©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of the Tertiary Structure and Function of Coagulation Factor IX by Magnesium(II) Ions (*)

Fujio Sekiya , Toshiko Yamashita , Hideko Atoda , Yutaka Komiyama (1), Takashi Morita (§)

From the (1)Department of Biochemistry, Meiji College of Pharmacy, Yato-cho, Tanashi, Tokyo 188 Department of Clinical Sciences and Laboratory Medicine, Kansai Medical University, Moriguchi, Osaka 570, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The indispensable role of Ca ions in the maintenance of the functional tertiary structures of vitamin K-dependent coagulation factors has been definitively established but the participation of Mg 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-stabilized conformation of coagulation factor IX undergoes a further conformational change upon binding of Mg 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 Ca ions, and Mg ions alone were ineffective. However, when added together with Ca ions, Mg ions at physiological concentrations greatly augmented the binding of these probes to factor IX; the required concentration of Ca ions was much reduced, and the affinity of each probe for factor IX was increased even in the presence of an excess of Ca ions. These results suggest the presence of a Mg-specific binding site that does not interact with Ca ions in factor IX. Furthermore, Mg ions potentiated the susceptibility of factor IX to activation by factor XIa, concomitant with their effect on the conformation. Similarly, the required Ca concentration was reduced by Mg ions, and the rate of conversion to factor IXa was increased by Mg ions in the presence of an excess of Ca ions. At a saturating concentration of Ca ions (5 mM), addition of 1 mM Mg reduced the apparent K value for factor IX from 0.31 to 0.18 µM, and in the presence of a physiological concentration of Ca ions (1 mM), the reduction in K by Mg ions was far more striking (from 0.91 to 0.24 µM). The apparent V values were hardly affected by Mg ions. Our present data reveal a hitherto novel physiological role of the Mg ions in plasma. Not only Ca ions but also Mg ions are important regulators of the stabilization of the native conformation of factor IX as well as of its efficient activation.


INTRODUCTION

Calcium is an element that is essential in the regulation of a great variety of biological processes. The metal is abundant in extracellular fluids. In blood plasma, calcium is present at concentrations of 2.2-2.6 mM (1.1-1.3 mM as free ions)(1) , and it plays an obligatory role in hemostasis. Calcium ions bind to a number of coagulation proteins and modulate their functions. Almost all the steps in blood coagulation require Ca ions, and chelating agents such as EDTA and citrate are very effective anticoagulants. Among the various proteins that participate in coagulation, vitamin K-dependent coagulation factors show strong dependence on Ca ions for maintenance of their functional conformation (for reviews, see Refs. 2 and 3). Each factor binds about 10 Ca ions at the N-terminal Gla domain, and Gla-independent Ca-binding sites have also been identified in some of these factors. These various proteins, namely prothrombin and factors VII, IX and X, and also the anticoagulant protein C, are synthesized as zymogens and are converted to active proteases via limited proteolysis catalyzed by specific activators. Each activated protease in turn activates another factor, constituting the cascade of coagulation. The protein conformations with bound Ca ions are essential for recognition by the respective activators. Moreover, the binding of these factors to procoagulant membranes rich in anionic phospholipids (e.g. the surface of activated platelets), which allows condensation of both activator and substrate at one site and thereby facilitates efficient activation, is also a Ca-dependent process.

Another alkaline-earth metal, magnesium, is also abundant in blood plasma (0.4-0.6 mM as free ions)(1) . While we know much about the role of Ca ions, the involvement of Mg ions in hemostasis has been poorly understood. Various polyvalent metal ions including Mg ions have been shown to interact with the Ca-binding sites in Gla domains of vitamin K-dependent coagulation factors (e.g. Refs. 4-7). According to previous studies, however, the affinity of Mg ions for these proteins is relatively weak and, so far as is known to date, Mg ions cannot be replaced Ca ions for the expression of biological activity. In 1980, Byrne et al.(8) reported that activation of factor IX by factor XIa was accelerated to some extent by the addition of Mg ions in the presence of a suboptimal concentration of Ca ions (1.25 mM). However, since only limited information about the structure of factor IX was available, the underlying molecular mechanism of this phenomenon was hard to explain at that time. Furthermore, the concentration of factor IX employed in that report was far beyond the physiological range and, therefore, it was unclear whether the observed phenomenon was physiologically meaningful. In the mid-1980s, Furie and co-workers (9-12) proposed the hypothesis that vitamin K-dependent coagulation factors undergo two sequential conformational changes upon binding to metal ions on the basis of observations with conformation-specific antibodies as structural probes. Their scheme can be summarized as follows: P P` P*. In this model, metal ions other than Ca ions (e.g. Mg ions) can elicit only a partial transition, from the metal-free form of the protein (P) to an intermediate form (P`) that is insufficient for the expression of biological activity. Transition to a final functional form (P*) is accomplished only by Ca ions at physiological concentrations. The consequences of this model are simple: only Ca ions are essential, and other cations are unnecessary for the regulation of the conformation and function of the coagulation factors. By contrast, we show in the present report that the Mg ion is another regulator of both the structure and the function of factor IX, which is crucial for normal hemostasis.

