A Modified Intrinsic Thrombin Generation Assay for Functional Assessment of Factor and Non-Factor Therapies in Hemophilia

Background/Objectives: Conventional Factor VIII (FVIII) and factor IX (FIX) assays may provide product and reagent-dependent results for modern hemophilia therapies. We developed a modified thrombin generation assay (TGAint) designed to enhance FVIII/FIX-dependent thrombin generation and evaluated its response to factor and non-factor therapies. Methods: TGA was modified by omitting exogenous tissue factor, using isolated platelets as a phospholipid surface, and inhibiting factor XI (FXI) activity with milvexian. The optimized assay used 10 × 109/L platelets and approximately 20% FXI activity. TGAint was evaluated using Advate, Adynovi, Altuvoct, Alprolix, and emicizumab. Results: Compared with standard TGAext, TGAint showed a longer lag time (+10.4 min; p = 0.0078), lower peak thrombin (−155.8 nM; p = 0.0156), and lower area under curve (AUC) (−2559 nM; p = 0.0156). Increasing factor concentrations generally increased peak thrombin and AUC, although responses differed among products. Factor recovery also varied substantially. Emicizumab at 55 μg/mL produced a distinct thrombin generation profile that could not be expressed as a single FVIII-equivalent activity. Conclusions: TGAint enables functional assessment of FVIII/FIX-dependent thrombin generation and may complement conventional factor assays by evaluating the overall hemostatic potential of factor and non-factor therapies. Further clinical validation is required.