KR102159436B1 - Composition for regulating blood coagulation comprising purified vesicles isolated from platelet for hemostasis - Google Patents

Abstract

The present invention relates to a composition for controlling blood coagulation comprising a platelet-derived purified product and various uses thereof. In the platelet-derived purified product of the present invention, mitochondrial DNA (mitochondrial DNA) has been removed, and proteins other than membrane proteins have been removed, and the secretion of inflammatory cytokines is remarkably lowered to promote hemostasis. It has an agglutination effect and it has been confirmed that the bleeding time can be shortened, so it can be usefully used in the related pharmaceutical industry.

Description

Composition for regulating blood coagulation comprising purified vesicles isolated from platelet for hemostasis}

The present invention relates to a composition for controlling blood coagulation comprising a platelet-derived purified product and various uses thereof.

Platelets are nucleus-free cell fragments present in mammalian blood and mediate clot formation and hemostasis. In addition, platelets play an important role in the repair and regeneration of connective tissue and release growth factors that promote wound healing. Platelets are terminal differentiation products of megakaryotic cells (MK), which are derived from pluripotent stem cells of the bone marrow. Platelets have an average lifespan of about 5 to 10 days, and their physiological blood concentration is normally between 150,000 and 450,000/μL. When a patient's concentration of circulating platelets is depleted below this physiological range, a condition known as thrombocytopenia follows. This condition is usually associated with bleeding and inadequate formation of hemostatic flocs, where bleeding is inversely proportional to platelet count. Blood transfusions are required to prevent platelet failure or a lack of platelets due to surgery. In addition, when a wound on the skin or blood vessels in the body occurs, the first step in healing the wound is platelet aggregation. During the aggregation process of platelets, as platelets themselves are activated, various types of cell membrane proteins must be activated to form aggregates, and various types of inflammatory cytokines are secreted within the activated platelets. The secreted inflammatory cytokines cause delay in wound healing time, and cause not only wounds but also inflammatory diseases (such as rheumatoid arthritis). Therefore, there is a need for research on particles that can be aggregated by cell membrane proteins without secreting inflammatory cytokines by removing nuclei and cytoplasmic components in platelets that secrete inflammatory cytokines.

Accordingly, in order to solve the above problem, the present inventors obtained a platelet-derived purified product from which mitochondrial DNA was removed from platelets and proteins excluding membrane proteins were removed. The platelet-derived purified product confirmed that the secretion of inflammatory cytokines was remarkably lowered, had an excellent aggregation effect, and could shorten the bleeding time, thereby confirming that it was possible to control blood coagulation, thus completing the present invention.

It is an object of the present invention to provide a composition for controlling blood coagulation, comprising, as an active ingredient, a purified product derived from platelets from which a platelet mitochondrial DNA and protein have been removed.

In addition, it is an object of the present invention to provide a composition for preventing bleeding and improving wounds, comprising as an active ingredient a purified product derived from platelets from which a platelet mitochondrial DNA and protein have been removed.

In addition, it is an object of the present invention to provide a pharmaceutical composition for preventing and treating bleeding diseases, comprising as an active ingredient a purified product derived from platelets from which mitochondrial DNA and protein of platelets are removed.

It is also an object of the present invention to provide a food composition for preventing and improving bleeding diseases, comprising as an active ingredient a purified product derived from platelets from which mitochondrial DNA and protein of platelets are removed.

In addition, the object of the present invention (a) removing mitochondrial DNA (mitochondrial DNA) from platelets; And (b) removing the protein except for the membrane protein (Membrane protein); it can provide a method for producing a composition for controlling blood coagulation comprising.

In order to achieve the above object, the present invention provides a composition for regulating blood coagulation, comprising, as an active ingredient, a purified product derived from platelets from which a platelet mitochondrial DNA and protein have been removed.

In addition, the present invention provides a composition for preventing bleeding and improving wounds, comprising as an active ingredient a purified product derived from platelets from which mitochondrial DNA of platelets and protein have been removed.

In addition, the present invention provides a pharmaceutical composition for preventing and treating bleeding diseases, comprising as an active ingredient a purified platelet derived from platelet mitochondrial DNA and protein removed.

In addition, the present invention provides a food composition for preventing and improving bleeding diseases, comprising as an active ingredient a purified product derived from platelets from which mitochondrial DNA and protein of platelets are removed.

In addition, the present invention (a) removing mitochondrial DNA (mitochondrial DNA) from platelets; And (b) removing proteins other than membrane proteins (Membrane protein).

