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.

Podoplanin and CLEC-2 drive cerebrovascular patterning and integrity during development

“…However, the mild hemorrhagic phenotype observed in NBEAL-2-deficient embryos, with ,20% exhibiting hemorrhaging, argues against a major role of platelet a-granule secretion in development and is consistent with the mild bleeding diathesis observed in adult NBEAL-2 mice. 32 We further considered a role for platelet-dense granules, which release a range of nonprotein molecules that can promote platelet activation, but saw no defect in vascular development 33 However, we did observe hemorrhaging in 75% of embryos where both a-granule and dense-granule secretion was abolished. These data strongly suggest a significant level of redundancy exists between different platelet activation pathways to compensate for the targeted loss of a single pathway.…”

Targeted downregulation of platelet CLEC-2 occurs through Syk-independent internalization

“…Platelet preparation, western blotting, flow cytometry, immunofluorescence staining, and in vitro differentiation of fetal liver cell-derived MKs were performed as previously reported 17,24,25 and are described in detail in the supplemental Methods.…”

Should any genetic defect affecting α‐granules in platelets be classified as gray platelet syndrome?

“…1). Studies on various Nbeal2 knockout mouse models confirm that a-granules fail to form in MKs and/or be translocated into proplatelets and newly formed platelets [15][16][17]. These mouse models of GPS highlight how platelet a-granule proteins intervene in tissue repair, cause an inflammatory state in the marrow and contribute to thromboinflammatory states in the circulation (e.g., cerebral ischemia) and facilitate cancer metastasis thereby further raising interest in the disease.…”

“…Emperipolesis of leukocytes by MKs was frequently observed. Intriguingly, mature MKs were visualized aligned in clusters along vascular sinuses (not seen in mouse models of GPS) [15]. Immunophenotyping failed to reveal defects within myeloid or erythroid lineages.…”