JPH0798840B2 - Urine-derived anti-blood pseudo-substance, method for producing the same, and pharmaceutical composition containing the same - Google Patents
Urine-derived anti-blood pseudo-substance, method for producing the same, and pharmaceutical composition containing the sameInfo
- Publication number
- JPH0798840B2 JPH0798840B2 JP1337187A JP33718789A JPH0798840B2 JP H0798840 B2 JPH0798840 B2 JP H0798840B2 JP 1337187 A JP1337187 A JP 1337187A JP 33718789 A JP33718789 A JP 33718789A JP H0798840 B2 JPH0798840 B2 JP H0798840B2
- Authority
- JP
- Japan
- Prior art keywords
- residue
- substance
- ala
- pro
- leu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
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Description
【発明の詳細な説明】 ［産業上の利用分野］ 本発明はヒト尿中の新規な抗血液凝固物質、その製法な
らびにそれを有効成分として含有することを特徴とす
る、血液凝固能異常に係わる疾患の予防剤及び治療剤に
関する。TECHNICAL FIELD The present invention relates to a novel anticoagulant substance in human urine, a method for producing the same, and abnormal blood coagulation ability characterized by containing it as an active ingredient. The present invention relates to preventive and therapeutic agents for diseases.
［従来技術］ 現在、抗血液凝固剤としてはヘパリンやアンチトロンビ
ンIIIが使用されている。また、血栓溶解剤としては、
尿または培養腎細胞から分離されたウロキナーゼや、β
溶連菌より抽出されたストレプトキナーゼなどが実用に
供されており、さらに最近では、組織プラスミノーゲン
アクチベーターも使用され始めている。[Prior Art] Currently, heparin and antithrombin III are used as anticoagulants. In addition, as a thrombolytic agent,
Urokinase isolated from urine or cultured kidney cells, β
Streptokinase extracted from streptococcus has been put to practical use, and more recently, tissue plasminogen activator has also begun to be used.
しかし、これらの物質は、出血傾向等の副作用を有し、
作用が抗血液凝固あるいは血栓溶解のいずれかに偏って
いる。However, these substances have side effects such as bleeding tendency,
The action is biased to either anticoagulation or thrombolysis.
基礎研究の分野で、近年、N.L.Esmonらにより、線溶を
促進するプロテインＣの活性化促進作用と血液凝固阻害
作用とを有する物質が家兎肺組織抽出物に存在すること
が報告され、トロンボモジュリンと命名された（J.Bio
l.Chem.257:859,1982）。トロンボモジュリンは血管内
皮細胞上に存在するトロンビンレセプターであり、トロ
ンボモジュリンと結合したトロンビンは血液凝固作用を
失い、トロンビン−トロンボモジュリン複合体はプロテ
インＣを活性化することにより抗凝固作用を示すことが
丸山らにより報告されている（J.Clin.Invest.75:987,1
985）。すなわち、トロンボモジュリンは血液凝固阻害
作用と線溶促進作用の両方の作用を発揮する可能性が有
り、臨床応用が期待されている。In the field of basic research, in recent years, NLEsmon et al. Reported that a substance having a protein C activation-promoting action that promotes fibrinolysis and a blood coagulation-inhibiting action is present in rabbit lung tissue extracts. Was named (J. Bio
l.Chem.257: 859,1982). According to Maruyama et al., Thrombomodulin is a thrombin receptor existing on vascular endothelial cells, thrombin bound to thrombomodulin loses blood coagulation action, and thrombin-thrombomodulin complex exhibits anticoagulation action by activating protein C. Reported (J.Clin.Invest.75: 987,1
985). That is, thrombomodulin may exert both an action of inhibiting blood coagulation and an action of promoting fibrinolysis, and clinical application is expected.
トロンボモジュリンはタンパク質であるから、臨床応用
にあたっては、抗原性の少ないヒト由来のものを用いる
べきである。現在までは、ヒトのトロンボモジュリンに
ついては、以下のような取得例が報告されている。な
お、分子量については、断りのない限りドデシル硫酸ナ
トリウム−ポリアクリルアミドゲル電気泳動（SDS-PAG
E）により、非還元状態での測定値を示した。Since thrombomodulin is a protein, human-derived one with low antigenicity should be used for clinical application. To date, the following cases of human thrombomodulin have been reported. Regarding the molecular weight, unless otherwise specified, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAG
E) shows the measured values in the non-reduced state.
H.H.Salemらは、ヒト胎盤よりトロンボモジュリンを精
製し、分子量75Kと報告した（J.Biol.Chem.259:12246,1
984）。丸山らは、ヒト肺よりトロンボモジュリンを精
製し、その性質は胎盤のものと同じであると報告した
（J.Clin.Invest.75:987,1985）。また、青木らは、ヒ
ト胎盤からトロンボモジュリンを精製し、分子量71Kと
報告した（Thromb.Res.37:353,1985および特開昭60-199
819号）。さらに、鈴木らは、ヒト血小板よりトロンボ
モジュリンを部分精製し、分子量を78Kと決めた上で、
電気泳動上の挙動、トロンビンとの親和性およびプロテ
インＣとの基質親和性より、血小板、胎盤および肺血管
内皮細胞のトロンボモジュリンは互いに等しい性質を持
つことを報告した（J.Biochem.104:628,1988）。HHSalem and colleagues purified thrombomodulin from human placenta and reported a molecular weight of 75K (J. Biol. Chem. 259: 12246,1.
984). Maruyama et al. Purified thrombomodulin from human lung and reported that its properties were similar to those of placenta (J. Clin. Invest. 75: 987, 1985). Aoki et al. Have purified thrombomodulin from human placenta and reported a molecular weight of 71K (Thromb. Res. 37: 353,1985 and JP-A-60-199.
No. 819). Furthermore, Suzuki et al. Partially purified thrombomodulin from human platelets, and after determining the molecular weight to be 78K,
It was reported that thrombomodulin in platelets, placenta and pulmonary endothelial cells have the same properties based on their electrophoretic behavior, affinity with thrombin and substrate affinity with protein C (J. Biochem. 104: 628, 1988).
また、前記のヒト・トロンボモジュリンと類似の性質を
有する物質については、以下のような存在例が報告され
ている。The following examples of the existence of substances having properties similar to those of human thrombomodulin have been reported.
H.Ishiiらはヒト血漿から部分精製し、分子量63Kと54K
のものが存在することを示した。また、尿中にも類似の
物質が存在することを示した（J.Clin.Invest.75:2178,
1985）。さらに、平本らは尿中には、分子量105K,63K,6
0K,33K,31Kおよび28K（何れも還元・非還元の別が不
明）のものが排泄されることを報告した（日本薬学会第
108年会講演要旨集,425頁，演題番号6F05,11-1,198
8）。その他、尿中からの取得例として、分子量200K,48
Kおよび40Kの混合物（特開昭63-30423号）、および、39
Kおよび31K（特開昭63-146896号）のものが報告されて
いる。H. Ishii et al. Have partially purified human plasma to obtain molecular weights of 63K and 54K.
Showed that there exists. It was also shown that similar substances are present in urine (J.Clin.Invest.75: 2178,
1985). Furthermore, Hiramoto et al. Have a molecular weight of 105K, 63K, 6 in urine.
It was reported that 0K, 33K, 31K and 28K (both reduced and non-reduced) are excreted (Japan Society of Pharmaceutical Sciences).
108th Annual Conference, 425 pages, No. 6F05,11-1,198
8). In addition, as an example of acquisition from urine, molecular weight 200K, 48
A mixture of K and 40K (JP-A-63-30423), and 39
Those of K and 31K (JP-A-63-146896) have been reported.
一方、遺伝子工学の手法により、鈴木らはヒト肺cDNAラ
イブラリーから、トロンボモジュリンの遺伝子をクロー
ニングし、全遺伝子構造を解明し、557残基のアミノ酸
配列を明らかにした（EMBO Journal 6:1891、1987）。
さらに、M.Zushiらは、トロンボモジュリン分子の一部
分に相当する種々のペプチドを遺伝子工学的に産生し、
そのプロテインＣ活性化能を測定することにより、トロ
ンボモジュリン様活性がアミノ末端から345-462番目の
アミノ酸残基に限局されており、その部分が１部でも欠
けると活性を失うことを示した（J.Biol.Chem.264:1035
1、1989および第12回国際血栓止血学会抄録334頁、演題
番号1039、1989）。On the other hand, Suzuki et al. Cloned the gene of thrombomodulin from a human lung cDNA library by genetic engineering, elucidated the entire gene structure, and revealed the amino acid sequence of 557 residues (EMBO Journal 6: 1891, 1987). ).
Furthermore, M. Zushi et al. Genetically produced various peptides corresponding to a part of the thrombomodulin molecule,
By measuring its protein C activating ability, it was shown that the thrombomodulin-like activity was localized at the amino acid residues 345-462 from the amino terminus, and even if even a part thereof was lost, the activity was lost (J .Biol.Chem.264: 1035
1, 1989 and 12th International Conference on Thrombosis and Hemostasis, page 334, presentation number 1039, 1989).
［発明が解決しようとする課題］ 従来報告されているヒト・トロンボモジュリンは、ヒト
胎盤、ヒト肺あるいはヒト血小板由来であり、これらを
用いて大量のトロンボモジュリンを得るのは原料の供給
の面から大変困難である。さらに、これらのトロンボモ
ジュリンを可溶化するには界面活性剤が必要であり、取
扱いに難点がある上、医薬品としても界面活性剤の混入
は好ましくない。従って、大量に入手が可能で、水に易
溶なトロンボモジュリン様物質が望まれる。[Problems to be Solved by the Invention] The previously reported human thrombomodulin is derived from human placenta, human lung or human platelets, and it is very difficult to obtain a large amount of thrombomodulin using these from the viewpoint of supply of raw materials. Is. Further, in order to solubilize these thrombomodulins, a surfactant is required, which is difficult to handle, and it is not preferable to mix the surfactant as a drug. Therefore, a thrombomodulin-like substance that can be obtained in a large amount and is easily soluble in water is desired.
また、従来のトロンボモジュリン様物質は、蛋白単位重
量当りのプロテインＣ活性化能や抗血液凝固活性が十分
高くないため、より生物活性が高く、医薬品として応用
した際により有用性の高い新規なトロンボモジュリン様
物質が望まれている。In addition, the conventional thrombomodulin-like substance has a higher biological activity because the protein C activating ability per unit weight of protein and the anticoagulant activity are not sufficiently high, and thus the novel thrombomodulin-like substance is more useful when applied as a drug. Material is desired.
一方、従来の尿からのトロンボモジュリン様物質の精製
方法では、アプロチニンやベスタチンなどの蛋白分解酵
素阻害剤を用いて分解を防止しているが、尿中にはこれ
らの蛋白分解酵素阻害剤では、事実上完全に阻害されな
いウロペプシンなどの酵素も含まれるため、精製過程に
おけるトロンボモジュリン様物質の分解を完全に阻止す
ることは困難であり、得られる収量が少ないという問題
点がある。On the other hand, in the conventional method for purifying a thrombomodulin-like substance from urine, degradation is prevented by using a protease inhibitor such as aprotinin or bestatin. Since an enzyme such as uropepsin that is not completely inhibited is also included, it is difficult to completely prevent the decomposition of the thrombomodulin-like substance in the purification process, and there is a problem that the yield obtained is small.
