WO2009123573A1 - Anticoagulants derived from naja nigricollis snake venom - Google Patents

Abstract

A polypeptide having anticoagulant activity is disclosed, the polypeptide having the amino acid sequence of SEQ ID NO: 1 and being derived from Naja nigricollis snake venom, or having an amino acid sequence sharing at least 90 percent sequence identity with the amino acid sequence of SEQ DD NO: 1.

Description

ANTICOAGULANTS DERIVED FROM NAJA NIGRICOLLIS SNAKE VENOM

Field of the Invention

The present invention relates to polypeptides, in particular polypeptides isolated from snake venom, and their use as anticoagulants .

Background to the Invention

The blood coagulation cascade is an innate response to vascular injury that results from a series of amplified reactions in which zymogens of serine proteases circulating in plasma are sequentially activated by limited proteolysis. This leads to the formation of a blood clot, thereby preventing the loss of blood (6, 7, 21) . The blood coagulation cascade can be divided into two pathways: the extrinsic pathway and the intrinsic pathway. Present evidence suggests that the intrinsic pathway plays an important role in the growth and maintenance of clot formation in the coagulation cascade, while the extrinsic pathway is critical for the initiation of clot formation (6) .

The extrinsic pathway is initiated when the tissue factor (TF) is exposed to blood due to vascular injury. The tissue factor forms a one to one complex with factor Vila (FVIIa) in the presence of calcium ion, also called the extrinsic tenase complex. The tissue factor-FVIIa complex then activates factor X (FX) by converting it to factor Xa (FXa) . The newly generated FXa then forms a one to one complex with factor Va

(FVa) in the presence of calcium ion and phospholipids. This complex is sometimes referred to as the prothrombinase complex

(6). The prothrombinase complex proteolytically cleaves prothrombin to produce thrombin (3) and the activation of thrombin leads to the formation of a fibrin clot and stops the bleeding. The tissue factor-FVIIa complex may also convert factor IX (FIX) to factor IXa (FIXa) . The activated FIXa forms a complex with factor Villa (FVIIIa) in the presence of calcium ions and phospholipids (3) and the complex converts FX to FXa. This leads to the formation of the prothrombinase complex and ultimately to the formation of fibrin. Any imbalance in the cascade could lead to either unclottable blood, resulting in excessive bleeding during injuries, or unwanted clot formation, resulting in death and debilitation due to vascular occlusion with the myocardial infarction, stroke, pulmonary embolism or venous thrombosis (21) .

Snake venoms are complex mixtures of pharmacologically active proteins and polypeptides. They play an important role in incapacitating and immobilizing prey. Toxins have therefore evolved to specifically target various critical points in the physiological systems of prey animals. Over the years, a number of toxins that affect blood coagulation have been isolated and characterized from various snake venoms. For example, the tetrameric anticoagulant hemextin AB complex from Hemachatus haemachatus venom is a specific inhibitior of coagulation serine protease factor Vila (3) . Again, prothrombin activators from Australian elapids are pro- coagulant proteins structurally and functionally similar to coagulation proteases, such as trocarin from Tropidechis carinatus, which is a homologue of factor Xa.

Generally, there are two types of the anticoagulants found in snake venom: enzymatic and non enzymatic anticoagulants. Those anticoagulants which are categorized as enzymatic anticoagulants are phospholipase A _2> metalloproteinase, serine proteases and L-amino acid oxidase. C-type lectin related proteins and three finger toxin are non enzymatic anticoagulants. Proteins/toxins from snake venoms have been used in the design and development of a number of therapeutic agents or lead molecules, particularly for cardiovascular diseases (22) . For example, a family of inhibitors of angiotensin-converting enzyme was developed based on bradykinin-potentiating peptides from South American snake venoms (10) .

An anticoagulant is a substance that inhibits coagulation; that is, it aims to stop blood from clotting. Anticoagulant mechanisms ensure careful control over coagulation and under normal conditions they prevail over the proagulant forces (5) . Anticoagulants are pivotal for the prevention and treatment of thromboembolic disorders and ~0.7% of the western population receives oral anticoagulant treatment (9). Heparin and coumarin are current clinically used anticoagulants. Heparin enhances the inhibition of thrombin and FXa by antithrombin III meanwhile coumarin inhibits activity of all vitamin K dependent proteins .

However, the non specific mode of action of these anticoagulants accounts for the therapeutic limitations in maintaining a balance between thrombosis and hemostasis (15) . These anticoagulants bring many adverse effects, including heparin-induced thrombocytopenia (HIT) , GI bleeding and allergies. Limitations of these existing anticoagulants have led to the development of newer anticoagulant therapies (13) that have been designed to target specific coagulation enzymes or steps in the coagulation pathway (13) . An ideal anticoagulant should have a high efficacy-to-safety index (13) .

Initiation of blood coagulation during injury or trauma is essential for the survival of the organism. However, the formation of unwanted clots has detrimental or debilitating effects and hence the need for anticoagulant therapies. Due to the narrow therapeutic window of existing anticoagulants, the invention of new anticoagulants that exhibit more specific action is needed. Summary of the Invention

We have purified and characterised an anticoagulant, called here "naniproin", from the crude venom of an elapid, Naja nigricollis (West-African Black-necked Spitting cobra) . We have surprisingly found that this protein induces potent anticoagulant activity and specifically inhibits factor Xa (FXa) to block the blood coagulation cascade. This protein addresses the need for anticoagulants that target specific components of the coagulation cascade, and thus may provide new and improved anticoagulant therapies.

In addition, we have shown that the anticoagulant activity of naniproin is mediated by inhibition of the prothrombinase complex. Further studies revealed that the protein inhibits serine protease FXa and not FVIIa. Thus, the anticoagulant function of naniproin is mediated by specific inhibition of FXa. FXa catalyses the formation of thrombin and it is the point in the coagulation cascade where the intrinsic and extrinsic pathways converge. FXa is therefore an attractive target for anticoagulant therapies. As naniproin specifically targets FXa this further underlines the potential importance of naniproin in new anticoagulant therapies.

The dissection approach was carried out to determine the specific site of action of the protein. Naniproin showed anticoagulant activity in both the prothrombin time assay and the stypven time assay but not in the thrombin time assay. In further studies naniproin showed no inhibition of the reconstituted extrinsic TF-FVIIa complex, which confirmed that this protein inhibits the prothrombinase complex. We found that naniproin specifically inhibits the prothrombinase complex.

To find out the target components in the prothrombinase complex, chromogenic substrate assays were carried out in the absence of different components. Naniproin inhibited activation of prothrombin by the fully assembled prothrombinase complex with an IC₅₀ of ~ 100 μM. When the phospholipids vesicles (PC: PS) were absent, naniproin inhibited prothrombin activation by FXa-FVa complex with an IC₅₀ of 400 nM and a Ki of 1.28 μM. When both phospholipid vesicles and cofactor (FVa) were absent, naniproin did not inhibit the amidolytic activity of FXa. Without being bound by theory, these results indicate that naniproin inhibits prothrombinase complex by interfering with the interaction between FXa and FVa, most likely by competing for the same binding site on FXa. Binding to FXa was further confirmed by carrying out isothermal titration calorimetric (ITC) studies on naniproin with FXa. We found that the binding association constant (K_a) of naniproin with FXa is 4.174 x 10⁵M^"1. When the same study was carried out with prothrombin no binding was observed.

Previously reported snake venom anticoagulants that inhibit the prothrombinase complex are not as specific for the prothrombinase complex as naniproin. For example, CM-IV, a strongly anticoagulant phospholipase A₂ from Naja nigricollis venom, prolongs coagulation by inhibiting two successive steps in the coagulation cascade. It inhibits the TF-FVIIa complex by both enzymatic and nonenzymatic mechanisms, and it inhibits the prothrombinase complex by a nonenzymatic mechanism (18) . Other examples include C-type lectin-like proteins (CLP) . These were among the first proteins isolated and characterized from snake venoms that bind to FX/FXa and FIX/FIXa with nanomolar and sub-nanomolar affinities and they exert their inhibitory activity through nonenzymatic mechanisms (25) . The binding interferes with calcium-dependent binding of FIX and FX to phospholipid membranes and hence exhibits anticoagulant effects. Atoda and Morita (1989) (2) have purified such proteins from Trimeresurus flavoviridis (Habu snake) venom and they also purified a FX-binding protein from Deinagkistrodon acutus venom (1) . In these cases, since the anticoagulant activity is targeted towards both the prothrombinase and intrinsic tenase complexes, it is not specific. Thus, previously reported snake venom anticoagulants may not be as therapeutically useful as naniproin.

In various aspects the invention relates to naniproin, variants thereof, and their use as anticoagulants. These and other aspects are discussed below.

In an aspect of the invention, there is provided a polypeptide comprising:

(i) the amino acid sequence of SEQ ID NO: 1; or

(ii) an amino acid sequence sharing at least 90 percent sequence identity with the amino sequence of SEQ ID NO: 1.

In a further aspect of the invention, there is provided a polypeptide comprising:

(i) an amino acid sequence sharing at least 60 percent sequence identity with the amino sequence of SEQ ID NO: 1;

(ii) a fragment of the amino acid sequence of SEQ ID NO: 1, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1, or

(iii) an amino acid sequence sharing at least 60 percent sequence identity with the fragment defined in (ii), wherein the amino acid sequence has at least 6 amino acids.

The polypeptide may have the ability to bind prothrombinase complex, e.g. the polypeptide may bind, e.g. specifically bind prothrombinase complex. The polypeptide may have the ability to bind FXa, e.g. the polypeptide may bind, e.g. specifically bind, FXa.

