. By "coagulopathy" is meant any disorder of blood coagulation. Preferably, said animal is a mammal, more preferably a human.

The term "therapeutically effective amount" refers to the amount of compound needed to treat or ameliorate a targeted disease or condition. The term "prophylactically effective amount" used herein refers to the amount of compound needed to prevent a targeted disease or condition. The exact dosage will generally be dependent on the patient's status as the time of administration. Factors that may be taken into consideration when determining dosage include the severity of the disease state in the patient, the general health of the patient, the age, weight, gender, diet, time and frequency of administration, drug combinations, reaction sensitivities and the patient's tolerance or response to therapy. The precise amount can be determined by routine experimentation, but may ultimately lie with the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg (mass of drug compared to mass of patient) to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones. The invention also provides the use of an EV445 protein, homologue or fragment, a fusion protein comprising an EV445 protein, homologue or fragment, a nucleic acid molecule comprising a nucleotide sequence encoding an EV445 or fragment thereof, or a pharmaceutical composition according to the invention in the manufacture of a medicament for treating or preventing a coagulopathy. Anticoagulants and thrombin inhibitors in particular have applications in the treatment and prevention of a wide range of diseases and conditions. The proteins, nucleic acid molecules and compositions of the invention may be used in any situation in which it is desired to induce anticoagulation to prevent or treat a coagulopathy.

The proteins, nucleic acid molecules and compositions of the invention may be used in the treatment or prevention of thromboembolic disorders or to promote prophylactic anticoagulation in patients undergoing surgery or prolonged periods of immobility who are at risk of venous thrombosis. They may also be used as an alternative anticoagulant to promote anticoagulation in patients with known or suspected heparin associated thromocytopaenia or for whom other anti-thrombin agents are contraindicated. The proteins, nucleic acid molecules and compositions of the invention may also be used in the treatment of disseminated intravascular coagulation.

A further example of a medical application of the EV445 proteins and fragments thereof, nucleic acid molecules and compositions of the invention is in the treatment or prevention of conditions caused by thrombin associated inflammation including, but not limited to, reperfusion injury, septic shock, adult respiratory distress syndrome and septicaemia.

The EV445 proteins and fragments thereof, nucleic acid molecules and compositions of the invention may also be used as an adjunct to the insertion of vascular or intravascular prostheses including stents, heart valves and vascular grafts; during percutaneous or transvascular interventions such as transluminal endoplasty or cardiac catheterisation; with the use extracorporeal circuits such as cardiopulmonary bypass or renal dialysis; or to treat any thromboembolic condition whilst oral anticoagulation is being established (including post myocardial infarction or uncontrolled atrial fibrillation).

According to a particular aspect of the invention, there is provided a method of treating an animal suffering from a condition caused by thrombin accumulation comprising administering to the animal a fusion protein comprising an EV445 protein, homologue or fragment thereof genetically or chemically fused to a therapeutic molecule, in a therapeutically effective amount. The invention also provides the use of a fusion protein comprising an EV445 protein, homologue or fragment thereof genetically or therapeutically fused to a therapeutic molecule in the manufacture of a medicament for treating a condition caused by thrombin accumulation. As indicated above, the EV445 protein is thought to inhibit thrombin activity by binding to it. This feature means that it can be used to convey the therapeutic molecule to the site of thrombin accumulation. Preferably, the therapeutic molecule is an anti-inflammatory agent or a thrombolytic agent. Preferably, the condition is a fibrin or a platelet thrombus.

The invention further provides for the use of an EV445 protein, homologue or fragment thereof, according to the above-described aspects of the invention as a diagnostic tool. Since the EV445 protein is thought to inhibit thrombin activity specifically by binding to it, it can be used to detect the presence of thrombin and hence to diagnose conditions caused by thrombin accumulation, such as a fibrin or platelet thrombus. The invention therefore provides a method of diagnosing a condition caused by thrombin accumulation comprising administering an EV445 protein, homologue or fragment thereof, according to the above-described aspects of the invention to a patient or to tissue isolated from a patient, and detecting the presence of said EV445 protein, homologue or fragment thereof, wherein the detection of said protein bound to thrombin is indicative of said disease or condition. Preferably, the EV445 protein, homologue or fragment thereof is in the form of a fusion protein comprising a marker domain, as described in more detail above, to facilitate detection. Preferably, the marker domain is a radiochemical tag so that detection can be carried out using known imaging methods. Preferably, the disease or condition is a fibrin or platelet thrombus. The present invention also provides the use of an EV445 protein, homologue or fragment thereof, according to the above-described aspects of the invention, as an anticoagulant. Preferably, the invention provides the use of an EV445 protein, homologue or fragment thereof as a thrombin inhibitor. The invention also provides the use of an EV445 protein, homologue or fragment thereof as an inhibitor of thrombin-stimulated platelet aggregation.

