By continuing to browse this site you agree to us using cookies as described in About Cookies
Notice: Wiley Online Library will be unavailable on Saturday 7th Oct from 03.00 EDT / 08:00 BST / 12:30 IST / 15.00 SGT to 08.00 EDT / 13.00 BST / 17:30 IST / 20.00 SGT and Sunday 8th Oct from 03.00 EDT / 08:00 BST / 12:30 IST / 15.00 SGT to 06.00 EDT / 11.00 BST / 15:30 IST / 18.00 SGT for essential maintenance. Apologies for the inconvenience.
Alan T Nurden, Emeritus Research Director CNRS, CRPP/PTIB, Hôpital Xavier Arnozan, 33604 Pessac, France. Tel.: +33 5 57 10 28 51; fax: +33 5 57 10 28 64. E-mail: Alan.Nurden@cnrshl.u-bordeaux2.fr
Summary. Genetic defects of platelet function give rise to mucocutaneous bleeding of varying severity because platelets fail to fulfil their haemostatic role after vessel injury. Abnormalities of pathways involving glycoprotein (GP) mediators of adhesion (Bernard–Soulier syndrome, platelet-type von Willebrand disease) and aggregation (Glanzmann thrombasthenia) are the most studied and affect the GPIb-IX-V complex and integrin αIIbβ3, respectively. Leukocyte adhesion deficiency-III combines Glanzmann thrombasthenia with infections and defects of kindlin-3, a mediator of integrin activation. Agonist-specific deficiencies in platelet aggregation relate to mutations of primary receptors for ADP (P2Y12), thromboxane A2 (TXA2R) and collagen (GPVI); however, selective abnormalities of intracellular signalling pathways remain better understood in mouse models. Defects of secretion from δ-granules are accompanied by pigment defects in the Hermansky–Pudlak and Chediak–Higashi syndromes; they concern multiple genes and protein complexes involved in secretory organelle biogenesis and function. Quebec syndrome is linked to a tandem duplication of the urokinase plasminogen activator (PLAU) gene while locus assignment to chromosome 3p has advanced the search for the gene(s) responsible for α-granule deficiency in the gray platelet syndrome. Defects of α-granule biosynthesis also involve germline VPS33B mutations in the ARC (arthrogryposis, renal dysfunction and cholestasis) syndrome. A mutation in transmembrane protein 16F (TMEM16F) has been linked to a defective procoagulant activity and phosphatidylserine expression in the Scott syndrome. Cytoskeletal dysfunction (with platelet anisotrophy) occurs not only in the Wiskott-Aldrich syndrome but also in filamin A deficiency or MYH9-related disease while GATA1 mutations or RUNX1 haploinsufficiency can affect expression of multiple platelet proteins.
This review explores recent advances in our understanding of the molecular basis of inherited disorders affecting platelet function [1,2]. These rare diseases give rise to bleeding syndromes of varying intensity. Spontaneous bleeding is mostly mucocutaneous in nature while excessive trauma-related haemorrhage is a feature of milder forms . Studies on these disorders have provided key information on the mechanisms of platelet function. Figure 1 updates the principal disorders affecting platelet surface constituents, while Fig. 2 is restricted to those affecting intracellular components. Classification of the disorders in groups related to abnormalities of platelet function is provided in Table 1.
Table 1. Classification of inherited disorders of platelet function
Group of abnormalities
Platelet count and morphology
Defective platelet function
Associated biological abnormalities
For an appraisal of the landmark studies giving rise to the original descriptions of the diseases contained in this Table the reader is referred to the reviews provided as refs [1–3,5,29,42,55,69,123–126]. VWD2B has been included as quite clearly in some patients an altered megakaryocytopoiesis contributes to the phenotype; some patients with VWD2B have been known in the past as the Montreal platelet syndrome (a term that should no longer be used).
BSS associates macrothrombocytopenia and decreased platelet adhesion to the subendothelium caused by quantitative or qualitative defects of the GPIb-IX-V complex [1,2]. The products of four separate genes (GPIBA, GPIBB, GP9 and GP5) assemble within the maturing megakaryocyte (MK) in the bone marrow to form GPIb-IX-V stabilised through cytoplasmic domains linked to the membrane cytoskeleton via filamin A (FlnA) . Mutations within GPIBA, GPIBB and GP9 interfere with biosynthesis and prevent formation and/or trafficking of the complex through the Golgi apparatus and endoplasmic reticulum of MKs . GPIbα contains the von Willebrand factor (VWF) binding site and two thrombin-binding sites located within the N-terminal domain. The additional absence of GPIbα-dependent binding of P-selectin, thrombospondin-1 (TSP1), factor VIIa, factor XI, factor XII, αMβ2 and high molecular weight kininogen may also contribute to the phenotype . Recent data suggest that bleeding results from the loss of high shear- and VWF-dependent platelet-to-platelet interactions in thrombus formation as well as a defective platelet attachment to VWF . In occasional rare variant forms of BSS, platelets express non-functional GPIbα. Hemizygous mutations in GPIBB can also cause BSS when associated with the DiGeorge/Velocardiofacial syndrome, a developmental disorder characterised by a hemizygous microdeletion at 22q11, the site of the GPIBB locus [1,2]. Mouse models of BSS show aberrant membrane development during MK maturation, impaired proplatelet formation and microtubule coil assembly as contributing to the phenotype [8–11].
