Red Blood Cell-derived Microparticles Research Articles

Red Cell Microparticles Suppress Hematoma Growth Following Intracerebral Hemorrhage in Chronic Nicotine-Exposed Rats

Spontaneous intracerebral hemorrhage (sICH) is a disabling stroke sub-type, and tobacco use is a prominent risk factor for sICH. We showed that chronic nicotine exposure enhances bleeding post-sICH. Reduction of hematoma growth is a promising effective therapy for sICH in smoking subjects. Red-blood-cell-derived microparticles (RMPs) are hemostatic agents that limit hematoma expansion following sICH in naïve rats. Considering the importance of testing the efficacy of experimental drugs in animal models with a risk factor for a disease, we tested RMP efficacy and the therapeutic time window in limiting hematoma growth post-sICH in rats exposed to nicotine. Young rats were chronically treated with nicotine using osmotic pumps. sICH was induced in rats using an injection of collagenase in the right striatum. Vehicle/RMPs were administered intravenously. Hematoma volume and neurological impairment were quantified ≈24 h after sICH. Hematoma volumes in male and female nicotine-exposed rats that were treated with RMPs at 2 h post-sICH were significantly lower by 26 and 31% when compared to their respective control groups. RMP therapy was able to limit hematoma volume when administered up to 4.5 h post-sICH in animals of both sexes. Therefore, RMPs may limit hematoma growth in sICH patients exposed to tobacco use.

Red Blood Cell Microparticles Limit Hematoma Growth in Intracerebral Hemorrhage.

Spontaneous intracerebral hemorrhage (sICH) is the deadliest stroke subtype with no effective therapies. Limiting hematoma expansion is a promising therapeutic approach. Red blood cell-derived microparticles (RMPs) are novel hemostatic agents. Therefore, we studied the potential of RMPs in limiting hematoma growth and improving outcomes post-sICH. sICH was induced in rats by intrastriatal injection of collagenase. RMPs were prepared from human RBCs by high-pressure extrusion. Behavioral and hematoma/lesion volume assessment were done post-sICH. The optimal dose, dosing regimen, and therapeutic time window of RMP therapy required to limit hematoma growth post-sICH were determined. We also evaluated the effect of RMPs on long-term behavioral and histopathologic outcomes post-sICH. RMP treatment limited hematoma growth following sICH. Hematoma volume (mm3) for vehicle- and RMP- (2.66×1010 particles/kg) treated group was 143±8 and 86±4, respectively. The optimal RMP dosing regimen that limits hematoma expansion was identified. RMPs limit hematoma volume when administered up to 4.5-hour post-sICH. Hematoma volume in the 4.5-hour post-sICH RMP treatment group was lower by 24% when compared with the control group. RMP treatment also improved long-term histopathologic and behavioral outcomes post-sICH. Our results demonstrate that RMP therapy limits hematoma growth and improves outcomes post-sICH in a rodent model. Therefore, RMPs have the potential to limit hematoma growth in sICH patients.

Red Blood Cell-Derived Microparticles Exert No Cancer Promoting Effects on Colorectal Cancer Cells In Vitro.

The biomedical consequences of allogeneic blood transfusions and the possible pathomechanisms of transfusion-related morbidity and mortality are still not entirely understood. In retrospective studies, allogeneic transfusion was associated with increased rates of cancer recurrence, metastasis and death in patients with colorectal cancer. However, correlation does not imply causation. The purpose of this study was to elucidate this empirical observation further in order to address insecurity among patients and clinicians. We focused on the in vitro effect of microparticles derived from red blood cell units (RMPs). We incubated different colon carcinoma cells with RMPs and analyzed their effects on growth, invasion, migration and tumor marker expression. Furthermore, effects on Wnt, Akt and ERK signaling were explored. Our results show RMPs do not seem to affect functional and phenotypic characteristics of different colon carcinoma cells and did not induce or inhibit Wnt, Akt or ERK signaling, albeit in cell culture models lacking tumor microenvironment. Allogeneic blood transfusions are associated with poor prognosis, but RMPs do not seem to convey tumor-enhancing effects. Most likely, the circumstances that necessitate the transfusion, such as preoperative anemia, tumor stage, perioperative blood loss and extension of surgery, take center stage.

