Rational design of a novel engineered factor X with chimeric activation peptide as bypassing agent for haemophilia.
Engineered factor X just got an upgrade. Researchers led by teams in Ferrara and Philadelphia created a novel factor X variant with a chimeric activation peptide, designed specifically to bypass the usual roadblocks in haemophilia A and B research. Standard replacement therapy can hit a wall when neutralizing antibodies show up. This new approach aims to sidestep that problem entirely.
J Thromb Haemost
by Tarantino R, Bunce MW, Tonetto E et al.
“Rational design of a novel engineered factor X with chimeric activation peptide as bypassing agent for haemophilia. Tarantino R(1), Bunce MW(2), Tonetto E(1), Testa MF(1), Castaman G(3), Bernardi F(1), Pinotti M(4), Camire RM(5), Branchini A(6). Author information: (1)Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy. (2)Division of Hematology and the Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. (3)Center for Bleeding Disorders, Department of Oncology, Careggi University Hospital, Florence, Italy. (4)Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy. Electronic address: pnm@unife.it. (5)Division of Hematology and the Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA; Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. Electronic address: rcamire@pennmedicine.upenn.edu. (6)Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy. Electronic address: brnlss@unife.it. BACKGROUND: While replacement therapy in Haemophilia A (HA) and B (HB) has provided remarkable improvements, the development of neutralizing antibodies remains a significant complication, with rational engineering of bypassing molecules being a relevant issue. OBJECTIVES: To develop a human factor X (FX) variant containing a FIX-derived chimeric activation peptide (AP) driving FVIII or FIX deficiency bypass. METHODS: Engineered variants were expressed and purified, and activation/activity properties were evaluated through functional assays, also in plasma with inhibitors from HA and HB patients. RESULTS: After screening a panel of recombinant variants, the most promising were purified for functional characterization. Unlike wild-type (FXWT), the bypassing (BP) variants FXBP4b and FXBP4c at physiological concentrations effectively normalized the clotting time in HA and HB plasma. Chromogenic assays confirmed that both variants preserved activation via the extrinsic tenase complex or by Russel Viper Venom (RVV)-X, while the intrinsic activation pathway was impaired, thus favouring their selective dose-dependent FXIa activation. In thrombin generation assays, FXBP4b-c restored the peak and the endogenous thrombin potential (ETP) in FX-deficient plasma. Remarkably, the FXBP4b outperformed FXWT in HA and HB plasma and showed a comparable or superior activity in comparison with B-domainless FVIII and FIX. Notably, FXBP4b significantly shortened the clotting time in plasma from patients with high-titer anti-FVIII or anti-FIX inhibitory antibodies, with efficacy levels comparable with those of well-established bypassing agents. CONCLUSIONS: These results support FXBP4b as a promising and versatile bypassing agent able to sustain coagulation across multiple haemophilic contexts including inhibitors presence, while maintaining favourable activation profiles. Copyright © 2026 The Author(s). Published by Elsevier Inc. All rights reserved.”
The team swapped in a chimeric activation peptide—borrowed from factor IX—onto factor X. The result: FXBP4b and FXBP4c variants that behave differently than wild-type factor X. At physiologically relevant levels, these new peptides normalized clotting time in haemophilia A and B plasma, even in the presence of high-titer anti-FVIII or anti-FIX inhibitors. That’s a big deal for anyone working on bypassing agents.
Key details for peptide researchers:
FXBP4b and FXBP4c kept their ability to be activated by the extrinsic tenase complex and Russell Viper Venom-X.
The intrinsic pathway was purposely impaired, making activation more selective and dose-dependent.
In thrombin generation assays, these variants restored both peak and endogenous thrombin potential in factor X-deficient plasma.
FXBP4b outperformed wild-type factor X and even matched or beat established bypassing agents like B-domainless FVIII and FIX.
Bottom line: FXBP4b looks like a versatile bypassing agent for research in haemophilia models, especially where inhibitors are present. It shows strong activity, maintains favorable activation profiles, and could open new doors for experimental design.
For more on how peptide modifications are reshaping haemostasis research, check the peptide research index. Want to compare sources for your next project? Browse the vendor directory.
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