The platelet cytoskeleton plays a pivotal role in maintaining and remodeling platelet morphology [10, 11]. It is organized based on actin filaments that are present in a mesh-like lattice throughout the cytoplasm, along with membrane cytoskeleton that lines the platelet membranes. The transmembrane receptors on the platelet membranes not only bind to the cytoskeletal proteins through their intracellular domains but also mediate signal transduction [12–15]. Microtubules contribute to the platelet production through proplatelet formation and regulate maturation and maintaining normal discoid shape of platelets [10, 11].

Congenital macrothrombocytopenia is a heterogeneous group of rare disorders, characterized by abnormally giant platelets and thrombocytopenia since birth [16]. The bleeding tendency varies from very mild in some to severe and life threatening in others. Recently, a significant progress has been made in the identification of new causative genes and the establishment of diagnostic tests for this group [6, 7]. In many of congenital macrothrombocytopenias, defects are associated with platelet cytoskeleton or adhesion receptors and their ligands. Thus, molecular diagnosis targeting subcellular localization or surface expression of defective gene product is available. On the other hand, the diagnosis of congenital thrombocytopenia with normal-sized platelets is more challenging. In many cases, the diagnosis is based solely on genetic analysis with conventional Sanger sequencing of potential candidate genes or recent high-throughput sequencing [17, 18]. This is because in this form of congenital thrombocytopenia, genetic defects are mostly in the transcription factors and thrombopoietin signaling regulating megakaryocyte commitment and growth, and therefore decreased megakaryocytes are the immediate cause for thrombocytopenia.

MYH9 disorders, the prototype of which is May-Hegglin anomaly (MHA), are autosomal dominant platelet disorders characterized by a triad of giant platelets, thrombocytopenia, and leukocyte inclusion bodies [19, 20]. In addition to MHA, Sebastian, Fechtner, and Epstein syndromes belong to MYH9 disorders. These disorders are caused by mutations in MYH9, the gene encoding for the non-muscle myosin heavy chain-IIA (NMMHC-IIA). From the first description a century ago, the hallmark of the disease is the presence of characteristic Döhle body-like leukocyte cytoplasmic inclusion bodies on conventionally stained peripheral blood smears [21, 22]. They are often faint or even unrecognized. Inclusion bodies are present in granulocytes but not in lymphocytes [23]. Detection of inclusion bodies is a prerequisite for a correct diagnosis, and failure in the detection often leads to misdiagnosis such as ITP. Inclusion bodies are present as a ribonucleoprotein complex consisting of MYH9 mRNA, clusters of ribosomes, and abnormally accumulated NMMHC-IIA protein [24]. MYH9 mRNA but not NMMHC-IIA is responsible for the morphological appearance/stainability of inclusion bodies on conventionally stained blood smears [25]. The rapid degradation of MYH9 mRNA accounts for the time-dependent decrease in the stainability of inclusion bodies.

Immunofluorescence analysis of neutrophil NMMHC-IIA localization has revolutionized the diagnosis of MYH9 disorders, and this analysis has become a gold standard for diagnosis of the disease [26–28]. The abnormal localization pattern can be classified according to the number, size, and shape of NMMHC-IIA aggregates, into types I, II, and III [26]. Type I comprises one or two large, intensely stained cytoplasmic NMMHC-IIA aggregates. Type II comprises up to 20 small cytoplasmic spots. Type III appears as speckled staining. The pattern of localization correlates with the site of MYH9 mutation. p.E1841K and frameshift and nonsense mutations in exon 40 are associated with type I localization. Missense mutations such as p.R1165C and p.D1424N are associated with type II localization. Normal NMMHC-IIA localization can exclude the diagnosis of MYH9 disorders [29]. Although MYH9 disorders were thought to be very rare, the advent of the immunofluorescence analysis has enabled the detection of even minute abnormal NMMHC-IIA aggregates and more precise diagnosis and classification; thus, MYH9 disorders are now known to be the most prevalent congenital thrombocytopenia. Patients with MYH9 disorders often develop non-hematological complications such as glomerulonephritis, sensorineural hearing loss, and cataracts. Because the onset and disease severity of non-hematological complications are related to the site of the MYH9 mutation, a genetic diagnosis is mandatory [27, 30].

