Thromb Haemost 2013; 110(02): 275-282
DOI: 10.1160/TH12-12-0953
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Plasma protein S residues 37–50 mediate its binding to factor Va and inhibition of blood coagulation

Mary J. Heeb
1   Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA
,
Rolf M. Mesters
1   Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA
2   Present address: Department of Medicine/Hematology and Oncology, University of Muenster, Muenster, Germany
,
José A. Fernández
1   Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA
,
Tilman M. Hackeng
1   Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA
3   Present address: Department of Biochemistry, University of Maastricht, Maastricht, The Netherlands
,
Ryon K. Nakasone
1   Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA
,
John H. Griffin
1   Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA
› Institutsangaben

Financial support: This work was supported in part by National Institutes of Health grants RO1 HL088375 (MJH), RO1 HL21544 and RO1 HL 031950 (JHG).
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Publikationsverlauf

Received: 27. Dezember 2012

Accepted after major revision: 04. Mai 2013

Publikationsdatum:
04. Dezember 2017 (online)

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Summary

Protein S (PS) is an anticoagulant plasma protein whose deficiency is associated with increased risk of venous thrombosis. PS directly inhibits thrombin generation by the blood coagulation pathways by several mechanisms, including by binding coagulation factors (F) Va and Xa. To identify PS sequences that mediate inhibition of FVa activity, antibodies and synthetic peptides based on PS sequence were prepared and employed in plasma coagulation assays, purified component prothrombinase assays, binding assays, and immunoblots. In the absence of activated protein C, monoclonal antibody (Mab) S4 shortened FXa-induced clotting in normal plasma but not in PS-depleted plasma. Mab S4 also blocked PS inhibition of FVa-dependent prothrombinase activity in purified component assays in the absence or presence of phospholipids and inhibited binding of PS to immobilised FVa. Epitope mapping identified N-terminal region residues 37–67 of PS as this antibody’s epitope. A peptide representing PS residues 37–50 inhibited FVa-dependent prothrombinase activity in a noncompetitive manner, with 50% inhibition observed at 11 µM peptide, whereas a peptide with a D-amino acid sequence of 37–50 was ineffective. FVa, but not FXa, bound specifically to the immobilised peptide representing residues 37–50, and the peptide inhibited binding of FVa to immobilised PS. These data implicate PS residues 37–50 as a binding site for FVa that mediates, at least in part, the direct inhibition of FVa-dependent procoagulant activity by PS.