Presentation Details
| Enhanced activation of FVIII positively impacts clot formation in vivo using hemophilia A mice Lacramioara Ivanciu1, 2, Mettine H.A.Bos1, Rodney M.Camire1, 2. 1The Children's Hospital of Philadelphia, Division of Hematology and the Raymond G.Perelman Center for Cellular and Molecular Therapeutics, Philadelphia, PA, USA.2University of Pennsylvania, Department of Pediatrics, Philadelphia, PA, USA |
Abstract
Background: Coagulation factor VIII (FVIII) circulates in blood as an inactive procofactor bound to von Willebrand Factor (vWF). Proteolytic cleavage by FXa and/or thrombin (Arg372, Arg740, and Arg1689) converts FVIII to its active form FVIIIa and released it from vWF. Biochemical studies have examined the roles of the individual FVIII activation sites; however, the importance of these cleavage sites in the regulation of FVIIIa cofactor activity in vivo is not understood. Objective: To better understand the role of individual activation sites to the development of FVIIIa cofactor activity and thus, clot formation at the site of vascular injury. Methods: We used a FVIII derivative, FVIII-2RKR that only comprises the Arg372 site to investigate the impact of eliminating Arg740 and Arg1689 on FVIII activity at the site of vascular injury using hemophilia A (HA) mouse models. Results: FVIII-2RKR is a heterodimer, that lacks the B- and a3 domains and thus cannot detectably bind to vWF. In a one-stage aPTT clotting assay, FVIII-2RKR demonstrated ~7-fold greater specific activity compared to FVIII-SQ. Consistent with this, FVIII-2RKR had a shortened lag time in a thrombin generation assay compared to FVIII-SQ. We initiated in vivo experiments with FVIII-2RKR using HA mice to explore its hemostatic performance using three injury models. In a ferric chloride (FeCl3)-induced injury model of the carotid artery two different experiments were conducted in which FVIII-2RKR (n=5; 1, 5 or 10 ug/kg) or FVIII-SQ (n=5; 5,10 or 50 ug/kg) were infused into HA mice either 10 minutes after or prior to injury. In either experiment, faster vessel occlusion was observed with FVIII-2RKR compared to FVIII-SQ reaching statistical significance at 5 ug/kg (p<0.004; 2RKR vs. SQ). Notably, infusion of FVIII-2RKR normalizes the time to occlusion using ~10-fold lower dose compared to FVIII-SQ. This is remarkable, considering that in vivo clearance of FVIII-2RKR (n=3, 40 ug/kg) was faster than FVIII-SQ due to impaired vWF binding. A FVIII variant (FVIII-2RKR-R372Q) that does not bind vWF and lacks procoagulant activity failed to induce vessel occlusion (n=5; 50 ug/kg). FVIII-2RKR (n=3; 1ug/kg) was also more efficacious in reducing bleeding in HA mice than FVIII-SQ (n=3; 1ug/kg) in a tail vein transection model. Based on biochemical experiments, the increased hemostatic potency of FVIII-2RKR is likely due to accelerated cleavage at Arg372 resulting in rapid FXa generation at the injury site. To test this further, we employed the cremaster muscle laser-induced injury model and compared the kinetics of thrombus formation in HA mice injected with FVIII proteins. Significantly greater accumulation of platelet and fibrin was observed with FVIII-2RKR than with FVIII-SQ (1ug/kg; n=3 mice; 15 injuries; p<0.0001). Quantitative analysis revealed that FVIII-2RKR increased platelet accumulation 2-3-fold and fibrin deposition 3-6-fold over FVIII-SQ. Conclusions: Overall, our data indicate that proteolysis of FVIII at Arg740 and Arg1689 does not contribute in a direct way to expression of cofactor activity. Rather these cleavages appear to somehow impair cleavage at Arg372. The work also shows that accelerated activation of FVIII leads to enhance hemostatic potential using multiple injury models in vivo.
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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.