Phospholipid binding of factor VIII in different therapeutic concentrates

Binding to anionic phospholipid (PL) is essential for the biological functi on 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 BI ACORE(TM) system which utilizes the Surface Plasmon Resonance (SPR) phenome non. A HPA sensor chip was employed on to which synthetic phospholipid unil amellar vesicles were adsorbed to form a 3:1 phosphatidylcholine: phosphati dylserine 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 th e 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 hi gh-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 ea ch product showed that the differences in the K-D values expressed were pri marily due to the differences in their respective k(on) values, although th e recombinant products showed changes in the k(off) values. The study showe d that the assessment of binding to PL by FVIII in concentrates was possibl e without prior purification and gave K-D values in the range reported prev iously for other methods. The difference between the products requires furt her investigation but may be partly due to other proteins present, in parti cular the content and quality of non Willebrand factor which is known to af fect PL binding of PVIII.