Platelet-derived extracellular vesicles in cardiovascular disease and treatment - from maintaining homeostasis to targeted drug delivery.

PURPOSE OF THE 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.

COVID-19 induces a hyperactive phenotype in circulating platelets.

Coronavirus Disease 2019 (COVID-19), caused by the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has affected over 30 million globally to date. Although high rates of venous thromboembolism and evidence of COVID-19-induced endothelial dysfunction have been reported, the precise aetiology of the increased thrombotic risk associated with COVID-19 infection remains to be fully elucidated. Therefore, we assessed clinical platelet parameters and circulating platelet activity in patients with severe and nonsevere COVID-19. An assessment of clinical blood parameters in patients with severe COVID-19 disease (requiring intensive care), patients with nonsevere disease (not requiring intensive care), general medical in-patients without COVID-19, and healthy donors was undertaken. Platelet function and activity were also assessed by secretion and specific marker analysis. We demonstrated that routine clinical blood parameters including increased mean platelet volume (MPV) and decreased platelet:neutrophil ratio are associated with disease severity in COVID-19 upon hospitalisation and intensive care unit (ICU) admission. Strikingly, agonist-induced ADP release was 30- to 90-fold higher in COVID-19 patients compared with hospitalised controls and circulating levels of platelet factor 4 (PF4), soluble P-selectin (sP-selectin), and thrombopoietin (TPO) were also significantly elevated in COVID-19. This study shows that distinct differences exist in routine full blood count and other clinical laboratory parameters between patients with severe and nonsevere COVID-19. Moreover, we have determined all COVID-19 patients possess hyperactive circulating platelets. These data suggest abnormal platelet reactivity may contribute to hypercoagulability in COVID-19 and confirms the role that platelets/clotting has in determining the severity of the disease and the complexity of the recovery path.

Markers of platelet activation foR identification of late onset sEpsis in infaNTs: PARENT study protocol.

BACKGROUND: Newborns are at high risk of sepsis. At present there is no definitive "rule in" blood test for sepsis at the point of clinical concern. A positive blood culture remains the gold standard test for neonatal sepsis, however laboratory markers that correlate prospectively with culture positive sepsis could aid clinicians in making decisions regarding administration of empiric antibiotic therapies. METHODS: This multi-site, prospective observational study will take place in two neonatal intensive care units (National Maternity Hospital and Rotunda Hospital, Dublin). Neonates born at less than 34 weeks will be enroled and informed consent obtained prior to late onset sepsis work up. If at any point subsequently during their neonatal intensive care stay they develop signs and symptoms of possible sepsis requiring blood culture, an additional sodium citrate sample will be obtained. Infants will be categorised into three groups as follows: (i) culture positive sepsis, (ii) culture negative sepsis where an infant receives 5 days of antibiotics (iii) non sepsis. Our primary outcome is to establish if differential platelet/endothelial activation can prospectively identify neonatal culture positive late onset sepsis. TRIAL REGISTRATION NUMBER: NCT05530330 IMPACT: Preterm infants are a high risk group for the development of sepsis which is a major cause of mortality in this population. Platelets have been associated with host response to invasive bacterial infections both in animal models and translational work. A positive blood culture is the gold standard test for neonatal sepsis but can be unreliable due to limited blood sampling in the very low birth weight population. This study hopes to establish if platelet/endothelial associated plasma proteins can prospectively identify late onset neonatal sepsis.

Platelets in pediatric and neonatal sepsis: novel mediators of the inflammatory cascade.

