Reagent and method for measuring blood coagulation-related enzyme or fibrinolysis-related enzyme in blood sample

Abstract

This reagent for measuring a blood coagulation-related enzyme or fibrinolysis-related enzyme using a blood sample comprises: a chromogenic peptide substrate for the enzyme; and an aliphatic hydrocarbon compound, aliphatic ether compound, or polyoxyalkylene compound, the compound having two or more hydroxyl groups, being liquid at normal temperature and normal pressure, and being water-soluble.

Description

Reagent for measuring blood coagulation-related enzyme or fibrinolysis-related enzyme in blood sample, and measurement method

[0001] The present invention relates to a reagent used for blood tests and the like. The present invention also relates to a measurement method using said reagent, more specifically to a measurement method for a blood coagulation-related enzyme or a fibrinolysis-related enzyme.

[0002] When measuring the activity of enzymes related to blood coagulation and fibrinolysis using a blood sample such as plasma, a chromogenic substrate as disclosed in Patent Document 1 is used. Enzyme activity is measured based on the change in absorbance caused by the decomposition of the substrate by an enzyme such as thrombin. However, a blood coagulation reaction occurs simultaneously with the substrate decomposition reaction, which also causes a change in absorbance, so the influence of background on enzyme activity measurement is unavoidable. Accordingly, conventionally, measurements have been carried out using fluorescent substrates, fibrin polymerization inhibitors, or the like that are less susceptible to the influence of absorbance changes caused by blood coagulation.

[0003] Japanese Patent No. 5192647 Specification

[0004] An object of the present invention is to provide a technique capable of performing high-precision measurement by reducing the influence of background caused by blood coagulation reaction in the measurement of a blood coagulation-related enzyme or a fibrinolysis-related enzyme in a blood sample using a chromogenic substrate.

[0005] The present inventors have conducted intensive studies to solve the above problem. As a result, they found that in the measurement of a blood coagulation-related enzyme or a fibrinolysis-related enzyme in a blood sample using a chromogenic substrate, adding a compound containing two or more hydroxyl groups such as glycerol can reduce the absorbance background associated with blood coagulation reaction and enable more accurate measurement, thus completing the present invention.

[0006] One aspect of the present invention relates to a reagent for measuring a blood coagulation-related enzyme or a fibrinolysis-related enzyme using a blood sample, the reagent comprising: a chromogenic peptide substrate for said enzyme; and a compound that is an aliphatic hydrocarbon compound, an aliphatic ether compound or a polyoxyalkylene compound having two or more hydroxyl groups, which is liquid at normal temperature and normal pressure and is water-soluble.

[0007] Another aspect of the present invention relates to a method for measuring blood coagulation-related enzymes or fibrinolysis-related enzymes in a blood sample, comprising the steps of adding to the blood sample a chromogenic peptide substrate of the blood coagulation-related enzyme or fibrinolysis-related enzyme, and a compound which is an aliphatic hydrocarbon compound, an aliphatic ether compound, or a polyoxyalkylene compound having two or more hydroxyl groups, which is liquid at room temperature and pressure and is water-soluble, and detecting the decomposition reaction of the substrate by the blood coagulation-related enzyme or fibrinolysis-related enzyme by absorbance measurement.

[0008] Another aspect of the present invention relates to a method for suppressing absorbance changes due to a blood coagulation reaction, comprising the step of adding to a blood sample a compound which is an aliphatic hydrocarbon compound, an aliphatic ether compound, or a polyoxyalkylene compound having two or more hydroxyl groups, is liquid at room temperature and pressure, and is water-soluble. In other words, the present invention relates to an agent for suppressing absorbance changes due to a blood coagulation reaction, comprising a compound which is an aliphatic hydrocarbon compound, an aliphatic ether compound, or a polyoxyalkylene compound having two or more hydroxyl groups, is liquid at room temperature and pressure, and is water-soluble.

