Number Of Platelet-derived Microparticles Research Articles

Platelet Exosome-Derived miR-223-3p Regulates Pyroptosis in the Cell Model of Sepsis-Induced Acute Renal Injury by Targeting Mediates NLRP3.

The present study investigated the roles and mechanisms of platelet-derived exosomes in sepsis-induced acute renal injury. The blood samples of septic patients and healthy controls were collected for clinical examination. The plasma levels of miR-223-3p and NLRP3 mRNA were analyzed by qRT-PCR and the serum IL-1β and creatinine levels were quantified by enzyme-linked immunosorbent assay (ELISA). C57BL/6 mice injected with LPS (lipopolysaccharide) were employed as the animal model for sepsis-induced acute renal injury. Human coronary artery endothelial cells (HCAECs) were treated with TNF-α as a cellular model for sepsis-induced endothelial damages. The number of PMP (platelet-derived microparticles) in patients with sepsis was increased. The level of miR-223-3p in the platelet exosomes isolated from the serum sample in patients with sepsis was significantly lower than that of the healthy controls. The level of miR-223-3p was also decreased in the platelet exosomes of mouse model with sepsis-induced acute renal injury. Downregulating miR-223-3p promoted sepsis-induced acute renal injury in mice model, while the administration of miR-223-3p reduced the inflammation in endothelial cells of sepsis-induced acute renal injury. NLRP3 (NLR Family Pyrin Domain Containing 3) was identified as one target of miR-223-3p in the platelet exosomes of sepsis-induced acute kidney injury. miR-223-3p attenuated NLRP3-induced pyroptosis in endothelial cell model of sepsis-induced acute kidney injury. Our data suggest that platelet exosome-derived miR-223-3p negatively regulates NLRP3-dependent inflammasome to suppress pyroptosis in endothelial cells. Decreased miR-223-3p expression promotes the inflammation in sepsis-induced acute renal injury. Targeting miR-223-3p may be developed into a therapeutic approach for sepsis-induced acute renal injury.

Rivaroxaban compared with low-dose aspirin in individuals with type 2 diabetes and high cardiovascular risk: a randomised trial to assess effects on endothelial function, platelet activation and vascular biomarkers

Aims/hypothesisIndividuals with type 2 diabetes mellitus and subclinical inflammation have stimulated coagulation, activated platelets and endothelial dysfunction. Recent studies with the direct factor Xa inhibitor rivaroxaban in combination with low-dose aspirin demonstrated a significant reduction of major cardiovascular events, especially in individuals with type 2 diabetes and proven cardiovascular disease. Therefore, we asked the question of whether treatment with rivaroxaban could influence endothelial function, arterial stiffness and platelet activation.MethodsWe conducted a multi-centre, prospective, randomised, open-label trial in 179 participants with type 2 diabetes (duration 2–20 years), subclinical inflammation (high-sensitivity C-reactive protein 2–10 mg/l) and at least two traits of the metabolic syndrome to compare the effects of the direct factor Xa inhibitor rivaroxaban (5 mg twice daily) vs aspirin (100 mg every day) on endothelial function (assessed by forearm occlusion plethysmography), skin blood flow (assessed by laser-Doppler fluxmetry), arterial stiffness (assessed by pulse wave velocity) and serum biomarkers of endothelial function and inflammation. Furthermore, we investigated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in platelets, the concentration of platelet-derived microparticles (PMPs) and the effects of isolated PMPs on HUVEC proliferation in vitro.ResultsRivaroxaban treatment for 20 weeks (n = 89) resulted in a significant improvement of post-ischaemic forearm blood flow (3.6 ± 4.7 vs 1.0 ± 5.2 ml/100 ml, p = 0.004), a numerically increased skin blood flow and reduced soluble P-Selectin plasma level vs aspirin. We did not find significant differences of arterial stiffness or further biomarkers. Neither rivaroxaban nor aspirin influenced VASP phosphorylation of platelets. The number of PMPs increased significantly with both rivaroxaban (365.2 ± 372.1 vs 237.4 ± 157.1 μl−1, p = 0.005) and aspirin (266.0 ± 212.7 vs 201.7 ± 162.7 μl−1, p = 0.021). PMPs of rivaroxaban-treated participants stimulated HUVEC proliferation in vitro compared with aspirin. Rivaroxaban was associated with a higher number of bleeding events.Conclusions/interpretationOur findings indicate that the direct factor Xa inhibitor rivaroxaban improved endothelial function in participants with type 2 diabetes and subclinical inflammation but also increased the risk of bleeding.Trial registration:ClinicalTrials.gov NCT02164578.FundingThe study was supported by a research grant from Bayer Vital AG, Germany.Graphical abstract

Platelet-Derived Procoagulant Microparticles as Blood-based Biomarker of Breast Cancer.