Sepsis, a dysregulated host response to infection, has been difficult to accurately define in children. Despite a higher incidence, especially in neonates, a non-specific clinical presentation alongside a lack of verified biomarkers has prevented a common understanding of this condition. Platelets, traditionally regarded as mediators of haemostasis and thrombosis, are increasingly associated with functions in the immune system with involvement across the spectrum of innate and adaptive immunity. The large number of circulating platelets (approx. 150,000 cells per microlitre) mean they outnumber traditional immune cells and are often the first to encounter a pathogen at a site of injury. There are also well-described physiological differences between platelets in children and adults. The purpose of this review is to place into context the platelet and its role in immunology and examine the evidence where available for its role as an immune cell in childhood sepsis. It will examine how the platelet interacts with both humoral and cellular components of the immune system and finally discuss the role the platelet proteome, releasate and extracellular vesicles may play in childhood sepsis. This review also examines how platelet transfusions may interfere with the complex relationships between immune cells in infection. IMPACT: Platelets are increasingly being recognised as important "first responders" to immune threats. Differences in adult and paediatric platelets may contribute to differing immune response to infections. Adult platelet transfusions may affect infant immune responses to inflammatory/infectious stimuli.

The role of the calibrated automated thrombogram in neonates: describing mechanisms of neonatal haemostasis and evaluating haemostatic drugs.

Premature infants are at high risk of haemorrhage and thrombosis. Our understanding of the differences between the neonatal and adult haemostatic system is evolving. There are several limitations to the standard coagulation tests used in clinical practice, and there is currently a lack of evidence to support many of the transfusion practices in neonatal medicine. The evaluation of haemostasis is particularly challenging in neonates due to their limited blood volume. The calibrated automated thrombogram (CAT) is a global coagulation assay, first described in 2002, which evaluates both pro- and anti-coagulant pathways in platelet-rich or platelet-poor plasma. In this review, the current applications and limitations of CAT in the neonatal population are discussed.Conclusion: CAT has successfully elucidated several differences between haemostatic mechanisms in premature and term neonates compared with adults. Moreover, it has been used to evaluate the effect of a number of haemostatic drugs in a pre-clinical model. However, the lack of evidence of CAT as an accurate predictor of neonatal bleeding, blood volume required and the absence of an evidence-based treatment algorithm for abnormal CAT results limit its current application as a bedside clinical tool for the evaluation of sick neonates. What is Known: • The Calibrated automated thrombogram (CAT) is a global coagulation assay which evaluates pro- and anti-coagulant pathways. • CAT provides greater information than standard clotting tests and has been used in adults to evaluate bleeding risk. What is New: • This review summarises the physiological differences in haemostasis between neonates and adults described using CAT. • The haemostatic effect of several drugs has been evaluated in neonatal plasma using CAT.

A review of the role of extracellular vesicles in neonatal physiology and pathology.

Extracellular vesicles (EVs) are cell-derived membrane-bound particles, extensively investigated across many fields to improve the understanding of pathophysiological processes, as biomarkers of disease and as therapeutic targets for pharmacological intervention. We aim to describe the current knowledge of EVs detected in the body fluids of human neonates, both term and preterm, from birth to 4 weeks of age. To date, EVs have been described in several neonatal body fluids, including cerebrospinal fluid, umbilical cord blood, neonatal blood, tracheal aspirates and urine. These studies demonstrate some important roles of EVs in the neonatal population, particularly in haemostasis. Moreover, some studies have demonstrated the pathophysiological mechanisms and the identification of potential biomarkers of neonatal disease. We must continue to build on this knowledge, evaluating the role of EVs in neonatal pathology, particularly in prematurity and during the perinatal adaption period. Future studies should use larger numbers, robust EV characterisation techniques and always correlate the findings to clinical outcomes. IMPACT: This article summarises the current knowledge of the effect of EVs in neonates. It describes the potential compensatory role of EVs in neonatal haemostasis. It also describes the role of EVs as mediators of pathology and as potential biomarkers of perinatal and neonatal disease.

Proteomic Analysis Reveals a Strong Association of ß-Catenin With Cadherin Adherens Junctions in Resting Human Platelets.