Platelet-type von Willebrand Disease (platelet-type VWD)
Platelet-type von Willebrand disease (VWD) is characterised by a gain-of-function phenotype with spontaneous binding of plasma VWF to platelets and increased platelet agglutination with low amounts of ristocetin in the presence of normal plasma. Heterozygous GPIBA mutations with autosomal dominant inheritance cause platelet-type VWD, with Gly233Val (or Ser) and Met239Val or (Ile) substitutions in the N-terminal domain; while a 27bp deletion in the macroglycopeptide-coding region of GPIBA implies that long-range conformational changes can also give rise to platelet-type VWD [1,2,12]. Interestingly, a knock-in mouse model with the Gly233Val mutation not only presents with a phenotype that mimics the human disorder but also exhibits increased bone mass . In the same model, spontaneous binding of VWF was shown to block the capacity of platelets to bind fibrinogen (Fg) when stimulated . Bleeding presumably results from this and an inhibited GPIbα, although ADAMTS13 (A Disintegrin And Metalloproteinase with TSP-1 repeats 13) cleavage of platelet-bound VWF multimers under shear may modulate the condition . Platelet size can be increased in platelet-type VWD and be associated with a moderate thrombocytopenia. The clinical condition resembles type 2B VWD and it is probably under-diagnosed.
Type 2B VWD (VWD2B)
Enlarged platelets and thrombocytopenia frequently occur in VWD2B (which can arise through certain heterozygous mutations in exon 28 of the VWF gene) . As with platelet-type VWD, inheritance is autosomal dominant. In one such family (carrying an Arg1308Pro VWF substitution), platelets showed signs of apoptosis, and culture of CD34+ cells from the peripheral blood resulted in an augmented surface expression of the mutant VWF on mature MKs that mediated interactions between proplatelets . This study was extended to nine patients with a total of seven different gain-of-function mutations and abnormal platelets typical of those in the circulation were produced ex vivo in MK cultures . Circulating platelet agglutinates in rare patients with VWD2B prompted comparison with the previously described phenotype of the Montreal platelet syndrome (MPS); this enigma was resolved when it was shown that patients in the founder kindred for MPS all have the common VWF Val1316Met mutation . Among the described characteristics of MPS were platelet calpain deficiency (probably due to autodegradation) and a reduced thrombin-induced aggregation; both findings are a likely consequence of the spontaneous binding of large VWF multimers to GPIbα. Significantly, lineage-specific mouse knock-in models of VWD2B show that abnormal plasma VWF (containing a VWD2B mutation) can broadly reproduce the human VWD2B phenotype whilst emphasising the haemostatic variability of the disease and a possible role for ADAMTS13-dependent modulation of disease severity [19,20].
Inherited variants of agonist receptors and signaling pathways
Deficient collagen receptor functions
Integrin α2β1 is a surface collagen receptor found in a wide variety of cell types. Although excessive bleeding linked to platelet α2β1 deficiency has been hinted at in the past, proof for a specific pathology is still lacking. Natural changes in receptor density given by single nucleotide polymorphisms (SNPs) in both coding and non-coding regions of the ITGA2 gene cause haplotype variability [21,22]. Rare patients lacking collagen-induced aggregation have platelets deficient in GPVI, a member of the immunoglobulin superfamily of cell membrane receptors naturally linked to FcRγ in the platelet membrane. Platelet expression of the GPVI-FcRγ complex is also greatly influenced by SNPs and epigenetic factors, and specific haplotypes are again linked to low platelet reactivity with collagen. For this receptor, acquired antibodies and possibly platelet activation itself can promote sheddase activity by multiple platelet-expressed proteinases causing loss of the GPVI ectodomain [22–25]. Only recently has the absence of collagen-induced platelet activation been linked to mutations in the GP6 gene [26,27]. In France, a young girl with a lifelong mild bleeding syndrome associates an Arg38Cys mutation in exon 3 of one allele with an insertion of 5 nucleotides in exon 4 of the second allele (leading to a premature stop codon and mRNA instability); the result was low expression of non-functional GPVI . In Belgium, a patient with strongly reduced platelet expression of non-functional GPVI has a combination of an out-of-frame 16-bp deletion and a Ser175Asn missense mutation .
Pathologies of ADP and ATP receptors
Platelets possess synergic receptors for ADP: P2Y1 mediates Ca2+ -mobilisation, shape change and starts aggregation, while P2Y12 promotes the formation of large and stable platelet aggregates. Both receptors belong to the seven transmembrane domain family of G-protein-linked receptors. Rare patients with an autosomal recessive hereditary disease and mild bleeding have much decreased and reversible platelet aggregation in response to ADP (despite a normal shape change and Ca2+-mobilisation). Studies from our laboratory helped identify P2Y12 as an ADP receptor when analysis of polymerase chain reaction products from the P2Y12 coding region of genomic DNA revealed a mutant allele at this locus . Strikingly in this pathology, platelets show identical functional changes to normal platelets treated with the anti-platelet drugs clopidogrel and prasugrel for which P2Y12 is the pharmacologic target . While an expressed but non-functional receptor associated with an Arg256Gln substitution has been reported, the majority of P2Y12 gene defects so far described either abolish P2Y12 expression or interfere with expression and/or prevent ligand binding; known mutations are listed in Table 1 of Cattaneo  and Watson et al. . Some subjects that are heterozygous for P2Y12 mutations have decreased sensitivity to ADP or have a secretory defect, while a Pro341Ala mutation in a PDZ domain interfered with receptor cycling and lead to intra-platelet accumulation (data reviewed in ). An enigma is that P2Y12 also regulates microglial activity by extracellular nucleotides, for although microglial chemotaxis is deficient in P2ry12(−/−) mice  no abnormalities in brain or central nervous system function have been reported in human patients with P2Y12 mutations. A synergistic role of ADP in the platelet functional response also means that platelets with dysfunctional P2Y12 show a decreased sensitivity to low doses of other agonists. P2X1, a purinergic receptor for ATP, modulates platelet intracellular Ca2+ levels in response to stimulation. A natural dominant negative P2X1 receptor due to deletion of a single amino acid has been described in a young girl with a bleeding syndrome . No human pathology of P2Y1 function has been reported despite the fact that mice deficient in this receptor have severe bleeding .
