Inherited Disorders of Platelet Function

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Inherited Disorders of Platelet Function Marco Cattaneo Unità di Medicina 2, ASST Santi Paolo e Carlo, Dipartimento di Scienze della Salute, Università degli Studi di Milano, Milan, Italy

INTRODUCTION 877 CLASSIFICATION OF INHERITED DISORDERS OF PLATELET FUNCTION 877 ABNORMALITIES OF THE PLATELET RECEPTORS FOR ADHESIVE PROTEINS 877 Abnormalities of the GPIb-IX-V Complex 877 Abnormalities of Integrin αIIbβ3 882 Abnormalities of Integrin α2β1 883 Abnormalities of GPVI 883 ABNORMALITIES OF THE PLATELET RECEPTORS FOR SOLUBLE AGONISTS 883 Abnormalities of the Platelet P2 Purinergic Receptors 883 Abnormalities of Other Platelet P2 Receptors 884 Defects of the Platelet Thromboxane A2 Receptor (TP) 884 Defects of the α2-Adrenergic Receptors 885 DEFECTS OF PLATELET GRANULES 885 Defects of the δ-Granules 885 Defects of the α-Granules 887 Defects of the α- and δ-Granules 889 DEFECTS OF SIGNAL TRANSDUCTION 890 Deficiency of Cytosolic Phospholipase A2α 890 Defects of Cyclooxygenase-1 (Aspirin-Like Defect) 890 Defects of Thromboxane Synthetase 890 Abnormalities of GTP-Binding Proteins 891 Defects in Phospholipase C Activation 891 CalDAG-GEFI Defect 891 Leukocyte Adhesion Deficiency-III (LAD-III) 891 Abnormality of GPVI/FcRc Signaling 892 Stormorken/York Platelet Syndrome 892 ABNORMALITIES OF MEMBRANE PHOSPHOLIPIDS 892 Scott Syndrome 892 MISCELLANEOUS DISORDERS OF PLATELET FUNCTION 892 Primary Secretion Defects 892 Other Platelet Abnormalities 893 INHERITED DEFECTS OF ADHESIVE PROTEINS, AFFECTING PLATELET FUNCTION 893 PREVALENCE AND DIAGNOSTIC EVALUATION OF INHERITED DISORDERS OF PLATELET FUNCTION 893 Prevalence 893 Diagnosis 893 TREATMENT OF INHERITED PLATELET FUNCTION DISORDERS 894 CONCLUSIONS 895 REFERENCES 895 Platelets. https://doi.org/10.1016/B978-0-12-813456-6.00048-5 Copyright © 2019 Elsevier Inc. All rights reserved.

INTRODUCTION When a blood vessel is injured, platelets adhere to the exposed subendothelium (platelet adhesion), are activated (platelet activation) and secrete their granule contents (platelet secretion), including some platelet agonists (adenosine diphosphate [ADP], serotonin), which, by interacting with specific platelet receptors, contribute to the recruitment of additional platelets to form aggregates (platelet aggregation). In addition, platelets play a role in the coagulation mechanism, providing the necessary surface of procoagulant phospholipids (platelet procoagulant activity). Inherited or acquired abnormalities of platelet number or function are associated with a heightened risk for bleeding, proving that platelets play an important role in hemostasis. Typically, patients with platelet disorders have mucocutaneous bleeding of variable severity and excessive hemorrhage after surgery or trauma. This chapter will review inherited disorders of platelet function.

CLASSIFICATION OF INHERITED DISORDERS OF PLATELET FUNCTION Inherited disorders of platelet function could be classified based on the functions or responses that are abnormal. However, since platelet functions are intimately related, a clear distinction between disorders of platelet adhesion, activation, secretion, aggregation and procoagulant activity is in many instances problematic. For example, platelets that are deficient in the glycoprotein (GP) Ib-IX-V complex, which is a receptor for von Willebrand factor (VWF), do not adhere normally to the subendothelium and for this reason are generally included in the group of abnormalities of platelet adhesion. However, they also do not undergo normal activation and aggregation at high shear, do not aggregate normally to thrombin, and display abnormal procoagulant responses. For this reason, this chapter will classify inherited disorders of platelet function based on abnormalities of platelet components that share common characteristics1: (1) platelet receptors for adhesive proteins; (2) platelet receptors for soluble agonists; (3) platelet granules; (4) signal transduction pathways; and (5) procoagulant phospholipids. Inherited disorders of platelet function that are less well characterized will be grouped into a 6th category of miscellaneous disorders. This classification approach is summarized in Table 48.1 and shown diagrammatically in Fig. 48.1. Table 48.2 lists the essential characteristics of the principal inherited disorders of platelet function. Several disorders of platelet function are characterized by thrombocytopenia and are therefore described also in Chapter 46.

ABNORMALITIES OF THE PLATELET RECEPTORS FOR ADHESIVE PROTEINS Abnormalities of the GPIb-IX-V Complex Bernard-Soulier Syndrome Bernard-Soulier syndrome (BSS, see also Chapter 46) is a quantitative or qualitative defect of the platelet GPIb-IX-V complex,

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TABLE 48.1 Inherited Disorders of Platelet Function 1. Abnormalities of the platelet receptors for adhesive proteins a. GPIb-IX-V complex (Bernard-Soulier syndrome, DiGeorge velocardiofacial syndrome, platelet-type von Willebrand disease) b. GPIIb-IIIa (αIIbβ3) (Glanzmann thrombasthenia) c. GPIa-IIa (α2β1) d. GPVI 2. Abnormalities of the platelet receptors for soluble agonists a. P2Y12 receptor b. Thromboxane A2 receptor c. α2-Adrenergic receptor 3. Abnormalities of the platelet granules a. δ-Granules (nonsyndromic δ-storage pool deficiency, Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, MRP4 deficiency, thrombocytopenia with absent radii syndrome, Wiskott-Aldrich syndrome, SFLN14-related disease, Familial platelet disorder with predisposition to AML) b. α-granules (gray platelet syndrome, GFI1B-related syndrome, GATA-1 related disease [X-linked macrothrombocytopenia with anemia/anisopoikilocytosis], SRC-related disease, ParisTrousseau syndrome, Jacobsen syndrome and other FLI1-related disorders, Arthrogryposis, renal dysfunction, and cholestasis [ARC] syndrome, Quebec platelet disorder, White platelet syndrome, Medich platelet disorder) c. α- and δ-granules (α,δ-storage pool deficiency, Familial platelet disorder with predisposition to AML, GFI1B-related syndrome) 4. Defects of signal transduction a. Abnormalities of the arachidonate/thromboxane A2 pathway (defects in phospholipase A2, cyclooxygenase, thromboxane synthetase) b. Abnormalities of GTP binding proteins (Gαq deficiency, Gαi1 defect, hyperresponsiveness of platelet Gsα) c. defects in phospholipase C activation (partial selective PLC-β2 isozyme deficiency) d. CalDAG-GEFI defect e. Leukocyte adhesion deficiency-III (LAD-III) f. Abnormality in GPVI/FcRc signaling g. Stormorken/York platelet syndrome 5. Abnormalities of membrane phospholipids a. Scott syndrome 6. Miscellaneous abnormalities of platelet function a. Primary secretion defects b. Other (osteogenesis imperfecta, Ehlers-Danlos syndrome, Marfan syndrome, hexokinase deficiency, glucose-6-phosphate deficiency)

which is formed by the products of four genes (GPIBA, GPIBB, GP9, and GP5), because GPIb consists of two subunits, GPIbα and GPIbβ (Chapter 10). The cytoplasmic domains of the four glycoproteins are linked to the membrane cytoskeleton through filamin A.2 The GPIbα-filamin A interaction seems to be important to secure normal platelet production and to maintain the mechanical stability of the plasma membrane under conditions of high shear.3,4 BSS is associated with defects in GPIBA, GPIBB, and GP9, while defects in GP5 do not lead to BSS, probably due to the fact that, while GPIbα, GPIbβ and GPIX assemble within the endoplasmic reticulum before exposure on the platelet membrane, GPV is not required for the expression of the complex.5 BSS is characterized by autosomal recessive inheritance in most cases, a prolonged bleeding time, variable degrees of thrombocytopenia, giant platelets and decreased platelet survival. The degree of thrombocytopenia may be overestimated when the platelet count is performed with automated counters, because giant platelets, which may be as frequent as 70%–80% in BSS patients, may reach the size of red blood cells (Fig. 48.2A) and, as a consequence, are not recognized as platelets by automated counters (Chapter 32).1,6

BSS is a relatively severe bleeding disorder with an estimated prevalence of approximately 1 case in 1 million people. Typical bleeding manifestations include epistaxes, gum bleeding, and postsurgical and posttraumatic bleeding. Pregnancy in BSS is associated with a high risk of serious bleeding for the mother and the neonate.7 Most heterozygotes, with few exceptions, do not have a bleeding diathesis.1,6 Typically, BSS platelets do not agglutinate when exposed to ristocetin or botrocetin, because GPIbα is unable to bind VWF. In contrast to von Willebrand disease (VWD), this defect is not corrected by the addition of normal plasma. The interaction of BSS platelets with the subendothelium is impaired at both high and low shear forces, although the defect is more pronounced at the high shear forces that are encountered in the normal microcirculation. Because VWF supports shear-induced platelet aggregation by interacting with GPIbα, thrombus formation on subendothelium at high shear forces is also defective. It has been shown that GPIbα contributes to platelet thrombus formation on subendothelium by adhesion mechanisms that are independent of the binding to VWF.8 The BSS platelet responses to physiological agonists are normal, with the exception of low concentrations of thrombin. GPIbα plays a critical role in the platelet aggregatory, secretory and procoagulant responses to thrombin, because the binding of thrombin to its high affinity binding sites on GPIbα accelerates the platelet response that is mediated by its two moderate affinity platelet receptors, protease-activated receptor (PAR)-1 and PAR-4 (Chapter 13).9–10 BSS platelets also display defective procoagulant activity,11 which is probably secondary to defective binding of high-molecular weight kininogen, factor XI, factor XII, P-selectin and Mac-112–15 and decreased fibrin polymerization, which is crucial for thrombin formation in platelet-rich plasma, in a VWF/GPIbα-dependent process.16 It has been hypothesized that GPIb-IX-V complex plays a central role of the in the platelet procoagulant activity that is independent of the binding properties of GPIbα.17 Diagnosis of BSS is based on the demonstration of GPIb-IXV deficiency by flow cytometry (Chapter 35) or immunoblotting. Heterozygotes usually have intermediate amounts of the GP complex and mild thrombocytopenia with few giant platelets. Molecular Defects. Each polypeptide of the GPIb-IX-V complex is encoded by a separate gene: GPIBA on chromosome 17, GPIBB on chromosome 22, and GP9 and GP5 on chromosome 3. Defects of GPIBA. Mutations causing complete absence of the glycoprotein include insertions and deletions followed by frameshifts; nonsense mutations are also frequent.6,13 Missense mutations have been described that can either lead to failure to translocate to the membrane or to the synthesis of dysfunctional proteins. Some variants with autosomal dominant inheritance have been described. In the p.Ala156Val mutation, also called the Bolzano variant, which is particularly common in Southern Italy, the binding of thrombin to platelets is conserved, while that of VWF is severely impaired.18 Heterozygous patients have mild macrothrombocytopenia and may have mild bleeding symptoms.19 Another BSS variant, associated with p.Leu57Phe mutation is characterized by increased susceptibility of platelets to proteolysis and slightly decreased platelet agglutination induced by ristocetin.20 A heterozygous mutation, c.A169C, resulting in an p.Asp41His substitution in GPIbα, was described in the affected members of two unrelated families with inherited thrombocytopenia. The molecular modeling suggests that the p.Asp41His substitution drastically disturbs the structure of the first portion of GPIbα N-terminal, directly involved in VWF binding.21 Defects of GPIBB. The majority of point mutations that have been identified in the GPIBB gene are mostly missense

Inherited Disorders of Platelet Function

ADP TxA2 5HT FIIa ...

FIIa

FV

TxA2 Adhesive protein

FXa FII

FVa

PHOSPHOLIPIDS Scott syndrome

RECEPTORS FOR SOLUBLE AGONISTS

RECEPTORS FOR ADHESIVE PROTEINS

P2Y12 defect TxA2 receptor defect

Glanzmann thrombasthenia

GRANULES α

48

Platelet

ADP

5HT

879

GRANULES SIGNAL TRANSDUCTION

δ

δ-Storage Pool Def. Hermansky-Pudlak S. Chediak-Higashi S.

δ

G proteins; PLC; PLA2; COX-1;…

α

Gray platelet syndrome Quebec platelet disorder

RECEPTORS FOR ADHESIVE PROTEINS Bernard-Soulier syndrome α2β1 defect

Adhesive protein

S

u

b

e

n

Platelet-type VWD GPVI defect

Adhesive protein

d

o

t

Adhesive protein

h

e

l

i

u

m

Fig. 48.1 Schematic representation of platelet structure and function, with the main inherited platelet function disorders divided into five categories. A sixth category, which is not indicated in this figure, includes other miscellaneous disorders (see text). Abbreviations: ADP, adenosine diphosphate; COX-1, cyclooxygenase-1; FII, factor II (prothrombin); FIIa, factor IIa (thrombin); FV, coagulation factor V; FXa, activated factor X; GP, glycoprotein; 5HT, 5-hydroxytryptamine (serotonin); PLA2, phospholipase A2; PLC, phospholipase C; TxA2, thromboxane A2; VWD, von Willebrand disease.

alterations, mainly affecting the extracellular regions of the glycoprotein. Nonsense and frameshift mutations within the extracellular or transmembrane domains have also been identified. The first patient with BSS associated with defects of GPIBB described in the literature suffered from a developmental disorder, the DiGeorge/velocardiofacial syndrome (see also Chapter 46), which was due to deletion at 22q11.2, including GPIBB, in one allele.13 The patient also had a mutation in the GPIBB promoter, within a binding site of the GATA-1 transcription factor in the other allele, which, in combination with the 22q11.2 deletion in the other allele, caused a severe deficiency of the GPIb-IX-V complex and a phenotype typical of BSS. Other mutations in GPIBB, associated with the DiGeorge syndrome, have been identified.13 In addition to the clinical features of BSS, these patients usually have cardiac defects, dysmorphic facial features, thymic hypoplasia, velopharyngeal insufficiency, which are distinctive features of the DiGeorge syndrome. It is important to emphasize that some patients with the 22q11.2 microdeletion displayed macrothrombocytopenia and bleeding tendency only, without the clinical features of the DiGeorge syndrome.22

A 4-year-old boy was described with a homozygous deletion comprising not only GP1BB but also the septin 5 gene (SEPT5), located 50 to GP1BB23; the patient presented with BSS, cortical dysplasia (polymicrogyria), developmental delay, and a platelet secretion defect, likely attributable to septin 5 deficiency, which surrounds platelet granules and may play a role in platelet secretion.24,25 Defects of GP9. Nonsense and missense mutations in GP9 have been reported.6,13 Of these the p.Asn45Ser mutation is particularly common in populations of Northern European origin.13 Two GP9 variants, c.230T> A (p.Leu77Gln) and c.255C> A (p.Asn85Lys) have been described in a patients with BSS.26

Platelet-Type von Willebrand Disease VWD is a disorder of primary hemostasis that is due to complete or partial defects of VWF, an adhesive protein that binds to platelet GPIbα and plays an essential role in platelet adhesion and aggregation under high shear forces (see below). Platelet-type VWD (PT-VWD, see also Chapter 46) is not due to a defect in VWF, but to a gain-of-function phenotype of

