Tissue Factor Residues That Modulate Magnesium-Dependent Rate Enhancements of the Tissue Factor/Factor VIIa Complex

Biochemistry. 2015 Aug 4;54(30):4665-71. doi: 10.1021/acs.biochem.5b00608. Epub 2015 Jul 27.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Substitution
  • Calcium / chemistry
  • Calcium / metabolism
  • Factor VIIa / chemistry*
  • Factor VIIa / genetics
  • Factor VIIa / metabolism
  • Factor X / chemistry
  • Factor X / genetics
  • Factor X / metabolism
  • Humans
  • Magnesium / chemistry*
  • Magnesium / metabolism
  • Multiprotein Complexes / chemistry*
  • Multiprotein Complexes / genetics
  • Multiprotein Complexes / metabolism
  • Mutation, Missense
  • Protein Structure, Tertiary
  • Thromboplastin / chemistry*
  • Thromboplastin / genetics
  • Thromboplastin / metabolism

Substances

  • Multiprotein Complexes
  • Factor X
  • Thromboplastin
  • Factor VIIa
  • Magnesium
  • Calcium