Figure 1. RT storage caused lesions to platelets and cold storage induced platelet activation.

A and B, Establishment of an assay detecting life span of transfused platelets. C57BL/6J mice were injected retro-orbitally with washed platelets (2.5 × 10⁸ per mouse) from C57BL/6-Tg(CAGEGFP)1Osb/J mice. Blood (<60 μl each time) was collected from mice and platelets were analyzed by flow cytometry. Quantitative results were expressed as percentage of survived transfused platelets (B) (percentage of GFP positive cells/percentage of GFP positive cells at 5 min after transfusion; mean ± SD; n=5). C, PRP from C57BL/6J mice was stored at RT for 24 h or 48 h. PRP was warmed in 37°C for 15 min and injected retro-orbitally injected to 8 weeks old C57BL/6J mice (2.5 × 10⁸ in 0.2 ml per mouse). Blood samples were collected at various time points after transfusion. Platelets were isolated by centrifugation and analyzed by flow cytometry. Survival of the transfused platelets was normalized to the survival of transfused fresh platelets at 5 min after transfusion (n=4~6). D, Washed platelets (3 × 10⁸/ml) from C57BL/6J mice were stored at RT or 4°C for 24h. Platelet counts were measured with a HEMAVET HV950FS multispecies hematology analyzer at indicated time and shown as relative platelet numbers to the basal platelet count. E and F, Washed platelets from C57BL/6J mice were stored at RT or 4°C for 24 h. Integrin αIIbβ3 activation was analyzed by FITC-labeled fibrinogen binding using flow cytometry. A representative flow cytometry plots (E) and quantification of fibrinogen (Fg) binding relative to RT (F) were shown. G and H, Washed platelets from C57BL/6J mice were stored at RT or 4°C for 24 h. The amount of serotonin (G) and PF4 (H) in supernatant were measured as described in the methods. I, Washed platelets from C57BL/6J mice were stored at 4°C for 24 h. Platelets were centrifuged and VWF in supernatant was detected by Western blot with a rabbit polyclonal antibody. J, Washed platelets from C57BL/6J mice were stored at RT or 4°C for indicated time and solubilized with SDS-PAGE sample buffer. Phosphorylation of Src was detected by Western blot with an antibody recognizing phosphorylated Src. An antibody against β-actin was used to verify equal loading. Shown is a representative figure of three independent experiments. * p<0.01 by unpaired Student’s t-test (n=3–4).