Congenital platelet disorders are rare and targeted treatment is usually not possible. Inherited platelet function disorders (iPFDs) can affect surface receptors and multiple platelet responses such as defects of platelet granules, signal transduction, and procoagulant activity. If iPFDs are also associated with a reduced platelet count (thrombocytopenia), it is not uncommon to be misdiagnosed as immune thrombocytopenia. Because the bleeding tendency of the different platelet disorders is variable, a correct diagnosis of the platelet defect based on phenotyping, function analysis, and genotyping is essential, especially in the perioperative setting. In the case of a platelet receptor deficiency, such as Bernard-Soulier syndrome or Glanzmann thrombasthenia, not only the bleeding tendency but also the risk of isoimmunization after platelet transfusions or pregnancy has to be considered. Platelet granule disorders are commonly associated with either intrinsically quantitative or qualitative granule defects due to impaired granulopoiesis, or granule release defects, which can also affect additional signaling pathways. Functional platelet defects require expertise in the clinical bleeding tendency in terms of the disorder when using antiplatelet agents or other medications that affect platelet function. Platelet defects associated with hematological-oncological diseases require comprehensive information about the patient including the clinical implication of the genetic testing. This review focuses on genetics, clinical presentation, and laboratory platelet function analysis of iPFDs with or without reduced platelet number. As platelet defects affecting the cytoskeleton usually show thrombocytopenia, but less impaired or normal platelet functional responses, they are not specifically addressed.

In this article, our goal is to offer an introduction and overview of the diagnostic approach to inherited platelet function defects (iPFDs) for clinicians and laboratory personnel who are beginning to engage in the field. We describe the most commonly used laboratory methods and propose a diagnostic four-step approach, wherein each stage requires a higher level of expertise and more specialized methods. It should be noted that our proposed approach differs from the ISTH Guidance on this topic in some points. The first step in the diagnostic approach of iPFD should be a thorough medical history and clinical examination. We strongly advocate for the use of a validated bleeding score like the ISTH-BAT (International Society on Thrombosis and Haemostasis Bleeding Assessment Tool). External factors like diet and medication have to be considered. The second step should rule out plasmatic bleeding disorders and von Willebrand disease. Once this has been accomplished, the third step consists of a thorough platelet investigation of platelet phenotype and function. Established methods consist of blood smear analysis by light microscopy, light transmission aggregometry, and flow cytometry. Additional techniques such as lumiaggregometry, immune fluorescence microscopy, and platelet-dependent thrombin generation help confirm and specify the diagnosis of iPFD. In the fourth and last step, genetic testing can confirm a diagnosis, reveal novel mutations, and allow to compare unclear genetics with lab results. If diagnosis cannot be established through this process, experimental methods such as electron microscopy can give insight into the underlying disease.

Introduction Inherited platelet disorders (IPD) are a heterogynous group of disorders affecting platelet number and function. Next generation sequencing (NGS) has been an important tool to analyze genetic alterations in multiple genes. However, platelet function analysis performed with light transmission aggregometry (LTA), flow cytometry, and microscopy are important to identify patients with an IDP and to characterize the platelet phenotype. We investigated a young girl, who presented with multiple hematoma and mild thrombocytopenia (Plt. 109 G/L). Case history revealed that a few of her mother´s relatives had suffered from leukemia as adults.

Glanzmann Thrombasthenia (GT) is an inherited platelet disorder caused by defects in platelet integrin αIIbβ3 (GPIIb/IIIa), which is a platelet receptor essential for the binding of fibrinogen. This can lead to severe bleeding, especially after trauma or perioperatively, and to microcytic anemia because of chronic blood loss. We report on a 40-year-old female patient with extensive bleeding complications and platelet antibody formation who presented in Homburg and Freiburg for extensive platelet function analyses and molecular genetic analyses. According to platelet aggregometry, the patient had previously been diagnosed with Glanzmann Thrombasthenia (GT). In addition, an MRI scan had been performed due to an unsteady gait and had revealed bilateral para-ophthalmic aneurysms of both internal carotid arteries (ICAs). Assuming a 5% rupture risk per 5 years for each aneurysm, the patient was offered and accepted endovascular treatment. Next-generation sequencing (NGS) panel analysis identified a previously undescribed homozygous one-base-pair deletion in ITGA2B, which leads to a loss of function of the αIIb-subunit of the receptor. This case illustrates the difficulties that can arise regarding the treatment of patients with rare platelet bleeding disorders, and supports the importance of continuous medical care by a specialized hemophilia center for these patients.

