Transcription factor 3 is dysregulated in megakaryocytes in myelofibrosis.

Transcription factor 3 (TCF3) is a DNA transcription factor that modulates megakaryocyte development. Although abnormal TCF3 expression has been identified in a range of hematological malignancies, to date, it has not been investigated in myelofibrosis (MF). MF is a Philadelphia-negative myeloproliferative neoplasm (MPN) that can arise de novo or progress from essential thrombocythemia [ET] and polycythemia vera [PV] and where dysfunctional megakaryocytes have a role in driving the fibrotic progression. We aimed to examine whether TCF3 is dysregulated in megakaryocytes in MPN, and specifically in MF. We first assessed TCF3 protein expression in megakaryocytes using an immunohistochemical approach analyses and showed that TCF3 was reduced in MF compared with ET and PV. Further, the TCF3-negative megakaryocytes were primarily located near trabecular bone and had the typical "MF-like" morphology as described by the WHO. Genomic analysis of isolated megakaryocytes showed three mutations, all predicted to result in a loss of function, in patients with MF; none were seen in megakaryocytes isolated from ET or PV marrow samples. We then progressed to transcriptomic sequencing of platelets which showed loss of TCF3 in MF. These proteomic, genomic and transcriptomic analyses appear to indicate that TCF3 is downregulated in megakaryocytes in MF. This infers aberrations in megakaryopoiesis occur in this progressive phase of MPN. Further exploration of this pathway could provide insights into TCF3 and the evolution of fibrosis and potentially lead to new preventative therapeutic targets.

What is the context? We investigated TCF3 (transcription factor 3), a gene that regulates megakaryocyte development, for genomic and proteomic changes in myelofibrosis.Myelofibrosis is the aggressive phase of a group of blood cancers called myeloproliferative neoplasms, and abnormalities in development and maturation of megakaryocytes is thought to drive the development of myelofibrosis.What is new? We report detection of three novel TCF3 mutations in megakaryocytes and decreases in TCF3 protein and gene expression in primary megakaryocytes and platelets from patients with myelofibrosis.This is the first association between loss of TCF3 in megakaryocytes from patients and myelofibrosis.What is the impact? TCF3 dysregulation may be a novel mechanism that is responsible for the development of myelofibrosis and better understanding of this pathway could identify new drug targets.

Non-severe burn injury causes sustained platelet hyperreactivity.

Individuals who present to a hospital for treatment of a burn of any magnitude are more frequently hospitalised for ischemic heart disease, even decades after injury. Blood platelets are key mediators of cardiovascular disease. To investigate platelet involvement in post-burn cardiovascular risk, platelet reactivity was assessed in patients at 2- and 6-weeks after non-severe (TBSA < 20%) burn injury, and in a murine model 30 days after 8% TBSA full-thickness burn injury. Platelets were stimulated with canonical agonists and function reported by GPIIb/IIIa PAC1-binding site, CD62P expression, and formation of monocyte-platelet aggregates. In vivo thrombosis in a modified Folts model of vascular injury was assessed. Burn survivors had elevated frequencies of circulating monocyte-platelet aggregates, and platelets were hyperreactive, primarily to collagen stimulation. Burn plasma did not cause hyper-reactivity when incubated with control platelets. Platelets from burn injured mice also demonstrated increased response to collagen peptides but did not show any change in thrombosis following vascular injury. This study demonstrates the persistence of a small but significant platelet hyperreactivity following burn injury. Although our data does not suggest this heightened platelet sensitivity modulates thrombosis following vascular injury, the contribution of sub-clinical platelet hyperreactivity to accelerating atherogenesis merits further investigation.

Mitochondrial gene expression is required for platelet function and blood clotting.

Platelets are anucleate blood cells that contain mitochondria and regulate blood clotting in response to injury. Mitochondria contain their own gene expression machinery that relies on nuclear-encoded factors for the biogenesis of the oxidative phosphorylation system to produce energy required for thrombosis. The autonomy of the mitochondrial gene expression machinery from the nucleus is unclear, and platelets provide a valuable model to understand its importance in anucleate cells. Here, we conditionally delete Elac2, Ptcd1, or Mtif3 in platelets, which are essential for mitochondrial gene expression at the level of RNA processing, stability, or translation, respectively. Loss of ELAC2, PTCD1, or MTIF3 leads to increased megakaryocyte ploidy, elevated circulating levels of reticulated platelets, thrombocytopenia, and consequent extended bleeding time. Impaired mitochondrial gene expression reduces agonist-induced platelet activation. Transcriptomic and proteomic analyses show that mitochondrial gene expression is required for fibrinolysis, hemostasis, and blood coagulation in response to injury.