Altered function of other receptors for primary agonists
A homozygous Arg60Leu substitution in the first cytoplasmic loop of the thromboxane A2 (TxA2) receptor (TxA2R) was described in a Japanese patient with increased trauma-related bleeding. Platelet aggregation was primarily defective to TxA2 (and its analogues) . The frequency of heterozygous expression of this mutation in several Japanese families suggested autosomal dominant inheritance, although for these subjects there was only a marginal clinical effect. When variant TxA2R was expressed in Chinese hamster ovary (CHO) cells, ligand binding was unaffected but signal transmission impaired . More recently, a heterozygous Asp304Asn substitution in TxA2R was discovered in a teenage boy with a history of severe nose bleeding. His platelets showed a selective loss in their response to the stable TxA2 mimetic, U45519 . However, his father from whom the mutation was inherited had no bleeding history raising the possibility of a second, unidentified defect in the propositus. An absent platelet response to adrenaline is common in routine screening of platelet function although its contribution as a cause of bleeding is still a matter of debate. Congenital deficiencies of the α2-adrenergic receptor associated with a decreased platelet response to adrenaline have been reported but such findings may reflect haplotype complexity within this gene [36,37]. Nevertheless, studies on mice point to a role for α2A-adrenergic receptors in thrombus stability . Other seven transmembrane domain receptor family members in human platelets are the thrombin receptors, protease-activated receptor (PAR)-1, and PAR-4. So far, no human pathologies of PAR receptors have been described.
Defects of intracellular signalling pathways
Pathologies involving the signal transduction pathways of platelets may be quite common. Here, defects of platelet aggregation affect some stimuli more than others and may be secretion-dependent. Little is known of their molecular basis largely due to the complexities of the platelet signalling pathways [39,40]. Table 1 of reference  describes the platelet phenotype from each of a selection of reports where key signalling proteins have been targeted in mice while Fig. 3 highlights proteins of major ‘inside-out’ and ‘outside-in’ signalling pathways that are candidates for dysfunction in platelet pathologies. Defects of G proteins have been reported in man; for example, decreased expression of Giα1 in a patient who associated chronic bleeding with a severely decreased aggregation with ADP (data reviewed in ). A platelet-specific defect of Gαq expression was reported in a patient with lifelong mucocutaneous bleeding and decreased platelet aggregation, calcium mobilisation and secretion to multiple agonists including ADP, thrombin and TxA2 (P2Y1, TxA2R and the PAR receptors are linked into Gαq) . Brief mention should also be made of congenital Gsα hypo- or hyperfunction syndromes . Gsα is linked to receptors for such agents as prostaglandin I2 (PGI2), PGE1 and pituitary adenylyl cyclase (AC) activating peptide (PACAP) and its stimulation leads to increased cAMP production. Patients with a partial trisomy 18p have three copies of the PACAP gene, elevated PACAP and strongly increased cAMP levels in a syndrome that associates severe neurological and other abnormalities with a pronounced bleeding tendency and mild thrombocytopenia. The complex imprinted gene cluster GNAS1 codes Gsα and genetic defects within this cluster (including an extra-large stimulatory Gsα isoform (XL-Gsα)) give rise to a range of disorders associated with a thrombotic phenotype and Gsα hyperfunction (data reviewed in ). Finally, platelet Gs hypofunction linked to a heterozygous mutation in a regulator of G-protein signalling (RGS) protein 2 results in different functional RGS2 isoforms with a stronger (and inhibitory) interaction with AC .
Some reports have identified lineage-specific defects in proteins essential for activation and secretion in platelets to transcription factor defects (see next section). Other reports, largely based on mouse models, suggest that defects in the formation of stable aggregates can result from deficiencies or the altered function of a range of proteins that are secreted from platelets or come into play after αIIbβ3 occupancy with fibrinogen (Fg) [39–41] (Fig. 3). The result is reduced outside-in signalling; a finding that may also be indicated by an abnormal clot retraction or an abnormal spreading of platelets on surface-bound Fg (or another adhesive protein), a simple test that is under-used in the diagnosis of platelet disorders.