Bernard-Soulier syndrome (BSS) is an autosomal recessive bleeding disorder characterized by giant platelets, thrombocytopenia, prolonged bleeding time, and absent ristocetin-induced platelet agglutination [31, 32]. This syndrome is caused by the deficiency of platelet glycoprotein (GP)Ib-IX-V complex, the platelet receptor for von Willebrand factor (VWF), due to compound heterozygous or homozygous mutations in the genes for GPIbα(GP1BA), GPIbβ(GP1BB), or GPIX(GP9). As a result of the absence of GPIb-IX-V complexes on the platelet membrane, platelets are unable to adhere to the vascular subendothelium. Thus, patients manifest a severe bleeding tendency from early childhood, primarily from mucocutaneous tissues; purpura, epistaxis, menorrhagia, and gingival bleeding are common, but hemarthroses and deep visceral hematomas are rare. The cytoplasmic domain of GPIbα, on the other hand, associates with actin via filamin A, and the defective linkage between GPIb-IX-V and cytoskeleton is the primary proposed mechanisms for the large platelet size in BSS [33, 34]. In addition, recent studies indicate that proplatelet formation is defective in megakaryocytes, suggesting defective interactions between GPIb-IX-V and filamin A-actin adversely affect both platelet morphogenesis and megakaryocytopoiesis [35].

Although BSS is a very rare disorder with an estimated prevalence of less than 1 in one million, the calculated frequency of BSS heterozygotes is 1 in 500, indicating that a sizable number of heterozygous carriers are present in the normal population [36, 37]. It is worth to note that although heterozygous BSS carriers are generally asymptomatic with mild or moderate thrombocytopenia, they have large platelets. Individuals with a heterozygous mutation have been often identified as having undifferentiated thrombocytopenia. In Italy, the Bolzano variant (GP1BA p. A172V) was found to be responsible for the autosomal dominant macrothrombocytopenia previously known as a Mediterranean macrothrombocytopenia [38, 39]. Likewise, GP1BB p. Y113C is prevalent in autosomal dominant macrothrombocytopenia in Japan [40, 41]. In addition, patients with DiGeorge/velocardiofacial syndrome due to a heterozygous chromosome 22q11.2 microdeletion, which includes the GP1BB gene, have macrothrombocytopenia [42, 43].

The classical diagnostic features are a prolonged bleeding time, moderate to severe thrombocytopenia, and giant platelets. Especially, giant platelets and the absence of ristocetin-induced platelet agglutination are the laboratory hallmarks of BSS. Flow cytometric determination of platelet GPIb/IX expression is a convenient method for a definite diagnosis. This also allows an evaluation of platelet size, and double labeling of GPIb/IX and GPIIb/IIIa permits determination of comparative expression levels. In a typical case of BSS, the fluorescence intensities of normal mouse IgG and anti-GPIIb/IIIa antibody are increased, which is reflected by giant platelets with a large surface area. In contrast, GPIbα, GPIbβ, and GPIX are all decreased. However, the abnormalities of the GPIb-IX-V complex are heterogeneous; residual amounts of the complex and/or some of the subunits are often present. Evaluating GPIbα, GPIbβ, and GPIX expression highlights selective deficiencies and candidate genes for investigation. Accordingly, the most deficient subunit in the platelets is often found to indicate the genetic basis: patients with residual GPIbβ and GPIX are often associated with a GP1BA mutation, and those with residual GPIbα and GPIbβ are often associated with a GP9 mutation [16, 36]. Patients with no substantial expression of each subunit usually have a GP1BB mutation, because GPIbβ is the critical subunit linking GPIbα and GPIX [37, 44].