さらに、トロンボモジュリンは糖タンパク質であり、遺
伝子工学の手法による合成法では、完全にヒトと同じ糖
鎖を持った分子を得ることができない。従って、その違
いが副作用など好ましくない性質をもたらす恐れがある
ため、より天然に近いトロンボモジュリンを得ることが
望まれている。Furthermore, thrombomodulin is a glycoprotein, and it is not possible to obtain a molecule having the same sugar chain as that of human by the synthetic method by the method of genetic engineering. Therefore, it is desired to obtain a more natural thrombomodulin because the difference may cause undesirable properties such as side effects.
［課題を解決するための手段］ 本発明者らは、ヒト尿中に存在する抗血液凝固物質の取
得法について鋭意検討した結果、既に報告されている尿
中トロンボモジュリン様物質とは分子量的に異なり、プ
ロテインＣの活性化能や血液凝固抑制作用が従来のトロ
ンボモジュリン様物質に比べて著しく高く、かつ、より
優れた薬理作用を有する、新たなトロンビン結合性の抗
血液凝固物質を精製・取得することに成功し、本発明を
完成した。[Means for Solving the Problems] As a result of intensive studies on the method for obtaining an anticoagulant present in human urine, the present inventors have found that it is different in molecular weight from the previously reported urinary thrombomodulin-like substance. , Purification and acquisition of a new thrombin-binding anti-coagulant substance, which has significantly higher protein C activating ability and blood coagulation inhibitory action than conventional thrombomodulin-like substances and has a superior pharmacological action And succeeded in completing the present invention.
以下に本発明を詳細に説明する。The present invention will be described in detail below.
本発明は、ヒト尿由来の新規なトロンビン結合性の抗血
液凝固物質とその製法、ならびにそれを有効成分として
含有することを特徴とする、血液凝固能異常に係わる疾
患の予防及び治療剤に関するものである。The present invention relates to a novel thrombin-binding anticoagulant derived from human urine, a method for producing the same, and a prophylactic and therapeutic agent for diseases associated with abnormal blood coagulation, which comprises the same as an active ingredient. Is.
本発明の抗血液凝固物質（以後本発明物質と呼び、後述
のTM1あるいはTM2を示す）は、ヒトの新鮮尿またはその
濃縮液を、pH8.3±0.3に調整し、析出した沈澱物を除去
した後に、pHを7.3±0.2に調整し、60±５℃、15±５分
間加熱処理を行った後、イオン交換クロマトグラフィ
ー、トロンビンをリガンドとしたアフィニティークロマ
トグラフィーおよびゲル濾過クロマトグラフィーのうち
から選ばれる少なくとも１つを用いて製造することがで
きる。The anticoagulant substance of the present invention (hereinafter referred to as “invention substance”, TM1 or TM2 described below) is used to adjust human fresh urine or its concentrated liquid to pH 8.3 ± 0.3 and remove the deposited precipitate. After that, the pH is adjusted to 7.3 ± 0.2 and heat treatment is performed at 60 ± 5 ° C. for 15 ± 5 minutes, and then selected from ion exchange chromatography, affinity chromatography using thrombin as a ligand, and gel filtration chromatography. Can be manufactured by using at least one of the above.
すなわち、健康な男子の新鮮尿をpH8〜９、好ましくpH
8.3±0.3に調整して一部の蛋白分解酵素を不活性化した
後、析出した沈澱物を除去し、必要に応じて分画分子量
１万〜４万の限外濾過膜等を用いて濃縮する。ついで、
pH5〜10、好ましくはpH7.3±0.2に調整した後に、蛋白
分解酵素を不活性化するため、50〜70℃で５〜45分間、
好ましくは60±５℃で15±５分間処理し、pH5.5〜7.5、
好ましくはpH6.5±0.2にコンディショニングした陰イオ
ン交換樹脂カラムに通して活性画分を吸着させる。つい
で、pH2〜4.5、好ましくpH4.0±0.05の緩衝液で活性画
分を溶出する。溶出液を、透析により脱塩あるいは分画
分子量１万〜４万の限外濾過膜で脱塩濃縮した後、トロ
ンビンをリガンドとしたアフィニティカラムに通し、0.
05〜0.8M NaCl、好ましく0.1〜0.7M NaClを含む洗浄液
で洗浄後0.9〜2.0M NaCl、好ましく1.0±0.05M NaClを
含む溶出液で活性画分を溶出する。溶出液を脱塩濃縮し
た後、必要に応じてトロンビンをリガンドとしたアフィ
ニティーカラムで上記と同様の操作を繰り返す。That is, healthy men's fresh urine is adjusted to pH 8-9, preferably pH
After adjusting to 8.3 ± 0.3 to inactivate some proteolytic enzymes, the precipitated precipitate is removed and, if necessary, concentrated using an ultrafiltration membrane with a molecular weight cutoff of 10,000 to 40,000. To do. Then,
After adjusting the pH to 5 to 10, preferably pH 7.3 ± 0.2, in order to inactivate the proteolytic enzyme, at 50 to 70 ℃ for 5 to 45 minutes,
It is preferably treated at 60 ± 5 ° C. for 15 ± 5 minutes, pH 5.5 to 7.5,
Preferably, the active fraction is adsorbed by passing through an anion exchange resin column conditioned to pH 6.5 ± 0.2. Then, the active fraction is eluted with a buffer having a pH of 2 to 4.5, preferably pH 4.0 ± 0.05. The eluate is desalted by dialysis or desalted and concentrated with an ultrafiltration membrane having a molecular weight cut-off of 10,000 to 40,000, and then passed through an affinity column using thrombin as a ligand,
After washing with a washing solution containing 05-0.8M NaCl, preferably 0.1-0.7M NaCl, the active fraction is eluted with an eluent containing 0.9-2.0M NaCl, preferably 1.0 ± 0.05M NaCl. After desalting and concentrating the eluate, the same operation as described above is repeated with an affinity column using thrombin as a ligand, if necessary.
次に脱塩濃縮した活性画分を、ゲル濾過カラムに通し
て、溶出される本発明物質（TM1およびTM2）に相当する
活性画分をそれぞれ採取する。これらの各画分を、それ
ぞれ必要に応じて繰り返しゲル濾過カラムに通し、活性
を有する画分を分取すれば、本発明物質はおのおの純粋
な形で得ることができる。Next, the desalted and concentrated active fractions are passed through a gel filtration column to collect the active fractions corresponding to the eluted substances of the present invention (TM1 and TM2). Each of these fractions can be repeatedly passed through a gel filtration column as needed to separate active fractions, whereby the substance of the present invention can be obtained in a pure form.
また、アフィニティーカラムにて溶出した画分を脱塩濃
縮後、非還元状態のSDS-PAGEで分離し、本発明物質をそ
れぞれ純粋な形で得ることもできる。Further, the fraction eluted with the affinity column can be desalted and concentrated, and then separated by non-reducing SDS-PAGE to obtain the substance of the present invention in a pure form.
一方、必要に応じ陽イオン交換クロマトグラフィー、吸
着クロマトグラフィーあるいは疎水クロマトグラフィー
を、陰イオン交換クロマトグラフィー、トロンビンをリ
ガンドとしたアフィニティークロマトグラフィーあるい
はゲル濾過クロマトグラフィー等の他に用いることもで
きる。On the other hand, if necessary, cation exchange chromatography, adsorption chromatography or hydrophobic chromatography can be used in addition to anion exchange chromatography, affinity chromatography using thrombin as a ligand, gel filtration chromatography and the like.
得られた本発明物質は、60±２℃で10時間処理をするこ
とによりウイルスを不活性し、医薬品としてより適した
状態にすることができる。The obtained substance of the present invention can inactivate the virus and be in a state more suitable as a medicine by treating at 60 ± 2 ° C. for 10 hours.
上述した精製過程で用いる陰イオン交換樹脂としては、
DEAEセルロース、DEAEセファロース、DEAEセルロファイ
ン、DEAEトヨパールなどがあり、トロンビンをリガンド
としたアフィニティカラムは、セルロース、アガロー
ス、デキストランなどの担体に臭化シアンを用いてトロ
ンビンを結合させた後、ジイソプロピルフルオロフォス
フェート（DFP）、フェニルメタンスルフォニルフロリ
ド（PMSF）などで処理したものを使用する。ゲル濾過用
の樹脂としては、セファクリルＳ−200、セファクリル
Ｓ−300、セファデックスG150、セファデックスG100、
セファデックスG200、トヨパールHW55、バイオゲルP10
0、バイオゲルP150、セファロース6Bなどを用いること
ができる。As the anion exchange resin used in the above-mentioned purification process,
There are DEAE cellulose, DEAE sepharose, DEAE cellulofine, DEAE Toyopearl, etc.Affinity column with thrombin as a ligand, thrombin is bound to a carrier such as cellulose, agarose, dextran using cyanogen bromide, and then diisopropyl fluorophos Use the one treated with Fate (DFP), Phenylmethanesulfonyl Fluoride (PMSF), etc. Resins for gel filtration include Sephacryl S-200, Sephacryl S-300, Sephadex G150, Sephadex G100,
Sephadex G200, Toyopearl HW55, Biogel P10
0, Biogel P150, Sepharose 6B and the like can be used.
上記の方法により、本発明物質をそれぞれ純粋な形で得
ることができる。本発明物質を得る過程で、類似した性
質を持つ別物質（TM3およびTM4）を得ることもできる。By the above method, the substance of the present invention can be obtained in pure form. In the process of obtaining the substance of the present invention, other substances (TM3 and TM4) having similar properties can be obtained.
かくして得られる本発明物質およびその類似物質は、次
の性質を有する。The substance of the present invention thus obtained and its analogues have the following properties.
（１） 分子量 TM1 72,000±3,000 TM2 79,000±3,000 TM3 94,000±3,000 TM4 114,000±3,000 測定方法:Laemmliの方法（Nature 227、680、1970）に
準じ、0.1％（W/V）SDSを含む7.5％ポリアクリルアミド
ゲルを用いた電気泳動により非還元状態で測定した。分
子量標準品として、分子量測定キット（生化学工業製、
ゲル濾過用）およびフォスフォリラーゼＢ（ベーリンガ
ーマンハイム社製）を用い、7mAで20時間電気泳動を行
った。(1) Molecular weight TM1 72,000 ± 3,000 TM2 79,000 ± 3,000 TM3 94,000 ± 3,000 TM4 114,000 ± 3,000 Measurement method: According to Laemmli's method (Nature 227, 680, 1970) 7.5% poly including 0.1% (W / V) SDS It was measured in a non-reducing state by electrophoresis using an acrylamide gel. As a molecular weight standard, a molecular weight measurement kit (Seikagaku Corporation,
Electrophoresis was performed at 7 mA for 20 hours using gel filtration) and phosphorylase B (Boehringer Mannheim).
（２） アミノ酸組成：（mol％） アスパラギン酸 9.5±2.0 トレオニン 4.0±1.5 セリン 5.1±1.5 グルタミン酸 10.9±2.5 プロリン 9.3±1.5 グリシン 11.0±3.0 アラニン 11.7±3.0 システイン 8.0±4.0 バリン 5.9±1.5 メチオニン 1.1±0.5 イソロイシン 2.8±1.5 ロイシン 7.5±2.0 チロシン 1.6±1.5 フェニルアラニン 3.7±1.5 ヒスチジン 2.5±1.0 リジン 0.8±0.5 アルギニン 4.6±1.5 測定方法：本発明物質（TM1およびTM2）1mgを用いてMoo
reらの方法（Methods in Enzy mol.6:819,1963）に準じ
て完全酸加水分解した後、アミノ酸分析装置（ベックマ
ン社製）によりアミノ酸組成分析を行った。(2) Amino acid composition: (mol%) Aspartic acid 9.5 ± 2.0 Threonine 4.0 ± 1.5 Serine 5.1 ± 1.5 Glutamic acid 10.9 ± 2.5 Proline 9.3 ± 1.5 Glycine 11.0 ± 3.0 Alanine 11.7 ± 3.0 Cysteine 8.0 ± 4.0 Valine 5.9 ± 1.5 Methionine 1.1 ± 0.5 Isoleucine 2.8 ± 1.5 Leucine 7.5 ± 2.0 Tyrosine 1.6 ± 1.5 Phenylalanine 3.7 ± 1.5 Histidine 2.5 ± 1.0 Lysine 0.8 ± 0.5 Arginine 4.6 ± 1.5 Measurement method: Inventive substances (TM1 and TM2) 1 mg Moo
After complete acid hydrolysis according to the method of Re et al. (Methods in Enzy mol. 6: 819, 1963), the amino acid composition was analyzed by an amino acid analyzer (manufactured by Beckman).
