RBC-derived Microparticles Research Articles

Analysis of non-physiological shear stress-induced red blood cell trauma across different clinical support conditions of the blood pump.

Systematic research into device-induced red blood cell (RBC) damage beyond hemolysis, including correlations between hemolysis and RBC-derived extracellular vesicles, remains limited. This study investigated non-physiological shear stress-induced RBC damage and changes in related biochemical indicators under two blood pump clinical support conditions. Pressure heads of 100 and 350mmHg, numerical simulation methods, and two in vitro loops were utilized to analyze the shear stress and changes in RBC morphology, hemolysis, biochemistry, metabolism, and oxidative stress. The blood pump created higher shear stress in the 350-mmHg condition than in the 100-mmHg condition. With prolonged blood pump operation, plasma-free hemoglobin and cholesterol increased, whereas plasma glucose and nitric oxide decreased in both loops. Notably, plasma iron and triglyceride concentrations increased only in the 350-mmHg condition. The RBC count and morphology, plasma lactic dehydrogenase, and oxidative stress across loops did not differ significantly. Plasma extracellular vesicles, including RBC-derived microparticles, increased significantly at 600min in both loops. Hemolysis correlated with plasma triglyceride, cholesterol, glucose, and nitric oxide levels. Shear stress, but not oxidative stress, was the main cause of RBC damage. Hemolysis alone inadequately reflects overall blood pump-induced RBC damage, suggesting the need for additional biomarkers for comprehensive assessments.

TMEM16F mediated phosphatidylserine exposure and microparticle release on erythrocyte contribute to hypercoagulable state in hyperuricemia

The link between hyperuricemia (HUA) and the risk of venous thromboembolism (VTE) has been well established. However, the mechanisms of thrombus generation and the effect of HUA on procoagulant activity (PCA) of erythrocytes remain unclear no matter in uremia or hyperuricemia. Here, phosphatidylserine (PS) exposure, microparticles (MPs) release, cytosolic Ca2+, TMEM16F expression, reactive oxygen species (ROS) and lipid peroxidation of erythrocyte were detected by flow cytometer. PCA was assessed by coagulation time, purified coagulation complex and fibrin production assays. The fibrin formation was observed by scanning electron microscopy (SEM). We found that PS exposure, MPs generation, TMEM16F expression and consequent PCA of erythrocyte in HUA patients significantly increased compared to those in healthy volunteers. Furthermore, high UA induced PS exposure, and MPs release of erythrocyte in concentration and time-dependent manners in vitro, which enhanced the PCA of erythrocyte and was inhibited by lactadherin, a PS inhibitor. Additionally, using SEM, we also observed compact fibrin clots with highly-branched networks and thin fibers supported by red blood cells (RBCs) and RBC-derived MPs (RMPs). Importantly, we demonstrated UA enhanced the production of ROS and lipid peroxidation and reduced the generation of glutathione (GSH) of erythrocyte, which enhanced TMEM16F activity and followed PS externalization and RMPs formation. Collectively, these results suggest that Ca2+-dependent TMEM16F activation may be responsible for UA-induced PS exposure and MPs release of RBC, which thereby contribute to the prothrombotic risk in HUA.

Morphological Characteristics, Hemoglobin Content, and Membrane Mechanical Properties of Red Blood Cell Delivery Systems.

Red blood cell (RBC)-based systems are under extensive development as platforms for the delivery of various biomedical agents. While the importance of the membrane biochemical characteristics in relation to circulation kinetics of RBC delivery systems has been recognized, the membrane mechanical properties of such carriers have not been extensively studied. Using optical methods in conjunction with image analysis and mechanical modeling, we have quantified the morphological and membrane mechanical characteristics of RBC-derived microparticles containing the near-infrared cargo indocyanine green (ICG). We find that these particles have a significantly lower surface area, volume, and deformability as compared to normal RBCs. The residual hemoglobin has a spatially distorted distribution in the particles. The membrane bending modulus of the particles is about twofold higher as compared to normal RBCs and exhibits greater resistance to flow. The induced increase in the viscous characteristics of the membrane is dominant over the elastic and entropic effects of ICG. Our results suggest that changes to the membrane mechanical properties are a result of impaired membrane-cytoskeleton attachment in these particles. We provide a mechanistic explanation to suggest that the compromised membrane-cytoskeleton attachment and altered membrane compositional and structural asymmetry induce curvature changes to the membrane, resulting in mechanical remodeling of the membrane. These findings highlight the importance of membrane mechanical properties as an important criterion in the design and engineering of future generations of RBC-based delivery systems to achieve prolonged circulation.

