International Journal of Laboratory Hematology The Official journal of the International Society for Laboratory Hematology

REVIEW

INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY

Update on the causes of platelet disorders and functional consequences K. FRESON*, A. WIJGAERTS*, C. VAN GEET* , †

*Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium † Department of Pediatrics, University of Leuven, Leuven, Belgium Correspondence: Kathleen Freson, Center for Molecular and Vascular Biology, KU Leuven, Campus Gasthuisberg, O&N1, Herestraat 49, Box 911, 3000 Leuven, Belgium. Tel.: +32 16 346017; Fax: +32 16 345990; E-mail: kathleen.freson@med. kuleuven.be doi:10.1111/ijlh.12213

Received 15 January 2014; accepted for publication 12 February 2014 Keywords Platelets, platelet Function, Megakaryocyte, genetics, Granulopoiesis

S U M M A RY

Platelets are derived from megakaryocytes in the bone marrow that create the cellular machinery the platelet needs to participate in the different processes of primary hemostasis including adhesion, activation and clot formation at the site of injury. Defects related to megakaryocyte differentiation, platelet formation, and/or platelet function can result in bleeding. Patients with thrombopathies can present with mucous membrane bleeding but may also present with bleeding following trauma or surgery. In this review, we have classified inherited platelet bleeding disorders (IPD) according to their underlying defective pathway: transcription regulation, TPO signaling, cytoskeletal organization, apoptosis, granule trafficking, and receptor signaling. Platelet function testing has provided insights into the underlying molecular defects that can result in bleeding. A major step forward was made during the last 3 years using new-generation genetic approaches that resulted in the discovery of novel genes such as NBEAL2, RBM8A, ACTN1, and GFI1B for the well-known IPD that cause gray platelet syndrome, thrombocytopenia-absent radius syndrome, and autosomal dominant thrombocytopenias, respectively. In the near future, it is expected that a similar approach will identify many novel genes that cause IPD of unknown etiology, which are common. The future challenge will be to use a functional, systems biology approach to study the genes mutated in IPD and determine their roles in megakaryocyte and platelet biology and pathology.

I N T R O D U C T I O N TO I N H E R E D P L AT E L E T DISORDERS Platelets play an essential role in primary hemostasis as their activation is needed to form a stable hemostatic clot at the site of the injured blood vessel wall. © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 313–325

Platelets are the smallest cells in the blood and have an average life span of about 10 days before removal by the spleen. Their bone marrow precursor cells, megakaryocytes, produce platelets via intermediate cytoplasmatic extensions called proplatelets to maintain a steady platelet count. This is a tightly regulated 313

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process involving the cytokine thrombopoietin (TPO) and some lineage-specific transcription factors [1]. Molecular abnormalities in megakaryopoiesis and platelet production lead to thrombocytopenia and sometimes also platelet function abnormalities. This condition can also be associated with a functional platelet defect. Functional platelet abnormalities can also occur in combination with a normal platelet count, in conditions with altered platelet adhesion, spreading, granule secretion, and/or aggregation [2]. The degree of thrombocytopenia and/or a type of functional platelet defect determines the severity of bleeding symptoms. Patients with inherited platelet defects (IPD) typically present with mucocutaneous bleeding symptoms such as easy bruising, purpura, gingival bleeding, and epistaxis, usually already obvious in early childhood [3]. Milder cases only have bleeding problems after trauma or surgery. Spontaneous life-threatening bleeding complications such as intracranial hemorrhage, gastrointestinal, or genitourinary bleeding are rare. Female patients can have menorrhagia and bleeding during pregnancy and labor. Bleeding questionnaires, such as the ISTH bleeding assessment tool (ISTH-BAT), have been developed to evaluate mostly mild bleeding symptoms in patients with suspected IPD [3]. A recent study showed that the ISTH-BAT is useful to document a lifelong bleeding history in patients, but the score proved not to be predictive of the presence of a platelet defect on lumiaggregometry in patients with suspected IPD [4]. It is important to examine the family history and assess for other clinical phenotypes, as IPD are often a component of a multisystem disorder. The presence of skeletal, renal, dysmorphic, ocular, audiological, neurological, endocrinological, cardiac, and immune problems associated with bleeding is suggestive of multisystem disorders. The first laboratory test for IPD is the measurement of platelet count with the mean platelet volume and a peripheral blood smear. Different laboratory investigations such as measurement of platelet aggregation tests, ATP secretion, flow cytometry to quantitate expression of platelet receptors, platelet adhesion by the platelet function analyzer (PFA-100â, Siemens Healthcare Diagnostics, Marburg, Germany), and electron microscopy have been standardized to further analyze defects in platelet function and/or morphology [5]. IPD are considered as a heterogeneous group of diseases that can be

inherited in an autosomal recessive, autosomal dominant, or X-linked manner. There are different ways to classify these disorders, but in this review, we have chosen to group the disorders according to the underlying defective pathway (Table 1).

