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Proceedings/Recueil Des Communications Research and Practice in Thrombosis and Haemostasis Année : 2019

Thrombin-mediated activation of factor VIII is insufficient to produce all necessary cofactor activity in vivo

Charlotte Kawecki
  • Fonction : Auteur
Ivan Peyron
  • Fonction : Auteur
Amine Bazaa
  • Fonction : Auteur
Vincent Muczynski
  • Fonction : Auteur
Stephen Ferrière
  • Fonction : Auteur
Cécile Loubière
  • Fonction : Auteur
Amélie Harel
  • Fonction : Auteur
Cécile Denis
Olivier Christophe
  • Fonction : Auteur
  • PersonId : 1351519
Peter J. Lenting

Résumé

Background Factor VIII (FVIII) is essential for optimal coagulation, enhancing Factor Xa (FXa) generation by factor IXa (FIXa). To become functional, FVIII requires proteolytic activation, involving cleavage of key residues within its heavy and light chain (HC & LC). Although well-studied in vitro, the relative roles of HC and LC cleavages in vivo remain elusive. Particularly, the contribution of FIXa and FXa to FVIII activation remains unknown. Aim To dissect the relative contribution of FVIII activation sites sensitive to thrombin, FIXa and/or FXa for FVIIIa cofactor activity in vivo. Methods FVIII mutants with defective HC and/or LC activation sites were generated. Pro-coagulant activity of these mutants was determined by measuring bleeding times in mice following hydrodynamic gene-transfer and was compared to in vitro activity values. Results Mutation of the FVIII-HC activation site (p.R372A) resulted in a complete lack of cofactor activity in a chromogenic assay, whereas 2-6% residual activity was detected using a one-stage and a thrombin generation assay. However, hemophilic mice expressing FVIII-p.R372A displayed similar blood loss as mock-treated mice. Combined mutations of proteolytic sites in FVIII-LC (p.R1689T-p.R1719T-p.R1721T) decreased FVIII activity by 85%. Indeed, expression of this mutant was associated with increased bleeding tendency. Finally, a mutant selectively lacking proteolytic sites for FIXa and FXa (p.R1719T-p.R1721T) was as active as WT-FVIII in vitro but proved inadequate to fully correct bleeding in FVIII-deficient mice (Figure 1). Conclusions Our data indicate that thrombin-mediated activation of FVIII is insufficient to induce full cofactor activity. Consequently, thrombin-activated FVIII fails to restore hemostasis in hemophilic mice. This points to a physiologically relevant role for FVIII-LC proteolysis by FIXa and/or FXa in the generation of FVIII pro-coagulant activity.

Domaines

Hématologie

Dates et versions

hal-04461945 , version 1 (16-02-2024)

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Charlotte Kawecki, Ivan Peyron, Amine Bazaa, Vincent Muczynski, Stephen Ferrière, et al.. Thrombin-mediated activation of factor VIII is insufficient to produce all necessary cofactor activity in vivo. Congress of the International Society of Thrombosis and Haemostasis, Research and Practice in Thrombosis and Haemostasis, 3, pp.1-891, 2019, ⟨10.1002/rth2.12229⟩. ⟨hal-04461945⟩
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