Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology, 2015.
Platelets are the smallest cellular component of circulating blood, and they are critically involved in hemostasis, thrombosis, and inflammation. Diverse pathological conditions impacting platelet production and/or clearance processes may lead to aberrant platelet counts, and pose health risks due to severe hemorrhages, thrombus formation, or impaired immune response. Current therapies for managing these abnormalities are not time- or cost-effective, and other conditions, such as infection and alloimmunization, limit their efficacy. Developing more effective therapies requires a better understanding of the molecular mechanisms underlying thrombopoiesis. Platelet production is a multistage process requiring megakaryocyte maturation and fragmentation in the bone marrow, triggered by an array of growth factors and cytokines. Neurotrophins are among the growth factors that exist in the bone marrow and act through binding tropomyosin receptor kinases (Trks) and/or low affinity receptor p75NTR.
In this study, we hypothesized that neurotrophin signaling regulates megakaryopoiesis and thrombopoiesis, and so might prove attractive as a therapeutic target. We have shown that TrkA is expressed by human megakaryocyte progenitors and mature megakaryocytes, and signaling induced by engagement of TrkA receptor with its cognate ligand, nerve growth factor (NGF), increases the number of both cell types, whereas this signaling reduces formation of platelet-like particles. In contrast, treatment with Trk receptor inhibitor, K252a, abrogates these effects and elevates the platelet-like particle counts. In order to study the regulatory role of TrkA signaling, we used Dami cells, a TrkA expressing megakaryocytic cell line, as a model. Modulation of TrkA signaling via K252a-mediated inhibition or through knocking out TrkA in Dami cells increases apoptotic cell death and platelet-like particle counts, which reveals that TrkA is important for megakaryocyte survival and interferes with platelet production. Interestingly, testing K252a in vivo does not consistently cause an increase in platelet counts in mice with either a normal or a thrombocytopenic state. Further studies with ex vivo differentiated mouse megakaryocytes demonstrated that NGF promotes megakaryocyte growth and inhibits platelet biogenesis as it does in human primary megakaryocytes and platelets but to a lower degree. Similarly, K252a-treatment decreases the number of both mouse megakaryocyte progenitors and mature megakaryocytes, however, there is no elevation in platelet-like particle counts after K252a treatment of these cells.
Overall, NGF/TrkA signaling enhances megakaryopoiesis in both human and mouse megakaryocytes; however, it negatively regulates thrombopoiesis. Manipulation of TrkA signaling by K252a treatment induces thrombopoiesis only in human cells but not in mouse cells, presumably due to species-specific differences in the cell intrinsic factors. With that, TrkA inhibition provides a new avenue for the treatment of thrombocytopenia.