[0009] According to the present invention, when measuring the activity of blood coagulation-related enzymes or fibrinolysis-related enzymes in a blood sample using a chromogenic substrate, the influence of background noise caused by the blood coagulation reaction can be reduced, enabling highly accurate measurements. Compounds such as glycerol suppress only the increase in turbidity due to the blood coagulation reaction and do not affect the enzymatic reaction rate of the peptide substrate, thus enabling the measurement of the activity of specific blood coagulation-related enzymes or fibrinolysis-related enzymes. Since there is no need to use fluorescent substrates or fibrin polymerization inhibitors, the measurement can be performed simply and inexpensively.

[0010] Graphs showing the time course of absorbance at 405 nm after adding glycerol of various concentrations to plasma. Graphs showing the effect of glycerol on the measurement of thrombin activity in plasma based on absorbance changes, with and without the addition of a synthetic substrate. Graphs showing the results of experiments in measuring thrombin activity in plasma based on absorbance changes, with varying concentrations of glycerol added to plasma. Graphs showing the effect of glycerol on plasmin measurement in plasma based on absorbance changes. Graphs showing the inhibitory effect of various compounds (added at a final concentration of 10%) on the increase in absorbance associated with blood coagulation. Graphs showing the effect of rivaroxaban on the absorbance curve of plasma with 10% glycerol + FXa substrate added. Graphs showing the effect of rivaroxaban on the absorbance curve of plasma with 10% ethylene glycol + FXa substrate added. Graphs showing the effect of rivaroxaban on the absorbance curve of plasma with 10% propylene glycol + FXa substrate added. Graphs showing the effect of rivaroxaban on the absorbance curve of plasma with FXa substrate. Graphs showing the effect of dabigatran on the absorbance curve of plasma with 10% glycerol + thrombin substrate. Graphs showing the effect of dabigatran on the absorbance curve of plasma with 10% ethylene glycol + thrombin substrate. Graphs showing the effect of dabigatran on the absorbance curve of plasma with 10% propylene glycol + thrombin substrate. Graphs showing the effect of dabigatran on the absorbance curve of plasma with thrombin substrate.

[0011] <Reagent> The reagent of the present invention is a reagent for measuring blood coagulation-related enzymes or fibrinolysis-related enzymes using a blood sample, and comprises a chromogenic peptide substrate of the enzyme, and a compound having two or more hydroxyl groups, which is an aliphatic hydrocarbon compound, an aliphatic ether compound, or a polyoxyalkylene compound, which is liquid at room temperature and pressure and is water-soluble.

[0012] <Blood Sample> The blood sample is not particularly limited as long as it is derived from blood collected from a living organism and contains blood coagulation-related enzymes and / or fibrinolysis-related enzymes, but plasma is preferred.

[0013] <Enzymes> Examples of blood coagulation-related enzymes include thrombin, coagulation factor VII (FVIIa), coagulation factor IX (FIXa), coagulation factor X (FXa), kallikrein, coagulation factor XI (FXIa), coagulation factor XIIa, coagulation factor III (FXIIIa), and activated protein C.

[0014] Examples of fibrinolysis-related enzymes include plasmin, tissue plasminogen activator (t-PA), and urokinase-type plasminogen activator (u-PA).

[0015] <Chromogenic Peptide Substrates> Chromogenic peptide substrates used as substrates for blood coagulation-related enzymes or fibrinolysis-related enzymes include substances having a structure in which a cleavable chromophore is bound to the C-terminus (carboxyl group terminus) of an oligopeptide portion containing the amino acid sequence recognized by the enzyme. By using such a substance, the chromogenic group is cleaved from the oligopeptide portion by the blood coagulation-related enzyme or fibrinolysis-related enzyme, exhibiting absorption at a specific wavelength, and enzyme activity can be measured using an absorption spectrophotometer.

[0016] Examples of chromophores conjugated to peptide substrates include para-nitroaniline (pNA), 5-amino-2-nitrobenzoic acid (ANBA), and their derivatives. These chromogenic groups may be bonded, for example, to the C-terminus of the oligopeptide moiety via an amide bond through an amino group.

[0017] The oligopeptide portion of the chromogenic peptide substrate can be selected depending on the type of enzyme. These are well known to those skilled in the art and can be selected from well known enzyme recognition sequences. For example, in the case of thrombin, Ala-Gly-Arg or Gly-Gly-Arg can be used. Alternatively, Msc-Val-Arg or Msc-Val-Arg (where Msc is methylsulfonylethyloxycarbonyl: European Patent Publication EP0802986B1) can also be used.

