JP6983420B2 - Red blood cell protectant - Google Patents

Description

The present invention relates to a red blood cell protectant. More specifically, it relates to an erythrocyte protectant containing a histidine-rich glycoprotein.

This application claims the priority of Japanese application Japanese Patent Application No. 2017-288738, which is incorporated herein by reference.

Blood products for transfusion include red blood cells, plasma, platelets, whole blood and the like. Red blood cell preparations are blood from which most of plasma, white blood cells, and platelets have been removed. When red blood cells (RBCs) are stored for a long period of time, the red blood cell membrane is destroyed and hemoglobin is released, so-called hemolysis occurs. For storage of blood cell concentrate, ACD solution containing acid-citrate-dextrose and CPD solution containing sodium citrate-sodium phosphate-dextrose have been used for a long time. Has been used. Furthermore, in recent years, adenine has been added to the preservation solution in order to supplement the adenine lost due to the deamination reaction. The storage temperature is 2 to 3 ° C., the effective period is 3 weeks after blood collection, and it is said that it can be extended to 6 weeks depending on the conditions. However, it was confirmed that when the refrigerated storage of red blood cells was continued, glucose consumption decreased and metabolic waste (that is, lactic acid and hydrogen ions) increased. Such a decrease in glucose metabolism causes depletion of adenosine triphosphate (ATP) and deterioration of erythrocytes. Development is underway to preserve red blood cells after they have been isolated from whole blood. For example, Adsol (trade name) (AS-1), Nutricel (trade name) (AS-3), Optosol (trade name) (AS-5), Erythro-sol (trade name) and the like can be mentioned. These ASs (AS-1, AS-2 and AS-3) contain saline solution, adenine, glucose, and small amounts of citric acid and / or mannitol as a "protective agent for cell membranes". There are disclosures of compositions and methods for the preservation of erythrocytes, including adenine and dextrose, at least one non-metabolic membrane-protecting sugar, and a pH buffer system (Patent Document 1).

Histidine-rich glycoprotein (HRG) is a plasma protein with a molecular weight of approximately 80 kDa identified by Heimburger et al (1972) in 1972. It is a high-histidine-containing protein composed of a total of 507 amino acids, of which 66 is histidine, which is synthesized mainly in the liver and is present in human plasma at a very high concentration of about 100 to 150 μg / mL. HRG is known to be involved in the regulation of the coagulation / fibrinolysis system and the regulation of angiogenesis (Non-Patent Document 1). In addition, methods of inhibiting angiogenesis by administration of HRG polypeptides, HRG polypeptides, antibodies and receptors that bind to HRG polypeptides, HRG-deficient plasma and polynucleotides, vectors encoding HRG polypeptides and host cells. The pharmaceutical compositions and products including the above are disclosed (Patent Document 2). Also, in the field of angiogenesis, there is a disclosure regarding the use of a substantially pure continuous polypeptide having anti-angiogenic activity containing a subfragment derived from the central region of HRG (Patent Document 3). Further, there is also disclosure of a neutrophil-vascular endothelial cell adhesion inhibitor containing HRG as an active ingredient (Patent Document 4).

However, the effect of HRG on the safety and stability of erythrocytes has not been reported. The development of further methods for protecting red blood cells is desired.

Blood, Vol.117, No.7, 2093-2101 (2011)

Special Table 2008-529550 Gazette Special Table 2004-527242 Gazette Special Table 2007-528710 Gazette Japanese Patent No. 5807937

An object of the present invention is to provide an erythrocyte protective agent that can store an erythrocyte-containing solution, for example, a erythrocyte concentrate, for a longer period of time and can be used more safely.

The inventors of the present application focused on the expression of phosphatidylserine (PS) on the surface of the erythrocyte membrane when erythrocytes are stored for a long period of time, and the expression of this PS causes the vascular endothelial cells of erythrocytes and collagen fibers to adhere to each other. As a result of diligent studies to solve the problem, we found for the first time that HRG can suppress PS expression on the cell surface, and completed the present invention.

That is, the present invention comprises the following.
1. 1. A red blood cell protectant containing HRG as an active ingredient.
2. 2. The erythrocyte protective agent according to item 1 above, wherein the erythrocyte protection suppresses the expression of phosphatidylserine on the surface of the erythrocyte membrane by HRG.
3. 3. The erythrocyte protective agent according to item 1 above, wherein the protection of erythrocytes reduces the concentration of calcium ions in the erythrocyte cells.
4. The erythrocyte protective agent according to item 1 above, wherein the protection of erythrocytes suppresses the release of antioxidant enzymes in erythrocyte cells.
5. A stabilizer for red blood cell-containing solutions containing HRG as an active ingredient.
6. A stabilizer for a erythrocyte-containing solution containing the erythrocyte protective agent according to any one of the above items 1 to 4.
7. A method for stabilizing a erythrocyte-containing solution, which comprises adding the stabilizer according to the above item 5 or 6 to the erythrocyte-containing solution.
8. A phosphatidylserine expression inhibitor on the surface of erythrocyte membrane containing HRG as an active ingredient.
9. A method for suppressing the expression of phosphatidylserine on the surface of an erythrocyte membrane, which comprises adding HRG to a erythrocyte-containing solution.