The identification of the EV445 protein will enable researchers to study the effects of anticoagulation and thrombin inhibition in particular. The present invention hence also includes the use of an EV445 protein, homologue or fragment thereof, a fusion protein comprising an EV445 protein, homologue or fragment thereof, or a nucleic acid molecule encoding an EV445 protein, homologue or fragment thereof, as a tool in the study of anticoagulation and the effects of anticoagulation, preferably as a tool in the study of thrombin inhibition and the effects of thrombin inhibition. In particular, the invention includes the use of an EV445 protein, homologue or fragment thereof, a fusion protein or a nucleic acid molecule, as described above, in the study of inhibition of thrombin- stimulated platelet aggregation and the effects of inihibition of thrombin-stimulated platelet aggregation. The invention also provides a method for inhibiting thrombin in a cell, tissue or non-human organism comprising adminstering to said cell, tissue or organism, an EV445 protein, homologue or fragment thereof, a fusion protein comprising an EV445 protein, homologue or fragment thereof, or a nucleic acid molecule encoding the EV445 protein, homologue or fragment thereof, according to the above-described aspects of the invention.

Various aspects and embodiments of the present invention will now be described in more detail by way of example. It will be appreciated that modification of detail may be made without departing from the scope of the invention. Figure Legends

Fig. 1. Inhibition of fibrin clot formation by SGE oi Amblyomma variegatum females in the thrombin time (TT), activated partial thromboplastin time (APTT, intrinsic pathway) and prothrombin time (PT, extrinsic pathway) assays. Results show the mean of duplicate values. In controls 150 mM NaCl was substituted for salivary glands. The clotting times for the controls were: 17 s (TT), 28 s (APTT), 15 s (PT).

Fig. 2. RP-HPLC chromatogram of an anion-exchange FPLC fraction of A. variegatum SGE eluted with 600 mM NaCl (Q-600). Peaks containing anticlotting activity in the TT assay are indicated. Percentage inhibition of clot formation as compared with the control is given in brackets; clotting time for control = 17 s. Protein concentration in fractions was <0.1 μg/μl. Numbers above the active peaks indicate retention times.

Fig. 3. RP-HPLC cliromatogram of crude SGE of A. variegatum (100 SG pairs) and the range of fractions containing anticoagulation activities. Protein concentrations in pooled fractions AV-I to AV-VIII ranged from 0.08 μg/μl (AV-I) to 1.39 μg/μl (AV-IV). The results of anticoagulation activities are presented as follows. TT assay (control clotting time - 19 s): NC - no clot after adding <0.01 μg fraction protein/50 μl plasma; *** prolongation of clot formation (>1 min) after adding <0.01 μg fraction protein/50 μl plasma; ** prolonged (> 40 s) after adding <0.01 μg fraction protein/50 μl plasma; * any inhibition of clot formation in comparison with the control. APTT assay (control clotting time - 40 s): NC - no clot after adding <0.01 μg fraction protein/50 μl plasma; ••• prolongation of clot formation (>1 min) after adding <0.01 μg fraction protein/50 μl plasma; •• prolonged (>1 min) after adding <0.1 μg fraction protein/50 μl plasma; • - any inhibition of clot formation in comparison with the control. PT assay (control clotting time - 15 s): oo prolongation of clot formation (>1 min) after adding <0.5 μg fraction protein 50 μl plasma; o any inhibition of clot formation in comparison with the control. ♦ Inhibition of platelet aggregation stimulated by thrombin.

Fig. 4. Purification of the thrombin inhibitor from A. variegatum SGE. A - second purification step, RP-HPLC chromatogram of fraction AV-III and range of fractions containing anticoagulation activities (dashed line); fractions indicated with asterisks contained specific anti-thrombin activity; protein concentrations in fractions ranged from 0.05 μg/μl to 0.17 μg/μl. B - third purification step, RP-HPLC of fraction with RT 23.083 min (from Fig. 4 A). C - third purification step, RP-HPLC of the purified inhibitor obtained from the fraction with RT 28.933 min (from Fig. 4 A).