Changes in von Willebrand Factor Multimers, Concentration, and Function During Pediatric Extracorporeal Membrane Oxygenation.

OBJECTIVES: To investigate changes in von Willebrand factor (VWF) concentration, function, and multimers during pediatric extracorporeal membrane oxygenation (ECMO) and determine whether routine monitoring of VWF during ECMO would be useful in predicting bleeding. DESIGN: Prospective observational study of pediatric ECMO patients from April 2017 to May 2019. SETTING: The PICU in a large, tertiary referral pediatric ECMO center. PATIENTS: Twenty-five neonates and children (< 18 yr) supported by venoarterial ECMO. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Arterial blood samples were collected within 24 hours pre-ECMO, daily for the first 5 days of ECMO, every second day until decannulation, and 24 hours post-ECMO. The STA R Max analyzer was used to measure VWF antigen (VWF:Ag) and ristocetin cofactor (VWF:RCo) activity. VWF collagen binding (VWF:CB) was measured using an enzyme-linked immunosorbent assay. VWF multimers were measured using the semi-automated Hydragel 11 VWF Multimer assay. Corresponding clinical data for each patient was also recorded. A total of 25 venoarterial ECMO patients were recruited (median age, 73 d; interquartile range [IQR], 3 d to 1 yr). The median ECMO duration was 4 days (IQR, 3-8 d) and 15 patients had at least one major bleed during ECMO. The percentage of high molecular weight multimers (HMWM) decreased and intermediate molecular weight multimers increased while patients were on ECMO, irrespective of a bleeding status. VWF:Ag increased and the VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios decreased while patients were on ECMO compared with the baseline pre-ECMO samples and healthy children. CONCLUSIONS: Neonates and children on ECMO exhibited a loss of HMWM and lower VWF:CB/VWF:Ag and VWF:RCo/VWF:Ag ratios compared with healthy children, irrespective of major bleeding occurring. Therefore, monitoring VWF during ECMO would not be useful in predicting bleeding in these patients and changes to other hemostatic factors should be investigated to further understand bleeding during ECMO.

Factors XI and XII in extracorporeal membrane oxygenation: longitudinal profile in children.

Background: Extracorporeal membrane oxygenation (ECMO) is used in children with cardiopulmonary failure. While the majority of ECMO centers use unfractionated heparin, other anticoagulants, including factor XI and factor XII inhibitors are emerging, which may prove suitable for ECMO patients. However, before these anticoagulants can be applied in these patients, baseline data of FXI and FXII changes need to be acquired. Objectives: This study aimed to describe the longitudinal profile of FXI and FXII antigenic levels and function before, during, and after ECMO in children. Methods: This is a prospective observational study in neonatal and pediatric patients with ECMO (<18 years). All patients with venoarterial ECMO and with sufficient plasma volume collected before ECMO, on day 1 and day 3, and 24 hours postdecannulation were included. Antigenic levels and functional activity of FXI and FXII were determined in these samples. Longitudinal profiles of these values were created using a linear mixed model. Results: Sixteen patients were included in this study. Mean FXI and FXII antigenic levels (U/mL) changed from 7.9 and 53.2 before ECMO to 6.0 and 34.5 on day 3 and they recovered to 8.8 and 39.4, respectively, after stopping ECMO. Function (%) of FXI and FXII decreased from 59.1 and 59.0 to 49.0 and 50.7 on day 3 and recovered to 66.0 and 54.4, respectively. Conclusion: This study provides the first insights into changes of the contact pathway in children undergoing ECMO. FXI and FXII antigen and function change during ECMO. Results from this study can be used as starting point for future contact pathway anticoagulant studies in pediatric patients with ECMO.

Platelet Phenotype and Function Changes With Increasing Duration of Extracorporeal Membrane Oxygenation.

