WO1996038585A1

WO1996038585A1 - Reagent for measuring blood coagulation activity

Info

Publication number: WO1996038585A1
Application number: PCT/JP1996/001488
Authority: WO
Inventors: Takashi Morita
Original assignee: Eisai Co., Ltd.
Priority date: 1995-06-01
Filing date: 1996-05-31
Publication date: 1996-12-05
Also published as: JPH08327631A

Abstract

Provided are a reagent for measuring blood coagulation activity mediated by blood coagulation factor IV, characterized in that the reagent contains Mg2+ ions, and a method of measuring blood coagulation activity mediated by blood coagulation factor IX, which comprises adding Mg2+ ions to a reaction solution for measuring the blood coagulation activity.

Description

DESCRIPTION

REAGENT FOR MEASURING BLOOD COAGULATION ACTIVITY

Field of the Invention

The present invention relates to a reagent for measuring blood coagulation activity mediated by blood coagulation factor IX, the reagent containing Mg²⁺ ions, as well as to a method of measuring blood coagulation activity mediated by blood coagulation factor IX, which comprises adding Mg²⁺ ions to a reaction solution for measuring the blood coagulation activity.

Prior Art

The blood has to be always fluid when it is in the blood vessels, whereas the blood must be immediately coagulated to stop bleeding when it is discharged outside the blood vessels. Thus, the blood has contradictory functions, which is called hemostatic balance. This is very important to maintain the life functions.

A role of the blood coagulation system is to immediately coagulate the blood, and various factors and ingredients serve in that role.

The blood coagulation reaction is a so-called "cascade" reaction in which an enzyme precursor (zymogen) of a serine protease undergoes restricted decomposition through a specific activator and an active protease is formed to activate the following zymogen. Blood coagulation factor IX is also called a Christmas factor which is a protease (precursor) that plays a central role in the blood coagulation system. The deficiency or abnormality thereof causes serious tendency of bleeding which is found in hemophilia B.

Among proteins that participate in the blood coagulation, vitamin K-dependent coagulation factors in which vitamin K is indispensable in biosynthesis thereof prothrombin (factor II), factor VII, factor IX, factor X have Ca²⁺-binding sites including a domain which is present at the N-terminal and which is rich in γ- carboxyglutamic acid (Gla) (this domain is called Gla domain), and these factors show strong dependence on Ca²⁺ ions for maintaining the functional conformation important to exhibit the functions (Blood, by Mann K.G. et al., 76, 1 - 16, 1990). The Ca²⁺ ions are bound to a blood coagulation factor, and change the conformation thereof into a form capable of being recognized through an activated protease, thereby controlling the coagulation reaction.

On the other hand, the participation of Mg²⁺ ions, the same alkaline-earth metal as Ca²⁺ ions, in the blood coagulation is almost unknown. For example, it has been reported that polyvalent metal ions including Mg²⁺ ions are bound to the Ca²⁺ binding sites of Gla domains of the vitamin K-dependent coagulation factors. However, it has been stated in the report that the affinity of Mg²⁺ ions for proteins is very weak and that Mg²⁺ ions cannot replace Ca²⁺ ions (J. Biol. Chem., by Church W. R. et al., 264, 17882 - 17887, 1989, Biochemistry, by Butenas S. et al., 33, 3449 - 3456, 1994, and Eur. J. Biochem., by Liebman H. A., 212, 339 - 345, 1993).

Accordingly, in the conventional system for measuring activity of blood coagulation factors, the role of Ca²⁺ ions have been well known, and Ca²⁺ ions have been added as a constituent of an agent for measuring blood coagulation activity, and the concentration of Ca²⁺ ions in the reaction solution has been usually adjusted at from 3 to 4 mM. Whereas the role of Mg²⁺ ions have been completely neglected, and Mg²⁺ ions in the reaction solution are limited to those derived from plasma sample .

The concentration of Mg²⁺ ions in plasma is usually between 0.4 and 0.6 mM. At the time of measurement, the plasma sample is diluted to between 5 and 10 times in a reagent solution; therefore, the concentration of the Mg²⁺ ions in the reaction solution is extremely lower than the physiological concentration thereof. This means that the data obtained by using the conventional reagent for measuring activity of blood coagulation in which factor IX participates is not given through the accurate measurement under true physiological conditions.

