Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Patent
1997-05-05
1999-08-31
Sayala, Chhaya D.
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
435214, 530384, A61K 3848
Patent
active
059451033
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to a method for the production of thrombin.
BACKGROUND ART
Thrombin is a serine protease having a molecular weight of about 34,000 and an isoelectric point of about 7.1, namely a proteolytic enzyme which exerts its action at the final stage of the blood coagulation process. That is, it acts upon fibrinogen to form fibrin, thereby generating its blood coagulation function.
Because of this, thrombin is used clinically as a topical styptic in the surgical field and as a styptic of upper gastrointestinal bleeding and the like in the field of internal medicine.
In the living body, thrombin is present in the form of prothrombin as its precursor and formed by receiving limited hydrolysis with activated factor X and the like. In general, thrombin is produced by firstly extracting and purifying prothrombin from human blood plasma as the material and then treating the thus purified prothrombin with throinboplastin and the like. In other words, conversion into thrombin is carried out using purified prothrombin.
Thus, it is the present situation that thrombin is produced through several steps, starting from (1) purification of prothrombin as described above, followed by (2) conversion of prothrombin into thrombin and then (3) purification of thrombin and other necessary steps.
Based on this method, various modification methods have been reported. For example, there are known methods in which blood plasma treated with citric acid is allowed to contact with an anion exchanger, the thus adsorbed prothrombin is converted into thrombin on said exchanger and then the converted product is eluted and recovered (U.S. Pat. No. 5,143,838, European Patent Publication No. 378798) and in which blood plasma is treated with low temperature ethanol and then with an anion exchanger, and the thus purified prothrombin is converted into thrombin which is Published Japanese Patent Application No. 3-128398 (U.S. Pat. No. 5,138,034, European Patent Publication No. 408029)!.
Of these steps, conversion into thrombin is effected for example by a method in which a snake venom is used or a method in which a high Patent Application No. 4-365481 and Unexamined Published Japanese Patent Application No. 5-194261 (European Patent Publication No. 543178)!, in addition to the aforementioned method in which thromboplastin is used. Also, Unexamined Published Japanese Patent Application No. 5-186369 (European Patent Publication No. 528701) and U.S. Pat. No. 5,143,838 describe about conversion of purified prothrombin into thrombin making use of calcium chloride.
In this connection, thromboplastin prepared from human placenta is mainly used for the aforementioned purpose, but it cannot always be said that it can be obtained in sufficient quantity because of a difficulty in preparing the material. The same problem also occurs in the case of the snake venom.
On the other hand, the spontaneous conversion with high concentration citric acid salt and the calcium chloride-aided conversion are still limited to a laboratory scale level and not established yet as industrial mass production methods.
In view of the above, the inventors of the present invention have attempted to develop a method for the industrial scale production of thrombin and accomplished the present invention as the result.
Accordingly, the object of the present invention is to provide a method in which the starting material for carrying out conversion of prothrombin into thrombin can be obtained easily and thrombin can be produced and purified in industrial scale.
DISCLOSURE OF THE INVENTION
The object of the present invention can be achieved by a method for the production of thrombin which comprises treating a prothrombin-containing aqueous solution with a Ca salt at 0 to 15.degree. C.
More illustratively, the thrombin production method of the present invention roughly comprises (1) preparation of prothrombin-containing aqueous solution, (2) conversion of prothrombin into thrombin and (3) purification of thrombin.
The prothrombin-containing aqueous so
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Hanada Shinichi
Honda Yoshinobu
Matsumoto Isahiko
Miyake Shoichi
Morisada Yasuaki
Sayala Chhaya D.
The Green Cross Corporation
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