According to the erythrocyte protective agent of the present invention, erythrocyte deterioration due to erythrocyte preservation can be suppressed. By adding HRG as an active ingredient to an existing blood storage solution or erythrocyte storage solution, or a blood storage solution or erythrocyte storage solution to be developed in the future, erythrocytes can be protected more effectively. ..

It is a figure which shows the result of the expression level of phosphatidylserine (PS) when each concentration Zn ^{2+ was added to a erythrocyte solution sample.} It was confirmed that PS was expressed on the surface of the erythrocyte membrane in a Zn ^{2+ concentration-dependent manner.} (Reference example 1) It is a figure which shows the result of having confirmed the effect of HRG or human serum albumin (HSA) added at the same time on the erythrocyte membrane surface PS whose expression is induced by Zn ^2+. The effect of Zn ²⁺ is not observed on ^{Ca 2+} and Mg ^2+. (Example 4) It is a result figure which confirmed the action of HRG or HSA added at the same time on the erythrocyte membrane surface PS whose expression is induced by Zn ^2+. It is a figure showing the result that HRG was observed to suppress PS expression more strongly than HSA. (Example 5) It is a result figure which confirmed the subsequent action of HRG or HSA on the erythrocyte membrane surface PS whose expression was induced by Zn ^{2+ in advance.} It is a figure showing the result that HRG was observed to suppress PS expression more strongly than HSA. (Example 6) It is a figure of the result of confirming the ability of HRG to suppress the adhesion of unstimulated erythrocytes to collagen I. It shows the result that erythrocytes were suppressed from adhering to collagen I in an HRG concentration-dependent manner. (Example 7) It is the result of stimulating erythrocytes in advance with Zn ²⁺ , then adding HRG, and examining the adhesive ability to vascular endothelial cells. It shows the result that erythrocytes were suppressed from adhering to vascular endothelial cells in an HRG concentration-dependent manner. (Example 8) It is a figure which shows the result of confirming the influence on the PS positive rate of the erythrocyte membrane surface when HSA was added to the erythrocyte preservation solution and stored at 4 degreeC for 21 days. (Example 9) It is a figure which shows the PS expression confirmation result of the CLP mouse erythrocyte membrane surface. It is a figure which shows the PS positive rate of the erythrocyte membrane surface, and the effect by HRG administration in vivo about the erythrocyte immediately after blood collection and the erythrocyte after incubating at 37 ℃ for 4 hours. (Example 10) It is a figure which shows the experimental result of the flow cytometry which confirmed the effect of HRG on the erythrocyte membrane surface PS whose expression was induced by Zn ^2+. (Example 11) It is a figure which shows the result of confirming the increase of intracellular calcium by Zn ^{2+ and the suppression by HRG (FIG. 10A).} FIG. 10A shows the result that the amount of calcium in erythrocyte cells increased in the group to which HRG was not added, but the free calcium concentration decreased in the group to which HRG was added. In addition, FIG. 10B shows the inhibitory effect of HRG on the aggregation of erythrocytes and the adhesion of aggregated erythrocytes to vascular endothelial cells by ^{Zn 2+ stimulation.} (Example 12) It is a figure which shows the result of having confirmed the release of peroxiredoxin 2 (Prx2) in the erythrocyte suspension sample stimulated with Zn ^{2+ stimulation or calcium ionophore.} FIG. 11A ^{shows an increase in Prx2 in the supernatant in a Zn 2+} concentration-dependent manner, and FIG. 11B shows an increase in Prx2 for a calcium ionophore-stimulated erythrocyte solution sample. (Reference example 2) It is a figure which shows the result of having confirmed the prx2 release by Zn ^{2+ and the inhibitory effect by HRG.} (Example 13) It is a figure which shows the result of having confirmed Prx2 in the obtained plasma by collecting blood 24 hours after CLP production from a CLP sepsis model mouse. It is a figure which shows the result of having confirmed that the plasma Prx2 is increased in the CLP mouse. (Reference example 3) It is a figure which shows the result of having confirmed the blood free hemoglobin, PS positive erythrocyte, the calcium concentration in a erythrocyte cell, and the Zn content in an organ tissue in a CLP sepsis model mouse. FIG. 14A shows a western blot of free hemoglobin and its quantification results, FIG. 14B shows the proportion of PS-positive erythrocytes immediately after blood sampling and after 4 hours of blood incubation, and FIG. 14C shows the concentration of free calcium in erythrocytes after 4 hours of incubation. Shown, FIG. 14D shows the Zn content of organ tissue. (Example 14)

The present invention relates to a red blood cell protectant, and more particularly to a red blood cell protectant containing a histidine-rich glycoprotein (HRG).

As used herein, the term "erythrocyte protectant" is used to prevent deterioration of erythrocytes in vitro, such as when erythrocytes contained in blood or erythrocytes separated from blood are suspended in a preservation solution or the like. A protective agent. The erythrocyte protectant of the present invention can contain a pharmacologically acceptable carrier. Examples of the pharmacologically acceptable carrier include excipients, disintegrants or disintegrant aids, binders, lubricants, coating agents, dyes, diluents, bases, solubilizers or solubilizers, and the like. Examples thereof include isotonic agents, pH adjusters, stabilizers, propellants, adhesives and the like.

