Erythrocyte Microparticles Research Articles

Age-Markers on the Red Blood Cell Surface and Erythrocyte Microparticles may Constitute a Multi-parametric Strategy for Detection of Autologous Blood Transfusion

BackgroundAutologous blood transfusion is one of the illicit strategies, banned by the World Anti-Doping Agency, to increase the levels of hemoglobin, with a consequent improvement in the delivery of oxygen to tissues. At present, this practice is detectable exclusively by the individual, longitudinal monitoring of hematological biomarkers, as in the hematological module of the Athlete Biological Passport; but this indirect approach may suffer from different confounding factors. We are presenting a multi-parametric, analytical strategy to detect autologous blood transfusions by targeting the modification of the red blood cells during storage. We focused on the assessment of “storage lesions”, targeting (i) membrane proteins: Glycophorin-A and Band 3 complex, (ii) biomarkers of oxidative stress: Peroxiredoxin-2, (iii) biomarkers of senescence: CD47 and Phosphatidylserine, (iv) erythrocytes microparticles.ResultsAll of the above markers were monitored, by immunological and flow cytofluorimetric methods, on samples of stored whole blood collected at different time intervals, and on fresh blood samples, collected for official doping control tests, mixed “ex vivo” to simulate an autotransfusion. Although anonymized before the delivery to the laboratory, it was possible to mix samples belonging to the same subject based on the “athlete biological passport” code. Our results showed that the irreversible alteration of RBCs morphology, the loss of membrane integrity, the occurrence of hemolysis phenomena, and, more in general, the “aging” of the erythrocytes during storage are closely related to: (i) the reduced concentration, on the erythrocyte membrane, of Band 3 protein (decrease of 19% and of 39% after 20 and 40 days of storage respectively) and of glycophorin A (− 47% and − 63% respectively); (ii) the externalization of phosphatidyl serine (with a five-fold increase after 20 days and a further 2× increase after 40 days); (iii) the reduced concentration of CD47; and (iv) increased levels of erythrocyte microparticles.ConclusionsThe most promising method to detect the presence of transfused blood in whole blood samples can be based on a multi-parametric strategy, considering jointly both protein expression on RBCs membranes and micro-vesiculation phenomena.

Shear stimulated red blood cell microparticles: Effect on clot structure, flow and fibrinolysis.

Microparticles (MPs) have activity in thrombus promotion and generation. Erythrocyte microparticles (ErMPs) have been reported to accelerate fibrinolysis in the absence of permeation. We hypothesized that shear induced ErMPs would affect fibrin structure of clots and change flow with implications for fibrinolysis. To determine the effect of ErMPs on clot structure and fibrinolysis. Plasma with elevated ErMPs was isolated from whole blood or from washed red blood cells (RBCs) resuspended in platelet free plasma (PFP) after high shear. Dynamic light scattering (DLS) provided size distribution of ErMPs from sheared samples and unsheared PFP controls. Clots were formed by recalcification for flow/lysis experiments and examined by confocal microscopy and SEM. Flow rates through clots and time-to-lysis were recorded. A cellular automata model showed the effect of ErMPs on fibrin polymerization and clot structure. Coverage of fibrin increased by 41% in clots formed from plasma of sheared RBCs in PFP over controls. Flow rate decreased by 46.7% under a pressure gradient of 10 mmHg/cm with reduction in time to lysis from 5.7 ± 0.7 min to 12.2 ± 1.1 min (p < 0.01). Particle size of ErMPs from sheared samples (200 nm) was comparable to endogenous microparticles. ErMPs alter the fibrin network in a thrombus and affect hydraulic permeability resulting in decelerated delivery of fibrinolytic drugs.

