DOG PLATELETS ACCUMULATE INTRACELLULAR FIBRINOGEN AS THEY AGE

The alpha granules of circulating platelets are dynamic structures tha t acquire endogenous and exogenous components by synthesis and uptake, respectively. The uptake of exogenous components is a result of eithe r receptor-mediated endocytosis or fluid-phase pinocytosis. Despite ma ny detailed studies on the function and content of alpha-granules, lit tle is known of the impact of platelet age on these organelles. In thi s report, we describe the use of platelet biotinylation to identify an d isolate aged platelets for the analysis of alpha-granule contents. W hen aged platelets were permeabilized and examined by flow cytometry u tilizing fluorescently labeled antibodies, two exogenously acquired pr oteins, fibrinogen, and immunoglobulin G, were found to increase signi ficantly with platelet age. The levels of intracellular fibrinogen wer e found to be elevated relative to control, 114 +/- 2% and 119 +/- 5% on days 4 and 5 postbiotinylation, respectively; the life span of dog platelets is 6.0 days. Intracellular immunoglobulin G content increase d similarly. Levels of two endogenously synthesized proteins, thrombsp ondin and beta-selectin, were not elevated in aged platelets. Confirma tion of the flow cytometric data was obtained by isolating aged, bioti nylated platelets by fluorescence-activated cell sorting and quantitat ing the fibrinogen levels with an ELISA assay. For platelets averaging 4.6 days of age, the fibrinogen level was elevated to 128 +/- 23% of the level for the entire platelet population. These data demonstrate t hat age-dependent changes in exogenously acquired alpha-granule protei ns do occur and that the uptake mechanism for these proteins is active through out the platelet life span. (C) 1994 Wiley-Liss, Inc.