Heterogeneity of Factor IX in Therapeutic Concentrates
In vivo and in vitro experience shows that factor IX concentrates are partially activated. A rabbit antibody to human factor IX was used to investigate the factor IX antigenic content and electrophoretic mobility of commercial products as well as experimental “activated products”.“Rocket” Immunoelectrophoresis of all concentrates showed a 1.5–3 fold increased antigenic content/unit factor IX clotting activity when compared to plasma. Two dimensional crossed Immunoelectrophoresis of standard factor IX preparation produced a single sharp peak whether electrophoresed in Ca or EDTA containing buffer. “Activated” preparations produced a dome shaped precipitin arc. The addition of plasma to factor IX concentrates yielded a marked shoulder only when the electrophoresis was done in EDTA. This effect could not be reproduced by the addition of antithrombin III (AT-III). The addition of plasma to the activated IX (IXa) revealed an even more pronounced heterogeneity whether in Ca or EDTA. The addition of AT-III produced a second precipitin peak when activated IX was electrophoresed in the presence of Ca++.These results indicate that at least three forms of factor IX exist in factor IX preparations. The absence of detectable AT-III reacting material in the regular IX preparations is a priori evidence of the absence of major amounts of IXa, whereas the presence of AT-III reacting material in the “activated” complex is evidence of biologically active material.