C L A S S I F I C AT I O N O F I N H E R I T E D P L AT E L E T D I S O R D E R S AC C O R D I N G TO D E F E C T I V E PAT H WAY Defective transcriptional regulation Defects in the X-linked transcription factor GATA1 that controls the development of erythroid and megakaryocytic cells are typically present as hemizygous loss-of-function mutations in the N-terminal zinc finger that impairs its binding to DNA or to the transcription cofactor FOG1 [6]. Patients have a variable degree of macrothrombocytopenia that can be associated with dyserythropoiesis, anemia, beta-thalassemia, or congenital erythropoietic porphyria depending on the missense variant. Their platelets are large, have a strongly reduced number of alpha granules, and are dysfunctional with reduced aggregation responses and abnormal membrane expression of the GPIb/IX/V receptor [6]. The FLI1 protooncogene encodes a member of the ETS family of winged helix-turn-helix transcription factors on chromosome 11q24.3. Chromosomal 11q23 deletions are described in Paris-Trousseau syndrome (also called Jacobson syndrome) [7]. A large study of 110 patients with 11q terminal deletions showed that nearly all patients (94%) have thrombocytopenia and platelet dysfunction with giant alpha granules. Other phenotypes comprised congenital heart defects, mental retardation, ophthalmologic, gastrointestinal, and genitourinary problems, gross and fine motor delays, and infections of the upper respiratory system in at least a large subset of patients [7]. Interestingly, a recent study showed that heterozygous variants in FLI1 and RUNX1 were found in patients with platelet dense granule secretion defects and mild thrombocytopenia [8]. RUNX1 is another transcription factor that functions as a master regulator of hematopoiesis. Autosomal dominant mutations in RUNX1 are found in patients with familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML) [9]. In © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 313–325

© 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 313–325

Regulation of TPO signaling

ANKRD26

RBM8A (Y14)

Thrombocytopenia 2

MIM188000

MIM274000

AD

AR

AR

Congenital amegakaryocytic thrombocytopenia Thrombocytopenia-absent radius syndrome

MPL (TPOR)

MIM601977/ 604498

Macrothrombocytopenia with dyserythropoiesis

GFI1B

AD

AD

MIM601399

Familial platelet disorder (FPD) with predisposition to AML

RUNX1 (CBFA2/ AML1)

MIM604383

AD

MIM188025/ 147791

Paris-Trousseau thrombocytopenia Jacobson syndrome

FLI1

X-linked

OMIM number MIM300367/ 314050

Macrothrombocytopenia with dyserythropoiesis/ anemia/Beta-thalassemia

GATA1 (ERYF1)

Transcription factors

Disorder name(s)

Gene name(s)

Class

Mode of inheritance

(micro)TP

TP (improves with age)

TP

MacroTP

TP

TP

MacroTP

Platelet count

11q deletions are associated with heart defects, mental retardation, ophthalmologic, gastrointestinal, and genitourinary defects, motor delays and infections Predisposition to AML

Abnormal erythropoiesis

Other clinical phenotypes

(continued)

Abnormal aggregations, dSPD, presence of MYH10 Abnormal erythropoiesis Abnormal aggregations, Reduced a granules Normal Predisposition for aplastic ultrastructure anemia or myelodysplastic syndrome Structural Absent radius and can be abnormalities associated with cow milk allergy, other bone abnormalities, renal and cardiac problems, short statures and risk for leukemia. Predisposition to acute Reduced a granules, low leukemia levels of GPalpha2

Abnormal aggregations Reduced a granules Giant a granules, dSPD, presence of MYH10

Platelet function defect

Table 1. Overview of IPD that cause a bleeding phenotype related to a defect in platelet count and/or function and genetic findings