TABLE 48.2 Essential Characteristics of the Principal Defects of Inherited Disorders of Platelet Function Platelet Size/ Morphology

Platelet Functional Abnormality

Associated Clinical Phenotypes

Decreased

Platelet size heterogeneity, large and giant platelets

Moderate mucocutaneous bleeding

Normal or decreased

Glanzmann thrombasthenia

Moderate to severe mucocutaneous bleeding

Normal (macrothrombocytopenia in a few reports)

Platelet size heterogeneity, large platelets Normal

Absent agglutination with ristocetin, but normal aggregation with all other agonists. Abnormalities of adhesion, shearinduced platelet aggregation, responses to thrombin, platelet procoagulant activity Increased affinity of GPIbα for VWF

α2β1 collagen receptor defect GPVI collagen receptor defect P2Y12 ADP receptor defect

Mild mucocutaneous bleeding Mild mucocutaneous bleeding Mild to moderate mucocutaneous bleeding

Normal

Normal

Normal

Normal

Normal

Normal

TXA2 receptor defect

Mild mucocutaneous bleeding

Normal

Normal

Nonsyndromic δstorage pool deficiency Hermansky-Pudlak syndrome

Mild to moderate mucocutaneous bleeding Mild to moderate mucocutaneous bleeding

Normal or decreased

Normal. Deficiency of δ-granules on EM Normal. Deficiency of δ-granules on EM

Chediak-Higashi syndrome

Mild to moderate mucocutaneous bleeding

Disorder

Bleeding Syndrome

Platelet Count

Bernard-Soulier syndrome

Moderate to severe mucocutaneous bleeding

Platelet-type VWD

Normal

Normal

Normal. Deficiency of δ-granules on EM; large peroxidasepositive granules in neutrophils

Absent aggregation with all agonists except ristocetin; defective clot retraction Decreased response to collagen Decreased response to collagen Small and rapidly reversible aggregation induced by ADP; impaired aggregation and secretion induced by other agonists Absent response to TXA2; impaired aggregation and secretion induced by other agonists Impaired aggregation and secretion induced by several agonists Impaired aggregation and secretion induced by several agonists

Impaired aggregation and secretion induced by several agonists

Inheritance

Gene Affected

DiGeorge velocardiofacial syndrome (associated with deletion of GPIbβ gene)

Autosomal recessive; rarely autosomal dominant

GPIBB GPIBB GP9

None

Autosomal dominant

GPIBA

None

Autosomal recessive

ITGA2B ITGB3

None

?

?

None

Autosomal recessive

GP6

None

Autosomal recessive; rarely autosomal dominant

P2Y12

None

Autosomal dominant

TXA2R

None

Autosomal recessive or dominant Autosomal recessive

?

Oculocutaneous albinism; ceroid-lipofuscin lysosomal storage disease; pulmonary fibrosis; granulomatous colitis

Oculocutaneous albinism; eczema; recurrent infections; lymphohistiocytosis

Autosomal recessive

HPS-1, AP3B1 (HSP-2), HPS-3, HPS-4, HPS5, HPS-6, HPS-7, BLOC1S3 (HPS-8), Pallidin (HPS-9) AP3D1 LYST

Wiskott-Aldrich syndrome

Mild to severe mucocutaneous bleeding

Decreased

Small platelets; deficiency of δgranules on EM

Impaired aggregation and secretion induced by several agonists

Familial platelet disorder with predisposition to AML (FPD/AML) Gray platelet syndrome

Mild to severe mucocutaneous bleeding

Decreased

Impaired aggregation and secretion induced by several agonists

Mild to moderate mucocutaneous bleeding

Decreased

Quebec platelet disorder

Moderate to severe musculoskeletal bleeding

Normal or decreased

Normal size platelets; deficiency of δ-granules, or of α- and δ-granules Platelet size heterogeneity, large platelets; gray platelets on smear; empty α-granules on EM Normal

Defects of signal transduction

Mild mucocutaneous bleeding

Normal or decreased

Normal

Scott syndrome

Moderate to severe musculoskeletal bleeding

Normal

Normal

Primary secretion defects

Mild to moderate mucocutaneous bleeding

Normal

Normal

Eczema; infections; immunodeficiency; high risk of autoimmune diseases and lymphoreticular malignancy Increased predisposition to develop acute myeloid leukemia

X-linked

WAS

Autosomal dominant

RUNX1

Heterogeneity of response to agonists: responses to ADP, thrombin or PAR-1 may be impaired

Myelofibrosis, idiopathic pulmonary fibrosis

Autosomal recessive or dominant

NBEAL2

Impaired platelet aggregation induced by epinephrine Impaired aggregation and secretion induced by several agonists

None

Autosomal dominant

? (see text)

None

Autosomal recessive or dominant

None

Autosomal recessive

PLA2G4A; Ptgs1 XLSα; PLC-β2; RASGRP2 FERMT3 … (see text) TMEM16F

None

?

Low expression of platelet procoagulant activity and microparticle release Impaired aggregation and secretion induced by several agonists

? (some of these patients may have heterozygous P2Y12 deficiency)

Abbreviations: EM, electron microscopy; GP, glycoprotein; PAR-1, protease-activated receptor 1; TXA2, thromboxane A2; VWD, von Willebrand disease; von Willebrand factor, VWF. Please refer to the text for the abbreviations of the listed genes.

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Abnormalities of Integrin αIIbβ3 Glanzmann Thrombasthenia

P P

P

(A)

P

P P P

P P

P P

P

(B) Fig. 48.2 (A) A peripheral blood smear from a patient with Bernard-Soulier syndrome, showing normally granulated platelets (P) of heterogeneous size. The giant platelet in the center of the figure is the size of a normal red blood cell. (B) A peripheral blood smear from a patient with gray platelet syndrome, showing platelets (P) of heterogeneous (but generally large) size and “gray” appearance, which is caused by the rarity of α-granules. (Picture provided by Alan D. Michelson, M.D., Boston Children’s Hospital and Harvard Medical School, Boston, USA.)

platelet GPIbα, which has an increased avidity for VWF, leading to the binding of the largest VWF multimers to resting platelets and their clearance from the circulation.1,6 Because the high molecular weight VWF multimers are the most hemostatically active, their loss is associated with bleeding risk. Platelets tend to be slightly increased in size and reduced in number, and display increased sensitivity to the agglutinating agent ristocetin. The clinical condition therefore resembles type 2B VWD, which is caused by a gain-of-function abnormality of the VWF molecule (see below). PT-VWD is an autosomal dominant disease, which is associated with amino acid substitutions occurring within the disulfide-bonded double loop region of GPIbα.27 The described mutations have been proposed to stabilize the loop conformation, leading to increased affinity for VWF. A similar phenotype is caused by a 27 base pair deletion in the macroglycopeptide-coding region of GPIBA; the mutation is thought to restrict the mobility of the extracellular domain, resulting in a gain-of-function.28 A patient with combined PT-VWD and type 2B VWD, caused by mutations in both GPIBA gene and VWF gene, has been described.29

Glanzmann thrombasthenia (GT) is an autosomal recessive disease that is caused by lack of expression or qualitative defects in one of the two GP forming the integrin αIIbβ3, which in activated platelets binds adhesive glycoproteins (e.g., fibrinogen, VWF, fibronectin) that bridge adjacent platelets, securing platelet aggregation. GT patients display a phenotype that is similar to that of BSS patients, albeit perhaps less severe, characterized by mucocutaneous bleeding and bleeding after trauma or surgery.1,30 Postpartum bleeding complications may be frequent and severe.31 Heterozygotes do not have a bleeding diathesis.1 The diagnostic hallmark of the disease is the lack or severe impairment of platelet aggregation induced by all agonists, but no impairment in ristocetin-induced platelet agglutination (see Chapter 34). Severe forms (previously called GT type-I) are characterized by lack of fibrinogen in the platelet α-granules (because platelet fibrinogen is acquired from plasma through an αIIbβ3-dependent uptake), while patients whose platelets have some residual, albeit very low, αIIbβ3 have normal fibrinogen content.32,33 Clot retraction is defective. GT platelets bind normally to the subendothelium, but fail to spread and to build up a thrombus. Some reports demonstrated impaired ability of GT platelets to generate thrombin and procoagulant microparticles.1 Diagnosis of GT is based on the presence of typical abnormalities of platelet aggregation (see above) and on the demonstration that αIIbβ3 is absent or severely reduced on the platelet membrane, using flow cytometry (Chapter 35) or Western blotting. Molecular Defects. The coding genes for the two glycoproteins, ITGA2B and ITGB3, colocalize to 17q21–23. ITGA2B spans 17 kb and is composed of 30 exons, while ITGB3 spans 46 kb and has 15 exons. The expression of ITGA2B is restricted to the megakaryocyte lineage, while the expression of ITGB3 also occurs in many other cell types as a component of the vitronectin receptor (αvβ3). Consanguinity is commonly associated with homozygous mutations. Mutations usually inhibit GP synthesis in the megakaryocytes or inhibit the transport of precociously formed complexes from the endoplasmic reticulum to the Golgi apparatus and/or their transport to the cell surface.34 Mutations in ITGA2B. Splice site mutations and nonsense mutations of ITGA2B, involving frameshifts and giving rise to truncated proteins are usually associated with severe forms of GT (type-I GT, according to early nomenclature).6 Missense mutations may give rise to less severe deficiency of the complex or to dysfunctional proteins.6 Mutations in ITGB3. Deletions, splice mutations and inversions of ITGB3, involving frameshifts and giving rise to truncated proteins are usually associated with severe forms of GT.6 The vitronectin receptor (αvβ3) shares the β3 subunit with αIIbβ3 and is therefore absent in GT patients with defects in β3, whereas its expression in patients with defects in αIIb may be increased. Despite the fact the vitronectin receptor is found in many cell types in addition to platelets and megakaryocytes, the phenotype of GT patients with β3 defects does not differ from that of other GT patients.6 Most variant forms of GT are linked to missense mutations of ITGB3, which are associated with impaired expression of the ligand-binding pocket, complex instability and defective signal transduction through β3. The first report of a GT variant described an p.Asp119Tyr substitution, which helped to identify an RGD binding site.6 Mutations in the cytoplasmic domain of β3 are linked to defective αIIbβ3 activation, confirming the important role of β3 in inside-out signaling.6

Inherited Disorders of Platelet Function

The β3 subunit is rich in disulfide bonds, and patients with cysteine mutations express low amounts of constitutively active αIIbβ3.6 A homozygous gain-of-function p.Cys560Arg mutation was associated with binding of fibrinogen to resting platelets.35 The patient had a bleeding diathesis, presumably because αIIbβ3 complexes are monovalently occupied by fibrinogen. Mutations in ITGB3 have been described in patients with inherited macrothrombocytopenia, without a full GT phenotype.36,37,38,39 Two large demographic studies have been published, highlighting the role of specific amino acids in structurefunction correlations of αIIbβ3.35,40

Abnormalities of Integrin α2β1 Two patients with mild bleeding disorders associated with deficient expression of the platelet receptor for collagen GPIa-IIa (integrin α2β1) and selective impairment of platelet responses to collagen have been described.41,42 The platelet defect spontaneously recovered after menopause in one patient,42 suggesting that α2β1 expression is under hormonal control. Definitive proof for a specific pathology is lacking.

Abnormalities of GPVI Selective defects of collagen-induced platelet aggregation have been described in some patients with mild to moderate bleeding disorders, characterized by deficiency of platelet GPVI, a member of the immunoglobulin superfamily of receptors, which mediates platelet activation by collagen (Chapter 11).1 However, in only some patients was the defect linked to mutations in the GP6 gene. The first patient was a 10-year-old girl with easy bruising since infancy, prolonged bleeding time despite a normal platelet count, no antiplatelet antibodies, and absent collageninduced platelet activation.43 GPVI quantification by flow cytometry evidenced an incomplete deficiency, while immunoblotting showed an abnormal migration of residual GPVI, and no FcRγ defect. GP6 DNA sequencing revealed a p. Arg38Cys mutation in one allele and an insertion of five nucleotides in exon 4 of the other allele, leading to a premature nonsense codon and absence of the corresponding mRNA. Introduction of the p.Arg38Cys mutation into recombinant GPVI-Fc resulted in abnormal protein migration and a loss of collagen binding.43 The second patient had a lifelong history of bleeding problems and structurally normal platelets, which did not respond to collagen, convulxin or the collagen-related peptide (CRP).44 Flow cytometry demonstrated an absent expression of GPVI whereas immunoblot analysis showed dramatically reduced levels of GPVI. The patient was found to be compound heterozygous for an out-of-frame 16-bp deletion and a missense mutation p.Ser175Asn in a highly conserved residue of the 2nd Ig-like GPVI domain. His parents are heterozygous carriers, without clinical bleeding problems. The mother carries the p. Ser175Asn mutation and displays a mild functional platelet defect.44 Finally, four unrelated patients from nonconsanguineous families who presented with mucocutaneous bleeding displayed an adenine insertion in exon 6 (c.711_712insA) of GP6, changing the reading frame and generating a premature “stop codon” in site 242 of the protein. Flow cytometry and immunofluorescence-confocal microscopy studies failed to detect GPVI on their platelets, which did not aggregate or secrete 14C-5-HT when stimulated by collagen, convulxin, or CRP. Heterozygous relatives had normal clinical and laboratory phenotypes.45

883

Under static conditions, GPVI-deficient murine platelets do not adhere to collagen, while under high shear stress conditions, GPVI likely acts in concert with the collagen-binding integrin α2β1 to allow for firm platelet adhesion. On the other hand, stable adhesion and spreading was severely hampered in GPVI-deficient platelets, illustrating the role of GPVI in postadhesion events leading to platelet activation and thrombus formation on collagen fibrils. Tail clipping experiments in GPVI-deficient mice, unexpectedly revealed only slightly prolonged bleeding times.

