Improved Procedures for the Purification of Selected Vitamin K-Dependent Proteins

Abstract

Improved methods are described to obtain bovine prothrombin, Factor IX, Protein C and autoprothrombin III (Factor X, Auto-III) in purified form. The prothrombin had a specific activity of 4340 Iowa units/mg. Theoretically, a preparation of clean thrombin should have a specific activity of 8200 U/mg, because 47.08% of the protein in prothrombin is lost when thrombin forms. Such thrombin preparations were obtained. The prothrombin concentration of bovine plasma is .apprx. 60 mg/l. Protein C [Factor XIV], the precursor of autoprothrombin II-A (Auto-II-A), was converted to Auto-II-A (Factor XIVa) by thrombin. Digesting purified Auto-III with purified thrombin removed a small glycopeptide from the COOH-terminal end of the heavy chain to yield Auto-IIIm. Auto-III .**GRAPHIC**. Auto-IIIm + peptide. Auto-IIIm was not converted to the active enzyme with thromboplastin and inhibited the activation of purified native Auto-III with thromboplastin. Auto-IIIm was not converted to the active enzyme when purified Factor VIII, purified Factor IXa, platelet factor 3 and Ca2+ were the procoagulants. The activation peptide released by RVV-X [Russel''s viper venom released active protein] from the NH2-terminal end of the heavy chain and the active enzyme (Auto-Cm) were purified. Auto-III was also activated with purified RVV-X. The same activation peptide was isolated, but Auto-C was obtained instead of Auto-Cm. Purified Factor IX developed anticoagulant activity when reacted with an optimum concentration of purified thrombin. A suitable reagent for the assay of Factor IX was prepared by removing prothrombin complex from anticoagulated bovine plasma and restoring the prothrombin and Auto-III concentration with use of the respective purified proenzymes.