Evidence that platelet buoyant density,but not size,correlates with platelet age in man

Following infusion of ⁵¹Cr-labeled autologous platelets into normal subjects, high-density (HD) and low-density (LD) platelet cohorts were isolated by prolonged centrifugation in isosmotic arabino-galactan (Stractan). Specific radioactivity of LD platelets declined rapidly post-infusion (T_1/2 = 1.5 days), but specific radioactivity of HD platelets remained constant or increased over a 3–4-day period and gradually declined for 6–7 days thereafter. These differences were exaggerated when platelet cohorts enriched in LD or HD cells by slow centrifugation in high-density albumin were labeled and transfused. Mean survival of a platelet cohort enriched with HD cells was significantly (P < 0.02) shorter (7.73 days) than that of a cohort enriched with LD cells (9.33 days). In normal subjects treated with aspirin, capacity for thromboxane synthesis was regained more rapidly (P < 0.05) in LD than in HD platelets. HD and LD platelets differed only slightly in mean volume (HD platelets = 7.57 μ³, LD platelets = 6.87 μ³, 0.05 < P < 0.01). We believe the most logical interpretation of these findings is that under normal conditions in man, newly formed platelets are less dense on the average than total platelets and become more dense as they age in the circulation. Thus, specific radioactivity of LD platelets declines rapidly as these platelets move into a more dense compartment and are replaced by newly formed, un-labelled cells; specific radioactivity of HD platelets remains constant or increases as labelled platelets enter this compartment in numbers equal to or greater than the number leaving it at the end of their life span. The similarity in mean volumes of LD and HD platelets suggests that platelet size is unrelated to platelet age under normal conditions.     

Increase in density and accumulation of serotonin by human aging platelets

⁵¹Cr-labeled autologous platelets were infused into splenectomized subjects and the specific radioactivities of high-density (HD) and low-density (LD) platelet subpopulations were determined sequentially in postinfusion samples. A rapid decrease in the specific radioactivity of LD cohorts (T 1/2 = 2.5 days) was observed, but the specific radioactivity of HD platelets remained constant or increased slightly during the first 4 days and then gradually declined for the next 5 days. No experimental artifacts during the platelet-labeling steps that could account for these results were demonstrated. These findings confirm previous observations in eusplenic individuals and support the hypothesis that human LD platelets are, on the average, younger than HD platelets. LD platelets contain 33.8 ± 13.5 ng serotonin (5HT)/10⁸ platelets and HD platelets 76.8 ± 9.5 ng 5HT/10⁸ platelets (P < 0.001). Sequential measurements of 5HT in PRP platelets were performed during the recovery phase of thrombocytopenia following splenectomy in patients with idiopathic thrombocytopenic purpura (ITP), a condition associated with aging of platelets in circulation. Presplenectomy platelet 5HT was 17.7 ng/10⁸ platelets and on days 1, 6, and 12 after surgery it increased to 18.1, 37.8, and 61.0 ng/10⁸ platelets (n = 7). When three healthy volunteers were given aspirin (500 mg/day) for up to 15 days, no significant change in the 5HT content of circulating platelets was observed. If aspirin blocks, at least partially, the secretory process in vivo without interfering the 5HT uptake by the platelets, this finding stands against the possibility that a net depletion of 5HT occurs during the life-span of normal human platelets. The observation that human HD platelets, enriched with older cells, contain more 5HT than LD platelets taken together with the parallel increase in platelet 5HT and age during the recovery from thrombocytopenia in ITP patients and the lack of effect of aspirin on platelet 5HT content, provides initial evidence that human platelets accumulate 5HT during their life-span in circulation.     

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.     

Further studies on the relationship between platelet buoyant density and platelet age

The relationship between platelet buoyant density and platelet age was investigated in eight human subjects submitted to an autologous chromium labeled platelet survival study. Platelets were isolated after isopycnic centrifugation using either discontinuous isoosmotic stractan gradients (five subjects), or various continuous and linear isoosmolar gradients (three subjects). A paradoxical radioactivity enrichment of the dense platelets and a premature loss of radioactivity in the light platelets were observed. These results are explained by a shift of the radioactivity distribution curve toward higher densities during the 3–4 days after platelet injection, while the standard deviation of the distribution was conserved throughout the platelet life span. These results suggest that young platelets are heterogeneous and slightly less dense than the total platelet population.     