なお、本方法で測定されるアスパラギン酸量は蛋白質中
のアスパラギンとアスパラギン酸量の和であり、グルタ
ミン酸量はグルタミンとグルタミン酸量の和である。ト
リプトファンは本法では測定できない。The amount of aspartic acid measured by this method is the sum of the amounts of asparagine and aspartic acid in the protein, and the amount of glutamic acid is the sum of the amounts of glutamine and glutamic acid. Tryptophan cannot be measured by this method.
本発明物質のTM1およびTM2は同一のアミノ酸組成を有す
る。TM1 and TM2 of the substance of the present invention have the same amino acid composition.
（３） 末端アミノ酸配列 本発明物質（TM1およびTM2）のＮ末端側およびＣ末端側
のアミノ酸配列の分析結果を下に示す。(3) Terminal amino acid sequence The analysis results of the amino acid sequences on the N-terminal side and the C-terminal side of the substances of the present invention (TM1 and TM2) are shown below.
Ｎ末端:Ala-Pro-Ala-Glu-Pro-Gln-Pro-Gly− −Gly-Sey-Gln-Cys-Val-Glu-His-Asp −Cys-Phe-Ala-Leu-Tyr-Pro-Gly-Pro −Ala-Thr-Phe-Leu− Ｃ末端：−Leu-Ala-Arg ［Ala:アラニン残基 Pro:プロリン残基 Glu:グルタミン酸残基 Gln:グルタミン残基 Gly:グリシン残基 Ser:セリン残基 Cys:システイン残基 Val:バリン残基 His:ヒスチジン残基 Asp:アスパラギン酸残基 Phe:フェニルアラニン残基 Leu:ロイシン残基 Tyr:チロシン残基 Thr:トレオニン残基 Arg:アルギニン残基をそれぞれ表わす］ 測定法：本発明物質各25mgをC.H.Hirsらの方法（Method
s in Enzymol.11、199、1967）に準じて還元カルボキシ
メチル化した後、脱塩し、アミノ酸配列分析用試料とし
た。N-terminal: Ala-Pro-Ala-Glu-Pro-Gln-Pro-Gly- -Gly-Sey-Gln-Cys-Val-Glu-His-Asp-Cys-Phe-Ala-Leu-Tyr-Pro-Gly- Pro-Ala-Thr-Phe-Leu- C-terminal: -Leu-Ala-Arg [Ala: Alanine residue Pro: Proline residue Glu: Glutamic acid residue Gln: Glutamine residue Gly: Glycine residue Ser: Serine residue Cys: Cysteine residue Val: Valine residue His: Histidine residue Asp: Aspartic acid residue Phe: Phenylalanine residue Leu: Leucine residue Tyr: Tyrosine residue Thr: Threonine residue Arg: Arginine residue] Measurement method: 25 mg each of the substance of the present invention is measured by the method of CH Hirs et al.
S. Enzymol. 11, 199, 1967), and then subjected to reductive carboxymethylation, desalted, and used as a sample for amino acid sequence analysis.
Ｎ末端アミノ酸配列分析は、気相アミノ酸配列自動分析
装置（アプライドバイオシステムズ社製、470A型）によ
り行い、Ｃ末端アミノ酸配列分析は、S.Yokoyamaらの方
法（Biochem.Biophys Acta.397:443、1975）に準じてカ
ルボキシペプチダーゼＰ（ペプチド研究所）処理して、
遊離するアミノ酸を高速液体クロマトグラフィーを用い
たアミノ酸分析装置（日本分光社製）により定量分析す
ることにより行った。The N-terminal amino acid sequence analysis is performed by an automatic gas phase amino acid sequence analyzer (Model 470A manufactured by Applied Biosystems), and the C-terminal amino acid sequence analysis is performed by the method of S. Yokoyama et al. (Biochem. Biophys Acta. 397: 443, 1975) and treated with carboxypeptidase P (Peptide Institute),
The liberated amino acid was quantitatively analyzed by an amino acid analyzer (manufactured by JASCO Corporation) using high performance liquid chromatography.
本発明物質のTM1およびTM2は同一のアミノ酸配列を有す
る。TM1 and TM2 of the substance of the present invention have the same amino acid sequence.
この結果から解るとおり、本発明物質のＮ末端アミノ酸
配列は、これまで明らかにされているヒト・トロンボモ
ジュリンの報告と全く一致するが、Ｃ末端側のアミノ酸
配列は、これまでの報告にあるものとは異なり、今回の
結果である−Leu-Ala-Argは鈴木らの報告の454-456番目
のアミノ酸残基の部分に該当する。すなわち、本発明物
質のＣ末端は、鈴木らの主張する活性最小単位の345-46
2番目のアミノ酸配列を有するペプチドのＣ末端より、
６アミノ酸残基短い部分に該当する。このことは、トロ
ンボモジュリン様物質の活性の発現には、彼らの主張す
るアミノ酸配列345-462残基の全てが必須でないことを
示している。またヒト・トロンボモジュリンにおいて
は、本発明物質のＣ末端から２番目に含まれている455
番目のAlaのみがValに変わった相同変異体が知られてお
り、本発明はこの変異体物質をも含む。As can be seen from these results, the N-terminal amino acid sequence of the substance of the present invention is completely in agreement with the previously reported reports of human thrombomodulin, but the C-terminal side amino acid sequence is as previously reported. In contrast, the present result, -Leu-Ala-Arg, corresponds to the portion of amino acid residues 454 to 456 in the report of Suzuki et al. That is, the C-terminal of the substance of the present invention is 345-46 which is the minimum activity unit claimed by Suzuki et al.
From the C-terminal of the peptide having the second amino acid sequence,
It corresponds to a short portion of 6 amino acid residues. This indicates that not all of their claimed amino acid sequences 345-462 are essential for the expression of thrombomodulin-like activity. In human thrombomodulin, the substance contained in the substance of the present invention is 455 second from the C terminus.
A homologous variant in which only the second Ala is changed to Val is known, and the present invention also includes this variant substance.
（４） 糖含量 TM1 中性糖 :5.5±1.0 アミノ糖:2.2±1.0 シアル酸:2.8±1.5 TM2 中性糖 :6.2±1.0 アミノ糖:3.1±1.0 シアル酸:3.8±1.5 測定方法：中性糖はフェノール硫酸法（Nature168:107,
1951）により、アミノ糖は本発明物質を4M HCl溶液中で
100℃、４時間処理後、Boasらの方法（J.Biol.chem.20
4:553,1953）に従い定量時の妨害物質を除去し、遠心濃
縮機による減圧乾固（60℃）を行って塩酸を除去した
後、Blixらの方法（Acta Chem.Scand.2:467,1948）（El
son-Morgan法のBlix変法）により、またシアル酸は0.1M
HCl溶液中で80℃、１時間処理後、Warrenらの方法（J.
Biol.Chem.234:1971,1959）により、それぞれ定量し
た。なお、結果は重量％で示した。(4) Sugar content TM1 Neutral sugar: 5.5 ± 1.0 Amino sugar: 2.2 ± 1.0 Sialic acid: 2.8 ± 1.5 TM2 Neutral sugar: 6.2 ± 1.0 Amino sugar: 3.1 ± 1.0 Sialic acid: 3.8 ± 1.5 Measuring method: Neutral Sugar is phenol sulfate method (Nature 168: 107,
1951), the amino sugars were treated with the substance of the present invention in a 4M HCl solution.
After treatment at 100 ° C for 4 hours, the method of Boas et al. (J. Biol. Chem.
4: 553,1953) to remove interfering substances at the time of quantification, and then perform vacuum drying (60 ° C) with a centrifugal concentrator to remove hydrochloric acid, followed by the method of Blix et al. (Acta Chem. Scand. 2: 467, 1948) (El
Blix of son-Morgan method) and sialic acid 0.1M
After treatment in an HCl solution at 80 ° C for 1 hour, the method of Warren et al. (J.
Biol.Chem.234: 1971,1959). The results are shown in% by weight.
（５） 紫外吸収 280nmにおける１％水溶液の1cm光路長の吸光度 TM1 7.7±1.0 TM2 6.7±1.0 測定方法：本発明物質の凍結乾燥品10mgを1mlの蒸留水
に溶解し、さらに蒸留水を用いて適当な倍率に希釈した
後、1cm光路長のセルを用いて、分光光度計（日立製作
所製:U−3200型）で波長280nmにおける吸光度を測定し
た。測定値に希釈倍率を乗じて、値を求めた。(5) Ultraviolet absorption Absorbance of 1% aqueous solution of 1% aqueous solution at 280 nm TM1 7.7 ± 1.0 TM2 6.7 ± 1.0 Measurement method: Dissolve 10 mg of the lyophilized product of the substance of the present invention in 1 ml of distilled water, and further use distilled water. After diluting to an appropriate magnification, the absorbance at a wavelength of 280 nm was measured with a spectrophotometer (U-3200 type manufactured by Hitachi, Ltd.) using a cell having an optical path length of 1 cm. The value was obtained by multiplying the measured value by the dilution ratio.
（６） 等電点 TM1 3.9±0.2 TM2 3.8±0.2 TM3 3.8±0.2 TM4 3.7±0.2 測定方法：アンフォライト（LKB社製、pH2.5〜4.5）を
用いた等電点電気泳動法により測定した。泳動条件は50
0Vで40時間とした。各フラクションについてpHを測定す
ると共に、透析した後に、別項に記載した方法によりプ
ロテインＣ活性化能を測定し、活性ピークの溶出される
pHを求めた。(6) Isoelectric point TM1 3.9 ± 0.2 TM2 3.8 ± 0.2 TM3 3.8 ± 0.2 TM4 3.7 ± 0.2 Measurement method: Measured by isoelectric focusing using amphorite (LKB, pH 2.5 to 4.5) . Running conditions are 50
It was 40 hours at 0V. The pH of each fraction is measured, and after dialysis, the protein C activation ability is measured by the method described in another section, and the activity peak is eluted.
The pH was determined.
（７） 安定性 本発明物質の安定性について検討した結果を第１表に示
した。(7) Stability Table 1 shows the results of studies on the stability of the substance of the present invention.
１から６の条件については、本発明物質TM1またはTM2を
60μg/mLの濃度で25℃,150分間処理した。また、７の条
件については本発明物質を各60μg/mLの濃度でpH7.5で
処理した。いずれの場合も処理後100倍に希釈してトロ
ンビン共存下でのプロテインＣの活性化能を測定した。
測定方法は、別項に述べる方法を用いた。活性残存率は
非処理検体を100％とした時の処理検体の残存活性で示
した。 For the conditions 1 to 6, the substance TM1 or TM2 of the present invention is used.
