Levels Of Platelet Microparticles Research Articles

Procoagulant Phospholipid Activity and MPV Values in Acute Ischemic Stroke

Ischemic stroke can be caused by atherothrombosis or embolism. Atherothrombosis occurs due to the rupture of an atherosclerotic plaque causing platelet activation. There are several markers of platelet activation, including platelet microparticles. The levels of platelet microparticles can be measured by examining procoagulant phospholipid (PPL) activity. It is not yet known exactly what activates thrombopoiesis, which can be assessed by an increase in Mean Platelet Volume (MPV). This study aimed to know whether there is an increase in PPL activity and MPV values in acute ischemic stroke, whether platelet activation is influenced by differences in ischemic stroke subtypes, and whether there is a relationship between PPL activity and MPV values in acute ischemic stroke. The study design was cross-sectional and involved 60 subjects. PPL activity in acute ischemic stroke (65.14±13.35 seconds) tends to be higher (shorter clotting time) than in healthy individuals (68.59±8.56 seconds), however, this difference was not statistically and clinically significant. The MPV value in acute ischemic stroke (9.83±0.72 fL) compared to healthy individuals (9.65±0.86 fL) however this difference was not statistically significant. PPL activity in the SAO subtype (61.66±1.31 seconds) tends to be higher than LAA (68.62±14.57 seconds), however, this difference was also not statistically significant. There was a weak correlation between PPL activity (seconds) and MPV value (fL) in acute ischemic stroke (r =0.34, p-value=0.03).

The study of platelet microparticles and P-selectin expression in patients with the peripheral arterial diseases

BACKGROUND: High platelet reactivity leads to the progression of atherosclerosis and its complications. Activated platelets adhere to the site of endothelium damage on the vessel wall and initiate the formation of an arterial thrombus, followed by acute ischemia of the organ. Biochemical and cellular marker such as platelet microvesicles and P-selectin can be analyzed using flow cytometry, which is based on a specific antigen-antibody interaction. Patients with peripheral arterial disease are at significantly greater risk of cardiovascular and cerebrovascular complications than individuals of the same age and sex. The use of antiplatelet agents is an important part of pathogenetic therapy and prevention of cardiovascular diseases and their complications.
 AIM: This study was to evaluate the level of platelet microparticles and P-selectin expression in patients with peripheral arterial disease receiving antiplatelet therapy.
 MATERIALS AND METHODS: The study included 49 people, which included three study groups: patients with obliterating disease of the arteries of the lower extremities (n = 14) on the background of long-term use (more than 14 days) of double (75 mg clopidogrel and 100 mg Acetylsalicylic acid) antiplatelet therapy, patients with COVID-19 (n = 15), who made up the positive control group in the determination of microparticles of platelet origin, and healthy volunteers (n = 20) without signs of acute respiratory disease, without a history of cardiovascular and thromboembolic episodes, not taking antiplatelet drugs. The functional activity of platelets was assessed by two methods: using light aggregometry and analysis of P-selectin expression on platelet surface by flow cytometry. The number of platelets microparticles in blood plasma was also determined using flow cytometry.
 RESULTS: A significant decrease in platelet aggregation was found in patients with peripheral arterial disease taking antiplatelet agents, compared with controls by used light aggregometry. Similar changes were obtained when analyzing the expression of P-selectin on platelets. A higher percentage of platelets microparticles with the CD9+CD41+ phenotype was found in patients with severe inflammation compared with peripheral arterial disease patients treated with antiplatelet agents and compared with the healthy controls.
 CONCLUSIONS: Thus, our study reflects the consistency of the results of three different laboratory tests in assessing the platelets reactivity in patients with the peripheral arterial diseases taking antiplatelet drugs.

