Phospholipid binding of factor VIII in different therapeutic concentrates

Abstract

Binding to anionic phospholipid (PL) is essential for the biological function of factor VIII (FVIII). We have developed a method to study the level of PL binding of FVIII in a variety of therapeutic concentrates, using the BIACORE^TM system which utilizes the Surface Plasmon Resonance (SPR) phenomenon. A HPA sensor chip was employed on to which synthetic phospholipid unilamellar vesicles were adsorbed to form a 3:1 phosphatidylcholine: phosphatidylserine lipid monolayer. Using this surface the interaction of unlabelled FVIII in concentrates was observed from which direct kinetic data (k_on, k_off and K_D values) were obtained in real‐time. Marked differences in the binding to PL, as measured by K_D values, between different products were observed. These fell into three categories: two recombinant FVIII products showed high affinities for PL with K_D values around 0.05–0.14 nm; four high‐purity plasma derived products, two prepared by monoclonal antibody and two prepared by ion‐exchange chromatography, had 6–8‐fold lower affinities, and two intermediate‐purity products had 34–60‐fold lower affinities with K_D values in the nm region. Measurements of k_on and k_off values for each product showed that the differences in the K_D values expressed were primarily due to the differences in their respective k_on values, although the recombinant products showed changes in the k_off values. The study showed that the assessment of binding to PL by FVIII in concentrates was possible without prior purification and gave K_D values in the range reported previously for other methods. The difference between the products requires further investigation but may be partly due to other proteins present, in particular the content and quality of von Willebrand factor which is known to affect PL binding of FVIII.