WO2015194165A1

WO2015194165A1 - Blood clotting accelerant, and agent for testing blood clot function using same

Info

Publication number: WO2015194165A1
Application number: PCT/JP2015/003003
Authority: WO
Inventors: 浩平白石; こずえ岡野
Original assignee: 学校法人近畿大学; 国立大学法人山口大学; 日油株式会社
Priority date: 2014-06-18
Filing date: 2015-06-16
Publication date: 2015-12-23
Also published as: JP6692023B2; JPWO2015194165A1

Abstract

　This blood clotting accelerant and agent for testing blood clot function include particles having oriented carboxyl groups on the surface thereof, or sponge-like titanium oxide particles.

Description

Blood coagulation promoter and blood coagulation function test drug using the same

The present disclosure relates to a blood coagulation promoter and a blood coagulation function test drug using the same.

フィ Fibrinogen in the blood becomes fibrin, and the blood is coagulated by generating a fibrin network. Blood coagulation is a complex mechanism involving various factors, and whether these mechanisms are functioning normally is determined by screening tests prior to surgery and screening tests for abnormal blood coagulation such as hemophilia. Etc. are important.

As a blood coagulation test, add calcium, which is factor IV of blood coagulation, to blood collected by adding an anticoagulant such as sodium citrate, and measure the time until fibrin is precipitated. There is. Calcium re-addition time is easy as an examination of the intrinsic coagulation system, but has problems such as low sensitivity and accuracy.

Activating partial thromboplastin time (APTT) is a test of the intrinsic coagulation system with improved sensitivity and accuracy. In APTT, blood coagulation reaction is promoted by adding a phospholipid called partial thromboplastin fraction together with calcium and an active agent composed of ellagic acid and the like. The examination of the exogenous coagulation system includes prothrombin time (PT) using a protein called tissue thromboplastin and calcium.

Various improvements have been made to the composition of the test agent for the purpose of improving the accuracy or sensitivity of the test relating to blood coagulation (see, for example, Patent Document 1).

JP 2009-058393 A

However, these test drugs basically use components derived from living bodies as drugs that promote blood coagulation, which is the main component. For this reason, the dispersion | variation in the measured value for every test | inspection agent and the dispersion | variation in the measured value for every lot are a problem.

In order to solve these problems, standardization using the international sensitivity index has been carried out, but a great effort is required to obtain the international sensitivity index. In addition, since there are many unstable components and changes with time are likely to occur, calibration is required every time at the site of use.

An object of the present disclosure is to realize a blood coagulation promoter exhibiting a stable blood coagulation action and a blood coagulation function test drug using the same.

The first aspect of the blood coagulation promoter of the present disclosure includes particles having carboxyl groups oriented on the surface.

In a first aspect of the blood coagulation accelerator, the surface density of carboxyl groups in the particles, 0.5 [mu] mol / m ² or more, can be 50 [mu] mol / m ² or less.

In the first embodiment of the blood coagulation promoter, the particles can be a resin.

In the first aspect of the blood coagulation promoter, the resin may include a monomer unit derived from a monomer having a carboxyl group.

In the first aspect of the blood coagulation promoter, the monomer having a carboxyl group can be at least one of itaconic acid, acrylic acid and methacrylic acid.

In the first aspect of the blood coagulation promoter, the resin can be a polymer containing 10 mol% or more of monomer units derived from a monomer having a carboxyl group.

In the first embodiment of the blood coagulation promoter, the particles may be cellulose having a carboxyl group.

In the first aspect of the blood coagulation promoter, the particles may have an amino group on the surface.

The second aspect of the blood coagulation promoter contains sponge-like titanium oxide particles.

In a second aspect of the blood coagulation accelerator, the particles of sponge-like titanium oxide, surface area 50 m ² / g or more, may be 1000 m ² / g or less.

In the first and second aspects of the blood coagulation promoter, the particles may have an average particle diameter of 10 nm or more and 10 μm or less.

The blood coagulation function test drug contains at least one of the blood coagulation promoters of the first aspect and the second aspect of the present disclosure.

