RECOMBINANT SERINE PROTEASES

Abstract

The present invention relates to recombinant proteins having serine protease polypeptides that have serine protease activity in the presence of a serine protease inhibitor and that are able to completely or partially reverse a serine protease inhibitor effect, for example in a subject treated with a serine protease inhibitor. More specifically, described herein are recombinant proteins and methods for completely or partially reversing an anti-coagulant effect of a coagulation inhibitor.

Claims

1. A recombinant protein comprising a serine protease, said serine protease comprising an insertion of an amino acid residue in an outer-surface peptide structure; wherein the serine protease polypeptide is not a coagulation factor X polypeptide or naturally processed or activated forms thereof.

2. The protein according to claim 1, wherein said peptide structure is a region of amino acid residues corresponding to the region of amino acid residues between His-450 and Asp-462 of SEQ ID NO: 1.

3. The protein according to claim 1, wherein the serine protease is selected from the group consisting of thrombin, coagulation factor XIa, trypsin and urokinase-type plasminogen activator; and wherein said peptide structure is: a region of amino acid residues between Gly-427 and Asp-462, of SEQ ID NO: 1 of thrombin; a region of amino acid residues between Val-463 and Asp-480 of SEQ ID NO: 2 of coagulation factor XIa; a region of amino acid residues between Leu-73 and Asp-107 of SEQ ID NO: 3 of trypsin; and a region of amino acid residues between Val-237 and Asp-275, of SEQ ID NO:4 of urokinase-type plasminogen activator.

4. The protein according to claim 1, wherein the insertion comprises 1-50 amino acid residues.

5. The protein according to claim 1, wherein the insertion comprises between 4 and 50 amino acid residues.

6. The protein according to claim 2, wherein the insertion of an amino acid residue is combined with a replacement of at least 5 amino acid residues in said region.

7. The protein according to claim 2, wherein said region, after insertion and/or replacement, has the amino acid sequence of: SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16 between His-450 and Asp-462 of SEQ ID NO:1; SEQ ID NO: 7 or SEQ ID NO: 8 between His-469 and Asp-480 of SEQ ID NO:2; SEQ ID NO: 9 or SEQ ID NO: 10 between His-96 and Asp-107 of SEQ ID NO:3; and and/or SEQ ID NO: 11 or SEQ ID NO: 12 between His-262 and Asp-275 of SEQ ID NO:4.

8. A recombinant protein comprising a serine protease polypeptide having an amino acid residue replacement on an amino acid residue position corresponding to Ile-542 of SEQ ID NO:1, wherein the serine protease polypeptide is not a coagulation factor X polypeptide or naturally processed or activated forms thereof.

9. A nucleic acid molecule comprising a DNA sequence that encodes the protein according to claim 1.

10. An expression vector comprising the nucleic acid molecule according to claim 9.

11. A host cell comprising the nucleic acid molecule according to claim 9.

12. A pharmaceutical composition comprising the protein according to claim 1 and a pharmaceutically acceptable carrier.

13. (canceled)

14. The protein according to claim 1, wherein the protein comprises thrombin or coagulation factor XIa.

15. A method of reversing an anti-coagulant effect of a coagulation inhibitor in a subject, said method comprising administering to said subject a therapeutically effective amount of the protein according to claim 1, wherein the protein comprises thrombin or coagulation factor XIa.

16. (canceled)

17. The protein according to claim 1, wherein the protein comprises trypsin.

18. A method of reversing the inhibition of peptide bond hydrolysis of a trypsin inhibitor in a subject, said method comprising administering to said subject a therapeutically effective amount of the protein according to claim 1 wherein the protein comprises trypsin.

19. (canceled)

20. (canceled)

21. The protein according to claim 1, wherein the protein comprises urokinase-type plasminogen activator.

Description

FIGURE LEGENDS

[0107] FIG. 1. The top panel displays the alignment of the region of amino acid residues from His-450 to Asp-462 in human prothrombin (SEQ ID NO: 1) with the region of amino acid residues corresponding to said region in human FXI (SEQ ID NO: 2), human trypsin (SEQ ID NO: 3) and human urokinase-type plasminogen activator (SEQ ID NO:4). The lower three panels display the newly generated A and B protein variants of human prothrombin, human FXI, human trypsin and human urokinase-type plasminogen activator having an insertion in the region of amino acid residues corresponding to said region of His-450 to Asp-462 in human prothrombin, between His-469 and Asp-480 for human FXI, between His-96 and Asp-107 for trypsin and between His-262 and Asp-275 for urokinase-type plasminogen activator. The conserved residues histidine and aspartic acid are highlighted.