OBJECTIVES: To investigate platelet pathophysiology associated with pediatric extracorporeal membrane oxygenation (ECMO). DESIGN: Prospective observational study of neonatal and pediatric ECMO patients from September 1, 2016, to December 31, 2019. SETTING: The PICU in a large tertiary referral pediatric ECMO center. PATIENTS: Eighty-seven neonates and children (< 18 yr) supported by ECMO. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Arterial blood samples were collected on days 1, 2, and 5 of ECMO and were analyzed by whole blood flow cytometry. Corresponding clinical data for each patient was also recorded. A total of 87 patients were recruited (median age, 65 d; interquartile range [IQR], 7 d to 4 yr). The median duration of ECMO was 5 days (IQR, 3-8 d) with a median length of stay in PICU and hospital of 18 days (IQR, 10-29 d) and 35 days (IQR, 19-75 d), respectively. Forty-two patients (48%) had at least one major bleed according to a priori determined definitions, and 12 patients (14%) had at least one thrombotic event during ECMO. Platelet fibrinogen receptor expression decreased (median fluorescence intensity [MFI], 29,256 vs 26,544; p = 0.0005), while von Willebrand Factor expression increased (MFI: 7,620 vs 8,829; p = 0.0459) from day 2 to day 5 of ECMO. Platelet response to agonist, Thrombin Receptor Activator Peptide 6, also decreased from day 2 to day 5 of ECMO, as measured by binding with anti-P-selectin, PAC-1 (binds activated GPIIb/IIIa), and anti-CD63 monoclonal antibodies (P-selectin area under the curve [AUC]: 63.46 vs 42.82, respectively, p = 0.0022; PAC-1 AUC: 93.75 vs 74.46, p = 0.0191; CD63 AUC: 55.69 vs 41.76, p = 0.0020). CONCLUSIONS: The loss of platelet response over time may contribute to bleeding during ECMO. These novel insights may be useful in understanding mechanisms of bleeding in pediatric ECMO and monitoring platelet markers clinically could allow for prediction or early detection of bleeding and thrombosis.

Immunophenotypic Analysis of Platelets by Flow Cytometry.

Platelets are small but very abundant blood cells that play a key role in hemostasis, contributing to thrombus formation at sites of injury. The ability of platelets to perform this function, as well as functions in immunity and inflammation, is dependent on the presence of cell surface glycoproteins and changes in their quantity and conformation after platelet stimulation. In this article, we describe the characterization of platelet surface markers and platelet function using platelet-specific fluorescent probes and flow cytometry. Unlike traditional platelet tests, immunophenotypic analysis of platelets by flow cytometry allows the analysis of platelet function in samples with very low platelet counts as often encountered in clinical situations. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Immunophenotyping of platelet surface receptors Alternate Protocol: Fix-first method for immunophenotyping of platelet surface receptors Basic Protocol 2: Determination of platelet activation using P-selectin expression and/or PAC1 binding Basic Protocol 3: Determination of procoagulant platelets using annexin V binding or antibodies specific for coagulation factor V/Va or X/Xa Support Protocol: Preparation of isolated platelets.

Corrigendum: Pediatric Burn Survivors Have Long-Term Immune Dysfunction With Diminished Vaccine Response.

[This corrects the article DOI: 10.3389/fimmu.2020.01481.].

Pediatric Burn Survivors Have Long-Term Immune Dysfunction With Diminished Vaccine Response.

Epidemiological studies have demonstrated that survivors of acute burn trauma are at long-term increased risk of developing a range of morbidities. The mechanisms underlying this increased risk remain unknown. This study aimed to determine whether burn injury leads to sustained immune dysfunction that may underpin long-term morbidity. Plasma and peripheral blood mononuclear cells were collected from 36 pediatric burn survivors >3 years after a non-severe burn injury (<10% total body surface area) and from age/sex-matched non-injured controls. Circulating cytokine and vaccine antibody levels were assessed using multiplex immunoassays and cell profiles compared using a panel of 40 metal-conjugated antibodies and mass cytometry. TNF-α (1.31-fold change from controls), IL-2 (1.18-fold), IL-7 (1.63-fold), and IFN-γ (1.18-fold) were all significantly elevated in the burn cohort. Additionally, burn survivors demonstrated diminished antibody responses to the diphtheria, tetanus, and pertussis vaccine antigens. Comparisons between groups using unsupervised clustering identified differences in proportions of clusters within T-cells, B-cells and myeloid cells. Manual gating confirmed increased memory T-regulatory and central memory CD4+ T-cells, with altered expression of T-cell, B-cell, and dendritic cell markers. Conclusions: This study demonstrates a lasting change to the immune profile of pediatric burn survivors, and highlights the need for further research into post-burn immune suppression and regulation.

