A HEMOSTATIC AGENT AND USES THEREOF

Abstract

An isolated recombinant soluble endothelial cell protein C receptor (r-sEPCR) for use as a hemostatic agent.

Claims

1. An isolated recombinant soluble endothelial cell protein C receptor (r-sEPCR) for use as a hemostatic agent.

2. The isolated recombinant soluble endothelial cell protein C receptor according to the use of claim 1, comprising SEQ ID NOs. 2 or 4, and a tag.

3. The isolated recombinant soluble endothelial cell protein C receptor according to the use of claim 2, wherein the tag is defined by SEQ ID NO. 14.

4. The isolated recombinant soluble endothelial cell protein C receptor according to claim 2 or claim 3, further comprising a linker between the tag and either SEQ ID NO. 2 or SEQ ID NO. 4.

5. The isolated recombinant soluble endothelial cell protein C receptor according to the use of any one of claims 1 to 4, further comprising a his-tag fused to the C-terminal end of the recombinant soluble endothelial cell protein C receptor via a peptide linker.

6. The isolated recombinant soluble endothelial cell protein C receptor according to the claim 5, wherein, the peptide linker is selected from SEQ ID NOs. 5 to 8 and 13.

7. The isolated recombinant soluble endothelial cell protein C receptor according to the use of any one of the preceding claims as defined by SEQ ID NO. 7 or SEQ ID NO. 8.

8. A nucleic acid encoding the recombinant soluble endothelial cell protein C receptor according to the use of any one of the preceding claims.

9. An expression vector comprising the nucleic acid of claim 8.

10. The expression vector of claim 9 selected from the group consisting of an adenovirus-associated virus (AAV) vector, a retroviral vector, an adenoviral vector, a plasmid, or a lentiviral vector.

11. The expression vector of claim 10, wherein the AAV vector comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVII, RhIO, Rh74 or AAV-2i8 AAV serotype.

12. The expression vector of claim 11, wherein the expression vector further comprises an intron, an expression control element, one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs) and/or a filler polynucleotide sequence.

13. The expression vector of claim 12, wherein the ITR comprises one or more ITRs of any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVII, RhIO, Rh74 or AAV-2i8 AAV serotypes, or a combination thereof.

14. The expression vector of any one of claims 9 to 13, further comprising an expression control element, wherein the expression control element comprises a constitutive or regulatable control element, or a tissue-specific expression control element or promoter.

15. The expression vector of claim 14, wherein the expression control element comprises an element that confers expression in liver.

16. The expression vector of claim 14 or claim 15, wherein the expression control element comprises a TTR promoter or mutant TTR promoter.

17. A pharmaceutical composition comprising the recombinant soluble endothelial cell protein C receptor according to the use of any one of claims 1 to 7 and a biologically acceptable carrier.

18. An isolated recombinant soluble endothelial cell protein C receptor according to the use of any one of claims 1 to 7 or the nucleic acid of claim 8 for use in a method of treating or preventing a hemostatic disorder.

19. The isolated recombinant soluble endothelial cell protein C receptor according to claim 18 for use in the method of claim 18, wherein the disorder is selected from the group consisting of hemophilia A, hemophilia B, FVII deficiency, Glanzmann's thrombasthenia, Bernard-Soulier syndrome, von Willebrand diseases, bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC) and over-anticoagulation treatment disorders.

20. The isolated the recombinant soluble endothelial cell protein C receptor according to claim 18 or claim 19 for use in the method of claim 18, wherein the recombinant soluble endothelial cell protein C receptor is fused to an antifibrinolytic protein.

21. The isolated the recombinant soluble endothelial cell protein C receptor according to claim 20 for use in the method of claim 20, wherein the antifibrinolytic protein includes wild type or engineered forms of soluble thrombomodulin, thrombin activatable fibrinolysis inhibitor, protease nexin-1, plasminogen activator 1, and plasminogen activator 2.

