| Project/Area Number |
63570581
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
Hematology
|
|---|
| Research Institution | The Tokyo Metropolitan Institute of Medical Science |
|---|
Principal Investigator |
YAMAZAKI Hiroh The Tokyo Metropolitan Institute of Medical Science, Vice Director, 副所長 (50013826)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
TSUKITA Shoichiro The Tokyo Metropolitan Institute of Medical Science, Department of Ultrastructur, 超微形態研究室, 室長 (50155347)
SUZUKI Hidenori The Tokyo Metropolitan Institute of Medical Science, Department of Cardiovascula, 循環器病研究部, 研究員 (30158977)
|
|---|
| Project Period (FY) |
1988 – 1989
|
| Project Status |
Completed (Fiscal Year 1989)
|
| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1989: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1988: ¥1,100,000 (Direct Cost: ¥1,100,000)
|
|---|
| Keywords | Platelets / Platelet activation / Platelet membrane glycoproteins / Rapid freeze-substitution method / Immunocytochemistry / alpha-granules / Cytoskeletons / Membrane associated structure / 急速凍結置換法 / a顆粒 / 微小管 / マイクロフィラメント / Triton X-100 / 裏打ち構造 |
|---|
| Research Abstract |
Association between cell membrane and membrane cytosckeleton of platelets during activation was observed under electron microscopy. Rapid freezesubstitution-method has an advantage to observe rapid changes preciously. However, membrane associated cytoskeletons can be observed only after a treatment of lysis of platelet membranes. Using a cross-linker and a solution for lysis and fixation that enable membrane bilayers to survive more efficiently, we examined the association and compared with the results which were obtained by the rapid freeze-substitution method.
After activation with 0.05 u/ml of thrombin, platelets were treated with 0.02% DTSP (dithiobis succinimidyl propinate, a cross-linker) for 10 min at 22ﾟC . They were simultaneously lysed and fixed by addition of an equal volume of a solution containing 0.2% Triton X-100, 2% glutaraldehyde, 10 mM EGTA and 100 mM lysine. They were immediately centrifuged to collect pellets. After embedding in Epon 812 or Lowicryl K4M, thin section
… More
s were prepared and processed for standard or immune-electron microscopy. In unstimulated platelets, the cyto skeletons remained as discoid shaped structures. About 1/3 of the whole cell membrane remained intact in the thin sections of-Triton X-100-insoluble residues. There were many amorphous structures with 10-70 nm in diameters distributed at 20-100 nm intervals on the surface of membrane. Platelet membrane glycoproteins, GPIIb/IIIa in which fibrinogen binding site is present, were detected on the amorphous structures by immunocytochemistry. Some of these structures appeared to penetrate the membrane. All the amorphous structures were connected to the membrane skeleton which was located just beneath the membrane and consisted of networks with microfilaments. The microfilaments were identified as actin filaments. After activation by thrombin, Platelets became spherical. Triton X-100-insoluble granules were centralized and surrounded by dense and thick networks of actin filaments. Myosin was detected in the actin filament net works. The cell membranes of stimulated platelets were also always associated with the membrane skeleton. Part of the membrane skeleton continuously connected to cytoplasmic actin filaments which were forming thick networks and surrounding the centralized granules. These results may demonstrate a pathway of signal transduction in platelet activation morphologically. Less
|
