Gray Platelet Research Articles

Successful hematopoietic engraftment with gray platelets after allogeneic hematopoietic stem cell transplantation from gray platelet syndrome donor

Gray platelet syndrome (GPS) is a rare inherited disorder characterized by the absence of α-granules and their constituents. It may be present with thrombocytopenia and bleeding tendency. Platelets have a large and gray appearance under light and electron microscope. A 19-year old female patient with her second relapse acute lymphoblastic leukemia had to be consolidated with allo-hematopoietic stem cell transplantation (HSCT) after achieving remission with induction chemotherapy. The only available and one mismatch compatible donor was her brother, who was previously diagnosed as GPS. Allogeneic HSCT was performed from her brother in spite of GPS, and successful neutrophil and platelet engraftment achieved at the 12th and 42nd day of reinfusion, consecutively. The engrafted and circulating thrombocytes were large and gray and had little or no α-granules under electron microscope. The patient was well with no major bleeding event and increased need for thrombocyte replacement until developing bronchiolitis obliterans organizing pneumonia and respiratory distress syndrome. Thereafter death occurred. This is the first case of successful thrombocyte engraftment with documented gray thrombocyte megakaryopoiesis after allogeneic HSCT from a GPS donor. The only noteworthy issue was the slight prolongation of engraftment.

The gray platelet syndrome

Le syndrome des plaquettes grises (SPG) est un syndrome hemorragique congenital plaquettaire. La transmission est le plus souvent recessive. Cliniquement le SPG se caracterise par des hemorragies « muqueuses » a type d’ecchymoses faciles, d’epistaxis, de gingivorragies ou de regles abondantes. On retrouve frequemment une splenomegalie, et le syndrome evolue souvent vers une myelofibrose progressive. Biologiquement, les plaquettes apparaissent pâles au frottis et la numeration des plaquettes montre une macrothrombocytopenie – deux criteres tres evocateurs. L’association avec des chiffres eleves de vitamine B12 plasmatique a recemment ete demontree. Le diagnostic repose sur l’absence de granules alpha a la microscopie electronique et la presence de nombreuses vacuoles – correspondant peut-etre a des granules alpha vides. Les examens plus pousses montrent des fonctions plaquettaires affectees partiellement, notamment l’agregation a la thrombine et au collagene. On retrouve toujours un deficit prononce en proteines secretrices alpha-granulaires (facteur plaquettaire 4, facteur de Willebrand…). Des proteines membranaires sont egalement affectees, notamment le recepteur membranaire plaquettaire du collagene I, le GPVI, clive par une activite protease inappropriee. Le SPG est essentiellement plaquettaire, avec une possibilite d’atteinte des polynucleaires neutrophiles chez certains malades. Aucune atteinte granulaire d’autres types cellulaires n’a ete rapportee – en particulier dans les cellules endotheliales de SPG, qui possedent des granules de Weibel-Palade normaux. Recemment, plusieurs equipes ont isole le gene responsable du SPG : il s’agit de NBEAL2, sur le chromosome 3p21, un membre de la famille des proteines neurobeachin-like, definies par leur homologie a la neurobeachin ou NBEA. Cette famille de proteines contient un domaine BEACH commun a de nombreuses proteines de trafic vesiculaire, dont la proteine LYST, issue du gene CHS, et dont les mutations entrainent le syndrome de Chediak-Higashi (entrainant notamment la formation de granules geants). Le mode d’action de NBEAL2 reste a elucider, mais il pourrait reguler positivement la formation des granules matures. NBEAL2 est exprimee dans de nombreux tissus, mais preferentiellement dans la lignee megacaryocytaire. L’analyse du gene NBEAL2 dans vingt-cinq familles de SPG, dans le monde entier, a permis de trouver des mutations differentes, se repartissant sur l’ensemble du gene, sans « points chauds » de mutations, et sans effet fondateur. Ces mutations entrainent soit un codon stop premature avec proteine tronquee (par deletion, anomalies d’epissage, decalage de phase de lecture), soit une substitution d’acide amine deletere. Les etudes futures permettront de mieux comprendre le role de NBEAL2 dans la biogenese des granules alpha, et de comprendre la physiopathologie du SPG (deficit des proteines alpha-granulaires, macrothrombocytopenie, myelofibrose ou encore deficit en GPVI) permettant d’envisager des therapeutiques specifiques.

