JPH0764877B2 - Anticoagulant and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel anticoagulant substance and a method for producing the same.

[Conventional technology]

Blood coagulation begins with the expression of thromboplastin activity,
It is believed that activation of factor X and factor V in blood, followed by activation of prothrombin to generate thrombin, which is completed by converting fibrinogen into fibrin.

In order to treat diseases caused by blood coagulation, it is effective to use substances that inhibit or inactivate various coagulation factors involved in this blood coagulation mechanism, that is, anticoagulants. This anticoagulant is currently heparin,
Heparin Coffe Actor-II, Antithrombin-III,
Known are α ₂ -macroglobulin, α ₁ -trypsin inhibitor, C ₁ -esterase inhibitor, protein C and the like. Recently, Chris PMReutelingsperger et al.
From the umbilical artery, a novel substance having a molecular weight of 32 kDa and having anticoagulant activity was found and reported [Eur. J. Biochem. 151 ,
625-629 (1985)].

[Problems to be solved by the invention]

However, the presence of many of these anticoagulants is merely confirmed, and only heparin is practically used as a medicine. However, since heparin has a side effect that causes a bleeding tendency, its use method and amount are extremely limited, and it is not satisfactory from the viewpoint of safety as an anticoagulant.

Further, the substance found by Reutering Spellger et al. Is different from the substance of the present invention as described later, and the activity is measured only in the state of a mixture, and it is not practically applicable.

Therefore, it has been desired to develop a further excellent anticoagulant.

[Means for solving problems]

Previously, the applicant has conducted extensive studies to provide an anticoagulant that is safe and has no side effects, and as a result, it contains a large amount of tissue thromboplastin, contains factor XIII, fibrinolytic inhibitory factor, etc., and is prone to thrombus formation. From the microsome fraction obtained by fractionating the precipitate obtained by centrifuging the human placenta homogenate and the supernatant part of the placenta, which is considered to be present in We found that a new anticoagulant can be obtained and applied for a patent (Japanese Patent Application No. 60-217512).

Furthermore, as a result of separating and purifying the centrifugally separated sediment, it was confirmed that a novel substance having a similar anticoagulant activity with different molecular weights was present, and the substance was successfully isolated, thus completing the present invention.

That is, the present invention provides a human placenta-derived novel anticoagulant having the following properties and a method for producing the same.

Molecular weight (SDS-polyacrylamide gel electrophoresis, reduced state and non-reduced state) 33,000 ± 2,000 Isoelectric point (isoelectric focusing column electrophoresis using amphilite) 6.2 ± 0.1 Stability a 50 ° C, heat treatment for 30 minutes Inactivation b Stable at pH 4 to 8.5 (37 ° C) c Stabilized in plasma at 37 ° C for 30 minutes i Extends calcium re-coagulation time Extends prothrombin time c Extends activated partial thromboplastin time Anticoagulation of the present invention Substance (hereinafter referred to as "the substance of the present invention")
Is prepared, for example, as follows.

First, placenta homogenate is prepared from human placenta and centrifuged. The homogenization operation is performed by removing amniotic membrane from the placenta, thoroughly washing with physiological saline, and using a Waring blender and polytron. The obtained homogenate is centrifuged to obtain a supernatant and a precipitate.

The placenta homogenate precipitate thus obtained is thoroughly washed with a buffer solution, centrifuged again, and the washed precipitate is separated and subjected to the subsequent extraction operation. That is, the washing precipitate is immersed in a buffer solution containing a chelating agent such as EDTA, EGTA, oxalic acid, citric acid, sodium nitrilotriacetate, and phosphoric acid, left overnight at 4 ° C to 8 ° C and then centrifuged. Collect the liquid and obtain the extract. In this case, the extraction can also be performed in the presence of a surfactant such as Triton X-100, Lubrol, SDS, deoxycholic acid and the like.

On the other hand, placenta homogenate supernatant is 50,000-100,000g
Ultracentrifugation is performed to obtain a microsome fraction, which is the sediment. From this microsome fraction, a chelating agent and / or a surfactant was extracted in the same manner as above, and ultracentrifugation was performed to collect the supernatant to obtain an extract containing the substance of the present invention. To be

The extract thus obtained is subjected to the ammonium sulfate fractionation. For ammonium sulfate fractionation, solid ammonium sulfate is added to the above extract solution so as to be 35% saturated, and the mixture is centrifuged to separate the supernatant. Then, ammonium sulfate is added to the supernatant so as to be 85% saturated, the mixture is centrifuged, and the precipitate is collected.