According to the blood coagulation promoter of the present disclosure, a stable blood coagulation action can be realized, and a stable blood coagulation function test drug can be realized.

1 (a) and 1 (b) are diagrams for explaining the promotion of blood coagulation by a surface in which carboxyl groups are oriented, and FIG. 1 (a) is a diagram showing a surface in which carboxyl groups are oriented. 1 (b) is a view showing a surface in which carboxyl groups are not oriented. FIG. 2 is an electron micrograph of plasma coagulated with particles having carboxyl groups oriented on the surface. FIG. 3 is an electron micrograph of plasma coagulated with particles having carboxyl groups oriented on the surface. FIG. 4 is an electron micrograph of plasma coagulated with sponge-like titanium oxide particles. FIG. 5 is an electron micrograph of plasma coagulated with particles having PMPC fixed on the surface. FIG. 6 is an electron micrograph of plasma coagulated by the addition of calcium.

The blood coagulation promoter of the present disclosure includes particles having carboxyl groups oriented on the surface. A blood coagulation promoter is a drug that promotes the coagulation reaction more than calcium alone by adding it together with calcium to whole blood or plasma containing an anticoagulant having a decalcification action. The particle having a carboxyl group oriented on the surface is a particle in which the carboxyl group is oriented outward and the carboxyl group is exposed on the particle surface.

Surface density of carboxyl groups is not particularly limited, 0.5 [mu] mol / m ² or more, preferably 1.0 [mu] mol / m ² or more, more preferably 5.0μmol / m ² or more, 50 [mu] mol / m ² or less, preferably 25μmol / M ² or less, more preferably 10 μmol / m ² or less. The amount of the carboxyl group can be determined by an electric conductivity titration method. The surface density of the carboxyl group can be obtained by dividing the amount of the carboxyl group by the value of the specific surface area measured by the BET (Brunauer, Emmet and Teller) method or the like.

The average particle diameter of the particles is not particularly limited, but may be 0.01 μm or more, preferably 0.05 μm or more, more preferably 0.1 μm or more, 10 μm or less, preferably 5 μm or less, more preferably 1 μm or less. . The average particle diameter can be measured by a laser diffraction particle size distribution measuring device or the like.

The particles may be spherical, flaky, scaly, or nanofibers. Further, it may be solid, hollow, or a porous shape having pores. The surface of the particles may be smooth or uneven.

The particles may be any particles as long as they can stably obtain the same characteristics. For example, industrially produced particles can be used. It may be derived from plants, animals or microorganisms as long as the same properties can be obtained stably. The particles may be an organic substance such as a resin or an inorganic substance such as silica. The particles may be solid or may be a material having a certain degree of fluidity such as hydrogel or liposome. Examples of industrially produced particles include synthetic polymers. The synthetic polymer can be, for example, a polymer including a monomer unit having a carboxyl group. For example, a polymer including monomer units derived from monomers such as acrylic acid, methacrylic acid, or itaconic acid may be used. The polymer may be a homopolymer composed of a monomer having a carboxyl group, or may be a binary or higher copolymer. The polymer may be cross-linked or non-cross-linked as long as it can be made into particles having a predetermined particle size.

The polymer may contain any other monomer unit as long as it contains a monomer unit having a carboxyl group. For example, the monomer unit derived from styrene may be included. It may contain a monomer unit derived from a hydrophobic monomer such as acrylic acid or methacrylic acid ester such as methyl acrylate or methyl methacrylate. Contains monomer units derived from hydrophilic monomers such as N-vinylpyrrolidone, 2-methacryloyloxyethyl phosphorylcholine (MPC), 2-dimethylaminoethyl methacrylate, 2-hydroxyethyl methacrylate, allylamine, or polyethylene glycol macromonomer You may go out. Moreover, the monomer unit which has a sulfonic acid group or a phosphoric acid group etc. may be included. In addition, a monomer unit having a functional group having a positive charge such as an amino group may be included instead of the functional group having a negative charge.