Introduction Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare inherited bone marrow failure syndrome presenting as an isolated thrombocytopenia at birth with development to pancytopenia due to exhaustion of hematopoietic progenitors. The disease is primarily caused by biallelic pathogenic variants in the thrombopoietin (TPO) receptor c-Mpl, encoded by the MPL-gene. TPO signaling is essential for adequate thrombopoiesis as well as hematopoietic stem cell homeostasis. Next generation sequencing (NGS) is an important tool to analyze genetic alterations in multiple genes associated with inherited thrombocytopenia.

Glanzmann thrombasthenia (GT) is a rare autosomal recessive inherited platelet disorder occurring frequently in populations with high incidence of consanguineous marriages. GT is characterized by quantitative and/or qualitative defect of the platelet αIIbβ3 (GPIIb/IIIa) receptor caused by pathogenic variants of the encoding genes: ITGA2B and ITGB3. Patients present with a moderate to severe bleeding tendency with normal platelet count. Platelets show reduced/absent aggregation for all agonists except ristocetin in light transmission aggregometry and reduced/absent αIIbβ3 expression in flow cytometry (FC). In this study, we investigated a cohort of 20 Pakistani patients and 2 families collected from the National Institute of Blood Disease, Karachi and Chughtai's Lab, Lahore. Platelet aggregation studies, FC (platelet CD41, CD61, CD42a, CD42b) and direct sequencing of the candidate genes were performed. All patients showed altered platelet aggregation, but normal agglutination after stimulation with ristocetin. Absent/reduced αIIbβ3 receptor expression was present in the platelets of 16 patients, in 4 patients expression was borderline/normal. Candidate gene sequencing identified pathogenic/likely pathogenic variants in 15 patients. Seven variants are novel. One patient with absent receptor expression remained without genetic finding. 13 (86.7%) of 15 patients stated consanguinity reflected by homozygosity finding in 14 (93.3%) patients.

Introduction Congenital thrombocytopenia can be associated not only with a reduced production, but also with an accelerated degradation of platelets. Sialic acid binds to platelet surface glycoproteins and is known to protect platelets from degradation via the Ashwell-Morell receptor. The GNE gene encodes an enzyme that initiates and regulates the biosynthesis of N-acetylneuraminic acid, a precursor of sialic acids. GNE mutations are autosomal recessive associated with adult-onset progressive GNE myopathy (with or without thrombocytopenia) and autosomal dominant with sialuria. Interestingly, so far only a few children (n<10) with biallelic GNE variants leading to isolated thrombocytopenia have been described. Recently, we identified compound heterozygous GNE variants in a young girl suffering from severe congenital thrombocytopenia. We showed decreased α2,3 sialic acid and increased terminal galactose and α2,6 sialic acid moieties of the girl´s platelets [1]. In the current study, we aimed to characterize the platelet defect in a family with two children (P1 and P2) affected by congenital thrombocytopenia. P1 is a newborn boy with thrombocytopenia since birth (lowest platelet count 6 x109/L) who needs weekly platelet transfusions. His 16-year-old sister (P2) presented with lifelong thrombocytopenia and suspected chronic immune thrombocytopenia. She received therapy with romiplostim which resulted in stable platelet counts (> 50 x109/L). The parents and two other siblings were clinically not affected.

Abstract Platelets play an important role regarding coagulation by contributing to thrombus formation by platelet adhesion, aggregation, and α-/δ-granule secretion. Inherited platelet disorders (IPDs) are a very heterogeneous group of disorders that are phenotypically and biochemically diverse. Platelet dysfunction (thrombocytopathy) can be accompanied by a reduction in the number of thrombocytes (thrombocytopenia). The extent of the bleeding tendency can vary greatly. Symptoms comprise mucocutaneous bleeding (petechiae, gastrointestinal bleeding and/or menorrhagia, epistaxis) and increased hematoma tendency. Life-threatening bleeding can occur after trauma or surgery. In the last years, next-generation sequencing had a great impact on unrevealing the underlying genetic cause of individual IPDs. Because IPDs are so diverse, a comprehensive analysis of platelet function and genetic testing is indispensable.