The obtained ammonium sulfate fraction is further subjected to known separation / purification means such as dialysis, ion exchange chromatography, gel filtration, adsorption chromatography, hydrophobic chromatography, isoelectric focusing column electrophoresis, lectin or antibody. The product of the present invention can be obtained by purification by using, for example, affinity chromatography using the above or a combination thereof. For example, the fraction obtained by fractionating the chelating agent extract with ammonium sulfate was thoroughly dialyzed, and the dialyzed solution was passed through DEAE-Toyopearl to concentrate the active fraction in the non-adsorbed portion, and Sephadex G-10
Subject to gel filtration using 0. After concentration, Mono-Q (Q-Sepharo
se; manufactured by Pharmacia) and eluted with a linear concentration gradient using sodium chloride to obtain the substance of the present invention.

Molecular weight measurement As a result of measurement by SDS-polyacrylamide gel electrophoresis (12% polyacrylamide gel; reduced state and non-reduced state), it was 33,000 ± 2,000.

Isoelectric point measurement Isoelectric point column electrophoresis (pH 3.
It was 6.2 ± 0.1 as a result of measuring by electrophoresing at 300 V for 48 hours at 5 to 10, 4 ° C). In addition, isoelectric focusing using a polyacrylamide gel with amphilite (pH 3.
As a result of measuring by migrating at 20 W for 1 hour at 5 to 9.5, 10 ° C), 6.4 was shown.

Stability test b Stability against heat treatment The anticoagulant activity after treatment for 30 minutes at each set temperature (0 to 80 ° C) was measured by prothrombin time, and the result was 50 ° C.
The activity completely disappeared at the above temperatures.

(2) pH stability Add various buffer solutions of pH 3.5 to 11.0, and add 1 at 4 ℃ or 37 ℃.
After treatment for 8 hours, the remaining anticoagulant activity was measured by prothrombin time. As a result, at 4 ° C, pH 3.5 to 10.0,
No decrease in the activity was observed in the range of 4-8.5 at 37 ° C.

(C) Stability in plasma As a result of measuring the residual anticoagulant activity by the calcium re-coagulation time method after adding the substance of the present invention to plasma and incubating at 37 ° C for 30 minutes, no decrease in activity was observed. .

Action (a) Action on recalcification time of calcium 100 μl of standard plasma (manufactured by Orso) and 100 μl of solution of the substance of the present invention
l was mixed and after 3 minutes 0.025M calcium chloride solution 100μ
1 was added and the coagulation time was measured. As a result, as shown in Table 1, a strong action of extending the recalcification time of calcium was recognized.

Effect on loprothrombin time (PT) 100 μl of PT reagent (Rioplastin: Mochida Pharmaceutical Co., Ltd.) diluted with 20 mM calcium chloride and 100 μl of the substance solution of the present invention were mixed, 100 μl of standard plasma was added after 3 minutes, and coagulation time Was measured. As a result, as shown in Table 2, a strong PT prolonging action was recognized.

(C) Effect on activated partial thromboplastin time (APTT) APTT reagent (active thrombofax; manufactured by Ortho) 10μ
1 and 90 μl of the substance solution of the present invention were mixed, and after 2 minutes, standard plasma was mixed.
Mix 100 μl. After 6 minutes, 100 μl of 0.025 M calcium chloride solution was added and the coagulation time was measured. as a result,
As shown in Table 3, a strong APTT prolonging action was recognized.

The substance obtained as described above is only described in the above-mentioned Reutering-Shelger et al. As the molecular weight measured by SDS-PAGE and the protein that is inactivated by heat treatment at 56 ° C. It is not clear because the substance has not been isolated and identified yet, but since the compound of the prior art is a substance extracted from the umbilical cord with Tris-hydrochloric acid buffer solution, and the isoelectric point is also different, It is judged to be different from this.

Examples of the dosage form when the substance of the present invention is used as an active ingredient of an anticoagulant include injections. It is preferable that the lyophilized powder is dissolved in distilled water for injection, physiological saline or the like for administration before use. A suitable site for administration is intravenous.

Although the dose varies depending on the severity of the disease, the body weight of the patient, etc., it is usually preferably 10 μg to 10 mg / kg · day.
It should be noted that the substance of the present invention is safe since no abnormality was observed within the above dose range. When the substance of the present invention is formulated as an injection, a stabilizer such as albumin, gelatin, mannitol, etc. can be added, which makes it possible to prevent decomposition, adsorption, etc. in the formulation process. Deactivation can be prevented, and the storage stability of the preparation is improved.

[Operation and effect of the invention]

The substance of the present invention has a strong anticoagulant activity, in particular, a state in which tissue thromboplastin activity is enhanced, that is, in a hypercoagulable state, it shows stronger activity, and thus anti-blood containing it as an active ingredient. Coagulants are safe with few side effects.