Urinary red blood cell-derived microparticles and phosphatidylserine-exposing red blood cells in glomerular and non-glomerular hematuria patients.

Distinguishing glomerular hematuria (GH) from non-glomerular hematuria (NGH) is important for treating the cause of hematuria. We aimed to determine red blood cell-derived microparticles (RMPs) and phosphatidylserine (PS)-exposing red blood cells (RBCs) and evaluate their use for diagnosing GH and NGH patients. All patients received a physical assessment and urological examination. Dysmorphic RBCs (dRBCs) and acanthocytes were examined using a light microscope. The urinary RMPs and PS-exposing RBCs were determined using flow cytometry. The ratio of RMPs to RBCs was higher in GH patients (n=29) than in NGH patients (n=29) (1.06 vs. 0.18). The value of the sum of the PS-exposing RBCs plus RMPs divided by the number of RBCs was higher in GH patients than in NGH patients (48.3% vs. 19.4%). The percentage of RBCs was higher in GH patients than in NGH patients (54.5% vs. 21.8%). Similarly, both the percentages of acanthocytes and of non-acanthocytes were higher in GH patients than in NGH patients (29% vs. 7.7% and 25.4% vs. 14.2%, respectively). The ROC-AUC of the number of PS-exposing RBCs plus RMPs divided by the number of RBCs was 0.9 (95% CI, 0.82-0.97), and the RMPs:RBCs ratio was 0.88 (95% CI, 0.79-0.98). The ROC-AUCs of the dRBCs and acanthocytes were 0.85 (95% CI, 0.78-0.95) and 0.88 (95% CI, 0.8-0.97), respectively. Patients with GH have higher numbers of urinary RMPs and PS-exposing RBCs. These parameters have the potential to be predictive tools for classifying GH in the future.

Membrane Cholesterol Enrichment of Red Blood Cell-Derived Microparticles Results in Prolonged Circulation.

Particles fabricated from red blood cells (RBCs) can serve as vehicles for delivery of various biomedical cargos. Flipping of phosphatidylserine (PS) from the inner to the outer membrane leaflet normally occurs during the fabrication of such particles. PS externalization is a signal for phagocytic removal of the particles from circulation. Herein, we demonstrate that membrane cholesterol enrichment can mitigate the outward display of PS on microparticles engineered from RBCs. Our in-vitro results show that the phagocytic uptake of cholesterol-enriched particles by murine macrophages takes place at a lowered rate, resulting in reduced uptake as compared to RBC-derived particles without cholesterol enrichment. When administered via tail-vein injection into healthy mice, the percent of injected dose (ID) per gram of extracted blood for cholesterol-enriched particles was ∼1.5 and 1.8 times higher than the particles without cholesterol enrichment at 4 and 24 h, respectively. At 24 h, ∼43% ID/g of the particles without cholesterol enrichment was eliminated or metabolized while ∼94% ID/g of the cholesterol-enriched particles were still retained in the body. These results indicate that membrane cholesterol enrichment is an effective method to reduce PS externalization on the surface of RBC-derived particles and increase their longevity in circulation.

Storage Duration and Red Blood Cell-Derived Microparticles in Packed Red Blood Cells Obtained from Donors with Thalassemia.

To address the effects of storage duration on red blood cell (RBC)-derived microparticles (RMPs) in packed RBCs from donors who have thalassemia. Packed RBCs were prepared according to laboratory routine. The quantity of RMPs was determined using FACSCalibur and counting beads. Across durations of storage, the packed RBCs from donors with thalassemia (n = 28) and healthy volunteers (n = 104) showed average RMPs to be 47,426 (10,139‒127,785) particles/μL vs 49,021 (13,033‒126,749) particles/μL, respectively (P = .63). The peak RMP levels in donors with thalassemia and healthy volunteers, respectively, were shown in products from storage days 34 and 38. Both groups showed a trend toward a positive association between RMP concentration and the duration of storage in packed RBC bags stored under blood bank conditions. Our results suggest that storage-induced RMP release has similar effects in stored packed RBCs obtained from both donors with thalassemia and healthy volunteers.

