JP2002195995A - Blood coagulation accelerant and container for blood test - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a blood coagulation accelerant with surpassing stability coagulating any blood, even containing heparin, in a short time and a container for blood test favorably-used not only for normal blood testing but also for blood serum sampling in blood testing of an artificial dialysis patient or a thrombosis patient subject to heparin administration. SOLUTION: One blood coagulation accelerant is provided with a peptide chain containing a hydrolase which enables hydrolysis of bond between Arg and an optional amino-acid residue and/or bond between Lys and an optional amino-acid residue, and an albumin and/or an amino methyl-cyclohexane carboxylic acid as a stabilizer for the hydrolase. The other blood coagulation accelerant contains an organic compound having an amine salt and/or a quaternary nitrogen. The container for blood test accommodates these blood coagulation accelerants.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a blood coagulation promoter, and more particularly to a blood coagulation promoter for promoting coagulation of a blood sample obtained from a patient receiving heparin administration, and a blood test container.

[0002]

2. Description of the Related Art Blood tests are generally performed for the purpose of preventing and diagnosing diseases. Most blood tests are serum tests. The serum required for the test is usually obtained by coagulating blood collected in a blood test container, separating it from blood clots having different specific gravities by centrifugation, and collecting the blood using a pipette or by decantation. Blood taken from a subject takes a relatively long time to coagulate, which is a problem when a patient must return to the hospital to know the result or when an urgent test needs to be performed. Therefore, conventionally, a coagulant capable of performing blood coagulation in a short time has been studied.

Another problem arises when handling blood samples from patients undergoing artificial dialysis or thrombosis. In such patients, heparin is administered to prevent thrombus, so that about 1 to 20 units of heparin is present per 10 ml of blood. This heparin binds to antithrombin III in blood and remarkably inhibits the action of thrombin. It is also said to inhibit the action of blood coagulation factors such as factor XII. Therefore, the conversion of fibrinogen to fibrin does not occur, and as a result, blood does not coagulate, and it becomes difficult to collect serum.

In order to solve these problems, for example, Japanese Patent Application Laid-Open No. 58-1460 discloses a blood collection tube using protamine sulfate or a thrombin-like enzyme contained in snake venom as a coagulation promoter. However, since protamine sulfate is a heparin neutralizing agent, the coagulation action is not sufficient, and the thrombin-like enzyme contained in snake venom has a heparin neutralization action and a coagulation action, but has insufficient stability.

[0005]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and has an object to coagulate blood, and even blood containing heparin, in a short time and to have excellent stability. An object of the present invention is to provide a blood coagulation accelerator and a blood test container.

[0006]

The blood coagulation promoter of the present invention contains a hydrolase and albumin and / or aminomethyl-cyclohexanecarboxylic acid (tranexamic acid) as a stabilizer for the hydrolase. .

In the first aspect of the present invention, the hydrolase is a protease, which is capable of hydrolyzing a bond between Arg and an arbitrary amino acid residue and / or a bond between Lys and an arbitrary amino acid residue in a peptide chain. Used. When the amount of the above-mentioned hydrolase is small, blood coagulation time may be prolonged or coagulation may be incomplete. When the amount is large, the test value may be adversely affected. Is preferred, and 0.5 to 50 units is more preferred.

Examples of the above-mentioned hydrolase include serine proteases such as trypsin, thrombin, and snake venom thrombin-like enzymes; thiol proteases such as cathepsin B and ficin; and metal proteases such as kininase I. In particular, serine proteases are preferable. Used for

In the present invention 1, in addition to the above-mentioned albumin and aminomethyl-cyclohexanecarboxylic acid, glycine, polyol and the like can be further used as a stabilizer for hydrolase. As a polyol, for example,
Glycols such as polyethylene glycol and polypropylene glycol, and saccharides such as glycerol, sucrose, dextrose, sorbitol, and gelatin.

When the amount of the stabilizer for the hydrolase is small, the effect of maintaining the stability of the hydrolase is insufficient, and when the amount is large, the test value may be adversely affected. 0.01 to 10 mg is preferable, and 0.05 to 5 mg is more preferable.