As used herein, the term "blood preservative solution" is a solution used for preserving blood, for example, a preservative solution used for blood products for transfusion, specifically, erythrocyte preparations, plasma preparations, and platelets. A preservative solution used for preparations and whole blood products. As used herein, the "blood storage solution" may be a storage solution known per se or any storage solution to be developed in the future. Specifically, it may be an ACD solution or a CPD solution. For example, it may be an additive solution for erythrocyte preservation (MAP solution) containing mannitol, adenine and phosphoric acid, or it may be a mixture of ACD solution or CPD solution and MAP solution. Examples of the composition of the MAP solution include D-mannitol, adenine, crystalline sodium dihydrogen phosphate, sodium citrate, citric acid, butou sugar, sodium chloride and the like.

As used herein, the term "erythrocyte preservation solution" is a solution used for storing red blood cells, for example, a preservation solution used for blood products for transfusion, specifically, storage used for red blood cell preparations and the like. A liquid solution. As used herein, the "erythrocyte preservation solution" may be a preservation solution known per se or any preservation solution developed in the future. Specifically, it may be an ACD solution, a CPD solution or a MAP solution, or may be a mixture of an ACD solution or a CPD solution and a MAP solution.

As used herein, the term "erythrocyte-containing solution" is not particularly limited as long as it is a solution contained in a solution in which red blood cells can be stored. For example, the above-mentioned blood storage solution containing red blood cells may be mentioned. An example of a red blood cell-containing solution is a red blood cell concentrate, which is a red blood cell preparation. The red blood cell concentrate is a dark red solution containing a small amount of CPD solution, which is a mixture of about 46 mL of MAP solution in the red blood cell layer from which most of leukocytes and plasma have been removed from 200 mL of human blood mixed with 28 mL of CPD solution. Is. In the examples described later, it may be referred to as a red blood cell solution sample.

As used herein, the term "stabilizer for erythrocyte-containing solution" refers to a erythrocyte-containing solution such as a red blood cell concentrate that is used to prevent deterioration of erythrocytes and stabilize them. The stabilizer for the erythrocyte-containing solution of the present invention can be prepared by adding HRG to a erythrocyte storage solution such as MAP solution. Specifically, a solution containing 10 to 100 μg / mL, preferably 50 to 100 μg / mL, and more preferably 100 μg / mL of HRG with respect to the erythrocyte storage solution is preferable. It is clear that the erythrocyte storage solution that can be used as the stabilizer for the erythrocyte-containing solution of the present invention is not limited to the MAP solution. The stabilizer of the erythrocyte-containing solution of the present invention can contain a pharmacologically acceptable carrier. Examples of the pharmacologically acceptable carrier include excipients, disintegrants or disintegrant aids, binders, lubricants, coating agents, dyes, diluents, bases, solubilizers or solubilizers, and the like. Examples thereof include isotonic agents, pH adjusters, stabilizers, propellants, adhesives and the like.

"HRG" as an active ingredient such as an erythrocyte protectant and a stabilizer for a erythrocyte-containing solution of the present invention is prepared by a method of isolating and purifying from a biological component, a method of preparing using a gene recombination technique, or a synthetic method. can do. For example, it can be purified / isolated from blood such as plasma and serum, and biological components such as spinal fluid and lymph. Suitable biological components are blood components such as plasma and serum. As a method for isolating and purifying from a biological component, a method known per se or any method developed in the future can be applied. For example, it can be prepared by passing plasma through an affinity column prepared using Ni-NTA (nickel-nitrilotriacetic acid) agarose resin.

As for the method for preparing HRG using the gene recombination technique, a method known per se or any method developed in the future can be applied. For example, a full-length cDNA encoding HRG or a cDNA encoding an active portion of HRG can be cloned into an expression vector and prepared. For example, it may be a protein that is biosynthesized from all or part of the nucleotide identified in GenBank Accession No. NM000412. For example, a full-length cDNA encoding the amino acid sequence of mature HRG (SEQ ID NO: 1) or a cDNA encoding a portion can be cloned into an expression vector and prepared. The HRG as the active ingredient of the present invention may be the whole HRG protein, or may be a partial protein or peptide having HRG activity. Further, it may contain a sugar chain or may not have a sugar chain added.

Amino acid sequence of mature HRG (SEQ ID NO: 1)
VSPTDCSAVEPEAEKALDLINKRRRDGYLFQLLRIADAHLDRVENTTVYYLVLDVQESDCSVLSRKYWNDCEPPDSRRPSEIVIGQCKVIATRHSHESQDLRVIDFNCTTSSVSSALANTKDSPVLIDFFEDTERYRKQANKALEKYKEENDDFASFRVDRIERVARVRGGEGTGYFVDFSVRNCPRHHFPRHPNVFGFCRADLFYDVEALDLESPKNLVINCEVFDPQEHENINGVPPHLGHPFHWGGHERSSTTKPPFKPHGSRDHHHPHKPHEHGPPPPPDERDHSHGPPLPQGPPPLLPMSCSSCQHATFGTNGAQRHSHNNNSSDLHPHKHHSHEQHPHGHHPHAHHPHEHDTHRQHPHGHHPHGHHPHGHHPHGHHPHGHHPHCHDFQDYGPCDPPPHNQGHCCHGHGPPPGHLRRRGPGKGPRPFHCRQIGSVYRLPPLRKGEVLPLPEANFPSFPLPHHKHPLKPDNQPFPQSVSESCPGKFKSGFPQVSMFFTHTFPK

