This review will discuss the molecular basis, diagnosis and treatment of inherited platelet disorders (IPDs) where abnormalities of platelet function and production give rise to largely mucocutaneous bleeding syndromes of variable intensity [1–5]. The characterization of IPDs has led to novel insights into the complex biology of megakaryopoiesis and platelet production and identified functionally important platelet receptors and intracellular signaling events that have pioneered current antithrombotic therapy.
Summary. Inherited diseases of the megakaryocyte lineage give rise to bleeding when platelets fail to fulfill their hemostatic function upon vessel injury. Platelet defects extend from the absence or malfunctioning of adhesion (GPIb-IX-V, Bernard–Soulier syndrome) or aggregation receptors (integrin αIIbβ3, Glanzmann thrombasthenia) to defects of primary receptors for soluble agonists, secretion from storage organelles, activation pathways and the generation of procoagulant activity. In disorders such as the Chediak–Higashi, Hermansky–Pudlak, Wiskott–Aldrich and Scott syndromes the molecular lesion extends to other cells. In familial thrombocytopenia (FT), platelets are produced in insufficient numbers to assure hemostasis. Some FT affect platelet morphology and give rise to the ‘giant platelet’ syndromes (e.g. MYH9-related diseases) with changes in megakaryocyte maturation within the bone marrow and premature release of platelets. Diseases of platelet production may also affect other cells and in some cases interfere with development and/or functioning of major organs. Diagnosis of platelet disorders requires platelet function testing, studies often aided by the quantitative analysis of receptors by flow cytometry and fluorescence and electron microscopy. New generation DNA-based procedures including whole exome sequencing offer an exciting new perspective. Transfusion of platelets remains the most common treatment of severe bleeding, management with desmopressin is often used for mild disorders. Substitute therapies are available including rFVIIa and the potential use of thrombopoietin analogues for FT. Stem cell or bone marrow transplanation has been successful for several diseases while gene therapy shows promise in the Wiskott–Aldrich syndrome.
Molecular basis of platelet disorders
Adhesion and the GPIb-von Willebrand factor axis
Bernard–Soulier syndrome (BSS) associates macrothrombocytopenia with decreased platelet adhesion due to an absence or nonfunctioning of GPIbα, a receptor for von Willebrand factor (VWF) exposed within vascular lesions. The products of four genes (GPIBA, GPIBB, GP9 and GP5) assemble within maturing MK in the marrow to form the GPIb-IX-V complex. Mutations within GPIBA, GPIBB and GP9 in BSS prevent formation or trafficking of the complex through endoplasmic reticulum (ER) and the Golgi apparatus . In rare variant forms, platelets express nonfunctional GPIbα; in platelet-type von Willebrand disease (VWD), specific GPIBA mutations lead to upregulated GPIbα function and a clinical condition resembling type 2B VWD where macrothrombocytopenia (and sometimes circulating platelet aggregates) due to activating mutations in exon 28 of the VWF gene may also affect megakaryopoiesis .
Inherited variants of agonist receptors, signaling pathways and secretion
The platelet-collagen interaction under flow is a multistep process involving α2β1 and GPVI which signals through the FcRγ-chain [2,6]. Like α2β1, GPVI density is under the control of SNPs and epigenetic factors; however, a loss in the collagen response due to mutations in GP6 occurs in rare families. Members of the seven transmembrane domain family of G-protein-linked receptors mediate platelet responses to soluble agonists. Rare patients with a decreased and reversible platelet aggregation to ADP have mutant alleles at the P2YR12 locus while a defective platelet aggregation to TXA2 is caused by mutations in TA2R. Significantly, these patients mimic the platelet function modifications achieved in anti-thrombotic therapy by clopidogrel (and prasugrel) and aspirin respectively. Decreased platelet aggregation to adrenaline is often seen in routine screening although its contribution to excessive bleeding is unclear. Abnormalities of signal transduction pathways into which surface receptors are locked mostly concern patients with mild bleeding while congenital deficiencies of metabolic pathways also lead to platelet function abnormalities [2,6,8–10].
IPDs of secretion (storage pool disease, SPD) cause selective defects in aggregation. SPD affecting dense granules, storage sites for serotonin, ADP and ATP, may be quite common and the granule deficiency severe or partial. When associated with abnormalities of other lysosome-related organelles they give clearly defined phenotypes [e.g. Hermansky–Pudlak (HPS) and Chediak–Higashi (CHS) syndromes] where melanosomal defects cause a lack of pigmentation of the skin and hair. Defects in at least 8 genes (HPS-1 through HPS-8) in HPS cause distinct subtypes with the encoded proteins interacting in complexes (BLOCS); the genetic defects disrupt these thereby affecting organelle biosynthesis and protein trafficking. In CHS, bleeding is associated with severe immunologic defects and progressive neurological dysfunction, a lymphoproliferative syndrome and an accelerated phase is seen in ∼90% of patients. Frameshift and nonsense mutations in the LYST gene result in a truncated CHS protein (LYST) with characteristic giant inclusion bodies in cells. Rare missense mutations may give a milder form .