Increased levels of circulating platelet microparticles as a risk of hypercoagulable state in β-thalassemia intermedia patients

Introduction Thromboembolism is a frequent complication in thalassemia. Platelet microparticles (PMPs) may have a role in the hypercoagulability in thalassemia. Objective To determine the levels of PMPs in β-thalassemia intermedia (β-TI) patients and whether increased levels of PMPs in these patients are correlated with coagulation parameters or not. Patients and methods Sixty-three β-TI patients, classified into 46 nonsplenectomized (NS) patients, 17 splenectomized (S) patients, and 20 age-matched and sex-matched volunteers as controls. For all, full medical history, through clinical examination, and laboratory investigations: complete blood count, prothrombin time, prothrombin concentration, activated partial thromboplastin time, FVIII, FXI, fibrinogen, D-dimer, and PMPs were detected by using flow-cytometry approach. Results PMPs were significantly higher in S and NS β-TI patients than controls (P<0.001, P=0.002), respectively. FVIII and D-dimer were significantly higher in S patients than controls (P=0.004, 0.048), respectively. Fibrinogen level was significantly lower in both NS and S β-TI patients than in controls (P<0.001). A significant positive correlation between PMPs and platelet count in S patients (r=0.549, P=0.023). Conclusion Thrombotic risk in β-TI patients is related to increased circulating PMPs and platelet count. Splenectomy was considered a risk factor of thrombosis in our study.

Increased Levels of Platelets and Endothelial-Derived Microparticles in Patients With Non-Valvular Atrial Fibrillation During Rivaroxaban Therapy.

It is known that atrial fibrillation (AF) is associated with the procoagulant state. Several studies have reported an increase of circulating microparticles in AF, which may be linked to a hypercoagulable state, atrial thrombosis and thromboembolism. We evaluated in our study alterations in both platelet (PMP, CD42b) and endothelial-derived (EMP, CD144) microparticle levels on anticoagulant therapy with rivaroxaban in nonvalvular AF. After administration of rivaroxaban, PMP levels were increased (median, [IQR] 35.7 [28.8-47.3] vs. 48.4 [30.9-82.8] cells/µL; P = 0.012), along with an increase in EMP levels (14.6 [10.0-18.6] vs. 18.3 [12.9-37.1] cells/µL, P < 0.001). In the multivariable regression analysis, the independent predictor of post-dose change in PMPs was statin therapy (HR −0.43; 95% CI −0.75,−0.10, P = 0.011). The post-dose change in EMPs was also predicted by statin therapy (HR −0.34; 95% CI −0.69, −0.01, P = 0.046). This study showed an increase in both EMPs and PMPs at the peak plasma concentration of rivaroxaban. Statins have promising potential in the prevention of rivaroxaban-related PMP and EMP release. The pro-thrombotic role of PMPs and EMPs during rivaroxaban therapy requires further study.

Thromboembolic Events in Patients With Left Ventricular Assist Devices Are Related to Microparticle-Induced Coagulation

Thromboembolic events (TEs) are a feared complication in patients supported by a continuous-flow left ventricular assist device (LVAD). The aim of the study was to analyze the role of circulating microparticles (MPs) in activating the coagulation system in LVAD patients, which might contribute to the occurrence of TEs. First, we analyzed the effect of LVAD support on endothelial function, on the levels of endothelial MPs (EMPs) and platelet MPs (PMPs), and on the procoagulative activity of circulating MPs (measured as MP-induced thrombin formation) before LVAD implantation, post-implantation, and at a 3 month follow-up (n = 15). Second, these parameters were analyzed in 43 patients with ongoing LVAD support who were followed up for the occurrence of TEs in the following 12 months. In patients undergoing LVAD implantation, the levels of PMPs and MP-induced thrombin formation increased post-LVAD implantation. The flow-mediated vasodilation (FMD) decreased, while the levels of EMPs increased post-LVAD implantation. TEs occurred in eight patients with ongoing LVAD support despite adequate coagulation. The levels of PMPs and MP-induced thrombin formation were higher in LVAD patients with TEs than in LVAD patients without TEs and were independent predictors for the risk of TEs under LVAD support. As conclusion, implantation of LVAD enhanced MP-induced coagulation, which was independently associated with the occurrence of TEs. These parameters may serve in risk stratification for early transplantation and individualized modification of standard LVAD therapy.

