title: The inhibition effect of anti-GPIIIa49-66 antibody on megakaryocyte differentiation.
abstract: We previously reported that patients with early-onset HIV-1 ITP developed a unique anti-platelet integrin GPIIIa antibody against the GPIIIa49-66 epitope. Anti-GPIIIa49-66 antibody-induced platelet fragmentation requires sequential activation of the platelet 12-lipoxygenase (12-LO) and NADPH oxidase to release reactive oxygen species (ROS). 12-LO is upstream of the NADPH oxidase pathway and 12(S)-HETE, the product of 12-LO, induces the same oxidative platelet fragmentation as anti-GPIIIa49-66. Since the megakaryocyte (MK) is the progenitor cell for platelets, we have investigated the effect of anti-GPIIIa49-66 on MK differentiation and, in particular, the potential role of anti-GPIIIa49-66 induced ROS in this process. We first show that polyclonal anti-GPIIIa49-66 antibody isolated from HIV-1 ITP patients inhibits MK proliferation 2.5-fold in in vitro culture of human cord blood CD34+ cells driven by thrombopoietin (TPO). We also observe a three-fold decrease in the number of MK colony-forming units in the presence of a human monoclonal anti-GPIIIa49-66 antibody. However, we could not detect ROS release in DCFH-loaded mouse megakaryoblastic cells L8057 treated with anti-GPIIIa49-66 antibody. In addition, 12(S)-HETE does not inhibit the in vitro differentiation of L8057 cells induced by TPO. In fact, we found a dose dependent increase in the percentage of CD41 positive cells (from 17.1% to 48.7%) in in vitro culture of L8057 cells treated with various concentrations of H2O2 (from 5 to 20 μM). We therefore conclude that the anti-GPIIIa49-66 antibody inhibits MK differentiation through β3 integrin signalling independent of ROS release.
authors.affiliation: Department of Medicine, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA. zongdong.li@nyumc.org
authors.forename: Ruimin
authors.initials: R
authors.lastname: Pan
authors.forename: Jianhui
authors.initials: J
authors.lastname: Wang
authors.forename: Michael A
authors.initials: MA
authors.lastname: Nardi
authors.forename: Zongdong
authors.initials: Z
authors.lastname: Li
mesh_terms: D019138:Antibodies, Blocking / Q000494:pharmacology; D001323:Autoantibodies / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D003602:Cytotoxicity, Immunologic / Q000187:drug effects / Q000502:physiology; D015658:HIV Infections / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology* / Q000472:pathogenicity; D006801:Humans; D039661:Integrin beta3 / Q000276:immunology / Q000378:metabolism; D008533:Megakaryocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D019138:Antibodies, Blocking; D001323:Autoantibodies; D039661:Integrin beta3; D010446:Peptide Fragments; D017382:Reactive Oxygen Species