Fc-independent immune thrombocytopenia via mechanomolecular signaling in platelets

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 that target the ligand-binding domain (LBD) of glycoprotein Ibα (GPIbα), 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 the 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, whereas 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.

Akt-mediated platelet apoptosis and its therapeutic implications in immune thrombocytopenia

“…However, it is intriguing that, unlike AN51 and SZ2, the anti-GPIbα antibody HIP1 could not activate Akt and induce Akt-mediated platelet activation and apoptosis. Similar effects on platelets have been reported with other anti-GPIbα antibodies (12,21). During the current submission, a report suggested that some anti-GPIbα antibodies, but not others, can exert a pulling force on GPIbα by cross-linking platelets under shear flow, which unfolds their mechanosensory domain, leading to platelet activation (21).…”

“…On the other hand, GPIbα desialylation was demonstrated to contribute to platelet clearance in an hepatocyte Ashwell-Morell receptordependent manner (20). Moreover, shear-induced unfolding of the GPIbα mechanosensory domain by anti-GPIbα monoclonal antibodies was found to trigger signaling, leading to platelet clearance (21). Therefore, while increasing evidence suggests that anti-GPIbα autoantibodies may induce platelet clearance via an Fc-independent manner, the mechanism for anti-GPIbα antibody-induced thrombocytopenia remains elusive.…”

Refrigeration-Induced Binding of von Willebrand Factor Facilitates Fast Clearance of Refrigerated Platelets

“…Botrocetin-induced binding of plasma VWF to platelets under physiological shear generates a pulling force on GPIbα and induces unfolding of its MSD, which was identified as a key step in the activation of GPIb-IX signaling and platelet clearance 15 . MSD unfolding on the platelet was detected using monoclonal antibody 5G6 that targets a 10-residue linear epitope in the MSD of human GPIbα 15, 16 . Since 5G6 is a conformation-insensitive monoclonal antibody and it binds the epitope peptide and native GPIb-IX with essentially the same affinity 20, 21 , an increase in 5G6 binding relative to the GPIbα expression level indicates an increased exposure of the 5G6 epitope in GPIbα on the platelet and is considered as an indicator of MSD unfolding 15 .…”

“…It was recently reported that binding of type 2B VWF, or botrocetin-induced binding of wild-type VWF, to platelets under physiological shear exerts a pulling force on GPIbα and induces unfolding of the MSD therein 15 . MSD unfolding thereafter induces GPIb-IX signaling, including the elevation of intracellular Ca 2+ ([Ca 2+ ] i ), phosphatidylserine (PS) exposure and platelet desialylation, and accelerates platelet clearance 15, 16 . In this study we report that refrigeration-induced VWF binding to platelet GPIbα also results in shear-dependent clearance signaling events, thereby facilitating platelet clearance upon transfusion.…”

Antibody‐mediated immunosuppression can result from RBC antigen loss independent of Fcγ receptors in mice

“…As antigen is removed on HOD RBCs as RBCs age over time, despite no difference in HEL antigen levels in vitro following incubation of HOD RBCs with anti‐HEL antibodies in the presence or absence of proteases, endothelial‐derived or other organ resident proteases, in addition to shear forces, may cumulatively impact HEL antigen levels following antibody engagement in vivo . Given that ligand has been shown to activate platelets via proteolytic cleavage of cell surface proteins in a shear‐dependent manner, it is possible that shear forces may potentially work synergistically with antibody to alter HOD cleavage site accessibility. In this setting, nonclassical type II Fc receptors outside the type I FcγR family, including CD23 and possible other lectin receptors, may engage IgG, facilitating shear‐dependent forces as antibody is tethered to the RBC through the HEL antigen.…”