Week 6 Coagulation Lecture 3 - Inherited Disorders of Platelets

Question 1

Clinical Features of Inherited Platelet Disorders

Answer 1

Most inherited platelet disorders present in childhood
Common clinical signs include:
- epistaxis
- petechiae
- ecchymoses
- gingival & cutaneous bleeding
- G.I. bleeding
- bleeding after trauma or surgery
Some platelet disorders have associated syndrome features that may suggest a specific diagnosis

Question 2

Platelet Disorders and their Related Features - Examples

Answer 2

Reduced or delayed pigmentation in Hermansky-Pudlak syndrome
Hearing loss, renal impairment and/or cataracts in MYH9-related disorders
Eczema, predisposition to infection and small platelets in X-linked thrombocytopenia and Wiskott Aldrich syndrome
Limb abnormalities in thrombocytopenia with absent radii syndrome

Question 3

Guidelines for Investigation of Inherited Platelet Disorders

Answer 3

Use needles between 19 & 21 gauge
The first 3-5 ml of blood should not be used
Blood should be collected into a 1/10 volume of trisodium citrate
All specimens must be maintained at room temperature

Question 4

Light Transmission Aggregometry

Answer 4

Measure the change in optical density (or light transmittance) over time of stirred PRP in cuvettes at 37C after addition of the agonists
The appropriate agonists must then be added directly to the PRP and not pipetted onto the side of the tube
Agonists are used to stimulate platelets under controlled conditions
Upon addition of agonists:
- platelets change shape from discs to more rounded forms with extended filipodia, resulting in a small decrease in light transmission
- followed by an increase in light transmission as the platelets aggregate
- secondary aggregation response observed with higher concentrations of ADP and epinephrine is due to TxA2 formation and secretion of granule contents
Platelet agglutination stimulated by ristocetin, which changes the conformation of plasma VWF allowing it to bind to GPIb– IX–V, is also measured by LTA

Question 5

Agonists Used for LTA

Answer 5

ADP
Epinephrine
Collagen (type I, tendon)
Arachidonic acid
Ristocetin

Question 6

When should Flow Cytometry be Used?

Answer 6

In the investigation or confirmation of GT, BSS and Scott syndrome
Investigate abnormalities in the collagen (GPVI and GPIa/IIa) and thrombin receptors (PAR-1)

Question 7

Measurement of Total & Released Nucleotides

Answer 7

When there is a high clinical suspicion of a platelet function defect, adenine nucleotides should be measured even if the aggregation is normal
If aggregation results suggest storage pool disease or a release defect, measure stored and released nucleotides

Question 8

First, Second and Third Step Tests for Platelet Physiology

Answer 8

First-step tests:
- blood smear
- LTA
- platelet granule release
- major platelet surface glycoproteins by flow cytometry
Second-step tests:
- LTA with an expanded agonist panel
- flow-cytometry with additional antibodies
- clot retraction
- measurement of serum TxB2 and TEM
Third-step tests:
- biochemical studies
- receptor binding assays
- molecular genetics

Question 9

Mechanisms of Disorders of Platelet Function

Answer 9

Abnormalities of receptors for adhesive proteins
Abnormalities of receptors for soluble agonists
Abnormalities of signal-transduction pathways
Abnormalities of platelet granules
Abnormalities of platelet cytoskeleton
Abnormalities of platelet phospholipids

Question 10

Abnormalities of Receptors for Adhesive Proteins

Answer 10

GPIb–IX–V complex
- Bernard–Soulier syndrome (BSS)
- ‘Platelet-type Von Willebrand Disease’
GPIIb-IIIa
- Glanzmann thrombasthenia (GT)

Question 11

Bernard-Soulier Syndrome

Answer 11

Characterised by thrombocytopenia, giant platelets and failure of platelets to undergo selective VWF-dependent platelet interactions due qualitative/quantitative abnormalities in the platelet GPIb-IX-V complex
- affects VWF-dependent adhesion of platelets to exposed vessel wall subendothelium
Significant mucocutaneous bleeding that presents in childhood
Results from mutations in the GP1BA, GP1BB, or GP9 genes

Question 12

Bernard-Soulier Syndrome - Results

Answer 12

FBP:
- moderate thrombocytopenia
- giant platelets
A prolonged closure time on the PFA-100 with both collagen-ADP and collagen-epinephrine cartridges
Platelet flow cytometry using monoclonal anti-CD42 antibodies measuring components of the GPIb–IX–V complex confirms the diagnosis

Question 13

Platelet-Type VWD (PT-VWD)

Answer 13

PT-VWD results from gain-of function mutations in the VWF binding domain of GPIbα, increasing its affinity for VWF
Promotes spontaneous interactions between VWF & GPIbα
May result in accelerated clearance of high molecular weight VWF multimers & platelets from the circulation
- => increased platelet aggregation response to low-dose ristocetin, loss of high molecular weight VWF multimers, circulating platelet aggregates & mild macrothrombocytopenia

Question 14

Glanzmann Thrombasthenia

Answer 14

Disorder caused by a quantitative or qualitative defect GPIIb/IIIa that impairs platelet aggregation
A classification based on the amount of GPIIb/IIIa on the platelet surface has been made:
- Type I contains 0-5%
- Type II 10-20%
- Type III 50-100% of the normal amount of GPIIb/IIIa
Caused by > 100 different mutations that alter the expression and/or function of the αIIbβ3 integrin complex at the platelet membrane
Caused by mutations in ITGA2B or ITGB3 genes encoding GPIIb and GPIIIa respectively

