title: Intramedullary megakaryocytes internalize released platelet factor 4 and store it in alpha granules.
abstract: BACKGROUND
  Megakaryocytes express and store platelet factor 4 (PF4) in alpha granules. In vivo, PF4 is a clinically relevant, negative regulator of megakaryopoiesis and hematopoietic stem cell replication. These findings would suggest a regulated source of free intramedullary PF4.
  
  
  OBJECTIVE
  Define the source of free intramedullary PF4 and its intramedullary life cycle.
  
  
  METHODS
  We interrogated both murine and human bone marrow-derived cells during megakaryopoiesis in vitro by using confocal microscopy and enzyme-linked immunosorbent assay. With immunohistochemistry, we examined in vivo free PF4 in murine bone marrow before and after radiation injury and in the setting of megakaryocytopenia and thrombocytopenia.
  
  
  RESULTS
  Exogenously added human PF4 is internalized by murine megakaryocytes. Human megakaryocytes similarly take up murine PF4 but not the related chemokine, platelet basic protein. Confocal microscopy shows that internalized PF4 colocalizes with endogenous PF4 in alpha granules and is available for release on thrombin stimulation. Immunohistochemistry shows free PF4 in the marrow, but not another alphagranule protein, von Willebrand factor. Free PF4 increases with radiation injury and decreases with megakaryocytopenia. Consistent with the known role of low-density lipoprotein receptor-related protein 1 in the negative paracrine effect of PF4 on megakaryopoiesis, PF4 internalization is at least partially low-density lipoprotein receptor-related protein 1 dependent.
  
  
  CONCLUSIONS
  PF4 has a complex intramedullary life cycle with important implications in megakaryopoiesis and hematopoietic stem cell replication not seen with other tested alpha granule proteins.
authors.affiliation: Department of Pediatrics Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.|Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors.forename: M P
authors.initials: MP
authors.lastname: Lambert
authors.affiliation: Departments of Pathology & Laboratory Medicine, and Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: R
authors.initials: R
authors.lastname: Meng
authors.affiliation: Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors.forename: L
authors.initials: L
authors.lastname: Xiao
authors.affiliation: Departments of Pathology & Laboratory Medicine, and Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.|Department of Pathology & Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors.forename: D C
authors.initials: DC
authors.lastname: Harper
authors.affiliation: Departments of Pathology & Laboratory Medicine, and Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.|Department of Pathology & Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors.forename: M S
authors.initials: MS
authors.lastname: Marks
authors.affiliation: Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.|Institute of Medical Biology, Polish Academy of Science, Lodz, Poland.
authors.forename: M A
authors.initials: MA
authors.lastname: Kowalska
authors.affiliation: Department of Pediatrics Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.|Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors.forename: M
authors.initials: M
authors.lastname: Poncz
mesh_terms: D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D003594:Cytoplasmic Granules / Q000378:metabolism* / Q000528:radiation effects; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008533:Megakaryocytes / Q000378:metabolism* / Q000528:radiation effects; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D010978:Platelet Factor 4 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism; D013921:Thrombocytopenia / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D034061:Thrombopoiesis; D013997:Time Factors; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D026503:Low Density Lipoprotein Receptor-Related Protein-1; C486645:Lrp1 protein, mouse; D011973:Receptors, LDL; D025521:Tumor Suppressor Proteins; D010978:Platelet Factor 4
keywords: diphtheria toxin; low density lipoprotein receptor-related protein-1; megakaryocytes; platelet factor 4; radiation injury; thrombocytopenia