The polypeptide may bind prothrombinase complex and/or FXa with a 1/Kd value and/or a Ka value of at least 1 x 10² M^"1, preferably at least 1 x 10³ M^"1, 1 x 10⁴ M^"1, 5 x 10⁴ M^"1, 6 x 10⁴ M^"1, 7 x 10⁴ M^"1, 8 x 10⁴ M^"1, 9 x 10⁴ M^"1, 1 x 10⁵ M^"1, 2 x 10^s M^"1, 3 x 10⁵ M^"1, 3.5 x 10⁵ M^"1, 3.6 x 10⁵ M^"1, 3.7 x 10⁵ M^"1, 3.8 x 10⁵ M^"1, 3.9 x 10⁵ M^"1, 4 x 10⁵ M^"1, or at least 4.1 x 10⁵ M^"1. For example, the polypeptide may bind prothrombinase complex and/or FXa with a 1/Kd and/or a Ka in the range 1 x 10² M^"1 to 1 x 10⁸ M^"1, 1 x 10³ M^"1 to 1 x 10⁷ M^"1, 1 x 10⁴ M^"1 to 1 x 10⁶ M^"1, 1 x 10⁵ M^"1 to 8 x 10^s M^"1, 2 x 10⁵ M^"1 to 6 x 10⁵ M^"1, or 4 x 10⁵ M^"1 to 5 x 10⁵ M^"1.

1/kd values, kd values and Ka values may be measured using isothermal titration calorimetry, as described in more detail below.

A polypeptide that "specifically binds" a particular target, such as prothrombinase complex and/or FXa, means that the polypeptide has specific binding affinity for the target, e.g. the polypeptide selectively binds to the target, e.g. the polypeptide preferentially binds to the target.

The polypeptide may be a three-finger toxin. Preferably, the polypeptide has anticoagulant activity. For example, the polypeptide may have the ability to inhibit blood coagulation, e.g. in a mammal. The polypeptide may reduce blood clotting, e.g. after the coagulation cascade has been activated. A reduction in blood clotting may be a reduction compared to blood clotting in the absence of the polypeptide, and may include one or more of a reduction in the number of blood clots, a reduction in the rate of blood clot formation, a reduction in the average size of blood clots, an increase in the time required for blood clot formation, and/or an increase in the time required for a blood clot to reach a particular size. Anticoagulant activity of the polypeptide may be observed and/or measured, for example, using the prothrombin time assay and/or the stypven time assay. Preferably, anticoagulant activity is measured by measuring inhibition of prothrombinase complex. These assays are described below. The polypeptide may have the ability to inhibit prothrombinase complex, e.g. activation of prothrombin. For example, the polypeptide may inhibit formation of the prothrombinase complex, e.g. by binding to FXa. Binding of the polypeptide to FXa may reduce the ability of FVa to bind to FXa, e.g. the polypeptide may compete with FVa for binding to FXa. The polypeptide may be capable of inhibiting, e.g. impairing, impeding, and/or reducing the activity, e.g. biological activity, of prothrombinase complex, e.g. by inhibiting formation of the prothrombinase complex. The polypeptide may inhibit binding of prothrombinase complex to prothrombin and/or thrombin. The polypeptide may inhibit the amidolytic activity of prothrombinase complex. In particular, the polypeptide may inhibit conversion of prothrombin to thrombin, e.g. such that the rate of conversion of prothrombin to thrombin is reduced compared to the rate of conversion in the absence of the polypeptide.

The polypeptide may have the ability to inhibit FXa. For example, the polypeptide may be capable of inhibiting, e.g. impairing, impeding, and/or reducing the activity, e.g. biological activity, of FXa. The polypeptide may inhibit binding of FXa to prothrombin and/or thrombin, e.g. by inhibiting binding of FVa to FXa. The polypeptide may inhibit the amidolytic activity of FXa, e.g. by inhibiting binding of FVa to FXa. In particular, the polypeptide may inhibit the conversion of prothrombin to thrombin, e.g. such that the rate of conversion of prothrombin to thrombin is reduced compared to the rate of conversion in the absence of the polypeptide.

Inhibition of the prothrombinase complex may be measured, for example, by reconstituting the prothrombinase complex, e.g. FVa, FXa, and PC: PS vesicles (phosphatidylcholine phosphatidylserine vesicles), and incubating with prothrombin. The rate of conversion of prothrombin to thrombin may be monitored by including a chromogenic thrombin susbstrate in the incubation, e.g. S-2238.

Likewise, inhibition of FXa may be measured by incubating FXa with prothrombin and monitoring conversion of prothrombin to thrombin.

An IC₅₀ value is the concentration of inhibitor at which a reaction is inhibited by 50% compared to the control reaction, i.e. the reaction in the absence of the inhibitor. IC₅₀ values may be measured by plotting a graph of activity, e.g. the rate of conversion of prothrombin to thrombin, against concentration of inhibitor polypeptide. Thus, the IC₅₀ value for inhibition of prothrombinase complex by the polypeptide is the concentration of polypeptide at which the rate of thrombin production is reduced by 50% compared to the rate of thrombin production in the absence of the polypeptide. Likewise, the IC₅₀ value for inhibition of FXa by the polypeptide is the concentration of polypeptide at which the rate of thrombin production is reduced by 50% compared to the rate of thrombin production in the absence of the polypeptide.

The polypeptide may inhibit prothrombinase complex in the presence or absence of PC: PS vesicles, with an IC₅₀ value of less than ImM, 900μM, 800μM, 700μM, 600μM, 500μM, 400μM, 300μM, 200μM, lOOμM, 50μM, 40μM, 30μM, 20μM, lOμM, 9μM, 8μM, 7μM, 6μM, 5μM, 4μM, 3μM, 2μM, lμM, 90OnM, 80OnM, 70OnM, 60OnM, 50OnM, 40OnM, 30OnM, 20OnM, 10OnM, 9OnM 85nM, 8OnM, 7OnM, 6OnM, or even 5OnM. For example, the polypeptide may inhibit prothrombinase complex with an IC₅₀ value in the range ImM to IpM, ImM to InM, 0.5mM to 10μM, lOOμM to InM, lOμM to InM, lμM to InM, lμM to 1OnM, or even 10OnM to 1OnM, e.g. in the presence of absence of PC: PS vesicles. The IC₅₀ value in the presence of PC: PS vesicles may be different to the IC₅₀ value in the absence of PC: PS vesicles.

The polypeptide may inhibit FXa with an IC₅₀ value of less than ImM, 900μM, 800μM, 700μM, 600μM, 500μM, 400μM, 300μM, 200μM, lOOμM, 90μM, 80μM, 70μM, 60μM, 50μM, 40μM, 30μM, 20μM, lOμM, 9μM, 8μM, 7μM, 6μM, 5μM, 4μM, 3μM, 2μM, lμM, 90OnM, 80OnM, 70OnM, 60OnM, 50OnM, 40OnM, 30OnM, 20OnM, 10OnM, 9OnM 85nM, 8OnM, 7OnM, 6OnM, or even 5OnM. For example, the polypeptide may inhibit FXa with an IC₅₀ value in the range ImM to IpM, ImM to InM, lOOμM to InM, or even lOμM to 1OnM, lOμM to 10OnM, or even 5μM to 50OnM.

The polypeptide may inhibit the prothrombinase complex, e.g. activation of prothrombin, with a Ki value of less than ImM, 900μM, 800μM, 700μM, 600μM, 500μM, 400μM, 300μM, 200μM, lOOμM, 90μM, 80μM, 70μM, 60μM, 50μM, 40μM, 30μM, 20μM, lOμM, 9μM, 8μM, 7μM, 6μM, 5μM, 4μM, 3μM, 2μM, lμM, 90OnM, 80OnM, 70OnM, 60OnM, 50OnM, 40OnM, 30OnM, 20OnM, 10OnM, 9OnM 85nM, 8OnM, 7OnM, 6OnM, or even 5OnM. Preferably the Ki value is determined in the absence of PC: PS veslicles.

The polypeptide may not have the ability to bind, e.g. the polypeptide may not specifically bind, to one or more components of the coagulation cascade other than the prothrombinase complex and/or FXa. For example, the polypeptide may not have the ability to bind to one or more components of the intrinsic pathway and/or to one or more components of the extrinsic pathway. Components of the intrinsic pathway to which the polypeptide may not bind include one or more of FXII, FXIIa, FXI, FXIa, FIX, FIXa, FVIII, FVIIIa, and the FIXa-FVIIIa complex. Components of the extrinsic pathway to which the polypeptide may not bind include one or more of FVII, FVIIa, and tissue factor. The polypeptide may also not bind to one or more components of the cascade after convergence of the intrinsic and extrinsic pathway, e.g. one or more of FV, FVa, prothrombin, thrombin, fibrinogen, fibrin, FXIII and FXIIIa.

The polypeptide may also not bind to any of protein C, active protein C, protein S and thrombomodulin.

An amino sequence that shares a particular sequence identity with the amino acid sequence of SEQ ID NO: 1 may share at least 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent sequence identity with the amino acid sequence of SEQ ID NO: 1. The sequence identity may be shared over at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 contiguous amino acids of the sequence of SEQ ID NO: 1. Preferably, the sequence identity is shared over the full- length sequence of SEQ ID NO: 1.

A fragment of the amino acid sequence of SEQ ID NO: 1 may have at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1. The fragment may have less than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 contiguous amino, acids of the amino acid sequence of SEQ ID NO: 1.

An amino acid sequence sharing at least a particular sequence identity with a fragment of the amino acid sequence of SEQ ID NO: 1 may share at least 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 percent sequence identity with the fragment. The sequence identity is shared over the full-length sequence of the fragment.

An amino acid sequence sharing at least a particular sequence identity with a fragment of the amino acid sequence of SEQ ID NO: 1 may have at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids . The amino acid sequence sharing at least a particular sequence identity with a fragment of the amino acid sequence of SEQ ID NO: 1 may have less than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids.

Preferably, the polypeptide has a molecular mass in the range 5000-8000 Da, 5500-8000, 6000-7500 Da, 6100-7400 Da, 6200-7300 Da, 6300-7200 Da, 6400-7100 Da, 6500-7000 Da, 6600-7000 Da, 6700-7000, 6800-6900 Da, 6850-6900. Most preferably, the molecular mass is within the range 6885-6890 Da. We have found that naniproin has a molecular mass of 6887.96 +/- 0.35 Da. The mass of the polypeptide may be measured, e.g. determined, using electrospray ionization mass spectrometry (ESI-MS), for example.