Figs 5 A, B. Complete amino acid sequence of EV445 determined by matrix-assisted laser desorption/ionisation reflectron time-of-flight MALDI-TOF mass spectrometer BIFLEX (Bruker-Franzen, Bremen, Germany). Sequencing results show that EV445 comprises 32 amino acid residues. A protein database search (FASTA search, version 3.3) revealed some similarity with Hirudin (amino acids at positions 19-28). A - Laser desorption mass spectrometry revealed a M/z signal of 3769.96 Da (monoisotopic mass = 3768.96 Da) in agreement with the sequence SDQGDVAEPKMHKT(GLUC)APPFDFEAIPEEYLDDES.

B - The M/z signal 3777.79 Da (monoisotopic mass = 3776.79 Da) was in agreement with the sequence SDQGDVAEPKMHKT(GLUC)APPFDFEAIPEEYLDDHS, where the glutamic acid at residue 31 of the peptide sequence shown in Fig. 5 A is replaced by a histidine residue.

Figs 6 A, B. Amino acid sequences of EV460 (A) and EV634 (B), determined by Edman degradation. EV460: at positions 4, 5 and 12 of the main sequence, the amino acids have been assigned as tentative because two (major signals) are present at each of these positions; residues (A) - 4, (D) -5 and (H) - 12 seem to be the most likely candidates. The signals (S) - 4, (G) - 5 and (P) - 12 are possibly the result of the presence of some contamination with one or more isoforms of the peptide. Amino acids in brackets () are tentatively assigned. The symbols ???-??? are used to indicate that the complete amino acid sequence of EV460 may be longer than the amino acid sequence shown. The sequence of EV460 shows a high degree of identity with the sequence of EV445.

EV634: at positions 14 no naturally occurring amino acid was identified. This residue is designated (Xaa). Signals are present just after N and near the T position. These signals suggest a derivative of T (possibly glycosylated T). Amino acids in brackets () are tentatively assigned. The symbols ???-??? are used to indicate that the complete amino acid sequence of EV634 may be longer than the amino acid sequence shown. The sequence of EV634 shows a high degree of identity with the sequence of EV460 and EV445.

Fig. 7. Progress curves showing substrate hydrolysis by bovine thrombin (thrombin - 0.028 U/well, substrate - 1.9 mM N-p-tosyl-gly-pro-arg-p-nitroanilide, 20 μl/well). Control - 150 mM NaCl, HPLC fractions from the third purification step (see Figs 4 B, C) - EV460 (-0.7 μg protein/well), EV634 (-0.5 μg protein well), EV445 (-0.5 μg protein/well).

Fig. 8. Effect of SGE and HPLC fractions derived from SGE of A. variegatum females on thrombin stimulated aggregation of human blood platelets. Crude SGE - dissected salivary glands were incubated at -90 °C for 5 min prior to homogenisation and assay; for fractions AV-II, AV-III see Fig. 2; for fraction AVI 6/3 see Fig. 9B. Fig. 9. Purification of the inhibitor of thrombin induced platelet aggregation from A. variegatum SGE. A - second purification step, RP-HPLC chromatogram of pooled fractions from the first purification with retention times 14-27 min. B - third purification step, RP-HPLC of fraction with RT 23.083 min (from A). Dotted line indicates range of fractions containing anticoagulant activities in the TT assay, arrow indicates fraction AVI 6/3, containing the inhibitor of platelet aggregation stimulated by thrombin. Note: In the second purification step, the fraction with retention time 23.083 min (see Fig. 4) contained two compounds, EV460 and EV634, that were purified and sequenced by Edman degradation. Both compounds are potent inhibitors of thrombin and their amino acid sequences show high identity to the antithrombin, EV445.

Fig. 10. Comparison of the effect of fraction AVI 6/3 (A) and recombinant Hirudin H0393 (Sigma) (B) on thrombin stimulated platelet aggregation. A (mm) = amplitude of aggregation curve at the 60^th second of stimulation.

Examples Preparation of salivary gland extracts (SGE)

Amblomma variegatum ticks originated from laboratory colonies. Adult ticks were used in all studies and rabbits were used as hosts. Partially fed females (day 9 after attachment) were removed from hosts and their salivary glands were dissected in 150 mM NaCl. Salivary glands were stored in 150 mM NaCl at -70°C. Prior to assays, salivary glands were homogenized and centrifuged in a bench-top microcentrifuge at 10,000 rpm for 15 min. The supernatant was collected and the pellet resuspended in 150 mM NaCl and recentrifuged. A sample of the preparations stored at -70°C was heated at ~90°C for 5 min, prior to homogenization, and than processed as described above. The supematants were pooled and used for coagulation assays. Purification of the thrombin inhibitor

The thrombin inhibitor was purified by a three-step chromatographic procedure. A total of 100 salivary gland pairs stored at -70°C in 150 mM NaCl were incubated in a water bath at ~90^°C for 5 min, homogenized and centrifuged in a bench-top microcentrifuge at 10,000 rpm for 15 min. The supernatant was collected and the pellet resuspended in 150 M NaCl and recentrifuged. The supematants (crude SGE) were pooled and filtered through a Millex-LG syringe driven filter unit (0.20 μm, 4 mm). Filtered SGE was further processed by HPLC. For purification of the inhibitor of thrombin stimulated platelet aggregation, a new SGE of 55 salivary gland pairs was prepared.

