Cell derived microparticles: method development, and clinical and experimental studies

Background Cell derived microparticles (MP) are released from the cell membrane upon activation or apoptosis. They resemble their parent cell by exposing similar proteins or surface receptors. This enables identification of their cellular origin. MP are considered to facilitate cross-talk between cells, and to be involved in coagulation, inflammation and vascular function. Elevated circulating MP have been shown in previous studies. Assessment of MP is, however, difficult due to methodological issues. Aims To evaluate pre-analytical procedures and a flow cytometric method for detection of microparticles. To study the effects of statin treatment and inflammation on phenotype and functional properties of microparticles. Methods and Results In Paper I we describe a flow cytometric method for measurements of platelet derived microparticles (PMP) exposing CD62P or CD142. Mean fluorescence intensity measurements were more reproducible than concentration measurements. The presence of platelet fragments could be detected with the peptide phalloidin. This approach can be used as a quality control of samples. Samples frozen and stored as platelet-free plasma generated lowest number of platelet fragments upon flow cytometric analysis. Using our flow cytometric protocol we found two times higher exposure of CD62P and CD142 on PMP in plasma from type 1-diabetes patients compared to healthy controls. In Paper II and III we investigated the effects of atorvastatin on MP. Nineteen patients with atherothrombotic disease were randomized to treatment with atorvastatin or placebo in a cross-over fashion. Thrombin generation and exposure of CD61, CD62P, CD142 and phosphatidylserine (PS) were assessed on PMP (Paper II). Endothelial derived MP (EMP) were assessed by CD144 or CD144+ CD142+ exposure (Paper III). During atorvastatin treatment both thrombin generation and exposure of CD61, CD62P, and CD142 on PMP decreased. No effect was seen on PS exposure. Furthermore, we demonstrated that MP enhanced thrombin generation through PS and CD142 exposure. Unexpectedly, circulating EMP measured as CD144 or CD144+ CD142+increased significantly during atorvastatin treatment. In Paper IV we investigated and characterized in vivo release of MP from 15 healthy volunteers administered lipopolysaccharide (LPS) in the presence of hydrocortisone with or without inhaled nitric oxide. MP from platelets (CD42a or CD41), endothelial cells (CD144 or CD62E) and monocytes (CD14) were studied. Nuclear content in MP was assessed (SYTO 13 binding) as well as HMGB1 exposure. Irrespective of treatment, LPS led to an increase in numbers of all MP, as well as the number of PMP and monocyte MP positive for anti-HMGB1 and SYTO 13. Conclusions We describe a flow cytometric method to measure MP in plasma, and we demonstrate that MP from platelets and endothelial cells respond differently to statin treatment, reflecting the complexity of MP formation. Furthermore, we show that experimental inflammation leads to elevated circulating MP, and that MP may be a source of extracellular HMGB1. MP may be used as biomarkers, an idea that deserves to be investigated more extensively in future studies

Fibrinolytic Activity of Recombinant Mutant Streptokinase

Background: Streptokinase is a bacterial protein produced by different beta hemolytic streptococci and widely used in thrombolytic treatment. The main disadvantage of using streptokinase is antibody formation which causes allergic reaction to neutralize effects of streptokinase therapy. Aim of this study was investigate of recombinant mutant streptokinase fibrinolytic activity.Materials and Methods: In this study recombinant mutant streptokinase without 42 amino acids from the C terminal region was purified by affinity S-Tag column chromatography and its fibrinolytic activity was studied.Results: The concentration of expressed and purified protein was 10 mg/ml. Its enzyme activity was assayed using zymography, radial caseinolytic activity and fibrin plate test methods and estimated quantitatively by casein digestion method compared to a commercial form.Conclusion: It was found that this product had the more volume and more enzymatic activity

