Membrane binding kinetics of factor VIII indicate a complex binding process.

Factor VIII functions as a component of the tenase enzyme complex upon phospholipid membranes. Factor VIII binds to phosphatidylserine-containing membranes and apparently provides high affinity binding sites for factor IXa upon these membranes. We have characterized the binding kinetics of human factor VIII with phosphatidylserine-containing membranes and directly compared the measured properties with those of factor V. The initial phase of association was evaluated in a stopped-flow apparatus by fluorescence energy transfer from aromatic residues in the protein to dansyl-labeled phosphatidylethanolamine in the vesicles. Association proceeded at an apparent second-order rate of 0.12 microM-1 s-1 for extruded phospholipid vesicles and 0.42 microM-1 s-1 for sonicated vesicles under pseudo-first-order conditions in which the phospholipid concentration determined the rate. Increased temperature resulted in more rapid association, and the effect decreased in the order extruded vesicles > sonicated vesicles > extruded vesicles of dioleoylphospholipids, indicating that the structure of the phospholipid membrane contributes to the activation energy of binding. The binding of fluorescein-labeled factor VIII to membranes supported on glass microspheres (lipospheres) was monitored by flow cytometry. Under conditions in which the factor VIII concentration determined the rate there was rapid initial association at 6.9 microM-1 s-1, accounting for half of the bound factor VIII, and a slower component of 0.87 microM-1 s-1, accounting for the other half. Likewise, the dissociation of factor VIII from liposphere membranes was biphasic with a faster component of 0.010 s-1 and a slower component of 0.0012 s-1. Rates of association and dissociation for factor V were similar to those for factor VIII and were biphasic. These results allow estimation of the size of the phospholipid sites that interact with factors VIII and V and suggest that both proteins bind to membranes via a multistep process in which rapid association is followed by a slower step yielding higher affinity binding.