The role of Rho GTPases in platelet biogenesis and function: Studies in conditional knock-out mice

血小板的前体细胞，巨核细胞（MK）的生物发生的分子机制是复杂的，知之甚少，但有一个无可争议的中心作用MK细胞骨架在这些过程中。此外，基本的血小板功能，如形状变化，颗粒释放和粘附/铺展在细胞外基质（ECM）成分严格依赖于微调的细胞骨架重排。Rho家族的小GTP酶在这些过程中起关键作用，但它们的确切功能和分子相互作用尚不清楚。我们第一个资助期的结果揭示了Rac 1，Cdc 42/RhoF，RhoA和RhoB在MK和血小板中的中央调节功能和部分冗余。此外，我们可以首次证明RhoA或Cdc 42中MK特异性缺陷诱导显著的巨噬细胞减少，这与骨髓中MK前所未有的定位缺陷相关。在RhoA缺陷小鼠中，约30%的MK具有血管内定位，并且这种缺陷通过伴随的Cdc 42缺陷或vWF受体复合物GPIb-IX的功能性阻断而恢复。这些和进一步的结果表明，RhoA和GPIb/Cdc 42是骨髓中巨核细胞生成/前血小板形成的中心对立调节因子。该轴也可以解释GPIb-IX缺陷患者中观察到的巨血小板减少症（Bernard Soulier综合征，BSS）。我们希望在体外和体内使用（条件性）基因敲除小鼠和造血干细胞中突变型GT3变体的病毒表达来测试这一假设。除其他技术外，分析还将涉及骨髓的活体双光子显微镜（2 P-IVM）和光片荧光显微镜（LSFM）以及血小板生物反应器中前血小板形成的体外研究。我们希望这些研究将提供根本上的新的见解，在骨髓中的巨核细胞生成/前血小板形成的分子机制，可能有助于更好地了解造血系统疾病。

The molecular mechanisms underlying the biogenesis of platelets by their precursor cells, megakaryocytes (MKs), are complex and poorly understood but there is an undisputed central role for the MK cytoskeleton in these processes. In addition, essential platelet functions, such as shape change, granule release and adhesion/spreading on extracellular matrix (ECM) components strictly depend on fine-tuned cytoskeletal rearrangements. Small GTPases of the Rho family are critically involved in these processes but their exact functions and molecular interplay are ill defined. The results from our first funding period revealed central regulatory functions and partial redundancies of Rac1, Cdc42/RhoF, RhoA, and RhoB in MKs and platelets. Furthermore, we could demonstrate for the first time that MK-specific deficiencies in RhoA or Cdc42 induce marked macrothrombocytopenias that are associated with unprecedented localisation defects of MKs in the bone marrow. In RhoA-deficient mice, approx. 30% of MKs had an intravascular localisation and this defect was reverted by a concomitant Cdc42-deficiency or functional blockade of the vWF receptor complex, GPIb-IX. These and further results indicate that RhoA and GPIb/Cdc42 are central opposing regulators of megakaryopoiesis/proplatelet formation in the bone marrow. This axis may also provide an explanation for the macrothrombocytopenia observed in GPIb-IX-deficient patients (Bernard Soulier Syndrome, BSS). We want to test this hypothesis in vitro and in vivo using (conditional) knock-out mice and viral expression of mutant GTPase variants in haematopoietic stem cells. In addition to other techniques, the analysis will involve intravital 2-photon microscopy (2P-IVM) and light sheet fluorescence microscopy (LSFM) of the bone marrow as well as ex vivo studies of proplatelet formation in a Platelet Bioreactor. We expect that these studies will provide fundamentally new insights into the molecular mechanisms underlying megakaryopoiesis/proplatelet formation in the bone marrow that may contribute to a better understanding of diseases of the haematopoietic system.