IN-VIVO BIOTINYLATION DEMONSTRATES THAT RETICULATED PLATELETS ARE THEYOUNGEST PLATELETS IN CIRCULATION

In both mice and humans, a subset of platelets can be identified that shows increased labeling with nucleic acid-specific fluorescent dyes, such as thiazole orange. Termed ''reticulated platelets,'' they have b een postulated to be platelets that have recently entered the circulat ion. Their numbers appear to reflect the rate of new platelet producti on in a number of clinical and experimental situations. To determine w hether reticulated platelets really are the youngest platelets in circ ulation and to estimate the length of time that they are identifiable after entering the circulation, we have employed a technique of ''in v ivo biotinylation'' in mice that labels the entire cohort of circulati ng cells with covalently bound biotin. Blood samples can then be doubl e-labeled with fluorescent avidin derivatives and thiazole orange, per mitting correlated measurement of both surface biotin content and nucl eic acid content. The biotinylation occurs rapidly, is complete within 30 minutes, is stable for several days, and does not appear to alter platelet function. The results show that within 24 hours after in vivo biotinylation, platelets appear in the circulation with decreased lev els of biotinylation and that these are the reticulated platelets. The estimated lifespan of reticulated platelets is 1.8 days, and the life span of all platelets by this method is 4.5 days, which is in agreemen t with estimates made by other methods.