Summary of the Invention

The present inventors have conducted assiduous studies with respect to blood coagulation factors, and have consequently found that the blood coagulation activity mediated by blood coagulation factor IX can be measured more accurately under conditions closer to physiolosical conditions by adding Mg²⁺ ions to a system for measuring activity of blood coagulation in which blood coagulation factor IX participates. This finding has led to the completion of the present invention.

That is, the present invention relates to a reagent for measuring blood coagulation activity mediated by blood coagulation factor IX, characterized in that the reagent contains Mg²⁺ ions, as well as to a method mediated by measuring blood coagulation activity of blood coagulation factor IX, which comprises adding Mg²⁺ ions to a reaction solution for measuring the blood coagulation activity.

The invention provides a blood coagulation activity determing agent, comprising such an agent and a compound to provide Mg²⁺, the activity being mediated, or involved, or required, or participated by blood coaguration factor IX.

During the studies with respect to the blood coagulation system, the present inventors have isolated a snake venom anticoagulant protein having activity of binding to factors IX and X, and have discovered that this protein recognizes conformational structures of Gla domains of factors IX and X bound to Ca²⁺ ions (Eur. J. Biochem., by Atoda H. et al., 224, 703 - 708, 1994). Then, they have examined the effects of Mg²⁺ ions on the binding of the snake venom anticoagulant protein to factors IX and X.

As a result, it has been found that the binding of the snake venom anticoagulant protein to factor IX is greatly increased in the presence of Ca²⁺ ions and Mg²⁺ ions compared to the case of the binding in the presence of Ca²⁺ ions alone, whereas the binding of the snake venom anticoagulant protein to factor X is not at all influenced by Mg²⁺ ions.

The present inventors have further studied the fact in which Ca²⁺ ions are effective for both factors IX and X. whereas Mg²⁺ ions are effective for factor IX alone, the effects (interrelation) of metal ion binding sites in anticoagulant protein and Mg²⁺ ions, and the effects of Mg²⁺ ions on the binding of conformational dependent or Ca²⁺-dependent anti-factor IX antibody to factor IX. Consequently, it has been found that the Mg²⁺ ions are not reactive for snake venom anticoagulant proteins but for factor IX.

With this phenomenon in mind, the relationship between Mg²⁺ ions and factor IX has been assiduously studied, and it has consequently been found that Mg²⁺ ions which have received no attention so far play an important role in the maintenance of the conformation of factor IX and in the development of physiological activity together with Ca²⁺ ions.

Conformational-dependent polyclonal antibodies against other blood coagulation factors having the Gla domain, human prothrombin, factor VII, factor X and protein C have been prepared, and the effects of Mg²⁺ ions on bindings of these factors to the antibodies have been examined. It has consequently been found that the bindings are all absolutely dependent on Ca²⁺ ions and that almost no affinities for the bindings are affected even if Mg²⁺ ions are co-existed. Thus, it has been found for the first time that Mg²⁺ ions are specifically effective for factor IX. This finding has elucidated the novel physiological role of Mg²⁺ ions in the blood coagulation system, showing the necessity for Mg²⁺ ions that have never been considered in the conventional reagent for measuring activity of blood coagulation in which factor IX participates.

Detailed Description of the Invention

The present invention will be described in detail below.

The present invention discloses, as demonstrated in Examples. that Mg²⁺ ions in a sufficient amount are required to activate factor IX. It relates to a reagent for measuring blood coagulation activity mediated by factor IX and a method of measuring this blood coagulation activity, wherein Mg²⁺ ions are freshly added besides Mg²⁺ ions derived from plasma samples.

The concentration of Mg²⁺ ions to be freshly added can be approximately 0.1 mM; it is preferably 0.3 mM or more, more preferably between 0.5 and 10 mM, still more preferably between 1 and 5 mM, most preferably between 1 and 3 mM. The reaction solution refers to a solution obtained by mixing a plasma sample with reagent ingredients, and the concentration of Mg²⁺ ions is a final concentration thereof. The amount of Mg²⁺ ions which are contained in the reagent of the present invention can be calculated based on the total volume of the reaction solution for measuring blood coagulation activity.

