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Ncomms8737.Pdf ARTICLE Received 20 Aug 2014 | Accepted 5 Jun 2015 | Published 17 Jul 2015 DOI: 10.1038/ncomms8737 OPEN Desialylation is a mechanism of Fc-independent platelet clearance and a therapeutic target in immune thrombocytopenia June Li1,2,3,*, Dianne E. van der Wal2,3,4,*, Guangheng Zhu2,3,*, Miao Xu1,2,3, Issaka Yougbare2,3,4,LiMa2,3,4, Brian Vadasz1,2,3, Naadiya Carrim2,3, Renata Grozovsky5, Min Ruan6, Lingyan Zhu6, Qingshu Zeng6, Lili Tao6, Zhi-min Zhai6, Jun Peng7, Ming Hou7, Valery Leytin1,2,3, John Freedman1,2,3,8, Karin M. Hoffmeister5 &HeyuNi1,2,3,4,8,9 Immune thrombocytopenia (ITP) is a common bleeding disorder caused primarily by autoantibodies against platelet GPIIbIIIa and/or the GPIb complex. Current theory suggests that antibody-mediated platelet destruction occurs in the spleen, via macrophages through Fc–FcgR interactions. However, we and others have demonstrated that anti-GPIba (but not GPIIbIIIa)-mediated ITP is often refractory to therapies targeting FcgR pathways. Here, we generate mouse anti-mouse monoclonal antibodies (mAbs) that recognize GPIba and GPIIbIIIa of different species. Utilizing these unique mAbs and human ITP plasma, we find that anti-GPIba, but not anti-GPIIbIIIa antibodies, induces Fc-independent platelet activation, sialidase neuraminidase-1 translocation and desialylation. This leads to platelet clearance in the liver via hepatocyte Ashwell–Morell receptors, which is fundamentally different from the classical Fc–FcgR-dependent macrophage phagocytosis. Importantly, sialidase inhibitors ameliorate anti-GPIba-mediated thrombocytopenia in mice. These findings shed light on Fc-independent cytopenias, designating desialylation as a potential diagnostic biomarker and therapeutic target in the treatment of refractory ITP. 1 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8. 2 Toronto Platelet Immunobiology Group, Toronto, Ontario, Canada M5B 1W8. 3 Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science of St. Michael’s Hospital, Toronto, Ontario, Canada M5B 1W8. 4 Canadian Blood Services, Ottawa, Ontario, Canada K1G 4J5. 5 Translational Medicine Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. 6 Department of Hematology, Anhui Medical University, Hefei 230032, China. 7 Department of Hematology, Qilu Hospital, Shandong University, Jinan 250012, China. 8 Department of Medicine, University of Toronto, Ontario, Canada M5S 1A8. 9 Department of Physiology, University of Toronto, Ontario, Canada M5S 1A8. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to H.N. (email: [email protected]). NATURE COMMUNICATIONS | 6:7737 | DOI: 10.1038/ncomms8737 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8737 mmune thrombocytopenia (ITP) is a common bleeding not been adequately studied. Whether antibody binding may also disorder characterized by increased destruction of autologous lead to deglycosylation and contribute to platelet clearance, and Iplatelets1–3. Low platelet counts increase the risk for bleeding, whether this may account for the differences in pathogenesis and which leads to severe intracranial haemorrhage in B5% of response to therapy in ITP has not been investigated. patients1–3. ITP patients live with the risk of fatal bleeding and In this study, by utilizing unique anti-GPIba and anti- many undergo long-term therapeutic regimens to manage platelet GPIIbIIIa monoclonal antibodies (mAbs) as well as human ITP counts, and suffer a marked decrease in quality of life4. First-line plasma samples, we show that significant platelet activation and treatments include immunosuppressive and immunomodulatory desialylation occurs predominantly in the presence of anti-GPIba, agents (that is, corticosteroids, intravenous immunoglobulin G but not anti-GPIIbIIIa antibodies in vitro and in vivo. (IVIG) and anti-RhD therapy). Splenectomy has to be considered Furthermore, anti-GPIba-mediated desialylation of platelets for patients with a persistent lack of response to treatment5. led to FcgR-independent clearance via hepatocytes. Most However, it is estimated that 15–25% of patients are inexplicably importantly, the use of sialidase inhibitors mitigated thrombo- refractory to first-line therapies and even splenectomy6. To date, cytopenia in a murine model of anti-GPIba-mediated ITP. Thus, there is no reliable measurement in the clinical setting to predict we identify a mechanism of platelet clearance previously the success or failure of any ITP treatment5,7. undescribed. In addition, we introduce a new diagnostic method Autoantibodies targeting platelet surface glycoprotein(s) (GP) to detect platelet desialylation in ITP and establish sialidase have been