Structural basis for the decreased procoagulant activity of human factor VIII compared to the porcine homolog

• Published in: The Journal of biological chemistry Vol. 266; no. 19; pp. 12481 - 12486
• Main Authors: LOLLAR, P, PARKER, E. T
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05.07.1991
• Subjects: Animals; Biological and medical sciences; Blood Coagulation; Blood coagulation. Blood cells; Chromatography, Affinity; Chromatography, Ion Exchange; Coagulation factors; Electrophoresis, Polyacrylamide Gel; Factor VIII - isolation & purification; Factor VIII - pharmacology; Fundamental and applied biological sciences. Psychology; Hemophilia A - blood; Humans; Hydrogen-Ion Concentration; Molecular and cellular biology; Recombinant Proteins - isolation & purification; Recombinant Proteins - pharmacology; Swine; Thrombin - metabolism; Human; Blood coagulation; Enzyme; Factor VIII; Primary structure; Thrombin; Activation; Pig; Vertebrata; Mammalia; Structure activity relation; Quaternary structure; Artiodactyla; Coagulation factor; Ungulata; Comparative study
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)98924-6

The stability of activated human and porcine factor VIII (fVIII) differ, but a direct comparison of their structural and functional properties has not been made. Highly purified, heterodimeric human recombinant and porcine plasma-derived fVIII were exchanged into a common buffer and some minor contaminants were removed by anion-exchange chromatography. The activations of human and porcine fVIII by thrombin were studied by a two-stage coagulation assay using human citrated plasma as the standard. The peak activation of porcine fVIII was 10-fold greater than human fVIII (1.1 x 10(6) unit/mg versus 1.1 x 10(5) unit/mg). The proteolytic fragmentation of fVIII by thrombin was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was not different between human and porcine fVIII, yielding previously identified bands corresponding to fragments A1, A2, A3-C1-C2, and the B domain. Following activation by thrombin, human fVIII was subjected to cation-exchange (Mono S) high performance liquid chromatography at pH 6.0 under conditions that yields stable, heterotrimeric (A1/A2/A3-C1-C2) porcine fVIIIaIIa (Lollar, P., and Parker, C.G. (1990) Biochemistry 28, 666-674). Coagulant activity was recovered in a single peak that was less than 0.5% that of porcine fVIIIaIIa (1.2 x 10(4) unit/mg versus 2.6 x 10(6) unit/mg). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the peak fraction revealed bands corresponding to the A3-C1-C2 and A1 fragments but only trace levels of the A2 fragment. In contrast, activation of human fVIII by thrombin followed by Mono S HPLC at pH 5.0 produced a peak with 10-fold greater activity (1.2 x 10(5) unit/mg) than at pH 6.0 and which contained significant amounts of the A2 fragment. We conclude that human fVIIIIIa, like porcine fVIIIIIa, is a heterotrimer and propose that its apparent decreased coagulant activity is due to weaker association of the A2 subunit.