61 - Oral Communication
Mechanisms of Disease I
Feb. 25, 2021, 1:45 p.m. - 3:15 p.m., Rome
von Willebrand factor (vWF) significantly modifies the sedimentation profile of FVIII-containing immune complexes (FVIII-IC) based on the type of anti-FVIII antibodies
O. Oleshko, U. Curth, A. Tiede, S. Werwitzke, Presenter: O. Oleshko (Hannover)
Background and Objective
Formation of anti-FVIII antibodies (inhibitors, Inh) is a severe complication of FVIII substitution therapy in haemophilia A. Inh can bind to FVIII thereby forming FVIII-IC with possibly harmful consequences like enhanced FVIII uptake by antigen-presenting cells in ongoing anti-FVIII immune reaction. There is a controversial debate about potential immuno-protective effects of vWF in regard to Inh development, but data about vWF impact on subsequent FVIII-IC formation are not available. This study is focused on the vWF influence on FVIII-IC characteristics using analytical ultracentrifugation (AUC).
Monoclonal IgG antibodies (mAb) GMA-8011 and GMA-8021 recognizing the C1 and A2 domain of FVIII, respectively, were used to generate FVIII-IC. 5 or 100 IU/ml of plasma-derived vWF were added to 100 IU/ml of recombinant human FVIII (rhFVIII) followed by incubation with 10 µg/ml Alexa Fluor 488 labeled mAb in order to investigate the impact of vWF on FVIII-IC formation. Samples were characterized using AUC with fluorescence detection (AU-FDS, Aviv Biomedical; ProteomeLab XL-I, Beckman Coulter) at 30,000 rpm and 20°C. Sedimentation velocity data were analyzed with a diffusion-deconvoluted differential sedimentation coefficient distribution model, c(s), in the program SEDFIT.
rhFVIII incubated with GMA-8011 formed complexes with sedimentation coefficients of 9.5 and 11.9 S (Fig. A, black trace). vWF substantially decreased FVIII-IC generation and increased the amount of free mAb at 5 and 100 IU/ml, consistent with the competitive binding of vWF and mAb to rhFVIII (Fig. A and D). In contrast, vWF did not interfere with FVIII-IC formation in the presence of GMA-8021 (Fig. B and D). Instead, faster sedimenting complexes (FSC) appeared, suggesting that FVIII-IC increased in size due to additional binding of vWF. GMA-8011 and GMA-8021 together, as a model of more complex immune reactions, resulted in FVIII-IC of heterogeneous size that were also substantially changed by the addition of vWF, as indicated by increased amounts of free mAb and FSC (Fig. C and D).
AUC allows studying FVIII-IC formation. vWF can interfere with FVIII-IC formation by preventing the interaction of some antibodies due to competitive binding or by increasing the total immune complex size. Variable effects can be expected in patients depending on anti-FVIII epitope distribution.