It was previously demonstrated that the WNT/ß-catenin pathway is present and active in platelets and established that the canonical WNT ligand, WNT-3a, suppresses platelet adhesion and activation. In nucleated cells, ß-catenin, the key downstream effector of this pathway, is a dual function protein, regulating the coordination of gene transcription and cell-cell adhesion. The specific role of ß-catenin in the anucleate platelet however remains elusive. Here, a label-free quantitative proteomic analysis of ß-catenin immunoprecipitates from human platelets is performed and nine co-immunoprecipitating proteins are identified. Three of the co-immunoprecipitating proteins (α-catenin-1, cadherin-6, and ß-catenin-interacting protein 1) are common to both resting and activated conditions. Bioinformatics analysis of proteomics data reveal a strong association of the dataset with both cadherin adherens junctions and regulators of WNT signaling. It is then verified that platelet ß-catenin and cadherin-6 interact and that this interaction is regulated by the activation state of the platelet. Taken together, this proteomics study suggests a novel role for ß-catenin in human platelets where it interacts with platelet cadherins and associated junctional proteins.

Platelet Releasate Proteome Profiling Reveals a Core Set of Proteins with Low Variance between Healthy Adults.

Upon activation, platelets release a powerful cocktail of soluble and vesicular signals, collectively termed the "platelet releasate" (PR). Although several studies have used qualitative/quantitative proteomic approaches to characterize PR; with debated content and significant inter-individual variability reported, confident, and reliable insights have been hindered. Using label-free quantitative (LFQ)-proteomics analysis, a reproducible, quantifiable investigation of the 1U mL-1 thrombin-induced PR from 32 healthy adults was conducted. MS proteomics data are available via ProteomeXchange, identifier PXD009310. Of the 894 proteins identified, 277 proteins were quantified across all donors and form a "core" PR. Bioinformatics and further LFQ-proteomic analysis revealed that the majority (84%) of "core" PR proteins overlapped with the protein composition of human platelet-derived exosomes. Vesicles in the exosomal-size range were confirmed in healthy-human PR and reduced numbers of similar-sized vesicles were observed in the PR of a mouse model of gray platelet syndrome, known to be deficient in platelet alpha-granules. Lastly, the variability of proteins in the PR was assessed, and reproducible secretion levels were found across all 32 healthy donors. Taken together, the PR contains valuable soluble and vesicular cargo and has low-population variance among healthy adults, rendering it a potentially useful platform for diagnostic fingerprinting of platelet-related disease.

Early onset preeclampsia is associated with an elevated mean platelet volume (MPV) and a greater rise in MPV from time of booking compared with pregnant controls: results of the CAPE study.

OBJECTIVE: To characterise Mean platelet volume (MPV) in patients with early onset preeclampsia (EOPE) and unaffected controls from time of first antenatal visit until the postpartum. MATERIALS AND METHODS: Retrospective secondary analysis of an observational study in an Irish tertiary referral centre with 9000 deliveries annually. The MPV of 27 women with EOPE was compared to 19 unaffected controls. The inclusion criteria for the disease state was the development of EOPE defined by the National Institute for Health and Care Excellence (NICE) guideline, as new onset hypertension presenting after 20 weeks and prior to 34 weeks with significant proteinuria. Between October 2013 and July 2015 we recruited 27 women with EOPE and 19 pregnant controls. Statistical analysis was performed using paired T-test of Mann-Whitney test where appropriate and a P-value <0.05 was deemed significant. RESULTS: At time of diagnosis and late in the third trimester MPV was significantly increased to 9.0 (±0.3) fL in cases of EOPE in comparison to 8.5 (±0.6) fL in normotensive controls (P<0.05). There was no significant difference during the first trimester or postpartum when comparing the MPV in EOPE to controls. CONCLUSION: Despite an increased MPV at time of diagnosis of EOPE this study did not demonstrate a potential use for increased MPV as a first trimester screening tool.

Comparative proteomic analysis of trophoblast cell models reveals their differential phenotypes, potential uses, and limitations.

Trophoblastic cell lines are widely used in in vitro studies of placental function as a surrogate for primary trophoblasts. To date, no reference proteomics dataset exists to directly compare the shared and unique characteristics of these cells. Here, we performed comparative proteomic profiling of the BeWo and HTR8/SVneo cell lines using label-free quantitative MS. A total of 1557 proteins were identified, which included 338 uniquely attributed to BeWo cells, and a further 304 specifically identified in HTR8/SVneo cells. Raw data are available via ProteomeXchange, identifier PDX005045. Of the 915 proteins expressed by both cell lines, 105 were of higher abundance in BeWo cells, while 199 proteins had a significantly higher expression in HTR8/SVneo cells. Comparative GO of unique and upregulated proteins revealed principal differences in cell junction/adhesion, catenin complex, spindle and microtubule associated complex, as well as cell differentiation. Our data indicate that BeWo cells express an epithelial proteome more characteristic of villous trophoblasts, whereas HTR8/SVneo cells embrace a mesenchymal phenotype, more characteristic of extravillous trophoblasts. This novel comparative proteomic profiling of these trophoblastic cell lines provides a useful platform for future investigations of placental function.

