JP4532975B2 - Platelet aggregation inhibitor and health food effective for platelet aggregation inhibition - Google Patents
Platelet aggregation inhibitor and health food effective for platelet aggregation inhibition Download PDFInfo
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- JP4532975B2 JP4532975B2 JP2004130706A JP2004130706A JP4532975B2 JP 4532975 B2 JP4532975 B2 JP 4532975B2 JP 2004130706 A JP2004130706 A JP 2004130706A JP 2004130706 A JP2004130706 A JP 2004130706A JP 4532975 B2 JP4532975 B2 JP 4532975B2
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- 229940127218 antiplatelet drug Drugs 0.000 title claims description 9
- 239000000106 platelet aggregation inhibitor Substances 0.000 title claims description 9
- 101000783577 Dendroaspis angusticeps Thrombostatin Proteins 0.000 title claims description 8
- 101000783578 Dendroaspis jamesoni kaimosae Dendroaspin Proteins 0.000 title claims description 8
- 235000013402 health food Nutrition 0.000 title claims description 8
- 206010050661 Platelet aggregation inhibition Diseases 0.000 title description 3
- 108010058861 Fibrin Fibrinogen Degradation Products Proteins 0.000 claims description 21
- 239000000208 fibrin degradation product Substances 0.000 claims description 21
- 108010073682 nattokinase Proteins 0.000 claims description 21
- 229940086319 nattokinase Drugs 0.000 claims description 20
- 108010073385 Fibrin Proteins 0.000 claims description 17
- 102000009123 Fibrin Human genes 0.000 claims description 17
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 claims description 17
- 229950003499 fibrin Drugs 0.000 claims description 17
- 208000010110 spontaneous platelet aggregation Diseases 0.000 claims description 13
- 239000007857 degradation product Substances 0.000 claims description 11
- 239000004480 active ingredient Substances 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 108010049003 Fibrinogen Proteins 0.000 claims description 5
- 102000008946 Fibrinogen Human genes 0.000 claims description 5
- 229940012952 fibrinogen Drugs 0.000 claims description 5
- 239000012466 permeate Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims 6
- 239000000706 filtrate Substances 0.000 claims 1
- 238000001914 filtration Methods 0.000 claims 1
- 230000001629 suppression Effects 0.000 claims 1
- 239000000243 solution Substances 0.000 description 11
- 230000002401 inhibitory effect Effects 0.000 description 10
- 239000002245 particle Substances 0.000 description 10
- 230000005764 inhibitory process Effects 0.000 description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerol Natural products OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- 238000004220 aggregation Methods 0.000 description 6
- 230000002776 aggregation Effects 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 239000002504 physiological saline solution Substances 0.000 description 6
- 210000004623 platelet-rich plasma Anatomy 0.000 description 6
- 230000015556 catabolic process Effects 0.000 description 5
- 238000006731 degradation reaction Methods 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 102000008186 Collagen Human genes 0.000 description 4
- 108010035532 Collagen Proteins 0.000 description 4
- 108010010803 Gelatin Proteins 0.000 description 4
- 239000002775 capsule Substances 0.000 description 4
- 229920001436 collagen Polymers 0.000 description 4
- 229920000159 gelatin Polymers 0.000 description 4
- 239000008273 gelatin Substances 0.000 description 4
- 235000019322 gelatine Nutrition 0.000 description 4
- 235000011852 gelatine desserts Nutrition 0.000 description 4
- 235000011187 glycerol Nutrition 0.000 description 4
- 239000000411 inducer Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 235000014113 dietary fatty acids Nutrition 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 229930195729 fatty acid Natural products 0.000 description 3
- 239000000194 fatty acid Substances 0.000 description 3
- -1 glycerin fatty acid ester Chemical class 0.000 description 3
- 239000007902 hard capsule Substances 0.000 description 3
- 210000002381 plasma Anatomy 0.000 description 3
- 239000007901 soft capsule Substances 0.000 description 3
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 108090000190 Thrombin Proteins 0.000 description 2
- 235000013871 bee wax Nutrition 0.000 description 2
- 239000012166 beeswax Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 244000309464 bull Species 0.000 description 2
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- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 229940083466 soybean lecithin Drugs 0.000 description 2
- 235000012424 soybean oil Nutrition 0.000 description 2
- 239000003549 soybean oil Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 229960004072 thrombin Drugs 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 229920002494 Zein Polymers 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000013557 nattō Nutrition 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 229940012957 plasmin Drugs 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000002537 thrombolytic effect Effects 0.000 description 1
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- 229940093612 zein Drugs 0.000 description 1
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Description
本発明は血小板凝集抑制剤、特にフィブリンをナットウキナーゼにより反応させて得られたフィブリン分解物を有効成分とする血小板凝集抑制剤、その保健食品に関する。 The present invention relates to a platelet aggregation inhibitor, in particular, a platelet aggregation inhibitor comprising as an active ingredient a fibrin degradation product obtained by reacting fibrin with nattokinase, and a health food thereof.
