A Practical Guide to Haemostasis

Platelet Function Testing: Introduction


An evaluation of patients with abnormal bleeding symptoms requires an objective assessment of the bleeding history, a physical examination, a careful drug history and followed by laboratory investigation.  

Quantitative and qualitative platelet disorders are common amongst patients with abnormal bleeding and may be clinically indistinguishable from other haemostatic disorders.  It is probable that because of the complexity of platelets and the difficulty in investigating them, that we under diagnose disorders of platelet function.

Laboratory tests for platelet disorders may include:
     - Assessing platelet number and size [Mean Platelet Volume [MPV]]
     - Assessing platelet morphology – blood film
     - Screening tests of platelet function e.g. Activated Clotting Time [ACT], Bleeding Time [BT] and PFA-100
     - Light Transmission Aggregometry [LTA] e.g. classical Born aggregometry
     - Impedance platelet aggregometry
     - Plateletworks - for monitoring the response of platelets to anti-platelet therapy
     - PFA-100
     - PFA-200
     - Global Thrombosis Test
     - TEG/ROTEM
     - Assessment of platelet nucleotides
     - Measurement of Thromboxane metabolites by ELISA
     - Flow cytometry e.g. to quantitate the presence or absence of platelet membrane glycoproteins
     - Mutational analysis

The aim of this section is introduce you to the complexities of investigating platelet function and to provide some help in how to investigate a patient with a suspected platelet function abnormality. 

There are some excellent reviews on the subject of platelets and these are referenced in the relevant sections.


This is not a comprehensive list of platelet function disorders but it is useful to have a scaffold that you can fit the various platelet function disorders into:

Disorder Possible Diagnoses
Abnormalities of the platelet receptors for adhesive proteins [Disorders of platelet adhesion] Bernard Soulier Syndrome [BSS]
  - BSS
  - Velo-Cardio-Facial [VCF] syndrome
Glanzmann's Thrombasthenia [GTT]
  - Congenital Afibrinogenaemia [although not a primary platelet disorder – fibrinogen is required for platelet-platelet interaction
Von Willebrand Disease
  - VWD or Platelet-type VWD
Collagen receptor defects
Abnormalities of the platelet receptors for soluble agonists [released during platelet activation] TxA2 receptor defects
α-adrenergic defects
P2Y12 receptor defects
  - Inherited deficiencies
  - Drugs e.g. Clopidogrel
Abnormalities of platelet granules Dense-granule deficiency
Inherited or acquired α-granule deficiency
Gray Platelet Syndrome
Quebec platelet disorder
Abnormalities of Platelet Secretion or the Signal Transduction Pathways [Impaired secretion of granule contents] Primary Secretion Defects
  - Defects in aggregation which are similar to those seen in Storage Pool Disorders [SPD] but normal granule contents and normal TxA2 generation
Defects in the agonist receptors on the surface of platelets
  - Epinephrine
  - Thromboxane
  - ADP
  - Collagen
Defects in G protein activation [defective intracellular signalling]
Abnormalities of Arachidonic Acid metabolism COX deficiency
Thromboxane synthase deficiency
Disorders of the platelet procoagulant mechanism Scott Syndrome
Platelet cytoskeletal defects MYH9-related disorders
Wiskott-Aldrich Syndrome

Click HERE to see a more comprehensive summary.

Classification of Platelet Disorders

There are a number of approaches to the classification of Platelet Disorders.

1. The table above provides a simple approach.

2. The diagram below outlines a flow chart for the investigation of a suspected platelet disorder based upon the Mean Platelet Volume [MPV]. This is not a comprehensive list as new variants are identified frequently.

Classification of Platelet Disorders using the MPV
Click HERE for an additional view of this image.

The table below summarises some of the disorders from the image above.  See Refs for additional information

Disorder Arises from
Paris-Trousseau Thrombocytopaenia
Jacobsen syndrome - the latter is also known as the 11q terminal deletion disorder.
Approximately 80% of cases of Jacobsen syndrome have Paris-Trousseau thrombocytopaenia.
Deletion of a variable region on chromosome 11q that contains the transcription factor FLI1. This has an important role in platelet development. Associated with moderate thrombocytopenia; absent collagen-induced platelet aggregation and large granules due to fusion of the α-granules in 1% to 5% of circulating platelets.
RUNX1 Thrombocytopaenia RUNX1 Thrombocytopaenia is a rare hereditary disorder that is associated with thrombocytopaenia, platelet dysfunction and an increased risk of myeloid malignancies.
May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome and Epstein’s syndrome Arise from mutations in the MYH9 gene that leads to the production of a non-functional myosin-9 protein that cannot properly interact with other subunits to form myosin IIA. Platelets, which only express myosin IIA, are most affected by a lack of functional myosin-9, accounting for the thrombocytopenia seen in individuals with MYH9-related disorders.
Gray Platelet Syndrome (GPS)   A rare disorder characterised by a mild-moderate thrombocytopaenia and a decrease of absence of the platelet a-granules and the proteins contained within the α-granules. Individuals with GPS are at risk of developing Myelofibrosis.
Arises from mutations in NBEAL2. NBEAL2 encodes a protein that is expressed in platelets and megakaryocytes and is required for the development of platelet α-granules.
Inheritance appears to be autosomal recessive.
ANKRD26-related thrombocytopenia   Thrombocytopenia caused by a mutation in the ANKRD26 gene and which appears to lead to defective protein-protein interaction and possibly disordered apoptosis.
Inheritance is autosomal dominant.
RUNX1 Germline Mutations