[0011] The blood coagulation promoter of the present invention 2 further contains an amine salt and / or an organic compound having a quaternary nitrogen. In the present invention, the organic compound having an amine salt and / or a quaternary nitrogen acts as a heparin neutralizing agent that adsorbs and neutralizes heparin and inactivates it. The amine constituting the amine salt may be any of primary, secondary and tertiary amines, and the acid constituting the amine salt may be any of inorganic acids and organic acids. Examples of the inorganic acid include hydrohalic acids such as hydrochloric acid, sulfuric acid, and sulfurous acid, and examples of the organic acid include formic acid and acetic acid. The organic residue of the amine salt is usually an alkyl group,
It may be a hydrocarbon group containing a different element such as an imino group or an ether group. The amine salt may be an inner salt. Specific examples of preferred amine salts include, for example, hexadecyldimethylamine hydrochloride represented by the formula (I) and tetradecyldi (aminoethyl) glycine represented by the formula (II).

[0012]

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[0013]

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Examples of the organic compound having a quaternary nitrogen include tetraalkylammonium. A compound having an aryl group instead of an alkyl group, or a hydrocarbon group containing a heterogeneous element such as an imino group or an ether group is used. May be a compound having Specific examples of preferred organic compounds having a quaternary nitrogen include, for example, dodecyltrimethylammonium chloride represented by the formula (III).

[0015]

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In addition to such relatively low molecular weight compounds, organic polymers having quaternary nitrogen can also be used. Examples of the organic polymer include a polycation having a repeating unit represented by the formula (IV).

[0017]

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(Where R _{1 to} R ₄ are hydrogen or an alkyl group, X is a halogen radical or an acid radical, Y is an alkylene group or-
Alkylene group -SO ₂ - shows the number of repetitions is 5-20
00. )

Among the compounds represented by the formula (IV), a polycation having a repeating unit represented by the formula (V) or (VI) is particularly preferable.

[0020]

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[0021]

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When the amount of the above-mentioned organic compound having a amine salt and / or quaternary nitrogen (neutralizing agent) decreases, heparin is not neutralized, so that blood containing heparin does not coagulate, and when the amount increases, the test value may be adversely affected. Therefore, 0.005 to 10 mg per ml of blood is preferable,
1-5 mg is more preferred.

The blood coagulation promoter of the present invention 1 or 2 is used at a rate of 1 × 10 ^-10 to 1 × 10 ^-1 g per ml of blood. If the amount decreases, the effect of promoting blood coagulation cannot be obtained, and if the amount increases, the effect corresponding to the amount used cannot be obtained, and there is a possibility that various effects may be exerted on blood tests.

The blood coagulation promoter may further contain an anti-radiation solvent and / or an anti-plasmin agent. Thus, the fibrin-degrading action of plasmin, which is generated antagonistically during the blood coagulation reaction, is inhibited. Therefore, blood coagulation is promoted, and the coagulation state can be stably maintained during coagulation. As the anti-radiation solvent and / or anti-plasmin agent, aprotinin, soybean trypsin inhibitor, ε-aminocaproic acid, p-aminomethylbenzoic acid, aminomethylcyclohexanecarboxylic acid, etc., which have been conventionally used clinically, alone or Used in combination. These are contained in the blood coagulation promoter in an amount that does not affect the clinical tests using the obtained serum. For example, aprotinin is about 100 to 600 KIU (unit) per ml of blood, and soybean trypsin inhibitor is about 500 to 4 KIU per ml of blood.
000 FU (unit), and ε-aminocaproic acid, p-aminomethylbenzoic acid, and aminomethylcyclohexanecarboxylic acid were all added at a rate of about 10 ⁻² to 10 ⁻⁸ g per ml of blood. Included in coagulation accelerators.