従来、ナットウキナーゼは、生体由来のプラスミンと同様にフィブリン（血栓）を直接分解する血栓溶解酵素でることは知られている（例えば、非特許文献１参照。）。 Conventionally, nattokinase is known to be a thrombolytic enzyme that directly degrades fibrin (thrombus) in the same manner as plasmin derived from living bodies (see, for example, Non-Patent Document 1).
又、ナットウキナーゼが血小板凝集抑制作用があることは本発明者のうち森山、及び高岡が発明し、出願した特願2003-111081に記載されている。
ナットウキナーゼがフィブリンを分解するということは前記のように知られていたが、しかし、この分解して出来た分解物がどのような作用、効果をもっているかは知られていなかった。
一方、前記のように、ナットウキナーゼが血小板凝集抑制作用をもつならばナットウキナーゼがフィブリンを分解して得られた分解物にも血小板凝集抑制作用があるのではないかと考えた。
このことに関し、先行技術を調査したが、ナットウキナーゼがフィブリンを分解して得られた分解物について血小板凝集抑制作用があるかどうか記載されている先行技術文献が存在しなかった。
It has been known that nattokinase degrades fibrin as described above, however, it has not been known what action and effect this degradation product has.
On the other hand, as described above, if nattokinase has a platelet aggregation inhibitory action, it was considered that a degradation product obtained by nattokinase degrading fibrin may also have a platelet aggregation inhibitory action.
Regarding this, prior art was investigated, but there was no prior art document describing whether or not a degradation product obtained by nattokinase degrading fibrin had a platelet aggregation inhibitory action.
この分解物は、ペプタイド（ペプチド）であって、酵素であるナットウキナーゼよりも安定しているため、製剤にするための製造が容易であり、長期保存が可能である。
したがって、当該分解物が血小板凝集抑制作用をもつということになれば、血小板凝集抑制剤、及びナットウキナーゼは納豆として、何百年も前から食されており、その安全性が証明されているゆえに、保健食品（健康食品）を得ることも出来る、と考えて本発明者等は鋭意研究を行い本発明を完成したものである。
Since this degradation product is a peptide (peptide) and is more stable than the enzyme nattokinase, it can be easily produced for preparation and can be stored for a long period of time.
Therefore, if the degradation product has an inhibitory action on platelet aggregation, platelet aggregation inhibitors and nattokinase have been eaten as natto for hundreds of years, and their safety has been proven. The present inventors have conducted intensive research and have completed the present invention, thinking that food (health food) can also be obtained.
したがって、本発明は、製剤にするための製造が容易であり、長期保存が可能な血小板凝集抑制剤、及び血小板凝集抑制に有効な保健食品を提供することを目的とする。 Therefore, an object of the present invention is to provide a platelet aggregation inhibitor that can be easily produced for preparation and can be stored for a long period of time, and a health food effective for inhibiting platelet aggregation.
