JPH03232900A - Human tissue factor-activating substance and its purification - Google Patents

Abstract

NEW MATERIAL:A human tissue factor-activating substance which can activate the VII factor in the coexistence of phospholipids and calcium ion to manifest a blood coagulation activity with the optimal pH of 7.5-8.5, capable of manifesting the activity in a pH range of 5.5-12, having a molecular weight of 54,000-60,000 and detectable in human urine. USE:A hemostatic agent for wounds and those of postsurgery, and a therapeutic agent for bleeders having VIII factor-neutralizing antibodies. PREPARATION:For example, human tissue factor(TF) apoprotein isolated from human placenta and purified by a conventional method is decomposed with CNBr and a fragment of the apoprotein on the amino group terminal side thereof is collected. The fragment is employed in an antigen to prepare a monoclonal antibody. The antibody is combined with an insoluble carrier and brought into contact with human urine to adsorb the TF in the human urine. The adsorbed TF is eluted with a 3MNaSCN solution, etc., to provide the human tissue factor-activating substance.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial field of application The present invention is directed to the use of tissue factor (T), which can be found in human urine.
The present invention relates to a novel substance having an activity similar to issue factor (hereinafter sometimes abbreviated as TF) and a method for purifying the substance.

More specifically, the present invention relates to a novel TF-like protein found in the human urine, which has the property of binding to a monoclonal antibody against TF derived from human placenta, and a method for purifying the same.

(b) Prior Art Human tissue factor (Tissue Factor) is also called tissue thromboplastin, and is known to play an important role as an initiator of extrinsic coagulation. That is, it is a substance that forms a complex with factor ① and activates factor X and factor 1X. Tissue factor is a glycolipid protein consisting of a lipid part and a protein part (apoprotein), and the presence of both is required for its activity to be expressed. Apoprotein is a glycoprotein with a molecular weight of around 50,000 and is considered to be a type of membrane protein. Purification of TF membrane protein is extremely difficult, and it has only recently been possible to isolate and purify it from placenta and brain tissue as membrane protein solubilization and purification techniques have advanced [L, V, M, Rag, S, 1 ．． Rapa
port, AnalBiochem, 165.36
5-370 (1987) 1° Furthermore, it has been reported that there are several types of placenta/brain-derived TF membrane proteins with different molecular weights [S, D, Carson.

S.E., Ross and R.A., Gramzi
nski Blood 71(2), 520-5
23 (1988)].

In intact cells, tissue factor is thought to exist with the surface covered in the cell membrane, and tissue factor is exposed on the cell surface and vasotropic coagulation is likely to occur when tissue or blood vessels are damaged, especially in cancer. Become. Mana TNF
(Tumor Necrosis Factor) and I
It has been reported that L (Inter Leukin), a cytotoxic compound, stimulates certain types of cells to express tissue factor activity on the cell surface [P, R, Conklin
g, C, S.

Greenberg, and J.B., Winb.
erg; Blood, 72. (1).

128-133 (see 1988+).

Recently, c
A DNA clone was isolated and the primary structure of the protein was revealed [E., 5picer, et aPro
c, Natl, Acad, Sci, USA
84 5148-5152 (1987+ reference).

However, the existence form of human tissue factor in blood or urine has not yet been reported.

(C) Object of the invention The present invention is to provide a novel human tissue factor active substance different from the above-mentioned tissue factor protein, and in particular, to provide a novel human tissue factor active substance that can be found in urine. It is about providing.

(d) Components of the Invention In other words, the present invention activates factor (I) in the coexistence of phospholipids and calcium ions to express blood coagulation activity, and the optimum pH is 7.5 to 8.5. Yes, and pH 5.5
It is possible to express activity in the range of ~12, and the molecular weight is 5.
4.000 to 60.000, a human tissue factor active substance that can not be found in human urine, and a method for its purification.

The human TF active substance of the present invention is a novel substance whose amino acid sequence is partially different from the tissue factor reported in the above-mentioned conventional literature.

A human TF active substance purified from human urine activates factor Ⅰ in the coexistence of phospholipids and calcium ions to express blood coagulation activity, and has an optimal PU of 7.5 to 8.5. It can exhibit activity in the pH range of 5.5 to 12, has a molecular weight in the range of 54,000 to 60,000, and further has a molecular weight of 36,000 due to cyanogen bromide.
It is a TF-like protein that is degraded into two fragments: ~40.000 and is, ooo ~20.000.