5613458 BLOOD MICROPARTICLES: A TOOL OF EVALUATING THE EFFECT OF TRANSFUSION IN SICKLE CELL DISEASE PATIENTS

Background: Blood microparticles (MPs) are phospholipid microvesicles derived from cell membranes. Typically, they range in size from 0.1 μm to 1.0 μm and they are released upon oxidative stress, cell activation, injury or apoptosis. MPs have acquired a biological and clinical interest and are recognized as entities able to modulate many biological functions. Literature suggests that they may have a role in the pathophysiology and downstream manifestations of sickle cell disease (SCD). Aims: This present study was carried out to determine MPs profiles in patients with SCD before and after transfusion and to compare these profiles with healthy subjects. Materials: Twenty healthy donors and twelve polytransfused patients with SCD recruited from National Bone Marrow Transplant Center were recruited in this study. None of the patients was transfused for at least 3 weeks before sampling. The blood samples obtained were analyzed for circulating MPs by flow cytometry. The cellular origin was determined by a multiple labeling with specific antibodies and dyes. Results: A high level of apoptotic, platelets, and endothelial MPs with the predominance of the number of erythrocyte MPs was detected in SCD patients compared to healthy donors. This suggests that erythrocytes MPs may contribute to thrombotic risk in sickle cell patients. Moreover, after transfusion most subjects register a decreased number of different types of MPs. Conclusion: Transfusion is an effective treatment option in patients with SCD, as it seems that it decreases the abnormally elevated levels of MPs.

Production of erythrocyte microparticles in a sub-hemolytic environment.

Microparticles are produced by various cells due to a number of different stimuli in the circulatory system. Shear stress has been shown to injure red blood cells resulting in hemolysis or non-reversible sub-hemolytic damage. We hypothesized that, in the sub-hemolytic shear range, there exist sufficient mechanical stimuli for red blood cells to respond with production of microparticles. Red blood cells isolated from blood of healthy volunteers were exposed to high shear stress in a microfluidic channel to mimic mechanical trauma similar to that occurring in ventricular assist devices. Utilizing flow cytometry techniques, both an increase of shear rate and exposure time showed higher concentrations of red blood cell microparticles. Controlled shear rate exposure shows that red blood cell microparticle concentration may be indicative of sub-hemolytic damage to red blood cells. In addition, properties of these red blood cell microparticles produced by shear suggest that mechanical trauma may underlie some complications for cardiovascular patients.

Use of Thrombodynamics for revealing the participation of platelet, erythrocyte, endothelial, and monocyte microparticles in coagulation activation and propagation.

Background and objectiveFor many pathological states, microparticles are supposed to be one of the causes of hypercoagulation. Although there are some indirect data about microparticles participation in coagulation activation and propagation, the integral hemostasis test Thrombodynamics allows to measure micropaticles participation in these two coagulation phases directly. Demonstrates microparticles participation in coagulation activation by influence on the appearance of coagulation centres in the plasma volume and the rate of clot growth from the surface with immobilized tissue factor.Methods: Microparticles were obtained from platelets and erythrocytes by stimulation with thrombin receptor-activating peptide (SFLLRN) and calcium ionophore (A23187), respectively, from monocytes, endothelial HUVEC culture and monocytic THP cell culture by stimulation with lipopolysaccharides. Microparticles were counted by flow cytometry and titrated in microparticle-depleted normal plasma in the Thrombodynamics test.ResultsMonocyte microparticles induced the appearance of clotting centres through the TF pathway at concentrations approximately 100-fold lower than platelet and erythrocyte microparticles, which activated plasma by the contact pathway. For endothelial microparticles, both activation pathways were essential, and their activity was intermediate. Monocyte microparticles induced plasma clotting by the appearance of hundreds of clots with an extremely slow growth rate, while erythrocyte microparticles induced the appearance of a few clots with a growth rate similar to that from surface covered with high-density tissue factor. Patterns of clotting induced by platelet and endothelial microparticles were intermediate. Platelet, erythrocyte and endothelial microparticles impacts on the rate of clot growth from the surface with tissue factor did not differ significantly within the 0–200·103/ul range of microparticles concentrations. However, at concentrations greater than 500·103/ul, erythrocyte microparticles increased the stationary clot growth rate to significantly higher levels than do platelet microparticles or artificial phospholipid vesicles consisting of phosphatidylcholine and phosphatidylserine.ConclusionMicroparticles of different origins demonstrated qualitatively different characteristics related to coagulation activation and propagation.

High Level of Circulating Microparticles in Patients with BCR/ABL Negative Myeloproliferative Neoplasm - a Pilot Study.