In the platelet-derived purified product of the present invention, mitochondrial DNA (mitochondrial DNA) has been removed, and proteins other than membrane proteins have been removed, and the secretion of inflammatory cytokines is remarkably lowered to aid in hemostasis. It has an agglutination effect and it has been confirmed that it can shorten the bleeding time to control blood coagulation, so it can be usefully used in the related pharmaceutical industry.

1 is a diagram showing a result of measuring a SEM image of platelets (A) and a result of measuring a TEM image of a purified product derived from platelets (B).
2 is a diagram showing the results of measuring the size and stability of the platelet-derived tablet of the present invention according to the concentration and time of a hypotonic buffer using DLS (Dynamic Light Scattering).
3 is a diagram showing the results of measuring the amount of nucleic acid in a platelet lysate and a platelet-derived vesicle.
4 is a diagram showing the results of confirming the protein bands of platelet lysates and platelet-derived vesicles.
Figure 5 is a diagram showing the size of the aggregates according to the confocal microscope (A), the presence or absence of thrombin and calcium chloride (B) for the aggregation effect of the platelet-derived tablet of the present invention over time.
6 is a diagram showing the results of confirming the secretion of platelets and inflammatory cytokines (interleukin-6 (IL-6), interleukin-1β (IL-1β)) of the platelet-derived purified of the present invention.
7 is a diagram illustrating the bleeding time of platelets and platelet vesicles.

The present invention provides a composition for controlling blood coagulation, comprising as an active ingredient a purified product derived from platelets from which mitochondrial DNA of platelets and protein have been removed.

The platelet-derived purified product may include a membrane protein, and specifically, the platelet-derived purified product may include a mitochondrial DNA and a membrane protein.

The composition has suppressed the expression of inflammatory cytokines, preferably interleukin-6 (IL-6), interleukin-1β (IL-1β)) secretion is low, does not cause an inflammatory reaction, shortens the wound recovery time I can make it. Therefore, the platelet-derived purified product of the present invention can overcome the problem of delaying the wound recovery time caused by inflammatory cytokines secreted from the platelets in the related art, thereby controlling blood coagulation.

In addition, the present invention provides a composition for preventing bleeding and improving wounds, comprising, as an active ingredient, a purified platelet derived from platelet mitochondrial DNA and protein removed.

In the present invention, the term "wound" refers to a physical rupture of continuity or integrity of a tissue structure, and "improving a wound" refers to restoration or control of tissue integrity. This may mean partial or complete restoration of tissue integrity, and thus wound improvement means facilitation, progression, acceleration or progression of one or more steps or processes involved in the wound healing or improvement process. In addition, the wound can be any internal wound or external wound, particularly a skin wound, in which the external structural integrity of the skin is maintained, such as a bruise or internal ulceration, and thus the tissue can be internal or external body tissue. For example, the tissue may be skin (eg, human skin), and the wound may be a skin wound such as a dermal or epidermal wound.

Types of wounds to which the composition for improving wounds of the present invention can be applied include, but are not limited to, cuts and lacerations, surgical incisions or wounds, punctures, abrasions, scratches, pressure wounds, abrasions ), friction wounds (e.g. diaper rash, friction blisters), pressure sore ulcers (e.g. bedsores or colostomy), thermal wounds (directly or through conduction, convection or radiation, and burns from heat and electricity sources) , Chemical wounds (such as acid or alkali burns), or pathogenic infections (such as viral, bacterial or fungal infections), including open or intact boils, skin rashes, blemishes and acne, ulcers, chronic wounds (diabetic-related wounds, Such as lower leg and foot ulcers, venous leg ulcers and bedsores), skin graft/transplant donor and recipient sites, abnormal immune responses, such as psoriasis and eczema, gastric or intestinal ulcers, oral wounds including mouth ulcers, damaged cartilage or Bone, amputated wounds, and corneal lesions.

In addition, the present invention provides a pharmaceutical composition for preventing and treating bleeding diseases, comprising as an active ingredient a purified platelet derived from platelet mitochondrial DNA and protein removed.

The bleeding disease is thrombocytopenia; Glanzmann's disease; Bernard-Soulier syndrome; Von Willebrand disease; Storage pool disease; Neocoagulosis; Severe liver disease; Traumatic blood loss; Bone marrow transplant; Hypothrombocytopenia; Cerebral hemorrhage; Platelet function disease; And at least one selected from the group consisting of congenital deficiency of factors V, VII, X, or XI, but is not limited thereto.