Platelet Microparticles Regulate Neutrophil Extracellular Traps in Acute Pancreatitis.

This study aimed to explore the clinical significance of the increase of platelet microparticles (PMPs) in acute pancreatitis (AP). Clinical data and plasma samples from patients with AP were collected, and healthy subjects were controls. The PMPs were detected by flow cytometry; meanwhile, the ability to promote neutrophil extracellular traps (NETs) formation was investigated. Neutrophils from healthy subjects were co-cultured with PMPs from AP patients. The NETs were visualized by confocal laser scanning microscopy. In the supernatant of cell co-culture, myeloperoxidase, neutrophil elastase, and histone H3 were detected by enzyme-linked immunosorbent assay. Patients with AP had elevated plasma levels of PMPs compared with controls; moreover, there were significantly higher PMPs levels in severe AP than mild AP and moderately severe AP. Healthy subjects' neutrophils were stimulated with PMPs from AP patients to release NETs. It was observed that NETs formed in AP group, but not in the controls. Correspondingly, there were higher levels of myeloperoxidase, neutrophil elastase, and histone H3 in AP group than in controls. The level of PMPs is a positive correlation with AP severity, which may be related to PMPs-NETs interaction. Platelet microparticles may be a potential predictor of severe AP and promising novel therapeutic target for AP.

The association between urinary levels of 1,3-butadiene metabolites, cardiovascular risk factors, microparticles, and oxidative stress products in adolescents and young adults

1,3-Butadiene (BD) is a synthetic colorless gas used in the production of synthetic rubber and polymers. Exposure to BD has been reported to increase oxidative stress and accelerate atherosclerosis in vitro and in animal studies. In occupational studies, BD exposure has been linked to cardiovascular disease (CVD). However, no previous research has been reported on whether BD exposure is associated with CVD risk factors and oxidative stress in the general population. We recruited 853 young participants to study the correlation between urinary levels of the BD metabolite, N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (DHBMA), CVD risk factors, serum levels of endothelial microparticles and platelet microparticles, and the urinary levels of 8-hydroxydeoxyguanosine (8-OHdG). The results showed the DHBMA levels were positively correlated with low-density lipoprotein-C, carotid intima-media thickness (CIMT), CD31+/CD42a − counts (endothelial apoptosis markers), and urinary 8-OHdG levels. Moreover, DHBMA levels were negatively correlated with CD62 P counts (platelet activation marker). The correlation between DHBMA, CIMT, and 8-OHdG was more evident when the levels of CD31+/CD42a − or CD62 P were above 50%. In conclusion, we reported that the urinary levels of DHBMA were associated with the lipid profile, CIMT, microparticles, and marker of oxidative stress in this young population. Future studies on BD exposure and atherosclerosis are needed.

Oral contraceptives increase platelet microparticle levels in normal-weight women with polycystic ovary syndrome.

Platelet microparticles (PMPs), which are microvesicles shed from platelets, participate in inflammation, vascular homeostasis, and thrombosis. PMPs are increased in obese women with polycystic ovary syndrome (PCOS). Agents that modulate hormonal aspects of PCOS could affect the levels of PMPs. The aim of the present study was to evaluate the effects of oral contraceptives (OCPs), antiandrogen, and metformin use for 6 and 12months on PMPs in normal-weight women with PCOS. Forty-five women with PCOS and 13 healthy women were recruited. Biochemical, hormonal, and clinical parameters were recorded. Women with PCOS received treatment with OCPs, OCPs+antiandrogens, or metformin, depending on their main complaint or clinical/biochemical findings. PMPs were measured at baseline and after 6 and 12months. At baseline, patients with PCOS had higher levels of PMPs than controls (p = 0.017), which increased after 6-month treatment with OCPs (p = 0.006). Subsequently, they decreased after 12-month treatment (p = 0.046). Metformin had no effect on PMP levels. In conclusion, PMP levels are increased in PCOS and further increase with OCP use. This effect could possibly contribute to the increased risk of venous thromboembolism associated with OCP use. However, further studies are needed to elucidate the exact role of PMPs in PCOS.

