Background While cardiopulmonary transition in neonates is well described, molecular changes which accompany it are not. The Extracellular Vesicles in Early preterm neonates and Thrombin generation (EVENT) study sought to explore molecular transition in preterm birth. How this differs from term-born neonates is not well described.Methods A post hoc evaluation of proteomic data generated from a subset of the prospective, single site observational EVENT study was performed comparing extracellular vesicle enriched plasma from term neonates (n=10) with preterm neonates (n=20) using a mass spectrometry, data dependent acquisition-based proteomic approach. Bioinformatics was performed using MaxQuant proteomics software and R statistical software.Results Substantial differences were found between the preterm and term extracellular vesicle proteome, with proteins involved in extracellular matrix biology, immune function and embryogenesis all differentially expressed. Preterm neonates could be identified by their proteomic profile alone, reflecting their distinct physiology.Conclusions These protein expression differences may underlie the pathogenesis of complications of prematurity, such as bronchopulmonary dysplasia, retinopathy of prematurity and intraventricular haemorrhage.

There is growing interest in the role of extracellular vesicles (EVs) in neonatal pathology. This study aimed to characterise circulating EVs following preterm birth. This single-centre prospective observational study included cord and postnatal plasma from preterm (n = 101) and full-term infants (n = 66). EVs were analysed using nanoparticle tracking analysis, flow cytometry, proteomics and procoagulant activity assay. We found changes in the concentration, size, cellular origin and proteomic content of circulating EVs in preterm infants during perinatal adaptation. To understand if these changes were related to prematurity or normal adaptation to extrauterine life, they were also investigated in term infants. There was a dramatic increase in the concentration of small and large EVs on Day 3 in the preterm group; specific subsets of platelet (CD42b+ and CD62P+), endothelial (VEGFR2) and tissue factor EVs were elevated. Differentially expressed proteins relating to haemostasis, pulmonary physiology and immunity were identified between Day 1 and 3 in preterm infants. These changes have never previously been described in a large cohort of preterm infants and differ from healthy term infants. These findings have major implications for future neonatal EV studies, particularly the timing of sample collection. Further work is required to understand the clinical implications of this unique EV profile following preterm birth.

Objective Clinical recruitment encompasses a significant challenge in multidisciplinary research, often acting as a bottleneck to timely completion due to slower-than-expected patient enrolment rates. Addressing this, enhanced communication within clinical departments is crucial. A quality improvement (QI) study was initiated in the Mater Misericordiae University Hospital (MMUH) to increase the slow recruitment rate of cancer-associated thrombosis patients to the EXPECT Study.Methods Process and stakeholder mapping as well as Plan Do Study Act (PDSA) cycles highlighted effective initiatives to increase recruitment rates to the study. The PDSA cycle 1 aimed at increasing clinical communication and study education through implementation of work package-1, which included engaging a clinical project sponsor to drive recruitment and increasing study awareness through educational talks/informative materials. The PDSA cycle 2 aimed to increase process efficiency and paired sample acquisition through implementation of work package-2, which included holding weekly QI meetings, building a strong multidisciplinary QI team and mapping the recruitment process. These efforts aimed to increase recruitment from one to four patients enrolled per month, with recruitment progress tracked with a run/bar chart over a 2 year period.Results The communication/education work package-1 initiatives increased the recruitment rate from one to two patients per month, with target enrolment met or exceeded 33% of the QI-project duration. Recruitment numbers nearly doubled in roughly half the timeframe, from 10 patients enrolled in the first 16 months to 18 patients enrolled in the 9 months of the QI study. Furthermore, a greater than threefold statistically significant increase in paired sample acquisition from 20% to 66% was documented following the execution of the second PDSA cycle, aimed at improving process efficiency.Conclusions This QI study highlights the need for a highly engaged study team, specifically the clinical project sponsor driving recruitment from a medical front-line perspective as well as a highly efficient recruitment process.

Extracorporeal Photopheresis (ECP) is a leukapheresis based treatment for Cutaneous T-Cell Lymphoma, which takes advantage of the cellular lethal effects of UVA light in combination with a photoactivated drug, 8-methoxypsoralen. 25% of patients treated with ECP do not respond to treatment, however the underlying mechanisms for this lack of response remain unknown. Platelets, a rich source of extracellular vesicles (EVs) and key mediators in thromboinflammatory oncological progression, as well as leukocytes, are both processed through ECP and are subsequently transfused back into the patient, delivering potent immunomodulation. The effect of exposing platelets and their EVs directly to Ultra Violet A light (UVA)/8-methoxypsoralen is currently unknown. Platelet-rich plasma (PRP) was isolated from healthy donors and exposed to UVA light and/or 8-methoxysporalen in vitro and platelet activation and aggregation was assessed. EV size and concentration were also characterised by Nanoparticle Tracking Analysis and Flow Cytometry. We found that UVA light and 8-methoxypsoralen treatment in vitro does not induce platelet aggregation or significantly alter levels of the platelet activation markers, soluble P-selectin or platelet factor 4, with circulating levels of small and large EV size and concentration remaining constant. Therefore, utilising the combination of UVA light and 8-methoxypsoralen used in ECP in vitro does not activate platelets or alter important circulating EVs. Further studies will be needed to validate if our observations are consistent in vivo.