Naniproin is a polypeptide of the invention. We obtained naniproin from the venom of Naja nigricollis, which is commercially available, and purified naniproin using size exclusion chromatography, cation-exchange chromatography, and reverse phase chromatography. The fractions containing naniproin were selected by assaying for anticoagulant activity using the prothrombin time clotting assay. These procedures are described below and are well known to the person skilled in the art.

Thus, polypeptides of the invention may be obtained, or may be obtainable, from the venom of Naja nigricollis . Preferably, the polypeptides of the invention are obtained, e.g. isolated, from the venom of Naja n±gricollis using chromatography, e.g. one or more of size exclusion chromatography, cation-exchange chromatography, and reverse phase chromatography. Polypeptides of the invention may be identified by assaying for anticoagulant activity, e.g. using the prothrombin time clotting assay.

Without being bound by theory, it is proposed that naniproin belongs to the three-finger family of snake venom proteins as evident from its NH₂-terminal sequence and characteristic beta- sheet structure, which we determined by Edman degradation protein chemistry and circular dichroism respectively.

We have also found that disulfide bonds are important for maintaining folding of naniproin. In particular, after reduction and alkylation the protein exhibited no anticoagulant activity and showed random coil in circular dichroism studies.

Preferably, the polypeptide includes at least two cysteine amino acids, e.g. at least 3, 4, 5, 6, 7, 8 or more cysteine amino acids. Preferably the polypeptide has at least 8 cysteine amino acids. Preferably, the polypeptide includes at least one disulfide bond. For example, the polypeptide may have at least 1, 2, 3, 4 or more disulfide bonds, e.g. in its active form, e.g. the form in which it exhibits anticoagulant activity. Preferably the polypeptide includes at least 4 disulphide bonds. In a further aspect of the invention, there is provided an isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide of the invention, e.g. when expressed in a host cell such as E. coli, as described below.

For example, the nucleic acid may comprise a nucleotide sequence that encodes a polypeptide comprising:

(i) the amino acid sequence of SEQ ID NO: 1; or (ii) an amino acid sequence sharing at least 90 percent sequence identity with the amino sequence of SEQ ID NO: 1.

The nucleic acid may comprise a nucleotide sequence that encodes a polypeptide comprising:

(i) an amino acid sequence sharing at least 60 percent sequence identity with the amino sequence of SEQ ID NO: 1;

(ii) a fragment of the amino acid sequence of SEQ ID NO: 1, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1, or

(iii) an amino acid sequence sharing at least 60 percent sequence identity with the fragment defined in (ii) , wherein the amino acid sequence has at least 6 amino acids .

Nucleic acids encoding polypeptides of the invention, e.g. naniproin, may be obtained by preparing a DNA library of the Naja nigricollis genome and using labelled probes that encode fragments of SEQ ID NO: 1. This procedure is well known the person skilled in the art, see for example Sambrook et al., "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1989. Alternatively, nucleic acids that encode polypeptides of the invention may be chemically synthesised based on the encoded amino acid sequence.

Small nucleotide sequences, e.g. that encode fragments of SEQ ID NO: 1, are useful as probes for detecting nucleic acid encoding naniproin or variants of naniproin, and also as primers for amplifying such nucleotide sequences, e.g. by polymerase chain reaction (PCR) . Methods of designing probes and their use in detecting nucleic acids of interest, and methods of designing primers and their use in amplifying nucleic acid are well known in the art. See for example Sambrook et al., "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1989.

The nucleic acid may also comprise a regulatory sequence, e.g. a promoter, that is operably linked to the nucleotide sequence that encodes the polypeptide of the invention.

The invention also includes vectors comprising a nucleic acid of the invention, which may include one or more elements that facilitate expression of nucleotide sequence in a host cell. A vector may include an element that allows the vector to replicate in a host cell and/or an element that allows selection of host cells that contain the vector, e.g. a marker gene. Methods of introducing nucleic acid such as vectors into cells, e.g. by transformation or transfection, are well known in the art.

The invention also includes host cells comprising a nucleic acid of the invention. The host cell may be a eukaryotic cell, e.g. a yeast or a CHO cell, or a prokaryotic cell, e.g. E. coli. The host cell may be a cell that is suitable for culturing, i.e. it may be a cell that is not part of a living human or animal body. The nucleic acid of the invention may be present in the host cell as part of the host cell genome. Alternatively, it may be present in the host cell as an autonomously replicating entity, e.g. a plasmid. Where the nucleic acid is present in the genome of the host cell it is preferably heterologous nucleic acid, i.e. nucleic acid that is not naturally present within the genome of the host cell. For example, the nucleic acid may be inserted into the genome by genetic engineering. In a further aspect, there is provided a method of producing a polypeptide of the invention, which method comprises the steps of:

(a) providing a host cell comprising a nucleotide sequence that encodes a polypeptide of the invention, and

(b) causing the host cell to express the polypeptide encoded by the nucleotide sequence.

The method may also comprise step (c) isolating the polypeptide and/or may also comprise, prior to step (a) , introducing the nucleic acid that encodes the polypeptide of the invention into the host cell, e.g. on a vector. The step of providing the host cell may also include culturing the host cell. Methods of culturing host cells to express polypeptides and methods of purifying polypeptides are well known in the art. The host cell may express the polypeptide under the control of a constitutive promoter or an inducible promoter.

In a further aspect, there is provided a polypeptide of the invention for use in a therapeutic method. In a further aspect, there is provided use of a polypeptide of the invention in the manufacture of a medicament for use in a therapeutic method.

In particular, the polypeptide may be for use as a blood anticoagulant .

The therapeutic method may be for preventing and/or inhibiting, e.g. suppressing, impairing, impeding, decreasing, and/or reducing, blood coagulation in a patient. For example, the therapeutic method may be for inhibiting the formation of blood clots. The method may be for reducing the rate at which blood clots form and/or the method may be for delaying the formation of blood clots. The therapeutic method may be for correcting an imbalance in the regulation of the blood coagulation cascade. In particular, the method may be for treating a patient suffering from a condition, e.g. a disease, that would benefit from treatment with an anticoagulant. For example, the method may be for treating a patient suffering from, or at risk of suffering from, a condition that involves the formation of unwanted blood clots, in particular blood clots that may have a debilitating effect on the health of the patient. The condition may be a condition in which the patient's blood has an increased tendency to clot, e.g. compared to a healthy individual. Conditions that may be treated by the polypeptides of the invention include, for example, thromboembolic disorders, vascular occlusion, thrombosis, deep vein thrombosis, venous thrombosis, pulmonary embolism, mycocardial infarction, and stroke.

The therapeutic method may be curative and/or prophylactic, e.g. it may be for treating a patient at risk of suffering from any of the above conditions. The therapeutic method may be for preventing, mitigating, alleviating, reversing, reducing, controlling, and/or curing any of the above conditions in a patient.

The therapeutic method may involve administering a polypeptide of the invention in combination with another compound, e.g. a compound having anticoagulant activity. For example, a polypeptide of the invention may be administered with Coumarin, Warfarin, Acenocoumarol, Phenprocoumon, Phenidione, Heparin, derivatives of heparin e.g. low molecular weight heparin, Fondaparinux, Idraparinux, Argatroban, Lepirudin, Bivalirudin, Ximelagatran, and Acetylsalicylic acid. Administering a polypeptide of the invention with one or more additional coagulant compounds may provide a synergistic effect, e.g. where the mechanism of action is different to the polypeptide of the invention. For example, Coumarin, Warfarin, Acenocoumarol, Phenprocoumon, and Phenidione are vitamin K inhibitors; Heparin and derivatives thereof activate antithrombin III which blocks thrombin from clotting blood; Argatroban, Lepirudin, Bivalirudin, Ximelagatran, inhibit thrombin directly; and Acetylsalicylic acid is thought to inhibit synthesis of prothrombin.

In a further aspect of the invention products containing a polypeptide of the invention and a compound selected from Coumarin, Warfarin, Acenocoumarol, Phenprocoumon, Phenidione, Heparin, derivatives of heparin e.g. low molecular weight heparin, Fondaparinux, Idraparinux, Argatroban, Lepirudin, Bivalirudin, Ximelagatran, and Acetylsalicylic acid are provided as a combined preparation for simultaneous, separate or sequential use in a method of inhibiting blood coagulation.

The polypeptides of the invention may be provided as a composition e.g. as a pharmaceutical composition. The composition may comprise a polypeptide of the invention and a pharmaceutically acceptable excipient, diluent and/or carrier. The composition may be for use as a blood anticoagulant.

In a further aspect, there is provided a pharmaceutical composition for use as a blood anticoagulant comprising a polypeptide of the invention.

In a further aspect, there is provided an anticoagulant comprising a polypeptide of the invention.

In a further aspect, there is provided a method of treating a patient suffering from, or at risk of, any of the conditions mentioned above, which method comprises administering a polypeptide of the invention to the patient. The method may also comprise administering a compound selected from the group consisting of Coumarin, Warfarin, Acenocoumarol, Phenprocoumon, Phenidione, Heparin and its derivatives including low molecular weight heparin, Fondaparinux, Idraparinux, Argatroban, Lepirudin, Bivalirudin, Ximelagatran, and Acetylsalicylic acid to the patient.

In a further aspect of the invention, use of a polypeptide as an anticoagulant in a method of medical treatment is provided, which treatment may be a therapeutic treatment.

In a further aspect, there is provided a kit for treating a patient suffering from, or at risk of, any of the conditions mentioned above, which kit comprises:

(i) a container comprising a composition, e.g. a pharmaceutical composition, which composition comprises a polypeptide of the invention, and optionally

(ii) instructions for administering the composition to the patient.

The patient to be treated may be any animal or human. The patient is preferably a mammal, more preferably a human. The patient may be male or female.

Variants of naniproin

Variant naniproin molecules, are polypeptides that do not include the exact amino acid sequence of SEQ ID NO: 1 or the naturally occurring full-length sequence of naniproin. The variant naniproin molecules of the present invention preferably have anticoagulation activity.

