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

BACKGROUND/OBJECTIVE: Type 2 diabetes is a major risk factor for atherosclerotic disease. It is well agreed that the reactivity of diabetic platelets is increased but how platelet reactivity regulates is unknown. In our laboratory, density separated platelets have been investigated extensively and high- and low-density platelets circulate in an activated state. The density distribution of circulating platelets is altered in diabetes type 2 as well. We hypothesize that such platelets modify whole blood (WB) in vitro α-thrombin-evoked (10 µM/mL) activity in type 2 diabetes. Thus, the study aims to identify features of circulating normal-sized density subpopulations affecting whole blood (WB) platelet reactivity in type 2 diabetes. PATIENTS AND METHODS: Patients with type 2 diabetes (n = 16) were enrolled. Their normal-sized platelets were divided into density subfractions (n = 16) using continuous polyvinylpyrrolidone-coated silica (Percoll™) gradients (density span, 1.090-1.040 kg/L) containing prostaglandin E1. The proportions (%) of such density-separated platelets expressing lysosomal-associated membrane protein 1 (LAMP-1) were analyzed using a flow cytometer. Further, determinations of WB ɑ-thrombin-evoked (10 U/mL) surface LAMP-1 (an assessment of lysosomal release), the fibrinogen (αIIbß3) receptor activity, annexin V (binds to exposed membrane phosphatidylserine), and mitochondrial transmembrane potentials (an estimate of organelle integrity) were performed. Surface LAMP-1 expressions of individual normal-sized platelet density subpopulations were stratified into equal-sized groups (n = 2) depending on reactivity, as judged from the ɑ-thrombin-induced WB activity markers. RESULTS: With some exceptions, the proportion of normal-sized circulating platelets showing spontaneous LAMP-1 was strongly associated with WB ɑ-thrombin-evoked (10 U/mL) surface LAMP-1 and αIIbß3 receptor activity. LAMP-1-expressing normal-sized platelets also displayed inverse associations with WB ɑ-thrombin-induced surface annexin V and mitochondrial damage, which are features of procoagulant platelets. CONCLUSIONS: From the current descriptive work only involving type 2 diabetes, it is impossible to judge whether the findings are features of the disease or if they occur in healthy individuals as well. However, the study describes LAMP-1 expressing subpopulations of circulating normal-sized platelets that associate with WB α-thrombin (10 U/mL) responses in vitro. Increased proportions of such platelets induced lysosomal release and αIIbß3 receptor activity, whereas lower proportions promoted WB agonist-induced procoagulant platelet creation. It is to hypothesize that the new described regulatory mechanism could in the future offer a possibility to influence platelet behavior in type 2 diabetes.Key messagesLysosomal exocytosis of circulating platelets influences reactivity, as determined by agonist-induced platelet reactions in vitroThus, the low release of lysosomes by normal-sized platelets in vivo increases agonist-evoked procoagulant platelet production.Higher lysosomal exocytosis of circulating normal-sized platelets promotes platelet aggregation and secretion.

Small procoagulant platelets in diabetes type 2.

BACKGROUND: Strong agonist provocation in vitro creates small procoagulant platelets characterized by down-regulated fibrinogen receptors as judged from surface αIIbß3 activation specific antibody (PAC-1). They further show increased surface Annexin V (binds to platelet membrane phosphatidylserine), lysosomal-associated membrane protein 1 (LAMP-1) (indicates lysosomal release) and exhibit disturbed mitochondria integrity as estimated from mitochondrial transmembrane potential changes. We postulated that some circulating platelets activate continuously thereby forming procoagulant populations in vivo. This study aimed to identify such platelets in diabetes type 2 a condition predisposing for thrombotic events. METHODS: A linear Percoll™ gradient covering the density span 1.090 to 1.040 kg/L was used to separate whole blood platelets from type 2 diabetic subjects (n = 12) into 17 density subpopulations. The gradient contained theophylline, prostaglandin E1 and EDTA to prevent platelet activation in vitro. A multi-colour flow cytometer was employed for analysing the characteristics mentioned above for all density separated small-sized platelet subfractions. RESULTS AND CONCLUSION: Small platelets were enriched in medium-dense subfractions (nos. 10-13) (1.065-1.053 kg/L). Their PAC-1 activities were significant lower (p < 0.001) as compared to other small-sized subpopulations. They further exposed enhanced surface Annexin V and LAMP-1 together with lower mitochondrial transmembrane potentials. In diabetes type 2 such small circulating platelets showed procoagulant features.

