VPB0207 - Factor VIII K1813A has high affinity for activated factor IX and enhances global coagulation function

Sunday, July 10, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: Virtual

Presenting Author(s)

Yuto Nakajima, MD PhD

assistant professor
Nara medical University
Kashihara, Nara, Japan

Co-Author(s)

Masahiro Takeyama, MD, PhD

Associate Professor
Nara Medical University
Kashihara, Nara, Japan
AO

Akihisa Oda

assistant professor
Department of Pediatrics, Nara medical university
Kashihara, Nara, Japan
KN

Keiji Nogami

Professor
Nara Medical University, Nara, Japan
Nara, Nara, Japan

Background: Factor (F) VIII functions as a cofactor in the tenase complex responsible for phospholipid surface-dependent conversion of FX to activated FX (FXa) by FIXa. Previous studies demonstrated the 1811-1818 region in FVIII A3 domain contributed to FIXa binding and that the binding affinity of K1813A/K1818A for FIXa was increased. We hypothesized that FVIII mutants with higher affinity for FIXa could enhance procoagulant activity.

Aims: To create gain-of-function FVIII mutant.

Methods: We prepared several FVIII mutants (K1813A, K1818A, and K1813A/K1818A) by BHK cells and evaluated these mutants by SPR-based assay, FVIII activity, thrombin generation assay (TGA), rotational thromboelastmetry (ROTEM) and hemophilia A mouse.

Results: The binding affinities of K1818A (Kd; 5.1±1.8 nM) and K1813A/K1818A (Kd; 4.7±1.2 nM) for FIXa on SPR-based assays were similar to wild type (WT) FVIII (Kd; 6.3±0.3 nM), however, that of K1813A (Kd; 3.9± 0.7 nM) was increased compared to WT. Peak FVIII activity of K1818A and K1813A/K1818A activated by thrombin was ~30% of WT. On the other hand, activation of K1813A by thrombin was comparable to those of WT, and decay rates of K1813A after peak thrombin activation was 2-fold slower than that of WT. As for FVIII stability at 55℃, the decay rate for K1813A was 2.5-fold improved relative to WT, indicating that K1813A was reduced in FVIII A2 dissociation. Tissue factor-triggered TGA revealed that 0.5 nM of K1813A showed similar peak thrombin and endogenous thrombin potential in 1 nM of WT. The ex vivo addition of K1813A (0.5nM) to whole blood from the patient with hemophilia A also showed almost same coagulation potential as that of WT (1nM) in ROTEM. In the tail clip assay of hemophilia A mouse, K1813A showed approximately 4-fold increased procoagulant function relative to WT.

Conclusion(s): K1813A enhanced global coagulation potential due to stability and the higher FIXa affinity.