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

Rho GTPases 在血小板生物发生和功能中的作用：条件性基因敲除小鼠的研究

• 批准号: 202079504
• 负责人: Professor Dr. Bernhard Nieswandt
• 金额: --
• 依托单位: Lehrstuhl für Experimentelle Biomedizin
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/202079504?language=en
• 关键词: role; Rho; GTPases; platelet; biogenesis

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.

巨核细胞(MKs)是血小板的前体细胞，其生物发生的分子机制复杂且知之甚少，但MK细胞骨架在这些过程中发挥着无可争辩的核心作用。此外，血小板的基本功能，如形状变化、颗粒释放和在细胞外基质(ECM)成分上的黏附/铺展，严格依赖于微调的细胞骨架重排。Rho家族的小分子GTP酶在这些过程中起着至关重要的作用，但它们的确切功能和分子间的相互作用尚不清楚。我们第一个资助期的结果显示，在巨噬细胞和血小板中，Rac1、CDC42/RhoF、RhoA和RhoB具有中央调控功能和部分冗余。此外，我们可以首次证明RhoA或CDC42中的MK特异性缺陷会导致明显的大血小板减少症，这与MK在骨髓中前所未有的定位缺陷有关。在RhoA缺陷小鼠中，大约。30%的MK有血管内定位，这一缺陷可通过伴随的CDC42缺乏或vWF受体复合体GPIB-IX的功能阻断而逆转。这些和进一步的结果表明，RhoA和GPIB/CDC42是骨髓中巨核细胞生成/原血小板形成的中心对立调节因子。这个轴也可以解释在GPIB-IX缺陷患者中观察到的大血小板减少症(Bernard Soulier综合征，BSS)。我们希望在体外和体内使用(有条件的)基因敲除小鼠和在造血干细胞中病毒表达突变的GTP酶变体来验证这一假说。除了其他技术外，分析还将涉及骨髓的活体双光子显微镜(2P-IVM)和光片荧光显微镜(LSFM)，以及对血小板生物反应器中原血小板形成的体外研究。我们期待这些研究将为骨髓中巨核细胞生成/血小板原形成的分子机制提供新的见解，这可能有助于更好地理解造血系统疾病。