Cell-Derived Microparticles in Blood Products from Blood Donors Deficient in Glucose-6-Phosphate Dehydrogenase.

To quantitate the microparticles (MPs) in whole blood and blood products obtained from blood donors who are deficient in glucose-6-phosphate dehydrogenase (G6PD). The current study analyzed whole blood and blood components prepared from 49 blood donors with G6PD deficiencies and 98 with G6PD-normal results. Packed red blood cells (PRBCs), platelet concentrate (PC), and plasma were prepared according to transfusion laboratory procedures. MP concentrations were determined using a flow cytometer. Blood components prepared from donors with G6PD deficiency were characterized by higher red blood cell-derived MP (RMP) concentration in PRBCs (25,526 vs 18,738 particles/µL) but lower concentrations of platelet-derived MPs (PMPs; in whole blood and PC), leukocyte-derived MPs (LMP; in whole blood and plasma) and total MP (in PC), compared with those from donors with G6PD-normal test results. These results suggest that differences in G6PD status may account for variation in RMP levels during processing.

Reduced lifespan of erythrocytes in Dahl/Salt sensitive rats is the cause of the renal proximal tubule damage

We studied the mechanisms of anemia and the influence of anemia on renal pathology in Dahl/Salt Sensitive (Dahl/SS) rat, a model of cardio-renal-anemia syndrome. Erythrocyte lifespan was shortened and associated with decreased hemoglobin level in the Dahl/SS rats given high-salt diet. Serum haptoglobin decreased, reticulocytes increased, and erythropoiesis in the bone marrow and extramedullary hematopoiesis in the spleen was markedly stimulated by increased serum erythropoietin in them. As a mechanism of hemolysis, we investigated the incidence of eryptosis, suicidal death of erythrocytes. Eryptosis was increased, and red blood cell-derived microparticles, small particle which are generated in hemolytic disease, were also increased in Dahl/SS rats fed with high-salt diet. Deposition of hemosiderin and mitochondrial morphologic abnormality, a sign of ferroptosis, in proximal renal tubules was associated with intravascular hemolysis. Treatment with deferasirox, an oral iron chelator, reduced the renal proximal tubular injury and the glomerular sclerosis in Dahl/SS rats fed with high-salt diet. In conclusion, reduced half-life of erythrocytes induced by hemolysis is the major cause of anemia in Dahl/SS rat. Iron accumulation induced by hemolysis causes renal proximal tubule injury and accelerates renal damage in this model.

Association of endothelial and red blood cell microparticles with acute myocardial infarction in Chinese: a retrospective study.

Mounting evidence suggests that endothelial cell-derived microparticles (EMPs) and red blood cell-derived microparticles (RMPs), which have procoagulant and vasoconstriction effects, are involved in the development of vascular acute cardiovascular events. The aim of this study was to analyze the circulating levels of EMPs and RMPs in patients with acute myocardial infarction (AMI), and to explore the correlations between EMPs and RMPs and the severity of coronary artery disease. Plasma samples from 110 patients with AMI and 57 non-coronary artery disease group (nonCAD) were collected in the present study. The flow cytometry was used to measure the EMPs (CD31) and RMPs (CD235a) qualitatively and quantitatively. The levels of EMPs and RMPs in the AMI group were higher than that in the Non-CAD group, yet no significant difference was found between STEMI and non-STEMI subjects. The levels of EMPs and RMPs in multi-vessel were higher than in the single-vessel l disease. In the Thrombolysis in Myocardial Infarction (TIMI) risk assessment, the levels of EMPs in the high-risk group were higher than that in both intermediate- and low-risk group. The low-risk group had the lowest EMP levels, the difference between the groups being statistically significant (P=0.001). No significant difference in RMP levels was noted upon TIMI stratification. According to the ROC curve analysis, the areas under the curve (AUC) of EMPs and RMPs were 0.706 and 0.668, respectively. The circulating levels of EMPs and RMPs in patients with AMI are elevated, and the level of EMPs is related to the degree of coronary artery disease and the prognosis risk. The EMPs are more likely to be potential biomarkers than RMPs to provide diagnostic value for AMI.