The mature HRG is composed of four major regions, the cystatin-like region 1, 2, the His / Pro region, and the C-terminal region, after being cleaved from the signal peptide by a proteolytic enzyme. The His / Pro region is very rich in proline and histidine residues, eg, in human form, it contains about 12 tandem repeats conserving the pentapeptide GHHPH (SEQ ID NO: 2). In another embodiment, it is specified by the amino acid sequence shown in positions 330 to 389 of the amino acid sequence specified by SEQ ID NO: 1.

The HRG used in the erythrocyte protective agent of the present invention has an action of suppressing the expression of phosphatidylserine (PS) on the surface of the erythrocyte membrane. PS on the surface of the erythrocyte membrane is stimulated by long-term storage of erythrocytes, under energy depletion conditions, or by Prostaglandin E ₂ (PGE ₂ ), Platelet activating factor (PAF), retinoic acid, or specific drugs. It is said that it appears when it occurs. It has also been suggested that the interaction between erythrocytes expressing PS and vascular endothelial cells damages vascular endothelial cells. The present invention extends to a PS expression inhibitor on the surface of a erythrocyte membrane containing HRG as an active ingredient, and also extends to a method for protecting erythrocytes, which comprises adding HRG to a erythrocyte-containing solution. Further, the present invention extends to a method for suppressing PS expression on the surface of an erythrocyte membrane, which comprises adding HRG to a erythrocyte-containing solution.

The HRG used in the erythrocyte protective agent of the present invention further has an action of lowering the intracellular calcium concentration and an action of suppressing the release of an important intracellular enzyme. An important intracellular enzyme includes an antioxidant enzyme, specifically peroxidase, and more specifically, peroxiredoxin (Prx). Prx has a strong antioxidant effect and is present in a large amount in the cell, so that it is an important antioxidant enzyme for maintaining the physiological function of the cell.

The present invention also extends to a method for stabilizing erythrocytes, which comprises adding the "erythrocyte protectant" or "stabilizer for erythrocyte-containing solution" of the present invention to an erythrocyte-containing solution. That is, it extends to a stabilization method using an erythrocyte protectant containing HRG as an active ingredient or a stabilizer of an erythrocyte-containing solution containing HRG as an active ingredient. By adding an erythrocyte protective agent to a erythrocyte-containing solution, for example, PS can be suppressed from being expressed on the surface of the erythrocyte membrane, or PS that has been expressed can also be suppressed. PS is a component of phospholipids and is usually retained in the inner lobe (cytoplasmic side) of the cell membrane by an enzyme called flippase. When a cell undergoes apoptosis, PS becomes exposed on the surface of the cell. In erythrocytes, when the nucleus is separated from the reticulocyte, the nucleus is said to expose PS on the cell surface. PS is a lipid expressed on the surface of apoptotic cells and is known to be exposed on the cell membrane only in dead cells. In erythrocytes as well, PS is considered to be exposed on the cell surface in the process of DNA degradation and erythrocyte nuclear decay in apoptosis. Therefore, it was considered that PS is exposed on the erythrocyte surface in the process of erythrocyte decay. Therefore, we confirmed the effect of HRG on the exposure of PS to the surface of erythrocytes, and since HRG can suppress the exposure of PS to the surface of erythrocytes, the erythrocyte storage solution containing HRG is the erythrocyte contained in the erythrocyte-containing solution. Can be used as a protective agent for red blood cells.

Hereinafter, the present invention will be described in more detail with reference to Reference Examples and Examples. The present invention is not limited by the following examples, etc., and can be appropriately modified and carried out within the range that can meet the purpose of the preceding and the following, and all of them are technically of the present invention. Included in the range.

(Example 1) Purification of human plasma-derived histidine-rich glycoprotein (HRG) In this example, human plasma-derived HRG was purified. Using human plasma (240 mL) as a starting material, HRG protein was purified using Ni-NTA affinity chromatography and high-performance liquid chromatography (anion exchange column (monodisperse hydrophilic polymer beads: Mono Q)). HRG purified samples were obtained with a molecular weight fraction of about 80 kDa.

(Example 2) Production of transgenic human HRG The recombinant human HRG was ^{CHO cultured with GIBCO (R)} Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F-12) containing 10% FCS. The HRG expression vector, transfection expression vector, and drug resistance gene expression vector were co-transfected into cells (Chinese Hamster Ovary cells) using FuGENE ™ -HD (gene transfer reagent) with a DNA content of 5: 4: 1. After gene transfer and culturing for 48 hours, puromycin (antibiotic) 10 μg / mL was added, and drug selective culture was performed for 3 weeks while exchanging the medium once every 3 days.

