Anticoagulant protein molecules and uses thereof

Abstract

Protein molecules comprising a variant factor Xa (fXa) or fragments thereof and the use of such protein molecules in the treatment of disease, such as a thrombin-mediated condition, methods of detecting the presence of fVa and a kit comprising the protein molecules of the invention.

Description

[0001] Anticoagulant Protein Molecules and Uses Thereof

[0002] Field of the Invention

[0003] This invention relates to protein molecules that bind to factor (f) Va and that modulate the generation of thrombin.

[0004] Introduction

[0005] Blood coagulation is a key process in the prevention of bleeding from damaged blood vessels (hemostasis). However, a blood clot that obstructs the flow of blood through a vessel (thrombosis) or breaks away to lodge in a vessel elsewhere in the body (thromboembolism) can be a serious health threat.

[0006] Thrombin is the enzyme that causes blood to clot. It is generated from its precursor prothrombin through the cleavage of two bonds by the enzyme complex known as prothrombinase. The protein components of prothrombinase, the cofactor fVa and the serine protease fXa, depend on the presence of Ca2+and a negatively-charged phospholipid (PL) membrane surface to assemble and function. In the absence of such surfaces, fXa and fVa do not assemble and prothrombin processing is inefficient. Hemostasis requires rapid and localized thrombin generation to activate platelets and convert fibrinogen into a fibrin mesh, the two components of a hemostatic clot. This process, however, must be regulated to limit how much thrombin is produced to avoid excessive blood coagulation, or thrombosis. Pathological blood coagulation is that which obstructs blood flow through a blood vessel.

[0007] A number of anticoagulant therapies are available to treat pathological blood coagulation. A common drawback of these therapies is an increased risk of bleeding (Mackman (2008) Nature 451 (7181): 914-918). Anticoagulant agents typically have a narrow therapeutic window between the dose that prevents thrombosis and the dose that induces bleeding. This window is often further restricted by variations in the response in individual patients.

[0008] All currently approved anticoagulants are small molecules (as opposed to proteins) with relatively short half-lives. As a result, clinically acceptable separation of the desirable effects on thrombosis and undesirable effects on hemostasis are typically achieved by careful doseselection (e.g. frequent administration of a low dose). The narrow therapeutic window often results in a dosing regimen where the agent is incompletely effective as an anti-thrombotic, but with an acceptable increase in risk of bleeding. The result is that current anticoagulants treatments are neither fully effective nor completely safe. Summary of the Invention

[0009] The present invention relates to protein molecules that modulate the production of thrombin by targeting the interaction between fXa and fVa, in a manner that preferentially inhibits thrombosis without promoting bleeding. Such protein molecules may be useful in the treatment and prevention of thrombosis, thromboembolism and other conditions mediated by pathological blood coagulation.

[0010] Modified protein molecules that bind fVa but not to the pro-cofactor fV and inhibit the formation and activity of the prothrombinase complex (agents according to the present invention) inhibit generation of thrombin without promoting or substantially promoting bleeding or hemorrhage, i.e. the protein molecules do not inhibit or substantially inhibit normal physiological responses to vascular injury (i.e. hemostasis). For example, hemostasis may not be inhibited or may be minimally inhibited by the protein molecules (i.e. inhibited to an insignificant extent which does not affect the well-being of patient or require further intervention). Bleeding may not be increased or may be minimally increased by the protein molecules.

[0011] Factor V is a well-understood component of the blood coagulation system (see for example Nesheim ME, Katzmann JA, Tracy PB, Mann KG. Factor V. Methods Enzymol. 1981 ;80 Pt 0:249-74 and Stefano Duga 1 , Rosanna Asselta, Maria Luisa Tenchini. Coagulation factor V. Int J Biochem Cell Biol. 2004 Aug;36(8):1393-9). It is a large protein synthesized principally by the liver, and secreted into the blood. It is produced and secreted as a single-chain precursor that has no ascribed biological activity. It consists of six separate domains: three A domains (A1 , A2 and A3), a B domain and two C domains (C1 and C2), in the order A1-A2-B-A3-C1-C2, from N-to-C terminus. The A domains mediate interactions with fXa and prothrombin; the B domain is responsible for conferring biological latency on the precursor protein fV and is excised upon activation to fVa; and the C domains are responsible for binding to the PL surface.

[0012] Factor V is activated to fVa by proteolytic excision of the B-domain by either fXa or by thrombin. The principal activator of fV is believed to be thrombin, which acts in a positive feedback to accelerate its own formation. Following the generation of small amounts of thrombin during the initiation stage of hemostasis, activation of fV by thrombin leads to a dramatic increase in the production of thrombin.

[0013] Consistent with an obligate role in hemostasis, homozygous deletion of Factor V in the mouse is incompatible with life (Cui, J., O’Shea, K.S., Purkayastha, A., Saunders, T.L. & Ginsburg, D. (1996) Fatal haemorrhage and incomplete block to embryogenesis in mice lacking coagulation factor V. Nature, 384, 66-68). Individuals who have very low levels of fV (due to genetic mutations) have a condition called Owren’s Parahemophilia (Asselta R, Peyvandi F. Factor V deficiency. Semin Thromb Hemost. 2009 Jun;35(4):382-9). Taken together, these observations demonstrate that fV (most likely due to the biological activity of fVa generated from it) is required for normal blood coagulation. However, the action of fVa also contributes to excessive production of thrombin that leads to thrombosis.

[0014] Thrombin activates fV to fVa by cleavage of the peptide bonds after residues 709, 1018 and 1545. This excises the B domain, leaving a heavy chain comprised of the A1 and A2 domains and a light chain comprised of the A3, C1 and C2 domains associated through non-covalent interactions. Residues 1 to 709 constitute the mature heavy chain of fVa and residues 1546 to 2196 constitute the mature light chain of fVa. The removal of the B domain results in the functional activation of fV by exposing the fXa binding site on the A2 and A3 domains. How the B-domain prevents binding of fXa to fV is not completely clear, but it requires the presence of acidic and a basic regions of the B-domain, so is not a simple case of steric hindrance (Rodney M Camire. A new look at blood coagulation factor V. Curr Opin Hematol. 2011 Sep;18(5):338- 42).

[0015] Under normal circumstances, the activity of the prothrombinase complex is terminated by further proteolytic cleavage of fVa C-terminal to residues 306 and 506 by the enzyme Activated Protein C (APC) (Esmon CT. The protein C anticoagulant pathway. Arterioscler Thromb. 1992 Feb; 12(2): 135-45). Individuals with variants in the gene encoding fV that confer resistance to APC cleavage (e.g. Factor V Leiden (Dahlback B. Early days of APC resistance and FV Leiden. Hamostaseologie. 2008;28(3):103-9); Factor V Cambridge (Williamson D, Brown K, Luddington R, Baglin C, Baglin T. Factor V Cambridge: a new mutation (Arg306-->Thr) associated with resistance to activated protein C. Blood. 1998 Feb 15;91 (4):1140-4); and Factor V Hong Kong (Chan WP, Lee CK, Kwong YL, Lam CK, Liang R. A novel mutation of Arg306 of factor V gene in Hong Kong Chinese. Blood. 1998 Feb 15;91 (4):1135-9)) display a pro-thrombotic phenotype. These observations provide further evidence that fVa plays a critical role in the balance between hemostasis and thrombosis.

[0016] To be useful in the present invention, the modified protein molecules must bind to fVa but not to any appreciable extent to fV, in a manner similar to fXa but in the absence of PL membranes. The modified protein molecules will thereby compete with fXa for binding to fVa. To date, no such fVa-specific protein molecule has been created and described in the scientific literature.

[0017] In some embodiments, a fVa-specific protein molecule may also bind to fVa from other nonhuman species. Factor Va sequences from other species are known in the art and available on public databases such as Genbank. The corresponding residues in fVa sequences from other species may be easily identified using sequence alignment tools. To be useful in the present invention, the fVa-selective protein molecules must additionally reduce or inhibit the function of the prothrombinase complex. For the avoidance of doubt, fVa- selective protein molecules that bind to fVa but have no effect on thrombin generation do not constitute agents according to the present invention. To inhibit the assembly and function of the prothrombinase complex, the fVa-selective protein molecules bind to fVa in a position that overlaps with that of fXa, and thereby competes.

[0018] In a preferred embodiment, the fVa-selective protein molecule no longer binds once fVa has been cleaved by APC.

[0019] In addition to the circulating pool of fV, fV and activated versions of fV are found in the secretory granules of platelets. Factor V is internalized from plasma into platelet-precursor cells, megakaryocytes, via the LRP-1 receptor (Bouchard BA, Meisler NT, Nesheim ME, Liu CX, Strickland DK, Tracy PB. A unique function for LRP-1 : a component of a two-receptor system mediating specific endocytosis of plasma-derived factor V by megakaryocytes. J Thromb Haemost. 2008 Apr;6(4):638-44). As a result, fV is present in both platelets (20%) and in plasma (80%). After uptake, fV is proteolytically processed inside the megakaryocyte / platelet, to yield a heterogeneous mixture that is active. The partially activated platelet pool of fV we denote as fVa*. The biological activity of fVa* is increased further upon exposure to thrombin, but only by 2-3 fold (compared to >100,000 fold increase for fV) ( D D Monkovic 1 , P B Tracy. Functional characterization of human platelet-released factor V and its activation by factor Xa and thrombin. J Biol Chem. 1990 Oct 5;265(28):17132-40). Thus, the pool of fV in platelets is pre-activated. Platelet fVa* is contained in alpha granules that are secreted at the site of vascular damage after platelets bind to extracellular collagen (Manon-Jensen T, Kjeld NG, Karsdal MA. Collagen- mediated hemostasis. J Thromb Haemost. 2016 Mar;14(3):438-48).

[0020] The importance of platelet-derived fVa* for hemostasis has been suggested by observation in humans. A patient has been described with normal levels of plasma fV, but a 50% reduction in platelet fVa* (presumably as a result of a defect in receptor-mediated uptake into megakaryocytes), who demonstrated a moderate bleeding phenotype (Platelet Factor V New York; Weiss et al 2001 AJH 2001 ;66:130). Cases of acquired fV deficiency have also been described. The development of fV inhibitors (antibodies against fV) is rare but occurs regularly, with an estimated annual incidence of 0.09-0.29 cases per million person-years (Favaloro EJ, Posen J, Ramakrishna R, Solatani S, McRae S, Just S, Aboud M, Low J, Gemmell R, Kershaw G, Coleman R,Deam M. Factor V inhibitors: rare or not so uncommon? A Multi-Laboratory Investigation Blood Coagul Fibrinolysis 2004;8: 637-47; and Ang A, Kuperan P, Ng C, Ng HJ. Acquired factor V inhibitor. A problem-based systematic review. J Thromb Haemost 2009; 101 : 852-9), and is usually associated with either aminoglycoside antibiotic administration, blood transfusions, cancer, autoimmune disorders or exposure to bovine thrombin (which contains trace amounts of bovine fV) during surgery. Nevertheless, there have been more than 150 case reports in the published literature of individual patients who have developed antibodies against fV (reviewed in P Knobl, K Lechner. Acquired factor V inhibitors. Baillieres Clin Haematol. 1998 Jun;11 (2):305-18).

[0021] In approximately 80% of the cases, the occurrence of fV inhibitory antibodies is associated with episodes of bleeding, ranging from mild to severe (Franchini M, Lippi G. Acquired factor V inhibitors: a systematic review. J Thromb Thrombolysis. 201 1 May;31 (4):449-57). The hallmark of circulating fV inhibitors is a prolongation of clotting time in PT (prothrombin time) and APTT (activated partial thromboplastin time), but not TT (thrombin time) assays using platelet poor plasma that is not corrected by 1 :1 mixture with pooled normal plasma. Importantly, the risk of bleeding and severity of bleeding does not correlate with the extent of prolongation of PT or APTT or with antibody titer. In addition, some patients with prolongation of both PT and APTT together with a high titer of fV antibody do not bleed.

[0022] How the fV inhibitory antibodies bind to fV is not known, but in most cases the result is the rapid and near complete reduction in the levels of fV in the plasma. It has been hypothesized that the complete loss of fV in the plasma is insufficient to cause bleeding due to the presence of the platelet pool of fVa*. Platelets contain fV and fVa* and the half-life of platelets in the blood is approximately 10 days. It has been suggested that the platelet pool of fV / fVa* is all that is required for hemostasis (Beth A Bouchard, John Chapin, Kathleen E Brummel-Ziedins, Peter Durda, Nigel S Key, Paula B Tracy. Platelets and platelet-derived factor Va confer hemostatic competence in complete factor V deficiency. Blood. 2015 Jun 4;125(23):3647-50).

[0023] The structure of blood clots has been extensively studied. It has recently been demonstrated that the core of a hemostatic clot is rich in platelets and is dense in structure, relative to the outer part of the clot (Stalker TJ, Traxler EA, Wu J, Wannemacher KM, Cermignano SL, Voronov R, Diamond SL, Brass LF. Hierarchical organization in the hemostatic response and its relationship to the platelet-signaling network. Blood. 2013 Mar 7;121 (10):1875-85). The differences in density are reflected in permeability or porosity. The core is impermeable to protein molecules of molecular weight ~50kDa and larger. Without being bound by any theory, the fVa* secreted by platelets at the initiation stage of hemostasis will be protected from protein molecules of sufficiently high molecular weight, whereas newly formed fVa in the outer layers of a clot will be accessible to the same protein molecules. A protein molecule of ~50kDa that binds to fVa and inhibits its interaction with fXa should preferentially inhibit the growth of a clot into the lumen of the blood vessel without affecting the stability of the core of the clot. Such a protein molecule would prevent excessive growth of the clot without affecting hemostasis. In the present invention, we demonstrate that a fVa binding protein molecule can inhibit the activity of the prothrombinase complex to deliver antithrombotic efficacy, without causing excessive bleeding in animal models. Such an agent may therefore be an effective treatment for pathological blood coagulation without causing an increase in risk of bleeding.

[0024] An aspect of the invention relates to a protein molecule comprising a variant human factor Xa (fXa) or a fragment thereof, wherein the protein molecule binds to fVa with higher affinity compared to the binding affinity of said protein molecule to fV, wherein the variant fXa or a fragment thereof comprises one or more amino acid substitutions compared to wild-type fXa.

[0025] An aspect of the invention relates to a pharmaceutical composition comprising a protein molecule according to the invention and a pharmaceutically acceptable excipient.

[0026] An aspect of the invention relates to a protein molecule or pharmaceutical composition according to the invention for use in therapy, such as for use in the treatment of a thrombin-mediated condition.

[0027] An aspect of the invention relates to a method of treating a thrombin-mediated condition comprising a therapeutically effective amount of the protein molecule or pharmaceutical composition according to the invention, to a subject in need thereof.

[0028] An aspect of the invention relates to the use of a protein molecule or pharmaceutical composition according to the invention in the manufacture of a medicament for the treatment of a thrombin- mediated condition.

[0029] An aspect of the invention relates to a method for reducing or inhibiting thrombin generation comprising: contacting a biological sample with a protein molecule according to the invention.

[0030] An aspect of the invention relates to a method for reducing or inhibiting coagulation comprising: contacting a biological sample with a protein molecule according to the invention.

[0031] An aspect of the invention relates to a method of detecting the presence of fVa in a biological sample comprising: contacting a biological sample with a protein molecule according to the invention, detecting binding of said protein molecule to fVa.

