Polychromatic emission in a wide energy range from InP-InAs-InP multi-shell nanowires.

InP-InAs-InP multi-shell nanowires (NWs) were grown in the wurtzite (WZ) or zincblende (ZB) crystal phase and their photoluminescence (PL) properties were investigated at low temperature (≈6 K) for different measurement geometries. PL emissions from the NWs were carefully studied in a wide energy range from 0.7 to 1.6 eV. The different features observed in the PL spectra for increasing energies are attributed to four distinct emitting domains of these nano-heterostructures: the InAs island (axially grown), the thin InAs capping shell (radially grown), the crystal-phase quantum disks arising from the coexistence of InP ZB and WZ segments in the same NW, and the InP portions of the NW. These results provide a useful frame for the rational implementation of InP-InAs-InP multi-shell NWs containing various quantum confined domains as polychromatic optically active components in nanodevices for quantum information and communication technologies.

Excitation energy-transfer in functionalized nanoparticles: Going beyond the Förster approach.

We develop a novel approach to treat excitation energy transfer in hybrid nanosystems composed by an organic molecule attached to a semiconductor nanoparticle. Our approach extends the customary Förster theory by considering interaction between transition multipole moments of the nanoparticle at all orders and a point-like transition dipole moment representing the molecule. Optical excitations of the nanoparticle are described through an envelope-function configuration interaction method for a single electron-hole pair. We applied the method to the prototypical case of a core/shell CdSe/ZnS semiconductor quantum dot which shows a complete suppression of the energy transfer for specific transitions which could not be captured by Förster theory.

Unintentional high-density p-type modulation doping of a GaAs/AlAs core-multishell nanowire.

Achieving significant doping in GaAs/AlAs core/shell nanowires (NWs) is of considerable technological importance but remains a challenge due to the amphoteric behavior of the dopant atoms. Here we show that placing a narrow GaAs quantum well in the AlAs shell effectively getters residual carbon acceptors leading to an unintentional p-type doping. Magneto-optical studies of such a GaAs/AlAs core-multishell NW reveal quantum confined emission. Theoretical calculations of NW electronic structure confirm quantum confinement of carriers at the core/shell interface due to the presence of ionized carbon acceptors in the 1 nm GaAs layer in the shell. Microphotoluminescence in high magnetic field shows a clear signature of avoided crossings of the n = 0 Landau level emission line with the n = 2 Landau level TO phonon replica. The coupling is caused by the resonant hole-phonon interaction, which points to a large two-dimensional hole density in the structure.

ATPergic signaling disruption in human sepsis as a potential source of biomarkers for clinical use.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated inflammatory response to infection. To date, there is no specific treatment established for sepsis. In the extracellular compartment, purines such as adenosine triphosphate (ATP) and adenosine play essential roles in the immune/inflammatory responses during sepsis and septic shock. The balance of extracellular levels among ATP and adenosine is intimately involved in the signals related to immune stimulation/immunosuppression balance. Specialized enzymes, including CD39, CD73, and adenosine deaminase (ADA), are responsible to metabolize ATP to adenosine which will further sensitize the P2 and P1 purinoceptors, respectively. Disruption of the purinergic pathway had been described in the sepsis pathophysiology. Although purinergic signaling has been suggested as a potential target for sepsis treatment, the majority of data available were obtained using pre-clinical approaches. We hypothesized that, as a reflection of deregulation on purinergic signaling, septic patients exhibit differential measurements of serum, neutrophils and monocytes purinergic pathway markers when compared to two types of controls (healthy and ward). It was observed that ATP and ADP serum levels were increased in septic patients, as well as the A2a mRNA expression in neutrophils and monocytes. Both ATPase/ADPase activities were increased during sepsis. Serum ATP and ADP levels, and both ATPase and ADPase activities were associated with the diagnosis of sepsis, representing potential biomarkers candidates. In conclusion, our results advance the translation of purinergic signaling from pre-clinical models into the clinical setting opening opportunities for so much needed new strategies for sepsis and septic shock diagnostics and treatment.

TLR4 expression and functionality are downregulated in glioblastoma cells and in tumor-associated macrophages: A new mechanism of immune evasion?