[0018] Examples of chromogenic peptide substrates for each enzyme are listed below. However, the list is not limited to those listed below; for example, variations in which pNA is replaced with other chromophores, or their salts, can also be used.

[0019] Examples of chromogenic peptide substrates for thrombin: H-β-Ala-Gly-Arg-pNA (CAS number: 858971-42-3) Pentapharm Pefachrome TG (β-Ala represents a β-alanine residue.) H-Gly-Gly-Arg-pNA HD-Phe-Pip-Arg-pNA (CAS number: 64815-81-2) (S-2238) (Pip represents a piperidine ring.) HD-CHA-Ala-Arg-pNA (CAS number: 143556-06-3) HD-CHA-Gly-Arg-pNA (CHA represents a 3-cyclohexylalanine residue.) HD-CHG-Ala-Arg-pNA HD-CHG-But-Arg-pNA (CHG represents a cyclohexylglycine residue.) Tos-Gly-Pro-Arg-pNA (Tos represents the tosyl[4-toluenesulfonyl] group.) CH3OCO-Gly-Pro-Arg-pNA (CH3OCO represents the methyloxylcarbonyl group.) H-Sar-Pro-Arg-pNA (Sar represents the sarcosine residue.)

[0020] An example of a chromogenic peptide substrate for blood coagulation factor VII (FVIIa): CH3SO2-D-CHA-Abu-Arg-pNA (Pentapharm). (CH3SO2 represents a methanesulfonyl group. Abu represents a 2-aminobutyric acid residue.)

[0021] An example of a chromogenic peptide substrate for blood coagulation factor IX (FIXa): CH3SO2-D-CHG-Gly-Arg-pNA (Pentapharm). (CHG represents a 3-cyclohexylglycine residue.)

[0022] Examples of chromogenic peptide substrates for blood coagulation factor X (FXa): Bz-Ile-Glu(γ-OR)-Gly-Arg-pNA S-2222 Sekisui Medical (Bz represents a benzoyl group. Glu(γ-OR) represents a glutamic acid residue in which the γ-carboxyl group of the side chain is alkyl esterified, with 50 mol% of R being hydrogen atoms and 50 mol% being methyl groups.) CH3OCO-D-CHA-Gly-Arg-pNA (CAS number: 80895-10-9) ZD-Arg-Gly-Arg-pNA (Z represents a benzyloxycarbonyl group.)

[0023] Chromogenic peptide substrates for blood coagulation factor 11 (FXIa): pyroGlu-Pro-Arg-pNA (pyroGlu represents a pyroglutamic acid residue), HD-Val-Leu-Arg-pNA, Z-Aad-Pro-Arg-pNA (Z represents a benzyloxycarbonyl group, Aad represents a 2-aminoadipic acid residue).

[0024] Examples of chromogenic peptide substrates for blood coagulation factor XIIa: HD-CHA-Gly-Arg-pNA (Pentapharm) HD-Pro-Phe-Arg-pNA (S-2302 Sekisui Medical Co., Ltd.)

[0025] Examples of chromogenic peptide substrates for blood coagulation factor 13 (FXIIIa): H-Tyr-Glu(pNA)-Val-Lys-Val-Ile-Gly-NH2 H-Tyr-Glu(pNA)-Val-Lys-Val-Ile-NH2

[0026] Examples of chromogenic peptide substrates for plasmin: HD-Val-Leu-Lys-pNA (CAS number: 63589-93-5) (S-2251 Sekisui Medical Co., Ltd.) pyroGlu-Phe-Lys-pNA

[0027] An example of a chromogenic peptide substrate for kallikrein is HD-Pro-Phe-Arg-pNA (S-2302, Sekisui Medical Co., Ltd.).

[0028] Examples of chromogenic peptide substrates for t-PA: CH3SO2-D-CHA-Gly-Arg-pNA (Pentapharm) HD-Ile-Pro-Arg-pNA

[0029] Examples of chromogenic peptide substrates for u-PA: pyroGlu-Gly-Arg-pNA (Pentapharm) (pyroGlu represents a pyroglutamic acid residue.) Bz-β-Ala-Gly-Arg-pNA

[0030] An example of a chromogenic peptide substrate for active protein C is HD-Lys(Cbo)-Pro-Arg-pNA (Pentapharm). (Lys(Cbo) represents a lysine residue with an ε-amino group protected by a carbamoyl group.)