This protein is about 36,000 molecules obtained by digestion with CNBr.
The molecular weight portion of is further reductively alkylated, and protease A
When sp-N is applied, about 12000 and about 2400
It is divided into fragments with a molecular weight of 0, and when the amino acid sequence of the fragments with a molecular weight of about 12,000 is analyzed, it is 1 2 3 4 5 6 7 from the N-terminus.
8 9 10 11Lys-G Iu
-GI y-A I a-A I a-I I e-
It was found that it had the sequence Leu-Phe-G I u-Pro-Th r.

In addition, this TF-like protein derived from human urine is similar to the TF-like protein derived from human placenta.
It has the property of being able to be similarly recognized by monoclonal antibody GX3, which was able to recognize F. The monoclonal antibody GX3 is an anti-tissue factor monoclonal antibody produced by a hybridoma deposited by the present inventors under the FERM deposit number FERM P-10505.

Furthermore, the above-mentioned TF-like protein has a solubility of about 50 μ/100 ml of saline in physiological saline after stirring at room temperature for 10 minutes and standing still at 37° C. for 1 hour.

The physicochemical properties of the human urine-derived TF active substance exhibiting such characteristics are as follows.

) Amino acid composition Amino acid Asp (and/or Asn) Gln (and/or Glu) et 1y is Content (wt%) 10-11 8-9 10-11 9-10 2-3 5-6 8-9 7- 8 6-7 4-5 7-8 1-2 2-3 8-9 3-4 3-4 Undetectable) (Undetectable) In addition, in the analysis of amino acid composition, urine TF active substances were
1 in 6N-HCI vapor containing % (V/V) phenol.
After hydrolysis at 10°C for 24 hours, an automatic amino acid analyzer (
PICO-TAG (manufactured by Waters) was used.

(2) Sugar content 25-30% by weight (3)
Isoelectric point 6,8~7.2 (4) Ultraviolet absorption spectrum I~le λmax=278nm (5) Specific activity (
Blood coagulation activity) Thromb, Haemosta! 1.36.90-
The activity is approximately 1/8 to 1/10 that of TF derived from human placenta purified by the method described in 103 (1976).

As the method for purifying the human TF active substance of the present invention described above, a monoclonal antibody against the human TF active substance of the present invention can be used. The antibody was prepared using the method of Köhler and Milstein [K'6hler &Milstein; Nat.
ure: 256.495-497 (1975)]
Accordingly, monoclonal antibodies produced from the created hybridoma cells can also be used. After binding the obtained monoclonal antibody to an insoluble carrier and contacting it with a liquid containing a human TF active substance, the insoluble carrier on which the antibody is immobilized is separated from the liquid and subjected to elution treatment of the human tissue factor active substance. A method for purifying a human tissue factor active substance is provided.

In this purification method, the insoluble carrier used to bind the anti-human TF active substance monoclonal antibody is not particularly limited and can be various types as long as it is a solid that is substantially insoluble in the eluent used in the elution process. materials such as sepharose, agarose,
polyacrylamide resin, cellulose and its derivatives,
Examples include dextran and maleic acid polymers. These materials can usually be used in the form of beads, fibers, powders, gels, and the like.

Methods for binding antibodies to these materials include a method of making an amide bond with the antibody using a carboxyl group or an amino group that may exist in these materials;
, a method of inducing CNBr, etc., and then binding an antibody [for example, Axen R, et al., Natur.
e, 214.1302-1304 (1967)].

The insoluble carrier to which the anti-human TF active substance monoclonal antibody has been bound in this way is packed into a column, for example, and a liquid containing the human TF active substance is poured onto the column, whereby the liquid is brought into contact with the insoluble carrier to which the antibody has been bound. let Then, according to a conventional melt processing method, for example, an eluent is poured from the top of the column, a fraction exhibiting human TF activity is collected from the eluate, and a human TF active substance is extracted from the fraction as described in, for example, George J, Broze et al., J.
, Biochem.

Chem, 260.10917-10920 (19
85), a substantially pure human TF active substance can be obtained.

Examples of the eluent that can be used in the above elution treatment include G
lycine-HCI (pH2,5), 3M
Na5CN (pH 7,4), 2.5 M NaI
(pH 7,5), 6M guanidine-HCl (pH
3,1), 8M urea < pH 7,0), 50%.

v/v, ethylene glycol (pH 11,5), etc. can be used.

