title: The Gla domain of factor IXa binds to factor VIIIa in the tenase complex.
abstract: During blood coagulation factor IXa binds to factor VIIIa on phospholipid membranes to form an enzymatic complex, the tenase complex. To test whether there is a protein-protein contact site between the gamma-carboxyglutamic acid (Gla) domain of factor IXa and factor VIIIa, we demonstrated that an antibody to the Gla domain of factor IXa inhibited factor VIIIa-dependent factor IXa activity, suggesting an interaction of the factor IXa Gla domain with factor VIIIa. To study this interaction, we synthesized three analogs of the factor IXa Gla domain (FIX1-47) with Phe-9, Phe-25, or Val-46 replaced, respectively, with benzoylphenylalanine (BPA), a photoactivatable cross-linking reagent. These factor IX Gla domain analogs maintain native tertiary structure, as demonstrated by calcium-induced fluorescence quenching and phospholipid binding studies. In the absence of phospholipid membranes, FIX1-47 was able to inhibit factor IXa activity. This inhibition is dependent on the presence of factor VIIIa, suggesting a contact site between the factor IXa Gla domain and factor VIIIa. To demonstrate a direct interaction we did cross-linking experiments with FIX1-479BPA, FIX1-4725BPA, and FIX1-4746BPA. Covalent cross-linking to factor VIIIa was observed primarily with FIX1-4725BPA and to a much lesser degree with FIX1-4746BPA. Immunoprecipitation experiments with an antibody to the C2 domain of factor VIIIa indicate that the factor IX Gla domain cross-links to the A3-C1-C2 domain of factor VIIIa. These results suggest that the factor IXa Gla domain contacts factor VIIIa in the tenase complex through a contact site that includes phenylalanine 25 and perhaps valine 46.
authors.affiliation: The Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec H3T 1A4, Canada. mark.blostein@mcgill.ca
authors.forename: Mark D
authors.initials: MD
authors.lastname: Blostein
authors.forename: Barbara C
authors.initials: BC
authors.lastname: Furie
authors.forename: Isabelle
authors.initials: I
authors.lastname: Rajotte
authors.forename: Bruce
authors.initials: B
authors.lastname: Furie
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000906:Antibodies / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015949:Factor IXa / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D015944:Factor VIIIa / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D014633:Valine / Q000378:metabolism
chemical_list: D000906:Antibodies; D003432:Cross-Linking Reagents; D009363:Neoplasm Proteins; D010649:Phenylalanine; D015944:Factor VIIIa; C053764:benzoylphenylalanine; D015949:Factor IXa; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant; D014633:Valine