Culture supernatants containing recombinant human HRG were collected. ^{QIAGEN (R)} Ni-NTA agarose gel (gel in which Ni-NTA is bound to Sepharose CL-6B support) pre-washed with 30 mL of PBS (-) is added to the culture supernatant and incubated at 4 ° C. for 2 hours. Then, the recombinant human HRG ^{was bound to QIAGEN (R)} Ni-NTA agarose gel. After transferring the QIAGEN ^(R) Ni-NTA agarose gel to a purification column, wash solution 1 (PBS (-) (pH 7.4) containing 30 mM Imidazole) and wash solution 2 (1 M NaCl + 10 mM PB (pH 7.4)). )), The column was washed sequentially with the washing liquid 3 (PBS (-) (pH 7.4)). Recombinant human HRG was eluted with PBS (-) (pH 7.4) containing 500 mM Imidazole at 4 ° C. The refined product confirmed HRG by Western blotting and protein staining after SDS-PAGE. The HRG prepared in this example was prepared by the method shown in Example 12 of International Application No. PCT / JP2016 / 79219.

(Example 3) Protective agent for packed red blood cells
D-mannitol (728.5 mg), adenine (7.0 mg), sodium dihydrogen phosphate (47.0 mg), sodium citrate hydrate (75.0 mg), citric acid hydrate (10.0 mg), glucose (10.0 mg) per 50 mL Add HRG and human serum albumin (HSA) prepared from human plasma to a commercially available erythrocyte storage solution MAP solution (MAP solution bag) containing 360.5 mg) and sodium chloride (248.5 mg) so that the final concentration is 100 μg / mL. Was used as a protective agent for the erythrocyte-containing solution.

(Reference Example 1) Induction of phosphatidylserine (PS) expression on the surface of erythrocyte membrane by ^{Zn 2+ In this reference example, the effect of Zn 2+ on} erythrocyte concentrate was confirmed.

Blood was collected in 10 mL each in a test tube containing 10% ACD solution as an anticoagulant, and centrifuged at 3000 rpm for 10 minutes to remove plasma and buffy coat to prepare a packed red blood cell solution, which was used as a red blood cell solution sample. ..

The expression rate of PS on the surface of erythrocytes was measured when ^{Zn 2+} was added to the prepared erythrocyte solution sample. After ^{adding Zn 2+} to 0, 5, 10, 15 and 20 μM and incubating at 37 ° C. for 1 hour, the PS expression level on the surface of erythrocytes was measured. For the PS expression level, 4 μL of FITC-labeled annexin V reaction solution (manufactured by Funakoshi) was added to 400 μL of the erythrocyte solution sample, the mixture was allowed to stand at room temperature for 15 minutes, 400 μL of 4% paraformaldehyde (PFA) was added, and the mixture was fixed and then analyzed by FACS. These operations were performed according to the instruction manual for the FITC-labeled Annexin V assay kit (manufactured by Funakoshi).

As a result of the above, it was confirmed that the PS expression rate on the surface of erythrocytes increased depending on the concentration of zinc added (Fig. 1).

(Example 4) Effect of HRG on erythrocyte PS expression-induced by metal ions HBSS (Hank's Balanced Salt Solutions) alone or ZnCl ₂ (20 μM) in HBSS was added to the erythrocyte solution sample prepared by the same method as in Reference Example 1. , MgCl ₂ (20 μM), CaCl ₂ (20 μM), ZnCl ₂ (20 μM) + HRG (100 μg / mL), ZnCl ₂ (20 μM) + HSA (100 μg / mL), HRG alone (100 μg / mL) or HSA alone (100 μg / mL) After incubating at 37 ° C. for 1 hour in a system containing mL), the PS expression level on the surface of erythrocytes was measured. The PS expression level was measured by the same method as in Reference Example 1.

As a result of the above ^{, PS was expressed on the surface of erythrocytes by the addition of Zn 2+} , but suppression of PS expression was observed by the addition of HRG or a system containing HSA (Fig. 2).

In effect this example for PS (Example 5) Zn ²⁺ by expression induced erythrocyte surface, the HSA or HRG, was confirmed the effect on PS expressed induced erythrocyte surface by Zn ^2+. _{PBS + ZnCl 2} (20 μM), HSA (1, 10, 100 μg / mL) + ZnCl ₂ (20 μM), HSA (100 μg / mL) + saline, HRG (1) in the erythrocyte solution sample prepared by the same method as in Reference Example 1. , 10, 100 μg / mL) + ZnCl ₂ (20 μM), HRG (100 μg / mL) + saline was incubated at 37 ° C. for 1 hour, and then the PS expression level on the surface of erythrocytes was measured. The PS expression level was measured by the same method as in Reference Example 1.

As a result of the above, in the _{group containing ZnCl 2} (20 μM), suppression of PS expression was observed at 1 μg / mL for HRG, whereas suppression of PS expression was not observed at 1 μg / mL for HSA (Fig. 3). .. As a result, it was observed that HRG suppresses PS expression more strongly than HSA.