前記目的を達成するため本発明の血小板凝集抑制剤は、
フィブリンをナットウキナーゼにより反応させて得られた分解物を有効成分とすることからなる。
又、フィブリンをナットウキナーゼにより反応させて得られた分解物の分子量が10,000ダルトン以下であるものを有効成分とすることからなる。
In order to achieve the above object, the platelet aggregation inhibitor of the present invention comprises:
It consists of using a degradation product obtained by reacting fibrin with nattokinase as an active ingredient.
Further, the active ingredient is a degradation product obtained by reacting fibrin with nattokinase and having a molecular weight of 10,000 daltons or less.
又、本発明の血小板凝集抑制に有効な保健食品は、フィブリンをナットウキナーゼにより反応させて得られた分解物を有効成分とすることからなる。
又、フィブリンをナットウキナーゼにより反応させて得られた分解物の分子量が10,000ダルトン以下であるものを有効成分とすることからなる。
Moreover, the health food effective for inhibiting platelet aggregation of the present invention comprises a degradation product obtained by reacting fibrin with nattokinase as an active ingredient.
Further, the active ingredient is a degradation product obtained by reacting fibrin with nattokinase and having a molecular weight of 10,000 daltons or less.
前記フィブリンがフィブリノーゲンとトロンビンを反応させて得られたことからなるのが好適である。 It is preferable that the fibrin is obtained by reacting fibrinogen and thrombin.
本発明において、分子量が10,000ダルトン以下としたのは製剤にするのに好適だからである。即ち、分子量が大きくなると安定性が悪くなり製剤に不適となるからである。 In the present invention, the molecular weight is 10,000 daltons or less because it is suitable for preparation. That is, when the molecular weight is increased, the stability is deteriorated and it is not suitable for the preparation.
本発明は以上の構成を有するので、製剤にするための製造が容易であり、長期保存が可能な血小板凝集抑制剤、及び血小板凝集抑制に有効な保健食品を提供することが出来るものである。 Since the present invention has the above-described configuration, it is possible to provide a platelet aggregation inhibitor that can be easily produced for preparation and can be stored for a long period of time, and a health food effective in inhibiting platelet aggregation.
以下、製造方法、加工例、実施例について述べる。
（製造方法）
100ml容三角フラスコに生理食塩水（0.85％NaCl）14ml及び9.6mg/ml フィブリノーゲン（シグマ製、フィブリノーゲン フラクションI Type I-S、牛血漿由来、PRODUCT NUMBER F-8630）4mlを量りとり、37±0.3℃の恒温水槽で10分間加温した後、20U/ml トロンビンを1ml加え、よく撹拌した。この液を37℃で20分間放置後、ナットウキナーゼ溶液1mlを加え、よく撹拌し、37±0.3℃で放置した。ナットウキナーゼ溶液を添加して20、40、60、80、100分後によく撹拌し、120分後に沸騰水中で10分間煮沸を行うことでフィブリン分解産物溶液（FDP溶液と略す）を調製した。FDP溶液はアドバンテック製ULTRA FILTER UNIT USY-5（分画分子量 50,000）で限外ろ過を行った。得られた透過液を更にULTRA FILTER UNIT USY-1（分画分子量 10,000）で限外ろ過し、分画分子量10,000以下のFDP溶液を得た。尚、全ての試薬類は0.85％生理食塩水で調製した。
Hereinafter, a manufacturing method, a processing example, and an example will be described.
