High-mobility group box 1 increases platelet surface P2Y12 and platelet activation in sickle cell disease.

Thrombosis and inflammation are intimately linked and synergistically contribute to the pathogenesis of numerous thromboinflammatory diseases, including sickle cell disease (SCD). While platelets are central to thrombogenesis and inflammation, the molecular mechanisms of crosstalk between the 2 remain elusive. High-mobility group box 1 (HMGB1) regulates inflammation and stimulates platelet activation through Toll-like receptor 4. However, it remains unclear whether HMGB1 modulates other thrombotic agonists to regulate platelet activation. Herein, using human platelets, we demonstrate that HMGB1 significantly enhanced ADP-mediated platelet activation. Furthermore, inhibition of the purinergic receptor P2Y12 attenuated HMGB1-dependent platelet activation. Mechanistically, we show that HMGB1 stimulated ADP secretion, while concomitantly increasing P2Y12 levels at the platelet membrane. We show that in SCD patients, increased plasma HMGB1 levels were associated with heightened platelet activation and surface P2Y12 expression. Treatment of healthy platelets with plasma from SCD patients enhanced platelet activation and surface P2Y12, and increased sensitivity to ADP-mediated activation, and these effects were linked to plasma HMGB1. We conclude that HMGB1-mediated platelet activation involves ADP-dependent P2Y12 signaling, and HMGB1 primes platelets for ADP signaling. This complementary agonism between ADP and HMGB1 furthers the understanding of thromboinflammatory signaling in conditions such as SCD, and provides insight for therapeutic P2Y12 inhibition.

Heme stimulates platelet mitochondrial oxidant production to induce targeted granule secretion.

Hemolysis, a pathological component of many diseases, is associated with thrombosis and vascular dysfunction. Hemolytic products, including cell-free hemoglobin and free heme directly activate platelets. However, the effect of hemolysis on platelet degranulation, a central process in not only thrombosis, but also inflammatory and mitogenic signaling, remains less clear. Our group showed that hemoglobin-induced platelet activation involved the production of mitochondrial reactive oxygen species (mtROS). However, the molecular mechanism by which extracellular hemolysis induces platelet mtROS production, and whether these mtROS regulate platelet degranulation remains unknown. Here, we demonstrate using isolated human platelets that cell free heme is a more potent agonist for platelet activation than hemoglobin, and stimulates the release of a specific set of molecules, including the glycoprotein thrombospondin-1 (TSP-1), from the α-granule of platelets. We uncover the mechanism of heme-mediated platelet mtROS production which is dependent on the activation of platelet toll-like receptor 4 (TLR4) signaling and leads to the downstream phosphorylation and inhibition of complex-V by the serine kinase Akt. Notably, inhibition of platelet TLR4 or Akt, or scavenging of mtROS prevents heme-induced granule release in vitro. Further, heme-dependent granule release is significantly attenuated in vivo in mice lacking TLR4 or those treated with the mtROS scavenger MitoTEMPO. These data elucidate a novel mechanism of TLR4-mediated mitochondrial regulation, establish the mechanistic link between hemolysis and platelet degranulation, and begin to define the heme and mtROS-dependent platelet secretome. These data have implications for hemolysis-induced thrombo-inflammatory signaling and for the consideration of platelet mitochondria as a therapeutic target in hemolytic disorders.

Doctors' engagement with a formal system of continuing professional development in Ireland: a qualitative study in perceived benefits, barriers and potential improvements.

OBJECTIVES: To examine experiences of participation in a mandatory system of continuing professional development (CPD) among doctors in Ireland, in order to identify areas for improvement. DESIGN: A qualitative cross-sectional design was used. PARTICIPANTS: 1408 participants (701 male, 707 female) were recruited via email from a population of 4350 doctors enrolled on a Royal College of Physicians of Ireland Professional Competence Scheme (PCS) for the 2017/2018 year, and completed an online survey as part of a larger study examining experiences and attitudes towards participation in PCS. A subset of the sample (434 participants) responded to an optional open-ended question about PCS participation. Responses to the open-ended question were analysed using thematic analysis. RESULTS: Thematic analysis resulted in five main themes relating to perceived barriers to PCS participation across a wide range of areas: 'Evidence of participation', 'The structure of PCS', 'Questioning the benefits of formal CPD', 'Workplace challenges' and 'Access issues'. CONCLUSIONS: Taken together, the five themes outlined in this study give a wide-ranging, in-depth picture of the challenges faced by Irish doctors, which expand on well-documented factors such as time constraints, to illustrate a series of complex, interacting factors. Some barriers, such as difficulty obtaining evidence of participation, may be relatively easily addressed. Others, such as issues with the way the PCS is structured, are more intractable, and require further research to understand more fully and develop appropriate solutions.

Primary thromboprophylaxis to prevent thrombotic events in pediatric oncology patients with a malignant mediastinal mass.

Children with malignant mediastinal masses have increased thrombotic events (TE). Eligible subjects with malignant mediastinal masses between January 2000 and December 2017 were evaluated for TE, with 19 among 76 subjects receiving enoxaparin thromboprophylaxis. There were 13 TEs among 76 subjects for an incidence of 17.1%. Mediastinal compression directly led to TE in 9.2% of subjects who also had statistically significant superior vena cava compression at diagnosis. Primary thromboprophylaxis did not significantly affect TE occurrence; however, larger studies are warranted to consider strategic thromboprophylaxis guided by radiological monitoring of dynamic vascular compression to improve TE outcomes.

Attitudes, Perceived Benefits, and Experiences of Engagement With Professional Competence Schemes for Doctors in Ireland: Findings From a National Survey.

