Contribution of factor VIII light-chain residues 2007–2016 to an activated protein C-interactive site

Masahiro Takeyama; Hironao Wakabayashi; Philip J. Fay

doi:10.1160/TH12-08-0561

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2013; 109(02): 187-198
DOI: 10.1160/TH12-08-0561

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Contribution of factor VIII light-chain residues 2007–2016 to an activated protein C-interactive site

Masahiro Takeyama

¹Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA

,

Hironao Wakabayashi

¹Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA

,

Philip J. Fay

¹Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA

› Author Affiliations

Abstract

Summary

Although factor (F) VIIIa is inactivated by activated protein C (APC) through cleavages in the FVIII heavy chain-derived A1 (Arg³³⁶) and A2 subunits (Arg⁵⁶²), the FVIII light chain (LC) contributes to catalysis by binding the enzyme. ELISA-based binding assays showed that FVIII and FVIII LC bound to immobilised active site-modified APC (DEGRAPC) (apparent K _d ~270 nM and 1.0 μM, respectively). Fluid-phase binding studies using fluorescence indicated an estimated K _d of ~590 nM for acrylodan-labelled LC binding to DEGR-APC. Furthermore, FVIII LC effectively competed with FVIIIa in blocking APC-catalysed cleavage at Arg³³⁶ (K _i = 709 nM). A binding site previously identified near the C-terminal end of the A3 domain (residues 2007–2016) of FVIII LC was subjected to Ala-scanning mutagenesis. FXa generation assays and western and dot blotting were employed to assess the contribution of these residues to FVIIIa interactions with APC. Virtually all variants tested showed reductions in the rates of APC-catalysed inactivation of the cofactor and cleavage at the primary inactivation site (Arg³³⁶), with maximal reductions in inactivation rates (~3-fold relative to WT) and cleavage rates (~3 to ~9-fold relative to WT) observed for the Met2010Ala, Ser2011Ala, and Leu2013Ala variants. Titration of FVIIIa substrate concentration monitoring cleavage by a dot blot assay indicated that these variants also showed ~3-fold increases relative to WT while a double mutant (Met2010Ala/Ser2011Ala) showed a >4-fold increase in K _m. These results show a contribution of a number of residues within the 2007–2016 sequence, and in particular residues Met2010, Ser2011, and Leu2013 to an APC-interactive site.

Keywords

Factor VIIIa - activated protein C - factor VIII mutants - proteolysis

Full Text

References

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