The content of the monomer unit having a carboxyl group in the polymer is not particularly limited, but can be 10 mol% or more, preferably 20 mol% or more, more preferably 50 mol% or more. Since the aggregation promoting effect is considered to be higher as the surface density of the carboxyl group is higher, a polymer in which all the monomer units are monomer units having a carboxyl group may be used. Further, the content of the monomer unit having a carboxyl group in the polymer may be 95 mol% or less, 90% or less, or 80% or less. By adjusting the content of the monomer unit having a carboxyl group, the density of the carboxyl group on the surface of the particle can be easily adjusted.

Instead of polymerizing a monomer having a carboxyl group to obtain a polymer having a carboxyl group, a carboxyl group may be introduced into the particle after forming the particle. For example, after forming particles made of a polymer having a terminal amino group, the amino group is reacted with a dicarboxylic acid such as oxalic acid or a tricarboxylic acid such as citric acid to thereby form particles having carboxyl groups oriented on the surface. It may be formed. In this case, amino groups may remain on the surface of the particles. Further, after forming a copolymer of maleic anhydride and styrene, maleic anhydride can be hydrolyzed to introduce a carboxyl group.

Natural polymers or modified ones may be used instead of synthetic polymers. For example, cellulose introduced with a carboxyl group can be used. As the cellulose, for example, cellulose nanofibers crushed so as to have a length of several μm to several tens of μm can be used. Cellulose can be crushed using physical methods, chemical methods, or both. Introduction of a carboxyl group into cellulose can be performed, for example, by oxidizing a hydroxyl group. Of these, oxidation using 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) is preferable. The introduction of the carboxyl group may be performed after pulverization of cellulose or before pulverization. Moreover, you may perform crushing and introduction | transduction of a carboxyl group simultaneously.

By adding particles having carboxyl groups oriented on the surface to blood together with calcium, blood coagulation can be promoted more than calcium alone. In the case of promoting blood coagulation, the concentration in the blood of particles having carboxyl groups oriented on the surface is not particularly limited, but is 0.5 μg / mL or more, preferably 5 μg / mL or more, more preferably 50 μg / mL or more, It can be 1000 μg / mL or less, preferably 500 μg / mL or less, more preferably 250 μg / mL or less. In this case, the concentration of calcium in the blood is 0.5 mmol / L or more, preferably 1 mmol / L or more, more preferably 5 mmol / L or more, 100 mmol / L or less, preferably 50 mmol / L or less, more preferably 25 mol. / L or less may be added. Calcium can be added to blood as an inorganic or organic salt.

Blood can be coagulated, whether it is whole blood or plasma. Blood can be coagulated even if it contains an anticoagulant having a decalcifying action such as citrate or ethylenediaminetetraacetic acid.

The blood coagulation promoter containing particles having carboxyl groups oriented on the surface can be used as a blood coagulation function test agent used for hemostasis function test and the like. Similar to the prothrombin time (PT) reagent or the activated partial thromboplastin time (APTT) reagent, the function of the coagulation system can be examined by measuring the coagulation time.

When used as a blood coagulation function test agent, the first reagent containing the blood coagulation promoter and the sample are mixed, then the second reagent containing calcium is mixed, and the coagulation time until fibrin is precipitated is measured. do it. The time until fibrin precipitates can be measured visually or by a change in absorbance.

The first reagent can be a suspension containing particles in which carboxyl groups are oriented. The density | concentration of the particle | grains in which the carboxyl group in the 1st reagent was orientated should just be a density | concentration which can be made into the suitable density | concentration which showed the final blood level previously. For example, the concentration of the particles in which the carboxyl group is oriented in the first reagent is 1.5 μg / mL or more, preferably 10 μg / mL or more, more preferably 50 μg / mL or more, particularly preferably 150 μg / mL or more, 3000 μg / mL. mL or less, preferably 1500 μg / mL or less, more preferably 1000 μg / mL or less, and particularly preferably 750 μg / mL or less. The second reagent is, for example, calcium 1.5 mmol / L or more, preferably 3 mmol / mL or more, more preferably 15 mmol / L or more, 300 mmol / L or less, preferably 150 mmol / mL or less, more preferably 75 mmol / L or less. It can be set as the solution containing.