22. A formulation comprising the isolated recombinant soluble endothelial cell protein C receptor according to the use of any one of claims 1 to 7 for use in the method as claimed in any one of claims 18 to 21.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The invention will be more clearly understood from the following description of an embodiment thereof, given by way of example only, with reference to the accompanying drawings, in which:—

[0057] FIG. 1 shows the recombinant expression and purification of human and murine sEPCR.sup.Snoop: (a) plasmids encoding soluble human and murine EPCR with a ‘SnoopTag’ and hexapeptide His-tag fused via a peptide linker GSGGSG to the C-terminal end of sEPCR (h-sEPCR.sup.Snoop and m-sEPCR.sup.Snoop) being used to generate lentivirus particles encoding h-sEPCR.sup.Snoop and m-sEPCR.sup.Snoop. h-sEPCR.sup.Snoop and m-sEPCR.sup.Snoop-encoding lentivirus particles were then used to transduce HEK293 cells and transduced cells isolated by puromycin selection. h-sEPCR.sup.Snoop and m-sEPCR.sup.Snoop-expressing HEK293 cells were expanded and incubated with serum-free medium for 5 days, after which the cell supernatant was collected. (b) h-sEPCR.sup.Snoop and m-sEPCR.sup.Snoop was then purified using nickel ion chromatography, characterised by immunoblotting with an anti-His tag antibody and quantified using human and murine EPCR ELISA.

[0058] FIG. 2 shows sEPCR-dependent recovery of thrombin generation in normal plasma in the presence of activated anticoagulant protein C pathway: sEPCR-dependent recovery of thrombin generation in normal plasma in the presence of activated anticoagulant protein C pathway. The ability of sEPCR to promote thrombin generation in normal platelet-poor plasma in the presence of either activated protein C (a & b) or soluble thrombomodulin (c & d) was assessed using calibrated automated thrombinography. Briefly, anionic phospholipid vesicles (phosphatidylcholine (PC):, phosphophatidylserine (PS):, phosphatidylethanolamine (PE):, 80:20:20% ratio, 4 μM) and soluble tissue factor (5 μM) was used to trigger thrombin generation. Activated protein C (2.5 nM) or thrombomodulin (5 nM) were added in the presence or absence of h-sEPCR.sup.Snoop (1 μM, b & d). Thrombin generation was measured using a Flouroskan Ascent plate reader and analysis of thrombin generation kinetic parameters in each assay was performed using dedicated Thrombinoscope software (thrombin peak, b & d).

[0059] FIG. 3 shows sEPCR-dependent recovery of thrombin generation in factor VIII-deficient plasma in the presence of activated anticoagulant protein C pathway: The ability of sEPCR to promote thrombin generation in platelet-poor factor VIII-deficient plasma in the presence of either activated protein C (a & b) or soluble thrombomodulin (c & d) was assessed using calibrated automated thrombinography. Briefly, anionic phospholipid vesicles (phosphatidylcholine (PC):, phosphophatidylserine (PS):, phosphatidylethanolamine (PE):, 80:20:20% ratio, 4 μM) and soluble tissue factor (5 μM) was used to trigger thrombin generation. Activated protein C (2.5 nM) or thrombomodulin (5 nM) were added in the presence or absence of h-sEPCR.sup.Snoop (1 μM, b & d). Thrombin generation was measured using a Flouroskan Ascent plate reader and analysis of thrombin generation kinetic parameters in each assay was performed using dedicated Thrombinoscope software (thrombin peak, b & d).

[0060] FIG. 4 shows that h-sEPCR.sup.Snoop suppresses APC binding to EPCR on endothelial cells. EA.hy926 endothelial cells were incubated with F.sub.c block for 10 minutes before adding RCR-252 (25 μg/mL), h-sEPCR.sup.Snoop (1 μM) or h-sEPCR-E86A.sup.Snoop (1 μM), an sEPCR variant unable to bind to PC/APC. The cells were then incubated with fluorescently labelled, biotinylated active-site blocked APC (50 nM). APC-conjugated streptavidin was also added for 30 minutes at 37° C. FIG. 4a(i) shows live endothelial cells in the absence of APC, whereas FIG. 4a(ii) shows APC bound to endothelial cells. FIG. 4a(iii) describes the binding of APC in the presence of an anti-EPCR monoclonal antibody (RCR-252) which blocks APC binding. Similarly, APC binding to endothelial cells (FIG. 4a(iv)) was completely impaired by the presence of h-sEPCR.sup.Snoop (FIG. 4a(v)), whereas the presence of an h-sEPCR.sup.Snoop mutant unable to bind to APC was unable to inhibit APC binding (FIG. 4a(vi)). The binding of APC to endothelial cells under each condition is summarised in FIG. 4b. Cells were then analysed using a FACSCanto II flow cytometer and data analysed using FlowJo software. Statistical analysis of experimental data was performed using one-way ANOVA and Tukey's multiple comparison post hoc analysis. ns signifies non-significant, *** signifies a p value of <0.001. This data indicates that h-sEPCR.sup.Snoop suppresses Protein C/APC binding to endothelial cells.

