Low-density Platelet Research Articles

Diabetes type 2: relationships between lysosomal exocytosis of circulating normal-sized platelets and in vitro ɑ-thrombin-evoked platelet responses

Background/objective Type 2 diabetes is a major risk factor for atherosclerotic disease. It is well agreed that the reactivity of diabetic platelets is increased but how platelet reactivity regulates is unknown. In our laboratory, density separated platelets have been investigated extensively and high- and low-density platelets circulate in an activated state. The density distribution of circulating platelets is altered in diabetes type 2 as well. We hypothesize that such platelets modify whole blood (WB) in vitro α-thrombin-evoked (10 μM/mL) activity in type 2 diabetes. Thus, the study aims to identify features of circulating normal-sized density subpopulations affecting whole blood (WB) platelet reactivity in type 2 diabetes. Patients and methods Patients with type 2 diabetes (n = 16) were enrolled. Their normal-sized platelets were divided into density subfractions (n = 16) using continuous polyvinylpyrrolidone-coated silica (Percoll™) gradients (density span, 1.090–1.040 kg/L) containing prostaglandin E1. The proportions (%) of such density-separated platelets expressing lysosomal-associated membrane protein 1 (LAMP-1) were analyzed using a flow cytometer. Further, determinations of WB ɑ-thrombin-evoked (10 U/mL) surface LAMP-1 (an assessment of lysosomal release), the fibrinogen (αIIbβ3) receptor activity, annexin V (binds to exposed membrane phosphatidylserine), and mitochondrial transmembrane potentials (an estimate of organelle integrity) were performed. Surface LAMP-1 expressions of individual normal-sized platelet density subpopulations were stratified into equal-sized groups (n = 2) depending on reactivity, as judged from the ɑ-thrombin-induced WB activity markers. Results With some exceptions, the proportion of normal-sized circulating platelets showing spontaneous LAMP-1 was strongly associated with WB ɑ-thrombin-evoked (10 U/mL) surface LAMP-1 and αIIbβ3 receptor activity. LAMP-1-expressing normal-sized platelets also displayed inverse associations with WB ɑ-thrombin-induced surface annexin V and mitochondrial damage, which are features of procoagulant platelets. Conclusions From the current descriptive work only involving type 2 diabetes, it is impossible to judge whether the findings are features of the disease or if they occur in healthy individuals as well. However, the study describes LAMP-1 expressing subpopulations of circulating normal-sized platelets that associate with WB α-thrombin (10 U/mL) responses in vitro. Increased proportions of such platelets induced lysosomal release and αIIbβ3 receptor activity, whereas lower proportions promoted WB agonist-induced procoagulant platelet creation. It is to hypothesize that the new described regulatory mechanism could in the future offer a possibility to influence platelet behavior in type 2 diabetes. Key messages Lysosomal exocytosis of circulating platelets influences reactivity, as determined by agonist-induced platelet reactions in vitro Thus, the low release of lysosomes by normal-sized platelets in vivo increases agonist-evoked procoagulant platelet production. Higher lysosomal exocytosis of circulating normal-sized platelets promotes platelet aggregation and secretion.

Alzheimer and platelets: Low-density platelet populations reveal increased serotonin content in Alzheimer type dementia

IntroductionAlzheimer's disease (AD) is a progressive form of dementia characterized by an increase in the toxic substance β-amyloid in the brain. Platelets display a substantial heterogeneity with respect to density. They further contain a substantial amount of β-amyloid precursor protein. Platelets take up and store serotonin (5-HT) that plays an important role in the pathogenesis of severe depression. The current study aims to investigate platelet serotonin content in different platelet density populations. Material and methodsThe study involved 8 patients (age 70±8 (SD) years) (3 females/5 males) with moderate AD. 6 healthy elderly subjects (age 66±9 (SD) years) (3 females/3 males) served as controls. The platelet population was divided into 17 subpopulations according to density, using a linear Percoll™ gradient. Platelets were counted in all fractions. After cell lysis an ELISA technique was employed to determine the 5-HT content in each platelet subfraction. ResultsThe two study groups did not differ significantly regarding platelet distribution in the gradients, but AD sufferers have a significantly higher 5-HT content (p<0.05) in the lighter platelet populations. DiscussionAD-type dementia proved to be associated with lighter platelets containing more 5-HT. It is possible that platelets from AD patients release less 5-HT. It is speculated that AD synapses are affected in a manner comparable to platelets, which could explain why 5-HT reuptake inhibitors are less effective in AD dementia.

