| Project/Area Number |
10557092
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 展開研究 |
|---|
| Research Field |
Hematology
|
|---|
| Research Institution | Kyushu University of Nursing and Social Welfare |
|---|
Principal Investigator |
FUNAKOSHI Takaynki Kyushu University of Nursing and Social Welfare, Professor, 看護福祉学部, 教授 (90150549)
|
|---|
| 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
|
|---|
| Project Period (FY) |
1998 – 2000
|
| Project Status |
Completed (Fiscal Year 2000)
|
| Budget Amount *help |
¥9,400,000 (Direct Cost: ¥9,400,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1999: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1998: ¥7,100,000 (Direct Cost: ¥7,100,000)
|
|---|
| Keywords | Annexin-V / Anticoagulant Peptide / Liposome Enclosure / Ca^<2+> Dependent Membran Binding Protein / Functional Site of Antcoagulation / Antithrombus Function |
|---|
| 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
… More
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
|
