Highly glycosylated human blood-clotting factor VIII fusion protein, and manufacturing method and application of same

Abstract

A highly glycosylated human blood-clotting factor VIII (FVIII) fusion protein, and a manufacturing method and application of same. The fusion protein comprises, from the N-terminus to the C-terminus, a human (FVIII), a flexible peptide connector, at least one rigid unit of a human chorionic gonadotropin β-subunit carboxyl terminal peptide, and a half-life extending portion (preferentially selected from a human IgG Fc variant). The fusion protein has a similar level of biological activity as a recombinant (FVIII) and an extended in vivo half-life, thereby improving pharmacokinetics and drug efficacy.

Claims

1. A fusion protein, wherein the fusion protein has the amino acid sequence of SEQ ID NO: 16.

2. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient and/or diluent, and an effective amount of a fusion protein of claim 1.

3. A method for preparing a fusion protein, including steps comprising: (a) introducing a DNA molecule encoding a fusion protein of claim 1 into a CHO cell to produce a CHO-derived cell line; (b) screening the cell strains of step (a) to obtain a high-yield cell strain expressing more than 1 IU/10.sup.6 (million) cells per 24 h in its growth medium; (c) culturing the cell strain obtained in step (b) to express the fusion protein; and (d) harvesting the fermentation broth of step (c) and isolating and purifying the fusion protein.

4. A method for treating hemorrhagic diseases in patients with congenital or acquired FVIII deficiency or for treating spontaneous or surgical bleeding in patients with hemophilia A, comprising administrating an effective amount of a fusion protein of claim 1 to the patient.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. The nucleotide sequence of FP-B in the Spel-EcoRI (the restriction sites are underlined) fragment in the pcDNA3 expression vector and the derived amino acid sequence according to an example of the present invention. Human FVIII consists of a signal peptide (amino acids 1-19, underlined with “ . . . ”) and a mature FVIII protein (amino acids 20-1457). The mature fusion protein comprises hFVIII (amino acids 20-1457), a flexible peptide linker (amino acids 1458-1484, underlined with “______”), a rigid CTP unit (amino acids 1485-1512, underlined with “______”), and a vFcγ2-3 variant (amino acids 1513-1735).

(2) FIG. 2. SEC-HPLC chromatograph of purified FP-B protein.

(3) FIG. 3. SDS-PAGE analysis of purified FP-B protein.

(4) FIG. 4. Bleeding volume (μl) for each mouse after tail transection. Note: *p<0.05, **p<0.01.

(5) FIG. 5. Bleeding time (s) for each mouse after tail transection. Note: *p<0.05, **p<0.01.

EXAMPLES

Example 1

Construction of an Expression Plasmid Encoding the FVIII Fusion Protein

(6) The gene sequences encoding the FVIII signal peptide, mature protein, flexible peptide linker, rigid CTP unit, and human IgG vFc variant were artificially codon-optimized for expression in CHO cells and artificially synthesized. The synthesized full-length DNA fragment of the fusion protein had a Spel restriction site at the 5′ end and a BamHl restriction site at the 3′ end. The full length DNA fragment was inserted into the corresponding restriction sites of the pUC57 transfer vector and verified by DNA sequencing.

(7) The full-length gene fragment of the fusion protein obtained above was cloned from an intermediate vector into the corresponding restriction sites of an expression plasmid PTY1A1 to construct a high expression plasmid of the fusion protein. The PTY1A1 plasmid was derived from pcDNA3.1 by modification. The PTY1A1 plasmid contained, but was not limited to, the following important expression elements: 1) a human cytomegalovirus early promoter and an enhancer needed for exogenous high-expression in mammalian cells; 2) double screening markers with kanamycin resistance in bacteria and G418 resistance in mammalian cells; 3) a murine dihydrofolate reductase (DHFR) gene expression cassette. When the host cell type was DHFR gene deficient, methotrexate (MTX) could co-amplify the fusion gene with the DHFR gene (see U.S. Pat. No. 4,399,216). The fusion protein expression plasmid was transfected into a mammalian host cell line. The preferred host cell line was the DHFR enzyme-deficient CHO cell line in order to achieve stable and high level of expression (see U.S. Pat. No. 4,818,679). Two days after transfection, the medium was replaced with a screening medium containing 0.6 mg/mL of G418. The cells were seeded in a 96-well plate at a certain concentration (5000-10000 viable cells/well) and were cultured for 10-14 days until large discrete cell clones appeared. The transfectants resistant to the selected antibiotic were screened by the ELISA assay. The wells producing high levels of the fusion protein were subcloned by limiting dilution on the 96-well culture plate.

