Identification of an inactivating cleavage site for alpha-thrombin on the heavy chain of factor Va.
Previous studies of factor (F)Va inactivation on human umbilical vein endothelial cells have shown that alpha-thrombin cleaves the heavy chain near the COOH-terminus to produce a M(r) 97,000 fragment containing the NH(2)-terminal portion of the heavy chain and a M(r) 8,000 peptide containing the rest of the molecule. The alpha-thrombin cleavage appeared to occur between amino acid residues 586 and 654 of FV. This region contains a consensus sequence for alpha-thrombin cleavage located at residues 640-644 (S-S-P-R-S). To test the hypothesis that alpha-thrombin cleaves the FVa heavy chain at Arg(643) and to evaluate the functional importance of this cleavage for FVa inactivation, site-directed mutagenesis was used to create recombinant FV molecules with mutations R(643) --> Q (FV(R643Q)) and R(643) --> A (FV(R643A)). All recombinant molecules were purified to homogeneity and assayed for activity following extended activation with alpha-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 FV(R643A) with alpha-thrombin no cleavage of the heavy chain at Arg(643) 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 Arg(643) by alpha-thrombin results in a partially inactive cofactor molecule and provides for an activated protein C (APC)-independent anticoagulant effect of alpha-thrombin.
Erdogan
Evrim
E
Department of Chemistry, Cleveland State University, 2351 Euclid Avenue, Science and Research Center SR 370, Cleveland, Ohio 44115, USA.
Bukys
Michael A
MA
Orfeo
Thomas
T
Mann
Kenneth G
KG
Kalafatis
Michael
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine; D001665:Binding Sites; D001780:Blood Coagulation Tests; D015943:Factor Va / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits; D013917:Thrombin / Q000378:metabolism / Q000502:physiology*
D021122:Protein Subunits; D015943:Factor Va; D001120:Arginine; D013917:Thrombin