Low-density platelet populations demonstrate low in vivo activity in sporadic Alzheimer disease

Platelets contain a substantial quantity of amyloid-precursor protein (APP) and β-amyloid. However, despite the large importance of APP and β-amyloid to dementia, little is known about platelets in sporadic Alzheimer dementia (AD). Furthermore, platelet heterogeneity influences human pathology and has been described to affect the progression of AD. This study investigated AD platelets with respect to density diversity and in vivo activity associated with density sub-fractions. We included 39 AD patients and used, as controls, 22 elderly individuals without apparent memory disorder. A continuous Percoll™ gradient covering the density span 1.04–1.09 kg/l provided the basis to divide platelets of whole blood into density fractions (n = 16). All platelet populations were evaluated accordingly. Platelet counts were determined electronically. A flow-cytometer was put to use to measure surface-bound fibrinogen as a measure of platelet in vivo activity. Samples obtained from patients diagnosed with sporadic AD contained platelets (fractions numbers 4–16) that circulated with significantly less surface-bound fibrinogen, i.e., their platelet activation in vivo was reduced, compared with controls. In particular, highly significant differences (p < 0.001) were obtained for the six less dense platelet populations (fractions numbers 11–16) when comparing sporadic AD with controls. In contrast, the densest AD platelets in fractions numbers 1–3 did not differ significantly from control cells with respect to in vivo platelet-bound fibrinogen. It is concluded that sporadic AD is characterized by lower density platelet populations that, while circulating, exhibited reduced activation. The clinical significance of this finding is unclear but these results suggest the importance of platelet heterogeneity in dementia as a topic for further investigation.

Identification of low-density platelet populations with increased reactivity and elevated α-granule content

The present study examines biochemical and functional characteristics of platelet density subpopulations together with their ability to mobilise intracellular fibrinogen when activated. Platelets from three healthy volunteers were investigated. The total platelet population was separated according to density in a linear Percoll™ gradient in a plasma-free milieu containing EDTA that binds soluble Ca 2+. Subsequently, platelets from each individual were divided according to density into 11 or 12 aliquots. In all fractions, we determined platelet count, intracellular P-selectin and the ADP-evoked platelet fibrinogen binding as a measure of platelet reactivity together with the platelet dense body content. The work demonstrates that platelets use stored intracellular fibrinogen when activated. It also shows that the platelet-fibrinogen binding can be initiated in a surrounding depleted of Ca 2+ and fibrinogen. Moreover, the study demonstrates subpopulations of light platelets having increased reactivity and more α-granules but less dense bodies. The biological significance of the findings needs to be elucidated.

Platelet sialic acid as a potential pathogenic factor in coronary heart disease

It was previously reported that, compared to healthy individuals, patients with coronary heart disease (CHD) exhibit a higher proportion of platelets with lower densities and higher propensity to aggregate. Reasons for this increased tendency to aggregate were unknown but appeared to be independent of the patient's age, gender, or smoking habits. Sialic acid ( N-acetyl-neuraminic acid), a negatively charged sugar and constituent of many glycoproteins and gangliosides, is known to confer the bulk of negative charges to mammalian cell surfaces. These negatively charged surfaces can help cells of the bloodstream to maintain a relative distance from each other due to repulsion of the same (negative) charges. In this study, we examined whether differences in platelet sialic acid are a potential pathogenic factor in patients with coronary heart disease. Upon isolating platelets, we found a significantly higher ( p<0.05) proportion of low density platelets in patients compared to healthy controls, which is in accordance with previously published data. We found significantly less ( p<0.05) sialic acid in platelets from patients compared to the control. Most of the platelet total sialic acid was susceptible to cleavage by neuraminidase, demonstrating sialic acid to be preferably localized at the outer platelet surface. We conclude that the lower sialic acid content found in platelets from CHD patients could represent a contributing factor for the observed higher aggregability of platelets from these patients. Due to the lower sialic acid content and resulting lower negative surface charge, less repulsion between the platelets could facilitate aggregation.

