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

• Applicant: Professor Dr. Harald Schulze
• Subject Area: Pediatric and Adolescent Medicine
• Term: from 2009 to 2013
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 104815969

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.

• DFG Programme: Research Grants