TL;DR
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.
Factor V (FV) links procoagulant amplification to anticoagulant feedback, but how FV limits tissue factor-initiated coagulation are not fully defined. We hypothesized that procofactor FV downregulates factor X (FX) activation by tissue factor:factor VIIa (TF:FVIIa), independently of tissue factor pathway inhibitor α (TFPIα), and that this mechanism is especially important in hemophilia. Thrombin generation was measured in FV/FVIII‑immunodepleted plasma and synthetic plasma while titrating FV, with TFPIα removed, blocked, or re-added. TF:FVIIa activation of FX was measured on phosphatidylserine‑containing or phosphatidylserine‑free liposomes with antibodies against the FV light chain and C2 domain. FV and TFPIα levels modulated thrombin generation in plasma from people with hemophilia A. In TF‑initiated coagulation lacking TFPIα, thrombin generation peaked at 2 nM FV and decreased as FV increased; at 20 nM FV (normal concentration), peak thrombin and thrombin generation rate were reduced by up to 50-80%, with larger effects at low FVIII. In purified TF:FVIIa assays, FV reduced FX activation by ~80% at physiologic concentration and inhibited FX activation on TF-expressing fibroblasts. Increasing PS content enhanced FX activation and increased the FV-sensitive component, while blocking the FV light chain or C2 domain partially relieved inhibition. In hemophilia A plasma, higher FV was associated with longer lag time and time-to-peak, and lower peak thrombin independent of TFPIα. Thus, FV is an endogenous anticoagulant that inhibits TF-initiated coagulation by limiting FX activation by TF:FVIIa through a membrane-dependent mechanism. This mechanism refines models of coagulation initiation and may help explain how FV variation contributes to bleeding and thrombosis.
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.
INTRODUCTION Various extended half-life recombinant factor VIII (EHL-FVIII) products have been designed to improve the pharmacokinetic properties of FVIII, allowing prolonged haemostatic coverage and reducing the injection burden in people with haemophilia A. Nevertheless, the influence of direct molecular attachment on fibrin clot formation and stability remains to be investigated. AIM To investigate the stability and architecture of fibrin clots in the presence of various types of EHL-FVIII. METHODS Three EHL-FVIII products with direct attachment modifications (damoctocog alfa pegol, efraloctocog alfa, and rurioctocog alfa pegol) and two standard FVIII (turoctocog alfa and rurioctocog alfa) were added to FVIII-deficient whole blood and plasma at various concentrations for functional comparison. Under whole-blood conditions, functional assays were performed using rotational thromboelastometry (ROTEM) and a microchip flow-chamber system (T-TAS). Under plasma-based conditions, fibrin fibres were directly observed by electron microscopy and coagulation function was assessed using clot waveform analysis (CWA). Anticoagulant and fibrinolytic activities were evaluated by CWA with the addition of activated protein C and tissue plasminogen activator, respectively. RESULTS At equivalent activity levels, none of the assays revealed significant differences among the three EHL products or the two standard products. All contributed comparably to fibrin clot formation and stability, as well as to anticoagulation and fibrinolysis functions. CONCLUSION Direct modification by PEGylation or IgG-Fc fusion to impart EHL characteristics preserves the functional properties of native FVIII. PLAIN LANGUAGE SUMMARY People with haemophilia A require treatment with factor VIII (FVIII) to prevent or control bleeding. Some FVIII products are designed to remain active in the body for a longer time, which can reduce the number of injections needed. This extended half-life is achieved by chemically or biologically modifying FVIII, for example by attaching polyethylene glycol (PEG) or the Fc portion of immunoglobulin G. These treatments are known as extended half-life FVIII (EHL-FVIII) products. However, it has not been fully established whether these modifications affect how blood clots form and remain stable. In this study, we compared three EHL-FVIII products with two standard FVIII products using FVIII-deficient blood and plasma. We evaluated clot formation and stability using several laboratory techniques, including whole-blood assays and scanning electron microscopy. We also examined potential differences in anticoagulant and fibrinolytic properties. At comparable FVIII activity levels, we observed no major differences between the EHL-FVIII products and the standard FVIII products in any of the assays performed. These findings suggest that PEGylation or Fc fusion, which are used to extend the half-life of FVIII, do not impair its functional properties related to fibrin clot formation and stability.
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.
Abstract Introduction Predicting bleeding in hemophilia A is difficult due to limitations of existing factor VIII (FVIII) activity assays, which correlate poorly with clinical bleeding tendency. Measurement of thrombin generation (TG) by calibrated automated thrombography (CT) may be an alternative to predict bleeding as it measures an individual’s complete plasma derived coagulation potential, rather than just focusing on one part of the coagulation cascade. However, lack of standardization limits its applicability. Objective To evaluate the ability of our standardized tissue factor (TF)/factor XI (FXI) a dual-activated thrombin generation assay (TGA) to assess TG across severe, moderate, and mild hemophilia A. Secondarily, we evaluate whether varying TG levels at similar FVIII concentrations translate to different clinical bleeding tendencies. Materials and Methods Plasma samples from 657 adult patients with hemophilia A of all severities included in the Hemophilia in the Netherlands 6 (HiN6) study were used. Samples were collected through standardized protocols from six Dutch hemophilia treatment centers, and stored in a centralized biobank. FVIII activity was centrally measured using one-stage assays. Thrombin generation was measured using our in-house TF/FXIa TGA protocol using 1 pm TF, 100 pm FXIa, and 30 μM phospholipids. Bleeding tendencies were analyzed based on patient questionnaires. Results The TF/FXIa dual-activated TGA protocol was able to measure thrombin generation across all severities of hemophilia A. Median peak heights (PH) were 119.5, 98.9, and 214.7 nM for severe, moderate, and mild, respectively. Moreover, the assay showed substantial variation in interindividual TG levels among patients with comparable FVIII activity levels (coefficient of variance of 99%, 83%, and 47% for severe, moderate, and mild hemophilia A, respectively). However, TG did not differentiate between patients with and without self-reported bleeding in this study. Conclusion The standardized TF/FXIa dual-activated TGA represents a tool for assessing individual coagulation potential in hemophilia A. More research is needed to further characterize the interindividual differences in thrombin generations at similar FVIII levels and possibly link these results to bleeding tendency.
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.
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