Kinetic and hydrodynamic analysis of blood clotting factor V-membrane binding

Publisher Website
Google Scholar
Abstract
The kinetics and hydrodynamic properties of [human] factor V-membrane interaction were characterized. Factor V bound to membranes containing acidic phospholipids with a high collisional efficiency. For membranes of 20% phosphatidylserine-80% phosphatidylcholine, an association rate constant of (1.13 .+-. 0.10) .times. 108 M-1 s-1 was obtained. These membranes contained .apprx. 20 factor V binding sites/vesicle of 3.6 .times. 106 daltons. This association rate represented about a 30% collisional efficiency. Dissociation of factor V was measured by a fluorescence energy transfer method with a dissociation rate constant of 0.0055 s-1 at 10.degree. C. The equilibrium Kd for binding to these membranes at 10.degree. C and 0.14 M ionic strength was 5 .times. 10-11 M. Ionic strength, pH, Ca and charge density in the membrane had large effects on the rate of factor V-membrane dissociation, indicating a strongly ionic interaction between protein and membrane. The association rate was nearly insensitive to ionic strength. The membrane-binding properties were relatively unchanged after thrombin digestion of factor V or after long-term protein storage which resulted in loss of procoagulant activity. Other proteins of the prothrombinase reaction greatly decreased the rate of factor Va-membrane dissociation. At protein saturation, factor V increased the hydrodynamic radius of phospholipid vesicles by 11.4 nm. Factor Va increased the hydrodynamic vesicle redius by only .apprx. 5 nm. The mass of membrane-bound protein was comparable for both proteins.