Aggregation defects secondary to mutations of transcription factors
Decreased platelet levels of protein kinase C (PKC)-θ (and mild thrombocytopenia) and phospholipase C-β2 were linked to aggregation and secretion defects in patients with defective plekstrin phosphorylation in response to thrombin (data reviewed in ) (Fig. 3). But reduced lineage-specific expression of proteins in conserved signalling pathways suggested altered MK gene transcription and a heterozygous nonsense mutation in the CBFA2 (core-binding factor 2) gene leading to a truncated RUNX1 (runt-related transcription factor 1) protein was identified in the patient with PKC-θ deficiency . Myosin-light chain phosphorylation was also decreased in this patient and platelet mRNA profiling showed that among multiple down-regulated genes was that encoding myosin regulatory light chain polypeptide (decreased ∼77-fold) . Also down-regulated, was the platelet 12-lipoxygenase gene (ALOX12), a direct transcriptional target of CBFA2 and responsible for a decreased production of 12-hydroperoxyeicosatetraenoic acid (12-HETE) in thrombin-activated platelets of the patient . Such results show how variations in transcription factor expression (or activity) have effects beyond platelet production. It should be emphasised that CBFA2 haplodeficiency is also a cause of familial thrombocytopenia associated with a predisposition to leukaemia . GATA-1 is another transcription factor that regulates the expression of multiple platelet proteins (including GPIbα and GPVI) as well as α-granule biogenesis (see Section on the gray platelet syndrome); collagen-induced platelet aggregation and GPVI-dependent tyrosine phosphorylations are particularly affected .
Patients with an inherited ‘aspirin-like’ platelet defect have dysfunctional arachidonic acid (AA) metabolism; mostly with autosomal dominant inheritance and a mild bleeding phenotype . Abrogated metabolism of AA to TxA2 has a number of potential causes including an abnormal release of AA from membrane phospholipids by phospholipase A2, and defects in its stepwise metabolism by prostaglandin G/H synthetase, cyclooxygenase-1 and thromboxane synthetase (data reviewed in ). However, their genetic basis remains unknown except for a recent report identifying mutations in the TBXAS1 gene encoding thromboxane synthetase in families with Ghosal hematodiaphyseal dysplasia, a syndrome where AA-dependent platelet dysfunction is associated with increased bone density . 12-Lipoxygenase dysfunction and disorders of glycogen-6 synthetase and ATP metabolism have all been reported to lead to platelet aggregation defects resembling those seen in storage pool disease.
Defects of secretion (storage pool disease, spd)
This heterogeneous collection of inherited disorders contains well-characterised examples of intracellular defects of platelets (Fig. 2); some disorders are due to defects in genes that encode a protein whose function extends to several cell types but where from a haemostasis point of view the defect mostly concerns secretion-dependent aggregation.
Defects of α-granules
Platelets contain storage pools of biologically active proteins that are either synthesised in MK (e.g. VWF), or endocytosed from plasma (e.g. Fg) and stored in α-granules. Early histochemical evidence suggested little overlap in the organisation of Fg and VWF in the α-granules and this heterogeneity was confirmed by immunofluorescent labelling [52,53]. Indeed, recent evidence suggests selective sorting with pro-angiogenic proteins (e.g. vascular endothelial growth factor, VEGF; basic fibroblast growth factor, bFGF) found in a different subpopulation of organelles from those that inhibit angiogenesis (e.g. TSP-1, endostatin) . The organelle membranes contain a variety of GP (e.g. P-selectin and CD63) that are translocated to the plasma membrane during secretion. Inherited loss or dysfunction of proteins whose major pool is plasmatic (e.g. factor V deficiency, fibrinogen in afibrinogenaemia, VWF in type 3 VWD) can be accompanied by parallel deficiencies in the α-granule pool. Only disorders unique to the α-granule pool will be described here.
Gray platelet syndrome (GPS) A usually mild bleeding disorder with predominantly autosomal recessive inheritance, GPS is characterised by the selective absence of all α-granule subpopulations and their contents [55,56]. Vestigial granules may be seen and the basic molecular defect likely involves packaging or storage of proteins during α-granule biogenesis. Other clinical features include the early onset of myelofibrosis and splenomegaly. GPS patients often have a moderate but progressive thrombocytopenia and platelets that are somewhat enlarged and vacuolated. A negative regulation of megakaryocytopoiesis by spontaneously released cytokines/chemokines and other α-granule proteins is likely. A novel recent finding is a high serum level of vitamin B12 . Secretion-dependent platelet aggregation tends to be reduced with thrombin-induced platelet aggregation particularly affected in some patients, in others it is the collagen response that is lacking. In a French patient this was accompanied by reduced GPVI expression, possibly due to cleavage by metalloproteases (MMPs) in the marrow or spleen . Interestingly, the endogenous pools of MMP-2 and MMP-9 and their inhibitors were normal in GPS platelets suggesting another unrecognised mechanism of storage and release of some biologically active proteins in platelets . The genetic defect responsible for GPS has remained an enigma, a report of X-linked GPS due to a GATA-1 Arg216Gln mutation is atypical, for this and other related GATA-1 mutations also give rise to dyserythropoietic anaemia a finding not characteristic of GPS . Recently, genome-wide linkage analysis of a large cohort of 25 GPS patients from 14 unrelated families spanning several continents has localised the GPS gene(s) to a 9.4-Mb interval on 3p21.1–3p22.1 including 197 protein-coding genes . Other variant disorders affecting α-granules include the White platelet syndrome and the Medich giant platelet disorder where platelets also have scroll-like membranous inclusions . Giant α-granules and defective secretion are also seen in the Paris–Trousseau syndrome linked to deletions on the long arm of chromosome 11 and a hemizygous deficiency of the transcription factor Friend leukaemia integration (Fli)-1 gene .
Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome Mutations in the gene encoding vacuolar sorting protein 33B (VPS33B), a regulator of SNARE-protein-dependent fusion, have been described in the ARC syndrome. Mainly affecting young children, platelet dysfunction and absent α-granules are associated with a multisystem disorder featuring renal tubular and other dysfunctions as well as bleeding. The defect extends to both stored and membrane components of α-granules .
Quebec platelet disorder (QPD) This autosomal dominant bleeding disorder with high penetrance is so far exclusive to French-Canadian families. The fact that bleeding responds to fibrinolytic inhibitors rather than platelet transfusions led to the discovery that the patients’ platelets possessed unusually large amounts of urokinase-type plasminogen activator (u-PA), a protein that is secreted upon platelet activation [63,64]. This protein is stored in α-granules; its excess over natural inhibitors results in plasmin generation and degradation of many otherwise normally expressed α-granule proteins (including P-selectin) while α-granule ultrastructure is preserved . Thrombocytopenia is sometimes observed in QPD; a platelet aggregation deficiency is most striking with epinephrine, the reason for this is unknown. Genetic marker analysis showed that QPD was linked to a 2-Mb region on chromosome 10q that included the PLAU gene encoding u-PA . No sequence alterations were found, but increased u-PA mRNA levels were confirmed during MK differentiation. Copy number variation was examined and a direct tandem duplication of a 78-kb genomic fragment that includes PLAU was detected that was exclusive to all family members with QPD . This is the first copy number defect attributed to an inherited platelet disorder.
Defects of dense (δ) granules
δ-granules are storage sites for serotonin, calcium (which gives them an intrinsic electron density and a dark appearance in electron microscopy), ADP and ATP, pyrophosphate and polyphosphate. Membrane markers include P-selectin, lysosomal-associated membrane protein (LAMP)-2 and LAMP-3 (CD63). While the roles of secreted serotonin and ADP are well known, only recently has the influence of pyrophosphate and polyphosphate on fibrin clot structure been recognised . Largely due to the abnormal secretion of ADP, SPD affecting δ-granules causes a defective secretion-dependent aggregation that affects the response to collagen in particular. The granule deficiency may be severe or partial, in some patients it may also extend to α-granules (αδ-storage pool deficiency). Little is known about the molecular basis of relatively common isolated deficiencies of platelet δ-granule secretion that can affect granule biogenesis or storage of selected substances. A recent study has highlighted a deficiency of multidrug-resistant protein 4 (MRP4) in two patients with δ-storage pool deficiency although its genetic basis was not resolved . When rare platelet deficiencies of δ-granules are associated with abnormalities of other lysosome-related organelles, they lead to clearly defined phenotypes . These include the Hermansky–Pudlak, Chediak–Higashi and Griscelli syndromes where defects also include a lack of pigmentation of the skin and hair.
Hermansky–Pudlak syndrome (HPS) Here, a bleeding diathesis is associated with albinism and impaired development of lysosome-related organelles including platelet δ-granule deficiency and abnormal melanosome formation. Albinism is accompanied by horizontal nystagmus with lateral eye movement accompanied by a decrease in pigmentation allowing iris transillumination . Additional features seen in some patients include ceroid-lipofuchsin storage in the reticulo-endothelial system; granulomatous colitis or fatal pulmonary fibrosis may also occur. HPS is common on the island of Puerto Rico where a 16-base duplication in exon 15 of the HPS-1 gene results in a frameshift and truncation of a 79 kDa protein with two membrane spanning domains. Defects in at least eight genes (HPS-1 through HPS-8) are now known to cause distinct HPS subtypes in man. The HPS proteins interact with each other in complexes termed BLOCS 1-3 (biogenesis of lysosome-related organelles complexes 1–3); the genetic defects disrupt these, thereby affecting organelle biosynthesis and protein trafficking [69,70]. In HPS-2, it is the beta3A subunit of the adaptor protein (AP)-3 complex that is abnormal . One manifestation of HPS-2 is an increased routing of lysosomal membrane proteins such as CD63 to the plasma membrane. HPS-2 is associated with innate immunity defects . Each of the HPS gene defects has a murine model (see Table 2 in ).
Chediak–Higashi syndrome (CHS) In CHS, a bleeding syndrome, δ-granule deficiency and decreased pigmentation is associated with the presence of giant inclusion bodies in a variety of granule-containing cells including platelets . Children have life-threatening infections; immunologic defects include neutropenia and a decreased function of natural killer cells. A fatal complication, lymphohistiocytosis (accelerated phase) occurs in 85% of patients with the uncontrolled proliferation of lymphocytes; there is progressive neurological dysfunction if the patient survives to adulthood . The CHS gene has been cloned and a series of frameshift and nonsense mutations described that result in a truncated CHS protein and a severe phenotype . Rare missense mutations may be associated with a milder form of the disease . The affected protein (lysosomal trafficking regulator, Lyst) is large with distinct structural domains including ‘BEACH’ and ‘HEAT’ suggestive of a function in membrane trafficking and organelle biogenesis.