Instead of adding calcium as the second reagent, a first reagent containing calcium may be prepared. In this case, the coagulation time may be measured by mixing the first reagent and the specimen.

It is considered that the particles having a carboxyl group oriented on the surface have a coagulation promoting function similar to that of tissue thromboplastin in PT reagent or partial thromboplastin fraction and activator in APTT reagent. Thus, the first reagent does not contain tissue thromboplastin or partial thromboplastin fractions but promotes blood coagulation. Platelets activated by bleeding or the like and exposed to phosphatidylserine promote blood coagulation. The particles having carboxyl groups oriented on the surface may promote the blood coagulation reaction by a mechanism similar to that of activated platelets exposed to phosphatidylserine. On the surface of the activated platelet cell membrane, phosphatidylserine is considered to be oriented and exposed outward.

As shown in FIG. 1 (a), on the surface where the carboxyl groups are oriented toward the outside and the carboxyl groups are exposed, coagulation active factors bound to the carboxyl groups are also arranged, so that the coagulation active complex can be easily formed. Can be formed. Thereby, it is considered that the coagulation reaction of blood is promoted. Therefore, it is considered that the particles in which the carboxyl groups are oriented toward the outside and the carboxyl groups are exposed on the particle surface can promote the blood coagulation reaction. On the other hand, on the surface where the carboxyl group is not oriented as shown in FIG. 1 (b), it is difficult to form a coagulation active complex even if a coagulation active factor binds to the carboxyl group. For this reason, in the case of the particle | grains in which the carboxyl group is not orientating toward the outer side, even if a carboxyl group exists, it is thought that the effect of promoting the coagulation reaction is hardly recognized.

A weakly acidic substance or a complex thereof may be added to the blood coagulation function test drug. For example, one or more of the following drugs can be added. Alginic acid, chitin (nano) fiber, chitosan (nano) fiber, ozone-oxidized cellulose (nano) fiber, natural rubber latex, poly (meth) acrylic acid, or a gelled product thereof. Sugar derivatives such as glucuronic acid, uronic acid, mannuronic acid, aldonic acid, aldaric acid, hyaluronic acid, or carboxylic acid-containing sugar chains. A dicarboxylic acid such as oxalic acid or malonic acid, an acidic protein, or an acidic peptide, a monoanion, or a polyanion. Nucleic acids such as deoxyribonucleic acid or ribonucleic acid, peptide nucleic acids, fumaric acid, maleic acid, phthalic acid, isophthalic acid, terephthalic acid, pyromellitic acid, citric acid, malic acid, or polybasic acids such as ethylenediaminetetraacetic acid. Phospholipids such as phosphatidylinositol or phosphatidylserine. Acidic amino acids such as aspartic acid or glutamic acid. Polyphenol, pyrogallol, pyrocatechol, tannic acid, epicatechin, epigallocatechin, ellagic acid, ellagic acid dihydrate, ascorbic acid, glutathione, α-tocopherol, butylhydroxyanisole, catechin, quercetin, uric acid, bilirubin and the like. Reaction mixture of terminal reactive polyethylene glycol and polystyrene or polyimine. Blood coagulation factors such as tissue factor or thrombin.

These agents may be added in a state of being bound to a carrier. As the carrier, for example, one or more of the following can be used. Metal particles such as gold or silver, inorganic particles such as kaolin, monolithic silica or colloidal silica, carbon particles such as carbon black, fullerene and fullerene nanotubes, inclusion compounds such as cyclodextrin or calixarene, pullulan, mannan, dextran or Polysaccharides such as amylopectin, liposomes, vesicles, calcium phosphate, hydroxyapatite, magnetic particles, multifunctional envelope-type nanostructures, polymer micelles, polyion complexes, polycyclic aromatic compounds, viruses, cells, microorganisms, brain sulfide, Or fibrin etc.

