抗血小板血栓剤の開発を目的とした血小板粘着反応の分子機作の解析

• Project/Area Number: 03670505
• Research Category: Grant-in-Aid for General Scientific Research (C)
• Allocation Type: Single-year Grants
• Research Field: Pediatrics
• Research Institution: Nara Medical University
• Principal Investigator: 藤村 〓博 (藤村 吉博) 奈良県立医科大学, 医学部, 助教授 (80118033)
• Project Period (FY): 1991 – 1993
• Project Status: Completed (Fiscal Year 1993)
• Budget Amount: ¥1,900,000 (Direct Cost: ¥1,900,000); Fiscal Year 1993: ¥600,000 (Direct Cost: ¥600,000); Fiscal Year 1992: ¥400,000 (Direct Cost: ¥400,000); Fiscal Year 1991: ¥900,000 (Direct Cost: ¥900,000)
• Keywords: botrocetin / von Willebrand factor / GPIb / GPII b / IIIa / dispase / bothrombin / シアール酸 / SS結合 / フオン ビルブランド因子 / GPIIb / リストセチン / ボトロセチン / ボトロンビン / 血小板粘着反応 / フォンビルブランド因子 / 抗GPIbモノクロナ-ル抗体

Research Abstract

1)Botrocetinの純化,モノクロナール抗体の作成とそのエピトープ解析:既に確立した方法(Biochemistry 30:1957-1964,1991)で数+mg単位のbotrocetin(von Willebrand因子に結合し,血小板凝集活性を示す蛇毒蛋白)を仰化し,ひき続き抗botrocetinモノクロナール抗体を作成したところ,BCI-7と名づけた抗体がこのvWF-botrocetin結合反応を特異的に阻害すること,この抗体のエピトープはbotrocetinのサブトライシン消化物の25KDdフラグメント上にあること。またBCI-7はヒト胎盤抽出液中の130KDd,〜30KDd,15kDdの三つのbandと交叉反応を当する事を確認し,現在これら蛋白の仰化・精製中である。
2)Bothrombinの純化・機能解析:Bothrops jararaca粗毒よりbothrombinと名づけたfibrinogen凝固蛋白を精製した。Bothrombinはヒトthrombinとは異なり,血小板を直接凝集せしめることはないが,外国製fibrinogen存在下にヒト血小板を凝集せしめる事,またこの凝集はPGE_1,EDTA,またGPII b/IIIaモノクロナール抗体CP-8で阻害され,さらにthrombinのGPIbへの結合を阻害するまえGPIbモノクロナール抗体LJ-Ib10で阻害される事より,bothrombinは先ずGPIbに結合し,更にGPII b/IIIaを活性化せしめるsignal transductorとしての作用を有する事を明らかにした(Biochemistry 33:1843-1849,1993)。
3)39/34・KDd-vWF dispase fragmentにおけるCys^<509>-Cys^<695>と糖鎖〓役割: GPIb結合ドメイン機能を有する最小限のvWF dispase fragment(Subunitのアミノ酸残基480/481-781)に還え・アルキル化処理あるいはノイラミニダーゼ処理(脱シアル酸化)を加え,^<125>I-vWFとGPIbの結合ならびに高ズリ応力下の血小板凝集がどのような作用を受けるかを検討し,39/34kDa-fragment内のCys^<509>-Cys^<695>のSS結合の存在とO-linked糖鎖の存在はvWF-GPIb結合反応を阻害する方向(downregulate)する事を発見した(Miura et al.Blood 投稿中.1994)。

