The inhibition effect of anti-GPIIIa49–66 antibody on megakaryocyte differentiation

Summary

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.

Keywords

Thrombocytopenia - HIV - autoantibody

^* These authors contributed equally to this work.

• References

• 1 Bierling P, Bettaieb A, Oksenhendler E. Human immunodeficiency virus-related immune thrombocytopenia. Semin Thromb Hemost 1995; 21: 68-75.
• 2 Najean Y, Rain JD. The mechanism of thrombocytopenia in patients with HIV infection. J Lab Clin Med 1994; 123: 415-420.
• 3 Karpatkin S, Nardi M, Green D. Platelet and coagulation defects associated with HIV-1-infection. Thromb Haemost 2002; 88: 389-401.
• 4 Nardi M, Tomlinson S, Greco MA. et al. Complement-independent, peroxide-induced antibody lysis of platelets in HIV-1-related immune thrombocytopenia. Cell 2001; 106: 551-561.
• 5 Nardi M, Feinmark SJ, Hu L. et al. Complement-independent Ab-induced peroxide lysis of platelets requires 12-lipoxygenase and a platelet NADPH oxidase pathway. J Clin Invest 2004; 113: 973-980.
• 6 Li Z, Nardi MA, Karpatkin S. Role of molecular mimicry to HIV-1 peptides in HIV-1-related immunologic thrombocytopenia. Blood 2005; 106: 572-576.
• 7 Li Z, Nardi MA, Wu J. et al. Platelet fragmentation requires a specific structural conformation of human monoclonal antibody against beta3 integrin. J Biol Chem 2008; 283: 3224-3230.
• 8 Stasi R, Willis F, Shannon MS. et al. Infectious causes of chronic immune thrombocytopenia. Hematol Oncol Clin North Am 2009; 23: 1275-1297.
• 9 Levy JA. HIV pathogenesis: knowledge gained after two decades of research. Adv Dent Res 2006; 19: 10-16.
• 10 Miguez-Burbano MJ, Jackson J, Jr. Hadrigan S. Thrombocytopenia in HIV disease: clinical relevance, physiopathology and management. Curr Med Chem Cardiovasc Hematol Agents 2005; 3: 365-376.
• 11 Karpatkin S. Autoimmune thrombocytopenias. Autoimmunity 2004; 37: 363-368.
• 12 Scaradavou A. HIV-related thrombocytopenia. Blood Rev 2002; 16: 73-76.
• 13 Passos AM, Treitinger A, Spada C. An overview of the mechanisms of HIV-related thrombocytopenia. Acta Haematol 2010; 124: 13-18.
• 14 Takahashi R, Sekine N, Nakatake T. Influence of monoclonal antiplatelet glycoprotein antibodies on in vitro human megakaryocyte colony formation and pro-platelet formation. Blood 1999; 93: 1951-1958.
• 15 Hasegawa Y, Nagasawa T, Kamoshita M. et al. Effects of anti-platelet glycoprotein Ib and/or IIb/IIIa autoantibodies on the size of megakaryocytes in patients with immune thrombocytopenia. Eur J Haematol 1995; 55: 152-157.
• 16 McMillan R, Wang L, Tomer A. et al. Suppression of in vitro megakaryocyte production by antiplatelet autoantibodies from adult patients with chronic ITP. Blood 2004; 103: 1364-1369.
• 17 Chang M, Nakagawa PA, Williams SA. et al. Immune thrombocytopenic purpura (ITP) plasma and purified ITP monoclonal autoantibodies inhibit mega-karyocytopoiesis in vitro. Blood 2003; 102: 887-895.
• 18 Alimardani G, Guichard J, Fichelson S. et al. Pathogenic effects of anti-glycoprotein Ib antibodies on megakaryocytes and platelets. Thromb Haemost 2002; 88: 1039-1046.
• 19 Kurata Y, Curd JG, Tamerius JD. et al. Platelet-associated complement in chronic ITP. Br J Haematol 1985; 60: 723-733.
• 20 Tsubakio T, Tani P, Curd JG. et al. Complement activation in vitro by antiplatelet antibodies in chronic immune thrombocytopenic purpura. Br J Haematol 1986; 63: 293-300.
• 21 Zauli G, Vitale M, Falcieri E. et al. In vitro senescence and apoptotic cell death of human megakaryocytes. Blood 1997; 90: 2234-2243.
• 22 Houwerzijl EJ, Blom NR, van der Want JJ. et al. Ultrastructural study shows morphologic features of apoptosis and para-apoptosis in megakaryocytes from patients with idiopathic thrombocytopenic purpura. Blood 2004; 103: 500-506.
• 23 McCrann DJ, Yang D, Chen H. et al. Upregulation of Nox4 in the aging vasculature and its association with smooth muscle cell polyploidy. Cell Cycle 2009; 8: 902-908.
• 24 McCrann DJ, Eliades A, Makitalo M. et al. Differential expression of NADPH oxidases in megakaryocytes and their role in polyploidy. Blood 2009; 114: 1243-1249.
• 25 O’Brien JJ, Spinelli SL, Tober J. et al. 15-deoxy-delta12,14-PGJ2 enhances platelet production from megakaryocytes. Blood 2008; 112: 4051-4060.
• 26 Moses A, Nelson J, Bagby GC. Jr. The influence of human immunodeficiency virus-1 on hematopoiesis. Blood 1998; 91: 1479-1495.
• 27 Zon LI, Arkin C, Groopman JE. Haematologic manifestations of the human immune deficiency virus (HIV). Br J Haematol 1987; 66: 251-256.
• 28 Prost S, Le Dantec M, Auge S. et al. Human and simian immunodeficiency viruses deregulate early hematopoiesis through a Nef/PPARgamma/STAT5 signaling pathway in macaques. J Clin Invest 2008; 118: 1765-1775.
• 29 Gibellini D, Vitone F, Buzzi M. et al. HIV-1 negatively affects the survival/maturation of cord blood CD34(+) hematopoietic progenitor cells differentiated towards megakaryocytic lineage by HIV-1 gp120/CD4 membrane interaction. J Cell Physiol 2007; 210: 315-324.
• 30 Umemoto T, Yamato M, Shiratsuchi Y. et al. Expression of Integrin beta3 is correlated to the properties of quiescent hemopoietic stem cells possessing the side population phenotype. J Immunol 2006; 177: 7733-7739.
• 31 Umemoto T, Yamato M, Shiratsuchi Y. et al. CD61 enriches long-term repopulating hematopoietic stem cells. Biochem Biophys Res Commun 2008; 365: 176-182.