Increased soluble GPVI levels in cirrhosis: evidence for early in vivo platelet activation.

Cirrhosis is a consequence of prolonged liver injury and is characterised by extensive tissue fibrosis: the deposition of collagen-rich extracellular matrix. The haemostatic balance is disordered in cirrhosis and coagulation activation appears to promote fibrosis. In spite of recent studies demonstrating a role for anticoagulant therapy in preventing cirrhosis progression, there has not been a change in clinical practice, suggesting that physicians are reluctant to anticoagulate patients with cirrhosis due to bleeding risks. Platelets play an important role in facilitating coagulation. Glycoprotein VI (GPVI) is a platelet-specific collagen receptor that is shed from the platelet surface in a metalloproteinase-dependent manner in response to GPVI ligation and coagulation activation. Our aim was to use soluble GPVI levels to determine whether there was evidence for collagen and coagulation-induced platelet activation in early, well-compensated cirrhosis. Plasma soluble GPVI levels were quantified in 46 patients with mixed aetiology cirrhosis and 55 healthy controls using an immunoassay. In the cirrhosis group, soluble GPVI levels were significantly increased (5.8 ± 4.4 ng/ml, n = 46) compared to healthy controls (3.3 ± 3.4 ng/ml, n = 55, p < 0.05). This increase in soluble GPVI levels was still evident when levels were adjusted for platelet count (Healthy controls; 0.015 ± 0.018 ng/106 platelets/ml vs. cirrhosis; 0.048 ± 0.04 ng/106 platelets/ml, p < 0.0001). This study provides evidence for early platelet activation in patients with well-compensated cirrhosis. This may have translational implications for prognosis, treatment, and risk stratification.

Analysis of the proteins associated with platelet detergent resistant membranes.

Proteomic studies have facilitated the identification of proteins associated with the detergent-resistant membrane (DRM) fraction in a variety of cell types. Here, we have undertaken label-free quantitative (LFQ) proteomic profiling of the proteins associated with detergent-resistant plasma and internal membranes from resting and activated platelets. One hundred forty-one proteins were identified and raw data is available via ProteomeXchange with identifier PXD002554. The proteins identified include a myriad of important platelet signaling and trafficking proteins including Rap1b, Src, SNAP-23, syntaxin-11, and members of the previously unattributed Ragulator complex. Mean LFQ intensities calculated across three technical replicates for the three biological donors revealed that several important platelet signaling proteins altered their detergent solubility upon activation, including GPIbα, GPIbß, Src, and 14-3-3ζ. Altered detergent solubility for GPIbα, following activation using a variety of platelet agonists, was confirmed by immunoblotting and further coimmunoprecipitation experiments revealed that GPIbα forms a complex with 14-3-3ζ that shifts into DRMs following activation. Taken together, proteomic profiling of platelet DRMs allowed greater insight in the complex biology of both DRMs and platelets and will be a useful subproteome to study platelet-related disease. All MS data have been deposited in the ProteomeXchange with identifier PXD002554 (http://proteomecentral.proteomexchange.org/dataset/PXD002554).

Nurse Engagement in Shared Governance and Patient and Nurse Outcomes.

