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Activated FVIII Inactivation Occurs Outside of the Intrinsic Xase Complex

A. Wilhelm1, L. George1,2,3

1The Children's Hospital of Philadelphia, Philadelphia, United States, 2The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Philadelphia, United States, 3Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States

Abstract Number: LPB0053

Meeting: ISTH 2021 Congress

Theme: Coagulation and Natural Anticoagulants » FVIII/IX

Background: The FVIIIa heterotrimer is stabilized by weak, electrostatic interactions. Inactivation of FVIIIa is due to either spontaneous A2-domain dissociation or activated protein C (APC) cleavage. Previous work demonstrated FIXa and lipids stabilize the A2-domain. Additionally, FIXa and FX have been shown to protect FVIIIa from APC cleavage. Importantly, many of these studies used FVIIIa concentrations above the Kd of A2-domain dissociation (200-300 nM) and FIXa concentrations where FVIIIa/FIXa is 100% bound. Collectively this suggests FVIIIa inactivation would be marginal when bound within the intrinsic Xase; however, given that the plasma concentration of FVIII (1nM) is below the Kd of FVIIIa-A2-domain dissociation and FIXa binding, the physiologic significance of these observations is unclear.

Aims: We sought to evaluate the stability and inactivation of FVIIIa within the intrinsic Xase.

Methods: FVIII was activated with thrombin and quenched with hirudin. FVIIIa was then incubated with active-site-blocked FIXa (FIXa-PPACK) at varying concentrations (0-5 nM), FX, and lipids in the presence or absence of APC and Protein S (PS). Inactivation of FVIIIa was then determined using a FXa generation assay. Samples were evaluated by western blot to identify APC cleavage fragments. FIXa-PPACK concentrations were chosen to approximate 0-100% bound FVIIIa according to the reported Kd of 2 nM (Table 1).

Results: When 90% FVIIIa was bound within the Xase, no loss in activity was observed with or without APC/PS present (Figure 1). In the absence of FIXa-PPACK, FVIIIa was sensitive to inactivation by A2-domain dissociation and APC cleavage (confirmed by western blot). When only 15% of FVIIIa was bound within the Xase, intermediate FVIIIa inactivation and Xase stabilizing effect were observed.

Residual FVIIIa inactivation over time in the presence or absence of APC and PS evaluated by FXa generation.

FVIIIa (nM) FIXa-PPACK (nM) FVIIIa bound (%)
2 0 0
2 0.5 12
2 5 90

Approximate fraction of FVIIIa bound within the intrinsic Xase enzyme complex assuming a Kd of 2 nM for FVIIIa and FIXa/lipids

Conclusions: ​​​​​Irrespective of the mechanism of inactivation, FVIIIa is remarkably stable when bound to FIXa-PPACK, FX, and lipids. These data support FVIIIa inactivation occurs largely or completely outside the intrinsic Xase enzyme complex.

To cite this abstract in AMA style:

Wilhelm A, George L. Activated FVIII Inactivation Occurs Outside of the Intrinsic Xase Complex [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/activated-fviii-inactivation-occurs-outside-of-the-intrinsic-xase-complex/. Accessed December 6, 2023.

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