At any rate, in the present invention. Mg²⁺ ions are added to a system or a reagent for measuring blood coagulation activity mediated by factor IX. Accordingly, the present invention includes not only the direct measurement of blood coagulation activity mediated by factor IX alone but also the measurement of the overall blood coagulation activity. For example, the addition of Mg²⁺ ions to a system for measuring blood coagulation activity mediated by factor IX and factors II. VII and X is included in the present invention too. In a reagent for measuring activity of blood coagulation in which factor IX is not considered to participate, even a trace amount of factor IX is often contained and affects a value of activity measured. The addition of Mg²⁺ ions to such a reagent is also included in the present invention. However, a reagent which is completely free from factor IX is not included in the present invention.

The reagent for measuring blood coagulation activity is described as follows. For example, in order to measure the blood coagulation activity of blood coagulation factor IX, the reagent usually contains ingredients necessary for blood coagulation except factor IX. That is, a reagent containing thromboplastin, plasma from which factor IX is specifically removed and which contains factor II, factor VII, factor X, phospholipids, fibrinogen, factor V and factor XIII required for blood coagulation, calcium and the like is taken up as the reagent for measuring blood coagulation activity.

The addition of Mg²⁺ ions refers to the addition of an Mg²⁺-ion source. Examples of the Mg²⁺-ion source include MgF₂, MgCl₂, MgBr₂, Mgl₂ and (CH₃CO₂)₂Mg.

In the present invention, Mg²⁺ ions, for example, as MgCl₂, are added to a solution of a reagent used to measure the prothrombin time, the partial prothromboplastin time or the activated partial thromboplastin time in the measurement of blood coagulation activity, which is now widely conducted in various clinical inspection rooms.

In order to improve activity of blood coagulation factor IX, a composition containing Mg²⁺ ions besides factor IX and Ca²⁺ ions can be also used in a measurement reagent.

According to the present invention, Mg²⁺ ions are added to a system for measuring blood coagulation activity, making it possible to stabilize the structure of blood coagulation factor IX and measure the blood coagulation activity more accurately under conditions closer to physiological conditions.

Brief Description of Drawings

Fig. 1 is a graph showing the effect of Mg²⁺ ions on the binding of factor IX to anti-factor IX-Ca²⁺ antibody.

The binding of bovine factor IX to anti-factor IX. Ca²⁺ antibody isolated was observed by ELISA in the presence of various concentrations of Ca²⁺ ions and Mg²⁺ ions.

Closed circles: Ca²⁺ ions alone, squares: Ca²⁺ ions and 0.3 mM Mg²⁺ ions, open circles: Ca²⁺ ions and 3 mM Mg²⁺ ions

Fig. 2 is a graph showing the effect of Mg²⁺ ions on the activation of factor IX by factor XIa.

Human factor IX (10 μg/ml ) and human factor XIa (2.5 ng/ml) were incubated at 37° C for 30 minutes in the presence of various concentrations of Ca²⁺ ions and Mg²⁺ ions, and the amount of factor IXa formed was determined. Closed circles: Ca²⁺ ions alone, open circles: Ca²⁺ ions and 1 mM Mg²⁺ ions

Fig. 3 is a graph showing the effect of Mg²⁺ ions on the activation of factor IX.

Human factor IX at various concentrations and human factor XIa (2.5 ng/ml) were incubated at 37° C for 30 minutes in the presence of 1 mM Ca²⁺ ions alone (●) or of 1 mM Ca²⁺ ions and 1 mM Mg²⁺ ions (O), and the amount of factor IXa formed was determined. The data obtained were analyzed through the Eadie-Hofstee plots [v = -K_m (v/s) +

V_max].

Closed circles: 1 mM Ca²⁺ ions alone, open circles: 1 mM

Ca²⁺ ions and 1 mM Mg²⁺ ions

Fig. 4 is a graph showing the time course of the activation of factor IX under physiological conditions.