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.

Influence of Cardiometabolic Risk Factors on Platelet Function.

Platelets are key players in the thrombotic processes. The alterations of platelet function due to the occurrence of metabolic disorders contribute to an increased trend to thrombus formation and arterial occlusion, thus playing a major role in the increased risk of atherothrombotic events in patients with cardiometabolic risk factors. Several lines of evidence strongly correlate metabolic disorders such as obesity, a classical condition of insulin resistance, dyslipidemia, and impaired glucose homeostasis with cardiovascular diseases. The presence of these clinical features together with hypertension and disturbed microhemorrheology are responsible for the prothrombotic tendency due, at least partially, to platelet hyperaggregability and hyperactivation. A number of clinical platelet markers are elevated in obese and type 2 diabetes (T2DM) patients, including the mean platelet volume, circulating levels of platelet microparticles, oxidation products, platelet-derived soluble P-selectin and CD40L, thus contributing to an intersection between obesity, inflammation, and thrombosis. In subjects with insulin resistance and T2DM some defects depend on a reduced sensitivity to mediators—such as nitric oxide and prostacyclin—playing a physiological role in the control of platelet aggregability. Furthermore, other alterations occur only in relation to hyperglycemia. In this review, the main cardiometabolic risk factors, all components of metabolic syndrome involved in the prothrombotic tendency, will be taken into account considering some of the mechanisms involved in the alterations of platelet function resulting in platelet hyperactivation.

Polyphenols: Modulators of Platelet Function and Platelet Microparticle Generation?

Platelets and platelet microparticles (PMPs) play a key role in the pathophysiology of vascular disorders such as coronary artery disease and stroke. In atherosclerosis, for example, the disruption of the plaque exposes endogenous agonists such as collagen, which activates platelets. Platelet hyper-activation and the high levels of PMPs generated in such situations pose a thrombotic risk that can lead to strokes or myocardial infarctions. Interestingly, dietary polyphenols are gaining much attention due to their potential to mimic the antiplatelet activity of treatment drugs such as aspirin and clopidogrel that target the glycoprotein VI (GPVI)–collagen and cyclooxygenease-1 (COX-1)–thromboxane platelet activation pathways respectively. Platelet function tests such as aggregometry and flow cytometry used to monitor the efficacy of antiplatelet drugs can also be used to assess the antiplatelet potential of dietary polyphenols. Despite the low bioavailability of polyphenols, several in vitro and dietary intervention studies have reported antiplatelet effects of polyphenols. This review presents a summary of platelet function in terms of aggregation, secretion, activation marker expression, and PMP release. Furthermore, the review will critically evaluate studies demonstrating the impact of polyphenols on aggregation and PMP release.

Hemocompatibility and safety of the Carmat Total Artifical Heart hybrid membrane

The Carmat bioprosthetic total artificial heart (C-TAH) is a biventricular pump developed to minimize drawbacks of current mechanical assist devices and improve quality of life during support. This study aims to evaluate the safety of the hybrid membrane, which plays a pivotal role in this artificial heart. We investigated in particular its blood-contacting surface layer of bovine pericardial tissue, in terms of mechanical aging, risks of calcification, and impact of the hemodynamics shear stress inside the ventricles on blood components. Hybrid membranes were aged in a custom-designed endurance bench. Mechanical, physical and chemical properties were not significantly modified from 9 months up to 4 years of aging using a simulating process. Exploration of erosion areas did not show risk of oil diffusion through the membrane. Blood contacting materials in the ventricular cavities were subcutaneously implanted in Wistar rats for 30 days as a model for calcification and demonstrated that the in-house anti-calcification pretreatment with Formaldehyde-Ethanol-Tween 80 was able to significantly reduce the calcium concentration from 132 μg/mg to 4.42 μg/mg (p < 0.001). Hemodynamic simulations with a computational model were used to reproduce shear stress in left and right ventricles and no significant stress was able to trigger hemolysis, platelet activation nor degradation of the von Willebrand factor multimers. Moreover, explanted hybrid membranes from patients included in the feasibility clinical study were analyzed confirming preclinical results with the absence of significant membrane calcification. At last, blood plasma bank analysis from the four patients implanted with C-TAH during the feasibility study showed no residual glutaraldehyde increase in plasma and confirmed hemodynamic simulation-based results with the absence of hemolysis and platelet activation associated with normal levels of plasma free hemoglobin and platelet microparticles after C-TAH implantation. These results on mechanical aging, calcification model and hemodynamic simulations predicted the safety of the hybrid membrane used in the C-TAH, and were confirmed in the feasibility study.