Objective:Breast cancer is the main cause of cancer death in women worldwide. Elevated plasma levels of circulating cell-derived microparticles (MPs) have been reported in various types of cancer, including breast cancer, with the ability to mediate inflammation and thrombosis. Microparticles are bioactive agents, and it has been suggested that MPs can be used as a diagnostic, prognostic, or therapeutic biomarker in various diseases. The aim of this study was to investigate the levels of platelet-derived MPs (PMPs) in breast cancer patients. Materials and Methods:In this case-control study, 30 patients with breast cancer and 20 normal subjects were sampled after obtaining written consent. MPs were isolated from blood samples by centrifugation technique. CD42b and annexin V markers were used respectively for counting PMPs and procoagulant MPs with flow cytometry. Results:Flow cytometry results showed that the number of PMPs and procoagulant annexin V positive MPs was significantly higher in the breast cancer patients than normal subjects (p<0.001). The number of the annexin V MPs differed significantly in patients with high tumor size (T2) compared to the patients with low tumor size (T1) and controls (p<0.001). Significant and positive correlations were found between PMP levels and tissue-based biomarkers, tumor grading, and distant metastasis (P<0.05). Tumor histological type did not correlate with the numbers of PMPs (p=0.065). Conclusion:Increased levels of PMPs and activity in terms of hemostasis and having a positive and significant relationship with tumor grading and metastasis may indicate the effective role of PMPs in the pathogenesis and prognosis of breast cancer.

Early Detection of Negative Smoking Impacts: Vascular Adaptation Deviation Based on Quantification of Circulated Endothelial Activation Markers.

IntroductionSmoking can cause vascular damage in the form of an inflammatory reaction characterized by endothelial activation. Endothelial activation forms a pathological adaptation pattern so that it can induce the atherogenesis process. Several markers, such as E-selectin, platelet-derived micro particles (PMPs) and hematopoietic stem cell (HSC) can identify the activation of endothelial in circulating blood. Therefore, the deviation of vascular adaptation due to smoking can be detected early through the feedback mechanism between E-selectin, PMPs, and HSC.PurposeThis study aims to analyze the initial picture of the negative impact of smoking on vascular adaptation by measuring E-selectin, PMPs, and HSC in the peripheral blood circulation. Participant criteria and methods: Peripheral blood samples (5 mL) were taken from each participant, both the smoking group (n = 30) and the non-smoker group (n = 31) to obtain peripheral blood mononuclear cells (PBMNC). PBMNC was isolated using ficoll-based gradient centrifugation. The flow cytometry assay method used to measure the E-selectin, PMPs and hematopoietic stem cells.ResultsThe mean of circulating E-selectin in smokers was higher than that of non-smokers. On the other hand, the average number of PMPs and HSCs in smokers was lower than non-smokers.ConclusionSmoking increases the risk of accelerated vascular block formation, as indicated by an increase in the amount of circulating E-selectin. The increase in E-selectin in the blood vessels mediates the increased adhesion of PMPs in the vascular area so that the number of circulating PMPs in smokers decreases. The decrease in circulating PMPs decreases the signal of vascular repair, which is characterized by a decline in the number of HSCs.

The Effects of the Combined Argatroban/Nitric Oxide-Releasing Polymer on Platelet Microparticle-Induced Thrombogenicity in Coated Extracorporeal Circuits.

