Microparticles in hemophilia - friend or foe? : to improve hemostasis or to induce atherothrombosis?

As one of the most well-known inherited bleeding disorders, hemophilia A (HA) is caused by the deficiency or dysfunction of coagulation factor VIII (FVIII). Patients with HA (PWHA) suffer from abnormal bleeding after injuries or surgeries, or even spontaneous bleeding in severe cases, particularly at joints and muscles. The main treatment for PWHA is FVIII replacement therapy. Patients with the same residual FVIII levels may present different clinical bleeding phenotypes, thus individualized adjustment of the treatment is necessary. Moreover, the risks of cardiovascular disease (CVD) are increased in middle-aged or elderly patients. The management of CVD in PHWA has become a new clinical challenge. Circulating microparticles (MPs) are small membrane vesicles that originate mostly from platelets in healthy individuals. MPs play important roles in hemostasis and thrombosis and are reported to participate in the development of CVD. However, the role of MPs in hemostasis of HA as well as in the development of CVD in PWHA remains unclear. The overall aim of this thesis was to investigate the role of MPs in HA. The first part focused on the effect of MPs on hemostasis of HA in vitro and in vivo. In the second part, the prevalence of CVD, the profiles of MPs, as well as their correlations in the middle-aged and elderly PWHA were investigated. In another study, fibrin formation for combinations of a new bispecific antibody and bypassing agents (aPCC or rFVIIa) was studied using in vitro approaches. In Paper I, we characterized the fibrin formation and fibrin clot structure in a human plasma model of severe HA after the addition of sequence-identical analogue (SIA) of emicizumab, alone or in combinations with bypassing agents (aPCC or rFVIIa). The combination of SIA and aPCC exhibited hypercoagulable patterns, suggesting that this combination might introduce thrombotic risk when used to treat PWHA. In Papers II and IV, the procoagulant effect of MPs in HA was studied using in vitro plasma models and in vivo mouse models of HA, respectively. MPs improved hemostasis and were found to be incorporated into the fibrin network in both in vitro and in vivo HA models. In Paper III, the prevalence of subclinical CVD in a Swedish PWHA (≥40 years old) cohort was determined using the advanced electrocardiography (A-ECG) technique, retrospectively. PWHA were found to have higher probabilities of developing CVD compared to the age-matched male control. In Paper V, a cross-sectional study with a Chinese cohort of PWHA treated on-demand (≥30 years old) was conducted. PWHA treated on-demand were not protected from developing subclinical CVD. No correlations between the CVD risks and MPs profiles were observed. In addition, the hypercoagulable state might lead to increased platelet activation in PWHA. Taken together, this thesis has provided new insights to understand the procoagulant effect of MPs in HA and confirmed that PWHA are not protected from developing CVD. MPs may modify the clinical bleeding phenotypes in PWHA. Moreover, MPs might be a potential biomarker for individualized therapy in PWHA, particularly for aging patients, who have increased risk of CVD

Increased Expression of Extracellular Vesicles Is Associated With the Procoagulant State in Patients With Established Rheumatoid Arthritis

This study sought to identify different subpopulations of extracellular vesicles (EVs) in plasma from female patients with established rheumatoid arthritis (RA) in relation to the activation of coagulation and fibrin formation in these patients. Forty women were included in the study, 20 patients and 20 age-matched healthy controls. The mean disease duration in patients was 13.0 (5.0-25.0) years, with medium to high disease activity despite ongoing treatment with low-dose prednisolone and methotrexate. There were no differences between the investigated groups regarding the presence of traditional cardiovascular risk factors. The concentration of phosphatidylserine-positive (PS+) EVs; platelet (CD42a(+)), leucocyte (CD45(+)), monocyte (CD14(+)), and endothelial (CD144(+))-derived EVs; and EVs-expressing tissue factor (CD142(+)), P-selectin (CD62P(+)), and E-selectin (CD62E(+)) were determined by flow cytometry analysis. Overall hemostasis potential (OHP) was assessed to follow the hemostatic disturbances, including the parameters for overall coagulation potential (OCP) and overall fibrinolytic potential (OFP). Fibrin clot turbidity was measured together with clot lysis time, and scanning electron microscopy was performed. Increased concentrations of PS+, CD42a(+), CD142(+), CD45(+), CD14(+), and CD62P(+) EVs were found in plasma from patients with RA compared to healthy controls, and the concentrations of PS+, CD42a(+), CD14(+), and CD62P(+) EVs were positively correlated with the inflammatory parameters in RA patients. Positive correlations were also found between the levels of PS+ and CD42a(+) EVs and OCP as well as between the levels of PS+, CD42a(+), and CD62P(+)EVs and OHP. The levels of PS+, CD42a(+), CD14(+), CD62P(+), and CD62E(+) EVs were negatively correlated with OFP. Elevated levels of circulating EVs of different cell origins were found in patients with established RA, in relation to the inflammatory burden and coagulation activation in the disease