Effect of hemodialysis on extracellular vesicles and circulating submicron particles

BackgroundAlthough hemodialysis is a highly effective treatment for diffusive clearance of low molecular weight uremic toxins, its effect on circulating extracellular vesicles and submicron particles is less clear. The purpose of this study was to examine the impact of hemodialysis on circulating levels of submicron particles.MethodsPlasma samples from patients were collected immediately before and after the mid-week hemodialysis session. Total submicron particles were assessed by nanoparticle tracking analysis and levels of endothelial (CD144+), platelet (CD41+), leukocyte (CD45+), and total (Annexin V+) membrane microparticles (MPs) were assessed by flow cytometry.ResultsTotal submicron particle number was significantly lower post-dialysis with reductions in particles < 40 nm, 40–100 nm, and 100–1000 nm in size. Circulating annexin V+ MPs, platelet MPs, leukocyte MPs, and endothelial MPs were all reduced following dialysis. Assessment of protein markers suggested that extracellular vesicles were not present in the dialysate, but rather adsorbed to the dialysis membrane.ConclusionsIn summary, hemodialysis is associated with reductions in circulating submicron particles including membrane MPs. Accordingly, there may be significant interdialytic variation in circulating submicron particles. Investigators interested in measuring extracellular vesicles in patients undergoing hemodialysis should therefore carefully consider the timing of biosampling.

Analysis of apoptotic, platelet-derived, endothelial-derived, and tissue factor-positive microparticles of children with acute lymphoblastic leukemia during induction therapy.

Thromboembolism is one of the most common complications during induction therapy of pediatric acute lymphoblastic leukemia (ALL). Procoagulant microparticles in the circulation may cause thromboembolic events. The aim of our study was to determine the levels of apoptotic, platelet-derived, endothelial-derived, and tissue factor-positive microparticles of children with ALL at diagnosis and during induction therapy. Sixteen precursor B-cell ALL cases and 30 healthy children between 1 and 18 years of age were included. Microparticle levels were analyzed from peripheral blood samples at initial diagnosis, on days 12 and 13 (before and after the first L-asparaginase administration), and on day 33 of ALL-BFM 2000 treatment protocol. Microparticle levels were analyzed by using flow cytometry. At initial diagnosis, platelet, endothelial-derived, and tissue factor-positive microparticle levels were significantly high in children with ALL. They increased significantly after prednisone and L-asparaginase administration. Apoptotic microparticle levels were not elevated at diagnosis, but remained high during all induction therapy period. None of the patients had evidence of thromboembolism during induction therapy. Our study demonstrated that children with ALL have increased levels of apoptotic, platelet-derived, endothelial-derived, and tissue factor-positive microprticles during induction therapy. Further studies are needed in larger groups of patients in order to evaluate the risk of elevated microprticles for development of thromboembolism during induction therapy period in children with ALL.