demonstrated to be the major factors responsible for inhibitors as a potential therapeutic for refractory ITP. platelet clearance2,8,9. Approximately 70–80% of patients have autoantibodies against GPIIbIIIa (integrin aIIbb3), 20–40% Results against the GPIb complex and some patients have auto- 11–13 Generation of novel anti-GPIba and anti-GPIIbIIIa antibodies. antibodies against both or other GPs . Platelet destruction To examine the effects of anti-GPIba and anti-GPIIbIIIa following autoantibody binding has generally been considered to antibodies on platelet function and clearance, we utilized gene- occur in the spleen, through binding of the Fc portion of deficient ( À / À ) mice to generate a panel of mouse anti-mouse- immunoglobulins on the platelet surface to FcgRIIa and FcgRIIIa 2 GPIba and -GPIIbIIIa mAbs, which possess cross-reactivity on tissue macrophages of the reticuloendothelial system . against other species, including tested human, rat and/or pig Accordingly, first-line therapies, such as IVIG and anti-Rh(D), antigens (Table 1). These are the first syngeneic anti-GPIba g target these Fc- and Fc R-dependent mechanisms to restore antibodies utilized in the study of ITP, thus circumventing platelet numbers10. Unexpectedly, we and others have identified a xenogeneic antibody complications. To our knowledge, this is novel mechanism of Fc-independent thrombocytopenia, in which also the first report that anti-GPIba antibodies can cross-react antibodies against GPIba, but not those against GPIIbIIIa, can with GPIba from different species. Further characterization of induce thrombocytopenia via their F(ab)2 (Fc independent) and these antibodies revealed that some of these mAbs affect risto- in FcgR À / À mice11,12. We further reported that most anti-GPIba cetin/botrocetin- or ADP/thrombin-induced platelet aggregation, antibody-mediated thrombocytopenia is resistant to IVIG 26 12 which are mediated by GPIba or GPIIbIIIa, respectively treatment . This is consistent with subsequent reports in 27–29 13–15 (Table 1) . Injection of these antibodies into mice induced humans, including our recent large patient cohort study . significant platelet decrease (thrombocytopenia) with no In addition, our retrospective studies suggest that ITP patients significant effects on red or white blood cell counts with anti-GPIba antibodies are also more likely to be refractory to (Supplementary Table 1). Thus, these mAbs may serve useful in 16 a steroid treatments . These data indicate that anti-GPIb the study of different animal models of human diseases, including antibodies are able to uniquely induce platelet clearance in an thrombosis and hemostasis, as well as other immune-mediated Fc-independent manner in murine models, which may also be thrombocytopenias. true in human ITP. However, the nature of this novel Fc-independent mechanism of platelet clearance is unknown. GPIIbIIIa and the GPIb complex are structurally and Anti-GPIba antibodies induce significant platelet activation. functionally distinct platelet receptors. Although different out- It has been previously reported that antibodies against GPIba side-in signalling pathways have been observed between these two may induce platelet activation25,30,31. Therefore, we investigated receptors following ligand stimulation17,18, the downstream whether our newly developed anti-GPIba mAbs may also affect effects of autoantibody binding have not been adequately platelet function. We incubated murine and human platelets studied. Thus, possible differences in pathogenesis and therapy with various anti-GPIba or anti-GPIIbIIIa mAbs of different between anti-GPIIbIIIa- and anti-GPIb-mediated ITP remain to immunoglobulin (Ig)G subclasses (Table 1). Three anti-GPIba be elucidated. mAbs (NIT A, NIT B and NIT F) were employed in our human As the second-most abundant platelet surface receptor, GPIba platelet studies as they were, to the best of our knowledge, the first is the largest subunit and possesses all known extracellular ligand- and only currently available mAbs that are cross-reactive to binding sites of the GPIb complex (that is, GPIb-IX-V). Binding human GPIba. of GPIba to the von Willebrand factor initiates GPIba outside-in Platelet granule secretion (surface expression of P-selectin) was signalling, which can subsequently activate GPIIbIIIa leading to significantly increased in the presence of all anti-GPIba mAbs platelet aggregation17,19. GPIba is also the most heavily tested in both mouse and human platelets (Fig. 1a,e). To glycosylated platelet surface protein with B60% carbohydrate circumvent biases stemming from the specific binding epitopes by weight20. It
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