JPS647788B2 - - Google Patents
Info
- Publication number
- JPS647788B2 JPS647788B2 JP56213389A JP21338981A JPS647788B2 JP S647788 B2 JPS647788 B2 JP S647788B2 JP 56213389 A JP56213389 A JP 56213389A JP 21338981 A JP21338981 A JP 21338981A JP S647788 B2 JPS647788 B2 JP S647788B2
- Authority
- JP
- Japan
- Prior art keywords
- immobilized
- blood
- activation
- reaction
- antithrombotic
- 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
Links
- 239000000463 material Substances 0.000 claims description 35
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 claims description 17
- 230000002785 anti-thrombosis Effects 0.000 claims description 16
- 239000003146 anticoagulant agent Substances 0.000 claims description 13
- 108010007568 Protamines Proteins 0.000 claims description 6
- 102000007327 Protamines Human genes 0.000 claims description 6
- 229940048914 protamine Drugs 0.000 claims description 6
- 108010033040 Histones Proteins 0.000 claims description 4
- 108010039918 Polylysine Proteins 0.000 claims description 4
- 229920000724 poly(L-arginine) polymer Polymers 0.000 claims description 4
- 108010011110 polyarginine Proteins 0.000 claims description 4
- 229920002704 polyhistidine Polymers 0.000 claims description 4
- 229920000656 polylysine Polymers 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 18
- 230000004913 activation Effects 0.000 description 13
- 230000015271 coagulation Effects 0.000 description 13
- 238000005345 coagulation Methods 0.000 description 13
- 239000008280 blood Substances 0.000 description 11
- 210000004369 blood Anatomy 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 239000008363 phosphate buffer Substances 0.000 description 9
- 239000011780 sodium chloride Substances 0.000 description 9
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 8
- 229920000669 heparin Polymers 0.000 description 8
- 229960002897 heparin Drugs 0.000 description 8
- 239000000499 gel Substances 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- -1 polyethylene Polymers 0.000 description 7
- 108010080865 Factor XII Proteins 0.000 description 5
- 102000000429 Factor XII Human genes 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 210000000056 organ Anatomy 0.000 description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 230000023555 blood coagulation Effects 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 239000004698 Polyethylene Substances 0.000 description 3
- 208000007536 Thrombosis Diseases 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 229920000573 polyethylene Polymers 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 2
- 239000005995 Aluminium silicate Substances 0.000 description 2
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 2
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 2
- 206010053567 Coagulopathies Diseases 0.000 description 2
- 102000009123 Fibrin Human genes 0.000 description 2
- 108010073385 Fibrin Proteins 0.000 description 2
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 108060005987 Kallikrein Proteins 0.000 description 2
- 102000001399 Kallikrein Human genes 0.000 description 2
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 2
- 108090000113 Plasma Kallikrein Proteins 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 108090000190 Thrombin Proteins 0.000 description 2
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 2
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 235000012211 aluminium silicate Nutrition 0.000 description 2
- 239000004019 antithrombin Substances 0.000 description 2
