Die Rolle des Zytoskeletts für die Thrombopoese: Untersuchungen zur transendothelialen Thrombozytopoese im Knochenmark

血小板是血液中的血球成分，对止血是必不可少的。它们的平均寿命为8-10天，因此，血小板由被称为巨核细胞(MK)的前体细胞持续补充。这些巨核细胞位于血窦中，它们通过内皮屏障将新生的血小板释放到血流中。由于血小板很容易获得，因此它们的细胞生物学特性总体上是很好的。相比之下，骨髓巨噬细胞属于骨髓中的稀有细胞，通常无法进行分析。如果产生针对血小板受体的交叉反应自身抗体，血小板会迅速从循环中清除，由此导致的血小板减少会导致严重或长期的出血，这种疾病被称为免疫性血小板减少症(ITP)。在这里，我们提出了一系列实验来分析两种不同的抗血小板抗体对巨核细胞生成的影响。我们将使用三种不同的ITP小鼠模型并分析骨髓MKs。冷冻切除使我们能够保存小鼠完整的股骨，而不会脱钙，随后用不同的抗体进行免疫组织化学染色。用激光共聚焦扫描免疫荧光显微镜对玻片进行分析，并通过数量、大小、形态及其与骨髓血窦的定位和结合来评价MK。此外，我们的目标是确定这些抗血小板抗体是如何与巨核祖细胞结合的，以及通过什么机制清除抗体。对于活体分析，我们利用了在GPIIb启动子下表达EYFP的小鼠品系，以便通过活体双光子显微镜很容易地检测到MKs。将耗竭抗体连接到第二种染料上，并在注射量子点后显示血管系统。这种方法允许在它们的自然三维栖息地检测MK。时间推移显微镜可以在6小时内检测到前血小板和前血小板的迁移和释放。我们期望这些实验将阐明不同的抗血小板抗体如何影响骨髓中的MK和循环中的血小板数量，以及新的拟血栓药物如何影响这一过程。血小板含有一环微管细丝(边缘带)，这是维持豆状形状所必需的。β1-微管蛋白是这些细丝中最重要的异构体。我们最近可以确定RanBP10是一种β1-微管蛋白结合蛋白，并建立了一个用于RanBP10消融的小鼠模型。缺乏RanBP10的小鼠有严重的出血问题，表明它是正常止血所必需的。在这里，我们提出了一系列实验，以进一步分析结构与功能的关系，以及体内实验，以确定在缺乏RanBP10的情况下，应用抗血小板抗体来去除血小板对体内巨核细胞和血小板生物生成的影响。

Platelets are corpuscular components in blood that are essential for hemostasis. They have an average life span of 8-10 days and platelets are therefore continuously replenished by precursor cells, designated megakaryocytes (MKs). These MKs are situated at sinusoids where they release nascent platelets across the endothelial barrier into the blood stream. As platelets are easily accessible, they are overall well-characterized regarding their cell biological properties. MKs, in contrast, belong to the rare cells in bone marrow and are typically not available for analysis. If cross-reacting autoantibodies against platelet receptors are generated, platelets are rapidly cleared from the circulation and the resulting thrombocytopenia can cause severe or prolonged bleeding episodes, a disorder referred to as immune thrombocytopenia (ITP).Here, we propose a series of experiments to analyze the consequences of two distinct anti-platelet antibodies on megakaryopoiesis. We will use three distinct mouse models for ITP and analyze bone marrow MKs. Cryosectioning allows us to preserve the complete femur bone of mice without decalcification and the subsequent immunohistochemical staining with different antibodies. Slides are analyzed by confocal laser scanning immunofluorescence microscopy and MKs evaluated by number, size, morphology and their localization and association to the bone marrow sinusoids. Furthermore, we aim to determine how these anti-platelet antibodies bind to the megakaryocytic progenitors and by what mechanisms the antibody is cleared. For the intravital analysis we take advantage of a mouse strain that expresses eYFP under the GPIIb promoter so that MKs can readily be detected by intravital two-photon microscopy. The depletion antibody is conjugated to a second dye and the vasculature visualized after injecting Quantum dots. This approach allows to detect MKs in their natural three-dimensional habitat. The migration and release of pre- and proplatelets can be detected by time-lapse microscopy over 6 hours. We expect that these experiments will shed some light on how different anti-platelet antibodies affect MKs in the bone marrow and the number of platelets in the circulation and how novel thrombomimetics might influence this process.Platelets harbor a cortical ring of microtubule filaments (marginal band) that are essential for maintaining the lentiform shape. Beta1-tubulin is the most important isoform in these filaments. We could recently identify RanBP10 as a beta1-tubulin binding protein and generated a mouse model for RanBP10 ablation. Mice lacking RanBP10 have a severe bleeding problem indicating that it is required for normal hemostasis. Here we propose a series of experiments to further analyze the structure-function relationship as well as in vivo experiments to determine how platelet depletion by application of anti-platelet antibodies affects megakaryopoiesis and platelet biogenesis in vivo in the absence of RanBP10.