The Nbeal2(-/-) mouse as a model for the gray platelet syndrome.

The gray platelet syndrome (GPS) is a rare, autosomal-recessive platelet disorder characterized by thrombocytopenia, large platelets lacking α-granules, and variable bleeding. GPS has been linked to mutations in the neurobeachin-like 2 gene (NBEAL2). We have recently characterized Nbeal2-deficient mice and shown that the absence of Nbeal2 results in defective protein sorting in megakaryocytes (MKs) and impaired α-granule biogenesis, a finding also seen for human MKs. In the mice, the lack of α-granules results in impaired aggregation, defective platelet adhesion to collagen under flow and reduced pro-coagulant activity; findings that translate into defective hemostasis and thrombosis in vivo indicating that α-granule secretion is critical for platelet plug stability. Furthermore, we revealed a role of α-granule proteins in ischemic stroke and wound healing. Thus, Nbeal2-deficient mice recapitulate the hallmarks of human GPS without showing its phenotypic heterogeneity and are a promising model to investigate the (patho-)physiological relevancy of α-granules.

Molecular Triggers of Granule Formation in Megakaryocytes and Platelets

AbstractAbstract SCI-34Platelet secretory granules develop within maturing bone marrow-resident megakaryocytes, where α-granules, δ-granules, and lysosomes are transported to extending proplatelets (1) and undergo further maturation after platelets are released into the circulation. Mature platelets contain 50 to 80 membrane-enclosed α-granules, three to eight dense (δ-) granules, and a few lysosomes. δ-granules store calcium, phosphate, ADP, ATP, and serotonin, which play important roles during platelet activation. α-granules store numerous soluble and membrane-bound proteins, including adhesion molecules, cytokines, chemokines, coagulation and fibrinolytic proteins, immunologic modulators, and an assortment of complement, growth, and pro- and antiangiogenic factors. These play important roles in clotting, angiogenesis, inflammation, wound healing, and bone remodeling, and provide defenses against infections. Insights into megakaryocyte and platelet δ-granule development have come from studying inherited δ-granule deficiencies such as Hermansky-Pudlak syndrome (HPS) and Chediak-Higashi syndrome (CHS; MIM214500), for which mouse models also exist. Several genes/proteins linked to the regulation of vesicle trafficking have been implicated in δ-granule formation. These include components of BLOC (biogenesis of lysosome-related organelles complex) protein complexes (BLOC-1, −2, and −3), known vesicle-trafficking proteins (VPS33A and the β3A and δ subunit of AP-3), and the BEACH domain, containing protein LYST. Less is known about α-granule development, in which two inherited disorders result in platelets lacking α-granules: ARC syndrome (Arthrogryposis, Renal dysfunction, and Cholestasis; MIM208085) and gray platelet syndrome (GPS; MIM139090). GPS is characterized by variable thrombocytopenia and large, gray-appearing platelets on blood smears, with α-granules and α-granule proteins markedly decreased or absent. We and others recently determined that GPS is caused by mutations in NBEAL2, encoding a BEACH protein (2, 3, 4). Our work has also shown that the large α-granule-deficient platelets in ARC syndrome can arise due to mutations in VPS33B, encoding the Sec1/Munc18 (SM) protein VPS33B involved in vesicular trafficking (5). SM proteins are known to interact with membrane-associated soluble N-ethylmaleimide-sensitive fusion (NSF)-attachment protein receptors (SNAREs) of the syntaxin subfamily. Recently we have also identified VPS16B as a VPS33B-binding protein. A patient with homozygous missense mutations in C14orf133, encoding VPS16B, has ARC syndrome, with platelets lacking α-granules and stored α-granule proteins. Thus VPS16B is also required for megakaryocyte and platelet α-granule formation, and, in contrast to GPS, in which platelets have α-granule membrane proteins such as P-selectin, VPS16 null platelets lack P-selectin. The observation that GPS and ARC platelets lack α-granules but contain δ-granules, while HPS platelets are devoid of δ-granules but contain α-granules, suggests there are distinct pathways for δ-granule and α-granule biogenesis in maturing megakaryocytes. Immunofluorescence microscopy suggests that VPS16B and VPS33B act along the trans-Golgi network/late endosome/α-granule vesicular trafficking pathway during formation of α-granules in megakaryocytes. It is predicted that complexes containing VPS33B and VPS16B facilitate docking and fusion of intracellular vesicles during α-granule formation, while NBEAL2 promotes the maturation of nascent α-granule vesicles. Disclosures:No relevant conflicts of interest to declare.