We previously isolated a novel type of anticoagulant protein that binds to the Gla domains of factors IX and X in a Ca-dependent fashion from snake venom(13, 14) , and we have recently found that this protein, designated IX/X-bp, recognizes the Ca-bound conformation of the Gla domains.()In this report, we now show that binding of IX/X-bp to factor IX, but not to factor X, is greatly augmented by physiological concentrations of Mg ions when added together with Ca ions. Furthermore, we demonstrate that not only Ca ions but also Mg ions are important for stabilization of the conformation as well as for expression of the biological activity of factor IX. Our discoveries highlight a novel physiological role of Mg ions in hemostasis.


EXPERIMENTAL PROCEDURES

Proteins

The following proteins were prepared by published methods; IX/X-bp(13) , bovine factors IX and X(15) , human factor IX (16), and human factor IXa(17) . All of the preparations were homogeneous as judged by SDS-polyacrylamide gel electrophoresis.

Human factor XIa was prepared using immunoaffinity chromatography by a modification of the method of isolation of the zymogen(18) . In brief, factor XI was activated by incubating 1 liter of plasma, anticoagulated with acid-citrate dextrose, with 25 g of Celite (Hyflo-super-cel; Wako Pure Chemicals, Osaka, Japan) at 37 °C for 10 min. Factor XIa absorbed to the Celite was eluted by stirring the filter cake in 100 ml of 20 mM Tris-HCl, 1.5 M NaCl, pH 7.5, for 18 h at ambient temperature. The eluate was passed through a column of soybean trypsin inhibitor-Sepharose to remove factor XIIa, after dialysis against 20 mM Tris-HCl, 140 mM NaCl, pH 7.5 (TBS).()The unbound fraction was then applied to a column of immobilized monoclonal antibody against human factor XI (KMXI-1; Ref. 19). Factor XIa was eluted with 0.02% NHOH, 0.15 M NaCl, pH 11. Trace amounts of high molecular weight contaminants were removed by gel filtration.

Binding of I-IX/X-bp

IX/X-bp was labeled with NaI (Du Pont NEN) by use of IODOBEADS (Pierce) in accordance with the manufacturer's instructions. The specific activity of the labeled protein was in the range of 1.0-2.0 10 cpm/µg. Binding assays were conducted as follows. Wells of breakable microtiter plates (Labsystems, Finland) were coated with a solution of 10 µg/ml factor IX or X in 50 µl of TBS at 4 °C overnight, and the remaining nonspecific binding sites were blocked by incubation with 1% bovine serum albumin (BSA, essentially fatty acid-free; Sigma) in TBS for 1 h. Each well was incubated with approximately 50,000 cpm of I-IX/X-bp (approximately 0.5 µg/ml) in 50 µl of TBS containing 1 mg/ml BSA and appropriate concentrations of Ca and Mg ions for 30 min at 37 °C. Wells were then washed twice with 200 µl of TBS plus metal ions at the same concentrations as those used for the incubation, cut into pieces, and counted for bound radioactivity in a counter. Magnesium ions were added as magnesium acetate, and acetate ions added as sodium acetate had no effect on the binding.

Equilibrium Dialysis

This was conducted at ambient temperature in a microvolume dialyzer with 250-µl cells (Hoffer Scientific Instrument, San Francisco, CA). Dialysis membranes were pretreated with a boiled solution of 0.1 M NaHCO, 2% EDTA and washed extensively with metal-free water prior to use. A 200-µl aliquot of a TBS solution of CaCl containing CaCl as a tracer (1,000,000 dpm/cell; Du Pont NEN) was dialyzed against 200 µl of 40 µM IX/X-bp in TBS for 20 h with constant rotation. Protein-bound Ca was quantified by liquid scintillation counting. In order to negate effects of possible contamination by metal ions, the buffer used was passed through a column of Chelex 100 (Bio-Rad) and the protein solution was dialyzed against a suspension of Chelex 100 (1 g/liter) in the buffer.

Polyclonal Antibodies

Antisera against bovine factor IX were raised in rabbits by immunizing them with the pure protein. Conformation-specific antibodies were prepared essentially by the method of Liebman et al.(11) . In brief, a pool of polyclonal antibodies was loaded onto an affinity gel of immobilized bovine factor IX which had been equilibrated with a buffer containing Ca ions. Anti-factor IX:Ca(II) antibodies,()which recognized factor IX with an absolute requirement for Ca ions, were eluted by changing the buffer to that containing Mg ions and no Ca ions. Anti-factor IX:Mg(II) antibodies, which recognized factor IX in the presence of various polyvalent metal ions including Mg, and metal-independent antibodies were obtained by elution with buffers containing EDTA and guanidine hydrochloride, respectively. Conformation-specific antibodies against other coagulation factors were also prepared by the same method. The nature of each antibody was confirmed by an enzyme-linked immunosorbent assay (ELISA) as described below.

Monoclonal Antibodies

A panel of monoclonal antibodies against human factor IX was prepared by the standard hybridoma technique. Clones producing antibodies reactive to the antigen were selected by ELISA. Characterization of the positive clones obtained was also performed by ELISA. The metal dependence of the binding was examined and preliminary epitope mapping utilizing various factor IX derivatives were performed. Among 119 positive clones, 46 clones exhibited Ca dependence for the binding to the antigen. Two clones for which binding was absolutely dependent on Ca ions, C1 (subclass IgG, ) and 2A2 (IgG, ), were selected for this study. The antibodies reacted with the isolated Gla domain fragment of factor IX (17) in the presence of Ca ions at millimolar concentrations.()