Reverse phase (RP-) HPLC

For purification and identification of anticoagulant compounds, SGE of Amblyomma variegatum ticks was used. The SGE samples were diluted in 500 μl of 10% acetonitrile (ACN) with 0.1% trifluoroacetic acid (TFA) (buffer A) and loaded onto a Beckman Instruments 126/168 DAD HPLC system. In the first purification, SGE was applied to a Vydac C-4, 250 x 4.6 mm ID, 5 μm particle size column, UV monitored at 210 nm and 220 nm. A gradient of 10-100 % ACN with 0.1% TFA, flow rate 1 ml/min and with 1% ACN/ min increments was used (e.g. Fig. 3). The active fraction from the first purification was further purified using a Beckman Ultrasphere C-18, 250 x 4.6mm ID, 5 μm particle size column and a gradient of 10-40% ACN with 0.1% TFA, flow rate 1 ml/min with 0.5% ACN/min increments, and monitored at 210 and 220 nm (Fig. 4 A and Fig. 9 A). For the third purification a Vydac C 18, 250 x 4.6 mm ID, 5 μm particle size column was used under the same conditions as for the second purification (Figs 4 B, C and Fig. 9 B). Fractions were collected and dried in a Savant Instruments Speed-Vac.

At the same time, fractions from ion exchange chromatography using the Bio-Rad FPLC system were purified and fractionated. The Microsep concentrated fractions SP-200, SP- 400, SP-600 and Q-600 were diluted with 10% ACN/ 0.1% TFA to a volume of 500 μl and applied to Vydac C-4 and Vydac C-18 columns and processed as described above (e.g. Fig. 2).

Ion-exchange chromatography

High resolution protein purification was performed using LPLC gradient system (BIO- RAD Biologic LP System). The SGE sample was diluted in 50 mM NaH₂PO₄ buffer, pH 7.4. The sample was manually injected onto 1 ml HiTrap SP (cation exchanger) and Q (anion exchanger) sepharose columns (Pharmacia/Biotech). Fractions of 3-20 ml were obtained over a gradient of NaCl (0.1 M-0.6 M) in 50 mM NaH₂PO buffer, pH 7.4. Fractions were concentrated, using a Microsep membrane (Flowgen) having a 1 kD molecular weight cut-off, to final volumes of 50-300 μl. Aliquots of the resulting fractions were analysed for anticoagulation activities and active fractions were further purified by RP-HPLC. Protein sequence analysis

Partial protein sequence analysis was performed by N-terminal Edman degradation using an automated sequencer (Model 494 Applied Biosystems). Further, the mass spectrum and complete amino acid sequence was measured on a matrix-assisted laser desorption/ionisation reflectron time-of-flight MALDI-TOF mass spectrometer BIFLEX (Bruker-Franzen, Bremen, Germany) equipped with a nitrogen laser (337 nm) and gridless delayed extraction ion source by Eurosequence (Groningen, the Netherlands).

A SGE preparation was cleaved with CNBr and 2% of this digest (in 50% acetic acid was dried and re-dissolved in 20 μl 0.1% TFA); 10 μl was desalted using "zip-tip" for MALDI analysis. The peptides were eluted directly onto the MALDI target using 1.5 μl of matrix solution (= 10 mg/ml, 2,5-dihydroxybenzoic acid in 0.1% TFA (v/v)/ acetonitrile, 1:1), followed by MALDI-MS analysis. Reagents, chemicals and materials were obtained from Applied Biosystems (Warrington, U.K and Foster City, CA, U.S.A).

Protein assays Protein concentrations were determined for SGE and SGE fractions by the method of Bradford (1976), adapted to 96-well microplates. Bovine serum albumin was used as a standard.

Coagulation assays

Human citrated plasma was provided by the Department of Haematology and Transfusiology of the Slovak Institute of Cardiovascular Diseases in Bratislava, Slovakia. Only plasma showing normal values was pooled. Plasma was frozen and stored at —20 °C for a maximum of one month.

