Metal ion-independent association of factor VIII subunits and the roles ofcalcium and copper ions for cofactor activity and inter-subunit affinity

Factor VIII circulates as a divalent metal ion-dependent heterodimer compri sed of alight chain (LC) and a heavy chain (HC). Reassociation of factor VI II subunits was assessed using fluorescence energy transfer where LC and HC were labeled with acrylodan (Ac; fluorescence donor) and fluorescein-5-mal eimide (Fl; fluorescence acceptor), respectively. The reduction of donor fl uorescence due to the acceptor was used as an indicator of binding. Subunit s associated with high affinity (K-d = 53.8 nM) in the absence of metal ion and presence of EDTA. However, this product showed no cofactor activity, a s measured by a factor Xa generation assay. In the presence of 25 mM Ca2+, no increase in the intersubunit affinity was observed (K-d = 48.7 nM) but s pecific activity of the cofactor was similar to 30% that of native factor V III. At saturating levels of Fl-HC relative to Ac-LC, donor fluorescence de creased to 79.3 and 73.5% of its original value in the absence and presence of Ca2+, respectively. Thrombin cleaved the heterodimers that were associa ted in the absence or presence of Ca2+ with similar efficiency, indicating that the lack of activity was not the result of a defect in activation. Cu2 + (0.5 muM) increased the intersubunit affinity by similar to 100 fold (K-d = 0.52 nM) and the specific activity to similar to 60% of native factor VI II. The former effect was independent of Ca2+, whereas the latter effect re quired Ca2+. These results indicate that the intersubunit association in fa ctor VIII is primarily metal-ion independent while divalent metal ions serv e specific roles. Ca2+ appears essential to promote the active conformation of factor VIII while Cu2+ primarily enhances the intersubunit affinity.