title: Differential contributions of Glu96, Asp102 and Asp111 to coagulation factor V/Va metal ion binding and subunit stability.
abstract: Blood coagulation FV (Factor V) is activated by thrombin-mediated excision of the B domain, resulting in a non-covalent heterodimer, FVa (activated FV). Previous studies implicated Glu96, Asp102 and Asp111 in the essential Ca2+-dependent FVa subunit interaction. In the present study, FV E96A, D102A and D111A were purified and evaluated for function, subunit dissociation and metal ion binding. Chromogenic and clotting assays in the presence of procoagulant vesicles showed that each variant was inhibited (approximately 20-40%). D111A was further inhibited (>90%) after cleavage by thrombin. Comparable function was observed on activated platelets. D111A inhibition correlated to spontaneous subunit dissociation and severely impaired Ca2+ binding. The Cu2+ interaction was also inhibited, suggesting interdependent Ca2+ and Cu2+ binding to FV. The parental FV (FV-810; wild-type human FV missing residues 811-1491) used here is fully active without proteolysis because the B domain is truncated. Therefore, a FVa-like functional configuration exists for intact D111A independent of normal metal ion interactions. Unlike D111A, the thrombin-mediated FVa derived from E96A and D102A had only moderately enhanced subunit dissociation upon chelation and had normal metal ion binding. For FV-810-, E96A- and D102A-derived FVa, loss of function after chelation significantly preceded subunit dissociation. This study defines the highly conserved segment spanning Glu96-Asp111 in FV as multifunctional. Of the three amino acids evaluated, Asp111 is essential and probably functions through direct and indirect effects on Ca2+ and Cu2+ interactions. Glu96 and Asp102 individually influence FV/FVa by more subtle effects, possibly at the metal ion-dependent subunit interface.
authors.affiliation: Canadian Blood Services, Research and Development, 2350 Health Sciences Mall, University of British Colombia, Vancouver, British Columbia, Canada V6T 1Z3.
authors.forename: Jina
authors.initials: J
authors.lastname: Song
authors.forename: Kimberley
authors.initials: K
authors.lastname: Talbot
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Hewitt
authors.forename: Ross T A
authors.initials: RT
authors.lastname: MacGillivray
authors.forename: Edward L G
authors.initials: EL
authors.lastname: Pryzdial
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D005165:Factor V / Q000737:chemistry* / Q000378:metabolism*; D015943:Factor Va / Q000737:chemistry* / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D055550:Protein Stability; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D001224:Aspartic Acid; D018698:Glutamic Acid; D015943:Factor Va; D003300:Copper; D005165:Factor V; D002118:Calcium