Impact of Trail Running Races on Blood Viscosity and Its Determinants: Effects of Distance.

Blood rheology is a key determinant of tissue perfusion at rest and during exercise. The present study investigated the effects of race distance on hematological, blood rheological, and red blood cell (RBC) senescence parameters. Eleven runners participated in the Martigny–Combes à Chamonix 40 km race (MCC, elevation gain: 2300 m) and 12 others in the Ultra-Trail du Mont Blanc (UTMB, 171 km, elevation gain: 10,000 m). Blood samples were collected before and after the races. After the UTMB, the percentage of RBC phosphatidylserine (PS) exposure was not affected while RBC CD235a levels decreased and RBC-derived microparticles increased. In contrast, after the MCC, RBC PS exposure increased, while RBC CD235a and RBC-derived microparticles levels were not affected. The free hemoglobin and hemolysis rate did not change during the races. RBC aggregation and blood viscosity at moderate shear rates increased after the MCC. RBC deformability, blood viscosity at a high shear rate, and hematocrit decreased after the UTMB but not after the MCC. Our results indicate that blood rheology behavior is different between a 40 km and a 171 km mountain race. The low blood viscosity after the ultra-marathon might facilitate blood flow to the muscles and optimize aerobic performance.

Post-translational modification as a response to cellular stress induced by hemoglobin oxidation in sickle cell disease

Intracellular oxidative stress and oxidative modification of sickle hemoglobin (HbS) play a role in sickle cell disease (SCD) pathogenesis. Recently, we reported that Hb-dependent oxidative stress induced post-translational modifications (PTMs) of Hb and red blood cell (RBC) membrane proteins of transgenic SCD mice. To identify the mechanistic basis of these protein modifications, we followed in vitro oxidative changes occurring in intracellular Hb obtained from RBCs and RBC-derived microparticles (MPs) from the blood of 23 SCD patients (HbSS) of which 11 were on, and 12, off hydroxyurea (HU) treatment, and 5 ethnic matched controls. We used mass spectrometry-based proteomics to characterize these oxidative PTMs on a cross-sectional group of these patients (n = 4) and a separate subgroup of patients (n = 2) studied prior to initiation and during HU treatment. Collectively, these data indicated that band-3 and its interaction network involved in MPs formation exhibited more protein phosphorylation and ubiquitination in SCD patients than in controls. HU treatment reversed these oxidative PTMs back to level observed in controls. These PTMs were also confirmed using orthogonal immunoprecipitation experiments. Moreover, we observed specific markers reflective of oxidative stress, including irreversible oxidation of βCys93 and ubiquitination of Hb βLys145 (and βLys96). Overall, these studies strongly suggest that extensive erythrocyte membrane protein phosphorylation and ubiquitination are involved in SCD pathogenesis and provide further insight into the multifaceted effects of HU treatment.

Innate coagulability changes with age in stored packed red blood cells

BackgroundPacked red blood cell (pRBC) units administered during resuscitation from hemorrhagic shock are of varied storage ages. We have previously shown that RBC-derived microparticles' impact on thrombogenesis. However, the impact of storage age on pRBC coagulability is unknown. Therefore, we sought to investigate the effect of storage age on innate coagulability and aggregability of stored pRBCs. MethodspRBCs prepared from male C57BL/6J mice were stored in Additive Solution-3 according to our standardized murine blood banking protocols for 14 days. Rotational thromboelastometry (ROTEM) was used to assess the innate coagulation status of fresh and 14-day old pRBCs. Viscoelastic coagulation parameters of clotting time (CT), clot formation time (CFT), alpha angle, and maximum clot firmness (MCF) were analyzed to determine coagulability. Plasma was added to the fresh pRBCs and 15-day old pRBCs to determine if the storage-associated coagulopathy was reversible with plasma. Statistical analyses were conducted with a Student's t-test. ResultsFifteen-day old pRBCs demonstrated a significant reduction in MCF (10.3 vs. 24.4 mm, P-value <0.001) and alpha angle (6.0 vs. 27.2 degrees, P-value <0.001) as well as significant prolongation of CFT and CT (1126.5 vs. 571.4 s, P-value <0.001) compared to fresh pRBCs. FFP addition to 15-day old and fresh pRBCs, demonstrated a significant reduction in MCF and persistent prolongation of CFT. This suggests that pRBCs lost coagulability as they aged and this deficit was not completely corrected by plasma administration. ConclusionsStorage duration may be an important factor in coagulation potential of pRBCs. Transfusion with older pRBCs may contribute to coagulopathy in massively transfused patients.