Research Products

• [Publications] Y.Fujimura et al: "The interaction of botrocetin with normal or variant von willebrand factor and its inhibition by monoclonal antibodies that block receptor binding" Thromb Haemostasis. 68. 464-469 (1992)
• [Publications] S.Goto et al: "Epinephrine augments von Willebrand factor dependent shear-induced platelet aggregation" Circulation. 86. 1859-1863 (1992)
• [Publications] Y.Usami et al: "Primary structure of two-chain botrocetin; a von Willebrand factor modulator purified from the venom of Bothrops jararaca" Proc Nat1 Acad Sci USA. 90. 928-932 (1993)
• [Publications] T.Matsui et al: "Human plasma a2-macroglobulin and von Willebrand factor possess covalently-linked ABO(H) blood group antigens in subjects with corresponding ABO phenotype" Blood. 82. 663-668 (1993)
• [Publications] E.Yoshida et al: "Alboaggregin-B and botrocetin, two snake venom proteins,with highly homologous amino acid sequences but totally distinct functions on von Willebrand factor binding to platelets" Biochem Biophys Res Communication. 191. 1386-1392 (1993)
• [Publications] S.Nishida et al: "Purification and characterization of bothrombin, a fibrinogen-clotting serine protease from the venom of Bothrops jararaca" Biochemistry. 33. 1843-1849 (1994)
• [Publications] 藤村吉〓・千谷晃一: "von Willebrand因子とその受容体" 金芳堂, 16 (1992)
• [Publications] Y.Fujimura and K.Titani: "Structure and function of von Willebrand factor in HEMOSTASIS AND THROMBOSIS" Churchill-Livingstone(London), 16 (1993)
• [Publications] Eri Yoshida et al.: "Alboaggregin B and Botrocetin,two snake venom proteins with highly homologous amino acid sequences but totallydistinct functions on von Willebrand factor binding to platelet" Biochemica Biophysics Reseurch Communication. (1993)
• [Publications] Taei Matsui et al.: "Human plasma α2-macroglobulin and uon Willebrand facter possess conalently-linked ABO(H)blood group antigens in subjects with corresponding ABO phenotypes" Blood. (1993)
• [Publications] Yoshiko Usami et al.: "Primary structure of two-chain botrocetir,a uon Willebrand factor modulator parified from the venon of Bothrops Jararac" Proceedings of National Acadeniy Science,USA. 90. 928-932 (1993)
• [Publications] Shinya Goto et al.: "Epinephrine augments von Willebrand facter dependent Shen-induced platelet aggregation" Circulation. 86. 1859-1863 (1992)
• [Publications] Yoshihiro Fujimura et al.: "The interaction of botrocetin with normal or variant von Willebrand factor (types IIA and IIB)and its inhibition by monoclonal antibodies that block receptor binding" Thrombosis and Haemostasis. 68. 464-469 (1992)
• [Publications] Kenji Nishio et al.: "Anti-platelet glycoprotein Ib monoclonal anti body (OP-F1) tolally abolisaes ristocetin-induced von Willebrand facter binding,but has mininal effect on the botrocetir-induced binding" Haemostasis. 21. 353-359 (1991)
• [Publications] Yoshihiro Fujimura and Koiti Titani: "Structure and function of von Willebrand factor-Hemostasir and Thronbosis(ed by A.L.Bloom et al)" Churchill-Livington(London), in press (1993)
• [Publications] 藤村 吉博,千谷 晃一: "von Willebrand 因子とその受容体" 宇宙堂 八木書店, 10 (1993)
• [Publications] K.Nishie,Y.Fujimura,S.Nishida,I.Takeda,A.Yoshioka,H.Fukui,Y.Tomiyama,Y.Kurata: "Antiplatelet glycoprotein Ib Monoclonal antibody (OpーF1) tolally abolisks listcetinーinduced von Willebrand factor binding,but has minimal effect on the botrocetinーinduced binding." Hemostasis. (1992)
• [Publications] Y.Fujimura,S.Miyata,S.Nishida,S.Miura,M.Kaneda,A.Yoshioka,H.Fukui,M.Katayama,E.G.D.Tuddenham,Y.Usami,K.Titani: "The interaction of botrocetin with nomal or variant von Willebrand factor (types IIA and IIB) and its inhibition by monoclonal antibodies that block receptor binding" Thronbosis and Haemostasis. (1992)
• [Publications] Y.Fujimura,K.Titani: "Structure and function of von Willebrand factor:Haemostasis and Thnombosis 3rd Editor" Chunchile Livingstone (London) Editors:A.L.Bloom,C.D.Foles,D.P.Thomas,E.G.D.Tuddenham, (1992)