Microparticles (MPs) are small (0.1-1 &#956;m) cell-derived vesicles released during activation or apoptosis, with a surface-exposed phosphatidylserine along with antigens indicating the cellular origin. The level of MPs is known to be elevated in thromboembolic diseases and malignancies; it is believed that MPs are not only amplifying but can also initiate the thrombogenesis processes. BCR/ABL negative myeloproliferative neoplasms (MPNs) are clonal haematopoietic diseases, which include polycythemia vera, essential thrombocythemia and primary myelofibrosis. One of the main problems of MPN patients is high risk and incidence of thrombosis which affect the survival, quality of life and life expectancy. The clinical significance of circulating MPs was assessed in a group of 179 patients with BCR/ABL-negative MPNs. Analysis of MPs was done using flow cytometry on 417 samples, and MPs procoagulation activity was performed using a functional assay called Zymuphen MP-activity (Hyphen Biomed, Neuville-sur-oise, France) on 274 samples. Significantly higher absolute and relative count of platelet MPs was found in MPN patients when compared with healthy group, respectively (p = 0.001, p = 0.043). Erythrocyte MPs were also significantly higher in MPN patients than in the healthy group (p &lt; 0.001). Procoagulation activity of MPs was as well significantly higher in patients compared to the control group (p &lt; 0.001). Patients with primary myelofibrosis had decreased absolute and relative count of platelet MPs compared to polycythemia vera and essential thrombocythemia patients, respectively (p = 0.008, p = 0.014). Presence of JAK2V617F mutation was associated with higher absolute and relative count of platelet MPs, respectively (p = 0.045, p = 0.029). Although some literature data support the hypothesis of a direct relation between MPs and thrombotic events in MPN patients, further studies are needed to evaluate the clinical implication of MPs in the hypercoagulation state of MPN patients.

GW28-e0041 The Comparison of the level of Erythrocyte Microparticles between Acute Coronary Syndrome and Stable Angina Pectoris Patients

GW28-e0041 The Comparison of the level of Erythrocyte Microparticles between Acute Coronary Syndrome and Stable Angina Pectoris Patients

OP.8A.01] NEWLY DIAGNOSED DIABETES MELLITUS IS ASSOCIATED WITH INCREASED LEVELS OF ERYTHROCYTE MICROPARTICLES

Objective: Patients with Diabetes Mellitus (DM) carry a high burden of cardiovascular damage and high risk of early thrombotic events. It is currently unknown how early this damage occurs and if it preexists in naive newly diagnosed DM patients. Design and method: We sought to determine the thrombotic tendency of patients with known cardiovascular risk factors. To this end we used sophisticated, novel markers of atherosclerotic and thrombotic damage: platelet (PLTs) and erythrocyte (RBCs) microparticles. Consecutive patients of the Micro- and Macro-vascular disease Clinic of Third Department of Internal Medicine at Papageorgiou General Hospital of Thessaloniki were included in the study. A thorough medical history was taken and a blood sample was drawn. A 24-hour urine collection was performed to assess the presence of microalbuminuria. Furthermore, PLTs and RBCs were determined using flow cytometry according to specific protocol. Cardiovascular risk factors were evaluated with emphasis on hypertension, newly diagnosed DM, dyslipidemia, smoking and body mass index. Results: Our study sample consisted of 45 patients aged 52.5 ± 11.9 years, 19 men: 26 women, with mean systolic / diastolic blood pressure: 132.7 ± 16.8 / 83.3 ± 13.7 mmHg. Newly-diagnosed DM was found in 19 patients, while untreated hypertension in 16. Multiple linear regression analysis showed that DM makes the strongest contribution to high RBCs values (beta: 0,673, p = 0,021) after the effect of cardiovascular risk factors is controlled for. No significant predictor of PLTs could be identified. Conclusions: In our study, RBCs strongly correlated with the presence of DM at the very early stages of the disease, despite the small sample size. Current knowledge suggests that RBCs consist a robust indicator of thrombotic tendency. The fact that PLTs did not correlate with the presence of DM in our study may, at least in part, reflect the multifactorial role of platelets not only in the thrombotic but also in the inflammatory process of atherosclerosis. Larger studies are needed to determine the value of PLTs and RBCs in patients with cardiovascular risk factors.