[0108] FIG. 2. The crystal structure of the argatroban-thrombin complex is shown (PDB 1DWC). The argatroban-contact residues (His57, Tyr60A, Lys60F, Leu99, Ile174, Glu192, Ser195, Asp189, Glu192, Gly216, Gly218, Gly226; chymotrypsin numbering (Bode, W. et al. 1989. EMBO J 8: 3467-3475)) and catalytic triad residues His57, Ser195, and Asp102 are shown as sticks. The His91 loop is highlighted and indicated with an arrow.

[0109] FIG. 3. The crystal structure of the compound 33-FXIa complex is shown (PDB 4X6P). Compound 33 is shown in a stick model, FXIa is shown in surface representation. The contact residues (His40, Leu41, Cys42, Cys58, Tyr58b, Tyr143, Ile151, Asp189, Lys192, Gly193, Asp 194, Ser195, Gly216, Gly218, Tyr228; chymotrypsin numbering (Bode, W. et al. 1989. EMBO J 8: 3467-3475)), the catalytic triad residues His57, Ser195, and the His91 loop are highlighted. The latter is additionally indicated with an arrow.

[0110] FIG. 4. The crystal structure of the melagatran-trypsin complex is shown (PDB 1K1P). The contact residues (Asp 189, Ser190, Gly216, and Gly21; chymotrypsin numbering (Bode, W. et al. 1989. EMBO J 8: 3467-3475)) and the catalytic triad residues His57, Ser195 are shown as sticks. The His91 loop is highlighted and indicated with an arrow.

[0111] FIG. 5. An alignment of the region of amino acid residues from His-450 to Asp-462 in human prothrombin (Thrombin, SEQ ID NO: 1) and the newly generated protein variants of human prothrombin (ISO1, ISO2, NSC, KL10, ALB) having an insertion in the region of amino acid residues corresponding to said region of His-450 to Asp-462 in human prothrombin. The conserved residues histidine at position 450 and aspartic acid at position 462 are highlighted.

[0112] FIG. 6. Inhibition of chromogenic thrombin activity by the direct thrombin inhibitor dabigatranserie 1. Peptidyl substrate conversion (S-2238; 100 M) by 5 nM plasma-derived thrombin (IIa; panel A), activated plasma-derived prothrombin (pd-IIa; panel B), recombinant thrombin (r-IIa; panel C), recombinant thrombin variant ISO1 (ISO1; panel D), or recombinant thrombin variant NSC (NSC; panel E) in the presence of increasing concentrations (20 nM-20 M) dabigatran. IC50 concentrations were obtained by fitting the S-2238 conversion (mOD/min) by nonlinear regression using the Graphpad Prism software suite. All data points represent the average of two independent experiments. Panel F: The substrate conversion (velocity) was plotted as the ratio of incubations in the absence of inhibitor. It is clearly shown in Panel F that the variants show a higher normalized velocity than the control.

[0113] FIG. 7. Inhibition of chromogenic thrombin activity by the direct thrombin inhibitor dabigatranserie 2. Peptidyl substrate conversion (S-2238; 100 M) by 5 nM plasma-derived thrombin (IIa; panel A), recombinant thrombin (r-IIa; panel B), recombinant thrombin variant ALB (ALB; panel C), recombinant thrombin variant KL10 (KL10; panel D), or recombinant thrombin variant ISO2 (ISO2; panel E) in the presence of increasing concentrations (20 nM-20 M) dabigatran. IC50 concentrations were obtained by fitting the S-2238 conversion (mOD/min) by nonlinear regression using the Graphpad Prism software suite. All data points represent the average of two independent experiments. Panel F: The substrate conversion (velocity) was plotted as the ratio of incubations in the absence of inhibitor. It is clearly shown in Panel F that the variants show a higher normalized velocity than the control.

[0114] FIG. 8. Crystal structure (PDB 1KTS) of thrombin in complex with dabigatran (Hauel et al., J Med Chem 45: 1757-1766 (2002)). The active site residues His406, Asp462, and Ser568 and the dabigatran interaction residues Tyr410, Leu459, Ile542, Asp562, Trp590, and Gly591 are shown in stick figures. The location of the S4 subpocket of the active site is marked by an oval, and residue Ile542 is indicated.

[0115] FIG. 9. Inhibition of chromogenic thrombin activity by the direct thrombin inhibitor dabigatranserie 3. Peptidyl substrate conversion (S-2238; 100 M) by 5 nM plasma-derived thrombin (panel A), recombinant thrombin (panel B), recombinant thrombin variant I542F (panel C), recombinant thrombin variant I542A (panel D), recombinant thrombin variant I542E (panel E), or recombinant thrombin variant I542S (panel F) in the presence of increasing concentrations (20 nM-20 M) dabigatran. IC50 concentrations were obtained by fitting the S-2238 conversion (mOD/min) by nonlinear regression using the Graphpad Prism software suite. All data points represent the average of two independent experiments.

