Department of Experimental Biomedicine, University Hospital and Rudolf Virchow Center, University of Würzburg, Würzburg, Germany.
J Thromb Haemost. 2014 Aug;12(8):1364-71. doi: 10.1111/jth.12623. Epub 2014 Jul 5.
Blood platelets are small anucleated cell fragments generated from bone marrow megakaryocytes (MKs) by a cytoskeleton-driven process. Thereby, mature MKs form long cytoplasmic protrusions (pro-platelets), which extend into the sinusoids within the bone marrow and finally release platelets. Podosomes are F-actin rich matrix contacts that have been suggested to play an important role in cell migration, but also in pro-platelet formation by MKs. Phospholipase D (PLD) has been proposed to contribute to the regulation of actin dynamics through the local generation of phosphatidic acid but its role in platelet formation is unknown.
We sought to investigate the significance of PLD in MK podosome formation and thrombocytopoiesis.
Podosome formation, spreading and ultra-structure of PLD single- and double-deficient MKs were analyzed using confocal and transmission electron microscopy.
Phospholipase D-deficient MKs displayed a highly altered ultra-structure in vivo and abnormal actin rearrangement, with almost abolished formation of podosomes upon spreading on collagen I in vitro. However, MK endomitosis and platelet production were not altered by PLD deficiency.
Together, our findings point to a specific function of PLD in actin dynamics as well as podosome formation and size determination in MKs on a collagen I matrix. The normal platelet number in PLD-deficient mice, however, suggests the existence of compensatory mechanisms in vivo that overcome the defective podosome formation observed in vitro.
血小板是由骨髓巨核细胞（MK）通过细胞骨架驱动过程产生的小型无核细胞碎片。由此，成熟的 MK 形成长的细胞质突起（前血小板），这些突起延伸到骨髓中的窦状隙中，最终释放血小板。Podosomes 是富含 F-肌动蛋白的基质接触点，被认为在细胞迁移中发挥重要作用，但也在 MK 的前血小板形成中发挥作用。磷脂酶 D (PLD) 通过局部产生磷脂酸被认为有助于调节肌动蛋白动力学，但它在血小板形成中的作用尚不清楚。
我们旨在研究 PLD 在 MK podosome 形成和血小板生成中的意义。
使用共聚焦和透射电子显微镜分析 PLD 单缺陷和双缺陷 MK 的 podosome 形成、扩展和超微结构。
PLD 缺陷的 MK 在体内表现出高度改变的超微结构和异常的肌动蛋白重排，在体外铺展在胶原蛋白 I 上时，podosome 的形成几乎被废除。然而，PLD 缺陷并不改变 MK 有丝分裂和血小板生成。
总之，我们的研究结果表明 PLD 在肌动蛋白动力学以及在胶原蛋白 I 基质上的 MK podosome 形成和大小决定中具有特定的功能。然而，PLD 缺陷小鼠的正常血小板数量表明体内存在补偿机制，可以克服体外观察到的缺陷 podosome 形成。