- 239000003114 blood coagulation factor Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000035602 clotting Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 229950003499 fibrin Drugs 0.000 description 2
- 230000003100 immobilizing effect Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 230000031915 positive regulation of coagulation Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 229960004072 thrombin Drugs 0.000 description 2
- 229960005356 urokinase Drugs 0.000 description 2
- 229920002554 vinyl polymer Polymers 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- DHKHKXVYLBGOIT-UHFFFAOYSA-N 1,1-Diethoxyethane Chemical compound CCOC(C)OCC DHKHKXVYLBGOIT-UHFFFAOYSA-N 0.000 description 1
- 229920000178 Acrylic resin Polymers 0.000 description 1
- 239000004925 Acrylic resin Substances 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 244000186140 Asperula odorata Species 0.000 description 1
- 229920000298 Cellophane Polymers 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 229920000219 Ethylene vinyl alcohol Polymers 0.000 description 1
- 235000008526 Galium odoratum Nutrition 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 244000043261 Hevea brasiliensis Species 0.000 description 1
- 108010077861 Kininogens Proteins 0.000 description 1
- 102000010631 Kininogens Human genes 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 229920000954 Polyglycolide Polymers 0.000 description 1
- 239000004642 Polyimide Substances 0.000 description 1
- 229920002367 Polyisobutene Polymers 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 229920001328 Polyvinylidene chloride Polymers 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 239000011354 acetal resin Substances 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000002429 anti-coagulating effect Effects 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 108010085937 benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide Proteins 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 229940019700 blood coagulation factors Drugs 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 229960000956 coumarin Drugs 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000003527 fibrinolytic agent Substances 0.000 description 1
- 230000003480 fibrinolytic effect Effects 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000009931 harmful effect Effects 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229920003052 natural elastomer Polymers 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- 229920001194 natural rubber Polymers 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 229920001084 poly(chloroprene) Polymers 0.000 description 1
- 229920003207 poly(ethylene-2,6-naphthalate) Polymers 0.000 description 1
- 229920002492 poly(sulfone) Polymers 0.000 description 1
- 229920002037 poly(vinyl butyral) polymer Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920002239 polyacrylonitrile Polymers 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 239000011112 polyethylene naphthalate Substances 0.000 description 1
- 229920000139 polyethylene terephthalate Polymers 0.000 description 1
- 239000005020 polyethylene terephthalate Substances 0.000 description 1
- 239000004633 polyglycolic acid Substances 0.000 description 1
- 229920001721 polyimide Polymers 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 229920000306 polymethylpentene Polymers 0.000 description 1
- 239000011116 polymethylpentene Substances 0.000 description 1
- 229920006324 polyoxymethylene Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 1