Platelets in myeloproliferative neoplasms have a distinct transcript signature in the presence of marrow fibrosis.

Marrow fibrosis is a significant complication of myeloproliferative neoplasms (MPN) that affects up to 20% of patients and is associated with a poor prognosis. The pathological processes that lead to fibrotic progression are not well understood, but megakaryocytes have been implicated in the process. The aim of this study was to determine whether platelets, derived from megakaryocytes, have transcriptomic alterations associated with fibrosis. Platelets from MPN patients with and without fibrosis and non-malignant control individuals were assessed using next generation sequencing. Results from the initial training cohort showed discrete changes in platelet transcripts in the presence of marrow fibrosis. We identified more than 1000 differentially expressed transcripts from which a putative 3-gene fibrotic platelet signature (CCND1, H2AX [previously termed H2AFX] and CEP55) could be identified. This fibrosis-associated signature was assessed blinded on platelets from an independent test MPN patient cohort. The 3-gene signature was able to discriminate between patients with and without marrow fibrosis with a positive predictive value of 71% (93% specificity, 71% sensitivity). This demonstrates that assessment of dysregulated transcripts in platelets may be a useful monitoring tool in MPN to identify progression to marrow fibrosis. Further, sequential monitoring could have clinical applications for early prediction of progression to fibrosis.

Antiplatelet Therapy Monitoring in Neonates and Children.

Antiplatelet agents are used for the prevention and treatment of thromboembolic disease in an increasingly complex population of pediatric patients. Despite their importance for clinical outcome, there is no consensus on the most effective monitoring strategies. This review describes the current state of knowledge focusing on antiplatelet therapy monitoring in children. The authors searched five databases (PubMed-NCBI, MEDLINE-OVID, SCOPUS-Elsevier, ScienceDirect, and Cochrane) from January 2000 to October 2017 using keywords selected a priori. Identified articles were sorted according to the antiplatelet agents administered, methods of antiplatelet monitoring, and outcome measures. Twenty studies were included, with 14 cohort studies, 3 randomized controlled trials, and 3 cross-sectional studies. Eleven different antiplatelet monitoring tools were used, with the most common being Light Transmission Aggregometry, Urinary Thromboxane, Thromboelastography with Platelet Mapping, and VerifyNow. In the majority of studies, antiplatelet therapy monitoring was used to describe adequacy or responsiveness to treatment based on laboratory cut-off values, which were not uniform and sourced from adult studies or extrapolated from test manuals. Several studies evaluated monitoring related to clinical outcome or adjusted therapy to reach predefined therapeutic targets. There was no single laboratory method found to be distinctly better for monitoring antiplatelet treatment. Associations between laboratory assays and clinical outcomes or assays and gold standard measurements were highly inconsistent. The current literature lacks consensus on clinical benefits and measurable effects of monitoring antiplatelet therapy in pediatric patients. This review highlights important areas for research required to determine the value of antiplatelet therapy monitoring in children.

Beneficial impacts of regular exercise on platelet function in sedentary older adults: evidence from a randomized 6-mo walking trial.