(Example 6) ^{Action on erythrocyte PS previously induced by Zn 2+ In} this example, the action of HSA or HRG on PS previously induced on the erythrocyte surface by ^{Zn 2+ was compared and examined.} _{ZnCl 2} (20 μM) was added to the erythrocyte solution sample prepared by the same method as in Reference Example 1 and incubated at 37 ° C. for 1 hour. Red blood cells treated with ZnCl ₂ (20 μM) were designated as RBC (Z). As a control _{, PBS was added instead of ZnCl 2} (20 μM) and incubated at 37 ° C. for 1 hour (RBC (s)).

For RBC (Z), each concentration of HRG or HSA was added and incubated at 37 ° C. for 15 minutes. The PS expression level on the surface of erythrocytes was measured by the same method as in Reference Example 1.

As a result of the above, regarding RBC (Z), PS expression suppression was observed at 100 μg / mL for HRG, whereas sufficient suppression of PS expression was not observed at 100 μg / mL for HSA (Fig. 4). As a result, it was observed that HRG suppresses PS expression more strongly than HSA.

(Example 7) Suppression of erythrocyte adhesion to collagen I by HRG In this example, the effect of HRG on erythrocyte adhesion to collagen I was confirmed. In this example, an erythrocyte solution sample prepared by the same method as in Reference Example 1 was used for examination.

^{Erythrocyte solution sample (erythrocyte count: 4 × 10 6} / mL) stored in PBS containing the above HRG (1, 10, 100 μg / mL) on a commercially available cell culture plate (Corning Biocoat Collagen I Cellware) coated with collagen I. ) Was sown and cultured at 37 ° C for 30 minutes. The control was erythrocyte seeded with erythrocytes stored only in PBS containing no HRG (PBS). As a result, it was observed that erythrocytes were suppressed from adhering to collagen I in an HRG concentration-dependent manner (Fig. 5).

(Example 8) Suppression of erythrocyte adhesion to collagen I by HRG In this example as well, a commercially available cell culture plate (Corning Biocoat Collagen I Cellware) coated with collagen I was subjected to the same method as in Example 6 or 7. A pre-zinc-stimulated erythrocyte solution sample (erythrocyte count: 4 × 10 ⁶ / mL) was seeded under HRG (1, 10, 100 μg / mL) dependence and cultured at 37 ° C. for 30 minutes. After that, the number of red blood cells adhered to the plate was counted by the same method as in Example 7. In this example, erythrocytes treated with PBS by the same method as in Example 6 were designated as RBC (s), and _{erythrocytes treated with PBS containing each concentration of ZnCl 2} were designated as RBC (Z). As a result, _{even in the group containing ZnCl 2} , erythrocytes were suppressed from adhering to collagen in an HRG concentration-dependent manner (Fig. 6). As a result, it was confirmed that when erythrocytes were stored in the group containing HRG, the adhesion to collagen was suppressed regardless of the presence or absence of ^{Zn 2+ (Fig. 6).}

(Example 9) Confirmation of erythrocyte protective effect of erythrocyte preservation solution In this example, the erythrocyte protective effect of HRG on a erythrocyte solution sample was confirmed. Add HRG or HSA to the MAP erythrocyte preservation solution so that the final concentration becomes 100 μg / mL, add the erythrocyte solution sample (erythrocyte count: 8 × 10 ⁹ / mL) prepared by the method shown below, and keep at 4 ℃ for 21 days. saved. Erythrocyte solution samples were taken on days 7, 14 and 21 of storage, and the proportion of PS-positive erythrocytes was calculated by FACS. As a result, it was confirmed that the proportion of PS-positive erythrocytes gradually increased according to the number of storage days, but the PS-positive rate was suppressed in the HRG-added group (Fig. 7).

The erythrocyte solution sample used in this example was prepared by the following method. Here, for preparing a sample of red blood cell solution, a bag for storing red blood cell concentrate (Terumo Corporation) equipped with a blood collection bag, a bag containing MAP solution, and a tube formed by connecting them was used.
(1) 2.7 mL of the MAP solution taken out from the MAP solution bag was added to the red blood cell concentrate storage bag (50 mL). 500 μL of HRG after filter sterilization was added to the above-mentioned bag for storing red blood cell concentrate and mixed so that the final concentrations of HRG and HSA were 100 μ / mL, respectively, to prepare a liquid for storing red blood cells.
(2) 80 mL of blood was collected per person in a test tube containing 8.8 mL of ACD solution, and the mixture was centrifuged at 3000 rpm for 10 minutes to remove plasma and buffy coat to prepare a packed red blood cell concentrate.
(3) 6.8 mL of the concentrated red blood cell solution prepared in (2) was added to the bag containing the red blood cell storage solution prepared in (1), and the tube at the tip was clipped and sealed. The bag was slowly inverted and mixed at 4 ° C. and stored with the tube of the bag containing the red blood cell concentrate facing up.
(4) When the red blood cell sample was taken out from the red blood cell storage solution bag after a certain period of time, the bag was overturned and mixed, a part of the tube portion was cut off, and the sample was taken. The tip of the tube was sealed each time for sampling over time.

(Example 10) Confirmation of PS expression on the surface of erythrocytes in sepsis model mice In this example, erythrocytes collected from cecal ligation and puncture (CLP) sepsis model mice (hereinafter, also referred to as “CLP mice”) The effect of HRG was confirmed.

