Ann Hematol (1992) 65:135-137 Annals of Hematology 9 Springer-Verlag 1992 Original article Platelet heterogeneity based on lactic dehydrogenase activity P. De Sole and B. Zappacosta Istituto di Fisiologia Umana, Servizio di Chimica Clinica, Catholic University, Rome, Italy Received December 31, 1991/Accepted July 9, 1992 Summary. Mean lactic dehydrogenase (LDH) activity of human platelets is about 5 nU/platelet, expressed on a cell volume basis, it is 0.5 nU/fl. Platelet separation by means of centrifugation on a Percoll discontinuous gradient gives different subpopulations with a clear-cut change of LDH at a mean platelet volume (MPV) of about 6 fl. The LDH content of platelets with a MPV > 6 fl is constant (about 0.5 nU/fl), while that of small platelets (3 < MPV < 6 fl) is inversely correlated with the MPV and reaches a value of 1.5-2.0 nU/fl. Because small platelets are granule-depleted, we suggest that platelet LDH activity be considered as an in vivo activa- tion index. Key words: Platelet - Lactic dehydrogenase Introduction Platelets are highly heterogeneous, both in volume and in density [4, 5, 12]. They can be separated, on the basis of their density, into different populations with biochemical and in vitro functional differences. The biological signifi- cance of platelet heterogeneity is a controversial topic and an updated discussion has recently been presented [8]. Some data in the literature seem to indicate that large, dense platelets are young platelets that become progres- sively small and light with age, by loosing portions of their cytoplasm during the activation process [3, 6, 7, 10]. Some other authors, however, have presented evidence that both size and density do not change with aging, but are related mainly to the megakaryocyte ploidy [2,9, 14, 15]. Unactivated platelets get their energy mainly from the glycolytic pathway and have a high lactic dehydrogenase (LDH) activity [1]. Moreover, we have shown that human Correspondence to: P. De Sole, Servizio di Chimica Clinica, Poli- clinico A. Gemelli, Lgo A. Gemelli 8, 00168 Rome, Italy platelets can vary their LDH content and, consequently, the rate of glycolytic flux [16]. In this paper we show that the LDH content of the platelet subpopulations obtained by Percoll sedimenta- tion is a nonlinear function of platelet volume. This find- ing sheds more light on platelet heterogeneity and can probably be used as an in vivo activation index. Materials and methods Subjects. Twelvenormal control subjects (20-33 years) were enrol- led in this study after their informed consent had been obtained. Chemicals. Digitonin (Carlo Erba, Milano, Italy) was prepared as a 0.02070 solution in water. Percoll was obtained from Pharmacia 0dppsala, Sweden). Stock isosmotic solution was obtained by adding 2 ml of 1.5 M NaC1 to 18 ml of PercoU; this was further diluted to 30%, 40~ 50070, and 60% (v/v) with saline solution to obtain gra- dients of different densities. Thereafter, 2 ml of each solution were carefully layered, one on the top of the other, in a plastic test tube [12]. Plateletpreparation, Citrate anticoagulated blood was centrifuged at 200xg and the platelets were isolated by repeated centrifugation and resuspension at 4~ in the incubation medium [16]. One milli- liter of the platelet suspension was layered on the Percoll gradient (8.0 ml) and centrifuged at 800 x g for 30 min at room temperature. After centrifugation, the first 1.0-ml aliquot was discarded and the further eight 1.0-ml aliquots were individually collected. The densi- ty values were controlled by means of the "Density Marker Beads" (Pharmacia). The mean values of the various aliquots were 1036, 1042, 1047, 1053, 1059, 1064, 1070, and 1076 mg/ml respectively. The platelet counts and the LDH measurements were done as previously reported [16], without any additional washing. No effect of Percoll on the LDH activity was found. The results for the platelet counts and the LDH measurements are the mean of the determinations performed on the different platelet subpopulations obtained from the 12 subjects studied. The analytical error for platelet count and LDH measurement was in all cases less than 5%. Results Figure 1 shows the platelet percent distribution as a func- tion of the different Percoll layers. More than 80~ of