The factor VIII (FVIII) B domain, a large and heavily glycosylated region, is crucial for FVIII secretion, although its structural and functional roles remain incompletely understood. Although the B domain is dispensable for cofactor activity, previous research hints at multiple, yet unverified, functional roles. Here, we used an integrative hybrid approach to generate detailed structural models of glycosylated and furin-cleaved full-length FVIII (Gly/FC-FL-FVIII) that include the B domain, using AlphaFold2 predictions. These models were further refined and validated with biophysical data from atomic force microscopy (AFM) and cryogenic electron microscopy of recombinant and plasma-derived FL-FVIII proteins. Additionally, we modeled a FL-FVIII-von Willebrand factor (VWF) complex with both the B domain and D'-D3 domains to investigate B-domain interactions during intracellular processing. Our results suggest that the B domain functions largely as an independent domain encircling the FVIII core. AFM studies revealed that VWF tails are associated with globular FVIII structures. Structural analysis indicated that B-domain glycosylation enhances stability, supports proper folding, and promotes efficient trafficking through the endoplasmic reticulum-Golgi pathway. We propose that, within the Golgi, the B domain may unfold around FVIII via furin cleavages, facilitating essential interactions with partners such as VWF. Our models also suggest that the B domain shields key FVIII epitopes, potentially reducing immune interference and inhibiting premature activation. This study presents a novel structural model of the FVIII B domain and FL-FVIII, offering critical insights into previously unexplored aspects of B-domain function and its essential role in FVIII biology.

Factor VIII (FVIII) circulates as an inactive procofactor and is converted to its active form (FVIIIa) by proteolytic cleavage at Arg372, Arg740, and Arg1689. Although these cleavage events are well characterized, their individual contributions to FVIII activation and hemostatic function remain incompletely defined. To address this, we engineered a FVIII variant (FVIII-2RKR) in which the B-domain and acidic region 3 region were replaced with a PACE/furin processing site, generating a molecule that requires only cleavage at Arg372 for conversion to FVIIIa. FVIII-2RKR exhibited markedly increased apparent activity in plasma-based assays, shortened thrombin generation lag time, and enhanced factor Xa (FXa) generation compared with B-domain-deleted FVIII, whereas both proteins were functionally equivalent following thrombin activation. FVIII-2RKR was cleaved more rapidly at Arg372 by thrombin and FXa, demonstrating that processing at Arg740 and Arg1689 regulates the rate of Arg372 cleavage rather than directly contributing to cofactor activity. In hemophilia A mice, FVIII-2RKR consistently enhanced early hemostatic function across ferric chloride, laser injury, and tail bleeding models, achieving improved efficacy at lower doses. Despite reduced half-life due to impaired von Willebrand factor (vWF) binding, FVIII-2RKR remained effective in vWF-deficient mice, indicating that vWF is not required for local FVIII function at sites of vascular injury. Together, these findings define distinct roles for FVIII activation cleavage sites and demonstrate that processing at Arg740 and Arg1689 regulates activation through Arg372, linking the rate of FVIII activation to hemostatic efficacy in vivo.

Hemophilia A is an inherited bleeding disorder caused by factor VIII (FVIII) deficiency, resulting in recurrent bleeding and progressive joint damage. Treatment has expanded beyond FVIII replacement to extended-half-life products, non-factor therapies, rebalancing agents, and gene therapy. These advances improve outcomes but introduce distinct long-term safety considerations.

Severe hemophilia A is exceptionally rare among preterm infants. Qwing to immature coagulation system and nonspecific bleeding manifestations, the condition is readily misdiagnosed as other neonatal hemorrhagic disorders, such as vitamin K deficiency, neonatal sepsis and thrombocytopenia. Delayed diagnosis will greatly increase the risk of severe complications, particularly intracranial hemorrhage. Herein, we report a case of genetically confirmed severe hemophilia A in a 34-week preterm infant without family history of hemophilia. Treatment comprised sequential adminnistration of coagulation factor VIII repalcement and emicizumab. We futher analyze the clinical characteristics, diagnostic difficulties and standardized management principles of hemophilia A in preterm infants, so as to improve clinicians' awareness, reduce misdiagnosis and missed diagnosis, and optimize the long-term prognosis of affected infants.