Correlation between platelet phenotype and NBEAL2 genotype in patients with congenital thrombocytopenia and -granule deficiency

The gray platelet syndrome is a rare inherited bleeding disorder characterized by macrothrombocytopenia and deficiency of alpha (α)-granules in platelets. The genetic defect responsible for gray platelet syndrome was recently identified in biallelic mutations in the NBEAL2 gene. We studied 11 consecutive families with inherited macrothrombocytopenia of unknown origin and α-granule deficiency. All of them underwent NBEAL2 DNA sequencing and evaluation of the platelet phenotype, including a systematic assessment of the α-granule content by immunofluorescence analysis for α-granule secretory proteins. We identified 9 novel mutations hitting the two alleles of NBEAL2 in 4 probands. They included missense, nonsense and frameshift mutations, as well as nucleotide substitutions that altered the splicing mechanisms as determined at the RNA level. All the individuals with NBEAL2 biallelic mutations showed almost complete absence of platelet α-granules. Interestingly, the 13 individuals assumed to be asymptomatic because carriers of a mutated allele had platelet macrocytosis and significant reduction of the α-granule content. However, they were not thrombocytopenic. In the remaining 7 probands, we did not identify any NBEAL2 alterations, suggesting that other genetic defect(s) are responsible for their platelet phenotype. Of note, these patients were characterized by a lower severity of the α-granule deficiency than individuals with two NBEAL2 mutated alleles. Our data extend the spectrum of mutations responsible for gray platelet syndrome and demonstrate that macrothrombocytopenia with α-granule deficiency is a genetic heterogeneous trait. In terms of practical applications, the screening of NBEAL2 is worthwhile only in patients with macrothrombocytopenia and severe reduction of the α-granules. Finally, individuals carrying one NBEAL2 mutated allele have mild laboratory abnormalities, suggesting that even haploinsufficiency has an effect on platelet phenotype.

The Medich giant platelet syndrome: Two new cases

Hypogranular platelet disorders in human subjects are relatively rare. They include the gray platelet syndrome, αδ storage pool deficiency, the Hermansky–Pudlak syndrome, and the white platelet syndrome. Perhaps the rarest of them all is the Medich giant platelet disorder. No additional cases of this condition have been reported since description of the first case in 2004. This study describes two children with thrombocytopenia and giant, hypogranular platelets found shortly after birth. Electron microscopic study of their platelets revealed sheets of membrane wrapped into tubes resembling scrolls. The scroll-like structures were open at both ends and often filled with glycogen particles. The abnormal structures are identical to those found in the initial case. As a result, the disorder can now be referred to as the Medich giant platelet syndrome.

International collaboration as a tool for diagnosis of patients with inherited thrombocytopenia in the setting of a developing country

Inherited thrombocytopenias (ITs) are heterogeneous genetic disorders that frequently represent a diagnostic challenge. The requirement of highly specialized tests for diagnosis represents a particular problem in resource-limited settings. To overcome this difficulty, we applied a diagnostic algorithm and developed a collaboration program with a specialized international center in order to increase the diagnostic yield in a cohort of patients in Argentina. Based on the algorithm, initial evaluation included collection of clinical data, platelet size, blood smear examination and platelet aggregation tests. Confirmatory tests were performed according to diagnostic suspicion, which included platelet glycoprotein expression, immunofluorescence for myosin-9 in granulocytes and platelet thrombospondin-1 and molecular screening of candidate genes. Thirty-one patients from 14 pedigrees were included; their median age was 32 (4-72) years and platelet count 72 (4-147)×10(9) L(-1). Autosomal dominant inheritance was found in nine (64%) pedigrees; 10 (71%) had large platelets and nine (29%) patients presented with syndromic forms. A definitive diagnosis was made in 10 of 14 pedigrees and comprised MYH9-related disease in four, while classic and monoallelic Bernard-Soulier syndrome, gray platelet syndrome, X-linked thrombocytopenia, thrombocytopenia 2 (ANKRD26 mutation) and familial platelet disorder with predisposition to acute myelogenous leukemia were diagnosed in one pedigree each. Adoption of an established diagnostic algorithm and collaboration with an expert referral center proved useful for diagnosis of IT patients in the setting of a developing country. This initiative may serve as a model to develop international networks with the goal of improving diagnosis and care of patients with these rare diseases.