It was treated at a concentration of 60 μg / mL at 25 ° C for 150 minutes. Further, for the condition of 7, the substance of the present invention was treated at a concentration of 60 μg / mL each at pH 7.5. In each case, after the treatment, it was diluted 100 times and the protein C activation ability in the presence of thrombin was measured.
As the measuring method, the method described in another section was used. The residual activity rate was indicated by the residual activity of the treated sample when the untreated sample was set to 100%.
本発明物質は還元剤中では失活したが、変成剤中（１％
SDS、6M塩酸グアニジンおよび8M尿素水溶液中）では安
定であった。また、pH2ならびにpH10の条件下と60℃、3
00分の加熱条件下では安定であった。The substance of the present invention was inactivated in the reducing agent, but in the denaturing agent (1%
It was stable in SDS, 6M guanidine hydrochloride and 8M aqueous urea solution. In addition, under conditions of pH2 and pH10 and 60 ℃, 3
It was stable under heating conditions of 00 minutes.
（８） 溶解性 本発明物質（TM1およびTM2）は、室温において少なくと
も30mg/mLの濃度まで蒸留水に溶解する。(8) Solubility The substances of the present invention (TM1 and TM2) are soluble in distilled water at room temperature to a concentration of at least 30 mg / mL.
以上のように、本発明物質は従来単離されたトロンボモ
ジュリン様物質とは異なる分子量を示す新規物質であ
る。また溶解するのに界面活性剤を必要としない点で従
来のトロンボモジュリンより有用な特徴を有する。As described above, the substance of the present invention is a novel substance having a molecular weight different from that of the conventionally isolated thrombomodulin-like substance. It also has a more useful feature than conventional thrombomodulin in that it does not require a surfactant to dissolve.
本発明物質は以下のような作用を有する。The substance of the present invention has the following actions.
（１） トロンビンに対する親和性（抗トロンビン作
用） ａ） DIP−トロンビンアガロースを用いるクロマトグ
ラフィー処理で、本発明物質TM1およびTM2はほぼ100％
吸着された。(1) Affinity for thrombin (antithrombin action) a) Chromatographic treatment using DIP-thrombin agarose shows that the substances TM1 and TM2 of the present invention are almost 100%.
Adsorbed.
ｂ） 牛トロンビン（1U/mL,持田製薬社製）100μＬと
本発明物質TM1またはTM2を含む溶液100μＬを混和し、3
7℃で30分間加温した後、ヒトフィブリノーゲン（2mg/m
L）100μＬを加え、コアギュロメーター（アメルング社
製）にて凝固時間を測定した。結果を第２表に示す。b) 100 μL of beef thrombin (1 U / mL, Mochida Pharmaceutical Co., Ltd.) was mixed with 100 μL of a solution containing the substance TM1 or TM2 of the present invention, and 3
After heating at 7 ℃ for 30 minutes, human fibrinogen (2mg / m
L) 100 μL was added, and the coagulation time was measured with a coagulometer (manufactured by Amelung). The results are shown in Table 2.
この結果に示されるように、本発明物質はトロンビンと
結合し、その凝固活性を著しく抑制する作用を有する。 As shown in these results, the substance of the present invention binds to thrombin and has an action of remarkably suppressing its coagulation activity.
第２表の結果は、本発明物質の抗トロンビン作用がすで
に報告されているヒト・トロンボモジュリンに比べ、数
十倍以上強いことを示している。このことは、例えば下
記のような比較によって裏付けることができる。The results in Table 2 show that the antithrombin action of the substance of the present invention is several tens of times stronger than that of human thrombomodulin which has already been reported. This can be supported by the following comparison, for example.
第３表に特開昭62-169728号公報記載のヒト胎盤由来ト
ロンボモジュリン様物質についての凝固時間の測定結果
を引用して示す。さらに、同公報の記載によれば、この
ヒト胎盤由来トロンボモジュリン様物質は既存のヒト・
トロンボモジュリンより２倍以上強力であるとの記載が
あり、第２表と第３表の比較から、本発明物質は既存の
トロンボモジュリン様物質あるいはトロンボモジュリン
よりも強力な抗トロンビン作用を有すると考えられる。Table 3 shows the measurement results of the coagulation time for the thrombomodulin-like substance derived from human placenta described in JP-A-62-169728. Further, according to the description of the publication, this human placenta-derived thrombomodulin-like substance is
It is described that it is more than twice as potent as thrombomodulin. From the comparison between Tables 2 and 3, it is considered that the substance of the present invention has a stronger antithrombin action than existing thrombomodulin-like substances or thrombomodulin.
また、ヒト尿中トロンボモジュリン様物質の抗凝固作用
についてのデータを特開昭63-30423号公報より引用して
第４表に示す。第２表と第４表との比較によっても、本
発明物質が既存のトロンボモジュリン様物質あるいはト
ロンボモジュリンよりも強力な作用を有することが明ら
かである。 In addition, data on the anticoagulant effect of a thrombomodulin-like substance in human urine are shown in Table 4 with reference to JP-A-63-30423. It is clear from the comparison between Tables 2 and 4 that the substance of the present invention has a stronger action than existing thrombomodulin-like substances or thrombomodulin.
（２） プロテインＣ活性化能 トロンビン共存下でのプロテインＣの活性化能を合成基
質Boc-Leu-Ser-Thr-Arg-MCA（財団法人蛋白質研究奨励
会ペプチド研究所製）を用い、ウサギトロンボモジュリ
ン（アメリカンダイアグノスティカ社製）を標準物質と
して測定した。すなわち、0.1M トリス塩酸緩衝液（pH
7.5）60μＬに牛トロンビン（持田製薬社製）10U/mL溶
液を20μＬ添加し、上記緩衝液で適当濃度（０〜15μg/
mL）に希釈したウサギ肺トロンボモジュリンもしくは本
発明物質（TM1またはTM2）10μＬを添加し、さらにヒト
プロテインＣ（アメリカンダイアグノスティカ社製）の
500μg/mL溶液を10μＬ添加する。37℃で30分間反応し
た後、反応液にヒトアンチトロンビンIII（ミドリ十字
社製）1U/mLとヘパリン（持田製薬社製）10U/mLの等量
混合液を150μＬ添加して混和後、さらに、37℃で15分
間反応する。ついで、反応液に前記合成基質0.1mM溶液
を250μＬ添加して、37℃で10分反応後に、20％酢酸溶
液500μＬを添加して反応を停止する。その後、反応液
を蛍光光度計（日立製作所製）を用いて、励起波長380n
m、発光波長460nmで蛍光強度を測定する。ウサギ肺トロ
ンボモジュリンについて得られた反応液の蛍光強度から
検量線を作成し、それを用いて、本発明物質のプロテイ
ンＣ活性化能をウサギ肺トロンボモジュリンに換算した
力価として算出した。その結果、TM1は、2.3mg力価/mg
蛋白、TM2は2.2mg力価/mg蛋白の比活性を有することが
わかった。なお、蛋白質濃度はLowryらの方法（J.Biol.
Chem.193:265,1951）に従って測定した。この結果に示
されるように、本発明物質はトロンビン共存下で著しい
プロテインＣ活性化能を有し、しかも、その作用は既存
のトロンボモジュリンに比べ、より強力であることが確
認された。 (2) Protein C activation ability The activation ability of protein C in the presence of thrombin was determined by using rabbit thrombomodulin using the synthetic substrate Boc-Leu-Ser-Thr-Arg-MCA (manufactured by Institute for Protein Research and Promotion, Peptide Institute). (American Diagnostica Inc.) was used as a standard substance. That is, 0.1 M Tris-HCl buffer (pH
7.5) Add 20 μL of 10 U / mL solution of beef thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) to 60 μL, and add an appropriate concentration (0-15 μg /
10 μL of rabbit lung thrombomodulin or the substance of the present invention (TM1 or TM2) diluted in (mL) and added with human protein C (manufactured by American Diagnostica Inc.)
Add 10 μL of 500 μg / mL solution. After reacting at 37 ° C for 30 minutes, 150 μL of an equal volume mixture of human antithrombin III (Midori Cross Co., Ltd.) 1 U / mL and heparin (Mochida Pharmaceutical Co., Ltd.) 10 U / mL was added to the reaction mixture and further mixed. Incubate at 37 ℃ for 15 minutes. Then, 250 μL of the 0.1 mM solution of the synthetic substrate is added to the reaction solution, and after reacting at 37 ° C. for 10 minutes, 500 μL of a 20% acetic acid solution is added to stop the reaction. Then, using a fluorometer (manufactured by Hitachi, Ltd.), the reaction liquid was excited at an excitation wavelength of 380 n.
Fluorescence intensity is measured at m and emission wavelength of 460 nm. A calibration curve was prepared from the fluorescence intensity of the reaction solution obtained for rabbit lung thrombomodulin, and the calibration curve was used to calculate the protein C activating ability of the substance of the present invention as a titer converted into rabbit lung thrombomodulin. As a result, TM1 has a titer of 2.3 mg / mg.
The protein, TM2, was found to have a specific activity of 2.2 mg titer / mg protein. The protein concentration was determined by the method of Lowry et al. (J. Biol.
Chem.193: 265,1951). As shown in these results, it was confirmed that the substance of the present invention has a remarkable protein C activating ability in the coexistence of thrombin, and its action is more potent than existing thrombomodulin.
（３） 抗血液凝固活性 健常人クエン酸添加乏血小板血漿100μＬと、本発明物
質を含む溶液（10〜1000μｇ力価/mL）10μＬとを混和
し、37℃で２分間加温した後、ヒトトロンビン（ミドリ
十字社製）（2U/mL）100μＬを加え、凝固時間を測定し
た。その結果を３例の平均値として第５表に示す。(3) Anticoagulant activity 100 μL of citric acid-poor platelet plasma of a healthy person and 10 μL of a solution containing the substance of the present invention (10 to 1000 μg titer / mL) were mixed and heated at 37 ° C. for 2 minutes, and then human. 100 μL of thrombin (manufactured by Green Cross) (2U / mL) was added, and the coagulation time was measured. The results are shown in Table 5 as the average value of 3 cases.
この結果に示されるように、本発明物質は強力な血液凝
固時間延長作用を示した。 As shown in these results, the substance of the present invention exhibited a strong blood coagulation time prolonging action.
次に本発明物質のin vivoにおける抗血液凝固作用を実
験例にて示す。Next, the in vivo anticoagulant effect of the substance of the present invention will be shown in Experimental Examples.
（実験例 １） ラットエンドトキシンDIC（汎発性血
管内凝固）モデルにおける有効性 吉川らの方法（日本血液学会雑誌、第45巻、第３号、63
3〜640頁、1982）に準じて実験を行なった。すなわち、
体重160〜200gの雌性ウィスター系ラットをペントバル
ビタールナトリウムにより麻酔し、生理食塩水に溶解し
たリポポリサッカライド（ディフコ社製）を、25mg/kg
の用量で、左大腿静脈より４時間かけて一定の速度で持
続注入してDICモデルを作成した。同時に本発明物質TM
1、あるいはヒト胎盤トロンボモジュリンの1.2mg蛋白/k
gを0.1％ヒト血清アルブミン及び0.14MのNaClを含む0.0
1Mリン酸緩衝液（pH7.0）（ヒト胎盤トロンボモジュリ
ンについては0.005％ルブロール含有）に溶解して４時
間かけて一定速度で持続注入した。持続注入前及び注入
終了後に頚静脈より血液を採取し、血小板数及び血漿フ
ィブリノーゲン量の測定を行った。対照群として、薬物
を含まない溶媒をリポポリサッカライドと同時にラット
に持続注入した。その結果を持続注入前の測定値に対す
る抑制率として第１図に示す。なお、縦軸の抑制率
（％）は次式で示される。(Experimental Example 1) Efficacy in rat endotoxin DIC (generalized intravascular coagulation) model Yoshikawa et al.'S method (Journal of the Hematological Society of Japan, Vol. 45, No. 3, 63)
Experiments were conducted according to pages 3 to 640, 1982). That is,
Female Wistar rats weighing 160-200 g were anesthetized with pentobarbital sodium, and 25 mg / kg of lipopolysaccharide (manufactured by Difco) dissolved in physiological saline.
