title: Conservative mutations in the C2 domains of factor VIII and factor V alter phospholipid binding and cofactor activity.
abstract: Factor VIII and factor V share structural homology and bind to phospholipid membranes via tandem, lectin-like C domains. Their respective C2 domains bind via 2 pairs of hydrophobic amino acids and an amphipathic cluster. In contrast, the factor V-like, homologous subunit (Pt-FV) of a prothrombin activator from Pseudonaja textilis venom is reported to function without membrane binding. We hypothesized that the distinct membrane-interactive amino acids of these proteins contribute to the differing membrane-dependent properties. We prepared mutants in which the C2 domain hydrophobic amino acid pairs were changed to the homologous residues of the other protein and a factor V mutant with 5 amino acids changed to those from Pt-FV (FV(MTTS/Y)). Factor VIII mutants were active on additional membrane sites and had altered apparent affinities for factor X. Some factor V mutants, including FV(MTTS/Y), had increased membrane interaction and apparent membrane-independent activity that was the result of phospholipid retained during purification. Phospholipid-free FV(MTTS/Y) showed increased activity, particularly a 10-fold increase in activity on membranes lacking phosphatidylserine. The reduced phosphatidylserine requirement correlated to increased activity on resting and stimulated platelets. We hypothesize that altered membrane binding contributes to toxicity of Pt-FV.
authors.affiliation: Department of Medicine, Veterans Administration Boston Healthcare System, Brigham and Women's Hospital, and Harvard Medical School, Boston, MA, USA. gary_gilbert@hms.harvard.edu
authors.forename: Gary E
authors.initials: GE
authors.lastname: Gilbert
authors.forename: Valerie A
authors.initials: VA
authors.lastname: Novakovic
authors.forename: Randal J
authors.initials: RJ
authors.lastname: Kaufman
authors.forename: Hongzhi
authors.initials: H
authors.lastname: Miao
authors.forename: Steven W
authors.initials: SW
authors.lastname: Pipe
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D017124:Conserved Sequence; D004546:Elapid Venoms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005165:Factor V / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010718:Phosphatidylserines / Q000737:chemistry / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
chemical_list: D004546:Elapid Venoms; D008563:Membrane Lipids; D010718:Phosphatidylserines; D010743:Phospholipids; C475049:Pseutarin C; D005165:Factor V; D005169:Factor VIII