Levels Of Microparticles In Patients Research Articles

Impact of splenectomy on circulating microparticles in patients with sickle cell anemia.

Circulating microparticles (MP) are being described as potential biomarkers for disease activity in a variety of conditions including sickle cell anemia (SCA). However, relatively little is known about the influence of spleen status on MP levels in patients with SCA. Using a prospective study design we characterize circulating MP in 144 patients with SCA in steady state by assessing their cellular origin and their relationships to spleen status defined by clinical and imaging findings. In addition, MP levels were studied according to demographic characteristics, clinical status, treatment modalities, and other hematological and biochemical parameters. Absolute plasma concentrations of MP were determined by flow cytometry. Patients with SCA displayed a 10-fold increase in levels of MP derived from red blood cell (RBC) and platelets (PLT) when compared to their healthy counterparts (p < 0.0001). Splenectomized patients with SCA have more pronounced levels of MPRBC and MPPLT, and remained elevated after several weeks of follow-up. Levels of MP were not significantly associated with spleen removal procedures, age, gender, clinical severity score, hydroxyurea therapy, hemoglobin F, and co-existence of glucose-6-phosphate dehydrogenase deficiency. Collectively, these results suggest that splenectomy affects circulating levels of MP regardless of the known SCA modifiers and correlates.

Preliminary study of microparticle coagulation properties in septic patients with disseminated intravascular coagulation.

BackgroundSepsis typically results in enhanced coagulation system activation and microthrombus formation. Microparticle (MP) production promotes coagulation and enhances pro-coagulation. This study investigated how circulating MP levels and tissue factor-bearing MP (TF+-MP) activity caused coagulation in patients with septic disseminated intravascular coagulation (DIC).MethodsThirty patients with septic DIC and 30 healthy controls were studied from December 2017 to March 2019. Patient blood samples were collected at enrolment (day 1) and on days 3 and 5; DIC scores and Sequential Organ Failure Assessment (SOFA) scores were recorded. TF+-MP activity was measured using TF-dependent factor Xa generation experiments. Circulating MP concentrations were determined by MP capture assay. Clotting factor activity, antithrombin level, soluble thrombomodulin, and serum tissue factor pathway inhibitor (TFPI) concentrations were measured.ResultsPatients with septic DIC had lower circulating MP levels than healthy control patients. Circulating MP levels in patients with septic DIC were positively correlated with DIC scores and negatively correlated with coagulation factors, but TF+-MP activity did not correlate with clotting factor levels and TFPI.ConclusionsIn patients with septic DIC, circulating MP levels are important in promoting coagulation activation and increasing clotting factor consumption. TF+-MP activity may not be the main form of active TF.

Circulating microparticles in patients with chronic hepatitis C and changes during direct-acting antiviral therapy.

Microparticles (MPs) are heterogeneous vesicles derived from membranes of different cells. Between 70 to 90% of MPs detected in blood originate from platelets. The release of MPs is associated with proinflammatory and procoagulant states. Elevated levels of MPs have been found in different diseases. We investigated MPs levels in patients with chronic hepatitis C (CHC) and changes in level during treatment using direct-acting antivirotics (DAA). Thirty-six patients with CHC and forty healthy volunteers were included in the study. Concentrations of MPs were determined indirectly by measuring their procoagulant activity in plasma at baseline, end of therapy (EOT), and 12 weeks after EOT when the sustained virological response was assessed (SVR12). All patients achieved SVR12, which was associated with rapid improvement of markers of liver damage and function as well as liver stiffness (P=0.002). MPs levels were significantly higher in CHC patients than in healthy volunteers (P<0.001). No statistically significant decrease was found observed between baseline and SVR12 (P=0,330). Analysis of subpopulations with minimal fibrosis F0-1 (P=0.647), advanced fibrosis F2-4 (P=0.370), women(P=0.847), men (P=0.164) and genotype 1 (P=0.077) showed no significant changes during the follow-up period. MPs levels are higher in CHC patients and remain elevated shortly after achieving SVR. Higher concentrations of MPs in plasma are probably caused by a chronic uncontrolled exaggerated inflammatory response caused by CHC. Longer observation would probably confirm the significance of MPs levels decrease because normalization of liver function, inflammation, and structure after SVR requires more than 12 weeks.