DIC model was created by continuous infusion from the left femoral vein for 4 hours at a constant rate. At the same time, the substance of the present invention TM
1, or 1.2 mg protein / k of human placental thrombomodulin
g containing 0.1% human serum albumin and 0.14M NaCl 0.0
It was dissolved in 1M phosphate buffer (pH 7.0) (containing 0.005% rubrol for human placental thrombomodulin) and continuously infused at a constant rate for 4 hours. Blood was collected from the jugular vein before and after continuous infusion, and the platelet count and plasma fibrinogen amount were measured. As a control group, a solvent containing no drug was continuously infused into rats simultaneously with lipopolysaccharide. The results are shown in FIG. 1 as the inhibition rate against the measured value before continuous infusion. The suppression rate (%) on the vertical axis is expressed by the following equation.
抑制率（％）＝〔（Ａ−Ｂ）÷Ａ〕×100 但し、A:コントロール群の減少量 B:投与群の減少量 DICの病態の指標である血小板数及び血漿フィブリノー
ゲン量の減少は、本発明物質の投与により顕著に抑制さ
れた。この作用は、ヒト胎盤トロンボモジュリンに比較
して明らかに強力なものであり、in vivoにおいても本
発明物質は既存のトロンボモジュリンよりも優れた抗凝
固活性を有することが示された。また、鈴木らが遺伝子
工学的に生産させたトロンボモジュリン様物質は、既存
の組織抽出トロンボモジュリンと同じ活性を示す（EMBO
Journal 6:1891,1987および特開平1-6219号）ことか
ら、本発明物質が、遺伝子工学的に合成されたトロンボ
モジュリン様物質よりも強力が活性を有することは自明
である。Inhibition rate (%) = [(A−B) ÷ A] × 100 where A: decrease in control group B: decrease in administration group The decrease in platelet count and plasma fibrinogen amount, which are indicators of DIC pathology, It was markedly suppressed by the administration of the substance of the present invention. This action is obviously stronger than that of human placental thrombomodulin, and it was shown that the substance of the present invention has an anticoagulant activity superior to that of existing thrombomodulin even in vivo. In addition, the thrombomodulin-like substance produced by Suzuki et al. Genetically shows the same activity as existing tissue-extracted thrombomodulin (EMBO
From Journal 6: 1891,1987 and JP-A No. 1-6219), it is obvious that the substance of the present invention has a stronger activity than the thrombomodulin-like substance synthesized by genetic engineering.
（実験例２） ラットトロンボプラスチンDIC（汎発性
血管内凝固）モデルにおける有効性 大野らの方法（Thrombosis Res.24:445,1981）に準じて
実験を行なった。すなわち、体重240〜270gの雄性ウィ
スター系ラットをカルバミン酸エチルにより麻酔し蒸留
水に溶解したトロンボプラスチン（シンプラスチン オ
ルガノテクニカ社製）を200mg/kgの用量で20分間持続注
入してDICモデルを作成した。本発明物質あるいは、ヒ
ト胎盤トロンボモジュリン0.25mg蛋白/kgを0.1％ヒト血
清アルブミン、0.14MNaCl及び0.01％ルブロールを含む
0.01Mリン酸緩衝液（pH7.0）に溶解して、トロンボプラ
スチン投与開始30分前より90分間かけて一定速度で持続
注入した。薬物持続注入前、及び注入終了１時間後に頸
静脈より血液を採取し、血小板数、及び血漿フィブリノ
ーゲン量の測定を行なった。対照群として薬物を含まな
い溶媒を薬物投与時と同様の方法で持続注入した。その
結果を対照群に対する抑制率として第２図に示す。な
お、縦軸の抑制率（％）は第１図の記載と同じ式で表わ
される。(Experimental example 2) Rat thromboplastin DIC (generic
Effectiveness in the intravascular coagulation model According to Ohno et al.'S method (Thrombosis Res. 24: 445, 1981).
An experiment was conducted. In other words, male wi
Star rats are anesthetized with ethyl carbamate and distilled
Thromboplastin (symplastin dissolved in water Oh
Lugano Technica's) 200 mg / kg dose for 20 minutes
I entered and created a DIC model. The substance of the present invention or
Placenta thrombomodulin 0.25 mg protein / kg 0.1% human blood
Contains Clear Albumin, 0.14M NaCl and 0.01% Lubrol
Dissolve in 0.01M phosphate buffer (pH 7.0) and
Continued at a constant rate for 90 minutes from 30 minutes before the start of stin administration
Injected. Neck before continuous drug infusion and 1 hour after the end of infusion
Blood was collected from the vein to determine the platelet count and plasma fibrino
The amount of x-ray was measured. Do not include drug as a control
A continuous solvent was continuously infused in the same manner as when the drug was administered. That
The results are shown in FIG. 2 as the inhibition rate relative to the control group. Na
The suppression rate (%) on the vertical axis is expressed by the same formula as that shown in FIG.
To be done.
DICの病態の指標である血小板数、及び血漿フィブリノ
ーゲン量の減少は、本発明物質の投与により顕著に抑制
された。この作用は、ヒト胎盤トロンボモジュリンに比
較して明らかに強力なものであり、in vivoにおいても
本発明物質は既存のトロンボモジュリンよりも優れた抗
凝固活性を有することが示された。The decrease in the platelet count and plasma fibrinogen amount, which are indicators of the pathological condition of DIC, was markedly suppressed by the administration of the substance of the present invention. This action is obviously stronger than that of human placental thrombomodulin, and it was shown that the substance of the present invention has an anticoagulant activity superior to that of existing thrombomodulin even in vivo.
（実験例 ３） マウスにおける急性毒性 本発明物質の急性毒性を調べた。10匹のddY系雄性マウ
スを用いて、本発明物質のTM1またはTM2を200mg力価/kg
の用量にて静脈内投与し、７日後までの観察を行なった
が、著明な毒性や死亡例は１例も認められなかった。(Experimental Example 3) Acute toxicity in mice The acute toxicity of the substance of the present invention was examined. Using 10 ddY male mice, TM1 or TM2 of the substance of the present invention is titrated to 200 mg / kg.
It was intravenously administered at a dose of 5 and observed for up to 7 days, but no significant toxicity or death was observed.
以上の説明及び実験結果から明らかなように、本発明物
質は強力な抗血液凝固作用を有し、in vivoにおいても
既存のトロンボモジュリンよりも優れた抗凝固活性を示
し、さらに毒性も低いことから、例えば、DIC、各種血
栓症、末梢血管閉塞症、心筋梗塞、脳梗塞、一過性脳虚
血発作、妊娠中毒症、肝不全、腎不全など、血液凝固能
異常に係わる疾患の治療および予防に有効である。As is clear from the above description and experimental results, the substance of the present invention has a strong anticoagulant activity, shows superior anticoagulant activity in vivo than existing thrombomodulin, and further has low toxicity, For example, for the treatment and prevention of diseases related to abnormal blood coagulation such as DIC, various thrombosis, peripheral vascular occlusion, myocardial infarction, cerebral infarction, transient cerebral ischemic attack, pregnancy toxemia, liver failure, renal failure, etc. It is valid.
本発明物質は、薬剤として一般的に用いられる適当な担
体または媒体、例えば滅菌水や生理食塩水、植物油、無
害性誘起溶媒等、さらには必要に応じて賦形剤、着色
剤、乳化剤、懸濁剤、安定化剤または保存剤等と適宜組
合せて、患者に効果的に投与するのに適した医薬用製剤
として、注射剤、吸入剤、座剤、好ましくは注射剤に調
製することができる。本発明物質を注射剤として用いる
場合には、一日１回ないし６回に分割して、一度にある
いは持続的に患者に投与される。その一日投与量は、本
発明物質0.05〜500mg力価、好ましくは0.1〜10mg力価で
あるが、患者の年齢、体重、症状等に応じて適宜増減す
ることが出来る。The substance of the present invention is a suitable carrier or medium that is generally used as a drug, such as sterile water or physiological saline, vegetable oil, a non-toxic inducing solvent, or the like, and if necessary, an excipient, a coloring agent, an emulsifier, or a suspending agent. Suitable combination with a suspending agent, a stabilizer, a preservative, etc., it can be prepared as an injection, inhalation, suppository, preferably injection as a pharmaceutical preparation suitable for effective administration to patients. . When the substance of the present invention is used as an injection, it is divided into 1 to 6 times a day and administered to a patient once or continuously. The daily dose is 0.05 to 500 mg titer of the substance of the present invention, preferably 0.1 to 10 mg titer, but it can be appropriately increased or decreased depending on the age, body weight, symptoms, etc. of the patient.
さらに、本発明の抗血液凝固物質は、人工血管、人工臓
器、カテーテルなどの医用器材の表面に架橋剤などを用
いて結合・吸着させて使用することができる。これによ
り、医用器材表面での血液凝固を防ぐことができる。Furthermore, the anticoagulant substance of the present invention can be used by binding and adsorbing it to the surface of medical equipment such as artificial blood vessels, artificial organs and catheters using a crosslinking agent or the like. This can prevent blood coagulation on the surface of the medical device.
［実施例］ 次に本発明物質の製造方法を実施例により具体的に示す
が、本発明は以下の実施例に限定されるものではない。[Examples] Next, the method for producing the substance of the present invention will be specifically described with reference to Examples, but the present invention is not limited to the following Examples.
（実施例 １） フェノール等の防腐剤を使用して採取した健康な男子の
新鮮尿100Lを、10％NaOHでpH8.5に調整し、析出した沈
殿物を除去した。次いで、尿のpHを4M HClでpH5.5に調
整した後、アクリロニトリル繊維で濾過し尿中のウロキ
ナーゼを吸着除去し、通過尿を分画分子量４万の限外濾
過膜を使用して脱塩濃縮した。pHを7.3に調整した後、6
0℃で15分間処理した。0.068M NaClを含有する0.05Mリ
ン酸緩衝液（pH6.5）で予めコンディショニングしてお
いたDEAEセルロース（ワットマン社製）の300mLカラム
に濃縮尿を通過させて活性画分を吸着させ、コンディシ
ョニングに使用したと同じ緩衝液750mLで洗浄した後、
0.05M NaClを含む酢酸緩衝液（pH4.0）で活性画分を溶
出した。(Example 1) 100 L of fresh male urine collected using a preservative such as phenol was adjusted to pH 8.5 with 10% NaOH, and the deposited precipitate was removed. Then, after adjusting the pH of urine to pH 5.5 with 4M HCl, it is filtered through acrylonitrile fiber to adsorb and remove urokinase in the urine, and the urine passing through is desalted and concentrated using an ultrafiltration membrane with a molecular weight cut-off of 40,000. did. After adjusting the pH to 7.3, 6
It was treated at 0 ° C. for 15 minutes. Concentrated urine was passed through a 300 mL column of DEAE cellulose (manufactured by Whatman) preconditioned with 0.05 M phosphate buffer (pH 6.5) containing 0.068 M NaCl to adsorb the active fraction for conditioning. After washing with 750 mL of the same buffer used,
The active fraction was eluted with an acetate buffer (pH 4.0) containing 0.05 M NaCl.