Clotting, anticoagulation, platelet consumption, and poor platelet function are major factors in clinical extracorporeal circulation (ECC). We have shown that nitric oxide-releasing (NOReL) coatings prevent thrombosis in a rabbit model of ECC without systemic anticoagulation. Nitric oxide-releasing prevents platelet adhesion and activation, resulting in preserved platelet count and function. Previous work has shown that activated platelets form platelet-derived microparticles (PMPs). These experiments were designed to determine if PMPs can identify platelet function during ECC. The objective of this study is to investigate the effects of NOReL on platelet activation and PMP formation during ECC. Uncoated ECCs, including with and without systemic heparin, and NOReL-coated ECCs, including DBHD/N2O2 and argatroban (AG)/DBHD/N2O2-coated ECCs without systemic heparin, were tested in a 4-hour rabbit thrombogenicity model. Before and after ECC exposure, platelets were stimulated with collagen, and PMPs were measured using flow cytometry. The uncoated ECCs clotted within the first hour, while the NOReL-coated ECCs circulated for 4 hours. During pre-ECC blood exposure, platelets stimulated with collagen produced PMPs. With post-ECC exposure, platelets from uncoated circuits generated less PMPs than baseline (mean ± SDs: 23246 ± 3611 baseline vs. 1300 ± 523 uncoated post circuit, p = 0.018) when stimulated with collagen. However, platelets from the AG/DBHD/N2O2-coated ECCs generated a greater number of PMPs as baseline values (23246 ± 3611 baseline vs. 37040 ± 3263 AG/DBHD/N2O2 post 4 hours circuit, p = 0.023). Blood exposure during ECC results in platelet activation and clotting in uncoated ECCs. The remaining circulating platelets have lost function, as demonstrated by the low PMP formation in response to collagen. AG/DBHD/N2O2-coated ECCs prevented significant platelet activation and clotting, while DBHD/N2O2 trended towards prevention of platelet activation. In addition, function of the circulating platelets was preserved, as demonstrated by PMP formation in response to collagen. These results indicate that PMPs may be an important measure of platelet activation during ECC. Platelet-derived microparticles may provide a simplified way to measure platelet function during clinical ECC.

Rapid isolation and purification of functional platelet mitochondria using a discontinuous Percoll gradient

The isolation of mitochondria is gaining importance in experimental and clinical laboratory settings. The mitochondrion is known as the powerhouse of the cell as it produces the energy to power most cellular functions but is also involved in many cellular processes. Of interest, mitochondria and mitochondrial components (i.e. circular DNA, N-formylated peptides, cardiolipin) have been involved in several human inflammatory pathologies, such as cancer, Alzheimer’s disease, Parkinson’s disease, and rheumatoid arthritis. Therefore, stringent methods of isolation and purification of mitochondria are of the utmost importance in assessing mitochondrial-related diseases. While several mitochondrial isolation methods have been previously published, these techniques are aimed at yielding mitochondria from cells types other than platelets. In addition, little information is known on the number of platelet-derived microparticles that can contaminate the mitochondrial preparation or even the overall quality and integrity of the mitochondria. In this project, we provide an alternate purification method yielding mitochondria of high purity and integrity from human platelets. Using human platelets, flow cytometry and transmission electron microscopy experiments were performed to demonstrate that the Percoll gradient method yielded significantly purified mitochondria by removing platelet membrane debris. Mitochondrial respiration following the substrate-uncoupler-inhibitor-titration (SUIT) protocol was similar in both the purified and crude mitochondrial extraction methods. Finally, the cytochrome c effect and JC-1 staining did not exhibit a significant difference between the two methods, suggesting that the mitochondrial integrity was not affected. Our study suggests that the Percoll discontinuous gradient purifies viable platelet-derived mitochondria by removing platelet-derived debris, including microparticles, therefore confirming that this isolation method is ideal for studying the downstream effects of intact mitochondria in mitochondrial-related diseases.

Differences in levels of platelet-derived microparticles in platelet components prepared using the platelet rich plasma, buffy coat, and apheresis procedures

BackgroundThere has been an increased interest in platelet-derived microparticles (PMPs) in transfusion medicine. Little is known about PMP status during the preparation of platelet concentrates for transfusion. AimThe aim of this study is to compare the PMP levels in platelet components prepared using the buffy coat (BC), platelet-rich plasma platelet concentrate (PRP-PC), and apheresis (AP) processes. MethodsPlatelet components were prepared using the PRP-PC and BC processes. Apheresis platelets were prepared using the Trima Accel and Amicus instruments. The samples were incubated with annexin A5-FITC, CD41-PE, and CD62P-APC. At day 1 after processing, the PMPs and activated platelets were determined using flow cytometry. ResultsBoth the percentage and number of PMPs were higher in platelet components prepared using the Amicus instrument (2.6±1.8, 32802±19036 particles/μL) than in platelet components prepared using the Trima Accel instrument (0.5±0.4, 7568±5298 particles/μL), BC (1.2±0.6, 12,920±6426 particles/μL), and PRP-PC (0.9±0.6, 10731±5514 particles/μL). Both the percentage and number of activated platelets were higher in platelet components prepared using the Amicus instrument (33.2±13.9, 427553±196965 cells/μL) than in platelet components prepared using the Trima Accel instrument (16.2±6.1, 211209±87706 cells/μL), BC (12.9±3.2, 140624±41003 cells/μL), and PRP-PC (21.1±6.3, 265210±86257 cells/μL). ConclusionsThe study suggests high variability of PMPs and activated platelets in platelet components prepared using different processes. This result may be important in validating the instruments involved in platelet blood collection and processing.