ACTN1 macrothrombocytopenia is recently identified by whole exome sequencing (WES) in families with dominant form of congenital macrothrombocytopenia in which no relevant mutations in the previously reported genes had been identified [45]. Following MYH9 disorders and heterozygous and homozygous Bernard-Soulier syndrome, ACTN1 macrothrombocytopenia represents the third most prevalent form of congenital macrothrombocytopenia. It is characterized by mild macrothrombocytopenia, with mild or no bleeding tendency without non-hematologic complications [45, 46]. ACTN1 gene codes α-actinin-1, of which dimers stabilize the actin filaments and contribute to actin cytoskeletal organization by cross-linking actin filaments [47]. Initially, ACTN1 mutations were identified within the functional actin-binding and calmodulin-like domains. They cause a disorganized actin-based cytoskeleton in megakaryocytes, resulting in the production of abnormally large proplatelet tips, which were reduced in number [45]. Recently, a p.L395Q mutation within the spacer rod domain, which is composed of spectrin-like repeats, has been found, suggesting that mutations not only affect actin-binding properties but also affect structural organization of actinin can cause ACTN1 macrothrombocytopenia [48].

Although α-actinin-1 binds to β-integrins and can mediate their signaling, there are no apparent abnormalities in platelet adhesion, aggregation, or clot retraction in ACTN1 macrothrombocytopenia [45, 46, 49]. Furthermore, there is no apparent abnormal α-actinin-1 localization in resting as well as surface-activated platelets. Accordingly, a molecular genetic analysis was only available for the diagnosis. However, a recent investigation suggested the immunofluorescence analysis to detect NMMHC-IIA in the granulomere zone in surface-activated platelets as a potential diagnostic test for ACTN1 macrothrombocytopenia [50].

It is well known that congenital deficiency of GPIIb/IIIa (integrin αIIbβ3), platelet receptor for fibrinogen, due to homozygous mutations in the genes for GPIIb (ITGA2B) or GPIIIa (ITGB3) results in bleeding disorder Glanzmann thrombasthenia [51]. It is also known that homozygous patients and heterozygous carriers have normal platelet count and morphology. However, this is not always the case. Recently, heterozygous, activating mutations in the juxtamembrane region of GPIIb and GPIIIa are found to cause congenital macrothrombocytopenia [52]. Membrane-proximal region of GPIIb/IIIa, especially a salt bridge between GPIIb R995 and GPIIIa D723, maintains the inactive conformation of the receptor [53]. Disruption of the electrostatic interaction produces constitutive, agonist-independent outside-in signaling of GPIIb/IIIa and phosphorylation of signaling proteins such as FAK and leads to perturbation of cytoskeletal reorganization and results in abnormal proplatelet formation [54, 55]. Focal adhesion kinase is known to inhibit Rho and may promote precocious proplatelet formation, which is common among MYH9 disorders [56, 57].

In GPIIb/IIIa macrothrombocytopenia, GPIIb/IIIa expression is decreased due to accelerated internalization of activated GPIIb/IIIa [58, 59]. While GPIIb/IIIa is constitutively activated, allowing binding of fibrinogen and the ligand-mimetic antibody PAC-1 to resting platelets, no expression of p-selectin is noted, and the platelets themselves are not activated [54, 55]. Thus, flow cytometry is useful for screening GPIIb/IIIa macrothrombocytopenia by detecting decreased GPIIb/IIIa, binding of PAC-1 and fibrinogen, and no expression of p-selectin. Actually, concomitant flow cytometry for GPIb/IX and GPIIb/IIIa can differentiate homozygous and heterozygous Bernard-Soulier syndrome and GPIIb/IIIa macrothrombocytopenia.

Von Willebrand disease (VWD) is the most common congenital bleeding disorder of primary hemostasis caused by qualitative or quantitative defects of von Willebrand factor (VWF) [60]. It is classified into three main types: type 1, quantitatively reduced VWF; type 2, qualitatively abnormal VWF; and type 3, absent VWF. Type 2 VWD is categorized into four subtypes: 2A, 2B, 2M, and 2N. Type 2B VWD is a rare autosomal dominant form (5–8% of all VWD) characterized by the increased affinity of mutant VWF to platelet GPIb due to mutations in the A1 domain of VWF that contains the GPIb-binding site. Enhanced VWF-GPIb interactions lead to spontaneous platelet aggregation in vivo and in vitro and result in thrombocytopenia and lack of high molecular weight multimers of VWF. Giant platelets and spontaneous platelet aggregates are often detected on peripheral blood smears [61]. Thrombocytopenia deteriorates during stressors, such as infection, surgery, and pregnancy, and thus patients are often misdiagnosed with ITP. This platelet phenotype was previously described as Montreal platelet syndrome, in which spontaneous platelet is noted, but is now known to be type 2B VWD due to p.V1316M mutation [62].

