title: Asymmetric processing of mutant factor X Arg386Cys reveals differences between intrinsic and extrinsic pathway activation.
abstract: Alterations in coagulation factor X (FX) activation, mediated by the extrinsic VIIa/tissue factor (FVIIa/TF) or the intrinsic factor IXa/factor VIIIa (FIXa/FVIIIa) complexes, can result in hemorrhagic/prothrombotic tendencies. However, the molecular determinants involved in substrate recognition by these enzymes are poorly defined. Here, we investigated the role of arginine 386 (chymotrypsin numbering c202), a surface-exposed residue on the FX catalytic domain. The naturally occurring FX386Cys mutant and FX386Ala variant were characterized. Despite the unpaired cysteine, recombinant (r)FX386Cys was efficiently secreted (88.6±21.3% of rFXwt) and possessed normal clearance in mice. rFX386Cys was also normally activated by FVIIa/TF and displayed intact amidolytic activity. In contrast, rFX386Cys activation by the FIXa/FVIIIa complex was 4.5-fold reduced, which was driven by a decrease in the kcat (1.6∗10(-4) s(-1) vs 5.8∗10(-4) s(-1), rFXwt). The virtually unaltered Km (70.6 nM vs 55.6nM, rFXwt) suggested no major alterations in the FX substrate exosite. Functional assays in plasma supplemented with rFX386Cys indicated a remarkable reduction in the thrombin generation rate and thus in coagulation efficiency. Consistently, the rFX386Ala variant displayed similar biochemical features suggesting that global changes at position 386 impact the intrinsic pathway activation. These data indicate that the FXArg386 is involved in FIXa/FVIIIa-mediated FX activation and help in elucidating the bleeding tendency associated with the FX386Cys in a rare FX deficiency case. Taking advantage of the unpaired cysteine, the rFX386Cys mutant may be efficiently targeted by thiol-specific ligands and represent a valuable tool to study FX structure-function relationships both in vitro and in vivo.
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Italy. Electronic address: marcello.baroni@unife.it.
authors.forename: M
authors.initials: M
authors.lastname: Baroni
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Italy; The Children's Hospital of Philadelphia, The University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19104, USA; The Center for Cell and Molecular Therapeutics, and Division of Hematology, The University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19104, USA; Department of Pediatrics, The University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: G
authors.initials: G
authors.lastname: Pavani
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Pinotti
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Branchini
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Bernardi
authors.affiliation: The Children's Hospital of Philadelphia, The University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19104, USA; The Center for Cell and Molecular Therapeutics, and Division of Hematology, The University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19104, USA; Department of Pediatrics, The University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: R M
authors.initials: RM
authors.lastname: Camire
mesh_terms: D000818:Animals; D001777:Blood Coagulation / Q000235:genetics*; D001780:Blood Coagulation Tests; D020134:Catalytic Domain; D015949:Factor IXa / Q000235:genetics / Q000378:metabolism; D015944:Factor VIIIa / Q000235:genetics / Q000378:metabolism; D005170:Factor X / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015951:Factor Xa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D015944:Factor VIIIa; D005170:Factor X; D015949:Factor IXa; D013917:Thrombin; D015951:Factor Xa; D008666:Metalloendopeptidases; C033858:russellysin
keywords: Activation determinant; Amino acid substitution; Coagulation serine proteases; Factor X; Factor X deficiency; Recombinant expression