K. FRESON, A. WIJGAERTS AND C. VAN GEET | INHERITED PLATELET DISORDERS 315

Apoptosis

MYH9 (NMHCIIA)

Cytoskeletal proteins

Thrombocytopenia 4

Autosomal dominant thrombocytopenia

ACTN1

CYCS

Wiskott-Aldrich syndrome

MYH9-related diseases (Sebastian/May Hegglin/ Fechtner/Epstein syndromes) Filaminopathy (cardiac valvular dysplasia/short bowel disease/ frontometaphyseal dysplasia/periventricular heterotopia/Melnick– Needles syndrome/ otopalatodigital syndromes/terminal osseaous dysplasia) Autosomal dominant macrothrombocytopenia

Disorder name(s)

WAS

TUBB1

FLNA (FLN1)

Gene name(s)

Class

Table 1. (Continued)

MIM612004

MIM615193

MIM301000

MIM613112

AD

AD

X-linked

AD

AD MIM605249/ 155100/ 153640/ 153650 X-linked MIM314400/ 300048/ 305620/ 300049/ 309350/ 311300/ 304120300244

OMIM number

Mode of inheritance

TP

MacroTP

MicroTP

Normal function and structure Normal function and structure

Normal function and structure Abnormal aggregations and fewer granules

Abnormal aggregation, secretion and adhesion, enlarged a granules

MacroTP

MacroTP

Defect in clot retraction

Platelet function defect

MacroTP

Platelet count

No

(continued)

Neutropenia with eczema, frequent infections, autoimmune pathologies and higher risk for malignancies No

No

D€ ohle-like inclusion in leukocytes, risk for deafness, cataract, and proteinuric nephropathy Wide-spectrum disease that can include periventricular heterotopia, skeletal dysplasia, mental retardation, cardiac valvular dystrophy, congenital intestinal pseudo-obstruction, and terminal osseous dysplasia

Other clinical phenotypes

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© 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 313–325

© 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 313–325

Normal

AR

AR

Chediak–Higashi syndrome MIM214500

Hermansky Pudlak syndromes types 1 to 9

MIM203300/ 608233/ 606118/ 614073/ 614074/ 614075/ 614076/ 614077/ 614171

HPS1 HPS2 (AP3B1) HPS3 HPS4 HPS5 (AIBP63) HPS6 HPS7 (DTNBP1) HPS8 (BLOC1S3) HPS9 (BLOC1S6) LYST

Normal

AR

MIM613404

VIPAS39 Arthrogryposis, renal (C14ORF133) dysfunction, and cholestasis syndrome

Normal

Normal

AR

MIM208085

Arthrogryposis, renal dysfunction and cholestasis syndrome

VSP33B

MacroTP

Platelet count

AR

Gray platelet syndrome

NBEAL2

Granule formation/ trafficking/ secretion

OMIM number

Mode of inheritance

MIM139090

Disorder name(s)

Gene name(s)

Class

Table 1. (Continued)

Abnormal aggregations, d-SPD

Abnormal aggregations, Reduced a granules Abnormal aggregations, Reduced a granules Abnormal aggregations, Reduced a granules, enlarged platelets Abnormal aggregations, d-SPD

Platelet function defect

(continued)

Albinism, defective phagocytosis, infections

Albinism, pulmonary fibrosis, ceroid accumulation, cholitis

Cerebral malformations, deafness, congenital heart disease, diabetes, dysmorphic features

Myelofibrosis

Other clinical phenotypes

K. FRESON, A. WIJGAERTS AND C. VAN GEET | INHERITED PLATELET DISORDERS 317

von Willebrand disease type 2B

Collagen receptor defect

GP6

MIM177820

Platelet-type von Willebrand disease

vWF

MIM231200

Bernard Soulier Syndrome

GP9 GP1BA GP1BB GP1BA

MIM614201

MIM613554

MIM273800

Glanzmann thrombastenia

ITGA2B ITGB3

Glycoprotein receptors and their signaling pathways

OMIM number

Disorder name(s)

Gene name(s)

Class

Table 1. (Continued)

AR

AD

AD

AR

AR (AD, see [26])

Mode of inheritance

Normal

MacroTP

MacroTP

Normal (only some mutations result in TP [26]) MacroTP

Platelet count

Aggregation defect for ristocetin Aggregation increased for ristocetin, loss of VWF multimers Aggregation increased for ristocetin, loss of VWF multimers Aggregation defect for collagen

