Structural and functional changes in platelets during activation observed by a rapid freeze-substitution method

电镜下观察血小板活化过程中细胞膜与膜细胞骨架的结合。快速冷冻置换法具有能准确观察快速变化的优点。然而，膜相关的细胞骨架只能在血小板膜裂解处理后观察到。用0.05u/ml凝血酶活化血小板后，用0.02%DTSP（dithiobissuccinimidylpropinate，a crosslinker）在22 ℃处理10分钟，观察血小板与血小板的结合情况，并与快速冷冻置换法进行比较。通过添加等体积的含有0.2% Triton X-100、2%戊二醛、10 mM EGTA和100 mM赖氨酸的溶液，同时对其进行裂解和固定。立即离心以收集沉淀。Epon 812或Lowicryl K4 M包埋后，薄切片 ...更多信息 制备并处理用于标准或免疫电子显微镜检查。在未受刺激的血小板中，细胞骨架保持为盘状结构。在TritonX-100不溶性残留物的薄切片中，约1/3的整个细胞膜保持完整。膜表面有许多直径为10-70 nm的非晶结构，每隔20-100 nm分布一个区域。通过免疫细胞化学在无定形结构上检测到血小板膜糖蛋白GPIIb/IIIa，其中存在纤维蛋白原结合位点。其中一些结构似乎穿透了膜。所有的无定形结构都与膜骨架相连，膜骨架位于膜的正下方，由微丝网络组成。微丝被鉴定为肌动蛋白丝。凝血酶活化后，血小板变成球形。Triton X-100不溶性颗粒集中，周围是致密而厚的肌动蛋白丝网络。在肌动蛋白丝网络中检测到肌球蛋白。受刺激血小板的细胞膜也总是与膜骨架相连。部分细胞膜骨架与细胞质肌动蛋白丝相连，形成粗大的网状结构，包围着集中的颗粒。这些结果可能从形态学上证实了血小板活化的信号转导途径。少

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