[0116] FIG. 10. Normalized inhibition of chromogenic thrombin activity by the direct thrombin inhibitor dabigatran. Peptidyl substrate conversion (S-2238; 100 M) by 5 nM plasma-derived thrombin (Ha), recombinant thrombin (r-IIa), recombinant thrombin variant I542A, recombinant thrombin variant I542E, recombinant thrombin variant I542F, or recombinant thrombin variant I542S in the presence of increasing concentrations (20 nM-20 M) dabigatran. The substrate conversion (velocity) was plotted as the ratio of incubations in the absence of inhibitor. All data points represent the average of two independent experiments. It is clearly shown that the variants show a higher normalized velocity than the control.

[0117] FIG. 11. An alignment of the region of amino acid residues from Cys-536 to Cys-550 in human prothrombin (Thrombin, SEQ ID NO:1), Cys-545 to Cys-560 in human factor XI (FXIa, SEQ ID NO:2), Cys-171 to Cys-185 in human trypsin-1 (Trypsin, SEQ ID NO:3), and Cys-345 to Cys-361 in human urokinase-type plasminogen activator (uPA, SEQ ID NO:4). The residues Ile-542 (SEQ ID NO:1), His-552 (SEQ ID NO:2), Gly-177 (SEQ ID NO:3), and Ser-353 (SEQ ID NO:4) are highlighted.

[0118]

TABLE-US-00002 TABLE 1 Amino 3- 1- Side-chain Side-chain Hydropathy Absorbance at Acid Letter Letter polarity charge (pH 7.4) index .sub.max(nm) .sub.max (10.sup.3 M.sup.1 cm.sup.1) Alanine Ala A nonpolar neutral 1.8 Arginine Arg R Basic polar positive 4.5 Asparagine Asn N polar neutral 3.5 Aspartic acid Asp D acidic polar negative 3.5 Cystine Cys C nonpolar neutral 2.5 250 0.3 Glutamic acid Glu E acidic polar negative 3.5 Glutamine Gln Q polar neutral 3.5 Glycine Gly G nonpolar neutral 0.4 Histidine His H Basic polar positive(10%) 3.2 211 5.9 neutral(90%) Isoleucine Ile I nonpolar neutral 4.5 Leucine Leu L nonpolar neutral 3.8 Lysine Lys K Basic polar positive 3.9 Methionine Met M nonpolar neutral 1.9 Phenylalanine Phe F nonpolar neutral 2.8 257, 206, 188 0.2, 9.3, 68.0 Proline Pro P nonpolar neutral 1.6 Serine Ser S polar neutral 0.8 Threonine Thr T polar neutral 0.7 Tryptophan Trp W nonpolar neutral 0.9 290, 219 5.8, 47.0 Tyrosine Tyr Y polar neutral 1.3 274, 222, 193 1.4, 8.0, 48.0 Valine Val V nonpolar neutral 4.2 indicates data missing or illegible when filed

EXAMPLES

Example 1: Material and Methods

Materials:

[0119] Direct serine protease inhibitors are obtained from Alsachim and Adooq, and corn trypsin inhibitor is from Haematologic Technologies. FXI-depleted and prothrombin-depleted human plasma, Neoplastin CI plus 10, and TriniCLOT automated APTT are obtained from Diagnostica Stago. The peptidyl substrates S-2238, S-2366, S-2444 and S-2222 are obtained from Chromogenix. All tissue culture reagents are from Life Technologies except insulin-transferrin-sodium selenite (ITS), which is from Roche. Calibrator and fluorescent substrate (FluCa) are from Thrombinoscope BV. Small unilamellar phospholipid vesicles (PCPS) composed of 75% (w/w) hen egg L-phosphatidylcholine and 25% (w/w) porcine brain L-phosphatidylserine (Avanti Polar Lipids) are prepared and characterized as described previously (Higgins et al. 1983. J Biol Chem 258: 6503-6508). General recombinant protein production and purification techniques are as described in Green and Sambrook, Molecular Cloning, 4.sup.th edition, July 2012.

