Cofactor-independent activity of FIX has previously been achieved in FIX-IDAV and FIX-FIAV variants containing L6F, V181I, E185D, K265A, and I383V substitutions. Cofactor-dependent FIX hyperactivity was attained through the V10K, R338L, and S377W (KLW) modifications. We evaluated whether combining IDAV/FIAV and KLW substitutions could enhance cofactor-independent activity and elucidated the biochemical mechanisms underlying FVIII-independent FIX-FIAV function. Recombinant FIX variants, expressed in HEK293 cells, were analyzed for FIX-specific and FVIII-equivalent clotting activities. Highly purified FIX-FIAV was characterized using purified component assays and FXIa-triggered thrombin generation in FVIII-deficient plasma. FIX-IDAV-KLW and FIX-FIAV-KLW exhibited 20-28-fold higher FIX-specific activity and a modest twofold increase in FVIII-equivalent clotting activity. Owing to thrombotic safety concerns, KLW combinations were not further pursued. FIX-FIAV displayed normal FIX-specific activity and fourfold enhanced FVIII-equivalent clotting activity. The FIAV substitutions did not alter activation by the extrinsic (TF-FVIIa) or intrinsic (FXIa) pathways, nor cofactor-dependent FX activation, but conferred FVIII-independent FX conversion. Kinetic analyses revealed a modified active site conformation with a trend toward reduced antithrombin inhibition. In FVIII-deficient plasma, FIX-FIAV dose-dependently increased FXIa-triggered thrombin generation by shortening the lag time and time to peak and shifted coagulation phenotype categories based on FVIII-equivalent thrombin generation (TG) activity from severe toward moderate/mild ranges in plasma-based models, without exceeding normal FVIII-equivalent TG activity. FIX-FIAV represents a FIX variant with FVIII-independent activity, normal FIX-specific activity, and reduced inactivation by antithrombin. Targeted modification of regions surrounding the FIXa active site enables allosteric activation, providing a potential foundation for novel bypassing strategies in hemophilia A therapy.

Hemophilia, a congenital deficiency of factor VIII (hemophilia A) or factor IX (hemophilia B), leads to recurrent bleeding episodes that may cause progressive joint damage and long-term disability. Traditional management relies on intravenous factor replacement therapy; however, limited half-life, immunogenicity, venous access challenges, and the burden of frequent infusions have prompted the development of extended half-life (EHL) factor products. Although EHL therapies represent an important advancement, they only partially reduce treatment burden and do not fully meet expectations for improved convenience and sustained bleed protection. Recent innovations are reshaping the therapeutic landscape. Nonfactor subcutaneous therapies such as anti-TFPI molecules, antithrombin reducing agents, and anti-protein C agents offer simplified administration with the potential for improved adherence. Gene therapy provides the prospect of a long-term therapeutic effect in selected patients. In Türkiye, hemophilia care remains largely factor-based; however, clinical trial participation and recent regulatory approvals for selected novel therapies have begun to expand real-world experience with these emerging treatment options. As global practice shifts toward individualized, less invasive treatment approaches, expanding availability of novel therapies and optimizing patient-specific treatment strategies will be essential. This review examines current and evolving treatment options, key challenges, and future opportunities shaping the trajectory of hemophilia management.

A deficiency and/or dysfunction of von Willebrand factor (VWF) or factor VIII (FVIII) results in the bleeding disorders of von Willebrand disease (VWD) and hemophilia A (HA), respectively. Whereas HA impacts coagulation, VWD primarily impairs hemostasis through defective platelet adhesion and aggregation. In addition, because VWF protects FVIII from proteolytic degradation, a deficiency in VWF can also reduce FVIII levels and affect coagulation. While regular prophylaxis to restore FVIII activity is the standard of care for severe HA, its use to correct the dual VWF/FVIII defect in severe VWD is less well established. Current treatment guidelines suggest the use of long-term prophylaxis rather than no prophylaxis in persons with VWD with a history of severe and frequent bleeds. In this narrative review, we discuss the barriers to the broader adoption of prophylaxis in persons with severe VWD and results of recent clinical studies that provide further evidence to support its use. A growing body of evidence suggests that prophylaxis should be established as the standard care for individuals with severe VWD and recurrent bleeding.