Von Willebrand disease (VWD) is the most prevalent congenital bleeding disorder. Diagnosis and classification of VWD is complex due to its heterogeneity regarding clinical manifestations and molecular genetic analysis. Genetic investigations became an inherent part of diagnosis and help distinguish different types/subtypes of VWD. Although many variants have been listed being causative for VWD, the genetic etiology remains undefined in a lot of patients. We report about two siblings with severely reduced values for von Willebrand factor collagen-binding activity (VWF:CB). Genetic analysis using panel sequencing identified a heterozygous non-synonymous single nucleotide variant in exon 30. At the protein level, the alteration (p.Ser1731Leu) is located in the A3 collagen-binding domain. The amino acid position is already known to be important for collagen binding because p.Ser1731Thr has been reported to affect the VWF:CB.

ACTN1-related thrombocytopenia (ACTN1-RT) is an inherited platelet disorder, normally associated with moderate/ mild macrothrombocytopenia and a mild bleeding diathesis.Whereas immunofluorescence studies describing the cytoskeleton organization have been reported several times, expression analyses of platelet CD62-P and CD63 using flow cytometry have rarely been performed.We report on familial thrombocytopenia, i.e. four patients -mother and three daughters with moderate/ mild thrombocytopenia who were identified as having a pathogenic variant (p.Arg46Trp) in ACTN1.Interestingly, in two of the four patients, flow cytometry analysis showed a profound decrease in surface expression of platelet activation markers CD62-P and CD63.The other two patients showed a mild reduction with different concentrations of thrombin and reached normal exposure after activation with 1 U/mL thrombin.Reduced expression of CD62-P and CD63 is hinting to an α-and δ-storage pool disease (α-/δ-SPD), respectively.Whereas a CD62-P reduction has been previously reported once, this -to our knowledge -is the first report of a CD63 reduction in association with an ACTN1-RT.Patients with platelet SPD are at higher risk for bleeding problems, especially after trauma or surgery.In summary, comprehensive investigation, including molecular genetic and platelet function analysis is important to classify the individual platelet defect.This may help to prevent unnecessary bleeding problems and to optimize perioperative management.

BACKGROUND:The transcription factor GATA1 is an essential regulator of erythroid cell gene expression and maturation and is also relevant for platelet biogenesis. GATA1-related thrombocytopenia (GATA1-RT) is a rare X-linked inherited platelet disorder (IPD) characterized by macrothrombocytopenia and dyserythropoiesis. Enlarged platelet size, reduced platelet granularity, and noticeable red blood cell anisopoikilocytosis are characteristic but unspecific morphological findings in GATA1-RT. OBJECTIVES:To expand the investigation of platelet phenotype of patients with GATA1-RT by light- and immunofluorescence microscopy on a blood smear. METHODS:We assessed blood smears by light- and immunofluorescence microscopy after May-Grünwald Giemsa staining using a set of 13 primary antibodies against markers belonging to different platelet structures. Antibody binding was visualized by fluorescently labeled secondary antibodies. RESULTS:We investigated 12 individuals with genetically confirmed GATA1-RT from 8 unrelated families. While confirming the already known characteristic of platelet morphology (platelet macrocytosis and reduced expression of markers for α-granules), we also found aggregates of nonmuscular myosin heavy chain II A (NMMIIA) in the erythrocytes in all individuals (1-3 aggregates/cell, 1-3 μm diameter). By systematically reanalyzing blood smears from a cohort of patients with 19 different forms of IPD, we found similar NMMIIA aggregates in the red blood cells only in subjects with GFI1B-related thrombocytopenia (GFI1B-RT), the other major IPD featured by dyserythropoiesis. CONCLUSION:Aggregates of NMMIIA in the erythrocytes associate with GATA1-RT and GFI1B-RT and can facilitate their diagnosis on blood smears. This previously unreported finding might represent a novel marker of dyserythropoiesis assessable in peripheral blood.

