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 Ca2+ ions that are critical for their folding and function, and most biochemical studies of blood clotting have employed supraphysiologic Ca2+ concentrations to ensure saturation of these domains with bound Ca2+. Recently, it has become clear that, at plasma concentrations of metal ions, Mg2+ actually occupies two or three of the divalent metal ion-binding sites in GLA domains, and that these bound Mg2+ ions are required for full function of these clotting proteins. In this study, we investigated how Mg2+ influences FVIIa enzymatic activity. We found that the presence of TF was required for Mg2+ 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 Mg2+. Several TF mutations, including those at residues G164, K166, and Y185, blunted the ability of Mg2+ 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 Mg2+-dependent manner (although effects of Mg2+ 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.
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