Efanesoctocog alfa (efa) is a recombinant coagulation factor VIII (FVIII) concentrate, engineered for improved extended half-life in hemophilia A treatment. Its design results in discrepancies in FVIII diagnostic tests, as has so far been demonstrated using spiked sample material (efa added to FVIII-deficient plasma). The aim of the present study was to evaluate FVIII assay discrepancies in post-infusion samples obtained from patients treated with efa. This retrospective analysis included 43 male patients on efa prophylaxis (26 on once weekly and 17 on twice weekly treatment scheme) summoned for determination of incremental recoveries. FVIII activity was measured at trough (3.5 or 7 days post-infusion) and peak levels (30 minutes post-infusion) using three diagnostic tests: two one-stage clotting assays (OSCAs) based on Actin FSL (AFSL) or Actin FS (AFS), and a chromogenic substrate assay (FVIII CSA). Incremental recoveries as determined by the recommended AFSL-based OSCA were found to be comparable between patient groups. At peak levels (64 to 214 IU/dL), comparable overestimation (1.9-fold) of plasma efa activity relative to Actin FSL-based OSCA was observed for both, the AFS-based OSCA and the FVIII CSA. In contrast, at trough levels (5 to 64 IU/dL), rate of overestimation relative to the AFSL-based OSCA results was found to be lower for the FVIII CSA (1.3-fold) when compared with the AFS-based OSCA (1.9-fold). Further analysis demonstrated different behavior of spiked and post-infusion samples within the FVIII CSA. Future studies will reveal underlying mechanisms and assess if drug-specific calibration will sufficiently correct for this phenomenon.

As assays tailored to Altuvoct are necessary, it is essential that every request for a FVIII level for monitoring of Altuvoct clearly identifies when it has been used for treatment of the patient and details the time of sampling relative to timing infusion.

BackgroundEmicizumab has emerged as a promising therapy for hemophilia A (HA), employing a bypassing mechanism to restore hemostasis. However, since the traditional factor assays cannot be used for measuring the effect of emicizumab, treatment monitoring is difficult.ObjectivesTo assess the impact of emicizumab on global hemostatic potential (OHP), as well as on chromogenic and modified one-stage (mOSA) FVIII assays calibrated for emicizumab in patient samples.MethodsPeripheral blood samples from patients with HA on emicizumab were utilized for a validation (n = 28) and a clinical cohort (n = 9). Emicizumab concentration was measured by chromogenic FVIII and mOSA (Actin FS, Actin FSL, and PTT-LA), while overall haemostatic potential (OHP) was used to assess global hemostasis.ResultsSignificantly lower emicizumab concentrations were observed using chromogenic FVIII assay compared to mOSA (P < .005, P < .005, and P < .005 for mOSA Actin FSL, mOSA Actin FS, and mOSA PTT-LA respectively) in the validation cohort. In the clinical cohort, emicizumab concentrations were significantly lower with chromogenic FVIII assay compared to mOSA Actin FS (P < .005) and mOSA PTT-LA (P = .009), but not mOSA Actin FSL. In the clinical cohort, no correlation was seen between OHP and emicizumab.ConclusionAccurate measurement of emicizumab concentration may have the potential to understand assay variability, which is important for informed clinical decision-making in HA patients. Our findings underscore the need for introducing new methods for measuring emicizumab concentration and suggest chromogenic-based methodologies as a viable option for mitigating assay interference.

The development of inhibitors to von Willebrand factor (VWF) is a rare but potentially serious complication of VWF replacement therapy in patients with von Willebrand disease (VWD). Patients who develop VWF inhibitors may become unresponsive and/or may develop severe anaphylactic reactions to VWF concentrates. Data on inhibitor development and management in VWD remain limited, and better understanding of inhibitor development is an important goal in VWD management. The WIL-31 study demonstrated the efficacy and safety of prophylaxis with wilate, a plasma-derived VWF/factor VIII (pdVWF/FVIII) concentrate, in children and adults with VWD of all types. The annualized bleeding rate (ABR) was reduced by 84% with wilate prophylaxis compared with on-demand treatment, and prophylaxis was well tolerated. No inhibitors developed during the WIL-31 study. Here, we report two brothers with type 3 VWD who at the 6-month visit were found to have VWF inhibitors, which on further investigation were found to have already been present before the study. Despite the presence of inhibitors, neither patient showed any clinical symptoms, and prophylaxis with wilate led to a ≥85% reduction in ABR in both boys compared with on-demand treatment.

This report concerns the preclinical development of TAK-754, an AAV8-based human factor VIII (FVIII) vector designed to deliver a codon-optimized and CpG-depleted B domain-deleted F8 transgene under the control of a liver-specific promoter for gene therapy in patients with hemophilia A. A dose-dependent increase in plasma FVIII activity was detected in FVIII knockout mice at a dose of 1.0 × 10[12] TAK-754 capsid particles (CP)/kg or higher. This increase was shown to be in accordance with a dose-dependent decrease in blood loss in a hemostatic efficacy assay. TAK-754 (3.1 × 10[12] CP/kg) mediated long-term and stable FVIII expression in immunologically tolerant transgenic human FVIII mice. Toxicology and biodistribution assessments with a single administration of TAK-754 ranging between 1.9 × 10[12] and 5.0 × 10[13] CP/kg were conducted in male C57BL/6J mice. The highest TAK-754 dose occurred without TAK-754-related adverse clinical signs. Biodistribution profiling showed predominant detection in the liver with a low occurrence of vector DNA in other tissues. Integration site analysis revealed minimal vector integration, with no observations of clonal outgrowth or preferred integrations in genes previously implicated in hepatocellular carcinoma formation within the observation period. These preclinical studies demonstrate a good safety and efficacy profile for TAK-754.