A wide range of biologically active proteins either synthesized in MK or endocytosed from plasma are stored in α-granules. Inherited diseases of the corresponding plasma proteins give specific deficiences (e.g. fibrinogen in afibrinogenaemia, VWF in type 3 VWD). The gray platelet syndrome (GPS) has a block in α-granule biogenesis and a general defect of protein packaging and storage. The affected gene is NBEAL2, a member of the neurobeachin-like gene family (see Diagnosis). Myelofibrosis in GPS is attributed to the spontaneous release from MK of newly synthesised growth factors. Mutations in VPS33B, encoding another regulator of α-granule biogenesis occur in children with the arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome. In the autosomal dominant Quebec platelet disorder (QPS), there is protease-related degradation of α-granule proteins (including P-selectin). The observation that bleeding responds to fibrinolytic inhibitors led to the discovery that QPS platelets possess excess urokinase-type plasminogen activator (u-PA). The genetic defect relates to an increased copy number of PLAU, the u-PA gene [2,12–15].
Glanzmann thrombasthenia (GT)
Here, platelets fail to aggregate due to quantitative or qualitative defects of the αIIbβ3 integrin. Upon platelet activation, αIIbβ3 binds Fg while VWF, fibronectin and vitronectin may also contribute to the protein bridges that mediate aggregation. Clot retraction and endocytosis of plasma Fg are also absent when αIIbβ3 deficiency is severe . GT is caused by mutations across the ITGA2B and ITGB3 genes. Nonsense and splice site mutations with frameshifts are common, as also are missense mutations causing amino acid substitutions. Although specific defects predominate in ethnic groups, mutations are mostly specific for each family; they either prevent subunit biosynthesis in MKs or inhibit transport of the precociously formed αIIbβ3 complexes from the ER to the Golgi apparatus and beyond . Analysis of GT is quite advanced and population studies are underway. The β3 subunit is also present in the vitronectin receptor (αvβ3) expressed in many tissues, but it is a minor component in platelets. In GT, αvβ3 is absent if the genetic lesion stops β3 production.
The first report of variant GT with expressed but nonfunctional integrin, described a D119Y substitution in β3, a mutation which identified a Fg-binding site. Studies on other variants revealed that the codon for R214 of ITGB3 is a mutational hotspot. A S752P substitution and a stop codon in the β3 cytoplasmic tail giving a truncated protein identified a signaling role for integrin cytoplasmic domains. Some Cys mutations in β3 that break disulfides lead to residual activated αIIbβ3 able to spontaneously bind Fg. Rare substitutions within transmembrane or intracytoplasmic domains of αIIb or β3 not only give partially activated αIIbβ3 but also altered megakaryopoiesis and platelet anisocytosis. Rare patients with LAD-III/variant syndrome show life-threatening GT-like bleeding and increased susceptibility to infections. These patients combine lymphocyte, neutrophil and platelet integrin dysfunction due to mutations in the kindlin-3 gene (FERMT3) which abolishes ‘inside-out’ integrin activation although allowing expression [16–20].
Caused by defective scrambling of phospholipids on blood cells including platelets, this disease exhibits decreased fibrin formation at sites of vascular injury. This is caused by a failure of factors Va and Xa to bind to the platelet membrane giving rise to a decreased conversion of prothrombin to thrombin. Procoagulant microparticle release is also defective. Mutations in the TMEM16F gene encoding transmembrane protein 16F, a protein that acts as a Ca2 + -activated chloride channel appears causative of this syndrome .
Familial thrombocytopenias (FT)
IPDs of platelet production often associate a low circulating platelet number with platelet morphological abnormalities; platelet dysfunction may also be present [1–4].