Erythrocyte Amyloid Beta Peptide Isoform Distributions in Alzheimer and Mild Cognitive Impairment.

INTRODUCTION: We recently showed that Amyloid Beta (Aß)40 accumulates in erythrocytes and possibly causes cell damage as evidenced by an increased number of assumed injured low-density (kg/L) erythrocytes. Furthermore, we have suggested a separation technique to isolate and concentrate such damaged red blood cells for subsequent analysis. OBJECTIVES: We isolated high- and low-density erythrocytes and investigated the accumulation patterns of the Aß peptides (Aß40, Aß42, and Aß43) in Alzheimer (AD), mild cognitive impairment (MCI), and Subjective Cognitive Impairment (SCI). METHODS: Whole blood was fractionated through a density gradient, resulting in two concentrated highand presumed injured low-density erythrocyte fractions. After cell lysis, intracellular Aß40, Aß42, and Aß43 were quantified by ELISA. RESULTS: In both high- and low-density erythrocytes, Aß40 displayed the lowest concentration in MCI, while it was equal and higher in AD and SCI. Aß40 was detected at a 10-fold higher level than Aß42, and in injured low-density erythrocytes, the lowest quantity of Aß42 was found in AD and MCI. Aß40 exhibited a 100-fold greater amount than Aß43, and lighter erythrocytes of MCI subjects displayed less intracellular Aß43 than SCI. CONCLUSION: Red blood cell accumulation patterns of Aß40, Aß42, and Aß43 differ significantly between AD, MCI, and SCI. The data must be verified through larger clinical trials. It is, however, tenable that Aß peptide distributions in erythrocyte subpopulations have the potential to be used for diagnostic purposes.

Alzheimer's Disease: Erythrocyte 2,3-diphosphoglycerate Content and Circulating Erythropoietin.

BACKGROUND: Alzheimer's Disease (AD) features the accumulation of ß-amyloid in erythrocytes. The subsequent red cell damage may well affect their oxygen-carrying capabilities. 2,3- diphosphoglycerate (2,3-DPG) binds to the hemoglobin thereby promoting oxygen release. It is theorized that 2,3-DPG is reduced in AD and that the resulting hypoxia triggers erythropoietin (EPO) release. METHODS & OBJECTIVE: To explore this theory, we analyzed red cell 2,3-DPG content and EPO in AD, mild cognitive impairment, and the control group, subjective cognitive impairment. RESULTS: We studied (i) 2,3-DPG in red cells, and (ii) circulating EPO in AD, and both markers were unaffected by dementia. Disturbances of these oxygen-regulatory pathways do not appear to participate in brain hypoxia in AD.

Amyloidogenic Nanoplaques in Blood Serum of Patients with Alzheimer's Disease Revealed by Time-Resolved Thioflavin T Fluorescence Intensity Fluctuation Analysis.

BACKGROUND: Biomarkers are central to current research on molecular mechanisms underlying Alzheimer's disease (AD). Their further development is of paramount importance for understanding pathophysiological processes that eventually lead to disease onset. Biomarkers are also crucial for early disease detection, before clinical manifestation, and for development of new disease modifying therapies. OBJECTIVE: The overall aim of this work is to develop a minimally invasive method for fast, ultra-sensitive and cost-effective detection of structurally modified peptide/protein self-assemblies in the peripheral blood and in other biological fluids. Specifically, we focus here on using this method to detect structured amyloidogenic oligomeric aggregates in the blood serum of apparently healthy individuals and patients in early AD stage, and measure their concentration and size. METHODS: Time-resolved detection of Thioflavin T (ThT) fluorescence intensity fluctuations in a sub-femtoliter observation volume element was used to identify in blood serum ThT-active structured amyloidogenic oligomeric aggregates, hereafter called nanoplaques, and measure with single-particle sensitivity their concentration and size. RESULTS: The concentration and size of structured amyloidogenic nanoplaques are significantly higher in the blood serum of individuals diagnosed with AD than in control subjects. CONCLUSION: A new method with the ultimate, single-particle sensitivity was successfully developed. The proposed approach neither relies on the use of immune-based probes, nor on the use of radiotracers, signal-amplification or protein separation techniques, and provides a minimally invasive test for fast and cost-effective early determination of structurally modified peptides/proteins in the peripheral blood, as shown here, but also in other biological fluids.

Non-coronary chest pain does not affect long-term mortality: a prospective, observational study using a matched population control.

