Influence of membrane composition on the enhancement of factor VIIa/tissue factor activity by magnesium ions

Abstract

Dear Sirs, A number of studies have shown that the γ-carboxyglutamate-rich (GLA) domains of vitamin K-dependent clotting proteins require Ca 2+ to fold properly and bind to membranes (1, 2). Although plasma contains about 1.25 mM free Ca 2+ and 0.5 mM Mg 2+ (3), in vitro assays of clotting factor function often employ supraphysiologic Ca 2+ concentrations (2.5-5 mM Ca 2+), and no Mg 2+. Sekiya et al. showed that Mg 2+ enhances factor IX (fIX) structure and function in combination with physiologic Ca 2+ concentrations (4, 5). Subsequent reports showed that, in the presence of plasma concentrations of Ca 2+ , Mg 2+ enhances the activity of factor VIIa (fVIIa) bound to tissue factor (TF) (6-10). The concept is that GLA domains typically bind seven or eight Ca 2+ when it is the only divalent metal ion present (at supraphysiologic Ca 2+ concentrations), but at plasma concentrations of Ca 2+ and Mg 2+ , two or three of these "calcium" binding sites are actually occupied by Mg 2+ , with functional consequences (11). Mg 2+ does not support clotting reactions in the absence of Ca 2+ (12), also consistent with the notion that only a subset of the metal ion binding sites in GLA domains can be productively occupied by Mg 2+ .

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

“…24 We also found that increasing the PS content of TF liposomes blunted the Mg 2+ response in these TF mutants. With several of these mutants (especially S162A), this blunting effect of PS was less extensive than that observed with WT TF.…”

“…Although Mg 2+ has been shown to modulate the enzymatic activity of FVIIa toward its cognate substrates, FIX 11 and FX, 13,24 and removal of the FX GLA domain abrogates this effect, 14 a thorough understanding of how each component of the TF/FVIIa/membrane complex responds to Mg 2+ has not yet been achieved. We therefore varied the constituents of this complex and examined the ability of Mg 2+ to modulate the rate of FX activation by FVIIa.…”

Structure–Function Relationship of the Interaction between Tissue Factor and Factor VIIa

“…1,52 The reasons for differential metal ion specificity of GLA domains remain unclear, but are likely due to differences in coordination geometries of each binding site along with the "hardness" properties of Ca 2þ versus Mg 2þ . 1,94 Mg 2þ has been demonstrated to modulate both the membrane binding 1 and enzymatic properties 95 of FVIIa and FIX. 96 One Ca 2þ binds to the EGF1 domain of FVIIa, for which Mg 2þ cannot substitute.…”

“…99,100 However, using the plasma concentrations of free Ca 2þ and Mg 2þ (1.25 and 0.6 mM, respectively) restores activity to maximal levels, indicating that Mg 2þ likely plays a role in vivo. 1,95,96,101 The GLA domains of both FVIIa and FX mediate enzymatic rate enhancements due to Mg 2þ , suggesting that conformational changes of the FX GLA domain upon Mg 2þ occupancy of metal-binding sites modify its interactions with TF. 101…”

Lipid specificity of the membrane binding domain of coagulation factor X

“…Each GLA residue carries a À2 charge, and typically coordinates one or more Ca 2+ (or, sometimes, Mg 2+ ) ions [17,18]. These ions are necessary for proper GLA domain folding, resulting in a motif where divalent metal ions and highly charged protein residues are packed into the protein's center, whereas three hydrophobic residues in the x-loop are exposed on the exterior [8,[19][20][21][22][23][24] (Fig.…”