OBJECTIVE: The objectives of this study were to examine differences in nurse engagement in shared governance across hospitals and to determine the relationship between nurse engagement and patient and nurse outcomes. BACKGROUND: There is little empirical evidence examining the relationship between shared governance and patient outcomes. METHODS: A secondary analysis of linked cross-sectional data was conducted using nurse, hospital, and Hospital Consumer Assessment of Healthcare Providers and Systems (HCAHPS) survey data. RESULTS: Engagement varied widely across hospitals. In hospitals with greater levels of engagement, nurses were significantly less likely to report unfavorable job outcomes and poor ratings of quality and safety. Higher levels of nurse engagement were associated with higher HCAHPS scores. CONCLUSIONS: A professional practice environment that incorporates shared governance may serve as a valuable intervention for organizations to promote optimal patient and nurse outcomes.

Canonical Wnt signaling in megakaryocytes regulates proplatelet formation.

Wnt signaling is involved in numerous aspects of vertebrate development and homeostasis, including the formation and function of blood cells. Here, we show that canonical and noncanonical Wnt signaling pathways are present and functional in megakaryocytes (MKs), with several Wnt effectors displaying MK-restricted expression. Using the CHRF288-11 cell line as a model for human MKs, the canonical Wnt3a signal was found to induce a time and dose-dependent increase in ß-catenin expression. ß-catenin accumulation was inhibited by the canonical antagonist dickkopf-1 (DKK1) and by the noncanonical agonist Wnt5a. Whole genome expression analysis demonstrated that Wnt3a and Wnt5a regulated distinct patterns of gene expression in MKs, and revealed a further interplay between canonical and noncanonical Wnt pathways. Fetal liver cells derived from low-density-lipoprotein receptor-related protein 6-deficient mice (LRP6(-/-)), generated dramatically reduced numbers of MKs in culture of lower ploidy (2N and 4N) than wild-type controls, implicating LRP6-dependent Wnt signaling in MK proliferation and maturation. Finally, in wild-type mature murine fetal liver-derived MKs, Wnt3a potently induced proplatelet formation, an effect that could be completely abrogated by DKK1. These data identify novel extrinsic regulators of proplatelet formation, and reveal a profound role for Wnt signaling in platelet production.

Extracellular vesicles in disorders of hemostasis following traumatic brain injury.

Traumatic brain injury (TBI) is a global health priority. In addition to being the leading cause of trauma related death, TBI can result in long-term disability and loss of health. Disorders of haemostasis are common despite the absence of some of the traditional risk factors for coagulopathy following trauma. Similar to trauma induced coagulopathy, this manifests with a biphasic response consisting of an early hypocoagulable phase and delayed hypercoagulable state. This coagulopathy is clinically significant and associated with increased rates of haemorrhagic expansion, disability and death. The pathophysiology of TBI-induced coagulopathy is complex but there is biologic plausibility and emerging evidence to suggest that extracellular vesicles (EVs) have a role to play. TBI and damage to the blood brain barrier result in release of brain-derived EVs that contain tissue factor and phosphatidylserine on their surface. This provides a platform on which coagulation can occur. Preclinical animal models have shown that an early rapid release of EVs results in overwhelming activation of coagulation resulting in a consumptive coagulopathy. This phenomenon can be attenuated with administration of substances to promote EV clearance and block their effects. Small clinical studies have demonstrated elevated levels of procoagulant EVs in patients with TBI correlating with clinical outcome. EVs represent a promising opportunity for use as minimally invasive biomarkers and potential therapeutic targets for TBI patients. However, additional research is necessary to bridge the gap between their potential and practical application in clinical settings.

The effect of UVA light/8-methoxypsoralen exposure used in Extracorporeal Photopheresis treatment on platelets and extracellular vesicles.

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.

Rivaroxaban in combination with low-dose aspirin is associated with a reduction in pro-inflammatory and pro-thrombotic circulating vesicle signatures in patients with cardiovascular disease.

