Collier Mary E W, Maraveyas Anthony, Ettelaie Camille
Dr. Mary Collier, Biomedical Section, Department of Biological Sciences, University of Hull, Hull, HU6 7RX, UK, Tel.: +44 1482 465528; Fax: +44 1482 465458, E-mail:
Thromb Haemost. 2014 Apr 1;111(4):647-55. doi: 10.1160/TH13-09-0769. Epub 2013 Nov 21.
We previously reported that the incorporation of tissue factor (TF) into cell-derived microvesicles (MVs) is regulated by the phosphorylation of the cytoplasmic domain of TF. Since the cytoskeletal protein filamin-A is known to bind to the cytoplasmic domain of TF in a phosphorylation-dependent manner, the involvement of filamin-A in the incorporation of TF into MVs was examined. Endothelial cells were transfected to express TF, whereas MDA-MB-231 cells were used to examine endogenously expressed TF. MV release was induced by activating protease-activated receptor-2 (PAR2). Partial suppression of filamin-A expression using two different filamin-A siRNA sequences resulted in significant reductions in the incorporation of TF antigen into MVs as determined by TF-ELISA and western blot analysis, and was reflected in reduced thrombin-generation and FXa-generation capacities of these MVs. Deletion of the cytoplasmic domain of TF also resulted in reduced incorporation of TF into MVs, whereas the suppression of filamin-A expression had no additional effect on the incorporation of truncated TF into MVs. Partial suppression of filamin-A expression had no effect on the number and size distribution of the released MVs. However, >90% suppression of filamin-A expression resulted in increased MV release, possibly as a result of increased instability of the plasma membrane and underlying cytoskeleton. In conclusion, the presence of filamin-A appears to be essential for the incorporation of TF into MVs following PAR2 activation, but is not required for the process of MV formation and release following PAR2 activation.
我们之前报道过，组织因子（TF）掺入细胞衍生微泡（MVs）的过程受TF胞质结构域磷酸化的调控。由于已知细胞骨架蛋白细丝蛋白A以磷酸化依赖的方式与TF的胞质结构域结合，因此研究了细丝蛋白A在TF掺入MVs过程中的作用。转染内皮细胞以表达TF，而使用MDA-MB-231细胞检测内源性表达的TF。通过激活蛋白酶激活受体-2（PAR2）诱导MV释放。使用两种不同的细丝蛋白A siRNA序列部分抑制细丝蛋白A的表达，导致通过TF-ELISA和蛋白质印迹分析测定的TF抗原掺入MVs的量显著减少，并且反映在这些MVs的凝血酶生成能力和FXa生成能力降低。删除TF的胞质结构域也导致TF掺入MVs的量减少，而抑制细丝蛋白A的表达对截短的TF掺入MVs没有额外影响。部分抑制细丝蛋白A的表达对释放的MVs的数量和大小分布没有影响。然而，>90%抑制细丝蛋白A的表达导致MV释放增加，这可能是由于质膜和潜在细胞骨架的稳定性增加所致。总之，细丝蛋白A的存在似乎是PAR2激活后TF掺入MVs所必需的，但不是PAR2激活后MV形成和释放过程所必需的。