The composition of the present invention may be used as a pharmaceutical composition, and the pharmaceutical composition may further include an adjuvant. Any of the adjuvants known in the art may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase its immunity.

The pharmaceutical composition according to the present invention may be prepared in a form in which an active ingredient is incorporated in a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention are not limited thereto, but lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.

The pharmaceutical compositions of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injectable solutions according to a conventional method. .

In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, in the active ingredient. It can be prepared by mixing and the like. Further, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, and other excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are commonly used diluents. I can. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like may be used. As a base for suppositories, witepsol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example, by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, and physical condition of the individual. It is obvious that the concentration of the active ingredient contained in the pharmaceutical composition can be selected in various ways depending on the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. When the concentration is less than 0.01 μg/ml, pharmaceutical activity may not appear, and when the concentration exceeds 5,000 μg/ml, toxicity to humans may occur.

In addition, the present invention provides a food composition for preventing and improving bleeding diseases, comprising as an active ingredient a purified product derived from platelets from which mitochondrial DNA and protein of platelets are removed.

In addition to containing the active ingredient, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as an additional ingredient, like a conventional food composition.

Examples of the above-described natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agents can be advantageously used as natural flavoring agents (taumatin), stevia extracts (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention can be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements, etc. There is this.

In addition, the food composition is an active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents and heavy weight agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid. And salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain flesh for the production of natural fruit juice and fruit juice beverages and vegetable beverages.

The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills. In the present invention, the term'health functional food composition' refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body pursuant to Health Functional Food Act No.6727, and with respect to the structure and function of the human body. It refers to ingestion for the purpose of obtaining useful effects for health purposes such as controlling nutrients or physiological effects. The health functional food of the present invention may contain ordinary food additives, and whether it is suitable as a food additive is determined according to the general rules and general test methods for food additives approved by the Food and Drug Administration, unless otherwise specified. It is judged according to the standards and standards. Examples of items listed in the'Food Additives Code' include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as reduced pigment, licorice extract, crystalline cellulose, high color pigment, and guar gum; Mixed preparations, such as a sodium L-glutamate preparation, an alkali additive for noodles, a preservative preparation, and a tar color preparation, etc. are mentioned. For example, in the health functional food in the form of a tablet, a mixture in which the active ingredient of the present invention is mixed with an excipient, a binder, a disintegrant, and other additives is granulated by a conventional method, and then compression molded by putting a lubricant, The mixture can be directly compression molded. In addition, the health functional food in the form of a tablet may contain a mating agent or the like if necessary. Among the health functional foods in the form of capsules, hard capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients in a conventional hard capsule, and the soft capsules contain the active ingredient of the present invention with additives such as excipients. The mixture mixed with can be prepared by filling a capsule base such as gelatin. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary. Ring-shaped health functional foods can be prepared by molding a mixture of the active ingredient of the present invention, excipients, binders, disintegrants, etc. by conventionally known methods, and can be coated with white sugar or other coating agents if necessary, Alternatively, the surface may be coated with a material such as starch or talc. Health functional foods in the form of granules can be prepared in granular form by a mixture of the excipients, binders, disintegrants, etc. of the active ingredients of the present invention by a known method, and if necessary, may contain flavoring agents, flavoring agents, etc. I can.

In addition, the present invention (a) removing mitochondrial DNA (mitochondrial DNA) from platelets; And (b) removing proteins other than membrane proteins (Membrane protein).

The present invention will be described in more detail through the following examples. However, the following examples are only for embodiing the contents of the present invention and the present invention is not limited thereto.

<Example 1> Platelet separation and platelet-derived purified product preparation

About 0.8-1.0 mL/mouse of blood was collected from the heart of 6-8 weeks old experimental mice. The collected blood was mixed with 0.8 mL of citrate-phosphate-dextrose buffer, and then inverted. Red blood cells and platelets + leukocyte layers were separated by centrifugation (100 xg, 20 min, 25 °C). The red blood cell layer settled with the pellet was discarded, and only the upper platelet layer was separated and placed in a new tube. The white blood cells in the upper layer were discarded through centrifugation (800 xg, 10 min, 25 °C), and the platelets settled into the pellet were resuspended in 500 μL of hypotonic buffer (20 mM Tris-HCl, 10 mM KCl, 2 mM MgCl ₂ ). . After that, the platelets were ultrasonically decomposed with a tip sonicator (total 40 sec, pulse on: 20 sec, pulse off: 10 sec, power 30%). In order to remove proteins other than the membrane protein, centrifugation was performed at 16000 xg, 20 min, 4°C. Discard the lower layer and transfer only the supernatant to a new tube. BCA assay is used to quantify the amount of platelets and protein derived from platelets.