Effect of intracoronary tirofiban bolus administration on platelet-derived microparticles and short-term clinical benefit in patients with acute ST-segment elevation myocardial infarction

Objective To investigate the effect of intracoronary tirofiban bolus administration on platelet-derived microparticles (PMPs) and its correlation with the short-term clinical benefit in patients with acute ST-segment elevation myocardial infarction (ASTEMI) undergoing emergency percutaneous coronary intervention (PCI). Methods A total of 90 patients with ASTEMI undergoing emergency PCI were selected and randomized into the intracoronary group (intracoronary tirofiban 10.00 μg/kg bolus within 1-3 min followed by intravenous continuous infusion at 0.15 μg·kg-1·min-1 for 36 h, n=30), intravenous group (intravenous tirofiban 10.00 μg/kg bolus within 1-3 min followed by intravenous continuous infusion at 0.15 μg·kg-1·min-1 for 36 h, n=30) and control group (without tirofiban administration, n=30). The 3 ml blood samples from coronary artery were obtained before and 10 min after tirofiban infusion. The 3 ml blood samples from radial artery were collected 24 hours after tirofiban infusion and 12 hours after drug withdrawal. The counts of PMPs were analyzed by flow cytometry. The thrombolysis in myocardial infarction (TIMI) flow grade classification and TIMI Myocardial Perfusion Grade in the culprit blood vessel after PCI, and the incidences of bleeding and major adverse cardiac events (MACE) within 30 days after surgery were recorded. Results There was no significant difference in baseline of PMPs among intracoronary group, intravenous group and control group (all P>0.05). The level of PMPs was decreased in the intracoronary and intravenous group as compared with the control group 〔(3.6±2.3)%, (5.1±2.7)% vs. (6.7±3.2)%, P 0.05), and PMPs levels were lower in intracoronary and intravenous group than in control group (both P 0.05). Immediately after PCI, the TIMI flow grade and TIMI myocardial perfusion grade in the culprit blood vessel in intracoronary group were superior to those in the intravenous group and control group (P 0.05). Conclusions The intracoronary versus intravenous tirofiban administration can effectively and immediately reduce the number of PMPs in patients with acute ST-segment elevation myocardial infarction undergoing emergency interventional treatment, quickly inhibit the activated platelets, and decrease the total major adverse cardiovascular events without increasing the risk of bleeding. Key words: Myocardial infarction; Blood platelets; Angioplasty, intervention, percutaneous coronary

Microparticle source and tissue factor expression in pregnancy.

Microparticles (MPs) bearing tissue factor (TF) are potent activators of the coagulation system. To investigate whether MPs originating from platelets or trophoblast cells contribute to coagulation changes in pregnancy, we aimed to characterize whether pregnancy, labor, and delivery are associated with changes in the origin and composition of circulating maternal MPs. We performed a prospective cohort study. Blood samples were collected in 20 non-pregnant women, 20 term pregnant women not in labor, and 20 term pregnant women in labor. Two samples were collected in the pregnant groups, the first prior to delivery and the second 1h after delivery. MPs from platelets and trophoblasts and MPs expressing TF were identified using flow cytometry. Comparisons were made among the non-pregnant and pregnant groups, non-laboring and laboring groups, and predelivery and postdelivery values within the pregnant groups. Pregnancy was not associated with changes in MP origin or number of TF-expressing MPs. Neither labor nor delivery was associated with changes in the percentage of trophoblast-derived MPs in the pregnant groups. The percentage of platelet-derived MPs among laboring women increased after delivery (8.5 vs. 20.5%, p = 0.02), although there was no difference with delivery in the non-laboring group. TF expression was not associated with delivery in either laboring or non-laboring women. In conclusion, pregnancy was not associated with changes in cell origin of circulating maternal MPs or in the number of TF-expressing MPs. However, labor and delivery appear to be associated with an increase in the number of platelet-derived MPs.