[0032] An aspect of the invention relates to a kit comprising the protein molecule according to the invention, and optionally instructions for use. Figures

[0033] The invention is further described in the following non-limiting figures.

[0034] Figure 1. Model of human prothrombinase complex based on crystal structure of pseutarin C (Lechtenberg BC, Murray-Rust TA, Johnson DJ, Adams TE, Krishnaswamy S, Camire RM, Huntington JA, 2013, Crystal structure of the prothrombinase complex from the venom of Pseudonaja textilis, Blood. 122(16):2777-83. And Pomowski A, Ustok Fl, Huntington JA, 2014, Homology model of human prothrombinase based on the crystal structure of Pseutarin C, Biol Chem. 395(10):1233-41 .). (A) Complex assembly is dependent on phospholipid membranes (depicted as grey and red sticks). The domains of fVa are colored blue for A1 , green for A2, yellow for A3, orange for C1 and red for C2. fXa is cyan.

[0035] Figure 2. Steady-state binding curves of fXa, truncated (EGF2-SP) and full-length HopD to fVa (left panel) and fV (right panel). (A) fXa binds to fVa with a Kd of 1 .4 |j.M and not at all to fV. (B) Truncated Hop D (comprised of the EGF2 and serine protease [SP] domains) binds to fVa and fV with identical high affinity (Kd or approximately 20nM; curve fits to fVa take into account a substantial non-specific component, unlike curves for fV which fit to a one site specific binding equation). (C) The addition of the Gia and EGF1 domain in full-length HopD has little influence on binding affinity for either fVa or fV.

[0036] Figure 3. Factor Xa and HopD domain organization, chimeras and mutations. (A) Wildtype human factor Xa (hfXa WT) and HopD share a domain organization of Gla-EGF1-EGF2- SP domains, from N- to C-terminus. Schematics are shown. The light chain, comprising Gla- EGF1-EGF2, is connected to the heavy chain, comprising the SP domain, via a disulfide bridge (dashed line). Each individual domain was swapped for that of HopD sequentially from the C- terminus, so that Gia EGF1 EGF2 SP represents wild-type HopD. Loop swap chimeras are represented, and the double (L23) and triple (L123) chimeras were also assessed with the swap of the entire EGF2 domain from HopD. (B) Sequence alignment of WT hfXa and EGF2 L123 from (A), with regions indicated, reveal a total of 45 amino acid changes (M45). (C) Sequence alignment of WT hfXa with M17.

[0037] Figure 4. Steady-state binding curves and calculated Kds (nM) for fXa / HopD chimeras to tVBD: (A) hfXa WT; (B) SP; (C) EGF2 SP; (D) EGF1 EGF2 SP; (E) WT HopD (Gia EGF1 EGF2 SP). Curves fit well to a one site specific binding equation due to the use of recombinant B- domainless fV (IVBD), which is as active as fVa.

[0038] Figure 5. Steady-state binding curves and calculated Kds (nM) for fXa / HopD chimeras and M17 vairant to fVBD: (A) hfXa WT and individual loop swaps, as indicated; (B) combined loop swaps L23 and L123; (C) full EGF2 swap with combined loop swaps L23 and L123; (D) M17. Figure 6. Raw data from Octet Red BLI instrument demonstrating a lack of interaction between M17 and fV. Streptavidin tips were loaded with M17 inhibited with biotinylated EGRCK and washed to baseline. Association was monitored in a well containing 64nM fV, followed by dissociation in buffer (inset). No evidence of association or dissociation was observed, indicating very weak or no binding.

[0039] Figure 7. Steady-state binding curves and calculated Kds (nM) for fXa / HopD variants to fVBD: (A) M13 variant with EGF2 and L23 mutations from M17, but without the 4 L1 mutations; (B) M7 variant with only the EGF2 mutations and a WT SP domain.

[0040] Figure 8. HopD competes with human fXa for fVa binding. (A) SDS gel of prothrombin processing by human prothrombinase (fXa and fVa with phospholipid vesicles and prothrombin) with increasing concentrations of EGRCK-inhibited truncated HopD (EGF2 SP; as indicated). (B) Densitometry of band H (heavy chain) plotted as fraction of H with no EGRCK-HopD (Fxn initial activity) against EGRCK-HopD concentration, as indicated. Calculated IC50 value was 3.6 nM. (C) Thrombin activity generation measured by ability to cleave chromogenic substrate S- 2238, with the value given as slope of absorbance units over time (mAU / min). Inhibition of thrombin activity generation with increasing concentration of EGRCK-HopD results in a fitted IC50 of 1.9 nM.

[0041] Detailed Description

[0042] The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0043] Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, molecular biology and protein and nucleic acid chemistry described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012).

[0044] Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0045] Protein Molecules

[0046] The present invention relates to a protein molecule comprising a variant factor Xa (fXa) or a fragment thereof, wherein the protein molecule binds to factor Va (fVa) with higher affinity compared to the binding affinity of said protein molecule to factor V (fV), wherein the variant fXa or a fragment thereof comprises one or more amino acid substitutions compared to wild-type fXa.

[0047] The protein molecules of the invention are designed to reduce coagulation with a number of advantageous effects. The protein molecules of the invention bind specifically to fVa, e.g., the molecules of the invention bind to fVa with high affinity and bind to fV with low affinity, in this way the molecules of the invention do not deplete fV from plasma or platelets. The molecules of the invention compete for the fXa binding site of fVa to inhibit thrombin generation. The molecules of the invention are also capable of producing an antithrombotic effect without increasing bleeding i.e., without perturbing hemostasis. Without wishing to be bound by theory the present inventors hypothesize that, wherein a hemostatic clot begins to form the platelet secreted fVa* would be protected from interacting with the protein molecule of the invention, however any newly generated fVa at the surface of the clot would more susceptible to inhibition by the protein molecule. This mode of action will reduce the risk of bleeding whilst reducing excessive thrombin generation.

[0048] Factor Xa is the active serine protease version of the zymogen fX and is composed of four domains: an N-terminal gamma-carboxyglutamic acid (Gia) domain which mediates the interaction of factor X / Xa with phospholipid membranes; two epidermal growth factor-like (EGF) domains, denoted EGF1 and EGF2; and a chymotrypsin family serine protease (SP) domain. In the presence of calcium ions, fXa forms a phospholipid-bound complex with fVa to form the ‘prothrombinase’ complex which activates prothrombin into thrombin. The molecules of the invention comprise variants of fXa. The term “factorXa (fXa)” as used herein refers to a molecule comprising a light chain such as SEQ ID NO: 3 or a fragment thereof and a heavy chain such as SEQ ID NO:4 or a fragment thereof.

[0049] SEQ ID NO: 3

[0050] ANSFLEEMKKGHLERECMEETCSYEEAREVFEDSDKTNEFWNKYKDGDQCETSPCQNQGK CKDGLGEYTCTCLEGFEGKNCELFTRKLCSLDNGDCDQFCHEEQNSVVCSCARGYTLADNG KACIPTGPYPCGKQTLERRKR SEQ ID NO:4

[0051] IVGGQECKDGECPWQALLINEENEGFCGGTILSEFYILTAAHCLYQAKRFKVRVGDRNTEQEE GGEAVHEVEVVIKHNRFTKETYDFDIAVLRLKTPITFRMNVAPACLPERDWAESTLMTQKTGIV SGFGRTHEKGRQSTRLKMLEVPYVDRNSCKLSSSFIITQNMFCAGYDTKQEDACQGDSGGP HVTRFKDTYFVTGIVSWGEGCARKGKYGIYTKVTAFLKWIDRSMKTRGLPKAKSHAPEVITSS PLK

[0052] The light chain comprises a gamma-carboxyglutamic acid (Gia) domain (SEQ ID NO: 5), EGF1 domain (SEQ ID NO: 6), and EGF2 domain (SEQ ID NO: 7). The heavy chain comprises a serine protease (SP) domain. Factor Xa can undergo autolysis to remove the peptide SHAPEVITSSPLK from the very C-terminus (the p-peptide), resulting in a new C-terminus ending with PKAK (Kinetics of Blood Coagulation Factor Xaa Auto proteolytic Conversion to Factor Xab J. Biol. Chem. Vol. 271 , No. 28, Issue of July 12, pp. 16621-16626, 1996 Edward L. G. Pryzdial^: and Garry E. Kessler), and recombinant versions are often expressed without the p-peptide.

[0053] The term variant fXa refers to a molecule capable of binding fVa, wherein the molecule comprises one or more amino acid modification, substitution, or deletion compared to the wildtype fXa sequence. The variant fXa may be a result of substituting, replacing, or modifying the original (e.g., wild-type or germline) amino acid, within a protein sequence, with a different amino acid. The process of substituting or replacing an amino acid can be done using standard techniques available to the skilled person, e.g., using recombinant DNA technology. Modification of an amino acid may be performed post-translationally and a variety of chemical or bioconjuagtion methods may be used to modify said amino acid. The amino acids are changed relative to the native (wild type I germline) sequence as found in nature in the wild type (WT). By “wild type” or “WT” herein is meant an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations. A wild-type protein or polypeptide has an amino acid sequence or a nucleotide sequence that has not been intentionally modified. In an embodiment the one or more amino acid modification, substitution, or deletion compared to the wild-type sequence enhances the affinity of the protein molecule for fVa compared to fV. In an embodiment the variant fXa comprises at least an EGF2 domain and a serine protease domain. In an embodiment the variant fXa comprises one or more amino acid modification, substitution, or deletion compared to the wild-type EGF2 domain as set out in SEQ ID NO: 7. In an embodiment the variant fXa comprises one or more amino acid modification, substitution, or deletion compared to the wild-type serine protease domain as set out in SEQ ID NO:4.

[0054] The term “amino acid” as used herein refers to one of the 20 naturally occurring (canonical) amino acids or any non-natural analogues (non-canonical amino acids) that may be present at a specific, defined position within a peptide sequence. “Amino acid” encompasses both naturally occurring and synthetic amino acids. Although in most cases, when the protein is to be produced recombinantly, only naturally occurring amino acids are used. The variant amino acid may comprise one of the twenty canonical amino acids. The variant amino acid may comprise a non- canonical amino acid, also known are non-natural amino acids for example hydroxyproline, hydroxylysine, phosphoserine, phosphothreonine, phosphotyrosine, N-acetyl lysine, methyllysine.

[0055] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Peptides, oligopeptides, dimers, multimers, and the like, are also composed of linearly arranged amino acids linked by peptide bonds, and whether produced biologically, recombinantly, or synthetically and whether composed of naturally occurring or non-naturally occurring amino acids, are included within this definition. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include co-translational and post-translational modifications of the polypeptide, such as, for example, disulfide-bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., cleavage by furins or metalloproteases and prohormone convertases (PCs)), and the like. Furthermore, for purposes of the present invention, a “polypeptide” encompasses a protein that includes modifications, such as deletions, additions, substitutions and post-translational modifications (generally conservative in nature as would be known to a person in the art), to the native sequence, as long as the protein maintains the desired activity. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts that produce the proteins, or errors due to PCR amplification or other recombinant DNA methods. Polypeptides or proteins are composed of linearly arranged amino acids linked by peptide bonds, but in contrast to peptides, have a well- defined conformation. The protein molecules of the invention are capable of binding to fVa, and as such may be referred to herein as “binding molecules”.

[0056] Proteins, as opposed to peptides, generally consist of chains of 50 or more amino acids. For the purposes of the present invention, the term “peptide” as used herein typically refers to a sequence of amino acids of made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds. Generally, peptides contain at least two amino acid residues and are less than about 50 amino acids in length.

[0057] In certain embodiments the variant fXa or fragment thereof comprises an EGF2 domain which is a variant of SEQ ID NO: 7, or a fragment thereof and / or a heavy chain serine protease (SP) domain which is a variant of SEQ ID NO: 4, or a fragment thereof. The EGF2 domain may comprise one or more amino acid substitution, modification and / or deletion. The heavy chain serine protease domain may comprise one or more amino acid substitution, modification and / or deletion. The variant fXa may comprise one or more amino acid substitution, modification and / or deletion present in the EGF2 domain compared to SEQ ID NO: 7 and / or one or more amino acid substitution, modification and / or deletion in the heavy chain SP domain compared to SEQ ID NO: 4.

[0058] The one or more amino acid substitution, modification and / or deletion may be present in specific regions of the heavy chain SP domain. For example the one or more amino acid substitution, modification and / or deletion may be present in region 1 of the heavy chain SP domain as defined by residues 112 to 123 of SEQ ID NO: 4, region 2 of the heavy chain SP domain as defined by residues 153 to 165 of SEQ ID NO: 4, region 3 of the heavy chain SP domain as defined by residues 222 to 235 of SEQ ID NO: 4. Region 1 of the heavy chain SP domain may be defined as the sequence RDWAESTLMTQK (SEQ ID NO: 11). Region 2 of the heavy chain SP domain may be defined as the sequence RNSCKLSSSFI (SEQ ID NO: 12). Region 3 of the heavy chain SP domain may be defined as the sequence TAFLKWIDRSMKTR (SEQ ID NO: 13). Region 1 of the heavy chain SP domain may also be referred to as residues 125 to 134 where the SP domain is numbered according to chymotrypsin numbering, also referred to as the 130-helix (Padmanabhan et al., J. Mol. Biol. (1993) 232, 947-966). Region 2 of the heavy chain SP domain may also be referred to as residues 165 to 175 when the SP domain is numbered according to chymotrypsin numbering, also referred to as the 170-helix. Region 3 of the heavy chain SP domain may also be referred to as residues 232 to 245 when the SP domain is numbered according to chymotrypsin numbering also referred to as the C-terminal helix and is part of the heparin binding site of fXa. Where chymotrypsin numbering has been used the first residue of SEQ ID NO: 4 is referred to as residue number 16, and loops were insertions are present relative to chymotrypsin are denoted with numbers followed by letters (e.g. 60, 60a, 60b, etc).

[0059] The present inventors have determined that the EGF2 domain and the SP domain are the most important domains for modulating interaction with fVa, as such the variant fXa may comprise an EGF2 domain and a serine protease domain. In some embodiments additional domains are present in the variant fXa or fragment thereof, for example the GLA domain and / or the EGF1 domain may be present. In an embodiment the variant fXa comprises a variant light chain (SEQ ID NO: 3) and / or a variant heavy chain (SEQ ID NO: 4), wherein the one or more amino acid substitution modification and / or deletion is present at one or more positions in the light chain and / or heavy chain.

