Abstract
BackgroundWashing of platelets is an important procedure commonly used for experimental studies, e.g. in cardiovascular research. As a known phenomenon, responsiveness to adenosine diphosphate (ADP) is reduced in washed platelets, although underlying molecular mechanisms—potentially interfering with experimental results—have not been thoroughly studied.ObjectivesSince ADP mediates its effects via three purinergic receptors P2Y1, P2X1 and P2Y12, their surface expression and function were investigated in washed platelets and, for comparison, in platelet-rich-plasma (PRP) at different time points for up to 2 hours after preparation.ResultsIn contrast to PRP, flow cytometric analysis of surface expression in washed platelets revealed an increase of all receptors during the first 60 minutes after preparation followed by a significant reduction, which points to an initial preactivation of platelets and consecutive degeneration. The activity of the P2X1 receptor (measured by selectively induced calcium flux) was substantially maintained in both PRP and washed platelets. P2Y12 function (determined by flow cytometry as platelet reactivity index) was partially reduced after platelet washing compared to PRP, but remained stable in course of ongoing storage. However, the function of the P2Y1 receptor (measured by selectively induced calcium flux) continuously declined after preparation of washed platelets.ConclusionIn conclusion, decreasing ADP responsiveness in washed platelets is particularly caused by impaired activity of the P2Y1 receptor associated with disturbed calcium regulation, which has to be considered in the design of experimental studies addressing ADP mediated platelet function.
Highlights
Platelets play an important physiological role in hemostasis, wound healing or inflammation [1, 2]
P2Y12 function was partially reduced after platelet washing compared to PRP, but remained stable in course of ongoing storage
Decreasing adenosine diphosphate (ADP) responsiveness in washed platelets is caused by impaired activity of the P2Y1 receptor associated with disturbed calcium regulation, PLOS ONE | DOI:10.1371/journal.pone
Summary
Platelets play an important physiological role in hemostasis, wound healing or inflammation [1, 2]. They are involved in pathophysiological processes, and in this regard, the analysis of platelet function is an important issue for the understanding of diseases like atherosclerosis, cancer or diabetes. It is often required to provide fresh and functionally intact platelets, e.g. for the evaluation of molecular and celluar mechanisms leading to thrombogenesis or for pharmacological testing of antiplatelet drugs. Washing of platelets is an important procedure commonly used for experimental studies, e.g. in cardiovascular research. Responsiveness to adenosine diphosphate (ADP) is reduced in washed platelets, underlying molecular mechanisms—potentially interfering with experimental results—have not been thoroughly studied
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