Gray Platelet Syndrome-Unusual Presentation with Spontaneous Splenic Rupture: A Case Report and Literature Review.

Gray platelet syndrome (GPS) is a rare hereditary hemorrhagic disorder characterized by macrothrombocytopenia and the absence of alpha-granules in platelets. Clinically, mild-to-moderate bleeding is the main manifestation, often accompanied by thrombocytopenia, splenomegaly, and myelofibrosis. Here, we present a case of a 15-year-old male patient with a history of hepatosplenomegaly, and thrombocytopenia for 8 years, who presented with sudden generalized abdominal pain. Despite initial suspicion of gastroenteritis, diagnostic imaging revealed an extensive hemoperitoneum. Subsequent genetic testing confirmed the diagnosis of GPS, which had not been previously identified. This case highlights the importance of considering inherited platelet disorders should be considered in adolescents with long-standing thrombocytopenia, and emphasizes the need for thorough evaluation in patients with suggestive symptoms.

Cell Surface Platelet Tissue Factor Expression: Regulation by P2Y12 and Link to Residual Platelet Reactivity.

BACKGROUND: ADP-induced platelet activation leads to cell surface expression of several proteins, including TF (tissue factor). The role of ADP receptors in platelet TF modulation is still unknown. We aimed to assess the (1) involvement of P2Y1 and P2Y12 receptors in ADP-induced TF exposure; (2) modulation of TFpos-platelets in anti-P2Y12-treated patients with coronary artery disease. Based on the obtained results, we revisited the intracellular localization of TF in platelets. METHODS: The effects of P2Y1 or P2Y12 antagonists on ADP-induced TF expression and activity were analyzed in vitro by flow cytometry and thrombin generation assay in blood from healthy subjects, P2Y12-/-, and patients with gray platelet syndrome. Ex vivo, P2Y12 inhibition of TF expression by clopidogrel/prasugrel/ticagrelor, assessed by VASP (vasodilator-stimulated phosphoprotein) platelet reactivity index, was investigated in coronary artery disease (n=238). Inhibition of open canalicular system externalization and electron microscopy (TEM) were used for TF localization. RESULTS: In blood from healthy subjects, stimulated in vitro by ADP, the percentage of TFpos-platelets (17.3±5.5%) was significantly reduced in a concentration-dependent manner by P2Y12 inhibition only (-81.7±9.5% with 100 nM AR-C69931MX). In coronary artery disease, inhibition of P2Y12 is paralleled by reduction of ADP-induced platelet TF expression (VASP platelet reactivity index: 17.9±11%, 20.9±11.3%, 40.3±13%; TFpos-platelets: 10.5±4.8%, 9.8±5.9%, 13.6±6.3%, in prasugrel/ticagrelor/clopidogrel-treated patients, respectively). Despite this, 15% of clopidogrel good responders had a level of TFpos-platelets similar to the poor-responder group. Indeed, a stronger P2Y12 inhibition (130-fold) is required to inhibit TF than VASP. Thus, a VASP platelet reactivity index <20% (as in prasugrel/ticagrelor-treated patients) identifies patients with TFpos-platelets <20% (92% sensitivity). Finally, colchicine impaired in vitro ADP-induced TF expression but not α-granule release, suggesting that TF is open canalicular system stored as confirmed by TEM and platelet analysis of patients with gray platelet syndrome. CONCLUSIONS: Data show that TF expression is regulated by P2Y12 and not P2Y1; P2Y12 antagonists downregulate the percentage of TFpos-platelets. In clopidogrel good-responder patients, assessment of TFpos-platelets highlights those with residual platelet reactivity. TF is stored in open canalicular system, and its membrane exposure upon activation is prevented by colchicine.

NBEAL2 deficiency in humans leads to low CTLA-4 expression in activated conventional T cells.

