Ca2+ Binding to Both the Heavy and Light Chains of Factor VIII Is

Biochemistry 2002, 41, 8485-8492

8485

Ca2+ Binding to Both the Heavy and Light Chains of Factor VIII Is Required for Cofactor Activity† Hironao Wakabayashi, Kyla M. Schmidt, and Philip J. Fay* Department of Biochemistry and Biophysics, UniVersity of Rochester School of Medicine, 601 Elmwood AVenue, Rochester, New York 14642 ReceiVed January 24, 2002; ReVised Manuscript ReceiVed April 3, 2002

Previously, we demonstrated that Ca2+ was necessary for the generation of cofactor activity following reconstitution of factor VIII from its isolated light chain (LC) and heavy chain (HC) but that Ca2+ did not affect HC-LC binding affinity (Wakabayashi et al. (2001) Biochemistry 40, 10293-10300). Titration of EDTA-treated factor VIII with Ca2+ followed by factor Xa generation assay showed a twosite binding pattern, with indicated high-affinity (Kd ) 8.9 ( 1.8 µM) and low-affinity (Kd ) 4.0 ( 0.6 mM) sites. Analysis by equilibrium dialysis using 45Ca and