title: Tissue Factor Residues That Modulate Magnesium-Dependent Rate Enhancements of the Tissue Factor/Factor VIIa Complex.
abstract: The blood coagulation cascade is initiated when the cell-surface complex of factor VIIa (FVIIa, a trypsin-like serine protease) and tissue factor (TF, an integral membrane protein) proteolytically activates factor X (FX). Both FVIIa and FX bind to membranes via their γ-carboxyglutamate-rich domains (GLA domains). GLA domains contain seven to nine bound Ca(2+) ions that are critical for their folding and function, and most biochemical studies of blood clotting have employed supraphysiologic Ca(2+) concentrations to ensure saturation of these domains with bound Ca(2+). Recently, it has become clear that, at plasma concentrations of metal ions, Mg(2+) actually occupies two or three of the divalent metal ion-binding sites in GLA domains, and that these bound Mg(2+) ions are required for full function of these clotting proteins. In this study, we investigated how Mg(2+) influences FVIIa enzymatic activity. We found that the presence of TF was required for Mg(2+) to enhance the rate of FX activation by FVIIa, and we used alanine-scanning mutagenesis to identify TF residues important for mediating this response to Mg(2+). Several TF mutations, including those at residues G164, K166, and Y185, blunted the ability of Mg(2+) to enhance the activity of the TF/FVIIa complex. Our results suggest that these TF residues interact with the GLA domain of FX in a Mg(2+)-dependent manner (although effects of Mg(2+) on the FVIIa GLA domain cannot be ruled out). Notably, these TF residues are located within or immediately adjacent to the putative substrate-binding exosite of TF.
authors.affiliation: †Department of Biochemistry and ‡Department of Chemistry, University of Illinois, Urbana, Illinois 61801, United States.
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Gajsiewicz
authors.affiliation: †Department of Biochemistry and ‡Department of Chemistry, University of Illinois, Urbana, Illinois 61801, United States.
authors.forename: Kristin M
authors.initials: KM
authors.lastname: Nuzzio
authors.affiliation: †Department of Biochemistry and ‡Department of Chemistry, University of Illinois, Urbana, Illinois 61801, United States.
authors.forename: Chad M
authors.initials: CM
authors.lastname: Rienstra
authors.affiliation: †Department of Biochemistry and ‡Department of Chemistry, University of Illinois, Urbana, Illinois 61801, United States.
authors.forename: James H
authors.initials: JH
authors.lastname: Morrissey
mesh_terms: D019943:Amino Acid Substitution; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D015942:Factor VIIa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005170:Factor X / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D013925:Thromboplastin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D046912:Multiprotein Complexes; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa; D008274:Magnesium; D002118:Calcium