Congenital platelet disorders and understanding of platelet function.

review

Congenital platelet disorders and understanding of platelet function Alan T. Nurden and Paquita Nurden L’Institut de Rhythmologie et Modelisation Cardiaque (LIRYC), Plateforme Technologique et d’Innovation Biomedicale, Hôpital Xavier Arnozan, Pessac, France

Summary Genetic defects of platelets constitute rare diseases that include bleeding syndromes of autosomal dominant, recessive or X-linked inheritance. They affect platelet production, resulting in a low circulating platelet count and changes in platelet morphology, platelet function, or a combination of both with altered megakaryopoiesis and a defective platelet response. As a result, blood platelets fail to fulfil their haemostatic function. Most studied of the platelet function disorders are deficiencies of glycoprotein mediators of adhesion and aggregation while defects of primary receptors for stimuli include the P2Y12 ADP receptor. Studies on inherited defects of (i) secretion from storage organelles (dense granules, a-granules), (ii) the platelet cytoskeleton and (iii) the generation of pro-coagulant activity have identified genes indirectly controlling the functional response. Signalling pathway defects leading to agonist-specific modifications of platelet aggregation are the current target of exome-sequencing strategies. We now review recent advances in the molecular characterization of platelet function defects. Keywords: platelets, bleeding disorders, mutations, haemostasis. This review of inherited disorders of platelet function will emphasize recent advances in the identification of megakaryocyte-expressed genes whose defects are at the origin of a bleeding syndrome (Nurden & Nurden, 2011; Bennett & Rao, 2012; Bunimov et al, 2013). Spontaneous bleeding is mostly mucocutaneous in nature; excessive trauma-related bleeding is a feature of milder forms. Treatment has been reviewed elsewhere (Seligsohn, 2012). We highlight altered platelet function through the modification of surface constituents and detail the increasing number of rare disease where the affected proteins

Correspondence: Alan T. Nurden, L’Institut de Rhythmologie et Modelisation Cardiaque (LIRYC), Plateforme Technologique et d’Innovation Biomedicale, H^ opital Xavier Arnozan, Avenue du Haut-Leveque, 33600 Pessac, France. E-mail: [email protected]

ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 165–178

are intracellular constituents involved in either signalling pathways or organelle biosynthesis. The principle disorders are listed in Table I. Familial thrombocytopenias (FT) have been reviewed elsewhere and will only be discussed when there is clear evidence for additional platelet function defects (Nurden & Nurden, 2012; Balduini et al, 2013).

Defects of platelet adhesion Abnormalities of the GPIb-IX-V complex Bernard-Soulier syndrome (BSS) (Online Mendelian Inheritance in Man [OMIM] reference 231200) combines a moderate to severe macrothrombocytopenia with a loss of von Willebrand factor (VWF)-dependent platelet adhesion to collagen under high flow (Lanza, 2006; Berndt & Andrews, 2011). The original identification of the absence of glycoprotein I (GPI) from BSS platelets was critical to identifying the role of GPIba in platelet adhesion and helped define the role of surface glycoproteins as key effectors of platelet function. GPIba is highly glycosylated with mucin-like O-linked oligosaccharide chains and extends well out from the platelet surface; it contains VWF and thrombin-binding sites within the concave-shaped N-terminal domain characterized by multiple leucine-rich repeats (LRR). The loss of these binding sites may account for the platelet adhesion defect as well as a poor platelet response to thrombin. The additional absence of extracellular binding sites on GPIba for P-selectin, thrombospondin-1, coagulation factors (F) XI and XII, integrin aMb2 (on white cells) and high molecular weight kininogen may additionally contribute to the phenotype of BSS (Berndt & Andrews, 2011). BSS platelets also have an increased tendency to expose phosphatidylserine (PS) when stimulated and, interestingly, have a greater tendency to show a collapsed mitochondrial inner membrane potential, an early sign of apoptosis (Rand et al, 2010). A decreased prothrombin consumption in BSS remains to be explained. The products of four genes (GP1BA, GP1BB, GP9 and GP5) assemble within maturing megakaryocytes (MKs) in the bone marrow to form GPIb-IX-V as present in the platelet membrane (Li & Emsley, 2013). Each subunit has a single transmembrane domain and a short cytoplasmic tail (with phosphorylation

First published online 29 November 2013 doi:10.1111/bjh.12662

Review Table I. Principle inherited defects of platelet function: genetic mutations and associated phenotype. Mutations of transcription factors that have a secondary influence on platelet function. Chromosomal location Inheritance

Syndrome

Gene mutation

Adhesion receptor Bernard-Soulier syndrome

GP1BA, GP1BB, GP9

17p13.2, 22q11.21 and 3q21.3 Autosomal recessive

GP1BA

17p13.2 Autosomal dominant 19q13.4 Autosomal recessive

Severely defective platelet adhesion to von Willebrand factor under flow. Reduced platelet aggregation to thrombin. Giant platelets Defective platelet adhesion. Enlarged platelets Absent or severely reduced platelet aggregation with collagen

3q25.1 Autosomal recessive 19p13.3 Autosomal recessive

Rapidly reversible and reduced platelet aggregation to ADP Defective platelet aggregation with arachidonic acid and thromboxane A2

7q34-q35 Autosomal recessive 1q25 Autosomal recessive

Defective platelet aggregation with arachidonic acid. Increased bone density Decreased platelet aggregation with ADP, collagen

HPS1, AP3B1, HPS3-6, DTNBP1, BLOC1S3, BLOC1S6 LYST (CHS1)

Found on chromosomes 1, 3, 22, 11, 10, 6, 19, 15 and 5 respectively. Autosomal recessive 1q41.3

Familial haemophagocytic lymphohistiocytosis types 3-5 Gray platelet syndrome

UNC13D, STX11, STXBP2

17q25.1, 6q24.2, 19p13.2 Autosomal recessive

Absence of dense-granules; decreased secretion-dependent platelet aggregation. Albinism (skin and eye), pulmonary fibrosis, ceroid accumulation, cholitis Absence of dense granules: decreased secretion-dependent platelet aggregation. Partial albinism, defective phagocytosis, infections, accelerated phase Abnormal platelet aggregation due to secretion defects

NBEAL2

3p21.31 Autosomal recessive

Arthrogryposis-renal dysfunction-cholestasis syndrome Quebec platelet syndrome

VPS33B, VIPAS39

15q26.1, 14q24.3 Autosomal recessive

PLAU Tandem duplication

10q22.2 Autosomal dominant

ITGA2B, ITGB3

17q21.31/32 Autosomal recessive

Quantitative or qualitative deficiencies of aIIbb3 and avb3. Absent platelet aggregation with all agonists. Clot retraction often reduced or absent.