(8) As shown in Table 1, the present invention constructed a series of hFVIII fusion proteins, which contained linkers of different lengths, rigid CTP units of different composition, and IgG Fc variant (vFc) elements of several different subtypes. To verify that at least one rigid CTP unit of different lengths could significantly improve the activity of the fusion protein, we constructed the fusion proteins, FP-A, FP-B, FP-C, FP-D and FP-E. The amino acids and coding nucleotides of FP-B were shown in FIG. 1. To verify the importance of the rigid CTP unit to the activity of the fusion protein, we also constructed the Fc fusion proteins FP-G and FP-H without the rigid CTP unit. The expression plasm ids were constructed as above. In addition, we also constructed FP-F that had the rigid CTP unit at the C-terminus of Fc to verify the importance of the position of the rigid CTP unit. See table 1 for details. The amino acid sequences for each component were shown in the sequence listings.

(9) TABLE-US-00003 TABLE 1 Compositions of various FVIII fusion proteins Code Composition of FVIII fusion protein series (from N to C terminus) FP-A FVIII-L3-CTP1-vFcγ1 FP-B FVIII-L2-CTP2-vFcγ2-3 FP-C FVIII-L5-CTP4-vFcγ4 FP-D FVIII-L1-CTP3-CTP3-vFcγ2-2 FP-E FVIII-L4-CTP3-vFcγ2-1 FP-F FVIII-L2-vFcγ2-3-CTP2 FP-G FVIII-L1-vFcγ2-3 FP-H FVIII-L4-vFcγ2-3

Example 2

Transient Expression and Activity Determination of Various Fusion Proteins

(10) Eight expression plasmids obtained in Example 1 were respectively transfected into 3×107 CHO-K1 cells using the DNAFect LT reagent (ATGCell) in a 30 mL shake flask, and the transfected cells were cultured in serum-free growth medium containing 1000 ng/mL of vitamin K1 for 5 days. The concentration of the fusion protein in the supernatant was measured and its activity was determined by the method described in Example 6 or 7. The ELISA results showed that the transient protein expression levels of the eight plasmids were similar under these conditions, but the coagulation activities of these fusion proteins showed large differences.

(11) We defined the molar specific activity of FP-A to 100%. The fusion protein FP-G secreted in the cell culture supernatant was mostly in the form of non-active aggregates. The FP-F and FP-H plasm ids expressed low-activity fusion proteins, with their activities being about 20.5% and 15.2% of that of FP-A, respectively. Similar to FP-G, most of the fusion proteins FP-F and FP-H were in the form of aggregates. Moreover, the fusion proteins FP-F, FP-G and FP-H were prone to degradation, showing poor stability. It was reported that the lipid binding region of FVIII (amino acids 2303-2332) was critical to its function, and small conformational changes in this region caused protein aggregation and led to loss of activity (Gilbert G E et al., Biochemistry, 1933,32(37): 9577-9585). Therefore, we speculated that the conformations of the lipid binding regions in the FVIII fusion proteins FP-F, FP-G and FP-H were changed due to influence of the C-terminal Fc ligands, which led to the aggregation of the proteins and significant reduction of the activities. The activities of FP-B, FP-C, FP-D and FP-E containing CTP were 113.4%, 96.0%, 87.4% and 93.7% of that of FP-A, respectively.