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.     

Heterogeneity of human whole blood platelet subpopulations. II. Use of a subhuman primate model to analyze the relationship between density and platelet age.

A subhuman primate model was developed to ascertain whether or not platelet heterogeneity could be explained by aging in the peripheral circulation. Density-dependent platelet cohorts, postulated to represent cells of different ages, were isolated on isosmolar arabinogalactan gradients and labeled with radiochromium. Mean platelet lifespan was measured for the different density cohorts, and simultaneous sequential density distribution analysis was performed to follow changes in cell density during aging. The average mean lifespan of light platelets was 74.6 hr, compared to 313.6 hr for heavy platelets. After injection, labeled light platelets were recovered only in the gradient light region, in contrast to labeled heavy platelets, which were initially restricted to the dense region and progressively migrated to the light region during their lifespan. This study supports the hypothesis that platelet age in unstressed primates correlates with cell density and provides a rationale for the use of "age-dependent" markers to estimate platelet turnover rates.     

Total sialic acid in human and canine platelets does not change with the platelet age.

Total platelet sialic acid (SA) was measured in three experimental conditions: (1) human and canine platelet density subpopulations obtained by centrifugation in arabinogalactan gradients, (2) circulating canine platelets during recovery from experimental immune and mechanical thrombocytopenias, and (3) platelets obtained from a patient with chronic immune thrombocytopenic purpura before and after splenectomy. The density of human and canine platelets is, in part, determined by their age. We found no significant differences in total SA between high-density (HD) and low-density (LD) platelets (9.32 +/- 2.0 vs. 9.55 +/- 1.3 micrograms/mg of platelet protein in dogs and 9.02 +/- 2.3 vs. 9.10 +/- 2.9 micrograms/mg in humans). In the human and canine thrombocytopenic models, the entrance of new platelets from the bone marrow is followed by their aging in the circulation. In these models, no significant changes in total SA content were detected in sequential measurements during the recovery of the thrombocytopenia. Accordingly, we conclude that total SA in human and canine platelets is unrelated to their age in circulation. These results do not support the notion that the loss of SA from membrane glycoproteins determines the recognition and removal of platelets from the circulation.     

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

Summary. 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> ID > 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.     

A simple,fluorescent method to internally label platelets suitable for physiological measurements

Current methods for studying platelet survival in vivo are limited by the use of radioisotopes, with their inherent safety and regulatory concerns, systemic drug administrations that produce biochemical modifications of platelet functions, or external labeling techniques, which may produce artifacts due to surface modifications. For these reasons, we sought to develop a simple, nonisotopic method for labeling platelets internally, thereby producing platelets more likely to have in vivo properties equivalent to native cells. Murine platelets in protein-free buffer were fluorescently labeled internally by incubation with 2.5 μM 5-chloromethyl fluorescein diacetate (CMFDA), and without washing, were injected into mice for platelet survival studies. CMFDA-labeled platelets were unactivated, as shown by minimal P-selectin expression. When tested in vitro for function by aggregometry, the response of CMFDA-labeled platelets to collagen and thrombin was identical to that of unlabeled platelets. Flow cytometric analysis demonstrated that CMFDA platelets were an intensely stained, unimodal population that was completely separated from unlabeled platelets. The mean half-life of labeled platelets in the murine circulation was 37.5 ± 4.5 hr (±1 SD), and the mean survival time was 3.1–3.3 days (n = 24), similar to results reported using ⁵¹Cr and ¹¹¹In. No evidence of in vivo transfer of dye from labeled platelets to unlabeled cells was observed. CMFDA produces a population of platelets that are nonradioactively, internally labeled with a highly fluorescent, stable product. The labeled platelets function equivalently to native platelets, as demonstrated by immunocytometry and aggregometry, and importantly, in vivo, by normal platelet survival. Am. J. Hematol. 56:17–25, 1997. © 1997 Wiley-Liss, Inc.     

