Figure 2. Cold storage-induced platelet activation and clearance required integrin activation, which involved the GPIb-IX signaling.

A, Washed platelets (3 × 10⁸/ml) from C57BL/6J mice were preincubated with vehicle or RGDS (1mM) for 15 min at RT and then stored at RT or 4°C for 24 h. 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. The statistical differences were examined by Student’s t-test. Values are means ± s.d. (n=3). *, p<0.05. B, Washed platelets from the C57BL/6-Tg(CAGEGFP)1Osb/J mice were preincubated with vehicle, RGDS (1 mM), or O-sialoglycoprotein endopeptidase (OSGE) for 15 min at RT and then stored at 4°C for 24 h. Platelets (2.5 × 10⁸ in 0.2 ml per mouse) were pre-warmed and injected retro-orbitally into C57BL/6J mice. 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). C, PRP from the C57BL/6-Tg(CAGEGFP)1Osb/J mice were preincubated with vehicle or RGDS (1 mM) for 15 min at RT and then stored at 4°C for 24 h. Platelets (2.5 × 10⁸ in 0.2 ml per mouse) were pre-warmed and injected retro-orbitally into C57BL/6J mice. Blood samples were collected at various time points after transfusion (n=4). D, Washed platelets (3 × 10⁸/ml) from integrin β3^−/− mice were stored at RT or 4°C for indicated time and platelet counts were measured. E, Washed platelets from the β3 deficient mice and WT controls were stored at 4°C for 24 h and then injected retro-orbitally into C57BL/6-Tg(CAGEGFP)1Osb/J mice. 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 maximal survival of transfused fresh platelets of the same genotype (n=4). Ratio of GFP negative platelets in total platelets in fresh group of the same genotype at 5 min after transfusion was set to 100% (survival 100%). F, Washed platelets from IL4Rα/GPIbα and WT controls were stored at 4°C for 24 h and then injected retro-orbitally into C57BL/6-Tg(CAGEGFP)1Osb/J mice. 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 WT platelets at 5 min after transfusion (n=4).