Level of Microparticles in Patients with Essential Thrombocythemia and Its Relation with JAK2V617F Mutation

To detect the levels of microparticles (MP) in plasma of patients with esseutial thrombo-cythermia(ET) and analyze the relationship between the JAK2V617F mutant and MP in ET patients. The numerical values of MPs were analysed by using flow cytometry. Venous blood of 56 ET patients and 28 healthy persons was collected in the morning and anticoagulated with sodium citrate (1∶9). The RMP, PMP, TF+MP and EMP were detected by FCM using phycoerythrin (PE)-conjugated monoclonal antibodies to CD235a for red blood cells, CD61 for platelets, CD142 for tissue factor (TF) and CD62E for endothelial cells, respectively. Forward scatter was set in scale using fluorescent microspheres of 0.8 μm. Standard fluorescent microbeads (0-0.8 μm) in diameter were used to set the microparticles gate. Genomic DNA was extracted from mononuclear cells by using a commercial DNA isolation kit and amplified by allele specific polymerase chain reaction (PCR). According to the size of molecular weight, the amplified products were separated by electrophoresis on a 2% agarose gel to screen 26 JAK2V617F mutations. The detection results showed that the MP levels in ET group were higher than those in normal control group: RMP (157.2±304.9/μl vs 21.3±18.4/μl), PMP (1378.9±2454/μl vs 113.8±97.1/μl), TF+MP (123±354.6/μl vs 21±15.7/μl) and EMP (1434.7±2601.9/μl vs 291.7±297.7/μl) (P<0.05). In 8 patients with thrombus, the levels of these four kinds of MP were higher than those of controls without thrombus: RMP (258.2±327.7/μl vs 55.6±63.8/μl), PMP (1853±1874.7/μl vs 444.7±712/μl), TF+MP (120.4±112.5/μl vs 60.3±128.3/μl) and EMP (1637.1±1563.5/μl vs 629.4±1000.2/μl) (P<0.05). The levels of those 4 kinds of MP in 17 patients with splenomegaly were significantly higher than those in 11 patients without splenomegaly: RMP (306.8±491.1/μl vs 59.3±51/μl), PMP (2944.8±3767.6/μl vs 334.8±420.2/μl), TF+MP (162.9±166.8/μl vs 31.0±28.7/μl) and EMP (3031.8±4048.8/μl vs 701.5±729.2/μl) (P<0.05). The level of other MP showed no difference between MPN patients with JAK2V617F mutation and without JAK2V617F mutation (P>0.05), except TF+MP (154.7±516.3/μl vs 100.5±126.6/μl) (P<0.05). The numerical values of MP detected are more in ET patients than those in healthy controls. The number of MP is higher in patients with thrombus than that without thrombus, so do in patients with splenomegaly and without splenomegaly. Patients with JAK2V617F mutation show higher number of TF+MP than that without JAK2V617F mutation. But the other three kinds of MP show no this difference.

Increased microparticle levels in middle-aged and elderly patients with insomnia may be involved in the pathogenesis of arteriosclerosis.

Insomnia may affect vascular factors and promote arteriosclerosis. Microparticles (MPs) are a heterogeneous group of bioactive small vesicles that can be found in blood and body fluids following activation, necrosis or apoptosis of virtually any eukaryotic cells. MPs are believed to participate in the pathogenesis of atherosclerosis. Few studies have been concerned with the microparticle level in patients with sleep disorder. The purpose of the present study is to measure the levels of endothelial microparticles (EMPs), platelet microparticles (PMPs) and leukocyte-derived microparticles (LMPs) in middle-aged and elderly patients with or without insomnia. Patients with insomnia (N.=30) and without insomnia (N.=18) were enrolled. The insomnia group covered patients with chronic insomnia (N.=16) and acute insomnia (N.=14). Levels of EMPs (CD31 +, CD62E +) and PMPs (CD41a +, CD42a +) and granulocyte-derived (CD11a +) MPs were measured. Flow cytometry was performed on the Beckman Coulter analyzer. Reference gate was defined for the level of MPs using 0.22-0.45-0.88μm microspheres, and the size gate for MPs was 0.5-1.0μm. Of all types of MPs detected, the levels of CD31 +MPs, CD62E +MPs and CD11a +MPs were significantly higher in the insomnia group than in the non-insomnia group (P<0.05). Besides, compared with acute insomnia, the levels of CD31 + MPs and CD11a +MPs were significantly higher in chronic insomnia (P<0.001). In insomnia patients, atherosclerosis progression may be increased by the CD31+ EMPs-mediated apoptosis and endothelial injury. The level of CD11a+ LMPs kept increasing as insomnia persisted, which may indicate atherosclerosis progression.

Urinary levels of podocyte-derived microparticles are associated with the progression of chronic kidney disease

Podocyte-derived microparticles (MPs) could be secreted from activated or apoptotic podocytes. An increased number of podocyte-derived MPs in the urine might reflect podocyte injury in renal diseases. This study aimed to observe the change of urinary podocyte-derived MP levels in patients with chronic kidney disease (CKD) and to further explore its correlation with the progression of CKD. A prospective, longitudinal study was conducted in eighty patients with biopsy-proven CKD. Podocyte-derived MPs (annexin V and podocalyxin positive) were detected by flow cytometry. The number of urinary podocyte-derived MPs was analyzed to evaluate the association with biochemical measurements and pathological glomerulosclerosis assessment. Patients with idiopathic membranous nephropathy (IMN) were followed up after the six-month treatment of prednisone combined with tacrolimus to evaluate the association of urinary podocyte-derived MP levels and the remission of IMN. The CKD patients had higher urinary podocyte-derived MP levels compared with healthy controls (HCs). Baseline urinary levels of podocyte-derived MPs were positively correlated with 24-hour proteinuria, while were inversely correlated with the percentage of global glomerulosclerosis. The urinary podocyte-derived MPs levels had good discrimination for glomerulosclerosis [area under curve (AUC), 0.66]. The urinary podocyte-derived MPs levels in IMN patients were significantly decreased accompanied with the recovery of abnormal clinical parameters after six-month treatment. The urinary levels of podocyte-derived MPs were closely associated with podocyte injury and glomerulosclerosis, which could be useful for monitoring disease activity in CKD patients. Urinary podocyte-derived MPs might be a non-invasive biomarker for the evaluation of early CKD progression.

