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.     

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.     

Platelet turnover and kinetics in immune thrombocytopenic purpura: results with autologous 111In-labeled platelets and homologous 51Cr- labeled platelets differ

Mean platelet survival and turnover were simultaneously determined with autologous 111In-labeled platelets (111In-AP) and homologous 51Cr- labeled platelets (51Cr-HP) in ten patients with chronic immune thrombocytopenic purpura (ITP). In vivo redistribution of the 111In-AP was quantitated with a scintillation camera and computer-assisted image analysis. The patients were divided into two groups: those with splenic platelet sequestration (spleen-liver 111In activity ratio greater than 1.4), and those with diffuse sequestration in the reticuloendothelial system. The latter patients had more severe ITP reflected by pronounced thrombocytopenia, decreased platelet turnover, and prominent early hepatic platelet sequestration. Mean platelet life span estimated with 51Cr-HP was consistently shorter than that of 111In-AP. Platelet turnover determined with 51Cr-HP was thus over-estimated. The difference in results with the two isotope labels was apparently due to greater in vivo elution of 51Cr. Although the limitations of the techniques should be taken into account, these findings indicate that platelet turnover is not always normal or increased in ITP, but is low in severe disease. We suggest that this may be ascribed to damage to megakaryocytes by antiplatelet antibody. The physical characteristics in 111In clearly make this radionuclide superior to 51Cr for the study of platelet kinetics in ITP.     

Thrombus radioimmunoscintigraphy: an approach using monoclonal antiplatelet antibody.

Thrombus detection and localization is of cardinal importance in clinical medicine. The currently available method using autologous 111In-labeled platelets is too lengthy and complex for everyday use. It requires careful separation of the platelets prior to labeling and visualization of the thrombus becomes possible only 24 hr after injection. An approach to thrombus imaging using a monoclonal antiplatelet antibody labeled with 111In or 123I is described. The antibody (7E3) prepared against human platelets inhibits the interaction between fibrinogen-coated beads and both human and dog platelets. 7E3 is an IgG1 that binds to the complexed glycoprotein IIb/IIIa. Ninety percent of a tracer dose of radiolabeled 7E3 binds to human platelets and 50% binds to dog platelets. In vitro studies showed that virtually all of the platelet-bound radioactivity becomes incorporated into clots formed by adding thrombin to whole blood. 7E3 was labeled with 111In by the cyclic anhydride diethylenetriaminepentaacetic acid method or by radioiodination with 123I. At a ratio of 1:50 (anhydride:7E3) the specific activity ranged between 10 and 40 muCi/micrograms (1 Ci = 37 GBq) without change in the antibody characteristics. In vivo studies in dogs were performed by preincubating for 1 hr the radiolabeled 7E3 with citrated blood or by directly injecting the radiolabeled 7E3 intravenously. Experimental thrombi were induced by transcatheter placement of copper coils into peripheral arteries and veins as well as in the superior vena cava and pulmonary artery. With gamma camera, visualization of venous and arterial thrombi as well as sites of intimal injury without visible thrombi, could be observed 1-1.5 hr after injection. There was no need for delayed imaging because of the fast clearance of radioactivity from the circulation nor was there need for blood pool subtraction. Two to 10-hr thrombi could be imaged but 48-hr thrombi were not detectable with this method. No change in platelet counts before and after the injection of labeled 7E3 nor increased bleeding tendency occurred. The advantages of this method are a shorter preimaging preparation time, faster visualization after injection, and no need for delayed imaging or subtraction techniques. For these reasons human investigations seem to be warranted.     

The effect of elemental diet on intestinal permeability and inflammation in Crohn's disease

This study examines whether treatment of acute Crohn's disease with an elemental diet improves intestinal integrity and inflammation as assessed by a 51Cr-labeled ethylenediaminetetraacetatic acid (EDTA) permeability test and the fecal excretion of 111In-labeled autologous leukocytes, respectively. Thirty-four patients with active Crohn's disease completed a 4-week treatment course with an elemental diet. Active disease was characterized by increased intestinal permeability [24-hour urine excretion of orally administered 51Cr-EDTA, 6.4% +/- 0.6% (mean +/- SE); normal, less than 3.0%] and by high fecal excretion of 111In-labeled leukocytes (14.2% +/- 1.1%; normal, less than 1.0%). Twenty-seven (80%) went into clinical remission, usually within a week of starting treatment. After 4 weeks of treatment, there was a significant decrease in both the urine excretion of 51Cr-EDTA (to 3.4% +/- 0.5%; P less than 0.01) and the fecal excretion of 111In (to 5.7% +/- 1.0%; P less than 0.001), indicating that such treatment is not just symptomatic. A framework for the mechanism by which elemental diet works, centering around the importance of the integrity of the intestinal barrier function, is proposed, and also appears to provide a logical explanation for some relapses of the disease.     