溶出液は、分画分子量３万の限外濾過膜で濃縮し、2M N
aOHでpH7.5に調整し、0.1M NaCl、1mMベンザミジン塩酸
塩および0.5mM CaCl2を含む0.02Mトリス塩酸緩衝液（pH
7.5）で予めコンディショニングしたDIP−トロンビン−
アガロースの2.5mLカラムを通過させて活性画分を吸着
させた。次いで、コンディショニングに使用したと同じ
緩衝液25mLで洗浄した後、1M NaCl、1mMベンザミジン塩
酸塩および0.5mM EDTAを含む0.02Mトリス塩酸緩衝液（p
H7.5）で溶出し、この溶出液をコンディショニングに使
用したと同じ緩衝液に対して透析後、再度前回と同様の
条件にコンディショニングしたDIP−トロンビン−アガ
ロースクロマトグラフィーで精製した。２回目のDIP−
トロンビン−アガロースクロマトグラフィーにおいても
同容のカラムを用い、コンディショニングで使用した緩
衝液10mLで洗浄した後に、10mLの0.8M NaCl、1mMベンザ
ミジン塩酸塩および0.5mM CaCl2を含む0.02Mトリス塩酸
緩衝液（pH7.5）で洗浄し、1M NaCl、1mMベンザミジン
塩酸塩および0.5mM EDTAを含む0.02Mトリス塩酸緩衝液
（pH7.5）で活性画分を溶出した。The eluate was concentrated with an ultrafiltration membrane with a molecular weight cut-off of 30,000 to obtain 2M N
Adjusted to pH 7.5 with aOH and added with 0.02M Tris-HCl buffer (pH: 0.1M NaCl, 1mM benzamidine hydrochloride and 0.5mM CaCl 2).
7.5) Pre-conditioned DIP-Thrombin-
The active fraction was adsorbed by passing through a 2.5 mL column of agarose. Then, after washing with 25 mL of the same buffer used for conditioning, 0.02 M Tris-HCl buffer containing 1 M NaCl, 1 mM benzamidine hydrochloride and 0.5 mM EDTA (p
H7.5), and the eluate was dialyzed against the same buffer used for conditioning and then purified again by DIP-thrombin-agarose chromatography conditioned under the same conditions as the previous time. Second DIP-
Using the same volume column in thrombin-agarose chromatography, after washing with 10 mL of the buffer used in conditioning, 10 mL of 0.8M NaCl, 1 mM benzamidine hydrochloride and 0.5 mM CaCl 2 0.02 M Tris-HCl buffer ( After washing with pH 7.5), the active fraction was eluted with 0.02 M Tris-HCl buffer (pH 7.5) containing 1 M NaCl, 1 mM benzamidine hydrochloride and 0.5 mM EDTA.
溶出液は、分画分子量３万の限外濾過膜で濃縮し、あら
かじめ0.14M NaClを含む0.01Mリン酸緩衝液（pH7.0）で
コンディショニングしておいたセファクリルＳ−300
（ファルマシアファインケミカル社製）の500mLカラム
でゲル濾過して、SDS-PAGEで分子量72,000±3,000およ
び79,000±3,000に相当する活性画分をそれぞれ採取し
た。各画分を、再度前回と同様の条件にコンディショニ
ングした同容のセファクリルＳ−300でゲル濾過して、
活性を有する画分を分画分取した。The eluate was concentrated with an ultrafiltration membrane with a molecular weight cut-off of 30,000, and was conditioned with 0.01M phosphate buffer (pH 7.0) containing 0.14M NaCl in advance on Sephacryl S-300.
Gel filtration was performed with a 500 mL column (Pharmacia Fine Chemical Co.), and active fractions corresponding to molecular weights of 72,000 ± 3,000 and 79,000 ± 3,000 were collected by SDS-PAGE. Each fraction was gel-filtered again with the same volume of Sephacryl S-300 conditioned under the same conditions as the previous time,
Fractions having activity were collected.
以上の製造方法により、最終的に得られた活性画分は、
ウサギ肺トロンボモジュリンに換算してTM1では247μｇ
力価、TM2では166μｇ力価であった。The active fraction finally obtained by the above production method,
Converted to rabbit lung thrombomodulin, TM1 is 247 μg
The titer was 166 μg for TM2.
この様にして得られた本発明物質、および参考例に示す
ヒト胎盤トロンボモジュリンの非還元状態でのSDS-PAGE
の結果を第３図に示す。図中に５、６および４の符号で
示すとおり、本発明物質であるTM1（第３図中５）およ
びTM2（同６）は、それぞれ胎盤トロンボモジュリン
（同４）とは明らかに異なる分子量を有する。またそれ
ぞれが単一バンドを示す。The substance of the present invention thus obtained and SDS-PAGE in the non-reducing state of human placental thrombomodulin shown in Reference Example
The results are shown in FIG. As indicated by reference numerals 5, 6 and 4 in the figure, TM1 (5 in FIG. 3) and TM2 (6) of the present invention each have a molecular weight distinctly different from placental thrombomodulin (4). . Each also represents a single band.
次に本発明物質を含有する製剤の実施例を示す。Next, examples of formulations containing the substance of the present invention will be shown.
（実施例 ２） TM1 20mg（力価） 精製ゼラチン 50mg リン酸ナトリウム 34.8mg 塩化ナトリウム 81.8mg マンニトール 25mg 上記成分を注射用蒸留水10mLに溶解し、無菌濾過した後
に1.0mLずつ無菌バイアルに分注し、凍結乾燥して、注
射用製剤を調製した。(Example 2) TM1 20 mg (titer) Purified gelatin 50 mg Sodium phosphate 34.8 mg Sodium chloride 81.8 mg Mannitol 25 mg The above components were dissolved in 10 mL of distilled water for injection, sterile filtered, and 1.0 mL each was dispensed into a sterile vial. It was freeze-dried to prepare an injectable preparation.
（実施例 ３） TM2 40mg（力価） アルブミン 20mg リン酸ナトリウム 34.8mg 塩化ナトリウム 81.8mg マンニトール 25mg 上記の各成分を秤量し、実施例２と同様の方法にて凍結
乾燥製剤を調製した。(Example 3) TM2 40 mg (potency) albumin 20 mg sodium phosphate 34.8 mg sodium chloride 81.8 mg mannitol 25 mg The above components were weighed and a freeze-dried preparation was prepared in the same manner as in Example 2.
（参考例） ヒト胎盤トロンボモジュリンの取得例 特開昭60-199819号の方法に準じ、ヒト胎盤より、トロ
ンボモジュリンを精製した。すなわち、ヒト胎盤12kg
（30個分）を、0.25Mシュークロース、1mMベンザミジン
塩酸塩を含む0.02Mトリス塩酸緩衝液（pH7.5）で洗浄し
た後、肉挽機にて破砕し、均質化した。均質化した懸濁
液を3000回転で40分間遠心分離し、得られた沈澱物を上
記緩衝液に懸濁させ、10分間撹拌後、再度遠心分離して
沈澱物を分取した。以上の操作を、１回あたり20Lの緩
衝液を用いて、合計３回繰り返し行い、分取した沈澱物
を、0.25Mシュークロース、1mMベンザミジン塩酸塩およ
び0.5％（V/V）トリトンX100（シグマ社製）を含む60L
の0.02Mトリス塩酸緩衝液（pH7.5）で抽出した。得られ
た抽出液中の総タンパク質量は46.7gであった（Lowry法
による、以下同じ）。粗抽出液60Lを、0.1M NaCl、0.5m
M CaCl2、1mMベンザミジン塩酸塩および0.5％（V/V）ト
リトンＸ−100を含む0.02Mトリス塩酸緩衝液（pH7.5）
でコンディショニングにしたDIP−トロンビン−アガロ
ースカラム（４φ×16cm）に吸着させ、コンディショニ
ングに用いたのと同じ緩衝液2Lで洗浄した。次いで、1M
NaCl、0.5mM EDTA、1mMベンザミジン塩酸塩および0.5
％（V/V）トリトンＸ−100を含む0.02Mトリス塩酸緩衝
液（pH7.5）で溶出した。溶出量は650mLであり、得られ
た蛋白質量は1.7gであった。この溶出液を限外濾過器
（ミリポア社製、分画分子量３万）を使用して脱塩濃縮
し、再度上記と同様にコンディショニングした同容のDI
P−トロンビン−アガロースカラムに吸着させた。次い
で、0.4M NaCl、0.5mM CaCl2、1mMベンザミジン塩酸塩
および0.5％トリトンＸ−100を含む150mLの0.02Mトリス
塩酸緩衝液（pH7.5）で洗浄した後、0.5mM EDTA、1mMベ
ンザミジン塩酸塩および0.5％トリトンＸ−100を含む0.
02Mトリス塩酸緩衝液（pH7.5）に、NaCl（0.4〜1M）を
加えた溶液を用いて、濃度勾配法により溶出し、30mL毎
に分画した。目的とする分画の液量は合計で1290mLで、
蛋白質量は68mgであった。次いで、この溶出液を限外濾
過器（ミリポア社製、分画分量３万）を使用して着塩濃
縮し、予め0.05％トリトンＸ−100、0.14M NaClを含む
0.01Mリン酸緩衝液（pH7.0）でコンディショニングした
Ｓ−300（ファルマシア社製）カラム（2.6φ×90cm）で
ゲル濾過し、目的とする画分を捕集した。取得した胎盤
トロンボモジュリンは蛋白質量として3.1mgであった。(Reference Example) Example of Obtaining Human Placental Thrombomodulin According to the method of JP-A-60-199819, thrombomodulin was purified from human placenta. That is, human placenta 12 kg
(30 pieces) were washed with 0.02 M Tris-HCl buffer (pH 7.5) containing 0.25 M sucrose and 1 mM benzamidine hydrochloride, and then crushed with a meat grinder and homogenized. The homogenized suspension was centrifuged at 3000 rpm for 40 minutes, the obtained precipitate was suspended in the above buffer solution, stirred for 10 minutes, and then centrifuged again to collect the precipitate. The above operation was repeated 3 times in total using 20 L of buffer solution each time, and the collected precipitates were mixed with 0.25 M sucrose, 1 mM benzamidine hydrochloride and 0.5% (V / V) Triton X100 (Sigma). 60L including)
It was extracted with 0.02 M Tris-HCl buffer (pH 7.5). The total protein amount in the obtained extract was 46.7 g (by Lowry method, the same applies hereinafter). 60 L of crude extract was added to 0.1 M NaCl, 0.5 m
0.02M Tris-HCl buffer (pH 7.5) containing M CaCl 2 , 1 mM benzamidine hydrochloride and 0.5% (V / V) Triton X-100
It was adsorbed on a DIP-thrombin-agarose column (4φ × 16 cm) conditioned by the above step, and washed with 2 L of the same buffer solution used for conditioning. Then 1M
NaCl, 0.5 mM EDTA, 1 mM benzamidine hydrochloride and 0.5
Elution was performed with 0.02 M Tris-HCl buffer (pH 7.5) containing 10% (V / V) Triton X-100. The elution amount was 650 mL, and the amount of protein obtained was 1.7 g. This eluate was desalted and concentrated using an ultrafilter (Millipore, cut-off molecular weight 30,000), and then conditioned again in the same manner as above to obtain the same volume of DI.
It was adsorbed on a P-thrombin-agarose column. Then, after washing with 150 mL of 0.02 M Tris-HCl buffer (pH 7.5) containing 0.4 M NaCl, 0.5 mM CaCl 2 , 1 mM benzamidine hydrochloride and 0.5% Triton X-100, 0.5 mM EDTA, 1 mM benzamidine hydrochloride was added. And 0.5% Triton X-100.