ELISA

Wells of microtiter plates were coated with 50 µl of protein solutions (1 µg/ml in TBS) for 2 h at ambient temperature with subsequent blocking by 1% BSA for 30 min. Coated wells were then incubated with 50 µl of a diluted solution of antibody in TBS containing 0.1% Tween-20 and various concentrations of Ca and Mg ions for 1 h, and the same buffers were used throughout the subsequent washing and incubation procedures. After two washes, the wells were reacted with 50 µl of peroxidase-conjugated goat antibodies against either rabbit IgG (for polyclonal antibodies) or mouse IgG (for monoclonal antibodies) (diluted 1:100; Kirkegaard and Perry Laboratories, Gaithersburg, MD) for 1 h. After extensive washing (five times), reaction with the antigen was visualized by incubation with 1 mg/ml o-phenylenediamine and 0.06% HO in 100 µl of 0.1 M citrate buffer, pH 5.5, and absorbance at 492 nm was recorded.

Quantification of Factor IXa

A two-stage clotting assay system was designed for the sensitive and selective quantification of factor IXa. A typical protocol was as follows. Human factor IX (10 µg/ml in 120 µl of TBS containing 1 mg/ml BSA and various concentrations of Ca and Mg ions) was incubated with 2.5 ng/ml factor XIa at 37 °C for 30 min. The reaction was stopped by the addition of 15 µl of a solution of EDTA/Ca/Mg; Ca and Mg ions were included so that all the samples in a given set of experiments would include the same amounts of metal ions at this step, and the concentration of EDTA was adjusted to neutralize both the Ca and Mg ions in the sample. Factor IXa in the samples was then quantified; one part of the sample (50 µl) was mixed with one part of plasma deficient in factor IX and one part of a suspension of phospholipids (phosphatidylcholine/phosphatidylserine (3:1, w/w; both from Sigma); 1 mg/ml in TBS) and equilibrated at 37 °C for 2 min. Fifty µl of 20 mM CaCl were then added, and the time required for clotting was measured in an Amelung Coagulometer KC 4A. The amount of factor IXa was determined by use of a standard curve that had been prepared with pure human factor IXa that contained the same amounts of EDTA/Ca/Mg as the tested samples; the logarithm of the clotting time was plotted against the logarithm of the concentration of standard factor IXa. The low concentrations of factor XIa and the rather long initial incubation period rendered the secondary activation of factor IX during the subsequent clotting assay negligible and allowed the accurate quantification of factor IXa. Note that sensitivity of the quantification is strongly dependent on the preparation of factor IX-deficient plasma, and the use of plasma with the lowest possible level of factor IX is necessary for the determination of extremely low levels of factor IXa. The preparations that we used were congenitally deficient human plasma (Sigma) or human plasma from which factor IX had been immunodepleted with monoclonal antibody (a gift from the Chemo-Sero Therapeutic Research Institute, Kumamoto, Japan). For the determination of kinetic parameters, concentrations of substrate ranging from 0.2 to 1.2 µM were utilized and the incubation period was set to ensure the linearity of the reaction. The data obtained were analyzed by construction of Eadie-Hofstee plots.


RESULTS

The snake venom anticoagulant IX/X-bp recognizes Ca-bound conformations of Gla domains in factors IX and X. Approximately 1 mM Ca ions is sufficient for binding to both factors(14) , consistent with the affinities of the Gla domains for Ca ions. By contrast, Mg ions alone were without effect (see Fig. 2). When Mg ions were added together with Ca ions, however, the extent of binding of IX/X-bp to factor IX was much increased (Fig. 1). In this experiment, the effect of the addition of Mg ions on the binding of I-IX/X-bp to solid-phase factor IX or X was investigated in the presence of 1 mM Ca ions. Upon the addition of Mg ions, the binding to factor IX was greatly enhanced whereas the binding to factor X was unchanged. The effect of further addition of Ca ions was modest for both proteins (data not shown). Since the same amount of Ca ions had little effect and since the effect of Mg ions was specific for factor IX, the observed phenomena appeared not to be due to a mere increase in the ionic strength of the incubation medium. Increase in the ionic strength of the medium tended, in fact, to inhibit the binding. In subsequent experiments, we focused our attention on the effects of Mg ions on factor IX. As is shown in Fig. 2, the Ca requirement curve for the binding to factor IX was shifted leftward, implying an enhancement of sensitivity to Ca ions by Mg ions. Furthermore, the augmentation was clearly seen even at saturating concentrations of Ca ions. The affinity of IX/X-bp for factor IX was increased by the addition of Mg ions, and the apparent K value for the binding of these proteins was decreased from 3.7 ± 1.6 10M (3 mM Ca alone; mean ± S.E., n = 4) to 1.3 ± 0.1 10M (3 mM Ca + 3 mM Mg), whereas that for factor X was unchanged (3.5 ± 0.6 10M and 3.5 ± 1.3 10M in the absence and presence of Mg ions, respectively). Half-maximal and maximal augmentation occurred at 0.3 and 3 mM Mg, respectively. It was revealed recently that IX/X-bp is capable of binding Ca ions and other polyvalent metal ions, and occupation of the two Ca-binding sites in IX/X-bp is a prerequisite for the recognition of Gla domains. It is unlikely, however, that the effect of Mg ions involves IX/X-bp directly, because the binding to factor X was unaffected by Mg ions. It seems that IX/X-bp does not interact with Mg ions; Ca binding to IX/X-bp was not altered at all by high concentrations of Mg ions (Fig. 3). These observations strongly suggest that factor IX is the molecule that interacts directly with Mg ions. The result that relatively low concentrations of Mg ions at saturating concentrations of Ca ions were still effective suggests that Mg ion(s) interact at specific site(s) that are separate from the putative Ca-binding sites in factor IX. The binding of Mg ion(s) to this specific site(s) appears to promote a further conformational change of factor IX, yielding a form distinct from the form that is achieved by binding of Ca ions alone.