Thrombin time (TT, thrombin activity assay), prothrombin time (PT, extrinsic pathway assay) and activated partial thromboplastin time (APTT, intrinsic pathway assay) were the assays used to determine the anticoagulant activities in tick SGE and SGE fractions. The assays were carried out with 50 μl citrated human plasma. The plasma was pre-incubated with a maximum of 5 μl (depending on the dilution) of the SGE or the same volume of 150 mM NaCl (control) at 37 °C for 1 min. After adding the corresponding reagent, the time required for the formation of the fibrin clot was determined in duplicate samples visually, using a stop watch. For the TT assay, 50 μl of pre-warmed Dade Thromboclotin reagent (lyophilized bovine thrombin, 2.5 NIH U/mL, Dade AG, Dϋdingen, Switzerland) were added to the pre- incubated sample. The reaction mixture was incubated until coagulation occurred.

In the PT assay, 100 μl of pre-warmed Dade Thromboplastin IS reagent (lyophilized thromboplastin from rabbit brain, 1.0 x 10² mol/L Ca²⁺, Dade International Inc., Miami, FL, USA) were added to the sample and the formation of the clot was timed.

In the APTT assay, 50 μl of pre-warmed Dade Actin FS Activated PTT reagent (purified soy phosphatides in 1.0 x 10⁴ M ellagic acid, Dade International Inc., Miami, FL, USA) was added to the pre-incubated plasma and incubated for 3 min. The reaction was started with the addition of 50 μl of pre-warmed 20 mM CaCl₂ and the time for the formation of the clot was determined.

Coagulation screening by thrombelastography (TEG)

The anticoagulation activity of crude SGE and fractions was confirmed at the Oxford

Haemophilia Centre, Churhill Hospital, Oxford. A panel of coagulation screening assays were performed to assess the haemostatic effects of crude and fractionated AV tick salivary gland extracts which included APTT, PT and TT as functional indices of the integrity of the coagulation pathways. These measurements are performed in citrated platelet poor plasma (PPP), and thus represent a non-physiological milieu in which to assess an agent's anticoagulant potential. However, whole blood testing systems like Thrombelastography (TEG, Haemoscope Inc., Skokie, IL, USA) was used to address this deficiency. This is a technique which permits coagulation monitoring in whole blood using viscoelastic assessment of clot formation as an endpoint. Basically, 10 μl each of SGE or 1 μl :200 μl and 1 μl :300 μl of each fraction were added to 290 μl of PPP, mixed and allowed to incubate for 5 min at 37°C prior to coagulation screening (APPT, PT, TT) using an MDA-180 analyser (Organon Teknika Ltd., Cambridge, UK). Simultaneously, 5 μl of extract was added to 335 μl of citrated whole blood, incubated for 5 min and the sample run on the TEG following recalcification. Clotting screen assays and TEG were classified as normal or abnormal relative to saline control.

Chromogenic substrate assays for thrombin The assays were adapted to 96-well microplates. For screening of anti-thrombin activity, 190 μl Tris buffer (50 mM Tris, 227 mM NaCl, pH 8.3, containing 0.1% BSA and 0.1% sodium azide), 10 μl Tris buffer containing 0.056 U thrombin (thrombin from bovine plasma, Sigma), 10 μl 150 mM NaCl (control) or the same volume of an aliquot of SGE or fraction were pipetted into a well and incubated at room temperature (~22°C) 10 min. Twenty μl of substrate (1.9 mM N-p-tosyl-gly-pro-arg-p-nitroanilide, Sigma, dissolved in redistilled water) were added.

Immediately after addition of the substrate, changes in absorbance were followed at 405 nm for 30 min using an ELISA reader (Multiskan RC, Labsystems).

Platelet aggregation

Materials: Tyrode's solution (pH 7.4) consisted of 136.9 mmol/1 NaCl, 2.7 mmol/1 KC1, 11.9 mmol/1 NaHCO₃, 0.4 mmol/1 NaH₂PO₄x2H₂O, 1 mmol/1 MgCl₂x6H₂O and 5.6 mmol/1 glucose. Hirudin (H0393, leech, recombinant, 7,000-14,000 units per mg protein) was from Sigma (Deisenhofen, Germany), human thrombin from Imuna (Sarisske Michal'any, Slovakia). All other chemicals of analytical grade were from available commercial sources. Isolation of blood platelets: Platelets were isolated from fresh blood of healthy male donors (20-50 years) who had not received any medication for at least 7 days. Blood samples (36 ml) were anticoagulated with 3.8% trisodium citrate (4 ml) and centrifuged at 200 g for 15 min. Platelet-rich plasma (PRP) was removed, mixed with a solution containing 4.5% citric acid and 6.6% glucose (50 μl/ml PRP) and centrifuged at 980 g for 10 min. Platelets were resuspended in an equal volume Tyrode's solution containing 5.4 mmol/1 EDTA, pH 6.5. After 10 min stabilisation, the suspension was centrifuged for 6 min at 980xg and platelets were resuspended in the same buffer without EDTA to obtain 2xl0⁵ platelets per 1 μl.

