PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Thromb Haemost. Author manuscript; available in PMC 2010 June 14.
Published in final edited form as:
PMCID: PMC2885445
NIHMSID: NIHMS205900

Contribution of magnesium in binding of factor IXa to the phospholipid surface: implications for vitamin K-dependent coagulation proteins

In the current schemes of coagulation, the intrinsic tenase complex consists of factor IXa, FVIIIa and Ca2+ assembled on the phospholipid (PL) membrane containing phosphatidylserine (PS) [1]. This complex activates FX to FXa, and the PS-containing PL membrane surface in vivo is provided by the activated platelets [1]. In virtually all investigations, saturating levels of Ca2+, ranging from 2 to 5 mM, are used to assemble the FIXa–FVIIIa–Ca2+–PL complex [1]. However, the physiologic concentration of Ca2+ in plasma is 1.1 mM [2], which is suboptimal and does not support complete occupancy of the Ca2+-binding sites in the γ-carboxyglutamic acid (Gla) domain of vitamin K-dependent clotting proteins [35]. However, plasma also contains 0.6 mM Mg2+ [2] and at this concentration, the Gla domain of prothrombin binds three Mg2+ [6]. Furthermore, although the Gla domain of prothrombin has seven Ca2+-binding sites under Mg2+-free conditions [7], it only binds four Ca2+ in the presence of Mg2+ [3]. Thus, the three Mg2+ in the Gla domain of prothrombin are not displaced by Ca2+. In agreement with this, the Gla domains of FVIIa and FXa each bind three Mg2+ [4,8], whereas FIXa Gla domain binds three Mg2+ when bound to its binding protein in the crystal structure [9], and apparently four in solution when not bound to any ligands [5]. Thus, with suboptimal Ca2+ concentrations, Mg2+ occupies specific sites in the Gla domains of vitamin K-dependent proteins and could provide support for PL binding. In this study, we investigated whether Mg2+ enhances FIXa binding to PL at physiologic concentrations of Ca2+; if so, it could facilitate assembly of the intrinsic tenase complex.

We measured dansyl-Glu-Gly-Arg (dEGR)–FIXa (FIXa with the active site blocked for stability) binding to PS/PC bilayers using surface plasmon resonance (SPR). In 1.1 mM Ca2+ (Fig. 1B), the kon for binding was (2.1 ± 0.91) × 103 M −1 s−1 (n = 3), koff was (3.9 ± 1.8) × 10−3 s−1 (n = 3), and Kd was 1.9 ± 0.23 μM. In 5 mM Ca2+ (Fig. 1C), the kon for binding was (4.1 ± 1.4) × 103 M −1 s−1 (n = 4), koff was (2.2 ± 1.0) × 10−3 s−1 (n = 4), and Kd was 0.54 ± 0.11 μM. In 1.1 mM Ca2+/0.6 mM Mg2+ (Fig. 1D), the kon for binding was (3.6 ± 1.2) × 103 M −1 s−1 (n = 4), koff was (2.4 ± 0.75) × 10−3 s−1 (n = 4), and Kd was 0.67 ± 0.18 μM. The kon and koff values were not notably affected when PL coupling to the L1 chip was varied from 4200 to 7200 relative units (RUs). In addition, we obtained Kd values using the equilibrium response (RUeq) for each concentration of dEGR–FIXa by fitting the data to the steady-state 1 : 1 interaction equation, RUeq = (RUmax × C)/(Kd + C), where C represents the concentration of dEGR–FIXa and RUmax the maximum binding capacity. The Kd obtained using the affinity equation was 3.61 ± 0.64 μM in 1.1 mM Ca2+ (Fig. 1B, inset), 0.87 ± 0.19 μM in 5 mM Ca2+ (Fig. 1C, inset), and 0.98 ± 0.17 μM in 1.1 mM Ca2+/0.6 mM Mg2+ (Fig. 1D, inset). Thus, the Kd values obtained using the kinetic or the affinity measurements were similar, indicating that the potential mass transport complications and the analyte (dEGR–FIXa) rebinding during the dissociation phase are minimal under our experimental conditions [12]. Moreover, the binding of dEGR–FIXa was ~ 3.7-fold weaker in 1.1 mM Ca2+ than in 5 mM Ca2+ or 1.1 mM Ca2+/0.6 mm Mg2+. Additionally, the binding of FIX in 5 mM or 1.1 mM Ca2+/0.6 mM Mg2+ was characterized by a Kd of 1.2 ± 0.10 μM (n = 3, data not shown), suggesting that FIX binds in a similar manner as dEGR–FIXa. Notably, our data for PL binding to FIX/FIXa obtained using SPR are in good agreement with the data obtained using light-scattering techniques [13,14].