[0031] For other examples of chromogenic peptide substrates specific to each blood coagulation-related enzyme or fibrinolysis-related enzyme, see, for example, U.S. Patent Publication US 4,508,644 and Japanese Patent Publication JP 07-076232. Alternatively, commercially available chromogenic peptide substrates from companies such as Pentapharm can also be used.

[0032] <Compounds Having Two or More Hydroxyl Groups> The reagents of the present invention include a compound (sometimes simply called a compound having two or more hydroxyl groups) which is an aliphatic hydrocarbon compound, an aliphatic ether compound, or a polyoxyalkylene compound having two or more hydroxyl groups, is liquid at room temperature and pressure, and is water-soluble. Here, "room temperature and pressure" means the conditions of "25°C, 1013 hPa". Also, "water-soluble" means having a solubility of 1 g / 100 mL or more in water at 25°C. The number of hydroxyl groups in the compound is preferably 2 to 5, more preferably 2 to 4, and even more preferably 2 to 3.

[0033] In aliphatic hydrocarbon compounds having two or more hydroxyl groups, the aliphatic hydrocarbon portion is preferably acyclic (straight-chain or branched-chain) and preferably saturated. The number of carbon atoms is preferably 2 to 6, and more preferably 2 to 4. Examples of such compounds include glycerol, ethylene glycol, and propylene glycol.

[0034] Aliphatic hydrocarbon compounds and aliphatic ether compounds having two or more hydroxyl groups include compounds having the structure (HO)R-O-R'(OH) (where R and R' are alkyl), where the total number of carbon atoms is preferably 4 to 12. Examples of such compounds include diethylene glycol and dipropylene glycol.

[0035] Examples of polyoxyalkylene compounds having two or more hydroxyl groups include polyethylene glycol and polypropylene glycol. For them to be liquid at room temperature and pressure and to be water-soluble, the molecular weight of polyethylene glycol is 106 to 1000, preferably 200 to 400, and the molecular weight of polypropylene glycol is 134 to 2000, preferably 200 to 600.

[0036] <Reaction-Promoting Enzyme> The reagent of the present invention may contain a reaction-promoting enzyme (also called a reaction-initiating enzyme) for promoting the reaction of the enzyme to be measured. The reaction-promoting enzyme can be an enzyme located upstream of the blood coagulation cascade with respect to the enzyme to be measured. By adding the reaction-promoting enzyme, the enzyme to be measured can be converted to an active form, and the enzymatic reaction of the enzyme to be measured is initiated or promoted. Depending on the type of enzyme, the reaction-promoting enzyme can be selected from enzymes located upstream of the enzyme to be measured among the enzymes that constitute the coagulation cascade. In the case of thrombin, examples include blood coagulation factor XIIa, blood coagulation factor XI (FXIa), blood coagulation factor X (FXa), blood coagulation factor IX (FIXa), blood coagulation factor VII (FVIIa), tissue factor, etc. In the case of blood coagulation factor X (FXa), examples include blood coagulation factor XIIa, blood coagulation factor XIIa, blood coagulation factor XIIa, tissue factor, etc. In the case of blood coagulation factor VII (FVIIa), examples include tissue factor, etc. In the case of blood coagulation factor IX (FIXa), examples include blood coagulation factor XI (FXIa) and blood coagulation factor VII (FVIIa). In the case of blood coagulation factor XIIa, examples include kallikrein. In the case of plasmin, examples include tissue plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). In the case of kallikrein, examples include blood coagulation factor XIIa. In the case of t-PA, examples include plasmin. In the case of u-PA, examples include plasmin and kallikrein. In the case of activated protein C, examples include thrombin and thrombomodulin.

[0037] In the reagent of the present invention, the substrate, compound, and optionally an accelerator may be pre-mixed, or they may be contained separately so that they are mixed at the time of use. That is, the reagent of the present invention may be a reagent kit containing a compound having two or more hydroxyl groups, a chromogenic peptide substrate, and optionally a reaction-promoting enzyme.