Among others, solutions containing chaotropic ions, e.g.
A 3M Na5CN pH 7.0 solution is preferred.

The present invention will be explained in more detail below.

0 A1 Monoclonal antibodies against the human TF-like protein of the present invention - Monoclonal antibodies against the TF-like protein of the present invention can also be obtained using tissue factor derived from human placenta as an antigen.

The human placenta-derived TF apoprotein was obtained by the method of Gonmori et al. [Gonmori H, et al;
b, Haemostas, 36.90-103 (
Human placenta was isolated and purified by [1976]. Using the TF obtained in this way, the method of Köhler and Milstein [K'6hler & Milstein・Na
ture: 256.495-497 (19751) to generate hybridoma cells and isolate and obtain the produced monoclonal antibodies.

Specifically, the Microtechnology Institute deposit number FEBM P-10505
(Antibody name; produces GX3) and FERM P-10
506 (antibody name: produces GX4) hybridoma cells and an anti-human TF monoclonal antibody having binding properties equivalent to the antibody.

B. Isolation and purification of TF-like protein from human urine As the antibody used when purifying the protein of the present invention from human urine, a monoclonal antibody against TF derived from human placenta can be used. Specifically, examples thereof include the antibody (GX3) produced by hybridoma cells with the FFE deposit number FROM P-10505 and an anti-human TF monoclonal antibody having binding properties equivalent to the antibody (GX3). The antibody can be chemically immobilized on an insoluble carrier such as sepharose agarose and used by packing it into a column or the like. To elute the target protein from the antibody-immobilized column,
In consideration of maintaining the activity of the protein, it is preferable to carry out the reaction at a pH around 7, and a solution containing chaotropic ions, such as a 3M Na5CN pH 7.0 solution, is preferable.

Alternatively, a glycine-HCl solution or the like can also be used if it is eluted under acidic conditions and immediately returned to neutrality with a Tris salt or the like.

Structural analysis of C5 urinary TF-like protein The protein obtained by elution from the antibody column is sparingly soluble in water, and must be solubilized for structural analysis. That is, after decomposing the protein with CNBr, the protein was denatured by heating in the presence of SDS and 2-mercaptoethanol, and then sugar chain cleaving enzyme 2 (N-glycanase) was applied to cleave the sugar chain from the protein portion. As a result, the protein was solubilized.

Molecular weight 36,000 and 18 with reverse phase column (C18 column)
000 fragments must be isolated and purified. After reducing the 36,000 fragment, it was digested with endoproteinase Asp-N, and the 1,200
0, 24,000, and 36,000 fragments were isolated.

The N-terminal amino acid sequence of each obtained fragment was determined using a gas phase fully automated amino acid sequencer.

The urinary TF-like protein in the present invention is a previously reported placenta-derived TF.
It is a novel protein present in urine that has a different amino acid sequence from membrane proteins. The content in urine is 2-3 μg according to the sandwich ELISA method using monoclonal antibodies.
/ml. The molecular weight of the protein is 54,000 to 58,000 under reducing and non-reducing conditions, which is equal to that of placenta-derived TF membrane protein. Furthermore, the protein of the present invention exhibits blood coagulating activity in the presence of calcium ions and phospholipids, and does not coagulate factor (I)-deficient plasma. That is, it is a substance that has TF activity.

]-3 (e) Effect of the invention The TF active substance derived from human urine having such characteristics can be used as a hemostatic agent after trauma or surgery, a therapeutic agent for hemophilia patients who have neutralizing antibodies against factor Ⅰ, etc. It may be useful as an initiator of blood coagulation.

(f) Examples Examples will be described in detail below.

Example 1 Preparation of a monoclonal antibody against human TF protein and examination of its properties By decomposing human placenta-derived TF apoprotein with CNBr, an amino-terminal fragment that does not contain a phospholipid-binding domain can be obtained. Each female Ba1b
/c mice (4 weeks old) 4 times at 14-day intervals for a total of 3 mice.
I was immunized. The first immunization is 50μ dissolved in physiological saline.
4 g of antigen is mixed with an equal volume of Freund's complete adjuvant and the emulsion is administered intraperitoneally. Second time.

For the third immunization, the same <50 μg of antigen was mixed with incomplete Freund's adjuvant and administered intraperitoneally. For the final immunization (4th time), 30 μg of antigen was additionally administered intravenously to the mouse. Lung cells from mice immunized 3 days after the final immunization were used for cell fusion.

