Kinetic and hydrodynamic analysis of blood clotting factor V-membrane binding.
The kinetics and hydrodynamic properties of factor V-membrane interaction were characterized. Factor V bound to membranes containing acidic phospholipids with a high collisional efficiency. For membranes of 20% phosphatidyl-serine-80% phosphatidylcholine, an association rate constant of (1.13 +/- 0.10) X 10(8) M-1 s-1 was obtained. These membranes contained about 20 factor V binding sites per vesicle of 3.6 X 10(6) 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 degrees C. The equilibrium dissociation constant for binding to these membranes at 10 degrees C and 0.14 M ionic strength was 5 X 10(-11) M. Ionic strength, pH, calcium, 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. In contrast, 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. In contrast, factor Va increased the hydrodynamic vesicle radius by only about 5 nm. The mass of membrane-bound protein was comparable for both proteins.
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D004735:Energy Transfer; D005165:Factor V / Q000378:metabolism*; D015943:Factor Va; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D009994:Osmolar Concentration; D013050:Spectrometry, Fluorescence
D008051:Lipid Bilayers; D015943:Factor Va; D005165:Factor V; D002118:Calcium