How to quantify microparticles in RBCs? A validated flow cytometry method allows the detection of an increase in microparticles during storage

The procoagulant and proinflammatory microparticles (MPs) released during storage of packed red blood cells (pRBCs) can potentially modify transfusion benefits. A robust method to quantify MPs in pRBCs is needed to evaluate their impact in clinical trials. The objective was to validate the preanalytic conditions required to prepare pRBC supernatant as well as a method to quantify and evaluate MP variations over 42 days of pRBC storage.A flow cytometry method with size-calibrated beads was developed and fully validated. Quantification of MPs in pRBCs (n = 109) was assessed during short-term (7 days) and long-term (42 days) storage at 4°C, during short-term storage (8 hours) at room temperature, and after 2 years frozen. Repeatability, reproducibility, and linearity of the quantification method were validated, and variations during conservation are presented. There was high variability in RBC (erythrocyte) MP (ERMP) and platelet MP (PMP) levels between RBC units, depending on the filter used for leukocyte reduction. During the 42 days of storage at 4°C, significant increases in ERMPs and PMPs occurred (from 58 to 138 ERMPs/µL from Day 2 to Day 42; p = 0.0002; and from 326 to 771 PMPs/µL from Day 2 to Day 42; p = 0.00026). We use a robust method to confirm that ERMPs and PMPs are present to various degrees in pRBCs and that storage for 42 days significantly increases their generation. This method is robust enough to allow MP quantification in pRBCs and is adapted to evaluate the clinical impact of transfused MPs in prospective clinical trials.

Circulating Erythrocyte Microparticles and the Biochemical Extent of Myocardial Injury in ST Elevation Myocardial Infarction

Objectives: Red blood cell microparticles (RBCm) have potential adverse vascular effects and they have been shown to be elevated in ST elevation myocardial infarction (STEMI). The purpose of this study is to investigate their relationship with biochemical infarct size. Methods: RBCm were quantified with flow cytometry in blood drawn from 60 STEMI patients after a primary angioplasty. The creatine kinase-myocardial brain fraction (CK-MB) was measured at predefined time points and the area under the curve (AUC) was calculated. Results: RBCm count was correlated with CK-MB AUC (Spearman's ρ = 0.83, p < 0.001). The CK-MB AUC values per RBCm quartile (lower to upper) were: 3,351 (2,452-3,608), 5,005 (4,450-5,424), 5,903 (4,862-10,594), and 8,406 (6,848-12,782) ng × h/ml, respectively. From lower to upper quartiles, the maximal troponin I values were: 42.2 (23.3-49.3), 49.6 (28.8-54.1), 59.2 (41.4-77.3), and 69.1 (48.0-77.5) ng/ml (p = 0.005). In multivariable analysis, RBCm remained a significant predictor of CK-MB AUC (standardized β = 0.63, adjusted p = 0.001). Conclusions: Erythrocyte microparticles appear to be related to the total myocardial damage biomarker output. The exact pathophysiologic routes, if any, for this interaction remain to be identified. However, these results suggest that erythrocytes may be a - thus far virtually ignored - player in the pathogenesis of ischemic injury.

EVALUATION OF RED BLOOD CELL MICROPARTICLES IN THALASSEMIA

Background: Thalassemia is a group of heterogeneous disorders with variable severity characterized by defect in globin chain synthesis due to genetic defect. Hypercoagulable state in thalassemia is well known complication affecting 1.1 % of patients. It is a multifactorial event with variable degree of severity and clinical presentation. Cell derived vesicles are very small vesicles < 1μm released from cells in different conditions and for different purposes. They play an important role in cell signaling, adhesion and coagulation. Objective: to evaluate erythrocyte microparticles in patients with thalassemia. Patients and methods: Blood samples were collected from 50 thalassemic patients and 50 normal individuals. Platelet poor plasma was separated and used for analysis of erythrocyte microparticles by flow cytometry using CD235a. Results: A highly significant difference was observed between all groups in basic hematological parameters. The level of microparticles was significantly higher in patients with thalassemia and was higher in adult than in children. This high level is positively correlated with HbF levels and negatively correlated with Hb levels and Hb A levels. Conclusion: The levels of erythrocyte microparticles were higher in patients with thalassemia which may contribute to the hypercoagulable state of the disease. Further studies on medications that inhibit microparticles formation may be of value in prevention and treatment of thrombosis caused by these vesicles.

Impact of microparticles derived from erythrocytes on fibrinolysis.

