Von Willebrand factor (vWF) Impairs FVIII-containing Immune Complex Formation and Attenuates Murine FVIII-specific Recall Response In vitro and In vivo

Objectives: Anti-factor VIII (FVIII) antibodies (Ab) neutralize the hemostatic activity of FVIII and modulate anti-FVIII immune responses via the formation of FVIII-containing immune complexes (FVIII-IC). In the circulation, vWF protects FVIII from proteolytic cleavage by proteinases and competes with anti-FVIII Ab for FVIII binding sites. In this study, we investigated the impact of vWF to influence FVIII-IC formation and thereby affecting FVIII-specific secondary immune response in hemophilia A (HA) mouse model.

Methods: Electron microscopy (EM), analytic ultracentrifugation (AUC), and Fc gamma receptor IIb (CD32) binding assays were used to characterize FVIII-IC in the presence or absence of vWF. FVIII-specific recall response of memory B cells (MBC) was studied in HA mice ex vivo and in vivo. Finally, the influence of vWF on FVIII-IC formation was investigated in samples from congenital HA patients with and without inhibitors.

Results: EM and AUC demonstrated that incubation of FVIII with anti-FVIII sera resulted in the formation of FVIII-IC. These were heterogeneous in size exhibiting sedimentation constants of up to 60 S. Pre-incubation of FVIII with vWF reduced FVIII-IC formation as demonstrated by increased amounts of free Ab and reduced detection of IC of up to 14 S. Addition of vWF to FVIII before incubation with anti-FVIII sera strongly decreased IC binding to CD32. 0.1 IU/ml vWF suppressed IC binding to CD32 by 50%. Increasing concentrations of vWF further reduced CD32 interaction and FVIII-IC binding was nearly abolished at 3 IU/ml vWF. vWF also reduced CD32 binding of FVIII-IC if it was added after complex formation. Differentiation of FVIII-specific MBC was strongly reduced in the presence of vWF demonstrated by decreased numbers of FVIII-specific antibody secreting cells detected in vitro. FVIII recall response in mice after adoptive transfer of FVIII-primed splenocytes was also reduced. Samples from HA patients with inhibitors, but not from HA patients without inhibitors, formed FVIII-IC upon addition of FVIII in a dose-dependent manner as indicated by CD32 binding. FVIII-IC formation was reduced by vWF to a variable degree in samples from HA patients with inhibitors.

Conclusions: FVIII and anti-FVIII antibodies formed FVIII-IC in both mouse and human plasma. Physical and functional properties of FVIII-IC were influenced by vWF that is able to decrease complex formation and prevents binding to Fc gamma receptor. These effects were associated with a decreased murine anti-FVIII recall response ex vivo and in vivo.

No conflict of interest has been declared by the author(s).