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A Practical Guide to Haemostasis


Platelet Function Testing:
Platelet Nucleotide Assays


Introduction

There are two separate nucleotide pools within platelets:
  - 60% is stored within the Dense granules and is not metabolically active
  - 40% constitutes the metabolic pool and provides the platelet with energy for its various activities.

There are differences in the relative concentrations of ADP and ATP in these two pools and exchange between the two pools is very slow:

  Metabolic Pool:          ATP:ADP ratio 8:1
  Dense Granules:        ATP:ADP ratio 2:3

Granules Composition
Dense Granules ADP [ADP is concentrated in Dense granules]
ATP/GDP/GTP
Serotonin
Ca2+/Mg2+

Dense granule release:
When platelets adhere to the damaged vascular endothelium, this leads to:
  → Activation of platelets through various intracellular signalling mechanisms
  → Release of alpha and dense granule contents including ADP and serotonin both of which lead to platelet activation
  → Generation and release of TxA2 [which binds to the Tx receptor]
  → Activation [conformational change in the GpIIb/IIIa receptor] facilitating  formation of the ‘tenase’ and ‘prothrombinase’ complexes
  → Exposure of anionic phospholipid - which allows formation of the 'tenase and 'prothrombinase' complexes
  → Generation of procoagulant microvesicles
Alpha Granules Fibrinogen/Fibronectin/VWF
Factor V
PF4
PDGF/TGFß


In Storage Pool Disorders [SPDs] in which there is a reduction or absence of the Dense bodies - this leads to a reduction in the total amount of ATP and ADP and a marked increase in the ratio of ATP to total platelet ADP.

Storage Pool Disorder

SPD Description
Dense granule deficiency Hermansky-Pudlak Syndrome – rare although amongst Puerto Ricans has a prevalence of 1:800
Chediak-Higashi syndrome
Idiopathic Dense Deficiency

Characteristic Features of Dense granule deficiency:

1. Light Transmission Aggregometry
Low dose ADP: Absent second wave aggregation [i.e. first wave only] with weak agonists.
High concentrations of ADP elicit full and irreversible aggregation.
Adrenaline: Absent second wave aggregation [i.e. first wave only] with adrenaline. However this also occurs in 10-15% of healthy individuals.
Collagen: Delayed and impaired response to collagen
Arachidonic Acid/Ristocetin: Impaired response to Arachidonic acid and Ristocetin

NB. LTA is relatively poor at detecting patients with SPD and measurement of platelet nucleotides, or Lumiaggregometry may be a more sensitive method for detecting these cases.

2. Marked reduction in both the content of ADP and the ADP:ATP ratio or an absence of ATP release [remember Dense granule release is a major amplification pathway for platelet activation and is necessary for the sustained activation of the GpIIb/IIIa receptor]

3. Decreased numbers of or absence of Dense granules on electron microscopy. 

4. Confirmation of Dense granule deficiency can also be made rapidly by detection of a reduction in mepacrine-labelled granules in platelets by flow cytometry.

NB. Disorders of platelet secretion and signal transduction can give similar LTA findings to SPD but platelet granules are present in normal numbers in these disorders – the problem lies with the release of the granule contents. Granule release is essential for second wave platelet aggregation and so whether the granules are deficient or not released properly – the effects seen in LTA are the same.

Acquired Disorders Myeloproliferative disorders [MPD]
DIC
Cardio-pulmonary bypass [CPB]
Thrombotic Thrombcytopenic Purpura [TTP]
Haemolytic Uraemic Syndrome [HUS]

 

Measuring Platelet Nucleotides

Technique  
Lumiaggregometry Lumiaggregometry is a modification of light transmission aggregometry which measures ATP release from the Dense granules, It is based on a bioluminescent determination of ATP in which the ATP reacts with luciferin and luciferase [firefly extracts] resulting in light emission. 
For measurement of aggregation, the lumiaggregometer uses an LED which emits light in the infrared range and changes in light transmission are detected by a phototransistor. For the measurement of luminescence resulting from ATP secretion, it uses a photomultiplier tube located at right angles to the light path of the LED.

Luciferin + ATP → Luciferyl Adenylate + Inorganic phosphate [PPi]


Luciferyl Adenylate + O2 → Oxyluciferin + AMP + LIGHT



Measurement of platelet secretion using the luciferin-luciferase reaction can also be evaluated with whole blood aggregometry.

Electron Microscopy: Dense granules are normally easily visible on EM and in Dense granule deficiency their absence is obvious .
HPLC Measuring platelet ADP and ATP content: Can be measured in a number of ways including HPLC.
Platelet Dense granule release Can also be measured by a 14C serotonin release assay.

 

Reference Ranges

Nucleotide Reference Range
Total platelet nucleotide content 5.5 - 9.6 nmol/108 platelets
ATP content of platelets 3.5 - 5.9 nmol/108 platelets
ADP content of platelets 1.9 - 3.8 nmol/108 platelets
Ratio 1.3 - 2.0
[>2 = abnormal]


What test next?

On the basis of an abnormal platelet nucleotides and additional platelet function tests, you should establish if this fits in with any recognisable disorder. Mutational analysis is now readily available to investigate abnormal platelet function in more detail.

A family pedigree should be constructed in all cases of a suspected platelet disorder - some of the rare platelet disorders are commoner in consanguineous relationships.