Characterization of the Clotting Activities of Structurally Different Forms of Activated Factor

Two structurally different forms of activated human Factor IX (Factor IXaa and IXa8) have been previously reported to have essentially identical clotting activity in vitro. Although it has been shown that activated Factor IXChapel Hills an abnormal Factor IX isolated from the plasma of a patient with mild hemophilia B, and normal Factor IXaa are structurally very similar, the clotting activity of activated Factor IXC,,,pe Hill iS much lower (approximately fivefold) than that of normal Factor IXaj. In the present study we have prepared activated Factor IX by incubating human Factor IX with calcium and Russell's viper venom covalently bound to agarose. Fractionation of the activated Factor IX by high-performance liquid chromatography demonstrated the presence of both Factors IXaa and IXMaf. On the basis of active site concentration, determined by titration with antithrombin III, the clotting activities of activated Factor IXO-ap.1 Hill and IXaa were similar, but both activities were <20% of the clotting activity of Factor IXaf. Activated Factor IX activity was also measured in the absence of calcium, phospholipid, and Factor VIII, by determination of the rate of Factor X activation in the presence of polylysine. In the presence of polylysine, the rates of Factor X activation by activated Factor IXChpe Hills Factor IXaa, and Factor IXa$ were essentially identical. We conclude that the clotting activity of activated Factor IXCh-.p. Hill is reduced when compared with that of Factor IXaft but essentially normal when compared with that of Factor IXaa.