(12) Based on the activity differences between FP-B, FP-F and FP-H, it could be understood that by only extending the length of the peptide linker, neither the activity of the fusion protein could be effectively improved, nor the problem of the fusion protein being prone to aggregation and degradation could be solved. The addition of the CTP unit resulted in a significant increase in the activity of the fusion protein FP-B. We speculated that the reasons were as follows. Overlong flexible peptide linkers gave FVIII higher flexibility, such that FVIII could rotate freely relative to the Fc domain. As a result, the three-dimensional structure of FVIII was located close to the Fc domain. On one hand, the addition of the rigid CTP unit between FVIII and Fc is equivalent to the addition of a rigid peptide linker, allowing the FVIII and Fc domains away from each other. More importantly, compared to the random coil of the flexible peptide linker, the rigid CTP peptide containing multiple glycosyl side chains could form a stable steric conformation, and effectively separate the different functional regions of the fusion protein. Thus, the FVIII and Fc portions were allowed to fold independently into correct three-dimensional conformations, maintaining high activities. We verified the correctness of this hypothesis by comparing the activities of FP-B and FP-F. The activity of FP-F was less than 20% of that of FP-B. In FP-F the rigid CTP unit was placed at the C-terminus of Fc, while in FP-B the rigid CTP unit was placed at the N-terminus of Fc. The above results demonstrated that the rigid CTP unit was critical to the activity of the fusion protein, and placing the rigid CTP unit at the N-terminus of Fc could effectively improve the activity of the fusion protein.

Example 3

Screening for Stably Transfected Cell Lines with High Expression of Fusion Proteins

(13) The expression plasmids of FP-A, FP-B, FP-C, FP-D and FP-E were transfected into mammalian host cell lines to express the FVIII fusion proteins. The preferred host cell was the DHFR-deficient CHO cell in order to maintain a stable high level of expression (U.S. Pat. No. 4,818,679). One preferred method of transfection was electroporation, and other methods might be used, including calcium phosphate co-deposition, liposome transfection, microinjection, etc. For the electroporation method, used was a Gene Pulser Electroporator (Bio-Rad Laboratories) set at 300 V voltage and 1050 μFd capacitance. 50 μg of Pvul linearized expression plasmid was added to 2 to 3×107 cells placed in a cuvette. After electroporation, the cells were transferred to a shake flask containing 30 mL of growth medium. Two days after transfection, the medium was replaced with a screening medium containing 0.6 mg/mL of G418. The cells were seeded in a 96-well plate at a certain concentration (5000-10000 viable cells/well) and were cultured for 10-12 days until large discrete cell clones appeared. The anti-human IgG Fc ELISA assay was used to screen the transfectants that were resistant to the selected drug. The quantitative determination of the fusion protein expression could also be performed using the anti-FVIII ELISA assay. Then wells producing high levels of fusion proteins were subcloned by limiting dilution.

(14) It was preferred to perform co-amplification by utilizing the DHFR gene which could be inhibited by the MTX drug to achieve higher level expression of the fusion protein. In growth medium containing increasing concentrations of MTX, the transfected fusion protein gene was co-amplified with the DHFR gene. The DHFR positive subclones were subjected to limiting dilution and transfectants capable of growing in medium containing up to 6 μM MTX was screened out by progressive pressure. The secretion efficiencies thereof were determined and the cell lines with high expression of exogenous proteins were screened out. The cell lines with a secretion efficiency of more than about 1 (preferably about 3) IU per 106 cells in 24 h were adapted to suspension culture using serum-free medium, and then the fusion protein was purified from the conditioned medium.

(15) In the examples below, FP-B was taken as an example to illustrate the method for fermentation and purification of the fusion protein. The methods for fermentation and purification of FP-A, FP-C, FP-D and FP-E were the same as that of FP-B, and would not be described here again.

Example 4

Production of the Fusion Protein

(16) The high expression cell strain obtained in Example 3 was first acclimated to serum-free medium in a petri dish and then transferred to a shake flask for suspension domestication. After the cells were adapted to these culture conditions, the cells were fed-batched in a 300 mL shake flask or cultured by replacing the medium daily to simulate a perfusion system. The CHO-derived cell strain expressing the fusion protein FP-B obtained from Example 3 was fed-batched in a 300 mL shake flask for 14 days, and the cumulative yield of the expressed recombinant fusion protein reached 200 mg/L, while the highest viable cell density could reach up to 15×106 cells/mL. 1000 mL shake flasks could be used for producing more fusion proteins. In another culture method, the above CHO-derived cell strain was cultured in a 100 mL shake flask with the medium changed daily. The expressed recombinant fusion protein reached a cumulative yield of about 20 mg/L per day. The highest viable cell density in the shake flask was up to 30×106 cells/mL. The biological activities of the recombinant fusion proteins produced by the above two methods were equivalent.

