Key Points• The platelet defect associated with Paris-Trousseau thrombocytopenia and Jacobsen syndrome is caused by an abnormal transcription factor FLI1.• FLI1 DNA-binding ETS domain mutations cause bleeding disorders with both autosomal dominant and recessive patterns of inheritance.Hemizygous deletion of a variable region on chromosome 11q containing FLI1 causes an inherited platelet-related bleeding disorder in Paris-Trousseau thrombocytopenia and Jacobsen syndrome. These multisystem disorders are also characterized by heart anomalies, changes in facial structure, and intellectual disability. We have identified a consanguineous family with autosomal recessive inheritance of a bleeding disorder that mimics Paris-Trousseau thrombocytopenia but has no other features of the 11q23 deletion syndrome. Affected individuals in this family have moderate thrombocytopenia; absent collagen-induced platelet aggregation; and large, fused a-granules in 1% to 5% of circulating platelets. This phenotype was caused by a FLI1 homozygous c.970C>T-point mutation that predicts an arginine-to-tryptophan substitution in the conserved ETS DNA-binding domain of FLI1. This mutation caused a transcription defect at the promoter of known FLI1 target genes GP6, GP9, and ITGA2B, as measured by luciferase assay in HEK293 cells, and decreased the expression of these target proteins in affected members of the family as measured by Western blotting of platelet lysates. This kindred suggests abnormalities in FLI1 as causative of Paris-Trousseau thrombocytopenia and confirms the important role of FLI1 in normal platelet development. (Blood. 2015;126(17):2027-2030

H eparin-induced thrombocytopenia (HIT) is a prothrombotic adverse reaction to heparin characterised by platelet-activating antibodies (almost exclusively IgG) that recognise and bind to platelet factor 4 (PF4)-heparin complexes. While antibody formation is common (up to 50% in the setting of cardiac surgery), only a small proportion of patients (0.2-3.0%) develop HIT (ie, thrombocytopenia and/or thrombosis). 1 Thrombosis can be arterial, venous or microvascular. Incidence of HIT varies depending on clinical setting (higher in surgical than medical, rarely in paediatric or obstetric), type of heparin (unfractionated heparin has higher rates than low molecular weight heparin), and dose (therapeutic v prophylactic). 2 PF4 tetramers (cationic) and heparin (anionic) associate by charge and hence certain molar concentrations (1:1 molar ratio) facilitate charge neutralisation, conformational change in PF4-exposing pathogenic epitopes, antibody binding and PF4-heparin-IgG complex formation. 3 PF4 can also bind to other polyanions, including glycosaminoglycans and chondroitin sulfate, and such interactions may play a role in the increasingly reported entity known as spontaneous HIT syndrome; that is, patients with clinical and laboratory features of HIT without exposure to heparin.Thrombocytopenia and thrombosis in HIT result from the binding of PF4-heparin-IgG complex to FcγRIIa receptors on the platelet surface. Subsequent cross-linking of the receptors leads to intense platelet activation, release of platelet granule content and procoagulant microparticles, thrombin generation, and activation of endothelial cells, neutrophils and monocytes. 4HIT is a clinicopathological entity and therefore requires integration of clinical and laboratory results. Accurate diagnosis is important and consultation with an appropriate specialist is recommended as delay in diagnosis and appropriate anticoagulant treatment is associated with an initial 6% daily risk of thromboembolism as well as amputation and death. 5 MethodsAustralian and New Zealand experts in the field of thrombosis and haemostasis who regularly diagnose and treat HIT syndrome were invited to a consensus statement development panel. This group, the Thrombosis and Haemostasis Society of Australia and New Zealand (THANZ) HIT Writing Group, represents THANZ members and received formal endorsement from the THANZ President. A comprehensive literature review, using common online databases, was undertaken for each subsection by at least two authors. Major computerised databases such as Cochrane, MEDLINE and EMBASE were utilised. These searches were further supplemented with review of reference lists and professional society information available from internet sources. A draft for each subsection, collated by its two authors after extensive literature review, was submitted to the lead author (JJ). Following this comprehensive review of the literature, a face-to-face meeting was held over 2 days to discuss specific questions and finalise a draft of the consensus statement. Durin...

