Journal of thrombosis and haemostasis : JTH · 2024
Lack of factor VIII detection in humans and dogs with an intron 22 inversion challenges hypothesis regarding inhibitor risk.
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Vir Pooja, Gunasekera Devi, Dorjbal Batsukh, McDaniel Dennis, Agrawal Atul, Merricks Elizabeth P, Ragni Margaret V, Leissinger Cindy A, Stering Allen I, Lieuw Kenneth, Nichols Timothy C, Pratt Kathleen P
Аннотация
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, F8B, is initiated from within F8-intron-22. F8B 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 F8B 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 FVIIIB 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.
OBJECTIVES: To determine if partial FVIII proteins are expressed from intron 22-inverted and/or F8B 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 FVIIIB 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.