INTRODUCTION: This study aimed to assess the attitudes, perceived benefits, and experiences of engaging with a formal system of continuing professional development (CPD) in the form of Professional Competence Schemes (PCSs) among doctors in Ireland. METHODS: The study utilized an anonymous online survey measure with both open-ended and Likert-scale questions. The measure examined general attitudes, perceived impact, and experiences of engagement with PCS. This study examines the scale items only. All 4350 doctors enrolled on a PCS were invited through email to complete the survey. One thousand four hundred eight doctors participated, indicating a response rate of 32%. RESULTS: Descriptive and inferential statistics (analysis of variance and Kruskal-Wallis) were performed on the scale items using IBM SPSS statistics, with group comparisons examining differences in responses according to gender and current primary role. A majority (80%) of respondents had positive overall attitudes to PCS. Most participants reported that CPD positively impacts their practice across a wide range of areas, particularly patient care. Most participants reported positive experiences engaging with PCS, although a minority reported difficulties, including lack of time to participate in and record CPD activities, difficulties obtaining evidence of participation, and feeling that their CPD activities are not easily captured by the PCS system. Significant differences in responses according to role were found for overall attitudes (confidence interval = 95%, P < .00) and overall experiences of PCS (confidence interval = 95%, P < .00) with nonconsultant hospital doctors emerging as a particularly vulnerable group with less positive attitudes and experiences. DISCUSSION: Across the board overall attitudes, perceived impact and experiences of PCS were positive, and indicate that doctors feel that PCS is a valuable and beneficial activity. However, a minority of respondents reported experiencing difficulties with engagement, and these areas of difficulty represent targets for future improvement.

Mitochondrial reactive oxygen species scavenging attenuates thrombus formation in a murine model of sickle cell disease.

BACKGROUND: Sickle cell disease (SCD) is characterized by hemolysis-associated platelet dysfunction that leads to increased risk of thrombosis and plays a role in the high morbidity and mortality of the disease. The mechanisms by which hemolysis induces platelet activation remain unclear. We recently demonstrated that patients with SCD showed increased platelet mitochondrial reactive oxygen species (mtROS) production that correlates with markers of hemolysis and platelet activation. Experiments in isolated platelets demonstrated that mtROS stimulated platelet activation. However, the role of hemolysis-induced mtROS in thrombus formation in vivo remains unclear. OBJECTIVES: Here, we hypothesize that scavenging of mtROS attenuates the propensity for thrombosis in mouse models of hemolysis. METHODS: We used models of hemolysate infusion into wildtype mice as well as the Berkley transgenic mouse model of SCD, a chronic mode of hemolysis, to test the effect of hemolysis on platelet mtROS production and thrombosis. RESULTS: We show that infusion of hemolysate in wildtype mice induces platelet mtROS production and decreases time to vessel occlusion in a model of ferric chloride-induced carotid artery thrombosis. Increased mtROS and propensity for thrombosis was also observed in the Berkley transgenic mouse model of SCD. Notably, treatment with mtROS scavengers decreased platelet mtROS levels and attenuated the propensity for thrombus formation in both models. CONCLUSIONS: These data demonstrate that mtROS significantly contribute to the mechanism of hemolysis-induced thrombosis in vivo and suggest a potential role for mitochondrially targeted antioxidant therapy in hemolysis and SCD-related thrombosis.

Atypical Outcomes for Hepatitis-associated Acquired Aplastic Anemia: 2 Case Studies and Review of the Literature.

There is little data specifically dedicated to the long-term outcomes of the hepatitis-associated variant of aplastic anemia (HAAA). A majority of patients with nonsevere (moderate) aplastic anemia progress to severe aplastic anemia, and severe aplastic anemia typically results in death if left untreated. We present 2 unique cases of HAAA that contribute to our knowledge of the natural history of this disease variant. One patient had moderate HAAA that never progressed to severe disease. The second patient had severe HAAA that spontaneously resolved without treatment. The rare possibility of moderate HAAA failing to progress to fulfill severe criteria, or of severe HAAA spontaneously improving, may complicate early treatment decisions for some patients.

Mitochondrial electron transport chain: Oxidative phosphorylation, oxidant production, and methods of measurement.

The mitochondrial electron transport chain utilizes a series of electron transfer reactions to generate cellular ATP through oxidative phosphorylation. A consequence of electron transfer is the generation of reactive oxygen species (ROS), which contributes to both homeostatic signaling as well as oxidative stress during pathology. In this graphical review we provide an overview of oxidative phosphorylation and its inter-relationship with ROS production by the electron transport chain. We also outline traditional and novel translational methodology for assessing mitochondrial energetics in health and disease.

Redox Signaling in Sickle Cell Disease.

Sickle cell disease (SCD) is characterized by chronic hemolysis and repeated episodes of vascular occlusion leading to progressive organ injury. SCD is characterized by unbalanced, simultaneous pro-oxidant and anti-oxidant processes at the molecular, cellular and tissue levels, with the majority of reactions tipped in favor of pro-oxidant pathways. In this brief review we discuss new findings regarding how oxidized hemin, hemolysis, mitochondrial dysfunction and the innate immune system generate oxidative stress while hemopexin, haptoglobin, heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) may provide protection in human and murine SCD. We will also describe recent clinical trials showing beneficial effects of antioxidant therapy in SCD.

Fatal Nongroupable Neisseria meningitidis Disease in Vaccinated Patient Receiving Eculizumab.

Patients receiving eculizumab have an increased risk for meningococcal disease, but most reported cases are attributable to encapsulated meningococcal strains. We describe a case in which a nongroupable meningococcal strain, which rarely causes disease in healthy persons, caused fatal disease in an eculizumab recipient despite meningococcal vaccination.