Department of Hematology/Oncology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
PLoS One. 2013 Jul 23;8(7):e69315. doi: 10.1371/journal.pone.0069315. Print 2013.
RhoA plays a multifaceted role in platelet biology. During platelet development, RhoA has been proposed to regulate endomitosis, proplatelet formation, and platelet release, in addition to having a role in platelet activation. These processes were previously studied using pharmacological inhibitors in vitro, which have potential drawbacks, such as non-specific inhibition or incomplete disruption of the intended target proteins. Therefore, we developed a conditional knockout mouse model utilizing the CRE-LOX strategy to ablate RhoA, specifically in megakaryocytes and in platelets to determine its role in platelet development. We demonstrated that deleting RhoA in megakaryocytes in vivo resulted in significant macrothrombocytopenia. RhoA-null megakaryocytes were larger, had higher mean ploidy, and exhibited stiff membranes with micropipette aspiration. However, in contrast to the results observed in experiments relying upon pharmacologic inhibitors, we did not observe any defects in proplatelet formation in megakaryocytes lacking RhoA. Infused RhoA-null megakaryocytes rapidly released platelets, but platelet levels rapidly plummeted within several hours. Our evidence supports the hypothesis that changes in membrane rheology caused infused RhoA-null megakaryocytes to prematurely release aberrant platelets that were unstable. These platelets were cleared quickly from circulation, which led to the macrothrombocytopenia. These observations demonstrate that RhoA is critical for maintaining normal megakaryocyte development and the production of normal platelets.
RhoA 在血小板生物学中发挥着多方面的作用。在血小板发育过程中，RhoA 被认为除了在血小板激活中发挥作用外，还可以调节核内有丝分裂、前血小板形成和血小板释放。这些过程之前是通过体外药理学抑制剂研究的，这些抑制剂具有潜在的缺点，如非特异性抑制或不完全破坏预期的靶蛋白。因此，我们利用 CRE-LOX 策略开发了一种条件性敲除小鼠模型，以特异性地在巨核细胞和血小板中敲除 RhoA，从而确定其在血小板发育中的作用。我们证明，在体内敲除巨核细胞中的 RhoA 会导致明显的巨血小板减少症。RhoA 缺失的巨核细胞体积更大，平均倍性更高，并且具有刚性膜和微吸管抽吸。然而，与依赖于药理学抑制剂的实验结果相反，我们没有观察到缺乏 RhoA 的巨核细胞中前血小板形成有任何缺陷。输注的 RhoA 缺失的巨核细胞迅速释放血小板，但在数小时内血小板水平迅速下降。我们的证据支持这样一种假设，即膜流变学的变化导致输注的 RhoA 缺失的巨核细胞过早释放不稳定的异常血小板。这些血小板很快从循环中清除，导致巨血小板减少症。这些观察结果表明，RhoA 对于维持正常巨核细胞发育和正常血小板的产生至关重要。