The collected mouse lung cells and syngeneic mouse myeloma cells (P3U1) were mixed at a ratio of approximately 5:1, and cell fusion was performed using 50% polyethylene glycol 1540 as a fusion promoter according to the method of Kohler and Milstein. went.

After fusion, the cells were mixed with RPMI 16 containing 10% bovine serum to give a cell concentration of IX106 cells/ml.
40 medium and dispensed into a 96-well microplate at 100 μρ per well.

Hybridomas (fused cells) are CO2 incubators.
(5% CO2, 37°C), hyboxanthin,
The medium was replaced with a medium containing aminopterin and thymidine (HAT medium), and the cells were grown in HAT5 medium to screen for hybridomas consisting of lung cells and myeloma cells.

Antibodies in cultured hybridoma slides were detected by ELISA using a microtiter plate adsorbed with human TF apoprotein purified from human placenta. Colony formation was observed in 669 wells out of a total of 1022 wells in which hybridomas were seeded, and among these, 3 wells were positive for producing antibodies that bind to human placenta-derived TF apoprotein.
There were 56 wells. (Table 1) Efficiency of cell fusion Four of these antibody-producing positive wells were repeatedly cloned twice by the limiting dilution method to obtain two monoclones. The obtained clone is 1
Suspend in 90% bovine serum solution containing 0% DMSO and store in liquid nitrogen. The monoclonal antibodies produced by each clone were purified from the ascites using a Protein A-Sepharose 4B column after the clones were grown intraperitoneally in Balb/c mice.

b) Class of purified monoclonal antibody The class of IgG of each clone purified from mouse ascites was determined by the Ophthalony method.

5μg of human TF apoprotein extracted and purified from human placenta
Adsorb onto a microtiter plate at a concentration of /ml,
After blocking with 1% BSA, monoclonal antibody solution diluted to an appropriate concentration <0.16-5.0μ
g/ml>. Next, add an alkaline phosphatase-labeled anti-mouse antibody and detect and examine the binding properties of the two monoclonal antibodies to human TF apoprotein.

8 The two types of monoclonal antibodies obtained showed strong binding to human TF apoprotein.

Add HCOOH to the human TF apoprotein (40 Mg) solution to make a 70% HCOOH solution. CNB in this solution
After adding r powder, dissolving and reacting at room temperature for 18 hours, HCOOH was evaporated (dry t+p). 40M
After dissolving the protein by adding 1 g of H20, the equivalent of 2 μg was reduced in the presence of 2-mercaptoethanol, and 5DS was purified using a gradient gel with a concentration of 4 to 20%.
- Polyacrylamide electrophoresis was performed (M, W,
36,000 and 18,000 fragments). For comparison, TF apoproteins (M, W,
54.000) was similarly reduced and electrophoresed.

After electrophoresis, proteins in the gel are electrically transferred to a nitrocellulose membrane using a plotting device (manufactured by Marizuru ■).

Two monoclonal antibodies (G
X3 and GX4) at a concentration of 2 μg/ml in 1% gelatin-TBS overnight at room temperature. 0.05
The nitrocellulose membrane was washed three times with 20% Tween-TBS and reacted with a 1% gelatin-TBS solution of peroxidase-labeled anti-mouse Ig antibody at room temperature for 4 hours. After washing, a 4chloro-1-naphthal substrate solution was added to develop the enzyme-labeled antibody bound to the nitrocellulose crotch. The protein bound to the monoclonal antibody could be detected as a dark blue band.

The binding characteristics of the two monoclonal antibodies obtained are shown in Table 3.

0 table ○: Strongly bonded.

Δ: Weakly bound ×: Not bound 2]-e) Effect of monoclonal antibodies on TF activity Each of the obtained monoclonal antibodies (GX3).

GX4) 50 Mg and 100 μg of human placenta-derived TF were mixed to make a 1 ml solution, reacted at 37°C for 1 hour, and then left at 4°C overnight. Of that, 200μρ is added to 100μ of human plasma.
In addition to g, react and measure the clotting time (PT). As a control, a rabbit antiserum against TF apoprotein was used instead of the monoclonal antibody. Measurement is Sysmex
Blood Coagulation Analysis manufactured by
The test was carried out using ZER CA-100.

Two types of monoclonal antibodies of the present invention (GX3).

GX4) had no effect on the coagulation activity of human placenta-derived TF.