Congenital thrombocytopenia can be associated not only with a reduced production, but also with an accelerated degradation of platelets. Sialic acid binds to platelet surface glycoproteins and protects platelets from degradation via the Ashwell-Morell receptor. The GNE gene encodes an enzyme that initiates and regulates the biosynthesis of N-acetylneuraminic acid, a precursor of sialic acids. According to OMIM alterations in GNE are autosomal recessive associated with adult-onset progressive GNE myopathy (with or without thrombocytopenia) and autosomal dominant with sialuria. In recent years, biallelic GNE mutations located mainly in the N-acetylmannosamine (ManNAc) kinase domain of GNE have been described in children/young adults with otherwise unexplained thrombocytopenia. Recently, we identified compound heterozygous GNE variants in a young girl (Pt.1) with life-threatening intracranial bleeding and severe congenital thrombocytopenia (lowest platelet count 5 x 10 9/L), who needed weekly platelet transfusions. We showed decreased α2,3 sialic acid and increased terminal galactose and α2,6 sialic acid moieties in the girl´s platelets. She received hematopoietic stem cell transplantation (HSCT) at 2,3 years, which normalized platelet counts and function. Follow-up data showed normalized platelet function (20 months after HSCT) and counts (20, 21, and 23 months after HSCT). Following HSCT, the platelet lectin profile was markedly different from before transplantation with an increase in α2,3 sialic acid recognizing lectins WGA and MAL I compared with data before transplantation, indicating that sialic acid moieties have been normalized. Additionally, we identified a biallelic GNE variant in two siblings (Pt.2 and Pt.3) affected by congenital thrombocytopenia. Pt.2 is a 1,8-year-old boy with thrombocytopenia since birth (lowest platelet count 6 x10 9/L) who needed weekly platelet transfusions. His 17-year-old sister (Pt.3) presented with lifelong thrombocytopenia and suspected chronic immune thrombocytopenia; however, therapy with immunoglobulin and steroids did not elevate the platelets. The parents and two other siblings were clinically not affected. Flow cytometry performed for the affected siblings showed severely decreased thrombin-induced platelet CD62 and CD63 exposure hinting at an impaired α- and δ-granule secretion. NGS identified a homozygous GNE variant (NM_001128227.3:c.1727G>C, p.Gly576Ala) with damaging prediction to be present in both siblings. The parents and two healthy siblings are heterozygous carriers of this variant. The nucleotide change c.1726G>C leading to Gly576Arg has been described compound heterozygous in a patient with GNE myopathy. For the sister (Pt.3) lectin binding analysis showed decreased α-2,3 sialylation (MAA) and increased terminal galactose (RCA120) expression on platelets. This pattern is consistent with loss of sialic acid synthesis and indicative of rapid platelet clearance. She had received therapy with romiplostim since she was 12, resulting in stable platelet counts (> 50 x10 9/L). At the age of 1 year, Pt.2 also started with romiplostim therapy. During the 8-month treatment, platelet counts were mainly stable above 30 x 10 9/L. Only two transfusions were necessary when platelet counts dropped to 18 and 11 x 10 9/L. In conclusion, this research elucidates novel associations between GNE mutations, specifically within the ManNAc kinase domain, and congenital thrombocytopenia. These findings were derived from studying three patients carrying such mutations, underscoring this discovery's potential broad relevance. Importantly, this alteration in the GNE gene, traditionally associated with GNE myopathy, prompts us to anticipate and closely monitor the potential future development of this neuromuscular condition in these patients. Of particular interest is the effectiveness of romiplostim in ameliorating thrombocytopenia in these cases. Both siblings in our study exhibited stable, acceptable platelet counts in response to this therapy, suggesting that romiplostim might offer a promising therapeutic strategy for managing congenital thrombocytopenia linked to GNE mutations.

