Painter R G, Gaarde W, Ginsberg M H
J Cell Biochem. 1985;27(3):277-90. doi: 10.1002/jcb.240270309.
When intact platelets are incubated at 37 degrees C with Concanavalin A (ConA), the two major surface membrane proteins GPIIb and III become associated with the Triton-insoluble cytoskeleton. Preincubation of platelets with a variety of metabolic inhibitors, including cytochalasin B, 2-deoxy-D-glucose, and antimycin A or lidocaine, had no effect on the ability of ConA to produce this effect. These results suggested that the ConA-induced anchorage of GPIIb and III to the Triton-insoluble cytoskeleton is a passive process requiring clustering of GPIIb-III molecules but not requiring the metabolic energy of an intact cell. This was supported by experiments that showed that ConA binding to plasma membrane-rich fractions at 37 degrees C could induce association of GPIIb and III with a sedimentable actin-rich, Triton-insoluble membrane matrix. Similar results were obtained when membranes were first isolated from ConA-treated cells. Adding DNAse I, an actin depolymerizing agent, into the Triton extraction buffer inhibited the ConA-induced sedimentation of GPIIb-III and actin by 50% in the presence of Mg2+-ATP. Treatment of ConA-treated membranes with dimethyl-3,3'-dithiobispropiomidate, a bifunctional, reducible protein crosslinking agent, produced Triton-insoluble crosslinked species of discrete molecular weights. When these cross-linked species were analyzed by SDS-PAGE in the presence of beta-mercaptoethanol, they were found to be composed of a 180-200 K dalton protein, GPIIb, GPIII, and actin. Crosslinking of these components was equally effective after Triton treatment and indicated as well that the species crosslinked in the intact membrane was stable after Triton extraction. Addition of crosslinker to membranes not treated with ConA produced similar crosslinked species. Analysis of their composition on reduced gels revealed that the amounts of GPIIb and III were reduced greatly (less than 10% of the total input GPIIb and III) but that the 180-200 k dalton protein and actin content were similar to that seen with ConA-treated membranes. These results are consistent with the notion that ConA clusters mobile, unanchored molecules of GPIIb-III (approximately 90-95% of the total) around a small fraction of IIb-III that is associated with a submembranous cytoskeleton.
当完整的血小板在37℃下与伴刀豆球蛋白A（ConA）一起孵育时，两种主要的表面膜蛋白糖蛋白IIb（GPIIb）和糖蛋白III（III）会与不溶于曲拉通的细胞骨架结合。用多种代谢抑制剂（包括细胞松弛素B、2-脱氧-D-葡萄糖、抗霉素A或利多卡因）对血小板进行预孵育，对ConA产生这种效应的能力没有影响。这些结果表明，ConA诱导的GPIIb和III锚定到不溶于曲拉通的细胞骨架是一个被动过程，需要GPIIb-III分子聚集，但不需要完整细胞的代谢能量。这得到了实验的支持，实验表明ConA在37℃下与富含质膜的组分结合可诱导GPIIb和III与可沉降的富含肌动蛋白、不溶于曲拉通的膜基质结合。当首先从ConA处理的细胞中分离膜时，也得到了类似的结果。在存在Mg2+-ATP的情况下，向曲拉通提取缓冲液中加入肌动蛋白解聚剂脱氧核糖核酸酶I（DNAse I）可使ConA诱导的GPIIb-III和肌动蛋白沉降减少50%。用双功能、可还原的蛋白质交联剂二甲基-3,3'-二硫代双丙酰胺处理ConA处理的膜，产生了具有离散分子量的不溶于曲拉通的交联物种。当在β-巯基乙醇存在下通过SDS-PAGE分析这些交联物种时，发现它们由180-200 kDa的蛋白质、GPIIb、GPIII和肌动蛋白组成。在曲拉通处理后，这些组分的交联同样有效，这也表明在完整膜中交联的物种在曲拉通提取后是稳定的。向未用ConA处理的膜中加入交联剂产生了类似的交联物种。在还原凝胶上分析它们的组成发现，GPIIb和III的量大大减少（不到总输入GPIIb和III的10%），但180-200 kDa蛋白质和肌动蛋白的含量与ConA处理的膜相似。这些结果与以下观点一致，即ConA在一小部分与膜下细胞骨架相关的IIb-III周围聚集可移动的、未锚定的GPIIb-III分子（约占总数的90-95%）。
