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

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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.