Absent aggregations

Platelet function defect

No

No

No

No

No

(continued)

Other clinical phenotypes

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Gs platelet defect related to bleeding

GNAS

MIM262890

MIM601709

MIM103580/ 603233/ 612463

AD

AD

AD (imprinted gene)

AR

AR

MIM600522

MIM609821

AR

AR

MIM231095

MIM614009

OMIM number

Mode of inheritance

Normal

Normal (TP in some patients)

Normal

Normal

Normal

Normal

Normal

Platelet count Aggregation defect for arachidonic acid and U46619 Aggregation defect for arachidonic acid Aggregation defect and dSPD Aggregation defect for ADP Abnormal aggregation inhibition test Degraded a granule proteins, decreased aggregation to epinephrine Decreased PS exposure

Platelet function defect

TP, thrombocytopenia; AD, autosomal dominant; AR, autosomal recessive; d-SPD, delta-storage pool disease.

Scott syndrome

ADP receptor defect

P2RY12

TMEM16F (ANO6)

Cytosolic phospholipase A2a deficiency

PLA2G4A

Quebec platelet disorder

Ghosal hematodiaphyseal dysplasia syndrome

TBXAS1

PLAU

Thromboxane receptor defect

TBXA2R

G proteincoupled receptors and their signaling pathways

Others (relation with coagulation pathway proteins)

Disorder name(s)

Gene name(s)

Class

Table 1. (Continued)

No

No

Short stature, mental disability and brachydactyly

No

Increased bone density

No

Other clinical phenotypes

K. FRESON, A. WIJGAERTS AND C. VAN GEET | INHERITED PLATELET DISORDERS 319

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addition to thrombocytopenia, platelets in FPD-AML are dysfunctional, with abnormalities evident in aggregation and ATP release assays [9]. Retained MYH10 protein expression in patients’ platelets was recently suggested as a diagnostic biomarker for underlying RUNX1 and FLI1 defects [10]. The most recent finding is the discovery of autosomal dominant mutations in GFI1B, another transcription factor important for megakaryocyte and erythrocyte development [11, 12]. Patients have mild clinical bleeding symptoms along with red cell anisopoikilocytosis and platelet abnormalities that include macrothrombocytopenia, almost absent alpha granules, and markedly decreased aggregation responses. Defective regulation of TPO signaling In addition to the lineage-specific transcription factors, megakaryopoiesis is tightly regulated by thrombopoietin (TPO). TPO binds to the c-MPL receptor and mediates not only the growth and differentiation of megakaryocytes but also the maintenance of stem cells. Congenital amegakaryocytic thrombocytopenia (CAMT) is an autosomal recessive disorder with isolated severe thrombocytopenia presented at birth due to MPL mutations [13]. Plasma TPO levels are high, and patients have strongly reduced megakaryocytes in their bone marrow. Some patients will progress to aplastic anemia or myelodysplastic syndrome. High TPO levels and defective TPO signaling were also detected in thrombocytopenia with absent radius (TAR) syndrome, but these patients have a normal MPL gene. Exome sequencing revealed recently that compound inheritance of a low-frequency noncoding SNP and a rare null allele in RBM8A, a gene encoding the exon-junction complex subunit member Y14, causes TAR syndrome [14]. It is not known why decreased levels of Y14 are associated with altered TPO signaling. TAR is a syndromic form of thrombocytopenia associated with absent radius, often an allergy to cow’s milk during childhood, and an increased risk for other bone defects, cardiac, or renal problems. Patients with autosomal dominant thrombocytopenia and genetic variants in the 50 UTR of the ANKRD26 gene have increased ankyrin repeat domain 26 protein levels [15]. It was recently shown that these variants result in loss of RUNX1 and FLI1 binding that