ABNORMALITIES OF THE PLATELET RECEPTORS FOR SOLUBLE AGONISTS Abnormalities of the Platelet P2 Purinergic Receptors Defects of the Platelet ADP Receptor P2Y12 P2Y12 deficiency. Inherited P2Y12 deficiency is an autosomal recessive disorder. The first patient with severe P2Y12 deficiency was described in 1992.46 He had a lifelong history of excessive bleeding, prolonged bleeding time (15–20 min), reversible aggregation in response to weak agonists and impaired aggregation in response to low concentrations of collagen or thrombin. However, the most typical feature was that ADP, even at very high concentrations (>10 μM), did not induce full and irreversible platelet aggregation. Other abnormalities of platelet function were: (i) no inhibition by ADP of prostaglandin (PG) E1-stimulated platelet adenylyl cyclase, but normal inhibition by epinephrine; (ii) normal shape change and borderline-normal mobilization of cytoplasmic Ca2+ induced by ADP; (iii) presence of approximately 30% of the normal number of binding sites for [33P]2MeSADP on fresh platelets47 or [3H]ADP on formalin-fixed platelets, which are associated with the ADP receptor P2Y1.48 Additional patients with severe P2Y12 deficiency, belonging to four kindreds, were subsequently described: one French man,48 two Italian sisters,49 a Japanese woman,50 and a British woman of Asian descent.51 Heterozygous P2Y12 deficiency is characterized by reversible platelet aggregation induced by ADP concentrations 10 μM and impaired platelet secretion induced by several agonists.49 Because the secretion defect in this patient’s platelets was not associated with impaired production of thromboxane (TX) A2 or low concentrations of platelet granule contents, it is very similar to that described in patients with an ill-defined and probably heterogeneous group of inherited defects of platelet secretion, sometimes referred to with the general term “primary secretion defect” (see below).49,51 The diagnosis of a P2Y12 defect should be suspected when ADP, even at relatively high concentrations (10 μM), is unable to induce full, irreversible platelet aggregation, while inducing normal shape change. Tests that evaluate the degree of inhibition of adenylyl cyclase by ADP, by measuring either the platelet levels of cyclic adenosine monophosphate (cAMP) or the phosphorylation of vasodilator-stimulated phosphoprotein (VASP)52 after the exposure of platelets to prostaglandin (PG) E1, should be used to confirm the diagnosis. The P2Y12 genes of the first patient and of the British patient of Asian descent displayed homozygous base pair deletions in the open reading frame, resulting in frameshifts and premature truncation of the protein.53 Molecular analysis of the P2Y12 gene of the two Italian sisters revealed an identical single bp deletion (c.378delC) occurring just beyond the coding sequence for the third transmembrane domain in P2Y12, resulting in a frame shift (p.Thr126fs) and premature truncation of the protein.53 As only alleles encoding the mutated DNA

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sequence were found by PCR analysis, the patients were considered homozygous for the 378delC mutation. However, a subsequent study revealed that they suffer from P2Y12 deficiency owing to haploinsufficiency and to the c.378delC mutation in their remaining allele.54 The Japanese patient is homozygous for a single nucleotide substitution in the transduction initiation codon (ATG to AGG).50 The molecular defect that is responsible for the severe P2Y12 deficiency of the French patient48 is less well defined.55 One mutant allele contains a deletion of 2 bp within the coding region, at amino acid 240, resulting in a frameshift and early truncation of the protein. Surprisingly, the other allele did not display any mutation. The findings that the patient’s platelets contained P2Y12 transcripts derived from the mutant allele only and that his daughter, who had a heterozygous phenotype, inherited the mutant allele from her father and a normal allele from her mother, suggest that this patient has an additional, as yet unknown, mutation that silences his normal allele. Inherited dysfunction of P2Y12. One patient has been described with a congenital bleeding disorder associated with abnormal P2Y12-mediated platelet responses to ADP, whose platelets display normal numbers of dysfunctional P2Y12 receptors.56 Platelets from this patient displayed severely impaired, albeit not completely absent P2Y12-dependent platelet function. Analysis of the patient’s P2Y12 gene revealed, in one allele, a G to A transition changing the codon for Arg256 in TM6 to Gln and, in the other, a C to T transition changing the codon for Arg265 in EL3 to Trp. Neither mutation interfered with the surface expression of the P2Y12 receptor but both altered receptor function, since ADP inhibited the forskolin-induced increase of cAMP markedly less in cells transfected with either mutant P2Y12 than in wild type cells.56 The important role of Arg265 in P2Y12 receptor function was highlighted in a later report of a family with normal expression of dysfunctional P2Y12, associated with heterozygous dominant negative p.Arg265Pro variant.57 A heterozygous, likely dominant negative point mutation in the same region of the molecule, which changed codon 258 coding for proline (CCT) to threonine (ACT) (p. Pro258Thr), was described in a patient with a mild bleeding disorder and severely impaired ADP-induced platelet aggregation.58 Since the proline to threonine substitution alters the protein hydrophobicity, size and rotational mobility, it is likely to affect the function of P2Y12. Finally, a heterozygous mutation, predicting a lysine to glutamate (p.Lys174Glu) substitution in P2Y12, was identified in one patient with mild type 1 VWD.59 Platelets from this patient showed reduced and reversible aggregation in response to ADP, up to 10 μM. The reduced response was associated with an approximate 50% reduction in binding of [3H]2MeS-ADP. Considering that Lys174 is situated in the second extracellular loop of P2Y12, adjacent to Cys175, which may be important for the expression of the ADP binding site receptor, and that a hemagglutinin-tagged p.Lys174Glu P2Y12 variant showed surface expression in Chinese hamster ovary cells, it is likely that the p.Lys174Glu mutation is responsible for disruption of the ADP binding site of the receptor. One patient displayed a heterozygous mutation (p.Pro341Ala) in the PDZ binding sequence of P2Y12, associated with reduced expression and compromised recycling of P2Y12.60 Mutations of Arg 122 (p.Arg122Cys and p.Arg122His) in two unrelated patients were associated with reduced P2Y12 function.61,62 Dysfunctional P2Y12, normal expression of the receptor but decreased affinity for its ligand, associated with homozygous p.His187Gln substitution was described in two brothers.63,64

Abnormalities of Other Platelet P2 Receptors A description of a patient with a history of bleeding following surgery and occasional weak ADP-induced platelet aggregation

was published in abstract form by Oury et al. in 1999.65 The defect was associated with normal P2Y1 encoding regions in the patient’s DNA, but reduced platelet levels of P2Y1 mRNA (75% of normal), suggestive of deficient P2Y1 gene transcription. However, no further details of this family have been published in a full article since this 1999 abstract. Oury et al. also described a patient with a severe bleeding diathesis associated with a naturally occurring dominant negative P2X1 mutant, lacking one leucine within a stretch of four leucine residues in the second trans-membrane domain (amino acids 351–354).66 However, the patient also displayed a severe defect of ADP-induced platelet aggregation that cannot be explained by the defect of P2X1, and could by itself account for the bleeding diathesis of the patient. Therefore, the relationship between genotype and phenotype in the patient described by Oury et al. remains unclear.

Defects of the Platelet Thromboxane A2 Receptor (TP) In 1981, three reports of impaired platelet responses to TXA2 in patients with bleeding disorders were published.67–69 The platelets from these patients could synthesize TXA2 from exogenous arachidonate, but were unable to undergo normal TXA2dependent aggregation and secretion in response to a variety of agonists. In one patient, the stable TXA2 mimetic U46619 was tested and found to be unable to elicit normal platelet responses,69 providing convincing evidence that his platelets had a defect at the receptor level. In 1993, a similar patient with a mild bleeding disorder was described, whose platelets did not undergo shape change, aggregation and secretion in response to the synthetic TXA2 mimetic STA2.70 Binding studies of radiolabeled TXA2 agonists and antagonists revealed that the patient platelets had normal number of TXA2 binding sites and normal equilibrium dissociation rate constants. Despite the normal number of TXA2 receptors (TP), TXA2-induced inositol 1,4,5-triphosphate formation, Ca2+ mobilization and guanosine-50 -triphosphate (GTP)ase activity were abnormal, suggesting that the abnormality of these platelets was impaired coupling between TP, G protein and phospholipase C (PLC). The platelet aggregation and secretion responses to several agonists were impaired. A similar patient, who was also affected by polycythemia vera, had previously been described by Ushikubi et al.71 These two last patients were subsequently found to have an Arg60 to Leu mutation in the first cytoplasmic loop of the TP,72 affecting both isoforms of the receptor.73,74 The mutant receptor expressed in Chinese hamster ovary cells showed decreased agonist-induced second messenger formation despite its normal ligand binding affinities. The mutation was found exclusively in the affected members of the two unrelated families and was inherited as an autosomal dominant trait. Although the heterozygous patients did not differ from the homozygous patients in terms of aggregation and secretion responses of platelets to TXA2, subsequent studies showed that in heterozygous patients, the mutant TP suppresses the wild-type receptormediated platelet aggregation and secretion by a mechanism independent of inhibition of PLC activation.75,76 A heterozygous p.Asp304Asn substitution in TP was described in a 14-year old boy experiencing severe nose bleeding. Platelet aggregation and ATP secretion induced by U46619 were reduced. The TP antagonist [3H]-SQ29548 showed an approximate 50% decrease in binding to the patient’s platelets, indicating that the mutation is associated with reduced ligand binding.77 A TP variant predicting a p.Asp42Ser substitution, associated with bleeding diathesis was described, which suggests a vital role for the highly conserved Asp42 residue for receptor structure and function.78 A heterozygous p.Trp29Cys

Inherited Disorders of Platelet Function

transition was associated with impaired TP receptor function, due to decreased surface receptor expression and ligand binding.79

Defects of the α2-Adrenergic Receptors In two families whose members had impaired platelet aggregation and secretion in response to epinephrine but normal responses to other agonists, the defect was a decreased number of platelet α2-adrenergic receptors.80,81 Surprisingly, in one family inhibition of platelet adenylyl cyclase by epinephrine was normal.80 Considering that impaired platelet responses to epinephrine are observed in a relatively high percentage of otherwise normal subjects (Chapter 34), the relationship between the described defects and bleeding manifestations still needs to be clarified.

DEFECTS OF PLATELET GRANULES Defects of platelet granules comprise a heterogeneous group of syndromic and nonsyndromic disorders, including deficiencies of the δ- and/or α-granules, or their constituents (δ-, α,δ-, and αstorage pool deficiency) and other, less common defects of the α-granules (see also Chapter 19).

Defects of the δ-Granules [Nonsyndromic] δ-Storage Pool Deficiency The term δ-storage pool deficiency, or δ-storage pool disease (δSPD), defines an inherited abnormality of platelets characterized by deficiency of dense granules in megakaryocytes and platelets. It is a relatively common disorder, which affects between 10% and 18% of patients with inherited abnormalities of platelet function.1,82 The inheritance is autosomal recessive in some families and autosomal dominant in others. δ-SPD is characterized by a bleeding diathesis of variable degree, mildly to moderately prolonged bleeding time, abnormal platelet secretion induced by several platelet agonists, impaired platelet aggregation and decreased platelet content of δ(dense)-granules.1,6 Although it is generally held that the platelet count in δ-SPD patients is normal, a recent report and review of the literature documented that mild thrombocytopenia may be observed in 20%–40% of patients.83 Studies of δ-granules with the uranaffin reaction (staining by uranyl ions of both the δ-granule membrane and core), with the fluorescence probe mepacrine (which concentrates in the δgranules), or by electron microscopy, revealed that platelets from patients with nonsyndromic δ-SPD have slightly reduced number of uranaffin-positive and mepacrine-positive granules, but a shift in uranaffin-positive distribution towards those lacking a dense core (“empty granules”), suggesting a more qualitative than quantitative type of δ-granule defect.1 In accordance with these findings, platelets from patients with nonsyndromic platelet δ-SPD had normal amounts of the δ-granule membrane protein granulophysin.1 It has been reported that δSPD is highly heterogeneous, with evidence of an underlying cytoskeleton defect.84 δ-SPD platelets have decreased levels of δ-granule constituents: ATP, ADP, serotonin, calcium and inorganic polyphosphate.1 ADP and ATP are normally contained in platelets in a metabolic pool, which represents about 1/3 of the total content, and in the δ-granules, which represent the storage pool. ADP is normally present in greater amounts in the storage pool, while the concentration of ATP is higher in the metabolic pool. The ratio of total ATP:ADP in normal platelets is <2.5:1, while that of the metabolic pool is about 10:1. Due to the deficiency

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of δ-granules in δ-SPD, the ratio of total ATP:ADP in platelets typically rises to >2.5–3:1.85 Platelets are the main storage site for serotonin in the human body. Normal platelets avidly take up serotonin from the bloodstream and store it in the δ-granules, where it is protected from the action of mitochondrial monoamino oxidases. When radioactive serotonin is incubated with normal platelets in vitro, >90% of it is rapidly incorporated into δ-granules. In contrast, when radioactive serotonin is incubated with δ-SPD platelets, the initial rate of uptake (through the platelet plasma membrane) is normal, but the saturation level is decreased, due to the catabolism of serotonin, resulting in the loss of the radioactive label from the platelets.86 In δ-SPD citrated platelet-rich plasma, primary aggregation induced by ADP or epinephrine and the agglutination response to ristocetin are normal, but the second wave of aggregation and the aggregation in response to collagen are generally absent or greatly reduced.87,88 The production of arachidonate metabolites can be defective after stimulation with epinephrine or collagen, but normal with arachidonate;89 however, the aggregation induced by sodium arachidonate or prostaglandin endoperoxides may be normal or decreased,88,89 depending on the severity of ADP deficiency in platelet granules.86 Normal responses to ADP or epinephrine have been observed in some patients,90 indicating that there is a large variability in platelet aggregation in patients with δ-SPD, which has been well documented in a large study of 106 patients with δ-SPD (inherited in 51 and acquired in 55), which showed that about 25% of the patients had normal aggregation responses to ADP, epinephrine and collagen, while only 33% had aggregation tracings “typical” for a platelet secretion defect.82 In agreement with these findings, it was later shown that, among 46 patients with prolonged bleeding times, normal VWF levels and normal platelet aggregation 17 (35%) had δ-SPD.91 High concentrations of thrombin induced a normal extent of aggregation of δ-SPD platelets, but the aggregates deaggregated more easily than normal,92 This defect was corrected by the addition of exogenous ADP immediately after thrombin stimulation, suggesting that released ADP plays a role in the stabilization of platelet aggregates.92 Other platelet function abnormalities described in δ-SPD patients include abnormal secretion of acid hydrolases,93 which was corrected by exogenous ADP,94 and defective aggregation at high shear.95,96 The in vitro interaction of δ-SPD platelets with the subendothelium was impaired in an experiment of perfusion of citrated blood through a chamber containing everted segments of rabbit aorta.97 Subsequent experiments performed at different flow conditions (shear rates varying from 650 to 3300/s) with nonanticoagulated blood showed that thrombus formation was decreased in δ-SPD patients in proportion to the magnitude of the granule defect.98 Weiss and Lages showed that the prothrombinase activity induced by collagen, thrombin, or collagen plus thrombin was impaired in δ-SPD platelets and was corrected by the addition of ADP.99 However, a previous study failed to demonstrate an abnormal procoagulant activity of δ-SPD platelets under slightly different experimental conditions.100 Abnormalities in coagulation and fibrinolysis in δ-SPD may be secondary to the deficiency in inorganic polyphosphate and contribute to the bleeding diathesis.101 Polyphosphate has procoagulant effects at different levels such as: activation of the contact pathway and factor V, interruption of tissue factor pathway inhibitor (TFPI), improving thrombin-activatable fibrinolysis inhibitor (TAFI), and modulating fibrin structure and lysis.102,103 Patients with δ-SPD have a mild to moderate bleeding diathesis, characterized mainly by mucocutaneous bleeding, such as epistaxis, menorrhagia and easy bruising. Patients with

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the most severe forms may also experience postsurgical hemorrhagic complications, especially after tooth extraction and tonsillectomy. Only one case with intracranial bleeding has been reported. Lumiaggregometry, which measures platelet aggregation and secretion simultaneously (see Chapter 34), is a more accurate technique than platelet aggregometry for diagnosing patients with δ-SPD and, more generally, with platelet secretion defects (see below). The diagnosis of δ-SPD is essentially based on the finding of defective platelet secretion induced by several agonists, decreased total platelet content of ADP and ATP, increase in the ATP/ADP ratio to >2.5–3.0, and normal serum concentration of the stable TXA2 metabolite, TXB2. Electron microscopy (Chapter 3)104 and methods involving the identification of mepacrine-loaded platelets by flow cytometry (Chapter 35)105 may also be useful in the diagnosis of this disorder. Molecular Defects. Molecular defects responsible for [nonsyndromic] δ-SPD have not been identified yet. Animal models identified RAB38,106 RAB27B,107 and SLC35D3108 as candidate genes for δ-SPD in humans. However, none of five patients with δ-SPD displayed mutations of the RAB27B gene,109 and none of 13 patients displayed mutations in the coding region of the SLC35D3 gene (Cattaneo M, unpublished observations).