Example 5

Purification and Qualitative Analysis of the Fusion Protein

(17) The invention mainly used a four-step chromatography procedure to purify the fusion protein FP-B, i.e., affinity chromatography, hydrophobic chromatography, anion exchange chromatography, and molecular sieve chromatography. In this example, the AKTA pure 25 M system (GE Healthcare, USA) was the instrument used for protein purification. The reagents used in this example were all purchased from Sinopharm Chemical Reagent Co., which were of analytical grade.

(18) Step 1, affinity chromatography: Sample capture, concentration and removal of part of contaminants were performed by using the alkali-resistant Protein A Diamond resin (Bestchrom, Shanghai) or other commercially available recombinant protein A affinity chromatography resins. The other resins included, for example, MabSelect (GE Healthcare), MabSelect SuRe (GE Healthcare), Toyopearl AF-rProtein A-650F (Tosoh Bioscience), rProtein A Beads (Smart-Lifesciences, Changzhou, China), MabPurix (Sepax Technologies), and Protein A Ceramic HyperD (Pall Life Sciences). The column was equilibrated at a linear flow rate of 50-100 cm/h with 3-5 column volumes (CVs) of equilibration buffer: 20 mM His-HCl, 150 mM NaCl, 5 mM CaCl2, 0.02% Tween-80, pH 6.8-7.2. The centrifuged fermentation supernatant was loaded onto the column at no more than 50000 IU protein/mL resin at a linear flow rate of 50-100 cm/h. After loading, the column was equilibrated with 3 to 5 CVs of the equilibration buffer at a linear flow rate of 50-100 cm/h to wash off unbound materials. The column was then washed with 3-5 CVs of decontamination buffer 1: 20 mM His-HCl, 2 M NaCl, 4 M urea, 5 mM CaCl2, 0.02% Tween 80, pH 6.8-7.2, at a linear flow rate of 50-100 cm/h to remove part of contaminants. The column was equilibrated with 3-5 CVs of the equilibration buffer at a linear flow rate of 50-100 cm/h. The column was further washed with 3-5 CVs of decontamination buffer 2: 20 mM His-HCl, 5 mM EDTA, 150 mM NaCl, 30% ethylene glycol, 5 mM CaCl2, 0.02% Tween 80, pH 6.8-7.2, at a linear flow rate of 50-100 cm/h to remove part of contaminants. The column was equilibrated with 3-5 CVs of the equilibration buffer at a linear flow rate of 50-100 cm/h. The target product was then eluted and collected with the elution buffer: 20 mM His-HCl, 5 mM CaCl2, 0.02% Tween 80, 50% ethylene glycol, pH 5.0 at a linear flow rate of not higher than 50 cm/h. Tris, pH 9.0 was added into the elute to adjust the pH to neutral (7.0-8.0).