Accordingly, it was confirmed that the platelet-derived purified product of the present invention was in a state in which the remaining nucleic acids except for DNA were removed, and the remaining proteins except for the cell membrane protein were also removed.

<Example 2> Platelet-derived purified product of the present invention TEM image confirmation

In order to confirm the platelet-derived purified product of the present invention, a platelet-derived purified solution diluted with distilled water to a protein standard of 100 μg/ml was placed on a 300-mesh copper grid, and then a 2% uranyl acetate solution was used. Dye the grid. After washing 3 times with DW, TEM was measured.

As shown in FIG. 1, as a result of measuring an SEM image of platelets (A), a size of about 2-3 μm was measured. As a result of TEM image measurement of the platelet-derived purified product of the present invention (B), it was confirmed that the size of the platelets was reduced by about 1/20-1/30 to a size of about 100 nm. In addition, it was confirmed that the platelet-derived purified product has a round shape similar to that of platelets.

<Example 3> Measurement of size and stability of platelet-derived tablets

After diluting with distilled water to a protein standard of 1 mg/mL, the platelet-derived purified product was measured in size by DLS (Dynamic Light Scattering). In addition, in order to measure the stability, a solution diluted with a hypotonic buffer at three concentrations (712, 1050 and 2536.1 μg/mL) was stored at 4° C. and measured by DLS over time.

As shown in Figure 2, the size of the platelet-derived purified product was measured to be about 121.4 ± 4.1 nm, similar to the TEM image result. As a result of measuring the stability at 4° C. for up to 96 hours for each of the three concentrations, it was confirmed that the maximum size of the platelet-derived tablet was 160.7 ± 1.9 nm at 72 hours at a concentration of 712 μg/mL. In addition, at a concentration of 1050 μg/mL, the size of the purified product derived from 156.6 ± 18.8 nm platelets at 96 hours was confirmed. In addition, at a concentration of 2536.1 μg/mL, the size was maintained within 200 nm at 146.1 ± 2.5 nm at 0 hours, thereby confirming stability at 4° C. for 96 hours.

Accordingly, it was confirmed that the platelet-derived purified product of the present invention maintains its size and has stability even under osmotic pressure conditions for a long time.

<Example 4> Measurement of the amount of nucleic acid in platelets and platelet-derived purified products

It was diluted with lysis buffer (50 mM tris-HCl, 15 mM NaCl, 10% SDS) to a protein standard of 5.5 μg/mL. Lamda DNA standard solution 2 μg/mL in the Picogreen dsDNA assay kit was taken as a standard (dilution buffer 1xTE) and lysis 5.5 μg/mL platelets and platelet-derived purified products were diluted 1/50 with 1xTE. For both the reference and sample, 50 μL/well was dispensed into a black well plate, and the picogreen dsDNA reagent stock was diluted 1/200, and 50 μL/well was dispensed. After storage at 25° C. for 2-5 minutes, the platelet was measured for fluorescence at 480 nm for excitation and 520 nm for emission.

As shown in FIG. 3, as a result of measuring the amount of nucleic acid in the platelet and platelet-derived purified product, it was confirmed that the amount of nucleic acid in the platelet-derived vesicle was reduced to 17.2 ± 20.7% compared to platelet lysate. The nucleic acid in platelets was found to be the mitochondrial DNA of platelets.

Therefore, it was confirmed that the platelet-derived purified product of the present invention had the platelet mitochondrial DNA removed.

<Example 5> Platelet and platelet-derived purified product SDS-PAGE

To examine the protein properties of platelet lysates and platelet-derived purified products, the SDS-PAGE gel band was checked. Specifically, each sample was put at a protein level of 10 μg per 10% SDS-PAGE gel well, treated for 40 minutes, stained with coomassie for 12 hours, and then bleached with DW for 12 hours.

As shown in Fig. 4, it was confirmed that the overall band tendency of the platelet lysate and the platelet-derived purified product was different. Proteins corresponding to each band were identified through the marker. The band at the position of about 60 kDa was observed to be thicker in the platelet-derived purified product compared to the platelet lysate as a GPVI cell membrane protein. In addition, the 180 kDa band is an actin binding protein and is present in the nucleus and cytoplasm. Therefore, it was observed that the band was thicker in the platelet lysate than in the platelet-derived purified product.

