| Project/Area Number |
15590198
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General physiology
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| Research Institution | Kinki University |
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Principal Investigator |
MATSUO Osamu Kinki University, School of Medicine Department of Physiology, Professor, 医学部, 教授 (40030879)
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| Co-Investigator(Kenkyū-buntansha) |
UESHIMA Shigeru Kinki University, Department of Agriculture, Professor, 農学部, 教授 (30193791)
OKADA Kiyotaka Kinki University, School of Medicine Department of Physiology, Lecturer, 医学部, 講師 (20185432)
KAWAO Naoyuki Kinki University, School of Medicine Department of Physiology, Assistant, 医学部, 助手 (70388510)
OKAMOTO Chikako Kinki University, School of Medicine Department of Physiology, Assistant, 医学部, 助手 (90368291)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | fibrinolytic system / vascular endothelial cell / tissue type plasminogen activator (t-PA) / t-PA receptor / 遺伝子組換え |
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| Research Abstract |
We demonstrated previously that tissue-type plasminogen activator (t-PA) bound to its specific receptor (t-PAR) on human umbilical vein endothlial cells (HUVEC) and that t-PAR interacted only with t-PA to form t-PA/ t-PAR complex. To investigate the function of t-PAR, we synthesized recombinant t-PAR (rt-PAR). Furthermore, the binding ability of t-PA to t-PAR on vascular endothelial cells was studied by using IAsys.
The cDNA coding region for t-PAR was cloned into glutathione S-transferase (GST) expression vector. The target protein was expressed as the fusion protein with GST. The mature rt-PAR was obtained from cleaving fusion protein by protease. The ligand blot analysis revealed that t-PA specifically bound to rt-PAR.
On the interaction analysis using IAsys, the binding signal of HUVEC to t-PA elevated in the cell concentration manner. It is considered that the t-PAR may concentrate t-PA on the surface of HUVEC and enhance the fibrinolytic properties around them.
When HUVEC were treated with EACA, the binding signal of HUVEC to t-PA decreased in an EACA dependent manner. It was suggested that the binding of t-PA to endothelial cell was mediated via lysine binding site in t-PA molecule. On the other hand, when the endothelial cell was treated with receptor associated protein, the binding parameter did not change. This result indicated that low density lipoprotein receptor related protein was not involved in the binding of t-PA to HUVEC. Furthermore, polyclonal antibody to annexin II and that to α ^-enolase did not interfere the interaction between t-PA and HUVEC. It concluded that t-PAR is the novel protein which is different from annexin II and α ^-enolase.
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