The impact of menopause on sexual function in women and their spouses

Background: The purpose of this study is to evaluate the impact of menopause on sexual function in women and their spouses. Methods: This is a cross-sectional study that was conducted from January 2018 to May 2019 in Rasht (North of Iran). The participants included 215 menopausal women and their spouses. Data were collected using the demographic questionnaire, the Female Sexual Function Index (FSFI) questionnaire, and the International Index of Erectile Function (IIEF) questionnaire. Results: On the basis of the FSFI and IIEF scores, 36.28% (78/215) women reported female sexual dysfunction (FSD) and 17.2% (37/215) men reported erectile dysfunction (ED) with 8.37% (N = 18) being mild, 5.58% (N = 12) mild to moderate, and 3.25% (N = 7) moderate ED. After adjusting differences in the female age distribution, the total score and scores of the IIEF subscales were also not significantly lower in the spouses of women with FSD than women without FSD. Conclusion: Although, significant correlations between male erectile function and menopausal female sexual function have not identified; but, low scores of the subscales of FSFI in female participants mostly impaired sexual satisfaction and overall satisfaction in their spouses

Phosphatidylserine positive microparticles improve hemostasis in in-vitro hemophilia A plasma models

Circulating microparticles (MPs) are procoagulant due to the surface containing phosphatidylserine (PS), which facilitates coagulation. We investigated if MPs improve hemostasis in HA plasma models. MPs isolated from pooled normal human plasma were added to severe, moderate and mild HA plasma models (0%, 2.5%, 20% FVIII). The MPs' effect on hemostasis was evaluated by calibrated automated thrombogram (CAT) and overall hemostasis potential (OHP) assays, while fibrin structure was imaged by standard confocal, stimulated emission depletion (STED) microscopy and scanning electron microscopy (SEM). MPs partially restored thrombin generation and fibrin formation in all HA plasma models. The procoagulant effect of MPs requires PS exposure, to a less extent of contact pathway activation, but not tissue factor exposure or in vitro stimulation of MPs. MPs partially normalized the fibrin structure, and using super-resolution STED, MPs attached to fibrin were clearly resolved. In summary, our results demonstrate that PS positive MPs could improve hemostasis in HA plasma models

Exploring the Midwives’ Experiences of Providing Delivery Care for Women with Suspected or Confirmed COVID-19 Infection: A Qualitative Study

Introduction: Midwives were at the forefront of caring for pregnant women during the COVID-19 pandemic, therefore, the present study was conducted with the purpose of exploring midwives’ experiences of providing delivery care for women with suspected or confirmed COVID-19 infection. Methods: In this qualitative study, 18 midwives working in the public hospitals affiliated with Guilan University of Medical Sciences (Iran). Who had experience in providing delivery care to women with suspected or confirmed COVID-19 infection were selected using purposive sampling. Data were collected via individual semi-structured interviews until reaching data saturation, and analyzed through conventional content analysis. Results: Data analysis led to the extraction of three main categories and six sub-categories. The main categories included "COVID-19 and organizational support" with two sub-categories including lack of resources/neglecting the role of midwives, "COVID-19 and positive achievements" with two sub-categories including professional resilience/turning threats into opportunities, "COVID-19 and informational support" with two sub-categories, including up-to-date training/empowering pregnant women. Conclusion: According to the findings, to realize and guarantee the provision of high-quality maternity care to pregnant women in dealing with epidemic diseases such as COVID-19 in the future, the attention of policymakers and healthcare service officials to the physical and psychological needs of midwives is necessary. Also, organizational and informational support, improving job satisfaction, and paying attention to the importance of midwives’ role in the medical team are recommended. Moreover, empowering pregnant women during epidemic diseases is essential

Augmented thrombin formation is related to circulating levels of extracellular vesicles exposing tissue factor and citrullinated histone-3 in anti-neutrophil cytoplasmic antibody-associated vasculitides