These drugs or drugs supported on a carrier can be added to the first reagent and mixed with the specimen together with the first reagent. Moreover, you may mix with a test substance separately from a 1st reagent. Further, it may be added to the second reagent.

The first reagent and the second reagent may further contain a component contained in a general test agent such as a preservative.

Spongy titanium oxide particles can be used as a blood coagulation promoter instead of particles having carboxyl groups oriented on the surface. Sponge-like titanium oxide particles are titanium oxide particles having a three-dimensional network structure. Sponge-like titanium oxide particles have a large number of Lewis acid sites on the surface. This Lewis acid site is considered to act in the same manner as the carboxyl group. The titanium oxide may be a rutile type or an anatase type.

Sponge-like titanium oxide particles are not particularly limited, but the surface area is 50 m ² / g or more, preferably 100 m ² / g or more, more preferably 200 m ² / g or more, 1000 m ² / g or less, preferably 800 m ² / g. More preferably 500 m ² / g or less.

The average particle diameter of the sponge-like titanium oxide particles is not particularly limited, but is 0.01 μm or more, preferably 0.05 μm or more, more preferably 0.1 μm or more, 100 μm or less, similarly to the particles having a carboxyl group on the surface. Preferably it is 50 micrometers or less, More preferably, it can be 10 micrometers or less.

The sponge-like titanium oxide particles are not particularly limited, and those formed by entanglement of fibrous titanium oxide so as to form pores of about several nm to 100 nm can be used. Moreover, what was formed by the hydrothermal synthesis method etc. can be used.

When the sponge-like titanium oxide particles are used as a blood coagulation function test agent in place of the particles having carboxyl groups oriented on the surface, the concentration of the sponge-like titanium oxide particles in the final blood is 0.2 μg / mL or more, Preferably, it can be 1 μg / mL or more, more preferably 2 μg / mL or more, 400 μg / mL or less, preferably 100 μg / mL or less, more preferably 40 μg / mL or less. The concentration of calcium in the blood is 0.5 mmol / L or more, preferably 1 mmol / L or more, more preferably 5 mmol / L or more, 100 mmol / L or less, preferably 50 mmol / L or less, more preferably 25 mol / L or less. can do. The concentration of the sponge-like titanium oxide particles contained in the first reagent is 0.6 μg / mL or more, preferably 3 μg / mL or more, more preferably 6 μg / mL or more, 1200 μg / mL or less, preferably 300 μg / mL. Hereinafter, it can be more preferably 120 μg / mL or less. About other conditions, it can be made to be the same as that of the particle | grains by which the carboxyl group was orientated on the surface.

It can also be a blood group coagulation function test agent containing both sponge-like titanium oxide and particles in which carboxyl groups are oriented.

Hereinafter, the blood coagulation agent of the present disclosure and the blood coagulation function test agent using the same will be described in more detail using examples.

<Functional group density>
The functional group density on the particle surface was measured by an electric conductivity titration method. A sodium hydroxide aqueous solution was used as a titrant for titration of carboxyl groups and sulfonic acid groups. Dilute hydrochloric acid was used for the titration of amino groups. The electrical conductivity was titrated under a nitrogen stream using a commercially available electrical conductivity meter (manufactured by Toa Decay Co., Ltd .: CM-60S). The surface area of the particle was calculated from the formula of the surface area of the sphere using the catalog value of the particle diameter. It was confirmed that the catalog value of the particle diameter almost coincided with the value measured by a laser diffraction light scattering photometer (manufactured by Shimadzu Corporation: SALD-2300).

