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Background: The recruitment of activated factor VIII (FVIII) at the surface of activated platelets is a key step toward the burst of thrombin and fibrin generation during thrombus formation at the site of vascular injury. It involves binding to phosphatidylserine and, possibly, to fibrin-bound α IIb β 3 . Seminal work had shown the binding of FVIII to resting platelets, yet without a clear understanding of a putative physiological relevance., Objectives: To characterize the effects of FVIII-platelet interaction and its potential modulation of platelet function., Methods: FVIII was incubated with washed platelets. The effects on platelet activation (spontaneously or triggered by collagen and thrombin) were studied by flow cytometry and light transmission aggregometry. We explored the involvement of downstream pathways by studying phosphorylation profiles (Western blot). The FVIII-glycoprotein (GP) VI interaction was investigated by ELISA, confocal microscopy, and proximity ligation assay., Results: FVIII bound to the surface of resting and activated platelets in a dose-dependent manner. FVIII at supraphysiological concentrations did not induce platelet activation but rather specifically inhibited collagen-induced platelet aggregation and altered glycoprotein VI (GPVI)-dependent phosphorylation. FVIII, freed of its chaperone protein von Willebrand factor (VWF), interacted in close proximity with GPVI at the platelet surface., Conclusion: We showed that VWF-free FVIII binding to, or close to, GPVI modulates platelet activation in vitro. This may represent an uncharacterized negative feedback loop to control overt platelet activation. Whether locally activated FVIII concentrations achieved during platelet accumulation and thrombus formation at the site of vascular injury in vivo are compatible with such a function remains to be determined., Competing Interests: Declaration of competing interests The authors declare no competing financial interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The development of an immune response against therapeutic factor VIII is the major complication in hemophilia A patients. Oligomannose carbohydrates at N239 and/or N2118 on factor VIII allow its binding to the macrophage mannose receptor expressed on human dendritic cells, thereby leading to factor VIII endocytosis and presentation to CD4+ T lymphocytes. Here, we investigated whether altering the interaction of factor VIII with mannose-sensitive receptors on antigen-presenting cells may be a strategy to reduce factor VIII immunogenicity. Gene transfer experiments in factor VIII-deficient mice indicated that N239Q and/or N2118Q factor VIII mutants have similar specific activities as compared to non-mutated factor VIII; N239Q/N2118Q mutant corrected blood loss upon tail clip. Production of the corresponding recombinant FVIII mutants or light chains indicated that removal of the N-linked glycosylation site at N2118 is sufficient to abrogate in vitro the activation of FVIII-specific CD4+ T cells by human monocyte-derived dendritic cells. However, removal of mannose-ending glycans at N2118 did not alter factor VIII endocytosis and presentation to CD4+ T cells by mouse antigen-presenting cells. In agreement with this, the N2118Q mutation did not reduce factor VIII immunogenicity in factor VIII-deficient mice. Our results highlight differences in the endocytic pathways between human and mouse dendritic cell subsets, and dissimilarities in tissue distribution and function of endocytic receptors such as CD206 in both species. Further investigations in preclinical models of hemophilia A closer to humans are needed to decipher the exact role of mannose-ending glycans in factor VIII immunogenicity., (Copyright © 2020 Delignat, Rayes, Dasgupta, Gangadharan, Denis, Christophe, Bayry, Kaveri and Lacroix-Desmazes.)