Expression and Purification of Thrombin:

[0120] Plasmids (pcDNA3.1(+)) encoding prothrombin variants A (prothrombin (SEQ ID NO:1) with between His-450 and Asp-462 the amino acid sequence of SEQ ID NO:5) and B (prothrombin (SEQ ID NO:1) with between His-450 and Asp-462 the amino acid sequence of SEQ ID NO:6) and wildtype prothrombin are introduced into HEK 293 cells using LipofectAMINE 2000 (Invitrogen) and pSV2neo as the selectable marker plasmid. High expressing clones are selected based on prothrombin-specific ELISA and PT clotting assays, essentially as described (Orcutt et al. 2004. J Biol Chem 279: 54927-54936). Selected clones are expanded into 10-stacked cell factories (Nalge-Nunc, Naperville, Ill.) and cultured in Dulbecco's modified Eagle's medium/F-12 media supplemented with 5 g/ml ITS and 10 g/ml Vitamin K. Conditioned media is collected for 5-6 days, centrifuged, and stored at 20 C. in the presence of 1 mM benzamidine.Prothrombin is purified from conditioned media employing Q-Sepharose FF (GE Healthcare), an HQ POROS matrix (Affinity Biologicals), and a ceramic hydroxyapatite matrix (Bio-Rad), essentially as described (Orcutt et al. 2004. J Biol Chem 279: 54927-54936). Purified prothrombin is stored at 20 C. in HBS containing 50% vol/vol glycerol. Protein purity is assessed by SDS-PAGE using pre-cast 4-12% gradient gels (Invitrogen) under reducing conditions followed by staining with Coomassie Brilliant Blue R-250. Thrombin is purified following preparative activation of prothrombin as described (Lundblad et al., 1976. Methods Enzymol 45: 156-176).

Expression and Purification of FXI:

[0121] Plasmids (pcDNA3.1(+)) encoding FXI variants A (FXI (SEQ ID NO:2) with between His-469 and Asp-480 the amino acid sequence of SEQ ID NO:7 and B (FXI (SEQ ID NO:2) with between His-469 and Asp-480 the amino acid sequence of SEQ ID NO:8 and wildtype FXI, fused via their C-terminus to the HPC4-antibody recognition sequence (amino acid sequence EDQVDPRLIDGK) to facilitate purification, are introduced into baby hamster kidney (BHK) cells using LipofectAMINE 2000 (Invitrogen) and pSV2neo as the selectable marker plasmid. High expressing clones are selected based on FXI-specific ELISA and APTT clotting assays, essentially as described for factor V (Toso et al. 2004. J Biol Chem 279: 21643-21650). Selected clones are expanded into triple flasks (Nalge-Nunc, Naperville, Ill.) and cultured in Dulbecco's modified Eagle's medium/F-12 media supplemented with 5 g/ml ITS and 1.0 mg/ml Albumax (Invitrogen). Conditioned media is collected for 5-6 days, centrifuged, and stored at 20 C. in the presence of 1 mM benzamidine.Conditioned media is thawed at 37 C., pooled, and loaded onto an anti-HPC4 Sepharose column equilibrated in 25 mM Tris, 0.05 M NaCl, 5 mM CaCl2, pH 7.4. The column is washed with the equilibration buffer, and then eluted with 25 mM Tris, 0.5 M NaCl, 5 mM EDTA, pH 7.4, followed by elution with 25 mM Tris, 2 M NaCl, 5 mM EDTA, pH 7.4. Fractions containing FXI activity are pooled, dialyzed versus 20 mM Hepes, 150 mM NaCl, pH 7.4, concentrated by ultrafiltration (Millipore), and the purified protein is stored at 80 C. Protein purity is assessed by SDS-PAGE using pre-cast 4-12% gradient gels (Invitrogen) under reducing conditions followed by staining with Coomassie Brilliant Blue R-250. Factor XIa is purified following preparative activation of FXI as described (Ogawa et al., 2005. J Biol Chem 280: 23523-23530).

Expression and Purification of Urokinase-Type Plasminogen Activator (uPA):

[0122] Plasmids (pcDNA3.1(+)) encoding human uPA variants A (uPA (SEQ ID NO:4) with between His-262 and Asp-275 the amino acid sequence of SEQ ID NO:11) and B (uPA (SEQ ID NO:4) with between His-262 and Asp-275 the amino acid sequence of SEQ ID NO:12) and wildtype uPA that are fused via their C-terminus to the HPC4-antibody recognition sequence (amino acid sequence EDQVDPRLIDGK) or to a His-tag (6His: HHHHHH or 12His: HHHHHHHHHHHH) to facilitate purification are introduced into mammalian cells (HEK 293 are BHK) using LipofectAMINE 2000 (Invitrogen) and pSV2neo as the selectable marker plasmid. High expressing clones are selected based on a human uPA-specific ELISA (R&D Systems). Selected clones are expanded into triple flasks (Nalge-Nunc, Naperville, Ill.) and cultured in Dulbecco's modified Eagle's medium/F-12 media supplemented with 5 g/ml ITS and 1.0 mg/ml Albumax (Invitrogen). Conditioned media is collected for 5-6 days, centrifuged, and stored at 20 C. in the presence of 1 mM benzamidine.