ANO6 (TMEM16F)

12p13.3 Autosomal dominant

Mutations of a Ca2+-dependent multi-pass protein. Much decreased PS exposure and microparticle release. Compromised procoagulant function of platelets and other blood cells

Platelet-type von Willebrand disease GPVI deficiency Receptors for soluble agonists P2Y12 ADP receptor deficiency Thromboxane A2 receptor deficiency Signalling pathways Thromboxane A synthase (Ghosal syndrome) Cytosolic phospholipase A2 Platelet secretion Hermansky-Pudlak syndrome

Chediak-Higashi syndrome

Platelet aggregation Glanzmann thrombasthenia

Procoagulant activity Scott Syndrome

166

GP6

P2YR12 TBXA2R

TBXAS1 PLA2G4A

Principle associated phenotype

Defective a-granule formation. Highly variable platelet aggregation response. Enlarged platelets. Defective wound healing. Splenomegaly. Myelofibrosis Absence of a-granules, decreased platelet function, large platelets, Renal dysfunction and cholestasis Increased urokinase-type plasminogen activator and degraded a-granule proteins. No platelet aggregation with epinephrine. Excessive fibrinolysis


Review and protein binding sites) and all extracellular domains contain at least one LRR. GPIba and GPIbb are disulphidelinked; otherwise the subunits associate through interactions between their transmembrane domains. GPIba, GPIbb and GPIX are in a 1:2:1 ratio while GPV has been claimed to associate either with one or two copies of GPIb-IX (McEwan et al, 2011). Mutations within GP1BA, GP1BB and GP9 but not GP5 cause BSS mostly by preventing the formation and/ or trafficking of the complex through the endoplasmic reticulum (ER) and Golgi apparatus as a result of a missing subunit or through an altered quaternary organization of GPIb-IX; new mutations are still being reported (McEwan et al, 2011; Savoia et al, 2011). A panoply of missense mutations is shown in Fig 1; deletions, insertions and nonsense mutations also feature as a cause of the disease (Lanza, 2006; Berndt & Andrews, 2011; Nurden & Nurden, 2012). The

absence of the interaction between GPIba and filamin A and/or other cytoskeletal components in the membrane cytoskeleton may account for the giant platelets with an altered organization of internal membrane systems and a modified proplatelet extrusion into the vascular sinus. In rare variant forms of BSS, platelets express non-functional GPIba caused by missense mutations (identified in colour in Fig 1) that occasionally may have autosomal dominant inheritance (Nurden & Nurden, 2012). Mostly, these affect VWF-binding domains at the N-terminal domain; epitopes that change function but not expression (McEwan et al, 2011). Abundant in Mediterranean countries is a heterozygous GP1BA Ala156Val mutation that is the cause of inherited macrothrombocytopenia but without platelet functional defects through a decreased ability of MKs to form proplatelets and those that are formed are morphologically

Fig 1. Mutations of the GP1BA, GP1BB and GP9 genes, encoding the GPIba, GPIbb and GPIX subunits of GPIb-IX-V, give rise to Bernard-Soulier syndrome (BSS) while rare mutations of GP1BA account for platelet-type von Willebrand disease (Plt-VWD). Mutations affecting GPV have yet to be reported. Here, we survey the panoply of mutations giving rise to both diseases. Missense mutations are numbered for the mature protein; the colour codes highlight genotype/phenotype relationships. In classic BSS the changed amino acid is in red; common mutations are in parentheses. The colour code distinguishes qualitative variants with GPIba expression but absent function, autosomal dominant heterozygous variants giving macrothrombocytopenia only (the asterisk identifies the common Bolzano variant), and hemizygous GPIbb mutants associated with a chromosome 22 microdeletion in the Di-George syndrome. Mutations of GP1BA that result in plt-VWD are in green. The mutation list has been updated from Lanza (2006) and Enayat et al (2012). Abbreviations: fs, frameshift; dup, duplication; del, deletion; ins, insertion; stop, stop codon. ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 165–178