Phosphatidylserine positive microparticles improve hemostasis in in-vitro hemophilia A plasma models

Circulating microparticles (MPs) are procoagulant due to the surface containing phosphatidylserine (PS), which facilitates coagulation. We investigated if MPs improve hemostasis in HA plasma models. MPs isolated from pooled normal human plasma were added to severe, moderate and mild HA plasma models (0%, 2.5%, 20% FVIII). The MPs' effect on hemostasis was evaluated by calibrated automated thrombogram (CAT) and overall hemostasis potential (OHP) assays, while fibrin structure was imaged by standard confocal, stimulated emission depletion (STED) microscopy and scanning electron microscopy (SEM). MPs partially restored thrombin generation and fibrin formation in all HA plasma models. The procoagulant effect of MPs requires PS exposure, to a less extent of contact pathway activation, but not tissue factor exposure or in vitro stimulation of MPs. MPs partially normalized the fibrin structure, and using super-resolution STED, MPs attached to fibrin were clearly resolved. In summary, our results demonstrate that PS positive MPs could improve hemostasis in HA plasma models

Molecular landscape and clinical significance of exon 11 mutations in KIT gene among patients with gastrointestinal stromal tumor: a retrospective exploratory study

ObjectiveThis aim of this study was to investigate the prognostic significance of KIT exon 11 mutation subtypes in patients with GISTs.MethodsA total of 233 consecutive patients diagnosed with GISTs at the First Affiliated Hospital of Zhengzhou University from January 2013 to August 2018 were included in this study. The prevalence and mutation landscape of exon 11 in KIT was presented. The clinicopathological characteristics and prognosis among the different mutation subtypes were analyzed. All the statistical analyses were performed by SPSS22.0.ResultsSomatic mutational analysis indicated that point mutations were the most frequently detected mutations followed by deletions & compound mutations and insertion and tandem duplication mutations in the stomach. Point mutations showed a low mitotic count and a high risk of recurrence, and deletions and compound mutations have a high mitotic count while insertions and tandem duplication mutations showed a low mitotic count with an intermediate recurrence risk. Point mutations and deletions frequently occurred in sequence region codons 550-560 of exon 11, while compound mutations, insertion, and tandem duplication were mainly detected in codons 557-559, 572-580, and 577-581, respectively. The multi-variation analysis demonstrated that tumor diameter and high recurrence risk groups had worse prognostic values. However, mutation types were not significant predictors of relapse-free survival (RFS) in GISTs. Survival analysis suggested no significant difference in RFS between the 557/558 deletion and the other deletions.ConclusionThis study suggested that mutations in exon 11 of the KIT gene were common with intermediate/high recurrence risk in GISTs patients. Tumor diameter ≥5 cm, and deletions mutations might predict a worse prognosis

Enhanced stability of M1 protein mediated by a phospho-resistant mutation promotes the replication of prevailing avian influenza virus in mammals

Avian influenza virus (AIV) can evolve multiple strategies to combat host antiviral defenses and establish efficient infectivity in mammals, including humans. H9N2 AIV and its reassortants (such as H5N6 and H7N9 viruses) pose an increasing threat to human health; however, the mechanisms involved in their increased virulence remain poorly understood. We previously reported that the M1 mutation T37A has become predominant among chicken H9N2 isolates in China. Here, we report that, since 2010, this mutation has also been found in the majority of human isolates of H9N2 AIV and its emerging reassortants. The T37A mutation of M1 protein enhances the replication of H9N2 AIVs in mice and in human cells. Interestingly, having A37 instead of T37 increases the M1 protein stability and resistance to proteasomal degradation. Moreover, T37 of the H9N2 M1 protein is phosphorylated by protein kinase G (PKG), and this phosphorylation induces the rapid degradation of M1 and reduces viral replication. Similar effects are also observed in the novel H5N6 virus. Additionally, ubiquitination at K187 contributes to M1-37T degradation and decreased replication of the virus harboring T37 in the M1 protein. The prevailing AIVs thereby evolve a phosphoresistant mutation in the M1 protein to avoid viral protein degradation by host factors, which is advantageous in terms of replication in mammalian hosts

