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

血小板是血液中的微粒成分，对止血至关重要。它们的平均寿命为8-10天，因此血小板不断被称为巨核细胞（mk）的前体细胞补充。这些mk位于窦状动脉，在那里它们释放新生血小板穿过内皮屏障进入血流。由于血小板很容易获得，它们的细胞生物学特性总体上很好地表征了。相比之下，mk属于骨髓中的稀有细胞，通常无法用于分析。如果产生针对血小板受体的交叉反应自身抗体，血小板迅速从循环中清除，由此产生的血小板减少可引起严重或长期出血发作，一种称为免疫性血小板减少症（ITP）的疾病。在这里，我们提出了一系列的实验来分析两种不同的抗血小板抗体对巨核生成的影响。我们将使用三种不同的ITP小鼠模型并分析骨髓mk。冷冻切片使我们能够保存完整的小鼠股骨骨，而不需要脱钙，随后用不同的抗体进行免疫组化染色。通过共聚焦激光扫描免疫荧光显微镜对载玻片进行分析，并通过数量、大小、形态及其与骨髓窦的定位和关联来评估mk。此外，我们的目标是确定这些抗血小板抗体如何与巨核细胞祖细胞结合，以及通过什么机制清除抗体。对于活体分析，我们利用在GPIIb启动子下表达eYFP的小鼠品系，这样mk可以很容易地通过活体双光子显微镜检测到。耗尽抗体与第二种染料结合，注射量子点后血管可见。这种方法可以在它们的自然三维栖息地中探测到mk。血小板前和血小板前的迁移和释放可以通过延时显微镜在6小时内检测到。我们期望这些实验将揭示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，以及新型血栓模拟物如何影响这一过程。血小板含有微管细丝的皮质环（边缘带），这对维持透镜状的形状至关重要。β -微管蛋白是这些纤维中最重要的同工异构体。我们最近鉴定出RanBP10是β -微管蛋白结合蛋白，并建立了RanBP10消融的小鼠模型。缺乏RanBP10的小鼠有严重的出血问题，这表明它是正常止血所必需的。在此，我们提出了一系列进一步分析结构-功能关系的实验和体内实验，以确定在缺乏RanBP10的情况下，应用抗血小板抗体消耗血小板如何影响体内巨核生成和血小板生物发生。

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.