Structural and Functional Studies of γ-Carboxyglutamic Acid Domains of Factor VIIa and Activated Protein C: Role of Magnesium at Physiological Calcium

Abstract

Crystal structures of factor (F) VIIa/soluble (s) tissue factor (TF), obtained under high Mg2+ (50 mM Mg2+/5 mM Ca2+), have three of seven Ca2+-sites in the γ-Carboxyglutamic Acid (Gla) domain replaced by Mg2+ at positions 1, 4 and 7. We now report structures under low Mg2+ (2.5 mM Mg2+/5 mM Ca2+) as well as under high Ca2+ (5 mM Mg2+/45 mM Ca2+). Under low Mg2+, four Ca2+ and three Mg2+ occupy the same positions as in high Mg2+ structures. Conversely, under low Mg2+, reexamination of the structure of Gla domain of activated Protein C (APC) complexed with sEPCR has position 4 occupied by Ca2+ and positions 1 and 7 by Mg2+. Nonetheless, in direct binding experiments, Mg2+ replaced three Ca2+-sites in the unliganded Protein C or APC. Further, the high Ca2+-condition was necessary to replace Mg4 in the FVIIa/sTF structure. In biological studies, Mg2+ enhanced phospholipid binding to FVIIa and APC at physiological Ca2+. Additionally, Mg2+ potentiated phospholipid-dependent activations of FIX and FX by FVIIa/TF, and inactivation of FVa by APC. Since APC and FVIIa bind to sEPCR involving similar interactions, we conclude that under the low Mg2+-condition, sEPCR binding to APC-Gla (or FVIIa-Gla) replaces Mg4 by Ca4 with an attendant conformational change in the Gla domain ω-loop. Moreover, since phospholipid and sEPCR bind to FVIIa or APC via the ω-loop, we predict that phospholipid-binding also induces the functional Ca4 conformation in this loop. Cumulatively, the data illustrate that Mg2+ and Ca2+ act in concert to promote coagulation and anticoagulation.

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

“…Sekiya and colleagues 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).…”

Quantifying vitamin K-dependent holoprotein compaction caused by differential γ-carboxylation using high-pressure size exclusion chromatography

“…It is important to note that all metal sites in the Gla domain will be filled by Ca 2+ at greater than 2 mM in the absence of Mg 2+ . The Mg 2+ -site 4 is predicted to switch to Ca 2+ upon binding of the Gla domain to phospholipid (PL) [38]. …”

“…3A: curve 6). At this supra-physiologic conditions, each X 2+ binding site in the core Gla domain has been previously reported to be occupied by Ca 2+ [38]. …”

Ca2+ switches the effect of PS-containing membranes on Factor Xa from activating to inhibiting: implications for initiation of blood coagulation

“…Mg 2+ ions at physiological concentrations (~0.6 mM) markedly enhance the biological activities of factor IX (28), and low Mg 2+ is consequently a significant contributor cardiovascular diseases (29). It is also reported that in the presence of 1.3 mM Ca 2+ (just above normal plasma range), the addition of 0.6 mM Mg 2+ leads to approximately a 5-fold faster rate of fX activation by fVIIa–soluble TF (sTF) (30), but that at low Ca 2+ concentration, Mg 2+ can alter the activity and structure of the fVIIa–sTF complex (31). These data suggest that both Mg 2+ and Ca 2+ are important in regulating coagulation and anticoagulation.…”