PB1051 - Tyrosine Under-Sulfation of Factor VIII Results in T-Cell Response and Decreased Affinity Towards Thrombin in a Model System

Background: Deficiency in coagulation factor VIII (FVIII) results in life-threatening bleeding (Hemophilia A) and is treated with infusions of therapeutic FVIII. Recombinant FVIII (rFVIII) products were shown to be more immunogenic and have a wider range of specific activities compared to plasma derived FVIII products. These correlate with structural differences in rFVIII, particularly, under-sulfation of specific tyrosines (Chun et al, 2021). Considering importance of tyrosine sulfation for FVIII activity (Michnick et al, 1994), we investigated whether under-sulfation of tyrosine residues affects FVIII function.

Aims: To elucidate potential effects of tyrosine under-sulfation in FVIII on its immunogenicity and interaction with thrombin.

Methods: Twenty-four 22-mer peptides representing the sulfated FVIII tyrosine residues containing both sulfated and the respective non-sulfated tyrosine residues were generated. Following ex vivo stimulation of human peripheral blood mononuclear cells (from 5 donors) with peptides, intracellular staining of CD4+ T-cells was used to measure interferon-γ (IFN-γ), a cytokine associated with T-cell activation. Concomitantly, a set of peptides (33-mers), corresponding to the A2/B domains junction of FVIII and having progressive decrease in the number of sulfated tyrosine residues, was tested for interactions with human α-thrombin using surface plasmon resonance (SPR).

Results: In the T-cell assay, both sulfated and non-sulfated peptides showed specific donor-dependent responses; in cells from at least one donor, the response for a non-sulfated peptide set was significantly higher than for the sulfated peptides. By SPR, the peptides with progressive decrease in the number of the sulfated tyrosine residues showed a progressive decrease in affinity for α-thrombin.

Conclusion(s): Altered tyrosine sulfation in the tested FVIII peptides resulted in T-cell response in culture and decreased affinity to thrombin. This indicates that under-sulfation of FVIII may decrease its rate of activation (i.e., potency of rFVIII products) and increase FVIII inhibitor development in some individuals with Hemophilia A.