A solution obtained by adding NaCl (0.4 to 1 M) to 02M Tris-hydrochloric acid buffer (pH 7.5) was used to elute by the concentration gradient method and fractionated every 30 mL. The total volume of the target fraction is 1290 mL,
The amount of protein was 68 mg. Next, this eluate was subjected to salt concentration using an ultrafilter (manufactured by Millipore, fractionation amount 30,000), and contained 0.05% Triton X-100 and 0.14M NaCl in advance.
Gel filtration was performed using an S-300 (Pharmacia) column (2.6φ × 90 cm) conditioned with 0.01 M phosphate buffer (pH 7.0) to collect the target fraction. The obtained placental thrombomodulin had a protein amount of 3.1 mg.
［発明の効果］ 本発明物質は、トロンビンと結合することにより、トロ
ンビンの作用を打ち消して血液凝固抑制および血小板凝
集抑制作用を発揮すると同時に、プロテインＣを活性化
してプロテインＣが有しているとされる血液凝固抑制作
用、血栓溶解作用をも発現するため、血栓形成抑制、血
栓溶解、抗DICなど、広範囲にわたる血液凝固能異常の
係わる疾患に対する予防及び治療効果が期待される。[Effects of the Invention] The substance of the present invention, when bound to thrombin, cancels the action of thrombin to exert blood coagulation-suppressing and platelet aggregation-suppressing actions, and at the same time, activates protein C and has the property that protein C has. Since it also exhibits a blood coagulation inhibitory action and a thrombolytic action, it is expected to have preventive and therapeutic effects on a wide range of diseases associated with abnormal blood coagulation such as thrombus formation inhibition, thrombolysis, and anti-DIC.
また、本発明物質は、従来単離精製されたことのない新
規な物質であり、過去に報告されているトロンボモジュ
リン様物質に比べてトロンビン結合能およびプロテイン
Ｃ活性化能が強力で、より少量で抗血液凝固作用ならび
にDICなどの病態モデルでの有効性を示す。in vivoにお
ける動物モデルでの有用性について本発明物質は、胎盤
トロンボモジュリンより優れている。従って、血液凝固
能異常のある血栓症やDICなどの疾患に対する予防薬あ
るいは治療薬として医薬品に応用した際に、従来の物質
よりもより強力な効果が期待でき、あるいは、より少量
の投与で同程度の効果が期待されるため、副作用発現の
危険性がより少なく、また、より経済的に使用すること
ができる。また、現在では治療が困難である疾患の治療
が可能になるなど、全く新しい効果も期待される。Further, the substance of the present invention is a novel substance that has never been isolated and purified, and has a stronger thrombin-binding ability and protein C-activating ability than a previously reported thrombomodulin-like substance, so that it can be used in a smaller amount. It shows anticoagulant activity and effectiveness in pathological models such as DIC. Regarding the utility in animal models in vivo, the substance of the present invention is superior to placental thrombomodulin. Therefore, when applied to medicines as a prophylactic or therapeutic drug for diseases such as thrombosis with abnormal blood coagulation ability and DIC, it can be expected to have a stronger effect than conventional substances, or with the administration of smaller doses. Since a certain degree of effect is expected, there is less risk of side effects and it can be used more economically. In addition, completely new effects are expected, such as the treatment of diseases that are currently difficult to treat.
また、本発明物質は、ヒトの尿から精製して得られる天
然の物質であるため、遺伝子工学の手法により得られる
トロンボモジュリン様物質において心配されるようなア
ナフィラキシーショックなどの副作用がなく、医薬品と
してより安全に使用することができる。Further, since the substance of the present invention is a natural substance obtained by purifying from human urine, there is no side effect such as anaphylactic shock, which is a concern in a thrombomodulin-like substance obtained by a genetic engineering technique, and it is more suitable as a pharmaceutical. Can be used safely.
さらに、尿由来の本発明物質は、胎盤・肺などの組織抽
出トロンボモジュリンの生体投与時の問題点である界面
活性剤を使用する必要がないため、医薬品としてより安
全に使用することができる。Furthermore, since the substance of the present invention derived from urine does not require the use of a surfactant which is a problem at the time of biological administration of tissue-extracted thrombomodulin such as placenta and lung, it can be more safely used as a pharmaceutical.
本発明の抗血液凝固物質は、上記のような医薬品として
の用途以外に、人工血管、人工臓器、カテーテルなどの
医用器材の表面に架橋剤などを用いて結合・吸着させ
て、血液凝固を防ぐ目的でも用いることができる。The anticoagulant substance of the present invention, in addition to its use as a drug as described above, prevents the blood coagulation by binding and adsorbing to the surface of medical instruments such as artificial blood vessels, artificial organs and catheters using a crosslinking agent or the like. It can also be used for purposes.
一方、本発明物質は、従来の方法に比べて大量に入手可
能なヒト尿を原料とし、しかも尿よりウロキナーゼ等の
有用物質を分離した後に目的物を分離精製できることか
ら、工業上極めて効率的に得ることが可能である。On the other hand, the substance of the present invention is made from human urine, which is available in a large amount as compared with the conventional method, as a raw material, and since the target substance can be separated and purified after separating useful substances such as urokinase from the urine, it is industrially extremely efficient. It is possible to obtain.
本発明の製造方法は、ベスタチンやアプロチニンなどの
蛋白分解酵素の阻害剤の代りに、アルカリ処理や熱処理
を行うことによって、目的物の分解を防ぐ方法を採用し
たため、従来完全には抑制されていなかった精製過程で
の分解が押えられ、新規な抗血液凝固物質を、より効率
良く精製・取得することができる。Since the production method of the present invention employs a method of preventing the decomposition of the target substance by performing alkali treatment or heat treatment instead of the inhibitor of proteolytic enzyme such as bestatin and aprotinin, it has not been completely suppressed in the past. In addition, decomposition in the purification process is suppressed, and a new anticoagulant substance can be purified and obtained more efficiently.
第１図は、本発明物質TM1、また参考例として取得した
ヒト胎盤トロンボモジュリンのラットエンドトキシシDI
Cモデルにおける有効性を検討した結果を示したグラフ
である。縦軸の抑制率（％）は次式で示される。 抑制率（％）＝〔（Ａ−Ｂ）÷Ａ〕×100 但し、A:コントロール群の減少量 B:投与群の減少量 第２図は、本発明物質TM1およびTM2、また参考例として
取得したヒト胎盤トロンボモジュリンのラットトロンボ
プラスチンDICモデルにおける有効性を検討した結果を
示したグラフである。なお、縦軸の抑制率（％）は第１
図と記載と同じ式で表される。第３図は、本発明物質TM
1およびTM2とヒト胎盤トロンボモジュリンの非還元状態
でのSDS-PAGEの泳動パターンを示した図面である。FIG. 1 shows the substance TM1 of the present invention and rat endotoxin DI of human placental thrombomodulin obtained as a reference example.
It is a graph showing the results of examining the effectiveness of the C model. The suppression rate (%) on the vertical axis is shown by the following equation. Inhibition rate (%) = [(A−B) ÷ A] × 100 where A: decrease amount of control group B: decrease amount of administration group FIG. 2 was obtained as substances of the present invention TM1 and TM2, and as reference examples. 3 is a graph showing the results of examining the efficacy of human placental thrombomodulin in a rat thromboplastin DIC model. Note that the suppression rate (%) on the vertical axis is the first
It is expressed by the same formula as the figure and description. Fig. 3 shows the substance TM of the present invention
1 is a drawing showing SDS-PAGE migration patterns of 1 and TM2 and human placental thrombomodulin in a non-reducing state.
フロントページの続き (72)発明者 大澤 伸雄 静岡県駿東郡長泉町中土狩564―５ ドミ ールＴ402号 (72)発明者 持田 英 東京都豊島区駒込２―５―４ (56)参考文献 特開 昭60−199819（ＪＰ，Ａ) 特開 昭63−146898（ＪＰ，Ａ) 国際公開87−50（ＷＯ，Ａ) Ｊ．Ｂｉｏｃｈｅｍ． Ｖｏｌ．104 （1988） Ｐ．628−632Front Page Continuation (72) Inventor Nobuo Osawa 56-5 Nakachikari, Nagaizumi-cho, Sunto-gun, Shizuoka Domele T402 (72) Inventor Hide Mochida 2-5-4 Komagome, Toshima-ku, Tokyo (56) References JP-A-60-199819 (JP, A) JP-A-63-146898 (JP, A) International publication 87-50 (WO, A) J. Biochem. Vol. 104 (1988) P. 628-632
Claims (9)
凝固物質。 a.トロンビンと親和性を有し、トロンビンのプロテイン
Ｃ活性化能を促進する作用を有する。 b.アミノ酸組成:mol（％）で表示 アスパラギン酸 9.5±2.1 トレオニン 4.0±1.5 セリン 5.1±1.5 グルタミン酸 10.9±2.5 プロリン 9.3±1.5 グリシン 11.0±3.0 アラニン 11.7±3.0 システイン 8.0±4.0 バリン 5.9±1.5 メチオニン 1.1±0.5 イソロイシン 2.8±1.5 ロイシン 7.5±2.0 チロシン 1.6±1.5 フェニルアラニン 3.7±1.5 ヒスチジン 2.5±1.0 リジン 0.8±0.5 アルギニン 4.6±1.5 ［Mooreらの方法に準じた完全酸加水分解によって得ら
れるアミノ酸組成のmol％］ c.末端アミノ酸配列： Ｎ末端： Ala-Pro-Ala-Glu-Pro-Gln-Pro-Gly− Gly-Sey-Gln-Cys-Val-Glu-His-Asp− Cys-Phe-Ala-Leu-Tyr-Pro-Gly-Pro− Ala-Thr-Phe-Leu− Ｃ末端： −Leu-Ala-Argまたは−Leu-Val-Arg ［Ala:アラニン残基 Pro:プロリン残基 Glu:グルタミン酸残基 Gln:グルタミン残基 Gly:グリシン残基 Ser:セリン残基 Cys:システイン残基 Val:バリン残基 His:ヒスチジン残基 Asp:アスパラギン酸残基 Phe:フェニルアラニン残基 Leu:ロイシン残基 Tyr:チロシン残基 Thr:トレオニン残基 Arg:アルギニン残基をそれぞれ表わす］ d.安定性： pH安定性:pH2〜10の範囲で安定。 熱安定性:60℃、300分処理で安定。 変性剤安定性:1％（W/V）ドデシル硫酸ナトリウム（SD
S）、6M塩酸グアニジンおよび8M尿素溶液中でそれぞれ
安定。 e.分子量:72,000±3,000 ［非還元状態でのSDS−ポリアクリルアミドゲル電気泳
