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WO1996038585A1 - Reactif permettant de mesurer l'activite de coagulation sanguine - Google Patents

Reactif permettant de mesurer l'activite de coagulation sanguine

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Publication number
WO1996038585A1
WO1996038585A1 PCT/JP1996/001488 JP9601488W WO9638585A1 WO 1996038585 A1 WO1996038585 A1 WO 1996038585A1 JP 9601488 W JP9601488 W JP 9601488W WO 9638585 A1 WO9638585 A1 WO 9638585A1
Authority
WO
WIPO (PCT)
Prior art keywords
ions
factor
blood coagulation
reagent
measuring
Prior art date
Application number
PCT/JP1996/001488
Other languages
English (en)
Inventor
Takashi Morita
Original Assignee
Eisai Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eisai Co., Ltd. filed Critical Eisai Co., Ltd.
Publication of WO1996038585A1 publication Critical patent/WO1996038585A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)
    • G01N2333/96441—Serine endopeptidases (3.4.21) with definite EC number
    • G01N2333/9645—Factor IX (3.4.21.22)

Definitions

  • the present invention relates to a reagent for measuring blood coagulation activity mediated by blood coagulation factor IX, the reagent containing Mg 2+ ions, as well as to a method of measuring blood coagulation activity mediated by blood coagulation factor IX, which comprises adding Mg 2+ ions to a reaction solution for measuring the blood coagulation activity.
  • 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.
  • 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.
  • vitamin K-dependent coagulation factors in which vitamin K is indispensable in biosynthesis thereof prothrombin (factor II), factor VII, factor IX, factor X have Ca 2+ -binding sites including a domain which is present at the N-terminal and which is rich in ⁇ - carboxyglutamic acid (Gla) (this domain is called Gla domain), and these factors show strong dependence on Ca 2+ ions for maintaining the functional conformation important to exhibit the functions (Blood, by Mann K.G. et al., 76, 1 - 16, 1990).
  • the Ca 2+ 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.
  • the concentration of Mg 2+ ions in plasma is usually between 0.4 and 0.6 mM.
  • the plasma sample is diluted to between 5 and 10 times in a reagent solution; therefore, the concentration of the Mg 2+ 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.
  • 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 Mg 2+ 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.
  • the present invention relates to a reagent for measuring blood coagulation activity mediated by blood coagulation factor IX, characterized in that the reagent contains Mg 2+ ions, as well as to a method mediated by measuring blood coagulation activity of blood coagulation factor IX, which comprises adding Mg 2+ 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 Mg 2+ , the activity being mediated, or involved, or required, or participated by blood coaguration factor IX.
  • 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 Ca 2+ ions (Eur. J. Biochem., by Atoda H. et al., 224, 703 - 708, 1994). Then, they have examined the effects of Mg 2+ ions on the binding of the snake venom anticoagulant protein to factors IX and X.
  • the present inventors have further studied the fact in which Ca 2+ ions are effective for both factors IX and X. whereas Mg 2+ ions are effective for factor IX alone, the effects (interrelation) of metal ion binding sites in anticoagulant protein and Mg 2+ ions, and the effects of Mg 2+ ions on the binding of conformational dependent or Ca 2+ -dependent anti-factor IX antibody to factor IX. Consequently, it has been found that the Mg 2+ ions are not reactive for snake venom anticoagulant proteins but for factor IX.
  • the present invention discloses, as demonstrated in Examples. that Mg 2+ 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 Mg 2+ ions are freshly added besides Mg 2+ ions derived from plasma samples.
  • the concentration of Mg 2+ 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 Mg 2+ ions is a final concentration thereof.
  • the amount of Mg 2+ 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.
  • Mg 2+ 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 Mg 2+ 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 Mg 2+ 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.
  • the reagent 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 Mg 2+ ions refers to the addition of an Mg 2+ -ion source.
  • Mg 2+ -ion source examples include MgF 2 , MgCl 2 , MgBr 2 , Mgl 2 and (CH 3 CO 2 ) 2 Mg.
  • Mg 2+ ions for example, as MgCl 2
  • a reagent used to measure the prothrombin time, the partial prothromboplastin time or the activated partial thromboplastin time 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.
  • a composition containing Mg 2+ ions besides factor IX and Ca 2+ ions can be also used in a measurement reagent.
  • Mg 2+ 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.
  • Fig. 1 is a graph showing the effect of Mg 2+ ions on the binding of factor IX to anti-factor IX-Ca 2+ antibody.
  • Fig. 2 is a graph showing the effect of Mg 2+ ions on the activation of factor IX by factor XIa.
  • Human factor IX (10 ⁇ g/ml ) and human factor XIa (2.5 ng/ml) were incubated at 37° C for 30 minutes in the presence of various concentrations of Ca 2+ ions and Mg 2+ ions, and the amount of factor IXa formed was determined. Closed circles: Ca 2+ ions alone, open circles: Ca 2+ ions and 1 mM Mg 2+ ions
  • Fig. 3 is a graph showing the effect of Mg 2+ 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 Ca 2+ ions alone ( ⁇ ) or of 1 mM Ca 2+ ions and 1 mM Mg 2+ ions (O), and the amount of factor IXa formed was determined.
  • Fig. 4 is a graph showing the time course of the activation of factor IX under physiological conditions.
  • Human factor IX (10 ⁇ g/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 Ca 2+ ions alone ( ⁇ ) or of 1 mM Ca 2+ ions and 1 mM Mg 2+ ions (O), and the amount of factor IXa formed was determined.
  • 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 Ca 2+ ions alone ( ⁇ ) or of Ca 2+ ions and Mg 2+ 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 Ca 2+ ions was set at 2.5 mM and that of Mg 2+ ions at 1 mM respectively.
  • 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.
  • a polyclonal antibody that recognizes bovine factor IX dependently on the conformation was isolated from a rabbit antiserum sensitized with bovine factor IX.
  • 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 Ca 2+ ions.
  • an antibody absolutely requiring Ca 2+ ions for the binding was eluted with TBS containing 5 mM Mg 2+ ions. Then, an antibody to be bound to factor IX in the presence of metal ions other than Ca 2+ ions, such as Mg 2+ ions (anti-factor IX-Mg 2+ 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.
  • TBS bovine serum albumin
  • the antibody was removed, and the residue was washed three times with 200 ⁇ l of TBS/Tween containing Ca 2+ ions and Mg 2+ 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 Ca 2+ ions and Mg 2+ ions at the same concentrations was added thereto in an amount of 50 ⁇ l/well. and the reaction was conducted for 1 hour.
  • reaction solution containing H 2 O 2 as an enzyme substrate and o- phenylenediamine as a color reagent [0.1 M citrate buffer containing 0.06% of H 2 O 2 and 1 mg/ml of o- phenylenediamine] was added thereto in an amount of 50 ⁇ l/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 ⁇ l/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.
  • the anti-factor IX- Ca 2+ antibody was bound to factor IX dependently on Ca 2+ ions, but this binding did not occur through Mg 2+ ions alone.
  • Mg 2+ ions were co-existent, the requirement for Ca 2+ ions was decreased, and the binding was greatly increased in the presence of excess Ca 2+ ions through Mg 2+ 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 Ca 2+ ions. In this case as well, the binding was greatly increased in the presence of 0.3 mM and 3 mM of Mg 2+ ions. The same results as shown in Fig. 1 were obtained.
  • the tertiary structure of the Gla domain of factor IX was much changed through Mg 2+ ions. That is, it is considered that unlike the conformation of the Gla domain of factor IX in the presence of either Ca 2+ ions or Mg 2+ ions, the conformation of the Gla domain of factor IX in the presence of Ca 2+ ions and Mg 2+ ions is maintained through not only Ca 2+ ions but also Mg 2+ ions.
  • 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 IXa ⁇ purified product containing Ca 2+ ions, Mg 2+ 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.
  • 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 ⁇ M.
  • 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.
  • 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

