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Fc-independent immune thrombocytopenia via mechanomolecular signaling in platelets

M. Edward Quach, Matthew A. Dragovich, Wenchun Chen, Anum K. Syed, Wenpeng Cao, Xin Liang, Wei Deng, Simon F. De Meyer, Guangheng Zhu, Jun Peng, Heyu Ni, Carolyn M. Bennett, Ming Hou, Jerry Ware, Hans Deckmyn, X. Frank Zhang and Renhao Li

Key points

  • Anti-GPIbα antibodies exert a pulling force on platelet GPIbα by crosslinking platelets under shear flow.

  • A mechanical feature of an anti-GPIbα antibody, rather than affinity or epitope, determines ability to induce Fc-independent clearance.

Abstract

Immune thrombocytopenia (ITP) is a prevalent autoimmune disease characterized by autoantibody-induced platelet clearance. Some ITP patients are refractory to standard immunosuppressive treatments such as intravenous immunoglobulin (IVIG). These patients often have autoantibodies targeting the ligand-binding domain (LBD) of glycoprotein (GP)Ibα, a major subunit of the platelet mechanoreceptor complex GPIb-IX. However, the molecular mechanism of this Fc-independent platelet clearance is not clear. Here, we report that many anti-LBD monoclonal antibodies such as 6B4, but not AK2, activated GPIb-IX in a shear-dependent manner and induced IVIG-resistant platelet clearance in mice. Single-molecule optical tweezer measurements of antibodies pulling on full-length GPIb-IX demonstrated that the unbinding force needed to dissociate 6B4 from LBD far exceeds the force required to unfold the juxtamembrane mechanosensory domain (MSD) in GPIbα, unlike the AK2-LBD unbinding force. Binding of 6B4, not AK2, induced shear-dependent unfolding of the MSD on the platelet, as evidenced by increased exposure of a linear sequence therein. Imaging flow cytometry and aggregometry measurements of platelets and LBD-coated platelet-mimetic beads revealed that 6B4 can sustain crosslinking of platelets under shear, while 6B4 Fab and AK2 cannot. These results suggest a novel mechanism by which anti-LBD antibodies can exert a pulling force on GPIb-IX via platelet crosslinking, activating GPIb-IX by unfolding its MSD and inducing Fc-independent platelet clearance.

  • Submitted May 15, 2017.
  • Accepted November 27, 2017.