Topological Studies of the Amino Terminal Modules of Vitamin K-dependent Protein S Using Monoclonal Antibody Epitope Mapping and Molecular Modeling

Tusar K. Giri; Bruno O. Villoutreix; Anders Wallqvist; Björn Dahlbäck; Pablo García de Frutos

doi:10.1055/s-0037-1615361

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1998; 80(05): 798-804
DOI: 10.1055/s-0037-1615361

Review Article

Schattauer GmbH

Topological Studies of the Amino Terminal Modules of Vitamin K-dependent Protein S Using Monoclonal Antibody Epitope Mapping and Molecular Modeling

Tusar K. Giri

¹Wright-Rieman Laboratories, Chemistry, Rutgers University, Piscataway, New Jersey, USA

,

Bruno O. Villoutreix

¹Wright-Rieman Laboratories, Chemistry, Rutgers University, Piscataway, New Jersey, USA

,

Anders Wallqvist

¹From the Department of Clinical Chemistry, Lund University, University Hospital, Malmö, Sweden

,

Björn Dahlbäck

¹Wright-Rieman Laboratories, Chemistry, Rutgers University, Piscataway, New Jersey, USA

,

Pablo García de Frutos

¹Wright-Rieman Laboratories, Chemistry, Rutgers University, Piscataway, New Jersey, USA

› Author Affiliations

Abstract

Summary

Protein S is an important anticoagulant protein acting as cofactor to activated protein C (APC) in the degradation of membrane-bound factors Va and VIIIa. Binding of protein S to the membrane depends on the Gla-domain, whereas sites for APC-interaction are located in the thrombin-sensitive region (TSR) and the first EGF domain. The aims of the present investigation were to localize the sites on protein S which are involved in APC-cofactor function and to elucidate possible orientations of the TSR in relation to the membrane. For these purposes, we determined the epitope for a calcium-dependent monoclonal antibody (HPS67) against the TSR, which inhibits APC cofactor activity even though it does not impede protein S binding to the membrane. HPS67 did not recognize wild-type mouse protein S but gained reactivity against a recombinant mouse protein in which G49 and R52 were mutated to R and Q (found in human protein S), respectively, suggesting these two residues to be part of a surface exposed epitope for HPS67. This information helped in the validation and refinement of the structural model for the Gla-TSR-EGF1-modules of protein S. The X-ray structure of a Fab-fragment mimicking HPS67 was docked onto the protein S model. The observation that HPS67 did not inhibit phospholipid binding of protein S has implications for the possible orientation of protein S on the membrane surface. In the proposed model for membrane-bound protein S, there is no contact between the TSR and the membrane. Rather, the TSR is free to interact with membrane-bound APC.

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References

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