Week 6 Coagulation Lecture 3 - Inherited Disorders of Platelets Flashcards
Clinical Features of Inherited Platelet Disorders
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
Platelet Disorders and their Related Features - Examples
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
Guidelines for Investigation of Inherited Platelet Disorders
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
Light Transmission Aggregometry
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
Agonists Used for LTA
ADP
Epinephrine
Collagen (type I, tendon)
Arachidonic acid
Ristocetin
When should Flow Cytometry be Used?
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)
Measurement of Total & Released Nucleotides
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
First, Second and Third Step Tests for Platelet Physiology
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
Mechanisms of Disorders of Platelet Function
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
Abnormalities of Receptors for Adhesive Proteins
GPIb–IX–V complex
- Bernard–Soulier syndrome (BSS)
- ‘Platelet-type Von Willebrand Disease’
GPIIb-IIIa
- Glanzmann thrombasthenia (GT)
Bernard-Soulier Syndrome
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
Bernard-Soulier Syndrome - Results
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
Platelet-Type VWD (PT-VWD)
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
Glanzmann Thrombasthenia
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
Glanzmann Thrombasthenia - Results
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
Abnormalities of Receptors for Soluble Agonists
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
Thromboxane-Prostanoid Receptor Defects
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
Abnormalities of Signal-Transduction Pathways
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
Abnormalities of Platelet Granules
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
Dense (δ) Granules
3-8/platelet
Storage sites for small molecules
- serotonin
- nucleotides ADP & ATP, GDP & GTP
- Ca2+
- magnesium
- polyphosphate
Hermansky-Pudlak Syndrome (HPS)
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
Chediak-Higashi Syndrome
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
α-Granules
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)
Gray Platelet Syndrome (GPS)
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
Abnormalities of Platelet Cytoskeleton
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
May-Hegglin Anomaly
Most common of the giant platelet syndromes
Results from point mutations in the MYH9 gene
Giant platelets & thrombocytopenia
Neutrophil inclusions (similar to Döhle bodies)
Wiskott-Aldrich Syndrome (WAS)
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
Abnormalities of Membrane Phospholipids
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