Granule exocytosis is required for platelet spreading: differential sorting of α-granules expressing VAMP-7

AbstractThere has been recent controversy as to whether platelet α-granules represent a single granule population or are composed of different subpopulations that serve discrete functions. To address this question, we evaluated the localization of vesicle-associated membrane proteins (VAMPs) in spread platelets to determine whether platelets actively sort a specific subpopulation of α-granules to the periphery during spreading. Immunofluorescence microscopy demonstrated that granules expressing VAMP-3 and VAMP-8 localized to the central granulomere of spread platelets along with the granule cargos von Willebrand factor and serotonin. In contrast, α-granules expressing VAMP-7 translocated to the periphery of spread platelets along with the granule cargos TIMP2 and VEFG. Time-lapse microscopy demonstrated that α-granules expressing VAMP-7 actively moved from the granulomere to the periphery during spreading. Platelets from a patient with gray platelet syndrome lacked α-granules and demonstrated only minimal spreading. Similarly, spreading was impaired in platelets obtained from Unc13dJinx mice, which are deficient in Munc13-4 and have an exocytosis defect. These studies identify a new α-granule subtype expressing VAMP-7 that moves to the periphery during spreading, supporting the premise that α-granules are heterogeneous and demonstrating that granule exocytosis is required for platelet spreading.

The York platelet syndrome: A fourth case with unusual pathologic features

The present report describes a fourth patient with platelet pathological features identical to those found in the first three cases with the York platelet syndrome (YPS), as well as other findings that suggest he may be a variant. His platelets contain the same giant opaque and target organelles found earlier, as well as enlarged organelles with a gray appearing matrix. It is possible that the giant structures have the same source, but are at different stages of development. The fourth patient has platelet pathology suggestive of other thrombocyte disorders. He has many large platelets and normal sized thrombocytes nearly devoid of alpha granules. As a result, he was originally thought to have the gray platelet syndrome. He also has significant numbers of platelets attached to platelets and platelets in platelets as seen in patients with the X-linked GATA-1 mutation. Some of the fourth YPS patient's platelets contained massive alpha granules suggesting the possibility of the Paris Trousseau Jacobson Syndrome. Yet, none of these other platelet disorders had giant dense organelles like those found in YPS thrombocytes. As a result, it is reasonable to include this child with the other three, and diagnose him as a patient with the YPS.

Inherited platelet disorders and oral health

Platelets play a key role in thrombosis and hemostasis. Accumulation of platelets at the site of vascular injury is the first step in the formation of hemostatic plugs, which play a pivotal role in preventing blood loss after injury. Platelet adhesion at sites of injury results in spreading, secretion, recruitment of additional platelets, and formation of platelet aggregates. Inherited platelet disorders are rare causes of bleeding syndromes, ranging from mild bruising to severe hemorrhage. The defects can reflect deficiency or dysfunction of platelet surface glycoproteins, granule contents, cytoskeletal proteins, platelet pro-coagulant function, and signaling pathways. For instance, Bernard-Soulier syndrome and Glanzmann thrombasthenia are attributed to deficiencies of glycoprotein Ib/IX/V and GPIIb/IIIa, respectively, and are rare but severe platelet disorders. Inherited defects that impair platelet secretion and/or signal transduction are among the most common forms of mild platelet disorders and include gray platelet syndrome, Hermansky-Pudlak syndrome, and Chediak-Higashi syndrome. When necessary, desmopressin, antifibrinolytic agents, and transfusion of platelets remain the most common treatment of inherited platelet disorders. Alternative therapies such as recombinant activated factor VII are also available for a limited number of situations. In this review, we will discuss the management of patients with inherited platelet disorders in various clinical situations related to dental cares, including surgical intervention.