- 239000004810 polytetrafluoroethylene Substances 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 229920002620 polyvinyl fluoride Polymers 0.000 description 1
- 239000005033 polyvinylidene chloride Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 229920003176 water-insoluble polymer Polymers 0.000 description 1
Description
本発明は抗血栓性材料に関し、詳しくは血液中
の内因性血液凝固因子の活性化を抑えた抗血栓性
材料に関する。
人工臓器やカテーテル等の使用時に生ずる内因
性血液凝固は、血液と異物表面との接触による凝
固系第XII因子の活性化により初期反応が開始さ
れ、凝固系因子のカスケード的活性化の結果、
Xaやトロンビン等が生成され、最終的にはフイ
ブリン網が形成されることにより生じる。
これらの活性化された凝固系因子は、血液中の
阻害物質であるアンチトロンビンによりその作
用が徐々に阻害されるが、ヘパリンの共存により
その阻害効果が著しく高められる、従つて、臨床
的に血液凝固の惹起が懸念される場合には、ヘパ
リンを投与することが常套手段となつている。こ
のため、人工心肺や人工腎臓等の人工臓器を利用
する場合には多量のヘパリンを投与することとな
るが、このような場合、出血傾向が顕著となる等
の多くの副作用を伴ない、これらの副作用は人工
臓器の長期使用により更に著しくなる。
そこで、近時においては、ヘパリンを適宜の水
不溶性高分子担体に固定化し、これに血液を接触
させることにより、ヘパリンを血液中に混入しな
い状態に保持しつつ、これに血液中のトロンビン
等を捕捉せしめて血液の凝固を防止する方法が提
案されているが、ヘパリンのモービリテイや血液
中への脱離等のために満足すべき抗凝血性が発現
されない。
一方、線溶系賦活化酵素であるウロキナーゼを
固定化し、生じたフイブリン網を溶解するように
した抗血栓材料も提案されているが、急激に生じ
るフイブリン塊等には効果が発揮されないままに
血栓が形成される問題がある。
そこで、本発明者らは、従来の抗血栓性材料に
おける上記した種々の問題を解決すべく鋭意研究
した結果、ある種の塩基性タンパク質を固定化す
れば、血液中の内因性凝固因子の活性化を阻害す
ることができ、かくしてすぐれた抗血栓性材料を
得ることができることを見出して、本発明に至つ
たものである。
従つて、本発明による抗血栓性材料は、プロタ
ミン、ヒストン、ポリリジン、ポリアルギニン及
びポリヒスチジンから選ばれる少なくとも一種の
塩基性タンパク質が固定化されていることを特徴
とする。
本発明において塩基性タンパク質が固定化され
る材料は、生理学的に許容し得る材料、即ち生体
に対して実質的に有害に作用せず、且つ、上記塩
基性タンパク質の少なくとも一種が固定化されれ
ばどのような材料でも用いることができるが、通
常、高分子材料が用いられる。このような高分子
重合体材料としては、例えばポリエチレン、ポリ
プロピレン、ポリイソブチレン、ポリメチルペン
テン、ポリスチレン、天然ゴム、クロロプレンゴ
ム、ポリ塩化ビニル、ポリフツ化ビニル、ポリテ
トラフルオロエチレン、ポリ塩化ビニリデン、ポ
リ酢酸ビニル、ポリビニルアルコール、エチレン
−ビニルアルコール共重合体、ポリアクリロニト
リル、ポリグリコール酸、ポリビニルピロリド
ン、ポリビニルホルマール、ポリビニルブチラー
ル、アセタール樹脂、アクリル樹脂、ポリアクリ
ルアミド、ポリカーボネート、ポリスルホン、ポ
リエチレンテレフタレート、ポリエチレンナフタ
レート、ポリアミド、ポリイミド、セルロース、
ニトロセルロース、セロハン、コラーゲン、ゼラ
チン、多糖類等の合成及び天然重合体を例示する
ことができる。
本発明の抗血栓性材料はこのような材料上にプ
ロタミン、ヒストン、ポリリジン、ポリアルギニ
ン及びポリヒスチジンから選ばれる少なくとも一
種の塩基性タンパク質が固定化されて形成されて
いる。材料への塩基性タンパク質の固定化の方法
は何ら制限されず、従来より知られているタンパ
ク質固定化方法を任意に用いることができ、例え
ば、共有結合法、イオン結合法等の適宜の方法が
用いられる。例えばカルボジイミド試薬或いはウ
ツドワード試薬と共に上記タンパク質を材料に反
応させれば容易に共有結合にて固定化され、ま
た、上記タンパク質の水溶液又は懸濁液に材料を
浸漬すればイオン結合にて固定化される。
材料に固定化される塩基性タンパク質の量につ
いては特に制限はないが、好ましくは材料の表面
の大部分乃至全部を被覆するように固定化され
る。また、本発明においては、材料には上記塩基
性タンパク質と共に他の抗凝血物質、例えばヘパ
リン、アンチトロンビンやウロキナーゼ等が固
定化されていてもよい。
一般に高分子材料と血液との接触によつて惹起
される血栓形成の一つの重要な原因は、高分子材
料表面における凝固系第XII因子の活性化である。
この第XII因子の詳細な活性化機序は未だ十分にな
されていないが、第XII因子の活性化にはプレカリ
クレインと高分子キニノーゲンの存在が必要であ
り、これら三者が材料の固相表面上である種の複
合体を形成して活性化反応に関与すると考えられ
ている。本発明による抗血栓材料は、何ら理論に
より制約を受けるものではないが、材料に固定化
された塩基性タンパク質が材料表面への上記三つ
の因子の少なくとも一つの付着を妨げて、活性化
反応に関与する複合体の形成を阻止することによ
つて、抗血栓性を発現するのであろう。
尚、塩基性タンパク質の代わりに塩基性アミノ
酸を担体に固定化しても、抗血栓性は発現され
ず、高分子量の塩基性タンパク質を固定化して初
めて抗血栓性が発現される。
本発明による抗血栓性材料は、使用目的に応じ
て、担体を予め所要形状に形成しておけば、粒子
状、シート状、管状等のいずれの形態でも調製で
きる。
本発明による抗血栓性材料は以上のように材料
表面に塩基性タンパク質が固定化されており、こ
の塩基性タンパク質が材料表面での血栓形成を未
然に阻止するので、血行回路、反応器等の直接血
液と接触する人工臓器に使用すれば、血液凝固系
の活性化に伴う血栓形成を効果的に阻止すること
ができる。
以下に実施例により本発明を説明するが、本発
明はこれら実施例により何ら制限されるものでは
ない。
実施例 1
(1) 固定化
材料としてアガロースゲル(CM−セフアロ
ーズCL−6B、Phamacia Fine Chemicals社)
を用い、この材料上にメチルアミン、グリシ
ン、リジン、アルギニン、ヒスチジン、アルブ
ミン、プロタミン、ヒストン、ポリリジン(分
子量約10000)、ポリアルギニン(分子量約
60000)又はポリヒスチジン(分子量約15000)
を固定化した。
固定化は次のようにして行なつた。即ち、材
料を2.0M塩化カリウム水溶液に懸濁、洗浄後、
蒸留水(PH4.5)で洗浄し、蒸留水(PH4.5)に
5g/50ml量で懸濁した。この懸濁液にN−エ
チル−N′−(3−ジメチルアミノプロピル)カ
ルボジイミド塩酸塩(EDC)の0.2M溶液(PH
4.5)50mlを混合し、更に上記アミノ酸又は塩
基性タンパク質10mg乃至1gを添加し、PHを
4.5に調整しつつ、室温で約4時間反応させた。
反応後、0.1M酢酸緩衝液(PH4.0)と0.1M
NaHCO3緩衝液(0.5M NaCl、PH8.3)で交互
に2回ずつ洗浄し、更に10mMリン酸緩衝液
(0.145M NaCl、PH7.4)にて洗浄し、この緩衝
液中に懸濁させて、4℃の温度で使用まで保存
した。
(2) 試験
(a) 上で得た各固定化ゲル10mg(乾燥重量)
と、0.2重量%のカオリンとを含有する10m
Mトリス−塩酸緩衝液(0.145M NaCl、PH
7.4)100μと、家兎クエン酸処理血漿100μ
に合成基質Carbobenzoxy−phenyl−