Increased Extracellular Nucleosome Levels and Microparticles in Atrial Fibrillation Patients Compared to the Age-Matched Normal Population

Introduction: Atrial fibrillation (AF) is currently the most common cardiac arrhythmia encountered in clinical practice and a major cause of morbidity and mortality among adults. It is estimated that atrial fibrillation affects approximately 2.7 million people in the United States. Extracellular plasma nucleosomes (PNs) are complexes of DNA and histones that are released during cell death. Histones have been shown to function as DAMPs when they are translocated from the nucleus to the extracellular space. It is hypothesized that these extracellular nucleosomes contribute to the observed hypercoagulable state and embolic complications in AF.Methods: The concentration of PNs in 48 plasma samples of atrial fibrillation patients and 38 aged-matched controls were measured using the Cell Death Detection ELISA PLUS assay (Roche Diagnostics, Mannheim, Germany) and functional microparticle levels were measured using an annexin binding method (Hyphen Biomedical, Paris, France).Results: When comparing the concentration of nucleosomes in plasma, the atrial fibrillation patients (306.1 ± 328.86 μg/mL) had much higher levels than the aged-matched controls (177.9 ± 146.7 μg/mL), (p = 0.0015). Functional microparticles were also markedly elevated in the AF patients (18.6 ± 4.1 nM) in comparison to the aged-matched controls (4.6 ± 3.1 nM), (p=0.001). A significant correlation was observed between the PN’s and microparticles, (p < 0.05).Conclusion: Both the microparticles and PNs were elevated in atrial fibrillation when compared to aged-matched controls, suggesting increased cell death resulting in the generation of these biomarkers. Extracellular nucleosomes function as DAMPs by binding to receptors and triggering the activation of multiple signal pathways which may contribute to the elevation of microparticles. Due to the involvement of inflammation and thrombosis in the pathology of atrial fibrillation, the simultaneously increased circulating nucleosome and microparticle levels in patients with AF implies their role in the pathogenesis of this syndrome. DisclosuresNo relevant conflicts of interest to declare.

Are Microparticle Levels Useful for Detecting Thrombosis in Patients with Ventricular Assist Devices?

Introduction: Ventricular assist devices (VADs) are increasingly being used in the clinical management of patients with heart failure. While effective, there are risks associated with the use of a VAD. One of the most serious risks is pump thrombosis characterized by an organized fibrin deposition in the pump, which requires clinical intervention and/or surgical replacement of the device. Due to the growing number of cardiac patients with an implanted VAD, it is important to understand the susceptibility to hemostatic dysregulation among these patients to prevent complications. In current clinical practice VAD pump thrombosis is detected through an elevation in plasma lactate dehydrogenase levels [LDH; >2x upper limit of normal (ULN)]. However, this method of detection is limited by an inability to detect a pump thrombus before it becomes clinically significant. Cellular microparticles (MPs) are submicron membrane-derived exocytotic vesicles that are derived from a variety of cell types by outward blebbing of the plasma membrane in response to activation stimuli. Blood cell-derived MPs can form in response to hemostatic activation, inflammation and altered rheology. The aim of this study was to determine whether levels of cellular MPs can be used more effectively than clinical parameters to predict thrombotic events in VAD patients.Methods: Blood samples were collected peri-operatively and long-term post-operatively (until transplant or expiration) during normal clinic visits from 13 consented patients who were implanted with a HeartMate II LVAD (Thoratec, Pleasanton, CA). Fresh whole blood collected in 3.2% sodium citrate was centrifuged for platelet poor plasma then ultracentrifuged (20,000 g, 90 min) to pellet MPs. Aliquots of the MP rich samples were stained with the dye PKH67 (to identify biological membranes) then separated into five tubes containing either: Tyrode's buffer (control), CD41-PE (platelets), CD45-PE [leukocytes (WBC]), CD146-PE [endothelium (EC)], or CD235-PE [erythrocytes (RBCs)]. Samples were analyzed on an EPICS XL flow cytometer (Beckman-Coulter, Miami, FL) to determine the presence and quantity of cellular MPs using an in-house assay. MPs were identified as PKH67(+) events with a size < 1µm based on size-defined polystyrene beads. Blood samples from 12 healthy individuals were processed in the same manner to establish normal MP levels. A database of clinical events in the LVAD patients was created from medical chart review and included LDH, platelet count, aPTT, D-dimer, fibrinogen and assessment of warfarin by INR. For each patient, clinical and MP data were graphed on a linear time scale; the day that the MP and the clinical values exceeded their ULN was identified and related to time of a clinical adverse event.Results: MP levels in patients without adverse events during the follow-up period (2/13) remained below the ULN of 500 microparticles. Patients with thrombotic episodes (11/13) had elevated levels (2000 to 9000 microparticles) a median 50 days (range 35-391 days) prior to LDH elevation; MP levels reverted to normal following resolution of the event. Platelet and RBC MPs were elevated earlier and to a greater extent than MPs from WBCs and ECs. Some patients (2/11) only had an elevation of RBC MPs. Platelet counts were elevated up to 50 days post-surgery (12/13 patients), but levels were normal at time of the thrombotic event. There was no obvious association with adverse events for INR time out of therapeutic range or any other clinical parameter.Discussion: Microparticle levels in VAD patients with thrombotic events were shown to cross the normal threshold weeks prior to the time when common clinical parameters indicated an abnormality, suggesting that changes in microparticle levels may be useful in predicting thrombotic events. Hypercoagulability in VAD patients can be influenced by a number of factors including exposure of blood to foreign surfaces, altered shear stresses, inflammation and infection. Data from this investigation warrants further study for the incorporation of microparticles into the clinical management of patients with implanted VADs and to determine whether the pattern of change in microparticle subtypes can identify the stimulus for pump thrombosis, leading to more effective prophylactic measures. DisclosuresNo relevant conflicts of interest to declare.