Graphene oxide-based biosensor for detection of platelet-derived microparticles: A potential tool for thrombus risk identification

We report here design of a graphene oxide-based electrochemical biosensor for detection of platelet-derived microparticles (PMPs), a major risk factor for arterial pro-thrombotic pathologies like acute myocardial infarction and stroke. Electrodes were fabricated with immobilized layers of graphene oxide and a specific antibody targeted against active conformation of integrin αIIbβ3 on PMP surface. Results showed progressive rise in impedance in Nyquist plots with increasing number of PMPs in analyte. The sensor was highly specific for PMPs and did not identify microparticles originating from other cells. Blood obtained from patients diagnosed with acute myocardial infarction exhibited significantly higher values of impedance, consistent with larger number of circulating PMPs in these patients, as compared to samples from healthy individuals, thus validating biosensor as a specific, sensitive, label-free and cost-effective tool for rapid point-of-care detection of PMPs at bedside. Our biosensor is most ideal for mass population screening programs at periphery-level healthcare units with limited resources. It is aimed at early detection of individuals having higher imminent cardiovascular risk, as well as for routine analysis, which in turn would contribute to better management and survival of screened ‘high-risk’ subjects.

Circulating microparticles in severe preeclampsia

The present study aimed to evaluate microparticles (MPs) from different sources in women with severe preeclampsia (PE) compared with normotensive pregnant women and non-pregnant women. This case–control study evaluated 28 pregnant women with severe PE, 30 normotensive pregnant women, and 29 non-pregnant women. MPs from neutrophils, endothelial cells, monocytes, platelets, leukocytes, erythrocytes, and syncytiotrophoblast were evaluated using flow cytometry. A higher total number of MPs were observed in women with severe PE compared with normotensive pregnant women and non-pregnant women (P=0.004 and P=0.001, respectively). MPs derived from erythrocytes were increased in women with severe PE compared with normotensive pregnant women (P=0.002). A trend towards association was observed between platelet count and the number of MPs derived from platelets (P=0.09) in severe PE group. A positive correlation was also found between the number of endothelial cell-derived MPs and the number of platelet-derived MPs, leukocyte-derived MPs, neutrophil-derived MPs, and lymphocyte-derived MPs (P<0.05) in severe PE pregnant women. MP counts can be increased in severe PE, and erythrocyte and endothelial cell-derived MPs seem to be associated to severe PE.

Increased levels of platelet activation markers are positively associated with carotid wall thickness and other atherosclerotic risk factors in obese patients

The role of platelets in the development of atherosclerosis and obesity-related prothrombotic state is still under investigation. In this cross-sectional cohort study, we measured the levels of different platelet activation markers and evaluated their relationship with carotid intima-media thickness (IMT) along with other atherosclerotic risk factors in obese patients with or without atherosclerotic co-morbidities. We enrolled 154 obese patients, including 98 with either hypertension, type 2 diabetes mellitus or dyslipidaemia, 56 without these co-morbidities and 62 age- and sex-matched healthy controls. Platelet P-selectin expression and the number of platelet-derived microparticles (PMPs) were measured by flow cytometry; soluble P-selectin levels were analysed by ELISA and Thr715Pro P-selectin polymorphism was determined by PCR-RFLP. Carotid IMT was examined by ultrasonography. The levels of platelet activation parameters were significantly elevated in all obese subjects with increased carotid IMT compared to healthy controls. There was no effect of Thr715Pro genotype on soluble P-selectin levels in obese individuals contrary to normal subjects. Significant and positive association was revealed between carotid IMT and platelet P-selectin (p<0.0001), soluble P-selectin (p=0.039) and PMP (p=0.0001) levels. After adjusting for multiple variables, independent association was found between soluble P-selectin and fibrinogen (p=0.007), PMP levels and body mass index (p<0.0001) as well as platelet P-selectin and carotid IMT (p=0.012) plus plasminogen activator inhibitor-1 (p=0.009). In conclusion, P-selectin and PMP levels showed positive associations with abnormal carotid IMT and other risk factors in obesity suggesting a critical role of enhanced platelet reactivity in atherosclerotic wall alteration.