Example 2 Detection of TF-like protein in human urine Monoclonal antibody GX3 was diluted with PBS (10mM phosphate buffer-0.15MN) to a concentration of 20Mg/ml.
The antibody 2 was diluted with aCl pH 7.4), added at 100 μρ to the wells of a microtiter plate, and incubated overnight to adsorb antibody 2 onto the solid phase. 1% BSA (bovine serum albumin)
Add 150 μρ/well of PBS containing PBS and leave at room temperature for 2 hours. Followed by 0.05% Tween 20 and 0.1
The cells were washed with PBS (washing buffer) containing % BSA. Next, human placenta-derived TF apoprotein was added to washing buffer at 25 ng/ml, 50 ng/ml, and 100 ng.
human urine was diluted 80 times and 40 times and added to each well at 100 μρ/well.
The reaction was carried out at 7°C for 1 hour. After washing three times with washing buffer, peroxidase-labeled monoclonal antibody G
X4 was diluted with a washing buffer to a concentration of 300 ng/ml, added at 100 μρ/well, and reacted at 37° C. for 1 hour. After washing three times with washing buffer, add 100 μρ/well of substrate solution (ABTS) and measure the absorbance at a wavelength of 415 nm.

Human TF was contained in the urine of healthy subjects at a concentration of 2 to 3 μg/ml. On the other hand, in the human urine of patients with renal failure, nephrotic syndrome, and various types of nephritis,
．． It was detected and quantified at cLg/ml, which was significantly higher than that of healthy individuals.

Example 3 Purification of TF-like protein from urine 500 ml of healthy human urine to which 20 units/ml tradiol (protease inhibitor) was added was filtered using filter paper (Toyo Roshi No. 2).
>, and then passed through an anti-TF monoclonal antibody column (antibody name GX3, column volume IQml, antibody binding amount 8mg) at a flow rate of 15ml/hr. Washing buffer A <20mM Tris-HCl, 0.5M
Contains NaCl pH 7, 60, 05% Tween 20 and 20 units/ml tradiol>200ml
After washing the column with washing buffer T-B (20
mM Tris-HCL O, 10M NaCl pH
7.6゜20 units/m1 (including Trasylol) 150
Wash with m1 and confirm that the absorbance A280 of the washed fraction at a wavelength of 280 nm is O. Then 3M N
A5CN pH 7.0 solution was passed through the column at a flow rate of 20 ml/hr to elute the urinary TF-like protein that had bound to the antibody column. Immediately after removing the N5SCN salt by dialysis, ProteinAssay reagent (Bio-Rad
The amount of protein was colorimetrically determined using dye reagent (manufactured by @), and the result was 591 μg. The column was washed with 6M guanidine-HCl pH 3.1 and then stored with 200 ml of wash buffer B. All of the above purification operations except protein concentration measurement were performed in step 4.
Performed at °C.

Example 4 Characteristics of purified urinary TF-like protein (1) Molecular weight assay The molecular weight of urinary TF-like protein was assayed by 5DS-polyacrylamide electrophoresis using a gel with a concentration of IO to 20%. After injecting 1 μg of protein into each lane, Coomassie staining was performed, and the molecular weight was calculated from the mobility.

(2) Measurement of blood coagulation activity Urine TF-like protein (1,25-2OAtg) and phospholipid 20
After mixing each μg, add CaCl2 solution to a final concentration of 2mM.
The mixture was added so as to give a reaction temperature of 30.degree. C. and reacted at 37.degree. C. for 30 minutes.

Add 200 μg of this reaction solution to 100 μg of human plasma and measure the clotting time (PT).The amount of protein added to the plasma (
Table 4 shows the relationship between coagulation time (in seconds) and coagulation time (in seconds). As a result, the molecular weight 5 of the urinary TF-like protein was 54,000 under reducing conditions, and it was confirmed that it had an activity of inducing blood coagulation.

Table 4 Activity of TF-like protein purified from urine Example 5 Effect of pH on expression of urinary TF-like protein activity 500μρ of human fresh plasma and 0.1M citrate buffer p)I3.0 or 0.05M)-Lis Buffer pH 9, 0, or 0.
1M sodium bicarbonate-NaoH buffer pH 12.5
was added, adjusted to various pH, and milliQH2
Use 0 to make it 550 μg.

