title: 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.
authors.affiliation: Research and Development Department, Canadian Blood Services, 1800 Alta Vista Drive, Ottawa, Ontario, Canada K1G 4J5.
authors.forename: Abed R
authors.initials: AR
authors.lastname: Zeibdawi
authors.forename: Jean E
authors.initials: JE
authors.lastname: Grundy
authors.forename: Bogna
authors.initials: B
authors.lastname: Lasia
authors.forename: Edward L G
authors.initials: EL
authors.lastname: Pryzdial
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics / Q000502:physiology*; D001665:Binding Sites; D002614:Chelating Agents / Q000494:pharmacology; D019281:Dimerization; D015943:Factor Va / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D018698:Glutamic Acid / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000378:metabolism; D021122:Protein Subunits; D013925:Thromboplastin / Q000378:metabolism
chemical_list: D002614:Chelating Agents; D010743:Phospholipids; D021122:Protein Subunits; D001224:Aspartic Acid; D018698:Glutamic Acid; D015943:Factor Va; D013925:Thromboplastin