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.

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.