Analysis of GTP-binding proteins, phosphoproteins, and cytosolic calcium in functional heterogeneous human blood platelet subpopulations

The biochemical basis for the functional heterogeneity of human blood platelets was investigated in terms of protein phosphorylation, cytoplasmic calcium ([Ca 2+] i), the ratio of 46 and 50 kDa vasodilatorstimulated protein (VASP), and GTP-binding proteins (G-proteins). Platelets were fractionated by density. Comparing resting low-density platelets (LDP) to high-density platelets (HDP) revealed higher phosphorylation of proteins in the 47, 31, and 24 kDa ranges. A higher phosphorylation of the 20 kDa protein in LDP compared to HDP was related to an enhanced [Ca 2+] i, an increased ADP-ribosylation of the inhibitory G-protein (G iα1–3) and rhoA, and a decreased ADP-ribosylation of the stimulatory G-protein (G sα). The differences in the ribosylation patterns of the subpopulations were not influenced by thrombin stimulation or exposure to prostaglandin E 1 (PGE 1). An 18 kDa phosphoprotein was more highly phosphorylated in resting HDP than in LDP. Thrombin exposure caused dephosphorylation of the 18 kDa phosphoprotein in the HDP, but generally increased phosphorylation of both HDP and LDP in the 47, 31, 24, and 20 kDa bands. Preincubation with prostaglandin E 1 or sodium nitroprusside diminished the subsequent thrombin-induced increase in phosphorylation, particularly in HDP. In unstimulated HDP, the 50 kDa VASP phospho form was enhanced, whereas in unstimulated LDP the 46 kDa VASP dephospho form was increased. Our findings suggest that the functional heterogeneity of platelets is partly derived from differences in signal transduction mechanisms reflected in varying phosphoprotein patterns and G-protein properties of platelet stimulatory and inhibitory pathways.

Involvement of guanylate cyclase and phosphodiesterases in the functional heterogeneity of human blood platelet subpopulations

Cyclic nucleotides are involved in the regulation of platelet activation, shape change, and aggregation. In this study we have investigated the role of guanylate cyclase and phosphodiesterase in three functional heterogeneous human blood platelet subpopulations separated according to density. In low-density platelets aggregation was enhanced and inhibited less when cyclic GMP was increased by sodium nitroprusside, compared to high-density platelets. Low-density platelets possessed a lower basal level of cyclic GMP and exhibited a smaller increase in cyclic GMP after stimulation with sodium nitroprusside. Cyclic GMP-dependent phosphodiesterase activity was similar in high, low, and intermediate-density platelets. In contrast, the activity of the cyclic AMP-dependent phosphodiesterase was higher in low-density compared to high and intermediate-density platelets. These results suggest that regulation of cyclic GMP and cyclic AMP levels plays an important role in the functional heterogeneity of human blood platelets.

Increased number of high sensitive platelets in hypercholesterolemia, cardiovascular diseases, and after incubation with cholesterol

The number of low density platelets was found to be increased in patients with hypercholesterolemia, as compared with the number in controls. The percentage increase of the low density platelet subpopulation was even more pronounced in patients with hypercholesterolemia when compared with that in patients suffering from myocardial infarction or angina. In vitro studies with control platelets incubated with cholesterol rich liposomes showed also an increase in the subpopulation of low density platelets. After incubation of control platelets with cholesterol rich liposomes, a higher membrane anisotropy and a higher cholesterol to phospholipid (C/P) molar ratio of the plasma membrane were found. Furthermore, cholesterol-enriched platelets were more sensitive upon thrombin stimulation. The results suggest that a shift of platelet subpopulations to a higher number of low density platelets could be caused by either the level of plasma cholesterol or an in-vitro incubation with cholesterol rich liposomes.

Daily variations of functional parameters and density distribution in human blood platelets.

Blood platelets play a critical role in the onset of myocardial infarction, which has been shown to have a circadian rhythmicity with a peak incidence in the morning. In an attempt to correlate platelet parameters with the outcome of cardiovascular diseases, we studied the daily (24-h) variation of the following platelet parameters: distribution pattern of functional heterogeneous platelet subpopulations; serotonin uptake; ketanserin binding; aggregation upon thrombin, serotonin, and ADP stimulation; and platelet count. Furthermore, we analyzed the tryptophan and serotonin concentrations in the blood samples. The percentage of less dense platelets, which represent the subpopulation with the highest preactivation, showed a rhythmicity period of 24 h and an acrophase at 21:18 h. The time course of intermediate and high density platelets was inverse to that of low density platelets. The serotonin uptake exhibited also a rhythmicity with a 24-h period. The acrophase was at 13:50 h. The aggregation curves were inverse to the ketanserin binding curves. The serotonin concentration exhibited a 12-h rhythmicity. The results obtained suggest that (a) changes in platelet activity are reflected by several parameters of platelet function that underlie daily variations; (b) the aggregation curves show a peak in the morning, with an additional peak in the afternoon; and (c) changes in the distribution pattern occur independently from variations in platelet functions like aggregation and serotonin binding.