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Review abnormal with enlarged terminal swellings (Balduini et al, 2009; Noris et al, 2012). It remains an enigma that the original BSS variant from Bolzano had homozygous GP1BA Ala156Val expression and an apparent autosomal recessive inheritance whereas Mediterranean macrothrombocytopenia has autosomal dominant expression of the same mutation. Single allele mutations in GP1BB cause BSS when associated with the DiGeorge/Velocardiofacial syndrome, a developmental disorder caused by a heterozygous microdeletion at 22q11 that results in the loss of GP1BB from the second allele. BSS is a relatively severe bleeding disorder, as shown in a systematic review of BSS in pregnancy with alloantibody formation a significant risk after platelet transfusion (Peitsidis et al, 2010; Nurden & Nurden, 2012). Platelet-type von Willebrand disease (platelet-type VWD) (OMIM 177820) is an autosomal dominant disease with mild to moderate bleeding and thrombocytopenia with enlarged platelets characterized by spontaneous VWF-binding to GPIba (Othman, 2011). Diagnosis is aided by increased platelet agglutination by low-dose ristocetin in the presence of normal plasma; this contrasts with BSS where ristocetin fails to act. A consequence is that functional high molecular weight VWF is removed from plasma and GPIba is monovalently blocked. A limited number of amino acid substitutions in the GPIba N-terminal domain (Fig 1) change the conformation of the so-called “thumb”, a disulphide-bonded loop that controls the accessibility of VWF multimers to their binding site. As a result, soluble VWF binds directly, changes also introduced by a long range p.Pro449_Ser457 deletion in the macroglycopeptide-coding region of GP1BA (Nurden & Nurden, 2011; Enayat et al, 2012). This clinical condition resembles type 2B VWD (VWD2B) and diagnosis requires care (Othman, 2011). Giant platelets, thrombocytopenia (sometimes severe) and even circulating platelet aggregates can occur in type 2B VWD, caused by mutations in exon 28 of the VWF gene. Culture of CD34+ cells from the peripheral blood of patients with type 2B VWD showed early association of the up-regulated VWF with GPIb; this led to an altered megakaryopoiesis and abnormal proplatelet production (Nurden et al, 2010). Mouse models for VWD2B involving gain-of-function VWF mutations have confirmed an altered platelet production, an increased platelet clearance and a marked reduction in thrombus formation and platelet adhesion in vivo with modulation of disease severity by ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type I motif, 13) (Golder et al, 2010; Rayes et al, 2010). Platelet-bound large VWF multimers are particularly susceptible to cleavage by this enzyme. It is fascinating that spontaneous binding of VWF to GPIba gives rise primarily to bleeding rather than thrombosis.

Deficient collagen receptor function Platelet-collagen interaction in flowing blood is a multistep process involving both integrin a2b1 and GPVI (GP6) that signals through the FcRc-chain, a process negatively regu168

lated by platelet-endothelial adhesion molecule-1 (PECAM1) (Varga-Szabo et al, 2008). Specific haplotypes in GP6 and ITGA2 provide wide variations in density of both receptors and it has been estimated, for example, that this can account for 40% variability in the GPVI signalling response when platelets bind to collagen (Kunicki et al, 2012; Bunimov et al, 2013). Patients with FT linked to heterozygous mutations in the 5′ untranslated (UTR) region of the ANKRD26 (ankyrin repeat domain 26) gene, encoding ankyrin (also known as thrombocytopenia 2 (THC2)) (OMIM 610855), a pro-apoptotic protein, can express very low levels of a2b1 otherwise there is no reported genetic defect causing loss of expression of this integrin (Noris et al, 2011). While the function of ankyrin in megakaryopoiesis and platelet function remains to be elucidated, it is of interest that both a2b1 conditional knockout mice and patients with ANKRD26 mutations have smaller platelets than usual (Kunicki et al, 2012; Habert et al, 2013). In contrast, initial reports described individuals in two unrelated families with a lifelong but mild bleeding syndrome and severely deficient collagen-induced platelet aggregation who were compound heterozygotes for mutations of GP6 causing loss of GPVI expression and/or function (OMIM 605546) (Dumont et al, 2009; Hermans et al, 2009). Quite recently, an inherited adenine insertion in exon 6 of GP6 (c.711_712insA) was shown to cause a frameshift and introduce a premature stop codon leading to a GPVI protein that was truncated before the transmembrane domain in several apparently unrelated patients with a mild bleeding disorder in Chile (Matus et al, 2013). Absent GPVI expression on the platelet surface led to a lack of aggregation and secretion to collagen, convulxin and collagen-related peptide. A peculiarity of GPVI is that acquired antibodies, extracellular proteases and even shear can induce sheddase activity by members of the ADAMTS family with concomitant loss of GPVI, a factor to take into account during diagnosis (Bender et al, 2010). Sheddase is a name given to cell-bound proteases that cleave membrane receptors close to their transmembrane domain with release of the extracellular domain. In summary, single nucleotide polymorphisms can influence collagen receptor reactivity but are not by themselves a cause of bleeding. A distinct genetic disease has so far only been shown for GP6, although mouse models point to smaller thrombi and reduced stable adhesion in thrombosis models when either Gp6 or Itga2 are deleted (Cosemans et al, 2013).

Variants of receptors for soluble agonists and of proteins in signalling pathways Pathology of ADP and thromboxane A2 (TXA2) receptors Defects of platelet aggregation to specific agonists are a frequent finding in patients with bleeding phenotypes, with many patients having as yet undefined abnormalities affecting Gi-receptor signalling, the TXA2 pathway or dense granule secretion (Nash et al, 2010; Dawood et al, 2012). Patients with ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 165–178

Review defects in genes coding for the seven transmembrane domain receptor-family members for soluble agonists are rare but have made a key contribution to unravelling the mechanisms of platelet activation. Platelets possess 2 classes of purinergic receptor for ADP: P2Y1 mediates Ca2+-mobilization, shape change and the initiation of the aggregation response while P2Y12 is responsible for aggregate growth and stabilization. Early studies highlighted a rare platelet disorder with mild bleeding characterized by a reduced and rapidly reversible platelet aggregation to high dose ADP and an inability of ADP to inhibit adenylate cyclase (data reviewed by Cattaneo & Gachet, 1998). In 1995, we characterized such a patient and showed a reduced binding of a radiolabelled ADP analogue and an inability of ADP to activate the aIIbb3 integrin (Nurden et al, 1995). A specific receptor defect was confirmed for this patient when analysis of polymerase chain reaction products from genomic DNA revealed a mutant allele at the P2RY12 locus (Hollopeter et al, 2001). This was important because the identification of this mutation not only identified a new disease (OMIM 600515); but concomitantly identified the receptor inhibited by anti-thrombotic drugs, such as clopidogrel and prasugrel, abundantly used in cardiovascular medicine. Several other such patients have now been described with mutations including nucleotide deletions in the open-reading frame, frameshifts resulting in premature protein truncation and missense mutations affecting ADP binding and even receptor trafficking (Cattaneo, 2011). These include a novel patient with a heterozygous cytoplasmic domain p.Pro341Ala mutation causing abnormal endosomal sorting and delayed Rab11-dependent recycling of platelet internal pools leading to a surface deficit of P2Y12 (Cunningham et al, 2013). An inherited defect of platelet aggregation to TXA2 was first reported in Japanese families with a mild bleeding disorder and linked to a p.Arg60Leu substitution in the first intracellular loop of the TXA2 receptor a-subunit (TPa); this resulted in impaired signal transmission and a loss of aggregation-induced by both arachidonic acid and U46619, a TXA2 receptor agonist (Hirata et al, 1996) (OMIM 188070). Other mutations of TBXA2R that disrupt both TPa function and receptor recycling have now been reported (Watson et al, 2010; Mumford et al, 2013). These mutations are especially important as physiologically, they mimic the effect of aspirin that blocks arachidonic acid metabolism by irreversibly inhibiting cyclooxygenase. A reduced platelet response to adrenaline is frequent in routine screening of patients and on occasion the response is absent, but its contribution as a cause of bleeding remains uncertain (Nash et al, 2010; Dawood et al, 2012).