Replacement therapy in severe hemophilia A leads to factor VIII (FVIII) inhibitors in 30% of patients. Factor VIII gene (F8) mutation type, a family history of inhibitors, ethnicity and intensity of treatment are established risk factors, and were included in two published prediction tools based on regression models. Recently investigated immune regulatory genes could also play a part in immunogenicity. Our objective is to identify bio-clinical and genetic markers for FVIII inhibitor development, taking into account potential genetic high order interactions. The study population consisted of 593 and 79 patients with hemophilia A from centers in Bonn and Frankfurt respectively. Data was collected in the European ABIRISK tranSMART database. A subset of 125 severely affected patients from Bonn with reliable information on first treatment was selected as eligible for risk stratification using a hybrid tree-based regression model (GPLTR). In the eligible subset, 58 (46%) patients developed FVIII inhibitors. Among them, 49 (84%) were "high risk" F8 mutation type. 19 (33%) had a family history of inhibitors. The GPLTR model, taking into account F8 mutation risk, family history of inhibitors and product type, distinguishes two groups of patients: a high-risk group for immunogenicity, including patients with positive HLA-DRB1*15 and genotype G/A and A/A for IL-10 rs1800896, and a low-risk group of patients with negative HLA-DRB1*15 / HLA-DQB1*02 and T/T or G/T for CD86 rs2681401. We show associations between genetic factors and the occurrence of FVIII inhibitor development in severe hemophilia A patients taking into account for high-order interactions using a generalized partially linear tree-based approach., Competing Interests: D. Bachelet, T. Albert, C. Mbogning, S. H¨assler, Y. Zhang, S. Schultze-Strasser, Y Repesse, J. Rayes, A. Pavlova, B. Pezeshkpoor, K. Liphardt, S. LacroixDesmazes, J. Oldenburg, P. Broët have nothing to declare. At the time of writing J. Davidson was employed by and held stocks/shares in GlaxoSmithKline. A.Hincelin-Mery is employed by Sanofi. P. D¨onnes is an employee of SciCross AB. Dr. K¨onigs reports grants and personal fees from Bayer Vital GmbH for research support and speakers bureau, grants and personal fees from Biotest AG for research support and speakers bureau, grants and personal fees from CSL Behring for research support and speakers bureau, personal fees from Grifols for speakers bureau, grants from Intersero for reasearch support, grants and personal fees from Pfizer for research support and speakers bureau, grants and personal fees from Shire for research support and speakers bureau, grants and personal fees from Sobi for research support and speakers bureau, personal fees from Roche for expert testimony, outside the submitted work. The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement no [115303], resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013)and EFPIA companies. J.E. Davidson, A. Hincelin-Mery: belong to EFPIA (European Federation of Pharmaceutical Industries and Association) member companies in the IMI JU and costs related to their part in the research were carried by the respective companies as in kind contributions under the IMI JU scheme. This does not alter his adherence to PLOS ONE policies on sharing data and materials. All the company-employed authors declare that this does not alter their adherence to PLOS ONE policies on sharing data and materials.

Hemophilia A is a rare hemorrhagic disorder caused by the lack of functional pro-coagulant factor VIII. Factor VIII replacement therapy in patients with severe hemophilia A results in the development of inhibitory anti-factor VIII IgG in up to 30% of cases. To date, immune tolerance induction, with daily injection of large amounts of factor VIII, is the only strategy to eradicate factor VIII inhibitors. This strategy is, however, efficient in only 60-80% of patients. We investigated whether blocking B-cell receptor signaling upon inhibition of Bruton tyrosine kinase prevents anti-factor VIII immune responses in a mouse model of severe hemophilia A. Factor VIII-naïve and factor VIII-sensitized factor VIII-deficient mice were fed with the selective inhibitor of Bruton tyrosine kinase, (R)-5-amino-1-(1-cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxyl] phenyl)-1H pyrazole-4-carboxamide (PF-06250112), to inhibit B-cell receptor signaling prior to challenge with exogenous factor VIII. The consequences on the anti-factor VIII immune response were studied. Inhibition of Bruton tyrosine kinase during the primary anti-factor VIII immune response in factor VIII-naïve mice did not prevent the development of inhibitory anti-factor VIII IgG. In contrast, the anti-factor VIII memory B-cell response was consistently reduced upon treatment of factor VIII-sensitized mice with the Bruton tyrosine kinase inhibitor. The Bruton tyrosine kinase inhibitor reduced the differentiation of memory B cells ex vivo and in vivo following adoptive transfer to factor VIII-naïve animals. Taken together, our data identify inhibition of Bruton tyrosine kinase using PF-06250112 as a strategy to limit the reactivation of factor VIII-specific memory B cells upon re-challenge with therapeutic factor VIII., (Copyright© 2019 Ferrata Storti Foundation.)