Proteomics applied to the study of platelet-related diseases: Aiding the discovery of novel platelet biomarkers and drug targets

Platelets play a fundamental role in hemostasis. Because they do not have a nucleus, proteomics is an ideal way to approach their biochemistry. Platelet proteomics is still a young field that emerged a decade ago. Initial platelet proteomic research focused on general proteome mapping followed by the exploration of sub-cellular compartments, the membrane proteome, and signaling pathways. The initial studies were later completed with the analysis of the platelet releasate and microparticle proteome. The success of these studies led to the application of platelet proteomics to the study of several pathologies where platelets play a fundamental role. Those include platelet-related disorders, such as storage pool disease, gray platelet syndrome, and Quebec platelet disorder; diseases where unwanted platelet activation is highly relevant, such as thrombosis and cardiovascular disease; and other diseases, such as cystic fibrosis, uremia, or Alzheimer's disease. In the present review article, we revise the most relevant proteomic studies on platelet-related diseases carried out to date, paying special attention to sample preparation requirements for platelet clinical proteomic studies. This article is part of a Special Issue entitled: Integrated omics.

Unusual Hematologic Disease Affecting Caucasian Children Traveling to Southeast Asia: Acquired Platelet Dysfunction with Eosinophilia

An 11-year-old American boy was staying with his family in Indonesia. He presented with a 5-month history of recurrent bruises and ecchymosis. A clinical diagnosis of acquired platelet dysfunction with eosinophilia was made when his full blood counts showed hypereosinophilia (7.4×109/L) with normal platelet count and gray platelets under the microscope. The diagnosis was supported by abnormal platelet aggregation tests consistent with a storage pool disorder. The bleeding symptoms and eosinophilia resolved a month later with a full course of antihelminthic therapy. Hematologists should be aware of this unusual disease in travelers returning from the Southeast Asia.

The Mystery of the Gray Platelets: Solved

The Mystery of the Gray Platelets: Solved

Pleiotropic platelet defects in mice with disrupted FOG1-NuRD interaction

AbstractUnderstanding platelet biology has been aided by studies of mice with mutations in key megakaryocytic transcription factors. We have shown that point mutations in the GATA1 cofactor FOG1 that disrupt binding to the nucleosome remodeling and deacetylase (NuRD) complex have erythroid and megakaryocyte lineages defects. Mice that are homozygous for a FOG1 point mutation (ki/ki), which ablates FOG1-NuRD interactions, have platelets that display a gray platelet syndrome (GPS)–like macrothrombocytopenia. These platelets have few α-granules and an increased number of lysosomal-like vacuoles on electron microscopy, reminiscent of the platelet in patients with GATA1-related X-linked GPS. Here we further characterized the platelet defect in ki/ki mice. We found markedly deficient levels of P-selectin protein limited to megakaryocytes and platelets. Other α-granule proteins were expressed at normal levels and were appropriately localized to α-granule–like structures. Treatment of ki/ki platelets with thrombin failed to stimulate Akt phosphorylation, resulting in poor granule secretion and platelet aggregation. These studies show that disruption of the GATA1/FOG1/NuRD transcriptional system results in a complex, pleiotropic platelet defect beyond GPS-like macrothrombocytopenia and suggest that this transcriptional complex regulates not only megakaryopoiesis but also α-granule generation and signaling pathways required for granule secretion.

Platelet Spreading Requires Exocytosis of a Subpopulation of α-Granules Expressing VAMP-7,

