Phospholipid binding of factor VIII in different therapeutic concentrates

Abstract

Summary. 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 af®nities 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 af®nities, and two intermediate-purity products had 34±60-fold lower af®nities 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 puri®cation 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.

Influence of the type of factor VIII concentrate on the incidence of factor VIII inhibitors in previously untreated patients with severe hemophilia A

“…43 Difference in PL binding to FVIII has been demonstrated between different types of FVIII concentrates; the highest PL affinity was associated with recombinant products and the lowest with intermediate purity plasma-derived concentrates. 44 The increase in PL affinity has been correlated with a higher frequency of inhibitor development. 45 Moreover, some recombinant FVIII molecules are unable to bind VWF, 46 possibly enhancing immunogenicity relative to plasma FVIII.…”

A solvent/detergent‐treated and 15‐nm filtered factor VIII: a new safety standard for plasma‐derived coagulation factor concentrates

“…Phospholipid (PL) binding of FVIII was assessed using the surface plasmon resonance (SPR) phenomenon as described elsewhere [24,27]. All SPR measurements were carried out on a BIAcore‐X‘ analytical system (BIAcore AB, Uppsala Sweden) with hydrophobic sensor chips (HPA, BIAcore AB) coated with synthetic phospholipid unilamellar vesicles of either phosphatidylcholine (PC) or a 3 : 1 ratio of PC : PS (PS, phosphatidylserine).…”

Alloantibodies to therapeutic factor VIII in hemophilia A: the role of von Willebrand factor in regulating factor VIII immunogenicity

“…54,[65][66][67][68] The exposed VWF-binding site on FVIII, incidentally, may be occupied by anionic phospholipids which were suggested to have additional implications for the immune response. 69,70 VWF inhibits uptake of FVIII in antigen presenting cells Qadura and colleagues were not able to confirm a protective effect of VWF in hemophilic mice, but found variant patterns of inflammatory mediators and of immune gene expression profiles in activated (CD11c + ) splenic dendritic cells (DCs) between pdFVIII-and rFVIII-treated hemophilic mice (type 1 vs. type 2 inflammatory response). 44 As the pdFVIII-treated mice in these experiments also developed a strong immune response to human VWF, these findings may have little relevance for the situation in humans.…”