Platelet activation, including the formation of monocyte platelet aggregates (MPAs), contributes to atherosclerosis, thrombus formation, and acute coronary syndromes. Regular participation in exercise can lower cardiovascular risk, but little is known regarding the impact of exercise training on platelet function. We investigated the effect of 6 mo of walking exercise on platelet function in sedentary older adults without significant cardiovascular disease. Twenty-seven participants were randomly allocated to 6 mo of either: no-exercise ( n = 13) or 3 × 50 min/wk of supervised center-based walking ( n = 14). Circulating and agonist-induced MPAs were assessed using flow cytometry before [ month 0 (0M)] and after [ month 6 (6M)] the intervention. Circulating MPAs increased from 0M (3.7 ± 1.0%) to 6M (4.7 ± 1.6%) in the no-exercise group ( P = 0.009), whereas a nonsignificant decrease was observed in the walking group (0M 4.3 ± 1.7 vs. 6M 3.7 ± 1.2 %, P = 0.052). The change in MPAs between groups was significant ( P = 0.001). There were no differences between groups in platelet responses to agonists across the interventions (all P > 0.05). Collectively, these data suggest that the absence of regular exercise may increase MPAs, which are cellular mediators involved in atherosclerosis, while regular walking inhibits such increases. The thrombotic function of platelets appears to be relatively unaltered by exercise training. This study provides novel data related to the cardioprotective effects associated with participation in exercise. NEW & NOTEWORTHY Monocyte-platelet aggregates contribute to atherosclerosis and exercise can lower cardiovascular risk. This is the first study to discover that a lack of regular physical activity is associated with increased monocyte-platelet aggregates over a 6-mo intervention period. In contrast, walking exercise inhibits increased monocyte-platelet aggregates in the circulation. This study highlights a novel pathway by which regular participation in exercise exerts its cardioprotective effects.

Acute Impact of Different Exercise Modalities on Arterial and Platelet Function.

PURPOSE: Acute coronary syndromes and ischemic stroke are associated with arterial events involving platelets, the endothelium, and atherosclerosis. Although regular physical activity is associated with lower risk of cardiovascular events and mortality, risk is transiently increased during and immediately after participation in an acute bout of exercise. No previous study has investigated the acute impact of exercise on platelet activation and arterial function in the same participants; it is also unknown if responses are dependent on exercise modality. We hypothesized that commonly adopted, yet physiologically distinct, modalities of exercise ("aerobic" vs "resistance") have differing effects on in vivo platelet activation and conduit artery diameter. METHODS: Eight apparently healthy middle-age (53.5 ± 1.6 yr) male subjects took part in four 30-min experimental interventions (aerobic exercise, resistance exercise, combined aerobic/resistance exercise, or no-exercise), in random order. Blood samples were collected, and the measurement of brachial artery diameter by ultrasound was performed before, immediately after, and 1 h after each intervention. Platelet activation was determined by the positive binding of antibodies to surface receptors exposed on activated platelets (anti-CD62P and PAC-1). RESULTS: Brachial artery diameter increased immediately after all three exercise modalities (P < 0.001) and remained above preexercise levels 1 h after resistance exercise and after combined aerobic/resistance exercise. No changes were observed in markers of in vivo platelet activation with any experimental protocol. CONCLUSIONS: These data suggest that postexercise enhancement in arterial function may mitigate the acute impact of exercise on platelet activation.

Relationship between monocyte-platelet aggregation and endothelial function in middle-aged and elderly adults.

Low-grade inflammation, endothelial dysfunction, and platelet hyper-reactivity to agonists are associated with an increased risk of cardiovascular events. In vitro and animal studies infer an inverse mechanistic relationship between platelet activation and the production of endothelium-derived nitric oxide and prostacyclin. This concept is supported by evidence of an inverse relationship between endothelial function and platelet activation in high-risk cardiac patients. The aim of this study was to investigate what relationship, if any, exists between platelet and endothelial function in healthy, middle-aged, and elderly adults. In 51 participants (18 male, 33 post menopausal female), endothelial function was assessed by flow-mediated dilation (FMD). Platelet function was assessed by flow cytometric determination of glycoprotein IIb/IIIa activation (measured by PAC-1 binding), granule exocytosis (measured by surface P-selectin expression), and monocyte-platelet aggregates (MPAs), with and without stimulation by canonical platelet agonists adenosine diphosphate (ADP), arachidonic acid (AA), and collagen. Correlation analysis indicated there was no significant (all P => 0.05) relationship between FMD and any marker of in vivo platelet activation (MPAs R = 0.193, PAC-1 R = -0.113, anti-CD62P R = -0.078) or inducible platelet activation by ADP (MPA R = -0.128, anti-CD62P R = -0.237), AA (MPA R = -0.122, PAC-1 R = -0.045, anti-CD62P R = -0.142), or collagen (MPA R = 0.136, PAC-1 R = 0.174, anti-CD62P R = -0.077). Our findings contrast with two previous studies performed in high-risk cardiac patients, which reported inverse relationships between platelet activation and endothelial function, suggesting that some compensatory redundancy may exist in the relationship between platelet and endothelial function in preclinical populations.

