BINDING OF BLOOD-COAGULATION FACTOR-VIII AND ITS LIGHT-CHAIN TO PHOSPHATIDYLSERINE PHOSPHATIDYLCHOLINE BILAYERS AS MEASURED BY ELLIPSOMETRY/

Factor VIII is a plasma protein which plays an essential role in the c oagulation system. When assembled with the enzyme Factor IXa on a phos pholipid membrane, it functions as a cofactor in the enzyme complex th at cleaves the zymogen Factor X to Factor Xa. We studied the binding o f both Factor VIII and the Factor VIII light chain to planar phospholi pid bilayers consisting of 25% dioleoylphosphatidylserine and 75% diol eoylphosphatidylcholine (PSPC) by ellipsometry. Equilibrium-binding st udies revealed that both Factor VIII and its light chain bind with hig h affinity to PSPC bilayers. The binding affinity of Factor VIII, with a dissociation constant K-d of 0.24 nM, was comparable with that of t he Factor VIII light chain (K-d 0.49 nM). Maximal binding was 2.3 mmol of protein per mol of PSPC for Factor VIII and 7.1 mmol of protein pe r mol of PSPC for the Factor VIII light chain. Adsorption kinetics of both Factor VIII and its light chain conformed to the classical Langmu ir adsorption model, yielding dissociation constants calculated from t he rates of adsorption that were similar to those obtained by equilibr ium-binding studies. In contrast, measurements of rates of desorption revealed a deviation from those expected for a single class of binding sites. The desorption rate of Factor VIII increased with increasing r esidence time on the lipid membrane. This indicates transition of Fact or VIII to a configuration with a lower binding affinity. As this time -dependent change in affinity could affect the validity of the measure ment of binding parameters, in particular equilibrium-binding determin ations carried out on a long timescale, binding affinity was also esti mated from adsorption kinetics at half-maximal surface coverage, a rel atively rapid procedure for the determination of the affinity. A K-d o f 0.087 nM was obtained under these conditions. Measurement of equilib rium binding to small PSPC vesicles, a system in which equilibrium is rapidly attained, resulted in similar binding parameters (K-d = 0.13 n M and a maximal binding of 2.8 mmol of protein per mol of PSPC). These data confirm the results of equilibrium binding to planar bilayers. T aken together, our results indicate that Factor VIII, by means of its 80 kDa light chain, binds to PSPC bilayers with a dissociation constan t below the concentration of Factor VIII in plasma and therefore may r eadily bind to exposed phospholipid membranes under physiological cond itions.