Identification of three novel pathogenic ITGA2B and one novel pathogenic ITGB3 mutations in patients with hereditary Glanzmann's thrombasthenia living in Eastern Turkey.

Glanzmann's thrombasthenia (GT) is an autosomal recessive disorder in which the underlying problem is the lack or dysfunction of the GpIIb/IIIa receptor on the platelet surface. The present study determines the genetic mutation typology and analyzes the association between mutation types and clinical findings in patients diagnosed with GT who were followed up in Department of Pediatric Hematology of the Yüzüncü Yil University School of Medicine. The medical charts of 17 patients who underwent therapy and that were followed up in the Department of Pediatric Hematology of the Yüzüncü Yil University Dursun Odabas Medical Center between January 2008 and April 2018 were reviewed retrospectively. Data on such patient characteristics as present genetic mutations, age, gender, age at diagnosis, presenting symptoms, clinical findings, and platelet count and volume were garnered from the patient records. Of the 12 patients identified with genetic mutations, six had the same type of mutation, while four were identified with novel mutations that have to date not been defined in literature. Of these four mutations, three were located in the ITGA2B gene and one in the ITGB3 gene. The present study identified no significant association between the genetic and clinical findings of the patients. Novel mutations were identified in four cases in the present study. No association was found between the mutation type, and the bleeding scores and bleeding phenotypes of the patients. Further studies involving a larger number of patients are required to determine the relationship between the genotypes and clinical findings in patients with GT.

Clinico-hematological and thromboelastographic profiles in glanzmann's thrombasthenia.

: Glanzmann's thrombasthenia is a rare inherited bleeding disorder characterized by the quantitative or qualitative defect of glycoprotein IIb/IIIa receptor on platelets which leads to ineffective aggregation. Light transmittance aggregometry is considered as the gold standard for diagnosis of Glanzmann's thrombasthenia. Thromboelastography (TEG) is a global hemostatic assay which measures clot formation, clot strengthening and fibrinolysis. This study evaluates the clinical, laboratory and TEG profiles in patients with Glanzmann's thrombasthenia. Bleeding score by (International Society on Thrombosis and Haemostasis) ISTH-bleeding assessment tool (bleeding score), laboratory tests to diagnose Glanzmann's thrombasthenia, and TEG parameters were correlated in 11 Glanzmann's thrombasthenia patients. Seventeen participants with normal bleeding score were included as controls. Bleeding score was increased in all patients. The highest bleeding score was in an adult female (26), whereas the lowest score (4) was in two children of less than 1 year. Majority of TEG parameters (except R-time) showed a statistically significant difference between Glanzmann's thrombasthenia patients and controls (K-time: P < 0.001, angle: P < 0.001, maximum amplitude: P < 0.001). The average time required to record the maximum amplitude was 23 min. Maximum amplitude was markedly reduced in all Glanzmann's thrombasthenia patients with an average of 20.9 mm (reference range 44-68 mm) having 100% sensitivity. The thromboelastographic profile of Glanzmann's thrombasthenia showed a consistently reduced maximum amplitude. Hence reduced maximum amplitude with a normal platelet count, significant bleeding score and prolonged bleeding time could potentially be used as a preliminary algorithm for the diagnosis of Glanzmann's thrombasthenia.

Successful Use of Hematopoietic Stem Cell Transplantation for 2 Pediatric Cases of Glanzmann Thrombasthenia and Review of the Literature.

Glanzmann thrombasthenia is a rare platelet disorder characterized by an abnormal integrin receptor on the surface of platelets that results in the failure of platelets to aggregate. Currently, curative therapy is allogeneic hematopoietic stem cell transplantation (HSCT). The authors report 2 patients with Glanzmann thrombasthenia who successfully underwent allogeneic HSCT from unrelated donors, including one using umbilical cord blood stem cells. Although both patients had evidence of engraftment, hematopoietic recovery, and normalization of platelet aggregation, they also experienced several post-transplant complications. Allogeneic HSCT carries a significant risk of morbidity and mortality that should be considered before proceeding with the transplant.

Impaired hemostatic activity of healthy transfused platelets in inherited and acquired platelet disorders: Mechanisms and implications.