Plasma membrane properties in heterogeneous human blood platelet subfractions modulate the cellular response at the second messenger level

Three subfractions of human blood platelets differing in density and function exhibited also differences when EPR (electron paramagnetic resonance) spin label measurements and fluorescence polarization were performed. The membrane anisotropy in the plasma membranes was higher in low density platelets as compared with that of platelets of intermediate and higher densities. This higher plasma membrane anisotropy correlated with a significantly higher cholesterol-phospholipid (C:P) molar ratio in the plasma membranes of low density platelets. As compared with the other platelet subfractions the low density platelets exhibited the smallest cAMP increase after activation with the aggregation inhibitor prostaglandin (PGE 1), and the highest percentage of inositol phosphate accumulation after thrombin stimulation. The results suggest a high correlation between functional parameters and the plasma membrane architecture of human blood platelets.

Correlation of membrane anisotropy with function in subpopulations of human blood platelets*

Human blood platelets were fractionated on a discontinuous Percoll gradient into high density (HD), intermediate (ID), and low density (LD) platelets. The subpopulations were characterized with regard to [14C]serotonin uptake and release, cAMP content, aggregation, and membrane anisotropy, which is inversely related to membrane fluidity. Membrane anisotropy, which was high in LD platelets, was found to decrease with increasing density (LD greater than ID greater than HD). LD platelets showed significant lower cAMP levels and [14C]serotonin uptake than the total platelet population (TPP) and ID and HD platelets. Upon ADP and serotonin stimulation the cAMP content was reduced in all platelet populations with the exception of HD platelets in which cAMP was unchanged. Upon thrombin stimulation the cAMP content was reduced only in TPP and LD platelet population and it was increased in HD platelet population. Thrombin activation changed the anisotropy only in LD platelets. Thrombin at a concentration of 0.001 U/ml reduced whereas 0.01 and 0.05 U thrombin/ml increased the membrane anisotropy significantly. As compared with TPP and the other subpopulations, LD platelets were most sensitive upon ADP and thrombin stimulated [14C]serotonin release as well as upon ADP, serotonin and thrombin induced aggregation. The findings suggest that the differing functional abilities of the platelet subpopulations are correlated to the various membrane anisotropies observed in these fractions.

Subcellular composition of platelet density subpopulations prepared using continuous Percoll gradients

Normal human platelets have been separated by density on continuous Percoll gradients and the subcellular composition of platelets of different density has been analysed. The number and concentration of dense granules increased significantly with platelet density, as did the concentrations of the dense granule constituents calcium and serotonin. The amount of serotonin per granule in the low density (LD) platelets was only two-thirds of the corresponding amount in the high density (HD) platelets. Platelets of all densities were able to sequester exogenous serotonin and release it in response to thrombin stimulation with similar efficiencies. The concentrations of the alpha-granule constituents von Willebrand factor and beta-thromboglobulin increased significantly with platelet density but the concentrations of the lysosomal enzyme beta-N-acetylglucosaminidase and total sialic acid did not differ significantly in the density subpopulations. The concentrations of the cytosolic enzymes lactate dehydrogenase and glucose-6-phosphate dehydrogenase were slightly higher in the LD population than in the other density subpopulations. The concentration of glycogen showed a marked positive relationship with platelet density and calculations suggested that glycogen was an important determinant of platelet density heterogeneity. The findings of the present study are compatible with recent suggestions that LD platelets may be slightly younger than HD platelets in normal human subjects.

Variation of Class I HLA Antigen Expression Among Platelet Density Cohorts: A Possible Index of Platelet Age?

Platelet alloantigens and other surface markers were studied in platelet cohorts of different mean density, using monoclonal and polyclonal probes. High density (HD) platelets expressed 12% more P1A1 molecules (46,942) than low density (LD) platelets (41,892). However, LD platelets carried 42% more HLA-A2 molecules (6,267 +/- 184) than HD platelets (4,406 +/- 232) (P less than .01) and 55% more class I HLA antigens (17,034 +/- 2,062 v 11,007 +/- 2,190) (P = .05). The platelet subpopulations did not differ in their content of glycoprotein (GP)IIb/IIIa complex or Baka antigen. The difference in expression of class I HLA antigens on HD and LD platelets is consistent with two possibilities: either class I HLA molecules are acquired from plasma or they are released into plasma as platelets age in circulation. Accordingly, class I HLA molecules may provide a useful marker of platelet age.

