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Although in vitro studies have demonstrated functional differences between young and old platelets, in vivo differences have not been precisely established. Therefore the in vivo hemostatic function of young and old platelets and the survival time have been examined in rabbits. The hemostatic function was measured by performing serial ear bleeding times in irradiation-induced thrombocytopenic rabbits. After irradiation with 930 rad the platelet count gradually diminished reaching a nadir (~20 × 103/μl) at 10 d. The platelets present in the circulation, 7-10 d after irradiation, were considered old platelets, and the platelets present after recovery, 11-14 d postirradiation, young platelets. The measurement of platelet size was consistent with the hypothesis that platelets become smaller with age: the mean size was 3.84 μm3 for old platelets and 5.86 μm3 for young platelets. Regression analysis of the relationship between the bleeding time and the platelet count in 18 rabbits showed a significantly different slope for rabbits with predominantly old platelets compared with rabbits with predominantly young platelets (P < 0.001). Young platelets were more effective giving much shorter bleeding times than old platelets at comparable platelet counts. Survival times of young and old platelets were measured using platelets harvested on day 8 postirradiation (old platelets) and day 12 postirradiation (young platelets) that were labeled and then reinjected into normal recipient animals. The mean platelet survival time, calculated by gamma function, of old platelets was 28.8 h; of young platelets, 87.4 h; and of normally circulating heterogeneous platelets, (normal platelets) 53.0 h. Notably, the survival of old platelets was found to be exponential, and of young platelets, linear. Analysis of the membrane glycoproteins in young, old and normal platelets indicated that there was no qualitative difference amongst the young, normal, and old platelets. The relative relationship among all the glycoprotein peaks was equal and the only changes observed were quantitative, with young platelets having significantly more membrane glycoprotein per cell than old platelets and normal platelets. Normal platelets had intermediate concentrations of each glycoprotein. These results demonstrate that young platelets are hemostatically more effective in vivo than old platelets. The data are compatible with the hypothesis that platelets age in the circulation by losing membrane fragments and then after becoming senescent, are removed from the circulation by a random process.