For example, a variant according to the invention preferably has a similar, or at least the same, anticoagulation activity as naniproin, e.g. as measured by inhibition of prothrombinase complex as described herein. A "similar ability" means that the anticoagulation activity of the variant is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100 percent of the ability of the anticoagulation activity of naniproin. Variants of naniproin molecules of the present invention may be:

(i) novel, naturally occurring, molecules, for example obtainable from Naja nigricollis or any other species of snake. Also included are natural biological variants (e.g. allelic variants or variants from snakes that are geographically separated and therefore unable to exchange genes) of naniproin.

(ii) artificial naniproin molecule derivatives, which can be prepared by the skilled person in the light of the present disclosure. Such derivatives may be prepared, for instance, by site directed or random mutagenesis, or by direct synthesis. Preferably, a variant nucleic acid, for example, is generated either directly or indirectly, e.g. via one or more amplification or replication steps from an original nucleic acid having all or part of the sequence shown herein.

Particularly included are truncated variants which include only a distinctive part or fragment, however produced, corresponding to a portion of the sequences described herein, for example functional parts of the polypeptide that have anticoagulation activity.

Also included are molecules which have been extended at their termini with non-naturally contiguous sequences, i.e. polypeptides of the invention may also comprise additional amino acids, additional domains, or may be conjugated to additional domains or other molecules. Additional amino acids, domains, or molecules conjugated to the polypeptide may provide an additional function, for example in assisting purification of the polypeptide. Examples of additional domains that may assist in purification of the polypeptide are 6-histidine tag, and glutathione S-transferase tag. Polypeptides may be fusion proteins, fused to a peptide or other protein, such as a label, which may be, for instance, bioactive, radioactive, enzymatic or fluorescent.

Variant polypeptides may comprise at least one modification compared to SEQ ID NO: 1, e.g. addition, substitution, inversion and/or deletion of one or more amino acids.

Purely as examples, conservative replacements which may be found in such polymorphisms may be between amino acids within the following groups: alanine, serine, threonine; glutamic acid and aspartic acid; arginine and leucine; asparagine and glutamine; isoleucine, leucine and valine; phenylalanine, tyrosine and tryptophan.

Production of variants of naniproin

Variants of naniproin may be produced by modifying SEQ ID NO: 1 and/ or by modifying the naturally occurring full-length amino acid sequence of naniproin.

The polypeptide of the invention may be a fragment of SEQ ID NO: 1 and/or a fragment of the naturally occurring full-lengh amino acid sequence of naniproin. Such fragments may be provided in isolated form, i.e. not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region. When comprised within a larger polypeptide, the fragment of the invention most preferably forms a single continuous region with one or two non-naturally contiguous sequences fused to it. Additionally, several fragments may be comprised within a single larger polypeptide.

Changes to nucleic acid sequences may be desirable for a number of reasons. For instance, they may introduce or remove restriction endonuclease sites or alter codon usage. Alternatively, changes to a sequence may produce a derivative by way of one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide.

Such changes may modify sites which are required for post translation modification, such as cleavage sites in the encoded polypeptide; motifs in the encoded polypeptide for glycosylation, lipoylation etc.. Leader or other targeting sequences (e.g. membrance or golgi locating sequences) may be added to the expressed polypeptide to determine its location following expression.

Other desirable mutations may be random or site directed mutagenesis in order to alter the activity, e.g. specificity, or stability of the encoded polypeptide. Changes may be by way of conservative variation, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine. As is well known to those skilled in the art, altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptides conformation.

Also included are variants having non-conservative substitutions. As is well known to those skilled in the art, substitutions to regions of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide's three dimensional structure. In regions which are critical in determining the peptides conformation or activity such changes may confer advantageous properties on the polypeptide. Indeed, changes such as those described above may confer slightly advantageous properties on the peptide e.g. altered stability or specificity.

The variants of the invention may also be created by chemical modification of naniproin. Methods for chemical modification of polypeptides are well known in the art.

Polypeptides of the invention may be obtained by expression of a nucleic acid that encodes the polypeptide using a suitable vector and host organism. Examples of suitable vectors and hosts are well known in the art (see e.g. Sambrook, J. et al . (1989) in: Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, New York) .

Polypeptides, and particularly fragments, of the invention may also be created using chemical synthesis by any suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution couplings. In conventional solution phase peptide synthesis, the peptide chain can be prepared by a series of coupling reactions in which the constituent amino acids are added to the growing peptide chain in the desired sequence. Many such methods are now commonplace to those skilled in the art.

The variants of the invention may also be created by modification, using molecular biological techniques, of the nucleic acid that encodes naniproin, or a nucleic acid that encodes a further variant or homologue of the invention. Molecular biological techniques are well known in the art. Thus, the invention provides a method of producing and/or identifying a variant of naniproin, which method comprises the steps of:

(i) providing a polypeptide comprising the amino acid sequence of SEQ ID NO: 1,

(ii) modifying the amino acid sequence of the polypeptide, and (iii) measuring the anticoagulation activity of the modified polypeptide, e.g. the ability of the modified polypeptide to inhibit prothrombinase complex and/or FXa, e.g. to inhibit binding of FVa to FXa, as described above.

The invention also provides a further method of producing and/or identifying a variant of naniproin, which method comprises the steps of:

(i) providing a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1,

(ii) modifying the nucleic acid sequence,

(iii) producing the polypeptide encoded by the modified nucleic acid sequence, and

(iv) measuring the anticoagulation activity of the polypeptide encoded by the modified nucleic acid sequence, e.g. the ability of the polypeptide to inhibit prothrombinase complex and/or FXa, e.g. to inhibit the binding of FVa to FXa, as described above.

Preferably, the variant has a similar, or at least the same, anticoagulation activity as naniproin.

Methods of purifying polypeptides from heterogenous mixtures are well known in the art (e.g. selective precipitation, proteolysis, ultrafiltration with known molecular weight cutoff filters, ion-exchange chromatography, gel filtration, etc.). Typical protocols are set out in "Protein Purification - principles and practice" Pub. Springer-Verlag, New York Inc (1982), and by Harris & Angal (1989) "Protein purification methods - a practical approach" Pub. O.U.P. UK, or references therein. Further methods which are known to be suitable for protein purification are disclosed in "Methods in Enzymology VoI 182 - Guide to Protein Purification" Ed. M P Deutscher, Pub. Academic Press Inc..

Antibodies to naniproin, or variants of naniproin, may also be used to screen for further variants of naniproin, which methods are well known to those skilled in the art.

The naniproin amino acid or nucleotide sequence may be used in a data-base (e.g. of ESTs, or STSs) search to find sequences that share a specified level of sequence identity, such as those which may become available in due course, and expression products of which can be tested for activity as described herein .

Alternatively, variants may be provided by standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined. DNA for probing may be prepared from RNA preparations from cells. Probing may optionally be done by means of so-called "nucleic acid chips", see Marshall & Hodgson (1998) Nature Biotechnology 16: 27-31, for a review.

Libraries of nucleic acid molecules may be created which may be screened for nucleic acids that encode a polypeptide of the invention. Such libraries may be created using the nucleic acid molecule that encodes naniproin. The naniproin nucleic acid molecule sequence may be mutated, for example using random PCR mutagenesis, or gene shuffling techniques. The resulting mutated nucleic acid molecules may be ligated into a vector. Libraries of polypeptides may be created, by- expressing the nucleic acid molecules in a suitable host.

Polypeptides and nucleic acids

In this specification, a polypeptide of the invention may be a polypeptide, protein, or peptide. The polypeptide may have been post-translationally modified.

A nucleic acid of the invention may be any nucleic acid (DNA or RNA) having a nucleotide sequence specified above or a nucleotide sequence that is complementary to any of the nucleotide sequences specified above. The nucleic acid of the invention may be an RNA transcript.

The polypeptides of the invention may be isolated polypeptides. The term "isolated polypeptide" refers to a polypeptide that has undergone some degree of isolation, e.g. by purification. For example, the concentration of the polypeptide, e.g. naniproin, relative to the other components of the Naja nigricollis venom may be greater than the concentration of the polypeptide relative to the other components in the naturally occurring venom. Preferably, the polypeptide is substantially free of the other components of the Naja nigricollis venom, for example 50, 60, 70, 80, 90, 95, 99, or even 100 percent free. For example, the polypeptide may be purified from the venom or crude extract by gel filtration, cation exchange chromatography and/or reverse phase chromatography.

The nucleic acids of the invention may be isolated nucleic acids. The term "isolated nucleic acid" refers to a nucleic acid that has undergone some degree of isolation, e.g. the nucleic acid is not present in a cell in which it naturally occurs . In this specification the term "operably linked" may include the situation where a selected nucleotide sequence and regulatory nucleotide sequence are covalently linked in such a way as to place the expression of a nucleotide sequence under the influence or control of the regulatory sequence. Thus a regulatory sequence is operably linked to a selected nucleotide sequence if the regulatory sequence is capable of effecting transcription of a nucleotide sequence which forms part or all of the selected nucleotide sequence. Where appropriate, the resulting transcript may then be translated into a desired protein or polypeptide.

Sequence identity

Percentage (%) sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical with residues in SEQ ID NO: 1 after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence identity is preferably calculated over the entire length of the respective sequences.

Where the aligned sequences are of different length, sequence identity of the shorter comparison sequence may be determined over the entire length of the longer given sequence or, where the comparison sequence is longer than the given sequence, sequence identity of the comparison sequence may be determined over the entire length of the shorter given sequence.

For example, where a given sequence comprises 100 amino acids and the candidate sequence comprises 10 amino acids, the candidate sequence can only have a maximum identity of 10% to the entire length of the given sequence. This is further illustrated in the following example:

(A) Given seq: XXXXXXXXXXXXXXX (15 amino acids) Comparison seq: XXXXXYYYYYYY (12 amino acids)

The given sequence may, for example, be that encoding SEQ ID NO: 1.