[0061] FIG. 5 shows that h-sEPCR.sup.Snoop suppresses APC generation on endothelial cells. Protein C (100 nM) and thrombin (5 nM) were added with h-sEPCR.sup.Snoop (1 μM) or h-sEPCR-E86A.sup.Snoop (1 μM) an sEPCR variant unable to bind to PC/APC, then incubated for 30 minutes. Thrombin activation was subsequently inhibited using hirudin and APC generation was measured with an APC-specific chromogenic substrate CS-21(66). These data indicate that h-sEPCR.sup.Snoop inhibits protein C activation on endothelial cells by the thrombin-thrombomodulin complex and that this activity is dependent on h-sEPCR.sup.Snoop binding to PC. The net effect of this restriction of APC generation. Statistical analysis of experimental data was performed using one-way ANOVA and Tukey's multiple comparison post hoc analysis. ns signifies non-significant, * signifies a p value of <0.05.

DETAILED DESCRIPTION OF THE DRAWINGS

[0062] Materials and Methods

[0063] Lentiviral Expression of Recombinant Human and Murine Soluble EPCR Variants (sEPCR.sup.Snoop)

[0064] Plasmids for the expression of recombinant human and murine sEPCR (SEQ ID NO. 1 and SEQ ID NO. 2, respectively) and of recombinant human and murine sEPCR with the SnoopTag sequence (h-sEPCR.sup.Snoop (SEQ ID NO. 5) and m-sEPCR.sup.Snoop (SEQ ID NO. 6), respectively) were synthesised and cloned into the lentiviral backbone plasmid pLJM1-Empty (Addgene Plasmid #91980). Both sEPCR constructs were generated with C-terminal hexapeptide His-tags and a ‘SnoopTag’ sequence (SEQ ID NO. 15) to facilitate fusion with additional protein components with the potential to modulate sEPCR half-life or functional activity. Lentiviral particles were prepared by seeding a 10 cm.sup.2 cell culture dish with 5×10.sup.6 Lenti-X HEK293T cells (TaKaRa Bio Cat #632180) in 8 mL of cell culture media and incubated overnight at 37° C., 5% CO.sub.2. Plasmids encoding h-sEPCR.sup.Snoop or m-sEPCR.sup.Snoop (7 μg) were mixed with distilled H.sub.2O to make a final volume of 600 μL and added to a Lenti-X packaging Single Shot vial (TaKaRa Bio Cat #631276), vortexed and incubated for 10 minutes at room temperature. The solution was then added dropwise to the dish of Lenti-X HEK293T cells, the media swirled gently and then incubated for 4 hrs at 37° C. 5% CO.sub.2 in a humidified atmosphere. An additional 6 mL of cell media was added and the plate was then incubated for a further 72 hrs for lentivirus particles to be produced. h-sEPCR.sup.Snoop and m-sEPCR.sup.Snoop lentivirus particles were harvested by removing 13 mL of virus-containing media from the cells and cellular debris cleared by centrifugation for 10 mins at 500×g. The viruses were then concentrated using Lenti-X concentrator solution (TaKaRa Bio Cat #631231). One volume of concentrator solution was mixed with three volumes of virus containing supernatant, incubated for 30 mins at 4° C. and centrifuged at 1500×g for 45 minutes. Supernatant was discarded and the virus particles re-suspended in 2 mL of media for transduction of HEK293 cells. Prior to this step, HEK293 cells were seeded at 2*10.sup.5 cells per well into a six well tissue culture plate and incubated overnight. To transduce HEK293 cells with the h-sEPCR.sup.Snoop and m-sEPCR.sup.Snoop lentiviral particles, cell culture media was removed from the cells and replaced with virus suspension (2 mL) and polybrene (Merck) was added (final concentration, 6 μg/mL) to aid transduction efficiency. Cells were incubated for 24 hrs at 37° C., 5% CO.sub.2 before the media was removed and replaced by fresh media. Transduced cells were incubated for a further 72 hrs to allow for plasmid integration. Successfully transduced cells were isolated by antibiotic selection using puromycin-containing cell media (0.6 μg/mL).