arginyl−4−methylco−umaryl−7−
amide(z−Phe−Arg−AMC)を加えた上
記と同じトリス−塩酸緩衝液1.0mlとをポリ
エチレン試験管に入れ、37℃の温度で45分間
反応させた後、17重量%酢酸水溶液2.0mlを
加えて反応を停止させ、螢光分光光度計(励
起波長380nm、反射波長460nm)にて遊離
したAmino−me−thyl−Coumarin(AMC)
の濃度を測定した。結果を第1表に示す。
(b) 上で得た各固定化ゲル20mg(乾燥重量)を
含有する50mMリン酸緩衝液(PH7.4)200μ
と、家兎クエン酸処理血漿100μと、上
記合成基質を含有する上記と同じリン酸緩衝
液1.0mlとをポリエチレン試験管に入れ、37
℃の温度で60分間反応させた後、17重量%酢
酸水溶液2.0mlを加えて反応を停止させ、(a)
と同様にして遊離AMC量を測定した。結果
を第1表に示す。
尚、本試験で用いた合成基質は凝固系第XII
因子に対するものではなく、カリクレインに
対するものであるが、第XII因子の活性化に伴
つてプレカリクレインも活性化されてカリク
レインとなることが知られており、従つて、
遊離AMC量によつて凝固系の活性化の程度
を知ることが
The present invention relates to an antithrombotic material, and more particularly to an antithrombotic material that suppresses the activation of endogenous blood coagulation factors in blood. Intrinsic blood coagulation that occurs when using artificial organs or catheters, etc., the initial reaction is initiated by the activation of coagulation system factor
It is caused by the generation of Xa, thrombin, etc., and ultimately the formation of a fibrin network. The action of these activated coagulation system factors is gradually inhibited by the inhibitory substance antithrombin in the blood, but the coexistence of heparin significantly enhances the inhibitory effect. When there is concern about the induction of coagulation, it is common practice to administer heparin. For this reason, when using artificial organs such as a heart-lung machine or an artificial kidney, large amounts of heparin must be administered; The side effects become more pronounced with long-term use of artificial organs. Therefore, in recent years, heparin has been immobilized on a suitable water-insoluble polymer carrier and blood is brought into contact with the carrier to keep heparin from being mixed into the blood while also removing thrombin, etc. in the blood. Although methods have been proposed for trapping heparin to prevent blood coagulation, satisfactory anticoagulant properties are not achieved due to heparin's mobility and desorption into the blood. On the other hand, an antithrombotic material has been proposed in which urokinase, a fibrinolytic system activating enzyme, is immobilized to dissolve the formed fibrin network. There are problems that form. Therefore, the present inventors conducted intensive research to solve the above-mentioned problems with conventional antithrombotic materials. As a result, the inventors found that if a certain type of basic protein is immobilized, the activity of endogenous coagulation factors in the blood can be improved. The present invention was achieved based on the discovery that it is possible to obtain an excellent antithrombotic material. Therefore, the antithrombotic material according to the present invention is characterized in that at least one basic protein selected from protamine, histone, polylysine, polyarginine, and polyhistidine is immobilized thereon. In the present invention, the material on which the basic protein is immobilized is a physiologically acceptable material, that is, a material that does not have a substantially harmful effect on living organisms, and on which at least one of the above basic proteins is immobilized. Although any material can be used, polymeric materials are usually used. Examples of such polymer materials include polyethylene, polypropylene, polyisobutylene, polymethylpentene, polystyrene, natural rubber, chloroprene rubber, polyvinyl chloride, polyvinyl fluoride, polytetrafluoroethylene, polyvinylidene chloride, and polyacetic acid. Vinyl, polyvinyl alcohol, ethylene-vinyl alcohol copolymer, polyacrylonitrile, polyglycolic acid, polyvinylpyrrolidone, polyvinyl formal, polyvinyl butyral, acetal resin, acrylic resin, polyacrylamide, polycarbonate, polysulfone, polyethylene terephthalate, polyethylene naphthalate, polyamide , polyimide, cellulose,