Tumor Electric Field Therapy (TEFT) disrupts mitosis in glioblastoma (GBM), but responses vary markedly among patients. In a retrospective cohort of TEFT-treated GBM, EGFR variant III (EGFRvIII) alteration is associated with shorter progression-free survival, prompting us to investigate a genotype-linked resistance mechanism. TEFT triggers a broadly shared bioenergetic stress response marked by activation of the AMPK-PPARα-CPT1A axis, whereas EGFRvIII primes IDO1 transcription through NF-κB. CPT1A further stabilizes IDO1 by promoting succinylation at lysine 377 through non-canonical LSTase-related activity, thereby limiting TRIM21-dependent ubiquitination and proteasomal degradation. Accumulated IDO1 increases kynurenine production and activates AhR, which upregulates DCLK1 and ARHGEF2 to preserve spindle organization and microtubule dynamics during electric-field exposure. Thus, EGFRvIII converts a general stress-adaptation pathway into a selective cytoprotective program. Genetic or pharmacological disruption of this pathway restores TEFT sensitivity in established and patient-derived GBM cells, organoids, and orthotopic models. Osimertinib suppresses the EGFRvIII-NF-κB-IDO1 arm and enhances TEFT efficacy, while exploratory clinical cases provide preliminary mechanism-informed support for the combination in recurrent EGFR-driven GBM. These findings define a genotype-field convergence mechanism linking metabolic adaptation to mitotic protection and support prospective evaluation of osimertinib plus TEFT.

BACKGROUND Acquired hemophilia A (AHA) is a rare but potentially life-threatening condition caused by autoantibodies against factor VIII. It often presents in older adults and its presentation can range from isolated lab abnormality of prolonged activated partial thromboplastin time (aPTT) without bleeding to mild bleeding or spontaneous vs disproportionate life-threatening bleeding. Delayed diagnosis is frequent due to the rarity of the condition and propensity to attribute bleeding to other common causes, such as falls. CASE REPORT A 92-year-old woman with recurrent ground-level falls presented with severe right lower extremity pain of several weeks' duration. She was not on any anticoagulant or anti-platelet agent. Computed tomography angiography demonstrated bilateral lower extremity intramuscular hematomas. She had 2 prior admissions for falls within 6 months, including a fall leading to intramuscular hematoma, which was conservatively managed without evaluation of coagulopathy. During current admission, prolonged aPTT prompted further workup, revealing low factor VIII activity of 5% and a high factor VIII inhibitor titer of 15.6 Bethesda Units, establishing the diagnosis of acquired hemophilia A. Bleeding control required sequential bypassing agents, corticosteroids, rituximab, cyclophosphamide, and eventually emicizumab. CONCLUSIONS This case highlights how recurrent or disproportionate intramuscular hematomas following even minor trauma, such as a ground-level fall, in the absence of risk factors such as anticoagulant use, may prompt evaluation for underlying coagulopathy such as AHA. Basic coagulation testing including prothrombin time and aPTT can help screen patients who warrant additional coagulopathy evaluation. Timely diagnosis is critical to avoid life-threatening bleeding complications.

Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively active EGFR deletion variant found in a subset of glioblastomas (GB). Its association with stem-like tumor populations is well documented, but the outcome of EGFRvIII expression may vary with the developmental and molecular state of the recipient cell. Here, we used human induced pluripotent stem cell (iPSC)-derived, glial fibrillary acidic protein (GFAP)-negative-induced neural stem cells (iNSCs) to explore how this receptor variant affects a defined neural stem/progenitor background. EGFRvIII was introduced either constitutively by lentiviral transduction or through a doxycycline-responsive Tet-On system. Both experimental approaches were associated with remodeling of the neural stem/progenitor marker profile, with reduced SRY-box transcription factor 2 (SOX2) expression in the inducible model and significant reductions in both SOX2 and nestin in the constitutive model. Constitutive expression was also associated with increased senescence-associated β-galactosidase (SA-β-Gal) activity, while doxycycline induction produced a distinct change in cell-number dynamics at low doxycycline concentration. Thus, in this experimental setting, EGFRvIII was linked to substantial remodeling of the SOX2+/nestin+ marker profile. These observations do not define a differentiation pathway, prove cellular senescence or malignant transformation, or identify the cell of origin of EGFRvIII-positive GB. Rather, they support the view that the cellular response to EGFRvIII should be considered in relation to the specific neural-glioblastoma precursor context.