Organ distribution and fate of human platelets: studies of asplenic and splenomegalic patients

Little is known about the organ distribution and fate of human platelets. We investigated the kinetics, organ distribution, and fate of autologous 111In-oxine-labeled platelets in 12 normal volunteers, four asplenic subjects, and four patients with splenomegaly. The initial recovery of infused 111In-platelets from the circulation was 97.8 +/- 9.8% (means +/- SD) for asplenic subjects and 26.3 +/- 5.9% for splenomegalic patients as compared to 59.2 +/- 9.3% for normal controls. The mean platelet survival times as derived from the multiple-hit model were 9.2 +/- 1.0 days for asplenics and 6.2 +/- 0.6 days for splenomegalic subjects (8.4 +/- 0.8 days for normals). At 30 min postinfusion, 79.4 +/- 19.2% of the infused 111In-platelets pooled in the spleen of splenomegalic subjects and 42.7 +/- 12.2% in normal controls. There was 7.1 +/- 2.0, 12.6 +/- 3.7, and 29.3 +/- 8.4% pooling in the liver of splenomegalic, normal, and asplenic subjects, respectively. At 10 days postinfusion, 37 and 24% of the 111In-platelets were sequestered in the spleen and liver of normal control subjects, respectively. Similar figures for splenomegalic subjects were 71 and 14%, respectively. In asplenic subjects, 89% was sequestered in the liver. We conclude that spleen and liver are the primary sites of platelet destruction, accounting for 61% of infused 111In-platelets in normal volunteers and 85% in splenomegalics, while the liver is the primary site of platelet destruction, accounting for 89% in asplenic subjects.     

Failure of ticlopidine to inhibit deposition of indium-111-labeled platelets on Dacron prosthetic surfaces in humans

In a randomized double-blind trial we sought to determine whether short-term therapy with ticlopidine (250 mg bid for 14 days) inhibited platelet deposition on Dacron aortic bifurcation grafts that had been in place a year or longer. A total of 10 men, 42 to 69 years old, underwent indium-111 platelet imaging during both placebo and drug phases of the trial at 24, 48, and 72 hr after the injection of labeled platelets. Platelet accumulation was quantitated by a graft/blood ratio that compared background-corrected activity of indium-111-labeled platelets in the graft with whole-blood activity of indium-111-labeled platelets. Additionally, blinded qualitative visual analysis of the unprocessed images was used to compare graft area activity with activity in adjacent native arteries. Ticlopidine significantly prolonged the template bleeding time from 5.3 +/- 0.5 to 17.1 +/- 3.1 min (+/- SEM) (p = .003). However, by quantitative analysis there was no significant reduction in platelet deposition in the graft during ticlopidine therapy compared with placebo at 24 hr (graft/blood ratio 2.3 +/- 0.4 vs 2.6 +/- 0.3), 48 hr (3.1 +/- 0.5 vs 3.2 +/- 0.4), or 72 hr (3.9 +/- 0.7 vs 4.0 +/- 0.6) after injection of labeled platelets. By visual analysis, nine patients had positive results for abnormal platelet deposition when on placebo that were unchanged when on ticlopidine. The tenth patient had an equivocal result for abnormal platelet deposition when on placebo and a negative result for abnormal platelet deposition when on ticlopidine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thrombocytopenia in dogs induced by granulocyte-macrophage colony- stimulating factor: increased destruction of circulating platelets