Platelet transfusions can fail to prevent bleeding in patients with inherited platelet function disorders (IPDs), such as Glanzmann's thrombasthenia (GT; integrin αIIbß3 dysfunction), Bernard-Soulier syndrome [BSS; glycoprotein (GP) Ib/V/IX dysfunction], and the more recently identified nonsyndromic RASGRP2 variants. Here, we used IPD mouse models and real-time imaging of hemostatic plug formation to investigate whether dysfunctional platelets impair the hemostatic function of healthy donor [wild-type (WT)] platelets. In Rasgrp2-/- mice or mice with platelet-specific deficiency in the integrin adaptor protein TALIN1 ("GT-like"), WT platelet transfusion was ineffective unless the ratio between mutant and WT platelets was ~2:1. In contrast, thrombocytopenic mice or mice lacking the extracellular domain of GPIbα ("BSS-like") required very few transfused WT platelets to normalize hemostasis. Both Rasgrp2-/- and GT-like, but not BSS-like, platelets effectively localized to the injury site. Mechanistic studies identified at least two mechanisms of interference by dysfunctional platelets in IPDs: (i) delayed adhesion of WT donor platelets due to reduced access to GPIbα ligands exposed at sites of vascular injury and (ii) impaired consolidation of the hemostatic plug. We also investigated the hemostatic activity of transfused platelets in the setting of dual antiplatelet therapy (DAPT), an acquired platelet function disorder (APD). "DAPT" platelets did not prolong the time to initial hemostasis, but plugs were unstable and frequent rebleeding was observed. Thus, we propose that the endogenous platelet count and the ratio of transfused versus endogenous platelets should be considered when treating select IPD and APD patients with platelet transfusions.

Acquired Glanzmann thrombasthenia: From antibodies to anti-platelet drugs.

In contrast to the inherited platelet disorder given by mutations in the ITGA2B and ITGB3 genes, mucocutaneous bleeding from a spontaneous inhibition of normally expressed αIIbß3 characterizes acquired Glanzmann thrombasthenia (GT). Classically, it is associated with autoantibodies or paraproteins that block platelet aggregation without causing a fall in platelet count. However, inhibitory antibodies to αIIbß3 are widely associated with primary immune thrombocytopenia (ITP), occur in secondary ITP associated with leukemia and related disorders, solid cancers and myeloma, other autoimmune diseases, following organ transplantation while cytoplasmic dysregulation of αIIbß3 function features in myeloproliferative and myelodysplastic syndromes. Antibodies to αIIbß3 occur during viral and bacterial infections, while drug-dependent antibodies reacting with αIIbß3 are a special case. Direct induction of acquired GT is a feature of therapies that block platelets in coronary artery disease. This review looks at these conditions, emphasizing molecular mechanisms, therapy, patient management and future directions for research.

Glanzmann Thrombasthenia in Children: Experience From a Tertiary Care Center in Southern India.

BACKGROUND: Glanzmann thrombasthenia (GT) is a globally rare inherited disorder of hemostasis. OBJECTIVES: To describe the clinical profile of GT in a tertiary care center in Southern India. METHODS: A retrospective chart review of all children with GT was performed between January 2005 and August 2017 in the Department of Paediatrics. RESULTS: A total of 48 patients (representing 43 families) were included. Median age at diagnosis was 2.75 years (interquartile range: 1.5 to 6.75). Two thirds had an onset of bleeding within the first 2 years of life. Sixty-seven percent were born out of consanguineous marriage. The common symptoms were epistaxis, gingival bleeding, and ecchymoses. Neonatal onset of bleeding manifested as purpura, epistaxis, and intracranial hemorrhage. Postsurgical bleeding and menorrhagia were unique presentations in adolescence. About 25% had life-threatening hemorrhage while 50% had growth retardation due to chronic anemia. CONCLUSIONS: GT is relatively more common in areas of Southern India due to the higher prevalence of consanguinity. Chronic anemia can contribute to growth stunting in these patients.

Missed at first Glanz: Glanzmann thrombasthenia initially misdiagnosed as Von Willebrand Disease.

Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder caused by a defect in platelet integrin αIIbß3. Given the rarity of the condition (1/1,000,000), assessment and diagnosis should be undertaken in a specialist centre. We report the case of a 34 year old woman with severe menorrhagia and a childhood diagnosis from another centre of Von Willebrand Disease. She had an extensive bleeding history, with epistaxis, menorrhagia and postoperative bleeding requiring multiple previous transfusions. Repeat haemostatic workup in our centre revealed normal Von Willebrand levels but abnormal platelet aggregation consistent with Glanzmann thrombasthenia. Antibody screening detected both anti-HLA and anti-αIIbß3 antibodies, complicating subsequent haemostatic management. This case highlights the importance of diagnostic accuracy, the potential negative sequelae of misdiagnosis and subsequent therapeutic interventions.

How we treat the platelet glycoprotein defects; Glanzmann thrombasthenia and Bernard Soulier syndrome in children and adults.

The inherited platelet glycoprotein deficiencies, Glanzmann thrombasthenia (GT) and Bernard Soulier syndrome (BSS) are rare but important long-term bleeding disorders. Once diagnosed, affected patients should be referred to a specialist centre for bleeding disorders for general advice and ongoing management. Patients do not require prophylactic treatment and so the management of GT and BSS focuses around prophylactic treatment prior to high risk procedures and treatment in response to non-surgical bleeding events and, in women, the management of menorrhagia and pregnancy. There is no consistent approach to the treatment or prevention of bleeding complications. Management must be tailored for each individual and the approach may not be the same for different events, even for the same patient, depending on the type of accident or invasive procedure, the extent of bleeding and the presence or not of platelet refractoriness.

Mass Cytometry Reveals Distinct Platelet Subtypes in Healthy Subjects and Novel Alterations in Surface Glycoproteins in Glanzmann Thrombasthenia.

Mass cytometry (MC) uses mass spectrometry to simultaneously detect multiple metal-conjugated antibodies on single cells, thereby enabling the detailed study of cellular function. Here, for the first time, we applied MC to the analysis of platelets. We developed a panel of 14 platelet-specific metal-tagged antibodies (targeting cluster of differentiation [CD] 9, CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CD107a, CD154, glycoprotein [GP] VI and activated integrin αIIbß3) and compared this panel with two fluorescence flow cytometry (FFC) panels (CD41, CD42b, and CD61; or CD42b, CD62P, and activated integrin αIIbß3) in the evaluation of activation-dependent changes in glycoprotein expression on healthy subject and Glanzmann thrombasthenia (GT) platelets. High-dimensional analysis of surface markers detected by MC identified previously unappreciated subpopulations of platelets in healthy donors. As expected, MC and FFC revealed that GT platelets had significantly reduced CD41, CD61, and activated integrin αIIbß3 surface expression. MC also revealed that surface expression of CD9, CD42a and CD63 were elevated, CD31, CD154 and GPVI were reduced and CD29, CD36, CD42b, CD62P and CD107a were similar on GT platelets compared to healthy donor platelets. In summary, MC revealed distinct platelet subtypes in healthy subjects and novel alterations in surface glycoproteins on GT platelets.

Clinical and molecular insights into Glanzmann's thrombasthenia in China.

Glanzmann's thrombasthenia (GT) is a rare bleeding disorder characterized by spontaneous mucocutaneous bleeding. The disorder is caused by quantitative or qualitative defects in integrin αIIbß3 (encoded by ITGA2B and ITGB3) on the platelet and is more common in consanguineous populations. However, the prevalence rate and clinical characteristics of GT in non-consanguineous populations have been unclear. We analyzed 97 patients from 93 families with GT in the Han population in China. This analysis showed lower consanguinity (18.3%) in Han patients than other ethnic populations in GT-prone countries. Compared with other ethnic populations, there was no significant difference in the distribution of GT types. Han females suffered more severe bleeding and had a poorer prognosis. We identified a total of 43 different ITGA2B and ITGB3 variants, including 25 previously unidentified, in 45 patients. These variants included 14 missense, 4 nonsense, 4 frameshift, and 3 splicing site variants. Patients with the same genotype generally manifested the same GT type but presented with different bleeding severities. This suggests that GT clinical phenotype does not solely depend on genotype. Our study provides an initial, yet important, clinical and molecular characterization of GT heterogeneity in China.

Reversal of Glanzmann thrombasthenia platelet phenotype after imatinib treatment in a pediatric chronic myeloid leukemia patient.