[0060] In some embodiments the variant fXa or fragment thereof comprises one or more of: a) an amino acid substitution at position 83 of SEQ ID NO: 3; b) an amino acid substitution at position 84 of SEQ ID NO: 3; c) an amino acid substitution at position 85 of SEQ ID NO: 3; d) an amino acid substitution at position 86 of SEQ ID NO: 3; e) an amino acid substitution at position 87 of SEQ ID NO: 3; f) an amino acid substitution at position 88 of SEQ ID NO: 3; g) an amino acid substitution at position 90 of SEQ ID NO: 3; h) an amino acid substitution at position 91 of SEQ ID NO: 3; i) an amino acid substitution at position 92 of SEQ ID NO: 3; j) an amino acid substitution at position 101 of SEQ ID NO: 3; k) an amino acid substitution at position 102 of SEQ ID NO: 3; l) an amino acid substitution at position 103 of SEQ ID NO: 3; m) an amino acid substitution at position 104 of SEQ ID NO: 3; n) an amino acid substitution at position 105 of SEQ ID NO: 3; o) an amino acid substitution at position 116 of SEQ ID NO: 4; p) an amino acid substitution at position 117 of SEQ ID NO: 4; q) an amino acid substitution at position 118 of SEQ ID NO: 4; r) an amino acid substitution at position 121 of SEQ ID NO: 4; s) an amino acid substitution at position 123 of SEQ ID NO: 4; t) an amino acid substitution at position 154 of SEQ ID NO: 4; u) an amino acid substitution at position 157 of SEQ ID NO: 4; v) an amino acid substitution at position 161 of SEQ ID NO: 4; w) an amino acid substitution at position 163 of SEQ ID NO: 4; x) an amino acid substitution at position 223 of SEQ ID NO: 4; or y) an amino acid substitution at position 229 of SEQ ID NO: 4.

[0061] Any combination of the aforementioned amino acid substitutions may be present in the variant fXa.

[0062] In some embodiments the variant fXa or fragment thereof comprises one or more of; a) an amino acid substitution at position 83 of SEQ ID NO: 3 to K; b) an amino acid substitution at position 84 of SEQ ID NO: 3 to V; c) an amino acid substitution at position 85 of SEQ ID NO: 3 to L; d) an amino acid substitution at position 86 of SEQ ID NO: 3 to Y or F; e) an amino acid substitution at position 87 of SEQ ID NO: 3 to Q; f) an amino acid substitution at position 88 of SEQ ID NO: 3 to S; g) an amino acid substitution at position 90 of SEQ ID NO: 3 to R; h) an amino acid substitution at position 91 of SEQ ID NO: 3 to A or V; i) an amino acid substitution at position 92 of SEQ ID NO: 3 to F; j) an amino acid substitution at position 101 of SEQ ID NO: 3 to K; k) an amino acid substitution at position 102 of SEQ ID NO: 3 to R; l) an amino acid substitution at position 103 of SEQ ID NO: 3 to V; m) an amino acid substitution at position 104 of SEQ ID NO: 3 to W or F; n) an amino acid substitution at position 105 of SEQ ID NO: 3 to S, F, R, L, V; o) an amino acid substitution at position 116 of SEQ ID NO: 4 to N; p) an amino acid substitution at position 117 of SEQ ID NO: 4 to E; q) an amino acid substitution at position 118 of SEQ ID NO: 4 to A; r) an amino acid substitution at position 121 of SEQ ID NO: 4 to K; s) an amino acid substitution at position 123 of SEQ ID NO: 4 to D; t) an amino acid substitution at position 154 of SEQ ID NO: 4 to H; u) an amino acid substitution at position 157 of SEQ ID NO: 4 to M; v) an amino acid substitution at position 161 of SEQ ID NO: 4 to D; w) an amino acid substitution at position 163 of SEQ ID NO: 4 to R; x) an amino acid substitution at position 223 of SEQ ID NO: 4 to R; or y) an amino acid substitution at position 229 of SEQ ID NO: 4 to K.

[0063] Any combination of the aforementioned amino acid substitutions may be present in the variant fXa. Where the amino acid substation is defined as a substitution at a certain position to a certain amino acid, this indicates that the wild-type or original amino acid has been replaced with the indicated amino acid.

[0064] In some embodiments the variant fXa or fragment thereof comprises one or more of; a) L83Kaccording to SEQ ID NO: 3; b) F84V according to SEQ ID NO: 3; c) T85L according to SEQ ID NO: 3; d) R86Y according to SEQ ID NO: 3; e) K87Q according to SEQ ID NO: 3; f) L88S according to SEQ ID NO: 3; g) S90R according to SEQ ID NO: 3; h) L91 A or L91V according to SEQ ID NO: 3; i) D92F according to SEQ ID NO: 3; j) H101 K according to SEQ ID NO: 3; k) E102R according to SEQ ID NO: 3; l) E103V according to SEQ ID NO: 3; m) Q104W or Q104F according to SEQ ID NO: 3; n) N105S, N105F, N105R, N105L, or N105V according to SEQ ID NO: 3; o) E116N according to SEQ ID NO: 4; p) S117E according to SEQ ID NO: 4; q) T118A according to SEQ ID NO: 4; r) T121 K according to SEQ ID NO: 4; s) K123D according to SEQ ID NO: 4; t) N154H according to SEQ ID NO: 4; u) K157M according to SEQ ID NO: 4; v) S161 D according to SEQ ID NO: 4; w) I163R according to SEQ ID NO: 4; x) A223R according to SEQ ID NO: 4; or y) D229K according to SEQ ID NO: 4.

[0065] Any combination of the aforementioned amino acid substitutions may be present in the variant fXa.

[0066] In an embodiment the variant fXa or fragment thereof comprises one or more of; a) an amino acid substitution at position 86 of SEQ ID NO: 3; b) an amino acid substitution at position 90 of SEQ ID NO: 3; c) an amino acid substitution at position 102 of SEQ ID NO: 3; d) an amino acid substitution at position 104 of SEQ ID NO: 3; e) an amino acid substitution at position 105 of SEQ ID NO: 3; f) an amino acid substitution at position 116 of SEQ ID NO: 4; g) an amino acid substitution at position 117 of SEQ ID NO: 4; h) an amino acid substitution at position 118 of SEQ ID NO: 4; i) an amino acid substitution at position 121 of SEQ ID NO: 4; j) an amino acid substitution at position 123 of SEQ ID NO: 4; k) an amino acid substitution at position 161 of SEQ ID NO: 4; l) an amino acid substitution at position 163 of SEQ ID NO: 4; or m) an amino acid substitution at position 223 of SEQ ID NO: 4.

[0067] Any combination of the aforementioned amino acid substitutions may be present in the variant fXa.

[0068] In an embodiment the variant fXa or fragment thereof comprises one or more of; a) an amino acid substitution at position 86 of SEQ ID NO: 3 to Y; b) an amino acid substitution at position 90 of SEQ ID NO: 3 to R; c) an amino acid substitution at position 102 of SEQ ID NO: 3 to R; d) an amino acid substitution at position 104 of SEQ ID NO: 3 to W or F; e) an amino acid substitution at position 105 of SEQ ID NO: 3 to S, F, R, L or V; f) an amino acid substitution at position 116 of SEQ ID NO: 4 to N; g) an amino acid substitution at position 117 of SEQ ID NO: 4 to E; h) an amino acid substitution at position 118 of SEQ ID NO: 4 to A; i) an amino acid substitution at position 121 of SEQ ID NO: 4 to K; j) an amino acid substitution at position 123 of SEQ ID NO: 4 to D; k) an amino acid substitution at position 161 of SEQ ID NO: 4 to D; l) an amino acid substitution at position 163 of SEQ ID NO: 4 to R; or m) an amino acid substitution at position 223 of SEQ ID NO: 4 to R.

[0069] In an embodiment the variant fXa or fragment thereof comprises ; a) one or more of S90R, L91 A, D92F, H101 K, E102R, E103V, N105S, according to SEQ ID NO: 3, E116N, S117E. T121 K, K123D, N154H, K157M, S161 D, I163R, A223R, D229K, according to SEQ ID NO: 4; b) one or more of S90R, E102R according to SEQ ID NO: 3, E116N, S117E, T1 18A, T121 K, K123D according to SEQ ID NO: 4; c) one or more of R86Y, S90R, E102R according to SEQ ID NO: 3, E1 16N, S1 17E, T1 18A, T121 K, K123D according to SEQ ID NO: 4; or d) one or more of R86Y, S90R, E102R, Q104W, Q105R according to SEQ ID NO: 3, E116N, S117E, T118A, T121 K, K123D according to SEQ ID NO: 4

[0070] In certain embodiments the variant fXa comprises one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or a fragment or portion thereof.

[0071] The protein molecules of the invention are capable of specifically binding fVa in the absence of phospholipid membranes. “Specifically binds", "specific binding" or "selective binding" means that the binding is selective for fVa, and can be discriminated from unwanted or non-specific interactions. The ability of the protein molecule to bind to a specific target molecule can be measured either through an enzyme-linked immunosorbent assay (ELISA), biolayer interferometry as used in the Examples or other techniques familiarto one of skill in the art, e.g., surface plasmon resonance (SPR) technique (analyzed on a BIAcore instrument), and traditional binding assays. The binding reaction may be shown with reference to a negative control test using a binding molecule of unrelated specificity.

[0072] In embodiments the protein molecule according to the invention, demonstrates negligible binding to fV. Negligible binding may be defined as binding that is at least 50-fold less, at least 40-fold less, at least 30-fold less, at least 20-fold less, at least 15-fold less than the binding affinity of said molecule to fVa.

[0073] In some embodiments the protein molecule does not bind to fV e.g., the protein molecule demonstrates undetectable levels of binding to fV when assessed using standard laboratory techniques such as those set out in the Examples (biolayer interferometry) or surface plasmon resonance.

[0074] In an embodiment the protein molecule binds fVa with a dissociation constant of 200 nM or lower. In an embodiment the protein molecule binds fVa with a dissociation constant of at 20 nM or lower. The protein molecule may bind fVa with a dissociation constant of 200 nM to 20 pM, 200 nM to 50 pM, 200 nM to 100 pM, 200 nM to 200 pM, 200 nM to 400 pM 200 nM to 500 pM, 200 nM to 600 pM 200 nM to 800 pM, 200 nM to 1 nM, 200 nM to 10 nM, 200 nM to 20 nM, 200 nM to 50 nM, 200 nM to 100 nM, 20 nM to 20 pM, 20 nM to 50 pM, 20 nM to 100 pM, 20 nM to 200 pM, 20 nM to 500 pM, 20 nM to 800 pM, or 20 nM to 1 nM.

[0075] In an embodiment the protein molecule binds fV with a dissociation constant of 200 nM or higher. The protein molecule may bind fV with a dissociation constant of 200 nM to 200 pM, 200 nM to 150 pM, 200 nM to 100 pM, 200 nM to 50 pM, 200 nM to 20 pM, 200 nM to 10 pM, 200 nM to 5 pM, 200 nM to 1 pM, 200 nM to 800 nM, 200 nM to 600 nM, 200 nM to 400 nM. In some embodiments the protein molecule demonstrates undetectable levels of binding to fV.

[0076] In an embodiment the protein molecule binds fVa with a dissociation constant of 200 nM or lower and binds fV with a dissociation constant of 200 nM or higher, wherein there is a 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25-fold difference between the dissociation constant of the protein molecule for fVa and the dissociation constant of the protein molecule for fV. In an embodiment there is a 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25-fold difference between the dissociation constant of the protein molecule for fVa and the dissociation constant of the protein molecule for fV, preferably a 10 to 20-fold difference for example a 15-fold difference.

[0077] As described above, an advantage of the present protein molecules is that they are able to bind fVa without the requirement of PL membrane surfaces. In embodiments the protein molecule binds to fVa with high affinity in the absence of PL membrane surfaces. In an embodiment the protein molecule binds to fVa with higher affinity compared to the binding affinity of said protein molecule to fV in the absence of PL membrane surfaces. In the absence of PL membrane surfaces is used herein to indicate that the protein molecule is not bound to PL membrane and / or binding to PL membranes is not a requirement in order for the protein molecule to bind fVa with high affinity. In some embodiments the protein molecule does not bind PL membranes.

[0078] The protein molecule may comprise an additional moiety or modifications. The additional moiety or modification may provide additional functionality for example to provide favorable pharmacokinetic properties to said molecule. The further moiety may be selected from a halflife extending moiety and / or a label.

[0079] In some embodiments, the protein molecule may comprise a half-life extension moiety. The inclusion of a half-life extension moiety suitably increases the half-life of the protein molecule when compared to the same protein molecule that does not include a half-life extension moiety.

[0080] The term "half-life" as used herein refers to the time taken for the serum concentration of the amino acid sequence, compound or polypeptide to be reduced by 50%, in vivo, for example due to degradation of the sequence or compound and / or clearance or sequestration of the sequence or compound by natural mechanisms.

[0081] Half-life may be increased by at least 1 .5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding protein molecule without such modification. For example, increased half-life may be more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding antibodies without such modification. The in vivo half-life of the protein molecule or fragment thereof of the invention a can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art. Halflife can for example be expressed using parameters such as the t1 / 2-alpha t1 / 2-beta and the area under the curve (AUC). It will be appreciated by a person skilled in the art that reference to the half-life of a protein molecule may also refer to the half-life of the compounds or pharmaceutical compositions of the invention (and may be used interchangeably herein).

[0082] The additional moiety may be selected from one or more half-life extending moieties for example, one or more PEG molecules, a liposome, an Fc domain, a serum albumin protein, or an antibody or antibody fragment that binds serum albumin. The serum albumin may be human serum albumin. The protein molecule may be conjugated or linked to the additional moiety in any suitable manner. For example, additional moieties may be conjugated or linked to the protein molecule via N-terminal extension, C-terminal extension, amino acid side chains such as those comprising primary amines (e.g. lysine side chain), or thiol groups (e.g. cysteine side chains). In some embodiments the protein molecule is provided as a fusion protein comprising the variant fXa and the additional moiety, for example wherein the additional moiety is a half-life extending moiety such as an Fc domain, a serum albumin protein, or an antibody or antibody fragment that binds serum albumin.

[0083] The half-life extension moiety may comprise an antibody and / or antigen-binding fragment, a protein, and or a polypeptide. For example, the half-life extension moiety may comprise a serum albumin protein or an antibody or antigen-binding fragment that binds human serum albumin. For example, the half-life extension moiety may comprise an antibody or antigen-binding fragment that binds human serum albumin, For example an antigen-binding fragment that binds human serum albumin.

[0084] The half-life extending moiety may comprise an Fc region or domain or fragment thereof. The Fc domain may be modified to improve the half-life of the protein molecule. In an embodiment the Fc domain comprises one or more amino acid modification or substitution which increases half-life of said protein molecule, suitable modifications are known in the art. For example, the Fc region may be modified to enhance interaction with FcRn receptor and thereby improve halflife of the protein molecule. IgG naturally persists for a prolonged period in the serum due to FcRn-mediated recycling, giving it a typical half-life of approximately 21 days. Half-life can be extended by engineering the pH-dependant interaction of the Fc domain with FcRn to increase affinity at pH 6.0 while retaining minimal binding at pH 7.4. The T250Q / M428L variant, conferred an approximately 2-fold increase in IgG half-life (assessed in rhesus monkeys), while the M252Y / S254T / T256E variant, gave an approximately 4-fold increase in IgG half-life (assessed in cynomolgus monkeys). Extending half-life may allow the possibility of decreasing administration frequency, while maintaining or improving efficacy. The Fc region may be modified to reduce interaction with FcyR receptor and thereby improve half-life of the bispecific molecule. An example of a modification that may be made to improve half-life is introduction of M252Y / S254T / T256E mutations in IgG 1 CH2 domain, this modification is commonly referred to as a “YTE” variant.

[0085] The additional moiety for example the half-life extending moieties may be conjugated or linked to the protein molecule or variant fXa in any suitable manner. In an embodiment one or more variant fXa may be conjugated or linked to the half-life extending moiety. For example, where an Fc domain is used as the half-life extending moiety, two variant fXa molecules may be conjugated to said Fc domain. The variant Fc domain may be conjugated or linked to the one or more variant fXa via the variant fXa C-terminus.