Temporal sequence of the human RBCs' vesiculation observed in nano-scale with application of AFM and complementary techniques

Based on the multimodal characterization of human red blood cells (RBCs), the link between the storage-related sequence of the nanoscale changes in RBC membranes in the relation to their biochemical profile as well as mechanical and functional properties was presented. On the background of the accumulation of RBCs waste products, programmed cell death and impaired rheological properties, progressive alterations in the RBC membranes including changes in their height and diameter as well as the in situ characterization of RBC-derived microparticles (RMPs) on the RBCs surface were presented. The advantage of atomic force microscopy (AFM) in RMPs visualization, even at the very early stage of vesiculation, was shown based on the results revealed by other reference techniques. The nanoscale characterization of RMPs was correlated with a decrease in cholesterol and triglycerides levels in the RBC membranes, proving the link between the lipids leakage from RBCs and the process of vesiculation.

Effects of repleting organic phosphates in banked erythrocytes on plasma metabolites and vasoactive mediators after red cell exchange transfusion in sickle cell disease.

Red blood cell (RBC) exchange (RCE) transfusion therapy is indicated for certain patients with sickle cell disease (SCD). Although beneficial, this therapy is costly and inconvenient to patients, who may require it monthly or more often. Identification of blood and plasma biomarkers that could improve or help individualise RCE therapy is of interest. Here we examined relevant blood and plasma metabolites and biomarkers of vasoactivity and RBC fragility in a pilot study of SCD patients undergoing RCE using either standard RBC units or RBC units treated with a US Food and Drug Administration (FDA)-approved additive solution containing phosphate, inosine, pyruvate, and adenine ("PIPA"). In this prospective, single-blind, cross-over pilot clinical trial, patients were randomised to receive either standard RBC exchange or PIPA-treated RBC exchange transfusion with each RCE session over a 6-month treatment period. Pre- and post-transfusion blood samples were obtained and analysed for RBC O2 affinity, ATP, purine metabolites, RBC microparticles, and cell free haemoglobin. Red blood cell O2 affinity was maintained after PIPA-RCE in contrast to standard RCE, after which P50 fell (net O2 affinity rose). Plasma ATP did not change significantly after RCE using either of the RBC unit types. Exchange transfusion with PIPA-treated RBC units led to modest increases in plasma inosine and hypoxanthine. Plasma cell free haemoglobin fell after either standard or PIPA-treated RBC exchange transfusion (novel findings), and to a similar extent. RBC-derived microparticles in the plasma fell significantly and similarly after both standard and PIPA-treated RCE transfusion. In summary, treatment of RBCs with PIPA prior to RCE elicited favourable or neutral changes in key metabolic and vascular biomarkers. Further study of its efficacy and safety is recommended and could ultimately serve to improve outcomes in chronically transfused SCD patients.

Impact of a 10km running trial on eryptosis, red blood cell rheology, and electrophysiology in endurance trained athletes: a pilot study.

Blood rheology is a key determinant of blood flow and tissue perfusion. There are still large discrepancies regarding the effects of an acute running exercise on blood rheological properties and red blood cell (RBC) physiology. We investigated the effect of a 10km running trial on markers of blood rheology and RBC physiology in endurance trained athletes. Blood was sampled before and after the exercise to measure lactate and glucose, hematological and hemorheological parameters (blood viscosity, RBC deformability, and aggregation), eryptosis markers (phosphatidylserine and CD47 exposure, RBC reactive oxygen species), RBC-derived microparticles (RBC-MPs), and RBC electrophysiological activity. Weight was measured before and after exercise. Peripheral oxygen saturation and heart rate were monitored before and during the trial. Blood lactate and glucose levels increased after exercise and subjects significantly lost weight. All athletes experienced a significant fall in oxygen saturation. Mean corpuscular volume (MCV) was increased from 95.1 ± 3.2 to 96.0 ± 3.3 and mean corpuscular hemoglobin concentration (MCHC) decreased after exercise suggesting a slight RBC rehydration. Exercise increased RBC deformability from 0.344 ± 0.04 to 0.378 ± 0.07, decreased RBC aggregates strength and blood viscosity, while hematocrit (Hct) remained unaffected. While RBC electrophysiological recording suggested a modulation in RBC calcium content and/or chloride conductance, eryptosis markers and RBC-MPs were not modified by the exercise. A 10km acute running exercise had no effect on RBC senescence and membrane blebbing. In contrast, this exercise increased RBC deformability, probably through rehydration process which resulted in a decrease in blood viscosity.