Abstract WP407: Therapeutic Window of Red Cell Microparticles in Limiting Hematoma Growth in a Rat Model of Intracerebral Hemorrhage

Spontaneous intracerebral hemorrhage (sICH) is a serious form of stroke without any effective therapy. Limiting hematoma growth is a promising therapeutic strategy. Published data showed that red blood cell-derived microparticles (RMPs) enhance both primary and secondary hemostasis. Previously, we observed that RMPs lower hematoma growth in a rat model of sICH. The goal of our present study was to determine the optimal therapeutic time window for RMP administration. Optimal therapeutic dose and treatment paradigm were used based on our previous studies. RMPs were prepared from human RBCs using high-pressure extrusion. sICH was induced in young male rats by injecting collagenase into the right striatum. Rats were treated with either saline (vehicle) or RMP (2.55х10 10 particles/kg, b.w. i.v.) starting at 2, 3, 4.5, or 6 h post-sICH induction. Rats were randomly assigned to each treatment group (n=10 for each RMP treatment time window, n=5 for each vehicle time window). Neurological scores (12 indicates maximum impairment) were evaluated ~24 h post-collagenase injection, and then, the rats were perfused using saline. Brains were collected and sectioned for hematoma volume assessment using ImageJ by a blinded investigator. The hematoma volumes (mm 3 ) of 2 (53±4, p<0.01), 3 (51±6, p<0.005), and 4.5 h (55±5, p<0.05) post-sICH RMP treatment groups were significantly lower than the vehicle group (72±4, pooled for all time windows). Similarly, the neurological scores in the 2 (8.8±0.5, p<0.001), 3 (9.0±0.3, p<0.001), and 4.5 h (8.8±0.4, p<0.001) post-sICH RMP treatment groups were significantly lower than the vehicle group (10.9±0.2). However, the hematoma volume (65±5) and neurological score (10.2±0.4) in the 6 h post-sICH RMP treatment group were not significantly different from the vehicle group. Our results indicate that RMPs were effective in lowering post-ICH hematoma volume when administrated up to 4.5 h after ICH induction. Together with our earlier results with optimal dose treatment paradigm and long-term outcome studies, these results further support the use of RMPs as a potential therapeutic option to lower hematoma growth in sICH patients.

PB2392 GLOBAL HEMOSTATIC CHANGES AND CIRCULATING MICROPARTICLES IN CHILDREN AND ADOLESCENTS WITH HEMOGLOBIN H AND BETA‐THALASSEMIA/HEMOGLOBIN E DISEASES

Background:Thalassemia is an inherited hemolytic anemia known to be associated with hypercoagulable state. Several factors including the abnormality of thalassemic red blood cell (RBC) membrane, increased platelet count, decreased anticoagulant proteins and increased circulating microparticles (MPs) contribute to the hypercoagulability in thalassemia. Global hemostatic changes as measured by rotatory thromboelastometry (ROTEM®) have been demonstrated in patients with beta‐thalassemia/hemoglobin E disease (beta‐thal/Hb E). However, there is little information in patients with hemoglobin H disease (Hb H) which is a less severe but highly prevalent non‐transfusion dependent thalassemia.Aims:To compare the global hemostatic changes and the levels of circulating MPs among children and adolescents with Hb H, beta‐thal/Hb E and healthy controls.Methods:A cross‐sectional study was conducted in 38 subjects with Hb H, 22 subjects with beta‐thal/Hb E and 12 healthy age‐matched controls. Complete blood count, ROTEM® and quantitation of circulating MPs [annexin V + MPs, red blood cell (RBC) derived MPs (RMPs) and platelet derived MPs (PMPs)] as analyzed by flow cytometry were performed in all subjects.Results:The age (range 5‐21 years) and percentage of male gender (range 50‐58%) among the Hb H, beta‐thal/Hb E and control groups were not statistically different (P = 0.383 and P = 0.798, respectively). The Hb levels were higher and the numbers of nucleated RBC (nRBC) were lower in the Hb H group than in beta‐thal/Hb E group. Overall, the ROTEM® parameters were not statistically different among the thalassemia and control groups. However, within the thalassemia subjects group, the maximum clot firmness (MCF) in splenectomized patients was significantly higher than non‐splenectomized subjects (P < 0.001). The levels of annexin V + MPs in both Hb H and beta‐thal/Hb E groups were significantly higher than the control group (P = 0.001). RMPs in beta‐thal/Hb E group were higher than the controls (P = 0.013) while there was no difference between the Hb H and control groups (P = 0.097). The level of circulating MPs was slightly higher in splenectomized subjects, but the differences were not statistically significant. Only the platelet count and nRBC, but not the circulating MPs, were significantly correlated with MCF in both INTEM (r = 0.620, P < 0.001 and r = 0.322, P = 0.006 respectively) and EXTEM (r = 0.567, P < 0.001 and r = 0.326, P = 0.005, respectively).Summary/Conclusion:The global hemostasis parameters as measured by ROTEM® were not different among the Hb H, beta‐thal/Hb E and control groups in this study. Hypercoagulable state was more commonly found in thalassemia subjects who were splenectomized, regardless of genotypes. The hypercoagulability was associated with higher nRBC and platelet count in the splenectomized group. Patients with Hb H had significantly higher circulating MPs levels than healthy controls although with the lesser extent than those seen in beta‐thal/Hb E.