Defects in intracellular signalling pathways Pathologies of signal transduction pathways concern patients with defects of platelet aggregation that affect some stimuli more than others; such disorders may be quite common (Nurden & Nurden, 2011; Bennett & Rao, 2012; Dawood ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 165–178

et al, 2012). Early studies highlighted patients with abnormalities of receptor-linked G-protein signalling, phospholipase C pathways, protein kinase C phosphorylation and Ca2+ mobilization; however, as gene sequencing was not available at the time, the genetic defect remains unknown (Bennett & Rao, 2012). Likewise, patients with purported congenital deficiencies of cyclooxygenase-1, prostaglandin H synthase-1, thromboxane synthase, phospholipase A2, lipoxygenase, glycogen synthase and ATP metabolism, gp91 phox deficiency associated with impaired isoprostane formation were all the object of initial reports largely based on platelet function testing (Pignatelli et al, 2011; Bennett & Rao, 2012). Two examples where specific gene mutations have now been described are (i) thromboxane synthase in Ghosal syndrome (linking defective arachidonic acid-induced platelet aggregation with an increased bone density) (Genevieve et al, 2008) and (ii) inherited cytosolic phospholipase A2a deficiency associated with impaired eicosanoid biosynthesis, small intestinal ulceration, and platelet dysfunction (Adler et al, 2008). Signalling defects may directly interfere with platelet activation pathways including aIIbb3 activation and fibrinogen binding or intervene secondarily by preventing secretion of ADP or formation and release of TXA2. A special category of patient with defects in the G-protein cascade involves second messengers or RGS (regulator of G protein signalling) proteins that affect cAMP levels (Louwette et al, 2012). RGS are multi-functional GTP-ase accelerating proteins that enhance GTP hydrolysis by G protein a-subunits and so intervene early in signalling cascades. The complex-imprinted gene cluster, GNAS (previously termed GNAS1), regulates Gsa. Direct genetic and epigenetic defects of GNAS include both Gsa hypofunction and a thrombotic phenotype associated with more generalized hormonal, skeletal defects and sometimes mental retardation (Van Geet et al, 2009; Louwette et al, 2012). A paternally inherited 36 bp insertion in the extra-large stimulatory Gsa isoform (XLas) is linked to the same receptors as Gsa and stimulates adenylate cyclase and is associated with Gs hyperfunction in platelets, leading to an increased trauma-related bleeding tendency but is also accompanied by neurological problems, growth deficiency and brachydactyly (Van Geet et al, 2009). Whether Gs defects occur in patients with platelet-specific bleeding disorders is as yet unknown. Identifying gene defects in the platelets of patients with discrete functional defects using a phenotypic approach has so far been mostly thwarted by the complexity of the signalling pathways and by the diversity of the platelet functional defects that have been described (see Watson et al, 2010; Dawood et al, 2012). However, the different platelet defects shown in mouse models with the deletion of specific genes coding for signalling proteins (Cosemans et al, 2013) points to the high likelihood that many such human defects remain to be discovered. Exome or whole genome sequencing now offer an alternative approach to their identification and exome sequencing has already been successfully applied to patients with familial 169

Review thrombocytopenias as well as to platelet secretory defects (Albers et al, 2011, 2012; Kunishima et al, 2013). However, care will be needed to distinguish between disease-causing mutations and single nucleotide polymorphisms (Bunimov et al, 2013). It should also be emphasized that signalling defects can be specific for MKs and platelets or extend to other cell types and be secondary to genetic defects of transcription factors, as we will show in later sections.

Defects of secretion (storage pool disease, SPD) Defects of dense (d) granules Dense granules are storage sites for serotonin, ADP, ATP and polyphosphate. SPD affecting dense granules is a common cause of defects of secretion-dependent platelet aggregation giving rise to a mild to moderate bleeding diathesis (Masliah-Planchon et al, 2012). Secretion deficiency may be severe or partial; in some patients it also extends to a-granules and may concern granule biogenesis and storage of constituents or the signalling pathways responsible for exocytosis. While a defective secretion from dense granules often gives reversible aggregation tracings with high dose ADP and a defective response with other agonists including collagen, a direct measure of secretion of nucleotides or serotonin is recommended to confirm the nature of the defect. ATP release can be assayed in parallel with aggregation in a Lumi-aggregometer (Chrono-Log, Havertown, PA, USA). A lack of dense granules is clearly identifiable by whole mount electron microscopy and, when associated with generalized abnormalities of lysosome-related organelles, can lead to clearly defined clinical phenotypes. This is so for the HermanskyPudlak (HPS, OMIM 203300) and Chediak-Higashi (CHS, OMIM 214500) syndromes where heterogeneous disorders of vesicle biogenesis and melanosomal defects also cause a lack of pigmentation of the skin and hair (Huizing et al, 2008; Masliah-Planchon et al, 2012). Oculocutaneous albinism is characteristic of HPS as is ceroid-lipofuchsin storage in the reticulo-endothelial system while granulomatous colitis, interstitial lung disease and fatal pulmonary fibrosis feature in some subtypes. Defects in nine genes (HPS1, AP3B1, HPS3-6, DTNBP1, BLOC1S3, BLOC1S6) cause distinct HPS subtypes in man (Huizing et al, 2008; Cullinane et al, 2011; Gochuico et al, 2012). A pathological 16-base duplication in exon 15 of the HPS1 gene predominates in Puerto Rican patients and gives a severe phenotype. HPS proteins interact with each other in complexes called BLOCS (biogenesis of lysosome related organelle complexes); genetic defects disrupt the processing of these and of SLC35D3 (a member of the solute carrier family of proteins) during dense granule biogenesis (Meng et al, 2012). The beta3A subunit of the adaptor protein-3 (AP-3) complex, encoded by AP3B1 is abnormal in HPS2 (OMIM 608233) a subtype associated with innate immunity defects (Gochuico et al, 2012). Interestingly, polyphosphates released from dense 170

