The indispensable role of Ca2+ ions in the maintenance of the functional tertiary structures of vitamin K-dependent coagulation factors has been definitively established but the participation of Mg2+ ions, another alkaline-earth metal that is present abundantly in blood plasma, in such a process is not yet understood. We show here that the Ca(2+)-stabilized conformation of coagulation factor IX undergoes a further conformational change upon binding of Mg2+ ions using three independent structural probes. The probes we used were (i) IX/X-bp, a snake venom anticoagulant that recognizes the Gla domains in coagulation factors IX and X, (ii) conformation-specific polyclonal antibodies against bovine factor IX, and (iii) monoclonal antibodies against the Gla domain of human factor IX. The binding of all these probes had an absolute requirement for Ca2+ ions, and Mg2+ ions alone were ineffective. However, when added together with Ca2+ ions, Mg2+ ions at physiological concentrations greatly augmented the binding of these probes to factor IX; the required concentration of Ca2+ ions was much reduced, and the affinity of each probe for factor IX was increased even in the presence of an excess of Ca2+ ions. These results suggest the presence of a Mg(2+)-specific binding site that does not interact with Ca2+ ions in factor IX. Furthermore, Mg2+ ions potentiated the susceptibility of factor IX to activation by factor XIa, concomitant with their effect on the conformation. Similarly, the required Ca2+ concentration was reduced by Mg2+ ions, and the rate of conversion to factor IXa was increased by Mg2+ ions in the presence of an excess of Ca2+ ions. At a saturating concentration of Ca2+ ions (5 mM), addition of 1 mM Mg2+ reduced the apparent Km value for factor IX from 0.31 to 0.18 microM, and in the presence of a physiological concentration of Ca2+ ions (1 mM), the reduction in Km by Mg2+ ions was far more striking (from 0.91 to 0.24 microM). The apparent Vmax values were hardly affected by Mg2+ ions. Our present data reveal a hitherto novel physiological role of the Mg2+ ions in plasma. Not only Ca2+ ions but also Mg2+ ions are important regulators of the stabilization of the native conformation of factor IX as well as of its efficient activation.
We recently showed that not only Ca 2؉ ions but also Mg 2؉ ions play a crucial role in stabilizing the native conformation of coagulation factor IX. We here report that Mg 2؉ ions at physiological concentrations greatly augment the biological activities of factor IX. In clotting assays with dialyzed plasma, addition of Mg 2؉ ions enhanced the apparent coagulant activity of factor IXa, while that of factor Xa was scarcely affected. Activation of factor X by factor IXa in the presence of factor VIIIa, phospholipids, and Ca 2؉ ions was accelerated by Mg 2؉ ions. It appeared that the cation increased the affinity between factor IXa and factor VIIIa, thereby increasing the apparent catalytic efficacy of the enzyme. We also evaluated the effect of Mg 2؉ ions in the coagulation pathway initiated by tissue factor and found that activation of factor IX by factor VIIa⅐tissue factor was accelerated by the cation. Consequently, clotting of normal plasma induced by factor VIIa⅐tissue factor was shortened by the cation, while no such effect was observed in plasma deficient in factor IX or VIII. These results indicate that the previously unrecognized plasma component, Mg 2؉ ions, plays crucial roles in blood coagulation and, moreover, that contributions of factors IX and VIII in the coagulation cascade have been seriously underestimated in previous investigations.The coagulation proenzyme factor IX plays a central role in hemostasis in concert with its essential procofactor factor VIII, as is apparent from the fact that genetic defects in these factors cause a life-threatening tendency to bleed, hemophilia (1). Once activated, factor IX forms a complex with the activated form of factor VIII (IXa⅐VIIIa) 1 on phospholipid membranes in the presence of Ca 2ϩ ions. The enzyme complex catalyzes efficient activation of factor X, and factor Xa in turn converts prothrombin to thrombin, which ultimately leads to successful formation of hemostatic plugs. In the classical cascade model of coagulation, factors VIII and IX are classified as components of the "intrinsic pathway" and are placed downstream of factor XI (Refs. 2 and 3; for recent review, see Refs. 4 and 5). However, it is now considered that the intrinsic pathway is insignificant in normal hemostasis, since individuals who lack initiators of this mechanism, i.e. factor XII (6), high molecular weight kininogen (7), and prekallikrein (8), are all asymptomatic. To today's best knowledge, the single mechanism that solely governs blood coagulation is the so-called "extrinsic pathway," which starts with the exposure of the extravascular component, tissue factor, to the bloodstream and formation of the active factor VIIa⅐tissue factor complex (VIIa⅐TF). The complex is able to activate factor X without a requirement for factors VIII and IX. Why, then, do hemophiliacs experience such severe hemorrhagic diatheses? This question was partly answered by the finding that VIIa⅐TF can activate not only factor X but also factor IX (9). Since then, evidence has accumulated for the indispensable ...
IX/X-bp, an anticoagulant protein isolated from the venom of the habu snake Trimeresurus flavoviridis, has a structure homologous to the carbohydrate-recognition domains of C-type (Ca(2+)-dependent) animal lectins, and it binds to the gamma-carboxyglutamic acid (Gla) domains of coagulation factors IX and X in a Ca(2+)-dependent fashion. In the present study, we elucidated the role of Ca2+ ions in this binding. The binding of 125I-labeled IX/X-bp to both coagulation factors required about 1 mM Ca2+ ions in this at pH 7.5. A decrease in the pH to 6.5 had a striking negative effect on the binding, and the Ca(2+)-requirement curve was shifted rightward. We investigated the binding of Ca2+ ions to IX/X-bp directly by equilibrium dialysis and identified two independent binding sites with different affinities. At pH 7.5, the apparent Kd values for these sites were 25 and 200 microM, respectively. When the pH was decreased to 6.5, the affinity of the high-affinity binding site was reduced only slightly but that of the low-affinity site was reduced considerably. Moreover, it was evident from observations of Ca(2+)-induced changes in the intrinsic fluorescence that IX/X-bp underwent a conformational change upon binding of Ca2+ ions.(ABSTRACT TRUNCATED AT 250 WORDS)
We demonstrated recently that coagulation factor IX has a specific binding site(s) for Mg 2÷ ions, independent of the Ca2÷-binding sites, and that binding of Mg 2+ ions is very important for expression of the functional conformation of this protein. We report here the localization of this Mg2+-specific binding site. We prepared three Gla-containing fragments of bovine factor IX, namely GIaEGFNc (residues 1-144+286-296), GlaEGFN (1-83) and the Gin domain peptide (1-46). Fragments GIaEGFN¢ and GIaEGFN retained the ability to undergo a conformational change upon binding of Mg z÷ ions in the presence of excess Ca 2+ ions. This change could be detected by a conformation-specific antibody. Furthermore, the Gla domain peptide was capable of binding Mg 2÷ ions, as determined by the metal ion-induced quenching of the intrinsic fluorescence. It appears that the Mg2÷-specific binding site of factor IX is located in the N-terminal Gla domain.
No abstract
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.