Potential of Core-Collapse Supernova Neutrino Detection at JUNO

JUNO is an underground neutrino observatory under construction in Jiangmen, China. It uses 20kton liquid scintillator as target, which enables it to detect supernova burst neutrinos of a large statistics for the next galactic core-collapse supernova (CCSN) and also pre-supernova neutrinos from the nearby CCSN progenitors. All flavors of supernova burst neutrinos can be detected by JUNO via several interaction channels, including inverse beta decay, elastic scattering on electron and proton, interactions on C12 nuclei, etc. This retains the possibility for JUNO to reconstruct the energy spectra of supernova burst neutrinos of all flavors. The real time monitoring systems based on FPGA and DAQ are under development in JUNO, which allow prompt alert and trigger-less data acquisition of CCSN events. The alert performances of both monitoring systems have been thoroughly studied using simulations. Moreover, once a CCSN is tagged, the system can give fast characterizations, such as directionality and light curve

Detection of the Diffuse Supernova Neutrino Background with JUNO

As an underground multi-purpose neutrino detector with 20 kton liquid scintillator, Jiangmen Underground Neutrino Observatory (JUNO) is competitive with and complementary to the water-Cherenkov detectors on the search for the diffuse supernova neutrino background (DSNB). Typical supernova models predict 2-4 events per year within the optimal observation window in the JUNO detector. The dominant background is from the neutral-current (NC) interaction of atmospheric neutrinos with 12C nuclei, which surpasses the DSNB by more than one order of magnitude. We evaluated the systematic uncertainty of NC background from the spread of a variety of data-driven models and further developed a method to determine NC background within 15\% with {\it{in}} {\it{situ}} measurements after ten years of running. Besides, the NC-like backgrounds can be effectively suppressed by the intrinsic pulse-shape discrimination (PSD) capabilities of liquid scintillators. In this talk, I will present in detail the improvements on NC background uncertainty evaluation, PSD discriminator development, and finally, the potential of DSNB sensitivity in JUNO

Real-time Monitoring for the Next Core-Collapse Supernova in JUNO

Core-collapse supernova (CCSN) is one of the most energetic astrophysical events in the Universe. The early and prompt detection of neutrinos before (pre-SN) and during the SN burst is a unique opportunity to realize the multi-messenger observation of the CCSN events. In this work, we describe the monitoring concept and present the sensitivity of the system to the pre-SN and SN neutrinos at the Jiangmen Underground Neutrino Observatory (JUNO), which is a 20 kton liquid scintillator detector under construction in South China. The real-time monitoring system is designed with both the prompt monitors on the electronic board and online monitors at the data acquisition stage, in order to ensure both the alert speed and alert coverage of progenitor stars. By assuming a false alert rate of 1 per year, this monitoring system can be sensitive to the pre-SN neutrinos up to the distance of about 1.6 (0.9) kpc and SN neutrinos up to about 370 (360) kpc for a progenitor mass of 30$M_{\odot}$ for the case of normal (inverted) mass ordering. The pointing ability of the CCSN is evaluated by using the accumulated event anisotropy of the inverse beta decay interactions from pre-SN or SN neutrinos, which, along with the early alert, can play important roles for the followup multi-messenger observations of the next Galactic or nearby extragalactic CCSN.Comment: 24 pages, 9 figure

Dependence of Parking Pricing on Land Use and Time of Day

A key strategy of sustainable transportation, parking pricing can directly contribute to decreased greenhouse gas emissions and air pollution. This paper describes an optimal structure of parking rates in terms of parking locations and time of day. A two-level parking model based on game theory is established using parking survey data collected in Beijing in 2014. The model was estimated based on Stackelberg game and the Nash equilibrium. Using the two-level parking model, the optimal structure of parking rates for inside/outside business zones and during peak/off-peak hours was calculated. In addition, the relationship between the government (which represents the public benefit) and car users, as well as the relationships among car users in the parking system were investigated. The results indicate that equilibrium among all of the agents in the parking system can be obtained using the proposed parking rate structure. The findings provide a better understanding of parking behavior, and the two-level parking model presented in the paper can be used to determine the optimal parking rate to balance the temporal and spatial distribution of parking demand in urban areas. This research helps reduce car use and the parking-related cruising time and thus contributes to the reduction of carbon emissions and air pollution