6 After mixing 10 μg of urine TF-like protein and 20 μg of phospholipid, 0
．． Add IM CaCl2 solution to a final concentration of 2mM and incubate at 37°C for 30 minutes. This reaction solution 2
Add 00 μl to 100 μr of the above pH-adjusted human plasma and measure the clotting time (PT). The relationship between pH and coagulation time is shown in Figure 1. As a result, the optimum pH for the urinary TF-like protein to exhibit blood coagulation activity is 7.5 to 8.5, and the pH range in which it can exhibit activity is 5.5 to 12.

Example 6 Structural analysis of urinary TF-like protein (1) Solubilization of urinary TF-like protein 100 μg of urinary TF-like protein (70% HCOOH solution; 1
Add CNBr to 00 μlJ) and incubate for 12 hours at room temperature (23°C). After drying, add 0.2M phosphate buffer - 0.15M Na1l pH 7.8 (0.10%
SDS, containing 5% 2-mercaptoethanol) at 10
After cooling to room temperature, add N-glycanase (Waters ■, 5 μg) to the protein solution.
Urine 7 TF-like protein was solubilized by adding 1 unit/ml) and reacting at 37°C for 2 days.

(2) Isolation of CNBr degradation fragments Add 40% distilled pure water containing 1% trifluoroacetic acid (TFA) to 100 μρ of the protein solubilized solution obtained in (1) above.
After adding 0μρ and mixing, add cps column (Waters 0
Sakaki, 300 A, reverse phase column). 0-60%
Using an acetonitrile concentration gradient of
00 fragment 39μg and molecular weight 18000 fragment 26μg
Do not isolate.

(3) The N-terminal amino acid sequence of the 12,000 fragments obtained by protease digestion was reduced and alkylated, and the 36,000 fragments (20 μg) were added to an aqueous solution with protease (Boehringer Mannheim ■, As
1 μg of p-Nl was added and reacted at 37° C. for 12 hours. After adding 1 portion of distilled pure water containing 0.1% TFA and 1% acetonitrile to the solution and mixing, Phenyl-5P
Passed through W column (TO8O■), molecular weight 12000,
Do not isolate the 24,000 and 36,000 fragments.

Using 1.8 μg (150 pmol) of the 12000 fragment, the N-terminal amino acid sequence was analyzed using 8 Protein Sequencer-47OA (Applied Biosystems), and the result was 23456 Lys-Glu-Gly-Ala-Ala-11eLe.
Identify the sequence of u-Phe-Glu-Pro-Thr.

On the other hand, 2.2 μg (92 pmo
l), and 3.4 μg of 36,000 fragments (94
pmol) sequencer, no amino acids were detected in any cycle in all the cycles, suggesting that the N-terminal amino acid was modified or blocked.

In addition, from the analysis of the amino acid sequences of the three fragments, the part with the above 11 amino acid sequence is shown in Figure 2.
It is thought that it exists in a location not shown in the schematic diagram.

Furthermore, the amino acid composition, sugar content, specific activity 1 isoelectric point, and λmax value of the TF active substance isolated from human urine were analyzed. They are summarized in Tables 5 and 6.

9 table Analysis value of urine TF active substance 0 table 6 Comparison of TF activity

[Brief explanation of drawings]

FIG. 1 shows the relationship between the activity expression of the urinary TF-like protein of the present invention and pH. FIG. 2 shows an overview of the overall structure of the urinary TF-like protein of the present invention.

Claims (4)

[Claims] (1) VII in the coexistence of phospholipids and calcium ions
Activate factors to express blood coagulation activity and maintain optimal pH
is 7.5 to 8.5, can express activity in the pH range of 5.5 to 12, and has a molecular weight of 54,000 to 12.
Human tissue factor active substance, which can be found in human urine, in the range of 60,000. (2) Molecular weight is 36,000-40 due to cyanogen bromide
2. The human tissue factor active substance according to claim 1, wherein the human tissue factor active substance is decomposed into two fragments of 1,000 and 18,000 to 20,000. (3) After bringing the anti-human tissue factor active substance monoclonal antibody bound to an insoluble carrier into contact with a liquid containing the human tissue factor active substance, the insoluble carrier on which the antibody is immobilized is separated from the liquid, and the human tissue factor active substance is separated from the liquid. 2. The method for purifying a human tissue factor active substance according to claim 1, wherein the human tissue factor active substance is subjected to an elution treatment. (4) The method according to claim 3, wherein the human tissue factor active substance-containing liquid is human urine.