Chronic Myelogenous Leukemia (CML) is a myeloproliferative neoplasm characterized by proliferation of Philadelphia positive clonal pluripotent hematopoietic cells. Bleeding is a rare presentation of CML that can occur due to platelet dysfunction. Both pre-treatment and post-treatment platelet function abnormalities in CML have been described in the literature. We describe a rare case of childhood CML who presented with mucocutateous bleeding manifestations. On laboratory workup, a Glanzmann Thrombasthenia (GT) like platelet phenotype was demonstrated along with confirmation of diagnosis of CML in chronic phase. The acquired nature of platelet function defect was confirmed by demonstrating recovery of platelet antigens glycoprotein IIb/IIIa after achieving complete hematological response with Imatinib. Due to presenting complaint of bleeding diathesis and absence of hepatosplenomegaly, the case was undiagnosed for CML until the patient reported to us. Careful evaluation of complete blood counts, peripheral blood picture and detailed laboratory workup was the window to proper diagnosis and treatment in this case.

Evaluation of platelet surface glycoproteins in patients with Glanzmann thrombasthenia: Association with bleeding symptoms.

BACKGROUND & OBJECTIVES: Glanzmann thrombasthenia (GT) is a rare, inherited autosomal recessive disorder characterized by qualitative or quantitative deficiency of integrin αIIbß3 [glycoprotein IIb (GPIIb)/IIIa, CD41/CD61] diagnosed by absent or reduced platelet aggregation to physiological agonists, namely, collagen, adenosine-di-phosphate, epinephrine and arachidonic acid. The objective of this study was to quantitate platelet surface GPs, classify GT patients and relate the results with the severity of bleeding and platelet aggregation studies. METHODS: Fifty one patients of GT diagnosed by platelet aggregation studies were evaluated for the expression of CD41, CD61, CD42a and CD42b on platelet surface by flow cytometry. The association between the clinical phenotype based on bleeding score and GT subtype on flow cytometric evaluation was assessed. RESULTS: Twenty four (47%) patients of GT were classified as type I (as CD41/CD61 were virtually absent, <5%), six (11.8%) patients as type II (5-20% CD41/CD61) and 21 (41.2%) as type III or GT variants as they had near normal levels of CD41 and CD61. Type III GT patients had significantly lower numbers of severe bleeders (P=0.034), but the severity of bleeding did not vary significantly in type I and II GT patients. In all GT patients, mean CD41 expression was found to be lower than mean CD61 expression (P=0.002). INTERPRETATION & CONCLUSIONS: Type I GT was found most common in our patients and with lowered mean CD41 expression in comparison with CD61. Type III GT patients had significantly lower numbers of severe bleeders, but the severity of bleeding did not vary significantly in type I and II GT patients.

Modeling Glanzmann thrombasthenia using patient specific iPSCs and restoring platelet aggregation function by CD41 overexpression.

Glanzmann thrombasthenia (GT) is a rare monogenic hemorrhagic disorder involving aggregation defect of non-nuclear platelets. In this study we generated induced pluripotent stem cells (iPSCs) from skin fibroblasts of a GT patient with complex heterogeneous mutations of ITGA2B gene. GT-iPSCs could be successfully differentiated into platelets (GT-iPS-platelets). GT-iPS-platelets were CD41-/CD42b+/CD61- and were platelet activation marker (PAC-1) negative after adenosine diphosphate (ADP) activation. Furthermore, GT-iPS-platelets were defective in platelet aggregation tests in vitro. Moreover, exogenous expression of the wild-type ITGA2B gene in GT-iPS platelets restored CD41 expression and normal platelet aggregation. Our study suggested that patient-specific iPSCs could be a potential target of stem cell based gene therapy for platelet diseases.

Novel mutations in RASGRP2, which encodes CalDAG-GEFI, abrogate Rap1 activation, causing platelet dysfunction.