Griscelli syndrome In this rare disease, patients belong to several subtypes with partial albinism and silver hair, neurological defects and/or lymphohistiocytosis . Major bleeding is rare and platelet δ-granules little studied. The major clinical difficulty is a fatal haemophagocytic syndrome caused by inappropriate lymphoid-cell activation and cytokine release. Griscelli disease is given by mutations in the genes encoding myosin Va, Rab27a (a small GTPase) and melanophilin . Only mutations in Rab27a are associated with immune deficiency. Differential diagnosis with HPS type II can be difficult. This is shown by the case of a child with bleeding and an impaired secretion-dependent platelet aggregation (and abnormal δ-granules) but who associated a heterozygous RAB27A mutation with a novel homozygous AP3B1 mutation . The patient developed fulminant haemophagocytic lymphohistiocytosis that was resistant to therapy.
Defects of the cytoskeleton
Wiskott–Aldrich syndrome (WAS)
This X-linked recessive disease combines thrombocytopenia and small platelets (microthrombocytopenia) with eczema, recurrent infections due to immune deficiency and an increased risk for autoimmune disorders and malignancy [1–3,75,76]. A milder form lacking the immune problems is known as hereditary X-linked thrombocytopenia. WAS platelets aggregate poorly and due to their small size have a reduced number of α-granules, δ-granules and mitochondria. T lymphocytes and leukocytes also show defective function. The WAS gene is composed of 12 exons and encodes a 502-amino acid protein (WASP). Genetic defects in WAS result either in the decreased expression of WASP or its absence. A large number of patients have been genotyped. For example, a recent European demographic study identified 87 affected males and 48 carrier females . Mutations were scattered throughout the gene; the most common sequence variations were missense mutations and small insertions followed by nonsense and splice site mutations, and insertions. Only one large deletion was seen. Generally in WAS, missense mutations accompanied by partially expressed and functional protein give rise to hereditary X-linked thrombocytopenia [75,76]. Mutations that lead to spontaneously activated WASP with increased actin polymerising activity cause an X-linked form of neutropenia with variable myelodysplasia . WASP is a key regulator of actin polymerisation in haematopoietic cells; it is involved in signal transduction with tyrosine phosphorylation sites and adapter protein function. WAS should be considered as a pathology of the membrane cytoskeleton. A lack of WASP compromises multiple aspects of normal cellular activity including proliferation, phagocytosis, immune synapse formation, adhesion and directed migration . It also leads to premature proplatelet formation in the marrow where a lack of actin-rich podosomes slows down MK migration to the vascular sinus . An altered in vivo activity with immunoglobulins also contributes to a decreased platelet survival, another characteristic of WAS.
Defects of the non-muscle myosin heavy chain IIA (NMMHCIIA) and filamin A (FlnA)
Autosomal dominant MYH9-related disease is best known for its macrothrombocytopenia associated with multiple phenotypic variations including different combinations of Döhle-like bodies in leukocytes, nephritis, sensorineural hearing loss and cataracts [reviewed in 79]. Strikingly, the same heterozygous mutations can be associated with different phenotypes suggesting that the diseases are not truly monogenic. Nevertheless, amino acid substitutions in the head domain (with Ca2+-ATPase activity) are more often associated with deafness and renal disease, while those affecting the rod (and myosin-IIA assembly) more frequently only have a haematological consequence . Haploinsufficiency may have a role, while other genetic and/or environmental factors probably also intervene to influence phenotype. Decreased myosin light chain phosphorylation and myosin-IIA function in MKs in MYH9-related disease appear to slow MK migration towards the sinusoids as well as blurring the signalling mechanism for proplatelet formation . This compromises the negative regulation exerted by MK interaction with type I collagen on proplatelet formation . The net result is an altered timing of proplatelet formation. MK-restricted MYH9 inactivation in mice resulted in macrothrombocytopenia but conserved platelet aggregation and secretion as in the human disease. Yet bleeding times were prolonged, shape change modest and clot retraction defective; the latter linked to a defective αIIbβ3-outside-in signalling, a finding associated with an impaired thrombus formation under flow .
Heterozygous mutations in the FLNA gene encoding the cytoskeletal protein FlnA are associated with a series of rare X-linked autosomal dominant diseases with a major feature being periventricular nodular heterotopia (PNH) . Significantly, FlnA is the attachment site for GPIbα in the platelet cytoskeleton; this helps maintain thrombus stability at high shear . Bleeding is an important feature for a cohort of patients with FLNA mutations as is thrombocytopenia and platelet anisotrophy . This again suggests a defective megakaryocytopoiesis, a hypothesis confirmed from studies on a conditional mouse model where FlnA null platelets also showed signalling defects and a reduced shear-dependent platelet accumulation on collagen .
Glanzmann thrombasthenia (GT)
In GT, platelets fail to aggregate due to quantitative or qualitative defects of the αIIbβ3 integrin (formerly called the GPIIb-IIIa complex) [1–3,88]. The most common platelet disorder, bleeding manifestations range from isolated epistaxis, gingival bleeding, menorrhagia, ecchymoses, easy bruising and bleeding after trauma or surgery, to repeated and spontaneous life threatening haemorrhage requiring transfusions or other treatments. Gastrointestinal bleeding is an increasingly noted complication particularly among elderly patients. Normal platelets have αIIbβ3 in a bent conformation but when platelets are stimulated it straightens, a process that accompanies exposure of determinants essential for the binding of Fg and other soluble adhesive proteins . The latter assure aggregation by cross-linking adjacent platelets. Surface αIIbβ3 also provides a liaison between fibrin and the intracellular cytoskeletal proteins that mediate clot retraction, a process that fails to occur in patients with severe deficiencies of αIIbβ3 (formerly called type I disease). Although GT platelets adhere to subendothelium after injury, platelet spreading on the exposed surface, a process that involves αIIbβ3, is defective in addition to thrombus build up .