Variation of class I HLA antigen expression among platelet density cohorts: a possible index of platelet age?

Platelet alloantigens and other surface markers were studied in platelet cohorts of different mean density, using monoclonal and polyclonal probes. High density (HD) platelets expressed 12% more P1A1 molecules (46,942) than low density (LD) platelets (41,892). However, LD platelets carried 42% more HLA-A2 molecules (6,267 +/- 184) than HD platelets (4,406 +/- 232) (P less than .01) and 55% more class I HLA antigens (17,034 +/- 2,062 v 11,007 +/- 2,190) (P = .05). The platelet subpopulations did not differ in their content of glycoprotein (GP)IIb/IIIa complex or Baka antigen. The difference in expression of class I HLA antigens on HD and LD platelets is consistent with two possibilities: either class I HLA molecules are acquired from plasma or they are released into plasma as platelets age in circulation. Accordingly, class I HLA molecules may provide a useful marker of platelet age.

Morphometric Analysis of Density Subpopulations of Normal Human Platelets

Platelets from seven normal subjects were fractionated on continuous Percoll density gradients and low density (LD), intermediate, and high density (HD) platelets were prepared for transmission electron microscopy followed by computerised morphometric analysis. Normal ultrastructural appearance and discoid shape were preserved by incubation of the platelets in nutrient medium at 37 degrees C immediately before fixation. HD platelet sections had a larger mean cross-sectional area but a lower ratio of the major to the minor axis compared to LD platelet sections. HD platelets contained more alpha granules, dense granules and mitochondria per square micron of section area than LD platelets. The percentage of section area occupied by open canalicular system was greater in the LD platelets while the percentage area occupied by glycogen fields was over ten-fold higher in the HD platelets. The mean cross-sectional areas of individual alpha granules and dense granules increased with density while the opposite trend was found for mitochondria. It is suggested that these ultrastructural differences mainly arise during thrombopoiesis and may indicate some functional specialization among platelets.

Monoamine Oxidase and Other Mitochondrial Enzymes in Density Subpopulations of Human Platelets

Normal human platelets have been separated according to density on continuous Percoll gradients and the platelet distribution divided into five fractions containing approximately equal numbers of platelets. The mean volumes and protein contents of the platelets in each fraction were found to correlate positively with density while the protein concentration did not differ significantly between the fractions. Four mitochondrial enzymes (monoamine oxidase, glutamate dehydrogenase, cytochrome oxidase and NADP-dependent isocitrate dehydrogenase) were assayed and their activities per unit volume were found to increase in a very similar monotonic fashion with platelet density. When MAO and GDH were assayed on the same set of density fractions the correlation between the two activities was very high (r = 0.94-1.00, p less than 0.001) and a similar close correlation was found between MAO and ICDH. The results support the hypothesis that high density platelets either have a higher concentration of mitochondria or have larger mitochondria than low density platelets.

The Relation of Platelet Density to Platelet Age: Survival of Low- and High-Density 111Indium-Labeled Platelets in Baboons

Abstract The relationship between platelet density and platelet age has been studied using continuous linear Percoll density gradients and 111In- labeling of autologous platelets in baboons. To investigate changes in platelet density during senescence in the circulation, baboons were infused with 111In-labeled autologous platelets, and blood was collected at one hour postinfusion and twice daily thereafter for six days. Platelets were isolated from these samples in high yield (greater than 95%) and separated in continuous linear Percoll density gradients following density equilibrium centrifugation. Although at one hour postinfusion the density distribution of radiolabeled platelets coincided closely with the distribution of the total platelet population, a detectable symmetrical shift toward higher densities was observed after five days. The relative specific radioactivity (RSR) of high-density platelets (1.064 to 1.067 g/mL) decreased at a slower rate than that of the total platelet population (platelets of all densities), whereas the RSR of low-density platelets (1.053 to 1.056 g/mL) showed a more immediate and rapid decrease. These results give rise to one of two interpretations: (1) low-density platelets have a shorter survival time than more dense platelets and are therefore cleared from the circulation at a faster rate, or (2) platelets of all densities increase in density upon aging in the circulation. To determine the explanation for changing RSR of different density fractions we studied the in vivo disappearance characteristics of low- and high-density 111In-labeled platelets. There were no significant differences between the mean survival times of low-density platelets (5.0 +/- 0.49 days, +/- 1 SD, n = 6), high-density platelets (4.9 +/- 0.56 days, n = 6), or control platelets representing platelets of all densities (4.9 +/- 0.38 days, n = 6). Although a slight increase in the density of all platelets during platelet senescence is indicated by these studies, we conclude that platelet density heterogeneity is not primarily a consequence of age-related changes in platelet density.