Loss of NBEAL2 function leads to grey platelet syndrome (GPS), a bleeding disorder characterized by macro-thrombocytopenia and α-granule-deficient platelets. A proportion of patients with GPS develop autoimmunity through an unknown mechanism, which might be related to the proteins NBEAL2 interacts with, specifically in immune cells. Here we show a comprehensive interactome of NBEAL2 in primary T cells, based on mass spectrometry identification of altogether 74 protein association partners. These include LRBA, a member of the same BEACH domain family as NBEAL2, recessive mutations of which cause autoimmunity and lymphocytic infiltration through defective CTLA-4 trafficking. Investigating the potential association between NBEAL2 and CTLA-4 signalling suggested by the mass spectrometry results, we confirm by co-immunoprecipitation that CTLA-4 and NBEAL2 interact with each other. Interestingly, NBEAL2 deficiency leads to low CTLA-4 expression in patient-derived effector T cells, while their regulatory T cells appear unaffected. Knocking-down NBEAL2 in healthy primary T cells recapitulates the low CTLA-4 expression observed in the T cells of GPS patients. Our results thus show that NBEAL2 is involved in the regulation of CTLA-4 expression in conventional T cells and provide a rationale for considering CTLA-4-immunoglobulin therapy in patients with GPS and autoimmune disease.

Immune dysregulation, autoimmunity, and granule defects in gray platelet syndrome.

Since the description of the first case with gray platelet syndrome (GPS) in 1971, this rare inherited platelet disorder has been the focus of extensive clinical and basic research. These studies have not only increased our knowledge about the clinical manifestations of GPS but also deepened our understanding of the biogenesis of platelet α-granules and their pathophysiology in hemostasis and thrombosis. The discovery of the causal gene, neurobeachin-like 2, in 2011 was a milestone in hematology. Following this was the rapid diagnosis and phenotyping of many new patients and the further development of experimental models to characterize the pathophysiological relevance of neurobeachin-like 2 in hemostasis and immunity. The impact of altered protein function on cells other than platelets became apparent, including defects in the granules of neutrophils and monocytes and changes in the transcriptomic and proteomic profiles of other immune cells such as T lymphocytes. Besides the previously recognized clinical manifestations of macrothrombocytopenia, splenomegaly, and early-onset bone marrow fibrosis, we now recognize that immunologic abnormalities, including autoimmune diseases and recurrent infections, affect a proportion of patients with GPS. There is a proinflammatory signature of the plasma in GPS, with quantitative alterations of multiple proteins, including many produced by the liver. This review will cover the classical features of GPS and then focus on additional clinical manifestations of immune dysregulation and cellular defects beyond platelets in patients with this rare disorder.

Syntaxin 12 and COMMD3 are new factors that function with VPS33B in the biogenesis of platelet α-granules.

Platelet α-granules regulate hemostasis and myriad other physiological processes, but their biogenesis is unclear. Mutations in only 3 proteins are known to cause α-granule defects and bleeding disorders in humans. Two such proteins, VPS16B and VPS33B, form a complex mediating transport of newly synthesized α-granule proteins through megakaryocyte (MK) endosomal compartments. It is unclear how the VPS16B/VPS33B complex accomplishes this function. Here we report VPS16B/VPS33B associates physically with Syntaxin 12 (Stx12), a SNARE protein that mediates vesicle fusion at endosomes. Importantly, Stx12-deficient MKs display reduced α-granule numbers and overall levels of α-granule proteins, thus revealing Stx12 as a new component of the α-granule biogenesis machinery. VPS16B/VPS33B also binds CCDC22, a component of the CCC complex working at endosome exit sites. CCDC22 competes with Stx12 for binding to VPS16B/VPS33B, suggesting a possible hand-off mechanism. Moreover, the major CCC form expressed in MKs contains COMMD3, one of 10 COMMD proteins. Deficiency of COMMD3/CCDC22 causes reduced α-granule numbers and overall levels of α-granule proteins, establishing the COMMD3/CCC complex as a new factor in α-granule biogenesis. Furthermore, P-selectin traffics through the cell surface in a COMMD3-dependent manner and depletion of COMMD3 results in lysosomal degradation of P-selectin and PF4. Stx12 and COMMD3/CCC deficiency cause less severe phenotypes than VPS16B/VPS33B deficiency, suggesting Stx12 and COMMD3/CCC assist but are less important than VPS16B/VPS33B in α-granule biogenesis. Mechanistically, our results suggest VPS16B/VPS33B coordinates the endosomal entry and exit of α-granule proteins by linking the fusogenic machinery with a ubiquitous endosomal retrieval complex that is repurposed in MKs to make α-granules.