<Evaluation of blood coagulation promoting function>
After adding 40 μL of a specimen and 40 μL of a reagent diluted to a predetermined magnification with ion-exchanged water into a well of a 96-well microplate, 40 μL of calcium chloride solution (0.25 mmol / L) is added. The mixture was stirred and reacted using a reader (PerkinElmer: 2030 ARVO X), and the absorbance at 630 nm was measured. Absorbance was measured until a plateau was reached. The time when the absorbance change was maximum was taken as the reaction time, and the absorbance reaching the plateau was taken as the final absorbance. The reaction time (corresponding to the calcium re-coagulation time) when physiological saline was added instead of the reagent was defined as the blank time, and the value obtained by dividing the reaction time by the blank time was defined as the reaction rate index. A smaller reaction rate index indicates that the coagulation reaction is promoted by the reagent. Further, the value obtained by dividing the final absorbance when the reagent was added by the final absorbance when physiological saline was added was defined as the absorbance change rate. The larger the absorbance change rate, the more the fibrin network grows. For all measurements, duplicate measurements were taken.

<Observation of fibrin network>
About the specimen which measured the coagulation reaction, formation of the fibrin network was confirmed by a focused ion beam scanning electron microscope (FIB-SEM, manufactured by FEI: Quunta3D FEG (using FIB, cryo apparatus, OmniProbe, Gas injection, EDAX)). .

<Sample>
The specimen was normal human plasma collected from a healthy person. Blood was collected using a vacuum blood collection tube (Becton Dickinson) containing a sodium citrate buffer and a 21 gauge blood collection needle. After blood collection, it was centrifuged at 3000 rpm for 10 minutes to separate plasma and blood cells.

<Reagent>
(Reagent 1)
A 2.5 w / v% suspension of resin particles (Poly Science: 15913-10) having an average particle diameter of 0.05 μm made of a copolymer of polystyrene, acrylic acid and methacrylic acid was used. The particle concentration was 3.6 × 10 ¹⁴ particles / mL. The surface density of the carboxyl group was 2.3 × 10 μmol / m ² .

(Reagent 2)
The same procedure as in Reagent 1 was repeated except that resin particles having an average particle diameter of 0.1 μm made of a copolymer of polystyrene, acrylic acid and methacrylic acid (Poly Science: 16688-15) were used. Particle concentration was 4.6 × 10 ¹³ cells / mL.

(Reagent 3)
The procedure was the same as that of Reagent 1 except that the resin particles were made of a copolymer of polystyrene, acrylic acid and methacrylic acid and had an average particle size of 0.5 μm (manufactured by Poly Science: 09836-15). The particle concentration was 3.6 × 10 ¹¹ particles / mL. The surface density of the carboxyl group was 1.5 × 10 μmol / m ² .

(Reagent 4)
The procedure was the same as that of Reagent 1 except that resin particles made of a copolymer of polystyrene, acrylic acid and methacrylic acid and having an average particle diameter of 1.0 μm (manufactured by Poly Science: 08226-15) were used. The particle concentration was 4.6 × 10 ¹⁰ particles / mL. The surface density of the carboxyl group was 8.0 × 10 μmol / m ² .

(Reagent 5)
Cellulose nanofibers (COOH-CNF) introduced with carboxyl groups were used. The particle concentration was 1 w / V%. The introduction of the carboxyl group was as follows. First, commercially available cellulose (manufactured by Nippon Paper Chemicals Co., Ltd .: cellulose powder KC Flock W-400G) was mercerized using sodium hydroxide. After this, 1 g of mercerized cellulose is dispersed in 100 mL of water, 25 mg of TEMPO catalyst, 0.25 g of sodium bromide (NaBr) and 9.27% sodium hypochlorite (NaClO) aqueous solution as an oxidizing agent 10 mL And oxidation treatment was performed at room temperature and pH 10 for 2 hours. Thereafter, the reaction was stopped by adding a small amount of ethanol, and centrifuged at 10,000 g for 10 minutes to remove impurities, and then methanol was further added to the supernatant to precipitate COOH-CNF. The resulting precipitate was further centrifuged to recover COOH-CNF. The recovered COOH-CNF was dried at 80 ° C. to 105 ° C. and then redispersed in water. The average particle diameter of COOH-CNF in water was 5.4 μm. The length of COOH-CNF was 2 μm to 5 μm and the width was 100 nm to 200 nm. The length and width of COOH-CNF were the average of the lengths of 10 COOH-CNFs in the field of view observed with an electron microscope (manufactured by JEOL Ltd .: Jsm-6510, magnification 10,000). The average particle diameter was measured with a laser diffraction light scattering photometer (manufactured by Shimadzu Corporation: SALD-2300). The surface density of the carboxyl group was 1.2 μmol / m ² .