(19) Step 2, hydrophobic chromatography: The Butyl Bestarose HP resin (Bestchrom, Shanghai) or other commercially available hydrophobic chromatography resins were used in the intermediate purification step to reduce the amount of aggregates. The other resins included Butyl Sepharose HP (GE Healthcare), Toyopearl Butyl-650 (Tosoh Bioscience), Butyl Beads 4FF (Smart-Lifesciences, Changzhou, China), Generic MC 30-HIC Butyl (Sepax Technologies), and Fractogel EMD Propyl (Merck). The elute of the first step affinity chromatography still contained a certain proportion of aggregates. The aggregates were formed due to a variety of reasons. Some of the aggregates contained proteins still in native conformation, while others contained proteins whose conformation had been changed. The aggregates in different conformational forms showed significant differences in the biological activity, leading to great interference in the activity analysis. Thus, after protein capture in the first purification step was completed, the aggregates needed to be removed next. After target protein aggregation, Non-aggregates and aggregates displayed different properties including the charge characteristics and hydrophobicity. The difference in hydrophobicity was used to separate the two. Since the last purification step was molecular sieve chromatography, the fusion protein captured in the first step affinity chromatography was further purified with Butyl HP to perform a second purification step in order to partially remove the aggregates, so that the content of aggregates was less than 10%. First, the column was equilibrated with 3-5 CVs of equilibration buffer: 20 mM His-HCl, 1.5 M NaCl, 5 mM CaCl2, 0.02% Tween 80, pH 6.8-7.2 at a linear flow rate of 50-100 cm/h. The affinity-captured sample was diluted twice with the equilibration buffer to reduce the organic solvent content, and then added to the sample was an equal volume of concentrated buffer: 20 mM His-HCl, 3 M NaCl, 5 mM CaCl2, 0.02% Tween 80, pH 6.8-7.2. The sample was then loaded onto the column at less than 20000 IU protein/mL resin. After loading, the column was washed with 3-5 CVs of the equilibration buffer at a linear flow rate of 50-100 cm/h, and washed with 3-5 CVs of wash buffer: 20 mM His-HCl, 1.5 M NaCl, 5 mM CaCl2, 0.02% Tween 80, 20% ethylene glycol, pH 6.8-7.2, to remove some of the aggregates. Finally, the target protein was eluted with the elution buffer: 20 mM His-HCl, 5 mM CaCl2, 0.02% Tween 80, 50% ethylene glycol, pH 6.8-7.2, eluting at a linear flow rate of not higher than 60 cm/h, and the eluted fractions were collected and analyzed by SEC-HPLC. The target fractions with the non-aggregates percentage greater than 90% were combined and subjected to the next step purification.

(20) Step 3, anion exchange chromatography: The Q-HP resin (Bestchrom, Shanghai) or other commercially available anion exchange chromatography resins were used in the intermediate purification step to separate structural variants and further remove contaminants such as HCP, DNA, etc. The other resins included Q HP (GE Healthcare), Toyopearl GigaCap Q-650 (Tosoh Bioscience), DEAE Beads 6FF (Smart-Lifesciences, Changzhou, China), Generik MC-Q (Sepax Technologies), Fractogel EMD TMAE (Merck), and Q Ceramic HyperD F (Pall Life Sciences). First, the column was washed with 3-5 CVs of equilibration buffer: 20 mM His-HCl, 200 mM NaCl, 5 mM CaCl2, 0.02% Tween 80, pH 6.8-7.2 at a linear flow rate of 50-100 cm/h. The target protein isolated by the second step hydrophobic chromatography was diluted twice for reducing the organic solvent content, and was loaded onto the column at less than 5000-10000 IU protein/mL resin. After loading, the column was washed with 3-5 CVs of the equilibration buffer at a linear flow rate of 50-100 cm/h, followed by elution with a linear gradient of salt concentration using the elution buffer: 20 mM His-HCl, 1 M NaCl, 5 mM CaCl2, 0.02% Tween 80, pH 6.8-7.2. The elution condition was a gradient from 0 to 100% elution buffer over 2 h at a linear flow rate of no higher than 50 cm/h. The eluted fractions were collected and analyzed for protein content, SEC-HPLC, activity and HCP content. After the protein concentration and activity were determined, the specific activity of the protein was calculated as about 10000 IU/mg.

(21) Step 4, molecular sieve chromatography: The Chromdex 200 prep grade resin (Bestchrom) or other commercially available molecular sieve resins (e.g., Superdex 200 from GE Healthcare) were used for separation, with the goal to reduce the aggregates content to <5% and further reduce the key contaminant content. The column was washed with 2 CVs of equilibration buffer: 20 mM His-HCl, 200 mM NaCl, 5 mM CaCl2, 0.02% Tween 80, pH 6.8-7.2 at a linear flow rate of 20-40 cm/h. The sample volume loaded was no more than 3% of the column volume. The protein sample was eluted at a linear flow rate of 20 cm/h, and the eluted fractions were collected and subjected to SEC-HPLC analysis followed by combining.

