| Co-Investigator(Kenkyū-buntansha) |
SHIMADA Hideaki University of Kumamoto, Educational Science, Associate Professor, 薬学部, 助手 (40226212)
TANAKA Haruhito , 教授
MIZOKAMI Hiroshi The Chemo-Sero-Therapeutic Research Institute, Department Head, 試作研究部, 部長
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| Research Abstract |
This research was performed to develop the effective antithrombus drag using the anticoagulant and antithrombus function of annexin-V and the peptides derived from the functional site of annexin-V.
1. Preparation of recombinant annexin-V : Complementary DNA (cDNA) encorded human placental annexin-V was inserterted to plasmid vector (pUC18) and cloning using yeast in the culture medium. The obtained recombinant annexin-V was separated from the culture medium by the method of (NH_4)_2SO_4 fractionation, DEAE-Sepharose chromatography, Gel Filtration on Sephadex G-75, high performance liquid chromatography (HPLC). This recombinant annexin-V obtained showed the same properties as an anticoagulant and identical amino acid sequence.
2. Chemically synthesis the peptides derived from the functional site of annexin-V : Histidine-containing peptides, SHLRKV and DHTLIR, corresponding to annexin-V residues 204-209 and 266-271, were included in the functional site of annexin-V and exhibited anticoagul
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ant activity. The similar anticoagulant peptides, HDTLIR, HDTQPRVLD, and terminal blocking peptides, Ac-HDTQPRVLD-NH_2, Ac-hDTQPRVLd-NH_2, and cyclic peptide Ac-cHDTQPRVLDc-NH_2 were synthesized and tested the anticoagulant activity. The terminal blocking peptides and cyclized peptide lost the anticoagulant activity.
3. Anticoagulant and antithrombus functions of enclosed annexin-V and anticoagulant peptides in liposomes : Annexin-V and anticoagulant peptides enclosed into liposomes resulted in the decreased the strength of the anticoagulant and antithrombus functions.
4. Antithrombus function : Pulmonary embolism model in mice were made with tissue factor injection from tail vein. Annexin-V and these anticoagulant peptides protected the tissue factor-induced pulmonary embolism in mice.
5. Side effect and Toxicity : Annexin-V and these anticoagulant peptides did not shows the side effect and toxicity to mouse, rat, ginea pig and rabbit by the infra-vascular injection.
6. Application to antithrombus drug : The strength of antithrombus functions of annexin-V and these peptides were in turn of Annexin-V>HDTQPRVLD>HDTLIR>DHTLIR>SHTLIR>liposome enclosure. This recombinant annexin-V seems to be most useful and safety antithrombus drug, and the other anticoagulant peptides were also seems to be useful and safety drags. Less
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