It has long been known that negatively charged membranes of erythrocyte-derived microparticles display procoagulant activity. However, relatively little is known about the possible fibrinolytic activity of such microparticles. This issue becomes particularly important during red blood cell storage, which significantly increases the number of microparticles. Whole blood was collected from 30 healthy donors. Microparticles were isolated on days 7, 14, 21, and 28 of erythrocyte storage. The effect of microparticles on the fibrinolytic activity of the donor plasma was determined by coagulation and optical (chromogenic substrate) methods. We demonstrated that erythrocyte microparticles had a prominent fibrinolytic activity which cleaves not only fibrin but also chromogenic substrates. Microparticles present fibrinolytic activity mainly due to the presence of plasminogen on them. Microparticles derived from erythrocytes significantly enhance cleavage of the chromogenic substrate by the streptokinase-plasminogen complex, but to a lesser extent accelerate euglobulin clot lysis time. Erythrocyte-derived microparticles display prominent fibrinolytic activity, which significantly decreases during storage of red blood cells.

Abstract 11761: Association of Red Blood Cell Microparticles With Biomarker-Defined Infarct Size in Acute Myocardial Infarction

Background: Red blood cell microparticles (RBCm) have drawn research attention as to their potential prothrombotic and vasoconstrictive effects in experimental settings. Our group has previously reported elevated RBCm counts in patients with ST-elevation myocardial infarction (STEMI) treated with primary percutaneous intervention (PCI). Methods: Circulating microparticles were quantified with flow cytometry in blood samples from consecutive STEMI patients after primary PCI. Creatine kinase myocardial-brain fraction (CK-MB) was measured every 6 hours during the first 3 days and every 12 hours thereafter until discharge. The area under the CK-MB concentration curve (CKMB-AUC), calculated with Simpson’s summing rule, was used as a biochemical measure of infarction size. Results: 51 STEMI patients (age 59.8±8.8 years) were enrolled. RBCm concentration was 33740±21169 /μl. The median values [25th-75th percentile] of CKMB-AUC according to RBCm quartile were (from the lower to the upper quartile) 3351 [2550-4060] ng.h.ml-1, 4883 [4179-5348] ng.h.ml-1, 6932 [5464-13229] ng.h.ml-1, and 8784 [7216-12605] ng.h.ml-1, respectively (Kruskal-Wallis p&lt;0.001) (see Figure). In a multivariable regression analysis, RBCm quartile remained associated with CKMB-AUC, after adjustment for total RBC count, age, serum creatinine, presence of diabetes and Killip class on admission (adjusted p&lt;0.001). Conclusions: Erythrocyte microparticles appear to be related to total myocardial damage biomarker output, which is a parameter known to reliably reflect infarct size in patients with STEMI. This finding may be explained by rheologic and vasoactive properties of RBCm (close interaction of the heavily oxidative heme with the endothelium, depletion of NO etc.), which may act deleteriously on coronary vasculature, resulting in increased myocardial damage.

Platelet and not erythrocyte microparticles are procoagulant in transfused thalassaemia major patients.

The level of circulating platelet-, erythrocyte-, leucocyte- and endothelial-derived microparticles detected by high-sensitivity flow cytometry was investigated in 37 β-thalassaemia major patients receiving a regular transfusion regimen. The phospholipid procoagulant potential of the circulating microparticles and the microparticle-dependent tissue factor activity were evaluated. A high level of circulating erythrocyte- and platelet-microparticles was found. In contrast, the number of endothelial microparticles was within the normal range. Platelet microparticles were significantly higher in splenectomized than in non-splenectomized patients, independent of platelet count (P<0·001). Multivariate analysis indicated that phospholipid-dependent procoagulant activity was influenced by both splenectomy (P=0·001) and platelet microparticle level (P<0·001). Erythrocyte microparticles were not related to splenectomy, appear to be devoid of proper procoagulant activity and no relationship between their production and haemolysis, dyserythropoiesis or oxidative stress markers could be established. Intra-microparticle labelling with anti-HbF antibodies showed that they originate only partially (median of 28%) from thalassaemic erythropoiesis. In conclusion, when β-thalassaemia major patients are intensively transfused, the procoagulant activity associated with thalassaemic erythrocyte microparticles is probably diluted by transfusions. In contrast, platelet microparticles, being both more elevated and more procoagulant, especially after splenectomy, may contribute to the residual thrombotic risk reported in splenectomized multi-transfused β-thalassaemia major patients.

