Platelet storage temperature and response to agonists in PRP and washed platelets (WPs). We obtained human platelets by apheresis and used platelets either fresh (green triangles) or stored for 5 days at either 4°C (blue squares) or 22°C (RT; red circles). Aggregation was induced by stimulation with 5 µg/mL of collagen, 20 µM of ADP, or 0.5 mM of arachidonic acid (AA; shown as maximum aggregation; mean ± standard error of the mean [SEM]; n = 6-7). (A) PRP: collagen, ADP, and AA (left) and representative aggregation traces (right). (B) WPs: collagen, ADP, and AA (left) and representative aggregation traces (right). (C) GPVI levels on platelets determined by flow cytometry with fluorochrome-conjugated anti-GPVI antibody (n = 7; left), and β1 integrin levels on platelets determined by flow cytometry with fluorochrome-conjugated β1 antibody (n = 7; right). (D) Separate cohort of healthy volunteers, whose platelets were stored for 7 days at RT or 4°C under the same conditions as described for the original cohort. GPVI levels were determined using liquid chromatography–tandem mass spectrometry. Results are shown as fold change from baseline (fresh; n = 5). (E) PRP was diluted with separately stored plasma and stimulated with 100 ng/mL of convulxin. We stained with antibodies against activated integrin (PAC-1) and P-selectin (anti-CD62P; shown as change in mean fluorescence intensity [MFI] ± SEM from unstimulated; n = 5). *P < .5; **P < .01; ***P < .001. ns, not significant.