Purification and Characterization of a Highly Purified Human Factor VIII Consisting of a Single Type of Polypeptide Chain

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 79; no. 23; pp. 7200 - 7204
• Main Authors: Fay, Philip J., Chavin, Stephen I., Schroeder, Duane, Young, Frank E., Marder, Victor J.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 01.12.1982; National Acad Sciences
• Subjects: Antibodies; Antigens; Biochemistry; Biological Assay; Blood plasma; Calcium; Chromatography; Electrophoresis; Elution; Factor VIII - isolation & purification; Gels; Humans; Molecular Weight; Sodium; Ungulates
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.79.23.7200

Human factor VIII was purified 350,000-fold (relative to plasma) from a commercial factor VIII concentrate. The procedure used standard protein separation techniques and was performed in the absence of protease inhibitors. The product has a specific activity of 4,900 units/mg, an activity-to-antigen ratio of 75:1 (unit/unit) and no more than 0.1% von Willebrand protein. Electrophoresis of the reduced protein in a denaturing polyacrylamide gel showed a single major band of Mr100,000. Procoagulant activity was eluted from a nondenaturing gel after electrophoresis in the region of the single major band. Thrombin converted the Mr100,000 polypeptide to a polypeptide of Mr75,000. The procoagulant activity was increased 10-fold by thrombin or factor Xaand was completely inhibited by activated protein C or factor VIII inhibitor plasma. This factor VIII preparation consists of a single high molecular weight polypeptide chain and has the highest specific activity thus far reported for human factor VIII.