Glioblastoma (GB) is the most common and aggressive form of primary brain tumor, in which the presence of an inflammatory environment, composed mainly by tumor-associated macrophages (TAMs), is related to its progression and development of chemoresistance. Toll-Like Receptors (TLRs) are key components of the innate immune system and their expression in both tumor and immune-associated cells may impact the cell communication in the tumor microenvironment (TME), further modeling cancer growth and response to therapy. Here, we investigated the participation of TLR4-mediated signaling as a mechanism of induced-immune escape in GB. Initially, bioinformatics analysis of public datasets revealed that TLR4 expression is lower in GB tumors when compared to astrocytomas (AST), and in a subset of TAMs. Further, we confirmed that TLR4 expression is downregulated in chemoresistant GB, as well as in macrophages co-cultured with GB cells. Additionally, TLR4 function is impaired in those cells even following stimulation with LPS, an agonist of TLR4. Finally, experiments performed in a cohort of clinical primary and metastatic brain tumors indicated that the immunostaining of TLR4 and CD45 are inversely proportional, and confirmed the low TLR4 expression in GBs. Interestingly, the cytoplasmic/nuclear pattern of TLR4 staining in cancer tissues suggests additional roles of this receptor in carcinogenesis. Overall, our data suggest the downregulation of TLR4 expression and activity as a strategy for GB-associated immune escape. Additional studies are necessary to better understand TLR4 signaling in TME in order to improve the benefits of immunotherapy based on TLR signaling.

Glycosylated hemoglobin and the risk of death and cardiovascular mortality in the elderly.

BACKGROUND AND AIMS: Glycosylated hemoglobin (HbA(1c)) has been associated with incident cardiovascular disease (CVD), but the findings are inconsistent. We tested the hypothesis that HbA(1c) may be associated with an increased risk of death and cardiovascular mortality in older adults. METHODS AND RESULTS: We evaluated the association between HbA(1c) with all-cause and cardiovascular mortality in 810 participants without a history of diabetes in a sub-study of the Cardiovascular Health Study (CHS), a community cohort study of individuals > or =65 years of age. Glycosylated hemoglobin was measured at baseline and all-cause and cardiovascular mortality was assessed during the follow-up period. The relation between baseline HbA(1c) and death was evaluated with multivariate Cox proportional hazards regression models. After a median follow-up of 14.2 years, 416 deaths were observed. The crude incidence rates of all-cause mortality across HbA(1c) groups were: 4.4% per year, 4.3% per year and 4.6% per year for tertile 1 (< or =5.6%), tertile 2 (5.61-6.20%) and tertile 3 (> or =6.21%), respectively. In unadjusted and fully adjusted analyses, baseline HbA(1c) was not associated with all-cause mortality and cardiovascular mortality (hazard ratio: 1.16 [95% confidence interval 0.91-1.47] and hazard ratio: 1.31 [95% confidence interval 0.90-1.93], respectively for the highest HbA(1c) tertile compared with the lowest). CONCLUSION: These results suggest that HbA(1c) does not significantly predict all-cause and cardiovascular mortality in non-diabetic community-dwelling older adults.

Immortalization of Mesenchymal Stromal Cells by TERT Affects Adenosine Metabolism and Impairs their Immunosuppressive Capacity.

Mesenchymal stromal cells (MSCs) are promising candidates for cell-based therapies, mainly due to their unique biological properties such as multipotency, self-renewal and trophic/immunomodulatory effects. However, clinical use has proven complex due to limitations such as high variability of MSCs preparations and high number of cells required for therapies. These challenges could be circumvented with cell immortalization through genetic manipulation, and although many studies show that such approaches are safe, little is known about changes in other biological properties and functions of MSCs. In this study, we evaluated the impact of MSCs immortalization with the TERT gene on the purinergic system, which has emerged as a key modulator in a wide variety of pathophysiological conditions. After cell immortalization, MSCs-TERT displayed similar immunophenotypic profile and differentiation potential to primary MSCs. However, analysis of gene and protein expression exposed important alterations in the purinergic signaling of in vitro cultured MSCs-TERT. Immortalized cells upregulated the CD39/NTPDase1 enzyme and downregulated CD73/NT5E and adenosine deaminase (ADA), which had a direct impact on their nucleotide/nucleoside metabolism profile. Despite these alterations, adenosine did not accumulate in the extracellular space, due to increased uptake. MSCs-TERT cells presented an impaired in vitro immunosuppressive potential, as observed in an assay of co-culture with lymphocytes. Therefore, our data suggest that MSCs-TERT have altered expression of key enzymes of the extracellular nucleotides/nucleoside control, which altered key characteristics of these cells and can potentially change their therapeutic effects in tissue engineering in regenerative medicine.