〔Example〕

 Next, the present invention will be described with reference to examples.

Example 1 (i) The membrane and the like were removed from 5 human placenta (about 2500 g),
Thoroughly wash with physiological saline and mince. In addition to this, 50 mM
Add 2 liters of Tris-HCl buffer (pH 7.4), crush with a Waring blender, and then add Polytron (Polytron).
tron). The homogenate obtained was 7,000 rpm 15
Centrifugation was carried out for a minute, and the precipitate was collected. Once again on the obtained sediment
2 l of 50 mM Tris-hydrochloric acid buffer (pH 7.4) was added, homogenized with Polytron, and centrifuged at 7,000 rpm for 15 minutes to obtain a washed precipitate. This operation was repeated several times to remove blood components to obtain 930 g of washed sediment.

(Ii) 50 mM containing 50 mM EDTA in 900 g of the precipitate obtained in (i)
Add about 2 liters of Tris-HCl buffer (pH 7.4) and homogenize with a Waring blender. 4 of this homogenate
The mixture was stirred overnight at ℃ and centrifuged at 7,000 rpm for 15 minutes to obtain 2 liter of extract.

(Iii) Solid ammonium sulfate was added to the extract obtained in (ii) to make it 35% saturated, allowed to stand at 4 ° C for 30 minutes to several hours, and then centrifuged at 7,000 rpm for 15 minutes to collect the supernatant. Ammonium sulfate was further added to this supernatant,
The mixture was made 85% saturated, left at 4 ° C. for 2 hours, and then centrifuged at 7,000 rpm for 15 minutes to collect the precipitate. Dissolve the obtained precipitate in a small amount of 20 mM Tris-hydrochloric acid buffer solution, and at 4 ℃ for the same buffer solution.
Then, it was dialyzed thoroughly overnight. The precipitate formed during dialysis was 7,00
It was removed by centrifugation at 0 rpm for 15 minutes to obtain 390 ml of dialysate.

(Iv) The obtained dialysate was added with 20 mM Tris-HCl buffer (pH
It was adsorbed on DEAE-Toyopearl (φ5.5 × 19 cm) equilibrated in 7.4) and thoroughly washed with the same buffer solution. The relevant active fraction was found in the non-adsorbed portion, and fractions 21 to 40 (400 ml) were collected.

(V) The obtained active fraction was concentrated using Diaflow Membrane Filter YM-10, and the concentrated solution was separated by Sephadex.
Apply the gel to G-100 (φ4.5 × 75cm) and elute with physiological saline to give a fraction of 8 ml. Active fraction 95-1
05 was collected and concentrated by ultrafiltration (8.2 ml).

(Vi) Divide the concentrated solution (8.2 ml) into several times and adsorb onto a Mono Q column (Q-Sepharose, FPLC) equilibrated with 50 mM Tris-HCl buffer (pH 8.0) and elute with a linear concentration gradient using sodium chloride. The active fraction was about 0.08.
It was eluted near the M sodium chloride concentration. All active fractions purified by FPLC were collected and concentrated by ultrafiltration to give 6.3 ml of the substance of the present invention (protein weight 7.8 mg, Lowry method)
Got

Claims (2)

[Claims] 1. A human placenta-derived anticoagulant having the following properties. Molecular weight (SDS-polyacrylamide gel electrophoresis, reduced state and non-reduced state) 33,000 ± 2,000 Isoelectric point (isoelectric focusing column electrophoresis using amphilite) 6.2 ± 0.1 Stability a 50 ° C, 30 minutes heat treatment Inactivation b Stable at pH 4 to 8.5 (37 ° C) c Stabilized in plasma at 37 ° C for 30 minutes i Extends calcium re-coagulation time Extends prothrombin time c Extends activated partial thromboplastin time 2. A human placenta is homogenized, the homogenate is subjected to centrifugation, the obtained centrifugation precipitate is extracted with a chelating agent, and the extract is separated and purified. A method for producing a human placenta-derived anticoagulant. Molecular weight (SDS-polyacrylamide gel electrophoresis, reduced state and non-reduced state) 33,000 ± 2,000 Isoelectric point (isoelectric focusing column electrophoresis using amphilite) 6.2 ± 0.1 Stability a 50 ° C, heat treatment for 30 minutes Inactivation b Stable at pH 4 to 8.5 (37 ° C) c Stabilized in plasma at 37 ° C for 30 minutes i Extends calcium re-coagulation time Extends prothrombin time c Extends activated partial thromboplastin time