[0065] sEPCR.sup.Snoop Protein Purification and Expression Analysis

[0066] Transduced HEK293 cells were seeded in Corning hyperflasks and grown to confluence, then incubated with serum-depleted cell culture medium for 4-5 days. Cell supernatants were then collected and recombinant h-sEPCR.sup.Snoop and m-sEPCR.sup.Snoop isolated from cell supernatants by nickel sepharose chromatography. Eluted fractions containing sEPCR.sup.Snoop were pooled and concentrated by tangential flow filtration. The presence of sEPCR.sup.Snoop was confirmed by immunoblotting and quantified using human and murine sEPCR ELISA (R & D Systems).

[0067] Assessment of sEPCR.sup.Snoop Inhibition of APC- or TM-Mediated Anticoagulant Activity

[0068] PPP-Reagent (Thrombinoscope) containing anionic phospholipid vesicles (phosphatidylcholine (PC):, phosphophatidylserine (PS):, phosphatidylethanolamine (PE):, 80:20:20% ratio, 4 μM) and soluble tissue factor, 5 μM) was used to trigger thrombin generation in platelet-poor pooled normal or factor VIII-deficient human plasma. A thrombin calibrator (Thrombinoscope) was added to enable thrombin quantification. 80 μL of plasma was added to each test well in addition to PPP-Reagent. Activated protein C or thrombomodulin were added as described (see FIGS. 2 and 3) in the presence of h-sEPCR.sup.Snoop (1 μM). To start the reaction, 20 μL Ca.sup.2+ containing saline buffer with a fluorogenic thrombin substrate was added. Thrombin generation was measured for 1 hour using a Flouroskan Ascent plate reader. Analysis of thrombin generation kinetic parameters was performed using Thrombinoscope software.

[0069] Assessment of In Vivo Haemostatic Activity of sEPCR in Murine Haemophilia a Model

[0070] The procoagulant properties of h-sEPCR.sup.Snoop can be analysed to determine whether they can be harnessed to drive coagulation in both normal and haemophilia plasma. m-sEPCR.sup.Snoop can be assessed for pro-haemostatic activity in vivo using a murine model of haemophilia A (FVIII.sup.−/− mice). FVIII.sup.−/− mice on a C57BL/6 background are already established within the Royal College of Surgeons in Ireland (RCSI). sEPCR can be evaluated for its ability to attenuate tail bleeding in FVIII.sup.−/− mice. Using these mice, a well-characterized assay of bleeding tendency that has been used previously can be utilised to monitor restoration of haemostatic activity (Aljamali M N, Margaritis P, Schlachterman A, et al. J Clin Invest 2008; 118(5):1825-1834). Briefly, tails of 8-10-week-old FVIII.sup.−/− mice are placed in saline for 10 minutes prior to injury. Bleeding is initiated by cutting 2.5 mm from the tip of the tail, whereupon the cut tail is immediately placed in saline. After 5 minutes, m-sEPCR.sup.Snoop (0.25-2 mg/kg) or vehicle (n=8 mice per group) is injected as a bolus into the carotid artery, then the tail placed in a new saline-containing container and the extent of bleeding monitored for 30 minutes. Total bleeding time is defined as the sum of the length of bleeding episodes from time of haemostatic agent injection until termination. Blood loss is determined by accumulation of haemoglobin in the saline from the time of treatment. Murine sEPCR (m-sEPCR.sup.Snoop) can be used here to limit the potential for artefactual species-specific differences in protein C/APC affinity.

[0071] Comparison of m-sEPCR.sup.Snoop Potency in Restoration of Haemostasis in Haemophilia A Mice Compared to Existing Commercially Available Haemostatic Agents.

[0072] The haemostatic potential of sEPCR.sup.Snoop compared to recombinant FVIII (the current prophylactic treatment for people with haemophilia A) and recombinant FVIIa (a commonly used pro-haemostatic ‘bypass’ agent) can be tested using the bleeding models described abobe. To achieve this, sEPCR.sup.Snoop can be compared to rFVIII and rFVIIa in their abilities to attenuate tail bleeding in FVIII.sup.−/− mice as described above. 5 minutes after tail clipping, m-sEPCR.sup.Snoop (1 mg/kg or alternative effective dose as determined in the previous experiment), rFVIII (50 IU/kg).sup.34, rFVIIa (1 mg/kg).sup.35 or vehicle (n=8 mice per group) is injected as a bolus into the carotid artery, the tail is then placed in a new saline-containing container, and the extent of bleeding monitored for 30 minutes and bleeding assessed as previously described.