[0086] The additional moiety may be a detectable or functional label. A label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorophores, fluorescers, radiolabels, enzymes, chemiluminescers, a nuclear magnetic resonance active label or photosensitizers. Thus, the binding may be detected and / or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance. The molecular label may be a fluorophore. Suitable fluorophores include fluorescein isothiocyanate, phycoerythrin, allophycocyaninlndocicarbocyanine, Indocarbocyanine, as well as those known by the trade names Alexa Fluor (such as 350, 405, 488, 532, 546, 568, 594, 647, 680, 700, 750) and DyLight (such as 405, 488, 550, 650, 680, 755, 800).

[0087] The molecular label may also be a biotin tag, derived from biotin. The labelling moiety may also be a radioisotope or a radioisotope containing moiety. Suitably, the labelling moiety is a positron emission tomography (PET) tracer. Suitable PET tracers include, for example, [18F] Fludeoxyglucose (18F) (FDG)-glucose analogue, [1 1 C] acetate, [11 C] methionine, [11 C] choline, copper Cu dotatate, [18F] EF5, [18F] fluciclovine, [18F] fluorocholine, [18F] fluoroethyl- L-tyrosine, [18F] fluoromisonidazole, [18F] fluorothymidine F-18, [64 Cu] Cu-ETS2, [68Ga] DOTA-pseudopeptides, [68Ga] DOTA-TATE and [68Ga] prostate-specific membrane antigen (PSMA). The additional moiety may also have the function of increasing the molecular weight of the protein molecule. Without wishing to be bound by theory it is hypothesized that increasing the molecular weight of the protein molecule may have advantageous effects, for example increasing the molecular weight may further restrict the protein molecule from entering the core of a clot. This results in the platelet secreted fVa* being less accessible to the protein molecule of the invention and not being inhibited, however any newly generated fVa at the surface of the clot would be inhibited by the binding protein molecule. This mode of action will reduce the risk of bleeding whilst reducing excessive thrombin generation. Additional moieties that may be used to increase the molecular weight of the protein molecule include but are not limited to one or more PEG molecules, a liposome, an Fc domain, a serum albumin protein, or a serum albumin binding molecule.

[0088] In some embodiments it may be advantageous to modify the variant fXa such that it is catalytically inactive. Any suitable method may be used to inactivate the variant fXa. For example, the catalytic triad of the serine protease may be modified by amino acid substitution, chemical modification or binding an inhibitor in the active site. In an embodiment the variant fXa comprises the amino acid substitution at position 185 according to SEQ ID NO: 4. In such an embodiment the catalytic serine has been substituted. Suitable amino acid substitutions include substituting the catalytic serine for A, R, N, D, E, Q, G, H, I, L, K, M, F, P, W, Y, or V. In an embodiment the amino acid substitution comprises S185A. Positions 185 may also be referred to as position 195 when using chymotrypsin numbering.

[0089] In an embodiment the variant fXa may be catalytically inactivated by the presence of a covalent inhibitor. Suitable covalent inhibitors include Glu-Gly-Arg-chloromethyl ketone (EGRCK) and D- Phe-Pro-Arg-chloromethyl ketone (PPACK). The covalent inhibitor may be bound in the active site of the variant fXa.

[0090] In an embodiment the variant fXa may comprise reduced affinity to tissue factor pathway inhibitor (TFPI) compared to wild-type fXa. Reduced affinity to TFPI may be achieved by introducing amino acid substitutions, modification and / or deletions which reduce TFPI affinity, suitable modifications are known in the art (A R Rezaie, C T Esmon. Contribution of residue 192 in factor Xa to enzyme specificity and function. J Biol Chem. 1995 Jul 7;270(27):16176-81 .). Suitable amino acid substitutions to reduce TFPI affinity include Q192E, wherein the position is numbered according to chymotrypsin numbering.

[0091] In an embodiment the variant fXa may comprise reduced reactivity with antithrombin (AT; ATIII; SERPINC1) compared to wild-type fXa. Reduced reactivity with antithrombin may be achieved by introducing amino acid substitutions, modification and / or deletions which reduce reactivity with antithrombin, suitable modifications are known in the art (Chandrashekhara Manithody, Likui Yang, Alireza R Rezaie. Role of basic residues of the autolysis loop in the catalytic function of factor Xa. Biochemistry. 2002 May 28;41 (21):6780-8). Suitable amino acid substitutions to reduce antithrombin reactivity include R150A or other substitutions, such as E, wherein the position is numbered according to chymotrypsin numbering.

[0092] An aspect of the invention relates to a nucleic acid that encodes the protein molecule of the invention as described above. The nucleic acid may encode the variant fXa as described above. In some embodiments the nucleic acid encodes a precursor molecule which is processed to form the variant fXa. In some embodiments the precursor molecule comprises a variant fX with a furin cleavage site and an activation peptide. The precursor molecule may be converted to the variant fXa by cleavage at the furin cleavage site and cleavage of the activation peptide. In some embodiments the nucleic acid encodes one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17.

[0093] An aspect of the invention relates to a pharmaceutical composition comprising a protein molecule as described herein and a pharmaceutically acceptable excipient.

[0094] The protein molecule of the invention may be provided as a pharmaceutical composition. The pharmaceutical composition may optionally comprise a pharmaceutically acceptable excipient, and / or adjuvant.

[0095] The pharmaceutical compositions described herein may comprise the protein molecule. The pharmaceutical compositions described herein can be administered by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitrial, intratumoural, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation, or topical, particularly to the ears, nose, eyes, or skin or by inhalation.

[0096] Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical, intra-articular or subcutaneous administration. In an embodiment, the compositions are administered parenterally.

[0097] The pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form. The term "carrier" refers to a diluent, adjuvant or excipient, with which a protein molecule of the present invention is administered. Suitable diluents, adjuvants and excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to an animal, the protein molecule of the present invention or compositions and pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when the drug antibody conjugates of the present invention are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0098] The pharmaceutical composition can be in the form of a liquid, e.g., a solution, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or for delivery by injection, infusion (e.g., IV infusion) or sub-cutaneous.

[0099] When intended for oral administration, the composition can be in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

[0100] As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following can be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, corn starch and the like; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the composition is in the form of a capsule (e. g. a gelatin capsule), it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

[0101] When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye / colorant and flavour enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.

[0102] Compositions can take the form of one or more dosage units. In specific embodiments, it can be desirable to administer the composition locally to the area in need of treatment, or by intravenous injection or infusion.

[0103] The amount of the protein molecule or pharmaceutical composition described herein that is effective / active in the treatment of a particular disease or condition will depend on the nature of the disease or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disease, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account.

[0104] Therapy

[0105] The protein molecule and pharmaceutical compositions of the invention have anticoagulant activity therefore they have use in therapy.

[0106] An aspect of the invention relates to a protein molecule or a pharmaceutical composition as described herein for use in the treatment of a thrombin-mediated condition.

[0107] An aspect of the invention relates to a method of treating a thrombin-mediated condition comprising a therapeutically effective amount of the protein molecule or pharmaceutical composition as described herein to a subject in need thereof.

[0108] An aspect of the invention relates to the use of a protein molecule or pharmaceutical composition as described herein, in the manufacture of a medicament for the treatment of a thrombin- mediated condition.

[0109] The molecules of the invention can be used to inhibit thrombin production and so have utility in thrombin-mediated conditions. The thrombin-mediated condition is selected from thrombosis, embolism, inflammation, clotting disorders, and thrombophilia. Thrombin-mediated conditions may also include thrombotic conditions such as thrombophilia, thrombotic stroke and coronary artery occlusion. In an embodiment the molecules of the invention have utility in stroke prevention in atrial fibrillation. Thrombin-mediated conditions may also include non-thrombotic conditions associated with thrombin activity, including inflammation, infection, tumour growth and metastasis, organ rejection and dementia (vascular and non-vascular, e.g. Alzheimer's disease) (Licari et al J Vet Emerg Crit Care (San Antonio). 2009 Feb; 19 (1) : 1 1-22; Tsopanoglou et al Eur Cytokine Netw. 2009 Dec 1 ;20 (4) :171-9).

[0110] The term “thrombosis” is used herein to refer to coagulation in excess of what is required for hemostasis (i.e. excessive coagulation), or which is not required for hemostasis (i.e. extrahemostatic or non-hemostatic coagulation). Thrombosis may include blood clotting within the blood vessel lumen. It is characterized by the formation of a clot (thrombus) that is in excess of requirement or not required for hemostasis. The clot may impede blood flow through the blood vessel leading to medical complications. A clot may break away from its site of formation, leading to embolism elsewhere in the circulatory system. In the arterial system, thrombosis is typically the result of atherosclerotic plaque rupture. In some embodiments, thrombosis may occur after an initial physiological hemostatic response, for example damage to endothelial cells in a blood vessel. In other embodiments, thrombosis may occur in the absence of any physiological hemostatic response.

[0111] Thrombosis may occur in “high-risk” individuals with an intrinsic tendency to thrombosis (i.e. thrombophilia) or in 'normal' individuals with no intrinsic tendency to thrombosis, for example in response to an extrinsic stimulus. The present molecules may be suitable for use in both “high- risk” and “normal” individuals.

[0112] Thrombosis and embolism may occur in any vein, artery or other blood vessel within the circulatory system and may include microvascular thrombosis. Thrombosis and embolism may be associated with surgery (either during surgery or afterwards) or the insertion of foreign objects, such as coronary stents, into a patient. For example, protein molecules as described herein may be useful in the surgical and other procedures in which blood is exposed to artificial surfaces, such as open heart surgery and dialysis.

[0113] In some embodiments the molecules of the invention are used in the treatment or prevention of thrombosis associated with atrial fibrillation. The term "atrial fibrillation” refers to a heart rhythm disorder that can result in a rapid and irregular heartbeat. When atrial fibrillation occurs, the heart beat becomes out of sync and the heart does not pump efficiently, this results in a disrupted blood flow within the chambers of the heart which can lead to clots. Clots that form in the heart due to atrial fibrillation are primarily composed of fibrin and are relatively platelet poor (Wysokinski WE, Owen WG, Fass DN, Patrzalek DD, Murphy L, McBane II RD. Atrial fibrillation and thrombosis:immunohistochemical differences between in situ and embolized thrombi. J Thromb Haemost 2004; 2: 1637-44.). It is possible for a clot formed due to atrial fibrillation to embolize and travel to the brain resulting in a stroke. The molecules of the invention may therefore be used to prevent and / or reduce the risk of clots forming due to atrial fibrillation. The molecules of the invention may therefore be used to prevent and / or reduce the risk of stroke due to atrial fibrillation.

[0114] Patients suitable for treatment as described herein include subject identified as having atrial fibrillation, a condition associated with an increased risk of ischemic stroke. Patients suitable for treatment as described herein include patients with conditions in which thrombosis is a symptom or a side-effect of treatment or which confer an increased risk of thrombosis or patients who are predisposed to or at increased risk of thrombosis, relative to the general population. For example, a protein molecule as described herein may also be useful in the treatment or prevention of venous thrombosis in cancer patients, and in the treatment or prevention of hospital-acquired thrombosis, which is responsible for 50% of cases of venous thromboembolism. Protein molecules as described herein may exert a therapeutic or other beneficial effect on thrombin-mediated conditions, such as thrombotic conditions, without substantially inhibiting or impeding hemostasis. For example, the risk of hemorrhage in patients treated with protein molecules of the invention may not be increased or substantially increased relative to untreated individuals.

[0115] Individuals treated with conventional anticoagulants, such as natural and synthetic heparins, warfarin, direct serine protease inhibitors (e.g. argatroban, dabigatran, apixaban, and rivaroxaban), hirudin and its derivatives (e.g. lepirudin and bivalirudin) cause bleeding. The risk of bleeding in patients treated with protein molecules as described herein may be reduced relative to individuals treated with conventional anticoagulants.

[0116] Methods

[0117] The protein molecules of the invention also have utility in certain methods for the inhibition of thrombin generation and coagulation, as well as methods for the detection of fVa.

[0118] An aspect of the invention relates to a method for reducing or inhibiting thrombin generation comprising: contacting a biological sample with a protein molecule described herein.

[0119] An aspect of the invention relates to a method for reducing or inhibiting coagulation comprising: contacting a biological sample with a protein molecule described herein.

[0120] The biological sample may be obtained from a subject. The biological sample may comprise a biological tissue sample, a biological fluid. A biological fluid may be, for example, blood, serum, lymph, urine, inflammatory exudate, cerebrospinal fluid, amniotic fluid, a tissue extract or homogenate, and the like. The methods for reducing or inhibiting thrombin generation and reducing or inhibiting coagulation may also be performed by introducing the protein molecule of the invention to a biological system, such as but not limited to an organ, tissue or cell. The methods may also be performed in vivo for example by introducing the protein molecule of the invention to an animal or human subject.

[0121] In some embodiments the methods of reducing thrombin generation and / or reducing coagulation may be performed on a blood sample obtained from a subject, which is then reintroduced to said subject. For example, these methods could be used as part of hemodialysis or extracorporeal membrane oxygenation. In such methods the blood sample may be introduced into an extracorporeal circulation system sequentially, separately or simultaneously to contacting with the protein molecule.

[0122] In the methods the protein molecule described herein competes with endogenous fXa for binding to fVa. By competing for binding with endogenous fXa this reduces the amount of prothrombinase formed and subsequently the amount of thrombin generated. As such the molecules of the invention act to reduce thrombin generation and reduce coagulation.

[0123] An aspect of the invention relates to a method of detecting the presence of fVa in a biological sample comprising; contacting a biological sample with a protein molecule described herein, detecting binding of said protein molecule to fVa.

[0124] The protein molecule used in the methods of the invention may comprises a detectable label. In particular it is advantageous forthe protein molecule used in the method of detection to comprise a detectable label. The detectable label may be any label described herein above. A label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorophores, fluorescers, radiolabels, enzymes, chemiluminescers, a nuclear magnetic resonance active label or photosensitizers. Thus, the binding may be detected and / or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance. The molecular label may be a fluorophore. Suitable fluorophores include fluorescein isothiocyanate, phycoerythrin, allophycocyanin, Indocicarbocyanine, Indocarbocyanine, as well as those known by the trade names Alexa Fluor (such as 350, 405, 488, 532, 546, 568, 594, 647, 680, 700, 750) and DyLight (such as 405, 488, 550, 650, 680, 755, 800).

[0125] The methods of the invention may be performed in vivo, in vitro or ex vivo. In some embodiments the methods are performed in vitro or ex vivo.

[0126] An aspect of the invention relates to a kit comprising the protein molecule described herein, and optionally instructions for use. The kit may contain materials useful for the treatment of the disorders described above is provided. The kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds the protein molecule of the invention which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).

[0127] The label or package insert indicates that the composition is used for treating the condition of choice, such as cancer. The kit may further contain a pharmaceutically- acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0128] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present disclosure, including methods, as well as the best mode thereof, of making and using this disclosure, the following examples are provided to further enable those skilled in the art to practice this disclosure. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present disclosure will be apparent to those skilled in the art in view of the present disclosure.

[0129] All documents mentioned in this specification are incorporated herein by reference in their entirety, including references to gene accession numbers, scientific publications and references to patent publications.