[0038] The reagent of the present invention may include instructions for use that describe how to use it, such as how to add it to a blood sample, and its applications in blood tests.

[0039] The reagent of the present invention may contain, as optional, other components in addition to a substrate, a compound containing two or more hydroxyl groups, and a reaction-promoting enzyme. Examples of other components include stabilizers for protein enzymes such as carbohydrates and glycerol, antifoaming agents, dispersion promoters, coenzymes, calcium, magnesium, zinc, and manganese.

[0040] The reagents of the present invention are used for measuring blood coagulation-related enzymes or fibrinolysis-related enzymes. Here, the measurement includes not only the measurement of activity but also the measurement of the abundance of the enzyme based on its activity. The reagents of the present invention are used in blood tests and the like. For example, if the reagent of the present invention contains a thrombin substrate, it can be used as a test or diagnostic reagent in a test method for measuring thrombin production in a blood sample. Examples of these test methods are described in European Patent Publication EP1367135A1 and European Patent Publication EP0420332B1. Also, for example, if the reagent of the present invention contains a plasmin substrate, it can be used as a test or diagnostic reagent in a test method for measuring plasmin production in a blood sample.

[0041] <Measurement Method> The measurement method of the present invention is a method for measuring blood coagulation-related enzymes or fibrinolysis-related enzymes in a blood sample, comprising the steps of adding to a blood sample a chromogenic peptide substrate of the blood coagulation-related enzyme or fibrinolysis-related enzyme, and a compound which is an aliphatic hydrocarbon compound, an aliphatic ether compound, or a polyoxyalkylene compound having two or more hydroxyl groups, which is liquid at room temperature and pressure and is water-soluble, and detecting the decomposition reaction of the substrate by the blood coagulation-related enzyme or fibrinolysis-related enzyme by absorbance measurement.

[0042] Here, blood samples, compounds containing two or more hydroxyl groups, and enzymes are as described in the section on reagents above.

[0043] A compound containing two or more hydroxyl groups is added to a blood sample at a concentration suitable for suppressing the change in absorbance accompanying coagulation, i.e., the increase in turbidity. That is, it is preferable to add the compound to a final concentration of, for example, 5 to 30 (w / w)%, 5 to 20 (w / w)%, 5 to 15 (w / w)%, or 7.5 to 15 (w / w)%, and addition at about 10 (w / w)% is particularly preferable. For example, when adding at a final concentration of 10 (w / w)%, 1 part of the compound containing two or more hydroxyl groups may be added to 9 parts of the blood sample and mixed.

[0044] The substrate may also be added at a concentration such that the enzyme reaction can be detected in the blood sample; for example, it is added to a final concentration of 0.01 to 2 mM, more preferably 0.05 to 1 mM, still more preferably 0.1 to 0.5 mM.

[0045] To promote the reaction, it is preferable to add the reaction-promoting enzyme described above. The reaction-promoting enzyme may be selected according to the type of the target enzyme to be measured, and added at a concentration required for promoting the reaction. In addition, when the blood sample has been subjected to anticoagulation treatment, it is preferable to add an agent that cancels anticoagulation at the start of measurement. For example, when the blood sample has been treated with citric acid, calcium may be added at the start of measurement.

[0046] Reaction conditions are not particularly limited as long as the enzyme reaction proceeds; the reaction is usually performed at 20 to 40°C, more preferably 30 to 40°C, and still more preferably 37°C.

[0047] The wavelength at which absorbance is measured depends on the structure of the chromophoric moiety contained in the substrate, so it can be appropriately selected according to the type of substrate. For example, in the case of pNA, the wavelength is around 380 to 410 nm, and 405 nm is more suitable.

[0048] For the measurement, the absorbance value based on substrate decomposition at a specific time point after a certain period of time has elapsed from the addition of the substrate, the compound, and if necessary the reaction-promoting enzyme may be measured; alternatively, the change in absorbance may be measured over time starting immediately after or after a certain period of time has elapsed from the addition of the substrate, the compound, and if necessary the reaction-promoting enzyme.