Linking the genetic background of patients with bleeding diathesis and altered platelet function remains challenging. We aimed to assess how a multiparameter microspot-based measurement of thrombus formation under flow can help identify patients with a platelet bleeding disorder. For this purpose, we studied 16 patients presenting with bleeding and/or albinism and suspected platelet dysfunction and 15 relatives. Genotyping of patients revealed a novel biallelic pathogenic variant in RASGRP2 (splice site c.240-1G>A), abrogating CalDAG-GEFI expression, compound heterozygosity (c.537del, c.571A>T) in P2RY12, affecting P2Y12 signaling, and heterozygous variants of unknown significance in the P2RY12 and HPS3 genes. Other patients were confirmed to have Hermansky-Pudlak syndrome type 1 or 3. In 5 patients, no genetic variant was found. Platelet functions were assessed via routine laboratory measurements. Blood samples from all subjects and day controls were screened for blood cell counts and microfluidic outcomes on 6 surfaces (48 parameters) in comparison with those of a reference cohort of healthy subjects. Differential analysis of the microfluidic data showed that the key parameters of thrombus formation were compromised in the 16 index patients. Principal component analysis revealed separate clusters of patients vs heterozygous family members and control subjects. Clusters were further segregated based on inclusion of hematologic values and laboratory measurements. Subject ranking indicated an overall impairment in thrombus formation in patients carrying a (likely) pathogenic variant of the genes but not in asymptomatic relatives. Taken together, our results indicate the advantages of testing for multiparametric thrombus formation in this patient population.

The GNE gene encodes an enzyme that initiates and regulates the biosynthesis of N-acetylneuraminic acid, a precursor of sialic acids. GNE mutations are classically associated with Nonaka myopathy and sialuria, following an autosomal recessive and autosomal dominant inheritance pattern. Reports show that single GNE variants cause severe thrombocytopenia without muscle weakness. Using panel sequencing, we identified two novel compound heterozygous variants in GNE in a young girl with life-threatening bleedings, severe congenital thrombocytopenia, and a platelet secretion defect. Both variants are located in the nucleotide-binding site of the N-acetylmannosamin kinase domain of GNE. Lectin array showed decreased α-2,3-sialylation on platelets, consistent with loss of sialic acid synthesis and indicative of rapid platelet clearance. Hematopoietic stem cell transplantation (HSCT) normalized platelet counts. This is the first report of an HSCT in a patient with an inherited GNE defect leading to normal platelet counts.

Hermansky-Pudlak syndrome (HPS), a rare heterogeneous autosomal recessive disorder, is characterized by oculocutaneous albinism (OCA) and a bleeding diathesis due to a defect regarding melanosomes and platelet delta (δ)-granule secretion. Interestingly, patients with HPS type 2 (HPS-2) or HPS type 10 (HPS-10) present additionally with an immunological defect. We investigated three patients (IP1, IP2, and IP3) who suffer from a bleeding diathesis. Platelet aggregometry showed impaired platelet function and flow cytometry revealed a severely reduced platelet CD63 expression hinting to either a defect of platelet delta granule secretion or a decreased number of delta granules in these patients. However, only IP3 presents with an apparent OCA. We performed panel sequencing and identified a homozygous deletion of exon 6 in DTNBP1 for IP3. Western analysis confirmed the absence of the encoded protein dysbindin confirming the diagnosis of HPS-7. Interestingly, this patient reported additionally recurrent bacterial infections. Analysis of lymphocyte cytotoxicity showed a slightly reduced NK-degranulation previously documented in a more severe form in patients with HPS-2 or HPS-10. IP1 is carrier of two compound heterozygous variants in the HPS3 gene (c.65C > G and c.1193G > A). A homozygous variant in HPS5 (c.760G > T) was identified in IP2. The novel missense variants were classified as VUS (variant of uncertain significance) according to ACMG guidelines. For IP1 with the compound heterozygous variants in HPS3 a specialized ophthalmological examination showed ocular albinism. HPS3 and HPS5 encode subunits of the BLOC-2 complex and patients with HPS-3 or HPS-5 are known to present with variable/mild hypopigmentation.