(Production method)
In a 100 ml Erlenmeyer flask, weigh 14 ml of physiological saline (0.85% NaCl) and 9.6 mg / ml fibrinogen (manufactured by Sigma, Fibrinogen Fraction I Type IS, derived from bovine plasma, PRODUCT NUMBER F-8630) at 37 ± 0.3 ° C. After warming for 10 minutes in a thermostatic water bath, 1 ml of 20 U / ml thrombin was added and stirred well. This solution was allowed to stand at 37 ° C. for 20 minutes, and then 1 ml of nattokinase solution was added, stirred well, and left at 37 ± 0.3 ° C. A nattokinase solution was added and stirred well after 20, 40, 60, 80, and 100 minutes, and after 120 minutes, boiling was performed in boiling water for 10 minutes to prepare a fibrin degradation product solution (abbreviated as FDP solution). The FDP solution was ultrafiltered with ULTRA FILTER UNIT USY-5 (molecular weight cut off 50,000) manufactured by Advantech. The obtained permeate was further ultrafiltered with ULTRA FILTER UNIT USY-1 (fraction molecular weight 10,000) to obtain an FDP solution having a fraction molecular weight of 10,000 or less. All reagents were prepared with 0.85% physiological saline.
（加工例）
前記フィブリン分解物乾燥粉末は、カプセル、錠剤、ドリンク、顆粒、ペーストなどの形態に加工できるものであり、以下加工例を示す。
(Processing example)
The fibrin degradation product dry powder can be processed into a form such as a capsule, a tablet, a drink, a granule, or a paste.
ソフトカプセルは、例えば、フィブリン分解物乾燥粉末を36.7mg、大豆レシチンを10mg、大豆油を133.3mg、ミツロウを15mg、グリセリン脂肪酸エステルを15mgの計210mgを混合乳化したものを内容液とし、ゼラチン100mg、グリセリン30mgの計130mgで構成された皮膜に充填し、総重量340mgのソフトカプセルが出来る。 Soft capsules, for example, 36.7 mg dry fibrin degradation powder, 10 mg soybean lecithin, 133.3 mg soybean oil, 15 mg beeswax, 15 mg glycerin fatty acid ester mixed and emulsified 210 mg total content gelatin, 100 mg gelatin, A capsule composed of 130 mg of glycerin 30 mg in total can be filled into a soft capsule with a total weight of 340 mg.
同様にハードカプセルの場合、フィブリン分解物乾燥粉末を36.7mg、デキストリンを209.8mg、ショ糖脂肪酸エステルを13.5mgの計260mgを混合したものを内容物とし、２号ゼラチンハードカプセル(70mg)に充填し、総重量330mgのハードカプセルが出来る。 Similarly, in the case of hard capsules, a mixture of 260 mg of 36.7 mg dry fibrin degradation powder, 209.8 mg dextrin, and 13.5 mg sucrose fatty acid ester is mixed, and filled into No. 2 gelatin hard capsule (70 mg), Hard capsules with a total weight of 330mg can be made.
腸溶カプセル（耐酸性コートを含む）は、例えば、フィブリン分解物乾燥粉末を36.7mg、大豆レシチンを10mg、大豆油を133.3mg、ミツロウを15mg、グリセリン脂肪酸エステルを15mgの計210mgを混合乳化したものを内容液とし、ゼラチン100mg、グリセリン30mgの計130mgで構成された皮膜に充填し、総重量340mgのソフトカプセルを作成する。これに、ゼインを30mgコーティングし、総重量370mgの腸溶カプセルを作成する。 Enteric capsules (including acid-resistant coats) are mixed and emulsified, for example, 36.7 mg dry fibrin degradation powder, 10 mg soybean lecithin, 133.3 mg soybean oil, 15 mg beeswax, 15 mg glycerin fatty acid ester, a total of 210 mg. The contents are filled into a film composed of a total of 130 mg of gelatin 100 mg and glycerin 30 mg to produce a soft capsule with a total weight of 340 mg. This is coated with 30 mg of zein to make an enteric capsule with a total weight of 370 mg.
その他、打錠品、ドリンク、顆粒、ペースト等に応用出来る。 In addition, it can be applied to tablets, drinks, granules, pastes, etc.