granules activate plasma FXII, addition of polyphosphates restored defective clotting in HPS implying that their deficit contributes to the bleeding syndrome (Muller et al, 2009). In CHS, bleeding is associated with severe immunological defects, with life-threatening infections and progressive neurological dysfunction if the patient survives to adulthood (Huizing et al, 2008). The immunodeficiency leads to the development of a lymphoproliferative syndrome and an accelerated phase in ~ 85% of patients. The hallmark of CHS is the presence of giant inclusion bodies in a variety of granule-containing cells including platelets. The gene responsible for CHS (LYST, lysosomal trafficking regulator) has been cloned and a series of frameshift and nonsense mutations result in truncated CHS protein and a severe phenotype (see Huizing et al, 2008). Rare missense mutations can be associated with a milder form of the disease. LYST is a large protein with distinct structural domains including ‘BEACH’ and ‘ARM/HEAT’ suggestive as for NEABL2 (see Section on gray platelet syndrome) of a function in membrane contact interactions and organelle protein trafficking. Patients with Griscelli syndrome (OMIM: 214450) have partial albinism and silver hair; different subtypes combine neurological defects and/or severe immunodeficiency with a defective cytotoxic lymphocyte activity. Mutations in the genes encoding myosin Va (MYOSA), Rab27a (RAB27A; a small GTPase), or melanophilin (MLPH) cause 3 subtypes of Griscelli syndrome but rarely bleeding while platelet secretory defects have yet to be described (Masliah-Planchon et al, 2012). Differential diagnosis with HPS type II can be difficult. In a new development, defective platelet secretion (dense granule, a-granule and lysosomal) despite normal granule cargo has been shown in familial haemophagocytic lymphohistiocytosis (FHL) types 3, 4 and 5, potentially lethal disorders of immune dysregulation caused respectively by defects in Munc (mammalian uncoordinated) 13–4 (UNC13D), syntaxin-11 (STX11) and Munc18b (STXBP2) coding genes (Sandrock et al, 2010; Al Hawas et al, 2012; Ye et al, 2012). Munc18b appears to be a partner of syntaxin-11 in platelet exocytosis. This work highlights how platelets may use similar secretory machinery as cytotoxic T lymphocytes and NK (natural killer) cells.

Defects of a-granules These are the storage site for many proteins including those synthesized in MK or endocytosed from plasma; most are biologically active and after secretion account for non-haemostatic roles of platelets as well as participating in haemostasis. In addition, the organelle membranes contain a variety of glycoproteins (e.g. P-selectin, CD40L and CD63) that are translocated to the plasma membrane during secretion. Specific deficiencies of a-granule-stored proteins also occur in inherited disorders of the corresponding plasma proteins (e.g. FV deficiency, fibrinogen in afibrinogenaemia, VWF in ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 165–178

Review type 3 VWD); these will not be discussed further in this review. Gray platelet syndrome (GPS) is a mild to moderate bleeding disorder that can, on occasion, be life-threatening and is characterized by a severe and specific deficiency of a-granules and their contents (Gunay-Aygun et al, 2010). The molecular defect involves packaging of proteins and a-granule biogenesis in MK. Clinical features whose extent varies between affected individuals includes macrothrombocytopenia, an early onset of myelofibrosis and enlarged spleens. Secretion-dependent platelet aggregation is diminished. Unlike for HPS, platelets from a GPS patient failed to spread when plated on polylysine, collagen or fibronectin showing that dense granule and agranule deficiencies have different effects (Peters et al, 2012). A low platelet expression of GPVI due to increased sheddase activity by members of the ADAMTS family has been reported in isolated cases (see Nurden & Nurden, 2011). Electron microscopy shows only vestigial a-granules in platelets and MKs, vacuoles are abundant in MKs and residual a-granule proteins can be detected in vacuoles. Emperipolesis (passage of white blood cells through MKs) is a feature. Unexpectedly, small vesicles containing tissue inhibitors of metalloproteases (TIMPs) were normally present in GPS platelets and such vesicles may represent a novel storage pool of proteins (Villeneuve et al, 2009). In 2011, three groups using new generation DNA or RNA sequencing technologies showed mutations in NBEAL2 (neurobeachin-like 2) in GPS (Albers et al, 2011; Gunay-Aygun et al, 2011; Kahr et al, 2011). NBEAL2 belongs to a gene family that includes LYST, previously identified as being responsible for CHS (see preceding section). NBEAL2 protein contains beige and BEACH domains (characteristic of the protein affected in the beige mouse model of CHS) and multiple WD40 domains (domains terminating in tryptophan (W) and aspartic acid (D)) and appears directly implicated in a-granule biogenesis in MKs. All published mutations (September 2013) in NBEAL2 giving rise to GPS are shown in Fig 2 and are related to NBEAL2 protein structure. Despite the uniformity of the GPS genotype with NBEAL2 mutated in

