Platelets are specialized anucleate cells that play an essential role in hemostasis, angiogenesis, immunity, and
inflammation. Thrombocytopenia (platelet counts <150x109/L) is a major clinical problem encountered across a
number of conditions including immune (idiopathic) thrombocytopenic purpura, myelodysplastic syndromes,
chemotherapy, surgery, and genetic disorders. The demand for platelets—and for an improved understanding
of their mechanistic formation—is at an all-time high. This program will use a multi-prong approach to
investigate megakaryocytes (MKs) to discover therapeutic strategies and molecular targets that drive
proplatelet formation and increase platelet counts. MKs are precursor cells that generate platelets by
remodeling their cytoplasm into beaded proplatelet processes, which function as the assembly lines for platelet
production. While we know that cytoskeletal mechanics power platelet production, many questions about
platelet biogenesis remain unanswered. We know that microtubule-based forces are critical for proplatelet
elongation; however, there is a surprising lack of understanding of the mechanisms that trigger platelet
production. We hypothesize that centrosome regulation, via super spindle formation and KIFC1 motor
involvement, is critical for the initiation of platelet production. We will use a novel high-content microscopy
screen to identify the small molecules and signaling pathways that drive platelet production. Using proplatelet
image analysis, we will test thousands of drug molecule candidates for their ability to stimulate or inhibit
platelet production; target pathway analysis, secondary screens, and dose-response curves will be established
to identify compound “hits.” While we know that proplatelet protrusions extend from bone marrow, breach the
endothelial barrier, and deposit platelets into the blood, we do not know how. Therefore, we will employ bio-
engineering and a unique microfluidic bone marrow on-a-chip to test the idea that actin-driven megakaryocyte
podosomes provide a mechanism to penetrate the endothelium. This chip will also be used to study how
organelles are transported into assembling platelets under physiological conditions, and to test the hypothesis
that super spindle assembly functions as a major transport hub for distributing these organelles. We will
determine if vascular thiol isomerases play a role in new platelet granule biology through investigating how
they are packaged, transported, and exocytosed from platelets. We expect that findings from this
investigation will 1) advance the understanding of the mechanisms that initiate and regulate platelet
formation, and 2) identify novel therapeutic targets and approaches to accelerate platelet production in
patients with thrombocytopenia. The R35 structure is necessary given the relative immaturity of the MK field
and will provide vital time and focus to expanding the current base of knowledge. This proposal will coordinate
a diverse group of collaborators, provide the field with novel data and theory, and support junior scientists with
consistent mentorship and proven leadership from a laboratory with broad ranging translational experience.