[0073] Assessment of h-sEPCR.sup.Snoop Inhibition of Protein C/APC Binding to EPCR on Endothelial Cells

[0074] EA.hy926 endothelial cells were incubated with FC block for 10 minutes before adding RCR-252 (25 μg/mL), h-sEPCR.sup.Snoop (1 μM) or h-sEPCR-E86A.sup.Snoop (1 μM), an sEPCR variant unable to bind to PC/APC. The cells were then incubated with fluorescently labelled, biotinylated active-site blocked APC (50 nM). APC-conjugated streptavidin was also added for 30 minutes at 37° C. Cells were then analysed using a FACSCanto II flow cytometer and data analysed using FlowJo software. Statistical analysis of experimental data was performed using one-way ANOVA and Tukey's multiple comparison post hoc analysis. ns signifies non-significant, *** signifies a p value of <0.001. This data indicates that h-sEPCR.sup.Snoop suppresses Protein C/APC binding to endothelial cells (see FIG. 4).

[0075] Assessment of h-sEPCR.sup.Snoop Inhibition of Protein C Activation by the Thrombin-Thrombomodulin Complex on Endothelial Cells Protein C (100 nM) and thrombin (5 nM) were added with h-sEPCR.sup.Snoop (1 μM) or h-sEPCR-E86ASnoop (1 μM) an sEPCR variant unable to bind to PC/APC, then incubated for 30 minutes. Thrombin activation was subsequently inhibited using hirudin and APC generation was measured with an APC-specific chromogenic substrate CS-21(66). These data indicate that h-sEPCR.sup.Snoop inhibits protein C activation on endothelial cells by the thrombin-thrombomodulin complex and that this activity is dependent on h-sEPCR.sup.Snoop binding to PC. The net effect of this restriction of APC generation. Statistical analysis of experimental data was performed using one-way ANOVA and Tukey's multiple comparison post hoc analysis. ns signifies non-significant, * signifies a p value of <0.05 (see FIG. 5).

[0076] Discussion

[0077] In FIG. 1a, we see a schematic diagram of sEPCR.sup.Snoop, comprised of human/murine EPCR truncated at the membrane spanning region (amino acids 1-219), a short linker region (GCSGCS (SEQ ID NO. 9)) and a SnoopTag (for fusion to other proteins-of-interest, SEQ ID NO. 14). In FIG. 1b, human and murine sEPCR.sup.Snoop expressed from HEK293 cells into serum-free supernatant and purified using Ni ion chromatography. Both human and murine sEPCR.sup.Snoop were visualised by Western blotting using an anti-His horseradish-peroxidase labelled antibody. These data indicate that the expressed sEPCR.sup.Snoop protein is expressed normally in a mammalian cell expression system and is present at the anticipated molecular weight.

[0078] FIG. 2 shows the ability of h-sEPCR.sup.Snoop to recover thrombin generation in normal platelet-poor plasma in the presence of either (a) APC or (b) TM. Briefly, co-incubation of h-sEPCR.sup.Snoop (1 μM) and (a) APC (blue line) is sufficient to overcome the anticoagulant activity of APC alone (gold line). Similarly, h-sEPCR.sup.Snoop (1 μM) co-incubated with TM (blue line) is sufficient to overcome the anticoagulant activity of TM alone (gold line). h-sEPCR.sup.Snoop restored peak thrombin generation in normal plasma incubated with either APC or TM (b & d). Importantly, h-sEPCR.sup.Snoop alone had no effect upon rate and size of thrombin generation in normal plasma. These data indicate that h-sEPCR.sup.Snoop can restore thrombin generation in the presence of activated protein C anticoagulant pathway in normal platelet-poor plasma, highlighting its potential as a novel haemostatic agent with application for the treatment of acquired bleeding disorders where the protein C pathway is activated. In addition, these data also indicate that h-sEPCR.sup.Snoop alone does not increase thrombin generation in the absence of APC generation, suggesting it is unlikely to possess deleterious prothrombotic properties.