RUNX1 encodes RUNX1 - a transcription factor that regulates the differentiation of hematopoietic stem cells into mature blood cells.
RUNX1 mutations are associated with thrombocytopaenia and an increased risk of haematological malignancies [AML or MDS].
Inheritance is autosomal dominant.
ETV6 Thrombocytopaenia ETV6 encodes a transcription factor ETV6 that is involved in the development/growth of a number of cells types including those of haematological origin. Mutations in ETV6 lead to thrombocytopenia and a predisposition to leukaemia.
TAR Syndrome TAR syndrome is due to mutations in the gene RBM8A and which encodes the protein - RNA-binding motif protein 8A. This protein appears to be involved in a number of intracellular functions including the transport of mRNA molecules from the nucleus of the cell to the sites of protein synthesis.
Inheritance is autosomal recessive.
GATA-1 mutations leading to X-linked thrombocytopaenia GATA-1 belongs to a family of X-linked transcription factors and has a fundamental role in the development of haematological cell lines. Mutations can lead to a variety of disorder including X-linked thrombocytopaenia.
Congenital amegakaryocytic thrombocytopenia [CAMT] arising from mutations in the MPL gene. CAMT is due to mutations in the MPL gene that encodes the Thrombopoietin (TPO) receptor (c-MPL) and which is expressed in hematopoietic stem cells and cells of the megakaryocyte lineage. Mutations affecting the c-MPL receptor can lead to a number of haematological problems depending upon the precise mutation.
Inheritance is autosomal recessive.
Congenital amegakaryocytic thrombocytopenia [CAMT] arising from mutations in the THPO gene. The THPO gene encodes the Thrombopoietin receptor and mutations have been associated with Congenital amegakaryocytic thrombocytopenia.
Inheritance is autosomal recessive.
Quebec Platelet Disorder Arises from a mutation in the PLAU gene that encodes urokinase plasminogen activator (uPA). The molecular defect of QPD leads to over-expression of uPA during megakaryopoiesis, generating platelets that contain active forms of uPA in the a-granules.
Inheritance is autosomal dominant.
FYB gene mutation [Rare] The FYB gene encodes the ADPA protein [Adhesion and Degranulation-Promoting Adaptor protein], a hematopoietic-specific protein involved that is involved in platelet activation, cell motility and proliferation. Mutation leads to thrombocytopaenia with a reduced MPV.
Inheritance is autosomal recessive.
GFI1B Mutations [Rare] GFI1B encodes the transcription factor GFI1B and which is involved in erythropoiesis and megakaryocyte development. Mutations lead to a macrothrombocytopaenia with a variable bleeding tendency. There is a reduction in platelet a-granule content, Fibrinogen and P-selectin.
Inheritance is autosomal dominant.
HOXA11 Mutations [Rare] HOXAII encodes a DNA-binding transcription factor. Mutations may lead to neonatal amegakaryocytic thrombocytopaenia and bone marrow failure in addition to various skeletal abnormalities.
Inheritance is autosomal dominant.
SLFN14-related thrombocytopenia The SLFN14 gene encodes SLFN14 (Schlafen Family Member 14) a protein which appears to be important in platelet formation and function,
Mutations in the SLFN14 gene leads to a moderate thrombocytopenia, enlarged platelets and decreased ATP secretion.
Inheritance is autosomal dominant.
SRC-related thrombocytopenia The SRC gene encodes a tyrosine kinase that is important in megakaryocyte and platelet signalling pathways. Mutations in the SRC gene have been associated with a macrothrombocytopaenia.
Inheritance is autosomal dominant.
PTPRJ-related thrombocytopaenia [Rare] The PTPRJ gene encodes the tyrosine phosphatase PTPRJ and which is expressed in platelets and megakaryocytes. Mutations lead to thrombocytopaenia, platelet dysfunction and small platelets through multiple defects in megakaryocyte biology.
Inheritance is autosomal recessive.
GALE Mutations [Rare] The GALE gene encodes the protein UDP-Galactose-4-Epimerase [GALE] and a mutation in this gene has been associated with a severe inherited thrombocytopenia possibly through disordered glycosylation and impaired megakaryocyte and platelet development.
Inheritance is autosomal recessive.