[0049] Based on the measurement results, enzyme activity and abundance can be calculated, allowing for the examination of coagulation and fibrinolysis properties in blood samples. This also makes it possible to estimate the presence or absence of disease and the use of anticoagulants.

[0050] <Method for suppressing absorbance changes due to blood coagulation reaction> The present invention also provides a method for suppressing absorbance changes due to blood coagulation reaction, comprising the step of adding a compound to a blood sample that is an aliphatic hydrocarbon compound, an aliphatic ether compound, or a polyoxyalkylene compound having two or more hydroxyl groups, is liquid at room temperature and pressure, and is water-soluble. The present invention also provides an agent for suppressing absorbance changes due to blood coagulation reaction, comprising a compound that is an aliphatic hydrocarbon compound, an aliphatic ether compound, or a polyoxyalkylene compound having two or more hydroxyl groups, is liquid at room temperature and pressure, and is water-soluble. Since it has become clear that the increase in absorbance associated with blood coagulation can be suppressed by adding a compound containing two or more hydroxyl groups, it can be used for purposes other than measuring blood coagulation-related enzymes or fibrinolysis-related enzymes using blood samples.

[0051] One aspect of the present invention relates to a microfluidic device for testing coated with the above-mentioned reagent. This device may consist of a microchip in which fine channels for transporting a sample are formed inside or on the surface of a substrate, and a reaction section is provided in a part of the channels in which the reagent of the present invention is held in a dry state (see, for example, WO2022 / 045355). A chromogenic peptide substrate, along with a compound such as glycerol, and optionally a reaction-promoting enzyme and a buffer, are pre-fixed to the reaction section by inkjet printing or spot coating. When a blood sample (whole blood or plasma) is introduced into the channel, the blood sample reaches the reaction section by capillary action or external drive, and the held reagent is rapidly redissolved, thereby initiating an enzymatic reaction between the blood coagulation-related enzyme or fibrinolysis-related enzyme in the blood sample and the chromogenic peptide substrate.

[0052] The structure of the microfluidic device can be configured such that an inlet for injecting a sample, a reaction chamber for mixing and reacting the reagent and the sample, and a detection unit for measuring the optical changes produced by the reaction are connected in that order, or the reaction unit and the detection unit are integrated. The detection unit has an observation window made of a translucent material and monitors changes in absorbance, fluorescence, or emission caused by a chromophore (e.g., p-nitroaniline) released from the substrate over time using an external optical sensor. This makes it possible to detect, quantify, or measure the activity of enzymes such as thrombin, factor Xa, and plasmin present in a blood sample by calculating enzyme activity kinetically (rate method) or from the total change after a certain period of time (endpoint method).

[0053] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the embodiments of the following examples. In the following examples, % refers to weight % (w / w).

[0054] For reference, citrated plasma was treated with 13 mM calcium chloride, 0.9 U / mL human α-thrombin, 8 mM HEPES, and glycerol at each final concentration. The time-dependent change in absorbance at 405 nm was then measured. The results are shown in Figure 1.

[0055] In plasma without glycerol, an increase in absorbance associated with blood coagulation was observed over time, even without a synthetic substrate. This increase in absorbance decreased as the glycerol concentration increased, and when 10% glycerol was added, no increase in absorbance was observed, completely suppressing the absorbance change due to blood coagulation.

[0056] Example 1 Next, the effect of glycerol was investigated with and without the addition of the thrombin synthesis substrate S-2238. To citrated plasma, calcium chloride at a final concentration of 13 mM, human α-thrombin at a final concentration of 0.9 U / mL, HEPES at a final concentration of 8 mM, S-2238 at a final concentration of 0.3 mmol / L, and glycerol at a final concentration of 10% were added, and the change in absorbance over time at 405 nm was measured. These results were compared with the case without S-2238.

[0057] As shown in the upper part of Figure 2, when the synthetic substrate S-2238 was not added, the absorbance increased without glycerol, whereas the increase in absorbance was suppressed when glycerol was added. As shown in the lower part of Figure 2, when the synthetic substrate was added, an increase in absorbance due to substrate decomposition was observed both with and without glycerol, which was thought to represent the amount of thrombin produced in the plasma sample. However, considering the results in the upper part of Figure 2 together, it was thought that the value without glycerol included background noise from the increase in absorbance due to blood coagulation. In contrast, when glycerol was added, it was thought that the value reflected only the reaction due to the generated thrombin.

