Background: Procoagulant factor VIII (FVIII) is an essential cofactor in the coagulation cascade. Deficiency of FVIII activity leads to hemophilia A. Recombinant FVIII Fc fusion protein (rFVIIIFc) was developed to extend factor half-life, thus decreasing treatment burden and increasing protection in patients. We have previously reported that rFVIIIFc, but not rFVIII, inhibits monocyte-derived osteoclast formation in vitro.

Aims: To further investigate the mechanisms by which rFVIIIFc interacts with blood monocytes to reduce their differentiation potential.

Methods: Primary human blood monocytes were treated with rFVIIIFc or rFVIII plus human IgG at various concentrations and then cultured for osteoclast differentiation in vitro. Multiple myeloid lineage markers were used to immunophenotype and distinguish differentiated monocytes and osteoclasts. The involvement of Fc or FVIII domains in mediating rFVIIIFc interaction with monocytes was probed using antibodies blocking each type of FcγRs, or anti-FVIII antibodies and von Willebrand factor (VWF) binding to various FVIII domains.

Results: We report a novel immune-regulatory effect of rFVIIIFc on inhibiting monocyte differentiation into osteoclasts through Fc and FVIII domains with a mean IC50 of 8nM. The cells differentiated from rFVIIIFc-treated monocytes were phenotypically distinct from osteoclasts and remained largely monocytic. As for the interactions between rFVIIIFc and monocytes modulating this phenotype, the Fc domain most effectively engaged FcγRII on the cell surface; C1 and C2 domains of FVIII were mapped to be required for interacting with monocytes, also evidenced by loss of the immune-regulatory effects of VWF-complexed rFVIIIFc.

Conclusions: These data suggest a “dual-touchpoints” model for rFVIIIFc interacting with monocytes. The FVIII portion interacts with monocytes via C1 and C2 domains and, in parallel, the Fc domain predominantly engages FcγRII on the same cell, subsequently reducing monocyte differentiation potential into osteoclasts. Therefore, rFVIIIFc may possess a biological activity unique from rFVIII which may reduce joint bone erosion and bone mass loss in hemophilia A patients.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/the-fc-and-c1-c2-domains-of-recombinant-factor-viii-fc-fusion-protein-engage-monocytes-in-an-fcgammarii-dependent-manner-to-reduce-their-differentiation-potential-into-osteoclasts/