[0123] Conditioned media is thawed at 37 C., pooled, and purified using an anti-HPC4 Sepharose essentially as described for the FXI variants. Alternatively, His-tagged uPA variants are purified using employing immobilized metal affinity chromatography. The purified protein is stored at 80 C., and protein purity is assessed by SDS-PAGE using pre-cast 4-12% gradient gels (Invitrogen) under reducing conditions followed by staining with Coomassie Brilliant Blue R-250. uPA variants are activated by human plasmin and purified employing Benzamidine-Sepharose.

Expression and Purification of Trypsin:

[0124] Plasmids (pcDNA3.1(+)) encoding human trypsinogen-1 variants A (trypsin (SEQ ID NO:3) with between His-96 and Asp-107 the amino acid sequence of SEQ ID NO:9) and B (trypsin (SEQ ID NO:3) with between His-96 and Asp-107 the amino acid sequence of SEQ ID NO:10) and wildtype trypsin (SEQ ID NO:3) in which the leader sequence is modified such that it lacks a trypsin-like enzyme cleavage site (see Patent EP 1141263 A1) and that are fused via their C-terminus to the HPC4-antibody recognition sequence (amino acid sequence EDQVDPRLIDGK) or to a His-tag (6His: HHHHHH or 12His: HHHHHHHHHHHH) to facilitate purification are introduced into HEK 293 cells using LipofectAMINE 2000 (Invitrogen) and pSV2neo as the selectable marker plasmid. High expressing clones are selected based on a human trypsinogen-specific ELISA (MyBioSource). Selected clones are expanded into triple flasks (Nalge-Nunc, Naperville, Ill.) and cultured in Dulbecco's modified Eagle's medium/F-12 media supplemented with 5 g/ml ITS and 1.0 mg/ml Albumax (Invitrogen). Conditioned media is collected for 5-6 days, centrifuged, and stored at 20 C. in the presence of 1 mM benzamidine.

[0125] Conditioned media is thawed at 37 C., pooled, and purified using an anti-HPC4 Sepharose essentially as described for the FXI variants. Alternatively, His-tagged trypsinogen variants are purified using employing immobilized metal affinity chromatography. The purified protein is stored at 80 C., and protein purity is assessed by SDS-PAGE using pre-cast 4-12% gradient gels (Invitrogen) under reducing conditions followed by staining with Coomassie Brilliant Blue R-250. Trypsin-1 variants are prepared from enterokinase-dependent cleavage of trypsinogen-1 variants and purified employing SP- or Benzamidine-Sepharose.

Example 2: Inhibition of Serine Proteases by Direct Serine Protease Inhibitors

[0126] Initially, the kinetics of peptidyl substrate hydrolysis (S-2388, S-2366, S-2444 or S-2222 as specific substrates for thrombin, FXIa, urokinase-type plasminogen activator or trypsin, respectively) are measured using increasing concentrations of substrate (10-500 M) in the presence of the aforementioned thrombin, FXIa, urokinase-type plasminogen activator or trypsin variants and wildtype. The ability of the direct serine protease inhibitors to bind and inhibit the serine proteases is tested by assessing the inhibitory constant (Ki) assuming classical competitive inhibition by initial velocity measurements of peptidyl substrate hydrolysis using increasing concentrations of direct inhibitor (1 nM-10 M) at fixed concentrations of peptidyl substrate (at or above the Km) and enzyme. All kinetic measurements are performed in 20 mM Hepes, 0.15 M NaCl, 0.1% (w/v) polyethylene glycol 8000, 2 mM CaCl2, pH 7.5.

