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

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

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

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