Administration of recombinant canine granulocyte-macrophage colony- stimulating factor (rcGM-CSF) to normal dogs in previous studies induced an increase in peripheral blood neutrophils and a dose- dependent decrease in platelet counts. In six dogs that received the highest tested dose of rcGM-CSF (50 micrograms/kg/d) for a minimum of 12 days, the mean nadir of the platelet count was 46,000/microL (range, 4,000 to 91,000/microL) on day 9 +/- 1.1 after starting therapy, compared with a mean baseline platelet count of 398,000/microL (range, 240,000 to 555,000/microL). In three dogs, survival of autologous 111In- labeled platelets was reduced from a mean of 4.9 days to 1.3 days during the administration of rcGM-CSF. Biodistribution studies with gamma camera imaging indicated that there was an increase in mean hepatic uptake during the administration of rcGM-CSF, from 15% to 44% of the total injected 111In-labeled platelets at 2 hours, whereas splenic uptake was not significantly changed. In contrast, in two evaluable dogs who were recipients of 111In-labeled platelets from matched allogeneic donors receiving rcGM-CSF, platelet survival was not reduced and no increased hepatic uptake was noted. A third dog became alloimmunized to the matched donor platelets and was not evaluable. Immunohistologic studies of liver and spleen were performed with monoclonal antibodies specific for canine gpIIb/IIIa and P-selectin in dogs treated with rcGM-CSF and compared with untreated controls. On treatment, a marked reduction of platelets in the red pulp of the spleen was evident, and in general, the presence of platelet antigen in the liver was unchanged. Therefore, platelets were not being sequestered, but destroyed in the liver and spleen. The platelet antigens, P-selectin and gpIIb/IIIa, were identified in association with Kupffer cells in the liver, but no difference in the number of distribution of these Kupffer cells was found between controls and rcGM- CSF-treated dogs. In the spleen during rcGM-CSF treatment, most platelet antigens were associated with large mononuclear cells in the marginal zone. During administration of rcGM-CSF, CD1c and CD11c expression was increased on Kupffer cells. Platelet P-selectin expression and binding of leukocytes to circulating platelets were unchanged from baseline studies with rcGM-CSF treatment. In conclusion, during the administration of rcGM-CSF to dogs, a local process in the liver and spleen is induced resulting in thrombocytopenia.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Low density lipoprotein receptor-independent hepatic uptake of a synthetic, cholesterol-scavenging lipoprotein: implications for the treatment of receptor-deficient atherosclerosis.

The metabolism of infused 111In-labeled phospholipid liposomes was examined in Watanabe heritable hyperlipidemic (WHHL) rabbits, which lack low density lipoprotein (LDL) receptors, and in normal control rabbits. The half-times (t1/2) for clearance of 111In and excess phospholipid from plasma were 20.8 +/- 0.9 hr and 20.3 +/- 4.6 hr in WHHL and 20.0 +/- 0.8 hr and 19.6 +/- 2.2 hr in the normal rabbits (means +/- SEM; n = 4). By 6 hr postinfusion, the plasma concentration of unesterified cholesterol increased by 2.2 +/- 0.23 mmol/liter in WHHL and 2.1 +/- 0.04 mmol/liter in normal rabbits, presumably reflecting mobilization of tissue stores. Disappearance of excess plasma cholesterol was greater than 90% complete in both groups of rabbits by 70 hr postinfusion. By quantitative gamma camera imaging, hepatic trapping of 111In-labeled liposomes over time was indistinguishable between the two groups. At autopsy, the liver was the major organ of clearance, acquiring 22.0% +/- 1.7% (WHHL) and 16.8% +/- 1.0% (normal of total 111In. Aortic uptake of 111In was less than 0.02%. Thus, mobilization of cholesterol and hepatic uptake of phospholipid liposomes do not require LDL receptors. Because phospholipid infusions produce rapid substantial regression of atherosclerosis in genetically normal animals, our results suggest that phospholipid liposomes or triglyceride phospholipid emulsions (e.g., Intralipid) might reduce atherosclerosis in WHHL rabbits and in humans with familial hypercholesterolemia.     

Should platelets be radiolabelled in plasma medium?

Because of the low labelling efficiency of platelets with In-111 oxine in plasma containing media, most investigators have labelled platelets in plasma-free media. However, labelling of platelets with In-111 oxine in plasma-free media may result in either: irreversible damage leading to rapid clearance from the circulation and permanent hepatic sequestration; or reversible damage with transient hepatic sequestration without affecting the in vivo survival. Since human platelets can be labelled with In-111 oxine in plasma in sufficient efficiency for routine clinical use, they should be labelled in plasma whenever possible. The use of newer In-111 chelates, tropolone and mercaptopyridine-N-oxide, which label platelets with high efficiency in plasma as well as in plasma-free media, may offer another alternative.     

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.     