inhibit ANKRD26 expression in normal conditions [16]. Increased ANKRD26 was associated with increased TPO signaling, but further studies are needed to clarify the platelet formation defect in these patients and/or their increased risk for developing leukemia. Platelets from these patients are relatively small and have reduced alpha granule numbers along with a reduced expression of the collagen receptor GPalpha2. The bleeding tendency is very mild. Defective cytoskeletal organization During platelet formation, megakaryocytes first undergo a dramatic rearrangement of the cytoskeleton that results in long, branching cytoplasmic extensions, called proplatelets, which fragment at their end to finally form platelets. Organelles and specific platelet granules need to be transported along these proplatelet extensions, over sizeable distances, before being loaded into nascent platelets. Microtubules, actin filaments, and other cytoskeletal proteins are essential to this process. Cytoskeletal proteins such as myosin heavy chain (MYH) and filamin (FLN) are abundantly present in platelets, important not only during their formation but also contributing to their structure after activation. MYH9-related diseases include the syndromes MayHegglin, Sebastian, Fechtner, and Epstein syndrome, which are characterized by autosomal dominant mutations in the MYH9 gene [17]. These diseases are syndromic and cause thrombocytopenia that can be associated with D€ ohle-like leukocyte inclusions and a variable risk to develop proteinuric nephropathy, deafness, and/or cataract. Platelets are giant and have a defect in clot retraction. FLNs stabilize actin filament networks. Mutations in the FLNA (filamin A) gene cause a spectrum of disorders, including brain malformations with periventricular nodular heterotopia as most frequent phenotype. FLNA is also the predominant isoform expressed in platelets, and patients with filaminopathy A typically have macrothrombocytopenia with enlarged alpha granules and heterogeneous abnormalities of platelet function, mainly affecting aggregations, secretions, and adhesion [18]. TUBB1 (tubulin beta 1) is the major tubulin isoform within platelet and megakaryocyte microtubules. An autosomal dominant missense TUBB1 variant was © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 313–325

K. FRESON, A. WIJGAERTS AND C. VAN GEET | INHERITED PLATELET DISORDERS

found to cause macrothrombocytopenia with an expected defect in platelet formation, but interestingly, no abnormalities of structure or aggregation responses [19]. Clinical bleeding problems were also not described for the propositus. Wiskott–Aldrich syndrome (WAS) is an X-linked disorder caused by mutations in the WAS gene [20]. The WAS protein interacts with the actin filament organization in hematopoietic stem cells and is important for normal platelet and neutrophil function. Patients have microthrombocytopenia with dysfunctional platelets and often neutropenia associated with eczema, immune dysfunction with high susceptibility to infections, autoimmune diseases, and malignancies. The most recent pathology related to a defective cytoskeletal organization is the autosomal dominant macrothrombocytopenia due to missense variants in the ACTN1 gene as discovered by exome sequencing [21]. ACTN (actin) isoforms are organized as antiparallel dimers with an actin-binding domain at the N terminus, through which they cross-link actin filaments into bundles. ACTN1-deficient patients have enlarged platelets with normal function and subcellular structures [21]. The bleeding tendency is mild. Defect in apoptosis Autosomal dominant mild thrombocytopenia was described in a large family from New Zealand due to a missense mutation in the somatic isoform of the cytochrome c (CYCS) gene. Patients have platelets of normal size and morphology, and only partially reduced platelet numbers, without prolonged bleeding episodes. Cytochrome c is a multifunctional protein with roles in electron transport, antioxidant defenses, and apoptosis, and interestingly, the mutation yields a cytochrome c variant with enhanced apoptotic activity in vitro [22]. Defects in platelet granule formation, trafficking, and secretion Three types of granules are present in platelets: dense granules (containing serotonin, ADP and ATP), alpha granules (containing many different proteins, including von Willebrand factor (VWF) and fibrinogen), and lysosomes. Deficiencies related to the granules (storage pool diseases; SPD) may cause a bleeding © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 313–325

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diathesis and are usually grouped according to the type of granule that is affected. For all the molecular defects described in this class, the molecular mechanism underlying the defective granule formation, trafficking, and/or secretion defect is not well understood. Next-generation sequencing techniques were used to finally identify the genetic defect for the gray platelet syndrome (GPS). This is an autosomal recessive disorder, characterized by macrothrombocytopenia and deficiency of alpha granules, and is caused by mutations in the NBEAL2 gene [23]. NBEAL2 is expected to regulate by a yet unknown mechanism the formation and trafficking of alpha granules. Platelets are dysfunctional and many patients develop myelofibrosis. Other proteins that were shown to be involved in platelet alpha granule formation are the vacuolar protein sorting-associated protein 33B (VPS33B) and the VPS33B interacting protein, apical–basolateral polarity regulator, Spe-39 (VIPAS39) [24]. Vesicle-mediated protein sorting plays an important role in segregation of intracellular molecules into distinct organelles. Autosomal recessive mutations in VPS33B or VIPAS39 result in arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome with platelet dysfunction and a paucity of alpha granules [24]. Patients typically suffer from cerebral malformations, deafness, congenital heart disease, diabetes, dysmorphic features, and a bleeding diathesis mainly after surgery or treatment with aspirin. Abnormal formation and/or secretion of dense granules is a hallmark of the Hermansky Pudlak syndromes (HPS) that can be caused by autosomal recessive mutations in a least one of the nine HPS genes [25]. HPS is also characterized by oculocutaneous albinism, and in some subtypes, patients suffer from colitis, interstitial lung disease, and fatal pulmonary fibrosis. HPS proteins interact with each other in complexes called BLOCS (biogenesis of lysosome related organelle complexes) that are important for dense granule formation. Platelets from HPS patients have delta-SPD with secretiondependent aggregation defects and a variable degree of bleeding. The lysosomal trafficking regulator (LYST) belongs to the same class as NBEAL2, and it is involved in formation of dense granules. Autosomal recessive