(i) Defects in transcription factors. Mutations in GATA-1 cause X-linked familial dyserythropoietic anemia and macrothrombocytopenia . Thrombocytopenia without anemia may be given by GATA-1 mutations that affect its interaction with FOG-1 but which allow GATA-1 binding to DNA. In contrast, substitutions in the N-terminal finger of GATA-1 that destabilize binding to palindromic DNA sites are associated with red cell abnormalities consistent with β-thalassemia. A low transcription of target genes such as those encoding GPIbβ and GPIX is a characteristic of GATA-1 pathologies and platelets also have fewer α-granules. Monoallelic mutations in RUNX1 (CBFA2, AML1) cause FT with a predisposition to acute myelogenous leukemia. Haplodeficiency and mutations interfering with DNA binding arrest MK maturation and give an expanded population of progenitor cells. Genes with decreased expression include those encoding myosin regulatory light chain polypeptide (MYL9), protein kinase C (PKC)-θ and platelet 12-lipoxygenase (ALOX12) .
In the TAR syndrome, a chromosome 1q21.1 deletion causes bone marrow failure and developmental defects. An 11q23 deletion in the autosomal dominant Jacobsen’s syndrome leads to congenital heart defects, trigonocephaly, facial dysmorphism, mental retardation and malfunctions of multiple organs. Thrombocytopenia or pancytopenia characterise the Paris–Trousseau variant with giant α-granules formed by fusion after MK maturation. Transient monoallelic FLI1 expression during early MK differentiation results in a subpopulation of immature cells that fail to reach the platelet production stage [reviewed in Ref. 2].
(ii) Congenital amegakaryocytic thrombocytopenia. Here, severe thrombocytopenia at birth rapidly develops into pancytopenia. Patients have low numbers of MKs in their marrow; abnormalities in the c-MPL gene mean that TPO is unable to fulfil its normal thrombopoietic role. Patients with early aplasia are more likely to have frameshift or nonsense mutations and a complete loss of c-MPL. Missense mutations with residual c-MPL are often associated with a slower progression of the disease.
(iii) Defects of the cytoskeleton and macrothrombocytopenia. MYH9-related diseases, affecting nonmuscle myosin heavy-chain IIA (myosin-IIA) show phenotypic variations associating macrothrombocytopenia with various combinations of Döhle-like bodies in leukocytes, nephritis, sensorineural hearing loss and cataracts . Platelets are sometimes giant with ultrastructural modifications that extend to MKs. Amino acid substitutions in the head domain with Ca2 + -ATPase activity are more likely associated with deafness and renal disease, while those affecting the rod or tail domain more frequently are restricted to a hematological consequence. Decreased myosin light chain phosphorylation and myosin-IIA function in MKs may affect MK migration and disturb the timing and extent of proplatelet formation. Macrothrombocytopenia may also occur in patients with mutations in FLNA encoding filamin A . These mutations give multiple defects including periventricular nodular heterotopia, an X-linked dominant disease. Filamin A is a cytoskeletal attachment site for GPIbα thereby underlining the importance of the VWF–GPIb–filamin A axis in MK development including the macrothrombocytopenia associated with the Bolzano GPIbα mutation.
(iv) Wiskott–Aldrich syndrome (WAS). This X-linked disease combines microthrombocytopenia with eczema, recurrent infections due to immune deficiency and a high incidence of autoimmunity and malignancy [reviewed in Ref. 2]. WAS platelets aggregate poorly and have a low granule number. Mutations in exons 1 and 2 can give hereditary X-linked thrombocytopenia, a milder form of the disease without infections, probably due to a high prevalence of missense mutations and residual protein. WASP is a key regulator of actin polymerization in hematopoietic cells; its deficiency induces premature proplatelet formation as a lack of actin-rich podosomes slows down MK migration to the vascular sinus.
(v) Other causes. Severe autosomal dominant thrombocytopenia with normal sized platelets is given by mutations in the 5′-untranslated region of ANKRD26, a gene involved in mitochondrial metabolism .
Diagnosis of a suspected IPD starts with the case history and physical examination of the patient [1,3–5]. IPDs mostly manifest early in life with bleeding immediately after injury, primarily in skin (petechiae), from mucous membranes and the nose. Some patients develop life-threatening blood loss in the gastrointestinal or genitourinary tracts while intracranial haemorrhaging can occur. Bleeding score questionnaires are useful to evaluate mild bleeding symptoms, particularly in children that have yet to be hemostatically challenged. Differentiating between mild chronic immune thrombocytopenia (ITP) and some forms of congenital thrombocytopenia can be difficult. If a family history of thrombocytopenia is present, a careful work-up is warranted to prevent inappropriate therapies (poorly chosen medication or splenectomy) while it is essential to compile a record of clinical complications such as bone marrow failure, oncological disorders, sensorial hearing loss, renal failure or others. For some patients, significant bleeding may only arise after surgery or trauma and a sufficient challenge to the hemostatic system. Similar bleeding patterns are found in type 1 or 2 VWD and therefore some IPDs can be wrongly diagnosed as VWD.