[0058] Example 2 Next, the same experiment as in Example 1 was conducted by changing the concentration of glycerol added to the plasma. As a result, as shown in Figure 3, it was found that adding 3.75% reduced the increase in absorbance associated with blood coagulation to less than half, 7.5% reduced it to a level where it could be almost ignored, and 10% reduced it completely.

[0059] Example 3 Next, the substrate was changed to the plasmin substrate S-2251, and plasmin was measured. To citrated plasma, calcium chloride at a final concentration of 13 mM, t-PA at a final concentration of 100,000 units / mL, HEPES at a final concentration of 8 mM, S-2251 at a final concentration of 0.3 mmol / L, and glycerol at each final concentration were added, and the change in absorbance over time at 405 nm was measured. These results were compared with the case where S-2251 was not added.

[0060] The results are shown in Figure 4. When t-PA is added in the absence of glycerol, turbidity (absorbance) increases due to blood coagulation, and then the coagulation dissolves due to plasmin activation, causing the turbidity to decrease (no substrate, no glycerol, 100,000 units of t-PA). When a plasmin substrate is added under the above conditions, the increase in turbidity due to coagulation and the color development of the substrate cannot be distinguished (S2251, no glycerol, 100,000 units of t-PA). In contrast, when glycerol is added at a final concentration of 10% under the above conditions, no turbidity occurs, and plasmin activity can be detected by the color development of the substrate (S2251, 10% glycerol, 100,000 units of t-PA). Under conditions without t-PA (S2251, no glycerol, no t-PA), there is no increase in absorbance at all, so the orange curve was confirmed to be due to plasmin activity.

[0061] Reference Example 2: Furthermore, we screened for compounds other than glycerol that can suppress the increase in absorbance due to blood coagulation. As a result of evaluation using the same system as in Reference Example 1, as shown in Figure 5, inhibitory effects were observed in propylene glycol, dipropylene glycol, polypropylene glycol, ethylene glycol, and diethylene glycol, and a weak effect was also observed in polyethylene glycol with an average molecular weight of 200.

[0062] Example 4: Calcium chloride at a final concentration of 13 mM, tissue factor at a final concentration of 5 pmol / L, HEPES at a final concentration of 8 mM, Xa substrate S-2222 at a final concentration of 0.12 mmol / L, and various compounds (10% glycerol, 10% ethylene glycol, 10% propylene glycol, or no compound) were added to citrated plasma, and the change in absorbance over time at 405 nm was measured. Rivaroxaban at various final concentrations (32, 64, 125, 250, 500 ng / mL) was also added to this system, and the change in absorbance over time was measured. The results are shown in Figures 6A-D. The results showed that in the case of no compound, the absorbance quickly reached its maximum and no change over time was observed. However, in the case of glycerol, ethylene glycol, or propylene glycol, a change in absorbance over time specifically reflecting the degradation of the Xa substrate was observed, and this value decreased depending on the amount of the anticoagulant rivaroxaban. This indicates that by adding the Xa substrate to a blood sample in the presence of glycerol, ethylene glycol, and propylene glycol, the activity of Xa can be specifically observed, and the effects of anticoagulants can also be evaluated.

[0063] Example 5: Calcium chloride at a final concentration of 13 mM, tissue factor at a final concentration of 5 pmol / L, HEPES at a final concentration of 8 mM, thrombin substrate Pefachrome TG (Pentafarm) at a final concentration of 2 mmol / L, and various compounds (10% glycerol, 10% ethylene glycol, 10% propylene glycol, or no compound) were added to citrated plasma, and the change in absorbance over time at 405 nm was measured. Dabigatran at various final concentrations (32, 64, 125, 250, 500 ng / mL) was also added to this system, and the change in absorbance over time was measured. The results are shown in Figures 7A-D. The results showed that in the case of no compound, the absorbance quickly reached its maximum and no change over time was observed. However, in the case of glycerol, ethylene glycol, or propylene glycol, a change in absorbance over time specifically reflecting the degradation of the thrombin substrate was observed, and this value decreased depending on the amount of the anticoagulant dabigatran. This indicates that by adding a thrombin substrate to a blood sample in the presence of glycerol, ethylene glycol, and propylene glycol, thrombin activity can be specifically observed, and the effects of anticoagulants can also be evaluated.

