Platelet nucleotides
Description:
Approximately 30% of ATP in platelets, and most of the ADP, is contained within the dense granules. The ratio of total ATP to ADP is <2.6 in normal platelets but in dense body defects the ratio is elevated because of the reduced or absent ADP. The nucleotides are measured in a luminometric technique as desecribed below.
Supernatant from the platelet lysate that contains platelet ATP is incubated with the enzyme luciferase and its substrate luciferin, which generates adenyl luciferin and light. The amount of light generated is directly proportional to the ATP concentration. The ADP from the dense bodies is unstable and cannot be measured directly so it is converted to ATP in a separate reaction using the enzyme pyruvate kinase (PK). The action of PK on its substrate phosphoenolpyruvate converts ADP to ATP and generates the product pyruvate. The ATP in this tube is then measured using the same reaction as before and the ATP level should be higher than that in the first. The ADP concentration is calculated by subtracting the first ATP result from the second whereby the ATP:ADP ratio can then be obtained.
Supernatant from the platelet lysate that contains platelet ATP is incubated with the enzyme luciferase and its substrate luciferin, which generates adenyl luciferin and light. The amount of light generated is directly proportional to the ATP concentration. The ADP from the dense bodies is unstable and cannot be measured directly so it is converted to ATP in a separate reaction using the enzyme pyruvate kinase (PK). The action of PK on its substrate phosphoenolpyruvate converts ADP to ATP and generates the product pyruvate. The ATP in this tube is then measured using the same reaction as before and the ATP level should be higher than that in the first. The ADP concentration is calculated by subtracting the first ATP result from the second whereby the ATP:ADP ratio can then be obtained.
Clinical details:
Platelets are anucleate fragments of the cytoplasm of their parent cell, the megakaryocyte. They circulate predominantly at the margins of blood vessels in a dormant, resting state, but are capable of a rapid and dramatic response to various stimuli arising from vessel trauma. They have a complex structure that facilitates their specialised functions, of which the main ones are listed below:
● Interaction with collagen-captured VWF to form the initial barrier to blood loss
● Propagate the clot via platelet aggregation
● Provide the platform for secondary haemostasis
● Localisation mechanisms
● Maintain endothelial junction integrity
Exposure of sub-endothelial collagen after vessel trauma promotes binding of VWF which tethers platelets via their GpIb receptor. Blood flow rolls the platelet over where it forms stable associations via separate collagen binding receptors which also serves to activate the platelet. Activated platelets change their shape to promote effective physical interaction and release of the contents of cytoplasmic granules which activate more platelets and promote platelet-to-platelet aggregation via fibrinogen bridging of receptors on adjacent platelets. Biochemical pathways are also activated to promote aggregation, and the phospholipid membrane re-organises to promote localisation of secondary haemostasis to stabilise the platelet plug.
Reduced platelet numbers, receptor deficiency/dysfunction, granule deficiency or granule content deficiency, biochemical abnormalilties and drug interactions can lead to bleeding disorders. Adenine nucleotides are found in platelet cytoplasm and dense granules and their measurement contributes to diagnosis of storage pool disorders affecting platelet function.
● Interaction with collagen-captured VWF to form the initial barrier to blood loss
● Propagate the clot via platelet aggregation
● Provide the platform for secondary haemostasis
● Localisation mechanisms
● Maintain endothelial junction integrity
Exposure of sub-endothelial collagen after vessel trauma promotes binding of VWF which tethers platelets via their GpIb receptor. Blood flow rolls the platelet over where it forms stable associations via separate collagen binding receptors which also serves to activate the platelet. Activated platelets change their shape to promote effective physical interaction and release of the contents of cytoplasmic granules which activate more platelets and promote platelet-to-platelet aggregation via fibrinogen bridging of receptors on adjacent platelets. Biochemical pathways are also activated to promote aggregation, and the phospholipid membrane re-organises to promote localisation of secondary haemostasis to stabilise the platelet plug.
Reduced platelet numbers, receptor deficiency/dysfunction, granule deficiency or granule content deficiency, biochemical abnormalilties and drug interactions can lead to bleeding disorders. Adenine nucleotides are found in platelet cytoplasm and dense granules and their measurement contributes to diagnosis of storage pool disorders affecting platelet function.
Units:
ATP & ADP: nmoles/108 platelets
Department:
Location:
Sample type and Volume required:
Contact laboratory
Turnaround time:
Contact laboratory
Contacts:
Diagnostic Haemostasis and Thrombosis Department
St Thomas': 020 7188 2797; Guy's: 020 7188 7188 ext. 53860
St Thomas' Hospital
North Wing - 4th and 5th Floors
Westminster Bridge Road
London SE1 7EH
Laboratory opening times
24/7
Guy's Hospital
Southwark Wing - 4th Floor
Great Maze Pond
London SE1 9RT
Outside core hours, contact Duty Haemostasis Biomedical Scientist
North Wing - 4th and 5th Floors
Westminster Bridge Road
London SE1 7EH
Laboratory opening times
24/7
Guy's Hospital
Southwark Wing - 4th Floor
Great Maze Pond
London SE1 9RT
Outside core hours, contact Duty Haemostasis Biomedical Scientist
Last updated: 07/08/2015