During initial screening, particularly important is measuring the platelet count and the mean platelet volume; while a peripheral blood smear is recommended for giant platelet syndromes as electronic counters may underestimate platelet numbers and size [24,27]. Measuring the Ivy bleeding time is no longer standard practice and some replace it by the platelet function analyzer (PFA-100). Laboratory investigation of platelet aggregation, ATP secretion and quantification of platelet receptors by flow cytometry are standard procedures. Often requiring specialist help, immunofluorescence (e.g. distinctive patterns for myosin-IIA in leukocytes are typical of MYH9 disease) and electron microscopy are often useful as an aid to diagnosis; while evaluating platelet adhesion and spreading on protein surfaces is informative especially if accompanied by a study of signalling pathways (phosphorylations, western blotting) [7,11,13,24,25,28]. Finally, flow chambers and computerized analysis of thrombus formation on protein-covered surfaces (e.g. Fg, VWF, collagen) under controlled flow, procedures often validated for platelets from genetically-modified mice, will fast become applicable to human pathology .
Isolated or broad spectrum IPDs
Platelets are easily obtainable and citrated platelet-rich plasma is mostly used to study platelet function under basal and activated conditions [3,5]. Algorithmns are being developed to permit step-by-step detection of specific pathologies. Defects in platelet adhesion, aggregation, G protein signaling, secretion and platelet production can result from mutations in platelet-specific genes leading to isolated thrombocytopathy or thrombocytopenia for which the main clinical feature is bleeding (e.g. BSS, GT, P2Y12 deficiency and other diseases as reviewed in Molecular basis of platelet disorders). In contrast, when mutations occur in widely expressed genes, patients usually develop a broader clinical phenotype with bleeding accompanied by neuropathology, endocrine dysfunction, other hematological and/or metabolic problems. Therefore, clinical investigation and platelet research go hand-in-hand to improve knowledge of broad phenotype mendelian disorders [10,30].
From candidate gene screening to new generation technologies for genetic diagnosis of IPD
In the past, platelets from patients with IPD were first functionally tested to gain information on the receptor and/or pathway likely to be defective before sequencing a single or a few genes (see Molecular basis of platelet disorders). Though major causes of IPD have been discovered via this approach over the past decades, the genetic basis for IPD in many patients remains unknown. The recent application of novel large scale screening methods in proteomics and transcriptomics [31,32], genome-wide association studies (GWAS)  and epigenetics  plus unprecedented large-scale cooperative efforts, have led to novel insights in platelet biology and of IPD complexity. Next generation sequencing approaches are expected to unravel IPDs that are already well phenotyped but which still have no identified genetic basis. The identification of NBEAL2 gene mutations as the causative factor for GPS via RNA or exome sequencing is the first such example. It is now clear that defective α-granule formation is caused by a protein family member of LYST and NBEA, proteins that are defective in platelets from patients with abnormal dense granules and broad spectrum IPD [11–14,35].
Advances in our knowledge of both the platelet genome and proteome will lead to further discoveries in MK and platelet biology. The use of novel ‘Omics’ technology via arrays or chips will potentially revolutionize not only current diagnosis of ‘classical’ IPDs through functional platelet testing but will additionally identify individuals with increased risk of bleeding previously hidden by their association with other clinical phenotypes. Discussion of the future of novel DNA-based diagnostic approaches for IPDs is particularly relevant as whole exome sequencing is fast becoming readily available and economically viable. These novel technologies will bring platelet function testing closer to the field of medical DNA sequencing . In the future, DNA sequencing will give clinicians important information regarding the IPD genotype of their patients and so improve early diagnosis and prognosis.
IPDs are rare disorders manifested by mild to severe mucocutaneous bleeding; for affected patients, e.g. GT or BSS, platelet transfusion is frequently needed for controlling spontaneous bleeding manifestations such as menorrhagia, epistaxis, and gastrointestinal bleeding, and is always needed when trauma occurs or surgery is performed. Childbirth is also a high-risk period [37,38]. For the mild to moderate bleeding entities, e.g. SPD, P2Y12 or TXA2 receptor defects, platelet transfusion is usually unnecessary. Transfusion of platelets should be used selectively and sparingly because of the substantial risk of alloimmunization against HLA antigens and/or platelet αIIbβ3 in GT and GPIb-IX-V in BSS that may lead to refractoriness to therapy [1,39]. To reduce the risk, HLA-matched single donors of platelets are recommended. If such donors are unavailable, leukocyte-depleted blood components should be used. Additional risks of platelet transfusion and blood component therapy are allergic reactions and transmission of infectious agents. Treatment modalities other than platelet transfusion comprise:
(i) Prevention. This includes vaccination against hepatitis B, avoidance of non-steroidal anti-inflammatory drugs, preservation of dental hygiene, correction of iron deficiency anemia and prenatal diagnosis when the mutation is known.