To coagulate blood, for example, the blood coagulation promoter may be added to blood collected in a container.
The blood test container of the present invention 3 is the same as that of the present invention 1 or 2
Is contained inside. The blood test container may be made of glass or resin. The blood coagulation promoter may be used, for example, in the form of a powder, or may be dissolved or dispersed in an appropriate solvent in advance. In order to avoid denaturation of the protein component by contacting the blood with a high concentration of a blood coagulation promoter, the blood coagulation promoter may be carried on a carrier having a large specific surface area and accommodated therein.

The carrier is not particularly limited as long as it does not adversely affect the blood test and has a large specific surface area. For example, a nonwoven fabric, a woven fabric, a resin bead, or the like is suitable. As a method of supporting the blood coagulation promoter on such a carrier, for example, a method of applying the solution or dispersion to the carrier, a method of immersing the carrier in the solution or dispersion, impregnating the carrier, and then drying. In addition, a method of preparing an aqueous dispersion of a blood coagulation accelerator containing an appropriate auxiliary such as gum arabic and rapidly freeze-drying the resultant to obtain a blood coagulation accelerator-carrying particulate material may be used.

A blood test container containing such a blood coagulation promoter (including a carrier having a blood coagulation promoter) can be used not only as a normal blood test container but also as a vacuum blood collection tube. The vacuum blood collection tube is prepared by containing the above blood coagulation accelerator inside a normal blood collection tube made of glass or synthetic resin, exhausting the blood collection tube, and sealing it with a highly sealable stopper made of butyl rubber or the like.

Since the blood coagulation promoter of the present invention contains a hydrolase such as serine protease, the activation of the blood coagulation factor is promoted, and blood coagulates in a short time. Further, when the blood coagulation promoter of the present invention 2 is added to blood containing heparin, heparin in the blood is adsorbed and neutralized by a neutralizing agent such as an amine salt and precipitates, so that heparin thrombin or factor XII is precipitated. No inhibitory effect on Thus, blood restores normal coagulation function. The time required for blood coagulation varies depending on the type and amount of the hydrolase and its stabilizer in the blood coagulation promoter, the neutralizing agent, the material of the container used, the amount of heparin in the blood, etc. It takes 10 to 20 minutes usually when using a container made of.

As described above, not only normal blood but also blood containing heparin can be coagulated within a short period of time, and the coagulated state can be stably maintained. Furthermore, since the separation of serum and blood clot is facilitated, blood clot components are not mixed in the separated and collected serum. Because the degree of contraction of the blood clot component is also sufficient,
Serum yield is also high.

[0030]

Next, embodiments of the present invention will be described. (Example 1) Thrombin (trade name:
Thrombin Mochida, Mochida Pharmaceutical Co., Ltd.), aminomethyl-cyclohexanecarboxylic acid as a stabilizing agent for hydrolase, 20,000 units / ml, 2000 m
An aqueous solution containing g was prepared and used as a blood coagulation promoter. Next, the blood coagulation promoter was sealed in a polyethylene wide-mouthed container and allowed to stand at 25 ° C. for one month, and then the following experiment was performed.

Polyethylene tube (16φ × 10m
In m), 10 ml of fresh human blood was prepared, and 20 μl of the above blood coagulation promoter was added dropwise thereto, followed by gentle stirring and standing at 25 ° C. At this time, the contents of thrombin and aminomethyl-cyclohexanecarboxylic acid per 1 ml of blood were 40 units and 4 mg, respectively.

Next, when the blood clotting time of the sample prepared as described above was measured, blood clotting was recognized within 5 minutes. Further, this sample was subjected to 10 rpm at 3000 rpm.
After centrifugation for minutes, clean serum was obtained in the upper layer and a clot was obtained in the lower layer.

(Example 2) Snake venom thrombin-like enzyme (trade name: Leptylase, manufactured by Tohyo Pharmaceutical Co., Ltd.) as a hydrolase, aminomethyl-cyclohexanecarboxylic acid as a stabilizer of hydrolase, 10 ml / ml
An aqueous solution containing 2,000 units and 2000 mg was prepared and used as a blood coagulation promoter. Next, the blood coagulation promoter was sealed in a polyethylene wide-mouthed container and allowed to stand at 25 ° C. for one month, and then the following experiment was performed.