Abstract 292: Microparticle-encapsulated miRNAs Are Associated With Peripheral Artery Disease

miRNAs are short, noncoding RNAs that circulate in plasma within membrane-bound vesicles, such as microparticles (MPs), or bound to proteins or lipoproteins. Circulating miRNAs are putative biomarkers for a wide range of diseases, including cardiovascular disease. Our group and others have evidence to suggest MP-encapsulated miRNAs, rather than total plasma miRNAs, may harbor more specific and functionally relevant biomarkers. We tested the hypothesis that MP-encapsulated miRNA levels would differ between patients with mild or severe peripheral artery disease (PAD) and healthy controls matched for age, gender, and race. Banked plasma stored at -80°C was obtained for 5 healthy subjects, 6 patients with mild PAD (ABI 0.8-1.0), and 10 patients with severe PAD (ABI &lt; 0.4). MPs were isolated using a standard differential centrifugation protocol and quantified by flow cytometry. Four miRNAs implicated in vascular disease (miR-126-3p, miR-126-5p, miR-21, and miR-222) were quantified with qRT-PCR. Relative expression for each miRNA (-2ΔCt) was calculated and divided the total number of MPs to determine miRNA expression per MP. There was a non-significant trend towards increased levels of MPs in patients with mild (19,462 MPs/μl ± 5,213) and severe PAD (21,688 MPs/μl ± 7,175) compared to healthy controls (7244 MPs/μl ± 919). MP levels of miR-126-5p were increased among healthy subjects compared to patients with mild and severe PAD (p = 0.003), while MP miR-126-3p levels were significantly lower in healthy subjects (p = 0.002). miR-21 and miR-222 did not differ significantly between groups. None of the four miRNAs distinguished mild versus severe PAD. In conclusion, our data suggests that MP-mediated transport of miR-126-5p and miR-126-3p is dysregulated in the setting of mild and severe vascular disease, and supports the concept that the plasma MP fraction may harbor disease-associated miRNA profiles.

Elevated circulating endothelial cell-derived microparticle levels in patients with liver cirrhosis: a preliminary report.

Aim of the studyTo determine plausible associations between liver cirrhosis and circulating endothelial cell-derived microparticles (EMPs), vascular endothelial growth factor (VEGF) levels and plasma nitric oxide (NO) metabolites.Material and methodsSixty patients with cirrhosis and 20 healthy control subjects were enrolled in the study. Circulating EMPs from platelet-poor plasma samples were examined by flow cytometry. These microparticles were categorized into endothelial cell-derived activated MPs (EMP-ac) (CD31+ CD42b– AN-V–) and endothelial cell-derived apoptotic MPs (EMP-ap) (CD31+ CD42b– AN-V+). Plasma VEGF levels were measured by enzyme-linked immunosorbent assay. Plasma NO metabolites (NOx–) levels were determined using a Greiss reaction method.ResultsCompared with the healthy control subjects, the patients with cirrhosis showed a significant increase in plasma levels of both phenotypes of EMPs. When the presence of ascites was considered, the plasma levels of EMP-ap were higher (p < 0.01), as well as NOx– (p < 0.05). EMP-ap positively correlated with VEGF level in all cirrhotic patients and this correlation was stronger in decompensated cirrhotic patients. In multivariate logistic regression analysis, the independent factors associated with the presence of ascites were high EMP-ap levels and elevated VEGF levels.ConclusionsElevated plasma levels of EMP-ap in addition to high levels of VEGF might be considered as valuable parameters for predicting the occurrence of ascites in cirrhotic patients.

