Livne A, Packham M A, Guccione M A, Mustard J F
Department of Biochemistry, University of Toronto, Ontario, Canada.
J Clin Invest. 1988 Feb;81(2):288-99. doi: 10.1172/JCI113320.
To investigate the association of lipid with the cytoskeleton of platelets during aggregation, rabbit and human platelets were isolated and labeled with [3H]palmitic acid; lipid extraction showed approximately 80% in phospholipid. Limited aggregation was induced with ADP or thrombin, and the cytoskeleton was isolated after lysis with 1% Triton X-100, 5 mM EGTA. Cytoskeleton from unactivated platelets had approximately 0.03% of the total label in the platelets, but after aggregation with ADP (2 microM) or thrombin (0.1 U/ml) for 20-30 s, 1.5-8% of the label was with the cytoskeleton. Fibrinogen enhanced aggregation and the association of label with the cytoskeleton; incorporation of label increased exponentially as aggregation proceeded, decreased exponentially during deaggregation, and appeared to be related to the number of sites of contact. Inhibitors that increase cyclic AMP inhibited aggregation and cytoskeletal labeling, but aspirin had no effect. Some experiments were done with DNase I and Ca2+ in the Triton X-100 lysis medium to cause actin depolymerization, under conditions in which the Ca2+-dependent protease activity was inhibited. This greatly reduced the association of label with the cytoskeleton at early time points, but when aggregation had proceeded further, a large proportion of the label was not dissociated by this treatment. These findings, electron microscopy, and the enrichment of the cytoskeleton of aggregated platelets with only some of the membrane proteins that were labeled by the 125I-lactoperoxidase method, indicated that with limited aggregation, the 3H-labeled lipid was mainly associated with the cytoskeleton and not with trapped membrane fragments resulting from incomplete lysis. Since the pattern of cytoskeleton labeling ([3H]palmitate) and the selective association of some membrane proteins with the cytoskeleton/lipid complex was the same with ADP and thrombin, the reactions must be dependent on aggregation and not on events associated with the release of granule contents.
为研究聚集过程中脂质与血小板细胞骨架的关联，分离了兔和人的血小板并用[3H]棕榈酸进行标记；脂质提取显示约80%存在于磷脂中。用ADP或凝血酶诱导有限聚集，并用1% Triton X - 100、5 mM EGTA裂解后分离细胞骨架。未活化血小板的细胞骨架含有血小板中总标记物的约0.03%，但在用ADP（2 microM）或凝血酶（0.1 U/ml）聚集20 - 30秒后，1.5 - 8%的标记物与细胞骨架结合。纤维蛋白原增强聚集以及标记物与细胞骨架的结合；随着聚集进行，标记物的掺入呈指数增加，在解聚过程中呈指数下降，且似乎与接触位点的数量有关。增加环磷酸腺苷的抑制剂抑制聚集和细胞骨架标记，但阿司匹林无作用。在Triton X - 100裂解培养基中用DNase I和Ca2+进行了一些实验，以在抑制Ca2+依赖性蛋白酶活性的条件下使肌动蛋白解聚。这在早期时间点大大降低了标记物与细胞骨架的结合，但当聚集进一步进行时，很大一部分标记物并未因这种处理而解离。这些发现、电子显微镜观察以及仅用125I - 乳过氧化物酶法标记的一些膜蛋白对聚集血小板细胞骨架的富集表明，在有限聚集时，3H标记的脂质主要与细胞骨架相关，而非与不完全裂解产生的截留膜片段相关。由于ADP和凝血酶作用下细胞骨架标记（[3H]棕榈酸盐）的模式以及一些膜蛋白与细胞骨架/脂质复合物的选择性结合相同，这些反应必定依赖于聚集，而非与颗粒内容物释放相关的事件。
