Identification of a protein S-interactive site within the A2 domain of the factor VIII heavy chain

Masahiro Takeyama; Keiji Nogami; Evgueni L. Saenko; Katsumi Nishiya; Kenichi Ogiwara; Midori Shima

doi:10.1160/TH09-03-0152

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2009; 102(04): 645-655
DOI: 10.1160/TH09-03-0152

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Identification of a protein S-interactive site within the A2 domain of the factor VIII heavy chain

Authors

Masahiro Takeyama

¹Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan
Keiji Nogami

¹Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan
Evgueni L. Saenko

²Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA
Katsumi Nishiya

¹Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan
Kenichi Ogiwara

¹Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan
Midori Shima

¹Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan

Abstract

Summary

We have recently demonstrated that protein S impairs the intrinsic tenase complex, independent of activated protein C, in competitive interactions between the A2 and A3 domains of factor VIIIa and factor IXa. In the present study, we have identified a protein S-interactive site in the A2 domain of factor VIIIa. Anti-A2 monoclonal antibody recognising a factor IXa-functional region (residues 484–509) on A2, and synthetic peptide inhibited the A2 binding to protein S by ∼60% and ∼70%, respectively, in solid-phase binding assays. The 484–509 peptide directly bound to protein S dose-dependently. Covalent cross-linking was observed between the 484–509 peptide and protein S following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide). The cross-linked adduct was consistent with 1:1 stoichiometry of reactants. Cross-linking formation was blocked by addition of the 484–497 peptide, but not by the 498–509 peptide. Furthermore, N-terminal sequence analysis of the 484–509 peptide-protein S adduct showed that three sequential residues (S488, R489, and R490) in A2 were not identified, suggesting that these residues participate in cross-link formation. Mutant A2 molecules where these residues were converted to alanine were evaluated for the binding of protein S. The S488A, R489A, and R490A mutants demonstrated ∼four-fold lower affinity than wild-type A2.These results indicate that the 484–509 region in the A2 domain of factor VIIIa, in particular sequential residues at positions 488–490, contributes to a unique protein S-interactive site.

Keywords

Factor VIII(a) - protein S - A2 domain - binding-site - EDC crosslinking

Full Text

References

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