Figure 2: Augmentation of binding of IX/X-bp to factor IX by Mg ions. Binding of I-IX/X-bp to solid-phase bovine factor IX was examined in the presence of Ca ions at indicated concentrations with (open circles) or without (closed circles) 3 mM Mg ions. Total radioactivity was 25,000 cpm. Other conditions were as in Fig. 1.




Figure 1: Effects of Mg ions on the binding of I-IX/X-bp to factors IX and X. Wells of microtiter plates coated with bovine factor IX (left) or factor X (right) were incubated with I-IX/X-bp in the presence of 1 mM Ca plus Mg ions at the indicated concentrations, as described under ``Experimental Procedures.'' Bound radioactivity is expressed as the percentage of the control value (1 mM Ca alone). Total radioactivities and control values for factors IX and X were 44,000 (total) and 2,200 and 10,500 (controls) cpm, respectively.




Figure 3: Absence of an effect of Mg ions on the binding of Ca ions to IX/X-bp. Binding of Ca ions to IX/X-bp was determined by equilibrium dialysis with Ca as the tracer as described under ``Experimental Procedures.'' Moles of bound Ca/mol of IX/X-bp (r) were plotted as a function of the concentration of Ca ions. Open circles indicate the results in the presence of 10 mM magnesium acetate. In the controls (closed circles), 30 mM sodium acetate was included to normalize the ionic strength.



To confirm our hypothesis we employed different probes, in an effort to exclude any possible effects of Mg ions on IX/X-bp. A pool of polyclonal antibodies raised against bovine factor IX was fractionated by affinity chromatography on immobilized factor IX according to the dependence on metal ions of the recognition of the antigen by the method of Liebman et al.(11) . Anti-factor IX:Ca(II) antibodies obtained in this way showed absolute Ca dependence for the binding, while Mg ions were ineffective, a feature identical to that originally described. The Ca-dependent binding of the antibodies to bovine factor IX was also potentiated by the addition of Mg ions (Fig. 4). Low concentrations of Mg ions augmented the binding in the presence of very high concentrations of Ca ions. Similar results were again obtained with Ca-dependent monoclonal antibodies against human factor IX (Fig. 5). The epitopes of these monoclonal antibodies were located within the Gla domain. Taking this result together with the results obtained with IX/X-bp, we can see that the tertiary structure of the Gla domain is greatly influenced by Mg ions. The conformation adopted by the Gla domain in the presence of both cations is apparently different from that adopted with either cation by itself.


Figure 4: Augmentation of the binding of anti-factor IX:Ca(II) antibodies to factor IX by Mg ions. Binding of isolated anti-factor IX:Ca(II) antibodies to bovine factor IX was determined by ELISA in the presence of Ca ions at the indicated concentrations, as detailed under ``Experimental Procedures.'' Closed circles, Ca alone; open squares, Ca plus 0.3 mM Mg; open circles, Ca plus 3 mM Mg.




Figure 5: Augmentation of the binding of Gla-domain-directed monoclonal antibodies to factor IX by Mg ions. Binding of two different monoclonal antibodies (right, C1; left, 2A2) to human factor IX was determined by ELISA in the presence of Ca ions at the indicated concentrations as in Fig. 4. Closed circles, Ca alone; open squares, Ca plus 0.3 mM Mg; open circles, Ca plus 3 mM Mg.



We also prepared conformation-specific polyclonal antibodies against human prothrombin, bovine factors VII and X, and bovine protein C by the same method as that employed for the preparation of anti-factor IX:Ca(II) antibodies, and we investigated the effects of Mg ions on the binding of the respective antibodies to these proteins. The antibodies again had absolute Ca dependence, and Mg ions alone were again ineffective. However, the Ca-dependent binding of these antibodies was barely affected by the addition of Mg ions, in contrast to the case with factor IX. Augmentation of the binding was not seen in the presence of excess Ca ions (5-10 mM) in each case. In the presence of suboptimal concentrations of Ca ions (of the order of 0.1 mM), slight leftward shifts in the Ca titration curves by Mg ions were seen in some cases, but these effects were negligible (data not shown). These results strongly suggest but do not prove that all of the Gla-containing coagulation proteases other than factor IX do not respond to Mg ions. It seems thus that factor IX is unique among various vitamin K-dependent coagulation factors in such a way that its conformation is stabilized not only by Ca ions but also by Mg ions.

