Abstract Number: PB1155
Meeting: ISTH 2020 Congress
Background: Previously, factor (F)IX variants have been generated that function independently of the cofactor VIIIa (FIX-IDAV, FIX-FIAV) or demonstrate FIX hyperactivity (FIX-KLW) and ameliorate the hemophilia phenotype in vitro/vivo [Quade-Lyssy et al. J.Thromb.Haemost. 2014].
Aims: Assess the FVIIIa-independent FIX activity of variants comprising the IDAV/FIAV and KLW modifications and evaluate the FVIII-bypass efficacy and biochemical characteristics.
Methods: Recombinant FIX variants were stably expressed, and (partially) purified variants were evaluated for specific clotting activity, kinetic tissue factor-factor VIIa (TF-FVIIa) activation, and FVIII-bypass efficacy.
Results: Specific FVIII clotting activity analysis of partially purified FIX variants demonstrated a ~2-fold increase in FVIII-like activity for FIX-IDAV-KLW and a ~2-fold reduction for FIX-FIAV-KLW relative to FIX-IDAV/FIX-FIAV. This indicates no distinct synergistic effect of the combined modifications on FVIII-like activity. Conversely, the FIX-IDAV/FIAV-KLW variants demonstrated a 9-14-fold increase in specific FIX clotting activity relative to wild-type FIX(-WT), analogous to the hyperactivity of FIX-KLW. FIX-FIAV displayed a favorable profile (high FVIII-bypass and normal FIX activity) and was selected for further evaluation following purification. TF-FVIIa activation revealed kinetic parameters like FIX-WT, and FIX-FIAV exhibited significant FVIII-like clotting activity (56±4U/mg) compared to FIX-WT (≤13U/mg). Values correlate with ≤28% of FVIII-independent activity for FIX-FIAV at FIX plasma levels (5ug/mL), confirming FIX-FIAV may enhance thrombin generation in FVIII deficiency. TF-initiated (0.5 or 1.0pM) thrombin generation in FVIII-immunodepleted plasma led to a reduced thrombin peak (88% or 81% reduction) relative to 100% FVIII. Adding FIX-FIAV (5ug/mL) partially restored these parameters, with a ~30% increase in thrombin peak and endogenous thrombin potential. FVIII-independent activity of FIX-FIAV in severe hemophilia A (HA) patient plasma w/wo an inhibitor provided up to 18% or 32% FVIII-like activity, demonstrating efficacy in the presence of FVIII inhibitors.
Conclusions: FIX-FIAV has a mechanism of activation capable of sustaining therapeutic levels of coagulation activity in FVIII deficiency, supporting its potential as a treatment for HA.
To cite this abstract in AMA style:Strijbis VJF, Cheung KL, Konstantinova P, Liu YP, van Deventer S, Bos MHA. Characterization of Factor IX-FIAV: A Factor IX Variant that Displays Cofactor-Independent Activity [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/characterization-of-factor-ix-fiav-a-factor-ix-variant-that-displays-cofactor-independent-activity/. Accessed January 28, 2022.
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