a large number of cases, it has been speculated that, genetically, GPS is a heterogeneous trait whose severity depends on the extent of the a-granule deficiency (Bottega et al, 2013). Quite recently, Nbeal2 / mice have been produced that confirm and extend the phenotype established for human GPS (Deppermann et al, 2013; Kahr et al, 2013). In particular, the mice showed defective thrombus formation and were protected from inflammatory brain infarction following focal cerebral ischemia while platelet-dependent procoagulant activity and tissue repair after injury was also impaired. Mutations of VPS33B, which encodes a regulator of soluble N-ethylmaleimide-sensitive factor activating receptor (SNARE)-dependent membrane fusion and of VIPAS39, encoding VPS33B-interacting protein, cause the arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome (OMIM 208085) (Smith et al, 2012). Mostly lethal for young children, ARC associates platelet dysfunction and low granule content with a multisystem disorder featuring renal tubular and other dysfunctions. The platelet defect extends to stored and membrane components of a-granules (Urban et al, 2012). The autosomal dominant Quebec platelet syndrome (QPS) is unique to French-Canadian families (Blavignac et al, 2011). Here, platelets show protease-related degradation of a-granule proteins (including P-selectin) despite normal a-granule ultrastructure. Thrombocytopenia is sometimes observed and there is a characteristic lack of a platelet aggregation response with epinephrine. Fibrinolytic inhibitors, not platelet transfusions, reduce bleeding due to the fact that platelets in QPS possess unusually large amounts of urokinase-type plasminogen activator (u-PA [PLAU]). This promotes intra-granular plasminogen generation and excessive fibrinolysis upon platelet secretion. The genetic basis of QPS is a tandem duplication of the u-PA gene, PLAU (Patterson et al, 2010).

Glanzmann thrombasthenia (GT) GT is a classic inherited platelet disorder; platelets fail to aggregate to all physiological agonists due to quantitative or

Fig 2. Gray platelet syndrome is determined by mutations that occur across the NBEAL2 gene. Mutations are summarized on this cartoon and related to both gene and protein structure; they are taken from the following publications (Albers et al, 2011; Gunay-Aygun et al, 2011; Kahr et al, 2011; Bottega et al, 2013). Abbreviations: fs, frameshift; dup, duplication; ConA, concanavalin A. ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 165–178

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Review qualitative defects of the integrin, aIIbb3. The original identification some 40 years ago of the absence of “GP II and GPIII” (subsequently called GPIIb and GPIIIa) from GT platelets was critical to uncovering the mechanism of platelet aggregation and was a key step in identifying the aIIbb3 integrin (Nurden & Caen, 1974; Coller & Shattil, 2008). In normal haemostasis, aIIbb3 on activated platelets binds fibrinogen and other adhesive proteins that link platelets together during aggregation. Other manifestations of GT include a defective platelet spreading on collagen, while clot retraction and aIIbb3-dependent fibrinogen storage in agranules are variably defective depending on the nature of the mutation. These manifestations give rise to a variable but sometimes severe bleeding disorder whose treatment with platelet transfusions can be compromised by alloantibody formation (Seligsohn, 2012). GT has been comprehensively dealt with in a series of recent reviews and only essential details will be repeated here (Nurden et al, 2011a, 2012, 2013). Direct sequencing of the ITGA2B and ITGB3 genes allows the detection of most mutations in GT. An autosomal recessive inheritance means that compound heterozygosity is common except in certain ethnic groups, such as the French Manouche gypsies where consanguinity is widespread. Genetic defects occur across both genes; the latter are closely located at 17q21-23. Nonsense mutations, splice site mutations resulting in frameshifts and missense mutations are all common (Fig 3). Missense mutations are highlighted in Fig 3 and are related to protein structure while a colour code distinguishes qualitative defects. Mostly missense mutations prevent subunit biosynthesis in MKs or inhibit transport of pro-aIIbb3 complexes from the ER to the Golgi apparatus and/or their export to the cell surface. Particularly abundant are mutations within the b-propeller region of aIIb and within the epithelial growth factor (EGF)-domains of b3 (for specific examples, see Mansour et al, 2011; Mor-Cohen et al, 2012). Interestingly, while mutations within many of the disulphides in the EGF domains of b3 severely interfere with aIIbb3 expression, some cysteine mutations are activating and allow the spontaneous binding of fibrinogen to residual integrin. Interestingly, an up-regulating b3Cys560Arg mutation continues to give rise to a severe bleeding syndrome with increased mortality when reproduced in mice (Fang et al, 2013). Analysis of GT is now advanced and population studies have started (Kannan et al, 2009). b3 also forms part of the vitronectin receptor (avb3) expressed in many tissues. It has but a minor presence in platelets. In GT, avb3 is absent if the genetic lesion affects b3 production although some missense mutations have differential effects on the ability of b3 to react with aIIb or av (Nurden et al, 2013). Surprisingly, patients with b3 gene defects do not have a distinctive phenotype for, unlike b3 / mouse models, clear evidence for abnormal vessel development, bone thickening, increased rates of abortion or of tumour development has not been forthcoming (reviewed in 172