Examples include synthetic and natural polymers such as nitrocellulose, cellophane, collagen, gelatin, and polysaccharides. The antithrombotic material of the present invention is formed by immobilizing at least one basic protein selected from protamine, histone, polylysine, polyarginine, and polyhistidine on such a material. The method of immobilizing the basic protein onto the material is not limited in any way, and any conventionally known protein immobilization method can be used. For example, an appropriate method such as a covalent bond method or an ionic bond method may be used. used. For example, if the above-mentioned protein is reacted with a material together with a carbodiimide reagent or Woodward reagent, it will be easily immobilized by covalent bonds, and if the material is immersed in an aqueous solution or suspension of the above-mentioned protein, it will be immobilized by ionic bonds. . Although there is no particular restriction on the amount of basic protein immobilized on the material, it is preferably immobilized so as to cover most or all of the surface of the material. Further, in the present invention, other anticoagulants such as heparin, antithrombin, urokinase, etc. may be immobilized on the material in addition to the above-mentioned basic protein. One important cause of thrombus formation, which is generally caused by contact between a polymeric material and blood, is activation of coagulation system factor XII on the surface of the polymeric material.
Although the detailed activation mechanism of factor XII has not yet been fully elucidated, activation of factor It is thought that the above-mentioned complex forms a certain type of complex and participates in the activation reaction. Although the antithrombotic material according to the present invention is not limited by any theory, it is believed that the basic protein immobilized on the material prevents at least one of the above three factors from adhering to the material surface and prevents the activation reaction. It may exert its antithrombotic properties by blocking the formation of the involved complexes. Incidentally, even if a basic amino acid is immobilized on a carrier instead of a basic protein, antithrombotic properties are not expressed, and antithrombotic properties are not expressed until a high molecular weight basic protein is immobilized. The antithrombotic material according to the present invention can be prepared in any form, such as particulate, sheet, or tubular, by forming the carrier into a desired shape in advance, depending on the purpose of use. As described above, the antithrombotic material according to the present invention has a basic protein immobilized on the material surface, and this basic protein prevents thrombus formation on the material surface, so it can be used in blood circulation circuits, reactors, etc. When used in artificial organs that come into direct contact with blood, it can effectively prevent thrombus formation due to activation of the blood coagulation system. The present invention will be explained below with reference to Examples, but the present invention is not limited to these Examples in any way. Example 1 (1) Immobilization Agarose gel (CM-Sepharose CL-6B, Pharmacia Fine Chemicals) was used as the material.