[0079] FIG. 3 also demonstrates that h-sEPCR.sup.Snoop can promote recovery of thrombin generation in the presence of either APC (a & b) or TM (c & d) in FVIII-deficient plasma that mimics plasma derived from people with severe haemophilia. Briefly, co-incubation of h-sEPCR.sup.Snoop and APC (blue line) is sufficient to overcome the anticoagulant activity of APC alone (gold line). In addition, h-sEPCR.sup.Snoop (1 μM) co-incubated with TM (blue line) is sufficient to overcome the anticoagulant activity of TM alone (gold line). h-sEPCR.sup.Snoop restored peak thrombin generation in both normal and FVIII-deficient plasma (b & d). Importantly, h-sEPCR alone had no effect upon rate and size of thrombin generation in FVIII-deficient plasma. These data indicate that h-sEPCR.sup.Snoop can restore thrombin generation in the presence of activated protein C anticoagulant pathway in platelet-poor FVIII-deficient plasma, highlighting its potential as a novel haemostatic agent with application for the treatment of haemophilia patients with anti-FVIII inhibitors. In addition, similar to experiments in normal platelet-poor plasma, these data also indicate that h-sEPCR.sup.Snoop alone does not increase thrombin generation in the absence of APC generation.

[0080] FIG. 4 shows that h-sEPCR.sup.Snoop suppresses APC binding to EPCR on endothelial cells, while FIG. 5 indicates that h-sEPCR.sup.Snoop inhibits protein C activation on endothelial cells by the thrombin-thrombomodulin complex and that this activity is dependent on h-sEPCR.sup.Snoop binding to PC. The net effect of this restriction of APC generation.

[0081] It has been previously shown that h-sEPCR.sup.Snoop effectively inhibits protein C activation triggered on human endothelial cells, suggesting it can reduce the rate of APC generation. Moreover, the inventors have shown here that sEPCR potently inhibited exogenously administered APC anticoagulant activity in normal pooled plasma. Importantly, h-sEPCR.sup.Snoop was also able to completely restore clotting activity in haemophilia A plasma in which endogenous APC was generated, that otherwise demonstrated severely impaired thrombin generation and clotting activity. h-sEPCR.sup.Snoop had no independent procoagulant activity in the absence of thrombomodulin or APC.

[0082] Furthermore, given that the mode of action of h-sEPCR.sup.Snoop is not directly tied to the correction of defective intrinsic tenase activity caused by missing or dysfunctional FVIII, h-sEPCR.sup.Snoop may also have utility as a general haemostatic agent in a number of other clinical contexts defined by dysregulated haemostasis.

[0083] The claimed invention acts as a soluble decoy receptor to prevent receptor or anionic phospholipid cell surface interactions with zymogen protein C (PC) and APC, respectively. The effect of this interaction is distinct from that described in WO 2014/085596. The claimed invention has shown that h-sEPCR.sup.Snoop binds PC or APC equally, and prevents interaction of PC or APC with EPCR on the endothelial cell surface (see FIG. 4). This activity is not described in WO 2014/085596, which binds solely to APC at a distinct molecular location. Inhibition of PC binding to endothelial cell surface EPCR by h-sEPCR.sup.Snoop slows generation of APC and in doing so, reduces anticoagulant activity (see FIG. 5). This activity is not described for in WO 2014/085596, which solely impairs APC proteolysis of its procoagulant substrate, activated factor V. sEPCR binding to APC prevents interaction with anionic cell surface phospholipids, preventing complex formation with APC cofactor protein S and activated factor V to attenuate APC anticoagulant activity.

[0084] The invention may be used as a means to treat bleeding in haemophilia patients with or without inhibitor antibodies directed against their replacement protein therapy. Furthermore, it may be used to prevent or treat bleeding in individuals with other inherited bleeding disorders (including, but not limited, to factor XI deficiency, factor V deficiency, factor FVII deficiency and factor X deficiency). The invention also illustrates (i) a large-scale recombinant expression system for sEPCR; (ii) an effective dose determination for m-sEPCR.sup.Snoop in restricting bleeding in pre-clinical haemophilia models; and (iii) shows a head-to-head pre-clinical efficacy assessment of sEPCR versus commercially available pro-haemostatic therapeutic agents. The invention could also be used as an emergency haemostatic agent that prevents bleeding following trauma or surgery, or to reverse anticoagulant therapy.

[0085] In the specification the terms “comprise, comprises, comprised and comprising” or any variation thereof and the terms “include, includes, included and including” or any variation thereof are considered to be totally interchangeable and they should all be afforded the widest possible interpretation and vice versa.

[0086] The invention is not limited to the embodiments hereinbefore described but may be varied in both construction and detail.