HRG (10 mg / kg), HSA (10 mg / kg) and PBS (100 μL) as a control were administered intravenously 10 minutes after CLP. Mice were anesthetized 24 hours after the operation, and 200 μL of blood was collected from the heart using 10% ACD solution as an anticoagulant. The collected blood was centrifuged at 400 g, and plasma and buffy coat were removed by suction. The obtained erythrocyte solution was washed with HBSS at 400 g three times for 5 minutes. 1 μL of washed erythrocytes was added to 1 mL of HBSS. 4 μL of FITC-conjugated Annexin V was added to the 400 μL suspension and incubated for 15 minutes at room temperature. Then, 400 μL of 4% PFA resin solution was added and fixed for 15 hours. After fixation, the number of PS-positive cells was measured by FACS. After adding 1 μL of separately washed erythrocytes to 1 mL of HBSS and incubating at 37 ° C. for 4 hours, FITC staining was performed in the same manner, and the number of PS-positive cells was measured.

As a result, it was confirmed that the PS positive rate immediately after blood sampling was about 2% in all groups, whereas the PS positive rate was suppressed in the HRG-administered group after incubation for 4 hours (). FIG. 8).

(Example 11) Zn ²⁺ -induced phosphatidylserine (PS) expression and suppression by HRG In this example, the PS expression level on the erythrocyte surface when HRG was added to a ^{Zn 2+ stimulated erythrocyte solution sample was confirmed.} .. To the erythrocyte solution sample prepared in Reference Example 1, Zn ²⁺ was added to 20 μM, HRG concentrations were added to 1, 10, and 100 μg / mL, and the mixture was incubated at 37 ° C. for 1 hour, and then erythrocytes. The PS expression level on the surface was measured by the same method as in Reference Example 1.

As a result of the above, the PS-expressing cell rate on the erythrocyte surface was 40% in the group without HRG, whereas the PS-expressing cell rate was suppressed to 10% or less in the group with HRG (1 μg / mL). It was confirmed that this was the case (Fig. 9).

(Example 12) ^{Increase of intracellular calcium by Zn 2+} and suppression by HRG In this example, the amount of calcium in erythrocyte cells when HRG was added to a ^{Zn 2+ stimulated erythrocyte solution sample was confirmed. Add Zn 2+} to the erythrocyte solution sample prepared in Reference Example 1 to 20 μM, add the HRG concentration to 1 μg / mL, and incubate at 37 ° C for 1 hour, and then the amount of calcium in the erythrocyte cells. Was measured.

As a result of the above, it was confirmed that the amount of calcium in the erythrocyte cells increased in the group to which HRG was not added, but the amount of calcium was suppressed in the group to which HRG was added (1 μg / mL) (FIG. 10A). In the group without HRG, erythrocytes aggregated and adhered to vascular endothelial cells, whereas in the group with HRG added, erythrocytes did not aggregate and adhesion to vascular endothelial cells was not observed (Fig.). 10B).

(Reference Example 2) Erythrocyte peroxiredoxin 2 release In this reference example, the amount of peroxiredoxin 2 (Prx2) released was confirmed for an erythrocyte solution sample stimulated with ^{Zn 2+ stimulation or calcium ionophore.} Prx is one of the peroxidases having a strong antioxidant effect, and since it is present in a large amount in the cell, it is an important antioxidant enzyme for maintaining the physiological function of the cell. By measuring the amount of Prx2 released from erythrocytes, it becomes an index for measuring the cytotoxicity of erythrocytes.

First, to the erythrocyte solution sample prepared in Reference Example 1, Zn ²⁺ was added to 1, 5, 10, 20 and 50 μM, and Prx2 in the erythrocyte solution sample supernatant after incubation at 37 ° C. for 1 hour was added. It was confirmed by electrophoresis under non-reducing conditions and reducing conditions. As a result, it was confirmed that Prx2 in the supernatant increased in a ^{Zn 2+ concentration-dependent manner (FIG. 11A).} Similarly, to the erythrocyte solution sample prepared in Reference Example 1, calcium ionophore (A23187) was added to 1, 5, 10 and 20 μM, and Prx2 in the erythrocyte solution sample supernatant after incubating at 37 ° C. for 1 hour was added. It was confirmed by electrophoresis under non-reducing conditions and reducing conditions. As a result, it was confirmed that Prx2 in the supernatant increased in a calcium ionophore concentration-dependent manner (FIG. 11B).

(Example 13) ^{Prx2 release by Zn 2+} and suppression by HRG In this example, the characteristics of Prx2 release from erythrocytes when HRG was added to a ^{Zn 2+ stimulated erythrocyte solution sample were confirmed.} Prx2 in the erythrocyte solution sample supernatant after incubating at 37 ° C. for 1 hour in a system in which Zn ²⁺ was added to the erythrocyte solution sample to 50 μM and further 100 μg / mL of HSA or HRG was added was used as a non-reducing condition. Confirmed by electrophoresis. As a result, a decrease in Prx2 was observed in the HRG-added group, and the effect was more remarkable than in the HSA-added group (Fig. 12).

(Reference Example 3) Confirmation of Prx2 in plasma of CLP mice In this reference example, blood was collected from CLP mice prepared by the method shown in Example 10 and Prx2 in the obtained plasma was confirmed.

