Abstract Number: PB0133
Meeting: ISTH 2021 Congress
Background: Factor X (FX) is proteolytically activated by the Tissue Factor (TF) and Factor VIIa (FVIIa) complex on membrane surfaces to initiate the clotting cascade. The N-terminal γ-carboxyglutamate-rich domain (Gla domain) of FX enables it to bind membranes. Besides membrane interactions, several studies have shown that the FX Gla-domain is also required for protein-protein interactions with the TF/FVIIa complex. However, the exact residues in FX-Gla domain engaging with (TF/FVIIa) remain unclear.
Aims: To elucidate the molecular interactions between FX Gla-domain residues and TF/FVIIa.
Methods: 12 FX Gla-domain predicted by molecular dynamics simulation studies to interact with phosphatidylserine (PS) headgroups were mutated and expressed in HEK293 cells. Influence of the FX Gla-domain mutations were tested on rates of FX activation by TF/FVIIa in solution or on PS-containing membrane surfaces. Further, membrane-specific interactions were evaluated by quantifying membrane binding affinities using Nanodiscs immobilized on microring resonators.
Results: Mutating Lys-9, Arg-15, Gla-25, Arg-28 or Gla-32 had little effect on the rates of FX activation by soluble TF/FVIIa complex, while mutating Gla-7, Gla-20 or Gla-29 reduced the rates by 50-80%. For membrane-bound TF/FVIIa, mutating Gla-7, Gla-14, Gla-19, Gla-20, Gla-25, Gla-29 or Gla-32 reduced FX activation rates by ~90% whereas mutating Lys-9, Lys-10, Arg-15 or Arg-28 reduced it by ~40%. Membrane binding affinity of FX was impaired upon mutating Lys-9, Lys-10, Gla-19, Arg-28 or Gla-32, while membrane binding was undetectable upon mutating Gla-7, Gla-14, Gla-20, Gla-25 or Gla-29. We found a positive correlation (R2=0.685) between FX membrane binding affinity and FX activation rates by membrane-bound TF/FVIIa.
Conclusions: Our study shows that Lys-9, Lys-10, Arg-15, Gla-19, Gla-25, Arg-28 and Gla-32 are most critical for the membrane binding affinity of the FX Gla-domain. However, Gla-7, Gla-14, Gla-20 and Gla-29 are crucial residues in FX Gla-domain that participate in both protein-membrane and protein-protein interactions with the TF/FVIIa.
To cite this abstract in AMA style:Paul D, Medfisch SM, Bailey RC, Morrissey JH. Mutagenesis Study Reveals Factor X Gla-domain Residues Critical for Initiation of the Clotting Cascade [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 1). https://abstracts.isth.org/abstract/mutagenesis-study-reveals-factor-x-gla-domain-residues-critical-for-initiation-of-the-clotting-cascade/. Accessed September 24, 2021.
« Back to ISTH 2021 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/mutagenesis-study-reveals-factor-x-gla-domain-residues-critical-for-initiation-of-the-clotting-cascade/