Nurden et al, 2011a). An unusually large number of incidents of deep vein thrombosis suggest that it may be an unexpected risk factor in GT (Nurden et al, 2013). This is intriguing in view of the fact that GT platelets tend to express lower amounts of PS when stimulated and therefore would be expected to generate less thrombin (Topalov et al, 2012). In variant GT, platelets express non-functional integrin; mostly, the mutations affect ITGB3 and amino acid substitutions (e.g. Asp119Tyr, Arg214Gln or Trp) affecting MIDAS (metal ion dependent adhesion site), ADMIDAS (adjacent to MIDAS) or SyMBS (synergistic metal ion binding site) domains (shown on Fig 3); these mutations have helped identify ligand binding sites in the activated integrin (reviewed by Coller & Shattil, 2008; Nurden et al, 2011a, 2012). Likewise, a Ser752Pro substitution in the cytoplasmic domain of b3, or stop codons leading to b3 being truncated within the cytoplasmic tail, have helped to identify domains involved in ‘inside-out’ signalling and activation of aIIbb3 through the binding of kindlin-3 and talin. Bleeding in dogs affected by heterozygous ITGA2B mutations preventing aIIbb3 expression has been corrected by lentivirus-mediated gene therapy, suggesting a promising strategy for the future treatment of patients with classic GT (Fang et al, 2011). An interesting new variant-type in GT is given by rare mutations of ITGA2B and ITGB3 that both variably affect aIIbb3 function and lead to moderate macrothrombocytopenia (Nurden et al, 2011a; Kashiwagi et al, 2013). These mutations mostly affect either cytoplasmic domains of both aIIb and b3 and especially the salt-bridge linking aIIbArg995 and b3Asp723 or membrane proximal residues in extracellular domains and favour at least partial spontaneous aIIbb3 activation. One recent study has proposed that the altered aIIbb3 activation state modifies MK crosstalk with matrix proteins causing interference with proplatelet production (Bury et al, 2012). Also to be mentioned is leucocyte adhesion deficiency-III (LAD-III) syndrome, in which life-threatening bleeding is associated with a high susceptibility for infections and poor wound healing in early life. The complex clinical features result from mutations in the FERMT3 gene, which encodes kindlin-3 that, together with talin, reacts with the b3 cytoplasmic tail to regulate integrin activation by breaking a salt link between aIIbR995 and b3D723. This key step is followed by activation and extension of the extracellular domain of aIIbb3 with exposure of fibrinogen-binding epitopes allowing aggregation (Coller & Shattil, 2008). Mutations in FERMT3 result in the loss of ‘inside-out’ integrin activation in platelets, white blood and endothelial cells (reviewed in Harris et al, 2013). Because LAD-III involves loss of function of both aIIbb3 and a2b1 integrins and affects several cell types it is a more severe disease than GT although again there is considerable inter-individual variation (Van de Vijver et al, 2012). ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 165–178

Review

I- (A)

(B)

II- (A)

(B)

Fig 3. Schematic representation of the ITGA2B (I) and ITGB3 (II) genes illustrating the wide spectrum of mutations that give rise to Glanzmann thrombasthenia (GT). Data were obtained from a survey of the literature and from consulting the GT database (http://sinaicentral.mssm.edu/intranet/research/glanzmann). In (A), the defects are designated a symbol according to their type. Mutations are found all along both genes. In (B), In view of space limitations, only missense mutations, identified by a single letter amino acid code, are aligned with respect to the structural domains of each subunit. Those which primarily prevent aIIbb3 expression are in black; mutations in blue are variant forms with expression of non-functional aIIbb3; in orange, the substitutions give activated integrin; while in mauve, mutations are associated with macrothrombocytopenia. Asterisks indicate the number of times the defect has been reported in apparently unrelated families. While missense mutations giving rise to classic GT and absent or much reduced aIIbb3 expression are widely distributed across both genes, variant forms are more likely to have ITGB3 gene defects. Figure reproduced from Nurden et al (2012) Understanding the genetic basis of Glanzmann thrombasthenia: implications for treatment. Expert Review of Hematology 5, 487–503, with permission.

Procoagulant activity Scott syndrome (OMIM 262890) is a rare inherited disorder caused by defective scrambling of phospholipids on blood cells (Lhermusier et al, 2011). It manifests by a decreased fibrin formation during shear-dependent adhesion of platelets to subendothelium. When activated, Scott platelets are unable to translocate PS to the outer phospholipid leaflet of the membrane bilayer; factors Va and Xa fail to bind leading to a decreased capacity of platelets to convert prothrombin ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 165–178

into thrombin. This lack of thrombin generation is sufficient to induce a bleeding syndrome. Physiological stimuli that induce PS translocation include a thrombin and collagen mixture, complement C5b-9 and high shear alone. Microvesiculation, a process that can be quantified by flow cytometry using fluorescein isothiocyanate-annexin V, accompanies PS expression and is also defective in Scott syndrome. The disease is caused by mutations in ANO6 (anoctamin 6, also known as TMEM16F) that encodes transmembrane protein 16F, a Ca2+-activated channel essential for Ca2+-dependent 173

Review PS exposure (Suzuki et al, 2010; Yang et al, 2012). Significantly, apoptosis-induced PS expression was also abnormal (Van Kruchten et al, 2013). In contrast to earlier reports, these authors also identified a residual PS exposure in Scott platelets induced by collagen/thrombin that appeared independent of ANO6 and was insensitive to inhibition of caspases and mitochondrial depolarization.

Platelet function abnormalities associated with familial thrombocytopenias Most often of autosomal dominant inheritance, FT frequently show altered platelet morphology but while platelet dysfunction has often been considered as secondary to the low platelet count, it can contribute to bleeding (FTs are reviewed in Nurden & Nurden, 2012; Balduini et al, 2011). A common cause of FT is an altered megakaryopoiesis resulting from transcription factor defects; abnormalities can extend to other marrow cells and may interfere with development, although among the affected genes are certain that play a role in platelet-vessel wall interactions. An example is X-linked familial thrombocytopenia (XLT) or XLT with thalassaemia (XLTT), which result from mutations in the GATA1 (GATA-binding protein 1 (globin transcription factor 1)) gene. One postulated cause for the altered function is a low transcription of GATA1 target genes, examples include a low density of GPIba and GPVI and a reduced platelet content of a-granules. The enlarged platelets aggregate poorly to collagen with a decreased tyrosine phosphorylation of GPVI-signalling proteins (Hughan et al, 2005). This finding has remained largely unexplained, but a direct role in platelet signalling may explain why GATA1 is retained in anucleate platelets. Another transcription factor present in platelets is STAT3 (signal transducer and activator of transcription3), which serves as a scaffold facilitating the catalytic interaction between SYK (spleen tyrosine kinase) and phospholipase Cc to enhance Ca2+-mobilization and platelet activation (Zhou et al, 2013). Collagen-induced platelet activation was much decreased when STAT3 was pharmacologically inhibited or deleted from platelets in mice. Thus, transcription factors can have secondary roles in platelets. Another gene unexpectedly involved in both thrombocytopenia and platelet dysfunction is GFI1B, which acts in both megakaryopoiesis and erythropoiesis. Genetic linkage analysis and new technology sequencing identified a single nucleotide insertion in GFI1B that predicted a frameshift mutation in a family with an autosomal dominant bleeding disorder combining FT and an impaired platelet aggregation response with ADP and arachidonic acid and a loss of the response with collagen (Stevenson et al, 2013). A protein essential to mention is RUNX1 (Runt-related transcription factor 1; also known as AML1, acute myeloid leukaemia 1). Together with its cofactor CBFB (core-binding factor b-subunit), RUNX1 is a master regulatory gene in haematopoiesis and is a common mutational target in human 174

