Role of magnesium in factor XIa catalyzed activation of factor IX: calcium binding to factor IX under physiologic magnesium.

Abstract

Factor IX (FIX) is a vitamin K-dependent protein that circulates in blood as a zymogen (57 000 Da) of the serine protease FIXa. It consists of an aminoterminal γ-carboxyglutamic acid (Gla) domain, two epidermal growth factor (EGF)-like domains, a connecting activation peptide segment, and a carboxyterminal catalytic domain [1]. The Gla domain binds eight calcium cations (Ca2+) that are coordinated to 12 γ-carboxyglutamate residues [2]. The EGF1 domain contains a single Ca2+-binding site that is coordinated to Asp47, Gln50, Asp64, and the backbone carbonyls of Gly48 and Asp65 [3]. The protease domain also contains a single Ca2+-binding site that is coordinated toGlu235, Glu245, and backbone carbonyls of residues 237 and 240 [4]. The importance of FIX in coagulation is inferred from the severe bleeding disorder accompanying its deficiency [1]. During the sustained phase of coagulation, FIX is activated by factor XIa (FXIa) requiring Ca2+ [1], and Mg2+ is thought to potentiate the rate of this reaction [5]. However, the number and location of Ca2+ binding sites in FIX that are substituted by Mg2+ under physiological conditions is not known. We measured Ca2+ binding to full-length human FIX and decarboxylated FIX in the absence and presence of physiologic concentrations (0.6 mM, [6]) of Mg2+. FIX in the absence of Mg2+ bound ~10.4 Ca2+ ions (Kd ~400 μM) with positive cooperativity for the initial 2–4 sites (Fig. 1A). In the presence of Mg2+, numbers of Ca2+ binding sites in FIX were reduced to ~7.1 (Fig. 1A) at saturating Ca2+ concentrations, and to ~5.8 at 1.1 mM physiologic Ca2+ concentration [6], respectively. Moreover, the Ca2+ binding affinity was increased (Kd ~200 μM) and the Ca2+-binding cooperativity was significantly altered in the presence of Mg2+ (Fig. 1A). Decarboxylated FIX in which Gla residues were converted to Glu [7] bound two Ca2+ ions (Kd ~200 μM) in the presence or absence of Mg2+ (Fig. 1A, inset). Fig. 1 Analyses of the Ca2+-binding and FIX activation kinetics. (A) Scatchard plots of Ca2+-binding to FIX. The moles of Ca2+ bound/mol of FIX (ν) are plotted against ν/Ca2+, where Ca2+ is the free ligand concentration. Ca2+ binding was measured ... Our current Ca2+ binding data are consistent with the known structural aspects of FIX [2–4] and represent an improvement over previously published reports [8,9]. Further, our data indicate that the EGF1 and the protease domain sites in FIX are unambiguously Ca2+-specific sites. Elegant studies from Hiskey’s laboratory have shown that the Gla domain of prothrombin binds three Mg2+ ions at 0.6 mM MgCl2 [10]. Additionally, in the presence of Ca2+, occupancy of these three sites would appear to be retained by Mg2+ [11]. In view of the new crystallographic data on FXa [12] and VIIa [13], these three Mg2+ sites in the prothrombin Gla domain most likely represent occupancy at positions 1, 4 and 7 (Fig. 1B). In FIX, there is an additional Ca2+ binding site (number 8 in Fig. 1B) involving Gla residues 36 and 40, which would also be occupied by Mg2+ at 0.6 mM MgCl2 [2]. Thus, at physiologic concentrations of Ca2+/Mg2+, we infer from our data (Fig. 1A) that sites 2, 3, 5 and 6 in the Gla domain of FIX are occupied by Ca2+, whereas the sites 1, 4, 7 and 8 are occupied by Mg2+. Further, based upon structural information [2] under conditions of excess Ca2+ (5 mM Ca2+/0.6 mM Mg2+) and/or ligand interaction, site 4 in the Gla domain would be occupied by Ca2+ instead of Mg2+ (Fig. 1A). Subsequent to identifying the location of the Mg2+ binding sites in FIX under physiologic conditions, we studied FIX activation by FXIa under the same conditions. Under low concentrations of Ca2+ (0.1–1.1 mM), Mg2+ decreased the Km by ~2-fold at each Ca2+ concentration used (Fig. 1C). Further, in the presence of Mg2+, the maximum rate of FIX activation was obtained at ~0.5 mM Ca2+, whereas in the absence of Mg2+ it was obtained at ~1.5 mM Ca2+ (Fig. 1C, inset). Thus, Mg2+ potentiates the activation of FIX at suboptimal concentrations of Ca2+. However, in contrast to a previous report [5], we did not observe Mg2+-induced increase in FIX activation rate at saturating Ca2+ concentrations (Fig. 1C). Although Mg2+ alone does not potentiate the activation of FIX by FXIa (Fig. 1C), it is able to fold the 14–47 residues (Fig. 1B) of the Gla domain [15]. This implies that Ca2+-specific dependent folding of the phospholipid binding region involving residues 1–11 is required for full participation of the Gla domain in this activation process [15–17].