% sequence identity = the number of identically matching amino acid residues after alignment divided by the total number of amino acid residues in the longer given sequence, i.e. (5 divided by 15) x 100 = 33.3%

Where the comparison sequence is longer than the given sequence, sequence identity may be determined over the entire length of the given sequence. For example:

(B)

Given seq: XXXXXXXXXX (10 amino acids) Comparison seq: XXXXXYYYYYYZZYZZZZZZ (20 amino acids)

Again, the given sequence may, for example, be that encoding SEQ ID NO: 1.

% sequence identity = number of identical amino acids after alignment divided by total number of amino acid residues in the given sequence, i.e. (5 divided by 10) x 100 = 50%.

Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ClustalW, T-coffee or Megalign (DNASTAR) software. When using such software, the default parameters, e.g. for gap and exention penalty, are preferably used. Preferably, sequences are aligned using ClustalW 1.82 software, and using the default parameters, e.g. DNA Gap Open Penalty = 15.0, DNA Gap Extension Penalty = 6.66, DNA Matrix = Identity, Protein Gap Open Penalty = 10.0, Protein Gap Extension Penalty = 0.2, Protein matrix = Gonnet, Protein/DNA ENDGAP = -1, and Protein/DNA GAPDIST = 4.

Identity of nucleic acid sequences may be determined in a similar manner involving aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity, and calculating sequence identity over the entire length of the respective sequences. Where the aligned sequences are of different length, sequence identity may be determined as described above and illustrated in examples (A) and (B) .

Hybridisation stringency

In accordance with the present invention, nucleic acid sequences may be identified by using hybridization and washing conditions of appropriate stringency.

Complementary nucleic acid sequences will hybridise to one another through Watson-Crick binding interactions . Sequences which are not 100% complementary may also hybridise but the strength of the hybridisation usually decreases with the decrease in complementarity. The strength of hybridisation can therefore be used to distinguish the degree of complementarity of sequences capable of binding to each other.

The "stringency" of a hybridization reaction can be readily determined by a person skilled in the art.

The stringency of a given reaction may depend upon factors such as probe length, washing temperature, and salt concentration. Higher temperatures are generally required for proper annealing of long probes, while shorter probes may be annealed at lower temperatures . The higher the degree of desired complementarity between the probe and hybridisable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so.

For example, hybridizations may be performed, according to the method of Sambrook et al., ("Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989) using a hybridization solution comprising: 5X SSC, 5X Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-42°C for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37°C in IX SSC and 1% SDS; (4) 2 hours at 42-65⁰C in IX SSC and 1% SDS, changing the solution every 30 minutes .

One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules is to calculate the melting temperature T_n, (Sambrook et al., 1989) :

T_n, = 81.5°C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/n

where n is the number of bases in the oligonucleotide.

As an illustration of the above formula, using [Na+] = [0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the T_m is 57°C. The T_m of a DNA duplex decreases by 1 - 1.5⁰C with every 1% decrease in sequence complementarity .

Hybridisation under high stringency conditions may involve performing the hybridisation at a temperature of Tm-15 or higher. Moderate stringency may be considered to be Tm-25 to Tm-15. Low stringency may be considered to be Tm-35 to Tm-25.

Accordingly, nucleotide sequences can be categorised by an ability to hybridise to a target sequence under different hybridisation and washing stringency conditions which can be selected by using the above equation. The T_m may be used to provide an indicator of the strength of the hybridisation.

The concept of distinguishing sequences based on the stringency of the conditions is well understood by the person skilled in the art and may be readily applied.

Sequences exhibiting 95-100% sequence complementarity are considered to hybridise under very high stringency conditions, sequences exhibiting 85-95% complementarity are considered to hybridise under high stringency conditions, sequences exhibiting 70-85% complementarity are considered to hybridise under intermediate stringency conditions, i.e. "stringent conditions", sequences exhibiting 60-70% complementarity are considered to hybridise under low stringency conditions and sequences exhibiting 50-60% complementarity are considered to hybridise under very low stringency conditions. Hybridisations performed at 42°C or higher may be considered to be under high stringency conditions.

Pharmaceutical compositions

Medicaments and pharmaceutical compositions according to aspects of the present invention may be formulated for administration by a number of routes, including but not limited to, parenteral, intravenous, intra-arterial, intramuscular, intratumoural, oral and nasal. The medicaments and compositions may be formulated in fluid or solid form. Fluid formulations may be formulated for administration by injection to a selected region of the human or animal body. Injectable formulations may comprise the selected compound in a sterile or isotonic medium. The polypeptides of the invention may be administered intravenously, or by intraperitoneal or intraventricular injection.

Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to the individual. The actual amount administered, and rate and time- course of administration, will depend on the nature and severity of the disease being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington' s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins .

Alternatively, targeting therapies may be used to deliver the active agent more specifically to a particular location, by the use of targeting systems such as antibody or cell specific ligands . Targeting may be desirable for a variety of reasons; for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage.

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

Brief Description of the Figures

Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

Figure 1.

Figure 1 shows purification of the novel anticoagulant protein. (AI) shows anticoagulant activity of crude venom. (A) shows size-exclusion chromatography (SEC) of the crude venom of N. nigricollis (100 mg in 1 itiL) on Superdex 30 column. (B) shows cation exchange chromatography of the strongly anticoagulant SEC fractions on an Uno S6 column. (C) shows the reverse-phase HPLC profile of fractions containing the novel anticoagulant protein on Jupiter C18 semipreparative column.

(D) shows elution profile of the anticoagulant protein on a narrow bore pepmap column (note that the single peak in the profile indicates, that the isolated protein is homogenous) .

(E) shows molecular weight of the anticoagulant protein, which was determined using ESI-MS. The protein has a molecular mass of 6887.96+ 0.35 Da. Note: in these Figures the horizontal bars and the arrow indicate the fractions containing the anticoagulant of interest and these fractions were used for further purification. Figure 2 .

Figure 2 shows N-terminal sequencing of naniproin. (A) shows the first 44 NH₂-terminal residues of the novel anticoagulant protein naniproin which were determined by Edman degradation. Conserved cysteine residues in the three-finger toxin family are shaded in black. (B) shows that reduction and alkylation of naniproin resulted in loss of the β-sheet structure, which became a random coil. (C) shows that the β-sheet structure of naniproin was lost when the temperature went beyond 6OC and naniproin became random coil. (D) shows anticoagulant activity of native protein, denatured and renatured protein and s- pyridylethylated protein.

Figure 3.

Figure 3 shows results of the dissection approach. (A) shows effect of the anticoagulant protein on Prothrombin time (•) , Stypven time (A.) and Thrombin time (■) (B) shows that anticoagulant protein showed no inhibition on the extrinsic tenase complex. (C) shows that the protein inhibits prothrombinase complex in a dose-dependant manner with an IC₅₀ of 100 uM.

Figure 4.

Figure 4 shows the specific binding site of the protein in the prothrombinase complex. (A) shows that when the phospholipids vesicles (PC: PS) were absent, naniproin inhibited prothrombin activation by FXa-FVa complex with an IC₅₀ of 400 nM. (B) shows a double reciprocal curve showing that naniproin competitively inhibited FVa with an average Ki of 1.28 μM. (C) shows that when both phospholipid vesicles and cofactor (FVa) were absent, naniproin did not inhibit the amidolytic activity of FXa on S2765 but increased the activation of prothrombin by FXa in a dose dependent manner. (D) The ITC result showed the protein binds to FXa via an exothermic reaction. Detailed Description of the Invention

Specific details of the best mode contemplated by the inventors for carrying out the invention are set forth below, by way of example. It will be apparent to one skilled in the art that the present invention may be practiced without limitation to these specific details.

Materials and Methods

Lyophilized crude W. nigricollis venom was obtained from Biotoxins Incorporated (St. Cloud, Florida). Thromboplastin with calcium (for prothrombin time assays), Russell's viper venom (for Stypven time assays) , thrombin reagent (for thrombin time assays) , 4-vinylpyridine, phosphatidylcholine

(PC) , and phosphatidylserine (PS) were purchased from Sigma Chemical Co (St Louis, MO) . β-Mercaptoethanol was purchased from Nacalai Tesque (Kyoto, Japan) . The chromogenic substrates, H-D-Isoleucyl-L-prolyl-L-arginine-pnitroaniline dihydrochloride (S-2288) , H-D-phenylalanyl-L-pipecolyl-L- arginine-p-nitroaniline dihydrochloride (S-2238), N-α- Benzyloxycarbonyl-Darginyl-L-glycyl-L-arginine-pnitroaniline- dihydrochloride (S-2765) were from Chromogenix (Milano, Italy) . All substrates were reconstituted in deionized water prior to use. Human plasma was donated by healthy volunteers. Recombinant human TF (Innovin) was purchased from Dade Behring

(M-arburg, Germany) . Human plasma was donated by healthy volunteers. All other chemicals and reagents used were of the highest purity available.

Proteins

Human plasma-derived FVIIa was a gift from the Factor VII Group (Kazuhiko Tomokiyo, Yasushi Nakatomi, Teruhisa Nakashima, and Soutatou Gokudan) of KAKETSUKEN and were purified as described (Wildgoose and Kisiel, 1989) . Pure bovine FXa, FVa and prothrombin were from Haematologic Technologies, Inc. (Essex Junction, Vermont, USA) . Purification

Naja nigricollls crawchawii (West Africa) venom (120mg in 2ml distilled water) was purified by size exclusion chromatography using a Superdex-30 gel filtration column (l.δxβOcm; Amersham Pharmacia, Uppsala Sweden) . The column was washed and pre- equilibrated with 5OmM Tris-HCl (pH 7.4). The proteins were eluted by using the same buffer with isocratic gradient at lml/min flow rate. The chromatography was carried out using the AKTA purifier system (Amersham Biosciences AB, Uppsala, Sweden) . Protein elution was monitored at 280nm. Individual peaks were collected and assayed for anticoagulant activity using the prothrombin time clotting assay.

The active peak from the size exclusion chromatography was collected and subjected to cation-exchange chromatography. UNO S6 (6ml column volume; Bio-Rad) was pre-equilibrated by buffer A (5OmM Tris-HCl; pH 7.4) and bound proteins were eluted using buffer B (5OmM Tris-HCl + 0.5M NaCl; pH 7.4) with a linear gradient and 3ml/min flow rate. Protein elution was monitored at 280nm. Individual peaks were collected and anticoagulant activity was tested using the prothrombin time clotting assay.