The relation of platelet density to platelet age: survival of low- and high-density 111indium-labeled platelets in baboons

The relationship between platelet density and platelet age has been studied using continuous linear Percoll density gradients and 111In-labeling of autologous platelets in baboons. To investigate changes in platelet density during senescence in the circulation, baboons were infused with 111In-labeled autologous platelets, and blood was collected at one hour postinfusion and twice daily thereafter for six days. Platelets were isolated from these samples in high yield (greater than 95%) and separated in continuous linear Percoll density gradients following density equilibrium centrifugation. Although at one hour postinfusion the density distribution of radiolabeled platelets coincided closely with the distribution of the total platelet population, a detectable symmetrical shift toward higher densities was observed after five days. The relative specific radioactivity (RSR) of high-density platelets (1.064 to 1.067 g/mL) decreased at a slower rate than that of the total platelet population (platelets of all densities), whereas the RSR of low-density platelets (1.053 to 1.056 g/mL) showed a more immediate and rapid decrease. These results give rise to one of two interpretations: (1) low-density platelets have a shorter survival time than more dense platelets and are therefore cleared from the circulation at a faster rate, or (2) platelets of all densities increase in density upon aging in the circulation. To determine the explanation for changing RSR of different density fractions we studied the in vivo disappearance characteristics of low- and high-density 111In-labeled platelets. There were no significant differences between the mean survival times of low-density platelets (5.0 +/- 0.49 days, +/- 1 SD, n = 6), high-density platelets (4.9 +/- 0.56 days, n = 6), or control platelets representing platelets of all densities (4.9 +/- 0.38 days, n = 6). Although a slight increase in the density of all platelets during platelet senescence is indicated by these studies, we conclude that platelet density heterogeneity is not primarily a consequence of age-related changes in platelet density.

Comparative Study of Size, Total Protein, Fibrinogen and 5-HT Content of Human and Canine Platelet Density Subpopulations

The size, total protein, fibrinogen and 5-HT content were evaluated in density subpopulations of human and canine platelets fractionated in linear arabinogalactan gradients. The methodology was assessed to ascertain that platelet separation was by density and to discard artifactual changes and platelet release during the procedure. EDTA or PGE1 increased the size of human PRP-platelets, but not of dog platelets. In humans, high density (HD) platelets were 1.26 times larger and contained 1.88 times more fibrinogen, 2.23 times more 5-HT and 1.37 times more protein than low density (LD) platelets; in dogs, these density cohorts did not differ in protein content, but LD platelets were 1.29 times larger and had 1.33 times more fibrinogen and 5-HT than HD platelets. These findings suggest that cell density is mostly dependent on the protein content per unit volume of platelets (and not on dense bodies). The differences in fibrinogen and 5-HT content between HD and LD cohorts in humans and dogs may be related to platelet age. The difference in volume between HD and LD platelets in dogs is of uncertain interpretation.

Survival of 111-indium platelet subpopulations of varying density in normal and post splenectomized subjects.

The present study was designed to investigate the survival of platelets of differing densities in normal and post-splenectomized subjects. Autologous platelets, labelled with 111In-oxine, were reinjected into normal subjects (n = 12); 63% were recovered in the circulation and their survival curve was linear with a T 1/2 of 4.5 d. When the platelets were layered onto a continuous Percoll gradient, they formed a band extending between 1.040 and 1.080 g ml-1. After fractionation of the gradient the specific radioactivity of 111In platelets recovered was measured. The specific activity of low density platelets (average 1.050 g ml-1) decreased rapidly with a T 1/2 of 2.0 d, whilst medium density platelets (average 1.060 g ml-1) survived with a T 1/2 of 4.5 d; high density platelets (average 1.073 g ml-1) exhibited a T 1/2 greater than 5.0 d. This latter population of high density platelets also showed a significant increase in specific activity on the first day following injection. In post-splenectomy subjects a similar relationship between density and 111In associated activity was observed but no increase in the specific activity of the dense platelets on day 1 was observed. We conclude that high density autologous 111In-platelets are preferentially retained in the spleen and have a more prolonged survival than those of lower density.