| Co-Investigator(Kenkyū-buntansha) |
FUJIMURA Yoshihiro Nara Medical College, Assistant Professor, 輸血部, 助教授 (80118033)
MATSUI Taei Fujita Health Univ. School of Medicine, Instructor, 医学部, 助手 (90183946)
OYAMA Fumitaka Fujita Health Univ. School of Medicine, Instructor, 医学部, 助手 (40194641)
SEKIGUCHI Kiyotoshi Fujita Health Univ. School of Medicine, Lecturer, 医学部, 講師 (50187845)
MIZUOCHI Tsuguo Fujita Health Univ. School of Medicine, Assistant Professor, 医学部, 助教授 (90133149)
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| Budget Amount *help |
¥25,100,000 (Direct Cost: ¥25,100,000)
Fiscal Year 1989: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1988: ¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 1987: ¥19,200,000 (Direct Cost: ¥19,200,000)
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| Research Abstract |
We have undertaken and accomplished the following research projects: 1) Modification of the purification procedure of human von Willebrand factor (vWF): We established a new purification procedure of human vWF using an immunoaffinity column with anti-vWF monoclonal antibody by which human vWF can be isolated more rapidly and in better yield than the previous procedures. 2) Determination of sugar chain structures of human vWF: 12 N-linked sugar chains of human vWF were released by hydrazinolysis, tritium-labeled and analyzed using various lectin columns and exoglycosidases. In addition to complex type bi-and tetra-antennary sugar chains previously reported, we discovered complex type tri-antennary, mixed type and bisecting glucosamine containing sugar chains. Recent results also showed that about 10 % of the sugar chains contains alpha- fucose linked to terminal galactose. 3) Identification of functional domains in human vWF: human-vWF binds platelet glycoprotein (GP) Ib, IIb/IIIa, coll
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agen, heparin, and coagulation factor VIII. We verified that a 52/48 kDa fragment (residues 449-728), obtained by trypsin and identified to bind GP Ib and collagen, also strongly binds heparin. In addition, we found that a 30 kDa fragment (residues 1-272), obtained also by trypsin, strongly binds factor VIII. 4) Epitope of anti-vWF monoclonal antibody NMC-4: using an immunoaffinity column with NMC-4 which represses ristocetin-induced platelet aggregation, we isolated a 97 kDa fragment from a tryptic digest of human vWF. The fragment was identified to be a homodimer of 52/48 kDa fragment (residues 449-728) containing GP Ib and collagen,binding sites. By western blotting, NMC-4 recognized also the monomeric form, but did not recognize the S- carboxymethylated fragment. 5) Purification and characterization of botrocetin: we isolated two kinds of botroceetin, the platelet coaggulutinin, from a snake venom (Bothrops jararaca) and verified their various properties. In addition, we obtained interesting results regarding differences in reaction mechanism between botrocetin and ristocetin. 6) Grafting of platelet binding site of vWF into other proteins: using protein engineering techniques, we inserted tetrapeptide Arg-Gly-Asp-Ser, the platelet GP IIb/IIIa binding site of vWF, into Protein A, a bacterial IgG binding protein, and obtained an artificial cell adhesive protein. Less
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