Generation and characterization of a human iPSC line SANi006-A from a Gray Platelet Syndrome patient.

Induced pluripotent stem cells (iPSCs) were generated from erythroblasts (EBLs) obtained from a patient diagnosed with Gray Platelet Syndrome (GPS), caused by compound heterozygous NBEAL2 mutations (c.6568delT and c.7937T>C). GPS is an autosomal recessive bleeding disorder characterized by a lack of α-granules in platelets and progressive myelofibrosis. EBLs were reprogrammed with CytoTune-iPS 2.0 Sendai Reprogramming Kit, where the generated iPSCs showed normal karyotype, expression of pluripotency associated markers and in vitro spontaneous differentiation towards the three germ layers. The generated iPSCs can be used to study GPS pathophysiology and the basic functions of NBEAL2 protein in different cell types.

Gray Platelet Syndrome Presenting With Pancytopenia, Splenomegaly, and Bone Marrow Fibrosis.

OBJECTIVES: Gray platelet syndrome (GPS) is a rare platelet storage pool disorder associated with a marked decrease or absence of platelet α-granules and their contents. It is characterized clinically by mild to moderate bleeding; moderate macrothrombocytopenia with large, agranular platelets; splenomegaly; and bone marrow fibrosis. Electron microscopy confirms markedly reduced or absent α-granules in platelets and megakaryocytes. The classic description of GPS is caused by homozygous mutations in NBEAL2 (neurobeachinlike 2). METHODS: A 28-year-old Hispanic man with a history of easy bruising and occasional episodes of epistaxis sought treatment for pancytopenia and splenomegaly. Peripheral blood smear and bone marrow analysis, electron microscopy, and next-generation sequencing were performed. RESULTS: Large and agranular platelets were present in the peripheral blood. There was bone marrow fibrosis. Electron microscopy of the platelets showed absence of α-granules. Next-generation sequencing revealed a germline apparently homozygous nonsense variant in the NBEAL2 gene: c.5674C>T, p.Gln1892X (p.Q1829X). CONCLUSIONS: The differential diagnosis of GPS includes a myeloid neoplasm such as myelodysplastic syndrome with bone marrow fibrosis. The availability of diagnostic genetic panels for hereditable platelet disorders can assist in the recognition of GPS and other platelet disorders. We also describe a previously unreported pathogenic germline homozygous nonsense variant in the NBEAL2 gene: c.5674C>T, p.Gln1892X (p.Q1829X) in a patient with GPS.

Neutrophil specific granule and NETosis defects in gray platelet syndrome.

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.

Platelet proteome and function in X-linked thrombocytopenia with thalassemia and in silico comparisons with gray platelet syndrome.

In X-linked thrombocytopenia with thalassemia (XLTT; OMIM 314050), caused by the mutation p.R216Q in exon 4 of the GATA1 gene, male hemizygous patients display macrothrombocytopenia, bleeding diathesis and a ß-thalassemia trait. Herein, we describe findings in two unrelated Swedish XLTT families with a bleeding tendency exceeding what is expected from the thrombocytopenia. Blood tests revealed low P-PAI-1 and P-factor 5, and elevated S-thrombopoietin levels. Transmission electron microscopy showed diminished numbers of platelet α- and dense granules. The proteomes of isolated blood platelets from 5 male XLTT patients, compared to 5 gender- and age matched controls, were explored. Quantitative mass spectrometry showed alterations of 83 proteins (fold change ≥±1.2, q< .05). Of 46 downregulated proteins, 39 were previously reported to be associated with platelet granules. Reduced protein levels of PTGS1 and SLC35D3 were validated in megakaryocytes of XLTT bone marrow biopsies by immunohistochemistry. Platelet function testing by flow cytometry revealed low dense- and α-granule release and fibrinogen binding in response to ligation of receptors for ADP, the thrombin receptor PAR4 and the collagen receptor GPVI. Significant reductions of a number of α-granule proteins overlapped with a previous platelet proteomics investigation in the inherited macrothrombocytopenia gray platelet syndrome (GPS). In contrast, Ca2+ transporter proteins that facilitate dense granule release were downregulated in XLTT but upregulated in GPS. Ingenuity Pathway Analysis showed altered Coagulation System and Protein Ubiquitination pathways in the XLTT platelets. Collectively, the results revealed protein and functional alterations affecting platelet α- and dense granules in XLTT, probably contributing to bleeding.

