title: Ca(2+) binding to both the heavy and light chains of factor VIII is required for cofactor activity.
abstract: Previously, we demonstrated that Ca(2+) 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 Ca(2+) did not affect HC-LC binding affinity (Wakabayashi et al. (2001) Biochemistry 40, 10293-10300). Titration of EDTA-treated factor VIII with Ca(2+) followed by factor Xa generation assay showed a two-site binding pattern, with indicated high-affinity (K(d) = 8.9 +/- 1.8 microM) and low-affinity (K(d) = 4.0 +/- 0.6 mM) sites. Analysis by equilibrium dialysis using (45)Ca and <400 microM free Ca(2+) verified a high-affinity binding (K(d) = 18.9 +/- 3.7 microM). Preincubation of either HC or LC with 6 mM Ca(2+) followed by reassociation with the untreated complementary chain in the presence of 0.12 mM Ca(2+) failed to generate significant cofactor activity (<0.5 nM min(-1) (nM LC)(-1)). However, pretreatment of both HC and LC with 6 mM Ca(2+) followed by reassociation (at 0.12 mM Ca(2+)) generated high activity (7.5 +/- 0.4 nM min(-1) (nM LC)(-1)). Progress curves for activity regain following factor VIII-Ca(2+) association kinetics fitted well to a series reaction scheme rather than one of simple association (p < 0.0001), suggesting a multistep process which may include a Ca(2+)-dependent conformational change. These results suggest that factor VIII contains two Ca(2+) binding sites with different affinities and that active factor VIII can be reconstituted from HC and LC only when both chains are preactivated by Ca(2+).
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, New York 14642, USA.
authors.forename: Hironao
authors.initials: H
authors.lastname: Wakabayashi
authors.forename: Kyla M
authors.initials: KM
authors.lastname: Schmidt
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism; D066246:ErbB Receptors / Q000737:chemistry; D005169:Factor VIII / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D003067:Coenzymes; D010743:Phospholipids; D021122:Protein Subunits; D011994:Recombinant Proteins; D005169:Factor VIII; D066246:ErbB Receptors; D002118:Calcium