The anticoagulant peak from the cation-exchange chromatography was further purified using reverse phase chromatography. A Jupiter C18 column was used and the column was equilibrated using Buffer A (0.1% Trifluoroacetic acid). The bound proteins were eluted with a linear gradient using buffer B (0.1% Trifluoroacetic acid + 80% acetonitrile) at 1.5ml/min flow rate. Individual peaks were collected, lyophilized, examined for anticoagulant activity. The peak of interest was rechromatographed on a narrow bore PepMap column using AKTA purifier system (Amersham Biosciences AB, Uppsala, Sweden) .

Electrospray Ionization Mass Spectrometry (ESI-MS)

The homogeneity and mass of the anticoagulant protein was determined by electrospray ionization mass spectrometry (ESI- MS) using a Perkin-Elmer Sciex API300 LC\MS system. Typically, reverse-phase HPLC fractions were directly used for analysis. Ion-spray, orifice, and ring voltages were set at 4600, 50, and 350 V, respectively. Nitrogen was used as a nebulizer and curtain gas. A Shimadzu LC-IOAD pump was used for solvent delivery (40% acetonitrile in 0.1% trifluoroacetic acid) at a flow rate of 50 μL/min. Full scan data was acquired over the ion range from 1000 to 2200 m/z with step size of 0.5 amu. BioMultiview software (PerkinElmer Life Sciences) was used to analyze and deconvolute raw mass spectra. The sample loop was pre-washed using methanol before sample loading.

Reduction and Pyridylethylation

The purified protein (3mg) was dissolved in ImI of 0.25mM Tris-HCl, ImM EDTA, and 6M guanidine-HCl (pH 8.5). 60μl of 2- mereptoethanol (20 μl/mg protein) was added and the solution was incubated under a nitrogen environment for 2 hours at 37C. 600μl of alkylating agent and 4-vinylpyridine (200μl/mg of protein) was subsequently added and the mixture was incubated under nitrogen for another 2 hours at room temperature. The S- pyridylethylated protein was separated from the reaction mixture by reverse-phase chromatography on an analytical Jupiter column (4.6 X 250mm) using a linear gradient of 80% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 0.5ml/min.

Enzymatic cleavage

The S-pyridylethylated protein was digested with the enzyme Lysyl endopeptidase (Lys C) . The enzyme was dissolved in 5OmM Tris HCl, 4M Urea and 5mM EDTA in pH7.5. The enzyme was added to the protein in the ratio of 1:100. The digestion was carried out at 37C for 1 hour.

Circular Dishroism (CD) Spectroscopy

Far-UV CD spectra (260-190nm) were recorded for varying concentrations of the anticoagulant protein (0.5mg/ml) in 5OmM Tris-HCl buffer (pH 7.4). The Jasco J-810 spectropolarimeter was used, and the instrument optics was flushed with 3OL of nitrogen gas/min. All measurements were carried out at rodm temperature using a 0.1cm path length stoppered cuvette. The spectra were recorded using a scan speed of 50nm/min, a resolution of 0.2nm, and a bandwidth of 2nm. A total of 5 scans were recorded and averaged for each spectrum, and the base line was subtracted. The CD spectra of the anticoagulant protein also were recorded under a different temperature. The CD spectra of S-pyridylethylated protein were also recorded.

Anticoagulant Activity

The anticoagulant activities of ZV. nigricollis venom and its fractions were determined by three clotting assays using a BBL Fibrometer .

Prothrombin Time

Prothrombin time clotting assay was measured according to the method of Quick (Quick, 1966) . Essentially, 100 μL of 50 mM Tris-HCl buffer (pH 7.4), 100 μL of plasma, and 50 μL of venom or its fractions were preincubated for 2 min at 37 ⁰C. Clotting was initiated by the addition of 150 μL of thromboplastin with calcium reagent, ensuring high tissue factor concentrations and a robust generation of FXa by TF- FVIIa. This completely masked the contribution of the FVIIIa- FIXa complex in the assay.

Stypyen Time

The Stypven time was measured according to the method of Hougie (Hougie, 1956) . Briefly, 100 μL of 50 mM Tris-HCl buffer (pH 7.4), 100 μL of plasma, 100 μL of Russell's viper venom (0.01 μg) , and 50 μL of venom or its fractions were preincubated for 2 min at 37 ⁰C. Clotting was initiated by the addition of 50 μL of 50 mM CaCl₂- Thrombin Time

The thrombin time was determined according to the method of Jim (Jim, 1957). 100 μL of 50 mM Tris-HCl buffer (pH 7.4), 100 μL of plasma, and 50 μL of venom or its fractions were pre-incubated for 2 min at 37 ⁰C. Clotting was initiated by the addition of standard thrombin reagent (0.01 NIH unit in 50 μL) . (National Institute of Health (NIH) standard (Lot J) is in common use for the calibration of commercial thrombin reagents. For more information, see Gaffney and Edgell, 1995; Whitton et al._r 2005.)

Preparation of PCPS Vesicles

Phospholipids vesicles of PC and PS in a ratio of 3:1 were prepared as described by Govers-Riemslag et al., 1992. Essentially, chloroform solutions of phospholipids were mixed and dried under nitrogen. The dried lipids were suspended in buffer, vortexed for 1 min, and subsequently sonicated for 10 min. All reactions were carried out at 37 ⁰C in buffer containing 50 mM Tris-HCl, 175 mM NaCl, 3 mM CaCl₂, and 1% bovine serum albumin (pH 8.0). Vesicles were isolated as the supernatant after a 10-min centrifugation at 11000 rpm.

Reconstitution of Extrinsic Tenase Complex

The TF-FVIIa complex was reconstituted by incubating 10 pM FVIIa with 70 pM recombinant human TF (Innovin) in buffer containing 50 mM Tris-HCL, 100 mM NaCl, 10 mM CaCl₂, and 1% bovine serum albumin (pH 8.0) for 10 min at 37 ⁰C. FX was added to the mixture to obtain a final concentration of 30 nM. After 15 min of incubation, the FXa generated by TF-FVIIa complex was assayed by measuring its amidolytic activity on 1 mM S-2288 in Buffer A at 405 nm. The inhibitory effect on extrinsic tenase activity was determined by adding the anticoagulant protein 15 min prior to FX addition. Reconstitution of Prothrombinase Complex

The FVa-FXa complex was reconstituted by incubating 0.5 nM FVa, 0.1 pM FXa, and PCPS vesicles in assay solution containing 50 mM Tris-HCl, 10 itiM CaCl₂, and 1% bovine serum albumin (pH 8.0) at 37°C. After 5 min, prothrombin (final concentration 15 μM) was added, followed by the addition of S- 2238 (1 mM) to determine the residual prothrombinase activity. The change in absorbance (A) as a function of time (t) was recorded at 405 ran using a thermostatically controlled microtiter plate reader (SLT Lab Instruments, model 340 ATTC) . Percent inhibition was calculated by taking the thrombin formed in the absence of the anticoagulant protein as 100%. IC₅₀ values (concentration of the anticoagulant protein required to inhibit 50% of thrombin production) were estimated from the dose-response curve.

Isothermal Titration Calorimetry (ITC)

The interaction of the anticoagulant protein with FXa was monitored with a VP-ITC titration calorimetric system (MicroCal, LLC, Northampton, MA) . The instrument was calibrated using the built in electrical calibration check. FXa (0.08 μM) in 5OmM Tris-HCl buffer and 1OmM CaCl₂ (pH 7.4) in the calorimetric cell was titrated with the anticoagulant protein (8.0 μM) dissolved in the same buffer in a 250μl injection syringe with continual stirring at 300rpm at 37C. All protein solutions were filtered and degassed prior to titration. The first injections presented defects in the base line, and these data were not included in the fitting process. The calorimetric data were processed and fitted to the single set of identifical sites model using MicroCal Origin (Version 7.0) data analysis software supplied with the instrument.

Anticoagulant activity of crude venom

N. nigricollis crude venom exhibited potent anticoagulant activity in the prothrombin time assay at 2mg/ml (Fig IA (I) ) . Purification

Purification of the anticoagulant protein from the crude venom was carried out firstly using the size-exclusion chromatography to separate the proteins by size (Fig IA) . Fractions in peak 3 and 4 exhibited anticoagulant activity in the prothrombin time clotting assay. All the fractions in these two peaks were pooled and fractioned using cation- exchange chromatography (Fig IB) . Peaks containing the anticoagulant protein (indicated by the horizontal bar) were further purified by reverse-phase HPLC (Fig 1C) . The peak in the RP-HPLC exhibiting the anticoagulant activity was collected and subjected to analytical column for testing the purity of the protein (Fig ID) . The homogeneity and mass of the individual proteins were determined by Electrospay ionization mass spectrometry (ESI-MS) . The result showed two peaks with mass/charge ratios of 5 and 6 and the calculated molecular mass was 6887.96 ± 0.35 Da (Fig IE) .

N-terminal sequencing

We determined the sequence of the first 44 amino acid residues of the anticoagulant protein using Edman degradation protein chemistry. The locations of the cysteine residues in the proteins were confirmed by sequencing the pyridylethylated protein. The identified protein shows similarity to cytotoxins, cardiotoxins, postsynaptic neurotoxins, and other members of the three-finger toxin family (Fig 2A) , but not to serine protease inhibitors, isolated from snake venoms (such as from the elapids Heiaachatus haemachatus (Ringhal's corbra) , Bungarus fasciatus (Banded krait) and Naja nivea (Cape cobra) ) (4) . Thus, the isolated protein belongs to the three-finger toxin family.