Platelet aggregation: Platelet aggregation was measured turbidimetrically in a dual channel aggregometer (Chrono-log aggrometer, USA). After 1 min stabilisation at 37 °C, a suspension of isolated platelets (450 μl) was incubated for 2 min with 20 μl test sample or hirudin ). Aggregation was initiated by addition of 20 μl thrombin (final concentration 0.05 NIH/ml), recorded by dual pen recorder and evaluated as the amplitude of the aggregation curve at the 60^th second of stimulation. To eliminate changes in aggregability not associated with the presence of tested inhibitors, each sample was run in parellel with a control and the antiaggregatory effect was expressed as percentage of inhibition. Example 1: Anticoagulation activities of crude SGE

A. variegatum SGE prolonged the fibrin clot formation in all coagulation assays (Fig. 1). In all assays, the increase in anticoagulant activity was dose dependent. Incubation of salivary glands for 5 min at 90 °C prior to homogenization and further processing did not affect the observed anticoagulant activities indicating that the anticoagulant factor(s) present in the SGE was heat stable. With the TEG assay, complete inhibiton occurred. Likewise, no clot formation was observed with the PT, APTT and TT assays (Table 1). To exclude the possibility that the anticoagulation factor present in A. variegatum SGE was interferring with fibrinogen conversion, a fibrinogen assay was performed with PNP as a control. A normal PNP value of 2.3 g/1 was obtained. The results of the TEG and TT assays indicate the presence of a potent anti-thrombin agent in AV SGE while the APTT (intrinsic pathwayVPT (extrinsic pathway) results might be due to an anti-FXa factor.

Example 2: Anticoagulation activities of SGE purified by ion-exchange FPLC and RP- HPLC Anticoagulation activities were detected in some ion-exchange FPLC fractions using standard clotting assays (Table 1). The strongest inhibition of both APTT and TT, and absence of anti-fibrinogen activity, occurred with the fraction eluted from the Q-column with 0.6 M NaCl (fraction Q-600). This indicates the presence of a specific potent anti- thrombin factor in the Q-600 fraction. Ion-exchange fractions showing anticoagulant activity were further processed using RP- HPLC and the resulting peaks were tested for anticoagulant activities. Strong inhibitory activity in the TT assay was detected in some peaks of fraction Q-600 (Fig. 2).

Example 3: Purification of the thrombin inhibitor (EV445)

Purification of the thrombin inhibitor from 100 pairs of A. variegatum salivary glands was achieved by a three-step purification procedure using RP-HPLC. The fractions obtained in the first step (-50 μl each) were pooled (Fig. 3). Protein concentrations of the fractions were determined and aliquots were assayed for anticoagulation activities (Fig. 3). Pooled fractions designated AV-III contained very potent inhibitory activity and were further purified (Fig. 4A). The resulting fractions were assayed for protein concentration and aliquots were tested in the coagulation screen assays and for specific anti-thrombin activity. Two fractions from the second purification step (RT 23.083 and 28.933 min) were subjected to further purification by RP-HPLC (Figs 4B, C). The resulting peaks contained most of the observed anticoagulation activities. Three peaks (EV460, EV634 and EV445) inhibited substrate hydrolysis in the chromogenic substrate assay for thrombin (Fig. 7).

The anticoagulant activity of the RP-HPLC fractions was confirmed at the Oxford Haemophilia Centre, Churhill Hospital, Oxford by TEG and clotting screening assays. EV445 was the most potent anticoagulant and inhibitor of thrombin in all screening assays (Table 2).

The partial sequence of the major component EV445 was determined by Edman degradation and the Mass spectrum and complete amino acid sequence were measured by MALDI-TOF. Laser desorption mass spectrometry revealed a M/z signal of 3769.96 Da (monoisotopic mass = 3768.96 Da) and a M/z signal of 3777.79 Da (monoisotopic mass = 3776.79 Da). Sequencing results presented in Figs 5 A, B show that EV445 comprises 32 amino acid residues. A protein database search (FASTA search, version 3.3) revealed some similarity with Hirudin (amino acids at positions 19-28).

