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
1991-08-12
1993-06-15
Russel, Jeffrey E.
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...
530413, 530417, C07K 320, A61K 3516
Patent
active
052199932
DESCRIPTION:
BRIEF SUMMARY
The present invention refers to a method of isolating and purifying the EPI protein.
BACKGROUND OF THE INVENTION
Blood coagulation is a complex process involving many activating and inactivating coagulation factors. Anticoagulant proteins are known to be important for regulation of the coagulation process. See B. Lammle and J. Griffin (Clinics in Haemotolog 14, p. 281-342, 1985) for a review on coagulation inhibitors and regulation of coagulation.
Thus heparin is used clinically to increase the activity of antithrombin III and heparin cofactor II. Antithrombin III is used for the inhibition of factor Xa and thrombin. Hirudin is used for the inhibition of thrombin Protein C may be used for the inhibition of factors V and VIII.
Coagulation can be initiated through the extrinsic pathway by the release of tissue factor (J.H. Morrissey et al.: Thromb Res 50, p. 481-93, 1988). Coagulation activation by the extrinsic pathway may be inhibited by different mechanisms (P.M. Sandset et al.: Thromb Res 47, p. 389-400, 1987; B.J. Warn-Cramer et al.: Thromb Res 4s, p. 11-22, 1987; S. Kondo and W. Kisiel: Blood 70, p. 1947-54, 1987; S.D. Carson: J. Biol Chem 262, p. 718-21, 1987; G.J. Broze et al.: Blood 77, p. 335-43, 1988; S. Kondo et al.: Thromb Res 4s, p. 449-59, 1987).
The basic trigger in many coagulation disorders is the release of tissue factor and thus activation of factor X by factor VII-tissue factor. During surgery, tissue factor is released and thrombi may be formed In heart attack a primary thrombus is formed and when this thrombus is released, tissue factor is exposed and coagulation is initiated resulting in a secondary, perhaps lethal thrombus. During sepsis, bacterial endotoxin induces the systemic release of tissue factor. This may lead to disseminated intravascular coagulation (DIC). DIC can be treated with antithrombin III (T.E. Emerson et al.: Circulatory Shock 21, p. 1-13, 1987) Which inhibits the late steps in the coagulation cascade. Activated Protein C which inhibits in the middle of the coagulation cascade can also be used for the treatment of DIC (F.B. Taylor et al.: J Clin Invest 79, p. 918-25, 1987).
Protein showing extrinsic coagulation Pathway Inhibitor (EPI) activity has been recovered and isolated from human cells. It is known that EPI inhibits factor VII-tissue factor catalyzed activation of FX. However, the exact mechanism by which EPI inhibits coagulation is not known. Human plasma contains 3 molecular species showing EPI activity. The molecular masses are>500 kDa, 200 kDa and 40 kDa respectively (P.M. Sandset et al.: Thromb Res 47, p. 389-400, 1987).
The object of the present invention is an improved method to isolate the protein EPI in concentrated or pure form.
DETAILED DESCRIPTION OF THE INVENTION
Definitions: EPI is Extrinsic coagulation Pathway Inhibitor. EPI is a protein which shows activity in the assay described by Sandset et al. (Throm Res 47, p. 389-400, 1987). One unit of EPI is the amount of EPI activity found i 1 ml of normal human plasma.
The EPI protein may be recovered from supernatants of cell lines using precipitation and affinity chromatography on factor Xa. However, this purification procedure cannot be used in large scale owing to the very limited availability of factor Xa (G.J. Broze and J.P. Miletich, Proc Natl Acad Sci
USA 84, p. 1886-1890, 1987).
It has now been found that a very efficient purification is obtained when the solution containing the EPI protein is applied to a matrix coupled with heparin.
The invention is based on the discovery that the EPI molecule contains a specific site having affinity to heparin. A column containing a matrix coupled with heparin will selectively bind the EPI protein.
A preferred matrix is heparin-Sepharose, from which the EPI protein may be eluted.
Thus it is possible to apply 100 vol. culture medium on 1 vol. heparin-Sepharose and 80% of the EPI activity will be found in the eluate.
On the other hand if an- or cation exchange is used the result will be poorer. 25 vol. of protein free culture medium applied on 1 vo
REFERENCES:
patent: 4721572 (1988-01-01), Jordan
Shing et al. Heparin Affinity: Purification of a Tumor-Derived Capillary Endothelical Cell Growth Factor, Science vol. 223, pp. 1296-1298, 1984.
Broze and Miletich, Isolation of Tissue Factor Inhibitor Produced by HepG.sub.2 hepatoma cells. PNAS vol. 84, pp. 1886-1890, 1987.
Cramer et al., Thrombosis Research, vol. 48, pp. 11-22 (1987).
Sandset et al., Thrombosis Research, vol. 47, pp. 389-400 (1987).
Carlsen Soren
Nordfang Ole
Novo Nordisk A S
Russel Jeffrey E.
Touzeau P. Lynn
Zelson Steve T.
LandOfFree
Method of recovering purified EPI protein from a solution especi does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method of recovering purified EPI protein from a solution especi, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method of recovering purified EPI protein from a solution especi will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1044048