Use of asplenic rabbits to demonstrate that platelet age and density are related

An experimental model using asplenic rabbits injected with radioactive amino acids has been developed to examine the density distribution of newly released platelets. Platelets from asplenic rabbits had a larger mean volume and greater protein content than those from asplenic animals. Radiolabel, indicative of new platelets, is preferentially incorporated into the most dense platelets during the early postinjection period. Platelets of intermediate density also demonstrated augmented early label incorporation compared to the lightest cells, In contrast, during the late postinjection phase, there is preferential labeling of the least dense platelets. The specific radioactivity of heavy and light platelets rises to approximately equal levels, and falls abruptly, but the peak activities occur at distinctly separate times consistent with the concept of platelet density modification during platelet aging. Although platelet biologic properties are broadly distributed, these studies support the concept that on the average young platelets have an increased density and become less dense as they circulate.     

In vitro function of platelet concentrates prepared after filtration of whole blood or buffy coat pools

BACKGROUND/METHOD: Data on the quality of platelet concentrates (PC) produced by the buffy coat method and stored beyond 5 days in plasma are limited. We therefore evaluated the quality of PCs prepared by leucocyte depletion of whole blood (Terumo WBSP, n = 10) or a buffy coat pool (Pall Autostop, n = 10), and stored for 7 days in plasma by assessing platelet parameters and markers of platelet activation. RESULTS: In both types of PC, levels of glucose decreased during storage but were not totally depleted (> 11 mM on day 7). In contrast, lactate levels increased on storage and was consistently < 20 mM throughout, with pH maintained at > 6.8 in all units. Hypotonic shock response scores were > 47% in all units at day 7. On day 1, markers of platelet activation were significantly higher in WBSP PC, but by day 7 were similar for percentage CD63+ and CD62P + (40%) with levels of platelet microparticles and annexin V binding two-fold higher in WBSP. The expression of CD61 did not alter during storage and the percentage of platelets expressing CD42b was > 88% in all units on day 7. RANTES (Regulated on activation, normal, T-cell expressed and secreted) and TGFbeta released from platelets by day 7 was < 800 ng/ml and 90 ng/ml, respectively. C3a(desarg) increased throughout storage in both types of PC, but without a commensurate increase in the terminal complex SC5b-9 or activation of factor XII. CONCLUSION: Our data indicates that the in vitro characteristics of PCs prepared using these methods is maintained over storage for 7 days in plasma and is not associated with significant deterioration of platelet function. ONE SENTENCE SUMMARY: In vitro function of platelet concentrates prepared by either filtration of whole blood, or pooled buffy coats.     

Survival of density subpopulations of rabbit platelets: use of 51Cr-or 111In-labeled platelets to measure survival of least dense and most dense platelets concurrently

The origin of the density heterogeneity of platelets was studied by measuring the survival of density subpopulations of rabbit platelets separated by discontinuous Stractan density gradient centrifugation. When a total population of 51Cr-labeled platelets was injected into recipient rabbits, the relative specific radioactivity of the most dense platelets decreased rapidly. In contrast, that of the least dense platelets had not changed 24 hr after injection, and then decreased slowly. To distinguish between the possibilities that most dense platelets are cleared from the circulation more quickly than least dense platelets or that platelets decrease in density as they age in the circulation, the concurrent survival of least dense and most dense platelets, labeled with either 51Cr or 111In-labeled total platelet populations, determined concurrently in the same rabbits, were identical, calculated from 1 hr values as 100%. However, the 1-hr recovery of 111In-labeled platelets was slightly but significantly less than that of 51Cr-labeled platelets. Therefore, we studied the survival of 51Cr-labeled least dense and 111In-labeled most dense platelets as well as that of 111In-labeled least dense and 51Cr-labeled most dense platelets. Mean 1-hr recovery of least dense platelets, labeled with either isotope (78% +/- 7%, SD) was similar to that of most dense platelets, labeled with either isotope (77% +/- 8%; SD). Mean survival of least dense platelets was 47.3 +/- 18.7 hr (SD), which was significantly less than that of most dense platelets (76.1 +/- 21.6 hr; SD) (p less than 0.0025). These results indicate that platelets decrease in buoyant density as they age in the circulation and that most dense platelets are enriched in young platelets, and least dense in old. Thus, the events that affect platelets as they age in the circulation contribute to platelet density heterogeneity, although they may not be the sole cause of it.     

