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

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