Serological evidence of infections and Type 2 diabetes: the MultiEthnic Study of Atherosclerosis.

AIMS: Prospective studies have identified chronic inflammation as a risk factor for Type 2 diabetes. However, it is not known whether infection by specific pathogens or having a greater 'pathogen burden' is associated with diabetes. The aim of this study was to examine the cross-sectional relation of seropositivity to five pathogens (Chlamydia pneumoniae, cytomegalovirus, Helicobacter pylori, hepatitis A virus, herpes simplex virus) and prevalent diabetes. METHODS: Baseline data from a random sample of MultiEthnic Study of Atherosclerosis (MESA) participants (n = 1000; age 45-84 years) were used. Diabetes was defined by American Diabetes Association 2003 criteria, and 'pathogen burden' by the number of pathogens (0-5) for which an individual was seropositive. Logistic regression was used to test differences in diabetes prevalence by seropositivity. Linear regression was used to explore associations between pathogen seropositivity and the inflammation markers C-reactive protein, interleukin-6 and fibrinogen. RESULTS: Diabetes prevalence was 12.7%, whereas seropositivity for C. pnuemoniae was 76%, cytomegalovirus 77%, H. pylori 45%, hepatitis A 58% and herpes simplex virus 85%. Seventy-two percent were seropositive for three or more pathogens. In crude analyses, the prevalence of diabetes was higher among those with a pathogen burden of three or more, and with seropositivity to cytomegalovirus, H. pylori, hepatitis A and herpes simplex virus. After adjustment for demographic covariates (particularly race), all associations became non-significant. Pathogen seropositivity was also not related to inflammation marker levels. CONCLUSIONS: Following demographic adjustments, no associations were observed between infection by several pathogens and diabetes status, suggesting no aetiological role for them in the occurrence of diabetes.

Validity of the single-particle approach for electron transport in quantum wires assisted by surface acoustic waves.

We study by means of time-dependent numerical simulations the quantum entanglement stemming from the Coulomb interaction between two electrons trapped in the minima of the piezoelectric potential generated by surface acoustic waves. We find that for particles captured in low-energy bound states the quantum correlations turn out to be negligible, thus validating a single-particle approach to the dynamics of such systems. At long times, for high-energy electrons, a substantial entanglement appears, which is an indicator of a mostly correlated dynamics.

HbA 1c as a risk factor for heart failure in persons with diabetes: the Atherosclerosis Risk in Communities (ARIC) study.

AIMS/HYPOTHESIS: Heart failure (HF) incidence in diabetes in both the presence and absence of CHD is rising. Prospective population-based studies can help describe the relationship between HbA(1c), a measure of glycaemia control, and HF risk. METHODS: We studied the incidence of HF hospitalisation or death among 1,827 participants in the Atherosclerosis Risk in Communities (ARIC) study with diabetes and no evidence of HF at baseline. Cox proportional hazard models included age, sex, race, education, health insurance status, alcohol consumption, BMI and WHR, and major CHD risk factors (BP level and medications, LDL- and HDL-cholesterol levels, and smoking). RESULTS: In this population of persons with diabetes, crude HF incidence rates per 1,000 person-years were lower in the absence of CHD (incidence rate 15.5 for CHD-negative vs 56.4 for CHD-positive, p<0.001). The adjusted HR of HF for each 1% higher HbA(1c) was 1.17 (95% CI 1.11-1.25) for the non-CHD group and 1.20 (95% CI 1.04-1.40) for the CHD group. When the analysis was limited to HF cases which occurred in the absence of prevalent or incident CHD (during follow-up) the adjusted HR remained 1.20 (95% CI 1.11-1.29). CONCLUSIONS/INTERPRETATIONS: These data suggest HbA(1c) is an independent risk factor for incident HF in persons with diabetes with and without CHD. Long-term clinical trials of tight glycaemic control should quantify the impact of different treatment regimens on HF risk reduction.

Ambulatory blood pressure monitoring phenotypes among individuals with and without diabetes taking antihypertensive medication: the Jackson Heart Study.