MYH9-related disorders (MYH9-RDs) caused by mutation of the MYH9 gene which encodes non-muscle myosin heavy-chain-IIA (NMMHC-IIA), an important motor protein in hemopoietic cells, are the most commonly encountered cause of inherited macrothrombocytopenia. Despite distinguishing features including an autosomal dominant mode of inheritance, giant platelets on the peripheral blood film accompanied by leucocytes with cytoplasmic inclusion bodies (döhle-like bodies), these disorders remain generally under-recognized and often misdiagnosed as immune thrombocytopenia (ITP). This may result in inappropriate treatment with corticosteroids, immunosupressants and in some cases, splenectomy. We explored the efficacy of next generation sequencing (NGS) with a candidate gene panel to establish the aetiology of thrombocytopenia for individuals who had been referred to our center from hematologists in the Australasian region in whom the cause of thrombocytopenia was suspected to be secondary to an inherited condition but which remained uncharacterized despite phenotypic investigations. Pathogenic MYH9 variants were detected in 15 (15/121, 12.4%) individuals and the pathogenecity of a novel variant of uncertain significance was confirmed in a further two related individuals following immunofluorescence (IF) staining performed in our laboratory. Concerningly, only one (1/17) individual diagnosed with MYH9-RD had been referred with this as a presumptive diagnosis, in all other cases (16/17, 94.1%), a diagnosis was not suspected by referring clinicians, indicating a lack of awareness or a failing of our diagnostic approach to these conditions. We examined the mean platelet diameter (MPD) measurements as a means to better identify and quantify platelet size. MPDs in cases with MYH9-RDs were significantly larger than controls (p < 0.001) and in 91% were greater than a previously suggested threshold for platelets in cases of ITP. In addition, we undertook IF staining in a proportion of cases and confirm that this test and/or NGS are satisfactory diagnostic tests. We propose that fewer cases of MYH9-RDs would be missed if diagnostic algorithms prioritized IF and/or NGS in cases of thrombocytopenia associated with giant platelets, even if döhle-like bodies are not appreciated on the peripheral blood film. Finally, our report describes the long-term use of a thrombopoietin agonist in a case of MYH9-RD that had previously been diagnosed as ITP, and demonstrates that treatment with these agents may be possible, and is well tolerated, in this group of patients.

Essentials Platelet function defects may cause atypical bleeding symptoms in immune thrombocytopenia (ITP).An isolated platelet defect of collagen‐induced aggregation was explored in a patient with ITP.ITP mediated by antibodies to glycoprotein (GP) VI curtail receptor function.Inclusion of GPVI in diagnostic antibody detection assays may improve their diagnostic utility. Idiopathic immune thrombocytopenia (ITP) is an autoimmune disorder characterized by relapsing/ remitting thrombocytopenia. Bleeding complications are infrequent with platelet counts above 30×109/L, and this level is commonly used as a threshold for treatment. The question of another/ co‐existent diagnosis or an alternate mechanism of platelet destruction arises when bleeding is experienced with platelet counts above this threshold. We report a case of anti‐GPVI mediated ITP that was diagnosed following investigations performed to address this key clinical question. A patient with ITP experienced exaggerated bruising symptoms despite a platelet count of 91×109/L. Platelet functional testing showed an isolated platelet defect of collagen‐induced aggregation. Next generation sequencing excluded a pathogenic variant of GP6, and anti‐GPVI antibodies that curtailed GPVI function were confirmed by extended platelet phenotyping. We propose that anti‐GPVI mediated ITP may be under‐recognized, and that inclusion of GPVI in antibody detection assays may improve their diagnostic utility and in turn, facilitate a better understanding of ITP pathophysiology and aid individualized treatment approaches.

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Morel-Kopp M-C. Receptor homodimerization plays a critical role in a novel dominant negative P2RY12 variant identified in a family with severe bleeding. J Thromb Haemost 2018; 16: 44-53. Essentials• Three dominant variants for the autosomal recessive bleeding disorder type-8 have been described. • To date, there has been no phenotype/genotype correlation explaining their dominant transmission.• Proline plays an important role in P2Y12R ligand binding and signaling defects.• P2Y12R homodimer formation is critical for the receptor function and signaling.Summary. Background: Although inherited platelet disorders are still underdiagnosed worldwide, advances in molecular techniques are improving disease diagnosis and patient management. Objective: To identify and characterize the mechanism underlying the bleeding phenotype in a Caucasian family with an autosomal dominant P2RY12 variant. Methods: Full blood counts, platelet aggregometry, flow cytometry and western blotting were performed before next-generation sequencing (NGS). Detailed molecular analysis of the identified variant of the P2Y12 receptor (P2Y12R) was subsequently performed in mammalian cells overexpressing receptor constructs. Results: All three referred individuals had markedly impaired ADP-induced platelet aggregation with primary wave only, despite normal total and surface P2Y12R expression. By NGS, a single P2RY12:c.G794C substitution (p.R265P) was identified in all affected individuals, and this was confirmed by Sanger sequencing. Mammalian cell experiments with the R265P-P2Y12R variant showed normal receptor surface expression versus wild-type (WT) P2Y12R. Agonist-stimulated R265P-P2Y12R function (both signaling and surface receptor loss) was reduced versus WT P2Y12R. Critically, R265P-P2Y12R acted in a dominant negative manner, with agonist-stimulated WT P2Y12R activity being reduced by variant coexpression, suggesting dramatic loss of WT homodimers. Importantly, platelet P2RY12 cDNA cloning and sequencing in two affected individuals also revealed three-fold mutant mRNA overexpression, decreasing even further the likelihood of WT homodimer formation. R265 located within extracellular loop 3 (EL3) is one of four residues that are important for receptor functional integrity, maintaining the binding pocket conformation and allowing rotation following ligand binding. Conclusion: This novel dominant negative variant confirms the important role of R265 in EL3 in the functional integrity of P2Y12R, and suggests that pathologic heterodimer formation may underlie this family bleeding phenotype.

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Development of SCC is a previously unreported adverse reaction to nilotinib.