The N-terminal amino acid sequence of naniproin is: LKCNRLIPPFWKTCPEGKNLCYKMTMRLAPKVPVKRGCIDVCPKSSLLIKYMCCTNDKCN (SEQ ID NO: 1) CD spectrometry

The purified protein exhibited negative minima at 215nm and positive maxima at 194nm. The protein showed a predominantly β-sheet structure (Fig 2B) . S-pyridylethylated anticoagulant protein exhibited a random-coil structure; meaning that the protein lost its β-sheet structure after reduction and pyridylethylation at the cysteine site (Fig 2B) . This indicated that disulfide bonds between the cysteine residues were important to maintain the structure of three finger toxin. The CD spectra of the protein in different temperature were also carried out (Fig 2C) . The result showed that the protein lost its predominantly β-sheet when the temperature went beyond 6OC; random coil was formed.

After denaturing and renaturing, the native protein the prothrombim time assay was carried out to test anticoagulant activity. S-pyridyethylated protein did not exhibit any anticoagulant activity. Thus, proper folding is important for the anticoagulant activity of the protein. (Fig 2D)

Dissection Approach

We employed three commonly used clotting time assays, prothrombin time assay, stypven time assay and thrombin time assay. These are based on the simple principle that initiating the cascade "upstream" from the inhibited step will result in elevated clotting times, while initiating the cascade "downstream" from the inhibited step will not affect the clotting time (17) . The protein showed anticoagulant activity for both of the prothrombin assay and stypven time assay (Fig 3A) . These results indicated that this protein maybe affect the extrinsic tenase complex and prothrombinase complex.

Effects on Reconstituted Extrinsic Tenase Complex The effect of the anticoagulant protein was monitored on reconstituted TF-FVIIa complex. The protein did not inhibit the amidolytic activity of the complex (Fig 3B) . A higher dosage of protein was used in this study in order to confirm that there was no inhibitory activity.

Effects on Reconstituted Prothrombinase Complex Next, we monitored the inhibitory activity of the protein on the amidolytic activity of the reconstituted prothrombinase complex. As observed in Figure 3C,- the protein inhibited the activity of prothrombinase complex in a dose-dependant manner with IC₅₀ (concentration of the inhibitor at which the inhibition is 50% of that of the control) of 80 DM. Thus, the anticoagulant activity of the protein is due to the specific inhibition of the prothrombinase complex.

Effects on the Different components of Prothrombinase Complex To find out the target components in the prothrombinase complex, chromogenic substrate assays were carried out in the absence of different components. Naniproin inhibited activation of prothrombin by the fully assembled prothrombinase complex with an IC₅₀ of ~ 100 μM. When the phospholipids vesicles (PC: PS) were absent, naniproin inhibited prothrombin activation by FXa-FVa complex with an IC₅₀ of 400 nM and a Ki of 1.28 μM. Lastly, when both phospholipid vesicles and cofactor (FVa) were absent, naniproin did not inhibit the amidolytic activity of FXa on S2765 but increased the activation of prothrombin by FXa in a dose dependent manner. Overall these results indicated that naniproin inhibited prothrombinase complex by interfering with the interaction between FXa and FVa. Naniproin most likely binds to FXa and competes with FVa for the same binding site. The absence of an effect on FXa small peptidyl substrate indicates that naniproin does not affect the integrity of the FXa active site pocket, while the increase in prothrombin activation is likely to be due to enhanced interaction of prothrombin with FXa caused by the binding of naniproin to FXa. These results indicate that naniproin acts as a cofactor to FXa but as an inhibitor of the prothrombinase complex.

Binding to FXa was further confirmed by carrying out isothermal titration calorimetric (ITC) studies on naniproin with FXa. We found that the binding association constant (K_a) of naniproin with FXa is 4.174 x 10⁵M^"1. When the same study was carried out with prothrombin no binding was observed.

Discussion

Initiation of blood coagulation during injury or trauma is essential for the survival of the organism (3) . However, the formation of unwanted clots has detrimental or debilitating effects and hence need for anticoagulant therapies (3) . Due to the narrow therapeutic window of existing anticoagulants, the invention of new anticoagulants that exhibits more specific action is needed. Because FXa plays a pivotal role in clot formation, it is an attractive drug target for the design and development of anticoagulant.

We have reported here the isolation and characterization of "naniproin" from the venom of Naja nigrϊcollis {Naja nigricollis factor Xa inhibitor) that induces potent anticoagulant activity. Naniproin belongs to the three finger toxin of snake venom. This protein exhibited the predominantly beta sheet CD structure. It is well known that disulfide bonds associated with cysteine residues are important structural units in proteins (8). The importance of the disulfide bonds was studied using the reduction and alkylation method. The reduced and alkylated protein exhibited no anticoagulant activity and showed random coil in the CD studies. This indicated the importance of the disulfide bond in maintaining the proper folding of the protein.

The dissection approach was carried out to determine the specific site of action of the protein. Naniproin showed anticoagulant activity in both prothrombin time assay and stypven time assay but not in the thrombin time assay. Thus, naniproin has no effect on the conversion of fibrinogen to fibrin, but it may affect either the prothrombinase complex alone or both entrinsic tenase complex and prothrombinase complex. Naniproin showed no inhibition on the reconstitution extrinsic TF-FVIIa complex which confirmed that this protein inhibited the prothrombinase complex. The protein specifically inhibited the prothrombinase complex with an IC₅₀ of lOOμM. To find out the target components in the prothrombinase complex, IC₅₀ studies of FXa, FXa-FVa and FXa-FVa-PCPS with the protein were carried out. Inhibition of the protein to FXa and FXa-FVa gave similar IC₅₀. Thus, this protein actually exhibit action on the FXa but not FVa. The inhibition of protein to FXa-FVa- PCPS is lower when compared to others. This suggested nonspecific binding of naniproin to the PCPS. The binding of the protein to FXa was further confirmed by carrying out the ITC study on the protein and FXa. The exothermic reaction with the binding affinity of 4.174 x 10⁵M^"1, shows the protein binds to the FXa. Same study was carried out with the prothrombin, no binding is showed.

Moreover, previously reported snake venom anticoagulants inhibiting the prothrombinase complex are not as specific as the protein discussed here. For example, CM-IV, a strongly anticoagulant phospholipase A₂ from Naja nigricollis venom, prolongs coagulation by inhibiting two successive steps in the coagulation cascade. It inhibits the TF-FVIIa complex by both enzymatic and nonenzymatic mechanisms (18), and it inhibits the prothrombinase complex by a nonenzymatic mechanism. Other examples include anticoagulant C-type lectin-like proteins (CLPs) . These were among the first proteins isolated and characterized from snake venoms that bind to FX/FXa and FIX/FIXa with nanomolar and sub-nanomolar affinities and exert their inhibitory activity through nonenzymatic mechanisms (25) . The binding interferes in calcium-dependent binding of FIX and FX to phospholipid membranes and hence exhibits anticoagulant effects. Atoda and Morita (1989) have purified such proteins from Trimeresurus flavoviridis (Habu snake) venom. They also purified a FX-binding protein from Deinagkistrodon acutus venom (2) . In these cases, since the anticoagulant activity is targeted towards both the prothrombinase and intrinsic tenase complexes, it is not specific.

Snake venoms also contain a number of isoforms of serine protease inhibitors, which inhibit the coagulation serine proteases (4). These proteins contain 57-60 amino acid residues and unlike three-finger toxins contain three disulfide bridges and belong to Kunitz/BPTI (bovine pancreatic trypsin inhibitor) family (19,20), although, these too are non-specific inhibitors.

Therefore, since the anticoagulant protein isolated in this study is a specific inhibitor of the prothrombinase complex, and also a natural inhibitor from snake venom, it may have greater thereapeutic value than previously characterized snake venom anticoagulants.

Snake venom inhibitors of prothrombinase complex have also been used to study the function and tertiary structure of coagulation factors (24) . For example, functional studies with CLPs have revealed that magnesium ions are critical components in the blood coagulation cascade system. The crystal structures of GIa domains of FX have recently been clarified in structural studies of complexes between the GIa domain of FX and venom CLP factor X-binding protein (23) . Hence, the purified protein may also be used to study the blood coagulation cascade. Further studies need to be carried out to further elucidate its properties . References

1. Atoda,H., Ishikawa,M., Mizuno,H., and Morita,T. (1998). Coagulation factor X-binding protein from Deinagkistrodon acutus venom is a GIa domain-binding protein. Biochemistry 37, 17361- 17370.

2. Atoda,H. and Morita,T. (1989). A novel blood coagulation factor IX/factor X-binding protein with anticoagulant activity from the venom of Trimeresurus flavoviridis (Habu snake) : isolation and characterization. Journal Of Biochemistry 106, 808-813.

3. Banerjee,Y., Mizuguchi, J. , Iwanaga,S., and Kini,R.M. (2005). Hemextin AB complex, a unique anticoagulant protein complex from Hemachatus haemachatus (African Ringhals cobra) venom that inhibits clot initiation and factor Vila activity. J. Biol. Chem. 280, 42601-42611.

4. Chen, C, Hsu, C. H., Su, N. Y., Lin, Y. C, Chiou,S.H., and Wu, S. H. (2001) . Solution structure of a Kunitz-type chymotrypsin inhibitor isolated from the elapid snake Bungarus fasciatus. J. Biol. Chem. 275, 45079-45087.

5. Dahlback,B. (2005). Blood coagulation and its regulation by anticoagulant pathways: genetic pathogenesis of bleeding and thrombotic diseases. J. Intern. Med. 257, 209-223.

6. Davie,E.W., Fujikawa, K., and Kisiel,W. (1991). The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 30, 10363-10370.

7. Davie,E.W. (1995) Thromb Haemostasis 74, 7-17

8. Debarbieux,L. and Beckwith,J. (1999). Electron avenue: pathways of disulfide bond formation and isomerization. Cell 99, 117-119.

9. Gustafsson, D. , Bylund,R., Antonsson, T. , Nilsson,I., Nystrom, J. E. , Eriksson, U., Bredberg, U. , and Teger-Nilsson, A. C. (2004). A new oral anticoagulant: the 50-year challenge. Nat. Rev. Drug Discov. 3, 649-659.

