Erythropoietin (EPO) stimulates proliferation of early-stage erythrocyte precursors and is widely used for the treatment of chronic anemia. However, several types of EPO-resistant anemia are characterized by defects in late-stage erythropoiesis, which is EPO independent. Here we investigated regulation of erythropoiesis using a ligand-trapping fusion protein (ACE-536) containing the extracellular domain of human activin receptor type IIB (ActRIIB) modified to reduce activin binding. ACE-536, or its mouse version RAP-536, produced rapid and robust increases in erythrocyte numbers in multiple species under basal conditions and reduced or prevented anemia in murine models. Unlike EPO, RAP-536 promoted maturation of late-stage erythroid precursors in vivo. Cotreatment with ACE-536 and EPO produced a synergistic erythropoietic response. ACE-536 bound growth differentiation factor-11 (GDF11) and potently inhibited GDF11-mediated Smad2/3 signaling. GDF11 inhibited erythroid maturation in mice in vivo and ex vivo. Expression of GDF11 and ActRIIB in erythroid precursors decreased progressively with maturation, suggesting an inhibitory role for GDF11 in late-stage erythroid differentiation. RAP-536 treatment also reduced Smad2/3 activation, anemia, erythroid hyperplasia and ineffective erythropoiesis in a mouse model of myelodysplastic syndromes (MDS). These findings implicate transforming growth factor-β (TGF-β) superfamily signaling in erythroid maturation and identify ACE-536 as a new potential treatment for anemia, including that caused by ineffective erythropoiesis.

New steps in the process of conversion of proplatelet extensions from megakaryocytes into mature platelets are defined.

TLR9 localizes to a novel intracellular compartment called the T granule to promote immune signaling by platelets.

No abstract

Proplatelet production represents a terminal stage of megakaryocyte development during which long, branching processes composed of platelet-sized swellings are extended and released into the surrounding culture. Whereas the cytoskeletal mechanics driving these transformations have been the focus of many studies, significant limitations in our ability to quantify the rate and extent of proplatelet production have restricted the field to qualitative analyses of a limited number of cells over short intervals. A novel highcontent, quantitative, live-cell imaging assay using the IncuCyte system (Essen BioScience) was therefore developed to measure the rate and extent of megakaryocyte maturation and proplatelet production under live culture conditions for extended periods of time. As proof of concept, we used this system in the present study to establish a mechanism by which trastuzumab emtansine (T-DM1), an Ab-drug conjugate currently in clinical development for cancer, affects platelet production. High-content analysis of primary cell cultures revealed that T-DM1 is taken up by mouse megakaryocytes, inhibits megakaryocyte differentiation, and disrupts proplatelet formation by inducing abnormal tubulin organization and suppressing microtubule dynamic instability. Defining the pathways by which therapeutics such as T-DM1 affect megakaryocyte differentiation and proplatelet production may yield strategies to manage drug-induced thrombocytopenias. IntroductionPlatelets are formed and released into the bloodstream by specialized precursor cells called megakaryocytes. During the final stages of development, megakaryocytes remodel their cytoplasm into long, microtubule-based proplatelet extensions from which platelets are produced. Each day, an adult human produces approximately 100 billion platelets 1 to maintain a circulating concentration of 1.5-4 ϫ 10 8 platelets/mL. Platelets are essential for blood clotting, and the rate of platelet production can increase by a factor of 10 or more when the demand for platelets suddenly increases. Conversely, when platelet numbers are very low, a patient is at serious risk of death from hemorrhage. Recent efforts to generate useful numbers of clinically viable platelets for infusion by reproducing elements of the BM vascular niche have had limited success. 2,3 Significant improvements in platelet quality and yield will require the ability to assay multiple individual components of the BM microenvironment empirically in high throughput for quantitative improvements in megakaryocyte maturation and proplatelet production before they are incorporated in scaled biomimetic systems.Drug-induced thrombocytopenia can be triggered by a wide range of medications through the inhibition of megakaryocyte development and platelet biogenesis and/or the clearance of platelets in the blood. 4,5 Approximately 10 persons per million in the United States and Europe are affected by drug-induced thrombocytopenia per year, which can range from mild to lifethreatening. 6 The slow recovery of platelet levels...