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.
Abstract
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.
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
The standardized TF/FXIa dual-activated TGA represents a tool for assessing individual coagulation potential in hemophilia A and showed substantial variation in interindividual TG levels among patients with comparable FVIII activity levels.
T. W. van de Berg, Alexandra C. A. Heinzmann, S. Thomassen et al.· TH Open· 0 citations
Targeted modification of regions surrounding the FIXa active site enables allosteric activation, providing a potential foundation for novel bypassing strategies in hemophilia A therapy.
V. Strijbis, K. Cheung, D. Gobbo et al.· Thrombosis Research· 0 citations
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.
N. Shimonishi, K. Nogami· Haemophilia· 0 citations
BACKGROUND
Bleeding disorders of unknown cause (BDUC) comprise a heterogeneous group of conditions characterized by clinically significant bleeding despite normal results on standard haemostatic investigations.
OBJECTIVES
To investigate tissue factor (TF)-dependent coagulation in patients with BDUC and to evaluate the in vitro ability of concizumab and recombinant activated factor VII (rFVIIa) to restore thrombin generation.
METHODS AND RESULTS
Thrombin generation was assessed using an assay designed to isolate TF-dependent coagulation by inhibiting contact pathway activation with corn trypsin inhibitor (CTI). A subgroup of patients with BDUC exhibited impaired thrombin generation under these conditions. Reduced thrombin generation was frequently associated with increased tissue factor pathway inhibitor (TFPI) activity, suggesting that excessive inhibition of TF-mediated coagulation contributes to the bleeding phenotype in a subset of patients. In vitro addition of concizumab or rFVIIa restored thrombin generation, indicating that defects in TF-dependent coagulation can be pharmacologically corrected.
CONCLUSIONS
These findings identify dysregulation of the TF-TFPI axis as a potential pathophysiological mechanism underlying bleeding in a subset of patients with BDUC and highlight TFPI inhibition and rFVIIa administration as potential therapeutic approaches. However, the absence of abnormalities in all patients supports the concept that BDUC represents a biologically heterogeneous group of disorders with multiple underlying mechanisms.
E. M. Manzano, P. Acuña, M. Martín Salces et al.· Journal of Thrombosis and Ha...· 0 citations
Venous thromboembolism (VTE) is multifactorial, and established hereditary risk factors explain only part of heritable risk. The protein C (PC) pathway is central to anticoagulant control, and common variants in thrombomodulin and endothelial protein C receptor genes (THBD, PROCR) have been proposed as modulators. In 73 participants, we used standardized in vivo coagulation activation with recombinant activated factor VII (15 µg/kg) and measured thrombin markers and activated protein C (APC) over 8 hours. Endothelial colony-forming cell-based ex vivo experiments were performed in 43 participants. In vivo, the APC area-under-the-curve (AUC)/thrombin-antithrombin complex (TAT) AUC ratio, reflecting the endogenous anticoagulant response relative to thrombin generation, provided the best model fit (adjusted R2=0.345). The APC response was lower in individuals with previous VTE (median 0.13 vs. 0.26; P=.009) and PROCR 655A>G carriers (0.11 vs. 0.26; P=.014), but higher in factor V Leiden (FVL) carriers (0.32 vs. 0.14; P=4.3×10-4) and THBD 1418C>T carriers (0.38 vs. 0.13; P=.032). The THBD 1418C>T effect was driven by reduced TAT AUC (30.6 vs. 82.7 pmol×h/L; P=.032), while the PROCR 655A>G effect reflected a lower APC AUC (8.6 vs. 10.7 pmol×h/L; P=0.04998). Ex vivo, the APC AUC/thrombin AUC ratio was lower with previous VTE and PROCR 655A>G, higher with FVL, and not associated with THBD 1418C>T. No in vivo or ex vivo association was observed for PROCR 4678C>G. This study provides in vivo evidence that common THBD and PROCR variants modulate PC pathway function, establishing the APC response as a sensitive endpoint for subtle genetic effects beyond FVL.
S. Reda, N. Schwarz, Sebastian Eckert et al.· Blood Advances· 0 citations