[0130] "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0131] The term “comprising” or “comprises” where used herein means including the component(s) specified but not to the exclusion of the presence of other components. The term “consisting essentially of’ or “consists essentially of’ means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components and the like.

[0132] The term “consisting of’ or “consists of’ means including the components specified but excluding other components.

[0133] Whenever appropriate, depending upon the context, the use of the term “comprises” or “comprising” may also be taken to include the meaning “consists essentially of’ or “consisting essentially of’, and also may also be taken to include the meaning “consists of’ or “consisting of’.

[0134] The optional features set out herein may be used either individually or in combination with each other where appropriate and particularly in the combinations as set out in the accompanying claims. The optional features for each aspect or exemplary embodiment of the invention, as set out herein are also applicable to all other aspects or exemplary embodiments of the invention, where appropriate. In other words, the skilled person reading this specification should consider the optional features for each aspect or exemplary embodiment of the invention as interchangeable and combinable between different aspects and exemplary embodiments.

[0135] Numbered Embodiments

[0136] 1 . A protein molecule comprising a variant factor Xa (fXa) or a fragment thereof, wherein the protein molecule binds to factor Va (fVa) with higher affinity compared to the binding affinity of said protein molecule to factor V (fV), wherein the variant fXa or a fragment thereof comprises one or more amino acid substitutions compared to wild-type fXa.

[0137] 2. The protein molecule according to embodiment 1 , wherein the variant fXa or fragment thereof comprises a variant EGF2 domain (SEQ ID NO: 7) and / or a variant heavy chain serine protease (SP) domain (SEQ ID NO: 4).

[0138] 3. The protein molecule of embodiment 2, wherein the one or more amino acid substitutions are present in the EGF2 domain (SEQ ID NO: 7) and / or the heavy chain SP domain (SEQ ID NO: 4).

[0139] 4. The protein molecule according to embodiment 3, wherein the one or more amino acid substitutions are present in one or more of the EGF2 domain (SEQ ID NO: 7), region 1 of the heavy chain SP domain as defined by residues 112 to 123 of SEQ ID NO: 4, region 2 of the heavy chain SP domain as defined by residues 153 to 165 of SEQ ID NO: 4, region 3 of the heavy chain SP domain as defined by residues 222 to 235 of SEQ ID NO: 4 5. The protein molecule of any preceding embodiment, wherein the variant fXa comprises a variant light chain (SEQ ID NO: 3) and / or a variant heavy chain (SEQ ID NO: 4)

[0140] 6. The protein molecule according to any one of embodiments 1 to 5, wherein the variant fXa or fragment thereof comprises one or more of: a) an amino acid substitution at position 83 of SEQ ID NO: 3; b) an amino acid substitution at position 84 of SEQ ID NO: 3; c) an amino acid substitution at position 85 of SEQ ID NO: 3; d) an amino acid substitution at position 86 of SEQ ID NO: 3; e) an amino acid substitution at position 87 of SEQ ID NO: 3; f) an amino acid substitution at position 88 of SEQ ID NO: 3; g) an amino acid substitution at position 90 of SEQ ID NO: 3; h) an amino acid substitution at position 91 of SEQ ID NO: 3; i) an amino acid substitution at position 92 of SEQ ID NO: 3; j) an amino acid substitution at position 101 of SEQ ID NO: 3; k) an amino acid substitution at position 102 of SEQ ID NO: 3; l) an amino acid substitution at position 103 of SEQ ID NO: 3; m) an amino acid substitution at position 104 of SEQ ID NO: 3; n) an amino acid substitution at position 105 of SEQ ID NO: 3; o) an amino acid substitution at position 116 of SEQ ID NO: 4; p) an amino acid substitution at position 117 of SEQ ID NO: 4; q) an amino acid substitution at position 118 of SEQ ID NO: 4; r) an amino acid substitution at position 121 of SEQ ID NO: 4; s) an amino acid substitution at position 123 of SEQ ID NO: 4; t) an amino acid substitution at position 154 of SEQ ID NO: 4; u) an amino acid substitution at position 157 of SEQ ID NO: 4; v) an amino acid substitution at position 161 of SEQ ID NO: 4; w) an amino acid substitution at position 163 of SEQ ID NO: 4; x) an amino acid substitution at position 223 of SEQ ID NO: 4; or y) an amino acid substitution at position 229 of SEQ ID NO: 4.

[0141] 7. The protein molecule according to any one of embodiments 1 to 6, wherein the variant fXa or fragment thereof comprises one or more of: a) an amino acid substitution at position 83 of SEQ ID NO: 3 to K; b) an amino acid substitution at position 84 of SEQ ID NO: 3 to V; c) an amino acid substitution at position 85 of SEQ ID NO: 3 to L; d) an amino acid substitution at position 86 of SEQ ID NO: 3 to Y or F; e) an amino acid substitution at position 87 of SEQ ID NO: 3 to Q; f) an amino acid substitution at position 88 of SEQ ID NO: 3 to S; g) an amino acid substitution at position 90 of SEQ ID NO: 3 to R; h) an amino acid substitution at position 91 of SEQ ID NO: 3 to A or V; i) an amino acid substitution at position 92 of SEQ ID NO: 3 to F; j) an amino acid substitution at position 101 of SEQ ID NO: 3 to K; k) an amino acid substitution at position 102 of SEQ ID NO: 3 to R; l) an amino acid substitution at position 103 of SEQ ID NO: 3 to V; m) an amino acid substitution at position 104 of SEQ ID NO: 3 to W or F; n) an amino acid substitution at position 105 of SEQ ID NO: 3 to S, F, R, L, V; o) an amino acid substitution at position 116 of SEQ ID NO: 4 to N; p) an amino acid substitution at position 117 of SEQ ID NO: 4 to E; q) an amino acid substitution at position 118 of SEQ ID NO: 4 to A; r) an amino acid substitution at position 121 of SEQ ID NO: 4 to K; s) an amino acid substitution at position 123 of SEQ ID NO: 4 to D; t) an amino acid substitution at position 154 of SEQ ID NO: 4 to H; u) an amino acid substitution at position 157 of SEQ ID NO: 4 to M; v) an amino acid substitution at position 161 of SEQ ID NO: 4 to D; w) an amino acid substitution at position 163 of SEQ ID NO: 4 to R; x) an amino acid substitution at position 223 of SEQ ID NO: 4 to R; or y) an amino acid substitution at position 229 of SEQ ID NO: 4 to K.

[0142] 8. The protein molecule according to any one of embodiments 1 to 7, wherein the variant fXa or fragment thereof comprises one or more of: a) an amino acid substitution at position 86 of SEQ ID NO:3; b) an amino acid substitution at position 90 of SEQ ID NO:3; c) an amino acid substitution at position 102 of SEQ ID NO:3; d) an amino acid substitution at position 104 of SEQ ID NO:3; e) an amino acid substitution at position 105 of SEQ ID NO:3; f) an amino acid substitution at position 116 of SEQ ID NO:4; g) an amino acid substitution at position 117 of SEQ ID NO:4; h) an amino acid substitution at position 118 of SEQ ID NO:4; i) an amino acid substitution at position 121 of SEQ ID NO:4; j) an amino acid substitution at position 123 of SEQ ID NO:4; k) an amino acid substitution at position 161 of SEQ ID NO:4; l) an amino acid substitution at position 163 of SEQ ID NO:4; or m) an amino acid substitution at position 223 of SEQ ID NO:4.

[0143] 9. The protein molecule according to any one of embodiments 1 to 8, wherein the variant fXa or fragment thereof comprises one or more of: a) an amino acid substitution at position 86 of SEQ ID NO: 3 to Y; b) an amino acid substitution at position 90 of SEQ ID NO: 3 to R; c) an amino acid substitution at position 102 of SEQ ID NO: 3 to R; d) an amino acid substitution at position 104 of SEQ ID NO: 3 to W or F; e) an amino acid substitution at position 105 of SEQ ID NO: 3 to S, F, R, L or V; f) an amino acid substitution at position 116 of SEQ ID NO: 4 to N; g) an amino acid substitution at position 117 of SEQ ID NO: 4 to E; h) an amino acid substitution at position 118 of SEQ ID NO: 4 to A; i) an amino acid substitution at position 121 of SEQ ID NO: 4 to K; j) an amino acid substitution at position 123 of SEQ ID NO: 4 to D; k) an amino acid substitution at position 161 of SEQ ID NO: 4 to D; l) an amino acid substitution at position 163 of SEQ ID NO: 4 to R; or m) an amino acid substitution at position 223 of SEQ ID NO: 4 to R.

[0144] 10. The protein molecule according to any one of embodiments 1 to 9, wherein the variant fXa or fragment thereof comprises: a) one or more of S90R, L91 A, D92F, H101 K, E102R, E103V, N105S, according to SEQ ID NO: 3, E116N, S117E. T121 K, K123D, N154H, K157M, S161 D, I163R, A223R, D229K, according to SEQ ID NO: 4; b) one or more of S90R, E102R according to SEQ ID NO: 3, E116N, S117E, T1 18A, T121 K, K123D according to SEQ ID NO: 4; c) one or more of R86Y, S90R, E102R according to SEQ ID NO: 3, E1 16N, S1 17E, T1 18A, T121 K, K123D according to SEQ ID NO: 4; or d) one or more of R86Y, S90R, E102R, Q104W, Q105R according to SEQ ID NO: 3, E116N, S117E, T118A, T121 K, K123D according to SEQ ID NO: 4

[0145] 11. The protein molecule according to any one of embodiments 1 to 10, wherein the protein molecule does not bind to fV or demonstrates negligible binding to fV.

[0146] 12. The protein molecule according to any one of embodiments 1 to 11 , wherein the protein molecule binds fVa with a dissociation constant of at 100 nM or lower.

[0147] 13. The protein molecule according to any one of embodiments 1 to 12, wherein the protein molecule binds fV with a dissociation constant of 500 nM or higher.

[0148] 14. The protein molecule according to any one of embodiments 1 to 13, wherein the protein molecule binds to fVa with high affinity in the absence of phospholipid membrane surfaces. 15. The protein molecule according to any one of embodiments 1 to 14, wherein the wherein the variant fXa or fragment thereof comprises an EGF1 domain and / or a gamma-carboxyglutamic acid (Gia) domain.

[0149] 16. The protein molecule according to any one of embodiments 1 to 16, further comprising a half-life extending moiety.

[0150] 17. The protein molecule according to embodiment 16, where in the half-life extending moiety is selected from one or more PEG molecules, a liposome, an Fc domain, a serum albumin protein, or an antibody or antibody fragment that binds serum albumin.

[0151] 18. The protein molecule according to embodiment 17, wherein the Fc domain comprises one or more amino acid modification or substitution which increases half-life of said protein molecule.

[0152] 19. The protein molecule according to any one of embodiments 1 to 18, wherein the variant fXa is catalytically inactive.

[0153] 20. The protein molecule according to embodiment 19, wherein the variant fXa comprises an amino acid substitution at position 185 according to SEQ ID NO: 4.

[0154] 21. The protein molecule according to embodiment 19, wherein the variant fXa comprises a covalent inhibitor in the active site, such as Glu-Gly-Arg-chloromethylketone (EGRCK) or D- Phe-Pro-Arg-chloromethylketone (PPACK).

[0155] 22. The protein molecule according to any one of embodiments 1 to 21 , wherein the variant fXa comprises reduced affinity to tissue factor pathway inhibitor (TFPI) compared to wild-type fXa.

[0156] 23. The protein molecule according to any one of embodiments 1 to 22, wherein the variant fXa comprises reduced reactivity to antithrombin compared to wild-type fXa.

[0157] 24. A pharmaceutical composition comprising a protein molecule according to any one of embodiments 1 to 23 and a pharmaceutically acceptable excipient.

[0158] 25. A protein molecule according to any one of embodiments 1 to 23 or a pharmaceutical composition according to embodiment 24 for use in therapy.

[0159] 26. A protein molecule according to any one of embodiments 1 to 23 or a pharmaceutical composition according to embodiment 24 for use in the treatment of a thrombin-mediated condition. 27. A method of treating a thrombin-mediated condition comprising a therapeutically effective amount of the protein molecule according to any one of embodiments 1 to 23, to a subject in need thereof.

[0160] 28. Use of a protein molecule according to any one of embodiments 1 to 23, in the manufacture of a medicament for the treatment of a thrombin-mediated condition.

[0161] 29. The protein molecule for use according to embodiment 26, the method of embodiment 27 or the use of embodiment 28, wherein the thrombin-mediated condition is selected from thrombosis, embolism, inflammation, clotting disorders, stroke associated with atrial fibrillation, thrombophilia, thrombotic stroke, coronary artery occlusion, infection, tumour growth, metastasis, organ rejection and dementia.

[0162] 30. A method for reducing or inhibiting thrombin generation comprising: contacting a biological sample with a protein molecule according to any one of embodiments 1 to 23.

[0163] 31 . A method for reducing or inhibiting coagulation comprising: contacting a biological sample with a protein molecule according to any one of embodiments 1 to 23.

[0164] 32. A method of detecting the presence of fVa in a biological sample comprising: contacting a biological sample with a protein molecule according to any one of embodiments 1 to 23, detecting binding of said protein molecule to fVa.

[0165] 33. The method according to embodiment 32, wherein the protein molecule comprises a detectable label.

[0166] 34. The method according to any one of embodiments 30 to 33, wherein the method is performed in vitro or ex vivo.

[0167] 35. A kit comprising the protein molecule according to any one of embodiments 1 to 23, and optionally instructions for use.

[0168] EXAMPLES

[0169] Introduction

[0170] Thrombin is generated from its circulating precursor prothrombin through the cleavage of two bonds by the enzyme complex known as prothrombinase. The assembly of the protein components of prothrombinase, the cofactor fVa and the serine protease fXa, is critically dependent on the presence of Ca2+and a negatively-charged phospholipid (PL) membrane surface (Figure 1). Activated PL membranes therefore play a critical role in regulating thrombin generation. In the absence of such surfaces, fXa and fVa do not assemble and prothrombin processing is inefficient. The PL-surface improves the affinity of the components by about 3- orders of magnitude. The substrate prothrombin also interacts with the PL surface.

[0171] A fXa orthologue from the venom of the Stephen’s Banded Snake (Hoplocephalus stephensii) known as ‘Hopsarin D’ (HopD) binds to human fVa with high affinity (Kd ~20nM; Figure 2) and activates prothrombin efficiently in the absence of PL membranes. It preserves the ordered cleavage of prothrombin, observed for fully assembled prothrombinase, with initial cleavage at Arg320 to form the active intermediate meizothrombin, followed by slower cleavage at Arg271 to release thrombin. HopD also binds to human fV with high affinity (Figure 2).

