Functional Assessment of Platelet Dense Granule ATP Release: Optimization Using Minute Blood Volumes

Objectives: This study was undertaken to explore the feasibility of assessing platelet dense granule release in response to platelet stimuli, using less than 1 mL of whole blood (WB). Methods: Optimization of the luciferin-luciferase (LL) assay for ATP release, together with additional modifications, was applied to 1:10 diluted WB. Results: LL assay optimization using nonstirred 1:10 diluted WB resulted in dense granule ATP release in response to thrombin receptor-activating peptide (TRAP) of similar magnitude to that observed using stirred platelet-rich plasma. Stirring of the 1:10 diluted WB restored collagen-induced dense granule secretion. Addition of lyophilized, formalin-fixed platelets, together with stirring, restored dense granule secretion responsiveness to ADP. TRAP, ADP, and collagen all stimulated ATP release in 1:10 diluted WB under the optimized conditions of this study at levels close to those observed using platelet-rich plasma. Blood sample reconstitution experiments offer hope that this assay may prove robust down to WB platelet counts as low as 50 x 10(3)/mu L. Conclusions: Platelet dense granule release in response to a number of classic stimuli, including ADP, was accomplished from less than 1 mL WB with minimal specimen processing, using widely available reagents and instrumentation.