BACKGROUND Previously, we reported that red blood cells (RBCs) stored in AS-5 accumulated proinflammatory substances during storage. We observed in those studies that supernates from nonleukoreduced (NLR) RBCs reduced mean anti-CD41a-fluorescein isothiocyanate (FITC) fluorescence on platelets (PLTs), indicative of decreased expression of glycoprotein (GP)IIb/IIIa on the PLT membrane. The objective of this study was to determine if supernates from stored RBCs impaired PLT aggregation as a consequence of reduction in GPIIb/IIIa expression. METHODS Leukoreduced (LR) and NLR RBC units were prepared in AS-5 and stored at 1 to 6 degrees C for 6 weeks. Supernates from RBC samples collected every 2 weeks were mixed with freshly collected type-matched blood and incubated for 30 minutes at 37 degrees C. PLTs in each incubated blood sample were evaluated for GPIIb/IIIa expression by flow cytometry and for aggregation response to collagen by whole blood aggregometry. RESULTS Supernates from stored NLR RBCs reduced CD41a-FITC fluorescence on PLTs by 15% to 31%. A reduction in fluorescence was induced by supernates of RBCs stored for 14 days and increased as storage time increased. Supernates from Day 42 NLR RBCs reduced the mean amplitude of PLT aggregation by 31% compared to Day 0 supernates and lengthened the time before onset of aggregation by 21%. In addition, amplitude correlated directly and lag time correlated inversely with CD41a-FITC fluorescence in all samples. Supernates from prestorage LR RBCs did not affect PLT CD41a-FITC fluorescence or aggregation response. CONCLUSIONS Substances that decrease expression of GPIIb/IIIa and inhibit PLT aggregation accumulate in NLR RBCs. Accumulation of this material is prevented by leukoreduction.