α-granule biogenesis: from disease to discovery.

Platelets are critical to hemostasis and thrombosis. Upon detecting injury, platelets show a range of responses including the release of protein cargo from α-granules. This cargo is synthesized by platelet precursor megakaryocytes or endocytosed by megakaryocytes and/or platelets. Insights into α-granule biogenesis have come from studies of hereditary conditions where these granules are immature, deficient or absent. Studies of Arthrogryposis, Renal dysfunction, and Cholestasis (ARC) syndrome identified the first proteins essential to α-granule biogenesis: VPS33B and VPS16B. VPS33B and VPS16B form a complex, and in the absence of either, platelets lack α-granules and the granule-specific membrane protein P-selectin. Gray Platelet Syndrome (GPS) platelets also lack conventionally recognizable α-granules, although P-selectin containing structures are present. GPS arises from mutations affecting NBEAL2. The GPS phenotype is more benign than ARC syndrome, but it can cause life-threatening bleeding, progressive thrombocytopenia, and myelofibrosis. We review the essential roles of VPS33B, VPS16B, and NBEAL2 in α-granule development. We also examine the existing data on their mechanisms of action, where many details remain poorly understood. VPS33B and VPS16B are ubiquitously expressed and ARC syndrome is a multisystem disorder that causes lethality early in life. Thus, VPS33B and VPS16B are clearly involved in other processes besides α-granule biogenesis. Studies of their involvement in vesicular trafficking and protein interactions are reviewed to gain insights into their roles in α-granule formation. NBEAL2 mutations primarily affect megakaryocytes and platelets, and while little is known about NBEAL2 function some insights can be gained from studies of related proteins, such as LYST.

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

There is much current interest in the role of the platelet storage pool of α-granule proteins both in hemostasis and non-hemostatic events. As well as in the arrest of bleeding, the secreted proteins participate in wound healing, inflammation, and innate immunity while in pathology they may be actors in arterial thrombosis and atherosclerosis as well as cancer and metastasis. For a long time, gray platelet syndrome (GPS) has been regarded as the classic inherited platelet disorder caused by an absence of α-granules and their contents. While NBEAL2 is the major source of mutations in GPS, other gene variants may give rise to significant α-granule deficiencies in platelets. These include GATA1, VPS33B, or VIPAS39 in the arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome and now GFI1B. Nevertheless, many phenotypic differences are associated with mutations in these genes. This critical review was aimed to assess genotype/phenotype variability in disorders of platelet α-granule biogenesis and to urge caution in grouping all genetic defects of α-granules as GPS. Am. J. Hematol. 91:714-718, 2016. © 2016 Wiley Periodicals, Inc.

Megakaryocytic emperipolesis and platelet function abnormalities in five patients with gray platelet syndrome.

The gray platelet syndrome (GPS) is a rare congenital platelet disorder characterized by mild to moderate bleeding diathesis, macrothrombocytopenia and lack of azurophilic α-granules in platelets. Some platelet and megakaryocyte (MK) abnormalities have been described, but confirmative studies of the defects in larger patient cohorts have not been undertaken. We studied platelet function and bone marrow (BM) features in five GPS patients with NBEAL2 autosomal recessive mutations from four unrelated families. In 3/3 patients, we observed a defect in platelet responses to protease-activated receptor (PAR)1-activating peptide as the most consistent finding, either isolated or combined to defective responses to other agonists. A reduction of PAR1 receptors with normal expression of major glycoproteins on the platelet surface was also found. Thrombin-induced fibrinogen binding to platelets was severely impaired in 2/2 patients. In 4/4 patients, the BM biopsy showed fibrosis (grade 2-3) and extensive emperipolesis, with many (36-65%) MKs containing 2-4 leukocytes engulfed within the cytoplasm. Reduced immunolabeling for platelet factor 4 together with normal immunolabeling for CD63 in MKs of two patients demonstrated that GPS MKs display an alpha granule-specific defect. Increased immunolabeling for P-selectin and decreased immunolabeling for PAR1, PAR4 and c-MPL were also observed in MKs of two patients. Marked emperipolesis, specific defect of MK alpha-granule content and defect of PAR1-mediated platelet responses are present in all GPS patients that we could study in detail. These results help to further characterize the disease.

A case of gray platelet syndrome masked by immune thrombocytopenia at presentation.

We report a case of gray platelet syndrome (GPS) associated with immune thrombocytopenia (ITP) at presentation. A 22-year-old male patient presenting with petechiae on his limbs was diagnosed with ITP due to a gradual decrease of his platelet count to a minimum of 26 × 10(9) /liter and an elevated platelet-associated IgG (PA-IgG) level in the absence of any other specific cause of thrombocytopenia. Administration of prednisolone increased his platelet count, but this dropped again to approximately 50 × 10(9) /liter as the dose was tapered, and remained at the same level after the treatment was terminated. Thirteen years later, we reassessed the cause of the thrombocytopenia because the PA-IgG level was found to be within the normal range. There were large hypogranular platelets on the blood film and a deficit of α-granules in the platelets on electron microscopy. On this basis, we diagnosed his thrombocytopenia as GPS. To our knowledge, this is the first report of a GPS case associated with ITP at presentation. This case illustrates the importance of carefully reviewing blood film results in the differential diagnosis of thrombocytopenia.

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.

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.

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.

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.

A Case of Chronic Thrombocytopenia in a 17-Year-Old Female.

Storage pool deficiency (SPD) is a group of rare platelet disorders that result from deficiencies in α-granules, δ-granules, or both. One type of α-SPD is gray platelet syndrome (GPS), caused by mutations in the neurobeachin-like 2 (NBEAL2) gene that results in a bleeding diathesis, thrombocytopenia, splenomegaly, and progressive myelofibrosis. Due to the lack of α-granules, platelets have a gray and degranulated appearance by light microscopy. However, definitive diagnosis of GPS requires confirmation of α-granule deficiency by electron microscopy. Treatment is nonspecific, with the conservative utilization of platelet transfusions being the most important form of therapy. We present a case of a 17-year-old female with a past medical history of thrombocytopenia, first identified at the age of five. Her clinical symptomatology included chronic fatigue, gingival bleeding, bruising, menorrhagia, and leg pain. This report will discuss both the clinical and the pathophysiologic aspects of this rare platelet disorder.

Super-resolution microscopy in the diagnosis of platelet granule disorders.

INTRODUCTION: Platelet granule deficiencies lead to bleeding disorders, but their specific diagnosis typically requires whole mount transmission electron microscopy, which is often not available and has a number of important limitations. We recently proposed the use of advanced forms of fluorescence microscopy - the so-called 'super-resolution' microscopies - as a possible solution. Areas covered: In this special report, we review the diagnosis of platelet granule deficiencies, and discuss how recent developments in fluorescence microscopy may be useful in improving diagnostic approaches to these and related disorders. In particular, we conclude that super-resolution fluorescence microscopy may have advantages over transmission electron microscopy in this application. Expert commentary: The value of the super-resolution microscopies has been amply demonstrated in research; however, their potential in diagnostic applications is ripe for further exploration. Hematology is a field particularly likely to benefit because of the relative simplicity of sample preparation. We anticipate that the costs of the necessary instrumentation will continue to fall rapidly, making these technologies widely accessible to clinicians.