Question 15

Glanzmann Thrombasthenia - Results

Answer 15

Patients have a normal platelet count, size & morphology
PFA-100 measurements will be significantly abnormal with prolonged closure both collagen-ADP and collagen-epi cartridges
The disease is characterised by severely impaired platelet aggregation induced by all agonists, except (high) ristocetin
Flow cytometry using antibodies to GPIIb (CD41) and GPIIIa (CD61) confirms the diagnosis

Question 16

Abnormalities of Receptors for Soluble Agonists

Answer 16

Soluble platelet agonists e.g. ADP, TxA2 act via G-protein coupled receptors
Several hereditary disorders have been described including:
- Thromboxane A2 (prostanoid TP) receptor defect
- ADP (P2Y12) receptor

Question 17

Thromboxane-Prostanoid Receptor Defects

Answer 17

TxA2 is formed during platelet activation from arachidonic acid due to the actions of phospholipase A2, COX-1 and thromboxane synthase
Normal [platelet] & morphology
Severely impaired aggregation with arachidonic acid or TxA2 analog agonists
Due to mutation in TBXA2R gene on chromosome 19p13.3

Question 18

Abnormalities of Signal-Transduction Pathways

Answer 18

Affect intra-platelet biochemical processes that are triggered by the interaction of agonists with platelet receptors and result in aggregation and secretion
Signal transduction mechanisms encompasses:
- mechanisms initiated by agonist-receptor interactions
- responses including G- protein activation, activation of phospholipase C (PLC) & phospholipase A2 (PLA2)
Include defects of:
- cytosolic phospholipase A2α
- cyclooxygenase 1
- phospholipase C-β2 deficiency
- thromboxane synthetase
- tyrosine phosphorylase
- PKA
- G proteins

Question 19

Abnormalities of Platelet Granules

Answer 19

δ-granules
- Hermansky-Pudlak syndrome
- Chediak-Higashi syndrome
- δ-storage pool deficiency
α-granules
- Gray platelet syndrome
- Quebec platelet disorder
- Paris-Trousseau-Jacobsen syndrome
α- and δ-granules
- α, δ-storage pool deficiency

Question 20

Dense (δ) Granules

Answer 20

3-8/platelet
Storage sites for small molecules
- serotonin
- nucleotides ADP & ATP, GDP & GTP
- Ca2+
- magnesium
- polyphosphate

Question 21

Hermansky-Pudlak Syndrome (HPS)

Answer 21

Characterised by oculocutaneous albinism and platelet δ-granule deficiency
LTA shows sub-normal aggregation in response to collagen
Lack of δ-granules can be identified by electron microscopy
Defects in nine genes (HPS1, AP3B1, HPS3-6, DTNBP1, BLOC1S3, BLOC1S6) cause distinct HPS subtypes in man

Question 22

Chediak-Higashi Syndrome

Answer 22

Caused by mutation of the LYST gene located on chromosome 1 encoding a lysosomal-trafficking regulator
Patients with CHS have oculo-cutaneous albinism, platelet δ-granule deficiency, variable presence of abnormal giant granules in nucleated blood cells and megakaryocytes & neutropenia
The platelet function abnormality is due to quantitative or qualitative abnormalities of δ-granules
LTA shows sub-normal aggregation in response to collagen

Question 23

α-Granules

Answer 23

Membrane enclosed structures
These are the storage site for >300 proteins that are either synthesised in megakaryocytes or endocytosed from plasma
Specific deficiencies of α-granule stored proteins may be associated with inherited deficiencies of the corresponding plasma proteins (e.g. FV in factor V deficiency, fibrinogen in afibrinogenaemia, VWF in type 3 VWD)

Question 24

Gray Platelet Syndrome (GPS)

Answer 24

Characterised by the appearance of large platelets lacking α-granules and their contents
- large grey platelets
NBEAL2 (neurobeachin-like 2) was identified as being causative of GPS

Question 25

Abnormalities of Platelet Cytoskeleton

Answer 25

Several abnormalities of the platelet cytoskeleton have been recognised:
1. MYH9-related disorders
- May-Hegglin anomaly
- Sebastian syndrome
- Fechtner syndrome
- Epstein syndrome
2. Wiskott–Aldrich syndrome
3. X-linked thrombocytopenia

Question 26

May-Hegglin Anomaly

Answer 26

Most common of the giant platelet syndromes
Results from point mutations in the MYH9 gene
Giant platelets & thrombocytopenia
Neutrophil inclusions (similar to Döhle bodies)

Question 27

Wiskott-Aldrich Syndrome (WAS)

Answer 27

X-linked recessive disorder characterised by thrombocytopenia and small platelets
Also eczema, recurrent infections due to immune deficiency and an increased risk for autoimmunity and malignancy
WAS platelets aggregate poorly & have a low granule number
Caused by mutations in the WAS gene encoding the WAS protein
Platelets have markedly reduced α-granules, δ-granules & mitochondria

Question 28

Abnormalities of Membrane Phospholipids

Answer 28

Scott syndrome
Due to mutation(s) in TMEM16F gene coding for transmembrane protein 16
In this disorder, activated platelets are unable to translocate phosphatidylserine to the outer phospholipid leaflet of the membrane bilayer
As a result Factors Va and Xa fail to bind, leading to a decreased capacity of the platelets to convert prothrombin into thrombin
This lack of thrombin generation results in a haemorrhage