CD235a (Glycophorin-A) Is the Most Predictive Value Among Different Circulating Cellular Microparticles in Thrombocytopenic Human Immunodeficiency Virus Type 1.

This study was conducted to assess different cellular microparticles (MPs) in thrombocytopenic human immunodeficiency virus type 1 and their significance as disease activity markers. Thirty-five thrombocytopenic human immunodeficiency diseases and 25 healthy controls with matched age and sex were selected. Viral load was quantitated by COBAS real-time polymerase reaction (PCR) assessment of absolute T-cell subsets CD4, CD8 as a disease progress marker. Platelet MPs, platelet-derived monocyte MPs (CD42a, CD61), erythrocyte MP (CD235a), monocytic MP (CD14), and platelet activity MPs (CD62P, PAC-1) were assessed by multicolor flow cytometry FACSCalibur, while platelet functions were assessed by platelet function analyzer (PFA-100). CD42a, CD61, and platelet activity index represented by PAC-1 and CD62. P-selectin in HIV-infected patient samples were significantly greater (P < 0.001) than among controls. There was a negative correlation between the proportion of PAC-1 and CD62 P-selectin-positive MPs and levels of CD4(+) T-cell counts (r = -0.403, P = 0.016; r = -0.438, P = 0.008), respectively. There was a negative correlation between collagen-ADP and levels of CD4(+) T-cell counts (r = -0.368, P = 0.03). There was a significant high expression level of CD14 monocyte MPs in patients than controls (P < 0.0001), overexpression of CD235a (P < 0.0001), and no correlation between CD14 and CD4, whereas there was a significant negative correlation with CD235a (r = -0.394, P = 0.019). A linear regression analysis of CD4 as a disease progression marker with other variable indicators in HIV patients showed that CD235a could be the most sensitive predictor similar to CD4. Different cellular MPs and platelets activated in HIV patients could have a role in thrombotic events in these patients.

Platelet-Independent Adhesion of Red Blood Cells to Von Willebrand Factor

Background/ObjectivesRed blood cell (RBC) transfusion can be lifesaving and is an essential therapy in conditions associated with tissue hypoxia due to anemia. However, recent clinical studies show that both the number of RBCs and the age of RBCs transfused are independent risk factors for an increase in transfusion related morbidity and mortality. It has been suggested that the so called “storage lesion” of RBCs, a reduction of quality of erythrocytes and changes in the erythrocyte concentrate storage medium, is the causal factor. Recently it has been shown that cold storage of erythrocytes induces microparticle formation. These erythrocyte microparticles are pro-coagulant and can cause thrombin formation. Another phenomenon of the storage lesion is the rapid and considerable loss of donor erythrocytes from the circulation of transfused patients. We wondered whether thrombin generated by transfused erythrocyte microparticles could contribute to red blood cell adherence to the vascular endothelium. Cytoadherence of red blood cells could contribute to the loss of circulating transfused red blood cells and vascular obstruction and could explain the observed transfusion associated complications in clinical practice.Methods/ResultsEmploying FACS analysis and a microparticle analyzer we showed that erythrocyte cold storage indeed induces microparticle formation. We confirmed the pro-coagulant properties of these microparticles using a chromogenic substrate specific for thombin and a thrombin-anti-thrombin complex ELISA. To determine whether thrombin could induce adhesion of red blood cells to endothelial cells, we cultured human umbilical vein endothelial cells in micro-perfusion chambers and used live-imaging to define the adherence potential of the erythrocytes to endothelial cells at post-capillary flow rate. Thrombin stimulation of the endothelial cells did increase erythrocyte adhesion to endothelial cells. Moreover, the adhesion of erythrocytes followed a pattern resembling platelets binding to von Willebrand factor (VWF). By using live immunofluoresence imaging we confirmed that the erythrocytes did bind to VWF secreted from endothelial cells. Since erythrocyte-VWF interactions may be mediated by platelets, we used fluorescence cell sorting to remove platelets and erythrocyte-platelet complexes from erythrocyte concentrates. The purified erythrocytes did also bind to VWF secreted by endothelial cells and thereby we confirmed that erythrocytes can bind to VWF in a platelet-independent fashion. We further analyzed the specificity of the erythrocyte-VWF interaction by using different protein coatings in micro-perfusion chambers. Erythrocytes did bind to recombinant high molecular weight VWF multimers. Furthermore, they adhered more potently to VWF when compared to fibrinogen or fibrin but showed little binding to fibronectin, collagen type I, or subendothelial extra-cellular matrix proteins.ConclusionOur results suggest that transfusion of RBCs is able to induce endothelial binding of erythrocytes based on a VWF-erythrocyte interaction. We propose that passive infusion of cold stored erythrocyte derived microparticles promotes thrombin generation which subsequently activates endothelial cells and induces VWF secretion. This results in binding of red blood cells to endothelial cells in a platelet-independent fashion which requires the presence of VWF. Based on our results we hypothesize that binding of erythrocytes to VWF may occlude micro-capillaries thereby contributing to transfusion associated complications. DisclosuresNo relevant conflicts of interest to declare.