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mutations in LYST are responsible for Chediak–Higashi syndrome (CHS), which is characterized by deficiency of platelet dense granules [23]. LYST patients have delta-SPD in addition to severe immunologic defects with life-threatening infections and progressive neurological dysfunction. Defective glycoprotein receptor signaling The two main platelet glycoprotein receptors GPIIbIIIa and GPIb-IX-V mediate platelet aggregation and adhesion, respectively. IPD related to mutations in genes that code for these receptors have widely been studied, and result is obvious spontaneous and trauma-related bleeding symptoms without other phenotypes. The two parts of the GPIIB-IIIa glycoprotein receptor are coded by genes ITGA2B and ITGB3, and autosomal recessive mutations in either one of these genes result in Glanzmann thrombastenia (GT), a classic IPD with absent platelet aggregations to all agonists except for ristocetin [26]. The clinical bleeding symptoms are highly variable according to the type of the mutations. Platelet counts are normal for GT except for a novel variant form due to specific autosomal dominant mutations of ITGA2B and ITGB3 that lead to mild thrombocytopenia. Bernard–Soulier syndrome (BSS) is caused by autosomal recessive mutations in any of the following genes: GP9, GP1BA, or GP1BB that code for different units of the GPIB-IX-V receptor that interacts with VWF via the GPIbalpha component [27]. Mutations in GP5 have not been described. BSS patients have macrothrombocytopenia and an absent platelet response for VWF and ristocetin. The severity of the bleeding phenotype depends on the type of mutation and the platelet count. The reason why the defective GPIb-IXV results in macrothrombocytopenia is not fully understood. Platelet-type von Willebrand disease (vWD) is an autosomal dominant disorder with mild macrothrombocytopenia and bleeding due to specific GP1AB mutations that result in increased binding of VWF for GPIbalpha [27]. A similar clearance of larger VWF multimers and platelets from the circulation is observed for vWD type2B patients. They also have macrothrombocytopenia and increased reactivity of GPIbalpha for VWF and ristocetin. In the latter,

autosomal dominant mutations are present in the A1 domain of VWF gene. Finally, mutations in the collagen glycoprotein VI receptor gene GP6 are very rare. Autosomal recessive GP6 mutations lead to a mild bleeding phenotype, an absent response to Horm collagen, convulxin, and the collagen-related peptide, but platelet count and morphology are normal [28]. Defective G protein-coupled receptor signaling Other types of receptors that are important for platelet function are the G protein-coupled receptors (GPCR) and their downstream direct or indirect effectors. These types of receptors are typically activated by soluble ligands to release the G protein and stimulate intracellular signaling. Many GPCRs, G proteins, and downstream molecules have been characterized in platelets, but this review will only focus on members that are linked to an IPD. Heterozygous loss-of-function mutations in the thromboxane A2 receptor gene (TBXA2R) are found in patients with platelet defects related to stimulations with arachidonic acid and U46619, as a dominant finding. Though in these carriers, heterozygosity for the TBXA2R mutation correlated with the platelet defect, there was no association with clinical bleeding problems [29]. Only one patient with a homozygous TBXA2R variant is described with both a platelet aggregation defect and clinical bleeding, and therefore, a second genetic hit is expected to explain bleeding in persons that are heterozygous for TBXA2R mutations. Platelet count and morphology is normal in these patients. Another thromboxane pathway defect with bleeding and abnormal aggregation responses for arachidonic acid was described for patients with Ghosal syndrome, an increased bone density disorder [30]. This condition is caused by autosomal recessive mutations in the TBXAS1 gene. Thromboxane synthase (TBXAS) is one of the terminal enzymes of the arachidonic acid cascade and converts prostaglandins H2 into thromboxane A2. The platelet defect is very mild as these patients don’t have a history of spontaneous or surgery-related bleeding symptoms. Cytosolic phospholipase A2alpha hydrolyzes arachidonic acid from cellular membrane phospholipids, thereby providing enzymatic substrates for the © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 313–325