We next examined the effects of Mg ions on function of factor IX. The inactive zymogen factor IX is converted to the active protease factor IXa by physiological activators factor XIa or factor VIIa/tissue factor/phospholipid complex(20) , or by the snake venom coagulant protease RVV-X(21) . The Ca-bound conformation of factor IX is considered to be essential for efficient activation by each of these activators. However, factor VIIa per se also requires Ca ions for the formation of the active complex with tissue factor and phospholipids. Such a requirement is also known for RVV-X, which is yet another metal-binding protein(22) . These features would clearly hinder evaluation of the effects of metal ions on the activation by these activators. By contrast, factor XIa itself does not need Ca ions, and the effects of Ca ions can be considered to be solely those exerted on factor IX(23) . We therefore employed factor XIa to examine whether the Mg-induced further conformational change in factor IX might affect its activation kinetics. Human factor IX was incubated with human factor XIa in the presence of Ca and Mg ions, and the amount of factor IXa generated was quantified as described under ``Experimental Procedures.'' Addition of Mg ions increased the rate of activation of factor IX in the presence of Ca ions, while Mg ions alone were ineffective (Fig. 6). In the presence of 1 mM Mg, the necessary Ca concentration was lowered by approximately 1 order of magnitude. The rate of activation in the presence of excess Ca ions, which reached a plateau value, was also increased by Mg ions. The shapes of curves and the range of effective Ca concentrations obtained here closely resemble those obtained with the conformation-specific probes (compare Fig. 6with Fig. 2, 4, and 5). It appears that the additional conformational change in factor IX induced by Mg ions renders the molecule susceptible to factor XIa. To gain further insight, we determined the kinetic parameters for the activation of factor IX in the absence and presence of 1 mM Mg ions; the results are summarized in . The parameters that we obtained are consistent with those in the literature, which were determined by a different method (23, 24). In the presence of a saturating concentration of Ca ions (5 mM), the K value was 0.31 µM, which is still higher significantly than the plasma concentration of factor IX (0.1 µM). Addition of Mg ions reduced the apparent K by a factor of 1.7, while the apparent V value was not altered. In the presence of a physiological concentration of Ca ions (1 mM), the effect of Mg ions was far more striking; the K was reduced from 0.91 to 0.24 µM (Fig. 7, ). Under these conditions, k was slightly (20%) reduced by Mg ions. These results mean that, if we assume that concentrations of free Ca ions and factor IX in the plasma are 1 mM and 0.1 µM, respectively, the initial velocity of factor IX activation under physiological conditions in the presence of Mg ions can be calculated to be 2.5 times higher than that in the absence of Mg ions, according to the Michaelis-Menten equation. With the results of this calculation in mind, we verified the significance of Mg ions by conducting an experiment in which physiological conditions were simulated. Factor IX at 10 µg/ml (close to the concentration in plasma) was activated in the presence of both cations at approximately physiological concentrations (1 mM Ca + 1 mM Mg), and the time course of activation was monitored. The activation proceeded efficiently, but when Mg ions were omitted the rate of activation was much lower (Fig. 8). These data strongly suggest that Mg ions, present in blood plasma at millimolar concentrations, are indeed involved in the activation of factor IX in vivo.


Figure 6: Acceleration of factor XIa-induced activation of factor IX by Mg ions. Human factor IX (10 µg/ml) was incubated with human factor XIa (2.5 ng/ml) for 30 min at 37 °C in the presence of Ca ions at the indicated concentrations, and then the amounts of factor IXa formed were measured as described under ``Experimental Procedures.'' Closed circles, Ca alone; open circles, Ca plus 1 mM Mg.




Figure 7: Effects of Mg ions on the kinetics of activation of factor IX. Human factor IX at various concentrations was activated by 2.5 ng/ml human factor XIa for 30 min at 37 °C in the presence of 1 mM Ca alone (closed circles) or 1 mM Ca plus 1 mM Mg (open circles). The data were analyzed by construction of Eadie-Hofstee plots (v = -K(v/s) + V).




Figure 8: Time course of the activation of factor IX under physiological conditions. Human factor IX (10 µg/ml) was incubated with human factor XIa (2.5 ng/ml) at 37 °C in the presence of either 1 mM Ca alone (closed circles) or 1 mM Ca plus 1 mM Mg (open circles) for indicated periods, and then the amounts of factor IXa formed were measured.




DISCUSSION

Factor IX plays a central role in blood coagulation, as is apparent from the fact that deficiency of this factor causes a severe bleeding disorder, hemophilia B(25) . It has been established that factor IX, similar to other vitamin K-dependent coagulation factors, has multiple binding sites for polyvalent metal ions, and the natural ligands are believed to be primarily Ca ions. The N-terminal Gla domain is the major Ca-binding site, and a dozen or so of Ca ions bind to this small segment with affinities comparable to the plasma concentration of Ca ions (K 1 mM)(3, 26, 27) . Binding of Ca ions to the Gla domain is essential for maintenance of the native functional conformation of factor IX, which is crucial for recognition and subsequent activation by specific activators. The active form (factor IXa) produced in this way also exhibits biological activity only when the Gla domain is associated with the full complement of Ca ions.

