Coagulation factor Va Glu-96-Asp-111: a chelator-sensitive site involved in function and subunit association

Abstract

Coagulation FVa (factor Va) accelerates the essential generation of thrombin by FXa (factor Xa). Although the noncovalent Ca2+-dependent association between the FVa light and heavy subunits (FVaL and FVaH) is required for function, little is known about the specific residues involved. Previous fragmentation studies and homology modelling led us to investigate the contribution of Leu-94-Asp-112. Including prospective divalent cation-binding acidic amino acids, nine conserved residues were individually replaced with Ala in the recombinant B-domainless FVa precursor (DeltaFV). While mutation of Thr-104, Glu-108, Asp-112 or Tyr-100 resulted in only minor changes to FXa-mediated thrombin generation, the functions of E96A (81%), D111A (70%) and D102A (60%) mutants (where the single-letter amino acid code is used) were notably reduced. The mutants targeting neighbouring acidic residues, Asp-79 and Glu-119, had activity comparable with DeltaFV, supporting the specific involvement of select residues. Providing a basis for reduced activity, thrombin treatment of D111A resulted in spontaneous dissociation of subunits. Since FVaH and FVaL derived from E96A or D102A remained associated in the presence of Ca2+, like the wild type, but conversely dissociated rapidly upon chelation, a subtle difference in divalent cation co-ordination is implied. Subunit interactions for all other single-point mutants resembled the wild type. These data, along with corroborating multipoint mutants, reveal Asp-111 as essential for FVa subunit association. Although Glu-96 and Asp-102 can be mutated without gross changes to divalent cation-dependent FVaH-FVaL interactions, they too are required for optimal function. Thus Glu-96-Asp-111 imparts at least two discernible effects on FVa coagulation activity.

The crystal structure of activated protein C-inactivated bovine factor Va: Implications for cofactor function

“…We believe that Ca 2ϩ orders a critical loop within the A1 domain to allow for constructive interactions between the A1 and A3 domains. This hypothesis is supported by mutational studies of residues within this loop, as exemplified by the E96A mutation in factor Va, where the two chains remain associated in the presence of Ca ϩ2 yet show a reduced cofactor activity (37). In our structure, Glu-96 does not participate in Ca ϩ2 binding but instead interacts with the A3 domain.…”

“…These ligands include the side chains of both Asp-111 and Asp-112, along with the main chain carbonyl oxygens of Lys-93 and Glu-108. Recent mutational data support a role for Ca 2ϩ binding in both factors Va and VIIIa at this site (37,38).…”

Residues 110–126 in the A1 Domain of Factor VIII Contain a Ca2+ Binding Site Required for Cofactor Activity

“…6). Recent data generated following site-directed mutagenesis within this region indicates that Glu 96 , Asp 102 , and Asp 111 appear to be crucial residues for the association of factor Va HC and LC (41), an interaction that is Ca 2ϩ -dependent in factor Va (17 …”

Activation of Factor v by Venom Proteases

“…that are necessary for the expression of the procoagulant function of factor V (Steen and Dahlbäck, 2002;Toso and Camire, 2004). Activated factor V, factor Va, consists of a 105 kDa heavy chain that is noncovalently bound in a calcium-dependent manner to a 71/74 kDa light-chain doublet, likely through calcium-induced exposure of interaction sites in the heavy-and light-chain domains of factor Va (Zeibdawi et al, 2004;Sörensen et al, 2004).…”