Acute impact of conventional and eccentric cycling on platelet and vascular function in patients with chronic heart failure.

Evidence-based guidelines recommend exercise therapy for patients with chronic heart failure (CHF). Such patients have increased atherothrombotic risk. Exercise can transiently increase platelet activation and reactivity and decrease vascular function in healthy participants, although data in CHF are scant. Eccentric (ECC) cycling is a novel exercise modality that may be particularly suited to patients with CHF, but the acute impacts of ECC cycling on platelet and vascular function are currently unknown. Our null hypothesis was that ECC and concentric (CON) cycling, performed at matched external workloads, would not induce changes in platelet or vascular function in patients with CHF. Eleven patients with heart failure with reduced ejection fraction (HFrEF) took part in discrete bouts of ECC and CON cycling. Before and immediately after exercise, vascular function was assessed by measuring diameter and flow-mediated dilation (FMD) of the brachial artery. Platelet function was measured by the flow cytometric determination of glycoprotein IIb/IIIa activation and granule exocytosis in the presence and absence of platelet agonists. ECC cycling increased baseline artery diameter (pre: 4.0 ± 0.8 mm vs. post: 4.2 ± 0.7 mm; P = 0.04) and decreased FMD%. When changes in baseline artery diameter were accounted for, the decrease in FMD post-ECC cycling was no longer significant. No changes were apparent after CON. Neither ECC nor CON cycling resulted in changes to any platelet-function measures (all P > 0.05). These results suggest that both ECC and CON cycling, at a moderate intensity and short duration, can be performed by patients with HFrEF without detrimental impacts on vascular or platelet function.NEW & NOTEWORTHY This is the first evidence to indicate that eccentric (ECC) cycling can be performed relatively safely by patients with chronic heart failure (CHF), as it did not result in impaired vascular or platelet function compared with conventional cycling. This is important, as acute exercise can transiently increase atherothrombotic risk, and ECC cycling is a novel exercise modality that may be particularly suited to patients with CHF.

Imaging flow cytometry in the assessment of leukocyte-platelet aggregates.

Platelets are subcellular blood elements with a well-established role in haemostasis. Upon activation platelets undergo granule exocytosis, resulting in α-granule P-Selectin being expressed on the cell membrane. This allows binding of activated platelets to P-Selectin glycoprotein ligand 1 (PSGL-1) expressing leukocytes, forming leukocyte-platelet aggregates (LPAs). Whole blood flow cytometry (FCM) has demonstrated that elevated circulating LPAs (especially monocyte LPAs) are linked to atherothrombosis in high risk patients, and that activated platelet binding influences monocytes towards a pro-adhesive and pro-atherogenic phenotype. However, a limitation of conventional FCM is the potential for coincident events to resemble LPAs despite no tethering. Imaging cytometry can be used to characterize LPA formation and distinguish circulating MPAs from coincidental events. Platelets and leukocyte subsets are identified by expression of surface markers (e.g. the lipopolysachharide receptor CD14 on monocytes, glycoprotein Ib CD42b on platelets). In conventional FCM, all events with both leukocyte and platelet characteristics are designated as LPAs. However, by using an 'internal' mask based on the brightfield image and the fluorescent platelet identifier, imaging flow cytometry is able to distinguish leukocytes with tethered platelets (genuine LPAs) from leukocyte with coincidental, untethered platelets nearby. Mechanisms (e.g. adhesion molecules) or consequences (e.g. signal transduction) can then be separately analysed in platelet tethered and untethered leukocytes. Imaging flow cytometry therefore provides a more accurate approach for both enumeration and analysis of LPAs than conventional FCM.

Impact of commonly prescribed exercise interventions on platelet activation in physically inactive and overweight men.