[0172] The present inventors set out to create a membrane-independent human fXa with mutations guided by sequence differences with HopD and a model of human prothrombinase (Figure 1). HopD and fXa share the domain organization of N-terminal gamma-carboxyglutamic acid (Gia) domain, two epidermal growth-factor like (EGF) domains, and a C-terminal serine protease (SP) domain (Figure 3). Full domain swap of the HopD SP domain modestly improved affinity for fVa. The EGF2-SP chimera bound to fVa with affinity indistinguishable to HopD itself (Figure 4), suggesting that most of the improved affinity is conferred by EGF2. As predicted from the model of prothrombinase, EGF1 and the Gia domains are relatively unimportant. Individual SP loops (L1 , L2 and L3; Figure 3) predicted to be in contact with fVa were swapped forthe corresponding sequence of HopD, with one loop (L1) contributing the most to improved binding, and accounting for an apparent cooperativity when combined with the swapping of the EGF2 domain (Figure 5). Further selection of residues predicted to be in contact with fVa and where the sequence change was not conserved reduced the number of mutations to 17 in total, with 4 in L1 , 4 in L2, 2 in L3 and 7 in EGF2 (Figure 3C). This variant, ‘M17’, binds to fVa with high affinity (Figure 5), indistinguishable from HopD. Surprisingly, unlike HopD, the M17 fXa variant was unable to bind to human fV in the absence of PL membranes (Figure 6).

[0173] Example 1. The contribution of individual fXa domains to the affinity forfVa

[0174] HopD and fXa share domain structure and considerable sequence identity. The percent identity for the individual domains are: Gia 64%; EGF1 68%; EGF2 39%; and SP 57%. To assess the importance of individual domains in contributing to the high affinity of HopD forfVa, each domain was swapped sequentially, beginning with the C-terminal SP domain (Fig. 3A). Affinity for fVa was assessed by fixing the fXa species to the tip of a biolayer interferometry (BLI) instrument and analyzing the binding of B-domainless fV (tVso and is functionally equivalent to fVa) at various concentrations (Figure 4). The response vs. concentration was fit to determine Kd. Wild- type fXa bound to VBD with a Kd of ~1.4 |j.M and HopD with a Kd of ~18 nM. Swapping of the entire SP domain improved the binding affinity by 3.2-fold to a Kd of ~400 nM. Surprisingly, the addition of EGF2 to the SP chimera improved affinity another 15-fold to a Kd of ~28 nM. Adding EGF1 from HopD to the chimera had little effect (1 .3-fold), as did the addition of the Gia domain (1.2-fold). The difference in Gibbs free energy of binding between WT fXa and HopD is -2.58 kcal / mol, 90% of which is provided by the EGF2 and SP domains. Efforts to improve the binding of fXa for fVa therefore focused on these two domains in which 133 residues are different between HopD and fXa.

[0175] Example 2. The contribution of individual SP loops to the affinity of fXa for fVa

[0176] A previously published model of human prothrombinase (Lechtenberg BC, Murray-Rust TA, Johnson DJ, Adams TE, Krishnaswamy S, Camire RM, Huntington JA, 2013, Crystal structure of the prothrombinase complex from the venom of Pseudonaja textilis, Blood. 122(16):2777-83) (Fig. 1) based on the crystal structure of the venom prothrombinase from the snake Pseudonaja textilis (pseutarin C)( Pomowski A, Ustok Fl, Huntington JA, 2014, Homology model of human prothrombinase based on the crystal structure of Pseutarin C, Biol Chem. 395(10):1233-41), was examined to identify candidate regions with in the SP domain to swap out for the sequence of HopD. Three regions were identified that are roughly centered on the 130-helix (L1), the 170- helix (L2), and the C-terminal helix (L3) (chymotrypsin numbering). L1 is predicted to bind at the gap between the A2 and A3 domains of fVa, and to interact with the a2-loop, L2 is predicted to interact with the A2 domain, and L3 with the highly acidic a2-loop (C-terminal of the A2 domain). L3 is part of the heparin binding site of fXa, and was shown to interact with the a2-loop in the structure of pseutarin C. L1 is composed of 14 residues, 5 of which are conserved, spanning from 124A to 135 (chymotrypsin numbering; residues 112 to 123 of SEQ ID NO: 4); L2 is 10 residues, of which 5 are conserved, spanning from 166 to 175 (chymotrypsin numbering; residues 153 to 165 of SEQ ID NO: 4); and L3 is 14 residues, of which 4 are conserved, spanning from 232 to 245 (chymotrypsin numbering; residues 222 to 235 of SEQ ID NO: 4).

[0177] The effect of individual loop swaps on affinity for IVBD was assessed by BLI, as above (Fig. 5. All loop swaps improved affinity, with L1 accounting for a 2.6-fold, L2 a 1.2-fold, and L3 a 1.5- fold decrease in Kd. Combining the three sets of mutations (L123) did not improve affinity beyond the effect of L1 on its own, and the L23 chimera was no better than either individual mutations. When the HopD EGF2 domain swap was added to the L23 chimera, affinity only improved by about 3.7-fold (Fig. 5C). When the HopD EGF2 domain swap was added to the L123 chimera, affinity was improved 15-fold (Fig. 5C). This suggests cooperativity between L1 and EGF2. The L123 / EGF2 chimera bound to tVso with a Kd of ~43 nM, representing 88% of the binding energy gained from swapping the entire SP and EGF2 domains. This chimera is comprised of 45 substitutions across three loops in the SP and the EGF2 domain (Fig. 3B). Example 3. M17 binds to fVa with high affinity

[0178] In order to reduce the number of mutations to just the key contact residues, the human prothrombinase model was examined to select only non-homologous mutations in the L123 / EGF2 fXa chimera predicted to be making contact with fVa. The model is not expected to be sufficiently accurate to predict actual side chain contacts, so this approach is agnostic with respect to whether a mutation was expected to favor or disfavor binding. Seven of the original 21 mutations in the EGF2 domain were chosen based on these criteria. In the SP domain, 4 of 9 mutations were selected in L1 , 4 of 5 in L2, and 2 of 10 in L3 (Fig. 3C). The resulting chimera had 17 mutations across the two domains and was denoted ‘M17’ fXa. As for the loop swap chimeras, we also made variants with subsets of mutations, including M7 which only contains the 7 EGF2 mutations and M13 which contains the L23 and EGF2 mutations. The affinities of M7, M13 and M17 for tVso were determined as above (Fig. 7). Once again, EGF2 was found to play a surprisingly large role in the interaction, with M7 binding with a Kd of 78 nM. Addition of L2 and L3 mutations to M7 (M13) had a modest effect (Kd of 48 nM); M17 was found to bind with high affinity, indistinguishable from the full domain-swap chimeras and HopD, with a Kd of 17 nM (Fig. 5D). The EGF2 / L1 variant (M1 1) is expected to be similar to M17.

[0179] Example 4. EGRCK-inhibited HopD competes with fXa for fVa binding

[0180] The ability of truncated HopD (EGF2-SP) to compete with human fXa was assessed by incubating increasing concentrations of EGRCK-inhibited HopD in a reaction mixture containing fXa, fVa, and PL, then adding recombinant S195A prothrombin and incubating for 60 minutes. Cleavage products were visualized by SDS-PAGE (Fig. 8A) and quantified by densitometry. The band corresponding to the heavy chain with no EGRCK-HopD was used to calculate initial activity and set to 1 , with increasing concentrations of EGRCK-HopD resulting in fraction inhibition. Fitting the fraction inhibition yielded a concentration required for half-inhibition (IC50) of 3.6 nM (Fig. 8B). A similar value of 1 nM was obtained using plasma-derived prothrombin and measuring chromogenic substrate cleavage by thrombin / meizothrombin (Fig. 8C). Active-site- inhibited HopD is a potent competitor for human fXa in the prothrombinase complex. The HopD used in this experiment was truncated (only EGF2-SP domains), so was an effective inhibitor of human prothrombinase even though it was missing the Gia domain and thus unable to bind to the PL membrane surface.

[0181] Methods

[0182] Expression and purification of proteins

[0183] Factors V and X were purified from plasma and activated as previously described (Nesheim ME, Katzmann JA, Tracy PB, Mann KG.

[0021] Factor V. Methods Enzym 1981 ; 80: 249-74). Wild-type prothrombin was purified from plasma and S195A prothrombin (chymotrypsin numbering) was produced recombinantly, both as previously described [20,26], Full-length HopD DNA (accession number AY940208.1) was synthesized (Gene Art), and the region encoding the protein including the prothrombin signal peptide for gamma carboxylation was cloned into the pCEP4 vector and stably transfected into HEK-EBNA cells. In order to facilitate removal of the activation peptide, the activation cleavage site was modified to that of human fX. Cells expressing HopD were grown in CD-CHO media and purified and activated as previously described

[0020] , Truncated HopD was produced in E. coli and refolded. PCR amplification of the HopD gene corresponding to the EGF2 and protease domain (residues 126 to 455) was performed using appropriate nucleotide primers. The resulting 1 .1 kb product was digested with EcoRI and Hindlll restriction enzymes and ligated into the pET23(+) vector (Novagen). pET23- HopD was transformed into the BL21 STAR (DE3) pLysS E. coli strain (Life Technologies) and expressed into inclusion bodies. Following cell lysis, the pellets were washed and then solubilized in 6 M guanidine hydrochloride with 100 mM Tris, pH 8.2, 20 mM EDTA, 15 mM reduced glutathione and 150 mM oxidized glutathione. The solubilized protein was dialyzed to remove the excess glutathione and refolded by dropwise addition into 1 L of 50 mM Tris-HCI, pH 8.5, 0.5 M L-arginine, 20 mM CaCh, and 0.5 mM L-cysteine. The refolded protein was concentrated and loaded onto a 5 mL HiTrap heparin Sepharose column (Cytiva) and eluted with a 0.25-1 M NaCI gradient over 5 column volumes. Fractions containing HopD were pooled and dialyzed into 20 mM Tris, pH 7.4, 250 mM NaCI. HopD was activated by RW-X treatment. Full-length and truncated human fX and variants thereof were produced in a similar manner.

[0184] Factor X chimeras and mutations

[0185] Primers were designed to introduce these mutations also had sequence specific overlapping regions with the adjacent fragment to enable correct assembly of the constructs using Klenow based ligation-independent assembly method (David, Md & Bailey, David & Yh, Mohamed. (2017). Klenow assembly method: seamless cloning). Fragments were amplified by PCR from a previously generated pCEP4-hfX plasmid using appropriate primers. All PCR procedures were conducted using Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs) as described by the manufacturer’s instructions. Post PCR reaction, each fragment was purified, Dpn1 digested, and gel extracted. Plasmid backbone was isolated by restriction digestion of a previously constructed pCEP4-hfX vector with Kpn1 / EcoRV restriction enzymes, a band (9149bp) containing the plasmid backbone was excised from an agarose gel, purified and used in the assembly procedure. All purified fragments and plasmid backbone was added to Klenow assembly mixture at equimolar amount (30ng), incubated at 37°C for 20 min followed by cooling on ice and transformation into E. coli competent cells (Max Efficiency DH5a competent cells, Invitrogen). Same method was used to generate individual and combined chimeric variants as well as point mutations. All of these constructs had the prothrombin pro and signal peptide to enable gamma-carboxylation (B C Furie, J V Ratcliffe, J Tward, M J Jorgensen, L S Blaszkowsky, D DiMichele, B Furie, The gamma-carboxylation recognition site is sufficient to direct vitamin K-dependent carboxylation on an adjacent glutamate-rich region of thrombin in a propeptide-thrombin chimera. J Biol Chem 1997 Nov 7;272(45):28258-62). Additionally, chimeric human factor X variants where constructs had HopD serine protease (SP), HopD EGF2-SP and HopD EGF1-EGF2-SP domains were generated using the same assembly method as described above. All plasmid sequences were confirmed by DNA sequencing prior to use in maxi-prep preparations for HEK-EBNA cell transfections.

[0186] Assessment of binding constants

[0187] Octet Red bio-layer interferometry was used to determine binding constants. All experiments were performed in binding buffer (20 mM HEPES pH 7.4, 150 mM NaCI, 2 mM CaCh, 0.1 mg / ml BSA, 0.1 % PEG8000) using Streptavidin biosensors. HopD (truncated or full-length) and fXa and variants were treated with biotinylated EGRCK, desalted and loaded onto the biosensors at 1 .0 pg / mL or 2.0 pg / ml. Remaining biotin binding sites on the biosensors were then quenched with biocytin (50 pg / mL). Human plasma derived fV and fVa, or recombinant B-domainless fV (tVso) (Toso R, and Camire R.M. Removal of B-domain Sequences from Factor V Rather than Specific Proteolysis Underlies the Mechanism by Which Cofactor Function Is Realized. The Journal of Biological Chemistry, Vol. 279, No. 20, pp. 21643-21650, 2004) were diluted in binding buffer prior to the experiment. Data traces and steady state responses were analyzed using forteBIO analysis software and Prism (GraphPad).

[0188] Chromogenic assay

[0189] Conditions for the thrombin generation assay were similar, with a reaction buffer of 20mM Tris,pH7.4 + 150mM NaCI + 5mM CaCI2 + 0.1 % PEG8000, and final concentrations of 1 nM plasma derived fXa, 20nM plasma derived fVa, 10pM PE:PC:PS(60:20:20) PL vesicles, and 1 pM plasma derived prothrombin. EGRCK-inhibited truncated HopD was added at the indicated concentrations. The order of addition was: prothrombin, EGRCK-HopD, fVa, PL (sit for 1 min), then fXa to initiate the reaction. Samples of 5p I were taken out at after 10min and added to 10OpI of 0.4mM S-2238 substrate and absorbance at 405nm was read for 1 minute on a plate reader. The slopes were plotted against EGRCK-HopD concentration to obtain a value for IC50.

[0190] Inhibition of human prothrombinase by EGRCK-inhibited HopD

[0191] Human plasma-derived prothrombin (5 pM) was incubated with 1 nM fXa, 20 nM fVa, 20 pM PC:PE:PS (60:20:20) PL vesicles in 20 mM Tris pH 7.4, 150 mM NaCI, 5 mM CaCI2at 25°C for 60 minutes. Increasing concentrations of EGRCK-inhibited truncated HopD (EGRCK-HopD) was added before initiating reactions by the addition of fXa. Samples (10 pL) of each reaction mixture were removed at each time point, immediately quenched by mixing with 5 pL of SDS- PAGE sample buffer containing 100 mM DTT and 100 pM AEBSF followed by heating to 95°C for 5 minutes. Reaction products were resolved on a 10% Bis-Tris gel run in MES buffer. Densitometry was performed on gel bands using the program GelQuant (BiochemLabSolution.com). Curves were fit using GraphPad to calculate IC50 values. References

[0192] 1 Davie E, Kulman J. An Overview of the Structure and Function of Thrombin. Semin Thromb Hemost 2006; 32: 003-15.

[0193] 2 Lane DA, Philippou H, Huntington JA. Directing thrombin. Blood 2005; 106: 2605-12.

[0194] 3 Spencer FA, Becker RC. The Prothrombinase Complex: Assembly and Function. J Thromb Thrombolys 1997; 4: 357-64.

[0195] 4 Skogen WF, Esmon CT, Cox AC. Comparison of coagulation factor Xa and des-(1-44)factor Xa in the assembly of prothrombinase. J Biol Chem 1984; 259: 2306-10.

[0196] 5 Krishnaswamy S. Prothrombinase complex assembly. Contributions of protein-protein and protein-membrane interactions toward complex formation. J Biol Chem 1990; 265: 3708-18.

[0197] 6 Persson E, Hogg PJ, Stenflo J. Effects of Ca2+ binding on the protease module of factor Xa and its interaction with factor Va. Evidence for two Gla-independent Ca(2+)-binding sites in factor Xa. J Biol Chem 1993; 268: 22531-9.