Mice were anesthetized 24 hours after CLP, and 200 μL of blood was collected from the heart using 10% ACD solution as an anticoagulant. Plasma was obtained by centrifuging the collected blood at 400 g, and Prx2 contained in the plasma was confirmed by electrophoresis under non-reducing conditions. As a result, Prx2 was observed in the plasma of CLP mice, but was hardly observed in SHAM (Fig. 13).

(Example 14) Confirmation with CLP mice In this example, free hemoglobin in blood collected from CLP mice prepared by the method shown in Example 10, PS on the surface of erythrocytes, concentration of free calcium in erythrocytes, and contained in various organs. The zinc content was confirmed.

A. HRG (20 mg / kg, iv), HSA (20 mg / kg, iv) and PBS (200 μl, iv) were administered to CLP mice 10 minutes after CLP induction, and the mice were anesthetized 24 hours after surgery, 10%. 200 μL of blood was collected from the heart using the ACD solution of the above as an anticoagulant. After plasma separation, plasma free hemoglobin levels in each group were detected by Western blotting. The results of Western blotting were quantified by densitometry and the bar graph below was created. In PBS-administered mice (controls) in CLP mice, an increase in plasma free hemoglobin was clearly observed, but in HRG-administered mice, the increase in free hemoglobin was strongly suppressed and decreased to the level of the sham surgery group (Fig. 14A).

B. HRG (20 mg / kg, i.v.), HSA (20 mg / kg) and PBSPBS (200 μl, i.v.) as a control were administered to CLP mice 10 minutes after CLP induction. Mice were anesthetized 24 hours after the operation, and 200 μL of blood was collected from the heart using 10% ACD solution as an anticoagulant. The collected blood was centrifuged at 400 g, and plasma and buffy coat were removed by suction. The obtained erythrocyte solution was washed with HBSS at 400 g three times for 5 minutes. 1 μL of washed erythrocytes was added to 1 mL of HBSS. 4 μL of FITC-conjugated Annexin V was added to a 400 μL suspension and incubated for 15 minutes at room temperature. Then, 400 μL of 4% PFA resin solution was added and fixed for 15 minutes. After fixation, the number of PS-positive cells was measured by FACS. After adding 1 μL of separately washed erythrocytes to 1 mL of HBSS and incubating at 37 ° C. for 4 hours, FITC staining was performed in the same manner, and the number of PS-positive cells was measured.

As a result, it was confirmed that the PS positive rate immediately after blood sampling was about 2% in all groups, whereas the PS positive rate was suppressed in the HRG-administered group after incubation for 4 hours (). FIG. 14B).

C. The intracellular calcium level was measured using the erythrocyte suspension (0 h) obtained in the experiment of B. Incubation was carried out for 30 minutes in a fluorescent probe Flou4-AM for intracellular free calcium measurement, and intracellular Ca ²⁺ levels in each group were quantified by FACS. As shown in the figure, the increase in intracellular calcium was observed in the PBS-administered mice of CLP mice as compared with the normal (intact) mice, but the increase was significantly suppressed in the HRG-administered mice (Fig. 14C).

D. Spleens, kidneys and lungs of CLP mice were harvested 24 hours later and homogenized with RIPA buffer. Centrifugal supernatant of homogenate ^{was measured using Metallo Assay Zn 2+} LS kit (Metallogenice, Chiba, Japan). ^{CLP mice had increased Zn 2+} content in the kidney and lung compared to intact mice, but there was no difference between the two in the spleen.

As described in detail above, according to the erythrocyte protective agent of the present invention, deterioration of erythrocytes due to storage can be suppressed. By adding HRG as an active ingredient to an existing erythrocyte preservation solution or blood preservation solution, or a erythrocyte preservation solution or blood preservation solution to be developed in the future, erythrocytes can be protected more effectively. .. This makes it possible to provide higher quality blood products for red blood cells and whole blood among blood products for blood transfusion.

Claims (9)

A erythrocyte protectant containing histidine-rich glycoprotein as an active ingredient. The erythrocyte protective agent according to claim 1, wherein the erythrocyte protection suppresses the expression of phosphatidylserine on the surface of the erythrocyte membrane by a histidine-rich glycoprotein. The red blood cell protective agent according to claim 1, wherein the protection of red blood cells reduces the calcium ion concentration in the red blood cell cells. The erythrocyte protective agent according to claim 1, wherein the protection of erythrocytes suppresses the release of antioxidant enzymes in erythrocyte cells. A stabilizer for erythrocyte-containing solutions containing histidine-rich glycoprotein as an active ingredient. A stabilizer for a erythrocyte-containing solution, which comprises the erythrocyte protective agent according to any one of claims 1 to 4. A method for stabilizing a erythrocyte-containing solution, which comprises adding the stabilizer according to claim 5 or 6 to the erythrocyte-containing solution. A phosphatidylserine expression inhibitor on the surface of erythrocyte membrane, which contains histidine-rich glycoprotein as an active ingredient. A method for suppressing the expression of phosphatidylserine on the surface of an erythrocyte membrane, which comprises adding a histidine-rich glycoprotein to a erythrocyte-containing solution.

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