Red blood cell and platelet microparticles in myocardial infarction patients treated with primary angioplasty

BackgroundRed blood cell and platelet microparticles (RBCm and PLTm, respectively) have drawn research attention as to their potential prothrombotic and vasoconstrictive effects in experimental settings. However, the relevance of circulating microparticles in clinical settings is largely undetermined. MethodsCirculating microparticles were quantified with a flow cytometric method in blood samples from consecutive STEMI patients after primary PCI. A matched cohort of healthy volunteers was used to derive reference values for comparison. STEMI patients were followed for 6months for a composite clinical endpoint. ResultsFifty-one STEMI patients (age 59.8±8.8years) and 50 controls (age 56.2±9.2years; p=0.155) were enrolled. RBCm concentration was 18,198±6062/μl in the reference cohort versus 33,740±21,169/μl in STEMI patients (p<0.001). RBCm count was not correlated to total RBCs (standardized beta 0.018; p=0.861). PLTm did not differ between groups (17,529±16,292/μl in STEMI patients versus 14,372±6211/μl in controls; p=0.203). RBCm c-statistic was 0.832 (95% confidence interval 0.720 to 0.944), while PLTm prognostic value was not statistically significant (c-statistic 0.614, 95% confidence interval 0.444 to 0.784). In the multivariate analysis, RBCm concentration was independently associated with the occurrence of the clinical endpoint, after adjustment for age, ejection fraction, serum creatinine and presence of diabetes (adjusted p=0.034). ConclusionsThe present study demonstrates for the first time that erythrocyte microparticles are elevated in patients with STEMI treated with primary PCI, with levels approximately double those measured in a reference population of healthy volunteers, and their concentrations appear to be positively associated with adverse clinical events.

Clinical significance of circulating blood and endothelial cell microparticles in sickle-cell disease

Increased thrombocyte activation leads to a higher likelihood of coagulation in sickle-cell disease. On the other hand, chronic inflammation and endothelial cell activation promote vaso-occlusion. The effect of circulating microparticles derived from erythrocytes, monocytes, thrombocytes, and endothelial cells on the vaso-occlusive process is unclear. This study aims to analyze the relationship between sickle-cell disease and miscellaneous organ complications by defining the circulating microparticles during the steady-state and painful crisis periods in 45 patients with sickle-cell disease. Microparticle analysis was conducted using an eight-parameter flow cytometric method, using CD61 PERCP, CD142PE, CD106 FITC, CD14 APC-H7, CD235a FITC, and Annexin-V APC monoclonal antibodies. Microparticle levels of sickle-cell patients were found to be significantly higher during both painful crisis and steady-state situations compared with the control group (for all, p<0.001). Among these microparticles, levels of erythrocyte microparticles (eMPs) were significantly higher during crisis than in the steady-state period (eMP steady state vs. painful crisis: 7.59±12.24 vs. 7.59±12.24, respectively; p<0.01). Microparticles, including eMPs, were not affected by hydroxyurea treatment. Their level did not reflect the high frequency of crisis (>3times/year). Thrombocyte microparticle levels were found to be higher in patients with nephropathia than in those without (48.05±40.23 vs. 7.67±6.75, respectively; p<0.049). Circulating microparticles seem to be involved in the pathogenesis of sickle-cell disease. eMPs may help with the management of crisis. Thrombocyte microparticles might predict renal damage induced by vaso-occlusion.