The importance of Ca ions has been definitively proven, as described above, but it is not yet understood whether Mg ions, another physiological constituent of blood plasma, also participate in coagulation. Various biochemical studies have been undertaken to demonstrate interactions between various metal ions and vitamin K-dependent coagulation factors, which include factor IX(4, 5, 6, 7, 8, 9, 10, 11, 12, 26, 27) . However, most studies have focused mainly on interactions with Ca ions, and little attention has been paid to the action of Mg ions. We have shown here that Mg ions also influence the tertiary structure of factor IX using three independent structural probes. The first probe was IX/X-bp, which recognizes the Gla domains of both factors IX and X from any species(13, 14) ; the second one was a preparation of conformation-specific polyclonal antibodies against bovine factor IX (anti-factor IX:Ca(II) antibodies; Ref. 11); and the third one was a preparation of monoclonal antibodies directed to the Gla domain of human factor IX. All of these probes recognized factor IX in the presence of millimolar concentrations of Ca ions, while Mg ions alone were ineffective. Since the Gla domain binds Ca ions with a K value of 1 mM and exhibits a dramatic conformational change upon the binding of Ca ions, we can conclude that these probes recognized the Ca-bound conformation of this module. Magnesium ions at relatively low concentrations augmented binding of these Gla-domain-directed probes even in the presence of excess Ca ions (Fig. 2, 4, and 5). These observations indicate that factor IX has specific binding site(s) for Mg ion(s) that do not interact with Ca ion(s) and, moreover, that the Gla domain that has already been filled with Ca ions undergoes a further conformational transition upon binding of Mg ion(s). Note that this conclusion does not necessarily imply that the Mg-specific site(s) is within the Gla domain or that Mg ions do not affect the shape and/or function of other modules that form the factor IX molecule (e.g. epidermal growth factor-like modules). The Mg-specific binding site(s) has not yet been identified, and we are currently attempting to localize it using protease-digested fragments of factor IX(28) . It is of interest to note that Amphlett et al.(26) reported previously that factor IX has a unique binding site for a Mn ion that cannot be filled by a Ca ion even at extremely high concentrations of Ca ions. It is possible that the natural ligand for this metal-binding site is a Mg ion. This issue awaits clarification.

It is particularly noteworthy that the effect of Mg ions seems to be specific for factor IX, since other coagulation factors, i.e. prothrombin, factors VII and X, and protein C, were not responsive to Mg ions. As is seen in Fig. 1, the binding of IX/X-bp to factor X was not affected by Mg ions. The Ca-dependent binding of conformation- specific polyclonal antibodies directed to these proteins was barely affected by Mg ions. Prendergast and Mann (29) reported previously that activation of prothrombin by prothrombinase complex (factor Xa plus factor Va, phospholipids, and Ca ions) was potentiated by Mg ions at millimolar concentrations when the concentration of Ca ions was low. We also examined the effect of Mg ions on the activation of prothrombin and obtained a similar result, but when the activation was monitored in the presence of physiological concentrations of Ca ions (>1 mM), the augmentation by Mg ions no longer occurred (data not shown). We conclude, therefore, that factor IX is the only molecule among various vitamin K-dependent coagulation factors whose conformation and function are regulated by Mg ions under physiological conditions. The conformation of (and probably the mechanism of the metal-ion-induced conformational change in) factor IX may differ somewhat from that of other Gla-containing coagulation factors.

Furie and co-workers (10, 11) postulated previously that there is a common mechanism for the metal-induced conformational transitions of vitamin K-dependent coagulation factors. Their hypothesis is based on observations with conformation-specific antibodies. They developed a method for fractionating antibodies according to the dependence on metal ions of the antigen recognition (as employed here to prepare anti-factor IX:Ca(II) antibodies). Two classes of metal-dependent antibodies have been described; one is specific for Ca-bound conformers (Ca(II) antibodies), and the other has lower specificity and recognizes the antigen in the presence of various polyvalent metal ions, such as Mg (Mg(II) antibodies)(10, 11) . The existence of two different classes of metal-dependent antibodies clearly indicates that these proteins can adopt at least three different conformations, depending on the presence of metal ions. Moreover, the full biological activities of the proteins are seen only in the presence of physiological concentrations of Ca ions, and other cations cannot be substituted. Hence, the conclusions of Furie and co-workers can be summarized as shown in Fig. 9(upper panel). In this model, the metal-free form of the protein (IX) is first converted to an intermediate, dysfunctional form (IX`) upon the binding of Mg ions (or other polyvalent cations including Ca ions when they are present at low concentrations). Only Ca ions at millimolar concentrations elicit the transition to final functional form (IX*). By contrast, our data show unequivocally that Mg ions actually play a crucial role in the regulation of the conformation and function of factor IX. From our present observations, we present an advanced model (Fig. 9, lower panel). In our model, Ca ions are essential but are insufficient to bring about a full conformational change, and both Ca and Mg ions are necessary. Since Mg ions are a normal constituent of plasma, the conformer IX should represent the physiological form. Furthermore, since the conformer IX is the preferred substrate for factor XIa as compared to the conformer IX*, Mg ions are also crucial for the function of factor IX. In addition, the function of Mg ions is cooperative with respect of Ca ions rather than additive. The leftward shifts in Ca titration curves ( Fig. 2and Fig. 4-6) clearly imply an increase in the affinity for Ca ions of this protein. The conformational rearrangement of factor IX induced by Mg ions appears to make the molecule better able to bind Ca ions. This point is extremely important if we consider the physiological level of the cations. The physiological level of free Ca ions (1-2 mM) is insufficient to bring about the full conformational change and concomitant expression of biological activity. When Mg ions are present, however, the folding of the factor IX molecule can be completed at this relatively low concentration of Ca ions, allowing factor IX to function with maximum potency.


Figure 9: The role of Ca and Mg ions in the regulation of the conformation of factor IX. Upper panel, the original hypothesis proposed by Liebman et al. The scheme presented in Ref. 11 has been modified to stress the action of Mg ions. Only Ca ions at physiological concentrations induce the conformational transition to the functional form (IX*), whereas Mg ions or any other polyvalent cations (or Ca ions at low concentrations) can induce a partial transition to a dysfunctional form (IX`). Lower panel, a modified hypothesis, based on the present results. The maximally active, functional form of factor IX (IX**), which is equivalent to the physiologically active conformer, is generated only when both Ca and Mg ions are present, and either cation alone can induce only a partial transition (to IX` or IX*).