Acquired Gray Platelet Syndrome Associated with Primary Myelofibrosis.

A 53-year-old man presented with uncontrolled bleeding caused by acquired platelet dysfunction accompanied by calreticulin-mutated primary myelofibrosis. Based on the detection of abnormal platelets, including large gray platelets, under light microscopy and the loss of the second wave of aggregation observed by light transmission aggregometry, the patient was diagnosed with platelet dysfunction accompanied by myeloproliferative neoplasms (MPNs). In addition, the absence of platelet α-granules was confirmed by electron microscopy. Therefore, this condition may be termed "acquired gray platelet syndrome." Acquired platelet dysfunction must be ruled out when abnormal platelets are observed in patients with MPNs.

Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome.

Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease.

The endoplasmic reticulum protein SEC22B interacts with NBEAL2 and is required for megakaryocyte α-granule biogenesis.

Studies of inherited platelet disorders have provided many insights into platelet development and function. Loss of function of neurobeachin-like 2 (NBEAL2) causes gray platelet syndrome (GPS), where the absence of platelet α-granules indicates NBEAL2 is required for their production by precursor megakaryocytes. The endoplasmic reticulum is a dynamic network that interacts with numerous intracellular vesicles and organelles and plays key roles in their development. The megakaryocyte endoplasmic reticulum is extensive, and in this study we investigated its role in the biogenesis of α-granules by focusing on the membrane-resident trafficking protein SEC22B. Coimmunoprecipitation (co-IP) experiments using tagged proteins expressed in human HEK293 and megakaryocytic immortalized megakaryocyte progenitor (imMKCL) cells established binding of NBEAL2 with SEC22B, and demonstrated that NBEAL2 can simultaneously bind SEC22B and P-selectin. NBEAL2-SEC22B binding was also observed for endogenous proteins in human megakaryocytes using co-IP, and immunofluorescence microscopy detected substantial overlap. SEC22B binding was localized to a region of NBEAL2 spanning amino acids 1798 to 1903, where 2 GPS-associated missense variants have been reported: E1833K and R1839C. NBEAL2 containing either variant did not bind SEC22B coexpressed in HEK293 cells. CRISPR/Cas9-mediated knockout of SEC22B in imMKCL cells resulted in decreased NBEAL2, but not vice versa. Loss of either SEC22B or NBEAL2 expression resulted in failure of α-granule production and reduced granule proteins in imMKCL cells. We conclude that SEC22B is required for α-granule biogenesis in megakaryocytes, and that interactions with SEC22B and P-selectin facilitate the essential role of NBEAL2 in granule development and cargo stability.

Correction of Severe Myelofibrosis, Impaired Platelet Functions and Abnormalities in a Patient with Gray Platelet Syndrome Successfully Treated by Stem Cell Transplantation.

Gray platelet syndrome (GPS) is an inherited disorder. Patients harboring GPS have thrombocytopenia with large platelets lacking α-granules. A long-term complication is myelofibrosis with pancytopenia. Hematopoietic stem cell transplant (HSCT) could be a curative treatment. We report a male GPS patient with severe pancytopenia, splenomegaly and a secondary myelofibrosis needing red blood cells transfusion. He received an HSCT from a 10/10 matched HLA-unrelated donor after a myeloablative conditioning regimen. Transfusion independence occurred at day+21, with a documented neutrophil engraftment. At day+ 180, we added ruxolitinib to cyclosporine and steroids for a moderate chronic graft versus host disease (GVHD) and persistent splenomegaly. At day+240 GVHD was controlled and splenomegaly reduced. Complete donor chimesrism was documented in blood and marrow and platelets functions and morphology normalized. At day+ 720, the spleen size normalized and there was no evidence of marrow fibrosis on the biopsy. In GPS, HSCT may be a curative treatment in selected patients with pancytopenia and myelofibrosis.