Presentation Details
| Intrinsic Xase Ligand Interactions Impact FVIIIa Regulation John J Morris1, 2, Nicole A Parsons2, Robert J Davidson1, Lindsey A George1, 2. 1Children's Hospital of Philadelphia, Philadelphia, PA, USA.2University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, USA |
Abstract
Background: Disease pathology resulting from states of deficient and excess (f)actor VIII, hemophilia A (HA) or prothrombotic risk, respectively, highlight the significance of maintaining activated FVIII (FVIIIa) function within a defined physiologic window. FVIIIa function is downregulated by spontaneous A2-domain dissociation and proteolytic cleavage by activated protein C (APC). Prior biochemical studies have demonstrated that A2 dissociation occurs rapidly with markedly slower kinetics of APC cleavage, supporting a lesser/marginal role of APC cleavage in FVIII/FVIIIa regulation. However, the contributions of these two mechanisms have historically been studied in purified systems, where the inherent instability of the A2 domain introduces experimental limitations. Further, these studies frequently lack FIXa and FX, which decrease A2 dissociation and APC cleavage, respectively. As a result, we hypothesize the physiologically relevant mechanisms that regulate FVIIIa are unclear, and the role of APC cleavage is underrepresented in prior work. Objectives: Evaluate how FIXa and FX ligand interactions with FVIIIa impact FVIIIa inactivation. Define how the fractional saturation of FVIIIa by FIXa impacts A2-domain stability. Demonstrate that APC cleavage meaningfully contributes to FVIIIa regulation in vivo. Methods: We generated a panel 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-QQVV). First, we studied FVIIIa inactivation with purified components varying the state of FVIIIa-FIXa saturation, ±APC, and ±FX to assess these ligand interactions on FVIIIa regulation. We then evaluated the impact of FIXa and FX on FVIIIa inactivation in plasma-based assays. Finally, we confirmed these findings in an in vivo tail clip injury model using HA/CD4KO mice. Results: In purified component assays, we found that the fractional saturation of FVIIIa by FIXa positively correlated with improved A2-domain stability, and the presence of FX reduced FVIII/FVIIIa APC cleavage, supporting that FVIIIa assembled in the intrinsic Xase is markedly stable. These results were replicated in thrombin generation assays where increasing FVIIIa saturation with FIXa improved A2-domain stability of FVIII-WT to that of an A2-stable FVIII variant, FVIII-VV, and the presence of FX blunted inactivation of FVIII-WT by APC, such that, in the presence of FX and APC, FVIII-WT activity was comparable to an APC-resistant FVIII variant, FVIII-QQ. The in vivo relevance of these findings was observed in a murine tail clip model. All FVIII variants with improved stability demonstrated improved hemostatic function compared to FVIII-WT (Figure 1A), suggesting that FVIIIa is not fully saturated with FIXa or FX in vivo. Notably, stabilizing the A2 domain had lesser hemostatic improvement (FVIII-VV, 2.4X) than inhibiting APC cleavage (FVIII-QQ, 5X) over FVIII-WT supporting that APC contributes to FVIIIa regulation in vivo. Administration of excess FX with FVIII demonstrated the same hemostatic effect as the corresponding protein with APC resistance, i.e., FVIII-WT and FVIII-VV hemostatic effect was analogous to FVIII-QQ and FVIII-QQVV, respectively (Figure 1B). Conclusions: These data suggest that both A2-domain dissociation and APC cleavage meaningfully contribute to FVIIIa regulation under physiologic conditions and highlight the potential benefit of FVIII transgenes resistant to inactivation for HA gene therapy.
No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, including photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author.
No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, including photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author.
Content Locked. Log into a registered attendee account to access this presentation.