Protein C activator, methods of preparation and use thereof
AssigneePentapharm AG
InventorsKurt F. Stocker, Lars G. Svendsen
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Abstract
A method and composition for assaying protein C is described. The method comprises reacting a protein C-containing medium with a protein C-activating activator preparation obtained from venom of the snake Agkistrodon contortrix, or venom of another snake species which undergoes an immunological cross-reaction with the venom of Agkistrodon contortrix, to cause maximum activation of protein C and subsequently determining the quantity of activated protein C, said quantity being proportional to the amount of protein C in said medium. Also disclosed is a method and composition for treating thrombotic disorders with the activator preparation and a method of obtaining the activator preparation by culturing of a cloned microorganism.
Claims18
An activator preparation which is capable of converting the zymogen protein C of men and mammals into a proteinase with protein C.sub.a -activity and which is obtainable from the venom of the snake Agkistrodon contortrix or from the venom of snake species which undergoes an immunological cross-reaction with the venom of Agkistrodon contortrix which comprises:
- (a) chromatography on an anion exchanger having the appropriate porosity for binding proteins,
- (b) elution with sodium phosphate buffer at neutral pH and increasing ionic strength,
- (c) removal of electrolytes from the active fractions by ultrafiltration, and
- (d) subsequent lyophilization.
An activator preparation which can be obtained in highly purified form which comprises:
- (a) dilution of the venom of the snake Agkistrodon contortrix or from the venom of snake species which undergoes an immunological cross-reaction with the venom of Agkistrodon contortrix in an aqueous medium,
- (b) removal of the undesired venom components from the solution either by fractionated alcohol precipitation, fractionated salt precipitation or heat treatment at an acid pH for the purposes of preparing a pre-purified venom fraction,
- (c) further purification of the obtained prepurified venom fraction by chromatography on an anion exchanger having the appropriate porosity for binding proteins,
- (d) elution with sodium phosphate buffer at neutral pH and increasing ionic strength,
- (e) further chromatography on a cation exchanger,
- (f) elution with a sodium acetate buffer at an acid pH,
- (g) concentration of the protein C-activating eluates by ultrafiltration,
- (h) removal of electroloytes and final purification of the concentrate by chromatography on a molecular sieve gel using diluted aqueous acetic acid as the eluent, and
- (i) subsequent lyophilization.
An activator preparation which is capable of converting the zymogen protein C of men and mammals into a proteinase with protein C.sub.a -activity comprising:
- (a) culturing a cloned microorganism containing at least one gene bearing the program for the biosynthesis of protein C-activator from the venom of the snake Agkistrodon contortrix or from the venom of snake species which undergoes an immunological cross-reaction with the venom of Agkistrodon contortrix;
- (B) isolating said protein C-activator from said culture.
Dependencies are inferred from the claim text.