(ii) Topical hemostatic measures. These can involve compression with gauze soaked with tranexamic acid, fibrin sealants containing tranexamic acid, acrylic splints for dental extraction, and packing for nose bleeds.
(iii) Antifibrinolytic agents (epsilon aminocaproic acid or tranexamic acid). Such agents are useful for prevention of bleeding following minor surgery, and can be employed as adjuncts of other treatment modalities such as recombinant factor VIIa (rFVIIa), 1-desamino-8D-arginine vasopressin (DDAVP), and platelet transfusion. Patients should be guided to start oral treatment with one of the antifibrinolytic agents whenever troublesome bleeding occurs and thereafter seek medical attention if necessary. Antifibrinolytic agents are essential for Quebec platelet syndrome.
(iv) Management by DDAVP. This agent increases the plasma concentrations of von Willebrand factor (VWF) and factor VIII. The mechanism of DDAVP action has been attributed to increased adhesiveness of platelets to the subendothelial matrix, and to augmented platelet aggregation at high shear rate resulting in shortening of bleeding time . Several small series of DDAVP-treated patients with variable inherited platelet dysfunctions have been reported. Entities for which unequivocal evidence indicates that bleeding time shortens after DDAVP include delta-storage pool diseases, disorders of granule secretion, signal transduction disorders, thromboxane receptor deficiency, May-Hegglin anomaly and patients with unexplained prolonged bleeding time. Equivocal evidence was provided for BSS, HPS and arachidonate metabolism defects. Patients with GT do not respond to DDAVP . Side effects of DDAVP administration include tachycardia, hypotension, facial flushing, headache, severe hyponatremia with seizures and arterial thrombosis.
(v) rFVIIa. GT patients have been treated for bleeding episodes by rFVIIa with partial success [42,43]. The mechanism by which rFVIIa arrests bleeding is probably related to increased thrombin generation by a tissue factor-independent process, enhanced adhesion of platelets to extracellular matrix and restoration of platelet aggregation [44–46]. The use of rFVIIa in patients with inherited platelet dysfunction has not been examined by randomized controlled studies. Among 59 GT patients in an international survey, 75% of 108 spontaneous bleeding episodes and 94% of 34 surgical procedures were manageable by rFVIIa. However, two patients who received a high dose of rFVIIa developed pulmonary embolism and a ureteric clot, respectively .
(vi) Female hormones. Menarche, particularly in patients with GT and BSS is frequently associated with excessive bleeding necessitating blood transfusions. This may result from prolonged estrogen stimulation of unovulatory cycles with extensive endometrial proliferation leading to breakthrough bleeding. Hemostasis in such cases can be achieved by intravenous infusion of high dose conjugated estrogen for 24–48 h followed by high doses of oral estrogen – progestin. Menorrhagia later in life is also frequent in patients with GT and BSS. If in such patients antifibrinolytic agents fail to decrease the blood loss, continuous oral contraceptives can be useful in eliminating menses and should be considered especially in women with anemia due to iron depletion. Depo-medroxyprogesterone acetate administered every three months is an alternative when combined oral contraceptives are contraindicated.
(vii) Use of TPO mimetics. Recent advances in raising the platelet count have included the use of the TPO mimetics, romiplostim and eltrombopag in chronic and refractory ITP. One group has shown that eltrombopag will raise the platelet count and protect patients with MYH9-related disease from the risk of bleeding . This raises the possibility of using TPO mimetics in inherited thrombocytopenias, for example, to increase the platelet count prior to surgery.
(viii) Hematopoietic stem cell (HSC) transplantation and gene therapy. To date, 14 patients with severe GT and 3 patients with BSS have been successfully transplanted with stem cells of HLA-identical siblings, matched unrelated donors, or matched family donors . Careful evaluation of the risk-benefit ratio of this procedure must be assessed in each individual. WAS is amenable to HSC gene therapy and genetically modified HSC have permitted its first successful use in two German patients with marked clinical improvement over 3 years .
We enter a new period with the identification of the molecular basis of most of the named platelet disorders with a defined phenotype. Diagnosis is being standardized and will undergo a revolution in the coming years with the application of new generation DNA typing probably identifying not only the genetic basis of a whole new range of platelet function and platelet production defects but also SNPs that control bleeding severity in what were otherwise thought to be monogenic disorders. This in turn will help personalize treatment for the individual patient.
The authors stated that they had no interests which might be perceived as posing a conflict or bias.