OC 73.5 - Delineating the Role of Physiologic Ligands on FVIIIa Inactivation

Background: FVIIIa is inactivated by A2-domain dissociation and APC/PS cleavage. Biochemical studies demonstrated rapid A2-dissociation with markedly slower kinetics of APC cleavage, supporting an anticipated lesser/marginal role of APC in FVIIIa regulation; however, we demonstrated APC contributes to in vivo FVIIIa regulation (Wilhelm et al., 2021). We hypothesized our in vivo studies captured FVIIIa interaction with physiologic ligands (e.g., FIXa) that impact FVIIIa regulation that are not/inadequately modeled in vitro.

Aims: Herein, we investigated the impact of FVIIIa saturation by FIXa on FVIIIa regulation.

Methods: We made a series of recombinant FVIII variants resistant to APC cleavage (FVIIIR336Q,R562Q; FVIII-QQ), A2-domain dissociation (FVIIID519V,E665V; FVIII-VV [Wakabayashi et al., JTH 2009]) or both (FVIII-VV/QQ). Proteins were characterized biochemically demonstrating, as expected, the same Km, < 2-fold Vmax variability and that stabilizing the A2-domain (FVIII-VV, FVIII-VV/QQ) improved specific activity 2-4-fold over FVIII-WT. We then developed a modified intrinsic Xase assay ±APC/PS (Figure 1) to study FVIIIa inactivation at varying states of FIXa saturation.

Results: FVIII variants with normal A2-domain stability (FVIII-WT and FVIII-QQ) demonstrated reduced rates of inactivation with increasing FIXa saturation wherein nearly 100% FIXa saturation resulted in undetectable FVIIIa inactivation by A2-domain dissociation. As expected, variants with A2-domain stability (FVIII-VV, FVIII-VV/QQ) demonstrated no detectable FVIIIa inactivation without APC/PS present (Figure 1A, C, E). With APC/PS present, the rate of APC inactivation of FVIII variants with intact APC cleavage sites (FVIII-VV, FVIII-WT) decreased with increasing FIXa saturation (Figure 1B, D, F). FVIII variants with mutated APC cleavage sites (FVIII-QQ, FVIII-VV/QQ) were not inactivated by APC/PS. Preliminary in vivo assessment demonstrated the following hemostatic function relationship between variant proteins FVIII-VV/QQ>FVIII-QQ>FVIII-VV>FVIII-WT.

Conclusion(s): Consistent with prior work, FVIIIa saturation by FIXa, impacts the rate and mechanism of FVIIIa inactivation. Specifically, FVIIIa saturated by FIXa is markedly stable, supporting improving FVIIIa affinity for FIXa could be exploited to develop novel HA therapeutics.