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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 a-thrombin-evoked (10 mu M/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 (TM)) gradients (density span, 1.090-1.040 kg/L) containing prostaglandin E-1. 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 alpha-thrombin-evoked (10 U/mL) surface LAMP-1 (an assessment of lysosomal release), the fibrinogen (a(IIb)beta(3)) 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 alpha-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 alpha-thrombin-evoked (10 U/mL) surface LAMP-1 and a(IIb)beta(3) receptor activity. LAMP-1-expressing normal-sized platelets als, Funding Agencies|OEstergoetland County Council, Sweden

Background: Strong agonist provocation in vitro creates small procoagulant platelets characterized by down-regulated fibrinogen receptors as judged from surface alpha(IIb)beta(3) 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 (TM) 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.

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.

Introduction: We recently showed that Amyloid Beta (A beta)(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 A beta peptides (A beta(40), A beta(42), and A beta(43) ) 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 high-and presumed injured low-density erythrocyte fractions. After cell lysis, intracellular A beta(40) , A beta 4(2), and A beta (43) were quantified by ELISA. Results: In both high- and low-density erythrocytes, A beta(40) displayed the lowest concentration in MCI, while it was equal and higher in AD and SCI. A beta(40) was detected at a 10-fold higher level than A beta(42), and in injured low-density erythrocytes, the lowest quantity of A beta(42) was found in AD and MCI. A beta(40) exhibited a 100-fold greater amount than A beta(43). and lighter erythrocytes of MCI subjects displayed less intracellular A beta(43) than SCI. Conclusion: Red blood cell accumulation patterns of A beta(40), A beta(42), and A beta(43) differ significantly between AD, MCI, and SCI. The data must be verified through larger clinical trials. It is, however, tenable that AP peptide distributions in erythrocyte subpopulations have the potential to be used for diagnostic purposes., Funding Agencies|Hultmans stiftelse for forskning och bistand; Konung Gustaf V och Drottning Victorias Stiftelse; Gun and Bertil Stohnes Stiftelse; Stiftelsen for Gamla Tjanarinnor; Margaretha af Ugglas Stiftelse; Hjarnfonden

Background: Alzheimers Disease (AD) features the accumulation of beta-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 amp; 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., Funding Agencies|Hultmans stiftelse for forskning och bistand; Konung Gustaf V och Drottning Victorias stiftelse; Hjarnfonden; Margaretha af Ugglas Stiftelse; Stiftelsen for Gamla Tjanarinnor; Gun and Bertil Stohnes stiftelse

Introduction: Serotonin (5-HT) is involved in the pathology of Alzheimers 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-beta 1-42 (A beta(42)), 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/A beta(42) 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., Funding Agencies|Konung Gustaf V och Drottning Victorias Stiftelse; Hultmans Stiftelse