Platelet Microparticles: A Tool to Predict Infarction Area in Rats

Background: Whether there is a quantitative correlation between platelet microparticles (PMPs)/calpain and infarction area is still unclear. Whether present antiplatelet agents can improve myocardial infarction by influencing PMPs need to be revealed. The object of our study was to answer those questions. Methods: Male Wistar rats were used for all studies. All rats were randomly divided into five groups: sham-operated group, myocardial infarction group (blank control group), aspirin intervention group, aspirin combined with clopidogrel intervention group, and aspirin combined with ticagrelor intervention group. Venous blood and hearts were collected at day 7 following MI. ELISA was applied to detect PMPs level. Infarction size was determined by TTC staining method. The comparisons of multiple means were tested with analysis of variance. And the two–two comparisons among the means were done by Student-Newman-Keuls and LSD method. Results: PMPs level and infarction area did not differ between aspirin combined with clopidogrel intervention group and aspirin combined with ticagrelor intervention group. However, significant differences were detected between any two other groups. PMPs were decreased more in dual antiplatelet intervention group. Pearson correlation analysis showed a strong correlation between PMPs and infarction area (r = 0.90) as well as calpain 10 and infarction area (r = 0.84). We created a regression model: y = 4.61 + 0.28*x (y: infarction area, x: PMPs) to assess myocardial infarction area by PMPs level. Conclusions: Antiplatelet agents may decrease infarction areas by modifying PMPs. There was a strong correlation between PMPs and infarction area. Therefore, PMPs could be used as a tool to assess infarction area.

Platelet microparticles level in juvenile idiopathic arthritis: A pediatric population-based cross-sectional study in a tertiary care center

Background: Platelet-derived microparticles (PMPs) are small vesicles that are released from the plasma membrane upon platelet activation, which are then involved in haemostasis, vascular health, and have recently been shown to be intimately involved in immune responses. Aims and Objectives: We aimed to evaluate the level of plasma Platelet-derived micro particles in children with JIA and to assess the relationship between PMP levels and disease activity in JIA. Materials and Methods: Children with JIA who fulfilled the International League of Associations for Rheumatology (ILAR) classification criteria for juvenile idiopathic arthritis were included. They were categorised into active disease group and inactive disease as assessed by Wallace criteria. Samples were run in Navios flow cytometer (Beckman Coulter). Platelet microparticles were identified by MPs positive for both Annexin V and CD41 antibodies. Results: Out of 26 children with JIA, 12 had active disease group and 14 had inactive disease as assessed by Wallace criteria. Mean PMP level was 83507 cells/μl and 34904 cells/μl in active and inactive disease respectively (P = 0.06). There was no significant correlation between PMP and CRP levels (P = 0.75 and r = 0 .102) or PMP and ESR levels (P = 0.56 and r = -0.186) in JIA children with active disease. Conclusion: PMP levels were significantly elevated in disease activity of JIA and could represent a new biomarker reflecting the state of cell activation in JIA. PMP role in the inflammatory processes needs to be further elucidated.

Cell Derived Microparticles (MP) in Different Phases of Thrombotic Thrombocytopenic Purpura (TTP) and Effect of Exchange Plasmapheresis (EPP) on Their Profiles

