Role of Mg2+ in Extrinsic and Intrinsic Coagulation Under Physiologic Conditions.
Authors: Agah Sayeh , Sutton Amanda , Velander William H , Bajaj S. Paul 
Published: April 2020

Abstract
Ca2+ is an obligatory factor for both the extrinsic and intrinsic pathways of coagulation. In majority of in vitro studies, investigators use saturating concentrations of Ca2+ (5 to 10 mM) for FVIIa/tissue factor (TF) activation of factor IX (FIX), and factor X (FX) (extrinsic coagulation), as well as for the activation of FIX by FXIa, FX by FIXa/FVIIIa, and prothrombin by FXa/FVa (intrinsic coagulation). However, the concentration of Ca2+ in plasma is only 1.1 mM, which is considerably below the saturating concentration needed for optimal coagulation. Importantly, plasma also contains 0.6 mM Mg2+ that could compensate for subsaturating concentrations of Ca2+ in promoting coagulation. Previous studies have attempted to clarify this concept in FIX, FX and prothrombin activation. However, these studies are sparse and in virtually all cases not detailed. We have systematically examined the role of plasma concentration of Mg2+ (in addition to the plasma concentration of Ca2+) in promoting all Ca2+ dependent steps of extrinsic and intrinsic coagulation and compared it with the saturating concentration of Ca2+. The Km (~ 100 nM) for activation of FIX by FXIa was similar in the presence of plasma concentrations of Ca2+/Mg2+ or 5 mM Ca2+. Furthermore, the Km and Vmax for the activation of FX and FIX by FVIIa/TF were essentially similar for both conditions. The Km and Vmax for the activation of FX by FVIIIa/FIXa ± phospholipid, and prothrombin by FXa/FVa ± phospholipid were also indistinguishable in these two different metal ion conditions. Notably, when only plasma concentrations of Ca2+ (1.1mM or 1.7mM) were used in all reactions mentioned above, coagulation proceeded at suboptimal rates. In further studies, we used Biacore to investigate the binding of FXIa and FVIII to FIXa, soluble TF to FVIIa, and FVa to FXa. Soluble TF, dansyl-Glu-Gly-Arg (dEGR)-IXa and dEGR-Xa were coupled to CM5 chips in the presence of 10 mM Ca2+. At 5 mM Ca2+, the binding of FXIa to dEGR-IXa was characterized by a Kd of ~40 nM, binding of FVIII to dEGR-IXa by a Kd of ~100 nM, and FVa to dEGR-Xa by a Kd of ~120 nM. In the presence of plasma concentrations of Ca2+ and Mg2+, binding constants were similar to those obtained in the presence of 5 mM saturating Ca2+ concentration. Additional 45Ca2+ binding studies using equilibrium dialysis and prothrombin fragment 1, dEGR-VIIa and decarboxylated dEGR-VIIa, FIX and decarboxylated FIX, and FX and decarboxylated FX, indicated that in the g-carboxyglutamic acid (Gla) domain, 2-3 Ca2+ binding sites (Shikimoto, et al., J. Biol. Chem. 278, 24090-24094, 2003; Wang, et al., Biochemistry42, 7959-7966, 2003; Bajaj, et al., J. Biol. Chem.281, 24873-24888, 2006) out of seven core divalent ion binding sites (Soriano-Garcia et al., Biochemistry31, 2554-2566, 1992) could be replaced by Mg2+. Conversely, Mg2+ could not displace the Ca2+ binding sites in the epidermal growth factor-like domain 1 (EGF1) and protease domains of FIX or FX. Overall these studies indicate that (1) saturating concentrations of Ca2+ used in in vitro investigations are valid representations of coagulation studies, except for that Mg2+ compensates for suboptimal concentrations of Ca2+ under physiological conditions; (2) two of the Ca2+-binding sites in the Gla domain (numbers 1 and 7, per Tulinsky numbering (Soriano-Garcia et al., Biochemistry31, 2554-2566, 1992)), and possibly a third site (number 4) are specific for Mg2+ under physiologic conditions; and (3) the Ca2+-binding sites in the EGF1 and protease domains are specific for Ca2+ and can not be occupied by Mg2+ under physiologic conditions. In conclusion, Ca2+ and Mg2+ act in concert to promote optimal coagulation under physiologic conditions. Mg2+ alone does not promote coagulation since it cannot bind to the Ca2+ specific sites in the Gla domain necessary for folding of the Gla domain omega loop.