Joseph Italiano Jr., PhD
Welcome to the Italiano Lab. Our research uses cell and molecular biology methods to address problems in megakaryocyte and platelet biology. The lab’s research focuses primarily on how blood platelets, which function as the band-aids of the bloodstream, are produced from megakaryocyte precursor cells. Specifically, the lab uses mouse megakaryocyte and human culture systems to study platelet production in vitro. Our primary methods include fluorescence microscopy, live cell imaging, molecular biology, biochemistry, electron microscopy, bioengineering, and knockout mice. Where possible, we attempt to study the dynamics of proteins in living megakaryocytes or reconstitute cellular process with cell extracts. Our lab has demonstrated that platelet formation follows a defined set of morphogenetic shape changes driven by forces derived from both microtubules and actin filaments. Current focuses include understanding the molecular signals that trigger platelet production, using biologically inspired engineering to establish how the bone marrow microenvironment influences platelet production, understanding how the cytoskeleton powers platelet production. We also have a major interest in understanding the non-hemostatic roles of platelets in health and disease. This includes establishing how platelets regulate new blood vessel growth, immunity, cancer, wound healing, and potentially aging.
Delineation of the Cytoskeletal Mechanisms that Trigger and Drive Proplatelet Production
The purpose of this project is to elucidate biochemical and cellular pathways that govern platelet release. Work from our and other laboratories has shown that megakaryocytes generate platelets by remodeling their cytoplasm into proplatelet extensions. In the past 20 years, we have made fundamental discoveries into the mechanics of platelet biogenesis, including identifying the cytoskeletal forces that power proplatelet elongation, defining the mechanics of organelle transport and packaging, and establishing new mechanisms of the final stages of platelet production.
A megakaryocyte forming proplatelets.
Proplatelet elongation from the megakaryocyte.
A High-Content Microscopy Screen to Identify Small Molecules that Regulate Proplatelet Initiation
We recently developed a high-content, quantitative, automated cell imaging assay using the Essen IncuCyte system to measure the rate and extent of proplatelet production in megakaryocyte cultures. We plan to extend this assay into a high-throughput screen to test thousands of bioactive small molecules to identify compounds that stimulate/inhibit platelet production. Target pathway analysis, secondary screens, and concentration-response curves will be established to identify compound “hits.” We will establish whether hits regulate in vivo platelet production and are efficacious in animal models. We expect that these studies will lead to the discovery of new drug candidates and novel signaling pathways that trigger platelet production.
Generation of Platelets In Vitro
Morbidity and mortality from bleeding due to low platelet count is a major clinical problem, and platelet transfusions are widely used for thrombocytopenia. In the U.S., platelet transfusions total well over 10 million units per year and the steady increase in demand continues to challenge the blood bank community. New strategies for generating platelets in vitro from non-donor-dependent sources are necessary to meet transfusion needs. Previously, in collaboration with Advanced Cell Technologies, we demonstrated that platelet-like particles can be generated from both embryonic stem cells as well as induced pluripotent stem cells. The lab has developed a bioreactor that uses biologically-inspired engineering to trigger megakaryocytes to make platelets. Our work has led to the development of important concepts and methodologies for generating in vitro platelets and the spinout of the biotechnology company, Platelet BioGenesis. Future studies will be aimed at understanding how the bone marrow vascular microenvironment influences platelet production.
Selinexor treatment inhibits thrombopoietin (TPO) signaling in vitro. Adapted from Machlus, et al., 2017.
Mechanisms of Drug-Induced Thrombocytopenia
Platelets are essential for blood clotting, and when their numbers are very low a patient is at serious risk of death from hemorrhage. Each day an adult produces approximately 100 billion platelets to maintain a circulating platelet concentration of 1.5-4 x 108 platelets/mL. These rates can increase by 10-fold or more when the demand for platelets suddenly increases. Medications are one source of thrombocytopenia. Typically, medications capable of causing clinically significant thrombocytopenia do so by one or both of the following mechanisms: decreased production of platelets in the bone marrow and/or increased destruction and clearance of platelets from peripheral blood. We have previously collaborated with Genentech to identify the mechanisms by which the anti-cancer drug, TDM-1 causes thrombocytopenia, as well as Karyopharm to establish how Selinexor causes thrombocytopenia. We are currently working with Ionis Pharmaceuticals to identify the mechanisms by which anti-sense oligonucleotide drugs cause platelet counts to drop.
Beyond the Band-Aids of the Blood
The fundamental role of platelets as the Band-Aids of the blood is well appreciated. However, more recently it has become increasingly apparent that platelets are not merely one-dimensional actors that only mediate thrombosis and hemostasis. Platelets play essential roles in wound healing, cancer, immunity, inflammation, liver regeneration, development of the lymphatic system, as well as rheumatoid arthritis. And the list of new platelet functions continues to grow. We like to think of platelets as the Swiss army knives of the blood. Platelets play important roles in modulating many biological processes, due in large part to their ability to adhere to various cell types. Furthermore, one of the most interesting characteristics of platelets is the large number of bioactive molecules taken up and stored in their alpha-granules (see inset), the molecules poised to be delivered to sites of vascular injury as part of the vascular release reaction. Given that each of us has roughly a trillion platelets in our blood, these cells represent one of the major transport and delivery systems in our body. We started working on the non-hemostatic role of platelets when a chance meeting with Judah Folkman, the father of the field of angiogenesis, led us to look at how platelets regulate neovascularization. Over the past several years, our interest in the other functions of platelets has expanded into studying platelet-based regulation of cancer as well as immunity. We now propose to leverage our expertise in the “other functions” of platelet biology to understand the role of platelets in in other processes, such as the pathogenesis of Alzheimer’s Disease, wound healing as well as ageing.
Electron Micrograph of a platelet.
Platelets labels with a fluorescent anti-beta-1 tubulin antibody.