Example 3: Thrombin Generation Assays

[0127] Thrombin generation is adapted from protocols earlier described (Hemker et al., 2003. Pathophysiol Haemost Thromb 33: 4-15). Briefly, thrombin generation curves are obtained by supplementing prothrombin- or FXI-deficient plasma with corn trypsin inhibitor (70 g/ml), PCPS (20 M) and substrate buffer (Fluca). Thrombin formation is initiated by the addition of 1 Unit (specific clotting activity) of a thrombin A or B variant, FXIa A or B variant, and wildtype thrombin or FXIa, which is premixed with the direct serine protease inhibitor. In an alternative set-up, the zymogen forms of the protein variants are assessed. To do so, prothrombin- or FXI-depleted plasma is supplemented with tissue factor (TF (Innovin), 2 or 20 pM final), corn trypsin inhibitor (70 g/ml), PCPS (20 M), the direct inhibitor, and 1 Unit (specific clotting activity) of recombinant prothrombin or FXI variant, respectively. Thrombin formation is initiated by the addition of Fluca to the plasma. Thrombin formation is determined every 20s for 30 minutes and corrected for the calibrator, using the Thrombinoscope software (Thrombinoscope BV). The lag time, mean endogenous thrombin potential (the area under the thrombin generation curve), time to peak, and peak thrombin generation are calculated from at least 3 individual experiments.

Example 4: Clot Lysis Time Assessment for Urokinase-Type Plasminogen Activator Variants

[0128] The clot lysis time is essentially assessed as described previously (Mosnier et al., 2001. Thromb Haemost 86: 1035-1039). Briefly, tissue factor (TF, Innovin) and PCPS are incubated at 37 C. for 1 hour in 25 mM Hepes, 137 mM NaCl, 3.5 mM KCl, 0.1% BSA, pH 7.4. The TF/PCPS mixture (0.5 pM/20 M final) is incubated with plasma (50% v/v), tPA (150 U/ml final), and CTI (70 g/ml final) for 10 minutes at 37 C. Coagulation is started with Ca2+ (17 mM final) that was pre-incubated at 37 C. The clot formation and the subsequent lysis are monitored by measuring the absorbance at 405 nm for 4 hours at 37 C. in a SpectraMax M2e microplate reader. The clot lysis time is defined as the average of the clear to turbid transition to the average of the turbid to clear transition, which is determined by a sigmoidal fit of the turbidity plots using GraphPad Prism 5.

Example 5: Recombinant Prothrombin Having an Insertion in Region His450-Asp462 of SEQ ID NO:1

Materials and Methods

[0129] The direct thrombin inhibitor dabigatran was obtained from Alsachim (France). Plasma-derived human prothrombin, plasma-derived human thrombin (alpha-thrombin, IIa), plasma-derived human factor Xa, plasma-derived human factor Va, and the thrombin inhibitor dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) were from Haematologic Technologies. Prothrombin-depleted human plasma and the prothrombin time clotting assay reagent STA-Neoplastine CI plus 10 were obtained from Diagnostica Stago. The peptidyl substrate S-2238 was obtained from Chromogenix (Instrumentation Laboratory). All tissue culture reagents were from Life Technologies (Thermo Fisher Scientific), except insulin-transferrin-sodium selenite (ITS), which is from Roche. General recombinant protein production and purification techniques were as described in Green and Sambrook, Molecular Cloning, 4th edition, July 2012. Small unilamellar phospholipid vesicles (PCPS) composed of 75% (w/w) hen egg L-phosphatidylcholine and 25% (w/w) porcine brain L-phosphatidylserine (Avanti Polar Lipids, Inc., US) were prepared and characterized as described previously (Higgins et al., J Biol Chem 258: 6503-6508 (1983)).

[0130] Plasmids (pcDNA3.1(+)) encoding wild-type prothrombin (SEQ ID NO:1) and prothrombin variants with between His-450 and Asp-462 the amino acid sequence of (i) SEQ ID NO:5 (prothrombin ISO1, derived from the homologous region in Pseudonaja textilis isoform factor X), (ii) SEQ ID NO:13 (prothrombin ISO2), (iii) SEQ ID NO:14 (prothrombin NSC, derived from the homologous region in Notechis scutatus venom factor X), (iv) SEQ ID NO:15 (prothrombin KL10, derived from the homologous region in human kallikrein 10), or (v) SEQ ID NO:16 (prothrombin ALB, derived from human albumin) were introduced into HEK 293 cells using LipofectAMINE 2000 (Invitrogen). High expressing clones were selected based on prothrombin-specific ELISA and prothrombin-time clotting assays, essentially as described in Orcutt et al., J Biol Chem, 279: 54927-54936 (2004). Selected clones were expanded into 175 cm.sup.2 flasks and cultured in Dulbecco's modified Eagle's medium/F-12 media supplemented with 5 g/ml ITS and 10 g/ml Vitamin K. Conditioned media was collected for 24 hours, concentrated using a 30 kDa spin-filter into HEPES-buffered saline, pH 7.5, and stored at 20 C. in 50% vol/vol glycerol. The prothrombin antigen concentration was determined using a paired antibody ELISA for the detection of prothrombin (CL20111K, Cedarlane Laboratories). The prothrombin activity was determined employing a prothrombin-specific one-stage prothrombin time clotting assay using the STA-Neoplastin CI plus 10 reagent in prothrombin-deficient plasma, employing known concentrations of prothrombin as standard. The specific activity (U/mg) was derived from the ratio of the prothrombin activity (U/ml) over the prothrombin antigen concentration (mg/ml).

