title: Identification of basic residues in the heparin-binding exosite of factor Xa critical for heparin and factor Va binding.
abstract: We recently demonstrated that a template mechanism makes a significant contribution to the heparin-accelerated inactivation of factor Xa (FXa) by antithrombin at physiologic Ca(2+), suggesting that FXa has a potential heparin-binding site. Structural data indicate that 7 of the 11 basic residues of the heparin-binding exosite of thrombin are conserved at similar three-dimensional locations in FXa. These residues, Arg(93), Lys(96), Arg(125), Arg(165), Lys(169), Lys(236), and Arg(240) were substituted with Ala in separate constructs in Gla domainless forms. It was found that all derivatives cleave Spectrozyme FXa with similar catalytic efficiencies. Antithrombin inactivated FXa derivatives with a similar second-order association rate constant (k(2)) in both the absence and presence of pentasaccharide. In the presence of heparin, however, k(2) with certain mutants were impaired up to 25-fold. Moreover, these mutants bound to heparin-Sepharose with lower affinities. Heparin concentration dependence of the inactivation revealed that only the template portion of the cofactor effect of heparin was affected by the mutagenesis. The order of importance of these residues for binding heparin was as follows: Arg(240) > Lys(236) > Lys(169) > Arg(165) > Lys(96) > Arg(93) >/= Arg(125). Interestingly, further study suggested that certain basic residues of this site, particularly Arg(165) and Lys(169), play key roles in factor Va and/or prothrombin recognition by FXa in prothrombinase.
authors.affiliation: Edward A. Doisy Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA. rezaiear@slu.edu
authors.forename: A R
authors.initials: AR
authors.lastname: Rezaie
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D015943:Factor Va / Q000378:metabolism*; D015951:Factor Xa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D015943:Factor Va; D006493:Heparin; D015951:Factor Xa; D002118:Calcium