Publisher Copyright: Copyright © 2023 Jonasdottir, Manojlovic, Vojinovic, Nordin, Bruchfeld, Gunnarsson, Mobarrez and Antovic.Objectives: To study circulating myeloperoxidase (MPO)-positive extracellular vesicles (MPO+EVs) exposing citrullinated histone-3 (H3Cit), tissue factor (TF), and plasminogen (Plg) in association to thrombin generation in patients with anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV). Methods: We have involved well-characterized patients with AAV together with population-based controls. Flow cytometry was used to assess the levels of MPO+EVs in citrated plasma. MPO+EVs were phenotyped by anti-MPO-antibodies together with anti-CD142 (anti-TF), anti-H3Cit, and anti-Plg antibodies. A modified Calibrated Automated Thrombogram (CAT) assay was utilized to measure thrombin generation in plasma initiated by EVs-enriched pellets. The activity of AAV was evaluated with the Birmingham Vasculitis Activity Score (BVAS). Results: This study comprised 46 AAV patients, 23 in the active stage of the disease and 23 in remission, as well as 23 age- and sex matched population-based controls. Augmented levels of all investigated MPO+ EVs were found in active AAV patients in comparison to the subgroup of patients in remission and controls. Thrombin generation, measured by endogenous thrombin potential (ETP) and peak of thrombin formation, was higher in plasma when triggered by EVs-enriched pellet from AAV patients. ETP and peak were associated with the levels of MPO+TF+ and MPO+H3Cit+ EVs. Additionally, MPO+TF+ EVs correlated with the disease activity evaluated with BVAS. Conclusion: Augmented thrombin generation is found in AAV patients regardless of disease activity and is associated with higher exposure of TF and H3Cit on MPO+EVs. This may contribute to the increased risk of thrombosis seen in AAV patients.Peer reviewe

Acute effects of electronic and tobacco cigarettes on vascular and respiratory function in healthy volunteers:a cross-over study

Objectives: To assess the acute effects of nicotine-containing electronic cigarettes versus tobacco smoking on vascular and respiratory function and circulating microparticles, particularly platelet microparticles (PMPs, biomarker of haemostasis/thrombosis) and endothelial microparticles (EMPs, biomarker of endothelial function). Methods: Heart rate (HR), blood pressure, reactive hyperaemia index (RHI, microvascular reactivity), augmentation index (arterial stiffness) and respiratory function were assessed in 20 smokers immediately before and after electronic cigarettes use and tobacco smoking. The number of microparticles was determined by flow cytometry using counting beads as a reference. Labelling with Annexin-V was used to detect the total microparticle fraction. EMPs were characterized as CD31+CD42− and PMPs as CD31+CD42+. Results: HR increased after electronic cigarettes use and tobacco smoking (P < 0.001), whereas blood pressure remained unchanged (P > 0.05). RHI (P = 0.006), augmentation index (P = 0.010) but not augmentation index standardized to HR 75 bpm (P > 0.05) increased with electronic cigarettes use but not with tobacco smoking. Following tobacco smoking, there was a significant increase in total microparticles (P < 0.001), EMPs (P < 0.001) and PMPs (P < 0.001). In contrast, electronic cigarettes were only associated with an increase in PMPs (P < 0.001), with no significant changes in the total microparticle fraction or EMPs (all P > 0.05). Peak expiratory flow significantly decreased following electronic cigarettes use (P = 0.019). Conclusion: Our results demonstrate that acute exposure to tobacco smoking as well as electronic cigarettes influences vascular and respiratory function. Where tobacco smoking significantly increased microparticle formation, indicative of possible endothelial injury, electronic cigarettes use induced vasoreactivity and decreased peak expiratory flow. These findings suggest that both electronic cigarettes and tobacco smoking negatively impact vascular function

Review of DC fault protection for HVDC grids

The development of modular multilevel converter (MMC) and the increased needs for long distance bulk power transmission using underground and subsea cables have promoted the rapid development and application of voltage source converter (VSC) based high-voltage DC (HVDC) systems. In this paper, recent advances in the area of DC fault protection in VSC based HVDC systems are reviewed. The main characteristics during DC faults are described and various converter topologies, which have DC fault blocking capability, are introduced and compared in terms of efficiency, cost, and control flexibility. The development of DC circuit breaker is introduced and various methods for DC fault detection and system level protection approaches for large scale HVDC grids are also discussed