Purpose of review Cardiovascular disease (CVD) remains a major global health burden. Rising incidences necessitate improved understanding of the pathophysiological processes underlying disease progression to foster the development of novel therapeutic strategies. Besides their well recognized role in CVD, platelet-derived extracellular vesicles (PEVs) mediate inter-organ cross talk and contribute to various inflammatory diseases. Recent findings PEVs are readily accessible diagnostic biomarkers that mirror pathophysiological disease progression but also may confer cardioprotective properties. Monitoring the effects of modulation of PEV signatures through pharmacotherapies has also provided novel insights into treatment efficacy. Furthermore, exploiting their inherent ability to infiltrate thrombi, atherosclerotic plaques and solid tumours, PEVs as well as platelet-membrane coated nanoparticles are emerging as novel effective and targeted treatment options for CVD and cancer. Summary Collectively, in-depth characterization of PEVs in various diseases ultimately enhances their use as diagnostic or prognostic biomarkers and potential therapeutic targets, making them clinically relevant candidates to positively impact patient outcomes.

The contents of the platelet releasate (PR) play significant roles in hemostasis, inflammation, and pathologic sequelae. Careful platelet isolation to ensure quiescence and subsequent activation is key to the successful generation of PR. Here, we describe steps to isolate and aggregate quiescent washed platelets from whole blood of a clinical patient cohort. We then detail the generation of PR from isolated human washed platelets under clinical conditions. This protocol allows the investigation of platelet cargoes released through various activation pathways.

We investigated endogenous levels of a novel peptide, pituitary adenylate cyclase activating polypeptide (PACAP), in the rat central nervous system. The amount of PACAP was measured by means of highly specific and sensitive sandwich-enzyme immunoassay. This assay system following HPLC analysis revealed that PACAP38 was a major portion of the total PACAP immunoreactivity and PACAP27 levels were negligibly low in the brain. Therefore, we measured the amount of PACAP38 in 62 regions punched out from frozen tissue sections. High amounts of PACAP38 were found in the lateral septal nucleus (intermediate part), diagonal band, central amygdaloid nucleus, several parts of the hypothalamus (suprachiasmatic, supraoptic, periventricular and arcuate nuclei), central gray, interpeduncular nucleus and dorsal raphe. The suprachiasmatic, paraventricular and periventricular hypothalamic nuclei showed the highest levels. A moderate amount of the peptide was observed in the lateral septal nucleus (dorsal part), medial septal nucleus, medial amygdaloid nucleus, thalamus (paraventricular, paratenial, central medial, ventromedial, reuniens and rhomboid nuclei), hypothalamus (lateral hypothalamic area and mammillary body), ventral tegmental area, interfascicular nucleus and in the locus coeruleus. Such a distribution of endogenous PACAP38 did not parallel the localization of PACAP binding sites which we had demonstrated recently. Moreover, the topographical distribution of PACAP38 observed in the present study differed from that of VIP which had been previously reported. The present results suggest that PACAP38 may have a neurotransmitter/neuromodulator role which is different from that of VIP in the central nervous system.