Hermansky-Pudlak Syndrome Hermansky-Pudlak syndrome (HPS, see also Chapter 19) is a rare, autosomal recessive disease of subcellular organelles of many tissues, involving abnormalities of melanosomes, platelet δ-granules and lysosomes.1 It is characterized by tyrosinasepositive oculocutaneous albinism, a bleeding diathesis due to δ-SPD and ceroid-lipofuscin lysosomal storage disease.110,111 The oculocutaneous albinism manifests as congenital nystagmus, iris transillumination, decreased visual acuity and various degrees of skin and hair hypopigmentation. Ceroid lipofuscin is a lipid-protein complex accumulating in lysosomal organelles, which is believed to be responsible for the development of progressive pulmonary fibrosis112 and granulomatous colitis113 in affected patients. Ten HPS subtypes are known.111,114–116 HPS-1 represents the most severe form of the disease, in which pulmonary fibrosis is particularly frequent. In albino patients, the absence of visible δ-granules in the platelet cytoplasm under the electron microscope (Chapter 3) and/or the deficiency of platelet adenine nucleotides is pathognomonic for the disease. The bleeding diathesis of HPS patients, similarly to that of other types of δ-SPD, manifests with easy bruising, epistaxes, gum bleeding, menorrhagia and postsurgical bleeding. Just as the degree of hypopigmentation is not uniform in all patients, the severity of the bleeding diathesis varies substantially. In one report, major bleeding, some of which was life-threatening, occurred in 40% of the studied patients.117 HPS is rare in the general population, but occurs with relatively high frequency in certain isolated groups, such as in the northwestern region of Puerto Rico, where its prevalence is 1 in 1800,118 and in an isolated village in the Swiss Alps.119 The number of δ-granules in platelets from seven HPS patients was markedly diminished when studied with electron microscopy, loading of the fluorescent probe mepacrine or the uranaffin reaction, indicating that the basic defect of HPS, at variance with that of isolated platelet δ-SPD, is a specific abnormality in organelle development which prevents the formation of an intact granule structure. In accordance with these findings, the platelet content of the δ-granule membrane protein granulophysin was shown to be very low in HPS patients.120 The bleeding time is prolonged in most, but not all HPS patients. A variable degree of abnormalities of tests of platelet

function can be observed in HPS patients, as in patients with isolated δ-SPD. The release of α-granule proteins induced by thrombin was impaired in one patient, and was normalized by the simultaneous stimulation of platelets with ADP.121 These findings are consistent with the demonstration that released ADP directly potentiates platelet secretion induced by U46619 or thrombin by interacting with the platelet P2Y12 receptor.49,122 Molecular Defects. The molecular basis for HPS has begun to be unraveled in the last decade (see also Chapter 19). To date, HPS has been associated with 10 human genes: HPS-1 (HPS subtype: HSP-1), AP3B1 (HSP-2), HPS-3 (HPS-3), HPS-4 (HPS-4), HPS-5 (HPS-5), HPS-6 (HPS-6), Dystrobrevin binding protein 1 (HPS-7), BLOC1S3 (HPS-8), Pallidin (HPS-9), AP3D1 (HPS-10).111,114–116 All known HPS proteins are components of one of four protein complexes: Biogenesis of Lysosome-related Organelles Complex (BLOC)-1 (HPS7, HPS8, HPS9); BLOC-2 (HPS3, HPS5, HPS6); BLOC-3 (HPS1, HPS4), and Adaptor Protein Complex-3 (HPS2, HPS10).115,116

Chediak-Higashi Syndrome Chediak-Higashi syndrome (CHS, see also Chapter 19) is a rare autosomal recessive disorder characterized by variable degrees of oculocutaneous albinism, very large peroxidase-positive cytoplasmic granules in a variety of hematopoietic (neutrophils) and nonhematopoietic cells, easy bruisability due to δSPD, and recurrent infections, associated with neutropenia, impaired chemotaxis and bactericidal activity, and abnormal natural killer (NK) cell function.111 About 85% of the patients may undergo an accelerated phase (lymphohistiocytosis), characterized by uncontrolled lymphoid infiltration of multiple organs. The syndrome is lethal, leading to death usually in the first decade of life. There is progressive neurological dysfunction in patients surviving to adulthood.111 The bleeding diathesis and the abnormalities of platelet aggregation and secretion are similar to those of other forms of δ-SPD.123 The levels of the δ-granule membrane protein granulophysin are very low in CHS platelets,120 as expected in platelets lacking δ-granules.124 The pathognomonic feature of CHS is peroxidase-positive granules that can be seen in polymorphonuclear leukocytes, as well as in megakaryocytes, neurons and other cells. Molecular Defects. CHS results from mutations in the lysosomal traffic regulator (LYST) gene,111,125 which encodes a large cytoplasmic protein, with distinct structural domains including BEACH and HEAT, involved in trafficking among lysosomes and δ-granules.126 Like HPS, CHS may prove to be a genetically heterogeneous disorder, with mutations at different loci resulting in a similar phenotype.

SFLN14-Related Disease Dominant mutations in schlafen 14 (SLFN14) were identified in 12 patients from 3 unrelated families, with inherited macrothrombocytopenia and decreased platelet ATP secretion.127 Electron microscopy revealed a reduced number of dense granules in affected patients platelets, correlating with decreased ATP secretion.127

Familial Platelet Disorder With Predisposition to AML (FPD/AML) Studies of a family that includes several members with autosomal dominant δ-SPD showed an association between δ-SPD

Inherited Disorders of Platelet Function

and the development of acute myelogenous leukemia;128 the hypothesis was therefore raised that a gene coding for a protein important for the formation of dense granules is located adjacent to a gene which, when abnormal, may predispose to the development of leukemia.129 This was likely the first description of a disorder that is now known as Familial Platelet Disorder with predisposition to AML (FPD/AML), which is due to variants the hematopoietic transcription factor RUNX1 (runtrelated transcription factor 1), resulting in inappropriate expression of downstream genes, including PF4130,131 (see also Chapter 46). Patients usually display moderate thrombocytopenia, but the platelet count may be normal.132 Platelet dysfunction is characterized by impaired platelet aggregation and secretion, which is due to abnormal secretory mechanism(s)133 associated in some, but not in all patients134 with deficiency of delta granules or alpha and delta granules.135,136 Decreased platelet pleckstrin phosphorylation and protein kinase C-θ (PKC-θ, gene PRKCQ) was described in some patients.137,138 PRKCQ is a direct transcriptional target of RUNX1.139 Myosin light chain phosphorylation was also decreased in this patient and platelet mRNA profiling showed that the gene encoding myosin regulatory light chain polypeptide was downregulated.140 Also downregulated was the platelet 12-lypoxygenase gene (ALOX12).141 RUNX1 haploinsufficiency usually leads to quantitative and qualitative platelet abnormalities, while a more complete gene deletion predisposes to leukemia.142

Thrombocytopenia with Absent Radii (TAR) Syndrome TAR syndrome is a developmental disorder characterized by bilateral absence of the radii with the presence of both thumbs and thrombocytopenia (<50 platelets  109/L), which is generally transient (see also Chapter 46).143 The thrombocytopenia may be present at birth or may develop within the first few weeks to months of life. In general, thrombocytopenic episodes decrease with age. The few studies on colony formation suggest that the thrombocytopenia could be due to a decreased response to thrombopoietin that affects both proliferation and differentiation. Cow’s milk allergy is common and can be associated with exacerbation of thrombocytopenia. Other anomalies of the skeleton (upper and lower limbs, ribs, and vertebrae), heart, and genitourinary system (renal anomalies and agenesis of uterus, cervix, and upper part of the vagina) can occur. Individuals with TAR syndrome display a microdeletion on chromosome 1q21 including the RBM8A gene144 and one of rare single nucleotide polymorphisms (SNPs) either at the 50 untranslated region (5’ UTR) or within the first intron of RBM8A,145,146 encoding for a protein designated Y14, a core subunit of the exon junction complex. The TAR syndrome can be either autosomal recessive or dominant. Poor response to collagen and absent secondary waves of aggregation in response to ADP or epinephrine, which are typical of defects of δ-granules, have been described in these patients.111

Wiskott-Aldrich Syndrome (WAS) WAS is an X-linked recessive disease of microthrombocytopenia, immunodeficiency, eczema recurrent infections due to immune deficiency and high risk of autoimmune diseases and lymphoreticular malignancy (see also Chapter 46).147 WAS is caused by mutations in the WAS gene, which encodes a 502 amino acid protein (the WAS protein [WASP]), which participates in innate and adaptive immunity through regulation of actin cytoskeleton-dependent cellular processes, including immune synapse formation, cell signaling, migration and cytokine release.148 Mutations causing the syndrome are distributed along the whole gene: the most common ones

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are missense mutations and small insertions; less common are nonsense and splice-site mutations, and insertions.149 Thrombocytopenia is associated with decreased platelet survival and with premature proplatelet formation in the bone marrow.150 Bleeding manifestations may be mild or severe. WAS patients have marked reduction in platelet δ-granules; more rarely, also in α-granules.111 One of the first diseases to be successfully treated by allogeneic hematopoietic stem cell transplantation, WAS is currently the subject of several phase I/II gene therapy trials for patients without HLA-compatible donors.151 The thrombopoietin receptor agonist eltrombopag has been successfully used in some patients with WAS, profound thrombocytopenia, and bleeding diathesis to increase the platelet count.152,153

Deficiency of Platelet MRP4 The multidrug resistance protein MRP4 (ABCC4), a member of the ATP-binding cassette (ABC) transporter superfamily is localized in the platelet plasma membrane and is highly concentrated in δ-granules.154 MRP4 is involved in the ATPdependent transport of a wide range of amphiphilic anions including steroid conjugates and eicosanoids, as well as cyclic nucleotides and nucleotide analogues and most likely ADP; in contrast, serotonin is not a substrate for MRP4.154 Two unrelated patients with a previously undescribed phenotype of human δ-SPD, characterized by selective deficiency of platelet adenine nucleotides and normal platelet serotonin content were identified. These patients displayed a selective defect of platelet MRP4, supporting the hypothesis that MRP4 plays a major role in platelet adenine nucleotide storage.155

Defects of the α-Granules Gray Platelet Syndrome The gray platelet syndrome (GPS, see also Chapter 46) owes its name to the gray appearance of the platelets in peripheral blood smears as a consequence of the rarity of platelet granules (Fig. 48.2B). Since its first description in 1971 by Raccuglia,156 >100 new cases have been reported in the literature.157–163 The inheritance pattern is autosomal recessive.157 Affected patients have a lifelong history of mucocutaneous bleeding, which may vary from mild to moderate in severity, prolonged bleeding time, mild thrombocytopenia, abnormally large platelets (Fig. 48.2B) and isolated reduction of the platelet α-granules content. Occasional patients may have more severe bleeding symptoms, including intracranial hemorrhage and postsurgical bleeding. Bone marrow examination reveals emperipolesis164,165 and mild to moderate myelofibrosis, which is progressive.37,166 and has been hypothetically ascribed to the action of cytokines that are present in abnormally high concentrations in the bone marrow, as a consequence of their loss from the hypogranular platelets and megakaryocytes,167 in combination with the presence of a proinflammatory phenotype of the bone marrow MKs, conferred by the deficiency of NBEAL2 (see later).168 Splenomegaly may be present156,166 and splenectomy may be followed by normalization of the platelet count, but not by an amelioration of the bleeding diathesis.166 High serum level of vitamin B12 is a consistent finding in GPS.163 Mouse models of GPS revealed that lack of α-granules is associated with abnormalities in the function of neutrophils and NK cells169 and defective arterial thrombus formation and protection from thrombo-inflammatory brain infarction170 and cancer metastasis.168 Gray platelets are severely and selectively deficient in soluble proteins contained in the α-granules, including platelet factor 4, β-thromboglobulin, VWF, thrombospondin, fibrinogen,

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fibronectin, immunoglobulins, albumin and others. The extent of the deficiency is more severe for proteins that are endocytosed from plasma, such as albumin and immunoglobulins, than for proteins that are synthesized by the megakaryocytes.171–174 In contrast to soluble proteins, and in contrast with ARC (see later), the α-granule membrane proteins are normal in GPS173,175–177 consistent with the demonstration of the presence of empty α-granules178 and the normal production of precursors of α-granules in GPS megakaryocytes.179 It is therefore conceivable that GPS platelets have defective targeting and packaging of endogenously synthesized proteins in platelet αgranules. This hypothesis is also consistent with the finding of increased plasma levels of β-thromboglobulin in GPS patients.180 The targeting defect seems to be specific for the megakaryocyte cell line, as GPS patients have normal WeibelPalade bodies, the endothelial cell storage granules equivalent to the platelet α-granules;181 however, decrease of secondary granules and secretory vesicles in polymorphonuclear neutrophils was described in some GPS patients.158,162 Circulating platelets are reduced in number, relatively large, vacuolated, and contain normal numbers of mitochondria, δ-granules, peroxisomes and lysosomes but specifically lack αgranules.182 The degree of thrombocytopenia is usually mild, but it may increase in severity with age.163 Platelet aggregation induced by ADP and adrenaline in citrated plasma are usually normal, but impaired aggregation responses induced by ADP or low concentrations of thrombin or collagen have been described in some patients.166,171,172,183 In one patient, defective aggregation induced by thrombin was associated with reduced expression of PAR-1,159 while in another patient, defective platelet response to collagen was associated to an acquired defect in GPVI expression.184 The secretion of 14C- serotonin was impaired in some patients,180 but not in others.183 Bevers et al. reported normal platelet prothrombinase activity in three patients with GPS.100 In contrast, another study showed that the collagen- plus thrombin-induced prothrombinase activity of platelets from a GPS patient was greatly impaired and was not corrected completely by the addition of exogenous factor Va.99 The results of the latter study are consistent with the demonstration that α-granule factor V bound to the surface of platelets that had been stimulated simultaneously by thrombin and collagen plays a unique role in generating prothrombinase activity.185 Genome-wide linkage analysis has mapped the GPS locus to within a 9.4 megabase region on chromosome 3p21,143,186 containing 197 coding genes, of which 69 have been completely or partially sequenced.163,186 Three independent groups identified mutations in the NBEAL2 gene as the genetic cause of GPS.187–189 GPS patients have biallelic mutations in NBEAL2;163 carriers of monoallelic mutations displayed platelet macrocytosis and significant reduction of the α-granule content, but normal platelet count.190 It has been reported that GPS can mimic autoimmune lymphoproliferative syndrome (ALPS), caused by dominant germline of somatic mutations of the FAS gene.191 Two siblings of a consanguineous family referred for suspected ALPS were found to harbor a homozygous nonsense NBEAL2 mutation (c.5299C >T p.Gln1767*): they had mild bleeding diathesis, macrothrombocytopenia, giant gray platelets, reduced CD62P expression, high vitamin B12 and soluble Fas ligand plasma levels. Yet, FAS mutations were absent.191 Three patients previosuly diagnosed GPS with NEABL2 mutations displayed similar features, including high plasma levels of sFASL.191 The NBEAL2 gene encodes a member of the family of BEACH (BEige And CHS [Chediak-Higashi Syndrome]) domain-containing proteins on chromosome 3 (3p21). The NBEAL2 (neurobeachin-like 2) protein contains a concanavalin A-like lectin domain, a pleckstrin homology domain, a BEACH domain and WD40 repeats.188 Like other BEACH

domain-containing proteins, NBEAL2 is likely involved in vesicular trafficking and may be critical for the development of α-granules. Proteomic analysis of sucrose-gradient subcellular fractions of platelets indicated that NBEAL2 localizes to the dense tubular system (endoplasmic reticulum) in platelets.187 It has been shown that NBEAL2 interacts with Dock7, Sec16a and Vac14, and that this interaction is disrupted in variants of NBEAL2, possibly accounting for the abnormal platelet formation in GPS.192

GFI1B-Related Syndrome Patients with autosomal dominant bleeding disorder, macrothrombocytopenia and α-granules deficiency associated with mutations in Growth Factor Independence 1b gene (GFI1B) have been described. GFI1B is a transcription factor that promotes erythroid and megakaryocytic proliferation and differentiation. The severity of the bleeding manifestation was variable in the members of the first family, described in 2013.193 A single nucleotide insertion was identified in GFI1B, predicting a frameshift mutation in the fifth zinc finger DNA-binding domain.193 A second report described another truncating mutation in GFI1B in a European family with bleeding diathesis of variable severity and macrothrombocytopenia. Since platelets had a gray appearance at morphological evaluation of a peripheral blood smear, the investigators considered this disorder an autosomal dominant form of GPS.194 Morphological evalution of the bone marrow revealed the presence of myelofibrosis, emperipolesis and pleomorphic megakaryocytes. Interestingly, the megakaryocyte marker CD34 was retained on platelets from several patients bearing GFI1B mutations,194–196 because GFI1B represses CD34 promoter;196 therefore, platelet CD34 expression can be considered an indicator of GFI1B mutations. Red cell anisopoikilocytosis was a common feature in some patients.193,195 Subjects bearing the p.Cys168Phe, which is predicted to disrupt the first nonDNA-binding zinc-finger domain, display macrothrombocytopenia only, without α-granule deficiency or bleeding symptoms.196 Patients with GFI1B mutations associated with combined alpha- and delta-granules deficiencies have been described197 (see later).