Endothelial and platelet microparticles in patients with antiphospholipid antibodies

BackgroundThe antiphospholipid syndrome (APS) is the association of thrombosis and recurrent pregnancy loss and/or pregnancy morbidity with persistent antiphospholipid antibodies (aPL). Previous studies of microparticles in patients with APS/aPL have mainly been small and findings, contradictory. ObjectivesTo quantify endothelial and platelet microparticle levels in patients with isolated antiphospholipid antibodies or primary antiphospholipid syndrome (PAPS). Patients/MethodsWe measured endothelial and platelet microparticle levels by flow cytometry in 66 aPL/PAPS patients and 18 healthy controls. ResultsLevels of circulating platelet (CD41 and CD61) and endothelial microparticles (CD51 and CD105) were significantly increased in patients with PAPS and aPL compared to healthy controls. There were correlations between platelet and endothelial microparticles levels in all patients with aPL. ConclusionsPlatelet and endothelial microparticles are increased in all patient groups within this cohort of patients aPL. Whether they may have a role in the pathogenesis of APS merits further study.

Dobutamine Stress–Induced Microparticle Release

Despite numerous advances in the diagnosis and treatment of cardiovascular disease (CVD), it remains a leading cause of morbidity and mortality in the developed world. CVD is the leading cause of death in the United States with total costs associated with CVD treatment reaching 17% of the national health expenditures.1 A substantial portion of the cost and time associated with CVD is in the administration of stress tests, including dobutamine stress echocardiogram (DSE), for noninvasive risk assessment of patients presenting with symptoms of obstructive coronary artery disease (CAD). Article, see p 109 Microparticles (MPs) are subcellular vesicles between 100 nm and 1 μm in diameter which are shed from the plasma membrane of activated or dying cells.2 Microparticles possess the procoagulant lipid phosphatidyl serine (PS) and cell surface proteins of their parent cells, which allow for efficient identification of cellular source via flow cytometry using fluorescently labeled annexin V, a PS binding protein, and receptor-specific antibodies.3 Microparticles contain cytokines and miRNA and act as local and systemic messengers. In addition, microparticle production is vital for removal of stressed-induced cellular by-products.4 Numerous studies have shown that microparticles activate and regulate many physiological and pathological processes.2,5 Microparticles likely play a significant role in CVD and CVD risk factors.3 Studies have shown increased microparticle levels in patients with diabetes mellitus and hypertension.6,7 Elevated microparticle levels have been correlated with a higher calculated 10-year Framingham CAD risk.8 Increases in microparticle levels have also been observed in patients with high risk coronary lesions and correlate …

Pre-Surgical Levels Of Circulating Cell-Derived Microparticles Are Most Significant Predictors Of Transfusion Requirement In Coronary Artery Bypass Graft Surgery

IntroductionBlood transfusion is associated with serious adverse events (SAE) such as poor surgical outcome, higher surgical mortality. The cost of transfusion is escalating due to increasing demand and limited blood supply. There is a worldwide growing interest in improving blood utilization methods and storage. Identification of reliable predictors of surgical bleeding is of upmost importance in order to reduce the number of unnecessary transfusions, save limited blood resources, and lower the related costs. Cell derived microparticles (MP) are small membrane vesicles of 0.1 µm in size, released during cell activation and apoptosis. They play an important role in hemostasis and thrombosis. MethodsData presented herein are from an NIH funded prospective randomized study to investigate role of cell derived MP in surgical complications in CABG. Of a total of 122 patients, 81 received transfusion during and/or after surgery (Tx group) whereas the remaining 41 did not (NoTx group). In addition to routine laboratory tests, special assays were performed pre-surgery including concentrations of MP from platelets (PMP), red cells (RMP), endothelia (EMP), and leukocytes (LMP). The two groups were compared with respect to pre-surgical variables in order to assess potential predictors of the need for surgery. ResultsThere were no significant differences between the two groups with respect to most of the pre-surgical demographic, clinical, and lab variables, except that Tx group was associated with higher incidence of female gender, type O blood group, and heparin use. On the other hand, the NoTx group was associated with higher pre-surgical levels of RMP and PMP. Using stepwise logistic regression analyses, four factors were identified as the most significant predictors of the need for transfusion. As depicted by an the areas under the curve (AUC) of the ROC curve, the 95% Confidence Interval of the AUC, and their corresponding p-value for each factor, were as follows.For the model combining all four variables, the AUC and its 95% CI were 0.86 [0.78 - 0.94]. In addition, analysis of post-surgery data revealed better outcomes for the NoTx group in terms of complications such as hours on mechanical ventilation (mean ± SD: 9.2 ± 7.0 vs 15.5 ± 17.1, p = 0.005), number of SAE other than death (0 vs 9 (11.1%), p =0.028), and number of in-hospital deaths (0 vs 5 (6.1%), p= 0.167).FactorAUC[95% C.I]p-valueHGB0.66[0.55 - 0.78]0.005aPTT0.68[0.57 - 0.79]0.015RMP0.69[0.58 - 0.80]0.005CD41+PMP0.77[0.67 - 0.86]<0.001 DiscussionThis is the first report to show that pre-surgical levels of circulating MP are highly significant predictors of need for transfusion in CABG. This finding is consistent with other evidence that MP play a major role in hemostasis. Implementation of pre-surgery MP assay will greatly imporove assessment of risk of bleeding in surgery, and requirements for transfusion. This could reduce the number of transfusions, with corresponding improvement in patient outcomes, as well as reduced costs and strains on limited blood supplies. Furthermore, it may be feasible to manipulate pre-surgical MP levels in patients to minimize bleeding and Tx requirements. This could be done by increasing rate of MP generation or retarding rate of clearance, or both. Alternatively, MP could be produced in vitro (autologous or not) for infusion during surgery to minimize blood loss. Such approach is in progress [Jy, et al Thromb Haemost, in press]. Disclosures:No relevant conflicts of interest to declare.

