title: A Glu113Ala mutation within a factor VIII Ca2+-binding site enhances cofactor interactions in factor Xase.
abstract: We recently identified an acidic-rich segment in the A1 domain of factor VIII (residues 110-126) that functions in the coordination of Ca(2+), an ion necessary for cofactor activity [Wakabayashi et al. (2004) J. Biol. Chem. 279, 12677-12684]. Mutagenesis studies showed that replacement of residue Glu113 with Ala (E113A) yielded a factor VIII point mutant possessing increased specific activity as determined by a one-stage clotting assay. Mutagenesis at this site suggested that substitution with relatively small, nonpolar residues was well tolerated, whereas replacement with a number of polar or charged residues appeared detrimental to activity. Ala substitution resulted in the greatest enhancement, yielding an approximately 2-fold increased specific activity. Time course experiments following reaction with thrombin revealed similar rates of activation and inactivation of E113A as observed for the wild type. Results from factor Xa generation assays showed minimal differences in kinetic parameters and factor IXa affinity for E113A and wild-type factor VIIIa when run in the presence of synthetic phospholipid vesicles, whereas factor VIIIa E113A displayed an approximately 4-fold greater affinity for factor IXa compared with factor VIIIa wild type in reactions run on the platelet membrane surface. This latter effect may be attributed, in part, to a 2-fold increased affinity of factor VIIIa E113A for the platelet membrane. Considering that low levels of factors VIIIa and IXa are generated during clotting in plasma, the increased cofactor specific activity observed for E113A factor VIII may result from its enhanced affinity for factor IXa on the physiological membrane.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, USA.
authors.forename: Hironao
authors.initials: H
authors.lastname: Wakabayashi
authors.forename: Ya-Chi
authors.initials: YC
authors.lastname: Su
authors.forename: Syed S
authors.initials: SS
authors.lastname: Ahmad
authors.forename: Peter N
authors.initials: PN
authors.lastname: Walsh
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001780:Blood Coagulation Tests; D001792:Blood Platelets / Q000378:metabolism; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015949:Factor IXa / Q000378:metabolism*; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000378:metabolism*; D010743:Phospholipids / Q000138:chemical synthesis / Q000378:metabolism; D015539:Platelet Activation / Q000235:genetics; D013917:Thrombin / Q000378:metabolism
chemical_list: D009363:Neoplasm Proteins; D010743:Phospholipids; D018698:Glutamic Acid; D005169:Factor VIII; D015949:Factor IXa; D013917:Thrombin; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant; D000409:Alanine; D002118:Calcium