Gray platelet syndrome and defective thrombo-inflammation in Nbeal2-deficient mice
Carsten Deppermann, Deya Cherpokova, Paquita Nurden, Jan-Niklas Schulz, Ina Thielmann, Peter Kraft, Timo Vögtle, Christoph Kleinschnitz, Sebastian Dütting, Georg Krohne, Sabine A. Eming, Alan T. Nurden, Beate Eckes, Guido Stoll, David Stegner, Bernhard Nieswandt
Carsten Deppermann, Deya Cherpokova, Paquita Nurden, Jan-Niklas Schulz, Ina Thielmann, Peter Kraft, Timo Vögtle, Christoph Kleinschnitz, Sebastian Dütting, Georg Krohne, Sabine A. Eming, Alan T. Nurden, Beate Eckes, Guido Stoll, David Stegner, Bernhard Nieswandt
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Research Article Hematology

Gray platelet syndrome and defective thrombo-inflammation in Nbeal2-deficient mice

Abstract

Platelets are anuclear organelle-rich cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity. The major platelet organelles, α-granules, release proteins that participate in thrombus formation and hemostasis. Proteins stored in α-granules are also thought to play a role in inflammation and wound healing, but their functional significance in vivo is unknown. Mutations in NBEAL2 have been linked to gray platelet syndrome (GPS), a rare bleeding disorder characterized by macrothrombocytopenia, with platelets lacking α-granules. Here we show that Nbeal2-knockout mice display the characteristics of human GPS, with defective α-granule biogenesis in MKs and their absence from platelets. Nbeal2 deficiency did not affect MK differentiation and proplatelet formation in vitro or platelet life span in vivo. Nbeal2-deficient platelets displayed impaired adhesion, aggregation, and coagulant activity ex vivo that translated into defective arterial thrombus formation and protection from thrombo-inflammatory brain infarction following focal cerebral ischemia. In a model of excisional skin wound repair, Nbeal2-deficient mice exhibited impaired development of functional granulation tissue due to severely reduced differentiation of myofibroblasts in the absence of α-granule secretion. This study demonstrates that platelet α-granule constituents are critically required not only for hemostasis but also thrombosis, acute thrombo-inflammatory disease states, and tissue reconstitution after injury.

Authors

Carsten Deppermann, Deya Cherpokova, Paquita Nurden, Jan-Niklas Schulz, Ina Thielmann, Peter Kraft, Timo Vögtle, Christoph Kleinschnitz, Sebastian Dütting, Georg Krohne, Sabine A. Eming, Alan T. Nurden, Beate Eckes, Guido Stoll, David Stegner, Bernhard Nieswandt

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Figure 3

Paucity of α-granules but unaltered ultrastructure in Nbeal2–/– MKs.

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Paucity of α-granules but unaltered ultrastructure in Nbeal2–/– MKs. (...
(A) Representative transmission electron microscopy images of mature bone marrow MKs. Normally developed demarcation membrane system with α-granules in a wild-type mouse (top left). Scale bar: 1 μm. The demarcation membrane system continues to be seen in Nbeal2–/– MKs (top right) but with a deficiency of α-granules and a markedly increased number of vacuoles in Nbeal2–/– MKs. Scale bar: 1 μm. Note the presence of a leukocyte within the cytoplasm of the mutant MK (bottom left). Scale bar: 3 μm. Protrusions from the MK cytoplasm and developing proplatelets could be clearly seen in Nbeal2–/– MKs (bottom right). Scale bar: 1 μm. VS, vascular sinus. (B) Analysis of actin (red) and tubulin-containing (green) structures in fetal liver cell–derived (FLC-derived) proplatelet-forming MKs by confocal microscopy. Nuclei were counterstained with DAPI (blue). Proplatelet formation was unaltered in FLC-derived MKs. Results are quantified as the percentage of proplatelet-forming MKs per visual field ± SD from ≥ 7 samples per group. Scale bar: 50 μm. (C) Distribution of VWF (green) in FLC- and bone marrow–derived MKs. Note absence of VWF staining in some Nbeal2–/– MKs and the accumulation of VWF in other mutant cells, in contrast to a uniform distribution in wild-type MKs. The actin cytoskeleton was visualized using phalloidin (red). Scale bar: 25 μm. (D) ELISA assay of plasma VWF, fibrinogen, and PF4 content. Data are presented as ΔOD 450 nm–OD 620 nm of 5 mice per group.