Claims for Patent: 5,066,787

Summary for Patent: 5,066,787

Title:	Blood coagulation inhibiting proteins, processes for preparing them and their uses
Abstract:	This invention discloses proteins which inhibit the coagulation of the blood, processes for preparing these proteins, and the use thereof.
Inventor(s):	Reutelingsperger; Christian P. M. (Maastricht, NL)
Assignee:	Boehringer Ingelheim International GmbH (Ingelheim am Rhein, DE)
Application Number:	07/123,970
Patent Claims:	1. A substantially purified anti-coagulant protein, having an anti-coagulation activity, belonging to the group of vascular anti-coagulants which do not inactivate the coagulation factors, wherein said anti-coagulant protein inhibits: (1) a modified prothrombin-time experiment; (2) a modified, activated, partial thromboplastin-time experiment; (3) a non-modified prothrombin-time experiment; (4) prothrombin activation by coagulation factor X.sub.a in the presence of negatively charged phospholipids and Ca.sup.2+ ; (5) intrinsic X-activation by factor IX.sub.a in the presence of negatively charged phospholipids and Ca.sup.2+ ; (6) prothrombin activation of isolated, stimulated blood platelets; (7) coagulation induced by blood vessel walls; and (8) coagulation-dependent aggregation of platelets; and wherein said anti-coagulant protein does not inhibit the biological and amidolytic activity of factors X.sub.a and II.sub.a, per se; and wherein said anti-coagulant protein inhibits coagulation induced by a vascular coagulant or by factor X.sub.a, but does not inhibit coagulation of thrombin. 2. The anti-coagulant protein of claim 1, characterized in that: (a) inhibition of coagulation by said anti-coagulant protein is dependent on phospholipid concentration, (b) said anti-coagulant protein does not hydrolyze phospholipids, and (c) inhibition of prothrombin activation, induced by said anti-coagulant protein by factor X.sub.a is dependent on phospholipid concentration. 3. The anti-coagulant protein of claim 1, characterized in that: (a) said anti-coagulant protein binds to negatively charged phospholipids in vesicles, liposomes or etherosomes, (b) said anti-coagulant protein binds to negatively charged phospholipids which are coupled with Spherocil, (c) said binding of said anti-coagulant protein to the negatively charged phospholipids is reversible, and (d) said anti-coagulant proteins can displace factor X.sub.a and prothrombin from a negatively charged phospholipid surface. 4. The anti-coagulant protein of claim 1, wherein said anti-coagulant protein has a molecular weight of approximately 70.times.10.sup.3 daltons. 5. The anti-coagulant protein of claim 1, wherein said anti-coagulant protein has a molecular weight of approximately 60.times.10.sup.3 daltons. 6. The anti-coagulant protein of claim 1, wherein said anti-coagulant protein has a molecular weight of approximately 34.times.10.sup.3 daltons. 7. The anti-coagulant protein of claim 1, wherein said anti-coagulant protein has a molecular weight of approximately 32.times.10.sup.3 daltons. 8. The anti-coagulant protein of claim 1, wherein said anti-coagulant protein is isolated from arteries. 9. The anti-coagulant protein of claim 1, wherein said anti-coagulant protein is isolated from highly vascularized tissue. 10. The anti-coagulant protein of claim 1, wherein said anti-coagulant protein is isolated from a tissue selected from the group consisting of human, bovine, murine, and equine tissue. 11. An anti-coagulant protein, obtainable by a process comprises: (a) homogenizing tissue, differentially centrifuging said homogenized tissue, and subjecting the supernatant liquid to one or more of the following purification treatments in any desired sequence: (b) precipitation with salt, (c) affinity chromatography, (d) ion exchange chromatography, (e) chromatography using a molecular sieve; wherein said anti-coagulant protein belongs to the group of vascular anti-coagulants which do not inactivate the coagulation factors. 12. The protein of claim 11, wherein said process additionally includes the purification of said anti-coagulant protein using immunoabsorption chromatography. 13. The protein of claim 11 wherein said process additionally includes the purification of said anti-coagulant protein using phospholipid vesicles. 14. The protein of claim 11 wherein in said process ammonium sulfate is used for the precipitatin in step (b), hydroxyapatite is used for chromatography in step (c), DEAE-Sephacel is used for chromatography in step (d), and Sephadex G-100 or G-75 is used for chromatography in step (e). 15. A substantially purified anti-coagulant protein, having an anti-coagulation activity, belonging to the group of vascular anti-coagulants which do not inactivate the coagulation factors, characterized in that: (1) said anti-coagulant protein is not a glycoprotein, (2) said anti-coagulant protein is not a phospholipase, (3) said anti-coagulant protein has an isoelectric point at pH 4.4-4.6, (4) the activity of said anti-coagulant protein at 56.degree. C. is thermally unstable, (5) the activity of said anti-coagulant protein in citrated plasma remains stable for hours at 37.degree. C., (6) the activity of said anti-coagulant protein is not completely destroyed by trypsin or chymotrypsin, (7) the activity of said anti-coagulant protein is not affected by collagenase or elastase, (8) said anti-coagulant protein binds to negatively charged phospholipids which can be found in vesicles, liposomes, or etherosomes, (9) said anti-coagulant protein binds to negatively charged phospholipids which are coupled to Spherocil, (10) the binding of said anti-coagulant protein to the negatively charged phospholipids is reversible and can be reversed by ethylenediamine tetraacetic acid (EDTA), (11) said anti-coagulant protein displaces factor X.sub.a and the prothrombin from a negatively charged phospholipid surface, (12) said anti-coagulant protein inhibits a modified prothrombin-time experiment, (13) said anti-coagulant protein inhibits a modified, activated, partial thromboplastin-time experiment, (14) said anti-coagulant protein inhibits a non-modified prothrombin-time experiment, (15) said anti-coagulant protein inhibits prothrombin activation by the coagulation factor X.sub.a in the presence of negatively charged phospholipids and Ca.sup.2+ in vitro, (16) said anti-coagulant protein does not inhibit biological and amidolytic activity of factors X.sub.a and II.sub.a, per se, (17) said anti-coagulant protein inhibits intrinsic X-activation by factor IX.sub.a in the presence of negatively charged phospholipids and Ca.sup.2+ in vitro, (18) said anti-coagulant protein inhibits prothrombin activation of isolated, stimulated blood platelets in vitro, (19) said anti-coagulant protein inhibits coagulation induced by walls of blood vessels in vivo, and (20) inhibition of prothrombin activation by factor X.sub.a induced by said anti-coagulant protein is dependent on phospholipid concentration and is reduced at high phospholipid concentrations. 16. A method of suppressing blood-clotting in a human which comprises administering to said human a therapeutically effective amount of the anti-coagulation protein of any one of claims 1, 2-9, 10 or 15 with a pharmaceutically inert carrier. 17. The method of claim 16 wherein said blood-clotting is associated with thrombosis formation. 18. A pharmaceutical composition which comprises anti-thrombosis forming amounts of the anti-coagulant protein of any one of claims 1, 2-9, 10 or 15 together with a pharmaceutically inert carrier.

Details for Patent 5,066,787

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2008-11-19