(Reagent 6)
An aqueous suspension of sponge-like titanium oxide having an average particle size of 0.1 μm (manufactured by Erscreen Tohoku Co., Ltd .: PW116-3) was used. The specific surface area of the sponge-like titanium oxide was 400 m ² / g. The particle concentration was 0.1 w / v%.

(Reagent 7)
Sponge-like titanium oxide used for reagent 6 was the same as reagent 5 except that it was heat-treated at 500 ° C. for 2 hours.

(Reagent 8)
The same procedure as in Reagent 1 except that resin particles having an amino group on the surface and an average particle size of 0.1 μm (Poly Science: 16586-5) was used. The particle concentration was 4.6 × 10 ¹³ particles / mL. The surface density of the amino group was 14 × 10 μmol / m ² .

(Reagent 9)
Reagent 1 was used except that resin particles having a sulfonic acid group on the surface and an average particle size of 0.5 μm (Poly Science Co., Ltd .: 19403-15) were used. The surface density of the sulfonic acid group was 0.64 × 10 μmol / m ² .

(Reagent 10)
The procedure was the same as that of Reagent 1 except that resin particles having a sulfonic acid group on the surface and having an average particle diameter of 1.0 μm (Poly Science, 19404-15) were used.

(Reagent 11)
The procedure was the same as that of Reagent 1 except that the resin particles were fixed with poly (2-methacryloyloxyethylphoshoryl choline) (PMPC). The resin particles were resin particles having an amino group bonded through a linker having 3 carbon atoms and having an average particle size of 0.1 μm (manufactured by Poly Science: 16586-5). The fixation of PMPC was performed via a succinimidyl group.

(Reagent 12)
It replaced with PMPC and was carried out similarly to the reagent 10 except having set it as the resin particle which fixed the block copolymer of MPC and acrylic acid shown in Formula 1. However, in Formula 1, m is 90 and n is 10 as preparation ratio.

(Reagent 13)
Instead of PMPC, the same procedure as in Reagent 10 was performed except that resin particles fixed with block copolymers of MPC, acrylic acid and dimethylsiloxane shown in Formula 2 were used. However, in Formula 2, as the preparation ratio, (m + n) is 50, l is 50, m is 90, and n is 10.

(Reagent 14)
An aqueous suspension of cellulose nanofiber (Sugino Machine Co., Ltd .: BiNFi-s) was used. The particle concentration was 0.1 w / v%.

(Reagent 15)
A commercially available PT reagent (Sysmex Corporation: Thrombocheck PT) was used after dissolving in purified water according to the dosage.

(Reagent 16)
A commercially available APTT reagent (manufactured by SIEMENS: ACTIN) was used.

Table 1 shows the reaction rate index for each reagent. The reaction rate indices at 100-fold dilution (250 μg / mL) for reagents 1 to 4 consisting of particles having carboxyl groups oriented on the surface were 0.59, 0.56, 0.49 and 0.60, respectively. . The reaction rate index at 100-fold dilution of the reagent 14 which is a commercially available PT reagent and the reagent 15 which is an APTT reagent was 0.55 and 0.49, respectively. Thus, the reaction rate index of Reagents 1 to 4 is comparable to that of commercially available PT reagents and APTT reagents. Therefore, Reagents 1 to 4 promote the coagulation of plasma in the same manner as commercially available PT reagents and APTT reagents, and have the same blood coagulation promoting function as these reagents. In addition, when the reagents 1 to 3 were diluted 10,000 times (2.5 μg / mL), the reaction rate index was smaller than 1, and blood coagulation was promoted.

