Functional Assessment of Platelet Dense Granule ATP Release

Abstract

Abstract 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 × 103/μL. 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.