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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   #
1   Department of Chemistry, Cleveland State University, Cleveland, Ohio, USA
,
Michael A. Bukys
1   Department of Chemistry, Cleveland State University, Cleveland, Ohio, USA
,
Thomas Orfeo
2   Department of Biochemistry, College of Medicine, University of Vermont, Burlington, Vermont, USA
,
Kenneth G. Mann
2   Department of Biochemistry, College of Medicine, University of Vermont, Burlington, Vermont, USA
,
Michael Kalafatis
1   Department of Chemistry, Cleveland State University, Cleveland, Ohio, USA
3   Department of Molecular Cardiology, Lerner Research Institute, The Cleveland Clinic, Cleveland, Ohio, USA
› Author AffiliationsFinancial support: This work was supported by Predoctoral Fellowship 0515132B from the American Heart Association Ohio Valley Affiliate (to EE), by Predoctoral Fellowship from the Molecular and Cellular Medicine Specialization at Cleveland State University (to MAB), and by R01 grants HL-34575 (to KGM) and HL-73343 (to MK) from the National Heart Lung and Blood Institutes.
Further Information

Publication History

Received 28 June 2007

Accepted after resubmission 24 August 2007

Publication Date:
30 November 2017 (online)

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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 Mr 97,000 fragment containing the NH2-terminal portion of the heavy chain and a Mr 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 (FVR643Q) and R643→A (FVR643A). All recombinant molecules were purified to homogeneity and assayed for activity following extended activation with α-thrombin. Under similar experimental conditions, appearance of the Mr 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 FVR643Q and FVR643A with α-thrombin no cleavage of the heavy chain at Arg643 was detected and no presence of the Mr 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

# Present address: ARUP Laboratories 500, Chipeta Way, Salt Lake City, UT 84108, USA.


 

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