Microparticles As Biomarkers Of Osteonecrosis Of The Hip In Sickle Cell Disease

IntroductionSickle cell disease (SCD) is the most common cause of osteonecrosis of the femoral head (ONFH) in children. ONFH is a debilitating condition that is associated with mobility limitations, chronic pain, and an impaired quality of life. While the mechanisms that cause ONFH remain unknown, ischemia from recurrent microvascular occlusion is likely to play a role. Vascular occlusion may result directly from obstruction by sickled cells, or indirectly via complex interdependent pathways characterized by sustained endothelial activation, chronic inflammation, and coagulation. Microparticles (MP) are small, cell membrane-derived vesicles generated in response to cellular activation, injury or apoptosis. MPs have emerged as potential modulators of inflammation and thrombosis and have been found to be elevated in patients with ONFH in the general population. ObjectiveThis pilot study examined whether microparticle levels in patients with SCD who have ONFH differ from SCD patients without ONFH, as well as healthy African American (AA) controls. MethodsSubjects were recruited at their baseline status and were excluded if they had been transfused within the past 30 days, hospitalized for a vaso-occlusive pain episode, acute chest syndrome, fever or surgery within the past 30 days, or had bony lesions of the femur or hip due to causes unrelated to SCD. For MP analysis, whole blood was collected in sodium citrate tubes and centrifuged for 15 minutes at 1500 x g at 20° C to generate platelet poor plasma. Aliquots of the plasma were immediately frozen and stored at -80° C until the time of MP analysis. 300 μl samples were diluted in PBS and centrifuged at 10000 x g for 1hr and the supernatant was centrifuged at 100,000 x g for 2 hr. The pellet was re-suspended in 1 mL of PBS and subjected to nanoparticle-tracking analysis to determine concentration and size. Additional laboratory biomarkers of inflammation and coagulation, including highly-sensitive C-reactive protein (hs-CRP), von Willebrand factor antigen (vWF Ag), tissue factor (TF), and D-dimer were analyzed for differences between groups. Analysis of variance was used to compare MP and biomarker levels between the three groups. The institutional review board at Children's Hospital & Research Center Oakland approved the study protocol and written informed consent was obtained from all participants. ResultsCharacteristics of the 30 subjects enrolled are shown in Table I.Total microparticle levels in ONFH(+) patients were 2.3-fold higher than in ONFH(-) patients, and 2.5-fold higher than in AA controls (Figure 1). Mean MP levels for ONFH(+) patients, ONFH(-) patients, and AA controls were 4.55 x 1010, 1.99 x 1010, and 1.85 x 1010, respectively. Microparticle levels in ONFH(-) SCD patients did not differ from AA controls. There were no statistically significant differences in hsCRP, vWF Ag, TF, or D-dimer levels between the ONFH(-) and ONFH(+) groups. [Display omitted] [Display omitted] ConclusionsThe results of this study demonstrate significantly elevated MP levels in individuals with SCD who have ONFH. Additional studies are needed to better understand the mechanistic effects of MPs on the development of ONFH and to determine whether MP levels may be useful as a predictive biomarker for early disease detection.This publication was supported by NIH/NCRR UCSF-CTSI Grant Number UL1 RR024131. Disclosures:No relevant conflicts of interest to declare.