Abstract 3248There has been recent controversy as to whether platelet α-granules represent a single, homogenous granule population or are composed of different subpopulations that contain different cargos and serve discrete functions. To evaluate this question, we have studied granule movement in spreading platelets. During platelet spreading, the majority of dense and α-granules coalesce in the central granulomere. However, some granules are observed in the platelet periphery. We evaluated whether platelets actively sort a specific subpopulation of α-granules to the periphery during spreading. Granules in spread platelets were stained with antibodies directed at vesicle-associated membrane proteins (VAMPs) −3, −7, and −8. We and others have shown that VAMP-3 and −8 function in granule exocytosis. VAMP-7 contains a profilin-like N-terminal extension capable of interacting with Arp2/3. Samples were imaged using confocal microscopy. Quantitative evaluation of antibody staining in the granulomere vs. the periphery demonstrated that 93+/−7% of VAMP-8, 86+/−14% of von Willebrand factor (an α-granule cargo), and 93+/−7% of serotonin (a dense granule cargo) localized to the central granulomere. In contrast, 77+/−8% of VAMP-7 localized to the periphery. To determine whether a VAMP-7+ α-granule subpopulation actively moves from the granulomere to the platelet periphery during spreading, we loaded platelet α-granules with Qdot 565 nanocrystals and performed time-lapse video microscopy of single platelets. While the majority of endocytosed nanocrystals remained in the central granulomere during spreading, a subpopulation of labeled granules actively moved from the granulomere towards the growing edge at the platelet periphery. Colocalization studies demonstrated that 73% of peripheral Qdot 565 nanocrystals colocalized with VAMP-7. Furthermore, 82% of granules expressing VAMP-7 colocalized with P-selectin (specific for α-granules). These results indicate that a subpopulation of VAMP-7+ α-granules actively moves to the platelet periphery during spreading. To determine whether α-granules are required for spreading, we evaluated spreading in platelets from a patient with gray platelet syndrome (GPS, a congenital absence of α-granules). Platelet surface area increased 4-fold with spreading in normal controls. In contrast, the increase in surface area of GPS platelets following plating for 15 min was minimal – suggesting that α-granules are required for platelet spreading. We also evaluated spreading in murine Jinx platelets, which lack functional Munc13-4. These platelets demonstrate normal morphology and intact proximal signaling, but have a severe granule exocytosis defect. Total surface area of Munc13-4 deficient platelets spread for 15 min was 47+/−3% that of wild-type controls. Platelet spreading was also evaluated using a grated optical biosensor capable of detecting membrane contact with its surface in real time. This technique showed that spreading was decreased in Munc13-4 deficient platelets to 48+/−17% of controls. These results identify a new α-granule subtype that expresses VAMP-7 and is required for platelet spreading. This observation supports the premise that α-granules are heterogeneous and demonstrates that different VAMP isoforms localize to functionally discrete α-granule subpopulations. Disclosures:Michelson:GLSynthesis: Research Funding; Lilly/Daiichi Sankyo: Data Monitoring Committee for clinical trial, Research Funding; Takeda: Research Funding.

Whole Blood Impedance Aggregometry: A New Tool for Severe Inherited Platelet Disorder Diagnosis?

Abstract 5266Diagnosis and characterization of platelet function disorders may be challenging. It requires multiple laboratory data including the assessment of platelet functions. Platelet function analysis is most commonly performed using light transmission aggregometry (LTA). LTA is a time-consuming method requiring centrifugation steps and large blood volumes. It is difficult to perform in children and in cases of thrombocytopenia. In contrast, platelet aggregation in whole blood using impedancemetry (WBI) is a fast method, allows omission of centrifugation steps and performance of platelet function studies under more physiological conditions with small samples size. It is based on the change of resistance proportional to the amount of platelets sticking to two electrodes where an alternating current is applied. Multiplate® (for “multiple electrode aggregometry”, Dynabite Medical) is a new generation of WBI aggregometer using diluted blood and single-use test cells containing twin electrodes that reduce the variation of results. We have already showed the good Multiplate® performance concerning ristocetin-induced platelet aggregation in a population of 30 patients with characterized von Willebrand disease (Valarche et al, 2011). Our aim in this ongoing study was to assess the performance of WBI in patients with inherited platelet function disorders. We tested 8 patients including 2 unrelated patients with Glanzmann Thrombasthenia (GT), 2 unrelated patients with Bernard-Soulier Syndrome (BSS), 1 patient with Gray Platelet Syndrome (GPS) and 3 patients from the same family with a platelet type von Willebrand disease (PTVWD). GT, BSS, and PTVWD diagnosis were confirmed using genotyping. BSS and GPS patients had chronic thrombocytopenia. GT, BSS, GPS and 1/3 PTVWD had platelet function tests with LTA in parallel. WBI was performed on heparinized whole blood diluted at ½ in NaCl at 37°C and triggered using high (0.77 mg/mL, WBI RH) and low (0.5 mg/mL, WBI RL) final ristocetin concentrations, ADP (6.5 Âμ Mol, WBI ADP) and collagen (3.2 Âμg/mL, WBI Coll). Results were expressed in arbitrary unit (AU) corresponding to the area under the aggregation curve observed during 6 min. Normal ranges indicated in brackets were based on the mean +/− 2 SD of 30 healthy volunteers’ results. Results highlighted in grey are those out of the normal ranges (Table 1).Table 1:Results of the 8 patients with inherited platelet disorders.PatientsPlatelet count (109/L)WBI RH (AU) [>500]WBI RL (AU) [<150]WBI ADP (AU) [>550]WBI Coll (AU) [>500]GT 116923441443GT 224955417ND7BSS 134371119129BSS 230254733582GPS7916217ND42PTVWD22099493ND338PTVWD231116560ND1092PTVWD2341174168ND852All patients except those with PTVWD had decreased results with WBI. However, as expected, patients with GT had flat traces using WBI ADP and WBI Coll but normal or only decreased curves (234 – 554 AU) using WBI RH. On the opposite, BSS patients had flat traces using WBI RH but detectable curves using WBI ADP (191 – 335 AU) despite decreased platelet count. The thrombocytopenic GPS patient has a flat trace using WBI Coll and decreased WBI RH (162 AU). Members of the PTVWD family had normal results except a slightly increased result with WBI RH in 1/3 patients. Finally, LTA results performed in 6/8 patients were all in accordance with those of the WBI. In conclusion, in 8 patients with well characterized inherited platelet disorders, WBI was able to detect all abnormalities except PTVWD. In such cases, different ristocetin concentrations use might be critical to increase sensitivity. In our hands, WBI was able to discriminate disorders involving platelet glycoprotein (GP) IIb-IIIa from GP Ib-IX-V: GT patients exhibited flat traces using WBI ADP and WBI Coll, whereas patients with BSS exhibited flat traces with ristocetin. These preliminary results need to be confirmed on a larger population of patients with various characterized platelet function disorders. They suggest that WBI using the Multiplate® analyzer, which is a fast, easy and blood-preserving test, could be a valuable extra step before or in addition to the classic LTA for the diagnosis of severe inherited platelet disorders. Disclosures:No relevant conflicts of interest to declare.