In addition to mutations in ITG2B or ITGB3 genes that cause defective αIIbß3 expression and/or function in Glanzmann's thrombasthenia patients, platelet dysfunction can be a result of genetic variability in proteins that mediate inside-out activation of αIIbß3 The RASGRP2 gene is strongly expressed in platelets and neutrophils, where its encoded protein CalDAG-GEFI facilitates the activation of Rap1 and subsequent activation of integrins. We used next-generation sequencing (NGS) and whole-exome sequencing (WES) to identify 2 novel function-disrupting mutations in RASGRP2 that account for bleeding diathesis and platelet dysfunction in 2 unrelated families. By using a panel of 71 genes, we identified a homozygous change (c.1142C>T) in exon 10 of RASGRP2 in a 9-year-old child of Chinese origin (family 1). This variant led to a p.Ser381Phe substitution in the CDC25 catalytic domain of CalDAG-GEFI. In 2 Spanish siblings from family 2, WES identified a nonsense homozygous variation (c.337C>T) (p.Arg113X) in exon 5 of RASGRP2 CalDAG-GEFI expression was markedly reduced in platelets from all patients, and by using a novel in vitro assay, we found that the nucleotide exchange activity was dramatically reduced in CalDAG-GEFI p.Ser381Phe. Platelets from homozygous patients exhibited agonist-specific defects in αIIbß3 integrin activation and aggregation. In contrast, α- and δ-granule secretion, platelet spreading, and clot retraction were not markedly affected. Integrin activation in the patients' neutrophils was also impaired. These patients are the first cases of a CalDAG-GEFI deficiency due to homozygous RASGRP2 mutations that are linked to defects in both leukocyte and platelet integrin activation.

The utility of International Society on Thrombosis and Haemostasis-Bleeding Assessment Tool and other bleeding questionnaires in assessing the bleeding phenotype in two platelet function defects.

The main objective of this study is to investigate the utility of International Society on Thrombosis and Haemostasis-Bleeding Assessment Tool (ISTH-BAT) in comparison with the condensed form of Molecular and Clinical Markers for the Diagnosis and Management of type 1 and WHO BATs, in assessing bleeding in two well known and clinically significant platelet function defects. Thirty-eight patients previously diagnosed with Glanzmann's thrombasthenia and 10 with Bernard-Soulier syndrome (BSS) were analyzed. Bleeding scores were significantly higher than that of controls using both electronic bleeding questionnaire (eBQ) and ISTH-BAT with no significant difference between both tools. ISTH-BAT had a sensitivity, specificity, positive predictive value and negative predictive value of 100%, 76.2%, 0.9 and 1. This was closely similar to eBQ. Both ISTH-BAT and eBQ are efficient in BSS and Glanzmann's thrombasthenia. However, given the ISTH recommendation, ISTH-BAT should be adopted. Larger study including other platelet defects will enhance its utility and support the integration of bleeding scores with standardized laboratory testing to allow for a universal diagnostic approach to patients with suspected bleeding disorders.

Coated platelets function in platelet-dependent fibrin formation via integrin αIIbß3 and transglutaminase factor XIII.

Coated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin α(IIb)ß3 activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin α(IIb)ß3 Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin α(IIb)ß3. Vice versa, star-like fibrin formation from platelets of a patient with deficiency in α(IIb)ß3(Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial α(IIb)ß3 activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin α(IIb)ß3.

The macromolecular architecture of platelet-derived microparticles.

Platelets are essential for hemostasis and wound healing. They are involved in fundamental processes of vascular biology such as angiogenesis, tissue regeneration, and tumor metastasis. Upon activation, platelets shed small plasma membrane vesicles termed platelet-derived microparticles (PMPs). PMPs include functional cell adhesion machinery that comprises transmembrane receptors (most abundant are the αIIbß3 integrins), cytoskeletal systems and a large variety of adapter and signaling molecules. Glanzmann thrombasthenia (GT) is a condition characterized by platelets that are deficient of the integrin αIIbß3 heterodimer. Here, we use cryo-electron tomography (cryo-ET) to study the structural organization of PMPs (in both healthy and GT patients), especially the cytoskeleton organization and receptor architecture. PMPs purified from GT patients show a significantly altered cytoskeletal organization, characterized by a reduced number of filaments present, compared to the healthy control. Furthermore, our results show that incubating healthy PMPs with manganese ions (Mn(2+)), in the presence of fibrinogen, induces a major conformational change of integrin receptors, whereas thrombin activation yields a moderate response. These results provide the first insights into the native molecular organization of PMPs.