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 8

Impaired dermal repair due to reduced myofibroblast activity in wounds of Nbeal2-deficient mice and severely diminished mature and pro–TGF-β in Nbeal2-deficient platelets.

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Impaired dermal repair due to reduced myofibroblast activity in wounds o...
(A–C) Representative sections of wounds at 7 days after injury. Wounds of Nbeal2-deficient mice display (A) reduced development of granulation tissue but similar epithelial wound closure (H&E staining), (B) reduced amounts of collagenous granulation tissue (central darker area in picrosirius red stain), (C) and severely diminished myofibroblast numbers per wound. Scale bar: 500 μm. (D) Area of granulation tissue is significantly reduced in mutants (n = 19) versus that in wild-type mice (n = 15). (E) Area occupied by αSMA-positive myofibroblasts is significantly diminished in mutants (n = 20) versus that in controls (n = 16). (F) Reduced amounts of mature TGF-β monomer (12 kDa) and of pro–TGF-β monomer (50 kDa) in lysates from Nbeal2-deficient versus wild-type platelets (corresponding to 6 × 106 platelets each). (G) Densitometric evaluation of TGF-β signals (12 + 50 kDa), normalized to β-actin signals. n = 4 for each genotype. *P < 0.05; ***P < 0.001.