Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Patent
1991-01-09
1993-03-30
Wax, Robert A.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
530380, 530413, C07K 320
Patent
active
051985349
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for preparing activated protein C (APC).
Protein C is a plasma glycoprotein whose active form is a serine protease (1, 2).
Human protein C is a protein of molecular weight 62,000 consisting of two chains: a heavy chain of molecular weight 41,000 bearing the active site and a light chain of molecular weight 21,000, linked via a disulphide bridge (2). Its plasma concentration is 3-5 mg/l.
Like other proteins, especially plasma proteins, this protein is a vitamin K-dependent factor. It is synthesized by the liver in the form of a precursor. The first 11 glutamic acids of the light chain are carboxylated at the gamma-position by a liver carboxylase having a vitamin K as a cofactor (3). These gamma-carboxy-glutamic residues are involved in an interaction with calcium ions (4). Since phosopholipids are negatively charged, calcium produces an ionic bridge between these compounds and the appropriate region of vitamin K-dependent factors The light chain possesses, moreover, a beta-hydroxyaspartic residue which also appears to be involved in the interaction with Ca.sup.++.
The proenzyme is converted to activated protein C (APC) by cleavage of a dodecapeptide in the N-terminal portion of the heavy chain (2). This reaction, which takes place in the microcirculation, is catalyzed in the presence of calcium by a 1:1 stoichiometric complex formed between thrombin and a protein located at the surface of the endothelial cells, thrombomodulin (5).
The active form has an anticoagulant action by inactivating the coagulation cascade cofactors V and VIII by limited proteolysis (6). The active enzyme also appears to increase fibrinolysis by activating tissue plasminogen activator inhibitor.
APC exercises its activity fully only in the presence of a cofactor, protein S, phospholipids and calcium. Protein S is also a vitamin K-dependent plasma glycoprotein. It is a single-chain protein, of MW 75,000, its plasma concentration being 25 mg/l. It circulates to the extent of 50% in free form and 50% in the form of a non-covalent complex with a protein of the complement system, "C4-Binding Protein" (C4BP) (7). When protein S is linked to C4BP, it cannot act as a cofactor (7).
In contrast to the other vitamin K-dependent enzymes, protein C is anticoagulant.
Congenital and acquired protein C deficiencies are known. In this case, as with AT III deficiencies, a tendency to repeated thrombotic accidents is observed.
The therapeutic use of the enzyme and the proenzyme is advantageous. Thus, protein C may be used for the treatment of protein C deficiencies, and especially in homozygotic neonates who develop purpura fulminans.
This often fatal disease is characterized by substantial thrombotic lesions which necessitate therapy with protein C-enriched plasma concentrates. The heterozygotic form can lead to mild to severe thrombotic episodes, or may be totally asymptomatic.
Both protein C and activated protein C may be employed for the prophylaxis and treatment of venous and arterial thromboses, pulmonary, cerebral and cardiac embolisms, CIVD and septic shock.
Activated protein C may also be used for the replacement of traditional anticoagulants (especially heparin) without their side effects in arterial or venous thromboses, surgery and phlebitis, and in the case of reocclusion of myocardial infarctions.
Great structural homology exists between certain vitamin K-dependant factors, and especially between factors VII, IX and X and protein C. They have, in addition, a very similar molecular weight (56,000, 57,000, 59,000 and 62,000, respectively).
The similarity of these molecular features hence causes difficulties in the isolation of these different factors.
The object of the present invention is specifically to overcome this obstacle and to propose a method enabling activated protein C to be obtained rapidly from a blood fraction enriched to a greater or lesser extent in protein C.
It was, in effect, demonstrated by the inventors that aprotinin, which is a polypeptide of animal origin of 58 amino acids, i
REFERENCES:
patent: 4604285 (1986-08-01), Smith et al.
patent: 4849403 (1989-07-01), Stocker et al.
Cooper, T. G., "The Tools of Biochemistry". 1977. John Wiley & Sons, New York. pp. 234-255.
Freifelder, D. "Physical Biochemistry. Applications to Biochemistry and Molecular Biology". 1976. W. H. Freeman & Co., San Francisco. pp. 204-205.
Espana et al. (Dec. 15, 1989). Aprotinin is a competitive inhibitor of activated protein C. Thrombosis Res. 56: 751-756.
Griffin, J. H. et al., Aprotinin (Trasylol) is a Competitive Inhibitor of Activated Protein C. Thrombosis and Haemostasis, Abstracts-XIIth Congress of the International Society of Thrombosis and Haemostasis, Tokyo, Japan, vol. 62, p. 385, abstract No. 1212 (Aug. 1989).
Thrombosis and Haemostasis, Abstracts-XIIth Congress of the International Society of Thrombosis and Haemostasis, Tokyo, Japan, vol. 62, p. 272, abstract Nos. 870 to 873 (Aug. 1989).
Chabbat Jacques
Steinbuch Marion
Taby Olivier
Ekstrom Richard C.
Fondation Nationale de Transfusion Sanguine
Wax Robert A.
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