Does endurance training improve red blood cell aging and hemorheology in moderate-trained healthy individuals?

ObjectiveTo examine the impact of a 6-week endurance training on red blood cell (RBC) aging and deformability of healthy participants to detect possible improved hemorheological and performance-related adaptations. MethodsA total of 31 participants (17 females and 14 males) performed a 6-week moderate training protocol (three 1-h running sessions per week at 70% of maximal heart rate). Blood was sampled before and after the training. RBCs from each participant were fractioned according to density and age into 4 RBC subfractions. Subfractions were examined for changes of RBC properties, including aging distribution, RBC deformability, RBC microparticles, and phosphatidylserine concentrations. RBC and plasma nitrite levels were measured as indicators of nitric oxide metabolism. ResultsAerobic performance, peak oxygen consumption, ventilatory thresholds, velocity at the aerobic–anaerobic threshold, and lactate at exhaustion improved after training. The relative amount of both young RBCs and old RBCs increased, and the amount of the main RBC fraction decreased. Phosphatidylserine externalization and RBC-derived microparticles decreased. Overall deformability expressed as shear stress required to achieve half-maximum deformation to theoretical maximal elongation index at infinite shear stress improved in unfractioned RBCs (p < 0.001). Nitrite decreased in total (p = 0.001), young (p < 0.001), main (p < 0.001), and old (p = 0.020) aged RBCs and in plasma (p = 0.002), but not in very old RBCs. ConclusionThese results indicate that non-endurance-trained healthy participants benefit from a regular moderate running training program because performance-related parameters improve and a younger RBC population with improved RBC properties is induced, which might support oxygen supply in the microcirculation.

Comparison of Phosphatidylserine-Exposing Red Blood Cells, Fragmented Red Blood Cells and Red Blood Cell-Derived Microparticles in β-Thalassemia/HbE Patients.

To determine the number and intensity of phosphatidylserine (PS) expression of the red blood cells (RBCs), fragmented RBCs, and RBC-derived microparticles (RMPs) in patients with β-thalassemia/hemoglobin (Hb)E. We used flow cytometry to determine the number and levels of PS expression. The number of PS-exposing RBCs was statistically significantly higher (P <.001) than that of PS-exposing fragmented RBCs or RMPs. In contrast, the intensity of PS expression was significantly higher (P <.001) in RMPs than in RBCs or fragmented RBCs. Our study showed a trend of association between RBC distribution width (RDW) and both the number of fragmented RBCs and RMPs and their intensity of PS expression. In β-thalassemia/HbE, PS-exposing RBCs, fragmented RBCs, and RMPs all differed in their numbers and their intensity of PS expression. The effects of these differences among PS-exposing populations on the pathophysiology of the disease require further investigation.

Microparticles formed during storage of red blood cell units support thrombin generation.

Intact red blood cells (RBCs) appear to support thrombin generation in in vitro models of blood coagulation. During storage of RBC units, biochemical, structural, and physiological changes occur including alterations to RBC membranes and release of microparticles, which are collectively known as storage lesion. The clinical consequences of microparticle formation in RBC units are unclear. This study was performed to assess thrombin generation via the prothrombinase complex by washed RBCs and RBC-derived microparticles as a function of RBC unit age. Well-characterized kinetic and flow cytometric assays were used to quantify and characterize microparticles isolated from leukocyte-reduced RBC units during storage for 42 days under standard blood banking conditions. Stored RBCs exhibited known features of storage lesion including decreasing pH, cell lysis, and release of microparticles demonstrated by scanning electron microscopy. The rate of thrombin formation by RBC units linearly increased during storage, with the microparticle fraction accounting for approximately 70% of the prothrombinase activity after 35 days. High-resolution flow cytometric analyses of microparticle isolates identified phosphatidylserine-positive RBC-derived microparticles; however, their numbers over time did not correlate with thrombin formation in that fraction. Red blood cell-derived microparticles capable of supporting prothrombinase function accumulate during storage, suggesting an increased potential of transfused units as they age to interact in unplanned ways with ongoing hemostatic processes in injured individuals, especially given the standard blood bank practice of using the oldest units available.