Human factor IX (10 μg/ml) and human factor XIa (2.5 ng/ml) were incubated at 37°C for various periods of time in the presence of 1 mM Ca²⁺ ions alone (●) or of 1 mM Ca²⁺ ions and 1 mM Mg²⁺ ions (O), and the amount of factor IXa formed was determined.

Closed circles: 1 mM Ca²⁺ ions alone, open circles: 1 mM Ca²⁺ ions and 1 mM Mg²⁺ ions

Fig. 5 is a graph showing the effect of Mg ions on the clotting time.

The active coagulation factors which had been diluted to various concentrations were added to plasmas dialyzed in the presence of Ca²⁺ ions alone (●) or of Ca²⁺ ions and Mg²⁺ ions (O), and the clotting time was measured. The logarithm of the clotting time was plotted against the logarithm of the active coagulation factors. In order to reproduce the physiological conditions, the amount of Ca²⁺ ions was set at 2.5 mM and that of Mg²⁺ ions at 1 mM respectively.

Closed circles: 2.5 mM Ca²⁺ ions alone, open circles: 2.5 mM Ca²⁺ ions and 1 mM Mg²⁺ ions

Examples

The present invention will be illustrated more specifically by referring to the following Examples. However, the present invention is not limited thereto.

Human and bovine factors IX, X and the like used in the following Examples were prepared by the known methods or bought as reagents (J. Biochem., by Hashimoto N. et al., 97, 1347 -1355, 1985, Anal. Biochem., by Miletich J. P. et al., 105, 340 - 350, 1980, and Biochem. Biophys. Res. Commun., by Morita T., 130, 841 - 847, 1985). Polyclonal antibodies and monoclonal antibodies of these factors were prepared by the known methods. General reagents, phosphatidylcholine, bovine serum albumin, factor IX-deficient plasma were bought as reagents.

Example 1

Effect of Mg²⁺ ions on binding of factor IX to anti-factor X antibody

To examine the effect of Mg²⁺ ions on binding of factor IX to anti-factor IX antibody, a polyclonal antibody that recognizes bovine factor IX dependently on the conformation was isolated from a rabbit antiserum sensitized with bovine factor IX. Using the polyclonal antibody prepared and a monoclonal antibody that recognizes the Gla domain of human factor IX dependently on Ca²⁺ ions, the binding to factor IX was observed by enzyme-linked immunosorbent assay (ELISA).

A conformational-dependent anti-factor IX polyclonal antibody was prepared by the modification of the method of Liebman et al. (J. Biol. Chem., by Liebman H. A. et al., 262, 7605 - 7612, 1987). A rabbit antiserum immunized with bovine factor IX was added to an affinity column of bovine factor IX (factor IX-Cellulofine) equilibrated with a Tris-buf fered saline (TBS: 20 mM Tris-HCl, 140 mM NaCl, pH 7.5) containing 5 mM Ca²⁺ ions. An antibody absolutely requiring Ca²⁺ ions for the binding (anti-factor IX-Ca²⁺ antibody) was eluted with TBS containing 5 mM Mg²⁺ ions. Then, an antibody to be bound to factor IX in the presence of metal ions other than Ca²⁺ ions, such as Mg²⁺ ions (anti-factor IX-Mg²⁺ antibody) and an antibody to be bound to factor IX independently on metal ions were eluted with TBS containing 5 mM EDTA and guanidine hydrochloride in sequence. The conformational-dependent polyclonal antibodies of the other coagulation factors were prepared in the above-mentioned manner.

The properties of these antibodies were identified through the following ELISA.