（実施例1）
実験方法：
フィブリン分解産物溶液を凍結乾燥したものを、生理食塩水で100mg、50mg、10mgになるように希釈した。
(Example 1)
experimental method:
The lyophilized fibrin degradation product solution was diluted to 100 mg, 50 mg, and 10 mg with physiological saline.
健常人、男：1 女：2を対象に採血して血小板凝集抑制能を測定した。血液採取はいずれも3.8％クエン酸ナトリウム加チューブを用い、21G針にて上腕正中静脈より行った。得られた血液検体は180×ｇ、10分間遠心分離し、その上清を多血小板血漿（PRP）とし、残りの検体は1600×ｇ、15分間遠心分離して、乏血小板血漿（PPP）とした。PRPをPPPにて希釈し、血小板数25±3×104／μlに調製して検体とした。凝集惹起剤にはコラーゲン（エム・シー・メディカル（株））を使用し、最終濃度は2μg／mlとした。
検体300μlに、フィブリン分解液24μl又は生理食塩水24μl（コントロール）を加え、１分間37℃で撹拌、インキュベート後、凝集惹起剤を36μl添加した。
Blood was collected from healthy subjects, males: 1 females: 2, and platelet aggregation inhibition ability was measured. All blood samples were collected from the midline brachial vein using a 21G needle using a 3.8% sodium citrate tube. The obtained blood sample is centrifuged at 180 × g for 10 minutes, and the supernatant is platelet rich plasma (PRP). The remaining sample is centrifuged at 1600 × g for 15 minutes to obtain platelet poor plasma (PPP). did. PRP was diluted with PPP, adjusted to a platelet count of 25 ± 3 × 10 4 / μl, and used as a specimen. Collagen (MC Medical Co., Ltd.) was used as the aggregation inducer, and the final concentration was 2 μg / ml.
To 300 μl of the sample, 24 μl of fibrin degradation solution or 24 μl of physiological saline (control) was added, stirred at 37 ° C. for 1 minute, incubated, and then 36 μl of an aggregation inducer was added.
血小板凝集測定には、レーザー散乱光を用いた粒子計測定型血小板凝集能測定装置（PA-20：興和（株））を用いた。PA-20は、直進する光が細かい粒子にあたって生じる散乱光強度が、粒子径の2乗に比例して増大するという原理を応用して開発された装置で、血小板凝集率のほかに血小板凝集塊の大きさとその生成数も算定できるものである。また、従来の吸光度法では、数千個の血小板からなる凝集塊が形成されてはじめて吸光度が低下したが、この装置では、血小板数10個からなる小凝集塊でも測定でき検出感度に優れている。なお、血小板凝集塊サイズは散乱光強度により、25ｍV＜（粒径9〜25μm）＜200ｍV、200ｍV＜中凝集塊(粒径25〜50μm)＜600ｍV、600ｍV＜大凝集塊（粒径50〜70μm）＜2,047ｍVの3つに分類した。［Hoshi K., Zhou X.,Terazono M., Satou Y.,Yamazaki M., Miyake F., Jpn. J. Clin. Pharmacol. Ther., 32, 223-230(2001)］。 For the platelet aggregation measurement, a particle meter measurement type platelet aggregation measuring apparatus (PA-20: Kowa Co., Ltd.) using laser scattered light was used. PA-20 is a device developed based on the principle that the intensity of scattered light generated by light traveling straight through fine particles increases in proportion to the square of the particle diameter. The size and number of generations can be calculated. In addition, in the conventional absorbance method, the absorbance decreased only after an aggregate consisting of several thousand platelets was formed, but this apparatus can measure even small aggregates consisting of 10 platelets and has excellent detection sensitivity. . The platelet aggregate size depends on the intensity of scattered light: 25 mV <(particle size 9-25 μm) <200 mV, 200 mV <medium aggregate (particle size 25-50 μm) <600 mV, 600 mV <large aggregate (particle size 50-70 μm) ) <3, 047 mV. [Hoshi K., Zhou X., Terazono M., Satou Y., Yamazaki M., Miyake F., Jpn. J. Clin. Pharmacol. Ther., 32, 223-230 (2001)].