Reagent 5 consisting of COOH-CNF showed a reaction rate index of 0.88 at 10-fold dilution (100 μg / mL) and 0.74 at 100-fold dilution (10 μg / mL). Thus, COOH-CNF also has a blood coagulation promoting function.

In addition, the reagent 6 composed of sponge-like titanium oxide showed a reaction rate index of 0.71 at 10-fold dilution (100 μg / mL) and 0.80 at 100-fold dilution (10 μg / mL). In addition, the reaction rate index of the reagent 7 made of heat-treated sponge-like titanium oxide was 0.70 at 10-fold dilution and 0.81 at 100-fold dilution, indicating almost the same reaction rate index as that without heat treatment. It was. Thus, the blood coagulation promoting function was recognized also about sponge-like titanium oxide particle. Furthermore, Reagents 6 and 7 had a reaction rate index smaller than 1 even at 1000-fold dilution (1 μg / mL), and promoted blood coagulation.

On the other hand, with regard to Reagents 8 to 14, the reaction rate index was 0.94 at a 100-fold dilution of Reagent 8 consisting of particles having amino groups on the surface, and a slight promotion of blood coagulation was observed. However, in all other cases, the reaction rate index was larger than 1, and the blood coagulation accelerating function was not observed when the surface did not contain particles having carboxyl groups oriented or sponge-like titanium oxide particles.

Table 2 shows the absorbance change rate for each reagent. In Reagent 1 to Reagent 8 having a reaction rate index of less than 1, Reagent 1 consisting of particles having an average particle diameter of 0.05 μm had an absorbance change rate of 1 or less at 100-fold dilution. Reagent 3 composed of particles having an average particle diameter of 0.5 μm and Reagent 4 composed of particles having an average particle diameter of 1.0 μm also had a slightly smaller absorbance change rate at 100-fold dilution than other reagents. In addition, in Reagent 9 to Reagent 14 having a reaction rate index of 1 or more, Reagent 9 composed of particles having an average particle diameter of 0.5 μm and Reagent 10 composed of particles having an average particle diameter of 1.0 μm are other reagents. The rate of change in absorbance at 100-fold dilution was slightly smaller than that of.

FIGS. 2 to 6 show SEM photographs of the fibrin network obtained when the reagents 1, 3, 6, and 11 were diluted 100 times and in the blank, respectively. In the case of reagent 1, fibrin fibers are finer than other reagents and blanks. However, reagent 1 also has a sufficient blood coagulation promoting function and is useful as a blood coagulation promoter.

The blood coagulation promoter of the present disclosure exhibits a stable blood coagulation function and is useful as a blood coagulation function test agent or the like.

Claims

Blood coagulation promoter containing particles with carboxyl groups oriented on the surface.
Surface density of carboxyl groups in the particles, 0.5 [mu] mol / m 2 or more and 50 [mu] mol / m 2 or less, the blood coagulation promoter according to claim 1.
The blood coagulation promoter according to claim 1 or 2, wherein the particles are made of a resin.
The blood coagulation promoter according to claim 3, wherein the resin contains a monomer unit derived from a monomer having a carboxyl group.
The blood coagulation promoter according to claim 4, wherein the monomer having a carboxyl group is at least one of itaconic acid, acrylic acid and methacrylic acid.
The blood coagulation promoter according to claim 4 or 5, wherein the resin is a copolymer containing 10 mol% or more of monomer units derived from the monomer having a carboxyl group.
The blood coagulation promoter according to claim 1 or 2, wherein the particles are cellulose having a carboxyl group.
The blood coagulation promoter according to any one of claims 1 to 7, wherein the particles have an amino group on the surface.
Blood coagulation promoter containing sponge-like titanium oxide particles.
The blood coagulation promoter according to claim 9, wherein the sponge-like titanium oxide particles have a surface area of 50 m 2 / g or more and 1000 m 2 / g or less.
The blood coagulation promoter according to any one of claims 1 to 10, wherein the particles have an average particle size of 10 nm or more and 10 µm or less.
A blood coagulation function test drug comprising the blood coagulation promoter according to any one of claims 1 to 11.