Polyethylene tube (16φ × 10m)
In m), 10 ml of fresh human blood was prepared, and 20 μl of the above blood coagulation promoter was added dropwise thereto, followed by gentle stirring and standing at 25 ° C. At this time, the contents of the snake venom thrombin and aminomethyl-cyclohexanecarboxylic acid per 1 ml of blood were 20 units and 4 mg, respectively. Hereinafter, the first embodiment will be described.
In the same experiment, blood coagulation was observed within 5 minutes.
A blood clot was obtained in the lower layer.

Example 3 Thrombin (trade name: Thrombin Mochida, manufactured by Mochida Pharmaceutical Co., Ltd.) as a hydrolase, and aminomethyl-cyclohexanecarboxylic acid, amine salt and / or quaternary nitrogen as a stabilizer for the hydrolase A polycation represented by the following formula (VII) as an organic compound having the following formula: 5000 units per ml, 125 mg,
An aqueous solution containing 50 mg was prepared and used as a blood coagulation promoter. Next, the blood coagulation promoter was sealed in a polyethylene wide-mouthed container and allowed to stand at 25 ° C. for one month, and then the following experiment was performed.

Polyethylene tube (16φ × 10m)
m) contains 1 human fresh blood containing 1 unit / ml heparin.
0 ml was prepared, and 20 μl of the blood coagulation promoter was added dropwise thereto, followed by gentle stirring and standing at 25 ° C. Blood 1 at this time
The contents of thrombin, aminomethyl-cyclohexanecarboxylic acid, and polycation represented by the following formula (VII) per ml were 10 units, 0.25 mg, and 0.1, respectively.
mg. Thereafter, an experiment was performed in the same manner as in Example 1 above. As a result, blood coagulation was observed within 5 minutes, and a clear serum was obtained in the upper layer and a clot was obtained in the lower layer by centrifugation.

[0037]

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Example 4 Snake venom thrombin-like enzyme (trade name: Leptylase, manufactured by Tohyo Pharmaceutical Co., Ltd.) as a hydrolase, and aminomethyl-cyclohexanecarboxylic acid, amine salt and / or Aqueous solutions containing 10,000 units, 500 mg, and 100 mg per ml of tetradecyldi (aminoethyl) glycine as an organic compound having a quaternary nitrogen were prepared, and used as a blood coagulation promoter. Next, the blood coagulation promoter was sealed in a polyethylene wide-mouthed container and allowed to stand at 25 ° C. for one month, and then the following experiment was performed.

Polyethylene tube (16φ × 10m)
m) contains 1 human fresh blood containing 1 unit / ml heparin.
0 ml was prepared, and 20 μl of the blood coagulation promoter was added dropwise thereto, followed by gentle stirring and standing at 25 ° C. Blood 1 at this time
The contents of thrombin, aminomethyl-cyclohexanecarboxylic acid, and tetradecyldi (aminoethyl) glycine per ml are 20 units, 1 mg, and 0.2 mg, respectively.
mg. Thereafter, an experiment was performed in the same manner as in Example 1 above. As a result, blood coagulation was observed within 5 minutes, and a clear serum was obtained in the upper layer and a blood clot was obtained in the lower layer by centrifugation.

(Example 5) An aqueous solution containing, as a hydrolase, a snake venom thrombin-like enzyme (trade name: Leptylase, manufactured by Tohoku Pharmaceutical Co., Ltd.) and glycine as a stabilizer for the hydrolase at 10,000 units and 500 mg per ml, respectively. Was prepared and used as a blood coagulation promoter. Next, the blood coagulation promoter was sealed in a polyethylene wide-mouthed container,
After standing at ℃ for one month, the following experiment was performed.

Polyethylene tube (16φ × 10m)
In m), 10 ml of fresh human blood was prepared, and 20 μl of the above blood coagulation promoter was added dropwise thereto, followed by gentle stirring and standing at 25 ° C. At this time, the contents of the snake venom thrombin and glycine per 1 ml of blood were 20 units and 1 mg, respectively. Hereinafter, when an experiment was performed in the same manner as in Example 1 above,
Blood coagulation was observed within 5 minutes, and further, by centrifugation, clean serum was obtained in the upper layer and a clot was obtained in the lower layer.