Thrombocytopenias Due to Gray Platelet Syndrome orTHC2Mutations

Over the last two decades the genetic causes of several Mendelian platelet disorders have been elucidated, while the genetics of many other thrombocytopenic conditions are still unresolved. Among those are the gray platelet syndrome (GPS) and the thrombocytopenia linked to the THC2 locus on human chromosome 10p11-12. GPS is an α-granule defect associated with the development of myelofibrosis and mild to moderate thrombocytopenia. Most forms of GPS are autosomal recessive, and recently, the recessive form of the disease was mapped to chromosome 3p21. THC2-linked thrombocytopenia is an autosomal dominant disorder in which affected family members have a mild reduction in platelet counts and occasional bleeding. Platelets in THC2-linked thrombocytopenia appear to be normal in size and function although bone marrow morphology reveals a lack of mature, polyploid megakaryocytes. To date, mutations in three different genes within the THC2 locus have been associated with congenital thrombocytopenia, including a mutation in MASTL. In this article, we summarize the recent discoveries in these two forms of thrombocytopenia, including the functional data that support a role for MASTL kinase in thrombopoiesis.

Gray platelet syndrome

Gray platelet syndrome

Exome sequencing identifies NBEAL2 as the causative gene for gray platelet syndrome.

Gray platelet syndrome (GPS) is a predominantly recessive platelet disorder characterized by a mild thrombocytopenia with large platelets and a paucity of α-granules; these abnormalities cause mostly moderate but in rare cases severe bleeding. We sequenced the exomes of four unrelated cases and identified as the causative gene NBEAL2, a gene with previously unknown function but a member of a gene family involved in granule development. Silencing of nbeal2 in zebrafish abrogated thrombocyte formation.

NBEAL2 is mutated in gray platelet syndrome and is required for biogenesis of platelet α-granules

Gray Platelet Syndrome (GPS) is an autosomal recessive bleeding disorder with large platelets that lack α-granules. We found that mutations of NBEAL2 (neurobeachin-like 2), encoding a BEACH/ARM/WD40 domain protein, cause GPS. We demonstrated that human megakaryocytes and platelets express a unique combination of NBEAL2 transcripts. Proteomic analysis of sucrose-gradient subcellular fractions of platelets indicated that NBEAL2 localizes to the dense tubular system (endoplasmic reticulum) in platelets.