Local increase in microparticles from the aspirate of culprit coronary arteries in patients with ST-segment elevation myocardial infarction

ObjectiveIt has been reported that the levels of procoagulant microparticles (MPs) are increased in patients with acute coronary syndromes and this may contribute to the formation of intracoronary thrombi. In the current study, we investigated the presence of locally elevated MPs within the culprit coronary arteries of patients with ST-segment elevation myocardial infarction (STEMI). MethodsThe study population consisted of 45 patients with STEMI who underwent primary percutaneous coronary intervention (PCI), and 16 control patients. Before and after PCI, blood samples were collected from the femoral artery and from the culprit coronary arteries. In controls, only peripheral blood was obtained. MPs were measured by a solid-phase capture assay using a commercial kit. The cell origins of MPs were determined by antigenic capture with specific antibodies. ResultsBaseline levels of MPs in patients with STEMI were higher than in controls. Before PCI, the levels of MPs were significantly higher in culprit coronary arteries than in peripheral arteries in STEMI patients (20.7 ± 15.5 vs. 14.6 ± 15.4 nM phosphatidylserine (PS) equivalent, p = 0.027). MPs from the culprit coronary artery were significantly reduced after PCI (20.7 ± 15.5 vs. 14.3 ± 14.9 nM PS equivalent, p = 0.010). Similarly, the locally increased levels of endothelial- and platelet-derived MPs within the culprit coronary arteries were significantly decreased after PCI. ConclusionLocally increased levels of MPs in culprit coronary arteries and their significant reduction after successful PCI suggest a potential role in coronary atherothrombosis in the early period of STEMI.

Circulating microparticle levels in patients with coronary artery disease: a new indicator of vulnerability?

This editorial refers to ‘Circulating CD31+/Annexin V+ microparticles correlate with cardiovascular outcomes’[†][1], by J.-M. Sinning et al. , on page 2034 Despite significant advances in medical and interventional management during the past decades, coronary artery disease (CAD) and its consequences, which include myocardial infarction, sudden cardiac death, and chronic cardiac failure, remain a major contributor to mortality in western countries. Lifestyle modifications and secondary prevention medications, such as statins or angiotensin-converting enzyme (ACE) inhibitors, have improved outcome, but some patients still experience adverse cardiovascular events as a consequence of a particular atherosclerotic disease evolution. Identification of these high-risk, vulnerable patients remains a challenging issue.1 Atherosclerosis is a systemic disease characterized by endothelial dysfunction and by local and general inflammation. It is aggravated by platelet activation, and its ultimate evolution is plaque rupture and thrombosis.2 Identification of vulnerable plaques cannot be achieved by conventional angiography and requires sophisticated and invasive investigations such as intravascular ultrasound, optical coherence tomography, or magnetic resonance imaging.1 Moreover, there is no real consensus regarding a simple definition of vulnerable plaque. Risk stratification of patients based on measurement of circulating biomarkers [high sensitivity C-reactive protein, soluble lipoprotein phospholipase A2, B-type natriuretic peptide (BNP), etc.] or non-invasive evaluation of vascular structure or function (carotid intima-media thickness, flow-mediated dilation, etc.) appear to be more efficient tools in many respects.1 However, none of the above-cited parameters appears to be perfectly appropriate in view of the required criteria (sensitivity, specificity, … [1]: #fn-2

Time course of plasma microparticle concentrations after acute spontaneous basal ganglia hemorrhage

To examine the changes in plasma microparticle (MP) levels in patients after intracerebral hemorrhage (ICH) and assess their association with outcome along with biological markers of the acute phase response. Thirty healthy controls and 86 patients with acute ICH were recruited. Plasma samples were obtained on admission and at days 1, 2, 3, 5, and 7 after ICH. MPs with procoagulant potential were measured with a prothrombinase assay. Plasma MP levels in patients were substantially higher than those in healthy controls during the 7-day period. Plasma MP levels were strongly associated with outcome and with biological markers of the acute phase response. Multivariate analysis showed baseline plasma MP level was a good predictor of 1-week mortality (odds ratio, 1.930; 95% confidence interval, 1.229-3.031; P=0.004). A receiver operating characteristic curve identified the plasma MP cutoff level (8.4 nmol/l phosphatidylserine equivalent) that predicted 1-week mortality with high sensitivity (90.6%) and specificity (68.5.0%) (P<0.001). Increased membrane microparticle levels occur after ICH and may contribute to the subsequent brain injury, in association with a poor clinical outcome.

High concentrations of procoagulant microparticles in the cerebrospinal fluid and peripheral blood of patients with acute basal ganglia hemorrhage are associated with poor outcome