methylamine, glycine, lysine, arginine, histidine, albumin, protamine, histone, polylysine (molecular weight approximately 10,000), polyarginine (molecular weight approximately
60,000) or polyhistidine (molecular weight approximately 15,000)
was fixed. Immobilization was performed as follows. That is, after suspending the material in a 2.0M potassium chloride aqueous solution and washing it,
It was washed with distilled water (PH4.5) and suspended in distilled water (PH4.5) in an amount of 5 g/50 ml. A 0.2M solution of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (PH
4.5) Mix 50ml, add 10mg to 1g of the above amino acid or basic protein, and adjust the pH.
While adjusting the temperature to 4.5, the reaction was carried out at room temperature for about 4 hours. After reaction, add 0.1M acetate buffer (PH4.0) and 0.1M
Wash alternately twice with NaHCO3 buffer (0.5M NaCl, PH8.3), further wash with 10mM phosphate buffer (0.145M NaCl, PH7.4), and suspend in this buffer. and stored at a temperature of 4°C until use. (2) Test (a) 10 mg of each immobilized gel obtained above (dry weight)
and 0.2% by weight of kaolin.
M Tris-HCl buffer (0.145M NaCl, PH
7.4) 100μ and 100μ of rabbit citrate-treated plasma
Synthetic substrate Carbobenzoxy-phenyl-
arginyl-4-methylco-umaryl-7-
amide (z-Phe-Arg-AMC) was added to 1.0 ml of the same Tris-HCl buffer as above in a polyethylene test tube, and after reacting for 45 minutes at a temperature of 37°C, 2.0 ml of a 17 wt% acetic acid aqueous solution was added. was added to stop the reaction, and Amino-me-thyl-Coumarin (AMC) was released using a fluorescence spectrophotometer (excitation wavelength 380 nm, reflection wavelength 460 nm).
The concentration of was measured. The results are shown in Table 1. (b) 200μ of 50mM phosphate buffer (PH7.4) containing 20mg (dry weight) of each immobilized gel obtained above.
, 100μ of rabbit citrate-treated plasma, and 1.0ml of the same phosphate buffer solution as above containing the above synthetic substrate were placed in a polyethylene test tube.
After reacting for 60 minutes at a temperature of °C, the reaction was stopped by adding 2.0 ml of a 17 wt% acetic acid aqueous solution, and (a)
The amount of free AMC was measured in the same manner as above. The results are shown in Table 1. In addition, the synthetic substrate used in this test is coagulation system XII.
Although it is not for factor XII but for kallikrein, it is known that prekallikrein is also activated and becomes kallikrein with the activation of factor XII, and therefore,
The degree of activation of the coagulation system can be determined by the amount of free AMC.
【表】
できる(三浦喜温ら、人工臓器、9、907
(1980))。
第1表から明らかなように、試験(a)におい
ては凝固系の活性化を顕著にするカオリンが
存在するにもかかわらず、本発明に従つて塩
基性タンパク質又は塩基性アミノ酸ポリマー
が固定化されている材料によれば、凝固系の
活性化が著しく抑制されている。また、試験
(b)においてはアガロースゲルにより凝固系の
活性化が惹起されやすいように系のイオン強
度を低くしたが、同様に本発明の固定化材料
によれば凝固系の活性化が著しく抑えられて
いる。この結果と対照的に、塩基性アミノ酸
モノマーの場合には凝固系の活性化を抑える
効果は全く認められない。
(c) 固定化ゲルの抗血栓性を凝固時間法により
評価した。
10mMリン酸緩衝液(0.145M NaCl、PH
7.4)に各固定化ゲル6mgを含有する懸濁液
100μ、リン脂質を10mg/100ml濃度で含有
する25mMCa2+溶液100μ、及びプラズマ
(クエン酸処理家兎全血を1500Gで15分間遠
心分離して得られる上澄液)100μをシリ
コン処理試[Table] Possible (Yoshion Miura et al., Artificial Organs, 9 , 907
(1980)). As is clear from Table 1, in test (a), despite the presence of kaolin, which significantly activates the coagulation system, basic proteins or basic amino acid polymers were not immobilized according to the present invention. According to the materials used, activation of the coagulation system is significantly suppressed. Also, exam
In (b), the ionic strength of the system was lowered so that activation of the coagulation system was easily induced by agarose gel, but similarly, activation of the coagulation system was significantly suppressed by the immobilization material of the present invention. . In contrast to this result, in the case of basic amino acid monomers, no effect on suppressing activation of the coagulation system was observed at all. (c) The antithrombotic properties of the immobilized gel were evaluated by clotting time method. 10mM phosphate buffer (0.145M NaCl, PH
7.4) Suspension containing 6 mg of each immobilized gel
100μ, 100μ of a 25mMCa 2+ solution containing phospholipids at a concentration of 10mg/100ml, and 100μ of plasma (supernatant obtained by centrifuging citrated rabbit whole blood at 1500G for 15 minutes) were added to the silicone treatment test.
