Identification of an inactivating cleavage site for α-thrombin on the heavy chain of factor Va

Evrim Erdogan; Michael A. Bukys; Thomas Orfeo; Kenneth G. Mann; Michael Kalafatis

doi:10.1160/TH07-06-0431

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2007; 98(05): 998-1006
DOI: 10.1160/TH07-06-0431

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Identification of an inactivating cleavage site for α-thrombin on the heavy chain of factor Va

Evrim Erdogan^#

¹Department of Chemistry, Cleveland State University, Cleveland, Ohio, USA

,

Michael A. Bukys

¹Department of Chemistry, Cleveland State University, Cleveland, Ohio, USA

,

Thomas Orfeo

²Department of Biochemistry, College of Medicine, University of Vermont, Burlington, Vermont, USA

,

Kenneth G. Mann

²Department of Biochemistry, College of Medicine, University of Vermont, Burlington, Vermont, USA

,

Michael Kalafatis

¹Department of Chemistry, Cleveland State University, Cleveland, Ohio, USA

³Department of Molecular Cardiology, Lerner Research Institute, The Cleveland Clinic, Cleveland, Ohio, USA

› Author Affiliations

Abstract

Summary

Previous studies of factor (F)Va inactivation on human umbilical vein endothelial cells have shown that α-thrombin cleaves the heavy chain near the COOH-terminus to produce a M_r 97,000 fragment containing the NH₂-terminal portion of the heavy chain and a M_r 8,000 peptide containing the rest of the molecule. The α-thrombin cleavage appeared to occur between amino acid residues 586 and 654 of FV. This region contains a consensus sequence for α-thrombin cleavage located at residues 640–644 (S-S-P-R-S). To test the hypothesis that α-thrombin cleaves the FVa heavy chain at Arg⁶⁴³ and to evaluate the functional importance of this cleavage for FVa inactivation, sitedirected mutagenesis was used to create recombinant FV molecules with mutations R⁶⁴³→Q (FV^R643Q) and R⁶⁴³→A (FV^R643A). All recombinant molecules were purified to homogeneity and assayed for activity following extended activation with α-thrombin. Under similar experimental conditions, appearance of the M_r 97,000 heavy chain fragment in the plasma and wild-type FVa molecules correlated with partial loss of cofactor activity, while following extended incubation of FV^R643Q and FVR⁶⁴³A with α-thrombin no cleavage of the heavy chain at Arg⁶⁴³ was detected and no presence of the M_r 97,000 heavy-chain fragment was noticed. Further, no loss in cofactor activity was observed using these mutant recombinant FVa molecules. Our data demonstrate that cleavage of FVa at Arg⁶⁴³ by α-thrombin results in a partially inactive cofactor molecule and provides for an activated protein C (APC)-independent anticoagulant effect of α-thrombin.

Keywords

Factor Va - α-thrombin - inactivation - heavy chain - coagulation

Full Text

References

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