Background Apoptosis plays an important role in further brain injury after intracerebral hemorrhage (ICH). Procoagulant microparticles (MPs) are shed from the plasma membrane of apoptotic cells. The objective of this study was to determine plasma and cerebrospinal fluid (CSF) levels of MPs in patients with spontaneous ICH and to correlate MP levels with Glasgow Coma Scale (GCS) scores, ICH volumes, presence of intraventricular hemorrhage (IVH), and survival rate. Methods Ten patients with suspicion of subarachnoid hemorrhage and 36 patients with spontaneous basal ganglia hemorrhage were included. Plasma and CSF samples were collected. Circulating MPs were obtained by double centrifugation and captured with annexinV. Their procoagulant potential was measured with a prothrombinase assay. Results Plasma or CSF MP levels in the ICH group were significantly higher than those in the control group (8.2 ± 3.0 vs 3.2 ± 1.7 nmol/L phosphatidylserine [PS] equivalent; P < .001 or 9.8 ± 3.7 vs 1.4 ± 0.6 nmol/L PS equivalent; P < .001). The MP levels were highly associated with GCS scores, ICH volumes, presence of IVH, and survival rate (all P < .05) in ICH. A receiver operating characteristic curve identified CSF and plasma MP cutoff levels that predicted 1-week mortality of patients with the high sensitivity and specificity values. Areas under curves (AUCs) of GCS scores and ICH volumes were larger than those of CSF and plasma MP levels, but only the difference between AUC of GCS scores and that of plasma MPs levels reached statistical significance ( P < .05). Conclusions High levels of procoagulant MPs are present in the CSF and peripheral blood of patients with ICH and may contribute to the pathogenesis of ICH. The levels of CSF and plasma MPs after spontaneous onset of ICH seem to correlate with clinical outcome in these patients. Taking clinical complexity into account, only plasma MP levels can be served as useful clinical markers for evaluating the prognosis of ICH.

E010 Increase in circulating microparticles in inflammatory bowel disease patients induces vascular alterations

Among the proposed hypothesis for inflammatory bowel disease (IBD) pathogenesis, a vascular cause has been suggested, mostly in reference to anatomic and pathological studies. We previously reported in small mesenteric arteries from Crohn's disease patients a balance between vasoconstrictor products from cyclooxygenase and unidentified vasodilatory products that maintained vascular reactivity in a physiological range despite an increase of circulatory cytokines in these patients (Tabernero et al., Circulation, 2003). Microparticles (MPs) are membrane vesicles released during cell activation and apoptosis. Elevated levels of circulating MPs have been detected in pathologic conditions and their increase has been associated with disease activity and/or prognosis. We investigated whether MPs from IBD patients may play a role in the regulation of endothelial function and vascular reactivity in this disease. Blood samples were obtained either from IBD patients as well as age and sex-matched healthy subjects. MPs concentration and origin were assessed by flow cytometry using specific antibodies. Male Swiss mice were injected intravenously with MPs from IBD patients or with saline solution and sacrified after 24 hours. Endothelial function and vascular reactivity were studied on aortic rings and small mesenteric resistance arteries (SMA) by myography and arteriography, respectively. Patients with IBD displayed increased circulating levels of MPs compared to healthy subjects including those from procoagulant, platelet and activated platelet, endothelial, leukocyte and erythrocyte origins. In aorta, MPs from IBD patients compared to saline significantly reduced both endothelium-dependent relaxation to acetylcholine and contraction to serotonin. In SMA, although MPs from IBD patients did not affect flow-induced dilation, a reduced NO-component that was completely compensated by the endothelium-derived hyperpolarizing factor-component of the response was highlighted. Besides, MPs from IBD did not affect myogenic tone in SMA. These results provide further evidence of increased circulating levels of MPs in patients with IBD. MPs origin may play a role in enhanced coagulation and inflammatory states reported in IBD patients. Finally, MPs from IBD patients influence both endothelial dysfunction and vascular hyporeactivity in the experimental model used. (Partially supported by Ferring France Laboratories).

Effect of the administration of n-3 polyunsaturated fatty acids on circulating levels of microparticles in patients with a previous myocardial infarction

Increased levels of microparticles exposing tissue factor circulate in the blood of patients with coronary heart disease, possibly disseminating their pro-thrombotic and pro-inflammatory potential. Because diets rich in n-3 (polyunsaturated) fatty acids have been associated with reduced incidence of coronary heart disease-related events, we investigated the in vivo effects of treatments with n-3 fatty acids on levels of circulating microparticles and their tissue factor- dependent procoagulant activity in patients with a previous myocardial infarction. Forty-six post-myocardial infarction patients were assigned to receive either 5.2 g of n-3 fatty acids daily (n=23) or an olive oil placebo (n = 23) for 12 weeks. Circulating microparticles were isolated from peripheral blood. The number of microparticles, their cellular source and tissue factor antigen were determined by flow cytometry, and their procoagulant potential assayed by a fibrin generation test. The total number of microparticles, endothelium-derived microparticles and microparticle tissue factor antigen were not significantly different between the two groups. However, the number of platelet-derived microparticles [from a median of 431 (126-1796, range) x 10(6)/L to a median of 226 (87-677, range)] x 10(6)/L and monocyte-derived microparticles [from a median of 388 (9-1681, range) x 10(6)/L to a median of 265 (7-984, range) x 10(6)/L] in plasma were significantly (p < 0.05) decreased by n-3 fatty acids, while they were unchanged in the placebo group. Total microparticle tissue factor-procoagulant activity was also reduced in the n-3 fatty acid group compared to that in the placebo group. Treatment with n-3 fatty acids after myocardial infarction exerts favorable effects on levels of platelet- and monocyte-derived microparticles, thus possibly explaining some of the anti-inflammatory and anti-thrombotic properties of these natural compounds.