Endocytosis of Red Blood Cell Microparticles by Pulmonary Endothelial Cells is Mediated By Rab5

Microparticles are submicron vesicles shed from aging erythrocytes as a characteristic feature of the red blood cell (RBC) storage lesion. Exposure of pulmonary endothelial cells to RBC-derived microparticles promotes an inflammatory response, but the mechanisms underlying microparticle-induced endothelial cell activation are poorly understood. In the present study, cultured murine lung endothelial cells (MLECs) were treated with microparticles isolated from aged murine packed RBCs or vehicle. Microparticle-treated cells demonstrated increased expression of the adhesion molecules ICAM and E-selectin, as well as the cytokine, IL-6. To identify mechanisms that mediate these effects of microparticles on MLECs, cells were treated with microparticles covalently bound to carboxyfluorescein succinimidyl ester (CFSE) and cellular uptake of microparticles was quantified via flow cytometry. Compared with controls, there was a greater proportion of CFSE-positive MLECs from 15 min up to 24 h, suggesting endocytosis of the microparticles by endothelial cells. Colocalization of microparticles with lysosomes was observed via immunofluorescence, indicating endocytosis and endolysosomal trafficking. This process was inhibited by endocytosis inhibitors. SiRNA knockdown of Rab5 signaling protein in endothelial cells resulted in impaired microparticle uptake as compared with nonsense siRNA-treated cells, as well as an attenuation of the inflammatory response to microparticle treatment. Taken together, these data suggest that endocytosis of RBC-derived microparticles by lung endothelial cells results in endothelial cell activation. This response seems to be mediated, in part, by the Rab5 signaling protein.

Exacerbation of oxidative stress during sickle vaso-occlusive crisis is associated with decreased anti-band 3 autoantibodies rate and increased red blood cell-derived microparticle level: a prospective study.

Painful vaso-occlusive crisis, a hallmark of sickle cell anaemia, results from complex, incompletely understood mechanisms. Red blood cell (RBC) damage caused by continuous endogenous and exogenous oxidative stress may precipitate the occurrence of vaso-occlusive crises. In order to gain insight into the relevance of oxidative stress in vaso-occlusive crisis occurrence, we prospectively compared the expression levels of various oxidative markers in 32 adults with sickle cell anaemia during vaso-occlusive crisis and steady-state conditions. Compared to steady-state condition, plasma levels of free haem, advanced oxidation protein products and myeloperoxidase, RBC caspase-3 activity, as well as the concentrations of total, neutrophil- and RBC-derived microparticles were increased during vaso-occlusive crises, whereas the reduced glutathione content was decreased in RBCs. In addition, natural anti-band 3 autoantibodies levels decreased during crisis and were negatively correlated with the rise in plasma advanced oxidation protein products and RBC caspase-3 activity. These data showed an exacerbation of the oxidative stress during vaso-occlusive crises in sickle cell anaemia patients and strongly suggest that the higher concentration of harmful circulating RBC-derived microparticles and the reduced anti-band 3 autoantibodies levels may be both related to the recruitment of oxidized band 3 into membrane aggregates.

Role of red blood cells in haemostasis and thrombosis.

In contrast to an obsolete notion that erythrocytes, or red blood cells (RBCs), play a passive and minor role in hemostasis and thrombosis, over the past decades there has been increasing evidence that RBCs have biologically and clinically important functions in blood clotting and its disorders. This review summarizes the main mechanisms that underlie the involvement of RBCs in hemostasis and thrombosis in vivo, such as rheological effects on blood viscosity and platelet margination, aggregation and deformability of RBCs; direct adhesion and indirect biochemical interactions with endothelial cells and platelets, etc. The ability of stored and pathologically altered RBCs to generate thrombin through exposure of phosphatidylserine has been emphasized. The procoagulant and prothrombotic potential of RBC-derived microparticles transfused with stored RBCs or formed in various pathological conditions associated with hemolysis has been described along with prothrombotic effects of free hemoglobin and heme. Binding of fibrinogen or fibrin to RBCs may influence their effects on fibrin network structure, clot mechanical properties, and fibrinolytic resistance. Recent data on platelet-driven clot contraction show that RBCs compressed by platelets pulling on fibrin form a tightly packed array of polyhedral erythrocytes, or polyhedrocytes, which comprises a nearly impermeable barrier important for hemostasis and wound healing. RBCs may perform dual roles, both helping to stem bleeding but at the same time contributing to thrombosis in a variety of ways.

