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 5

Nbeal2–/– platelets show defective adhesion, thrombus formation, and PS exposure under flow.

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Nbeal2–/– platelets show defective adhesion, thrombus formation, and PS...
(A) Severely impaired adhesion and thrombus formation of Nbeal2–/– platelets on collagen under flow at a shear rate of 1,700 s–1. Representative phase-contrast (bright field [BF]) and fluorescence images are shown as well as mean surface coverage and relative platelet deposition, as measured by integrated fluorescent intensity (IFI) per mm2 ± SD (n = 4 mice per group). Scale bar: 50 μm. (B) Representative phase-contrast (BF) and fluorescence images of Nbeal2–/– platelets stained with Annexin A5–DyLight 488 perfused over a collagen-coated surface at a shear rate of 1,000 s–1. Scale bar: 65 μm. (C) Procoagulant index represents the ratio of Annexin A5–positive cells to surface coverage (n = 5 mice per group). (D) Highly diluted washed platelets were stimulated with 1 μg/ml convulxin, 20 μg/ml CRP, or 20 μg/ml CRP plus 0.1 U/ml thrombin, and the percentage of Annexin A5–positive cells was determined by flow cytometry (n = 5). Data are representative of 3 independent experiments. *P < 0.05; ***P < 0.001.