Circulating platelet and erythrocyte microparticles in young children and adolescents with sickle cell disease: Relation to cardiovascular complications

Sickle cell disease (SCD) is characterized by a complex vasculopathy, consisting of endothelial dysfunction and increased arterial stiffness, with a global effect on cardiovascular function. The hypercoagulable state may result from chronic hemolysis and circulating cell-derived microparticles (MPs) originating mainly from activated platelets and erythrocytes. We measured the levels of platelet and erythrocyte-derived MPs (PMPs and ErMPs) in 50 young SCD patients compared with 40 age- and sex-matched healthy controls and assessed their relation to clinicopathological characteristics and aortic elastic properties. Patients were studied stressing on the occurrence of sickling crisis, transfusion history, hydroxyurea therapy, hematological, and coagulation profile as well as flow cytometric expression of PMPs (CD41b+) and ErMPs (glycophorin A+). Echocardiography was performed to assess aortic stiffness and distensibility, left ventricular function and pulmonary artery pressure. Both PMPs and ErMPs were significantly elevated in SCD patients compared with control group (p < 0.001). SCD patients had significantly elevated d-dimer and von Willebrand factor antigen (vWF Ag) levels with lower antithrombin III compared with controls (p < 0.001). Aortic stiffness index and pulmonary artery pressure were significantly higher in SCD (p < 0.001), whereas aortic strain and aortic distensibility were significantly lower (p < 0.001) compared with controls. MPs levels were significantly increased in SCD patients with pulmonary hypertension, acute chest syndrome, and stroke as well as those who had history of thrombosis or splenectomy (p < 0.001). Also, patients in sickling crisis during the study had higher PMPs and ErMPs levels than those in steady state (p < 0.001). Patients on hydroxyurea therapy had lower MPs levels than untreated patients (p < 0.001). PMPs and ErMPs were positively correlated with disease duration, transfusion index, white blood cell count, HbS, markers of hemolysis, serum ferritin, D-dimer, and vWF Ag, whereas negatively correlated with hemoglobin and HbF levels (p < 0.05). Both PMPs and ErMPs levels were positively correlated with aortic stiffness, pulmonary artery pressure, and tricuspid regurgitant velocity (p < 0.05) while negatively correlated with aortic distensibility. We suggest that PMPs and ErMPs overproduction may be considered a potential biological marker for vascular dysfunction and disease severity in SCD and may be implicated in the pathogenesis of coagulation abnormalities encountered in those patients. Their levels are closely related to sickling crisis, pulmonary hypertension, markers of hemolysis, fibrinolysis, and iron overload. Therefore, quantification of MPs in SCD may provide utility for identifying patients who are at increased risk of thrombotic events or cardiovascular abnormalities and would help to monitor response to hydroxyurea therapy.

Erythrocyte microparticles can induce kidney vaso-occlusions in a murine model of sickle cell disease

AbstractPatients with sickle cell disease suffer from painful crises associated with disseminated vaso-occlusions, increased circulating erythrocyte microparticles (MPs), and thrombospondin-1 (TSP1). MPs are submicron membrane vesicles shed by compromised or activated cells. We hypothesized that TSP1 mediates MP shedding and participates in vaso-occlusions. We injected TSP1 to transgenic SAD mice with sickle cell disease and characterized circulating phosphatidylserine+ MPs by FACS. TSP1 stimulated MPs in plasma and initiated vaso-occlusions within minutes. In vitro, TSP1 triggered rapid erythrocyte conversion into spicule-covered echinocytes, followed by MP shedding. MP shedding was recapitulated by peptides derived from the TSP1 carboxyterminus. We purified MPs shed by erythrocytes in vitro and administered them back to SAD mice. MPs triggered immediate renal vaso-occlusions. In vitro, MPs triggered the production of radical oxygen species by endothelial monolayers, favored erythrocyte adhesion, and induced endothelial apoptosis. MPs also compromised vasodilation in perfused microvessels. These effects were inhibited by saturating MP phosphatidylserine with annexin-V, or with inhibitors of endothelial ROS production. We conclude that TSP1 triggers erythrocyte MP shedding. These MPs induce endothelial injury and facilitate acute vaso-occlusive events in transgenic SAD mice. This work supports a novel concept that toxic erythrocyte MPs may connect sickle cell anemia to vascular disease.