DIAPH1-related thrombocytopaenia [Rare] The DIAPH1 gene encodes the protein - Rho-effector diaphanous-related formin 1 (DIAPH1) and mutations in the gene have been associated with a macrothrombocytopaenia possibly due to reduced pro-platelet formation.
Inheritance is autosomal dominant.
ACTN1-related thrombocytopaenia The ACTN1 gene encodes the α-actinin-1 (ACTN1) protein. This protein has multiple roles in different cells types.
Platelet size in individuals carrying the mutation is variable and appears to be related to the specific underlying mutation.
Inheritance is autosomal dominant.
ITGA2B & ITGB3-related thrombocytopaenia The genes ITGA2B & ITGB3 encode for the components of the GPIIb/IIIa complex. Mutations lead to activation of the complex, which affects platelet cytoskeleton.
TUBB1-related thrombocytopenia The TUBB1 gene encodes the protein TUBB1 [β1 tubulin] which is a component of the microtubules expressed in megakaryocytes and platelets. Mutations disrupt microtubule assembly and result in defective pro-platelet formation. 
Inheritance is autosomal dominant.
TRPM7-related thrombocytopenia The TRPM7 gene encodes TRPM7, a protein that is involved in regulating calcium and magnesium homoeostasis and a kinase that modulates the activity of non-muscle myosin IIA. Mutations of the gene lead to impaired pro-platelet formation and a macrothrombocytopenia.
TPM4-related thrombocytopenia The gene TPM4 encodes the protein Tropomyosin 4. Tropomyosins are cytoskeletal proteins that are found in megakaryocytes and platelets.
Mutations that impair Tropomyosin 4 lead to a macrothrombocytopenia.
Inheritance is autosomal dominant.
CYCS-related thrombocytopenia The CYCS gene encodes Cytochrome c, a mitochondrial protein. Mutations appear to lead to thrombocytopenia by inducing the premature release of platelets by a proplatelet-independent mechanism.
Inheritance is autosomal dominant.
Stormorken syndrome A rare disorder caused by a mutation in the STIM1 gene which encodes the protein STIM1. The STIM1 protein regulates the entry of calcium ions into cells and in Stormorken syndrome a gain-of-function mutation leads to a protein that is constantly active leading to an influx of calcium ions and the premature destruction of platelets.
Inheritance is autosomal dominant.
York platelet syndrome A rare disorder characterised by a mitochondrial myopathy and disordered platelet structure and  function.  The disorder arises from a gain-of-function mutation in the STIM1 gene – see also Stormorken syndrome.
Platelets range in size from normal to very large.
Inheritance is autosomal dominant.
FLNA-related thrombocytopenia The FLNA gene located on the X chromosome, encodes Protein Filamin A - an important part of the cell cytoskeleton. Mutations in the FLNA gene result in defective proplatelet formation and a macrothrombocytopaenia.
MASTL-related thrombocytopaenia The MASTL [microtubule-associated serine/threonine-like kinase] gene encodes a microtubule associated serine/threonine kinase.  Mutations of the MASTL gene may lead to a thrombocytopaenia.
Inheritance is autosomal dominant.
PRKACG-related thrombocytopenia The PRKACG gene encodes the γ-catalytic subunit of a cAMP-dependent protein kinase and data suggests that the PRKACG protein plays a role in proplatelet formation.  Mutations in the PRKACG gene appear to affect both platelet formation and activation.
Inheritance is autosomal recessive.
IKZF5-related thrombocytopaenia IKZF5 is a novel transcriptional regulator of megakaryopoiesis and mutations in the IKZF5 gene have recently been shown to be associated with Thrombocytopaenia.
Inheritance is autosomal dominant.

Click HERE for a PDF that summarises some of the more common inherited disorders of platelet numbers/function and tests of platelet function.


3. Norris et al - Ref 28 - have proposed classifying the inherited disorders that affect platelet numbers and/or function into 3 groups:

Group   Examples
1 Disorders which affect only the platelet. Bernard Soulier Syndrome
Gray Platelet Syndrome etc
2 Disorders in which the platelet phenotype is associated with additional congenital defects. Wiskott-Aldrich Syndrome
TAR Syndrome etc
3 Disorders of platelet function but which are associated with an increased risk of additional disorders during life. MYH9-related disorders


See Norris et al [Ref 28] for additional information.