Duration of the effect of aspirin on the synthesis of thromboxane by density subpopulations of rabbit platelets stimulated with thrombin

The controversy concerning the relationship between platelet buoyant density and platelet age is unresolved. Our earlier results with rabbit platelets indicate that the most-dense subpopulations are enriched in young platelets and that some platelets become less dense as they age. Other investigators have concluded that platelets either do not change in density upon aging or become more dense. In the present experiments, rabbit platelets were separated on discontinuous gradients of Stractan. Most-dense platelets synthesized significantly more thromboxane B2 (TXB2) (1.27 ng per 10(6) platelets) in response to thrombin (0.75 U/mL) than did least-dense platelets (0.70 ng per 10(6) platelets), indicating that the arachidonate pathway in most-dense platelets is more active than in least-dense platelets. After aspirin administration to rabbits, most-dense platelets recovered their ability to synthesize thromboxane B2 significantly more quickly than did least-dense platelets. Because the platelet cyclooxygenase that is responsible for TXB2 formation is permanently inhibited by aspirin, it is only the new platelets entering the circulation that will be able to form TXB2. These results indicate that, at least in rabbits, the most-dense platelets are enriched in young platelets, and that platelets decrease in density as they age in the circulation.     

The quantitation of platelet-associated IgG on cohorts of platelets separated from healthy individuals by buoyant density centrifugation

Evidence suggests that as platelets age in the circulation they become progressively smaller and less dense through the loss of protein. The smallest, least dense platelets have a significantly shortened survival, but the mechanism of clearance of these platelets is not known. To evaluate whether the binding of IgG could play a role in the clearance of senescent platelets, we measured platelet size, total protein, and platelet-associated IgG on subpopulations of platelets isolated from 6 healthy individuals using a discontinuous iso-osmotic arabinogalactan (stractan) gradient. There was a close correlation between density, size, and total protein content (r greater than 0.9) for all platelet fractions. There was also a relationship between the amount of platelet-associated IgG (PAIgG), total protein, and platelet size (r greater than 0.9) for the first 3 progressively less dense platelet factions. However, the fourth platelet fraction containing the smallest, least dense, and on current evidence, oldest platelets had very elevated amounts of IgG. This amount was approximately 10 times higher than the mean platelet IgG for the same individual and was similar to the amount of PAIgG found on platelets from patients with immune thrombocytopenia. A progressive increase in the ratio of PAIgG measured after platelet solubilization to PAIgG measured on intact platelets was also noted for the first three populations, indirectly suggesting that platelets clear IgG from their surface during aging. Increased binding of IgG to senescent platelets may mediate their destruction.     

Platelet survival in patients with beta-thalassemia

Thromboembolic events associated with significant morbidity and mortality have been observed in patients with beta-thalassemia major (TM). These include arterial as well as venous thrombosis and the development of early arteriosclerosis. To elucidate the possibility that TM patients may develop a hypercoagulable state we carried out a study of platelet kinetics on ten patients with TM and four patients with thalassemia intermedia (TI). Autologous platelets were labeled with indium-111-oxine, and the platelet lifespan (PLS) was determined. A significant shortening of PLS was observed in 13 out of 14 patients examined. The mean PLS (+/- 1 SD) in ten patients (8 TM, 2 TI) who underwent splenectomy was 107 +/- 36 hr (control splenectomized 248 + 51 hr) (P less than .001) and in four nonsplenectomized patients (2 TM, 2 TI) was 102 +/- 64 hr (control 224 + 23 hr) (P less than .01). The short PLS in addition to reported findings of increased circulating platelet aggregates and the decreased response of TM platelets to aggregating agents suggests in vivo platelet activation in thalassemic patients.     

Kinetics of platelet density subpopulations in splenectomized mongrel dogs

Autologous ⁵¹Cr-platelet kinetic studies were performed in splenectomized mongrel dogs. Mean survival time of PRP-platelets was 5.4 ± 1.5 (SD) days (n = 6). The curves, though slightly curvilinear, showed mostly a linear type of decay, denoting that platelet removal from the circulation is mainly determined by aging of the cells. High-density (HD) and low-density (LD) platelet cohorts were isolated in Stractan gradients from samples drawn daily after infusion of labeled platelets. Specific radioactivity in HD cohorts declined rapidly postinfusion (T1/2 = 1.3 days), but specific radioactivity in LD platelets increased for 2 days and steadily declined for 4 days thereafter (n = 6). Labeled HD platelets, comprising 11.7% of the total population, lived significantly longer in circulation than LD platelets (19.1% of the total population) (n = 3). The patterns of decay of the radioactivity, however, do not have all the characteristics of pure age-cohort survival curves; 3.7 days after the infusion of labeled HD platelets, the specific radioactivity in LD cohorts was six times higher than on day 1, but attained only 20% of the initial specific radioactivity in HD platelets. After the infusion of labeled LD platelets no radioactivity was recovered in circulating HD cohorts. These findings indicate that mongrel dog platelets decrease in density with aging, but also that platelet density heterogeneity is in part determined during the thrombopoietic process. These data are consistent with those of other authors in rabbits and rhesus monkeys, but contrast with the observations that platelets in humans, baboons, and Macaca fasicularis monkeys increase in density with age, suggesting that the displacement of platelets toward compartments of either higher or lower density depends on the species under study.     