Factor IX is converted to the active protease either by factor XIa (the intrinsic pathway) or by factor VIIa/tissue factor (the extrinsic pathway). Factor IXa then forms a complex with factor VIIIa and anionic phospholipids in the presence of Ca ions and, in turn, activates factor X. Although it has long been known that factor VIIa/tissue factor can directly activate factor X, it was shown recently and unequivocally that factor IX is the preferred substrate of this activator; the value of k/K for factor IX is approximately 10 times higher than that for factor X(30) . It now seems conceivable that the primary substrate of factor VIIa/tissue factor in vivo is factor IX, and that the activation of factor X is a secondary event, which, in large part, is mediated by factor IXa/factor VIIIa subsequent to the exposure of tissue factor to the bloodstream. On the other hand, the contribution of factor XI (and its activator factor XII) to the initiation of coagulation is considered to be relatively small, at least under normal physiological conditions (31). This notion is supported by a number of clinical investigations that indicate that abnormalities in factor VIII or IX result in a severe tendency to hemorrhage, hemophilia, whereas persons who have defects in the initiators of the intrinsic pathway (i.e. activators of factor XI, factor XII(32) , and high molecular weight kininogen (33, 34)) are asymptomatic. Defects in factor XI do, however, cause mild but significant hemorrhagic tendencies (35, 36). These contradictory observations can be explained by the recently identified positive feedback system for factor XI activation that is mediated by thrombin (37, 38). Factor XI may not be involved in the initiation of coagulation but should play an important role in the amplification phase subsequent to initiation via the mechanism associated with the extrinsic pathway. Lawson et al. (39) recently reported that tissue factor-induced thrombin generation in a reconstituted system that included purified coagulation factors was indeed potentiated in the presence of factor XI. However, they did not include Mg ions in the reaction mixture. The significance of factor XI might thus have been underestimated as can be deduced from our present observations. In this regard, our finding that Mg ions facilitate efficient activation contribute to a much better understanding of the physiological significance of this process.

The effects of Mg ions on the activation of factor IX by factor VIIa/tissue factor and on the activation of factor X by factor IXa remain to be determined. It seems likely that both of these processes are positively modulated by Mg ions since these two processes are also strongly dependent on the correct folding of the Gla domain of factor IX/IXa, and since Mg ions do affect the tertiary structure of this module. We are currently investigating these issues. Our preliminary results indicate that the time required for factor IXa-induced clotting is considerably decreased by the addition of Mg ions, while factor Xa-induced clotting is not affected.()These results imply that the activation of factor X by factor IXa is also accelerated by Mg ions.

In conclusion, we have revealed a novel physiological function of Mg ions. Magnesium ions appear to be another important regulator of hemostasis. At present, there is a vast literature about the role of Ca ions in blood coagulation but we believe that most studies (in particular, those with reconstituted systems composed of purified components) have to be re-examined in view of the demonstrated effects of Mg ions. Our findings should contribute to a further understanding of the mechanism of coagulation, the process that is one of the major subjects of today's medical sciences.

  
Table: Kinetic parameters of activation of factor IX by factor XIa

The steady-state kinetics of activation of factor IX were analyzed as described under ``Experimental Procedures.'' The data are means ± S.E. of three independent determinations. The k values were calculated on the assumption that all the active sites of factor XIa were intact. The data of Sinha et al. (23) were obtained by determination of the release of H-labeled activation peptide from factor IX.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel./Fax: 81-424-21-0429.

F. Sekiya, T. Yamashita, and T. Morita, submitted for publication.

The abbreviations used are: TBS, Tris-buffered saline; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay.

Terminology of the conformation-specific antibodies follows the original description (11).

H. Atoda and T. Morita, unpublished observation.

F. Sekiya, T. Yamashita, and T. Morita, unpublished observation.


ACKNOWLEDGEMENTS

We thank Tomohiro Nakagaki of The Chemo-Sero Therapeutic Research Institute for providing factor IX-deficient plasma. We also acknowledge the technical assistance of Shun Igarashi, Hiromi Nakahata, and Daisuke Yamada.