Fig. 1
Contribution of Mg2+ to binding of dansyl-Glu-Gly-Arg (dEGR)–FIXa to the phospholipid (PL) surface, determined using surface plasmon resonance. Phosphatidylcholine (PC) and phosphatidylserine (PS) were obtained from Avanti Polar Lipids. Liposomes ...

Next, we studied the Mg2+ dependence of dEGR–FIXa (400 nM) binding to PL by measuring the equilibrium response signal at different concentrations of Ca2+ (Fig. 1E). In the absence of Mg2+, dEGR–FIXa required > 0.5 mM Ca2+ to depict noticeable binding to the PL. This Ca2+ requirement was most diminished at the physiologic concentration of 0.6 mM Mg2+. Furthermore, binding of PL to dEGR–FIXa reached a plateau value at ~ 2 mM Ca2+ in the absence of Mg2+, whereas it reached a plateau value at ~ 1 mM Ca2+ in the presence of 0.6 mM Mg2+. Moreover, Mg2+ did not enhance PL binding to dEGR–FIXa at saturating Ca2+ concentrations (Fig. 1E). Thus, it would appear that Mg2+ and Ca2+ act in concert to promote optimal PL binding to FIX/FIXa under physiologic conditions.

It has been reported that Mg2+ increases the affinity between FIXa and FVIIIa [15]. However, data to support such a conclusion are lacking. Remarkably, Mg2+ did not enhance the rate of FX activation by FIXa/FVIIIa unless PL was included in the system [15]. In fact, the previous observations [15] can now be explained by our data provided in this report. Our data indicate that Mg2+ binding to the specific sites in the C-terminal half of the FIX/FIXa Gla domain prepares it for Ca2+ binding to the N-terminal half of the Gla domain. The occupancy of the Ca2+-specific sites then results in folding of the ω-loop (residues 1–11) that is suitable for PL binding. Thus, Mg2+ promotes FIXa binding to PL, where it encounters FVIIIa for enhanced activation of FX; this would represent an indirect FVIIIa potentiation effect [15]. Cumulatively, we conclude that Mg2+ promotes PL binding to all vitamin K-dependent clotting proteins [13,14,16,17]. Mn2+, presumably by binding to the Mg2+-specific sites [18], can also enhance Ca2+-dependent binding of PL to the Gla domains of vitamin K-dependent proteins [13]. However, the Mn2+ concentration in plasma or serum is ~ 100 nM [19], which is extremely low for it to substitute forMg2+under physiologic conditions.

Acknowledgments

This work was supported by NIH grants HL 78944 and HL 36365.

Footnotes

Disclosure of Conflict of Interests

The authors state that they have no conflict of interest.