GATA-1-Related Disease (X-Linked Macrothrombocytopenia With Anemia/Anisopoikilocytosis) A family with thrombocytopenia, large platelets, appearing gray on blood smear segregating as a sex-linked trait was described.198 Linkage analysis revealed a 63 cM region on the X chromosome between markers G10578 and DXS6797 which segregated with the platelet phenotype and included the GATA1 gene. Sequencing of GATA1 revealed a G to A mutation at position 759, corresponding to amino acid change p. Arg216Gln. This mutation was previously described as a cause of X-linked thrombocytopenia with thalassemia (XLTT),198,199 Given the differences with GPS in terms of type of inheritance and presence of abnormalities of red blood cells, it appears that GATA1 mutations are not responsible for a subtype of GPS, as initially suspected.198 This topic is also discussed in Chapter 46.

SRC-Related Disease The SRC gene encodes the prototype proto-oncogene tyrosine kinase SRC, which has an important role in human cancer. A large pedigree has been described with autosomal dominant, gain-of-function variant of SRC (p.Glu527Lys), which was associated with thrombocytopenia, normal platelet size, paucity of α-granules, myelofibrosis, complete edentulism in young adulthood, osteoporosis and mild facial dysmorphism.200

Inherited Disorders of Platelet Function

Paris-Trousseau Syndrome, Jacobsen Syndrome, and Other FLI1-Related Disorders Paris-Trousseau syndrome (PTS) and Jacobsen Syndrome (JS, now termed 11-q terminal deletion disorder) (see also Chapter 46) are related disorders that associate with a mild hemorrhagic diathesis and are characterized by inherited thrombocytopenia, normal platelet life span and increased number of marrow megakaryocytes, many of which have signs of abnormal maturation and intramedullary lysis. A fraction of the circulating platelets has giant α-granules, which are unable to release their content upon platelet stimulation with thrombin. A deletion of the distal part of one chromosome 11 [del (11)q23.3➔qter] involving two Ets transcriptional factors, ETS1 and FLI1, was found in the affected patients.201–203 While the platelet defect is predominant in PTS, JS, which is characterized by a larger deletion of chromosome 11, has a more severe phenotype, which also includes congenital heart defects, mental retardation, gross and fine motor delays, trigonocephaly, facial dysmorphism, and ophthalmologic, gastrointestinal, and genitourinary problems.204 Two FLI1 variants (c.1010G >A and c.1033A> G) were identified in patients with unexplained thrombocytopenia. Platelets from these patients displayed an aggregation defect, impaired secretion of ATP and CD63 expression upon TRAP stimulation, reduced uptake and secretion of mepacrine; platelet dense granules were nearly absent in platelets, some of which displayed giant α-granules and vacuoles.205 Other FLI1 variants were described in patients with variable platelet count and abnormal ATP secretion (no information of platelet granules was provided).206

Arthrogryposis, Renal Dysfunction, and Cholestasis (ARC) Syndrome Arthrogryposis multiplex congenita, renal dysfunction, and cholestasis (ARC) is an autosomal recessive condition associated with abnormal bleeding, failure to thrive, cerebral malformations, hypotonia, ichthyosis, renal tubular dysfunction, congenital heart disease, hepatic cholestasis. Arthrogryposis (joint contractures), sensorineural deafness and death within the first year of life.207,208 Platelets from patients with the ARC syndrome display reduced platelet aggregation induced by arachidonate and ADP,207 structural abnormalities, including increased platelet size, a pale appearance in blood films, elevated numbers of δ-granules, and completely absent α-granules—similar to those observed in GPS platelets. At variance with GPS platelets, both soluble and membrane-bound αgranule proteins are severely decreased or undetectable in ARC platelets, suggesting that in ARC the formation of precursor alpha-granules is defective, while in GPS cargo packaging and/or retention and granule maturation are abnormal.207,208 Mutations affecting VPS33B, a Sec1/Munc18 protein, have been linked to the ARC syndrome,207 in approximately 75% of patients.209 The remaining ARC patients displayed mutations in VPS16B (vacuolar protein sorting 16 homologue B), which is a VPS33B binding protein.208 VPS33B and VPS16B are ubiquitously expressed and are involved in many biological processes in addition to α-granules biogenesis, accounting for the severe phenotype of ARC.

Quebec Platelet Disorder The Quebec platelet disorder (QPD) is an autosomal dominant qualitative platelet abnormality that occurs in French Canadians, characterized by abnormal proteolysis of α-granule proteins, severe deficiency of platelet factor V, deficiency of multimerin, reduced to normal platelet counts and markedly

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decreased platelet aggregation induced by epinephrine.210,211 Multimerin, one of the largest proteins found in the human body, is present in platelet α-granules and in endothelial cell Weibel-Palade bodies.212–214 Multimerin binds factor V and its activated form, factor Va. Multimerin deficiency in patients with QPD is probably responsible for the defect in platelet factor V, and other proteins stored in α-granules, as a consequence of increased generation of plasmin. This is associated with increased expression and storage of active urokinase-type plasminogen activator (u-PA) in platelets in the setting of normal to increased u-PA in plasma.215–217 Other α-granule proteins are degraded along with factor V in QPD, including VWF, fibrinogen, osteonectin, fibronectin, P-selectin and thrombospondin while platelet factor 4, β-thromboglobulin, albumin, IgG, CD63 and external membrane glycoproteins are not affected, indicating that there is restriction in the platelet proteins degraded.218,219 In addition, electron microscopic and immunoelectron microscopy studies indicated preserved α-granular ultrastructure and normal to reduced labeling for platelet αgranule proteins, suggesting that the pathologic proteolysis of α-granule proteins is not secondary to a defect in targeting proteins to α-granules.219 Patients with QPD experience severe posttraumatic and postsurgical bleeding complications, joint bleeds, large bruises that are unresponsive to platelet transfusion, but are well controlled by the administration of antifibrinolytic agents.220 Genetic marker analyses indicated that QPD was significantly linked to a 2-Mb region on chromosome 10q containing the urokinase plasminogen activator gene (PLAU). However, analysis of PLAU by sequencing and Southern blotting excluded mutations within PLAU and its known regulatory elements as the cause of QPD. Analyses of uPA mRNA indicated that QPD distinctly increased transcript levels of the linked PLAU allele with megakaryocyte differentiation.221 Copy number variation was then investigated and a direct tandem duplication of a 78-kb genomic segment that includes PLAU was detected. This mutation was specific to the examined 38 family members with QPD, as it was not present in any unaffected family members (n ¼ 114), unrelated French Canadians (n ¼ 221), or other persons tested (n ¼ 90).222

Other Inherited Abnormalities of the Platelet α-Granules The White Platelet Syndrome is an inherited, autosomal dominant macrothrombocytopenia, characterized by mild to moderate bleeding symptoms, prolonged bleeding times, poor responses to all aggregating agents and unique structural abnormalities of the platelets, which was described in a large family of Minnesota.223 Structural abnormalities, which were seen in a fraction of circulating platelets included: presence of fully developed Golgi complexes, reduced α-granule content, cytoplasmic sequestration by residual dense tubular system membranes, autodigestion, larger than normal mitochondria and half normal-sized dense bodies (Chapter 3). The Medich Platelet Syndrome has been described in three unrelated patients and is characterized by bleeding diathesis, macrothrombocytopenia, markedly decreased α-granules but normal dense granules. The platelet population is heterogeneous, including platelets of normal size and content of cytoplasmic organelles. At variance with GPS platelets, the patient’s platelets did not contain large amounts of empty vacuoles without granule contents, but contained membranous cigar-shaped inclusions.224,225

Defects of the α- and δ-Granules α,δ-Storage pool deficiency is a heterogeneous inherited disorder of platelet secretion characterized by deficiencies of both α- and δ-granules.226,227 It is important to note that blood

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samples should be collected in sodium citrate for measurement of platelet granule contents, because platelets from some individuals may undergo degranulation in vitro when blood is collected into EDTA, thereby artifactually resembling α,δSPD. The phenotypic heterogeneity of this disorder is illustrated by the finding that the platelet content of P-selectin was normal in three members of a family with mild α,δ-SPD, while it was approximately halved in a patient with severe α,δ-SPD.228 Approximately 80% of platelets from the patient with severe α,δ-SPD expressed little or no P-selectin after stimulation, whereas the remaining 20% expressed normal amounts. This heterogeneity was not found among platelets of the three patients with mild α,δ-SPD. Compared to δ-SPD platelets, which have a normal density, α,δ-SPD platelets show a shift to the left of the density distribution, suggesting that αgranules are a major determinant of platelet density.229 A report documented severe α,δ-SPD in four members in three generations of the same family and suggested that in this disorder, the α-granules and dense bodies become connected to channels of the open canalicular system (OCS) and lose their contents to the exterior without prior activation of the platelets.230 Combined α- and δ-granules deficiencies have been reported in some patients displaying mutations in GFIB1 gene136,197 or in RUNX1.135,136 The clinical picture and the platelet aggregation abnormalities are similar to those of patients with GPS or δ-SPD. As in GPS, the platelet prothrombinase activity in response to collagen plus thrombin was impaired in a patient with severe α,δSPD and was not completely corrected by added factor Va.99 Platelet thrombus formation on everted rabbit vessel segments under various flow conditions was severely impaired,98 as was the production of arachidonate metabolites after stimulation with arachidonate, epinephrine or collagen.89

DEFECTS OF SIGNAL TRANSDUCTION Deficiency of Cytosolic Phospholipase A2α Cytosolic phospholipase A2α (cPLA2α) hydrolyzes arachidonic acid from cellular membrane phospholipids, thereby providing enzymatic substrates for the synthesis of eicosanoids, such as prostaglandins and leukotrienes. A 45-year-old patient was described with a life-long history of occult gastrointestinal blood loss, chronic anemia, iron deficiency, and frequent bouts of abdominal pain as a child and young adult.231 Repeated episodes of acute gross gastrointestinal bleeding late in his fourth decade and multiple episodes of small bowel perforation required five surgical interventions between 38 and 45 years of age. Surgical exploration of the small intestine and intraoperative endoscopy revealed multiple recurrent ulcerations. Levels of TXB2 and 12-hydroxyeicosatetraenoic acid produced by platelets and leukotriene B4 released from calcium ionophore-activated blood were markedly reduced, indicating defective enzymatic release of the arachidonic acid substrate for the corresponding cyclooxygenase and lipoxygenases. Platelet aggregation and degranulation induced by ADP or collagen were diminished but were normal in response to arachidonic acid. Two heterozygous single base pair mutations and a known single nucleotide polymorphism were found in the coding regions of the patient’s PLA2G4A genes (p.Ser111Pro + Arg485His; p.Lys651Arg). The total PLA2α activity in sonicated platelets was diminished, and the urinary metabolites of prostacyclin, PGE2, PGD2, and TXA2 were also reduced. Apart from the bleeding episodes from the GI ulcerations, the patient did not experience abnormal bleeding in other sites. Two nonidentical twins were subsequently described with PLA2α deficiency, who, in addition to severe TxA2-dependent

abnormalities of platelet function and GI bleeding also experience a generalized bleeding diathesis, which cause iron deficiency in anemia in one.232 They also had mild factor XI deficiency, which could have contributed to their bleeding tendency. They were homozygous for 1723G >C transition in PLA2G4A gene, which changed the codon for Asp575 to His, which conferred a high degree of instability to the variant molecule, thus explaining its severe deficiency.233 A homozygous 4 bp deletion (g.155574_77delGTAA) in the PLA2G4A gene, located in the splice donor site directly after exon 17 was described in two siblings, who displayed similar GI symptoms and defective platelet function, with no bleeding diathesis.234

Defects of Cyclooxygenase-1 (Aspirin-Like Defect) Patients with inherited abnormalities in cyclooxygenase-1 (or prostaglandin synthase-1), the enzyme catalyzing the first step in prostaglandin synthesis from arachidonate, have been described.1 The platelets from these patients have the same functional defect of normal platelets that have been treated with aspirin, which irreversibly acetylates the platelet cyclo-oxygenase: impaired aggregation and secretion induced by ADP, epinephrine, collagen or arachidonic acid, normal responses to TXA2/endoperoxides analogs, and absent platelet TXA2 production. The actual concentration of cyclooxygenase-1 antigen in platelet lysates, measured with an immunoassay, was found to be defective in some patients only.235,236 It has therefore been proposed that the platelet cyclooxygenase-1 defect can be subdivided in type 1, characterized by undetectable levels of the enzyme protein, and type 2, characterized by the presence of normal levels of a dysfunctional protein. However, before a diagnosis of type 2 cyclooxygenase-1 deficiency can be safely made, caution should be taken to rule out the possibility of surreptitious or inadvertent ingestion of acetylsalicylic acid, which is contained in several generic medications. In one patient, the synthesis of both platelet TXA2 and vessel wall PGI2 were measured and found to be severely impaired.237 Since that patient had a mild bleeding diathesis, the finding suggested that the contemporary abolition of TXA2 and PGI2 synthesis results in a hemorrhagic diathesis, rather than in a thrombotic tendency, as had been previously hypothesized. Heterozygous nonsynonymous variant in the signal peptide of cyclooxynease-1 (c.50C > T; p.Pro17Leu), associated with impaired platelet aggregation and TXA2 production was described in members of a family with a bleeding diathesis.238 Some members of the same family had mild hemophilia A: the severity of the bleeding phenotype of hemophilia A was more severe in those patients who also carried the cyclooxygenase-1 variant.238

Defects of Thromboxane Synthetase Two reports of patients with inherited defects of platelet thromboxane synthetase have been published.239,240 Defreyn et al. described three family members of three successive generations with moderate bleeding tendency, markedly prolonged bleeding time, absent aggregation induced by arachidonic acid, and monophasic aggregation induced by ADP or epinephrine. The platelet production of TXB2 was decreased, while that of PGF2α, PGE2 and PGD2 were increased. The plasma levels of the PGI2 metabolite 6-keto PGF1α were also increased. These findings are compatible with a partial platelet thromboxane synthetase defect and reorientation of cyclic endoperoxide metabolism to increased production of the inhibitory prostaglandins PGI2 and PGD2, which would contribute, in conjunction with the reduced synthesis of TXA2, to the abnormality of primary hemostasis.