Factor IX diluted with TBS to 1 μg/ml was adsorbed on a microtiter plate for 2 hours, and the solution was then removed. The residue was blocked with 100 μl of TBS containing 1 mg/ml of bovine serum albumin (BSA). Each well was washed three times with 200 μl of TBS. Subsequently, each of the antibodies which had been diluted to an appropriate concentration with TBS/Tween (TBS containing 0.1% (v/v) of Tween 20), and various concentrations of Ca²⁺ ions and Mg²⁺ ions was added thereto in an amount of 50 μl/well, and the reaction was conducted at 37° C for 2 hours. The antibody was removed, and the residue was washed three times with 200 μl of TBS/Tween containing Ca²⁺ ions and Mg²⁺ ions at the same concentrations. Then, a peroxidase-labeled anti-rabbit IgG antibody (for polyclonal antibody) or anti-mouse IgG antibody (for monoclonal antibody) which had been diluted to an appropriate concentration with TBS/Tween containing Ca²⁺ ions and Mg²⁺ ions at the same concentrations was added thereto in an amount of 50 μl/well. and the reaction was conducted for 1 hour. After the enzyme-labeled antibody was removed, the residue was washed five times with 200 μl of TBS/Tween containing various concentrations of Ca²⁺ ions and Mg²⁺ ions. Subsequently, a reaction solution containing H₂O₂ as an enzyme substrate and o- phenylenediamine as a color reagent [0.1 M citrate buffer containing 0.06% of H₂O₂ and 1 mg/ml of o- phenylenediamine] was added thereto in an amount of 50 μl/well, and the reaction was conducted for 30 minutes by shielding the light. 4 N sulfuric acid was added to the reaction mixture in an amount of 50 μl/well to terminate the reaction. The absorbance of 492 nm was then measured using a microtiter plate reader (MPR-4A manufactured by Tosoh Corp.). All of the procedures were conducted at room temperature. The buffers used were all those which had been treated through a column of Chelex.

Consequently, as shown in Fig. 1, the anti-factor IX- Ca²⁺ antibody was bound to factor IX dependently on Ca²⁺ ions, but this binding did not occur through Mg²⁺ ions alone. When Mg²⁺ ions were co-existent, the requirement for Ca²⁺ ions was decreased, and the binding was greatly increased in the presence of excess Ca²⁺ ions through Mg²⁺ ions.

The above-mentioned test was conducted by changing the probe to the monoclunal antibody. Two types of the antibodies used are those which recognize the Gla domain of human factor IX bound to Ca²⁺ ions. In this case as well, the binding was greatly increased in the presence of 0.3 mM and 3 mM of Mg²⁺ ions. The same results as shown in Fig. 1 were obtained.

Thus, it was clarified that the tertiary structure of the Gla domain of factor IX was much changed through Mg²⁺ ions. That is, it is considered that unlike the conformation of the Gla domain of factor IX in the presence of either Ca²⁺ ions or Mg²⁺ ions, the conformation of the Gla domain of factor IX in the presence of Ca²⁺ ions and Mg²⁺ ions is maintained through not only Ca²⁺ ions but also Mg²⁺ ions.

Reference Example 1

Effect of Mg²⁺ ions on other Gla-containinα coagulation proteins

In order to examine the effect of Mg²⁺ ions on Gla- containing coagulation proteins other than factor IX. conformation-dependent polyclonal antibodies against proteins, namely human prothrombin. bovine factor VII, bovine factor X and bovine protein C were prepared by the same method as the anti-factor IX·Ca²⁺ antibodies, and the effect of Mg²⁺ ions on bindings of the proteins to the polyclonal antibodies was observed.

All of the bindings were absolutely dependent on Ca²⁺ ions, and no binding occurred in the presence of Mg²⁺ ions alone. However, unlike the case of factor IX, the affinity for the binding was almost unchanged in the presence of Ca²⁺ ions and Mg²⁺ ions. The binding was not at all influenced by Mg²⁺ ions in the presence of excess Ca²⁺ ions (from 5 to 10 mM). In factor X and protein C, the decrease in the requirement for Ca²⁺ ions by Mg²⁺ ions was observed, but the change was negligible compared to the case of factor IX. This result strongly suggests that the Gla-containing coagulation proteins other than factor IX are not influenced by Mg²⁺ ions.

Example 2

Effect of Mg²⁺ ions on the function of factor IX

The effect of Mg²⁺ ions on the activation of factor IX corresponding to the change in the conformation of factor IX was examined using factor XIa as a factor IX activator.

