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Analysing Platelet Function in Blood Donors: Selection of Subjects for Functional Genetic Studies
Author(s) -
Garner S. F.,
Jones C. I.,
Angenent W.,
Bernard A.,
Carr P.,
Hogwood J.,
Rankin A.,
Stephens J.,
Tom B. D.,
Walton J.,
Dudbridge F.,
Ouwehand W. H.,
Goodall A. H.
Publication year - 2006
Publication title -
transfusion medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.471
H-Index - 59
eISSN - 1365-3148
pISSN - 0958-7578
DOI - 10.1111/j.1365-3148.2006.00693_14.x
Subject(s) - platelet , apyrase , fibrinogen , adenosine diphosphate , whole blood , medicine , platelet activation , hirudin , aspirin , immunology , pharmacology , endocrinology , andrology , thrombin , platelet aggregation
The objective of this study; performed as part of the BLOODOMICS project, was to develop a methodology to characterise the platelet response phenotype following agonist induced activation, which would then be used to test a cohort of healthy blood donors. Method The study consisted of three phases. Phase 1 evaluated a range of platelet function assays in 46 individuals to determine reproducibility and feasibility for a large cohort study. Calcium flux, flow cytometric detection of fibrinogen and Annexin V binding and P‐selectin expression, and aggregometry were used to measure responses to three concentrations of adenosine diphosphate (ADP) and cross linked collagen related peptide (XL‐CRP). From this study, whole blood flow cytometric measurement of fibrinogen binding and P‐selectin expression in response to a single sub‐maximal dose of ADP (10–7 M ) and XL‐CRP (0.1 μg mL ‐1 ) were selected for use in phase 2, during which the platelet responses were measured in 506 blood donors attending the Cambridge Centre. In these assays inhibitors of thrombin (hirudin), thromboxane (aspirin), and ADP (apyrase–XL‐CRP stimulated samples only) were included to ensure activation was pathway specific. Results There was a wide range of response to both XL‐CRP (fibrinogen binding 3.1%–84.4% +ve; P‐selectin expression 6.2%–90.3% +ve) and ADP (fibrinogen binding 3.5%–78.8% +ve; P‐selectin expression 2.8%–54.1% +ve). Three groups of individuals were selected; two groups with responses to both agonists and both endpoints that were at the ‘Extreme Ends’ of the normal range, (15 low and 16 high responders) and a third group of 16 identified as having mid‐range responses. In phase 3 these 47 donors were re‐tested to confirm the reproducibility of the original classification. There was a high level of concordance between the two visits for the fibrinogen‐CRP, fibrinogen‐ADP, P‐Selectin‐CRP, and P‐Selectin‐ADP assays ( P = 0.851, 0.794, 0.855 and 0.527 respectively). Conclusion We have demonstrated that different measures of the platelet response to two separate agonists are highly variable between healthy subjects, but are consistent over time within an individual. This standard method for defining platelet functional phenotype is being used in the BLOODOMICS programme to define the association between genetic variation and the risk of atherothrombosis.