(Example 6) Snake venom thrombin-like enzyme (trade name: Leptylase, manufactured by Tohoku Pharmaceutical Co., Ltd.) as a hydrolase, and polyethylene glycol 6000 as a hydrolase stabilizing agent containing 10,000 units / 500 mg / ml. An aqueous solution was prepared to be used as a blood coagulation promoter. Next, the blood coagulation promoter was sealed in a polyethylene wide-mouthed container and allowed to stand at 25 ° C. for one month, and then the following experiment was performed.

Polyethylene tube (16φ × 10m
In m), 10 ml of fresh human blood was prepared, and 20 μl of the above blood coagulation promoter was added dropwise thereto, followed by gentle stirring and standing at 25 ° C. At this time, the contents of the snake venom thrombin and polyethylene glycol 6000 per ml of blood were 2 respectively.
0 units and 1 mg. Hereinafter, when an experiment was performed in the same manner as in Example 1 above, blood coagulation was observed within 5 minutes,
Further, by centrifugation, clean serum was obtained in the upper layer and a clot was obtained in the lower layer.

(Example 7) Snake venom thrombin-like enzyme (trade name: Leptylase, manufactured by Tohoku Pharmaceutical Co., Ltd.) as a hydrolase, and glycerol as a stabilizer of the hydrolase
An aqueous solution containing 10,000 units and 500 mg per 1 ml was prepared and used as a blood coagulation promoter. Next, the blood coagulation promoter was sealed in a polyethylene wide-mouth container,
After standing at 25 ° C. for one month, the following experiment was performed.

Polyethylene tube (16φ × 10m)
In m), 10 ml of fresh human blood was prepared, and 20 μl of the above blood coagulation promoter was added dropwise thereto, followed by gentle stirring and standing at 25 ° C. At this time, the contents of the snake venom thrombin and glycerol per 1 ml of blood were 20 units and 1 mg, respectively. Thereafter, an experiment was performed in the same manner as in Example 1 above. As a result, blood coagulation was observed within 5 minutes, and a clear serum was obtained in the upper layer and a clot was obtained in the lower layer by centrifugation.

(Comparative Example 1) Thrombin (trade name: Thrombin Mochida, manufactured by Mochida Pharmaceutical Co., Ltd.) as a hydrolase, and polyamine represented by the above formula (VII) as an organic compound having an amine salt and / or a quaternary nitrogen. 1 ml of each cation
An aqueous solution containing 50 mg / 5000 units was prepared and used as a blood coagulation promoter. Next, the blood coagulation promoter was sealed in a polyethylene wide-mouthed container and allowed to stand at 25 ° C. for one month, and then the following experiment was performed.

Polyethylene tube (16φ × 10m)
m) contains 1 human fresh blood containing 1 unit / ml heparin.
0 ml was prepared, and 20 μl of the blood coagulation promoter was added dropwise thereto, followed by gentle stirring and standing at 25 ° C. Blood 1 at this time
The content of thrombin per ml and the content of the polycation represented by the above formula (VII) were 10 units and 0.1 mg, respectively.
Met. Hereinafter, when an experiment was performed in the same manner as in Example 1 above, no blood coagulation was observed even after 30 minutes or more, and when centrifugation was performed, fibrin showing insufficient coagulation was observed in the serum of the upper layer. Was done.

Comparative Example 2 1 ml of thrombin (trade name: Thrombin Mochida, manufactured by Mochida Pharmaceutical Co., Ltd.) was used as a hydrolase.
An aqueous solution containing 20,000 units per unit was prepared and used as a blood coagulation promoter. Next, the blood coagulation promoter was sealed in a polyethylene wide-mouthed container, and allowed to stand at 25 ° C. for one month.
The following experiment was performed.

