Lack of factor VIII detection in humans and dogs with an intron 22 inversion challenges hypothesis regarding inhibitor risk

Pooja Vir; Devi Gunasekera; Batsukh Dorjbal; Dennis McDaniel; Atul Agrawal; Elizabeth P Merricks; Margaret V Ragni; Cindy A Leissinger; Allen I Stering; Kenneth Lieuw; Timothy C Nichols; Kathleen P Pratt

doi:10.1016/j.jtha.2024.08.007

Lack of factor VIII detection in humans and dogs with an intron 22 inversion challenges hypothesis regarding inhibitor risk

J Thromb Haemost. 2024 Sep 2:S1538-7836(24)00493-8. doi: 10.1016/j.jtha.2024.08.007. Online ahead of print.

Affiliations

¹ Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA; The Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, Maryland, USA.
² Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA; Biological Instrumentation Center, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA.
³ Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
⁴ University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
⁵ Department of Medicine, Louisiana Center for Bleeding and Clotting Disorders, Tulane University School of Medicine, New Orleans, Louisiana, USA.
⁶ Walter Reed National Military Medical Center, Bethesda, Maryland, USA.
⁷ Walter Reed National Military Medical Center, Bethesda, Maryland, USA; Department of Pediatrics, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA.
⁸ Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA. Electronic address: Kathleen.pratt@usuhs.edu.

PMID: 39233012
DOI: 10.1016/j.jtha.2024.08.007

Abstract

Background: Almost half of severe hemophilia A (HA) cases are caused by an intron 22 inversion (Int22Inv) mutation, which truncates the 26-exon F8 messenger RNA (mRNA) after exon 22. Another F8 transcript, F8_B, is initiated from within F8-intron-22. F8_B mRNA consists of a short exon spliced to exons 23 to 26 and is expressed in multiple human cell types. It has been hypothesized that Int22Inv patients have self-tolerance to partial factor (F)VIII proteins expressed from these 2 transcripts. FVIII is expressed in endothelial cells, primarily in the liver and lungs. Several studies have reported FVIII expression in other cell types, although this has been controversial.

Objectives: To determine if partial FVIII proteins are expressed from intron 22-inverted and/or F8_B mRNA and if FVIII is expressed in nonendothelial cells.

Methods: A panel of FVIII-specific antibodies was validated and employed to label FVIII in cells and tissues and for immunoprecipitation followed by western blots and mass spectrometry proteomics analysis.

Results: Immunofluorescent staining localized FVIII to endothelial cells in liver sections from non-HA but not HA-Int22Inv dogs. Neither FVIII nor FVIII_B was detected in human peripheral blood mononuclear cells, B cell or T cell lines, or cell lines expanded from peripheral blood mononuclear cells, whereas FVIII antigen and activity were readily detected in primary nonhemophilic liver sinusoidal endothelial cells.

Conclusion: If FVIII is expressed in nonendothelial cells or if partial FVIII proteins are expressed in HA-Int22Inv, the concentrations are below the detection limits of these sensitive assays. Our results argue against promotion of immune tolerance through expression of partial FVIII proteins in Int-22Inv patients.

Keywords: factor VIII; factor VIII coagulant antigen; hemophilia A; vascular endothelial cells.