Neutrophil specific granule and NETosis defects in gray platelet syndrome

Abstract

Gray platelet syndrome (GPS) is an autosomal recessive bleeding disorder characterized by a lack of α-granules in platelets and progressive myelofibrosis. Rare loss-of-function variants in neurobeachin-like 2 (NBEAL2), a member of the family of beige and Chédiak-Higashi (BEACH) genes, are causal of GPS. It is suggested that BEACH domain containing proteins are involved in fusion, fission, and trafficking of vesicles and granules. Studies in knockout mice suggest that NBEAL2 may control the formation and retention of granules in neutrophils. We found that neutrophils obtained from the peripheral blood from 13 patients with GPS have a normal distribution of azurophilic granules but show a deficiency of specific granules (SGs), as confirmed by immunoelectron microscopy and mass spectrometry proteomics analyses. CD34+ hematopoietic stem cells (HSCs) from patients with GPS differentiated into mature neutrophils also lacked NBEAL2 expression but showed similar SG protein expression as control cells. This is indicative of normal granulopoiesis in GPS and identifies NBEAL2 as a potentially important regulator of granule release. Patient neutrophil functions, including production of reactive oxygen species, chemotaxis, and killing of bacteria and fungi, were intact. NETosis was absent in circulating GPS neutrophils. Lack of NETosis is suggested to be independent of NBEAL2 expression but associated with SG defects instead, as indicated by comparison with HSC-derived neutrophils. Since patients with GPS do not excessively suffer from infections, the consequence of the reduced SG content and lack of NETosis for innate immunity remains to be explored.

A Deep Dive into the Pathology of Gray Platelet Syndrome: New Insights on Immune Dysregulation

“…thrombin, protease-activated receptor 1 (PAR1)-and PAR4-AP (activating peptide) was a consistent finding in several studies 1,22,39 and seems to be a common defect in GPS patients, which may be paralleled by a reduction in PAR1 and PAR4 expression in megakaryocytes and platelets. 22,39 Although the total P-selectin pool may be normal or mildly reduced in GPS platelets, 1 P-selectin was found to be constitutively expressed on the platelet surface in one study, 18 whereas upregulation of P-selectin exposure in response to agonists, was markedly impaired, both in this work 18 and in a separate study. 39 Mice lacking NBEAL2 display platelet functional abnormalities, [30][31][32] delayed arterial thrombus formation 30 and protection from thrombo-inflammatory brain injury following focal cerebral ischemia.…”

“…Interestingly, however, neutrophil extracellular trap (NET) formation was blunted or substantially reduced in response to classic NET inducers, such as PMA, urate crystals and Candida albicans, which engage different NET-triggering pathways. 18 Considering that upstream steps of NET formation, such as ROS production and myeloperoxidase and elastase reactivity were not altered in GPS neutrophils, this finding suggests that, in addition to azurophilic granules, specific granules may play a previously unrecognized role in NET formation. This granule subset may be particularly important in the late phases of NETosis, as reflected by defective membrane rupture and chromatin web release, whereas initial steps such as chromatin decondensation and cell rounding proceeded normally.…”

“… 8 In some pedigrees, three different variants have been identified and the pathogenic role of each one of them is not clear. 6 , 8 , 10 , 18 Current gene curation efforts for platelet disorders, such as those carried out recently for Glanzmann thrombasthenia, 20 will contribute to accurate interpretation of variants. Heterozygous NBEAL2 variants may be found in unaffected carriers, who may display a mild decrease in α-granule content, although they are not thrombocytopenic.…”

“…This granule subset may be particularly important in the late phases of NETosis, as reflected by defective membrane rupture and chromatin web release, whereas initial steps such as chromatin decondensation and cell rounding proceeded normally. 18 It remains to be explored whether a particular specific granule component is responsible for this function. Increased susceptibility to infections, particularly mild upper respiratory infections and otitis media, has been previously pointed out in isolated case reports, 3 , 11 , 25 , 46 and was recently reported to affect 17% of GPS patients in the large international cohort mentioned above.…”

Inherited Platelet Disorders: An Updated Overview

“…This is a progressive syndrome, such that usually evolves towards severe thrombocytopenia during adolescence and adulthood, accompanied by a gradual degree of myelofibrosis and, in some cases, splenomegaly. Very recently, these patients have been reported to exhibit leukopenia, predisposition to autoimmune diseases, defective NETosis and to developing autoantibodies [ 51 , 53 , 158 , 159 ].…”