Pleomorphic Structures in Human Blood Are Red Blood Cell-Derived Microparticles, Not Bacteria.

BackgroundRed blood cell (RBC) transfusions are a common, life-saving therapy for many patients, but they have also been associated with poor clinical outcomes. We identified unusual, pleomorphic structures in human RBC transfusion units by negative-stain electron microscopy that appeared identical to those previously reported to be bacteria in healthy human blood samples. The presence of viable, replicating bacteria in stored blood could explain poor outcomes in transfusion recipients and have major implications for transfusion medicine. Here, we investigated the possibility that these structures were bacteria.ResultsFlow cytometry, miRNA analysis, protein analysis, and additional electron microscopy studies strongly indicated that the pleomorphic structures in the supernatant of stored RBCs were RBC-derived microparticles (RMPs). Bacterial 16S rDNA PCR amplified from these samples were sequenced and was found to be highly similar to species that are known to commonly contaminate laboratory reagents.ConclusionsThese studies suggest that pleomorphic structures identified in human blood are RMPs and not bacteria, and they provide an example in which laboratory contaminants may can mislead investigators.

Platelet and red blood cell interactions and their role in rheumatoid arthritis.

Cytokines, lymphocytes, platelets and several biomolecules have long been implicated in the pathology of rheumatoid arthritis (RA), and the influences of antibody production and tagging, and cytokine, chemokine and enzyme production at specific rheumatoid joints were thought to be exclusive to the advancement of disease parameters. Another role player in RA is red blood cells (RBCs) which, of late, have been found to be involved in RA pathobiology, as there is a positive correlation between RBC counts and joint pathology, as well as with inflammatory biomarkers in the disease. There is also an association between RBC distribution width and the incidence of myocardial infarction amongst RA patients, and there is a change in the lipid distribution within RBC membranes. Of late, certain RBC-associated factors with previously obscure roles and cell-derived particles thought to be inconsequential to the other constituents of plasma were found to be active biomolecular players. Several of these have been discovered to be present in or originating from RBCs. Their influences have been shown to involve in membrane dynamics that cause structural and functional changes in both platelets and RBCs. RBC-derived microparticles are emerging entities found to play direct roles in immunomodulation via interactions with other plasma cells. These correlations highlight the direct influences of RBCs on exacerbating RA pathology. This review will attempt to shed more light on how RBCs, in the true inflammatory milieu of RA, are playing an even greater role than previously assumed.

T lymphocyte responses to RBC-derived microparticles are APC-dependent (P1040)

Abstract Microparticles (MPs) generated in stored blood are thought to cause immunomodulatory effects in blood recipients. MPs are small double membrane vesicles derived from leukocytes, platelets or other cells. We hypothesized that RBC-derived MPs would inhibit T cell responses. MPs were isolated from stored RBCs using differential centrifugation. PBMCs were cultured +/-MPs, supernatants were collected on day one, and PBMC survival was tested on day seven. PBMCs or purified T cells were stained with CFSE and cultured for seven days with PHA+/-MP. We found that MPs alone induced pro-inflammatory cytokine secretion and enhanced survival of T cells, B cells, and NK cells to a similar degree, but did not induce T cell proliferation. MPs augmented PHA-induced proliferation of CD4+ and CD8+ T cells in PBMC cultures, and blockade of CD40 or CD40L accessory molecules prevented the augmentation effect of MPs on T cell proliferation. Furthermore, stimulation of purified T cells with PHA+/-MP did not induce T cell proliferation, and addition of MP-exposed and washed monocytes to purified T cells recapitulated augmentation of T cell proliferation. In summary, addition of RBC-MPs to PBMCs improved survival of unstimulated cells and augmented mitogen driven T cell responses in an APC-dependent manner. These results suggest that, contrary to our hypothesis, MPs found in transfusion products may bias the recipient immune system in a pro-inflammatory fashion.

