Thrombin generation assay using factor XIa to measure factors VIII and IX and their glycoPEGylated derivatives is robust and sensitive

Abstract

To cite this article: Waters EK, Hilden I, Sørensen BB, Ezban M, Holm PK. Thrombin generation assay using factor XIa to measure factors VIII and IX and their glycoPEGylated derivatives is robust and sensitive. J Thromb Haemost 2015; 13: 2041-52.Summary. Background: Conventional coagulation factor assays are associated with certain limitations, as they do not always reflect the clinical heterogeneity of bleeding in hemophilic patients or correctly reflect the individual patient response to treatment with bypassing agents or novel factor concentrates. The thrombin generation assay (TGA) is currently being assessed as a possible method for characterizing bleeding phenotypes in individuals with hemophilia. Objectives: This study assessed the robustness and sensitivity of the TGA for measuring the activity of recombinant factor VIII (rFVIII), recombinant factor IX (rFIX) and their glycoPEGylated derivatives, N8-GP and N9-GP, in vitro. Methods: Factor-deficient plasma was spiked with 0.13-130 IU dL À1 rFVIII or N8-GP (hemophilia A [HA] plasma), or rFIX or N9-GP (hemophilia B [HB] plasma). A calibrated automated thrombogram triggered with tissue factor (TF) or activated FXI (FXIa) was used to measure thrombin generation over time. Endogenous thrombin potential, peak thrombin, velocity index, lag time and time to peak thrombin were analyzed. Results: FXIa-triggered assays were not affected by glycoPEGylation and were sufficiently sensitive to differentiate between spiked samples mimicking severe and moderate HB and HA; TF-triggered assays were not sufficiently sensitive for this distinction in HA. Both FXIa-triggered and TF-triggered assays had an acceptable level of variability (≤ 20%), although TF-triggered assays were associated with greater variability. Conclusions: FXIa-triggered TGA reactions produced more robust and sensitive results than TF-triggered TGA reactions, and have the potential for use in monitoring patients treated with glycoPEGylated or non-PEGylated coagulation factor concentrates. These promising results merit confirmation with clinical samples to correlate in vitro and in vivo data.

Optimization of the thrombin generation test components to measure potency of factor VIII concentrates

“…In addition, we found that FXIa-activated TGT is equally suitable for evaluation of FIX-deficiency and the assignment of potency of FIX-containing concentrates (manuscript in preparation). In agreement with our study, Waters et al found that a FXIa-activated TGT is more robust and sensitive than traditional TF-triggered TGT when applied for evaluation of FVIII-and FIX-spiked haemophilia A and B plasma samples [44]. Unlike Waters et al who added FXIa to the lipid reagent, we found that our TGT assay variant can be adapted from a commercially available TGT kit by spiking the substrate and calcium chloride reagent, e.g.…”

Concizumab, an anti‐tissue factor pathway inhibitor antibody, induces increased thrombin generation in plasma from haemophilia patients and healthy subjects measured by the thrombin generation assay

“…The thrombin generation assay (TGA) is a global haemostatic assay that provides a more complete picture of the coagulation process [8,9].…”

Evaluating the thrombin generation profiles of four different rFVIII products in FVIII‐deficient plasma using FIXa and FXIa activation

“…By studying these two activators in parallel, the differences in TGA sensitivity can be teased out. The concentrations used for FIXa and FXIa TGA activation in this study (ie 5 nmol/L FIXa and 0.22 nmol/L FXIa) have been studied individually by members of our research team . The FIXa concentration used in this study was significantly lower compared to data from McIntosh et al (14 nmol/L FIXa), while the FXIa concentration was higher than that studied by Jha et al who used 15‐100 pM FXIa + 0.5 pM TF for TGA activation.…”

“…In haemophilia A plasma, activating the TGA using the intrinsic pathway with either FIXa or FXIa can make the assay highly sensitive to FVIII concentrations < 1%. Previous studies have shown that using either FIXa or FXIa activation, the TGA was more accurate and sensitive to FVIII levels well below 1% . However, no studies have compared FIXa and FXIa TGA activation in a parallel fashion.…”