BACKGROUND: Cell derived MPs are small membrane vesicles released during cell activation or apoptosis. They express an inside-out membrane, which exposes the negatively charged phospholipid layer outside, allowing clotting factors to be anchored and generate thrombin. Among various species of MPs, RMP (red cell MPs), PMP (platelet MPs), LMP (leukocytes), EMP (endothelial cells) are of special interest. They play an important role in hemostasis, thromboses, and inflammation. MPs mirror early injury of parent cells and are sensitive early biomarkers of underlying disorders.TTP is a microangiopathy mediated by antibody-induced depletion of ADAMTS13, a von Willebrand factor-cleaving protease. Endothelial injury promotes platelet clumping and formation of platelet rich microthrombi in the microcirculation. Subsequent platelet sequestration leads to impaired microcirculation, thrombocytopenia and red cell fragmentation with a microangiopathic hemolytic anemia. Exchange plasmapheresis (EPP) is the standard therapy. It removes antibodies to ADAMTS13, replacing it with ADAMTS13 rich plasma. It is possible that EPP removes thrombogenic MPs to improve the clinical course of TTP. In this study, we investigated MP profiles in active and remission phase of TTP and the effect of EPP on MP profiles. We also aimed to determine if MP profiles may be a useful measure for monitoring clinical course and tracking progress of therapy.METHODS: A retrospective study was conducted evaluating MP assays in patients with TTP. MP profiles were reviewed in acute and remission phases of TTP. Acute phase was defined as thrombocytopenia, clinical evidence of microangiopathy and hemolytic anemia and low ADAMTS13 activity. Remission was defined as sustained normalization of laboratory parameters and no further microangiopathy for at least one month. Patients were studied longitudinally, with MP assays before and after EPP. EMP were measured by CD31+/CD42b− (EMP31), CD62E+ (EMP62); PMP by CD31+/CD42b+ (PMP42) and CD41+ (PMP41). All were measured in platelet-poor plasma by flow cytometry. All MP data are presented in units of x105/µL. The differences in MP patterns among TTP patients in active and remission phases of disease, as well as the effect of EPP on MP profiles were assessed.RESULTS: Among 20 patients with TTP, 8 (40%) were in acute phase and 12 (60%) in remission. An average of 10.7 EPP were performed. The average platelet count prior to EPP was 50.6x103/µL, which increased to 248 x103/µL following the last EPP. ADAMTS13 activity was generally <10% at the onset.For patients in the acute phase, PMPs were low: PMP41 0.32 (±0.11), PMP42 3.11 (±2.56), but consistently increased following EPP to the point of statistical significance at the last treatment: PMP41 1.26 (±0.71), p=0.005 and PMP42 7.65 (±5.03), p=0.039. Moreover, levels were higher in the remission phase for both PMPs, but only statistically significant for PMP41: 1.39, p=0.034.Conversely, EMP62E was initially elevated on presentation, but declined with successive EPP: 6.07 (±3.02) prior to initiation of EPP to 4.5 (±3.1) upon the last day of EPP. Furthermore, this pattern continued into remission, with EMP62E of 2.72 (±2.11), p=0.009. Similar to the trend in EMP62E, RMP was elevated in the acute phase (25.1 [±18.5]) before steadily declining with EPP (19.65 [±12.53]). This progressive drop in RMP persisted for those in remission phase of TTP 10 (±5.42), p=0.015.DISCUSSION/CONCLUSION: Taken collectively, cell derived MP's were found to reflect disease activity and response to therapy.There was a linear correlation between PMP levels and platelet count. Low PMPs in the acute phase reflects the initial thrombocytopenia characteristic of TTP. When EPP was initiated, the disease improved and there was a rise in PMP which mirrored the rise in platelets. This rise was sustained in the remission phase.Conversely, EMP62E and RMP are increased in acute phase TTP due to endothelial activation, microthrombi deposition and red cell fragmentation. After EPP the disease is quiescent, hence the progressive decline of EMP62E and RMP.These results show promising utility of cell derived MP profiles as a clinical tool to surveil chronic TTP patients and to predict disease relapse at an early stage. After EPP is initiated, change in MP Profiles may be used to monitor response to therapy and determine the appropriate time to wean EPP. DisclosuresNo relevant conflicts of interest to declare.

Platelet microparticles contribute to aortic vascular endothelial injury in diabetes via the mTORC1 pathway.