Platelet size and age determine platelet function independently

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.     

99mTc体外标记血小板的方法探讨及家兔体外和在体实验研究

目的探讨利用酒石酸亚锡溶液作为还原剂9,9mTc直接标记血小板的可行性和影响因素,探讨还原剂的最适pH值。方法实验兔在左右髂动脉处置入血管支架,3 d后抽静脉血,分离血小板,分别使用pH为1、2、3、45、的酒石酸亚锡溶液作为还原剂9,9mTcO4体外标记血小板,测量标记后的血小板悬浮液的放射活度及上清液的放射活度,计算出相应的血小板标记率,测定标记后的血小板聚集功能;另一方面,将标记后的血小板重新输入兔子,进行体外核素显像,观察血小板的聚集和黏附功能。结果当酒石酸亚锡溶液pH为3、亚锡溶液亚锡离子浓度为4.5 mmol/L时,血小板标记率最高,为(29.7±3.6)%,并能保持血小板部分活性,标记后的血小板聚集功能仍能达到(42.5±2.5)%,亦具有黏附能力。结论经过本研究改进的、在国内现有实验条件下利用99mTc标记血小板的方法,具有标记率较高、稳定、血小板活性受影响小、简便廉价等优点,可以作为体外标记血小板方法的一种可选方案。     

Radioactive Diisopropyl Fluorophosphate as a Platelet Label: An Evaluation of in Vitro and in Vivo Technics

A technic for the in vitro labeling of human platelets with DFP³² is presented, critically evaluated, and compared to in vivo methods employing DFP³² and to in vitro methods using Cr⁵¹. The initial recovery of platelets labeled in vitro with DFP³² averaged 79 per cent, but the survival curve was characterized by an irreversible initial loss of platelet radioactivity. Experiments in which platelets were simultaneously labeled in vitro with both DFP³² and Cr⁵¹ suggest that this is not due to elution of DFP³². The survival curve of platelets labeled in vivo with DFP³² shows an initial transient reduction in platelet radioactivity. It is suggested that both of these aberrations in initial survival are the result of platelet injury by DFP³². Significant "tailing" was observed in the survival curves obtained with DFP³², and possible explanations of this phenomenon are discussed. DFP³²-labeled platelets circulating after 5 hours apparently survive normally and disappear from the circulation as a rectilinear function over the next 6-8 days. Although both in vitro and in vivo labeling methods employing DFP³² provide a meaningful approximation of platelet lifespan, the initial and terminal aberrations of the survival curves greatly complicate further interpretation. Dextran had no detectable effect on platelet survival, and epinephrine, Mecholyl, and cutaneous vasodilatation did not alter the platelet count or the specific activity of circulating labeling platelets in human subjects. The problem of initial platelet survival and the question of an extravascular or marginal platelet pool is discussed in the light of these data.

Submitted on May 24, 1966 Accepted on November 8, 1966     

P物质前体与血小板活化及血小板参数的相关性

目的探讨P物质前体(Pro-SP)与血小板活化及血小板参数的相关性。方法总共纳入心内科患者76例,根据急性心肌梗死诊断标准将患者分为非急性心肌梗死组(39例)和急性心肌梗死组(38例)。采用流式细胞仪测定血小板-单核细胞聚集体(PMA)形成率评估血小板活化程度,酶联免疫吸附实验测定Pro-SP浓度。比较Pro-SP与PMA形成率及血小板参数相关性,以及两组间Pro-SP差异。结果 Pro-SP与PMA形成率(Pearson相关系数为0. 407,P0. 001)、血小板分布宽度(Pearson相关系数为0. 269,P=0. 018)呈正相关,与血小板总数、血小板平均容积、血小板比积、大血小板比例不相关(P0. 05)。多元线性回归分析显示Pro-SP是PMA形成率的独立影响因素,急性心肌梗死组Pro-SP较非急性心肌梗死组显著升高(P0. 05)。结论 Pro-SP与血小板活化正相关,且与血小板分布宽度呈正相关,与血小板总数、血小板平均容积、血小板比积、大血小板比例不相关(P0. 05)。     