A polyethylene tube (16φ × 10m)
In m), 10 ml of fresh human blood was prepared, and 20 μl of the above blood coagulation promoter was added dropwise thereto, followed by gentle stirring and standing at 25 ° C. At this time, the content of thrombin per 1 ml of blood was 40 units. Hereinafter, when an experiment was performed in the same manner as in Example 1 above, no blood coagulation was observed even after 30 minutes or more, and fibrin showing insufficient coagulation was found in the serum of the upper layer after centrifugation. Was done.

Example 8 20 μl of the blood coagulation promoter obtained in Example 1 was placed in a polyethylene tube (1).
(6φ × 10 mm) and dried to prepare a blood test container. After the blood test container was allowed to stand at 25 ° C. for one month, the following experiment was performed. 10 ml of human fresh blood was prepared, placed in the blood test container, gently stirred, and allowed to stand at 25 ° C. At this time, the contents of thrombin and aminomethyl-cyclohexanecarboxylic acid per 1 ml of blood are the same as in Example 1. Thereafter, when an experiment was performed in the same manner as in Example 1 above, blood coagulation was observed within 5 minutes, and a clear serum was obtained in the upper layer and a blood clot was obtained in the lower layer by centrifugation.

Example 9 20 μl of the blood coagulation accelerator obtained in Example 2 was placed in a polyethylene tube (1).
(6φ × 10 mm) and dried to prepare a blood test container. After the blood test container was allowed to stand at 25 ° C. for one month, the following experiment was performed. 10 ml of human fresh blood was prepared, placed in the blood test container, gently stirred, and allowed to stand at 25 ° C. At this time, the contents of the snake venom thrombin and aminomethyl-cyclohexanecarboxylic acid per 1 ml of blood are the same as in Example 2. Hereinafter, the first embodiment will be described.
As a result, blood coagulation was observed within 5 minutes, and a clear serum was obtained in the upper layer and a blood clot was obtained in the lower layer by centrifugation.

Example 10 20 μl of the blood coagulation accelerator obtained in Example 3 was applied to a polyethylene tube (16φ × 10 mm) and dried to prepare a blood test container. After the blood test container was allowed to stand at 25 ° C. for one month, the following experiment was performed. 1 unit / m of heparin
10 ml of fresh human blood containing l was prepared, placed in the blood test container, gently stirred, and allowed to stand at 25 ° C. At this time, the contents of thrombin, aminomethyl-cyclohexanecarboxylic acid, and the polycation represented by the above formula (VII) per 1 ml of blood are the same as in Example 3. Hereinafter, when an experiment was performed in the same manner as in Example 1 above,
Blood coagulation was observed within 5 minutes, and centrifugation
Clean serum was obtained in the upper layer and a clot was obtained in the lower layer.

Example 11 20 μl of the blood coagulation promoting agent obtained in Example 4 was applied to a polyethylene tube (16 × 10 mm) and dried to prepare a blood test container. After the blood test container was allowed to stand at 25 ° C. for one month, the following experiment was performed. 1 unit / m of heparin
10 ml of fresh human blood containing l was prepared, placed in the blood test container, gently stirred, and allowed to stand at 25 ° C. The contents of thrombin, aminomethyl-cyclohexanecarboxylic acid, and tetradecyldi (aminoethyl) glycine per 1 ml of blood at this time are the same as those in Example 4. Thereafter, an experiment was conducted in the same manner as in Example 1 above. Blood coagulation was observed within 5 minutes, and a clear serum was obtained in the upper layer and a blood clot was obtained in the lower layer by centrifugation.

Example 12 20 μl of the blood coagulation accelerator obtained in Example 5 was applied to a polyethylene tube (16 × 10 mm) and dried to prepare a blood test container. After the blood test container was allowed to stand at 25 ° C. for one month, the following experiment was performed. 10 ml of fresh human blood
This was prepared, placed in the blood test container, gently stirred, and allowed to stand at 25 ° C. At this time, the contents of snake venom thrombin and glycine per 1 ml of blood are the same as in Example 5. Thereafter, an experiment was conducted in the same manner as in Example 1 above. Blood coagulation was observed within 5 minutes, and a clear serum was obtained in the upper layer and a blood clot was obtained in the lower layer by centrifugation.