Laser desorption mass spectrometry of EV460 revealed two components (M/z 3953.54 and 3409.57 Da), and three components (M/z 3680.23, 3368.94 and 3173.62 Da) in EV634. Partial amino acid sequences were determined by Edman degradation (Figs 6 A, B). EV460 and EV634 comprise 28 and 29 amino acid residues, respectively. A protein database search (FASTA search, version 3.3) again revealed some similarity with Hirudin.

Example 4: Specificity of inhibition Incubation of bovine thrombin with its chromogenic substrate resulted in an increase in absorbance at 405 nm. The reaction was inhibited by A. variegatum crude SGE, fractions from the second purification step (Fig. 4 A) and by the fractions from the third purification step. The results indicate a specific inhibition of thrombin by the purified inhibitors EV445, EV460 and EV634 (Fig. 7). Example 5: Inhibition of thrombin stimulated platelet aggregation

Figure 8 shows dose response curves for crude Amblyomma variegatum SGE and HPLC fractions in a human platelet aggregation assay and, for comparison, a dose response curve for the leech derived thrombin inhibitor Hirudin. Crude SGE of A. variegatum and HPLC fractions from the first and third purification step (Figs 3, 9 B) inhibited aggregation of human blood platelets stimulated with thrombin (Fig. 8). Decreased amplitude of aggregation curves (reduced extent of aggregation) as well as prolonged onset of aggregation (reduced availability of platelets to aggregation) were observed in the presence of these inhibitors. Crude SGE, and fractions AV-II and AV-III decreased aggregation with a similar potency (percentages of inhibition for 0.05, 0.1 and 0.5 μg/ml sample protein concentrations are given in parentheses): crude SGE (66, 75 and 92%), AV-II (57, 67 and 80%), AV-III (56, 67 and 83%). Hirudin was more effective, producing 69, 91 and 91% inhibition at the respective concentrations. However, the most pronounced inhibition of aggregation was observed in the presence of fraction AV 16/3. At concentrations 0.0005, 0.001 and 0.005 μg/ml it reduced aggregation by 36, 49 and 61%, i.e. AV 16/3 was active at 100-times lower concentrations than the other samples tested. It is highly probable that the platelet aggregation inhibitor is identical with the previously purified antithrombin or one of its isoforms (most probably EV460 or EV634).

In addition, these data show that the dose response curve for Hirudin is steeper in the mid section than for the Amblyomma variegatum fractions, in particular than that of fraction 16/3. In general, the steeper the mid section of a dose response curve, the less the therapeutic margin of safety because a small increment in dose may produce a large increase in effect. Therapeutic products derived from Amblyomma variegatum saliva may therefore display advantages relative to Hirudin.

Table 1. Anticoagulant activities of Amblyomma variegatum salivary gland derived - FPLC fractions. PNP=pooled normal plasma, TEG= thromboelastography, PT= prothrombin time, APTT= activated partial thromboplastin time, TT= thrombin time, FIB= fibrinogen conversion assay.

Table 2. Anticoagulant activities of Amblyomma variegatum salivary gland derived — HPLC fractions. TEG= thromboelastography; PT= prothrombin time; APTT= activated partial thromboplastin time; TT= thrombin time; PNP= pooled normal plasma; r= r phase, the period of time of latency from the time that blood was placed in the TEG until the initial fibrin formation; k= k phase, is a measure of the speed to reach a certain level of clot strength.

References

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Claims

Claims:

I. A EV445 protein comprising the amino acid sequence given in Figure 5A or a homologue thereof, or a fragment of said EV445 protein or homologue, wherein said EV445 protein, homologue or fragment possesses anticoagulant activity.

2. A EV445 protein homologue according to claim 1 comprising the amino acid sequence given in Figure 5B, or a fragment of said EV445 protein homologue.

3. A EV445 protein homologue according to claim 1 comprising the amino acid sequence given in Figure 6A (EV460), or a fragment of said EV445 protein homologue.

4. A EV445 protein homologue according to claim 1 comprising the amino acid sequence given in Figure 6B (EV634), or a fragment of said EV445 protein homologue.

5. A EV445 protein, homologue or fragment thereof according to any one of claims 1 to 4 wherein said anticoagulant activity is anti-thrombin activity.

6. An antibody which binds to an EV445 protein, homologue or fragment thereof according to any one of claims 1 to 5.

7. A fusion protein comprising an EV445 protein, homologue or fragment thereof according to any one of claim 1 to 6 that is genetically or chemically fused to one or more peptides or polypeptides.