Ambulatory blood pressure monitoring (ABPM) can detect phenotypes associated with increased cardiovascular disease (CVD) risk. Diabetes is associated with increased CVD risk but few data are available documenting whether blood pressure (BP) phenotypes, detected by ABPM, differ between individuals with versus without diabetes. We conducted a cross-sectional analysis of 567 participants in the Jackson Heart Study, a population-based study of African Americans, taking antihypertensive medication to evaluate the association between diabetes and ABPM phenotypes. Two clinic BP measurements were taken at baseline following a standardized protocol. ABPM was performed for 24 h following the clinic visit. ABPM phenotypes included daytime, sustained, nocturnal and isolated nocturnal hypertension, a non-dipping BP pattern, and white coat, masked and masked isolated nocturnal hypertension. Diabetes was defined as fasting glucose ⩾126 mg dl-1, haemoglobin A1c ⩾6.5% (48 mmol mol-1) or use of insulin or oral hypoglycaemic medications. Of the included participants (mean age 62.3 years, 71.8% female), 196 (34.6%) had diabetes. After multivariable adjustment, participants with diabetes were more likely to have daytime hypertension (prevalence ratio (PR): 1.32; 95% confidence interval (CI): 1.09-1.60), masked hypertension (PR: 1.46; 95% CI: 1.11-1.93) and masked isolated nocturnal hypertension (PR: 1.39; 95% CI: 1.02-1.89). Although nocturnal hypertension was more common among participants with versus without diabetes, this association was not present after adjustment for daytime systolic BP. Diabetes was not associated with the other ABPM phenotypes investigated. This study highlights the high prevalence of ABPM phenotypes among individuals with diabetes taking antihypertensive medication.

Diabetes and the risk of infection-related mortality in the U.S.

OBJECTIVE: To determine whether diabetes predicts infection-related mortality and to clarify the extent to which this relationship is mediated by comorbid conditions that may themselves increase risk of infection. RESEARCH DESIGN AND METHODS: We performed a retrospective cohort study using the Second National Health and Nutrition Examination Survey Mortality Study of 9,208 adults aged 30-74 years in 1976-1980. We defined demographic variables, diabetes, cardiovascular disease (CVD), and smoking by self-report; BMI, blood pressure, and serum cholesterol from baseline examination; and cause-specific mortality from death certificates. RESULTS: Over 12-16 years of follow-up, 36 infection-related deaths occurred among 533 adults with diabetes vs. 265 deaths in 8,675 adults without diabetes (4.7 vs. 1.5 per 1,000 person-years, P < 0.001). Diabetes (RR 2.0, 95% CI 1.2-3.2) and congestive heart failure (2.8, 1.6-5.1) were independent predictors of infection-related mortality after simultaneous adjustment for age, sex, race, poverty status, smoking, BMI, and hypertension. After subdividing infection-related deaths into those with (n = 145) and without (n = 156) concurrent cardiovascular diagnoses at the time of death, diabetic adults were at risk for infection-related death with CVD (3.0, 1.8-5.0) but not without CVD (1.0, 0.5-2.2). CONCLUSIONS: These nationally representative data suggest that diabetic adults are at greater risk for infection-related mortality, and the excess risk may be mediated by CVD.

Impaired posthypoxic relaxation in single cardiac myocytes: role of intracellular pH and inorganic phosphate.

OBJECTIVE: The aim was to examine the effects of alterations in intracellular pH and inorganic phosphate concentration (known to influence myofilament kinetics and to change rapidly during hypoxia) on cell contraction, relaxation, and the Ca2+ transient in normoxic and hypoxic myocytes. METHODS: Single adult rat ventricular myocytes were electrically stimulated (0.2 Hz) and cell length (photodiode array), intracellular Ca2+ (indo-1 fluorescence), or intracellular pH (SNARF-1 fluorescence) measured. Hypoxia was induced in a special open chamber in which a laminar layer of argon prevented the back diffusion of atmospheric oxygen. RESULTS: Electrically stimulated contraction was preserved during exposure to hypoxia. At reoxygenation 10 minutes later the time from the stimulus to the peak of contraction (TPK) increased by 30(SEM 9)% and the time from the peak of contraction to 50% recovery of cell length (RT50) increased by 59(13)% relative to prehypoxic values (n = 8). These changes were not accompanied by a change in the kinetics of the Ca2+ transient. pHi fell from a baseline of 7.33(0.04) to 7.25(0.03) during hypoxia and then overshot to 7.44(0.03) at reoxygenation (n = 5). Since an intracellular alkalosis can slow myofilament relaxation, proton extrusion routes were blocked to examine posthypoxic relaxation in the absence of an alkalosis. Despite inhibition of the pHi overshoot, posthypoxic relaxation remained impaired. Intracellular inorganic phosphate levels were manipulated in two protocols (2-deoxyglucose to "trap" phosphate and Tris(hydroxymethyl)-aminomethane to buffer phosphate) and both TPK and RT50 increased in normoxic cells. Having established that these two interventions, which would be expected to decrease intracellular inorganic phosphate, result in a slowing of relaxation, myocytes were first phosphate loaded (exposed to 5.0 mM phosphate) and then made hypoxic and reoxygenated after 10 min to blunt the expected fall in phosphate accompanying reoxygenation. This led to a reduction in the slowing of contraction and relaxation following reoxygenation [TPK increased by 7(5)% and RT50 by 17(9)%, n = 8; p < 0.05 v cells studied in control buffer]. CONCLUSIONS: Impaired posthypoxic relaxation is not the result of changes in pHi but is attenuated by phosphate loading of cells and may be due to a rapid decrease in intracellular phosphate accompanying the resynthesis of high energy phosphates at reoxygenation.