Therefore, it was confirmed that the platelet-derived purified product of the present invention was obtained by removing proteins other than cell membrane proteins.

<Example 6> Measurement of the aggregation effect of platelet-derived tablets

When aggregates such as platelets exist, it was confirmed that the platelet-derived purified product of the present invention also aggregated.

Specifically, 712 μg/mL of the prepared platelet-derived purified product was used as a platelet-derived purified product using CMTPX, known as a cell tracker, and stained with 3 μM of CMTPX at 37° C. for 40 minutes, followed by 5 mM calcium chloride and 0.3 U/mL. Add to the concentration of thrombin in mL. The platelet-derived purified product was placed in a 96-well plate, stored in a CO ₂ incubator for 0, 3, 24 and 48 hours, and then images were measured with a confocal microscope. After preparing a concentration of platelet-derived purified product 712, 2536 μg/mL stained by the same method, stained with 3 μM CMTPX at 37° C. for 40 minutes, then added to a thrombin concentration of 0.3 U/mL, 0, 4 After being stored in a CO ₂ incubator for ₁ day, the images were measured with a confocal microscope. In addition, the aggregation effect was also confirmed by DLS.

As shown in Fig. 5A, no fluorescence intensity was observed at 0 hours, and aggregates having a size of several microns were observed from 3 hours. It was confirmed that the aggregates grew to a size of several millimeters from 24 hours and 48 hours.

As shown in FIG. 5B, in the absence of calcium chloride, the size of the platelet-derived tablet hardly changed even when the concentration of thrombin increased. On the other hand, it was confirmed that the size of the platelet-derived purified product increased with time when calcium chloride was present. Even when the concentration of thrombin was 0, it increased, but the amount of change was relatively smaller than when there were 0.3, 3, and 30 U/mL of thrombin. The thrombin concentration of 0.3 U/mL was 3.5 ± 1.2 μm, which was the greatest increase after 48 hours. In addition, at the thrombin concentration of 3 and 30 U/mL, the largest size was measured at 24 hours as 2.5 ± 1.1 μm in both.

It is confirmed that, in the platelet-derived purified product of the present invention, calcium chloride is introduced into the platelet-derived purified product in the presence of calcium chloride. As a result, it was confirmed that the calcium ions introduced into the platelet-derived purified product activated αIIbβ3, one of the cell membrane proteins, causing an aggregation effect. On the other hand, the reason that the aggregate size of the platelet-derived purified product did not change even when the amount of thrombin was increased is that there is no calcium ion to activate αIIbβ3.

Therefore, it was confirmed that the platelet-derived purified product of the present invention formed an aggregate even if the remaining proteins except for the cell membrane protein were also removed, and exhibited the same aggregation effect as the conventional platelets (in a state in which nothing was removed).

<Example 7> Measurement of inflammatory cytokines (interleukin-6(IL-6), interleukin-1β(IL-1β)) secretion

The amount of inflammatory cytokines IL-6 and IL-1β secreted in platelets and platelet-derived purified products were measured. After resuspending 5 x 10 ⁸ platelets in RPMI media containing 10% FBS, 0 and 0.5 U/mL of thrombin were added and placed in a 24-well plate. The platelet-derived purified product treatment group and platelet vesicle of the present invention were placed. Each of the treatment groups was diluted 1/2 with RPMI containing 10% FBS concentrated twice with speed vac with the same number. Thrombin was added to 0, 0.5 U/mL to the platelet-derived purified product in RPMI, and placed in a 24-well plate. After incubation for 4 days in a CO ₂ incubator at 37° C., platelets and platelet-derived purified products were removed by centrifugation (16000 rcf, 20 min, 4° C.), and IL-6 and IL-1β were measured by ELISA.

As shown in Fig. 6A, the amount of IL-6 secretion in the platelet-derived purified product of the present invention was 14.5 ± 11.9 pg/mL in the presence of thrombin and 15.8 ± 9.2 pg/mL in the absence of thrombin.

On the other hand, it was confirmed that the amount of IL-6 secretion from platelets increased by about 2.0 times to 49.0 ± 4.7 pg/mL in the absence of thrombin and 99.1 ± 39.4 pg/mL in the presence of thrombin.

As shown in Fig. 6B, the amount of IL-1β secretion in the platelet-derived purified product of the present invention was found to be 1.9 ± 1.3 pg/mL in the absence of thrombin and 4.4 ± 1.4 pg/mL in the absence of thrombin.