Claims

1. A reagent for measuring blood coagulation-related enzymes or fibrinolysis-related enzymes using a blood sample, comprising: a chromogenic peptide substrate of the enzyme; and a compound having two or more hydroxyl groups, which is a liquid at room temperature and pressure and is water-soluble.

2. The reagent according to claim 1, wherein the compound is one or more selected from the group consisting of glycerol, propylene glycol, dipropylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, and polyethylene glycol.

3. The reagent according to claim 1, wherein the blood coagulation-related enzyme is thrombin, blood coagulation factor VII (FVIIa), blood coagulation factor IX (FIXa), blood coagulation factor X (FXa), blood coagulation factor XI (FXIa), blood coagulation factor XIIa, blood coagulation factor III (FXIIIa), kallikrein, or activated protein C, and the fibrinolysis-related enzyme is plasmin, tissue plasminogen activator (t-PA), or urokinase-type plasminogen activator (u-PA).

4. The reagent according to claim 1, further comprising a reaction-promoting enzyme.

5. The reagent according to claim 4, wherein if the enzyme is thrombin, the reaction-promoting enzyme is coagulation factor XIIa, coagulation factor XI (FXIa), coagulation factor X (FXa), coagulation factor IX (FIXa), coagulation factor VII (FVIIa), or tissue factor; if the enzyme is coagulation factor X (FXa), the reaction-promoting enzyme is coagulation factor XIIa, coagulation factor XIIa, coagulation factor XI (FXIa), coagulation factor IX (FIXa), coagulation factor VII (FVIIa), or tissue factor; and if the enzyme is plasmin, the reaction-promoting enzyme is tissue plasminogen activator (t-PA).

6. A method for measuring blood coagulation-related enzymes or fibrinolysis-related enzymes in a blood sample, comprising the steps of adding to a blood sample a chromogenic peptide substrate of the blood coagulation-related enzyme or fibrinolysis-related enzyme, and a compound having two or more hydroxyl groups, which is an aliphatic hydrocarbon compound, an aliphatic ether compound, or a polyoxyalkylene compound, is liquid at room temperature and pressure, and is water-soluble, and detecting the decomposition reaction of the substrate by the blood coagulation-related enzyme or fibrinolysis-related enzyme by absorbance measurement.

7. The method according to claim 6, wherein the compound is one or more selected from the group consisting of glycerol, propylene glycol, dipropylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, polyethylene glycol, and erythritol.

8. The method according to claim 6, wherein the blood coagulation-related enzyme is thrombin, blood coagulation factor VII (FVIIa), blood coagulation factor IX (FIXa), blood coagulation factor X (FXa), blood coagulation factor XI (FXIa), blood coagulation factor XIIa, blood coagulation factor III (FXIIIa), kallikrein, or activated protein C, and the fibrinolysis-related enzyme is plasmin, tissue plasminogen activator (t-PA), or urokinase-type plasminogen activator (u-PA).

9. The method according to claim 6, wherein the reaction is accelerated by adding a reaction-promoting enzyme to the enzyme.

10. The method according to claim 6, wherein the compound is added at a final concentration of 5 to 30 (w / w)%.

11. The method according to claim 6, wherein the blood sample is plasma.

12. A method for suppressing absorbance changes due to blood coagulation reaction, comprising the step of adding to a blood sample a compound which is an aliphatic hydrocarbon compound, an aliphatic ether compound, or a polyoxyalkylene compound having two or more hydroxyl groups, is liquid at room temperature and pressure, and is water-soluble.

13. An agent for suppressing absorbance changes based on blood coagulation reactions, comprising a compound having two or more hydroxyl groups, an aliphatic hydrocarbon compound, an aliphatic ether compound, or a polyoxyalkylene compound, which is liquid at room temperature and pressure and is water-soluble.

14. A microfluidic device for testing coated with the reagent described in any one of claims 1 to 5.

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