Platelet microparticles (PMPs) are closely associated with diabetic macrovascular complications. The present study aimed to investigate the effects of PMPs in diabetes on aortic vascular endothelial injury and to explore the underlying mechanisms. Peritoneal injection of streptozotocin was used to generate a diabetic rat model in vivo, and human umbilical vein endothelial cells (HUVECs) treated with PMPs were used in vitro. PMP levels in the circulation and aorta tissues were time-dependently increased in streptozotocin-induced diabetic rats at weeks 4, 8, and 12 (P < 0.05). Aspirin significantly inhibited the PMP levels at each time point (P < 0.05). In diabetic rats, the endothelial nitric oxide levels were decreased significantly combined with increased endothelial permeability. PMPs were internalized by HUVECs and primarily accumulated around the nuclei. PMPs inhibited endothelial nitric oxide levels to about 50% and caused approximately twofold increase in reactive oxygen species production. Furthermore, PMPs significantly decreased the endothelial glycocalyx area and expression levels of glypican-1 and occludin (P < 0.05). Interestingly, the PMP-induced endothelial injuries were prevented by raptor siRNA and rapamycin. In conclusion, increased PMPs levels contribute to aortic vascular endothelial injuries in diabetes through activating the mTORC1 pathway.

A11518 Hemodialysis reduces levels of circulating microparticles in individuals with hypertension

Objectives: Individuals with end stage kidney disease (ESKD) and hypertension are at increased risk of cardiovascular complications. Circulating microparticles (MPs) are putative biomarkers of vascular injury and circulating levels of MPs are increased in both hypertension and ESKD. The purpose of this study was to determine the effect of hemodialysis on circulating MPs in individuals with and without hypertension. Methods: 37 individuals with hypertension and 17 normotensive individuals on conventional 3 times weekly hemodialysis were studied (age 61 ± 2 years). Plasma samples from were collected in a non-fasting state immediately before and after hemodialysis at the mid-week hemodialysis session. An FX 800 hemodiafilter (Fresenius Medical Care) was used. Levels of endothelial (CD144 + ), platelet (CD41 + ), leukocyte (CD45 + ) and total (Annexin V + ) microparticles were assessed by flow cytometry. Results: Across all participants, significant reductions were seen in circulating annexin V + ve MPs (3x107vs1.6x107, P < 0.01), platelet MPs (1.8x107vs1.0x107, P < 0.001), leukocyte microparticles (2.4x106vs 1.8x106, P < 0.05), and endothelial MPs (8.4x105vs5.9x105, P < 0.01). There were no differences in pre-dialysis or post-dialysis MP levels (total, endothelial, leukocyte, or platelet) between hypertensive and non-hypertensive individuals and the inter-dialytic increases in MP levels were not significantly different between hypertensive and non-hypertensive individuals. Conclusion: Hemodialysis is associated with reductions in circulating platelet, leukocyte, and endothelial MPs. There may be significant interdialytic variation in MP levels and this should be an important consideration in studying MPs as biomarkers in ESKD. The presence of hypertension does not appear to alter inter-dialytic increases in circulating MPs.

Analytical Considerations in Nanoscale Flow Cytometry of Extracellular Vesicles to Achieve Data Linearity.

Platelet microparticles (PMPs) and their abundance in the blood are a prognostic biomarker in thrombotic disorders and cancer. Nanoscale flow cytometry (nFC) is ideal for high-throughput analysis of PMPs but these clinical assays have not been developed previously. This article demonstrates that nFC is a suitable technology to enumerate PMPs present in plasma samples in a clinical setting. nFC was performed using the Apogee A50-Micro instrument. Instrument settings and acquisition parameters were developed with the use of fluorescent beads and plasma samples. Sample preparation and handling was also optimized. nFC allows for linear detection of particles between approximately 200 and 1,000 nm based on calibration beads and was dependent on dilution factor and flow rate. Linearity in event analysis as samples became more diluted was lost when events approximately 100 nm were gated while linearity was maintained despite dilution of sample in events larger than 200 nm in diameter. Higher flow rates lead to an under-estimation of events analysed per microlitre of analyte and this was more pronounced when plasma samples were not diluted more than 1/20×. nFC offers multi-parametric analysis of PMPs when optimal calibration of acquisition and sample processing settings is performed. Analysis of plasmas from metastatic prostate cancer patients and leukaemia patients revealed that PMP levels were larger than 100 nm and were equally abundant in patients that responded to or failed androgen deprivation therapy or between patients representing different stages of leukaemia.