Metal Ion-independent Association of Factor VIII Subunits and the Roles of Calcium and Copper Ions for Cofactor Activity and Inter-Subunit Affinity

• Published in: Biochemistry (Easton) Vol. 40; no. 34; pp. 10293 - 10300
• Main Authors: Wakabayashi, Hironao, Koszelak, Mary E, Mastri, Maria, Fay, Philip J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 28.08.2001
• Subjects: 2-Naphthylamine - analogs & derivatives; Calcium - chemistry; Calcium - metabolism; Cations, Divalent - chemistry; Cations, Divalent - metabolism; Coenzymes - chemistry; Coenzymes - metabolism; Copper - chemistry; Copper - metabolism; Dimerization; Electrophoresis, Polyacrylamide Gel; Energy Transfer; Factor VIII - chemistry; Factor VIII - metabolism; Factor Xa - metabolism; Fluoresceins; Fluorescent Dyes; Humans; Kinetics; Protein Subunits; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Spectrometry, Fluorescence; Thrombin - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi010353q

Factor VIII circulates as a divalent metal ion-dependent heterodimer comprised of a light chain (LC) and a heavy chain (HC). Reassociation of factor VIII subunits was assessed using fluorescence energy transfer where LC and HC were labeled with acrylodan (Ac; fluorescence donor) and fluorescein-5-maleimide (Fl; fluorescence acceptor), respectively. The reduction of donor fluorescence due to the acceptor was used as an indicator of binding. Subunits 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, as 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 specific activity of the cofactor was ∼30% that of native factor VIII. At saturating levels of Fl-HC relative to Ac-LC, donor fluorescence decreased to 79.3 and 73.5% of its original value in the absence and presence of Ca2+, respectively. Thrombin cleaved the heterodimers that were associated 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 μM) increased the intersubunit affinity by ∼100 fold (K d = 0.52 nM) and the specific activity to ∼60% of native factor VIII. The former effect was independent of Ca2+, whereas the latter effect required Ca2+. These results indicate that the intersubunit association in factor VIII is primarily metal-ion independent while divalent metal ions serve specific roles. Ca2+ appears essential to promote the active conformation of factor VIII while Cu2+ primarily enhances the intersubunit affinity.