Inherited Disorders of Platelet Function

Abnormalities of GTP-Binding Proteins Four patients were described with abnormal thrombin-induced liberation of 3H-arachidonic acid from prelabeled platelets. TXB2 production after stimulation with ADP or thrombin was impaired, while it was normal with arachidonic acid.241 Subsequent studies of one of these patients showed that his platelets contained normal amounts of PLA2, while agonistinduced Ca2+ mobilization, G protein activation and immunological Gaq levels were reduced.242 Immunoblot analysis of Gα subunits in the patient’s platelet membranes showed a decrease in Gαq (<50%) but not Gαi, Gαz, Gα12, and Gα13. Gαq mRNA levels were decreased by >50% in platelets, but not in neutrophils,242 which had normal responses and normal levels of the protein, suggesting a hematopoietic lineage-specific defect, possibly due to defects in transcriptional regulation or mRNA stability.243 A patient with a bleeding diathesis and severely impaired platelet responses to the weak agonists ADP and epinephrine had markedly reduced platelet levels of Gi1, a minimally expressed species in human platelets.244 This finding is somewhat surprising, considering that Gi1 has not been recognized to have a role in platelet function and that studies with Gi1 knockout mice have so far not revealed any obvious physiologic abnormalities. Further studies are needed to elucidate this issue. Patients with a bleeding syndrome had a polymorphism of the gene encoding the extra-large stimulatory G-protein αsubunit (XLSα), associated with hyperresponsiveness of platelet Gsα and enhanced intraplatelet cAMP generation.245,246 The functional polymorphism in these patients involves the imprinted region of the XLSα gene, a phenomenon not described previously for platelet disorders but already known for defects expressing phenotypically in other tissues. Gs hyperactivity, in combination with abnormalities of the platelet cytoskeleton and reduced platelet collagen reactivity were described in patients with Duchenne muscular dystrophy.247 Three patients were described with a platelet Gs signaling defect caused by a heterozygous variant of Regulator of Gprotein signaling (RGS) 2, which negatively regulates Gs signaling by inhibiting the activation of adenylyl cyclase.248

Defects in Phospholipase C Activation In 1989, Rao et al. described a 42-year-old white woman and her 23-year-old son who had a mild hemorrhagic diathesis; their platelets underwent abnormal secretion and aggregation induced by ADP, epinephrine, PAF, arachidonic acid and the calcium ionophore A23187.249 In both patients, the platelet ADP and ATP contents and TXA2 synthesis were normal, while the concentration of cytoplasmic Ca2+ [Ca2+]i in resting platelets, peak [Ca2+]i concentrations stimulated by ADP, PAF, collagen, the prostaglandin endoperoxides analog U46619 and thrombin were impaired. Subsequent studies indicated that, upon platelet activation, the formation of inositol 1,4,5trisphosphate and diacylglycerol, and the phosphorylation of pleckstrin were abnormal.250 These data suggested that the patient platelets had a defect in PLC activation. This hypothesis was confirmed by the finding that platelets from one of these patients had a selective decrease in one of the seven PLC isoforms present in platelets, PLC-β2, suggesting that this isoenzyme may have an important role in platelet activation.251 The decreased PLC-β2 protein levels were associated with normal coding sequence but reduced PLC-β2 mRNA levels in platelets, but not in neutrophils, providing evidence for a lineage (platelet)-specific defect in PLC-β2 gene expression.252 In 1997, Mitsui et al. described a patient with a mild bleeding disorder since early childhood, which was characterized a

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prolonged bleeding time, defective platelet aggregation responses to U46619 and arachidonic acid, despite normal binding of [3H]-labeled U46619. Normal GTPase activity was also induced in the patient’s platelets by stimulation with U46619. However, inositol 1,4,5-triphosphate formation was not induced by U46619, suggesting that the patient’s platelets had a defect in phospholipase C activation beyond TXA2 receptors.253

CalDAG-GEFI Defect CalDAG-GEFI is the exchange factor that plays a fundamental role in Ca2+-dependent activation of Ras-proximate-1 (Rap1) in platelets, which is followed by αIIbβ3 activation, binding of adhesive proteins and platelet aggregation254 (see Chapters 12 and 18). In 2014, Canault et al. described three siblings from consanguineous parents, with life-long bleeding diathesis and platelet function abnormalities associated with inherited dysfunctional CalDAG-GEFI.255 Platelet aggregation was defective, except when induced by high concentrations of collagen or of a thrombin receptor activating peptide (TRAP). A similar platelet phenotype was later described by Lozano et al. in two patients with bleeding diathesis and mutations in the RASGRP2 gene, which encodes for CalDAG-GEFI.256 All patients had moderate/severe mucocutaneous bleeding episodes, which occasionally caused iron-deficiency anemia and required red blood cell and/or platelet transfusions. Interestingly, the severity of the bleeding diathesis seemed to decrease in adult life. The only relevant difference between these two series of patients is that, while the patients described by Canault et al. did not display abnormal integrin-dependent leukocyte function, β2 integrin activation was impaired in stimulated neutrophils from the patients described by Lozano et al. Deficiency of CalDAG-GEFI is expected to affect leukocyte function, in addition to platelet function, because Rap1 activation is important also for leukocyte integrin activation: indeed, neutrophils from CalDAGGEFI-deficient mice failed to adhere firmly to stimulated venules and to migrate into sites of inflammation.257 One possible explanation for these discrepancies is that structural domains important for leukocyte functions could be spared by the patients’ homozygous c.G742T mutation of RASGRP2 gene, which was not associated with defective expression of the protein, but with a defective, mutant protein (p.Gly248Trp) in Canault’s patients. In contrast, the patients described by Lozano et al. had severely reduced expression of CalDAG-GEFI, similarly to KO mice.258 Neutrophil β2 integrin activation was not impaired in a patient with compound heterozygous CalDAG-GEFI mutations, p.Lys309X and p.Leu360del.259 High-throughput sequencing and phenotype data from 2042 patients with unexplained bleeding revealed that 11 patients displayed 11 different, previously unreported biallelic RASGRP2 variants, including five high-impact variants predicted to prevent CalDAG-GEFI expression and six missense variants affecting the CalDAG-GEFI CDC25 domain.260 Additional patients with novel mutations in RASGRP2 have more recently been described.261,262

Leukocyte Adhesion Deficiency-III (LAD-III) LAD-III is associated with severe defects in leukocyte and platelet β1, β2, and β3 integrin activation despite normal integrin expression. Platelets of LAD-III patients fail to aggregate because of an impaired activation of the integrin β3, leading to Glanzmann thrombasthenia-like bleeding symptoms. FERMT3, encoding for kindlin-3, was identified as candidate gene for LAD-III.263 Kindlin-3, a member of an important family of focal adhesion proteins contains a FERM domain located at the carboxyl terminus that bind to β-integrin cytoplasmic

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tails and cooperate with talin in integrin activation. It is restricted to hematopoietic cells and is abundantly expressed in megakaryocytes and platelets. Several reports demonstrate that LAD-III is indeed caused by mutations in Kindlin3, affecting integrin activation.263–268 Despite the obvious qualitative similarities, the collagen-induced platelet aggregation defect in LAD-III appears more severe than in GT, because small platelet aggregates can form in GT that are mediated by integrin α2β1, which, like other integrins, is functionally defective in LAD-III: this could explain the more severe bleeding phenotype of LAD-III compared to GT.269

Abnormality of GPVI/FcRc Signaling A 60-year-old female patient with a history of immune disorders, excessive bleeding from childhood and a life-threatening hemorrhage posttrauma had normal platelet aggregation to ADP, thrombin receptor-agonist peptide or ristocetin/VWF was normal, but platelet aggregation to GPVI agonists, collagen, CRP, or convulxin, was defective. Both GPVI/FcRc expression and ligand-induced GPVI ectodomain shedding were normal, confirming expression of functional GPVI/FcRc, but suggesting a signaling defect downstream of Syk.270

Stormorken/York Platelet Syndrome Stormorken syndrome, first described in 1985, is an autosomal dominant disorder characterized by bleeding tendency, abnormal platelet function, thrombocytopenia, anemia, asplenia, tubular aggregate myopathy, muscle fatigue, congenital miosis, and ichthyosis.271 Resting platelets from affected patients display full procoagulant activity, microvesicles, abnormal clot retraction and aggregation and secretion, especially when induced by collagen.272 Thrombus formation on purified human collagen type III under standardized flow conditions was reduced at shear rates of 650 and 2600 s1, whereas platelet adhesion to the collagen surface was higher than normal.272 Misceo et al. reported the presence of a heterozygous missense, dominant gain-of-function mutation in the gene encoding for stromal interaction molecule 1 (STIM1) exon 7 (c.910C> T; p. Arg304Trp) in six patients from four unrelated families.273 STIMI1 binds the plasma membrane protein ORAI1, a Ca2+ release-activated calcium (CRAC) channel, which mediates store-operated calcium entry (SOCE) in cells.273 Platelets from these patients displayed increased cytoplasmic Ca2+ levels and attenuated SOCE, suggesting that the mutation results in constitutive activation of the ORAI1 calcium channel. As a consequence of high Ca2+, platelets were in a preactivated state with high exposure of aminophospholipids and microparticles; however, they were much less responsive to in vitro stimulation, especially by collagen. Therefore, the findings of Misceo et al. confirm the platelet phenotype that had been described in the first family by Stormorken et al.271,272 The p.Arg304Trp mutation was described in other unrelated families.274, 275 In seven patients from four unrelated families with York platelet syndrome, Markello et al. identified the p.Arg304Trp mutation and the Ile115Phe mutation that had previously been described in patients with tubular aggregate myopathy-1 (TAM1).276 Therefore, it appears that Stormorken syndrome and York syndrome are strictly related or even the same clinical entity277 (see also Chapter 46).

membranes of blood cells, including platelets,278,279 leading to reduced thrombin generation and defective wound healing. The asymmetric phospholipid distribution in plasma membranes is normally maintained by energy-dependent lipid transporters that translocate different phospholipids from one monolayer to the other against their respective concentration gradients. When cells are activated, or enter apoptosis, lipid asymmetry can be perturbed by other lipid transporters, which shuttle phospholipids nonspecifically between the two monolayers. This exposes phosphatidylserine (PS) at the cells’ outer surface and in cell-derived microvesicles, which, by providing a catalytic surface for interacting coagulation factors, promotes thrombin generation (Chapters 21 and 22). Therefore, the abnormality of this process accounts for the bleeding diathesis of Scott syndrome patients, which is characterized by bleeding episodes following trauma or surgery, epistaxis, postpartum bleeding, which can be extremely severe and longlasting menorrhagia, resulting in iron-deficiency anemia. Scott syndrome is transmitted as an autosomal recessive trait.280 Prothrombin consumption during clotting of whole blood is defective in Scott syndrome and clotting time after recalcification of kaolin-activated platelet-rich plasma in the presence of Russell’s viper venom is prolonged. In contrast, platelet count and structure are normal, and no abnormalities of platelet secretion, aggregation, metabolism, granule content, or platelet adhesion to subendothelium have been described.278 The diagnosis can be based on the demonstration of defective PS exposure on activated platelets, measured by annexin-V binding in a flow cytometry assay.281 Although Scott syndrome was originally described as an isolated disorder of platelet procoagulant activity, the underlying defect in Ca2+-induced lipid scrambling is not restricted to platelets but is also evident in erythrocytes and Epstein-Barr virus-transformed B-lymphocytes.282,283 Suzuki et al. showed that the Ca2+ Activated Cl Channel Transmembrane Protein 16 (TMEM16F), known also as Anoctamin 6 (ANO6) is an essential component for the Ca2+dependent exposure of PS on the cell surface.284 When a mouse B-cell line, Ba/F3, was treated with a Ca2+ ionophore under low-Ca2+ conditions, it reversibly exposed PS. Using this property, a Ba/F3 subline was established that strongly exposed PS by repetitive fluorescence-activated cell sorting. A complementary DNA library was constructed from the subline, and a cDNA that caused Ba/F3 to expose PS spontaneously was identified by expression cloning. Wild-type TMEM16F was localized on the plasma membrane and conferred Ca2+-dependent scrambling of phospholipids. A patient with Scott syndrome was found to carry a mutation at a splice-acceptor site of the gene encoding TMEM16F, causing the premature termination of the protein.284 Two different mutations were subsequently identified in another patient with Scott Syndrome: a transition at the first nucleotide of intron 6 (IVS6_1G3A), disrupting the donor splice site consensus sequence of intron 6, and a singlenucleotide insertion in exon 11 (c.1219insT, cDNA numbering from the ATG), predicting a frame shift and premature termination of translation at codon 411.285 Two sisters with bleeding diathesis had ANO6 deficiency due to the association of a deletion of the encoding gene and a nonsense variation (c.889C >T (p.Arg297*).286

ABNORMALITIES OF MEMBRANE PHOSPHOLIPIDS

MISCELLANEOUS DISORDERS OF PLATELET FUNCTION

Scott Syndrome

Primary Secretion Defects

Scott syndrome is a rare bleeding disorder associated with the maintenance of the asymmetry of the lipid bilayer in the

The term Primary Secretion Defect indicates all those ill-defined abnormalities of platelet secretion not associated with platelet

Inherited Disorders of Platelet Function

granules deficiencies.287 The term was later used to indicate the platelet secretion defects not associated with platelet granule deficiencies and abnormalities of the arachidonate pathway,49 or, more in general, all the abnormalities of platelet function associated with defects of signal transduction. With the progression of our knowledge in platelet pathophysiology, this heterogeneous group, which lumps together the majority of patients with inherited disorders of platelet function, will become progressively thinner, losing those patients with better defined biochemical abnormalities responsible for their platelet secretion defect. An example is given by patients with heterozygous P2Y12 deficiency who were included in this group of disorders until their biochemical abnormality was identified.49 In 1981, Wu et al. described a large family with an inherited bleeding disorder associated with defective platelet secretion, despite normal platelet granule contents and normal TXA2 production.288 Although the platelets of these patients might have had an abnormality of TP receptors, because they did not respond to a PGH2 analog, their biochemical abnormality has not been further elucidated. Hardisty et al. later reported a patient with similar platelet abnormalities, including the lack of response to PGH2 and TXA2.289 Some abnormalities of platelet secretion are described in patients with psychiatric disorders, such as attention deficit disorder290 and conduct disorder.291 These reports emphasize the role of platelets as a model for neurons in functional disorders.