The emerging roles of eryptosis in liver diseases.

Erythrocytes undergo programmed cell death, similar to apoptosis, known as eryptosis. This process is a result of several factors including hyperosmolarity, oxidative stress, and exposure to xenobiotics, and is characterized by the breakdown of membrane phospholipid asymmetry, the clustering of band 3, and the generation of red blood cell-derived microparticles. Under pathological conditions, the liver is the primary site of erythrocyte clearance and plays an important role in iron recycling. Phosphatidylserine exposure and band-3 clustering on eryptotic erythrocytes represent mainly pro-phagocytic signals. Further, the percentage of eryptotic erythrocytes is enhanced in the circulating blood of patients with hepatic failure, hyperbilirubinemia, and nonalcoholic steatohepatitis. In this review, we concentrate on recent progress regarding the pathophysiological roles of eryptosis in liver diseases.

Abstract WP460: Use of Red Cell Microparticles to Limit Hematoma Growth Following Intracerebral Hemorrhage

Currently there is no effective treatment for spontaneous intracerebral hemorrhage (sICH). Limiting hematoma extension in sICH is an attractive therapeutic target. Earlier studies demonstrated that red blood cell-derived microparticles (RMP) enhance platelet function and accelerate coagulation, augmenting both primary and secondary hemostasis. In the present study, we determined the efficacy of RMP in reducing hematoma expansion in a rat model of collagenase-induced sICH. RMPs were prepared from human RBCs using a high-pressure extrusion method. At ~24 hours post-collagenase injection, neurological scores were evaluated, rats were sacrificed, and the hematoma areas on images of brain sections were measured. In an earlier study (presented in an abstract form) we determined optimal RMP dose required to lower hematoma growth following sICH. The hematoma volume was lower by 17, 40 (p<0.001 vs placebo group), and 6% in low dose (7.19 x 10 9 particles / kg b.w.), medium dose (2.55 x 10 10 particles / kg b.w.), and high dose (9.48 x 10 10 particles / kg b.w.) treated groups when compared to placebo group, respectively. For those experiments, total RMP dose was delivered over 2 hours using continuous infusion starting 1 h post-collagenase injection. In the present study we evaluated optimal treatment paradigm using optimal protective (medium) dose as identified above. We evaluated the efficacy of RMP when delivered starting at 1 h post-collagenase injection either via four bolus injections (6.375 x 10 9 particles / kg b.w. per injection) 30 min apart (paradigm B) and when delivered using continuous infusion (2.55 x 10 10 particles / kg b.w.) over 1 hour (paradigm C), and compared with when RMP dose was delivered (2.55 x 10 10 particles / kg b.w.) over 2 hours using continuous infusion (paradigm A). We observed that hematoma volume (mm 3 ) for vehicle, and paradigm A, B and C was 71 ± 3 (n = 35), 43 ± 2 (n = 10, p<0.001 vs vehicle), 60 ± 4 (n = 15, p = 0.057 vs vehicle), and 61 ± 4 (n = 15, p = 0.073 vs vehicle), respectively. We observed that neurological score was lower (all p<0.001) in paradigm A (9.3 ± 0.4, n = 10), B (8.7 ± 0.3, n = 15), and C (9.5 ± 0.4, n = 15) groups as compared to placebo group (10.9 ± 0.1, n = 35). Our results indicate that RMPs have potential to lower hematoma growth in sICH patients.