leukaemia explaining why mutations in RUNX1 give rise to FT with a predisposition to haematological malignancies (Bluteau et al, 2012). Inactivating or dominant-negative mutations interfere with DNA binding; an arrest of MK maturation promotes an expanded population of progenitor cells. Platelet aggregation and secretion are impaired due to a decreased expression of a series of genes potentially affecting platelet function including protein kinase-h and platelet 12-lipoxygenase (ALOX12); another finding is a decreased phosphorylation of signalling and cytoskeletal proteins (Jalagadugula et al, 2011). Defects in cytoskeletal proteins, a frequent source of macrothrombocytopenia, can also perturb platelet function. The most-studied example is MYH9-related disease (MYH9-RD; myosin heavy chain 9-RD) (OMIM 160775) caused by decreased expression and/or function of non-muscle myosin heavy-chain IIA (or myosin-9) (Nurden & Nurden, 2012; Balduini et al, 2013). Mutations affecting the head domain (with Ca2+-ATPase activity) favour deafness and renal disease in later life, while those affecting the rod (and myosin-IIA assembly) are more likely to have only a haematological consequence (Balduini et al, 2011). Decreased myosin light chain phosphorylation and myosin function in MKs slow their migration towards the sinusoids as well as blurring the signalling mechanism for proplatelet formation (Pecci et al, 2011). In view of these motility defects it is not surprising that an absence of clot retraction is a major reported functional defect of platelets in MYH9-related disease. Myosin-9 is able to interact with both GPIba and aIIbb3, showing the close inter-relationship between the major surface receptors and the cytoskeleton. Two other cytoskeletal proteins with similar properties are filamin A and a-actinin. X-linked mutations in FLNA, encoding filamin A, result in a variety of developmental defects with abnormal neuronal migration resulting in periventricular nodular heterotopia (PNH). Filamin A is another attachment site for both GPIba and aIIbb3 in the platelet cytoskeleton and we have described how FLNA mutations can also give rise to macrothrombocytopenia (Nurden et al, 2011b). Significantly, FLNA mutations are associated with different functional impacts especially with regard to thrombus growth under flow (Berrou et al, 2013). In a new development, Kunishima et al (2013) have reported mutations in ACTN1, coding for a-actinin, in a series of Japanese families with congenital macrothrombocytopenia. This is of particular interest because a-actinin has been shown to regulate integrin-dependent force transmission to the extracellular matrix, suggesting a novel role in megakaryopoiesis and thrombus stability (Roca-Cusachs et al, 2013). Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disease combining thrombocytopenia and small platelets with eczema, recurrent infections, an increased risk for autoimmunity and malignancy (Thrasher & Burns, 2010; Mahlaoui et al, 2013). A milder form without immune problems is known as hereditary X-linked thrombocytopenia (XLT). The ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 165–178

Review small platelets aggregate poorly and have a low granule number without the causal mechanisms being truly defined. T lymphocytes show defective function. The WAS gene is composed of 12 exons, genetic defects result either in the decreased expression of WAS protein or its absence, the latter being predictive of a more severe disease. Missense mutations affecting the N-terminal Ena Vasp homology 1 domains predominate in hereditary XLT probably because of residual protein expression (Mahlaoui et al, 2013). WAS protein is a key regulator of actin polymerization in haematopoietic cells; it is involved in signal transduction with tyrosine phosphorylation sites and adapter protein function. WAS protein also induces ectopic proplatelet formation in the marrow where a lack of actin-rich podosomes retards MK migration to the vascular sinus. Subsequently, it has been shown that podosomes are pivotal for MK motility and remodelling of the extracellular matrix and react with multiple matrix components (Schachtner et al, 2013). One of the first disorders to be treated with haematopoietic stem cell transplantation, WAS is now the subject of several phase I/II gene therapy trials (Aluti et al, 2013).

We have also minimized discussion of disorders where the primary cause of bleeding is a low platelet count. Much remains to be done both in terms of diagnosis and understanding of the molecular basis of mild bleeding disorders associated with specific defects of signalling pathways but where precautions need to be taken in case of surgery or childbirth. Whole exome sequencing and other new generation technologies will help resolve the missing pieces of the puzzle and lead the way towards DNA analysis becoming the first step in diagnosis (Albers et al, 2011, 2012; Kunishima et al, 2013). Gene polymorphisms identified by candidate gene and genome-wide association studies contribute to phenotypic variation within a disease by down-regulating platelet function as well as affecting such parameters as platelet size or count (Faraday et al, 2011; N€ urnberg et al, 2012; Bunimov et al, 2013). Such approaches may also help define secondary genomic regulators that help determine bleeding tendency for wide ranges are seen within all of the disorders described in this review. After the identification of the primary gene defect in each family, the next step will be to determine what makes one patient bleed more than another.

Conclusions This review has provided an up-to-date assessment of the genetics of inherited disorders of platelet function. Due to space restrictions, single case reports have mostly been omitted, as have many historical reports with no recent update.

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