Other Platelet Abnormalities Montreal platelet syndrome (MPS),292 hitherto described in only one kindred, is an inherited thrombocytopenia associated with mucocutaneous bleeding, giant platelets, and spontaneous platelet aggregation in vitro. These are features shared with some forms of type 2B VWD; however, the MPS kindred had not been investigated for VWD until recently. Jackson et al. found that all affected MPS family members had borderline to normal VWF antigen, discrepantly low ristocetin cofactor activity and loss of plasma, but not platelet, high molecular weight VWF multimers. In addition, they were heterozygous for the previously reported V1316M type 2B VWD mutation. Based on these findings, it was determined that MPS patients actually are type 2B VWD patients with the V1316M VWF mutation.293 Platelet function abnormalities have been reported in osteogenesis imperfecta, Ehlers-Danlos syndrome, Marfan syndrome, hexokinase deficiency and glucose-6-phosphate deficiency1.

INHERITED DEFECTS OF ADHESIVE PROTEINS, AFFECTING PLATELET FUNCTION The interaction of platelets with the vessel wall that brings about the formation of a hemostatic plug is secured by the interaction of adhesive proteins, such as VWF and fibrinogen, with specific platelet receptors. Therefore, inherited abnormalities of primary hemostasis due to impaired platelet interaction with the vessel wall are caused not only by defects of the platelet receptors, but also by inherited defects of adhesive proteins, such as VWD and afibrinogenemia. VWD is a bleeding disorder caused by inherited defects in the concentration, structure, or function of VWF, which mediates platelet-vessel wall and platelet-platelet interaction, especially at high shear rates, and binds coagulation factor VIII. VWD is classified into three primary categories. Type 1 includes partial quantitative deficiency; type 2 includes qualitative defects; and type 3 includes virtually complete deficiency of VWF. Inherited VWD has been recently reviewed elsewhere.294

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Inherited fibrinogen disorders can be classified into qualitative and quantitative anomalies: dysfibrinogenemia is characterized by normal circulating levels of fibrinogen with abnormal function; hypofibrinogenemia and afibrinogenemia are characterized by reduced or absent fibrinogen in the circulation, respectively, while hypodysfibrinogenemia is defined by reduced fibrinogen with reduced function. All these disorders are due to mutations in one of the three fibrinogen genes, FGA, FGB and FGG, which are clustered in a region of 50 kb on the long arm of human chromosome 4. Inherited fibrinogen disorders have recently been reviewed.295 Patients with afibrinogenemia display markedly prolonged bleeding times and defective platelet aggregation, which is a consequence of the lack of fibrinogen binding to activated platelets. Correction of the bleeding time with transfusion of fresh frozen plasma persisted far beyond the disappearance of transfused fibrinogen from plasma and was associated with its concentration in platelet α-granules.33

PREVALENCE AND DIAGNOSTIC EVALUATION OF INHERITED DISORDERS OF PLATELET FUNCTION Prevalence The prevalence of inherited platelet disorders among the general population is unknown, but it might be much higher than generally assumed, probably as high as VWD. These disorders are more commonly diagnosed in women, likely because the added hemostatic challenges of menstruation and childbirth affect the burden of living with a bleeding disorder and the likelihood of referral for evaluation.296 Disorders that affect platelet secretion, either due to platelet granule defects or to abnormalities of signal transduction, are by far the most common inherited disorders of platelet function.

Diagnosis A careful assessment of the bleeding history is extremely important for diagnosing platelet function disorders. The risk of excessive bleeding after significant hemostatic challenges (major and minor surgery, trauma) is increased; typically the abnormal bleeding occurs with very rapid onset. The severity of the bleeding episodes varies with the severity and nature of the defect: BSS, GT, LAD-III, QPD, and Scott syndrome are usually associated with the most severe bleeding manifestations. With milder disorders, the bleeding risk is lower and, as a result, abnormal bleeding may not occur with all challenges. Other, partially unknown factors contribute to the bleeding risk, because the severity of the bleeding history may vary widely among patients with the same defect. The most common spontaneous bleeding manifestations include easy bruising and mucocutaneous hemorrhages, such as epistaxes, gum bleeding and menorrhagia. Although QPD and Scott syndrome may be associated with some “platelet-type” bleeding, they are usually characterized by delayed onset bleeding after hemostatic challenges and “coagulation-type” bleedings (e.g., joint bleeds). Bleeding scores have limited diagnostic utility for inherited platelet function disorders,297 but the family histories may be helpful. Global tests of primary hemostasis, such as the bleeding time and the platelet function analyzer (PFA)-100 are not very sensitive and highly nonspecific and are therefore not very useful.298,299 The diagnostic laboratory assessment appropriate for evaluation of a suspected inherited platelet disorder is complex and should be done in specialized laboratories.300 A two-step diagnostic strategy is suggested. The first step, based on screening

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tests, should help in raising a diagnostic hypothesis, while the second step, based on specific tests, should confirm the diagnostic hypothesis. The first step should include an assessment of blood counts, a careful evaluation of the blood smear, an evaluation of platelet size and measurement of platelet aggregation and secretion. Light transmission aggregometry (LTA) (see also Chapter 34) is a time-consuming and technically challenging technique that is affected by many preanalytical and analytical variables, which must be carefully controlled for by expert personnel. The Subcommittee on Platelet Physiology of the Scientific and Standardization Committee of the International Society on Thrombosis and Hemostasis published official guidelines for the standardization of the variables affecting LTA, which should be followed in order to harmonize the procedures across different laboratories worldwide.301,302 As already mentioned, LTA is relatively insensitive to the most common inherited disorders of platelet function, which involve abnormalities of platelet secretion. Therefore, laboratory tests that measure platelet aggregation and secretion simultaneously, such as lumiaggregometry, should be preferred to traditional, light transmission aggregometry.303,304 Impedance aggregometry allows the measurement of platelet aggregation in whole blood, because it is sensitive to the increase in electrical impedance that is caused by the formation of a platelet aggregate on platinum electrodes upon the addition of a platelet agonist to a diluted whole blood sample, in which the electrodes are immersed (see Chapter 34). A relatively recently introduced instrument, the Multiplate, is less technical demanding and time consuming than LTA; however, it is sensitive to many variables, including the platelet count, which significantly affects the results even for slight variations within the normal range, and the hematocrit.305 In addition, Multiplate has drawbacks that theoretically hamper its validity as a diagnostic test for platelet function disorders: it does no give information on the change in shape of platelets upon their stimulations with agonists and on the reversibility of platelet aggregates, which is an important hallmark of some defects of platelet function. Indeed, it has been shown that Multiplate is much less sensitive in identifying patients with mild/moderate platelet function defects than LTA306 or lumiaggregometry307 while it is equally effective in diagnosing severe defects, such as GT.308 The flow cytometer (see also Chapter 35) allows the measurement of platelet reactivity in vitro and the detection of activated platelets, platelet-leukocyte aggregates and plateletderived microparticles in the circulation.309 Flow cytometry allows the study of platelet secretion (CD62P, CD63, mepacrine from preloaded platelets)310 and platelet procoagulant activity.281 Platelet aggregation can be measured by flow cytometry either using a dual color assay,311 or by evaluating a surrogate end-point, such as the exposure of activated GPIIb-IIIa receptor or its binding of fibrinogen.312 The technique allows the detection of defects of membrane glycoproteins, such as GPIIb-IIIa, GPIb, GPVI, on resting platelets, to confirm the diagnosis of specific platelet disorders that can be suspected based on well-defined abnormalities of platelet aggregation. Finally, it may be particularly useful for studying pediatric patients, from whom only small amounts of blood samples can be drawn, or patients with moderate/severe thrombocytopenia who cannot be studied using LTA.313–315 The first step in the diagnostic work-up of patients with platelet function disorders should also include clot retraction, which, in addition to giving important information on platelet function, allows one to save patient serum in which TXB2 can be measured to rule out surreptitious intake of nonsteroidal antiinflammatory drugs and/or to diagnose inherited abnormalities of the arachidonate pathway of platelet activation. Additional tests to be performed in the first diagnostic step have been proposed.316

The second step in the laboratory evaluation of platelet function disorders include specific tests (e.g., flow cytometry, Western blotting, immunoprecipitation analyses, measurement of platelet granule content and eicosanoids, electron microscopy, etc.) and are used for the workup of suspected deficiencies/dysfunction, based on the results of tests used in the first diagnostic step. Molecular and cytogenetic tests are helpful to confirm conditions with characterized genetic and/or cytogenetic abnormalities.299, 316 A DNA-based diagnostic approach has a potentially very important role in the investigation of patients with platelet function disorders. Several studies have already been published, reporting interesting and very promising results.317–321

TREATMENT OF INHERITED PLATELET FUNCTION DISORDERS General principles of treatment of platelet function defects are: avoidance of major body contact activities; avoidance of drugs that interfere with platelet function (e.g., aspirin and other nonsteroidal antiinflammatory drugs) and other components of the hemostatic system; avoidance of intramuscular injections; good dental care (to minimize gum bleeding and avoid tooth extraction); local hemostatic measures; oral iron (to prevent or treat iron deficiency secondary to blood loss); oral contraceptive pills may be necessary for the control of menorrhagia; immunizations can be given subcutaneously with direct pressure using ice; consideration should be given to the wearing of a medical bracelet or necklace with the words “platelet function defect” inscribed or carrying an information card to that effect.322 Genetic counseling can also be provided.6 The management of patients with inherited defects of platelet function is focused on preventing bleeding with major and minor hemostatic challenges, and controlling major hemorrhagic events. A recent investigation showed that severe bleeds requiring blood transfusions were observed in 50% of deliveries in GT, but not other types of platelet function defects; prophylaxis with platelet transfusion did not prevent bleeding in some GT patients.323 Minor hemorrhagic events, such as bruising, do not require treatment. Four treatment options are available: platelet transfusions, desmopressin (DDAVP), fibrinolytic inhibitors and recombinant Factor VIIa (rFVIIa). Platelet transfusions (Chapter 64) should be reserved for patients with serious bleeding unresponsive to medical therapies or the most severe platelet function defects, such as BSS, GT and Scott syndrome. They should be used also to treat bleeding episodes in patients with platelet-type VWD, in whom infusion of VWF concentrates or desmopressin, which are useful in patients with type 2B VWD, would exacerbate the condition. Limited efficacy in patients with GT could be caused by competitive interference of diseased platelets with exogenous, normal platelets.324 In individuals with deficient platelet membrane glycoproteins, there is an increased risk of alloimmunization from platelet transfusion therapy that can limit future responses to platelet transfusion. This risk appears to be higher for GT than for BSS.296 Desmopressin (Chapter 62) can be used in the management of the more common, less severe platelet disorders and for milder bleeding manifestations. Desmopressin shortens the prolonged bleeding times in most patients with defects of platelet function. However, the evidence on its clinical effectiveness in the prophylaxis and treatment of bleeding in these patients is based on case reports and clinical experience. Desmopressin can be given intravenously, subcutaneously or intranasally. Common side effects of desmopressin include facial flushing, temporary fluid retention (regardless of the route of administration), and, in some individuals, mild headaches.

Inherited Disorders of Platelet Function

Fibrinolytic inhibitors (epsilon-aminocaproic acid and tranexamic acid) are useful as adjunctive therapy for preventing and controlling bleeding with dental extractions or oral/nasal surgery. A short course (5–7 days) may be helpful for recurrent epistaxes. Fibrinolytic inhibitors should not be used to treat hematuria and they should be avoided for operative procedures associated with thrombotic high risks (e.g., orthopedic surgery). Fibrinolytic inhibitor drugs are the only therapy helpful to prevent and control bleeding in QPD.296 Recombinant FVIIa (Chapter 63) can be used for management of serious bleeding in patients with BSS or GT who no longer respond to platelet transfusions because of alloimmunization.296 Medical therapy with oral contraceptives and fibrinolytic inhibitors are usual first line therapies in women with inherited disorders of platelet function with menorrhagia, and fibrinolytic inhibitors may be useful when oral contraceptives are discontinued to attempt pregnancy. Intrauterine devices, designed for reducing menstrual blood loss are another option to consider, particularly when individuals are intolerant of medical therapies and do not wish to become pregnant. Although desmopressin nasal spray can reduce menorrhagia symptoms, side effects and tolerance are common with repeated dosing. So desmopressin therapy for menorrhagia is often restricted to managing days of heavier menstrual flow in individuals with inadequate responses to other therapies.296 When severe bleeding cannot be adequately controlled by other measures, bone marrow transplantation has been used in children with severe diseases such as Chediak-Higashi syndrome, WAS and, occasionally, GT.325–327 However, benefitto-risk ratio should be carefully calculated in each individual patient, due to the risks associated with the procedure.328 The potential role of gene therapy for inherited platelet function defects is discussed in Chapter 67.

CONCLUSIONS The prevalence of inherited platelet disorders among the general population is unknown, but it might be much higher than generally assumed, probably at least as high as VWD. The severity and type of bleeding is influenced by the severity and nature of the defect. Mucocutaneous bleeding is typical, including easy bruising, epistaxis and menorrhagia. Postsurgical or posttraumatic bleedings typically occur with a rapid onset. In severe forms, involving abnormalities of platelet coagulant activity (e.g., Scott syndrome), bleeding manifestations that are more typical of disorders of coagulation, such as hematoma and hemarthrosis, may occur. A two-step diagnostic strategy is recommended. The first step, based on screening tests, should help in raising a diagnostic hypothesis, while the second step, based on specific tests, should confirm the diagnostic hypothesis. In the first step, laboratory tests that measure platelet aggregation and secretion simultaneously, such as lumiaggregometry, should be preferred to traditional, light transmission aggregometry. The management of patients with inherited defects of platelet function is focused on preventing bleeding with major and minor hemostatic challenges, and controlling major hemorrhagic events. Minor hemorrhagic events, such as bruising, do not require treatment. Four treatment options are available: platelet transfusions, DDAVP) fibrinolytic inhibitors and rFVIIa. Platelet transfusions should only be used to treat serious bleeding unresponsive to medical therapies or the most severe platelet function defects, such as BSS, GT and Scott syndrome. Recombinant FVIIa can be used for management of serious bleeding in patients with BSS or GT who no longer respond to platelet transfusions because of alloimmunization.

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Fibrinolysis inhibitors are the only therapy helpful to prevent and control bleeding in the QPD.