次の式を用い血小板抑制率を計算した。
血小板抑制率（％）＝（１−X／Y）×100
X：フィブリン分解物添加後のコラーゲンを添加した散乱強度又はOD（吸光度）
Y：フィブリン分解物添加前のコラーゲンを添加した散乱強度又はOD
[Moriyama H., Iizuka T., Nagai M., Hoshi K., Biol. Pharm. Bull., 26, 1361-1364(2003).]
The platelet inhibition rate was calculated using the following formula.
Platelet inhibition rate (%) = (1−X / Y) × 100
X: Scattering intensity or OD (absorbance) with added collagen after addition of fibrin degradation product
Y: Scattering intensity or OD with added collagen before addition of fibrin degradation product
[Moriyama H., Iizuka T., Nagai M., Hoshi K., Biol. Pharm. Bull., 26, 1361-1364 (2003).]
実験の結果
下記の表１は透過率（ＯＤ）を用い抑制率を示しており、コラーゲンによる凝集惹起を高い濃度のフィブリン分解物により（6.70mg/ml）血小板の凝集を顕著に抑制したことを示している。また、抑制率はフィブリン分解物の濃度に依存することも判明した。
さらに、下記の表２は血小板凝集塊のサイズ分布を示しているが、高い濃度のフィブリン分解物を添加したサンプルでは大凝集塊の発現率が低く、逆に小凝集塊の発現率が高く、フィブリン分解物には大凝集塊の形成を抑えることが明らかになった。この結果、フィブリン分解物には血小板抑制作用があることが明らかになった。
（実施例2）
実験方法：
フィブリン分解産物溶液を減圧乾燥し粉にしたものを、生理食塩水で100mg、50mg、10mgになるように希釈した。
(Example 2)
experimental method:
The fibrin degradation product solution was dried under reduced pressure and powdered, and diluted with physiological saline to 100 mg, 50 mg, and 10 mg.
健常人、男：1 女：2を対象に採血して血小板凝集抑制能を測定した。血液採取はいずれも3.8％クエン酸ナトリウム加チューブを用い、21G針にて上腕正中静脈より行った。得られた血液検体は180×ｇ、10分間遠心分離し、その上清を多血小板血漿（PRP）とし、残りの検体は1600×ｇ、15分間遠心分離して、乏血小板血漿（PPP）とした。PRPをPPPにて希釈し、血小板数25±3×104／μlに調製して検体とした。凝集惹起剤にはADP[エム・シー・メディカル（株）]を使用し、最終濃度は5μMとした。
検体300μlに、フィブリン分解液24μl又は生理食塩水24μl（コントロール）を加え、１分間37℃で撹拌、インキュベート後、凝集惹起剤を36μl添加した。
Blood was collected from healthy subjects, males: 1 females: 2, and platelet aggregation inhibition ability was measured. All blood samples were collected from the midline brachial vein using a 21G needle using a 3.8% sodium citrate tube. The obtained blood sample is centrifuged at 180 × g for 10 minutes, and the supernatant is platelet rich plasma (PRP). The remaining sample is centrifuged at 1600 × g for 15 minutes to obtain platelet poor plasma (PPP). did. PRP was diluted with PPP and adjusted to a platelet count of 25 ± 3 × 10 4 / μl to prepare a specimen. ADP [MC Medical Co., Ltd.] was used as the aggregation inducer, and the final concentration was 5 μM.
To 300 μl of the sample, 24 μl of fibrin degradation solution or 24 μl of physiological saline (control) was added, stirred for 1 minute at 37 ° C., incubated, and 36 μl of an aggregation inducer was added.
