Phillips D R, Jennings L K, Edwards H H
J Cell Biol. 1980 Jul;86(1):77-86. doi: 10.1083/jcb.86.1.77.
Membrane glycoproteins that mediate platelet-platelet interactions were investigated by identifying those associated with the cytoskeletal structures from aggregated platelets. The cytoskeletal structures from washed platelets, thrombin-activated platelets (platelets incubated with thrombin in the presence of mM EDTA to prevent aggregation) and thrombin- aggregated platelets (platelets activated in the presence of mM Ca(++) were prepared by first treating platelet suspensions with 1 percent Triton X-100 and 5 mM EGTA and then isolating the insoluble residue by centrifugation. The readily identifiable structures in electron micrographs of the residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue by SDS gel electrophoresis showed that it consisted primarily of three proteins: actin (mol wt = 43,000), myosin (mol wt = 200,000) and a high molecular weight polypeptide (mol wt = 255,000) which had properties indentical to actin-binding protein (filamin). When platelets are activated with thrombin in the presence of EDTA to prevent aggregation, there was a marked increase in the amount of insoluble precipitate in the subsequent Triton extraction. Transmission electron microscopy showed that this residue not only contained the random array of actin filaments as seen above, but also organized structures from individual platelets which appeared as balls of electron-dense filamentous material approximately 1mum in diameter. SDS polyacrylamide gel analysis of the Triton residue of activated platelets showed that this preparation contained more actin, myosin and actin-binding protein than that from washed platelets plus polypeptides with mol wt of 56,000 and 90,000 and other minor polypeptides. Thus, thrombin activation appeared to increase polymerization of actin in association with other cytoskeletal proteins into structures that are observable after Triton extraction. The cytoskeletal structures from thrombin-aggregated platelets were similar to those from thrombin-activated platelets, except that the structural elements from individual platelets remained aggregated rather than randomly dispersed in the actin filaments. This suggested that the membrane components that mediate the direct interaction of platelets were in Triton residue from aggregated platelets. Only a small percentage of the membrane surface proteins and glycoproteins were found in the cytoskeletal structures from either washed platelets or thrombin-activated platelets. In contrast, the aggregated cytoskeletal structures from thrombin-aggregated platelets contained membrane glycoproteins IIb (26 percent of the total in pre-extracted platelets) and III (14 percent), suggesting that one or both of these glycoproteins participate in the direct interaction of platelets during aggregation.
通过识别与聚集血小板的细胞骨架结构相关的膜糖蛋白，对介导血小板 - 血小板相互作用的膜糖蛋白进行了研究。洗涤血小板、凝血酶激活血小板（在含有毫摩尔浓度乙二胺四乙酸（EDTA）的情况下与凝血酶孵育以防止聚集的血小板）和凝血酶聚集血小板（在毫摩尔浓度钙离子（Ca(++)）存在下激活的血小板）的细胞骨架结构，首先用1% Triton X - 100和5毫摩尔浓度乙二醇双四乙酸（EGTA）处理血小板悬浮液，然后通过离心分离不溶性残渣来制备。洗涤血小板残渣的电子显微镜照片中易于识别的结构具有肌动蛋白丝的形状和尺寸。对洗涤血小板的这种残渣分析表明它主要由三种蛋白质组成：肌动蛋白（分子量 = 43,000）、肌球蛋白（分子量 = 200,000）和一种高分子量多肽（分子量 = 255,000），其性质与肌动蛋白结合蛋白（细丝蛋白）相同。当在EDTA存在下用凝血酶激活血小板以防止聚集时，随后的Triton提取中不溶性沉淀物的量显著增加。透射电子显微镜显示，这种残渣不仅含有如上述所见的随机排列的肌动蛋白丝，还含有来自单个血小板组织化的结构，这些结构表现为直径约1微米的电子致密丝状物质球。对激活血小板的Triton残渣进行SDS聚丙烯酰胺凝胶分析表明，该制剂比洗涤血小板含有更多的肌动蛋白、肌球蛋白和肌动蛋白结合蛋白，再加上分子量为56,000和90,000的多肽以及其他次要多肽。因此，凝血酶激活似乎增加了肌动蛋白与其他细胞骨架蛋白的聚合，形成了Triton提取后可观察到的结构。凝血酶聚集血小板的细胞骨架结构与凝血酶激活血小板的相似，只是来自单个血小板的结构元件保持聚集状态，而不是随机分散在肌动蛋白丝中。这表明介导血小板直接相互作用的膜成分存在于聚集血小板的Triton残渣中。在洗涤血小板或凝血酶激活血小板的细胞骨架结构中，仅发现一小部分膜表面蛋白和糖蛋白。相比之下，凝血酶聚集血小板的聚集细胞骨架结构含有膜糖蛋白IIb（占提取前血小板总量的26%）和III（14%），这表明这些糖蛋白中的一种或两种在聚集过程中参与了血小板的直接相互作用。