An appropriate concentration of human factor IX and 2.5 ng/ml of human factor XIa were reacted in TBS containing BSA in the presence of various concentrations of Ca²⁺ ions and Mg²⁺ ions at 37° C for an appropriate period of time. After the completion of the reaction, Ca²⁺ ions and Mg²⁺ ions at such concentrations that the total concentrations of metal ions became identical in all of the samples and ethylenediaminetetraacetic acid (EDTA) at a concentration capable of completely neutralizing the metal ions were added thereto to terminate the reaction. The amount of factor IXa formed by the activation of factor IX was determined through the following coagulation assay.

Fifty microliters of factor-IX-deficient plasma, 50 μl of the above-obtained solution and 50 μl of a phospholipid [obtained by diluting 1 mg/ml of a mixture of phosphatidylcholine and phosphatidylserine at a ratio of 3:1 (w/w) with TBS] were incubated at 37° C for 2 minutes. Fifty microliters (final concentration 5 mM) of TBS containing 20 mM Ca²⁺ ions were added to start the coagulation at 37°C. The clotting time was measured using an Amelung Coagulometer KC 4A.

The amount of factor IXa formed was calculated using a standard curve. The standard curve was obtained by conducting coagulation assay with a human factor IXaβ purified product containing Ca²⁺ ions, Mg²⁺ ions and EDTA at the same concentrations as in the actual sample, and plotting the logarithm of the clotting time against the logarithm of the concentration of factor IXa.

In this test system, factor XIa was used at as low a concentration as possible and a considerably long reaction time was employed in the first reaction. Under such reaction conditions, the secondary activation of factor IX could be neglected during the second coagulation assay, making it possible to accurately determine the amount of factor IXa. In order to determine quite a low concentration of factor IXa, it is advisable that plasma used contains a very small amount of factor IX. Human plasma congenitally deficient in factor IX and plasma from which factor IX had been removed with a monoclonal antibody were used in this test.

Kinetic parameters were calculated by changing the concentration of factor IX in the range of from 0.2 to 1.2 μM. The reaction time was set within such a range as to ensure the linearity of the activation of factor IX. The data obtained were analyzed by the Eadie-Hofstee plots. The buffers used in all of the procedures were those which had been treated with a column of Chelex.

Consequently, as shown in Fig. 2, when Ca²⁺ ions and Mg²⁺ ions were co-existed, the amount of factor IXa formed was increased. On the other hand, when only Mg²⁺ ions were present, factor IX was not activated. When 1 mM Mg²⁺ ions were co-existed, the requirement for Ca²⁺ ions was lowered by 10 times. Even in the presence of excess Ca²⁺ ions in which the rate of activation reached a plateau value, the amount of factor IXa formed was further increased by Mg²⁺ ions. The shapes of curves and the range of Ca²⁺ concentrations required in Fig. 2 closely resemble those in Fig. 1 obtained with respect to the effect of Mg²⁺ ions on the structure. This shows that since the structure of factor IX was changed by Mg²⁺ ions, it was liable to be recognized by factor XIa.

For clarifying the details of the effect of Mg²⁺ ions, kinetic parameters of the activation of factor IX were determined in the presence or absence of 1 mM Mg²⁺ ions. The results are shown in Table 1. The results obtained in the absence of Mg²⁺ ions are approximately consistent with those in the literature which were

obtained by a different method. Even when Ca²⁺ ions were present at a concentration (5 mM) by which the maximum rate of activation was reached, the K_m value was 0.31 μM which was higher than that (approximately 0.1 μM) in plasma of factor IX. When Mg²⁺ ions were co-existed, the V_max value remained unchanged, but the K_m value was decreased to 0.6 time. In the presence of a physiological concentration (1 mM) of Ca²⁺ ions, the effect of Mg²⁺ ions was more increased, and the K_m value was reduced from 0.91 μM to 0.24 μM as shown in Fig. 3 and Table 1. Meanwhile, the K_cat value was slightly decreased (~20%) by Mg²⁺ ions. On the basis of these results, the initial rate of

activation under physiological conditions (assuming that the concentration of factor IX in plasma was 0.1 μM and the concentration of free Ca²⁺ ions was 1 mM) was

calculated according to the Michaelis-Menten equation. As a result, the initial rate of activation was 2.5 times higher in the presence of Ca²⁺ ions and Mg²⁺ ions than in the presence of Ca²⁺ ions alone.

