Identification of membrane proteins mediating the interaction of human platelets.
Identification of membrane proteins mediating the interaction of human platelets.
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DOI：
10.1083/jcb.86.1.77
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发表时间：
1980-07
期刊：
影响因子：
--
通讯作者：
Edwards HH
中科院分区：
文献类型：
--
作者：
Phillips DR;Jennings LK;Edwards HH
介导血小板-血小板相互作用的膜糖蛋白通过鉴定与聚集的血小板的细胞骨架结构相关的膜糖蛋白进行了研究。通过首先用1%Triton X-100和5 mM EGTA处理血小板悬浮液，然后通过离心分离不溶性残余物，制备来自洗涤的血小板、凝血酶活化的血小板（在mM EDTA存在下与凝血酶一起孵育以防止聚集的血小板）和凝血酶聚集的血小板（在mM Ca（++）存在下活化的血小板）的细胞骨架结构。在电子显微镜下，洗涤后的血小板残留物的结构很容易辨认，具有肌动蛋白丝的形状和尺寸。对来自洗涤血小板的这种残留物的分析具有肌动蛋白丝的形状和尺寸。SDS凝胶电泳分析表明，该蛋白主要由三种蛋白质组成：肌动蛋白（mol wt = 43，000）、肌球蛋白（mol wt = 200，000）和一种高分子量多肽（mol wt = 255，000），该多肽具有与肌动蛋白结合蛋白（细丝蛋白）相同的性质。当在EDTA存在下用凝血酶活化血小板以防止聚集时，在随后的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，该残留物不仅包含如上所述的肌动蛋白丝的随机阵列，而且还包含来自单个血小板的有组织的结构，其表现为直径约1 μ m的电子致密丝状材料的球。SDS-聚丙烯酰胺凝胶分析表明，活化血小板中含有较多的肌动蛋白、肌球蛋白和肌动蛋白结合蛋白，而洗涤血小板中含有较多的分子量为56，000和90，000的多肽和其它次要多肽。因此，凝血酶活化似乎增加了肌动蛋白与其他细胞骨架蛋白的聚合成Triton提取后可观察到的结构。凝血酶聚集的血小板的细胞骨架结构与凝血酶活化的血小板相似，不同之处在于单个血小板的结构元素保持聚集，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组分在聚集血小板的Triton残基中。只有一小部分的膜表面蛋白和糖蛋白被发现在细胞骨架结构从洗涤血小板或凝血酶活化血小板。相比之下，凝血酶聚集的血小板聚集的细胞骨架结构包含膜糖蛋白IIb（占预提取血小板总量的26%）和III（14%），这表明这些糖蛋白中的一种或两种参与血小板聚集过程中的直接相互作用。
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.