Identification of valid endogenous control genes for determining gene expression in C6 glioma cell line treated with conditioned medium from adipose-derived stem cell.

INTRODUCTION: There is growing evidence that mesenchymal stem cells (MSCs) can be important players in the tumor microenvironment. They can affect the glioma progression through the modulation of different genes. This modulation can be evaluated through a very useful model, treating the tumor cells with MSC-conditioned medium. However, for an accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is a prerequisite. METHODS: We performed a systematic review in an attempt to find a reference gene to use when analyzing gene expression in C6 glioma cells lines. Considering that we were not able to find a reference gene originated by an appropriate validation, in this study we evaluated candidate genes to be used as reference gene in C6 cells under different treatments with adipose-derived stem cells conditioned medium (CM-ADSCs). ß-actin (ACTB); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine-guanine phosphoribosyltransferase I (HPRT-1); TATA box binding protein (TBP) and beta-2-microglobulin (B2M) were evaluated by real-time reverse transcription PCR (RT-qPCR). The mean Cq, the maximum fold change (MFC) and NormFinder software were used for reference gene evaluation and selection. RESULTS: The GAPDH and ACTB genes have been the most widely used reference genes to normalize among the different investigated genes in our review, however, controversially these genes underwent a substantial variability among the genes evaluated in the present work. Individually, TBP gene was more stable when compared with other genes analyzed and the combination of TBP and HPRT-1 was even more stable. CONCLUSION: These results evidence the importance of appropriate validation of reference genes before performing qPCR experiments. Besides, our data will contribute with researchers that work analyzing the role of ADSCs in glioma microenvironment through gene expression.

Hydrolysis of NADP+ by platelet CD38 in the absence of synthesis and degradation of cyclic ADP-ribose 2'-phosphate.

CD38 is a multifunctional cell surface ectoenzyme that catalyzes both the synthesis of cyclic ADP-ribose from NAD+ and its hydrolysis to ADP-ribose. In this work, we investigated the metabolism of NADP+ by CD38 expressed on human platelets. Incubation of either platelet membranes or intact cells with NADP+ resulted in the rapid and time-dependent accumulation of ADP-ribose 2'-phosphate that paralleled the consumption of the substrate. However, under the same conditions, synthesis of cyclic ADP-ribose 2'-phosphate was not observed. By immunoprecipitation experiments, we identified CD38 as the enzyme responsible for the observed NADP+ glycohydrolase activity. The lack of detection of cyclic ADP-ribose 2'-phosphate was not due to its rapid hydrolysis, since direct incubation of platelet membranes with cyclic ADP-ribose 2'-phosphate did not result in the formation of ADP-ribose 2'-phosphate. By contrast, the same membrane samples expressed a significant ability to hydrolyze cyclic ADP-ribose to ADP-ribose. The absence of cyclic ADP-ribose 2'-phosphate hydrolase activity was also confirmed using high concentrations of substrate and by analysing both intact Jurkat T-lymphocytes and immunoprecipitated CD38. These results indicate that CD38, which is a multifunctional enzyme towards NAD+, displays exclusively a NADP+ glycohydrolase activity and is unable to catalyze both the synthesis and the hydrolysis of cyclic ADP-ribose 2'-phosphate.

Cytoskeleton-dependent inhibition of the ADP-ribosyl cyclase activity of CD38 in thrombin-stimulated platelets.