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.

Physiologic Impact of Circulating RBC Microparticles upon Blood-Vascular Interactions.

Here, we review current data elucidating the role of red blood cell derived microparticles (RMPs) in normal vascular physiology and disease progression. Microparticles (MPs) are submicron-size, membrane-encapsulated vesicles derived from various parent cell types. MPs are produced in response to numerous stimuli that promote a sequence of cytoskeletal and membrane phospholipid changes and resulting MP genesis. MPs were originally considered as potential biomarkers for multiple disease processes and more recently are recognized to have pleiotropic biological effects, most notably in: promotion of coagulation, production and handling of reactive oxygen species, immune modulation, angiogenesis, and in initiating apoptosis. RMPs, specifically, form normally during RBC maturation in response to injury during circulation, and are copiously produced during processing and storage for transfusion. Notably, several factors during RBC storage are known to trigger RMP production, including: increased intracellular calcium, increased potassium leakage, and energy failure with ATP depletion. Of note, RMP composition differs markedly from that of intact RBCs and the nature/composition of RMP components are affected by the specific circumstances of RMP genesis. Described RMP bioactivities include: promotion of coagulation, immune modulation, and promotion of endothelial adhesion as well as influence upon vasoregulation via influence upon nitric oxide (NO) bioavailability. Of particular relevance, RMPs scavenge NO more avidly than do intact RBCs; this physiology has been proposed to contribute to the impaired oxygen delivery homeostasis that may be observed following transfusion. In summary, RMPs are submicron particles released from RBCs, with demonstrated vasoactive properties that appear to disturb oxygen delivery homeostasis. The clinical impact of RMPs in normal and patho-physiology and in transfusion recipients is an area of continued investigation.

Microparticles from stored red blood cells promote a hypercoagulable state in a murine model of transfusion

BackgroundRed blood cell-derived microparticles are biologically active, submicron vesicles shed by erythrocytes during storage. Recent clinical studies have linked the duration of red blood cell storage with thromboembolic events in critically ill transfusion recipients. In the present study, we hypothesized that microparticles from aged packed red blood cell units promote a hypercoagulable state in a murine model of transfusion. MethodsMicroparticles were isolated from aged, murine packed red blood cell units via serial centrifugation. Healthy male C57BL/6 mice were transfused with microparticles or an equivalent volume of vehicle, and whole blood was harvested for analysis via rotational thromboelastometry. Serum was harvested from a separate set of mice after microparticles or saline injection, and analyzed for fibrinogen levels. Red blood cell-derived microparticles were analyzed for their ability to convert prothrombin to thrombin. Finally, mice were transfused with either red blood cell microparticles or saline vehicle, and a tail bleeding time assay was performed after an equilibration period of 2, 6, 12, or 24 hours. ResultsMice injected with red blood cell-derived microparticles demonstrated an accelerated clot formation time (109.3 ± 26.9 vs 141.6 ± 28.2 sec) and increased α angle (68.8 ± 5.0 degrees vs 62.8 ± 4.7 degrees) compared with control (each P < .05). Clotting time and maximum clot firmness were not significantly different between the 2 groups. Red blood cell-derived microparticles exhibited a hundredfold greater conversion of prothrombin substrate to its active thrombin form (66.60 ± 0.03 vs 0.70 ± 0.01 peak OD; P < .0001). Additionally, serum fibrinogen levels were lower in microparticles-injected mice compared with saline vehicle, suggesting thrombin-mediated conversion to insoluble fibrin (14.0 vs 16.5 µg/mL, P < .05). In the tail bleeding time model, there was a more rapid cessation of bleeding at 2 hours posttransfusion (90.6 vs 123.7 sec) and 6 hours posttransfusion (87.1 vs 141.4 sec) in microparticles-injected mice as compared with saline vehicle (each P < .05). There was no difference in tail bleeding time at 12 or 24 hours. ConclusionRed blood cell-derived microparticles induce a transient hypercoagulable state through accelerated activation of clotting factors.