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synthesis of eicosanoids, such as prostaglandins and leukotrienes. Inherited cytosolic phospholipase A2a deficiency is caused by autosomal recessive mutations in the PLA2G4A gene and is associated with impaired eicosanoid biosynthesis, small intestinal ulceration, and platelet dysfunction with reduced aggregation and ATP secretion assays [31]. P2Y12 deficiency is an autosomal recessive bleeding disorder, characterized by mutation in the P2RY12 gene, bleeding, a prolonged bleeding time and reversible aggregation in response to weak agonists, impaired aggregation toward low concentrations of collagen and thrombin, and a severely impaired response to ADP [32]. The extra-large stimulatory G protein alpha (Gs) subunit (XLsalpha) is coded by the imprinted gene cluster GNAS from the paternal allele [33]. Patients with a 36 bp insertion and two basepair substitutions flanking this insertion in the paternally inherited XL-GNAS1 exon 1 have an enhanced trauma-related bleeding tendency, a variable degree of mental retardation, and brachydactyly. The platelet aggregation inhibition test to evaluate Gs function showed Gs hyperfunction and enhanced cAMP generation upon stimulation of Gs-coupled receptors. Some other types of IPD Quebec platelet syndrome and Scott syndrome are very rare IPD that cannot be classified under the previous sections. Both are very closely linked to the interplay between platelets and the fibrinolytic or coagulation system, respectively. Quebec platelet disorder is unique to FrenchCanadian families and is an autosomal dominant bleeding disorder associated with a unique gainof-function defect in fibrinolysis [34]. It is caused by a tandem duplication of the PLAU gene that codes for urokinase-type plasminogen activator (uPA). Platelets from these patients have protease-related degradation of alpha granule proteins but structurally normal granules and show a decreased aggregation response to epinephrine. Only some patients have thrombocytopenia, but a potential pathogenic effect of uPA overexpression on megakaryopoiesis is possible.

© 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 313–325

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Scott syndrome is an IPD characterized by loss of the capacity of platelets to externalize negatively charged phosphatidylserine (PS) [35]. Patients have normal platelet counts and size, and their aggregation findings are normal. The syndrome is caused by autosomal dominant mutations in the anoctamin ANO6 gene that codes for transmembrane protein TMEM16F [34]. As Scott syndrome platelets fail to expose PS, factors Va and Xa fail to bind leading to a decreased capacity of platelets to convert prothrombin into thrombin that results in a bleeding syndrome.

FUTURE DIRECTIONS FOR RESEARCH R E L AT E D TO I N H E R I T E D P L AT E L E T DISORDERS Though the list of genes responsible for IPD is rapidly growing, most patients with IPD still receive no genetic diagnosis, especially for patients with platelet secretion defects (storage pool diseases), which is a relatively frequent type of IPD, most genes are yet to be discovered. The identification of molecular defects in patients with IPD has improved our understanding of normal megakaryocyte and platelet biology, as well as mechanisms of hemostasis and thrombosis. These insights are also important for the development of novel therapeutic drugs for patients with thrombosis and thrombocytopenia. The utilization of next-generation sequencing approaches will potentially revolutionize not on the current diagnosis via functional platelet testing of ‘classical’ IPD but possibly also the rapid identification of individuals with increased risk of bleeding often hidden but in association with other clinical phenotypes. Future medical DNA sequencing is also expected to give clinicians important information regarding the genetic phenotype of the IPD in their patients to improve early diagnosis.

AC K N OW L E D G E M E N T S This research was supported by research grants G.0B17.13N from Fund for Scientific Research-Flanders (F.W.O.-Vlaanderen, Belgium). C.V.G. is holder of a clinical-fundamental research mandate of the F.W.O.-Vlaanderen and of the Bayer and Norbert Heimburger (CSL Behring) Chairs.

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