Loss of G6b-B Confines Megakaryocyte Differentiation Potential and Promotes Osteosclerosis in Mice

I.C. Becker^1,2,3, G. Manukjan², Z. Nagy^2,1, M. Haffner-Luntzer⁴, M. Englert^2,1, T. Heib^2,1, T. Voegtle^2,1, G. Carina^2,1, R. Bharti¹, S. Dietrich¹, K. Mott², J. Heck^2,1, N. Schlegel², T. Heckel¹, D. Stegner^2,1, I. Pleines^2,1, A. Ignatius⁴, H. Schulze², B. Nieswandt^2,1

¹University of Wuerzburg, Wuerzburg, Germany, ²University Hospital Wuerzburg/Institute of Experimental Biomedicine, Wuerzburg, Germany, ³Boston Children's Hospital, Boston, United States, ⁴University Medical Center Ulm, Ulm, Germany

Abstract Number: OC 29.2

Meeting: ISTH 2021 Congress

Theme: Platelets and Megakaryocytes » Megakaryocytes and Thrombopoiesis

Background: Megakaryocytes (MKs) develop from hematopoietic stem cells (HSCs) in a complex differentiation process including endomitosis and cytoplasmic maturation, which eventually enables them to release platelets into the bloodstream. MK differentiation is governed by a complex interplay between extracellular signals and the temporal expression of lineage-specific transcription factors. The most important signaling pathway regulating HSC commitment towards the MK lineage is driven by thrombopoietin (TPO) and involves a phosphorylation cascade downstream of the TPO receptor c-Mpl. The immunoreceptor tyrosine-based inhibition motif receptor G6b-B (encoded by Mpig6b) is specifically expressed in the MK- and platelet lineage and its loss was previously shown to result in macrothrombocytopenia and myelofibrosis in mice, however, its role in TPO-driven MK differentiation remained elusive so far.

Aims: Our aim was to identify the role of G6b-B during MK maturation.

Methods: MK function, bone and marrow morphology in point-mutated Mpig6bmut mice lacking G6b-B were analyzed in vitro and ex vivo by immunofluorescence microscopy, flow cytometry, immunosorbent assays and micro-computed tomography.

Results: Mpig6b^mut mice presented with myelofibrosis and sex-specific differences in bone marrow (BM) morphology including a progressive osteosclerosis. MKs derived from Mpig6bmut mice exhibited a transcriptional and cytoplasmic maturation block in conjunction with markedly decreased GATA-1 levels and impaired TPO signaling resulting in the accumulation of immature MKs in the BM. Analysis of MK-derived cytokines revealed enhanced levels of transforming growth factor beta β1 in BM plasma of Mpig6bmut mice due to an increased release and cleavage of the latent protein, which promoted myelofibrosis progression and osteoblast proliferation.

Conclusions: Our findings uncover an unexpected essential role of G6b-B during MK differentiation and establish the receptor not only as a key regulator of platelet reactivity and a biomarker for myelofibrosis, but also a potential target to interfere with disease progression.
Proposed model of G6b-B-dependent TPO signaling: G6b-B- and subsequent Shp2-phosphorylation promote TPO signaling resulting in enhanced gene transcription and increased GATA-1 levels. Lack of G6b-B and reduced Shp2 activity leads to reduced TPO signaling and impaired MK lineage differentiation. Red arrows: activation; black arrows: interaction or translocation.

To cite this abstract in AMA style:

Becker IC, Manukjan G, Nagy Z, Haffner-Luntzer M, Englert M, Heib T, Voegtle T, Carina G, Bharti R, Dietrich S, Mott K, Heck J, Schlegel N, Heckel T, Stegner D, Pleines I, Ignatius A, Schulze H, Nieswandt B. Loss of G6b-B Confines Megakaryocyte Differentiation Potential and Promotes Osteosclerosis in Mice [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/loss-of-g6b-b-confines-megakaryocyte-differentiation-potential-and-promotes-osteosclerosis-in-mice/. Accessed September 21, 2023.

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