Elevated Circulating Platelet-Derived Microparticles in Ph1 Negative Chronic Myeloproliferative Disorders.

Abstract 1906Poster Board I-929Microparticles (MP) are plasma membrane vesicles bearing potent procoagulant proteins and released into the circulation by various blood and endothelial cells during cellular activation and apoptosis. Their concentration has been shown markedly increased in vascular and thromboembolic diseases and in several types of cancer. Essential Thrombocythemia (ET), Polycythemia Vera (PV) and Primary Myelofibrosis (PM) are Bcr-Abl-negative chronic myeloproliferative disorders (MPD) with common biological and clinical features, especially thrombocytosis and more or less effective megakaryocytic proliferation. ET, PV and PMF are also characterized by an increased risk of thrombotic complications. Given the involvement of megacaryocytopoiesis in this group of diseases, we chose to focus on circulating platelet-derived microparticles (PMP) whose count can be established by flow cytometry. Our goals were to look for an increased release of PMP in MPD and if so, try to establish a correlation between thrombotic events recorded retrospectively and PMP level. Patients and methods:Plasma samples were collected from 85 patients with MPD (40 ET, 28 PV, 17 PMF) at any time of the disease course except blastic phase and 31 healthy age-paired controls ; the quantity of PMP (positive for the platelet marker CD41 and the microparticle marker AnnexinV) was measured by flow cytometric analysis using the FC500 cytometer from Beckmann-Coulter™. Pre-analytic and testing procedures complied with the recommendations of the ISTH Standardization Subcommittee and besides, our laboratory participates in a multicenter program to standardize the enumeration of cellular microparticles. Results:The number of PMP is significantly higher in ET, PV and MFP patients (median: 4862 MPP/mL, 3289 MPP/mL and 6114 MPP/mL respectively) than in controls (median 1310 MPP/mL) [p=0.000018, p=0.0013, p=0.0009]. Neither in MPD patients nor in control group PMP correlates with platelet count and appears thus as an independent parameter. ROC method allows to fix a cut-off at 3121 MPP/mL to discriminate MPD from controls. This value has a sensibility of 75 % and a specificity of 94%. Interestingly, the total number of PMP is not different between untreated patients (mean=7246 MPP/mL) and those who received myelosuppressive therapy (mean=6320 MPP/mL). Regarding thrombotic events, we failed to demonstrate any significant difference between patients with previous thrombosis and those without; however, the small size of the series and the retrospective assessment are possible bias. Conclusion:Patients with Phi negative MPDs have a high number of circulating PMP, irrelevant to platelet count or treatment by Hydroxy-urea or aspirin. A role for microparticles in the development of these diseases and in the occurrence of thrombotic complications can be hypothesized but requires further studies -;preferably prospective- on larger cohorts of patients. Disclosures:Charpentier:Schering-Plough: Research Funding.

Lactadherin as a Probe for Phosphatidylserine Exposure and as An Anticoagulant for the Procoagulant Activity in the Study of Stored Platelets.

Abstract 3150Poster Board III-87 BackgroundPhosphatidylserine (PS) can support coagulant reactions. However, it is uncertain how the location and extent of PS exposure to the membranes of stored platelets affect such reactions. We compared annexin V with lactadherin as a way of detecting how of PS exposure influences the procoagulant properties of stored platelets in platelet concentrates (PCs). MethodPS exposure and the relevant procoagulant activity (PCA) of platelets in 5 different PCs were investigated by flow cytometry, confocal microscopy, coagulation time analysis and enzymatic assays. PS exposure was separately measured using annexin V and lactadherin, respectively. ResultsExposure of PS to stored platelets promoted thrombin formation. A progressive increase in PS exposure was detected by flow cytometry. Moreover, using lactadherin, we identifed higher levels of PS exposure on the platelets and platelet-derived microparticles (PMPs) compared to detection using annexin V. The percentage of PS-positive cells was 0.02 % by annexin V versus 0.3 % by lactadherin on day 0, 7.5 % by annexin V versus 12.3 % by lactadherin on day 5, and 29 % by annexin V versus 44.3 % by lactadherin on day 9. Rare microparticles (MPs) were released from fresh platelets, and, the number of PMPs increased approximately 2-fold on day 5 and further progressively increased. Using lactadherin and platelets in the earlier stage of storage, confocal microscopy revealed earlier and localized PS exposure based on plasma membrane staining. For later storage platelets, increased levels of PS-positive platelets and PMPs were clearly detected by both annexin V and lactadherin. Thirty-two nM lactadherin or annexin V prolonged coagulation time 2.4 fold versus 2 fold. The productions of thrombin and intrinsic/extrinsic factor Xase were approximately inhibited 85 % and 60 % by lactadherin and annexin V, respectively. ConclusionPS exposure was localized to the cellular rims, blebbing vesicles and thin elongated filopodia-like areas on banked platelets. Furthermore, lactadherin provides a more accurate measurement of PS exposure and the relevant with PCA, which is an important factor to consider for transfusion medicine. Our findings of elevated PS-positive platelets and PMPs indicate that platelets should not be stored for extended periods of time. DisclosuresNo relevant conflicts of interest to declare.