BACKGROUND: Chest pain assumed to be of non-coronary origin (NCCP) may be linked to enhanced mortality due to coronary heart disease (CHD). The aim of this study was to follow NCCP patients, as defined in primary care, with respect to mortality and long-term morbidity of CHD. We further examined if NCCP associates with risk factors for CHD. METHODS: Patients consulting general practitioners (GPs) in 1998-2000 in three primary care centers in the southeast Sweden for chest pain regarded as NCCP were compared with controls matched for age, gender and residential area. Causes of death were gathered from registry data and death certificates. In 2005 a postal questionnaire was distributed to the survivors to collect demographic and clinical data. If participants had CHD diagnosed by a physician prior to inclusion they were excluded. RESULTS: Patients with NCCP (n = 382) and population controls (n = 746) did not differ with respect to mortality and incidence of CHD. The NCCP group reported more ongoing chest pain (OR 3.34 95 % CI 2.41-4.62), they more often had elevated blood pressure (OR 1.86 95 % CI 1.32-2.60), consumed more ß-blockers (p < 0.001), aspirin (p = 0.013), thiazides (p = 0.004) and long-acting nitrates (p = 0.002). They further had more remedies for acid-related disorders (p = 0.014) and obstructive pulmonary disease (p < 0.001). CONCLUSIONS: The study suggests that individuals with chest pain judged by GPs to be NCCP do not develop CHD more frequently than population controls. It is evident that NCCP often lasts for many years and that the condition associates with hypertension.

Association of Platelet Serotonin Levels in Alzheimer's Disease with Clinical and Cerebrospinal Fluid Markers.

INTRODUCTION: Serotonin (5-HT) is involved in the pathology of Alzheimer's disease (AD). OBJECTIVE: We aimed to measure 5-HT level in platelets in AD and explore its association with cerebrospinal fluid (CSF), AD biomarkers (amyloid-ß 1-42 (Aß42), total tau (t-tau), and phosphorylated tau (p-tau)), and clinical symptoms. METHODS: 15 patients with AD and 20 patients with subjective cognitive impairment (SCI) were included. 5-HT metabolites were measured, in a specific fraction, using high performance liquid chromatography with electrochemical detection (HPLC-ECD). RESULTS: Significantly lower 5-HT concentrations were observed in AD patients compared to SCI patients both after normalization against total protein (p = 0.008) or platelet count (p = 0.019). SCI patients with lower 5-HT level have higher AD CSF biomarkers, total tau (p = 0.026) and tau/Aß42 ratio (p = 0.001), compared to those with high 5-HT levels. CONCLUSION: AD patients have reduced platelet 5-HT levels. In SCI, lower 5-HT content was associated with a higher AD-CSF biomarker burden.

Platelets and Inflammatory Parameters Do Not Affect Long-Term Survival after Acute Stroke.

RATIONALE: According to literature, the inflammatory response and platelets are associated with coronary heart disease mortality. In this study, we examine if similar relationships exist after acute cerebral infarctions. DESIGN: Between 2005 and 2007, individuals (n = 61) hospitalized with acute stroke were investigated 2.1 ± .3 (SD) days after hospital admission. After 9.3 ± .7 (SD) years, 29 patients (age 79 ± 8 [SD]; 12 women) had died. They were compared with survivors (age 69 ± 9 [SD]; 9 women) with respect to inflammatory parameters and platelet features such as activity and reactivity. RESULTS AND CONCLUSION: Inflammation and platelets at the acute event do not forecast long-term survival of stroke sufferers.

Platelets, gender and acute cerebral infarction.

OBJECTIVE: Platelets may well be significant in the pathogenesis of cerebral infarction. Platelets vary substantially according to gender. The scope of our current work is to establish if female and male stroke sufferers differ regarding platelet reactivity. PATIENTS AND METHODS: 73 Consecutive individuals stricken by acute ischemic cerebral infarction (31 females, 42 males) participated. All stroke subtypes were included. Platelet counts was determined electronically. Platelet reactivity i.e. the presence of surface-bound fibrinogen following provocation was analyzed with a flow cytometer. ADP (1.7 µmol/L) and a thrombin receptor agonist (TRAP-6) (57 µmol/L) were the agonists used. RESULTS: Female stroke sufferers had higher platelet counts (p = 0.013) but their platelets were less reactive. The p values were (p = 0.038) and (p = 0.016) for ADP and TRAP-6, respectively. CONCLUSION: The current study demonstrates that women suffering acute cerebral infarction have less reactive platelets. It is concluded that gender affects platelets. Our study indicates that it may be beneficial to individualize platelet inhibition of stroke sufferers according to gender.

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

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

Alzheimer's disease and granulocyte density diversity.