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Introduction: Hypercoagulability, thrombosis and microvascular dysfunction are hallmarks of the Philadelphia chromosome-negative myeloproliferative neoplasms (MPN). Elevated peripheral blood cell counts appear to contribute to thrombotic risk but the precise underlying mechanisms remain to be fully elucidated. The interplay between haemostatic and pro-inflammatory pathway activity ('thrombo-inflammation') has emerged in recent years as a source of hypercoagulability in MPN. Platelets are recognised as being mediators of thrombo-inflammation in other diseases and may also be effectors of pro-inflammatory/pro-coagulant activity in MPN. We hypothesized that the platelet proteome is altered in MPN and that its characterisation would reveal insights into the pathophysiology of the disease and the associated thrombotic risk. Aim: To determine if differences exist in the pattern of platelet protein expression in patients with polycythaemia vera (PV) and essential thrombocythaemia (ET) in contrast to healthy donors. Methods: 62 patients (ET, n=38; PV, n=24) and 9 healthy volunteers were recruited at Papa Giovanni XXIII Hospital, Bergamo, Italy and the Mater Misericordiae University hospital, Dublin, Ireland. Platelets were isolated from platelet rich plasma, washed in Krebs Ringer buffer and resuspended at 1x 109 platelets/mL in phosphate buffered saline. Whole platelets were lysed in RIPA buffer and differential proteomic signatures established using label-free quantification (LFQ) mass spectrometry (MS), where platelet lysate proteins were double digested using the commercially available PreOmics kit and analysed in a Bruker TimsTOF mass spectrometer connected to a EvoSep liquid chromatography system. Identified peptides were searched against a human FASTA using MaxQuant. For statistical analysis, proteins identified in a minimum of 70% of samples in at least one group were included. Differences in protein expression were determined using an unpaired t-test with a false discovery rate of 5% and a minimal fold change of 0.1 within the Perseus software; p- values below 0.05 were considered significant. Results: 2,180 proteins from platelet lysates were quantified across all patient and control samples. In MPN platelet lysates, 49 proteins were found to be differentially expressed in comparison to controls (p< 0.05) (Figure 1), including increased expression of markers of platelet activity such as vesicle-associated membrane protein 7 (VAMP-7; regulator of granule exocytosis and actin cytoskeleton activity) and CD109 (a GPI-linked glycoprotein expressed by activated platelets). Bioinformatical analysis revealed a cohort of mitochondrial, cytoskeletal & ribosomal proteins as well as potential effectors of thrombopoiesis and thrombo-inflammation which were significantly upregulated in the MPN cohort. Strikingly, protein disulfide-isomerase (PDI, a member of the thioredoxin superfamily of redox proteins) was increased in MPN lysates. PDI has been shown to contribute to thrombosis, platelet activation and platelet-neutrophil interactions in vivo; plasma levels of PDI have been shown to be increased in MPN and are associated with thrombosis risk. Conclusion: The platelet proteome is altered in MPN and is suggestive of an activated platelet phenotype with over-expression of proteins with known pro-coagulant activity. To our knowledge the over expression of PDI in MPN platelets has not previously been described. The identification of differentially expressed proteins, such as PDI, provides mechanistic insights into the pathophysiology underlying the substantial burden of arterial and venous thrombosis in MPN and may also assist in the identification of novel therapeutic targets. Additional proteomic analysis of platelet releasate and plasma extracellular vesicles is currently ongoing and may provide additional pathophysiological insights. Figure 1: Volcano plot of MPN versus control platelet lysate proteomes (x-axis, t-test difference between the mean log2 of the LFQ values; y-axis, the negative log transformed p-value) representing the proteins significantly altered in MPN. Black hyperbolic curves show the threshold for statistical significance. 36 proteins were found to be increased in MPN platelet lysates (including CD109, PDIA4 and VAMP-7) in comparison to control lysates (red) while 13 proteins were decreased in MPN platelet lysates (blue). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Background : COVID-19 confers an increased risk of thrombosis however the mechanisms underlying this coagulopathy and the optimal approach to thromboprophylaxis are unknown. Thrombotic risk is likely greatest among patients with severe COVID-19 requiring critical organ support however patients with moderate disease may be at risk and might also benefit from intensified thromboprophylaxis. Aims : To characterise plasma thrombin generation (TG) in patients with COVID-19 of moderate severity, treated with pharmacological thromboprophylaxis. Methods : Blood was collected from individuals admitted to hospital with COVID-19 of moderate severity (not requiring critical care support) and a group of age-matched patients admitted with infective/ inflammatory illness (negative for COVID-19). All subjects received standard dose low molecular weight heparin (LMWH) thromboprophylaxis with samples taken at time of predicted trough levels (confirmed by measuring anti-FXa activity). TG in platelet-poor plasma was determined by calibrated automated thrombography in the presence/absence of tissue factor (TF) (ppp-LOW reagent, 1 pM TF & 4 μM phospholipid;MP-reagent, 4 μM phospholipid;Thrombinoscope BV™). Results : Fourteen COVID-19 positive subjects and 11 hospitalised COVID-19 negative controls were recruited. Mean trough plasma anti-Xa activity was similar in both groups (0.06 vs 0.04 IU/mL;P = 0.2). In the presence of TF, mean endogenous thrombin potential was significantly higher in the COVID group in comparison to controls (1929 ± 119.7 vs 1528 ± 138.9 nM∗min;P = 0.02). Peak thrombin was also higher in COVID-19 (267.3 ± 22.2 vs 208.6 ± 17.8 nM;P = 0.06). Despite increased TG overall, lagtime to TG was significantly prolonged in COVID-19 (8.1 ± 0.5 vs 6.2 ± 0.5 mins;P = 0.02). No difference in any parameter of TG was observed between groups in the absence of TF. Conclusions : Despite pharmacological thromboprophylaxis plasma TG is enhanced in COVID-19. The underlying mechanisms remain to be elucidated. Specific clinical implications of increased TG despite pharmacological thromboprophylaxis have yet to be determined although clinical trials evaluating intensified anticoagulant regimens in a similar population are ongoing.