10. Higuchi, S. ,Murayama,N. , Saguchi,K. ,Ohi, H. , Fujita, Y. , Camargo, A. C. , O gawa, T . ,

11. Desshimaru,M. , and Ohno,M. (1999) Immunopharmacology 44, 129-135

12. Hirsh,J., Dalen,J., Anderson, D. R. , Poller,L., Bussey,H., Ansell,J., and Deykin,D. (2001a). Oral anticoagulants: mechanism of action, clinical effectiveness, and optimal therapeutic range. Chest 119, 8S-21S. 13. Hirsh,J., 0'Donnell,M. , and Weitz, J. I . (2005). New anticoagulants. Blood 105, 453-463.

14. Hirsh,J., Warkentin, T. E. , Shaughnessy, S .G. , Anand, S.S., Halperin, J. L. , Raschke,R. , Granger, C, Ohman,E.M., and Dalen,J.E. (2001b) . Heparin and low-molecular-weight heparin: mechanisms of action, pharmacokinetics, dosing, monitoring, efficacy, and safety. Chest 119, 64S-94S.

15. Hirsh, J. , and Weitz, J.I. (1999) Lancet 353, 1431-1436

16. Kerns, R. T., Kini,R.M., Stefansson, S . , and Evans, H. J. (1999). Targeting of venom phospholipases : the strongly anticoagulant phospholipase A(2) from Naja nigricollis venom binds to coagulation factor Xa to inhibit the prothrombinase complex. Arch. Biochem. Biophys . 369, 107-113.

17. Kini.R.M, Banerjee Y. Dissection approach: a simple strategy for the identification of the step of action of anticoagulant agents in the blood coagulation cascade. J Thromb Haemost 2004

18. Kini,R.M. and Evans, H. J. (1995c). The role of enzymatic activity in inhibition of the extrinsic tenase complex by phospholipase A2 isoenzymes from Naja nigricollis venom. Toxicon 33, 1585-1590.

19. Laskowski,M. , Jr. (1986). Protein inhibitors of serine proteinases—mechanism and classification. Advances In Experimental Medicine And Biology 255, 1-17.

20. Laskowski,M. , Jr. and Kato,I. (1980). Protein inhibitors of proteinases. Annual Review Of Biochemistry 49, 593-626.

21. Mann, K. G., Butenas,S., and Brummel,K. (2003). The dynamics of thrombin formation. Arterioscler . Thromb. Vase. Biol. 23, 17-25.

22. Markland, F. S. (1998). Snake venoms and the hemostatic system. Toxicon 36, 1749-1800.

23. Mizuno,H., Fujimoto, Z . , Koizumi, M., Kano,H., Atoda,H., and Morita,T. (1997). Structure of coagulation factors IX/X-binding protein, a heterodimer of C-type lectin domains. Nature Structural Biology 4, 438-441.

24. Morita,T. (2004). Use of snake venom inhibitors in studies of the function and tertiary structure of coagulation factors. Int. J. Hematol. 79, 123-129.

25. Morita,T. (2005). Structures and functions of snake venom CLPs

(C-type lectin-like proteins) with anticoagulant-, procoagulant-, and platelet-modulating activities. Toxicon 45, 1099-1114. 26. Stefansson,S., Kini,R.M., and Evans, H. J. (1990). The basic phospholipase A2 from Naja nigricollis venom inhibits the prothrombinase complex by a novel nonenzymatic mechanism. Biochemistry 29, 7742-7746.

Claims

Claims

1. A polypeptide comprising:

(i) the amino acid sequence of SEQ ID NO: 1; or (ii) an amino acid sequence sharing at least 90 percent sequence identity with the amino sequence of SEQ ID NO: 1.

2. A polypeptide according to claim 1, wherein the polypeptide has anticoagulant activity.

3. A polypeptide according to claim 1 or claim 2, wherein the amino acid sequence defined in (ii) shares said sequence identity with the full-length amino acid sequence of SEQ ID NO: 1.

4. A polypeptide comprising:

(i) an amino acid sequence sharing at least 60 percent sequence identity with the amino sequence of SEQ ID NO: 1;

(ii) a fragment of the amino acid sequence of SEQ ID NO: 1, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1; or

(iii) an amino acid sequence sharing at least 60 percent sequence identity with the fragment defined in (ii) , wherein the amino acid sequence has at least 6 amino acids; and wherein the polypeptide has anticoagulant activity.

5. A polypeptide according to claim 4, wherein the amino acid sequence defined in (i) shares said sequence identity with the full-length amino acid sequence of SEQ ID NO: 1.

6. A polypeptide according to claim 4 or claim 5, wherein the amino acid sequence defined in (iii) shares at least 90 percent sequence identity with the fragment defined in (ii) .

7. A polypeptide according to any one of claims 1 to 6, wherein the polypeptide inhibits formation of the prothrombinase complex.

8. A polypeptide according to any one of claims 1 to 7, wherein the polypeptide binds FXa.

9. A polypeptide according to claim 8, wherein the polypeptide binds FXa with a 1/Kd value of at least 1 x 10² M^"1.

10. A polypeptide according to claim 8, wherein the polypeptide binds FXa with a 1/Kd value of at least 1 x 10⁵ M^"1.

11. A polypeptide according to any one of claims 1 to 10, wherein the polypeptide inhibits activation of prothrombin.

12. A polypeptide according to claim 11, wherein the polypeptide inhibits activation of prothrombin with an IC₅₀ of less than 500μM.

13. A polypeptide according to claim 11, wherein the polypeptide inhibits activation of prothrombin with an IC₅₀ of less than 200μM.

14. A polypeptide according to any one of claims 1 to 13, wherein the polypeptide has a molecular mass in the range 5000-8000 Da.

15. A polypeptide according to any one of claims 1 to 14, wherein the polypeptide is obtainable from the venom of Naja nigricollis .

16. A polypeptide according to any one of claims 1 to 15, wherein the polypeptide has at least two cysteine amino acids.

17. A polypeptide according to any one of claims 1 to 16, wherein the polypeptide has at least one disulfide bond.

18. An isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide comprising:

(i) the amino acid sequence of SEQ ID NO: 1; or (ii) an amino acid sequence sharing at least 90 percent sequence identity with the amino sequence of SEQ ID NO: 1.

19. An isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide comprising:

(i) an amino acid sequence sharing at least 60 percent sequence identity with the amino sequence of SEQ ID NO: 1;

(ii) a fragment of the amino acid sequence of SEQ ID NO: 1, which fragment has at least 6 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1; or

(iii) an amino acid sequence sharing at least 60 percent sequence identity with the fragment defined in (ii) , wherein the amino acid sequence has at least 6 amino acids; and wherein the polypeptide has anticoagulant activity.

20. An isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide as defined in any one of claims 1 to 17.

21. A method of producing a polypeptide of the invention, which method comprises the steps of:

(a) providing a host cell comprising a nucleotide sequence as defined in any one of claims 18 to 20; and

(b) causing the host cell to express the polypeptide encoded by the nucleotide sequence.

22. A polypeptide according to any one of claims 1 to 17 for use in therapy.

23. A composition comprising a polypeptide as defined in any one of claims 1 to 17 and a pharmaceutically acceptable excipient, diluent and/or carrier.

24. A polypeptide or composition according to claim 22 or claim 23, wherein the polypeptide or composition is for use as a blood anticoagulant.

25. A polypeptide or composition according to any one of claims 22 to 24, wherein the polypeptide or composition is for use in treating a patient suffering from, or at risk of suffering from, a condition that involves the formation of unwanted blood clots .

26. Use of a polypeptide as defined in any one of claims 1 to 17 in the manufacture of a medicament for use as a blood anticoagulant .

27. Use according to claim 26, wherein the medicament is for treating a patient suffering from, or at risk of suffering from, a condition that involves the formation of unwanted blood clots.

28. A polypeptide, composition, or use according to any one of claims 22 to 27, wherein the polypeptide, composition or medicament is for treating a condition selected from the group consisting of vascular occlusion, thrombosis, deep vein thrombosis, venous thrombosis, pulmonary embolism, mycocardial infarction, and stroke.

29. A polypeptide, composition, or use according to any one of claims 22 to 28, wherein the polypeptide, composition or medicament is for administration with a compound selected from the group consisting of Coumarin, Warfarin, Acenocoumarol, Phenprocoumon, Phenidione, Heparin, Fondaparinux, Idraparinux, Argatroban, Lepirudin, Bivalirudin, Ximelagatran, and Acetylsalicylic acid.

30. Products containing a polypeptide as defined in any one of claims 1 to 17 and a compound selected from the group consisting of Coumarin, Warfarin, Acenocoumarol, Phenprocoumon, Phenidione, Heparin, Fondaparinux, Idraparinux, Argatroban, Lepirudin, Bivalirudin, Ximelagatran, and Acetylsalicylic acid, as a combined preparation for simultaneous, separate or sequential use in treating a patient suffering from, or at risk of suffering from, a condition that involves the formation of unwanted blood clots.

31. Use of a polypeptide as defined in any on of claims 1 to 17 as a blood anticoagulant in a method of medical treatment.

32. A method of treating a patient suffering from, or at risk of suffering from, a condition that involves the formation of unwanted blood clots, which method comprises administering a polypeptide as defined any one of claims 1 to 17 to the patient .

33. A method according to claim 32, wherein the condition is selected from the group consisting of vascular occlusion, thrombosis, deep vein thrombosis, venous thrombosis, pulmonary embolism, mycocardial infarction, and stroke.

34. A method according to claim 32 or claim 33, wherein the method comprises administering a compound selected from the group consisting of Coumarin, Warfarin, Acenocoumarol, Phenprocoumon, Phenidione, Heparin, Fondaparinux, Idraparinux, Argatroban, Lepirudin, Bivalirudin, Ximelagatran, and Acetylsalicylic acid to the patient.

35. A kit for treating a patient suffering from, or at risk of suffering from, a condition that involves the formation of unwanted blood clots, which kit comprises:

(i) a container comprising a composition comprising a polypeptide as defined in any one of claims 1 to 17; and optionally

(ii) instructions for administering the composition to the patient.

36. A polypeptide, composition, use, method or kit according to any one of claims 25 to 35, wherein the patient is a human.

37. An anticoagulant comprising a polypeptide as defined in any one of claims 1 to 17.