Inhibition of Thrombin by the Direct Thrombin Inhibitor Dabigatran

[0131] Plasma-derived prothrombin (Haematologic Technologies) or recombinant wild-type prothrombin or the recombinant prothrombin variants described in the previous paragraph (ISO1, ISO2, NSC, KL10, and ALB) (125 nM) were activated into thrombin by incubations with prothrombinase (1 nM factor Xa, 50 nM factor Va, 50 M PCPS, 5 mM calcium) in the presence of 10 M DAPA during 5 minutes at ambient temperature. Samples were subsequently quenched in EDTA (25 mM final) and diluted to 5 nM (final) in buffer containing EDTA (50 mM), NaCl (150 mM), 0.1% PEG8000 and HEPES (20 mM), pH 7.5. The ability of the direct thrombin inhibitor dabigatran to bind and inhibit activated plasma-derived prothrombin (pd-IIa) or recombinant activated prothrombin (r-IIa) or the recombinant activated prothrombin variants was tested by assessing the half maximal inhibitory concentration (IC50) by initial velocity measurements of the peptidyl substrate S-2238 hydrolysis using increasing concentrations of dabigatran (20 nM-20 M) at a fixed concentration of S-2238 (100 M). Residual chromogenic activity towards the peptidyl substrate was determined during 10 minutes in a microplate reader (SpectraMax M2e, Molecular Devices) set at A405 nm. IC50 concentrations were obtained by fitting the S-2238 conversion (mOD/min) by nonlinear regression using the Graphpad Prism 6 software suite. The same experiment was performed using plasma-derived thrombin (IIa, Haematologic Technologies) as a control.

Results

[0132] The results of these experiments are displayed in Table 2 and FIGS. 6 and 7. From all this it follows that an insertion in the claimed amino acid residue region of prothrombin provides for desensitization towards direct thrombin inhibitors such as dabigatran, while still having clotting potential or a coagulant effect.

TABLE-US-00003 TABLE 2 Specific Dabigatran Prothrombin Activity Chromogenic Inhibition Variant 10.sup.3 U/mg Activity % IC.sub.50 (M) IIa n.d. 92 0.05 r-IIa 3.88-4.15 96 0.06 ISO1 0.41 39 2.02 ISO2 0.50 70 1.36 NSC 0.14 4 2.55 KL10 0.69 43 1.70 ALB 1.17 30 3.80

[0133] Table 2 shows the characteristics of the different prothrombin variants. The plasma-derived thrombin is indicated as IIa and the recombinant wild-type thrombin as r-IIa. The specific activity (U/mg) is derived from the ratio of the prothrombin activity (U/ml), determined using a prothrombin-specific one-stage prothrombin-time clotting assay, over the prothrombin antigen concentration (mg/ml). The percentage (%) of chromogenic activity denotes the S-2238 conversion of each prothrombin variant related to a standard curve of purified plasma-derived thrombin. The half maximal inhibitory concentration (IC50) displays the concentration of dabigatran required to inhibit 50% of the chromogenic activity of 5 nM thrombin variant.

Example 6. Recombinant Prothrombin Having an Amino Acid Residue Replacement or Substitution at Position Ile-542 of SEQ ID NO:1

Materials and Methods

[0134] The direct thrombin inhibitor dabigatran was obtained from Alsachim (France). Plasma-derived human prothrombin, plasma-derived human thrombin (alpha-thrombin, IIa), plasma-derived human factor Xa, plasma-derived human factor Va, and the thrombin inhibitor dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) were from Haematologic Technologies. Prothrombin-depleted human plasma and the prothrombin time clotting assay reagent STA-Neoplastine CI plus 10 were obtained from Diagnostica Stago. The peptidyl substrate S-2238 was obtained from Chromogenix (Instrumentation Laboratory). All tissue culture reagents were from Life Technologies (Thermo Fisher Scientific), except insulin-transferrin-sodium selenite (ITS), which was from Roche. General recombinant protein production and purification techniques were as described in Green and Sambrook, Molecular Cloning, 4th edition, July 2012. Small unilamellar phospholipid vesicles (PCPS) composed of 75% (w/w) hen egg L-phosphatidylcholine and 25% (w/w) porcine brain L-phosphatidylserine (Avanti Polar Lipids, Inc., US) were prepared and characterized as described previously (Higgins et al., J Biol Chem 258: 6503-6508 (1983)).