【表】
験管に入れ、室温で緩やかに振とうしながら
反応させて凝固時間を測定した。結果を第2
表に示す。塩基性タンパク質及び塩基性アミ
ノ酸ポリマーの固定化された材料のみが血液
凝固を阻害することが明らかである。
参考例
本発明の抗血栓性材料は、材料に固定化された
塩基性タンパク質に凝固系第XII因子、プレカリク
レイン又は高分子キニノーゲンの少なくとも一種
が結合し、これによつて凝固系の活性化が阻害さ
れると考えられるので、この点を明らかにした。
プラズマ(クエン酸処理した家兎全血を1500G
で15分間遠心分離して得られる上澄液)400μ
と、10mMリン酸緩衝液(0.145M NaCl、PH7.4)
に固定化プロタミンゲルを6mg(乾燥重量)/
100μの量で懸濁せた懸濁液400μと、10mM
リン酸緩衝液(0.145M NaCl、PH4.5)400μと
をポリスチレン試験管に入れ、室温で10分間振と
うした後、700Gで5分間遠心分離して上澄液○イ
を得た。次に、上記遠心分離後の固定化プロタミ
ンゲルに2.0M塩化ナトリウム1.0mlを加え、室温
で10分間振とうした後、700Gで5分間遠心分離
して上澄液○ロを得た。
上記上澄液○イ及び○ロを用い、次の反応系1〜3
を調製した。
反応系 1
上澄液○イ200μ、10mMリン酸緩衝液(PH7.4)
550μ及び1.0M NaCl250μ
反応系 2
上澄液○ロ200μ、10mMリン酸緩衝液(PH7.4)
750μ及び1.0M NaCl50μ
反応系 3
上澄液○イ200μ、上澄液○ロ200μ及び10mM
リン酸緩衝液(PH7.4)600μ
これらの各反応系に0.2mMの合成基質z−Phe
−Arg−MCAを含有する10mMリン酸緩衝液
(PH7.4)1.0mlを加え、表面で凝固系の活性化が
顕著に起こるガラス管内で37℃の温度で30分間反
応させた後、17重量%酢酸3.0mlを加えて反応を
停止させた。この後、生成AMC量を前記と同様
にして測定した。結果を第3表に示す。
以上の結果から、プロタミン固定化ゲルにより
第XII因子、プレカリクレイン又は高分子キニノー
ゲンの少なくとも一種が除かれたプラズマ上澄液
(反応系1)と、プロタミン固定化ゲルから2.0M
NaClによつて遊離された因子(反応系2)とは、
それらのみによつては凝固系を活性化しないが、
反応系1及び2を共存させることにより上記三因
子をすべて共存させた場合に凝固系が活性化され
た(反応系3)。[Table] The sample was placed in a test tube and allowed to react with gentle shaking at room temperature, and the clotting time was measured. Second result
Shown in the table. It is clear that only immobilized materials of basic proteins and basic amino acid polymers inhibit blood coagulation. Reference Example In the antithrombotic material of the present invention, at least one of coagulation system factor This point has been clarified because it is thought that this may be inhibited. Plasma (1500G of citric acid-treated rabbit whole blood)
Supernatant obtained by centrifugation for 15 minutes) 400μ
and 10mM phosphate buffer (0.145M NaCl, PH7.4)
6 mg (dry weight) of immobilized protamine gel/
Suspension 400μ in an amount of 100μ and 10mM
400μ of phosphate buffer (0.145M NaCl, PH4.5) was placed in a polystyrene test tube, shaken at room temperature for 10 minutes, and then centrifuged at 700G for 5 minutes to obtain a supernatant. Next, 1.0 ml of 2.0 M sodium chloride was added to the immobilized protamine gel after centrifugation, and after shaking at room temperature for 10 minutes, centrifugation was performed at 700 G for 5 minutes to obtain a supernatant liquid. Using the above supernatant liquids ○A and ○B, perform the following reaction systems 1 to 3.