8. A fusion protein according to claim 7 wherein said EV445 protein, homologue or fragment thereof is genetically or chemically fused to a therapeutic molecule.

9. A fusion protein according to claim 8 wherein said therapeutic molecule is an anti- inflammatory agent or a thrombolytic agent.

10. A fusion protein according to claim 7 wherein said EV445 protein, homologue or fragment thereof is genetically or chemically fused to a marker domain.

I I. A fusion protein according to claim 10 wherein said marker domain is a radiochemical tag.

12. A nucleic acid molecule comprising a nucleotide sequence encoding an EV445 protein, homologue or fragment thereof according to any one of claims 1 to 5.

13. An antisense nucleic acid molecule which hybridises under high stringency hybridisation conditions to a nucleic acid molecules according to claim 12.

14. A vector comprising a nucleic acid molecule according to claim 12 or claim 13.

15. A host cell comprising a nucleic acid molecule according to claim 12, an antisense nucleic acid molecule according to claim 13 or a vector according to claim 14.

16. A method for preparing an EV445 protein, homologue or fragment thereof according to any one of claims 1 to 5 comprising culturing a host cell according to claim 15 under conditions whereby said protein is expressed and recovering said protein thus produced.

17. A composition comprising an EV445 protein, homologue or fragment thereof, according to any one of claims 1 to 5, a fusion protein according to any one of claims 7 to 11, or a nucleic acid molecule according to claim 12 in conjunction with a pharmaceutically acceptable carrier.

18. A EV445 protein, homologue or fragment thereof according to any one of claims 1 to 5, a fusion protein according to any one of claims 7 to 11, or a nucleic acid molecule according to claim 12 for use in therapy.

19. A method of treating an animal suffering from a coagulopathy or preventing an animal developing a coagulopathy comprising administering to said animal an EV445 protein, homologue or fragment thereof according to any one of claims 1 to 5, a fusion protein according to any one of claims 7 to 11, a nucleic acid molecule according to claim 12 or a composition according to claim 17 in a therapeutically or prophylactically effective amount.

20. A method according to claim 19 wherein said coagulopathy is a condition caused by thrombin accumulation and said method comprises administering to said animal a fusion protein according to claim 8 or 9 or a composition comprising such a fusion protein as recited in claim 17 in a therapeutically effective amount.

21. Use of an EV445 protein, homologue or fragment thereof according to any one of claims 1 to 5, a fusion protein according to any one of claims 7 to 11, a nucleic acid molecule according to claim 12 or a composition according to claim 17 in the manufacture of a medicament for treating or preventing a coagulopathy.

22. Use according to claim 21 wherein said coagulopathy is a condition caused by thrombin accumulation and a fusion protein according to claim 8 or claim 9 or a composition comprising such a fusion protein, as recited in claim 17 is used in the manufacture of the medicament.

23. A method according to claim 20 or use according to claim 22 wherein said condition caused by thrombin accumulation is the presence in the body of a thrombus or a platelet fibrin.

24. Use of an EV445 protein, homologue or fragment thereof according to any one of claims 1 to 5 or a fusion protein according to claims 10 to 11 as a diagnostic tool.

25. A method of diagnosing a disease or condition caused by thrombin accumulation comprising administering an EV445 protein, homologue or fragment thereof, according to any one of claims 1 to 5 or a fusion protein according to claims 10 to 11 to a patient or to tissue isolated from the patient, and detecting the presence of said EV445 protein, homologue or fragment thereof bound to thrombin, wherein the detection of said EV445 protein, homologue or fragment thereof bound to thrombin is indicative of said disease or condition.

26. A method according to claim 25 wherein said disease or condition is a fibrin or platelet thrombus.

27. Use of an EV445 protein, homologue or fragment thereof, according to any one of claims 1 to 5 as an anticoagulant, preferably as a thrombin inhibitor.

28. Use of an EV445 protein, homologue or fragment thereof according to any one of claims 1 to 5, a fusion protein according to any one of claims 7 to 11 or a nucleic acid molecule according to claim 12 as a tool in the study of anticoagulation and the effects of anticoagulation, preferably as a tool in the study of thrombin inhibition and the effects of thrombin inhibition.

29. A method for inhibiting thrombin in a cell, tissue or non-human organism comprising administering to said cell, tissue or organism, an EV445 protein, homologue or fragment thereof according to any one of claims 1 to 5, a fusion protein according to claim 7 or claim 8, or a nucleic acid molecule according to claim 9.