Influence of intravascular molecular weight of hydroxyethyl starch on platelets

Abstract: Complications concerning the blood coagulation have been observed repeatedly after administration of highly substituted, high molecular weight hydroxyethyl starch (HES), but it has not been examined as to how intravascular molecular weight and degree of substitution of HES influence platelet number and volume after repeated administration. Thirty patients with cerebrovascular diseases were treated for 10 days with hemodilution. 500 to 1500 ml of HES 200/0.62 (n = 10), HES 200/0.5 (n = 10) or HES 40/0.5 (n = 10) were infused daily. During the first days, the number of platelets was not lowered beyond the dilution effect, but at the end of the therapy the number of platelets had increased in all 3 groups beyond the initial value. Platelet volume was lowered significantly in the 3 groups. HES 200/0.62 caused the largest drop in platelet volume (-10%, p<0.01). A possible explanation could be that HES macromolecules are attached to platelets or are phagocytized by them. The larger platelets are then broken down and, to compensate the loss, more thrombocytes are released. A correlation between the molecular weight of HES and the breakdown rate of the platelets can be suspected, because HES 200/0.62 had the highest intravascular mean molecular weight (121 kD) and the largest effect on platelet volume.     

Platelet Heterogeneity

Human platelets were separated into 2 density populations by repeated centrifugations of platelet-rich plasma at increasing gravitational force. The heaviest platelet fraction was rich in larger platelets. The lightest platelet fraction was rich in smaller platelets. In both fractions and in the platelet button, lipid peroxidation (malonaldehyde-MDA-production after addition of thrombin) was measured at basal condition, on the 1st, 3rd, 5th, 7th and 9th day after aspirin ingestion. At basal conditions and after ingestion of aspirin, MDA production was higher in the heavy-large platelets than in light-small ones, but a parallel increase of MDA production was observed in the light and in the heavy population and in the platelet button. The data are not compatible with the hypothesis that platelet density and size are age-related. Aspirin inhibits platelet lipid peroxidation by permanently acetylating their cyclooxygenase and if the heaviest platelets were the young ones, lipid peroxidation should reappear sooner in them.     

Properties of the exchangeable splenic platelets released into the circulation during exercise-induced thrombocytosis

The human spleen normally retains about one-third of the body's platelets in an exchangeable pool which can be released into the circulation by alpha-adrenergic stimulation. Some previous investigators concluded that the splenic platelet population was enriched in a subpopulation of large, young, dense platelets (megathrombocytes) but more recent research suggests that platelet size, age, and density are largely independent variables. In this investigation the properties of the splenic platelets were studied after their release into the circulation by acute strenuous exercise in 11 normal subjects. The exercise caused a rise in mean platelet count from 245 +/- 49 to 328 +/- 71 x 10(9)/L--a net increase of 24 +/- 6% after correction for haemoconcentration. The mean platelet volume (MPV) of citrated platelets increased from 6.38 +/- 0.78 to 6.59 +/- 0.68 fL after exercise (P less than 0.01)--a rise of 3.7 +/- 4.1% suggesting that the MPV of the splenic platelet population was about 20% greater than that of the normal circulating population. The age distribution of the platelets was studied by measuring the platelet monoamine oxidase (MAO) activity several days after irreversible inhibition by tranylcypromine, when the young platelets had normal MAO activity but the older platelets had only 20% of normal activity. The mean platelet MAO activity did not change after exercise, indicating that the age distributions of the circulating and splenic populations were very similar. The platelet contents of several putative markers of platelet age (sialic acid, serotonin, beta-thromboglobulin, beta-N-acetylglucosaminidase) were also unchanged after exercise. Modal platelet density decreased slightly but not significantly after exercise. The splenic platelet population has a larger MPV but appears to have similar age and density distributions to the basal circulating population.