was prepared. Reaction system 1 Supernatant liquid 200μ, 10mM phosphate buffer (PH7.4)
550μ and 1.0M NaCl 250μ Reaction system 2 Supernatant ○○ 200μ, 10mM phosphate buffer (PH7.4)
750μ and 1.0M NaCl50μ Reaction system 3 Supernatant ○I 200μ, supernatant ○Ro 200μ and 10mM
Phosphate buffer (PH7.4) 600μ Add 0.2mM synthetic substrate z-Phe to each of these reaction systems.
- Add 1.0 ml of 10 mM phosphate buffer (PH7.4) containing -Arg-MCA and react for 30 minutes at a temperature of 37 °C in a glass tube where the activation of the coagulation system occurs on the surface. The reaction was stopped by adding 3.0 ml of % acetic acid. Thereafter, the amount of AMC produced was measured in the same manner as above. The results are shown in Table 3. From the above results, we found that the plasma supernatant (reaction system 1) from which at least one of factor XII, prekallikrein, or polymeric kininogen was removed by the protamine-immobilized gel, and
The factor released by NaCl (reaction system 2) is
Although they alone do not activate the coagulation system,
By coexisting reaction systems 1 and 2, the coagulation system was activated when all three factors were allowed to coexist (reaction system 3).
Claims (1)
ルギニン及びポリヒスチジンから選ばれる少なく
とも一種の塩基性タンパク質が固定化されている
ことを特徴とする抗血栓性材料。1. An antithrombotic material characterized in that at least one basic protein selected from protamine, histone, polylysine, polyarginine, and polyhistidine is immobilized.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP56213389A JPS58118763A (en) | 1981-12-30 | 1981-12-30 | Anti-thrombotic material |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP56213389A JPS58118763A (en) | 1981-12-30 | 1981-12-30 | Anti-thrombotic material |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS58118763A JPS58118763A (en) | 1983-07-14 |
JPS647788B2 true JPS647788B2 (en) | 1989-02-10 |
Family
ID=16638386
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP56213389A Granted JPS58118763A (en) | 1981-12-30 | 1981-12-30 | Anti-thrombotic material |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS58118763A (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5114413A (en) * | 1990-06-08 | 1992-05-19 | Wisconsin Alumni Research Foundation | Methods to make and use proteinaceous material present in kinin-free high molecular weight kininogen |
US5188618A (en) * | 1991-05-03 | 1993-02-23 | Thomas Bruce W | Thrombus-mobilizing thoracostomy tube |
FR2679772B1 (en) * | 1991-08-02 | 1995-05-19 | Peters Sa | EMBOLS IN NON-RESORBABLE PARTICLES COATED WITH HEMOSTATIC MATERIAL. |
US7169404B2 (en) | 2003-07-30 | 2007-01-30 | Advanced Cardiovasular Systems, Inc. | Biologically absorbable coatings for implantable devices and methods for fabricating the same |
US8916188B2 (en) | 2008-04-18 | 2014-12-23 | Abbott Cardiovascular Systems Inc. | Block copolymer comprising at least one polyester block and a poly (ethylene glycol) block |
-
1981
- 1981-12-30 JP JP56213389A patent/JPS58118763A/en active Granted
Also Published As
Publication number | Publication date |
---|---|
JPS58118763A (en) | 1983-07-14 |
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