Role of Mg²⁺ in Extrinsic and Intrinsic Coagulation Under Physiologic Conditions.

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 K_m (~ 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 K_m and V_max for the activation of FX and FIX by FVIIa/TF were essentially similar for both conditions. The K_m and V_max 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 K_d of ~40 nM, binding of FVIII to dEGR-IXa by a K_d of ~100 nM, and FVa to dEGR-Xa by a K_d 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 (