Procoagulant activity in patients with sickle cell trait

Patients with sickle cell trait (STr) are usually considered to be asymptomatic. However, complications, including hypercoagulability, increased risk of venous thromboembolism and the exertional exercise syndrome with rhabdomyolysis and sudden death, have been described. The exact cause of these adverse events is unclear. We have investigated two patients, a set of monozygotic twins with STr, to establish their procoagulant activity status as a potential indicator of thrombotic risk. In-vivo thrombin generation was assessed by the measurement of prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin complexes (TAT). D-dimer was used as a marker of fibrinolytic activity. The potential to generate thrombin was determined using an ex-vivo thrombin generation test (TGT). The impact of red blood cell (RBC)-derived microparticle shedding and RBC rheology were examined. TAT (>60 μg/l) and F1 + 2 (948 pmol/l) were markedly elevated in patient 2 but within the normal reference range in patient 1 (TAT = 2.5 μg/l; F1 + 2 = 138 pmol/l). D-dimer levels (0.9 mg/l FEU) were similarly elevated in both patients. TGT peak thrombin and endogenous thrombin potential (ETP) were elevated to similar degrees in both patients. Flow cytometric analysis for RBC-derived microparticles showed that both patients had elevated levels on two occasions. RBC deformability, blood viscosity and RBC aggregation were normal and similar in both patients. The results demonstrated different coagulation activity in the patients with one patient in a prothrombotic state, suggesting that there may be two levels of hypercoagulability in STr. Measurement of such differences would allow for separation of high and low-risk patients from serious complications.

RBC-Derived Microparticles Evolve Over RBC Storage Time and Boost T Cell Mitogen Responses

Abstract 1256 Introduction:It is widely believed that transfusion of younger blood carries less risk of adverse reactions compared to older blood, attributed to a red cell “storage lesion”. One potential effect of RBC transfusion is immune modulation, and we tested whether cell-derived microparticles (MPs) could modulate T cell responses. MPs are small double membrane vesicles with an approximate size of 0.1–1.2 μm, released into circulation during activation, stress, necrosis or apoptosis or can be generated during the storage of blood products. MPs express different surface markers depending on their cell of origin and may contain RNA, DNA or cellular proteins. Here we tracked the cells of origin for MPs found in RBC units throughout the storage period. Furthermore, we hypothesized that RBC-derived MPs would suppress T cell immune responses. Methods:RBC units were split into 35 ml in replicate small transfer bags and aged at 4°C. MPs were isolated using differential centrifugation with a final speed of 100,000g. Panels including 17 fluorochrome-conjugated antibodies were used identify MPs derived from RBCs, platelets, lymphocytes subpopulations, monocytes and granulocytes for four different RBC units. CFSE staining was used to measure T cell proliferation after stimulation with PHA plus or minus MP addition using MPs from six different units. Results:Two platelet markers in our panel (CD41a and CD42a) increased from day 0 through 21 of storage. The mean percentage of CD41a+ MPs increased from 3.9% at day 0 to 20% at day 21 (P<0.05) and decreased to 4.3% at day 42. CD42a+ MPs increased from 3.3% at day 0 to 13% at day 21 (P<0.05) and decreased to 1.9% at day 42. The percentages of CD14+ MPs (monocyte marker) ranged from 7.0% at day 0 to 12% at Day 42 and CD108a+ MPs (RBC marker) showed a trend to increasing from 1.7% at day 0 to 27.1% at day 42 (P>0.05). The percentage of CD3 (T lymphocyte marker) and CD16 (from NK cells and other cell types), CD19 (B cell marker), CD152 and CD154 (costimulatory molecules) percentages were <5% at baseline and remained low throughout storage.Contrary to our hypothesis, CD4+ and CD8+ T lymphocytes showed augmented proliferation when PBMCs were stimulated with MPs plus PHA relative to PHA alone. Mean percentage of CFSElow (proliferated) CD4+ T cells was 42% for PHA stimulated vs. 62% for PHA+MP stimulated PBMCs (p=0.018). CD8+ T cells showed similar differences (52 vs. 75% CFSElow, p=0.03). In the three samples studied longitudinally, the MP-induced augmentation was not significantly different for day 0 vs. day 42 MPs. Addition of MPs to PBMCs without mitogen did not induce any de novo proliferation. Conclusion:RBC-derived MPs appear to be predominantly derived from RBCs, platelets, and monocytes, and their composition can change with RBC unit storage time. Surprisingly, RBC-derived MPs augmented mitogen-stimulated CD4+ and CD8+ T cell responses. Further work will be required to determine the mechanism of enhancement and requirement of antigen presenting cells in the MP-induced enhancement of T cell responses. These findings illustrate how complicated and pleiotropic effects of transfusion related immune modulation can be. Disclosures:No relevant conflicts of interest to declare.