Aug 2026· Thrombosis Research· Vol 264, pp.
109803
· 0 citations· 67 references
Medicine
TL;DR
Targeted modification of regions surrounding the FIXa active site enables allosteric activation, providing a potential foundation for novel bypassing strategies in hemophilia A therapy.
Abstract
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.
Factor VIII Aurora (FVIII-R571S) is the first described naturally occurring enhanced-potency FVIII variant identified in a patient with recurrent thrombosis and early mortality. The patient's plasma exhibited increased procoagulant activity and reduced responsiveness to activated protein C (APC). To define the mechanism, we generated recombinant FVIII-R571S and performed in vitro and in vivo studies. Consistent with the clinical phenotype, FVIII-R571S demonstrated a 6-fold increase in one-stage assay activity, while chromogenic substrate assay activity was comparable to wild-type FVIII (FVIII-WT). This discrepancy was explained by biochemical studies showing that activated FVIII-R571S (FVIIIa-R571S) has 10-20-fold higher affinity for FIXa; notably, the chromogenic assay is insensitive to differences in FVIIIa-FIXa affinity. Additional analyses demonstrated that FVIII-R571S is inactivated by APC and protein S analogous to FVIII-WT, indicating that the variant is not intrinsically APC-resistant. However, the increased affinity of FVIIIa-R571S for FIXa confers FIXa-dependent reduced A2-domain dissociation and APC-mediated inactivation in purified and plasma-based studies. These enhanced biochemical properties translated in vivo to a more potent procoagulant phenotype in hemophilia A mice. In the tail clip assay, FVIII-R571S exhibited a 4-5-fold increase in potency compared to FVIII-WT. In a thrombosis model, FVIII-R571S promoted significantly increased platelet and fibrin accumulation relative to FVIII-WT at equivalent antigen levels. Collectively, these data demonstrate that the prothrombotic phenotype of FVIII-R571S is driven by increased FIXa affinity, which enhances FVIIIa-FIXa complex assembly and function. This same mechanism confers reduced A2 dissociation and functional APC resistance, providing a unifying explanation for the observed gain-of-function phenotype.
Johnathan J Morris, Robert J. Davidson, Connor T Watson et al.· Blood· 0 citations
BACKGROUND
Factor XI (FXI) occupies a unique and clinically significant niche, bridging the tissue factor-driven and contact-driven coagulation pathways. Irrespective of the trigger, activated factor XI (FXIa) contributes to clotting by activating factor IX (FIX). Biochemical studies established that this reaction requires the membrane-binding FIX-Gla domain to engage an exosite on the FXIa Apple 3 (A3) domain, that only become available upon FXI activation. Structural data for FXIa, FIX, FIXaβ, and the FXIa:FIX complex are lacking; current understanding relies on zymogen FXI crystal structures and homology modeling of FXIa after kallikrein.
OBJECTIVES
Elucidate the high-resolution structure of FXIa in functionally relevant conformation in complex with FIX.
METHODS
We utilized cryogenic electron microscopy (cryo-EM) to determine the structures of human FXIa in complex with its full-length substrate, FIX, and activated product, FIXaβ.
RESULTS
We report the first cryo-EM structures of FXIa in complex with its substrate, FIX, and activated FIX (FIXaβ). The structures capture a functionally relevant conformational change in the FXIa catalytic domain and reveals the first view of the entire FIX and FIXaβ. Critically, we visualize the FIX-Gla domain precisely docked to the FXIa-A3 exosite on both subunits of the FXIa dimer. We also define the first step of proteolysis, visualizing the FIX Arg145 inserted into the primary specificity pocket of FXIa.
CONCLUSIONS
The structures define the full FXIa:FIX interface providing a structural template for understanding the sequential activation of FIX and for developing a new class of selective allosteric antithrombotic agents.
Bassem M Mohammed, Samantha Deavila, Tristan Friet et al.· Journal of Thrombosis and Ha...· 0 citations
FV is an endogenous anticoagulant that inhibits TF-initiated coagulation by limiting FX activation by TF:FVIIa through a membrane-dependent mechanism, which refines models of coagulation initiation and may help explain how FV variation contributes to bleeding and thrombosis.
M. Jewell, Christine H Baird, D. Thornhill et al.· Blood· 0 citations
BACKGROUND
FIX exhibits significant extravascular distribution and binds type IV collagen in the subendothelial extracellular matrix in the extravascular space, which influences its function and pharmacokinetics (PK). Many hemophilia B patients carry missense F9 mutations that produce circulating, dysfunctional FIX protein (cross-reacting material; CRM). In murine hemophilia models, the presence (CRM+) or absence (CRM-) of dysfunctional FIX modifies the PK and hemostatic efficacy of exogenous FIX. We hypothesized that the PK parameters of EHL FIX products that have a significant (Fc-FIX) or negligible (PEG-FIX) extravascular distribution might be differently influenced by the presence of endogenous FIX antigen in the vascular and extravascular space.
AIM
To investigate the association of F9 variants and individual PK profile of patients treated with EHL FIX concentrates.
METHODS
Individual PK profiles were extracted from the WAPPS-Hemo database, and causal F9 mutations were sourced from linked databases. Differences in PK parameters between CRM+ and CRM- groups were analyzed.
RESULTS
Seventy individuals treated with EHL FIX, with known PK profile and gene mutation were included. Among rFIX-Fc recipients, CRM+ patients had significantly lower Clearance, longer Terminal half-life, higher In vivo recovery, and smaller Volume of distribution. No significant PK differences were found between CRM+ and CRM- patients receiving N9-GP.
CONCLUSIONS
The pharmacokinetics of EHL FIX products are influenced by their ability to access the extravascular space. For products with substantial extravascular distribution, patient CRM status further modulates pharmacokinetic behaviour.
D. Matino, Z. Al-Housni, B. Samelson-Jones et al.· Journal of Thrombosis and Ha...· 0 citations
Data indicate that platelets support greater activity of FVIII than PLV through stabilization against dissociation of the A2 domain and protection from degradation by APC.
Valerie A Novacovic, Jialan Shi, G. Gilbert· Blood Advances· 0 citations
INTRODUCTION
Hemophilia A (HA) management has progressed significantly, yet standard and extended half-life (EHL) clotting factors present persistent limitations, including intravenous burden and suboptimal trough levels.
AREAS COVERED
This narrative review highlights recently approved agents and phase III candidates for HA. In replacement therapy, efanesoctocog alfa maintains normal-to-near-normal factor VIII (FVIII) levels weekly by bypassing endogenous von Willebrand factor dependence. In non-replacement therapies, the focus centers on rebalancing agents - the anti-tissue factor pathway inhibitor (TFPI) monoclonal antibodies concizumab and marstacimab, and the antithrombin-targeting small interfering RNA (siRNA) fitusiran - as well as next-generation FVIII-mimetics like denecimig. With this objective, a structure literature search of the main original manuscripts and reviews published in English in major medical databases, including PubMed, EMBASE, and Cochrane Library from 2016 to now was conducted.
EXPERT OPINION
While these innovators promise near-zero bleeding rates and subcutaneous convenience, they introduce unique challenges. Rebalancing therapies present potential thromboembolic risks, complex breakthrough bleed protocols, and standard laboratory assay interference (requiring antithrombin monitoring or specialized assays). Critical goals for contemporary hemophilia management include tailoring therapies through multidisciplinary collaboration, monitoring subclinical joint disease via point-of-care ultrasound, and implementing standardized, real-world protocols for emergency hemostasis.
A. Giuffrida, Daniele Focosi, Marco Bodini et al.· Expert Opinion on Pharmacoth...· 0 citations