REFERENCES
  1. Paterson, C. R.(1983) Essentials of Human Biochemistry, pp. 237-248, Churchill Livingstone, Edinburgh
  2. Furie, B., and Furie, B. C.(1988) Cell53, 505-518 [Medline] [Order article via Infotrieve]
  3. Mann, K. G., Nesheim, M. E., Church, W. R., Haley, P., and Krishnaswamy, S.(1990) Blood76, 1-16 [Abstract]
  4. Bloom, J. W., and Mann, K. G.(1978) Biochemistry17, 4430-4438 [Medline] [Order article via Infotrieve]
  5. Sperling, R., Furie, B. C., Blumenstein, M., Keyt, B., and Furie, B. (1978) J. Biol. Chem.253, 3898-3906 [Medline] [Order article via Infotrieve]
  6. Church, W. R., Boulanger, L. L., Messier, T. L., and Mann, K. G.(1989) J. Biol. Chem.264, 17882-17887 [Abstract/Free Full Text]
  7. Butenas, S., Lawson, J. H., Kalafatis, M., and Mann, K. G.(1994) Biochemistry33, 3449-3456 [Medline] [Order article via Infotrieve]
  8. Byrne, R., Amphlett, G. W., and Castellino, F. J.,(1980) J. Biol. Chem.255, 1430-1435 [Free Full Text]
  9. Liebman, H. A., Limentani, S. A., Furie, B. C., and Furie, B.(1985) Proc. Natl. Acad. Sci. U. S. A.82, 3879-3883 [Abstract]
  10. Borowski, M., Furie, B. C., Bauminger, S., and Furie, B.(1986) J. Biol. Chem.261, 14969-14975 [Abstract/Free Full Text]
  11. Liebman, H. A., Furie, B. C., and Furie, B.(1987) J. Biol. Chem.262, 7605-7612 [Abstract/Free Full Text]
  12. Liebman, H. A.(1993) Eur. J. Biochem.212, 339-345 [Abstract]
  13. Atoda, H., and Morita, T.(1989) J. Biochem.106, 808-813 [Abstract]
  14. Atoda, H., Yoshida, N., Ishikawa, M., and Morita, T.(1994) Eur. J. Biochem.224, 703-708 [Abstract]
  15. Hashimoto, N., Morita, T., and Iwanaga, S.(1985) J. Biochem.97, 1347-1355 [Abstract]
  16. Miletich, J. P., Broze, G. J., and Majerus, P. W.(1980) Anal. Biochem.105, 340-350 [Medline] [Order article via Infotrieve]
  17. Morita, T., and Kisiel, W.(1985) Biochem Biophys, Res. Commun.130, 841-847 [Medline] [Order article via Infotrieve]
  18. Komiyama, Y., Nishikado, H., Masuda, M., Egawa, H., Kobayashi, N., Kobatake, S., Matsuura, S., and Murata, K.(1988) Thrombos. Res.50, 329-334 [Medline] [Order article via Infotrieve]
  19. Komiyama, Y., Masuda, M., Murakami, T., Egawa, H., Nishikado, H., and Murata, K.(1989) Thrombos. Res.55, 527-536 [Medline] [Order article via Infotrieve]
  20. , B., and Rapaport, S. I.(1977) Proc. Natl. Acad. Sci. U. S. A.74, 5260-5264 [Abstract]
  21. Lindquist, P. A., Fujikawa, K., and Davie, E. W.(1978) J. Biol. Chem.253, 1902-1909 [Medline] [Order article via Infotrieve]
  22. Amphlett, G. W., Byrne, R., and Castellino, F. J.,(1982) Biochemistry21, 125-132 [Medline] [Order article via Infotrieve]
  23. Sinha, D., Seaman, F. S., and Walsh, P. N.(1987) Biochemistry26, 3768-3775 [Medline] [Order article via Infotrieve]
  24. Walsh, P. N., Bradford, H., Sinha, D., Piperno, J. R., and Tuszynski, G. P.(1984) J. Clin. Invest.73, 1392-1399 [Medline] [Order article via Infotrieve]
  25. Thompson, A. R.(1986) Blood67, 565-572 [Medline] [Order article via Infotrieve]
  26. Amphlett, G. W., Byrne, R., and Castellino, F. J.(1978) J. Biol. Chem.253, 6774-6779 [Abstract]
  27. Bajaj, S. P.(1982) J. Biol. Chem.257, 4127-4132 [Free Full Text]
  28. Valcarce, C., Persson, E., Astermark, J., hlin, A.-K., and Stenflo, J.(1993) Methods Enzymol.222, 416-435 [Medline] [Order article via Infotrieve]
  29. Prendergast, F. G., and Mann, K. G.(1977) J. Biol. Chem.252, 840-850 [Abstract]
  30. Komiyama, Y., Pedersen, A. H., and Kisiel, W.(1990) Biochemistry29, 9418-9425 [Medline] [Order article via Infotrieve]
  31. Zur, M., and Nemerson, Y.(1987) in Haemostasis and Thrombosis, 2nd Ed.. (Bloom, A. L., and Thomas, D. P., eds) pp.148-164, Churchill Livingstone, Edinburgh
  32. Hoak, J. C., Swenson, L. W., Warnar, E. D., and Connor, W. E.(1966) Lancet ii, 884-886
  33. Colman, R. W., Bagdasarian, A., Talamo, R. C., Scott, C. F., Seavey, M., Guimaraes, J. A., Pierce, J. V., and Kaplan, A. P.(1975) J. Clin. Invest.56, 1650-1662 [Medline] [Order article via Infotrieve]
  34. Wuepper, K. D., Miller, D. R., and Lacombe, M. J.(1975) J. Clin. Invest.56, 1663-1672 [Medline] [Order article via Infotrieve]
  35. Rosenthal, R. L., Dreskin, O. H., and Rosenthal, N.(1953) Proc. Soc. Exp. Biol. Med.82, 171-174
  36. Asakai, R., Chung, D. W., Ratnoff, O. D., and Davie, E. W.(1989) Proc. Natl. Acad. Sci. U. S. A.86, 7667-7671 [Abstract]
  37. Naito, K., and Fujikawa, K.(1991) J. Biol. Chem.266, 7353-7358 [Abstract/Free Full Text]
  38. Gailani, D., and Broze, G. J., Jr.(1991) Science253, 909-912 [Medline] [Order article via Infotrieve]
  39. Lawson, J. H., Kalafatis, M., Stram, S., and Mann, K. G.(1994) J. Biol. Chem.269, 23357-23366 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.