The immunogenicity of therapeutic factor VIII (FVIII) in patients with hemophilia A has been puzzling scientific and clinical communities for more than 3 decades. Indeed, the development of inhibitory antibodies to FVIII remains a major clinical challenge and is associated with enormous societal costs. Thus, the reasons for which a presumably innocuous, short-lived, intravenously administered glycoprotein triggers such a deleterious, long-lasting neutralizing immune response is an enigma. This review does not pretend to bring an answer to this challenging question. It will however summarize the latest findings regarding the molecular interactions at play in the recognition of FVIII by the immune cells, the validity of the proposed risk factors for FVIII alloimmunization, and the different solutions that allow induction of FVIII-specific tolerance in preclinical models of hemophilia A., Competing Interests: None., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Development of neutralizing antibodies against therapeutic Factor VIII (FVIII) is the most serious complication of the treatment of hemophilia A. There is growing evidence to show the multifactorial origin of the anti-FVIII immune response, combining both genetic and environmental factors. While a role for the complement system on innate as well as adaptive immunity has been documented, the implication of complement activation on the onset of the anti-FVIII immune response is unknown. Here, using in vitro assays for FVIII endocytosis by human monocyte-derived dendritic cells and presentation to T cells, as well as in vivo complement depletion in FVIII-deficient mice, we show a novel role for complement C3 in enhancing the immune response against therapeutic FVIII. In vitro , complement C3 and its cleavage product C3b enhanced FVIII endocytosis by dendritic cells and presentation to a FVIII-specific CD4 + T-cell hybridoma. The C1 domain of FVIII had previously been shown to play an important role in FVIII endocytosis, and alanine substitutions of the K2092, F2093 and R2090 C1 residues drastically reduce FVIII uptake in vitro Interestingly, complement activation rescued the endocytosis of the FVIII C1 domain triple mutant. In a mouse model of severe hemophilia A, transient complement C3 depletion by humanized cobra venom factor, which does not generate anaphylatoxin C5a, significantly reduced the primary anti-FVIII immune response, but did not affect anti-FVIII recall immune responses. Taken together, our results suggest an important adjuvant role for the complement cascade in the initiation of the immune response to therapeutic FVIII., (Copyright© 2018 Ferrata Storti Foundation.)

The development of an immune response against therapeutic factor VIII is the major complication in hemophilia A patients. Oligomannose carbohydrates at N239 and/or N2118 on factor VIII allow its binding to the macrophage mannose receptor expressed on human dendritic cells, thereby leading to factor VIII endocytosis and presentation to CD4+ T lymphocytes. Here, we investigated whether altering the interaction of factor VIII with mannose-sensitive receptors on antigen-presenting cells may be a strategy to reduce factor VIII immunogenicity. Gene transfer experiments in factor VIII-deficient mice indicated that N239Q and/or N2118Q factor VIII mutants have similar specific activities as compared to non-mutated factor VIII; N239Q/N2118Q mutant corrected blood loss upon tail clip. Production of the corresponding recombinant FVIII mutants or light chains indicated that removal of the N-linked glycosylation site at N2118 is sufficient to abrogate in vitro the activation of FVIII-specific CD4+ T cells by human monocyte-derived dendritic cells. However, removal of mannose-ending glycans at N2118 did not alter factor VIII endocytosis and presentation to CD4+ T cells by mouse antigen-presenting cells. In agreement with this, the N2118Q mutation did not reduce factor VIII immunogenicity in factor VIII-deficient mice. Our results highlight differences in the endocytic pathways between human and mouse dendritic cell subsets, and dissimilarities in tissue distribution and function of endocytic receptors such as CD206 in both species. Further investigations in preclinical models of hemophilia A closer to humans are needed to decipher the exact role of mannose-ending glycans in factor VIII immunogenicity. [ABSTRACT FROM AUTHOR]