(22) The SEC-HPLC purity analyses and SDS-PAGE electrophoresis of the purified FP-B were shown in FIGS. 2 and 3, respectively. The SEC-HPLC results showed that the purity of the main peak of the purified fusion protein FP-B was above 97%. The band patterns of the SDS-PAGE electrophoresis appeared as expected. The non-reducing electrophoresis lane contained a band for the unprocessed fusion protein FP-B (390 kDa), a band for HC-LC-L-CTP-Fc:LC-L-CTP-Fc (300 kDa), of which one heavy chain fragment of FVIII was lost during electrophoresis, a band for the dimer (LC-L-CTP-Fc)2 (210 kDa), of which the two heavy chain fragments were lost during electrophoresis, and a band for the heavy chain fragment HC (90 kDa). The reducing electrophoresis lane contained a band for HC-LC-L-CTP-Fc (190 kDa), a band for LC-L-CTP-Fc (105 kDa), and a band for the single chain HC (90 kDa).

Example 6

Indirect Determination of In Vitro Activity of the Fusion Protein by the Chromogenic Substrate Assay

(23) The activity of the FVIII fusion protein could be determined by the chromogenic substrate assay. In this example the Chromogenix Coatest SP FVIII kit (Chromogenix, Ref. K824086) was used and the assay principle was as follows. When activated by thrombin, FVIIIa bound to FIXa in the presence of phospholipid and calcium ions to form an enzyme complex, which in turn activated factor X into its active form, Xa. The activated factor Xa then decomposed its specific chromogenic substrate (Chromogenix S-2765), releasing the chromophore pNA. The amount of pNA produced was measured at 405 nm, and thus the activity level of FXa which was directly proportional to the amount of pNA was obtained. As the amount of factor IXa and factor X in the assay system was excessive and constant, the activity of FXa was only directly related to the amount of FVIIIa. The specific activities of the FVIII fusion proteins were about 6000-10000 IU/mg as determined by this assay.

Example 7

Direct Determination of the Biological Activity of the Fusion Protein by the Clotting Assay

(24) The clotting assay for determining the biological activity of FVIII was based on the property of FVIII to correct the prolonged clotting time of FVIII-deficient plasma. Using the Coagulation Factor VIII Deficient Plasma kit (Cat. No. OTXW17) of the German company Siemens, the method for determining the FVIII activity was as follows. First, the FVIII standard with a known potency from National Institutes for Food and Drug Control (China) was diluted to 10 IU/mL with 5% FVIII-deficient plasma, which was then further diluted 10 times, 20 times, 40 times, and 80 times, respectively. The activated partial thromboplastin time (APTT) was determined by an automatic hemagglutination analyzer (CA500, Sysmex). A standard curve was established with the FVIII standard by plotting a linear regression of the logarithm of the potencies (IU/mL) of the FVIII standard solutions vs. the logarithm of their corresponding clotting times (s). Then the test sample was properly diluted and mixed with the FVIII-deficient substrate plasma to perform the APTT assay. The potency of the test FVIII sample (IU/mL) could be calculated by substituting the clotting time into the standard curve equation. Thus the specific activity of the test FVIII sample could be calculated in the unit of IU/mg. The specific activities of the FVIII fusion proteins were about 6000-10000 IU/mg as determined by this assay.

Example 8

Hemostatic Effect of Fusion Proteins on Acute Hemorrhage in Hemophilia A Mice

(25) We evaluated the hemostatic activity of the fusion protein FP-B prepared in Example 5 in a VIII factor gene-knockout homozygous HemA mouse tail clip bleeding model. Male HemA mice (8-12 weeks old, Shanghai Model Organisms Center, Inc.) were adaptively fed for one week, and then randomly divided into 6 groups. In addition, one group of HemA mice was set up as negative control, and another group of normal C57 mice was set up as positive control. To the 8 groups, different active doses of the fusion protein FP-B or the control drug Xyntha (Pfizer) were given by a single tail vein injection. Table 2 showed experimental design and animal grouping.