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.

Portable dynamic light scattering instrument and method for the measurement of blood platelet suspensions

No routine test exists to determine the quality of blood platelet transfusions although every year millions of patients require platelet transfusions to survive cancer chemotherapy, surgery or trauma. A new, portable dynamic light scattering instrument is described that is suitable for the measurement of turbid solutions of large particles under temperature-controlled conditions. The challenges of small sample size, short light path through the sample and accurate temperature control have been solved with a specially designed temperature-controlled sample holder for small diameter, disposable capillaries. Efficient heating and cooling is achieved with Peltier elements in direct contact with the sample capillary. Focusing optical fibres are used for light delivery and collection of scattered light. The practical use of this new technique was shown by the reproducible measurement of latex microspheres and the temperature-induced morphological changes of human blood platelets. The measured parameters for platelet transfusions are platelet size, number of platelet-derived microparticles and the response of platelets to temperature changes. This three-dimensional analysis provides a high degree of confidence for the determination of platelet quality. The experimental data are compared to a matrix and facilitate automated, unbiased quality testing.

P-Selectin- and CD63-Exposing Platelet Microparticles Reflect Platelet Activation in Peripheral Arterial Disease and Myocardial Infarction

Platelet-derived microparticles (PMPs) are generally considered a marker of platelet activation in cardiovascular disease. We studied the extent to which PMP subpopulations parallel platelet activation in vitro and in vivo. Using flow cytometry, we analyzed PMP subpopulations from resting and activated platelets in vitro (n = 6) as well as from plasma samples of patients with stable angina, peripheral arterial disease, or myocardial infarction [non-ST-elevation (NSTEMI) and ST-elevation (STEMI)] and from older, age- and sex-matched and young healthy individuals [n = 10 for all groups except NSTEMI (n = 11)]. Coagulation markers prothrombin fragment F(1 + 2) and thrombin-antithrombin complexes were determined by ELISA. The PMP-associated fraction of soluble (s)P-selectin was estimated by ELISA. In vitro, stimulation of platelets with thrombin receptor-activating peptide (15 micromol/L) or the calcium ionophore A23187 (2.5 micromol/L) increased fractions of both platelets and PMPs exposing P-selectin or CD63 (P <0.001 for all). Whereas the number of PMPs released by A23187-stimulated platelets increased significantly (P <0.001), the number of PMPs released from thrombin receptor-activating peptide-stimulated platelets remained constant (P >0.05). Ex vivo, numbers of circulating PMPs were comparable in all groups. Compared with young persons, P-selectin-exposing PMPs were increased in older persons (P = 0.02) and were further increased in patients with NSTEMI (P = 0.007) and STEMI (P = 0.045). CD63-exposing PMPs were increased in patients with peripheral arterial disease (P = 0.041), NSTEMI (P = 0.001), and STEMI (P = 0.049). Subpopulations exposing P-selectin or CD63 correlated with each other (r = 0.581; P <0.001), but neither correlated with the plasma concentrations of F(1 + 2) or thrombin-antithrombin complexes. The PMP-associated fraction of sP-selectin constituted only 2.2 (4.7)% [mean (SD)] of total sP-selectin. PMP subpopulations reflect platelet activation status better than the total number of PMPs. Increased concentrations of circulating PMP subpopulations are found in aging, and further increases are encountered in peripheral arterial disease and myocardial infarction.