A method for the separation of platelets on the basis of their size has been developed using counterflow centrifugation. Platelets were separated, free of plasma proteins and other cells, into seven subpopulations. The smallest-sized platelets, designated as Fraction 1, had a mean platelet volume (MPV) of 3.94 +/- 0.60 micrometer 3 (SD). Each successive fraction had a progressively larger MPV. The MPV for the largest-sized platelets, designated Fraction 7, was 8.19 +/- 0.64 micrometer 3. The MPV for the original platelets prior to fractionation was 6.57 +/- 0.61 micrometer 3. The mean density of Fraction 1 platelets was 1.067 +/- 0.002 g/cm3, while Fraction 7 had a mean density of 1.072 +/- 0.001 g/cm3. Transmission electron microscopy demonstrated that Fraction 1 had 4.3 +/- 0.9 dense bodies per platelet, and Fraction 7 had 12.6 +/- 2.4 dense bodies per platelet. Platelet LDH activity showed that the Fraction 1 platelets had 4.77 +/- 0.92 iu per 10(10) platelets; Fraction 7 platelets had 14.88 +/- 1.23 iu per 10(10) platelets. The LDH activity in the platelets before separation into subpopulations was 9.47 +/- 1.45 iu per 10(10) platelets. Platelet function was measured by ADP-induced aggregation, serotonin uptake, and thrombin-induced release. Progressively more rapid and more complete aggregation was observed as the platelet size increased over the seven fractions. Serotonin uptake was 4.2 times greater in the Fraction 7 platelets than in the Fraction 1 platelets. Quantitative release of serotonin following thrombin stimulation was significantly greater in the larger-sized platelets than in the smaller-sized platelets. The observed differences in platelet aggregation, dense body content, LDH activity, and serotonin uptake and release suggest that large platelets may be functionally more important than smaller platelets.

This study was undertaken to examine the interaction of platelet size and age in determining in vitro platelet function. Baboon megakaryocytes were labeled in vivo by the injection of 75Se- methionine. Blood was collected when the label was predominantly associated with younger platelets (day 2) and with older platelets (day 9). Size-dependent platelet subpopulations were prepared on both days by counterflow centrifugation. The reactivity of each platelet subpopulation was determined on both days by measuring thrombin-induced aggregation. Platelets were fixed after partial aggregation had occurred by the addition of EDTA/formalin. After removal of the aggregated platelets by differential centrifugation, the supernatant medium was assayed for remaining platelets and 75Se radioactivity. Comparing day 2 and day 9, no significant difference was seen in the rate of aggregation of a given subpopulation. However, aggregation was more rapid in the larger platelet fractions than in the smaller ones on both days. A greater percentage of the 75Se radioactivity appeared in the platelet aggregates on day 2 than on day 9. This effect was independent of platelet size, as it occurred to a similar extent in the unfractionated platelets and in each of the size-dependent platelet subpopulations. The data indicate that young platelets are more active than older platelets. This study demonstrates that size and age are both determinants of platelet function, but by independent mechanisms.

Most biologists working today have not considered the problem of how signal transduction events, which commit cells to energetically demanding processes such as growth and division, are connected to cellular metabolism. The primary reason for this is that we have believed for the last 30 or more years that the metabolism of cells is a homeostatic, self-regulating process that does not depend on any extracellular input. The traditional view is that a mammalian cell decides to take up nutrients whenever its bioenergetic and synthetic reserves are depleted. However, a considerable body of evidence now exists that challenges the notion that the nutrient uptake and metabolism of metazoan cells are cell-autonomous.

The relationship between platelet size and in vivo aging was investigated in the baboon using size-dependent platelet subpopulations separated by counterflow centrifugation. The separation characteristics, size, lactate dehydrogenase (LDH) activity, and dense- body content of the baboon platelet subpopulations were similar to those previously observed in studies of human platelets. Three independent labeling techniques were used: (1) in vivo labeling with 75Se-methionine, (2) in vitro labeling with 51Cr, and (3) in vivo labeling with 14C-serotonin. Maximal incorporation of all three labels showed a close correlation between the mean platelet volume (MPV) of each fraction and the platelet radioactivity. The onset of incorporation and rate of accumulation of 75Se-methionine were comparable in all fractions when corrected for differences in volume, suggesting that platelet size heterogeneity was present from the time of release of the platelets from the bone marrow. Survival studies using 51Cr and 14C-serotonin showed no translocation of the label from one fraction to another in the circulation over time. In vivo survival values for the three radionuclides showed a slight but significant correlation between the lifespan and the MPV of the fractions. The data suggest that large platelets were not younger platelets, but rather platelets with a longer life-span. Platelet size heterogeneity is the result of production factors in the bone marrow and not maturation in the circulation.