The GATA1 transcription factor is essential for normal erythropoiesis and megakaryocytic differentiation. Germline GATA1 pathogenic variants in the N-terminal zinc finger (N-ZF) are typically associated with X-linked thrombocytopenia, platelet dysfunction, and dyserythropoietic anemia. A few variants in the C-terminal ZF (C-ZF) domain are described with normal platelet count but altered platelet function as the main characteristic. Independently performed molecular genetic analysis identified a novel hemizygous variant (c.865C>T, p.H289Y) in the C-ZF region of GATA1 in a German patient and in a Spanish patient. We characterized the bleeding and platelet phenotype of these patients and compared these findings with the parameters of two German siblings carrying the likely pathogenic variant p.D218N in the GATA1 N-ZF domain. The main difference was profound thrombocytopenia in the brothers carrying the p.D218N variant compared to a normal platelet count in patients carrying the p.H289Y variant; only the Spanish patient occasionally developed mild thrombocytopenia. A functional platelet defect affecting αIIbβ3 integrin activation and α-granule secretion was present in all patients. Additionally, mild anemia, anisocytosis, and poikilocytosis were observed in the patients with the C-ZF variant. Our data support the concept that GATA1 variants located in the different ZF regions can lead to clinically diverse manifestations.

Inherited platelet disorders (IPDs) constitute a large heterogeneous group of rare bleeding disorders. These are classified into: (1) quantitative defects, (2) qualitative disorders, or (3) altered platelet production rate disorders or increased platelet turnover. Classically, IPD diagnostic is based on clinical phenotype characterization, comprehensive laboratory analyses (platelet function analysis), and, in former times, candidate gene sequencing. Today, molecular genetic analysis is performed using next-generation sequencing, mostly by targeting enrichment of a gene panel or by whole-exome sequencing. Still, the biochemical and molecular genetic characterization of patients with congenital thrombocytopathias/thrombocytopenia is essential, since postoperative or posttraumatic bleeding often occurs due to undiagnosed platelet defects. Depending upon the kind of surgery or trauma, this bleeding may be life-threatening, e.g., after tonsillectomy or in brain surgery. Undiagnosed platelet defects may lead to additional surgery, hysterectomy, pulmonary bleeding, and even resuscitation. In addition, these increased bleeding symptoms can lead to wound healing problems. Only specialized laboratories can perform the special platelet function analyses (aggregometry, flow cytometry, or immunofluorescent microscopy of the platelets); therefore, many IPDs are still undetected.

Objective Hermansky-Pudlak syndrome (HPS) is a heterogeneous group of 10 rare autosomal recessive multisystem disorders. HPS-associated genes encode components of four proteins complexes: BLOC-1, 2, 3 (biogenesis of lysosome‐related organelles complex 1-3) and the transporter complex AP-3 (adaptor protein-3). The main symptoms are bleeding tendency and oculocutaneous albinism. In HPS1 and HPS2 pulmonary fibrosis and granulomatous colitis can occur, in HPS2 and HPS10 immune deficiency is associated. Milder phenotypes have been described for HPS3, HPS5 and HPS6. The symptoms are caused by malfunction of lysosome related organelles: platelet δ-granules, melanosomes, lamellar bodies in lung cells and lytic and azurophil granules in neutrophiles. Identification of the HPS subtype is important for prognosis, clinical management and treatment options. We investigated 5 patients and 8 family members to identify the underlying disease.

Hermansky-Pudlak syndrome (HPS) is a heterogeneous disorder combining oculocutaneous albinism (OCA) and a platelet function disorder of varying severity as its most prominent features. The genes associated with HPS encode for different BLOC- (biogenesis of lysosome-related organelles complex) complexes and for the AP-3 (adaptor protein-3) complex, respectively. These proteins are involved in maturation, trafficking, and the function of lysosome-related organelles (LROs) such as melanosomes and platelet δ-granules. Some patients with different types of HPS can develop additional complications and symptoms like pulmonary fibrosis, granulomatous colitis, and immunodeficiency. A new type of HPS has recently been identified associated with genetic alterations in the BLOC1S5 gene, which encodes the subunit Muted of the BLOC-1 complex. Our aim was to unravel the genetic defect in two siblings with a suspected HPS diagnosis (because of OCA and bleeding symptoms) using next generation sequencing (NGS). Platelet functional analysis revealed reduced platelet aggregation after stimulation with ADP and a severe secretion defect in platelet δ-granules. NGS identified a novel homozygous essential splice site variant in the BLOC1S5 gene present in both affected siblings who are descendants of a consanguine marriage. The patients exhibited no additional symptoms. Our study confirms that pathogenic variants of BLOC1S5 cause the recently described HPS type 11.