血小板凝集測定には、レーザー散乱光を用いた粒子計測定型血小板凝集能測定装置（PA-20：興和（株））を用いた。PA-20は、直進する光が細かい粒子にあたって生じる散乱光強度が、粒子径の2乗に比例して増大するという原理を応用して開発された装置で、血小板凝集率のほかに血小板凝集塊の大きさとその生成数も算定できるものである。また、従来の吸光度法では、数千個の血小板からなる凝集塊が形成されてはじめて吸光度が低下したが、この装置では、血小板数10個からなる小凝集塊でも測定でき検出感度に優れている。なお、血小板凝集塊サイズは散乱光強度により、25ｍV＜（粒径9〜25μm）＜200ｍV、200ｍV＜中凝集塊(粒径25〜50μm)＜600ｍV、600ｍV＜大凝集塊（粒径50〜70μm）＜2,047ｍVの3つに分類した。［Hoshi K., Zhou X.,Terazono M., Satou Y.,Yamazaki M., Miyake F., Jpn. J. Clin. Pharmacol. Ther., 32, 223-230(2001)］。 For the platelet aggregation measurement, a particle meter measurement type platelet aggregation measuring apparatus (PA-20: Kowa Co., Ltd.) using laser scattered light was used. PA-20 is a device developed based on the principle that the intensity of scattered light generated by light traveling straight through fine particles increases in proportion to the square of the particle diameter. The size and number of generations can be calculated. In addition, in the conventional absorbance method, the absorbance decreased only after an aggregate consisting of several thousand platelets was formed, but this apparatus can measure even small aggregates consisting of 10 platelets and has excellent detection sensitivity. . The platelet aggregate size depends on the intensity of scattered light: 25 mV <(particle size 9-25 μm) <200 mV, 200 mV <medium aggregate (particle size 25-50 μm) <600 mV, 600 mV <large aggregate (particle size 50-70 μm) ) <3, 047 mV. [Hoshi K., Zhou X., Terazono M., Satou Y., Yamazaki M., Miyake F., Jpn. J. Clin. Pharmacol. Ther., 32, 223-230 (2001)].
次の式を用い血小板抑制率を計算した。
血小板抑制率（％）＝（１−X／Y）× 100
X：フィブリン分解物添加後のADPを添加した散乱強度又はOD（吸光度）
Y：フィブリン分解物添加前のADPを添加した散乱強度又はOD
[Moriyama H., Iizuka T., Nagai M., Hoshi K., Biol. Pharm. Bull., 26, 1361-1364(2003).]
The platelet inhibition rate was calculated using the following formula.
Platelet inhibition rate (%) = (1−X / Y) × 100
X: Scattering intensity or OD (absorbance) with addition of ADP after addition of fibrin degradation product
Y: Scattering intensity or OD with ADP added before fibrin degradation product
[Moriyama H., Iizuka T., Nagai M., Hoshi K., Biol. Pharm. Bull., 26, 1361-1364 (2003).]
実験結果：
前記表１は透過率（ＯＤ）を用い抑制率を示しており、ＡＤＰによる凝集惹起を高い濃度のフィブリン分解物により（6.70mg/ml）血小板の凝集を顕著に抑制したことを示している。また、抑制率はフィブリン分解物の濃度に依存することも判明した。
Experimental result:
Table 1 shows the inhibition rate using the transmittance (OD), which indicates that the aggregation of ADP was remarkably suppressed by the high concentration fibrin degradation product (6.70 mg / ml). It was also found that the inhibition rate depends on the concentration of fibrin degradation product.
さらに、下記の表３は血小板凝集塊のサイズ分布を示しているが、高い濃度のフィブリン分解物を添加したサンプルでは血小板の大凝集塊の発現率が低く、逆に小凝集塊の発現率が高く、フィブリン分解物には大凝集塊の形成を抑えることが明らかになった。この結果、フィブリン分解物には血小板抑制作用があることが明らかになった。
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