Analysing platelet surface GPs by flow cytometry or Western blotting will identify αIIbβ3 deficiency. Direct sequencing of exons and splice sites of the ITGA2B and ITGB3 genes will lead to the detection of most mutations. ITGA2B spans 17 kb and is comprised of 30 exons, ITGB3 spans 46 kb and has 15 exons; they colocalise to 17q21–23. High-resolution melting analysis is a rapid and inexpensive new method for following mutations within families or indeed screening for GT . Genetic defects occur along the length of both genes. Nonsense and splice site mutations with frameshifts are common, as also are small insertions or deletions while missense mutations give amino acid substitutions (see the GT database: http://sinaicentral.mssm.edu/intranet/research/glanzmann) (illustrated for ITGB3 in Fig. 4). Consanguinity is commonly associated with homozygous mutations and accounts for the increased frequency of GT in some ethnic groups . But generally, mutations are 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/or their export to the cell surface . Sometimes, sufficient mutated αIIbβ3 is processed to allow at least a partial clot retraction and uptake and storage of plasma Fg into α-granules (another task of αIIbβ3). Analysis of GT is now quite advanced and the first large demographic studies have been reported [93,94].
The β3 subunit is also a component of the vitronectin receptor (αvβ3) expressed on many cells, including endothelial cells, osteoclasts, fibroblasts, monocytes and activated B lymphocytes. It has but a minor presence in platelets. In GT, αvβ3 is absent if the genetic lesion stops β3 production. However, for missense mutations the situation is less clear-cut as some β3 mutations have different effects on αIIbβ3 compared to αvβ3 . Mouse models of β3 deficiency suggest accelerated angiogenesis (linked to enhanced VEGF-flk1 signalling in endothelial cells), facilitated tumour growth (with defective macrophage-related tumour suppression), age-dependent osteosclerosis (and bone thickening) and diet-induced vascular inflammatory events including increased atherosclerosis [96–99]. Whether patients with β3 gene defects have a distinctive phenotype remains undetermined, for the incidence of bleeding is comparable and no evidence for abnormal vessel development, increased rates of abortion or of susceptibility to cancer has as yet been forthcoming. Interestingly, Coller et al. performed bone density measurements on five women from the Iraqi-Jew GT patient cohort lacking αIIbβ3 and αvβ3; a cohort subsequently shown to be completely deficient in β3 due to an out-of-frame deletion (c.2031–2041del) leading to premature termination of β3 . Their data showed no evidence for increased bone thickening. Horton et al. generated osteoclasts from the peripheral blood of the Iraqi-Jewish GT patients. While the osteoclasts indeed lacked αvβ3, they exhibited a two- to four-fold increase in α2 and β1 integrin expression. This was proposed to partially compensate for the αvβ3 deficiency with an accompanying decrease in bone resorption of 44% and 59% in pit number and depth, respectively. It was suggested that integrin expression on the cells was sufficient to allow bone resorption to proceed albeit to a submaximum extent and to account for an absence of osteopetrosis. However whether an increased α2β1 expression was a direct result of the absence of β3 or a natural consequence of their ITGA2 or ITGB1 haplotypes determining α2β1 density was not investigated .
Interestingly, most variant forms of GT are linked to mutations within ITGB3 (Fig. 4). The first report of variant GT with platelets expressing non-functional integrin described an Asp119Tyr substitution in β3, a mutation that helped to identify an RGD-binding site (data reviewed in ). The codon for Arg214 of ITGB3 is a mutational hotspot and substitution of this amino acid occurring within the MIDAS (metal ion-dependent adhesion site) domain prevented the expression of the ligand-binding epitopes on platelet stimulation. Interestingly, clot retraction and Fg storage in α-granules was also abrogated. A Ser752Pro substitution in the cytoplasmic domain of β3, or a stop codon leading to a truncated protein containing only the first 8 of the 47 amino acids normally present in the β3 cytoplasmic domain confirmed their role in ‘inside-out’ signalling and activation of αIIbβ3 [1,88]. However, these mutations permitted clot retraction and Fg storage. In contrast, a defective spreading and focal adhesion kinase (FAK) phosphorylation of heterologous cells transfected with αIIbβ3(Ser752Pro) or αIIbβ3(Arg724Ter) confirmed a role for the cytoplasmic domains in ‘outside-in’ signalling possibly in coordination with FcγRIIA [103,104].
The β3 subunit is rich in disulphide bonds, and several patients with β3 cysteine mutations express low amounts of constitutively active αIIbβ3 (reviewed in ). One such patient with a homozygous Cys560Arg in β3 has platelets that express about 20% of the normal levels of αIIbβ3; these were able to spontaneously bind Fg . Platelet aggregation and clot retraction are both severely reduced but the α-granule store of Fg is present. The activated phenotype was reproduced on CHO cells expressing recombinant αIIbβ3(Cys560Arg). Significantly, when Cys560 was substituted by any of 12 different amino acids, αIIbβ3 always spontaneously bound Fg [105,106]. Thus, breaking the disulphide was the crucial event. The presence of surface-bound Fg is unique to this patient so far and may contribute to increased platelet consumption. This situation recalls platelet-type VWD where normal VWF multimers spontaneously bind to a mutated GPIbα subunit and block its function (see earlier Section). These results elegantly confirm how the change from a bent to a straightened conformation of αIIbβ3 is influenced by the removal of constraints within the tertiary structure of ‘resting’β3 .
