Factor VIIIa cofactor activity shows enhanced ionic strength sensitivity in the absence of phospholipid

摘要

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 absence of PL, promoting a reduced k_(cat) for the cofactor-dependent generation of factor Xa.