[0198] 7 Krishnaswamy S, Mann KG, Nesheim ME. The prothrombinase-catalyzed activation of prothrombin proceeds through the intermediate meizothrombin in an ordered, sequential reaction. J Biological Chem 1986; 261 : 8977-84.

[0199] 8 Bos MHA, Camire RM. Procoagulant Adaptation of a Blood Coagulation Prothrombinase-like Enzyme Complex in Australian Elapid Venom. Toxins 2010; 2: 1554-67.

[0200] 9 Markland FS. Snake venoms and the hemostatic system. Toxicon 1998; 36: 1749-800.

[0201] 10 Kini RM, Rao VS, Joseph JS. Procoagulant Proteins from Snake Venoms. Pathophysiol Haemost Thromb 2002; 31 : 218-24.

[0202] 11 Pierre LSt, Masci PP, Filippovich I, Sorokina N, Marsh N, Miller DJ, Lavin MF. Comparative Analysis of Prothrombin Activators from the Venom of Australian Elapids. Mol Biol Evol 2005; 22: 1853-64.

[0203] 12 REZA MA, LE TNM, SWARUP S, KINI RM. Molecular evolution caught in action: gene duplication and evolution of molecular isoforms of prothrombin activators in Pseudonaja textilis (brown snake). J Thromb Haemost 2006; 4: 1346-53.

[0204] 13 RAO VS, JOSEPH JS, KINI RM. Group D prothrombin activators from snake venom are structural homologues of mammalian blood coagulation factor Xa. Biochem J 2003; 369: 635- 42.

[0205] 14 Masci PP, Whitaker AN, Jersey J de. Purification and characterization of a prothrombin activator from the venom of the Australian brown snake, Pseudonaja textilis textilis. Biochem Int 1988; 17: 825-35.

[0206] 15 Joseph JS, Chung MC, Jeyaseelan K, Kini RM. Amino acid sequence of trocarin, a prothrombin activator from Tropidechis carinatus venom: its structural similarity to coagulation factor Xa. Blood 1999; 94: 621-31. 16 Rao VS, Kini RM. Pseutarin C, a prothrombin activator from Pseudonaja textilis venom: its structural and functional similarity to mammalian coagulation factor Xa-Va complex. Thromb Haemost 2002; 88: 611-9.

[0207] 17 Lechtenberg BC, Murray-Rust TA, Johnson DJD, Adams TE, Krishnaswamy S, Camire RM, Huntington JA. Crystal structure of the prothrombinase complex from the venom of Pseudonaja textilis. Blood 2013; 122: 2777-83.

[0208] 18 Bos MHA, Boltz M, Pierre LSt, Masci PP, Jersey J de, Lavin MF, Camire RM. Venom factor V from the common brown snake escapes hemostatic regulation through procoagulant adaptations. Blood 2009; 114: 686-92.

[0209] 19 Pomowski A, Ustok Fl, Huntington JA. Homology model of human prothrombinase based on the crystal structure of Pseutarin C. Biol Chem 2014; 395: 1233-41 .

[0210] 20 Ustok Fl, Huntington JA. Mapping the Prothrombin Binding Site of Pseutarin C by Site- directed PEGylation. Blood 2022; 139: 2972-82.

[0211] 21 Tans G, Govers-Riemslag JW, Rijn JL van, Rosing J. Purification and properties of a prothrombin activator from the venom of Notechis scutatus scutatus. J Biol Chem 1985; 260: 9366-72.

[0212] 22 Newell-Caito JL, Laha M, Tharp AC, Creamer JI, Xu H, Maddur AA, Tans G, Bock PE. Notecarin D Binds Human Factor V and Factor Va with High Affinity in the Absence of Membranes*. J Biol Chem 2011 ; 286: 38286-97.

[0213] 23 Weinstein SA, Williams V, White J. Preliminary characteristics of the prothrombin converting enzyme from venom of Stephen’s banded snake (Hoplocephalus stephensii). Toxicon 2001 ; 39: 1937-9.

[0214] 24 Nesheim ME, Katzmann JA, Tracy PB, Mann KG.

[0021] Factor V. Methods Enzym 1981 ; 80: 249-74.

[0215] 25 Buddai SK, Toulokhonova L, Bergum PW, Vlasuk GP, Krishnaswamy S. Nematode Anticoagulant Protein c2 Reveals a Site on Factor Xa That Is Important for Macromolecular Substrate Binding to Human Prothrombinase*. J Biol Chem 2002; 277: 26689-98.

[0216] 26 Mann KG, Elion J, Butkowski RJ, Downing M, Nesheim ME. Prothrombin. Methods Enzym 1981 ; 80 Pt C: 286-302.

[0217] 27 Venkateswarlu D, Krishnaswamy S, Darden TA, Pedersen LG. Three-dimensional solution structure of Tropidechis carinatus venom extract trocarin: a structural homologue of Xa and prothrombin activator. Mol Model Annu 2002; 8: 302-13.

[0218] 28 Thorelli E, Kaufman RJ, Dahlback B. Cleavage Requirements for Activation of Factor V by Factor Xa. EurJ Biochem 1997; 247: 12-20.

[0219] 29 Bos MHA, Camire RM. Blood coagulation factors V and VIII: Molecular Mechanisms of Procofactor Activation. J Coagul Disord 2010; 2: 19-27.

[0220] 30 Rezaie AR, Esmon CT. Contribution of Residue 192 in Factor Xa to Enzyme Specificity and Function *. J Biol Chem 1995; 270: 16176-81. 31 Kim PY, Nesheim ME. Further Evidence for Two Functional Forms of Prothrombinase Each Specific for Either of the Two Prothrombin Activation Cleavages*. J Biol Chem 2007; 282: 32568-81.

[0221] Toso R, and Camire R.M. Removal of B-domain Sequences from Factor V Rather than Specific Proteolysis Underlies the Mechanism by Which Cofactor Function Is Realized. The Journal of Biological Chemistry, Vol. 279, No. 20, Issue of May 14, pp. 21643-21650, 2004

[0222] Bailey DMD, Mohamed MYH. The Klenow Assembly Method (KAM) for the seamless cloning of overlapping double stranded DNA fragments: very cheap alternative to the GibsonAssembly. https : / / openwetware.org / mediawiki / index.php?title=Klenow_Assembly_Method:_Seamless_clo ning&oldid=1071130. Accessed 08 May 2019.

[0223] B C Furie , J V Ratcliffe, J Tward, M J Jorgensen, L S Blaszkowsky, D DiMichele, B Furie, The gamma-carboxylation recognition site is sufficient to direct vitamin K-dependent carboxylation on an adjacent glutamate-rich region of thrombin in a propeptide-thrombin chimera. J Biol Chem 1997 Nov 7;272(45):28258-62.

[0224] Sequences

[0225] SEQ ID NO:1 P00742 is the full transcript of human fX, including signal and pro regions: >sp|P00742|FA10_HUMAN Coagulation factor X OS=Homo sapiens OX=9606 GN=F10 PE=1 SV=2

[0226] MGRPLHLVLLSASLAGLLLLGESLFIRREQANNILARVTR-ANSFLEEMKKGHLERECMEE TCSYEEAREVFEDSDKTNEFWNKYKDGDQCETSPCQNQGKCKDGLGEYTCTCLEGFEGKN CELFTRKLCSLDNGDCDQFCHEEQNSWCSCARGYTLADNGKACIPTGPYPCGKQTLERR KRSVAQATSSSGEAPDSITWKPYDAADLDPTENPFDLLDFNQTQPERGDNNLTRIVGGQE CKDGECPWQALLINEENEGFCGGTILSEFYILTAAHCLYQAKRFKVRVGDRNTEQEEGGE AVHEVEWIKHNRFTKETYDFDIAVLRLKTPITFRMNVAPACLPERDWAESTLMTQKTGI VSGFGRTHEKGRQSTRLKMLEVPYVDRNSCKLSSSFIITQNMFCAGYDTKQEDACQGDSG GPHVTRFKDTYFVTGIVSWGEGCARKGKYGIYTKVTAFLKWIDRSMKTRGLPKAKSHAPE VITSSPLK

[0227] > WT construct - SEQ ID NO:2

[0228] ANSFLEEMKKGHLERECMEETCSYEEAREVFEDSDKTNEFWNKYKDGDQCETSPCQNQGK CKDGLGEYTCTCLEGFEGKNCELFTRKLCSLDNGDCDQFCHEEQNSVVCSCARGYTLADNG KACIPTGPYPCGKQTLERRKRSVAQATSSSGEAPDSITWKPYDAADLDPTENPFDLLDFNQT QPERGDNNLTRIVGGQECKDGECPWQALLINEENEGFCGGTILSEFYILTAAHCLYQAKRFKV RVGDRNTEQEEGGEAVHEVEVVIKHNRFTKETYDFDIAVLRLKTPITFRMNVAPACLPERDWA ESTLMTQKTGIVSGFGRTHEKGRQSTRLKMLEVPYVDRNSCKLSSSFIITQNMFCAGYDTKQ EDACQGDSGGPHVTRFKDTYFVTGIVSWGEGCARKGKYGIYTKVTAFLKWIDRSMKTRGLPK AKSHAPEVITSSPLK >hfXa Light Chain - SEQ ID NO: 3 ANSFLEEMKKGHLERECMEETCSYEEAREVFEDSDKTNEFWNKYKDGDQCETSPCQNQGK CKDGLGEYTCTCLEGFEGKNCELFTRKLCSLDNGDCDQFCHEEQNSVVCSCARGYTLADNG KACIPTGPYPCGKQTLERRKR

[0229] >hfXa Heavy Chain (serine protease domain) - SEQ ID NO: 4

[0230] IVGGQECKDGECPWQALLINEENEGFCGGTILSEFYILTAAHCLYQAKRFKVRVGDRNTEQEE GGEAVHEVEVVIKHNRFTKETYDFDIAVLRLKTPITFRMNVAPACLPERDWAESTLMTQKTGIV SGFGRTHEKGRQSTRLKMLEVPYVDRNSCKLSSSFIITQNMFCAGYDTKQEDACQGDSGGP HVTRFKDTYFVTGIVSWGEGCARKGKYGIYTKVTAFLKWIDRSMKTRGLPKAKSHAPEVITSS PLK

[0231] Region 1 of the heavy chain SP domain (SEQ ID NO: 11), Region 2 of the heavy chain SP domain (SEQ ID NO: 12), and Region 3 of the heavy chain SP domain (SEQ ID NO: 13) are highlighted in underline.

[0232] >hGla SEQ ID NO:5

[0233] ANSFLEEMKKGHLERECMEETCSYEEAREVFEDSDKTNEFWNKYK

[0234] >hEGF1 - SEQ ID NO:6

[0235] DGDQCETSPCQNQGKCKDGLGEYTCTCLEGFEGKNCEL

[0236] >hEGF2 - SEQ ID NO:7

[0237] FTRKLCSLDNGDCDQFCHEEQNSWCSCARGYTLADNGKACIPTGPYPC

[0238] >hAct_pept - SEQ ID NO:8

[0239] GKQTLERRKRSVAQATSSSGEAPDSITWKPYDAADLDPTENPFDLLDFNQTQPERGDNNLTR

[0240] >hSP - SEQ ID NO:9

[0241] IVGGQECKDGECPWQALLINEENEGFCGGTILSEFYILTAAHCLYQAKRFKVRVGDRNTEQEE GGEAVHEVEVVIKHNRFTKETYDFDIAVLRLKTPITFRMNVAPACLPERDWAESTLMTQKTGIV SGFGRTHEKGRQSTRLKMLEVPYVDRNSCKLSSSFIITQNMFCAGYDTKQEDACQGDSGGP HVTRFKDTYFVTGIVSWGEGCARKGKYGIYTKVTAFLKWIDRSMK

[0242] >hSP_C-term - SEQ ID NO:10

[0243] TRGLPKAKSHAPEVITSSPLK

[0244] >construct M17 - SEQ ID NO: 14

[0245] ANSFLEEMKKGHLERECMEETCSYEEAREVFEDSDKTNEFWNKYKDGDQCETSPCQNQGK CKDGLGEYTCTCLEGFEGKNCELFTRKLCRAFNGDCDQFCKRVQSSVVCSCARGYTLADNG KACIPTGPYPCGKQTLERRKRSVAQATSSSGEAPDSITWKPYDAADLDPTENPFDLLDFNQT

[0246] QPERGDNNLTRIVGGQECKDGECPWQALLINEENEGFCGGTILSEFYILTAAHCLYQAKRFKV

[0247] RVGDRNTEQEEGGEAVHEVEVVIKHNRFTKETYDFDIAVLRLKTPITFRMNVAPACLPERDWA

[0248] NETLMKQDTGIVSGFGRTHEKGRQSTRLKMLEVPYVDRHSCMLSSDFRITQNMFCAGYDTK

[0249] QEDACQGDSGGPHVTRFKDTYFVTGIVSWGEGCARKGKYGIYTKVTRFLKWIKRSMKTRGL PKAKSHAPEVITSSPLK

[0250] >construct M7 - SEQ ID NO: 15

[0251] ANSFLEEMKKGHLERECMEETCSYEEAREVFEDSDKTNEFWNKYKDGDQCETSPCQNQGK

[0252] CKDGLGEYTCTCLEGFEGKNCELFTRKLCRLDNGDCDQFCHREQNSVVCSCARGYTLADNG

[0253] KACIPTGPYPCGKQTLERRKRSVAQATSSSGEAPDSITWKPYDAADLDPTENPFDLLDFNQT

[0254] QPERGDNNLTRIVGGQECKDGECPWQALLINEENEGFCGGTILSEFYILTAAHCLYQAKRFKV

[0255] RVGDRNTEQEEGGEAVHEVEVVIKHNRFTKETYDFDIAVLRLKTPITFRMNVAPACLPERDWA

[0256] NEALMKQDTGIVSGFGRTHEKGRQSTRLKMLEVPYVDRNSCKLSSSFIITQNMFCAGYDTKQ

[0257] EDACQGDSGGPHVTRFKDTYFVTGIVSWGEGCARKGKYGIYTKVTAFLKWIDRSMKTRGLPK AKSHAPEVITSSPLK

[0258] >construct M8 - SEQ ID NO: 16

[0259] ANSFLEEMKKGHLERECMEETCSYEEAREVFEDSDKTNEFWNKYKDGDQCETSPCQNQGK

[0260] CKDGLGEYTCTCLEGFEGKNCELFTYKLCRLDNGDCDQFCHREQNSVVCSCARGYTLADNG

[0261] KACIPTGPYPCGKQTLERRKRSVAQATSSSGEAPDSITWKPYDAADLDPTENPFDLLDFNQT

[0262] QPERGDNNLTRIVGGQECKDGECPWQALLINEENEGFCGGTILSEFYILTAAHCLYQAKRFKV

[0263] RVGDRNTEQEEGGEAVHEVEVVIKHNRFTKETYDFDIAVLRLKTPITFRMNVAPACLPERDWA

[0264] NEALMKQDTGIVSGFGRTHEKGRQSTRLKMLEVPYVDRNSCKLSSSFIITQNMFCAGYDTKQ

[0265] EDACQGDSGGPHVTRFKDTYFVTGIVSWGEGCARKGKYGIYTKVTAFLKWIDRSMKTRGLPK AKSHAPEVITSSPLK

[0266] >construct M10 - SEQ ID NO: 17

[0267] ANSFLEEMKKGHLERECMEETCSYEEAREVFEDSDKTNEFWNKYKDGDQCETSPCQNQGK

[0268] CKDGLGEYTCTCLEGFEGKNCELFTYKLCRLDNGDCDQFCHREWRSVVCSCARGYTLADN

[0269] GKACIPTGPYPCGKQTLERRKRSVAQATSSSGEAPDSITWKPYDAADLDPTENPFDLLDFNQ

[0270] TQPERGDNNLTRIVGGQECKDGECPWQALLINEENEGFCGGTILSEFYILTAAHCLYQAKRFK

[0271] VRVGDRNTEQEEGGEAVHEVEVVIKHNRFTKETYDFDIAVLRLKTPITFRMNVAPACLPERDW

[0272] ANEALMKQDTGIVSGFGRTHEKGRQSTRLKMLEVPYVDRNSCKLSSSFIITQNMFCAGYDTK

[0273] QEDACQGDSGGPHVTRFKDTYFVTGIVSWGEGCARKGKYGIYTKVTAFLKWIDRSMKTRGL PKAKSHAPEVITSSPLK

[0274] SEQ ID NO:18 human fXa, minus signal and pro regions:

[0275] ANSFLEEMKKGHLERECMEE TCSYEEAREVFEDSDKTNEFWNKYKDGDQCETSPCQNQGKCKDGLGEYTCTCLEGFEGKN

[0276] CELFTRKLCSLDNGDCDQFCHEEQNSWCSCARGYTLADNGKACIPTGPYPCGKQTLERR

[0277] KRSVAQATSSSGEAPDSITWKPYDAADLDPTENPFDLLDFNQTQPERGDNNLTRIVGGQE

[0278] CKDGECPWQALLINEENEGFCGGTILSEFYILTAAHCLYQAKRFKVRVGDRNTEQEEGGE

[0279] AVHEVEWIKHNRFTKETYDFDIAVLRLKTPITFRMNVAPACLPERDWAESTLMTQKTGI

[0280] VSGFGRTHEKGRQSTRLKMLEVPYVDRNSCKLSSSFIITQNMFCAGYDTKQEDACQGDSG

[0281] GPHVTRFKDTYFVTGIVSWGEGCARKGKYGIYTKVTAFLKWIDRSMKTRGLPKAKSHAPE VITSSPLK

[0282] SEQ ID NO: 19 EGF2-123 amino acid sequence

[0283] ANSFLEEMKKGHLERECMEETCSYEEAREVFEDSDKTNEFWNKYKDGDQCETSPCQ

[0284] NQGKCKDGLGEYTCTCLEGFEGKNCELFTRKLCRAFNGNCWHFCKRVQSETQCSCA

[0285] ESYRLGVDGHSCIPTGPYPCGKQTLERRKRSVAQATSSSGEAPDSITWKPYDAADL

[0286] DPTENPFDLLDFNQTQPERGDNNLTRIVGGQECKDGECPWQALLINEENEGFCGGT

[0287] ILSEFYILTAAHCLYQAKRFKVRVGDRNTEQEEGGEAVHEVEWIKHNRFTKETYD

[0288] FDIAVLRLKTPITFRMNVAPACLPTADFANEVLMKQDSGIVSGFGRTHEKGRQSTR

[0289] LKMLEVPYVDRHTCMLSSDFRITQNMFCAGYDTKQEDACQGDSGGPHVTRFKDTYF

[0290] VTGIVSWGEGCARKGKYGIYTKVSRFIPWIKKIMSLKGLPKAKSHAPEVITSSPLK

Claims

CLAIMS1 . A protein molecule comprising a variant factor Xa (fXa) or a fragment thereof, wherein the protein molecule binds to factor Va (fVa) with higher affinity compared to the binding affinity of said protein molecule to factor V (fV), wherein the variant fXa or a fragment thereof comprises one or more amino acid substitutions compared to wild-type fXa.

2. The protein molecule according to claim 1 , wherein the variant fXa or fragment thereof comprises a variant EGF2 domain (SEQ ID NO: 7) and / or a variant heavy chain serine protease (SP) domain (SEQ ID NO: 4).

3. The protein molecule of claim 2, wherein the one or more amino acid substitutions are present in the EGF2 domain (SEQ ID NO: 7) and / or the heavy chain SP domain (SEQ ID NO: 4).

4. The protein molecule according to claim 3, wherein the one or more amino acid substitutions are present in one or more of the EGF2 domain (SEQ ID NO: 7), region 1 of the heavy chain SP domain as defined by residues 112 to 123 of SEQ ID NO: 4, region 2 of the heavy chain SP domain as defined by residues 153 to 165 of SEQ ID NO: 4, region 3 of the heavy chain SP domain as defined by residues 222 to 235 of SEQ ID NO:

45. The protein molecule of any preceding claim, wherein the variant fXa comprises a variant light chain (SEQ ID NO: 3) and / or a variant heavy chain (SEQ ID NO: 4)6. The protein molecule according to any one of claims 1 to 5, wherein the variant fXa or fragment thereof comprises one or more of: a) an amino acid substitution at position 83 of SEQ ID NO: 3; b) an amino acid substitution at position 84 of SEQ ID NO: 3; c) an amino acid substitution at position 85 of SEQ ID NO: 3; d) an amino acid substitution at position 86 of SEQ ID NO: 3; e) an amino acid substitution at position 87 of SEQ ID NO: 3; f) an amino acid substitution at position 88 of SEQ ID NO: 3; g) an amino acid substitution at position 90 of SEQ ID NO: 3; h) an amino acid substitution at position 91 of SEQ ID NO: 3; i) an amino acid substitution at position 92 of SEQ ID NO: 3; j) an amino acid substitution at position 101 of SEQ ID NO: 3; k) an amino acid substitution at position 102 of SEQ ID NO: 3; l) an amino acid substitution at position 103 of SEQ ID NO: 3; m) an amino acid substitution at position 104 of SEQ ID NO: 3; n) an amino acid substitution at position 105 of SEQ ID NO: 3;o) an amino acid substitution at position 116 of SEQ ID NO: 4; p) an amino acid substitution at position 117 of SEQ ID NO: 4; q) an amino acid substitution at position 118 of SEQ ID NO: 4; r) an amino acid substitution at position 121 of SEQ ID NO: 4; s) an amino acid substitution at position 123 of SEQ ID NO: 4; t) an amino acid substitution at position 154 of SEQ ID NO: 4; u) an amino acid substitution at position 157 of SEQ ID NO: 4; v) an amino acid substitution at position 161 of SEQ ID NO: 4; w) an amino acid substitution at position 163 of SEQ ID NO: 4; x) an amino acid substitution at position 223 of SEQ ID NO: 4; or y) an amino acid substitution at position 229 of SEQ ID NO: 4.

7. The protein molecule according to any one of claims 1 to 6, wherein the variant fXa or fragment thereof comprises one or more of: a) an amino acid substitution at position 83 of SEQ ID NO: 3 to K; b) an amino acid substitution at position 84 of SEQ ID NO: 3 to V; c) an amino acid substitution at position 85 of SEQ ID NO: 3 to L; d) an amino acid substitution at position 86 of SEQ ID NO: 3 to Y or F; e) an amino acid substitution at position 87 of SEQ ID NO: 3 to Q; f) an amino acid substitution at position 88 of SEQ ID NO: 3 to S; g) an amino acid substitution at position 90 of SEQ ID NO: 3 to R; h) an amino acid substitution at position 91 of SEQ ID NO: 3 to A or V; i) an amino acid substitution at position 92 of SEQ ID NO: 3 to F; j) an amino acid substitution at position 101 of SEQ ID NO: 3 to K; k) an amino acid substitution at position 102 of SEQ ID NO: 3 to R; l) an amino acid substitution at position 103 of SEQ ID NO: 3 to V; m) an amino acid substitution at position 104 of SEQ ID NO: 3 to W or F; n) an amino acid substitution at position 105 of SEQ ID NO: 3 to S, F, R, L, V; o) an amino acid substitution at position 116 of SEQ ID NO: 4 to N; p) an amino acid substitution at position 117 of SEQ ID NO: 4 to E; q) an amino acid substitution at position 118 of SEQ ID NO: 4 to A; r) an amino acid substitution at position 121 of SEQ ID NO: 4 to K; s) an amino acid substitution at position 123 of SEQ ID NO: 4 to D; t) an amino acid substitution at position 154 of SEQ ID NO: 4 to H; u) an amino acid substitution at position 157 of SEQ ID NO: 4 to M; v) an amino acid substitution at position 161 of SEQ ID NO: 4 to D; w) an amino acid substitution at position 163 of SEQ ID NO: 4 to R; x) an amino acid substitution at position 223 of SEQ ID NO: 4 to R; or y) an amino acid substitution at position 229 of SEQ ID NO: 4 to K.

8. The protein molecule according to any one of claims 1 to 7, wherein the variant fXa or fragment thereof comprises one or more of: a) an amino acid substitution at position 86 of SEQ ID NO:3; b) an amino acid substitution at position 90 of SEQ ID NO:3; c) an amino acid substitution at position 102 of SEQ ID NO:3; d) an amino acid substitution at position 104 of SEQ ID NO:3; e) an amino acid substitution at position 105 of SEQ ID NO:3; f) an amino acid substitution at position 116 of SEQ ID NO:4; g) an amino acid substitution at position 117 of SEQ ID NO:4; h) an amino acid substitution at position 118 of SEQ ID NO:4; i) an amino acid substitution at position 121 of SEQ ID NO:4; j) an amino acid substitution at position 123 of SEQ ID NO:4; k) an amino acid substitution at position 161 of SEQ ID NO:4; l) an amino acid substitution at position 163 of SEQ ID NO:4; or m) an amino acid substitution at position 223 of SEQ ID NO:4.

9. The protein molecule according to any one of claims 1 to 8, wherein the variant fXa or fragment thereof comprises one or more of: a) an amino acid substitution at position 86 of SEQ ID NO: 3 to Y; b) an amino acid substitution at position 90 of SEQ ID NO: 3 to R; c) an amino acid substitution at position 102 of SEQ ID NO: 3 to R; d) an amino acid substitution at position 104 of SEQ ID NO: 3 to W or F; e) an amino acid substitution at position 105 of SEQ ID NO: 3 to S, F, R, L or V; f) an amino acid substitution at position 116 of SEQ ID NO: 4 to N; g) an amino acid substitution at position 117 of SEQ ID NO: 4 to E; h) an amino acid substitution at position 118 of SEQ ID NO: 4 to A; i) an amino acid substitution at position 121 of SEQ ID NO: 4 to K; j) an amino acid substitution at position 123 of SEQ ID NO: 4 to D; k) an amino acid substitution at position 161 of SEQ ID NO: 4 to D; l) an amino acid substitution at position 163 of SEQ ID NO: 4 to R; or m) an amino acid substitution at position 223 of SEQ ID NO: 4 to R.

10. The protein molecule according to any one of claims 1 to 9, wherein the variant fXa or fragment thereof comprises: a) one or more of S90R, L91 A, D92F, H101 K, E102R, E103V, N105S, according to SEQ ID NO: 3, E116N, S117E. T121 K, K123D, N154H, K157M, S161 D, I163R, A223R, D229K, according to SEQ ID NO: 4;b) one or more of S90R, E102R according to SEQ ID NO: 3, E116N, S117E, T1 18A, T121 K, K123D according to SEQ ID NO: 4; c) one or more of R86Y, S90R, E102R according to SEQ ID NO: 3, E1 16N, S1 17E, T1 18A, T121 K, K123D according to SEQ ID NO: 4; or d) one or more of R86Y, S90R, E102R, Q104W, Q105R according to SEQ ID NO: 3, E116N, S117E, T118A, T121 K, K123D according to SEQ ID NO:

411. The protein molecule according to any one of claims 1 to 10, wherein the protein molecule does not bind to fV or demonstrates negligible binding to fV.

12. The protein molecule according to any one of claims 1 to 11 , wherein the protein molecule binds fVa with a dissociation constant of at 100 nM or lower.

13. The protein molecule according to any one of claims 1 to 12, wherein the protein molecule binds fV with a dissociation constant of 500 nM or higher.

14. The protein molecule according to any one of claims 1 to 13, wherein the protein molecule binds to fVa with high affinity in the absence of phospholipid membrane surfaces.

15. The protein molecule according to any one of claims 1 to 14, wherein the wherein the variant fXa or fragment thereof comprises an EGF1 domain and / or a gamma-carboxyglutamic acid (Gia) domain.

16. The protein molecule according to any one of claims 1 to 16, further comprising a half-life extending moiety.

17. The protein molecule according to claim 16, where in the half-life extending moiety is selected from one or more PEG molecules, a liposome, an Fc domain, a serum albumin protein, or an antibody or antibody fragment that binds serum albumin.

18. The protein molecule according to claim 17, wherein the Fc domain comprises one or more amino acid modification or substitution which increases half-life of said protein molecule.

19. The protein molecule according to any one of claims 1 to 18, wherein the variant fXa is catalytically inactive.

20. The protein molecule according to claim 19, wherein the variant fXa comprises an amino acid substitution at position 185 according to SEQ ID NO: 4.

21. The protein molecule according to claim 19, wherein the variant fXa comprises a covalent inhibitor in the active site, such as Glu-Gly-Arg-chloromethylketone (EGRCK) or D-Phe-Pro-Arg- chloromethylketone (PPACK).

22. The protein molecule according to any one of claims 1 to 21 , wherein the variant fXa comprises reduced affinity to tissue factor pathway inhibitor (TFPI) compared to wild-type fXa.

23. The protein molecule according to any one of claims 1 to 22, wherein the variant fXa comprises reduced reactivity to antithrombin compared to wild-type fXa.

24. A pharmaceutical composition comprising a protein molecule according to any one of claims 1 to 23 and a pharmaceutically acceptable excipient.

25. A protein molecule according to any one of claims 1 to 23 or a pharmaceutical composition according to claim 24 for use in therapy.

26. A protein molecule according to any one of claims 1 to 23 or a pharmaceutical composition according to claim 24 for use in the treatment of a thrombin-mediated condition.

27. A method of treating a thrombin-mediated condition comprising a therapeutically effective amount of the protein molecule according to any one of claims 1 to 23, to a subject in need thereof.

28. Use of a protein molecule according to any one of claims 1 to 23, in the manufacture of a medicament for the treatment of a thrombin-mediated condition.

29. The protein molecule for use according to claim 26, the method of claim 27 or the use of claim 28, wherein the thrombin-mediated condition is selected from thrombosis, embolism, inflammation, clotting disorders, stroke associated with atrial fibrillation, thrombophilia, thrombotic stroke, coronary artery occlusion, infection, tumour growth, metastasis, organ rejection and dementia.

30. A method for reducing or inhibiting thrombin generation comprising: contacting a biological sample with a protein molecule according to any one of claims 1 to 23.31 . A method for reducing or inhibiting coagulation comprising: contacting a biological sample with a protein molecule according to any one of claims 1 to 23.

32. A method of detecting the presence of fVa in a biological sample comprising:contacting a biological sample with a protein molecule according to any one of claims 1 to 23, detecting binding of said protein molecule to fVa.

33. The method according to claim 32, wherein the protein molecule comprises a detectable label.

34. The method according to any one of claims 30 to 33, wherein the method is performed in vitro or ex vivo.

35. A kit comprising the protein molecule according to any one of claims 1 to 23, and optionally instructions for use.

Images