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196. Rabbolini DJ, Morel-Kopp MC, Chen Q, Gabrielli S, Dunlop LC, Chew LP, Blair N, Brighton TA, Singh N, Ng AP, Ward CM, Stevenson WS. Thrombocytopenia and CD34 expression is decoupled from α-granule deficiency with mutation of the first growth factor-independent 1B zinc finger. J Thromb Haemost 2017;15(11):2245–58. 197. Ferreira CR, Chen D, Abraham SM, Adams DR, Simon KL, Malicdan MC, Markello TC, Gunay-Aygun M, Gahl WA. Combined alpha-delta platelet storage pool deficiency is associated with mutations in GFI1B. Mol Genet Metab 2017; 120(3):288–94. 198. Tubman VN, Levine JE, Campagna DR, et al. X-linked gray platelet syndrome due to a GATA1 Arg216Gln mutation. Blood 2007; 109:3297–9. 199. Thompson AR, Wood WG, Stamatoyannopoulos G. X-linked syndrome of platelet dysfunction, thrombocytopenia, and imbalanced globin chain synthesis with hemolysis. Blood 1977;50 (2):303–16. 200. Turro E, Greene D, Wijgaerts A, Thys C, Lentaigne C, Bariana TK, Westbury SK, Kelly AM, Selleslag D, Stephens JC, Papadia S, Simeoni I, Penkett CJ, Ashford S, Attwood A, Austin S, Bakchoul T, Collins P, Deevi SV, Favier R, Kostadima M, Lambert MP, Mathias M, Millar CM, Peerlinck K, Perry DJ, Schulman S, Whitehorn D, Wittevrongel C, Consortium BRIDGE-BPD, De Maeyer M, Rendon A, Gomez K, Erber WN, Mumford AD, Nurden P, Stirrups K, Bradley JR, Raymond FL, Laffan MA, Van Geet C, Richardson S, Freson K, Ouwehand WH. A dominant gain-of-function mutation in universal tyrosine kinase SRC causes thrombocytopenia, myelofibrosis, bleeding, and bone pathologies. Sci Transl Med 2016;8 (328):328ra30. 201. Breton-Gorius J, Favier R, Guichard J, et al. A new congenital dysmegakaryopoietic thrombocytopenia (Paris-Trousseau) associated with giant platelet alpha-granules and chromosome 11 deletion at 11q23. Blood 1995;85:1805–14. 202. Hart A, Melet F, Grossfeld P, et al. Fli-1 is required for murine vascular and megakaryocytic development and is hemizygously deleted in patients with thrombocytopenia. Immunity 2000; 13:167–77. 203. Favier R, Jondeau K, Boutard P, et al. Paris-Trousseau syndrome: clinical, hematological, molecular data of ten new cases. Thromb Haemost 2003;90:893–7. 204. Grossfeld PD, Mattina T, Lai Z, et al. The 11q terminal deletion disorder: a prospective study of 110 cases. Am J Med Genet A 2004;129:51–61. 205. Saultier P, Vidal L, Canault M, Bernot D, Falaise C, Pouymayou C, Bordet JC, Saut N, Rostan A, Baccini V, Peiretti F, Favier M, Lucca P, Deleuze JF, Olaso R, Boland A, Morange PE, Gachet C, Malergue F, Faur
e S, Eckly A, Tr
egouët DA, Poggi M, Alessi MC. Macrothrombocytopenia and dense granule deficiency associated with FLI1 variants: ultrastructural and pathogenic features. Haematologica 2017;102(6):1006–16. 206. Stockley J, Morgan NV, Bem D, Lowe GC, Lordkipanidz
e M, Dawood B, Simpson MA, Macfarlane K, Horner K, Leo VC, Talks K, Motwani J, Wilde JT, Collins PW, Makris M, Watson SP. Daly ME; UK genotyping and phenotyping of platelets study group. Enrichment of FLI1 and RUNX1 mutations in families with excessive bleeding and platelet dense granule secretion defects. Blood 2013;122(25):4090–3. 207. Lo B, Li L, Gissen P, Christensen H, et al. Requirement of VPS33B, a member of the Sec1/Munc18 protein family, in megakaryocyte and platelet alpha-granule biogenesis. Blood 2005;106(13): 4159–66. 208. Urban D, Li L, Christensen H, Pluthero FG, Chen SZ, Puhacz M, Garg PM, Lanka KK, Cummings JJ, Kramer H, Wasmuth JD, Parkinson J, Kahr WH. The VPS33B-binding protein VPS16B is required in megakaryocyte and platelet α-granule biogenesis. Blood 2012;120(25):5032–40. 209. Cullinane AR, Straatman-Iwanowska A, Seo JK, Ko JS, Song KS, Gizewska M, Gruszfeld D, Gliwicz D, Tuysuz B, Erdemir G, Sougrat R, Wakabayashi Y, Hinds R, Barnicoat A, Mandel H, Chitayat D, Fischler B, Garcia-Cazorla A, Knisely AS, Kelly DA, Maher ER, Gissen P. Molecular investigations to improve diagnostic accuracy in patients with ARC syndrome. Hum Mutat 2009; 30(2):E330–7.

Inherited Disorders of Platelet Function 210. Tracy PB, Giles AR, Mann KG, et al. Factor V (Quebec): a bleeding diathesis associated with a qualitative platelet factor V deficiency. J Clin Invest 1984;74:1221–8. 211. Hayward CP, Rivard GE, Kane WH. An autosomal dominant, qualitative platelet disorder associated with multimerin deficiency, abnormalities in platelet factor V, thrombospondin, von Willebrand factor, and fibrinogen, and an epinephrine aggregation defect. Blood 1996;87:4967–78. 212. Hayward CPM, Warkentin TE, Horsewood P, Kelton JG. Multimerin: a series of large, disulfide-linked multimeric proteins within platelets. Blood 1991;77:2556–60. 213. Hayward CPM, Bainton DF, Smith JW, et al. Multimerin is found in the α-granules of resting platelets and is synthsized by a megakaryocytic cell line. J Clin Invest 1993;91:2630–9. 214. Hayward CPM, Hassell JA, Denomme GA, et al. The cDNA sequence of human endothelial cell multimerin: a unique protein with RGDS, coiled-coil, and EGF-like domains and a carboxylterminus similar to the globular domain of complement C1q and collagens type VIII and X. J Biol Chem 1995;270:19217–24. 215. Kahr WH, Zheng S, Sheth PM, et al. Platelets from patients with the Quebec platelet disorder contain and secrete abnormal amounts of urokinase-type plasminogen activator. Blood 2001; 98:257–65. 216. Sheth PM, Kahr WH, Haq MA, et al. Intracellular activation of the fibrinolytic cascade in the Quebec platelet disorder. Thromb Haemost 2003;90:293–8. 217. Diamandis M, Adam F, Kahr WH, et al. Insights into abnormal hemostasis in the Quebec platelet disorder from analyses of clot lysis. J Thromb Haemost 2006;4:1086–94. 218. Janeway CM, Rivard GE, Tracy PB, Mann KG. Factor V Quebec revisited. Blood 1996;87:3571–3578. 219. Hayward CPM, Cramer EM, Kane WH, et al. Studies of a second family with the Quebec platelet disorder: evidence that the degradation of the α-granule membrane and its soluble contents are not secondary to a defect in targeting proteins to α-granules. Blood 1997;89:1243–53. 220. McKay H, Derome F, Haq MA, et al. Bleeding risks associated with inheritance of the Quebec platelet disorder. Blood 2004; 104:159–65. 221. Diamandis M, Paterson AD, Rommens JM, et al. Quebec platelet disorder is linked to the urokinase plasminogen activator gene (PLAU) and increases expression of the linked allele in megakaryocytes. Blood 2009;113(7):1543–6. 222. Paterson AD, Rommens JM, Bharaj B, et al. Persons with Quebec platelet disorder have a tandem duplication of PLAU, the urokinase plasminogen activator gene. Blood 2010;115(6):1264–6. 223. White JG, Key NS, King RA, Vercellotti GM. The white platelet syndrome: a new autosomal dominant platelet disorder. Platelets 2004;15:173–84. 224. White JG. Medich giant platelet disorder: a unique alpha granule deficiency I. Structural abnormalities. Platelets 2004;15:345–53. 225. Gunning W, Dole M, Brecher M, White JG. The Medich giant platelet syndrome: two new cases. Platelets 2013;24(2):107–12. 226. Weiss HJ, Lages B, Vicic W, et al. Heterogeneous abnormalities of platelet dense granule ultrastructure in 20 patients with congenital storage pool deficiency. Br J Haematol 1993;83:282–95. 227. Weiss HJ, Witte LD, Kaplan KL, et al. Heterogeneity in storage pool deficiency: Studies on granule-bound substances in 18 patients including variants deficient in α-granules, platelet factor 4, β-thromboglobulin, and platelet-derived growth factor. Blood 1979;54:1296–319. 228. Lages B, Shattil SJ, Bainton DF, Weiss HJ. Decreased content and surface expression of alpha-granule membrane protein GMP-140 in one of two types of platelet alpha delta storage pool deficiency. J Clin Invest 1991;87:919–20. 229. Vicic WJ, Weiss HJ. Evidence that platelet alpha-granules are a major determinant of platelet density: studies in storage pool deficiency. Thromb Haemost 1983;50:878–80. 230. White JG, Keel S, Reyes M, Burris SM. Alpha-delta platelet storage pool deficiency in three generations. Platelets 2007;18:1–10. 231. Adler DH, Cogan JD, Phillips 3rd JA, et al. Inherited human cPLA (2alpha) deficiency is associated with impaired eicosanoid biosynthesis, small intestinal ulceration, and platelet dysfunction. J Clin Invest 2008;118(6):2121–31. 232. Faioni EM, Razzari C, Zulueta A, Femia EA, Fenu L, Trinchera M, Podda GM, Pugliano M, Marongiu F, Cattaneo M. Bleeding

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PART IV Disorders of Platelet Number and/or Function mild bleeding disorder: deficiency of the inositol 1,4,5 triphosphate formation despite normal G-protein activation. Thromb Haemost 1997;77:991–5. Stefanini L, Paul DS, Robledo RF, et al. RASA3 is a critical inhibitor of RAP1-dependent platelet activation. J Clin Invest 2015;125 (4):1419–32. Canault M, Ghalloussi D, Grosdidier C, et al. Human CalDAGGEFI gene (RASGRP2) mutation affects platelet function and causes severe bleeding. J Exp Med 2014;211(7):1349–62. Lozano ML, Cook A, Bastida JM, Paul DS, Iruin G, Cid AR, AdanPedroso R, Ramón González-Porras J, Hernández-Rivas JM, Fletcher SJ, Johnson B, Morgan N, Ferrer-Marin F, Vicente V, Sondek J, Watson SP, Bergmeier W, Rivera J. Novel mutations in RASGRP2, which encodes CalDAG-GEFI, abrogate Rap1 activation, causing platelet dysfunction. Blood 2016;128(9):1282–9. Bergmeier W, Goerge T, Wang HW, et al. Mice lacking the signaling molecule CalDAG-GEFI represent a model for leukocyte adhesion deficiency type III. J Clin Invest 2007;117(6):1699–707. Cattaneo M. Inherited CalDAG-GEFI deficiency. Blood 2016;128 (9):1165–7. Kato H, Nakazawa Y, Kurokawa Y, Kashiwagi H, Morikawa Y, Morita D, Banno F, Honda S, Kanakura Y, Tomiyama Y. Human CalDAG-GEFI deficiency increases bleeding and delays αIIbβ3 activation. Blood 2016;128(23):2729–33. Westbury SK, Canault M, Greene D, Bermejo E, Hanlon K, Lambert MP, Millar CM, Nurden P, Obaji SG, Revel-Vilk S, Van Geet C, Downes K, Papadia S, Tuna S, Watt C, NIHR BioResource-Rare Diseases Consortium, Freson K, Laffan MA, Ouwehand WH, Alessi MC, Turro E, Mumford AD. Expanded repertoire of RASGRP2 variants responsible for platelet dysfunction and severe bleeding. Blood 2017;130(8):1026–30. Bermejo E, Alberto MF, Paul DS, Cook AA, Nurden P, Sanchez Luceros A, Nurden AT, Bergmeier W. Marked bleeding diathesis in patients with platelet dysfunction due to a novel mutation in RASGRP2, encoding CalDAG-GEFI (p.Gly305Asp). Platelets 2018;29(1):84–6. Sevivas T, Bastida JM, Paul DS, Caparros E, Palma-Barqueros V, Coucelo M, Marques D, Ferrer-Marín F, González-Porras JR, Vicente V, Hernández-Rivas JM, Watson SP, Lozano ML, Bergmeier W, Rivera J. Identification of two novel mutations in RASGRP2 affecting platelet CalDAG-GEFI expression and function in patients with bleeding diathesis. Platelets 2018;29(2):192–5. Malinin NL, Zhang L, Choi J, et al. A point mutation in KINDLIN3 ablates activation of three integrin subfamilies in humans. Nat Med 2009;15(3):313–8. Jurk K, Schulz AS, Kehrel BE, et al. Novel integrin-dependent platelet malfunction in siblings with leukocyte adhesion deficiency-III (LAD-III) caused by a point mutation in FERMT3. Thromb Haemost 2010;103(5):1053–64. McDowall A, Svensson L, Stanley P, et al. Two mutations in the KINDLIN3 gene of a new leukocyte adhesion deficiency III patient reveal distinct effects on leukocyte function in vitro. Blood 2010;115(23):4834–42. Svensson L, Howarth K, McDowall A, et al. Leukocyte adhesion deficiency-III is caused by mutations in KINDLIN3 affecting integrin activation. Nat Med 2009;15(3):306–12. Meller J, Malinin NL, Panigrahi S, Kerr BA, Patil A, Ma Y, Venkateswaran L, Rogozin IB, Mohandas N, Ehlayel MS, Podrez EA, Chinen J, Byzova TV. Novel aspects of Kindlin-3 function in humans based on a new case of leukocyte adhesion deficiency III. J Thromb Haemost 2012 Jul;10(7):1397–408. Harris ES, Smith TL, Springett GM, Weyrich AS, Zimmerman GA. Leukocyte adhesion deficiency-I variant syndrome (LAD-Iv, LADIII): molecular characterization of the defect in an index family. Am J Hematol 2012;87(3):311–3. van de Vijver E, De Cuyper IM, Gerrits AJ, Verhoeven AJ, Seeger K, Guti
errez L, van den Berg TK, Kuijpers TW. Defects in Glanzmann thrombasthenia and LAD-III (LAD-1/v) syndrome: the role of integrin β1 and β3 in platelet adhesion to collagen. Blood 2012;119(2):583–6. Dunkley S, Arthur JF, Evans S, et al. A familial platelet function disorder associated with abnormal signalling through the glycoprotein VI pathway. Br J Haematol 2007;137(6):569–77. Stormorken H, Sjaastad O, Langslet A, et al. A new syndrome: thrombocytopathia, muscle fatigue, asplenia, migraine, dyslexia and ichthyosis. Clin Genet 1985;28:367–74.

272. Stormorken H, Holmsen H, Sund R, et al. Studies on the haemostatic defect in a complicated syndrome: an inverse Scott syndrome platelet membrane abnormality? Thromb Haemost 1995;74:1244–51. 273. Misceo D, Holmgren A, Louch WE, Holme PA, Mizobuchi M, Morales RJ, De Paula AM, Stray-Pedersen A, Lyle R, Dalhus B, Christensen G, Stormorken H, Tjønnfjord GE, Frengen E. A dominant STIM1 mutation causes Stormorken syndrome. Hum Mutat 2014 May;35(5):556–64. 274. Nesin V, Wiley G, Kousi M, Ong EC, Lehmann T, Nicholl DJ, Suri M, Shahrizaila N, Katsanis N, Gaffney PM, Wierenga KJ, Tsiokas L. Activating mutations in STIM1 and ORAI1 cause overlapping syndromes of tubular myopathy and congenital miosis. Proc Natl Acad Sci USA 2014;111(11):4197–202. 275. Morin G, Bruechle NO, Singh AR, Knopp C, Jedraszak G, Elbracht M, Br
emond-Gignac D, Hartmann K, Sevestre H, Deutz P, H
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324. Al-Battat S, Rand ML, Bouskill V, Lau W, Blanchette VS, Kahr WHA, Rivard GE, Carcao MD. Glanzmann thrombasthenia platelets compete with transfused platelets, reducing the haemostatic impact of platelet transfusions. Br J Haematol 2018;181 (3):410–3. 325. Haddad E, Le Deist F, Blanche S, et al. Treatment of ChediakHigashi syndrome by allogenic bone marrow transplantation: report of 10 cases. Blood 1995;85:3328–33. 326. Filipovich AH, Stone JV, Tomany SC, et al. Impact of donor type on outcome of bone marrow transplantation for Wiskott-Aldrich syndrome: Collaborative study of the International Bone Marrow

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