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. 1995 Sep 1;310(Pt 2):539–545. doi: 10.1042/bj3100539

Binding of blood coagulation factor VIII and its light chain to phosphatidylserine/phosphatidylcholine bilayers as measured by ellipsometry.

J Spaargaren 1, P L Giesen 1, M P Janssen 1, J Voorberg 1, G M Willems 1, J A van Mourik 1
PMCID: PMC1135928  PMID: 7654192

Abstract

Factor VIII is a plasma protein which plays an essential role in the coagulation system. When assembled with the enzyme Factor IXa on a phospholipid membrane, it functions as a cofactor in the enzyme complex that cleaves the zymogen Factor X to Factor Xa. We studied the binding of both Factor VIII and the Factor VIII light chain to planar phospholipid bilayers consisting of 25% dioleoylphosphatidylserine and 75% dioleoylphosphatidylcholine (PSPC) by ellipsometry. Equilibrium-binding studies revealed that both Factor VIII and its light chain bind with high affinity to PSPC bilayers. The binding affinity of Factor VIII, with a dissociation constant Kd of 0.24 nM, was comparable with that of the Factor VIII light chain (Kd 0.49 nM). Maximal binding was 2.3 mmol of protein per mol of PSPC for Factor VIII and 7.1 mmol of protein per mol of PSPC for the Factor VIII light chain. Adsorption kinetics of both Factor VIII and its light chain conformed to the classical Langmuir adsorption model yielding dissociation constants calculated from the rates of adsorption that were similar to those obtained by equilibrium-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 residence time on the lipid membrane. This indicates transition of Factor VIII to a configuration with a lower binding affinity. As this time-dependent change in affinity could affect the validity of the measurement of binding parameters, in particular equilibrium-binding determinations carried out on a long timescale, binding affinity was also estimated from adsorption kinetics at half-maximal surface coverage, a relatively rapid procedure for the determination of the affinity. A Kd of 0.087 nM was obtained under these conditions. Measurement of equilibrium binding to small PSPC vesicles, a system in which equilibrium is rapidly attained, resulted in similar binding parameters (Kd = 0.13 nM 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. Taken together, our results indicate that Factor VIII, by means of its 80 kDa light chain, binds to PSPC bilayers with a dissociation constant below the concentration of Factor VIII in plasma and therefore may readily bind to exposed phospholipid membranes under physiological conditions.

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Selected References

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