Stimulation of human platelets with thrombin caused a 42% inhibition of the ADP-ribosyl cyclase activity of membrane CD38. This effect was mediated by the activation of the platelet thrombin receptor rather than by proteolysis of CD38, and was not due to a different distribution of the synthesised nucleotide or to a reduced accessibility of CD38 to the substrate. The inhibitory effect of thrombin required actin polymerisation and was not observed when interaction of CD38 with the cytoskeleton was prevented by cytochalasin D. Finally, we analysed whether cADPR could play a role as a Ca2+-mobilising agent in human platelets. Using saponin-permeabilised cells, we found that unlike IP3, cADPR did not induce any release of Ca2+ from intracellular stores. These results indicate that the enzymatic activity of membrane CD38 can be modulated by platelet activation, and that the function of this glycoprotein is probably not related to Ca2+ mobilisation.

Thrombin induces the association of cyclic ADP-ribose-synthesizing CD38 with the platelet cytoskeleton.

The effect of platelet stimulation on the subcellular localization of CD38, a membrane glycoprotein that catalyses the synthesis of cyclic ADP-ribose from beta-NAD+ was investigated. Treatment of human platelets with thrombin caused the association of about 40% of the total ADP-ribosyl cyclase activity with the cytoskeleton, through the translocation of the CD38 molecule from the Triton X-100-soluble to the insoluble fraction. The interaction of CD38 with the cytoskeleton was a specific and reversible process, mediated by the binding to the actin-rich filaments and was inhibited by treatment of platelets with cytochalasin D. This event was regulated by integrin alphaIIb beta3 and platelet aggregation as it was prevented by the inhibition of fibrinogen binding and was not observed in platelets from a patient affected by Glanzmann thrombasthenia. These results demonstrate that the subcellular localization of CD38 can be influenced by platelet stimulation with physiological agonists, and that membrane CD38 can interact with intracellular proteins.

The platelet cytoskeleton regulates the aggregation-dependent synthesis of phosphatidylinositol 3,4-bisphosphate induced by thrombin.

Pretreatment of intact platelets with cytochalasin D prevented actin polymerization and cytoskeleton reorganization induced by thrombin, but did not affect platelet aggregation. Under these conditions, synthesis of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) stimulated by thrombin was strongly inhibited, while production of phosphatidic acid was unaffected. The inhibitory effect of cytochalasin D was not observed when platelet aggregation was prevented by the RGDS peptide. We also found that cytochalasin D did not affect PtdIns(3,4)P2 synthesis induced by concanavalin A (ConA), which is known to occur through an aggregation-independent mechanism. Moreover, thrombin, but not ConA, induced the translocation of phosphatidylinositol 3-kinase to the cytoskeleton. This process was equally inhibited by both the RGDS peptide and cytochalasin D. These results demonstrate that the cytoskeleton represents a functional link between thrombin-induced aggregation and synthesis of PtdIns(3,4)P2.

Agonist-induced actin polymerization is required for the irreversibility of platelet aggregation.

Cytochalasin D was used to investigate the role of intracellular cytoskeleton in the stabilization of platelet aggregation induced by strong platelet agonists. Incubation of gel-filtered platelets with increasing concentrations of cytochalasin D resulted in a dose-dependent inhibition of actin polymerization and association of actin-binding proteins with the Triton X-100-insoluble material induced by the thromboxane analogue, U46619, and the thrombin receptor activating peptide, TRAP. The same concentrations of cytochalasin D did not significantly inhibit platelet aggregation promoted by the two agonists. The addition of the chelating agent EDTA to fully aggregated platelets, that had been treated with cytochalasin D, resulted in the rapid and almost complete disaggregation. EDTA did not cause disaggregation of control, solvent-treated, aggregated platelets. The degree of platelet disaggregation induced by EDTA was dependent on the dose of cytochalasin D used, and was correlated with the inhibition of the cytoskeletal reorganization. Aggregation of cytochalasin D-treated platelets stimulated with U46619 or TRAP was also reverted by the addition of the tetrapeptide RGDS or the fibrinogen gamma-chain dodecapeptide, which competitively interfere with fibrinogen binding to the glycoprotein IIb-IIIa complex. These results indicate that the intracellular cytoskeleton plays an essential role in the stabilization of the fibrinogen-platelet interaction, and is necessary for the irreversibility of platelet aggregation induced by strong agonists.