On the other hand, it was confirmed that the IL-1β secretion of platelets increased by about 2.1 times to 7.1 ± 0.7 pg/mL in the absence of thrombin and 15.2 ± 3.7 pg/mL in the presence of thrombin.

Therefore, it was confirmed that the amount of secretion of IL-6 and IL-1β in platelets by thrombin was the lowest in the platelet-derived purified product of the present invention and the highest in the platelet-treated group.

Accordingly, it was confirmed that the increase in the secretion of inflammatory cytokines in the platelets by thrombin was the highest. This indicates that when the platelets are activated by thrombin, cytokines are secreted by α-granule in the platelets. On the other hand, in the case of the platelet-derived purified product of the present invention, since various organelles including α-granule in the platelets are removed, the secretion of inflammatory cytokines is small, so that the inflammatory reaction does not occur, thus lowering the inflammatory reaction and not delaying wound healing. It was confirmed not.

<Example 8> Measurement of bleeding time

After preparing the platelet-derived purified product and platelets of the present invention, protein was quantified by BCA assay. In order to inject 600 μg/kg into ICR (Institute of Cancer research) mice 6 weeks old (22-24 g), the platelet-derived tablets and platelets of the present invention were diluted, and the control, platelets and platelets-derived tablets of the present invention were 1xPBS containing 74.3% hypotonic buffer was prepared. The sample was administered by tail vein injection to the mouse, and then left for 30 minutes. After 30 minutes, 2 mm of the tip of the tail was cut, and the bleeding time was measured by immersing in 14 ml 1xPBS warmed at 37°C.

As shown in FIG. 7, when the platelet-derived purified product of the present invention was injected, it was confirmed to be 39.0 ± 14.2 seconds, and it was confirmed that the bleeding time was reduced to 57.1% compared to the control group and 51.3% compared to the platelets. On the other hand, when platelets were injected, it was confirmed that the bleeding time was not significantly reduced at 80.0 ± 17.0 seconds compared to the control group (1xPBS containing 74.3% hypotonic buffer, 91.0 ± 15.7 seconds).

Therefore, it was confirmed that the platelet-derived purified product of the present invention has a small size and a large surface area even when the same amount is injected, thereby significantly reducing the bleeding time.

Claims (8)

delete delete delete As a composition for preventing bleeding and improving wounds, comprising a platelet-derived tablet,
The platelet-derived purified product is made of membrane protein and DNA, and has a size of 100-200 nm,
The platelet-derived purified product may include proteins other than membrane proteins; Nucleic acids other than DNA; And mitochondrial DNA (mitochondrial DNA) is removed, bleeding prevention and wound improvement composition. It contains a purified product derived from platelets,
The platelet-derived purified product is made of membrane protein and DNA, and has a size of 100-200 nm,
The platelet-derived purified product may include proteins other than membrane proteins; Nucleic acids other than DNA; And the mitochondrial DNA (mitochondrial DNA) is removed,
Thrombocytopenia; Glanzmann's disease; Bernard-Soulier syndrome; Von Willebrand disease; Storage pool disease; Neocoagulosis; Severe liver disease; Traumatic blood loss; Bone marrow transplant; Hypothrombocytopenia; Cerebral hemorrhage; Platelet function disease; And Factors V, VII, X, or a pharmaceutical composition for the prevention and treatment of bleeding diseases consisting of congenital deficiency of XI. delete Including a purified product derived from platelets,
The platelet-derived purified product is made of membrane protein and DNA, and has a size of 100-200 nm,
The platelet-derived purified product may include proteins other than membrane proteins; Nucleic acids other than DNA; And the mitochondrial DNA (mitochondrial DNA) is removed,
Thrombocytopenia; Glanzmann's disease; Bernard-Soulier syndrome; Von Willebrand disease; Storage pool disease; Neocoagulosis; Severe liver disease; Traumatic blood loss; Bone marrow transplant; Hypothrombocytopenia; Cerebral hemorrhage; Platelet function disease; And a food composition for preventing and improving bleeding diseases consisting of congenital deficiency of factors V, VII, X, or XI. (a) proteins other than platelet membrane proteins; Nucleic acids other than DNA; And removing mitochondrial DNA, thereby obtaining a platelet-derived purified product consisting of a membrane protein and DNA; And
(b) obtaining that the platelet-derived tablet has a size of 100-200 nm; containing, a method for preparing a composition for preventing bleeding and improving wounds.

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