Microparticles from stored red blood cells enhance procoagulant and proinflammatory activity.

The pathomechanisms of morbidity due to blood transfusions are not yet entirely understood. Elevated levels of red blood cell-derived microparticles (RMPs) are found in coagulation-related pathologies and also in stored blood. Previous research has shown that RMPs mediate transfusion-related complications by the intrinsic pathway. We hypothesized that RMPs might play a role in post-transfusion thrombotic complications by enhancing procoagulant activity also through the extrinsic pathway of coagulation. In this laboratory study, blood from 18 healthy volunteers was stimulated with microparticles from expired stored red blood cells. Various clotting parameters were recorded. Flow cytometry, enzyme-linked immunosorbent assays, and real-time polymerase chain reaction were used to investigate possible mediating mechanisms. The addition of RMPs shortened the clotting time from 194 to 161 seconds (p < 0.001). After incubation with RMPs, there was increased expression of tissue factor (TF) on monocytes and in plasma. TF messenger RNA expression increased in a time-dependent and concentration-dependent manner. There was a significant induction of interleukin-1β and interleukin-6. After stimulation with RMPs, there was a significant increase in the number of activated platelets, an increased percentage of PAC-1/CD62P (procaspase activating compound-1/platelet surface P-selectin) double-positive platelets, and an increased number of platelet-neutrophil duplets and platelet-monocyte duplets, indicating enhanced interaction of platelets with neutrophils and monocytes. Levels of CXCL-8 (C-X-C motif chemokine ligand 1) and interleukin-6 were significantly higher after treatment with RMPs. Our results suggest that RMPs trigger coagulation through TF signaling, induce the secretion of proinflammatory cytokines, and induce cell-cell interaction between platelets and neutrophils. Thus, under certain conditions, RMPs could play a role in post-transfusion complications through these mechanisms.

Influence of red blood cell-derived microparticles upon vasoregulation.

Here we review recent data and the evolving understanding of the role of red blood cell-derived microparticles (RMPs) in normal physiology and in disease progression. Microparticles (MPs) are small membrane vesicles derived from various parent cell types. MPs are produced in response to a variety of stimuli through several cytoskeletal and membrane phospholipid changes. MPs have been investigated as potential biomarkers for multiple disease processes and are thought to have biological effects, most notably in: promotion of coagulation, production and handling of reactive oxygen species, immune modulation, angiogenesis, and in apoptosis. Specifically, RMPs are produced normally during RBC maturation and their production is accelerated during processing and storage for transfusion. Several factors during RBC storage are known to trigger RMP production, including: increased intracellular calcium, increased potassium leakage, and energy failure with ATP depletion. Of note, RMP composition differs from that of intact RBCs, and the nature and composition of RMP components are affected by both storage duration and the character of storage solutions. Recognised RMP bioactivities include: promotion of coagulation, immune modulation, and promotion of endothelial adhesion, as well as influence upon vasoregulation via nitric oxide (NO) scavenging. Of particular relevance, RMPs are more avid NO scavengers than intact RBCs and this feature has been proposed as a mechanism for the impaired oxygen delivery homeostasis that has been observed following transfusion. Preliminary human studies demonstrate that circulating RMP abundance increases with RBC transfusion and is associated with altered plasma vasoactivity and abnormal vasoregulation. In summary, RMPs are submicron particles released from stored RBCs, with demonstrated vasoactive properties that appear to disturb oxygen delivery homeostasis. The clinical impact of RMPs in transfusion recipients is an area of continued investigation.

The number of red blood cell-derived microparticles in predicting periprocedural adverse effects in acute STsegment elevation myocardial infarction patients

The role of the circulating number of Red Blood Cell (RBC) Microparticles (MPs) as predictive biomarker in acute myocardial infarction patients after primary Percutaneous Coronary Intervention (PCI) is widely argued.

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.