Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1997-08-18
1999-10-26
Carlson, Karen Cochrane
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435212, 530412, 530413, 530856, C12N 948, C12N 950, A23J 100
Patent
active
059726814
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a novel calcium-requiring prothrombin activator derived from snake venom.
BACKGROUND ART
In the body, the blood circulates under a certain balance between the coagulation system and the fibrinolytic system.
In the second phase of blood clotting, prothrombin (Factor II), which is one of the blood coagulation factors, is converted into thrombin by the action of thromboplastin (Factor III) in the presence of calcium ions (Ca.sup.2+ ions: Factor IV). In the third phase, fibrinogen (Factor I) is acted upon by the thrombin and changed into strands of fibrin, whereby the blood clotting is completed.
Anticoagulant therapy is given to patients suffering from ischemic heart diseases or patients having a heart valve prosthesis and for the monitoring of the therapy, prothrombin time is measured. The measurement of prothrombin time is carried out by measuring the time required for coagulation by adding thromboplastin, which is an exogenous substance causing blood clotting, to a specimen, whereby the extrinsic coagulation pathway activity can be studied. This method is used for total measurement of extrinsic coagulation factors (Factors I, II, V, VII and X).
Thromboplastin used as a reagent for the above measurement is prepared from an organ-extracted phospholipid so that it differs in the sensitivity according to the products or batches. The measured value therefore does not accurately reflect the amount of coagulation factors in the specimen, which tends to heighten the frequency of hemorrhage caused by excessive administration of an oral anticoagulant and has come to be a serious clinical problem (Poller L. "Progress in standardization in anticoagulant control", Hematol. Rev. 1, 225-241(1987); Latallo Z S, Thomson J M and Poller L, "An evaluation of chromogenic substrates in the control of oral anticoagulant therapy", Br. J. Haematol, 47, 307-318(1981); Poller L. and Taberner D A, "Dosage and control of oral anticoagulants", An international survey, Br. J. Haematol. 51, 479-485(1982)).
An object of the present invention is to overcome the above-described problem by finding a novel prothrombin activator, applying it to the measurement of prothrombin in the blood and thereby providing a reagent for accurate measurement. An another object of the present invention is to apply the reagent to thrombin-related diseases.
DISCLOSURE OF THE INVENTION
Prothrombin activators which have been separated or identified to date from the snake venom can be classified into three types (Rosing, J. and Tans, G., Toxicon, 30, 1515-1527(1992)). The enzymes belonging to Group 1 are metalloproteases whose action on prothrombin is independent of any plasma or exogenous cofactors. Those belonging to Group 2 are Gla-containing, factor Xa-like serine proteases that require factor Va, phospholipids and Ca.sup.2+ ions. Those belonging to Group 3 are hybrid enzymes comprising factor Xa-like catalytic subunits and factor Va-like regulatory subunits and similar to those belonging to Group 2, require phospholipids and Ca.sup.2+ ions.
It is widely known (T. Morita et al., Methods Enzymol., 80, 303-311, 1981) that metalloproteases which activate prothrombin are present in the venom of Echis carinatus. The present inventors thought out various devices for assay conditions. Under those conditions, the inventors screened prothrombin activators in the venom of Echis carinatus and succeeded in isolating two calcium-requiring prothrombin activators different from metalloproteases such as ecarin which are known to date. Those two activators were designated as carinactivase-1 and carinactivase-2 (which will hereinafter be abbreviated as "CA-1" and "CA-2"), respectively. As a result of investigation on their activity, it has been found that although ecarin, which is an enzyme known to date, reacts with PIVKA-II (abnormal blood coagulation factor (protein induced by Vitamin K absence or antagonist) of Factor II (prothrombin)) and normal prothrombin, the activators of the present invention do not react with PI
REFERENCES:
patent: 5453370 (1995-09-01), Triplett et al.
Poller, L., "Progress in Standardisation in Anticoagulant Control," Hematology Review, vol. 1 (1987) pp. 225-241.
Latallo, et al., An Evaluation of Chromogenic Substrates in the Control of Oral Anticoagulant Therapy, British Journal of Haematology, vol. 47 (1981) pp. 307-318.
Poller, et al., "Dosage and control of oral anticoagulants," British Journal of Haematology, vol. 51 (1982) pp. 479-485.
Rosing, et al., "Structural and Functional Properties of Snake Venom Prothrombin Activators," Taxicom, vol. 30, No. 12 (1992) pp.g 1515-1527.
Morita, et al. "Prothrombin Activator from Echis carinatus Venom," Methods in Enzymology, vol. 80 (1981) pp. 303-311.
Speijer, et al., "Platelet Procoagulant Properties Studied with Snake Venom prothrombin Activators," Thrombosis and Haemostasis, vol. 57, No. 3 (1987) pp. 349-355.
Pirkle, et al. "Thrombin-Like Enzymes of Snake Venoms," Thrombosis Research, vol. 8 (1976) pp. 619-627.
Carlson Karen Cochrane
Eisai Co. Ltd.
Schnizer Holly
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