Strikingly, mutations at Arg995 in αIIb and Asp723 in β3 lead to platelet anisotrophy and thrombocytopenia without giving a full GT phenotype [108,109]. Significantly, Arg995 and Asp723 form a salt linkage binding the cytoplasmic tails of αIIbβ3 together and helping to keep the integrin in a bent resting state. Mutations weakening this link (if not abolishing it) increase the activation state of αIIbβ3 and interfere with megakaryocytopoiesis by down-regulating RhoA signalling; other mutations affecting platelet production involve extracellular but membrane proximal domains of β3 (data reviewed in ). A novel integrin β3 extracellular mutation with dominant inheritance, an in-frame deletion producing the loss of amino acids 647–686, has been recently reported to associate macrothrombocytopenia and platelet dysfunction in two Italian families . Whether this mutation has long-range repercussions on the β3 intracytoplasmic domain is unknown.
Also to be mentioned are the very rare patients with leukocyte adhesion deficiency-III (LAD-III) syndrome in which life-threatening bleeding is associated with increased infections and poor wound healing in early life. Infections range from bacterial pneumonia and early septicaemia to fungal disease. The complex clinical features combine lymphocyte, neutrophil and platelet integrin dysfunction due to mutations in the kindlin-3 gene that abolishes ‘inside-out’ integrin activation although allowing their expression . The activation of β1, β2 and β3 integrins are all compromised even though kindlin-1 and kindlin-2 can take over in some cell types. Interestingly, kindlin-3 has been found to function in endothelial cells, leaving open the possibility that endothelial cell defects can contribute to the bleeding .
The Scott syndrome is a rare inherited disorder caused by defective scrambling of phospholipids on blood cells including platelets [1,2]. The disease is manifested by decreased fibrin formation at sites of vascular injury due to the inability of activated cells to generate a procoagulant surface. Scott platelets when stimulated by a thrombin plus collagen mixture are unable to translocate phosphatidylserine (PS) to the outer phospholipid leaflet of the membrane bilayer, with the result that factors Va and Xa fail to bind, leading to a decreased capacity of the platelets to convert prothrombin into thrombin. This lack of thrombin generation is sufficient to induce a bleeding syndrome. Microparticle release, a process that can be readily measured by flow cytometry using FITC-annexin V, accompanies PS expression and is also defective in Scott syndrome . The detection in a Scott syndrome patient of a heterozygous missense mutation in the ATP-binding cassette transporter A1 (ABCA1), implicated in the exofacial transport of PS, was thought to offer clues on the basis of this disease even though similar genetic defects are seen in Tangier disease, basically a disease of cholesterol and phospholipid transport . Nonetheless, while platelet secretion is impaired in Tangier disease, activation-dependent changes in PS distribution had previously been shown to be normal both in patients and mice deficient in ABCA1 . Another explanation for the molecular defect in Scott syndrome has recently been forthcoming with the report of a mutation at a splice-acceptor site of the gene encoding transmembrane protein 16F (TMEM16F) in the initially reported American patient . This mutation caused premature termination of a protein that acts as a Ca2+-activated chloride channel. Additional transfection studies using a mouse B-cell line, led the authors to conclude that TMEM16F is an essential and newly identified component for the Ca2+-dependent exposure of PS at the platelet surface. Its relationship to other phospholipid scramblases and to Ca2+-sensing STIM1 and the store-operated Orai1 channels remain to be determined .
Conclusions and a look towards the future
It is clear that the last few years have seen considerable advances in our understanding of the molecular causes of inherited disorders of platelet function. Gene sequencing has become standard practice and large demographic studies are underway particularly in GT. The net is closing in on the enigmatic disorders such as GPS and the Scott syndrome whose mutated genes have resisted efforts to identify them; while the next few years will see the redoubling of efforts to elucidate the gene defects known to cause dysfunctional signalling pathways and a selective platelet functional dysfunction to individual agonists. Such disorders may be numerous and involve combinatorial haplotypes as well as individual gene defects. We have illustrated how transcription factor defects can have multiple secondary effects on platelet function; while abnormalities of microRNA regulatory pathways in MKs could well affect protein expression as recently shown for vesicle-associated membrane protein 8/endobrevin . Future studies will involve continuing the selective phenotypic approach to gene mapping so nicely described by Watson et al.. But more and more, new generation sequencing approaches will permit the mass screening of patients. The results of the human genome project and current knowledge of the MK transcriptome facilitate the application of transcription profiling (and microarray pull-down assays) or whole exome analysis to platelet disorders [120,121]. As the complexity of the platelet signalling pathways is unravelled, then a comprehensive platelet proteome database and a map of the protein interactome showing the functional consequences of all defects will be established .
We acknowledge support from the French Health Ministry for the CRPP and to the GIS Maladies Rares. We thank all founder members of the CRPP Reference Centres (Marie Dreyfus, Rémi Favier, Claude Négrier, Nicole Schlegel, Pierre Sié) and also those who have since become associated (Marie Christine Alessi, Yves Gruel, Thomas Lecompte, and Jean-François Schved) and who have helped develop a National French network for the study and care of patients with inherited platelet diseases.
Disclosure of Conflict of Interest
The authors state that they have no conflict of interest.