Example 13 20 μl of the blood coagulation accelerator obtained in Example 6 was applied to a polyethylene tube (16φ × 10 mm) and dried to prepare a blood test container. After the blood test container was allowed to stand at 25 ° C. for one month, the following experiment was performed. 10 ml of fresh human blood
This was prepared, placed in the blood test container, gently stirred, and allowed to stand at 25 ° C. The contents of the snake venom thrombin and polyethylene glycol 6000 per ml of blood at this time are the same as in Example 6. Thereafter, an experiment was conducted in the same manner as in Example 1 above. Blood coagulation was observed within 5 minutes, and a clear serum was obtained in the upper layer and a blood clot was obtained in the lower layer by centrifugation.

Example 14 20 μl of the blood coagulation promoter obtained in Example 7 was applied to a polyethylene tube (16 × 10 mm) and dried to prepare a blood test container. After the blood test container was allowed to stand at 25 ° C. for one month, the following experiment was performed. 10 ml of fresh human blood
This was prepared, placed in the blood test container, gently stirred, and allowed to stand at 25 ° C. The contents of the snake venom thrombin and glycerol per 1 ml of blood at this time are the same as in Example 7. Thereafter, an experiment was conducted in the same manner as in Example 1 above. Blood coagulation was observed within 5 minutes, and a clear serum was obtained in the upper layer and a blood clot was obtained in the lower layer by centrifugation.

(Comparative Example 3) 20 μl of the blood coagulation accelerator obtained in Comparative Example 1 was placed in a polyethylene tube (1).
(6φ × 10 mm) and dried to prepare a blood test container. After the blood test container was allowed to stand at 25 ° C. for one month, the following experiment was performed. 10 ml of fresh human blood containing 1 unit / ml of heparin was prepared, placed in the blood test container, gently stirred, and allowed to stand at 25 ° C. Thrombin per ml of blood at this time, the above formula (VII)
The content of the polycation represented by was 10 units and 0.1 mg, respectively. Hereinafter, when an experiment was performed in the same manner as in Example 1 above, no blood coagulation was observed even after 30 minutes or more, and fibrin showing insufficient coagulation was found in the serum of the upper layer after centrifugation. Was done.

Comparative Example 4 20 μl of the blood coagulation accelerator obtained in Comparative Example 2 was placed in a polyethylene tube (1).
(6φ × 10 mm) and dried to prepare a blood test container. After the blood test container was allowed to stand at 25 ° C. for one month, the following experiment was performed. 10 ml of human fresh blood was prepared, placed in the blood test container, gently stirred, and allowed to stand at 25 ° C. At this time, the content of thrombin per 1 ml of blood is the same as in Comparative Example 2. Hereinafter, when an experiment was carried out in the same manner as in Example 1 above, no blood coagulation was observed even after 30 minutes or more, and when centrifugation was performed, fibrin showing insufficient coagulation in the serum of the upper layer was found. Admitted.

[0059]

The blood coagulation accelerator and the blood test container of the present invention are as described above, and coagulate not only normal blood but also heparin-containing blood in a short time and have excellent stability. . Furthermore, since the coagulation state is kept stable and the separation of serum and blood clot is facilitated, blood clot components are not mixed in the separated and collected serum. Since the degree of contraction of the blood clot component is sufficient, the serum yield is also high. Therefore, the blood coagulation accelerator and the blood test container of the present invention are suitably used not only for normal blood tests, but also for fractionation of serum in blood tests of dialysis patients and thrombosis patients receiving heparin administration. .

Claims (3)

[Claims] 1. A hydrolase capable of hydrolyzing a bond between Arg and an arbitrary amino acid residue and / or a bond between Lys and an arbitrary amino acid residue in a peptide chain, and a stabilizer for the hydrolase A blood coagulation promoter comprising albumin and / or aminomethyl-cyclohexanecarboxylic acid as a component. 2. The blood coagulation promoter according to claim 1, further comprising an organic compound having an amine salt and / or a quaternary nitrogen. 3. A blood test container containing the blood coagulation promoter according to claim 1 or 2 therein.