Mn2+ binding to factor VIII subunits and its effect on cofactor activity.
Metal ions, such as Ca2+ and Mn2+, are necessary for the generation of cofactor activity following reconstitution of factor VIII from its isolated light chain (LC) and heavy chain (HC). Titration of EDTA-treated factor VIII with Mn2+ showed saturable binding with high affinity (K(d) = 5.7 +/- 2.1 microM) as detected using a factor Xa generation assay. No significant competition between Ca2+ and Mn2+ for factor VIII binding (K(i) = 4.6 mM) was observed as measured by equilibrium dialysis using 20 microM Ca2+ and 8 microM factor VIII in the presence of 0-1 mM Mn2+. The intersubunit affinity measured by fluorescence energy transfer of an acrylodan-labeled LC (fluorescence donor) and fluorescein-labeled HC (fluorescence acceptor) in the presence of 20 mM Mn2+ (K(d) = 53.0 +/- 17.1 nM) was not significantly different from the affinity value previously obtained in the absence of metal ion (K(d) = 53.8 +/- 14.2 nM). The sensitization of phosphorescence of Tb3+ bound to factor VIII subunits was utilized to detect Mn2+ binding to the subunits. Mn2+ inhibited the phosphorescence of Tb3+ bound to HC and LC, as well as the HC-derived A1 and A2 subunits with a relatively wide range of estimated inhibition constant values (K(i) values = 169-1147 microM), whereas Ca2+ showed no effect on Tb3+ phosphorescence. These results suggest that factor VIII cofactor activity can be generated by Mn2+ binding to site(s) on factor VIII that are different from the high-affinity Ca2+ binding site. However, like Ca2+, Mn2+ did not alter the affinity for HC and LC association. Thus, Mn2+appears to generate factor VIII cofactor activity by a similar mechanism as observed for Ca2+following its association at nonidentical sites on the protein.
Wakabayashi
Hironao
H
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, New York, 14642, USA.
Zhen
Zhu
Z
Schmidt
Kyla M
KM
Fay
Philip J
PJ
D001665:Binding Sites; D001667:Binding, Competitive; D001780:Blood Coagulation Tests; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent; D019281:Dimerization; D015949:Factor IXa / Q000378:metabolism; D005169:Factor VIII / Q000737:chemistry / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008163:Luminescent Measurements; D008345:Manganese / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D013725:Terbium / Q000037:antagonists & inhibitors / Q000378:metabolism
D002413:Cations, Divalent; D021122:Protein Subunits; D013725:Terbium; D008345:Manganese; D005169:Factor VIII; D005170:Factor X; D015949:Factor IXa; D015951:Factor Xa; D002118:Calcium