(26) TABLE-US-00004 TABLE 2 Animal grouping regarding the hemostatic effect of fusion proteins in HemA mice Group Quantity number Group Mouse type (each) Dosage 1 HA control HemA 6 Physiological saline group mice 2 C57 control C57 mice 8 Physiological saline group 3 FP-B-270 HemA 9 FP-B, 270 IU/kg mice 4 Xyntha-270 HemA 11 Xyntha, 270 IU/kg mice 5 FP-B-90 HemA 9 FP-B, 90 IU/kg mice 6 Xyntha-90 HemA 9 Xyntha, 90 IU/kg mice 7 FP-B-30 HemA 11 FP-B, 30 IU/kg mice 8 Xyntha-30 HemA 10 Xyntha, 30 IU/kg mice

(27) Before administration, each of the mice were anesthetized by injecting intraperitoneally with 1.0% pentobarbital sodium (Sigma) at a dose of 0.1 mL/10 g, and then placed on a 37° C. heating pad to maintain body temperature. The tail of the mouse was immersed in warm water at 37° C. for 10 min to expand the tail vein, and then the corresponding dose in Table 2 was administered. 10 min after administration, the tail was cut off at 1.5 cm from the tail tip, and the tail was rapidly immersed in about 13 mL of preheated saline contained in a centrifuge tube. Started timing. If bleeding stops within 30 min, recorded the bleeding time and volume. If the bleeding time was more than 30 min, recorded it as 30 min. Bleeding volume (mL)=(weight of centrifuge tube after blood collection (g)−weight of centrifuge tube before blood collection (g))/1.05. After 30 min, removed the tail from the tube containing saline. Within 24 h, observed and recorded recurrent bleeding every 10 min and recorded the number of surviving mice. All data were expressed as mean±standard error (x±SEM). The t-test was used to compare the experimental groups. The analysis software was GraphPad Prism 5.0. P<0.05 was considered statistically significant.

(28) FIGS. 4 and 5 showed statistical analyses of bleeding time and volume for each group of the animals. 10 min after giving the HemA mice 270 IU/kg of FP-B, the bleeding time and volume of the FP-B-270 group were close to those of the C57 control group. The procoagulant effect of FP-B was evident, indicating that FP-B could be used as an effective coagulation agent for acute hemorrhage in patients with coagulation factor deficiencies such as hemophilia. When the mice were given 90 IU/kg of FP-B, the bleeding time and volume of the FP-B-90 group were also close to those of the C57 control group. There was no significant difference in bleeding volume between the HemA mice given the same amount of active dose of FP-B and Xyntha, but the bleeding time of the FP-B group at each dose was slightly less than that of the Xyntha group, indicating that FP-B may have a certain efficacy advantage compared to Xyntha. Compared with the 30 IU/kg FP-B group, the 90 IU/kg FP-B group showed a significantly shorter bleeding time (p<0.05), and the 270 IU/kg FP-B group showed a significantly shorter bleeding time (p<0.05) and also a significantly reduced bleeding volume (p<0.05). This indicated that the fusion protein FP-B had a dose-effect relationship on the hemostasis of acute hemorrhage in HemA mice (see Table 3 for details).

(29) According to the postoperative recovery, when given the same amount of active dose of FP-B and Xyntha, the FP-B group at each dose had a higher mouse survival rate than the Xyntha group at a same active dose, indicating that the fusion protein FP-B had a more lasting effect than Xyntha (see Table 3).

(30) TABLE-US-00005 TABLE 3 Bleeding time, volume, recurrent bleeding and survival rate statistics for each group of HemA mice after tail transection C57 HA FP-B Xyntha control control Group 270 IU/kg 90 IU/kg 30 IU/kg 270 IU/kg 90 IU/kg 30 IU/kg group group Bleeding 189.2 ± 48.9 313.4 ± 68.2 456.5 ± 95.3 161.7 ± 37.2 336.4 ± 61.1 459.4 ± 56.7 209.3 ± 38.8  760.5 ± 38.9 volume (μL) Bleeding   160 ± 23.4   269 ± 46.2   904 ± 218.3   210 ± 23.3   448 ± 176.7  1224 ± 213.4   310 ± 66.7 1800b ± 0.0 time (s) 24-48 h 100% 100% 64%a 91% 78% 50%a 100% 17% survival rate Note: aBecause there were mice dying within 24-48 h, so the 48 h survival rate was presented. bIf the bleeding time was more than 30 min, recorded it as 1800 s.
All documents mentioned in the present invention are hereby incorporated by reference to the same extent as if each of the documents is individually recited for reference. It is to be understood that various changes and modifications may be made by those skilled in the art upon reading the above teachings of the present invention, which also fall within the scope of the claims appended hereto.