Research
Deficient platelet production, due to genetic causes, secondary to cancer therapy, or from unknown etiology, poses significant risks of mortality, mostly due to bleeding. Our primary research interests are associated with deciphering the cellular mechanisms and proteins regulating normal and abnormal blood platelet production and function.
Platelet production
Blood platelets are produced in the bone marrow by megakaryocytes in unique processes that require polyploidisation and extensive membrane rearrangements. These include the formation of the demarcation membrane system, the surface-connected membrane reservoir for future platelets. As megakaryocytes mature, the demarcation membrane system initiates as a single plasma membrane invagination that extends between the lobes of the polyploid nucleus and further expands into its mature form by addition of intracellular membrane materials that are biosynthesized in the endoplasmic reticulum or transported from the Golgi apparatus. The precise molecular mechanisms responsible for these unique membrane rearrangements remain poorly understood. Our current research is focused on the role of receptor-mediated endocytosis in platelet production and function, focusing on the F-BAR protein PACSIN2 and the large GTPase dynamin 2 (DNM2).
PACSIN2
PACSIN2 belongs to the F-BAR family of proteins that bind lipid bilayers to generate membrane tubular invaginations in cells. Single nucleotide variants in PACSIN2 have been associated with altered platelet count and mean platelet volume. We have identified PACSIN2 as an internal component of the initiating demarcation membrane system in megakaryocytes, where its membrane tubulation activity is regulated by the cytoskeletal and scaffolding protein filamin A (Begonja, Pluthero et al. Blood. 2015;126(1):80-88), a critical regulator of platelet production and function (Falet et al. J Exp Med. 2010;107(9):1967-1979; Begonja et al. Blood. 2011;118(8):2285-2295). Pacsin2–/– mice display mild thrombocytopenia and severe thrombus formation defects due to hyperactive platelet integrin β1 (Biswas, Boyd et al. J Thromb Haemost. 2023;21(12):3619-3632).
DNM2
Through its role in membrane fission, DNM2 is involved in a wide range of cellular functions, including endocytosis and vesicle transport. DNM2 mutations have been associated with Charcot-Marie-Tooth disease, centronuclear myopathy, and T-cell acute lymphoblastic leukemia in humans and Dnm2 deletion results in early embryonic lethality in mice. Dnm2fl/fl Pf4-Cre (Dnm2Plt–/–) mice specifically lacking DNM2 in the platelet lineage develop a severe macrothrombocytopenia (Bender, Giannini et al. Blood. 2015;125(6):1014-1024). Dnm2Plt–/– megakaryocytes accumulate arrested clathrin-coated vesicles that alter their demarcation membrane system, demonstrating that DNM2-dependent endocytosis is required for normal megakaryocyte development. Dnm2Plt–/– mice also display a profound bleeding diathesis due to impaired platelet hemostatic function (Eaton et al. Haematologica. 2020;105(5):1414-1423).
Myelofibrosis
Dnm2Plt–/– mice develop hallmarks of myelofibrosis, such as massive megakaryocyte hyperplasia, hematopoietic stem and progenitor cell proliferation, bone marrow fibrosis, extramedullary hematopoiesis, and severe splenomegaly (Bender, Giannini et al. Blood. 2015;125(6):1014-1024). The phenotype is reminiscent of patients with myelofibrosis that is characterized by uncontrolled JAK/STAT signaling in hematopoietic stem cells. At the cellular level, Dnm2Plt–/– platelets are unable to endocytose the thrombopoietin receptor Mpl, leading to elevated circulating thrombopoietin levels stimulating hematopoietic stem cells (Eaton et al. Front Oncol. 2022;12:959806).
In summary, our research addresses the gap of knowledge on receptor-mediated endocytosis in platelet production and function. We anticipate that findings obtained from these studies will yield better understanding to obtain basic science information on how membrane rearrangements contribute to platelet production and function, and to develop treatments for effective re-establishment of platelet production in the setting of thrombocytopenia.