Abstract Number: LPB0053
Meeting: ISTH 2021 Congress
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.
|FVIIIa (nM)||FIXa-PPACK (nM)||FVIIIa bound (%)|
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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