The data in Table 1 were average ± SE values obtained by three measurements. The K_cat value was calculated on the assumption that all of the binding sites of factor XIa were retained.

The data of Sinha et al . (Biochem., by Sinha D, et al., 26, 3768 - 375, 1987) are also shown in Table 1. They measured and analyzed the amount of ³H-labeled activation peptide released from factor IX.

Example 3

Effect of Mg²⁺ ions on the time course of activation of factor IX

In consideration of the effect of Mg²⁺ ions on the initial rate of activation of factor IX calculated, the physiological phenomenon was simulated to ensure the significance of Mg²⁺ ions in plasma. Factor IX (10 μg/ml - concentration in plasma) was activated in the presence of 1 mM Ca²⁺ ions and 1 mM Mg²⁺ ions (close to physiological release concentrations), and the time course of activation was observed. The activation proceeded efficiently in the presence of Mg²⁺ ions (namely under physiological

conditions). However, when Mg²⁺ ions were removed, the rate of activation was notably decreased as shown in Fig. 4. This result proves that Mg²⁺ ions present in plasma in the order of mM have quite a significant role in the activation of factor IX in vivo.

Example 4

Effect of Mg²⁺ ions on clotting time

The participation of Mg²⁺ ions in the coagulation of plasma was actually examined under more physiological conditions. Factor Xa, IXa or XIa was added to human plasma from which metal ions contained had been removed through dialysis against TBS in the presence of

phospholipids and physiological concentrations of Ca²⁺ ions and Mg²⁺ ions to start the coagulation. The clotting time was measured, and the effect of Mg²⁺ ions on the rate of formation of thrombin was examined.

Plasma in bloods which had been offered by peoples of the institute were used in this test. The bloods were collected with citric acid, and centrifuged at room temperature for 10 minutes at 3,000 rpm to obtain

platelet-deficient plasma. In order to completely remove metal ions and citric acid, the plasma was dialyzed against TBS (2 liters x 2) for 1 hour, and were then immediately stored at -80°C.

Fifty microliters of the thus-treated plasma. 50 μl of active coagulation factor which had been diluted with TBS containing BSA at various concentrations and 50 μl of phospholipids were incubated at 37°C for 2 minutes. To the culture were added 50 μl (final concentration; 2.5 mM or 1 mM) of TBS containing 10 mM Ca²⁺ alone or 10 mM Ca²⁺ ions and 4 mM Mg²⁺ ions to start the coagulation. The clotting time was then measured.

Consequently, as shown in Fig. 5, when the

coagulation was started with factor Xa , no effect of Mg²⁺ ions was observed. However, when the coagulation was started with factors IXa and XIa. the clotting time was shortened in the presence of Mg²⁺ ions. The above- mentioned results prove that Mg²⁺ ions accelerate not only the activation of factor IX by factor XIa but also the activation of factor X by factor IXa. Meanwhile, it is considered that Mg²⁺ ions do not participate in the activation of prothrombin by factor Xa.

Claims

Claim

1. A reagent for measuring blood coagulation activity mediated by blood coagulation factor IX,

characterized in that said reagent contains Mg²⁺ ions.

2. The reagent as claimed in claim 1, which

contains Mg²⁺ ions such that the concentration of the Mg²⁺ ions reaches at least 0.3 mM when the Mg²⁺ ions are added to a reaction solution for measuring the blood coagulation activity.

3. The reagent as claimed in claim 1, which

contains Mg²⁺ ions such that the concentration of the Mg²⁺ ions reaches from 1 to 3 mM when the Mg²⁺ ions are added to a reaction solution for measuring the blood coagulation activity.

4. A method of measuring blood coagulation activity mediated by blood coagulation factor IX, characterized by adding Mg²⁺ ions to a reaction solution for measuring the blood coagulation activity.

5. The method as claimed in claim 4, wherein the Mg²⁺ ions are added such that the concentration of the Mg²⁺ ions reaches at least 0.3 mM.

6. The method as claimed in claim 4, wherein the Mg²⁺ ions are added such that the concentration of the Mg²⁺ ions reaches from 1 to 3 mM.