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Abstract

L'invention concerne un réactif permettant de mesurer l'activité de coagulation sanguine induite par le facteur IV de coagulation sanguine, ledit réactif étant caractérisé en ce qu'il contient des ions Mg2+. L'invention concerne également un procédé permettant de mesurer l'activité de coagulation sanguine induite par le facteur IX de coagulation sanguine, ce procédé consistant à ajouter des ions Mg2+ à une solution réactive pour mesurer l'activité de coagulation sanguine.
PCT/JP1996/001488 1995-06-01 1996-05-31 Reactif permettant de mesurer l'activite de coagulation sanguine WO1996038585A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP7134998A JPH08327631A (ja) 1995-06-01 1995-06-01 血液凝固活性測定試薬
JP7/134998 1995-06-01

Publications (1)

Publication Number Publication Date
WO1996038585A1 true WO1996038585A1 (fr) 1996-12-05

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2908892A1 (fr) * 2006-11-21 2008-05-23 Hyphen Biomed Soc Par Actions Procede pour le dosage chromogenique de l'activite du facteur viii:c et necessaire de dosage chromogenique de l'activite du facteur viii:c,dans les plasmas ou les fractions therapeutiques
WO2012066260A1 (fr) * 2010-11-18 2012-05-24 Lfb Biomedicaments Determination du pouvoir thrombogene d'immunoglobulines humaines

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991002813A1 (fr) * 1989-08-17 1991-03-07 Baxter International Inc. Analyse chromogene du facteur ix

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991002813A1 (fr) * 1989-08-17 1991-03-07 Baxter International Inc. Analyse chromogene du facteur ix

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
F. SEKIYA ET AL.: "Magnesium (II) is a crucial constituent of the blood coagulation cascade.", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 271, no. 15, 12 April 1996 (1996-04-12), pages 8541 - 8544, XP002014455 *
F. SEKIYA ET AL.: "Regulation of the terciary structure and function of coagulation factor IX by Magnesium (II) ions.", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 270, no. 24, 16 June 1995 (1995-06-16), pages 14325 - 14331, XP002014454 *
S. J. FREEDMAN ET AL.: "Structure of the metal-free gamma carboxyglutamic acid-rich membrane binding region of factor IX by two-dimensional NMR spectroscopy.", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 270, no. 14, 7 April 1995 (1995-04-07), pages 7980 - 7987, XP002014453 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2908892A1 (fr) * 2006-11-21 2008-05-23 Hyphen Biomed Soc Par Actions Procede pour le dosage chromogenique de l'activite du facteur viii:c et necessaire de dosage chromogenique de l'activite du facteur viii:c,dans les plasmas ou les fractions therapeutiques
WO2012066260A1 (fr) * 2010-11-18 2012-05-24 Lfb Biomedicaments Determination du pouvoir thrombogene d'immunoglobulines humaines
FR2967781A1 (fr) * 2010-11-18 2012-05-25 Lfb Biomedicaments Determination du pouvoir thrombogene d'immunoglobulines humaines
US9891238B2 (en) 2010-11-18 2018-02-13 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Determination of the thrombogenic power of human immunoglobulins

Also Published As

Publication number Publication date
JPH08327631A (ja) 1996-12-13

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