BACKGROUND: Despite secondary prevention with aspirin, patients with stable cardiovascular disease (CVD) remain at elevated long-term risk of major cardiovascular events (MACE). The COMPASS double-blind randomized clinical trial demonstrated that aspirin plus low-dose Rivaroxaban (COMPASS regime) significantly decreased the incidence of MACE by 24% compared with aspirin alone. However, the mechanisms underlying these potential synergistic/non-antithrombotic effects remain elusive. Extracellular vesicles (EVs) are crucial messengers regulating a myriad of biological/pathological processes and are highly implicated in CVD. We hypothesised that circulating EV profiles reflect the cardioprotective properties of the COMPASS regime. PATIENTS/METHODS: A cohort of stable CVD patients (n=40) who participated in the COMPASS trial and were previously randomised to receive aspirin, were prospectively recruited and assigned a revised regimen of open-label aspirin plus Rivaroxaban. Blood samples were obtained at baseline (aspirin only) and 6-months follow-up. Plasma EV concentration, size and origin were analysed by NTA and flow cytometry. EVs were enriched by ultracentrifugation for proteomic analysis. RESULTS: The COMPASS regime fundamentally altered small (<200nm) and large (200-1000nm) EV concentration and size, compared to aspirin alone. Crucially, levels of platelet-derived, and myeloperoxidase-positive EVs became significantly decreased at follow-up. Comparative proteomic characterisation further revealed a significant decrease in highly pro-inflammatory protein expression at follow-up. CONCLUSIONS: The observed changes in EV subpopulations, together with the differential protein expression profiles, suggest amelioration of an underlying pro-inflammatory and pro-thrombotic state upon dual therapy, which may be of clinical relevance towards understanding the fundamental mechanism underlying the reported superior cardiovascular outcomes associated with this antithrombotic regimen.

Proteomic analysis of extracellular vesicle cargoes mirror the cardioprotective effects of rivaroxaban in patients with venous thromboembolism.

BACKGROUND: Venous thromboembolism (VTE) remains a significant cause of morbidity and mortality worldwide. Rivaroxaban, a direct oral factor Xa inhibitor, mediates anti-inflammatory and cardiovascular-protective effects besides its well-established anticoagulant properties; yet, these remain poorly characterized. Extracellular vesicles (EVs) are considered proinflammatory messengers regulating a myriad of (patho)physiological processes and may be highly relevant to the pathophysiology of VTE. The effects of Rivaroxaban on circulating EVs in VTE patients remain unknown. We have established that differential EV biosignatures are found in patients with non-valvular atrial fibrillation anticoagulated with Rivaroxaban versus warfarin. Here, we investigated whether differential proteomic profiles of circulating EVs could also be found in patients with VTE. METHODS AND RESULTS: We performed comparative label-free quantitative proteomic profiling of enriched plasma EVs from VTE patients anticoagulated with either Rivaroxaban or warfarin using a tandem mass spectrometry approach. Of the 182 quantified proteins, six were found to be either exclusive to, or enriched in, Rivaroxaban-treated patients. Intriguingly, these proteins are involved in negative feedback regulation of inflammatory and coagulation pathways, suggesting that EV proteomic signatures may reflect both Rivaroxaban's anti-coagulatory and anti-inflammatory potential. CONCLUSIONS: These differences suggest Rivaroxaban may have pleiotropic effects, supporting the reports of its emerging anti-inflammatory and cardiovascular-protective characteristics relative to warfarin.

Measurement of bone density around the Oxford medial compartment knee replacement using iDXA. A precision study.

The goal of this study was to evaluate whether the Lunar iDXA densitometer can accurately measure the bone mineral density (BMD) around the tibial component of the Oxford unicompartment knee replacement (UKR). Both knees in 20 patients were measured 3 times in the supine position with repositioning between each scan. We chose 7 regions of interest to evaluate the bone density around the implant. Small but significant differences between the implant and nonimplanted knee were noticed with the nonimplanted knee having slightly higher BMD and bone mineral content (BMC) in areas 1-3 (p≤0.001) and area 6 (p=0.002). There was higher BMD in area 4 (p=0.028). The precision for BMD in the 7 areas of interest in the implanted knee varied between 0.55% and 4.04% and BMC between 1.8% and 5.3%. There was no significant difference in the precision between the nonimplanted and implanted knees. Prospective serial measurements around the Oxford UKR using iDXA will be able to assess specific areas of stress shielding and potential implant stability, which is likely to help predict the survival of the implant.

An optimized protocol to isolate quiescent washed platelets from human whole blood and generate platelet releasate under clinical conditions.

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.