Fox J E, Boyles J K, Reynolds C C, Phillips D R
J Cell Biol. 1984 Jun;98(6):1985-91. doi: 10.1083/jcb.98.6.1985.
The extent of actin polymerization in unstimulated, discoid platelets was measured by DNase I inhibition assay in Triton X-100 lysates of platelets washed at 37 degrees C by gel filtration, or in Triton X-100 lysates of platelets washed at ambient temperatures by centrifugation in the presence of prostacyclin. About 40% of the actin in the discoid platelets obtained by either method existed as filaments. These filaments could be visualized by electron microscopy of thin sections. Similar results were obtained when the actin filament content of discoid platelets was measured by sedimentation of filaments from Triton X-100 lysates at high g forces (145,000 g for 45 min). However, few of these filaments sedimented at the lower g forces often used to isolate networks of actin filaments from cell extracts. These results indicate that actin filaments in discoid cells are not highly crosslinked. Platelets isolated by centrifugation in the absence of prostacyclin were not discoid, but were instead irregular with one or more pseudopodia. These platelets also contained approximately 40-50% of their actin in a filamentous form; many of these filaments sedimented at low g forces, however, indicating that they were organized into networks. The discoid shape of these centrifuged platelets could be restored by incubating them for 1-3 h at 37 degrees C, which resulted in the reversal of filament organization. High g forces were then required for the sedimentation of the actin. Approximately 80-90% of the actin in platelets washed at 4 degrees C was filamentous; this high actin filament content could be attributed to actin polymerization during the preparation of the platelets at low temperatures. These studies show that platelet activation involves mechanisms for the structural reorganization of existing filaments, in addition to those previously described for mediating actin polymerization.
通过DNase I抑制试验，在37℃下经凝胶过滤洗涤的血小板的Triton X - 100裂解物中，或在环前列腺素存在下于室温经离心洗涤的血小板的Triton X - 100裂解物中，测量未刺激的盘状血小板中肌动蛋白聚合的程度。通过这两种方法获得的盘状血小板中，约40%的肌动蛋白以细丝形式存在。这些细丝可通过薄切片的电子显微镜观察到。当通过在高重力（145,000 g，45分钟）下从Triton X - 100裂解物中沉淀细丝来测量盘状血小板的肌动蛋白细丝含量时，也得到了类似的结果。然而，在常用于从细胞提取物中分离肌动蛋白细丝网络的较低重力下，这些细丝很少沉淀。这些结果表明，盘状细胞中的肌动蛋白细丝交联程度不高。在没有环前列腺素的情况下通过离心分离的血小板不是盘状的，而是不规则的，有一个或多个伪足。这些血小板中也约有40 - 50%的肌动蛋白呈细丝状；然而，其中许多细丝在低重力下沉淀，表明它们被组织成了网络。将这些离心后的血小板在37℃下孵育1 - 3小时，可恢复其盘状形状，这导致细丝组织发生逆转。然后需要高重力才能使肌动蛋白沉淀。在4℃下洗涤的血小板中，约80 - 90%的肌动蛋白是细丝状的；这种高肌动蛋白细丝含量可归因于在低温下制备血小板过程中的肌动蛋白聚合。这些研究表明，血小板活化除了涉及先前描述的介导肌动蛋白聚合的机制外，还涉及现有细丝结构重组的机制。