Hemophilia is an X-linked hereditary bleeding disorder caused by loss-of-function mutations in the genes encoding coagulation factors, leading to excessive bleeding and potentially being life-threatening. Currently, regular treatment for hemophilia is the infusion of recombinant blood coagulation factors. This approach is not only costly but can also give rise to complications such as the development of neutralizing antibodies (Nabs), which impede the therapeutic outcome. Hemophilia is a monogenic disease, making gene therapy a promising curative measure that includes gene addition and gene editing approaches. Adeno-associated virus (AAV) vectors have gained massive attention as the premier delivery vehicle for clinical gene therapy due to their diverse tissue tropisms dictated by the natural and engineered AAV capsids, their excellent safety profile since AAVs do not cause any known human diseases, low immunogenicity, and long-lasting gene expression. Several AAV-based gene addition therapies have been approved for hemophilia B and hemophilia A, and dozens of similar AAV-based clinical trials are underway. Gene editing technologies, like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), have shown immense potential in the treatment of genetic diseases. CRISPR/Cas9-mediated transgene integration provides critical advantages over regular gene insertion therapy by enabling site-specific, targeted genomic integration rather than random integration. Clinical trials focusing on AAV-CRISPR/Cas9-mediated knock-in of human genes represent the frontier of in vivo genetic medicine, and one such prominent ongoing clinical trial for hemophilia B is designed to insert the human Factor IX gene into hepatocytes. This review outlines recent advances and feasible strategies of CRISPR/Cas9-based genome editing for hemophilia therapy, highlights the potential applications of next-generation gene editing and vector delivery technologies, and offers new insights for the advantages, limitations, and future directions for these novel treatment modalities.

Hemophilia A (HA) is caused by factor VIII (FVIII) deficiency and requires lifelong prophylaxis. Although activated FVIII (FVIIIa)-mimetic antibodies have enabled subcutaneous administration, thereby reducing treatment burden compared with intravenous FVIII replacement, an oral therapy remains a significant unmet need for individuals with HA. Here, we describe Inno8, a novel bispecific single-chain antibody fragment comprising two variable domains of heavy-chain antibodies that bridges coagulation factors IXa and X to mimic FVIIIa function and is engineered for oral delivery. Inno8 was generated from llama and alpaca immunizations and optimized through iterative structure-guided and machine learning-assisted engineering to enhance FVIIIa-mimetic activity, reduce the isoelectric point (pI) to improve oral bioavailability, and extend systemic exposure through site-specific fatty acid conjugation. Formulations containing permeation enhancer SNAC and solubilizing agent niacinamide were evaluated in preclinical models. Procoagulant activity was assessed in HA plasma and whole blood using thrombin generation assays and thromboelastography. Inno8 demonstrated, on average, 90‑fold greater in vitro potency than a sequence‑identical emicizumab analogue. Reducing the pI increased oral exposure ~10-fold in rats, and a tablet formulation containing SNAC and niacinamide demonstrated significant oral bioavailability (0.215% with 4-hour post dose fasting) and a 115-hour half-life in beagle dogs. Together, these findings support Inno8 as a potent, orally deliverable FVIIIa-mimetic and provide a framework for engineering oral, long-acting antibody-based therapies. Inno8 is currently in clinical evaluation, with first-in-human dosing completed (VOYAGER1, NCT06649630). Pending further clinical evaluation, Inno8 may represent an advance in biologics development and a potential non-invasive prophylactic therapy for individuals with HA.