動法（PAGEにより測定）］ f.等電点:3.9±0.21. An anticoagulant derived from human urine having the following properties. a. It has an affinity with thrombin and has an action of promoting the protein C activating ability of thrombin. b. Amino acid composition: displayed in mol (%) Aspartic acid 9.5 ± 2.1 Threonine 4.0 ± 1.5 Serine 5.1 ± 1.5 Glutamic acid 10.9 ± 2.5 Proline 9.3 ± 1.5 Glycine 11.0 ± 3.0 Alanine 11.7 ± 3.0 Cysteine 8.0 ± 4.0 Valine 5.9 ± 1.5 Methionine 1.1 ± 0.5 Isoleucine 2.8 ± 1.5 Leucine 7.5 ± 2.0 Tyrosine 1.6 ± 1.5 Phenylalanine 3.7 ± 1.5 Histidine 2.5 ± 1.0 Lysine 0.8 ± 0.5 Arginine 4.6 ± 1.5 [mol% of amino acid composition obtained by complete acid hydrolysis according to the method of Moore et al. ] C. Terminal amino acid sequence: N-terminal: Ala-Pro-Ala-Glu-Pro-Gln-Pro-Gly- Gly-Sey-Gln-Cys-Val-Glu-His-Asp- Cys-Phe-Ala-Leu- Tyr-Pro-Gly-Pro- Ala-Thr-Phe-Leu- C-terminal: -Leu-Ala-Arg or -Leu-Val-Arg [Ala: Alanine residue Pro: Proline residue Glu: Glutamic acid residue Gln: Glutamine residue Gly: Glycine residue Ser: Serine residue Cys: Cysteine residue Val: Valine residue His: Histidine residue Group Asp: Aspartic acid residue Phe: Phenylalanine residue Leu: Leucine residue Tyr: Tyrosine residue Thr: Threonine residue Arg: Represents arginine residue] d. Stability: pH stability: pH 2 to 10 range And stable. Thermal stability: stable at 60 ° C for 300 minutes. Denaturant stability: 1% (W / V) sodium dodecyl sulfate (SD
S), stable in 6M guanidine hydrochloride and 8M urea solutions. e. Molecular weight: 72,000 ± 3,000 [SDS-polyacrylamide gel electrophoresis in non-reducing state (measured by PAGE)] f. Isoelectric point: 3.9 ± 0.2
尿由来の抗血液凝固物質。 a.糖含量（重量％）： 中性糖:5.5±1.0 ［フェノール硫酸法で測定］ アミノ糖:2.2±1.0 ［Elson-Morgan法（Blix変法）で測定］ シアル糖:2.8±1.5 ［Warren法で測定］ b.紫外吸収:7.7±1.0 ［280nmにおける１％水溶液の1cm光路長の吸光度］2. The anticoagulant derived from human urine according to claim 1, which has the following properties. a. Sugar content (% by weight): Neutral sugar: 5.5 ± 1.0 [Measured by phenol-sulfuric acid method] Amino sugar: 2.2 ± 1.0 [Measured by Elson-Morgan method (modified Blix method)] Sial sugar: 2.8 ± 1.5 [Warren Method] b. Ultraviolet absorption: 7.7 ± 1.0 [Absorbance of 1% aqueous solution of 1% aqueous solution at 280 nm]
凝固物質。 a.トロンビンと親和性を有し、トロンビンのプロテイン
Ｃ活性化能を促進する作用を有する。 b.アミノ酸組成:mol（％）で表示 アスパラギン酸 9.5±2.0 トレオニン 4.0±1.5 セリン 5.1±1.5 グルタミン酸 10.9±2.5 プロリン 9.3±1.5 グリシン 11.0±3.0 アラニン 11.7±3.0 システイン 8.0±4.0 バリン 5.9±1.5 メチオニン 1.1±0.5 イソロイシン 2.8±1.5 ロイシン 7.5±2.0 チロシン 1.6±1.5 フェニルアラニン 3.7±1.5 ヒスチジン 2.5±1.0 リジン 0.8±0.5 アルギニン 4.6±1.5 ［Mooreらの方法に準じた完全酸加水分解によって得ら
れるアミノ酸組成のmol％］ c.末端アミノ酸配列： Ｎ末端： Ala-Pro-Ala-Glu-Pro-Gln-Pro-Gly− Gly-Sey-Gln-Cys-Val-Glu-His-Asp− Cys-Phe-Ala-Leu-Tyr-Pro-Gly-Pro− Ala-Thr-Phe-Leu− Ｃ末端： −Leu-Ala-Argまたは−Leu-Val-Arg d.安定性： pH安定性:pH2〜10の範囲で安定。 熱安定性:60℃、300分処理で安定。 変性剤安定性:1％（W/V）ドデシル硫酸ナトリウム（SD
S）、6M塩酸グアニジンおよび8M尿素溶液中でそれぞれ
安定。 e.分子量:79,000±3,000 ［非還元状態でのSDS-PAGEにより測定］ f.等電点:3.8±0.23. An anticoagulant derived from human urine having the following properties. a. It has an affinity with thrombin and has an action of promoting the protein C activating ability of thrombin. b. Amino acid composition: displayed in mol (%) Aspartic acid 9.5 ± 2.0 Threonine 4.0 ± 1.5 Serine 5.1 ± 1.5 Glutamic acid 10.9 ± 2.5 Proline 9.3 ± 1.5 Glycine 11.0 ± 3.0 Alanine 11.7 ± 3.0 Cysteine 8.0 ± 4.0 Valine 5.9 ± 1.5 Methionine 1.1 ± 0.5 Isoleucine 2.8 ± 1.5 Leucine 7.5 ± 2.0 Tyrosine 1.6 ± 1.5 Phenylalanine 3.7 ± 1.5 Histidine 2.5 ± 1.0 Lysine 0.8 ± 0.5 Arginine 4.6 ± 1.5 [mol% of amino acid composition obtained by complete acid hydrolysis according to the method of Moore et al. ] C. Terminal amino acid sequence: N-terminal: Ala-Pro-Ala-Glu-Pro-Gln-Pro-Gly- Gly-Sey-Gln-Cys-Val-Glu-His-Asp- Cys-Phe-Ala-Leu- Tyr-Pro-Gly-Pro- Ala-Thr-Phe-Leu- C-terminal: -Leu-Ala-Arg or -Leu-Val-Arg d. Stability: pH stability: Stable in the range of pH 2-10. Thermal stability: stable at 60 ° C for 300 minutes. Denaturant stability: 1% (W / V) sodium dodecyl sulfate (SD
S), stable in 6M guanidine hydrochloride and 8M urea solutions. e. Molecular weight: 79,000 ± 3,000 [Measured by SDS-PAGE in non-reducing state] f. Isoelectric point: 3.8 ± 0.2
尿由来の抗血液凝固物質。 a.糖含量（重量％）： 中性糖:6.2±1.0 ［フェノール硫酸法で測定］ アミノ糖:3.1±1.0 ［Elson-Morgan法（Blix変法）で測定］ シアル糖:3.8±1.5 ［Warren法で測定］ b.紫外吸収:6.7±1.0 ［280nmにおける１％水溶液の1cm光路長の吸光度］4. The anticoagulant derived from human urine according to claim 3, which has the following properties. a. Sugar content (% by weight): Neutral sugar: 6.2 ± 1.0 [Measured by phenol-sulfuric acid method] Amino sugar: 3.1 ± 1.0 [Measured by Elson-Morgan method (modified Blix method)] Sial sugar: 3.8 ± 1.5 [Warren Method] b. Ultraviolet absorption: 6.7 ± 1.0 [Absorbance of 1% optical path length of 1% aqueous solution at 280 nm]
澱物を除去した後に、pHを7.3±0.2に調整し、60±５
℃、15±５分間加熱処理を行った後、トロンビンをリガ
ンドとして用いるアフィニティクロマトグラフィー、イ
オン交換クロマトグラフィーおよびゲル濾過クロマトグ
ラフィーのうちから選ばれる少なくとも１つを用いるこ
とを特徴とする請求項１〜４のいずれかに記載のヒト尿
由来の抗血液凝固物質の製造方法。5. Human urine is adjusted to pH 8.3 ± 0.3, the deposited precipitate is removed, and then the pH is adjusted to 7.3 ± 0.2 to obtain 60 ± 5.
After heat treatment at 15 ° C. for 15 ± 5 minutes, at least one selected from affinity chromatography using thrombin as a ligand, ion exchange chromatography, and gel filtration chromatography is used. 4. The method for producing a human urine-derived anticoagulant substance according to any one of 4 above.
物質を有効成分として含有することを特徴とする、血液
凝固能異常に係わる疾患の予防及び治療剤。6. A preventive and / or therapeutic agent for a disease associated with abnormal blood coagulation ability, which comprises the human urine-derived anticoagulant substance according to claim 1 as an active ingredient.
物質を有効成分として含有することを特徴とする、血液
凝固能異常に係わる疾患の予防及び治療剤。7. A preventive and / or therapeutic agent for a disease associated with abnormal blood coagulation ability, which comprises the human urine-derived anticoagulant substance according to claim 2 as an active ingredient.
物質を有効成分として含有することを特徴とする、血液
凝固能異常に係わる疾患の予防及び治療剤。8. A prophylactic and therapeutic agent for diseases associated with abnormal blood coagulation ability, which comprises the human urine-derived anticoagulant substance according to claim 3 as an active ingredient.
物質を有効成分として含有することを特徴とする、血液
凝固能異常に係わる疾患の予防及び治療剤。9. A preventive and therapeutic agent for diseases associated with abnormal blood coagulation ability, which comprises the human urine-derived anticoagulant substance according to claim 4 as an active ingredient.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1337187A JPH0798840B2 (en) | 1988-12-27 | 1989-12-26 | Urine-derived anti-blood pseudo-substance, method for producing the same, and pharmaceutical composition containing the same |
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33041388 | 1988-12-27 | ||
| JP63-330413 | 1988-12-27 | ||
| JP28524089 | 1989-11-01 | ||
| JP1-285240 | 1989-11-01 | ||
| JP1337187A JPH0798840B2 (en) | 1988-12-27 | 1989-12-26 | Urine-derived anti-blood pseudo-substance, method for producing the same, and pharmaceutical composition containing the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03218399A JPH03218399A (en) | 1991-09-25 |
| JPH0798840B2 true JPH0798840B2 (en) | 1995-10-25 |
Family
ID=27337153
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1337187A Expired - Fee Related JPH0798840B2 (en) | 1988-12-27 | 1989-12-26 | Urine-derived anti-blood pseudo-substance, method for producing the same, and pharmaceutical composition containing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0798840B2 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5834028A (en) * | 1993-12-17 | 1998-11-10 | Mochida Pharmaceutical Co., Ltd. | Soluble thrombomodulin-containing composition |
| MY148565A (en) | 2006-10-06 | 2013-04-30 | Asahi Kasei Pharma Corp | Agent for therapy and/or improvement of disseminated intravascular coagulation |
| US8258269B2 (en) | 2007-03-23 | 2012-09-04 | Asahi Kasei Pharma Corporation | Method for producing high-purity soluble thrombomodulin |
| EP3150628A1 (en) | 2010-04-30 | 2017-04-05 | Asahi Kasei Pharma Corporation | Highly-purified soluble thrombomodulin |
| PT2781221T (en) | 2011-11-15 | 2020-06-01 | Asahi Kasei Pharma Corp | Medicine for treatment and/or improvement of sepsis |
| CN107987157A (en) * | 2017-12-19 | 2018-05-04 | 江苏艾迪药业有限公司 | It is a kind of can industrialized production people source blood clotting regulatory protein preparation method |
| ES2986586T3 (en) | 2018-10-22 | 2024-11-12 | Asahi Kasei Pharma Corp | Drug for the treatment and/or improvement of septicemia associated with coagulation abnormality |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60199819A (en) * | 1984-03-23 | 1985-10-09 | Kowa Co | Thrombin binding substance and preparation thereof |
| JPS63146898A (en) * | 1986-07-15 | 1988-06-18 | Kowa Co | Thrombin-linking substance and production thereof |
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1989
- 1989-12-26 JP JP1337187A patent/JPH0798840B2/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| Ｊ．Ｂｉｏｃｈｅｍ．Ｖｏｌ．１０４（１９８８）Ｐ．６２８−６３２ |
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