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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

Dense granules contain:

• ADP [ADP is concentrated in dense granules]

• ATP/GDP/GTP

• Serotonin

• Ca²⁺/Mg²⁺

Alpha-granules contain:

• Fibrinogen/fibronectin/VWF

• Factor V

• PF4

• PDGF/TGFß

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 is 8:1

  Dense Granules:        ATP:ADP ratio is 2:3

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

Principles

Dense granule release:

When platelets adhere to the damaged vascular endothelium, this leads to:

   → Activation of platelets through various intracellular signaling mechanisms
   → Release of alpha and denise granule contents including ADP and serotonin both of which lead to platelet activations
   → 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

Storage Pool Disorders [SPDs]

Dense granule deficiency:

• Hermansky-Pudlak Syndrome – rare although amongst Puerto Ricans has a prevalence of 1:800

• Chediak-Higashi syndrome

• Idiopathic Dense Deficiency

Acquired Disorders

• Myeloproliferative disorders [MPD]
• DIC
• CPD
• Thrombotic Thrombcytopenic Purpura [TTP]
• Haemolytic Uraemic Syndrome [HUS]

Characteristic Features of dense granule deficiency:

• Absent second wave aggregation [i.e. first wave only] with weak agonists:

• Low dose ADP.  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.

• Delayed and impaired response to collagen

• Impaired response to arachadonic acid and ristocetin

• 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.

• 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]

• Decreased numbers of or absence of dense granules on electron microscopy. 

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

• 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.

Measuring Platelet Nucleotides

1. 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.

2. Electron Microscopy: Dense granules are normally easily visible on EM and in Dense granule deficiency their absence is obvious .

3. Measuring platelet ADP and ATP content: Can be measured in a number of ways including HPLC.

4. Platelet dense granule release can also be measured by a ¹⁴C serotonin release assay.

Reference Ranges

Useful Links & References

1. Hardisty, R.M., Disorders of platelet secretion. Baillieres Clin Haematol, 1989. 2(3): p. 673-94.

2. Lages, B. and H.J. Weiss, Heterogeneous defects of platelet secretion and responses to weak agonists in patients with bleeding disorders. Br J Haematol, 1988. 68(1): p. 53-62.

3. Wall, J.E., et al., A flow cytometric assay using mepacrine for study of uptake and release of platelet dense granule contents. Br J Haematol, 1995. 89(2): p. 380-5.

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.