Platelet aggregates on peripheral blood smears, which are comprised of several to dozens of platelets, are suggestive of type 2B VWD and platelet-type (pseudo) VWD (PT VWD) [61, 63]. While type 2B VWD and PT VWD are characterized by increased ristocetin sensitivity, they can be differentiated by mixing studies of ristocetin-induced platelet agglutination using patient platelets in normal control plasma and normal control platelets in patient plasma. When an unexpected low platelet count and platelet aggregates on a smear are obtained in individuals without bleeding tendency, EDTA-induced pseudothrombocytopenia should be considered [64]. EDTA-induced pseudothrombocytopenia is a common phenomenon caused by in vitro platelet clumping in the presence of EDTA anticoagulant, with a prevalence of as much as 0.1–0.2%. Accurate platelet counts can be usually obtained without anticoagulant or collecting in other anticoagulants such as sodium citrate or heparin.

Gray platelet syndrome (GPS) is an autosomal recessive bleeding disorder that is characterized by macrothrombocytopenia and lack of platelet α-granules [65]. Due to the absence of α-granules and their constituents, platelets appear gray or colorless on May-Grünwald-Giemsa or Wright’s stained blood smears. Since α-granules contain fibrinogen and VWF as well as other adhesive molecules and coagulation factors, the condition is closely associated with bleeding tendency. Furthermore, α-granule contents such as platelet-derived growth factor and transforming growth factor-β are synthesized but not properly stored within the granules and released from megakaryocytes into the bone marrow space; myelofibrosis is present in most cases. Although biochemical studies have failed to reveal any genetic alterations, using WES and RNA sequencing, Albers et al., Gunay-Aygun et al., and Kahr et al. simultaneously and independently identified homozygous or compound heterozygous mutations in NBEAL2 [66–68]. NBEAL2 contains the Beige and Chediak-Higashi (BEACH) domain, which is critical in vesicle trafficking and also present in LYST, a gene responsible for Chediak-Higashi syndrome.

Platelet α-granule deficiencies are also found in ARC syndrome (arthrogryposis-renal dysfunction-cholestasis syndrome) due to homozygous mutation in VPS33B and VIPAS39, which belong to the Sec1/Munc18 protein family that regulates vesicle formation [69]. Partial deficiency but not complete loss of α-granules can occur in other congenital macrothrombocytopenias due to mutations in GATA1 and GFI1B [70, 71]. GATA-1 is an X-linked megakaryocyte and erythroid-specific transcription factor required for normal growth and differentiation of both lineages [72]. Defective GATA-1 function due to GATA1 missense mutations causes reduced transcription of its target genes, suggesting that NBEAL2 may also be dysregulated. Dyserythropoiesis due to diminished or unbalanced synthesis of globin chains is observed in GATA1 macrothrombocytopenia [73].

Growth factor independence 1b (GFI1B) is a transcriptional repressor containing six zinc finger domains and regulates erythropoiesis and megakaryocytopoiesis [74]. In a family with autosomal dominant macrothrombocytopenia with platelet α-granule deficiencies, genome-wide linkage analysis and candidate gene sequencing led to the identification of a nonsense mutation in GFI1B zinc finger 5, which is critical for DNA binding [75]. Functional analyses suggested a dominant negative effect. Subsequently, two other null GFI1B mutations that also disrupt zinc finger 5 were identified in patients with autosomal dominant macrothrombocytopenia, a decrease in α-granules, and red blood cell anisopoikilocytosis [70, 76]. GfI1b mutants dominantly affect the terminal maturation of megakaryocytes and subsequent release of platelets with persistent CD34 expression and decreased CD42b expression.