The exercise paradox infers that, despite the well-established cardioprotective effects of repeated episodic exercise (training), the risk of acute atherothrombotic events may be transiently increased during and soon after an exercise bout. However, the acute impact of different exercise modalities on platelet function has not previously been addressed. We hypothesized that distinct modalities of exercise would have differing effects on in vivo platelet activation and reactivity to agonists which induce monocyte-platelet aggregate (MPA) formation. Eight middle-aged (53.5 ± 1.6 years) male participants took part in four 30 min experimental interventions (aerobic AE, resistance RE, combined aerobic/resistance exercise CARE, or no-exercise NE), in random order. Blood samples were collected before, immediately after, and 1 h after each intervention, and incubated with one of three agonists of physiologically/clinically relevant pathways of platelet activation (thrombin receptor activating peptide-6 TRAP, arachidonic acid AA, and cross-linked collagen-related peptide xCRP). In the presence of AA, TRAP, and xCRP, both RE and CARE evoked increases in MPAs immediately post-exercise (P < 0.01), whereas only AA significantly increased MPAs immediately after AE (P < 0.01). These increases in platelet activation post-exercise were transient, as responses approached pre-exercise levels by 1 h. These are the first data to suggest that exercise involving a resistance component in humans may transiently increase platelet-mediated thrombotic risk more than aerobic modalities.

An active, collaborative approach to learning skills in flow cytometry.

Advances in science education research have the potential to improve the way students learn to perform scientific interpretations and understand science concepts. We developed active, collaborative activities to teach skills in manipulating flow cytometry data using FlowJo software. Undergraduate students were given compensated clinical flow cytometry listmode output (FCS) files and asked to design a gating strategy to diagnose patients with different hematological malignancies on the basis of their immunophenotype. A separate cohort of research trainees was given uncompensated data files on which they performed their own compensation, calculated the antibody staining index, designed a sequential gating strategy, and quantified rare immune cell subsets. Student engagement, confidence, and perceptions of flow cytometry were assessed using a survey. Competency against the learning outcomes was assessed by asking students to undertake tasks that required understanding of flow cytometry dot plot data and gating sequences. The active, collaborative approach allowed students to achieve learning outcomes not previously possible with traditional teaching formats, for example, having students design their own gating strategy, without forgoing essential outcomes such as the interpretation of dot plots. In undergraduate students, favorable perceptions of flow cytometry as a field and as a potential career choice were correlated with student confidence but not the ability to perform flow cytometry data analysis. We demonstrate that this new pedagogical approach to teaching flow cytometry is beneficial for student understanding and interpretation of complex concepts. It should be considered as a useful new method for incorporating complex data analysis tasks such as flow cytometry into curricula.

Comprehensive Assessment of the Hemostatic System in Polycystic Ovarian Syndrome.

Polycystic ovarian syndrome (PCOS) affects 12 to 19% of women and has reproductive and metabolic features (endothelial dysfunction, increased diabetes, and cardiovascular risk factors). It also appears to have altered coagulation and fibrinolysis with a prothrombotic state with epidemiological evidence of increased venous thromboembolism. We aimed to comprehensively assess hemostasis in women with PCOS versus control women. In an established case-control cohort of lean, overweight, and obese women with (n = 107) and without PCOS (n = 67), with existing measures of plasminogen activator inhibitor 1 (PAI-1), asymmetric dimethylarginine (ADMA), hormonal, and metabolic markers, we also assessed prothrombin fragments 1 and 2 (PF1 & 2), plasminogen, tissue plasminogen activator (tPA), and thrombin generation (TG). Higher levels of ADMA (0.70 vs. 0.39 µmol/L, p < 0.01), PAI-1 (4.80 vs. 3.66 U/mL, p < 0.01), and plasminogen (118.39 vs. 108.46%, p < 0.01) were seen in PCOS versus controls, and persisted after adjustment for age and body mass index (BMI). PF1 & 2 was marginally lower (180.0 vs. 236.0 pmol/L, p = 0.05), whereas tPA and TG were not different between groups, after adjustment for age and BMI. Significant relationships were observed between hormonal and metabolic factors with ADMA and PAI-1. We demonstrate impaired fibrinolysis in PCOS. In the context of abnormal endothelial function and known hormonal and metabolic abnormalities, this finding may underpin an increased risk of cardiovascular disease and venous thrombosis in PCOS.