[0135] Plasmids (pcDNA3.1(+)) encoding wild-type prothrombin (SEQ ID NO:1) and prothrombin variants in which the Isoleucine at amino acid residue position 542 of SEQ ID NO:1 were replaced by Alanine (I542A, no side-chain), Serine (1542S, small side-chain), Phenylalanine (1542F, large bulky side-chain), or Glutamic acid (I542E, charged side-chain) were introduced into HEK 293 cells using LipofectAMINE 2000 (Invitrogen). High expressing clones were selected based on prothrombin-specific ELISA and prothrombin-time clotting assays, essentially as described in Orcutt et al., J Biol Chem, 279: 54927-54936 (2004). Selected clones were expanded into 175 cm.sup.2 flasks and cultured in Dulbecco's modified Eagle's medium/F-12 media supplemented with 5 g/ml ITS and 10 g/ml Vitamin K. Conditioned media was collected for 24 hours, concentrated using a 30 kDa spin-filter into HEPES-buffered saline, pH 7.5, and stored at 20 C. in 50% vol/vol glycerol. The prothrombin antigen concentration was determined using a paired antibody ELISA for the detection of prothrombin (CL20111K, Cedarlane Laboratories). The prothrombin activity was determined employing a prothrombin-specific one-stage prothrombin time clotting assay using the STA-Neoplastin CI plus 10 reagent in prothrombin-deficient plasma, employing known concentrations of prothrombin as standard. The specific activity (U/mg) was derived from the ratio of the prothrombin activity (U/ml) over the prothrombin antigen concentration (mg/ml).

Inhibition of Thrombin by the Direct Thrombin Inhibitor Dabigatran

[0136] Recombinant wild-type prothrombin or the recombinant prothrombin variants (1542S, I542A, I542F and I542E) (125 nM) were activated into thrombin by incubations with prothrombinase (1 nM factor Xa, 50 nM factor Va, 50 M PCPS, 5 mM calcium) in the presence of 10 M DAPA during 5 minutes at ambient temperature. Samples were subsequently quenched in EDTA (25 mM final) and diluted to 5 nM final in buffer containing EDTA (50 mM), NaCl (150 mM), 0.1% PEG8000 and HEPES (20 mM), pH 7.5. The ability of the direct thrombin inhibitor dabigatran to bind and inhibit recombinant activated prothrombin (r-IIa) or the recombinant activated prothrombin variants was tested by assessing the half maximal inhibitory concentration (IC50) by initial velocity measurements of the peptidyl substrate S-2238 hydrolysis using increasing concentrations of direct inhibitor (20 nM-20 M) at a fixed concentrations of S-2238 (100 M). Residual chromogenic activity towards the peptidyl substrate was determined during 10 minutes in a microplate reader (SpectraMax M2e, Molecular Devices) set at A405 nm. IC50 concentrations were obtained by fitting the S-2238 conversion (mOD/min) by nonlinear regression using the Graphpad Prism 6 software suite. The same experiment was performed using plasma-derived thrombin (IIa, Haematologic Technologies) as a control.

Results

[0137] The results of these experiments are displayed in Table 3 and FIGS. 9 and 10. From all this it follows that a replacement, substitution or mutation at amino acid residue position Ile-542 of SEQ ID NO:1 provides for desensitization towards direct thrombin inhibitors such as dabigatran, while still having clotting potential or a coagulant effect.

TABLE-US-00004 TABLE 3 Specific Dabigatran Prothrombin Activity Chromogenic Inhibition Variant 10.sup.3 U/mg Activity % IC.sub.50 (M) IIa n.d. 92 0.05 r-IIa 3.88-4.15 96 0.06 I542S 0.30 183 0.73 I542A 0.40 127 0.79 I542F 0.74 156 0.90 I542E 0.24 119 1.32

[0138] Table 3 shows the characteristics of the prothrombin variants. The plasma-derived thrombin is indicated as IIa and the recombinant wild-type thrombin as r-IIa. The specific activity (U/mg) is derived from the ratio of the prothrombin activity (U/ml), determined using a prothrombin-specific one-stage prothrombin-time clotting assay, over the prothrombin antigen concentration (mg/ml). The percentage (%) of chromogenic activity denotes the S-2238 conversion of each prothrombin variant related to a standard curve of purified plasma-derived thrombin. The half maximal inhibitory concentration (IC50) displays the concentration of dabigatran required to inhibit 50% of the chromogenic activity of 5 nM thrombin variant.