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.

PlateletSeq: A novel method for discovery of blood-based biomarkers.

Platelets are small circulating fragments of cells that play important roles in thrombosis, haemostasis, immune response, inflammation and cancer growth. Although anucleate, they contain a rich RNA repertoire which offers an opportunity to characterise changes in platelet gene expression in health and disease. Whilst this can be achieved with conventional RNA sequencing, a large input of high-quality RNA, and hence blood volume, is required (unless a pre-amplification step is added), along with specialist bioinformatic skills for data analysis and interpretation. We have developed a transcriptomics next-generation sequencing-based approach that overcomes these limitations. Termed PlateletSeq, this method requires very low levels of RNA input and does not require specialist bioinformatic analytical skills. Here we describe the methodology, from sample collection to processing and data analysis. Specifically, blood samples can be stored for up to 8 days at 4 °C prior to analysis. Platelets are isolated using multi-step centrifugation and a purity of ≤ 1 leucocyte per 0.26x106 platelets is optimal for gene expression analysis. We have applied PlateletSeq to normal adult blood samples and show there are no age-associated variations and only minor gender-associated differences. In contrast, platelets from patients with myeloproliferative neoplasms show differences in platelet transcript profiles from normal and between disease subtypes. This illustrates the potential applicability of PlateletSeq for biomarker discovery and studying platelet biology in patient samples. It also opens avenues for assessing platelet quality in other fields such as transfusion research.

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.

Vaccine-induced immune thrombosis and thrombocytopenia syndrome following adenovirus-vectored severe acute respiratory syndrome coronavirus 2 vaccination: a novel hypothesis regarding mechanisms and implications for future vaccine development.

We hypothesize that thrombosis with thrombocytopenia syndrome recently described after administration of adenovirus-vectored vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurs as a result of the unique properties of the adenovirus vectors, which can have widespread biodistribution throughout the body. The antigen is delivered to megakaryocyte cells, which act as part of the primary immune system and distribute the antigen within progeny platelets, also a key component of the immune system. The interaction of the antigen induces preformed antiplatelet factor 4 (PF4) antibodies to bind to PF4-heparan sulfate complexes in the absence of exogenous heparin, at sites where the heparan sulfate concentration in the vascular glycocalyx is optimal for complex formation, causing thrombosis and thrombocytopenia as observed clinically. This hypothesis is testable in cell culture and animal models, and potentially in vivo, and if proven correct has significant implications for vaccine development and our understanding of the links between the coagulation and immune systems.

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.

Whole blood flow cytometry protocol for the assessment of platelet phenotype, function, and cellular interactions.

Platelets are a key component of the hemostatic system and their roles in inflammation via interactions with leukocytes have also gained attention in recent years. Changes in platelet phenotype and function can cause bleeding and/or thrombosis and, as such, monitoring platelet-specific changes is crucial to assessing hemostasis in the clinical setting. Currently, available platelet function tests such as platelet aggregometry and thromboelastography require a large volume of blood, which is a major limitation for the pediatric population. Whole blood flow cytometric analysis of platelets is increasingly utilized in recent years, primarily due to the sensitivity of this method, but also because it only requires a small amount of blood with minimal sample manipulation. We have developed a whole blood flow cytometry methodological approach that enables the assessment of platelet phenotype, function, and their interactions with monocytes and neutrophils.

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.

Guidelines for panel design, optimization, and performance of whole blood multi-color flow cytometry of platelet surface markers.

Flow cytometry is a valuable tool in determining the phenotype and function of platelets accurately. The emergence of platelet flow cytometry in recent years provides an attractive alternative to other platelet analytical techniques, with advantages such as requiring small volumes and being highly sensitive to minimal changes in receptor function and expression. Here we present a methodical approach encompassing the stages in the development and optimization of platelet flow cytometry panels based on our extensive experience in this area.

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.

Measuring the performance of patient-specific solutions for minimally invasive transforaminal lumbar interbody fusion surgery.

Pre-surgical planning using 3D-printed BioModels enables the preparation of a "patient-specific" kit to assist instrumented spinal fusion surgery. This approach has the potential to decrease operating time while also offering logistical benefits and cost savings for healthcare. We report our experience with this method in 129 consecutive patients undergoing minimally invasive transforaminal lumbar interbody fusion (MIS TLIF) over 27 months at a single centre and performed by a single surgeon. Patient imaging and surgical planning software were used to manufacture a 3D-printed patient-specific MIS TLIF kit for each patient consisting of a 1:1 scale spine BioModel, stereotactic K-wire guide, osteotomy guide, and retractors. Pre-selected pedicle screws, rods, and cages were sourced and supplied with the patient-specific kit. Additional implants were available on-shelf to address a size discrepancy between the kit implant and intraoperative measurements. Each BioModel was used pre-operatively for surgical planning, patient consent and education. The BioModel was sterilised for intraoperative reference and navigation purposes. Efficiency measures included operating time (153 ±â€¯44 min), sterile tray usage (14 ±â€¯3), fluoroscopy screening time (57.2 ±â€¯23.7 s), operative waste (19 ±â€¯8 L contaminated, 116 ±â€¯30 L uncontaminated), and median hospital stay (4 days). The pre-selected kit implants exactly matched intraoperative measurements for 597/639 pedicle screws, 249/258 rods, and 46/148 cages. Pedicle screw placement accuracy was 97.8% (625/639) on postoperative CT. Complications included one intraoperative dural tear, no blood products administered, and six reoperations. Our experience demonstrates a viable application of patient-specific 3D-printed solutions and provides a benchmark for studies of efficiency in spinal fusion surgery.

Platelet Phenotype and Function in the Setting of Pediatric Extracorporeal Membrane Oxygenation (ECMO): A Systematic Review.

Background: Despite increasing technical improvement and extracorporeal membrane oxygenation (ECMO)-related knowledge over the past three decades, morbidity and mortality associated with bleeding and clotting complications remain high in pediatric patients undergoing ECMO. Platelets, a key element of the coagulation system, have been proposed to be the main cause of coagulopathy in the setting of ECMO. This systematic review aims to summarize and discuss the existing knowledge of platelet phenotype and function in the pediatric ECMO population. Methods: A systematic review was conducted for the Embase, Medline, and PubMed databases following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Results: The detailed study selection process yielded a total of 765 studies and only 3 studies that fulfilled the selection criteria were included in this review. Techniques used to assess platelet function in the three existing studies included platelet aggregometry, flow cytometry, and thromboelastography-platelet mapping. The finding that is common to the three studies is reduced platelet function in pediatric patients during ECMO either compared to before the initiation of ECMO or in non-survivors compared to survivors. Two studies demonstrated reduced platelet aggregation that are irreversible by platelet transfusion during ECMO. Two studies reported bleeding events and mortality in children on ECMO and none of the studies investigated thrombotic events. Conclusions: This systematic review demonstrates the extremely limited information available for platelet phenotype and function in the pediatric ECMO population. Evidence from the existing literature suggests reduced platelet aggregation and increased platelet activation in children during ECMO. However, this needs to be interpreted with care due to the limitations associated with the techniques used for platelet function testing. Furthermore, the association between platelet dysfunction and clinical outcomes in the pediatric ECMO population remains elusive. Multiple research gaps have been identified when it comes to the knowledge of platelet phenotype and function of children on ECMO, highlighting the need for robust, well-designed studies in this setting.

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.