BACKGROUND: The current study investigates circulating eosinophils and neutrophils in Alzheimer's (AD) type dementia with respect to density (kg/L). The existence of ß-amyloid plaques in the brain is a feature of AD. Sporadic scientific reports indicate that the disease affects circulating neutrophils. In contrast, numerous publications investigate inflammatory reactions in AD brains. Locally, the plaques evoke a substantial inflammatory response involving activated microglia and astrocytes. METHODS: Subjects with probable AD (n = 39) were included and compared with elderly individuals (n = 22) lacking apparent memory problems. We sampled 10 mL venous blood in citrate. Granulocytes were separated according to density in linear Percoll™ gradients. Subsequently, the gradients were divided into density subfractions (n = 16). In every fraction, determination of eosinophil and neutrophil counts was carried out. RESULTS: AD sufferers displayed less granulocytes in fractions nos. 13-15 containing light cells. For these fractions, the P-values proved to be (P < 0.001; not significant; P = 0.03) and (P = 0.01; P = 0.01; not significant), for eosinophils and neutrophils, respectively. CONCLUSIONS: The present work describes that less circulating light granulocytes are a feature of AD demented individuals. It is to hypothesize that it is a sign of impaired granulocyte turnover and cell damage. It is concluded that AD affects inflammatory cells in the periphery and that the behaviour of granulocytes in dementia is worthwhile further studies.

P-selectin paradox and dementia of the Alzheimer type: circulating P-selectin is increased but platelet-bound P-selectin after agonist provocation is compromised.

OBJECTIVE: Knowledge concerning the neurobiological importance of platelets in Alzheimer's disease (AD) is sparse. P-selectin, which is located together with ß-amyloid precursor proteins in platelet α-granules, is also found in endothelial cells. Upon activation, P-selectin is relocated to cell surfaces where it acts as a receptor. Subsequently, the protein is cleaved from the membrane, to then be circulated. We investigated P-selectin behavior in AD dementia. METHODS: We recruited 23 persons diagnosed moderate AD and 17 healthy elders without obvious memory problems. Circulating P-selectin was analyzed using an ELISA technique and flow cytometry was used to measure surface-bound P-selectin. The latter measure was carried out without provocation (platelet activity) and after in vitro agonist stimulation (platelet reactivity). A thrombin-receptor activating peptide (TRAP-6) (74 µmol/L)) was used as a platelet agonist. RESULTS: Soluble P-selectin was augmented in AD (p = 0.019) but platelet membrane-attached P-selectin did not differ from controls. AD diagnosis was associated with less surface-bound P-selectin after provocation. Significant results were obtained when 74 µmol/L TRAP-6 was used as a platelet agonist (p = 0.0008). CONCLUSION: This study describes apparently paradoxical P-selectin reactions in moderate AD. While soluble P-selectin was higher in the disease group, membrane-attached P-selectin without agonist stimulation was no different between the disease and control groups. In contrast, AD was linked to lower platelet reactivity. The current findings encourage further research into this P-selectin paradox and its relevance for AD and, perhaps, other types of dementia as well.

Platelet density distribution in essential thrombocythemia.

Essential thrombocythemia (ET) is characterized by high platelet counts and a slightly increased bleeding risk. Why severe hemorrhage does not occur more frequently is not known. Variations of platelet density (kg/l) depend mainly on cell organelle content in that high-density platelets contain more α and dense granules. This study compares ET patients (n = 2) and healthy volunteers (n = 2) with respect to platelet density subpopulations. A linear Percoll™ gradient containing prostaglandin E(1) was employed to separate platelets according to density. The platelet population was subsequently divided by density into 16 or 17 subpopulations. Determination of platelet counts was carried out. In each density fraction, platelet in vivo activity, i.e. platelet-bound fibrinogen, was measured using a flow cytometer. To further characterize platelet subpopulations, we determined intracellular concentrations of CD40 ligand (CD40L) and P-selectin in all fractions. Patients and controls demonstrated similar density distributions, i.e. 1 density peak. High-density platelets had more surface-bound fibrinogen in conjunction with signs of platelet release reactions, i.e. with few exceptions they contained less CD40L and P-selectin. Peak density platelets showed less surface-bound fibrinogen. These platelets contained less CD40L and P-selectin than nearby denser populations. The light platelets had more surface-bound fibrinogen than peak platelets together with elevated concentrations of CD40L. In ET, the malignant platelet production could exist together with platelets originating from normal megakaryocytes. It is also possible that clonal megakaryocytes produce platelets covering the entire density span. The 'normal' density distribution offers a tenable explanation as to why serious bleedings do not occur more frequently.