In vivo biotinylation studies: specificity of labelling of reticulated platelets by thiazole orange and mepacrine

Animal in vivo biotinylation studies have demonstrated that thiazole orange (TO) labels the youngest cells in the circulation. TO has since been widely used for the measurement of reticulated platelets. As recent findings suggest that at high concentrations TO also labels platelet dense granules non-specifically, the value of previous work is unclear. Mepacrine also labels platelet dense granules and can detect storage pool defects. In this study, a mouse in vivo biotinylation model was used to determine the specificity of TO and mepacrine staining on platelets recently released into the circulation. The mean life span of biotin/TO (low), biotin/TO (high) and mepacrine/TO dual-positive platelets was 1.4 d (SD 0.5), 2.2 d (SD 0.2) and 2.3 d (SD 0.3) respectively (n = 6) compared with a life span for biotin-positive platelets of 4.9 d (SD 1.6). TO (low), TO (high) and mepacrine labelled 8.0% (SD 3.1), 43.9% (SD 8.3) and 40.0% (SD 9.9) of the total platelet population respectively (results of 30 samples from six mice), which decreased to 6.8% (SD 3. 9), 26.6% (SD 6.9) and 25.7% (SD 10.6) after thrombin degranulation. The shorter life span and lack of thrombin sensitivity of TO (low)-positive platelets, suggests that TO (low) measures reticulated platelets specifically. The comparative life spans and thrombin sensitivity of TO (high) and mepacrine-positive platelets suggest that TO (high) labels platelet dense granules. These data also suggest that dense granules are lost during platelet ageing.     

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.     

Morphological and kinetic abnormalities of platelets in hypercholesterolemic rabbits

Hypercholesterolemia (HC = hypercholesterolemia or hypercholesterolemic) was produced in rabbits by feeding them diets supplemented with cholesterol and peanut oil. Platelet counts and volumes, white cell counts, reticulocyte counts, and hematocrits were determined at intervals for 8-12 weeks in blood from HC animals and controls on a normal rabbit diet. Microthrombocytosis was a consistent occurrence in the presence of HC, developing as early as 2 weeks into the diet. Microthrombocytosis was generally associated with normal platelet counts, but mild thrombocytosis occurred late in the diet at the time of the highest levels of serum cholesterol (greater than 1300 mg/dl). Platelets from HC rabbits were morphologically normal by transmission electron microscopy. Survivals of 51Cr-labeled platelets from HC and non-HC rabbits were measured in HC and non-HC recipients. The results identified an intrinsic defect in the ability of HC platelets to survive in the circulation. They also confirmed previous findings of an environmental defect in HC that causes shortened platelet survival.     

Phospholipid transfer between plasma and platelets in vitro

Washed rabbit platelets were resuspended in plasma in which all of the major phospholipids had been isotopically labeled by injection of 32PO4 into rabbits. At certain time intervals during a 6-hr incubation at 37 degrees C, aliquots were removed from the incubation mixture and the platelets were isolated and subjected to lipid extraction and phospholipid analysis. A continuous rise in platelet non-lipid-bound and lipid-bound radioactivity was observed through-out the incubation period. Two platelet phospholipids, lecithin and lysolecithin, were significantly labeled, whereas little or no labeling of the other phospholipids was found. There was no detectable change in total or individual platelet phospholipid content. At 6 hr, 4% of total platelet phospholipid, 43% of platelet lysolecithin, and 7% of platelet lecithin were labeled. Platelets incubated in plasma from rabbits with diet- induced hyperlipidemia took up and incorporated significantly more label into their phospholipids than did platelets in normal plasma. Labeling of both platelet lysolecithin and lecithin could be due to uptake and metabolism of plasma lysolecithin by platelets. However, labeling of platelet lecithin could at least in part be the result of direct exchange of this phospholipid with the plasma. Uptake and incorporation of endogenous plasma lysolecithin by platelets and, possibly, direct exchanged of platelet lecithin may be important mechanisms in the modification by plasma lipids of platelet membrane phospholipid fatty acid composition and platelet function.