title: Factor VIIIa cofactor activity shows enhanced ionic strength sensitivity in the absence of phospholipid.
abstract: Factor VIIIa, a cofactor for the protease factor IXa, is a trimer of A1, A2 and A3-C1-C2 subunits. In the absence of phospholipid (PL), the k(cat) for factor VIIIa-dependent, factor IXa-catalyzed conversion of factor X was markedly less than that observed in the presence of PL (approx. 150 min(-1)) and decreased as the ionic strength of the reaction increased. At low salt concentration, the k(cat) (5.5 min(-1)) was approx. 8-fold greater than observed at near physiologic ionic strength (0.7 min(-1)). However, this level of salt showed minimal effects on the intermolecular affinities of factor VIIIa (or isolated A2 subunit) for factor IXa or on the K(m) for factor X. Alternatively, the association of A2 subunit with A1 subunit was sensitive to increases in salt and paralleled the reduction in k(cat) observed with factor VIIIa. This instability was not observed in PL-containing reactions. Fluorescence energy transfer between acrylodan-A2 and fluorescein-A1/A3-C1-C2 dimer showed a requirement for both PL and factor IXa for maximal association of A2 with dimer. These results indicate that in the presence of factor IXa, the salt-dependent dissociation of factor VIIIa subunits is significantly enhanced in the absence of PL, promoting a reduced k(cat) for the cofactor-dependent generation of factor Xa.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, NY 14642, USA. philip_fay@urmc.rochester.edu
authors.forename: P J
authors.initials: PJ
authors.lastname: Fay
authors.forename: M
authors.initials: M
authors.lastname: Mastri
authors.forename: M E
authors.initials: ME
authors.lastname: Koszelak
mesh_terms: D015949:Factor IXa / Q000737:chemistry; D015944:Factor VIIIa / Q000737:chemistry*; D005170:Factor X / Q000737:chemistry; D015951:Factor Xa / Q000737:chemistry; D005453:Fluorescence; D007700:Kinetics; D009994:Osmolar Concentration; D010743:Phospholipids / Q000032:analysis; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D010743:Phospholipids; D011994:Recombinant Proteins; D015944:Factor VIIIa; D005170:Factor X; D015949:Factor IXa; D015951:Factor Xa