References

1. Mann KG. Thrombin formation. Chest. 2003;3(Suppl):4S–10S. [PubMed]
2. Wang S, McDonnell EH, Sedor FA, Toffaletti JG. pH effects on measurements of ionized calcium and ionized magnesium in blood. Arch Pathol Lab Med. 2002;126:947–50. [PubMed]
3. Prendergast FG, Mann KG. Differentiation of metal ion-induced transitions of prothrombin fragment 1. J Biol Chem. 1977;252:840–50. [PubMed]
4. Bajaj SP, Schmidt AE, Agah S, Bajaj MS, Padmanabhan K. High resolution structures of p-aminobenzamidine- and benzamidine-VIIa/soluble tissue factor: unpredicted conformation of the 192–193 peptide bond and mapping of Ca2+, Mg2+, Na+, and Zn2+ sites in factor VIIa. J Biol Chem. 2006;281:24873–88. [PubMed]
5. Agah S, Bajaj SP. Role of magnesium in factor XIa catalyzed activation of factor IX: calcium binding to factor IX under physiologic magnesium. J Thromb Haemost. 2009;7:1426–8. [PMC free article] [PubMed]
6. Deerfield DW, 2nd, Olson DL, Berkowitz P, Byrd PA, Koehler KA, Pedersen LG, Hiskey RG. Mg(II) binding by bovine prothrombin fragment 1 via equilibrium dialysis and the relative roles of Mg(II) and Ca(II) in blood coagulation. J Biol Chem. 1987;262:4017–23. [PubMed]
7. Soriano-Garcia M, Padmanabhan K, de Vos AM, Tulinsky A. The Ca2+ ion and membrane binding structure of the Gla domain of Ca-prothrombin fragment 1. Biochemistry. 1992;31:2554–66. [PubMed]
8. Wang SX, Hur E, Sousa CA, Brinen L, Slivka EJ, Fletterick RJ. The extended interactions and Gla domain of blood coagulation factor Xa. Biochemistry. 2003;42:7959–66. [PubMed]
9. Shikamoto Y, Morita T, Fujimoto Z, Mizuno H. Crystal structure of Mg2+- and Ca2+-bound Gla domain of factor IX complexed with binding protein. J Biol Chem. 2003;278:24090–4. [PubMed]
10. Erb EM, Chen X, Allen S, Roberts CJ, Tendler SJ, Davies MC, Forsén S. Characterization of the surfaces generated by liposome binding to the modified dextran matrix of a surface plasmon resonance sensor chip. Anal Biochem. 2000;280:29–35. [PubMed]
11. Mathur A, Bajaj SP. Protease and EGF1 domains of factor IXa play distinct roles in binding to factor VIIIa. Importance of helix 330 (helix 162 in chymotrypsin) of protease domain of factor IXa in its interaction with factor VIIIa. J Biol Chem. 1999;274:18477–86. [PubMed]
12. Kortt AA, Gruen LC, Oddie GW. Influence of mass transfer and surface ligand heterogeneity on quantitative BIAcore binding data. Analysis of the interaction of NC10 Fab with an anti-idiotype Fab′ J Mol Recogonit. 1997;10:148–58. [PubMed]
13. Nelsestuen GL, Kisiel W, Di Scipio RG. Interaction of vitamin K dependent proteins with membranes. Biochemistry. 1978;17:2134–8. [PubMed]
14. Beals JM, Castellino FJ. The interaction of bovine factor IX, its activation intermediate, factor IX alpha, and its activation products, factor IXa alpha and factor IXa beta, with acidic phospholipid vesicles of various compositions. Biochem J. 1986;236:861–9. [PubMed]
15. Sekiya F, Yoshida M, Yamashita T, Morita T. Magnesium (II) is a crucial constituent of the blood coagulation cascade. Potentiation of coagulant activities of factor IX by Mg2+ ions. J Biol Chem. 1996;271:8541–4. [PubMed]
16. Freedman SJ, Blostein MD, Baleja JD, Jacobs M, Furie BC, Furie B. Identification of the phospholipid binding site in the vitamin K-dependent blood coagulation protein factor IX. J Biol Chem. 1996;271:16227–36. [PubMed]
17. Erb EM, Stenflo J, Drakenberg T. Interaction of bovine coagulation factor X and its glutamic-acid-containing fragments with phospholipid membranes. A surface plasmon resonance study. Eur J Biochem. 2002;269:3041–6. [PubMed]
18. Bajaj SP, Byrne R, Nowak T, Castellino FJ. Interaction of manganese with bovine factor X. J Biol Chem. 1977;252:4758–61. [PubMed]
19. Arnaud J, Bourlard P, Denis B, Favier AE. Plasma and erythrocyte manganese concentrations. Influence of age and acute myocardial infarction. Biol Trace Elem Res. 1996;53:129–36. [PubMed]