Arp2/3 complex is required for actin polymerization during platelet shape change
Arp2/3 complex is required for actin polymerization during platelet shape change
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DOI：
10.1182/blood.v99.12.4466
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发表时间：
2002-06-15
期刊：
影响因子：
20.3
通讯作者：
Bearer, EL
中科院分区：
文献类型：
--
作者：
Li, Z;Kim, ES;Bearer, EL
当血小板被凝血酶受体激活肽（TRAP）激活或在玻璃上扩散时，会发生一系列肌动蛋白依赖的形态变化。肌动蛋白的聚合导致丝状足、板足和应力纤维的顺序形成，但调节这种聚合的分子机制尚不清楚。Arp2/3复合体在体外进行肌动蛋白聚合，在细胞内也可以完成这一功能。为了检验Arp2/3是否调控血小板肌动蛋白聚合，我们使用重组Arp2蛋白（rArp2）生成Arp2特异性抗体（alphaArp2）。通过芘测定，完整的和Fab片段的alphaArp2抑制了trap刺激的血小板提取物中肌动蛋白聚合活性。通过添加rArp2蛋白，抑制被逆转。为了测试Arp2/3抑制对特定肌动蛋白结构形成的影响，我们设计了一种新的方法来渗透静息血小板，同时保持它们在接触玻璃时的粘附能力和形成丝状足和板足的能力。Arp2/3的抑制将血小板冻结在圆形，激活的早期阶段，在丝状足和板足形成之前。经形态计量学分析，经alphaArp2治疗后，圆形期血小板比例从未治疗组的2.85%上升至63%。这种效应也出现在Fab片段上，并通过添加rArp2蛋白而逆转。通过对不同扩散阶段血小板的免疫荧光检测，在丝状足和板足中发现了Arp2/3复合物。这些结果表明，TRAP刺激激活皮层的Arp2/3复合体会引发肌动蛋白丝的爆炸性聚合，这是所有随后的肌动蛋白依赖事件所必需的。（Blood. 2002;99:4466-4474） (C) 2002年由美国血液学会出版。
Platelets undergo a series of actin-dependent morphologic changes when activated by thrombin receptor activating peptide (TRAP) or when spreading on glass. Polymerization of actin results in the sequential formation of filopodia, lamellipodia, and stress fibers, but the molecular mechanisms regulating this polymerization are unknown. The Arp2/3 complex nucleates actin polymerization in vitro and could perform this function inside cells as well. To test whether Arp2/3 regulated platelet actin polymerization, we used recombinant Arp2 protein (rArp2) to generate Arp2-specific antibodies (alphaArp2). Intact and Fab fragments of alphaArp2 inhibited TRAP-stimulated actin-polymerizing activity in platelet extracts as measured by the pyrene assay. Inhibition was reversed by the addition of rArp2 protein. To test the effect of Arp2/3 inhibition on the formation of specific actin structures, we designed a new method to permeabilize resting platelets while preserving their ability to adhere and to form filopodia and lamellipodia on exposure to glass. Inhibition of Arp2/3 froze platelets at the rounded, early stage of activation, before the formation of filopodia and lamellipodia. By morphometric analysis, the proportion of platelets in the rounded stage rose from 2.85% in untreated to 63% after treatment with alphaArp2. This effect was also seen with Fab fragments and was reversed by the addition of rArp2 protein. By Immunofluorescence of platelets at various stages of spreading, the Arp2/3 complex was found in filopodia and lamellipodia. These results suggest that activation of the Arp2/3 complex at the cortex by TRAP stimulation initiates an explosive polymerization of actin filaments that Is required for all subsequent actin-dependent events. (Blood. 2002;99:4466-4474) (C) 2002 by The American Society of Hematology.